lurk " ,lrl, I’ll! ‘ ‘ ,hlrvnl {I'IEIIP / 'Wi-Q' 3 -. ”’3'9‘15‘7‘913 it»? r13 3’“? f ‘ Michigan State ‘ Pniversity This is to certify that the Dif fer ent ial dissertation entitled susceptibility of immature rat testis to doxorubicin, procarbazine, cytosine arabinoside, cyclophosphamide and vincristine at four critical stages of testicular maturation presented by Romona Jean Haebler ‘ has been accepted towards fulfillment of the requirements for Doctor of Philosophy degreein Pathology A? 15421;“? LU I AéZL‘fLé/V’ Major professor R. W. Leader, DVM Date 11/5/86 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 bV1531_J RETURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from w your record. FINES will be charged if book is returned after the date stamped below. DIFFERENTIAL SUSCEPTIBILITY OF IMMATURE RAT TESTIS TO DOXORUBICIN, PROCARBAZINE, CYCLOPHOSPHAMIDE, VINCRISTINE AND CYTOSINE ARABINOSIDE AT FOUR CRITICAL STAGES OF TESTICULAR MATURATION By Romona Jean Haebler A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1986 5/3)“:— 94 4 3 ABSTRACT DIFFERENTIAL SUSCEPTIBILITY OF THE IMMATURE RAT TESTIS TO DOXORUBICIN, PROCARBAZINE, CYCLOPHOSPHAMIDE, VINCRISTINE AND CYTOSINE ARABINOSIDE AT FOUR CRITICAL STAGES OF DEVELOPMENT By Romona Jean Haebler The three major cell types of the testis, i.e., spermatogenic, Leydig and Sertoli cells, change markedly during postnatal maturation. Therefore, susceptibility to tissue toxicity may vary with age at the time of chemical exposure and the mechanism of the agent. To define tissue susceptibility of the developing testis, groups of rats were treated at four critical postnatal stages with one of five anticancer drugs (doxorubicin, procarbazine, cyclophosphamide, vincristine, or cytosine arabinoside) selected for their mechanisms of action. A single dose of one of these agents was administered to male Sprague Dawley rats at either 6, 16, 24 or 45 days of age. Observations were made at 3, 7 and 14 days following treatment (Phase I) and at 80 and 129 days of age independent of treatment age (after 5 weeks and 12 weeks of serial mating once the animals reached 45 days of age; Phase II). Semi-thin sections of glycol methacrylate or ePon embedded testicular tissues were evaluated. The following physio— logical indicators of tissue activity were measured: androgen-binding Protein for Sertoli cells, androgen-responsiveness for Leydig cellS, and Sperm head counts for germinal epithelium. Serial mating was used to determine the onset of reproductive capacity and to identify spec1- fic spermatogenic cell effects. Tissue susceptibility to tox1c1ty was related to the developmental stage of the rat and type 0f anticancer ROMONA JEAN HAEBLER drug administered. The morphological, biochemical and functional indi- cators were integrated in order to assess testicular toxicity in detail. Differential susceptibility of the immature testis to treatment with doxorubicin was clearly demonstrated. Animals treated at 6 days of age were most severely affected in all reproductive endpoints measured. Spermatogonia, stem cells, and Sertoli cells were clearly damaged. As a result, there was essentially no sperm production and the animals were sterile. Animals treated at 16 days of age had severe reproductive im- pairment and damage was likely due to disruption of the blood-testis barrier. Only minimal reproductive effects occurred in animals treated Q l at 24 and 45 days of age. Damage to the reproductive system produced by procarbazine and vincristine were also related to age at treatment, but effects were more severe as age at exposure increased. There were not clear reproductive target organ effects in animals treated with cyclo- phosphamide or cytosine arabinoside. To my parents, Eva and Ariel Haebler, who have given me strength and taught me the beauty of life. And to my dogs, Drena, Greloda, and Tuco. ii ACKNOWLEDGMENTS I wish to convey my deep appreciation to all who have helped and supported me during my years in the Department of Pathology. First I would like to express my gratitude to Dr. Robert Leader, my major pro- fessor, who has encouraged me every step of the way; and to Dow Chemi— cal and members of the Toxicology Research Division, who provided both monetary support and scientific guidance. I would also like to express my sincere appreciation to Dr. Robert Dixon, Dr. Rudolf Bechter, and Dr. Robert Ettlin who provided me with the opportunity to do my research at the National Institute of Environmental Health Sciences. Further, I would also like to recognize Dr. Jerry Hook and the members of my committee, Dr. Richard Dukelow, Dr. Stuart Sleight, Dr. Shirley Siew, Dr. David McConnell, and Dr. Richard Kociba for their encouragement and valuable guidance. I would like to recognize the ex— tremely valuable scientific training I received from Dr. Lonnie Russell, Southern Illinois University, in interpretation of testicular morpho- logy, and Dr. Tom Lobl, the Upjohn Company, in androgen binding protein assays. I would also like to thank the late Dr. Sergio Fabro and Dr. William Jaffurs of the Columbia Hospital for Women, Washington, D.C. for welcoming me into their reproductive toxicology research unit and providing me with full use of their pathology laboratory. Very special thanks go to Don Ehreth, Acting Assistant Administra— tor for Research and Development, Environmental Protection Agency, (my boss), for allowing me the time and giving me constant support in pre- paring my final dissertation. Also at EPA, my gratitude to Roxanne Settle for her patience, time and typing abilities in helping me prepare the manuscript. Finally, I would like to express my deepest appreciation to Dr. Lourens Zaneveld for his support, scientific advice, encouragement, pa- tience, occasional silence, and outstanding typing ability. RJH iv TABLE 9E CONTENTS CHAPTER I. Literature Review A. Normal Testicular Structure and Function 1. Mature Testis 2. Sertoli Cells 3. Blood - Testis Barrier 4. Germinal Epithelium 5. Leydig Cells 6. Endocrine Control of the Testis B. Maturation of Testis: Birth to Reproductive Capacity 1. Morphology at Birth 2. Sertoli Cells 3. Germinal Epithelium 4. Leydig Cells C. Chemical Injury of the Testis l. Mechanisms 2. Effect of Chemicals on the Immature Testis 3. Doxorubicin 4. Procarbazine S. Cyclophosphamide 6. Vincristine 7. Cytosine Arabinoside II. Rationale for Present Investigation PAGE 10 11 11 ll 14 IS l6 I6 20 22 23 m III IV. TABLE OF CONTENTS (Continued) Experimental Design A. B. C. Age at Exposure: Pilot Study I and II Test Chemicals Study Design Materials and Methods A. Animals and Housing Conditions B. Exposure: Route, Dose and Time C. Hematology D. Gross Examination and Tissue Preparation E. Testicular Tissue Preparation 1. Glutaraldehyde Perfusion 2. Bouin's Fixative F. Measurement of Spermatid Reserves and Sperm Head Counts G. Androgen Binding Protein Measurement H. Mating Studies I. Statistical Design Results A. Results of Pilot Studies B. Low Dose Exposure C. Hematology D. Controls 1 and 2 E. Doxorubicin 1. Clinical Signs vi M 25 25 26 28 35 35 36 38 39 39 41 41 42 42 42 44 44 51 51 51 56 56 CHAPTER TABLE OF CONTENTS (Continued) 2. Gross Necropsy 3. Body Weight; Testicular and Epididymal Weights 4. Morphologic Evaluation 5. Serial Mating Data 6. Functional and Biochemical Data Procarbazine 1. Clinical signs 2. Gross Necropsy 3. Body Weight; Testicular and Epididymal Weights 4. Morphologic Evaluation 5. Serial Mating Data 6. Functional and Biochemical Data Cyclophosphamide 1. Clinical Signs 2. Gross Necropsy 3. Body Weight; Testicular and Epididymal Weights 4. Morphologic Evaluation 5. Serial Mating Data 6. Functional and Biochemical Data Vincristine 1. Clinical Signs 2. Gross Necropsy 3. Body Weight; Testicular and Epididymal Weights M 56 57 61 78 79 90 9o 90 90 134 134 134 134 w VI. VII. TABLE OF CONTENTS gContinued) 4. Morphologic Evaluation 5. Serial Mating Data 6. Functional and Biochemical Data I. Cytosine Arabinoside 1. Clinical Signs 2. Gross Necropsy 3. Body Weight; Testicular and Epididymal Weights 4. Morphologic Evaluation 5. Serial Mating Data 6. Functional and Biochemical Data Discussion A. Doxorubicin B. Procarbazine C. Cyclophosphamide D. Vincristine E. Cytosine Arabinoside Bibliography Vita viii PAGE 135 141 143 143 143 143 149 149 149 150 159 159 166 171 175 178 180 193 — 1 LIST OF TABLES tan Bags 1. Test Chemicals and Mechanisms of Action 27 2, Phase II: Time Schedule and Animal Ages 33 3. Indicators of Toxicity 34 4. Test Chemicals and Dose 37 5. Four Critical Stages of Differentiation 49 6. Fertility Data in Control 1 Animals 53 7. Fertility Data in Control 2 Animals 55 8. Effect of Doxorubicin (3 mg/kg) on Body Weight 58 9. Effect of Doxorubicin (3 mg/kg) on Testicular Weight 59 10. Effect of Doxorubicin (3 mg/kg) On Epididymal Weight 60 ll. Fertility Data in Doxorubicin Treated Animals 81 at 6 Days of Age (3 mg/kg) 12. Fertility Data in Doxorubicin Treated Animals 82 at 16 Days of Age (3 mg/kg) 13. Fertility Data in Doxorubicin Treated Animals 83 at 24 Days of Age (3 mg/kg) 14. Fertility Data in Doxorubicin Treated Animals 84 at 45 Days of Age (3 mg/kg) 15. Effect of Doxorubicin (3 mg/kg) on Spermatid 85 Reserves in Testis, Sperm Head Counts in Epididymis and on Androgen Binding Protein (ABP) Animals treated at 6, 16, 24 or 45 Days of Age and Sacrificed 5 or 12 Weeks After Start of Serial Mating. 16. Effect of Procarbazine (200 mg/kg) on Body Weight 92 17. Effect of Procarbazine (200 mg/kg) on Testicular 93 Weight 18. Effect of Procarbazine (200 mg/kg) on Epididymal 94 Weight ix — ‘ LIST OF TABLES Continued) Table Egg; 19. Fertility Data in Procarbazine Treated Animals 113 at 6 Days of Age (200 mg/kg) 20. Fertility Data in Procarbazine Treated Animals 114 at 16 Days of Age (200 mg/kg) 21. Fertility Data in Procarbazine Treated Animals 115 at 24 Days of Age (200 mg/kg) 22. Fertility Data in Procarbazine Treated Animals 116 at 45 Days of Age (200 mg/kg) 23. Effect of Procarbazine (200 mg/kg) on Spermatid 117 Reserves in Testis, Sperm Head Counts in Epididymis and on Androgen Binding Protein (ABP). Animals Treated at 6, 16, 24 or 45 Days of Age and Sacri- ficed 5 or 12 Weeks After the Start of Serial Mating. 24. Effect of Cyclophosphamide (80 mg/kg) on Body 124 Weight 25. Effect of Cyclophosphamide (80 mg/kg) on 125 Testicular Weight 26. Effect of Cyclophosphamide (80 mg/kg) on 126 Epididymal Weight 27. Fertility Data in Cyclophosphamide Treated Animals 129 at 6 Days of Age (80 mg/kg) 28. Fertility Data in Cyclophosphamide Treated Animals 130 at 16 Days of Age (80 mg/kg) 29. Fertility Data in Cyclophosphamide Treated Animals 131 at 24 Days of age (80 mg/kg) 30. Fertility Data in Cyclophosphamide Treated Animals 132 at 45 Days of Age (80 mg/kg) 31. Effect of Cyclophosphamide (80 mg/kg) on Spermatid 133 Reserves in Testis, Sperm Head Counts in Epididymis and on Androgen Binding Protein (ABP). Animals Treated at 6, 16, 24 or 45 Days of Age and Sacri- ficed After 5 or 12 Weeks of Serial Mating. 32. Effect of Vincristine (0.6 mg/kg) on Body Weight 136 F< Fe Am An h LIST OF TABLES Continued Table Egg; 33. Effect of Vincristine (0.6 mg/kg) on Testicular 137 Weight 34. Effect of Vincristine (0.6 mg/kg) on Epididymal 138 Weight 35. Fertility Data in Vincristine Treated Animals 144 at 6 Days of Age (0.6 mg/kg) 36. Fertility Data in Vincristine Treated Animals 145 at 16 Days of Age (0.6 mg/kg) 37. Fertility Data in Vincristine Treated Animals 146 at 24 Days of Age (0.6 mg/kg) 38. Fertility Data in Vincristine Treated Animals 147 at 45 Days of Age (0.6 mg/kg) 39. Effect of Vincristine (0.6 mg/kg) on Spermatid 148 Reserves in Testis, Sperm Head Counts in Epididymis and on Androgen Binding Protein (ABP). Animals Treated at 6, 16, 24 or 45 Days of Age and Sacrificed at 5 or 12 Weeks After Serial Mating. 40. Effect of Cytosine Arabinoside (600 mg/kg) on 151 Body Weight 41. Effect of Cytosine Arabinoside (600 mg/kg) on 152 Testicular Weight 42. Effect of Cytosine Arabinoside (600 mg/kg) on 153 Epididymal Weight 43. Fertility Data in Cytosine Arabinoside Treated 154 Animals at 6 Days of Age (600 mg/kg) 44. Fertility Data in Cytosine Arabinoside Treated 155 Animals at 16 Days of Age (600 mg/kg) 45. Fertility Data in Cytosine Arabinoside Treated 156 Animals at 24 Days of Age (600 mg/kg) 45. Fertility Data in Cytosine Arabinoside Treated 157 Animals at 45 Days of Age (600 mg/kg) xi TABLE OF CONTENTS (Continued Table Page 47. Effect of Cytosine Arabinoside (600 mg/kg) on 158 Spermatid Reserves in Testis, Sperm Head Counts in Epididymis and on Androgen Binding Protein (ABP). Animals Treated at 6, 16, 24 or 45 Days of Age and Sacrificed After 5 or 12 Weeks of Serial Mating. Figgre Ley‘ Test ref] A fe (Mag Test comp mato acte Rela Cell Test Days bule Sia. size 180x Test 6 Da nife hypo bula CIEa #- w raw Base 1. Phase I 29 2. Phase II 30 3. Diagramatic Representation of the Technique for 40 Perfusing Testes with Fixative. 4. Testicular Tissue of 6 Day Old Rat. Solid seminiferous 45 cords contain only Sertoli cells, Spermatogonia, and gonocytes, the primordial germ cells. Leydig cells, located in clumps in the interstitium, have fetal char acteristics evidenced by abundant, pale, billowy cyto plasm (Magnification: 360x). 5. Testicular Tissue of 16 Day Old Rat. Spermatogonia have 45 divided mitotically to produce primary spermatocytes and Leydig cells are rare (Magnification: 360x). 6. Testicular Tissue of 24 Day Old Rat. Spermatids exist, 47 reflecting completion of the second meiotic division. A few Leydig cells have differentiated to adult form (Magnification: 360x). 7. Testicular Tissue of 45 Day Old Rat. Spermatogenesis is 47 complete with the production and release of mature sper- matozoa; Sertoli cells and Leydig cells have adult char- acteristics (Magnification: 360x). 8. Relative Proliferative Activity of Sertoli Cells, Leydig 50 Cells and Germinal Epithelium During Sexual Maturation. 9. Testicular Tissue of Rat Treated with Doxorubicin at 6 64 Days of Age and Sacrificed at 80 Days of Age. All tu- bules were atrophic with severe germinal cell hypopla— sia. Tubular lumens had not formed and Leydig cell size and cellular density were increased (Magnification: 180x). 10. Testicular Tissue of Rat Treated with Doxorubicin at 66 6 Days of Age and Sacrificed at 129 Days of Age. Semi- niferous tubules were atrophic, germinal epithelium hypoplastic, Sertoli cell cytoplasm occluded some tu- bular lumens and Leydig cell size and density was in- creased (Magnification: 180x). xiii , . blag 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. LIST OF FIGURES Continued Testicular Tissue of Control Rat Sacrificed at 129 Days of Age. A Stage VIII tubule from a normal animal illustrates normal tubular size, germinal cell population, tubular lumen formation and Leydig cell size and density (Magnification: 180x). Testicular Tissue of Rat Treated with Doxorubicin at 16 Days of Age and Sacrificed at 80 Days of Age. Germinal cell hypoplasia was severe with spermatogenic arrest in most tubules; early sper- matids were frequently the most mature cell type (Magnification: 180x). Testicular Tissue of Rat Treated with Doxorubicin at 16 Days of Age and Sacrificed at 80 Days of Age. Architectural disorganization was extensive, many germ cells were degenerative or necrotic (Magnifica— tion: 180x). Testicular Tissue of Rat Treated with Doxorubicin at 16 Days of Age and Sacrificed at 129 Days of Age. Architectural disruption and spermatogenic arrest of the seminiferous epithelium was evidenced by almost total absence of the spermatocyte and early sperma tid layers and no late Spermatids (Magnification: 180x). Testicular Tissue of Rat Treated with Doxorubicin at 24 Days of Age and Sacrificed at 129 Days of Age. Testicular morphology was normal (Magnification: 180x). Testicular Tissue of Rat Treated with Doxorubicin at 45 Days of Age and Sacrificed at 129 Days of Age. Testicular morphology was normal (Magnification: 180x). Fertility and Sperm Counts of Animals Treated with Doxorubicin. Total Implants of Animals Treated with Doxorubicin. Live Implants and Resorptions of Animals Treated with Doxorubicin. Androgen Binding Protein of Animals Treated with Doxorubicin. 69 69 73 76 76 86 87 88 89 Fig_u e LIST OF FIGURES Continued Figure Page 21. Testicular Tissue of Rat Trated with Procarbazine 96 at 6 Days of Age and Sacrificed at 80 Days of Age. Severe architectural disruption was present with loss of most of the spermatogonial and spermatocyte layers. Sertoli cell cytoplasm was vacuolated and filled with debris (Magnification: 360x). 22. Testicular Tissue of Rat Treated with Procarbazine 98 at 6 Days of Age and Sacrificed at 129 Days of Age. Testicular morphology was similar to that of control animals (Figure 23), indicating recovery of the toxic effects of procarbazine on the germinal epithelium (Magnification: 180x). 23. Testicular Tissue of Control Rat Sacrificed at 129 98 Days of Age. Testicular tissue from a control ani— mal with normal morphology (Magnification: 180x). 24. Testicular Tissue of Rat Treated with Procarbazine 101 at 16 Days of Age and Sacrificed at 80 Days of Age. Severe germinal hypoplasia was evidenced by the presence of only a few spermatogonia and spermato— cytes. These remaining germinal cells were degenerative and Sertoli cells were structurally abnormal (Magnification: 180x). 25. Testicular Tissue of Rat Treated with Procarbazine 102 at 16 Days of Age and Sacrificed at 129 Days of Age. Testicular tissue was morphologically nor- mal (Magnification: 180x). 26. Testicular Tissue of Rat Treated with Procarbazine 102 at 16 Days of Age. Most tubules were atrophic, with severe germinal cell hypoplasia and sperma- togenic arrest (Magnification 180x). 27. Testicular Tissue of Rat Treated with Procarbazine 106 at 24 Days of Age and Sacrificed at 129 Days of Age. Though most tubules were morphologically nor— mal, a few tubules were severely damaged with al- most total absence of germinal epithelial cells. Early Spermatids were most mature cell type pre- sent (Magnification: 180x). 28. Testicular Tissue of Rat Treated with Procarbazine 106 at 45 Days and Sacrificed at 80 Days of Age. Se- vere germinal hypoplasia involved primarily sper- matocytes and early Spermatids with a complete ab- sence of late Spermatids (Magnification: 360x). XV _T_ab_le 29. 30. 31. 32. 33. 34. LIST OF FIGURES Continued Testicular tissue of Rat Treated with Procarbazine at 45 Days of Age and Sacrificed at 129 Days of Age. Many tubules remained severely damaged, often with essentially a 'Sertoli—cell' only pattern with oc- casional spermatogonia. These tubules were atro- phic with spermatogenic arrest (Magnification: 180x). Testicular Tissue of Rat Treated with Procarbazine at 45 Days of Age and Sacrificed at 129 Days of Age. Many seminiferous tubules have marked de- pletion of Spermatid cell layer. Remaining sper- matocytes were often swollen, degenerative and lo- cated near the adluminal border (Magnification: 360x). Fertility and Sperm Counts of Animals Treated with Procarbazine Total Implants of Animals Treated with Procar- bazine. Live Implants and Resorptions of Animals Treated with Procarbazine Androgen Binding Protein of Animals Treated with Procarbazine 108 118 119 120 121 I. LITERATURE REVIEW A. Normal Testicular Structure and Function LW The testis has two primary functions: the production of spermatozoa and the secretion of androgens. Formation of these products is localized within two separate compartments: the seminiferous tubules produce sperm; and Leydig cells, located in the interstitial tissue, secrete androgens. Although these pro- ducts are produced by two distinct populations of cells, the testis must be considered as one functional unit. The testis of the rat is physically located outside the body cavity in a sac like structure, the scrotum. The parenchyma of the testis con~ sists of interstitial tissue and seminiferous tubules. The in- terstitium, along with Leydig cells, contains blood vessels, lym— phatics, nerves and connective tissue. The seminiferous tubules contain two types of epithelial cells, Sertoli cells and germi— nal epithelial cells, as well as a surrounding tubular wall con- sisting of four distinct layers (Steinberger and Steinberger, 1975). Tubular lumens contain newly-released spermatozoa and the seminiferous fluid. 2. Sertoli Cells Sertoli cells are tall columnar cells evenly distributed among the germinal epithelium of the seminiferous tubules. Nuclei are generally basilar. The cytoplasm extends from the basement membrane to the tubular lumen and is highly variable in shape (Dym, 1973). In the adult, this population of cells is 'iq'zi: f0] the men the: reSL mato uli 1 reac 1981 ture and. Sert Ster Seer. flui Prot MulL them intr. with —7—" 2 stable with no evidence of proliferation (Clermont and Perey, 1957; Steinberger and Steinberger, 1971; and Ritzen g; $1., 1981). The Sertoli cell has many functions. The H-Y antigen on the Sertoli cell surface, in particular, controls the differen- tiation of the gonad during embryological development (Wachtel _£ _1., 1977). The blood-testis barrier is maintained through formation of inter-Sertoli cell occluding junctions. Therefore, the seminiferous epithelium is partitioned into a basal compart- ment containing spermatogonia and early spermatocytes and an ad- luminal compartment containing the more developed germinal epi— % thelium (Dym and Fawcett, 1970; Fawcett g; g1., 1970). As a result, Sertoli cells control the environment surrounding sper- matocytes and Spermatids since all nutrients and hormonal stim- uli must first pass through Sertoli cell cytoplasm before reaching cells within the adluminal compartment (Ritzen g; gl., 1981). Sertoli cells play an active role in the release of ma- ture spermatozoa and phagocytosis of sloughed residual bodies and any abnormal germ cells (Carr 2; al., 1968; Fawcett, 1975). Sertoli cells are capable of some, though probably limited, steroid metabolism (Fawcett, 1975; Ritzen, 1981). These cells secrete as well as maintain the ionic gradient of seminiferous fluid (Waites, 1977; Ritzen g; g1., 1981) and secrete several proteins including androgen binding protein (ABP), inhibin, and Mullerian inhibiting hormone (MIH). Although the functions of these proteins are not fully understood, ABP is thought to be an intracellular carrier of testosterone and dihydrotestosterone within the Sertoli cell, or to store androgenic hormones within mair ment (Set by i. Spec blom totai (Set< barrj (Okun Plete lar v inter nent and F 3 the seminiferous tubules and epididymis, and to carry testos- terone from the testis to the epididymis (Fritz g; g1., 1976; Hagenas et 1., 1975; Hansson et __ __ 1., 1973; Sanborn g; g1., 1975; Ritzen g; 1., 1981). Inhibin is thought to inhibit the release of follicle stimulating hormone (FSH) by the anterior pituitary (Steinberger and Steinberger, 1976; 1977); MIH sup- presses the formation of the female internal genitalia during fetal development (Jost g; g1., 1974). 3. Blood-Testis Barrier The function of the blood-testis barrier is two-fold: it maintains a special fluid environment in the adluminal compart- ment of the tubules which is favorable to spermatogenesis (Setchell, 1970) and protects the testis from autoimmune disease by isolation of spermatozoal antigen (Johnson, 1972). A special environment can be created due to the ability of the blood-testis barrier to determine the rate of entry into or the total exclusion of substances from the seminiferous tubules (Setchell and Waites, 1975). Transport across the blood-testis barrier depends primarily on molecular size and lipid solubility (Okumura g; _1., 1975; Dixon and Lee, 1980). Although an incom- plete system of tight junctions between myoid cells in the tubu- lar wall of the'rat creates a partial permeability barrier, inter-Sertoli cell tight junctions constitute the primary compo- nent of the blood-testis barrier (Fawcett g3 g1., 1970; and Dym and Fawcett, 1970). As the germinal epithelium develops, it ‘L 4—4 tub prn and base mito meiot the c tiate Chang; ment c the ma for pa: 1975). defined Cyclical clermOnt —:———H ‘ A. must pass from the basal to the adluminal compartment while maintaining the blood—testis barrier. Russell (1978) has postulated that a permeability barrier forms below leptotene spermatocytes prior to the dissolution of the tight junctions above, creating an intermediate compartment which functions as a transit chamber. 4. Germinal Epithelium Spermatogenesis is the process of development and matura- tion of the germ cells in the epithelium of the seminiferous tubules. This is an active, orderly process and involves four primary cell types: spermatogonia, spermatocytes, Spermatids and spermatozoa. The spermatogonia, which lie adjacent to the basement membrane, are the most primitive cell type and divide mitotically to form spermatocytes. These cells then undergo two meiotic divisions to form the haploid Spermatids. Then, through the complicated process of Spermiogenesis, Spermatids differen- tiate into spermatozoa. Spermiogenesis involves structural change of the nucleus, formation of new organelles and develop— ment of the motility apparatus. Spermatids become spermatozoa, the male gametes, which are then released into the tubular lumen for passage to the epididymis (Steinberger and Steinberger, 1975). The tubular germinal epithelium is organized in well- defined cellular associations which follow one another in a cyclical pattern called the spermatogenic cycle (Leblond and Clermont, 1952). This occurs because several spermatogonia in Spe‘ Vat. ten< Spe- mar: Spe: tic undl Spe: latt — r 5 a local area of the tubule divide spontaneously and the cells that are produced develop according to a similar timetable. Thus, each tubular cross-section contains four or five types of cells at predictable stages of maturation. The rat has 14 dif- fering cellular associations (Perey g; g1., 1961). Since these stages have different morphologic characteristics (Clermont, 1962; Clermont, 1963; Clermont and Huckins, 1961), stages of the cycle can be identified histologically. The duration of spermatogenesis is the time from first commitment of spermato- gonia to release of mature spermatozoa into the tubular lumen. In the rat, type A spermatogonia divide mitotically to produce intermediate and type B spermatogonia. Type B cells are the immediate mitotic precursors of the primary spermatocytes, which undergo the first meiotic division. There are several types of primary spermatocytes which can be differentiated based on their nuclear chromatin characteristics. The first primary spermatocytes are termed resting or preleptotene. When acti— vated, these resting cells sequentially form leptotene, zygo- tene, pachytene, and diplotene spermatocytes, which finally form two secondary spermatocytes. During this process of primary spermatocyte development, gene shuffling occurs. Diplotene pri- mary spermatocytes then divide meiotically to produce secondary spermatocytes. Secondary spermatocytes undergo the second meio- tic division to form the haploid Spermatid. The Spermatids then undergo the complex process of Spermiogenesis to form the mature spermatozoa. During Spermiogenesis, many types of early and late spermatids can be identified (Perey gt gl., 1961). As the ad‘ Sill Val aut P0P ing tio 196 min Cyt Spe all bee tel- —7——" 6 germ cells develop through these different stages of the sper- matogenic process, different cell types undergo varying bio- chemical processes. The replication of spermatogonia neces- sitates intensive DNA, RNA, and protein synthesis. Early pri- mary spermatocytes preparing for meiosis undergo considerable DNA synthesis. Protein synthesis is high in preleptotene and pachytene primary spermatocytes and elongated Spermatids. RNA synthesis is particularly high in late spermatocytes, secondary spermatocytes, and early Spermatids (Ettlin and Dixon, 1985). Since spermatogonia divide throughout the lifetime of an adult male, there must be a mechanism for replenishing their supply. Steinberger and Steinberger (1975) reviewed the many variations of stem cell renewal postulated by a number of authors. In addition to renewal of spermatogonia, there is a population of reserve stem cells that become active only follow- ing injury to the germinal epithelium, at which time they func- tion to repopulate the tubules (Clermont and Bustos—Obregon, 1968). The inter—Sertoli cell tight junctions separate the ger- minal cells into two compartments with the leptotene spermato— cyte being the cell type that crosses this barrier. Since these spermatocytes are the last germ cells capable of DNA synthesis, all DNA synthesis involved in production of the male gamete has been completed prior to entry into the adluminal compartment. 5. Leydig Cells Prominent clusters of Leydig cells are located in the in- terstitial tissue of the testis. The only known physiological _ 4____J due the 1973 back hypot testi integ tary l resPOr the hy gonado ing h01 diate S ‘ . 7 function of these cells is the production of steroids, particu- larly androgens. Testosterone, the primary androgen with biolo- gical activity, regulates the development and function of the male reproductive tract and external sex characteristics (Christensen, 1975). Testosterone, together with follicle sti- mulating hormone (FSH), stimulates spermatogenesis at puberty but it appears that testosterone alone is sufficient to maintain germ cell development and differentiation. Luteinizing hormone (LH) produced by the anterior pituitary regulates testosterone synthesis by the Leydig cells and can a1— so produce an increase in total Leydig cell numbers, most likely due to stimulation of differentiation from stem cells rather than from mitotic divisions of mature Leydig cells (Christensen, 1975; Christensen and Peacock, 1980). Testosterone has a feed— back inhibitory effect on LH synthesis and secretion. 6. Endocrine Control of Testicular Function A complex interaction among the central nervous system, the hypothalamus and the pituitary gland (adenohypophysis) controls testicular function. The hypothalamus is primarily a center for integration of input from the CNS (light, olfaction), the pitui- tary gland (gonadotropins) and the testis (testosterone). In response, gonadotropin releasing hormone (GnRH) is released by the hypothalamus and, in turn, stimulates the secretion of the gonadotropins: luteinizing hormone (LH) and follicle stimulat- ing hormone (FSH) from the pituitary. These gonadotropins me- diate spermatogenesis and androgen biosynthesis. The testis has ing feel ca1< quil its Ste: tioi men; 16W 0811: (Od. Sit , . 8 negative feedback control over the hypothalamo-pituitary axis (Gay and Dever, 1971; Swerdloff and Walsh, 1973). Testosterone inhibits LH synthesis and secretion, and may partially control FSH. Inhibin, a substance believed to be produced by the Sertoli cells, may contribute to the control of FSH (Baker gg al., 1976; Braunstein and Swerdloff, 1977; Franchimont g; al., 1977). LH acts on the Leydig cell to stimulate steroidogenesis; LH action on the seminiferous tubule is indirect through the influence of testosterone. FSH acts primarily on the semini- ferous tubule and may possibly act to enhance LH stimulation of testosterone secretion (Odell gg al., 1973). At the tubule, FSH binds to Sertoli cells and stimulates protein synthesis includ— ing the production of ABP (Means g; al., 1976). FSH also af- fects the Sertoli cell cytoskeleton and controls intracellular calcium (Means g; _1., 1978). At puberty, spermatogenesis re- quires both FSH and testosterone. Testosterone is necessary for its initiation and FSH for its completion (Steinberger, 1971; Steinberger g; _l., 1973). Subsequently, testosterone stimula- tion alone appears to be sufficient to maintain spermatogenesis. Puberty, or sexual maturation, involves both the develop- ment of the physical characteristics of the adult male as well as the capacity to reproduce. Both increased gonadotropin levels as well as increased gonadal sensitivity to gonadotropins cause an elevation of testosterone secretion during puberty (Odell and Swerdloff, 1976). FSH rises prior to LH and may sen- sitize the Leydig cell to LH stimulation. ing sux 1ag proI mari are and] tatic Pr0mi cells during Steinb, the for Ween d Critic.1 which i: bular 1t B. Maturation of Immature Testis l. Morphology at Birth The testis of the fetal rat differentiates on day 14 of gestation (Franchi and Mandl, 1964). Solid sex cords develop which contain gonocytes, the primordial germ cells, and support- ing cells, the precursors of Sertoli cells. Interstitial tissue surrounding the sex cords contains Leydig cells, as well as col- lagen fibers, blood vessels, lymphatics, and nerves. Gonocytes cease mitotic activity at 18 days of gestation and remain in prolonged interphase until four to five days of age. At birth, gonocytes are large round cells located pri- marily in the central area of the sex cords. Supporting cells are smaller and located along the basement membrane (Clermont and Perey, 1957). Leydig cells differentiate on day 17 of ges- tation (Lording and deKretser, 1972) and at birth are found in prominent clusters throughout the interstitial areas. 2. Sertoli Cells From birth until approximately 15 days of age, Sertoli cells of the rat undergo the only period of mitotic division during the lifetime of the animal (Clermont and Perey, 1957; Steinberger and Steinberger, 1971). Structural maturation and the formation of inter—Sertoli cell tight junctions occur be- tween days 16 through 19. These junctional complexes play a critical role in the formation of the blood-testis barrier, which is first detectable during this same time interval. A tu- bular lumen in the seminiferous cords appears concurrently with germ only birt sion Cells Sperm divid Primal Cycle daY-01 to the Cytes. form Se tion of divisio] mi°gene5 SPErm by — r 10 the development of the tight junctions (Vitale g; $1,. 1973), and is thought to be due to the initiation of fluid production by the Sertoli cells (Ritzen g3 g1., 1981). Sertoli cells continue to mature until about 45 days of age when they attain all the structural and functional characteris- tics of typical Sertoli cells found in the mature testis (Cler- mont and Perey, 1957). 3. Germinal Epithelium Clermont and Perey (1957), Franchi and Mandl (1964), and Sapsford (1962) characterized the progressive development of the germinal epithelium of the rat in detail. Gonocytes are the only type of germinal epithelial cell in the sex cords from birth until about four days of age when they begin mitotic divi- sion to form Type A spermatogonia. By six days of age, Type A cells are common and occasional Type B and intermediate type spermatogonia can be identified, a few of which may be actively dividing. At nine days of age, gonocytes disappear and a few primary spermatocytes develop. Four successive stages of the cycle of the seminiferous epithelium can be identified in 18— day—old animals, with the most mature cells having progressed to the pachytene stage of meiotic prophase in primary spermato- cytes. By 26 days, primary spermatocytes complete meiosis to form secondary spermatocytes and a few of these undergo forma- tion of Spermatids, thus having completed the second meiotic division. Following the first appearance of Spermatids, sper— miogenesis progresses with the formation and release of mature sperm by 45 days of age. Cell: (Chrj dUrin rapid 4. C. l. 11 usage—11s Leydig cells of the rat undergo biphasic development: a fetal phase lasting from the 17th day of gestation to the second post-natal week, and an adult phase lasting from the third post- natal week onward. Since the time interval between the two gen— erations is very short, there may be some overlap between the populations (Lording and deKretser, 1972). Fetal Leydig cells are functional and produce peak testos- terone levels at 18.5 days of gestation, the critical time for Wolffian duct development. Although the cell numbers and tes- tosterone levels decrease thereafter, the remaining cells main- tain a low but measurable level of testosterone in the plasma (Resko g; _l., 1968). Leydig cell numbers begin to increase again at about 20 days after birth and reach maximal numbers at 50 days (Knorr g; _1., 1970). Reappearance of the Leydig cells is due both to differentiation from fibroblast-like "stem" cells and also by division of some mature Leydig cells (Christensen, 1975). Testosterone levels remain relatively low during the time of increasing Leydig cell numbers, then rise rapidly to adult levels by 60 days of age (Knorr g; gl., 1970). Chemical Injury of the Testis Mechanisms In spite of the blood-testis barrier, the complexity of testicular structure and function make this organ susceptible to damage by a number of mechanisms. Hyperthermia (produced by pyrexia), local inflammation, cryptorchidism or environmental dam pox: Free of h John Cale] tein, tion (Leat Saase Cells Cause 1962; Steir ti0n. of ti Leydj 12 factors can cause aspermia and increased mutation rates due to altered blood or lymph flow, gas tension, testicular fluids, specific metabolic pathways and enzyme systems (VanDemark and Free, 1970). Intermediate-type germinal epithelial cells, es- pecially pachytene spermatocytes and young Spermatids, are most susceptible (Chowdbury and Steinberger, 1964). Exposure to temperatures of less than 0°C for extended periods of time damages the testis due to reduced blood flow resulting in hy- poxia as well as decreased androgen production (VanDemark and Free, 1970). High altitudes alter spermatogenesis as a result of hypoxia and reduced atmospheric pressure (Cockett and Johnson, 1970). Improper nutrition caused by starvation, caloric restriction, insufficient quality and quantity of pro- tein, vitamin or mineral deficiencies can alter testicular func- tion due primarily to disruption of the endocrine system (Leathem, 1970). Ischemia resulting from organic vascular di- sease, vasoactive agents or cadmium is especially damaging to cells just prior to or during mitotic activity, possibly be- cause of interference with DNA synthesis (Boccabella g3 g1., 1962; Lee and Dixon, 1973; Aberg and Wahlstrom, 1972; Steinberger and Dixon, 1959). The testis is especially susceptible to injury by radia- tion. Though most of the damage involves the genetic material of the germinal epithelium, some disruption of Sertoli cell and Leydig cell function may occur independent of nucleic acid changes. Intermediate and Type B spermatogonia are most div tes. ing may Some tis 1 keton Burek lar f ferin germll iCals have 1 wallei ments damage by Whi know. ——7 __.._......... l3 sensitive but the effect is reversible unless exposure is suffi— cient to damage stem cells. The immature testis also is sensi- tive to radiation (Reviewed by Ellis, 1970). Neurogenic damage to the testis can result from central nervous system disorders which interrupt hypothalamic or pitui- tary function or by injury to peripheral nerves. Neuropathy of these local nerves causes degeneration of the most rapidly dividing germinal cells and vasodilation of the vessels of the testis and epididymis with the majority of damage likely result- ing from hypoxia secondary to vascular stagnation. Recovery may result from reinnervation of blood vessels (Hodson, 1970). Some chemical agents suspected of indirectly damaging the tes- tis following injury to peripheral nerves are methyl-N-butyl ketone, N—hexane and acrylamide (Krasavage g; g1., 1980; Burek pp g1., 1979). Chemicals may also cause a more direct change in testicu- lar function either by altering hormone stimulation or by inter- fering with the function of the Leydig cells, Sertoli cells or germinal epithelium. Current and comprehensive reviews of chem- icals which cause toxic effects in the male reproductive system have recently been published by Ettlin and Dixon (1985) and Waller pp Q1. (1985). The scientific literature clearly docu— ments that exposure to many chemical toxicants can directly damage the various cell types of the testis, but the mechanisms by which many of these agents cause this injury are often un- known. fur W8) 8 t anm val. addi on t tiOna feren Pendh been c may be ing te F1 DiXOHy fi 14 Cell death or dysfunction may be due to disruption of one or more of the vital processes of a given cell type, including DNA or RNA synthesis, protein synthesis and secretion, enzyme action, steroidogenesis or microtubule formation and integrity. Susceptibility of a particular cell type to a given agent is determined by whether or not those processes disrupted by that toxicant are necessary for its survival and function. Of course, the number of agents capable of disrupting testicular function is many times greater than the number of vital path- ways that can be interrupted (Russell gp _1., 1981). Disruption of these vital processes may be due either to direct effects of a toxicant on a given cell type or indirectly through damage to another cell type on which the first may depend for its survi- val. For example, a toxicant which injures Sertoli cells will additionally damage the germinal epithelial cells which depend on these Sertoli cells for their support (Russell g; g1., 1981). 2. Effect of Chemicals on the Immature Testis From birth to puberty and maturity, the structure and func- tional activity of the germ cells change and there may be dif- ferential susceptibility of the testis to a given toxicant de— pending on age of exposure. As yet, little definitive work has been done to evaluate the possibility that the immature testis may be more (or less) susceptible to toxicants capable of caus- ing testicular injury. From the reviews by Waller g; _1., 1985 and Ettlin and Dixon, 1985, it is clear that there is an obvious deficiency in uatl wide. 566m: tumor inter RNA In. — ‘ 15 the information available concerning the susceptibility of the immature testis to these chemicals. Of the many compounds re- viewed, only nine had been tested for their effect on the imma— ture testis: cadmium (Cd), ethylene dibromide (EDB), monosodium glutamate (MSG), nitrosoureas, procarbazine, cyclophosphamide, vincristine, and cytosine arabinoside. Even these studies were limited and primarily involved human clinical observations in the case of the last four anticancer agents. From a clinical standpoint, testicular injury of children caused by anticancer agents is of particular interest because such therapy may result in remission of the disease but cause damage to the reproductive system. Very little is known about the specific injury to the immature testis that is caused by such agents. It was the objective of this research to study the effects of these agents on the immature testis using the rat as the animal model. The following is a brief overview of our pre- sent knowledge regarding the anticancer agents that were eval- uated in the present study, primarily emphasizing their known effects on the immature testis. 3. Doxorubicin (Adriamycinz Doxorubicin (adriamycin), an anthracycline antibiotic, is widely used for a variety of leukemias and solid tumors, and seems to be particularly helpful in the treatment of malignant tumors in children (Bonadonna pp g1., 1969). The drug is an intercalating agent, and induces functional changes in DNA and RNA metabolism (DiMarco et al., 1975). Inhibition of DNA and the (Spi Vers (Dec fECt: matul germi of pr invol. —: .—, 16 RNA polymerase have been reported (Zunino g; g1., 1975; Goodman pp a1., 1977). Mutagenic activities 1p y1§£g and lg 2129 have been reported by McCann g; _1., (1975); Meier and Schmid (1976), Au g5 Q1. (1981), and Au and Hsu (1980). Parvinen and Parvinen (1978) showed that after doxoru- bicin treatment, DNA and RNA synthesis was inhibited in pre- mitotic and premeiotic stages of spermatogenesis in rats. Doxo— rubicin also killed stem cells and differentiating spermatogonia in mice (Lu and Meistrich, 1979), eventually leading to testicu— lar atrophy (Au and Hsu, 1980). No studies appear to have been performed to evaluate the effect of doxorubicin on the immature testis. In clinical use for humans, the most commonly used dosage is 60-75 mg/m2 as a single intravenous dose administered at 21 day intervals. Procarbazine Procarbazine is a potent anticancer drug frequently used in the treatment of Hodgkin's disease and malignant lymphomas (Spivack, 1974). Procarbazine is known to have a variety of ad- verse biological effects. Mutagenic (Wild, 1978), carcinogenic 1., 1974; Sieber g; g1., 1978) and teratogenic ef- (Deckers g; fects (Chaube and Murphy, 1969) have been reported. Testes of mature monkeys treated with procarbazine prior to puberty had germinal aplasia (Sieber g; g1., 1978). The mode of action of procarbazine, however, is not fully understood but seems to involve disturbance of DNA, RNA and protein synthesis (Lee and (11 t1 the tee Cyc Che dis 17 Dixon, 1978). Procarbazine is an alkylating agent (Weinkam and Shiba, 1978) and may act by a variety of mechanisms such as formation of hydrogen peroxide and other breakdown products, followad by degradation of DNA (Berneis g5 g1., 1963), amino- methylation of cellular macromolecules (Weitzel g; g1., 1964) or transmethylation, especially onto the guanine of transfer RNA (Kreis, 1971). Procarbazine also inhibits DNA polymerase, DNA dependent RNA polymerase and the cellular uptake of nucleosides; thus, the synthesis of DNA, RNA and proteins is decreased (Weitzel g; g1., 1968). There have been a few reports on mor- phological effects of procarbazine using both light microscopy (Hilscher and Reschelt, 1968; Heese, 1972; Meyhofer, 1973) and electron microscopy (Parvinen, 1979; Russell pp _1., 1983a;b). However, so far no detailed assessment has been published about the time course of procarbazine-induced alterations in the testis including repair processes over a whole spermatogenic cycle. In clinical use for humans, procarbazine is administered orally at a dosage of 4-6 mg/kg daily; duration of administra— tion is determined by severity of secondary leukopenia or throm- bocytopenia. 5. Cvcluur L ide (Cytoxanl Cyclophosphamide, in addition to being the most widely—used chemotherapeutic agent in the treatment of various neoplastic diseases, is also used as an immunosuppressive drug in a va- riety of non—malignant diseases (IARC, 1975). It is also the d) hi Re ti. Que Ci? wi‘ tht , , 18 compound that has been studied in most detail for its effect on the male reproductive tract. Following cyclophosphamide ther- apy, mature males were found to be oligospermic or aspermic and to have histologic evidence of testicular atrophy (Fairly pp g1., 1972; Quershi g; _1., 1972; Knorr g; _1., 1970). The ger- minal epithelium appeared to be selectively damaged with no apparent injury to Sertoli cells or Leydig cells and there was an associated increase in plasma levels of FSH and occasionally LH (Etteldorf g; _1., 1976). An increased incidence of gonadal dysfunction was associated with prolonged therapy, especially at higher dose levels (Etteldorf g; _1., 1976; Hsu g; _1., 1979). Regeneration of seminiferous epithelium occurred in some pa- tients after discontinuation of therapy (Knorr g; g1., 1970; Quershi g; _1., 1972). Cyclophosphamide is used in the treatment of several di- seases in the prepubescent male and, as in the adults, testi- cular damage was noted. This was first reported by Hyman and Gilbert (1972) who found severe seminiferous tubular atrophy with "Sertoli cell only" pattern and interstitial fibrosis at the autopsy examination of testes from an eight-year-old boy who had received cyclophosphamide; additional reports of retrospec- tive clinical cases soon followed (Arneil, 1972; Rapola pp g1., 1973). Evidence of testicular injury in patients treated prior to or during puberty and examined after puberty included oligo- spermia, aspermia, testicular atrophy, histologic damage of the germinal epithelium and elevated plasma FSH and/or LH levels 1., 1974; Pennisi g; _1., 1975; Lentz g; _1., 1977). (Penso g; l9 Severity of gonadal injury was not always clearly associated with dose or duration of therapy and damage to the testis was postulated to vary with the stage of maturation during exposure _1., 1975). (Rapola et p1,, 1973; Penso et 1., 1974; Pennisi pp Lendon pp _1. (1978), however, found no correlation between the \ degree of injury and age during exposure. Leydig cells showed no histologic evidence of damage, and testosterone as well as the response to human chorionic gonadotropin (HCG) stimulation was generally nominal in these patients, and as a result there was no maturational delay (Pennisi pp _1., 1975; Shalet pp _1., 1981). Recovery of the germinal epithelium and attainment of the ability to reproduce occurred in many patients and varied with time since cessation of therapy (Pennisi pp _1., 1975; Kirkland pp p1., 1976). Since the disease process itself may influence testicular morphology or function (Shalet pp p1., 1981), evaluation in animal models is necessary to clearly de- termine specific drug effects. Cyclophosphamide is a derivative of nitrogen mustard and depends on 1p 2129 activation to form reactive metabolites with alkylating and cytotoxic capabilities (IARC, 1975). Exposure of animals to cyclophosphamide has produced histologic damage to the germinal epithelium, sperm abnormalities, increased mutation rates and unscheduled DNA synthesis in germ cells (Lee and Dixon, 1972b; wyrobeck and Bruce, 1975; Sotomayor and Cumming, 1975; Schmid and Zbinden, 1979). Though the exact mechanism of action has not been established, the cross-linking of DNA is a major possibility (IARC, 1975). Furthermore, Lee and Dixon neoI ment bin; Pr0( aplé test C€11 tern 11.... . 20 (1972b) postulate that, since alkylating agents can also react with thiols, phosphate esters, ribonucleic acid components and proteins, the actual mechanism may depend on the relative sensi- tivity of the biochemical processes which determine the differ- entiation and replication of certain cell types. These investi- gators found that spermatids were the most susceptible cell type of the germinal epithelium to the toxic effects of cyclophospha- mide followed by minor damage to spermatogonia and no effect on spermatocytes. Thus, both replicating and non—replicating cells were damaged. Cyclophosphamide is administered either orally or intrave- nously in human clinical medicine. Dosage varies depending on the route; maintenance therapy is 1-5 mg/kg per 03 daily, 10-15 mg/kg intravenously every 7—10 days or 3-5 mg/kg intrave- nously twice weekly. Vincristine Vincristine, a vinca alkaloid, is used for both its anti- neoplastic as well as immunosuppressive properties in the treat- ment of many diseases. A group of pubertal boys receiving com— bination therapy consisting of mechlorethamine, vincristine, procarbazine and prednisone had histologic evidence of germinal aplasia, elevated FSH and LH serum levels, reduced serum tes- tosterone and gynecomastia. There was thus evidence of Leydig cell damage as well as tubular injury (Sherins pp p1., 1978). Prepubescent boys who received the same therapy were de- termined to have no apparent testicular injury based on normal dea por int] 19N and bly trat tera< (Parr from and d Skele c0mm — ‘ 21 FSH and LH levels and absence of gynecomastia. However, though elevated FSH levels are usually consistent with severe tubular damage, normal FSH levels were frequently seen in the presence of significant but not yet severe damage (Lentz pp p1., 1977). Another possible explanation is that pubertal age during therapy may determine the extent of dysfunction. A group of boys who received prednisone, vincristine, methotrexate and 6—mercapto- purine before, during and after puberty had no testicular dam— age (Blatt pp p1., 1981). Exposure of laboratory animals to vinca alkaloids, vincris- tine and vinblastine, has provided evidence for toxic damage to the testis. Exposure of rats and mice to vincristine and vin- blastine resulted in mitotic and meiotic arrest followed by cell death of the respective cell types and sloughing of the apical portions of Sertoli cell cytoplasm along with related germ cells into the tubular lumens (Lee and Dixon, 1972a; Parvinen pp p1., 1978; Russell pp p1,, 1981). Vinca alkaloids are known to cause metaphase arrest (Lee and Dixon, 1972a). These compounds prevent microtubule assem- bly by binding to tubulin resulting in intracytoplasmic seques- tration of microtubular protein into crystals and may also in- teract with tubulin nonspecifically as alkaloid cations (Parvinen pp p1., 1978). Mitotic and meiotic arrest resulted from disruption of the spindle apparatus (Russell pp p1., 1981) and destruction of microtubules resulted in loss of the cyto- skeleton of Sertoli cells followed by destabilization of its contact with germ cells and their premature release. Sertoli tur EXp Spe ter fre pla 200 tot 22 cells appeared to be able to regenerate their apical cyto— plasm and repopulate their processes with microtubules (Russell pp p1., 1981). Vincristine is administered to humans intravenously at weekly intervals. The usual dose for children is 2 mg/m2 and for adults 1.4 mg/mz. LEW Cytosine arabinoside is a cytotoxic agent used alone or in combination with other drugs in the treatment of acute leukemias in both young and adult patients (Pratt and Ruddon, 1979). Cytosine arabinoside caused significant damage to the germinal epithelium of young men, but did not injure Leydig cells (Lendon _p p1., 1978; Shalet pp p1., 1981). The major mechanism of action of cytosine arabinoside is thought to be the inhibition of DNA polymerase which would in turn block DNA synthesis (Reviewed by Lee and Dixon, l972d). Exposure of adult male rats to cytosine arabinoside damaged only spermatogonial cells in S—phase, thus further supporting the above-proposed mechanism (Lee and Dixon, 1972d). For human clinical use, cytosine arabinoside is adminis- tered only intravenously or subcutaneously. This compound is frequently used in a combination regimen with other antineo- plastic agents. When used as the only therapeutic agent, 200 mg/m2 is administered by continuous infusion for 5 days; total dose is 1000 mg/mz. This course is repeated every week. The gems of the development Yet, as evi involves ef thorough in' the testis ‘ ation; 2) W] 0f the test; whether the specific age ferences in mature testj 101mg. Chi] medicine (PE 1978)_ Comp found to Car (Friedman it MSG (Lamper into the env BP’ a flame with dibl‘oxno tis (Blum an due to envir. Chemical SP1 home 0,. 0n t] The structure and function of the testis, unlike most other or- gans of the body, changes markedly during maturation, including unique developmental processes that occur at no other time following puberty. Yet, as evidenced from the previous discussion, most available knowledge involves effects only in the sexually mature male. There have been no thorough investigations concerning: 1) whether the susceptibility of the testis to certain agents and mechanisms of damage varies with matur- ation; 2) whether a given agent and mechanism affects the specific cells of the testis differentially at the various stages of maturation; or 3) whether the immature testis can recover following damage caused by a specific agent and/or mechanism. A thorough understanding of any dif- ferences in the effects of a chemical on the immature as compared to the mature testis is critically important to the health and safety of the young. Children are exposed to many chemical toxicants used in clinical medicine (Penso pp p1., 1974; Pennisi pp 1., 1975; Sherins pp p1., 1978). Compounds common in a child‘s environment have recently been found to cause toxic injury to the mature testis, e.g., caffeine (Friedman pp 1., 1979), Vitamin A (Lamano-Carvalho et al., 1978) and MSG (Lamperti and Blaha, 1980). New synthetic compounds introduced into the environment of children may be potentially harmful, e.g., tris BP, a flame retardant used in children's sleepwear, was contaminated with dibromocloropropane, a compound capable of damaging the mature tes- tis (Blum and Ames, 1977). Accidental exposure to chemicals could occur due to environmental sources in hazardous waste sites, explosions or Chemical spills, or exposure to toxic agents intended for use in the home or on the farm. 23 I isms of . the heme; a chemica ing accid In ceptibilil and method 1) FiVe this study b of their C11 been studied 24 If the susceptibility of the immature testis to varying mechan- isms of damage can be determined, it will then be possible to evaluate the benefit/risk potential prior to intentional exposure of a child to a chemical or to anticipate the possible damage which may occur follow- ing accidental exposure. In order to address this question of differential testicular sus— ceptibility, the present studies were performed. Studies were designed and methods selected to provide knowledge in five key areas: 1) To determine if a particular toxicant with a known mechanism of action differentially affects the testis at specific critical stages of development. 2) To determine the specific cell type damaged and to evaluate both the extent of morphological damage as well as dysfunction. 3) To evaluate the ability of the specific cell types to recover. 4) To detect any delay in maturation resulting from exposure to toxicants prior to puberty. 5) To determine relationships or interdependence of damaged cell types. Five known chemotherapeutic (anticancer) agents were used for this study because of their known effect on the mature testis, because of their clinical interest and because their mechanism of action has been studied. fol dif hol llI the I I. EXPERIMENTAL DESIGN Age at Exposure The selection of specific time points for exposure was im- portant to this study since it was necessary that critical stages of maturation of the individual cell types as well as the time of onset of reproductive capacity could be clearly identified. For this reason, two pilot studies were performed. My; Since the literature described several populations as well as strains of rats, a morphologic analysis of a single population of Sprague Dawley rats was made to validate the time sequence of events. Male rats of l, 3, 6, 9, 12, 15, 18, 24, 30, 36 and 45 days of age (three animals per group) were sacrificed by decapitation following ether anesthesia. Each animal per age group was from a different litter. Body weights, testicular size and weights were recorded. Testes were fixed in Bouin's and washed in 70% alco- hol. Tissue was embedded in paraffin and stained with hematoxy- lin and eosin and a separate section was embedded in glycol me- thacrylate, sectioned at 2u and stained with toluidine blue. Pilot Study II According to the literature, Spermiogenesis in the rat is complete and spermatozoa are released from the seminiferous tu- bule for the first time at approximately 45 days of age. Prior to ejaculation, further maturation in the epididymis requires one 25 mal nal cag tim wee aft 26 to two weeks (Galbraith g; _1., 1982). Therefore, a male rat should be capable of impregnating a female at eight to nine weeks of age. This is considered the onset of reproductive capacity. This means that ejaculated sperm are capable of fertilization and production of a viable conceptus. This term differs from "puberty" which is a much broader concept and in- volves both the onset of the ability to reproduce as well as development of the physical male secondary sexual characteris- tics. To verify the time of onset of reproductive capacity, 10 male six-week-old Sprague Dawley rats were serially mated to sex- ually mature females for four weeks. A single male was housed in a cage and at weekly intervals a virgin female was put into the cage. The two animals were allowed to cohabitate for one week, time for a full estrus cycle in the female. At the end of each week, that female was replaced with a new female. At 10 days after breeding, the females were sacrificed by decapitation fol- lowing ether anesthesia and their uteri were examined for viable implantation sites. Test Chemicals The number of agents capable of disrupting testicular func- tion has been estimated to be many times greater than the number of vital pathways that can be interrupted (Russell gt al., 1981). In this study, five chemicals (Table l) were selected based on their specific mechanisms of action. All five chemicals are chemotherapeutic agents. Because of their specific use in clini- cal medicine, their chemical structure, mechanism of action and Table l, cyto Cycl Doxo: Proce Vincr Table 1. Test Chemicals and Mechanisms of Action Test Chemicals Mechanisms of Action Cytosine arabinoside Inhibits DNA polymerase and RNA function Cyclophosphamide Alkylating agent, cross links DNA Doxorubicin Intercalates DNA and inhibits RNA function Procarbazine Inhibits DNA, RNA and protein synthesis Vincristine Inhibits microtubule function, alters mitosis and meiosis Table 1. Test Chemicals and Mechanisms of Action W " ‘* isms of Action Cytosine arabinoside Inhibits DNA polymerase and RNA function Cyclophosphamide Alkylating agent, cross links DNA Doxorubicin Intercalates DNA and inhibits RNA function Procarbazine Inhibits DNA, RNA and protein synthesis Vincristine Inhibits microtubule function, alters mitosis and meiosis at log tes wid fere Mate ages stud: toxh recox singl and s mals 28 toxicity are well documented in the literature. By understanding the mechanism of action of the agent and the maturational events at the time of exposure, a better understanding of the physio- logical response of the testis should be possible. Thus, if testicular susceptibility to (a) given mechanism(s) can be deter- mined, it may in the future be possible to postulate about the potential effects of other compounds which have (a) similar mech— anism(s) of action. Animals were injected once intraperitoneally with either a high or low dose of a single chemical at four dif- ferent days of age. (Details of dose and route are discussed in Materials and Methods.) Study Design Based on the pilot studies and published literature, four ages were selected for exposure: 6, 16, 24, and 45 days. The study was designed in two phases to optimize evaluation of acute toxic effects separately from long-term toxicity and ability to recover. During Phase I (Figure 1), animals were treated with a single dose injected intraperitoneally at the four selected ages, and sacrificed at either 3, 7, or 14 days post treatment. Ani- mals received either a high or low dose of one of the selected compounds. The primary purpose of Phase I was to evaluate acute toxic effects. At sacrifice, the age of animals in the various groups differed, so specific effects on age related endpoints (e.g., morphology of testis) could not be compared between age groups. However, by having equal intervals between treatment and sacrifice, it was possible to compare within an age group the AGE 0—1 AGE IN DAYS AT TREATMENT A3 I I I v A5 a I _| First Second Third Sacrifice Sacrifice Sacrifice I j l j 0 3b 7b 14" Days After Treatment Gross lesions Acute Bod - - - . . y weight and weight gain 322”” Organ weight Microscopic lesions a) N = 9 per treatment group and 9 per control group b) N = 3 per treatment group and 3 per control group Figure 1. Phase I SERIALMATINGWEEK:° 1 2 3 4 (5) 6 7 8 9 10 11 ® G—>L———, .. n .—>‘———1"‘ n 45 "—“‘ HRST b SECOND f f Q A} SACRIFICE SACRIFICEC AGE IN DAYS ATTREATMENT l l r r 1 0 5 10 15 20 ANIMAL AGE (WEEK) Long Term Gross lesions Toxicity and Body weight and weight gain Recovery Organ weight Data Microscopic lesions Sperm counts Serial Mating Onset of Reproductive Capacity Data Fertility, Fecundity Genotoxicity a) N = 15 per treatment group and 15 per control group b) N = 5 per treatment group and 5 per control group C) N = Figure 2. Phase II 10 per treatment group and 10 per control group mai sac Stui afti Sac: 31 impact of acute toxicity and ability to recover and between age groups the general susceptibility to toxic effects could be determined. During Phase II (Figure 2), the primary goal was to evaluate long-term toxicity and recovery data and to evaluate the animals' ability to reproduce. Therefore, the design of Phase II differed from Phase I. Animals were treated at the same days of age, either 6, I6, 24, or 45. Animals were treated with high dose only during Phase II. Each group was then allowed to mature to 45 days of age, the time when the first mature spermatozoa should be released. In each age group 15 animals were treated. Ten animals per group, all starting at 45 days of age, were ser- ially mated for 12 weeks (See Materials and Methods for details). The males that were serially mated were sacrificed after 12 weeks. At this time, all animals were 129 days of age. The re- maining five animals per group Were not serially mated and were sacrificed after five weeks of the beginning of the serial mating study when all animals were 80 days of age. Although the time after treatment at the beginning of serial mating and at the two sacrifice points during Phase II differed between groups (Table 2), all animals at these time points were the same day of age. This allowed a direct comparison of all parameters measured be- tween animals treated at the four different critical ages of de- velopment. The endpoints measured during Phases I and II were selected as specific indicators of toxicity (Table 3). (Not all endpoints were measured in each animal at every sacr1f1ce p01nt. Details are in Materials and Methods.) Endpoints and indicators 32 were chosen to provide information in three specific areas: 1) general toxicity; 2) structure and function of the three major cell types of the testis; Sertoli cells, Leydig cells and germinal epithelium; and 3) the integrative function of the male system which allows successful reproduction. Table 2. 1 Age At 1 0f Treatn (Days) 33 Table 2. Phase II: Time Schedule and Animal Ages Time After Treatment (Days) Age At Time of Treatment (Days) At Beginning of 5 Week Sacrifice 12 Week Serial Mating (Animal Age: Sacrifice (Animal Age: 80 Days) (Animal Age: 45 Days) 129 Days) 6 39 74 123 16 29 64 113 24 21 56 105 45 O 35 84 BwUfldfiDH. HON “mg HOUSE bHUHVADIH “0 ”“883 um QM.” .H 34 mcoflhwuommu rmmoa hogan 3H8 63m 33 bananas fiwoomwo o>flbsnonmmm 3500 noon in unsung Ea Engage maggot 5 magma mam seams gasses mSOflon camoomouuflz €28me seams fluwmp Ea massages humane: mmouw nag ufiomEm n88? fifloflxos no €38.85 3 magma #550356 cowumgmon 0305.0 use 8200 3mm “x8 mmooosm £033.:ng Amuommum towns." Em 683 Eflmfifim Bags at flounwm 53g confiseoummm mg 3.9.8953.“ng Rhona MO #030 “8.35 Ea pumice mflmwcwmmomwoogmwmm montage 58% mammfimsm nae .8305 :8 38.4mm Ems 538m 94.6.3 5% 3.80 was soflbm cog behooves 33380.6 at 33:8 3205.2 $338 BEES. 05% 292 033030.9me coflpoflmfl Em £395 .3338 ufinmz cameo fiflummufi sammo magnmuogfi guano mmonw 334.088 How Dogma anir ace] ding hOus. Stud: the dim A11 .3 IV. MATERIALS AND METHODS Animals and Housing Conditions We The rat was selected as the experimental animal because the cell kinetics and physiology of the germ cells are the most ex- tensively investigated and well-understood of any mammalian spe- cies. Furthermore, the animal is relatively inexpensive, easy to maintain under laboratory conditions and is available from reli- able commercial suppliers. Source Sprague-Dawley rats, CD strain, were purchased from Charles River Laboratories, Kingston, North Carolina. The incoming ani- mals were examined for health status by a veterinarian at the National Institute of Environmental Health Sciences, Research Triangle Park, NC, and immediately afterward transferred to an animal room at Duke University in Durham, NC, where they were acclimated for three days prior to the beginning of the study. Housing All animals were housed in plastic cages with corncob bed- ding (Bed-O—Cobs, The Andersons, Maumee, OH). Females were housed two per cage when not being mated. During the mating study, males were housed with one female at a time. Pups under the age of 24 days remained with their dam and littermates in in— dividual cages and all other males were housed two per cage. All animals were observed daily for signs of toxicity or disease. 35 i" <2 mal ting ink. mic pour sing iti: ity pro com irr 0n] th is Feed NIH 31 pellets and water were available ad libitum (formula for NIH 31 pellets available in Knapka, 1983). Control of Environment Temperature: 70 i 2°F Photoperoid: 12 light: 12 dark (0600-1800) Identification of Animals Males were housed 2 per cage. Identification for each ani- mal was recorded on the outside of the cage and animals were dis— tinguished from each other by markings on the tail with indelible ink. Exposure: Route, Dose and Time Each male received a single dose of one of the test com- pounds listed in Table 4 via intraperitoneal (IP) injection. A single dose was used because it best allowed evaluation of in- itial toxic effects, secondary or long-term effects and the abil- ity of specific cell types to recover. Use of IP administration provided assurance that the animal received the total dose. All compounds were dissolved in ultrapure, triple distilled water at a concentration so that all animals received 4 ml/kg body weight irrespective of the dose administered. Control animals received only water. All compounds were obtained from the Drug Distribu- tion Bank, National Cancer Institute, Bethesda, MD. Two dose levels were selected following preliminary range finding studies. The high dose was determined to be the maximum tolerated dose Table 4. Test 5 Ultrapur Cytosine Cyclopho, DOXO rub it Procarba Vinerist Table 4. Test Chemicals and Dose Test Chemicals Lot No. Source Dose mg/kg/bw (low/high) Ultrapure water Harleco Kingston, NC Cytosine arabinoside 2774-43-21 ICN 300/600 Costa Mesa, CA Cyclophosphamide MAS 52 Mead Johnson 40/80 Evansville, IN Doxorubicin 10082015 Farmitalia 1.5/3.0 Milan, Italy Procarbazine 061066 Hoffman-LaRoche 100/200 Nutley, NJ Vincristine 67522 Mario Negri 0.3/0.6 Milan, Italy gn ym for mal Dur blo, mem ted (Hb) Smea euth 38 that did not cause overt systemic toxicity such as alopecia, growth retardation, anorexia or listlessness. Low dose was one half the high dose. During Phase I, animals were exposed to either high or low dose of a selected compound. During Phase II, only high doses were administered. Body weights were recorded for all animals at exposure. The number of animals per exposure group varied. In Phase I, groups of three males were treated at either 6, 16, 24, or 45 days of age for sacrifice periods of 3, 7 and 14 days post exposure. During Phase II, groups of 15 males were treated at either 6, 16, 24, or 45 days. Of these, three to five animals were sacrificed after five weeks of serial mating and 10 animals after 12 weeks of serial mating. Hematology Blood was collected from the abdominal aorta of animals greater than 44 days of age and by intracardiac puncture in younger animals just prior to euthanasia. Analyses were per- formed in the Hematology Laboratory, Division of Laboratory Ani- mal Resources, Duke University Medical Center, Duke University, Durham, NC. Tests included: white blood cell (wbc) and red blood cell (rbc) counts by direct measurement by Coulter ZBI; mean corpuscular volume (mcv) and hematocrit (pcv) were calcula- ted from data measurements from the Coulter ZBI and hemoglobin (Hb) was analyzed by a Coulter Hemoglobinometer. Bone marrow smears were taken from the femur of all animals immediately after euthanasia and stained with Wright's stain. 39 Gross Examination and Tissue Preparation Immediately prior to sacrifice, body weights were recorded for all animals. Animals were sacrificed by two methods depending on the fixative method to be used for testicular tis- sue. Animals less than 45 days were decapitated, older animals were anesthesized with phenobarbital and bled during perfusion fixation. Complete gross examinations were performed on all ani- mals. Organ weights were recorded for testis and epididymis. Sections of kidney, liver, lung, prostate, seminal vesi- cles and epididymis were immersed in 10% neutral buffered forma- lin, embedded in paraffin and stained with haematoxylin and eosin. One testis and one epididymis of animals 45 days and older were preserved and frozen at -80°C for later measure- ment of Spermatid reserves and sperm head counts. Testicular Tissue Preparation 1. Glutaraldehyde Perfusion All animals from Phase I more than 45 days of age and three per group in Phase II were anesthetized with phenobarbital (50 mg/kg) administered IP. The technique of retrograde perfusion through the abdominal aorta as described by Vitale g; _l., (1973) was slightly modified (Figure 3). Perfusion with 0.9% saline for approximately two minutes removed blood from the vasculature. The left spermatic cord was clamped and the testis and epididymis were removed and frozen at —80°C for further analysis. Gluta- raldehyde (5% in 0.2 M cacodylate buffer at pH 7.4) was then per— fused through the abdominal aorta of each rat for 30 minutes. Figure This i Vitale diaPhre Curv SUrroun collect illfllsiol —__—". , 40 Four transverse slices of 1 mm thickness were cut and placed in gluta— raldehyde. Tissue blocks 1x1x2 mm were washed three times for 15 min- utes with 0.2 cacodylate buvver (pH 7.4). Tissues were then post fixed for 90 minutes in a solution of 1% osmium tetroxide and 1.5% potassium ferrocyanide (Russell and Burguet, 1977). Cairo: A. Sup an’eric A llioiumbar A Right lnt. Spermciic A Figure 3. Diagramatic Representation of the Technique for Perfusing Testes with Fixative This is a modified version of the procedure originally described by Vitale thfll" 1973. 1) Ligature is prepared blindly around big sub— diaphragmatic vessels and tightened at the beginning of the perfusion. 2) Curved hemostat slightly lifts the aorta which is cleaned from the surrounding connective tissue. 3) Point where the aorta is incised for collection of blood and insertion of a 19—gauge needle connected to an infusion set. (Ettlin and Dixon, 1985). 41 They were then washed three times for five minutes in cacodylate buffer and dehydrated and infiltrated with epon. Sections were cut at one micron and stained with toluidine blue. In addition, a large section of each perfused testis was em- bedded in glycol methacrylate (GMA), sectioned at two microns and stained with toluidine blue. 2. Bouin's Fixation Testes from all animals less than 45 days and all unper- fused animals in the Phase II study were immersed in Bouin‘s fix— ative for 24 hours and cut in 5 mm sections. They were then im- mersed in a series of three washes of 70% alcohol and embedded in glycol methacrylate. Two micron sections were stained with to- luidine blue. Measurement of Spermatid Reserves and Sperm Head Counts Spermatid reserves in testes and sperm head counts in epi- didymides were determined using the modified method of Robb gt al., (1978), as described by Lee and Russell (1985). After weighing testes and cauda epididymides individually they were placed in scintillation vials with CTC buffer and the tissue was finely minced. Tissue was then incubated at room temperature for 3.5 hours on a rotary shaker at 400 rpm. Following incubation, a Hepes-Triton x 100 reagent was added and tissue was homogenized with the SEM—microhomogenizer for two minutes at 10,000 rpm. Spermatid reserves and sperm heads were then counted in a Makler Chamber. Was ‘ feta; 42 Androgen Binding Protein Androgen binding protein (ABP) was measured using the dex- tran—coated charcoal method (DCC) of Musto and Bardin (1976). The binding of 3H-DHT (New England Nuclear, Boston MA) was determined in triplicate in the cytosol of the caput epididymidis of each animal. A pool-standard cytosol was prepared and frozen in portions at the beginning of the experiments; ABP was determined in this standard cytosol in each experiment as an internal reference. The purity of the testosterone (Sigma, Inc, St Louis, MO) and 3H-DHT was determined by thin layer chromato- graphy before the stock solutions were made up. Protein concentrations in the cytosolic preparations were determined according to the method of Lowry gt g1.,(1951). Mating Studies Reproductive function was assessed by serial mating of 10 males per age group in Phase II. When the animals were 45 days old, they were caged individually, each with one virgin female. The females were replaced at weekly intervals for 12 weeks. One week after being separated from the males, the females were sacrificed and their uteri examined visually for implants, resorp- tions and viable fetuses. A male was regarded as fertile if the corresponding female had one or more viable implants. Statistical Design The statistical significance of the differences in fertility was determined by using the Fisher exact test (Siegel, 1956). The fetal mortalities, litter sizes, body weights, testis weights, — t 43 epididymal weights, implantations, sperm head counts, Spermatid reserves, and ABP data were all analyzed using the Mann-Whitney U-test (Siegel, 1956). All tests were two—sided; the level of significance was p < 0.05. rel ger Lor rel. the V. Results Results of Pilot Studies 1. Pilot Study I Based on the data obtained and structural and functional events observed by others, four critical ages were identified: Dgy_§, gonocytes, the primordial germ cells, and Sertoli cell precursors, were actively dividing mitotically and Leydig cells had fetal characteristics (Figure 4); Day 15, Sertoli cells were forming tight junctional complexes, a critical part of the blood- testis barrier, spermatogonia were dividing mitotically to produce primary spermatocytes and Leydig cell numbers were at their nadir (Figure 5); Day 24, Spermatids had formed, thus reflecting both mitosis and meiosis, and adult Leydig cells were beginning to dif- ferentiate (Figure 6); Day 45, Sertoli cells had attained all adult characteristics, mature spermatozoa had developed and there was a full complement of adult Leydig cells (Figure 7). Thus, ex— posure during these four time periods should allow detection of any differential susceptibility of the immature rat testis to chemical toxicants (Table 5). Figure 8 is a representation of the relative proliferative activity of Sertoli cells, Leydig cells and germinal epithelium during sexual maturation based on data from Lording and deKretser (1972) and Clermont and Perey (1957). The relative proliferative rates at times of treatment are listed in the insert of Figure 8. 44 45 Figure 4. Testicular Tissue of 6 Day Old Rat. Solid seminiferous cords contain only Sertoli cells, spermatogonia, and gonocytes, the primordial germ cells. Leydig cells, located in clumps in the interstitium, have fetal char— acteristics evidenced by abundant cytoplasm (Magnifica— tion: 360x). 2, "'"*‘\‘-n—‘_.—___ A Figur Figure 5. Testicular Tissue of 16 Day Old Rat. Spermatogonia have d1v1ded mitotically to produce primary spermatocytes and Leydig cells are rare (Magnification: 360x). Figure id seminiferous togonia, and aydig cells, ave fetal char— asm (Magnifica- matogonia have rmatOCYtes and x). Figure 5. Figure 6. Figure 7. 47 Testicular Tissue of 24 Day Old Rat. Spermatids exist, reflecting completion of the second meiotic division. A few Leydig cells have differentiated to adult form (Magnification: 360x). Testicular Tissue of 45 Day Old Rat. Spermatogenesis is complete with the production and release of mature spermatozoa; Sertoli cells have adult characteristics (Magnification: 360x). 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Q ~ Amaflmuop How uxmu mmmv sOHuwHDumz Hmnxum wcwHDQ aghastufldm Hmswfiuoo ace mflmo menses 6:8 2330. do sun/3.2a mfiumtwmfloum 3.32% .w 333m 35.2.50“. m>:.(..u& \onocq \\S\o\ 3‘th bexw3 1 so 1 0.0 1 ad a 3 1 W A _ a u .d _ a u 0 _ H” n n .4. _ 13 n a I w u A _ a V D H... M In. IA _ _ . _ _ . _ . u 2mm 39330 _. _ 5.. IIIIIIII _ _ sneezed _ -oxwxfimw _ . _ _ _ _ . _ _ . _ _ . 51 2. Pilot Study II During the first two weeks of serial mating all females had corpora lutea, but no implantations or resorptions. During week three of serial mating (males 54 days of age), 5 of 10 females had implantations with no resorptions, one female had both implanta- tions and resorptions and four females had neither implantations or resorptions, but did have corpora lutea on both ovaries. In week 10 of serial mating, (males 70 days of age), all females had implantations, none had resorptions. Thus, the strain of males used reached sexual maturity as expected and therefore had to be releasing their first spermatozoa from the seminiferous epithe- lium at about 45 days of age. B. Low Dose Exposure During Phase I, low dose exposure produced only minimal treatment-related effects with procarbazine and none with the other 4 components. Therefore, these results are not discussed and the following data relate only to the high dose tested. C. Hematology Blood parameters were measured on all high dose animals in Phase I. No statistically significant differences from control animals were detected. Since hematologic examinations were per- formed to detect acute, primarily systemic toxicity and no effects were found, hematologic evaluations were not done during Phase II. D. Controls 1 and 2 Due to the extremely large number of animals involved in Phase II, it was necessary to perform the study in two parts. 52 During Part 1, animals were treated with cyclophosphamide, cyto- sine arabinoside and vincristine, and there were age matched con- trols: Control 1. During Part 2, animals were treated with doxorubicin and procarbazine and there was another population of age matched controls: Control 2. Within Control 1 and Control 2, the results of the four control groups treated with the water vehicle at the age of 6, 16, 24 and 45 days did not differ signi— ficantly. Serial mating and biochemical data were therefore pooled. Control 1 (Table 6): Two of the 40 control animals were fer- tile within the first week of serial mating. The number of fer- tile males increased rapidly during the next two weeks and was stable thereafter, representing normal reproductive capacity. During weeks one to four, the time when the onset of reproduc— tive capacity should occur, 60.0% of the matings led to litters with viable implants. Once fertility was established (weeks 5-12), 87.8% of the 320 matings of the 40 males were successful. During the onset of reproductive capacity, the mean value of to- tal implants per litter produced each week increased from 2.5 in the first week to 11.5 in the third week, and stayed between 11.5 and 13.9 thereafter. The average number of resorptions per lit— ter was one, at the most, for the whole mating study. The mean number of viable implants per litter increased from 0.5 in week 1 to 9.7 in week 2. The weekly mean values of viable implants per litter were between 11.3 and 13.7 thereafter. [4.14: rite... 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H309 H953 Hog 3.5.35 m 83> 83...: 38.82.... 3.380 030.8%. 5.3 03 mo 33th snags.“ H 305200 . 3 no mHQmB 54 Control 2 (Table 7): Five of the 40 control animals were fertile within the first week of serial mating. The number of fertile males increased rapidly during the next three to four weeks, and was stable thereafter, representing normal reproduc— tive capacity. During the onset of reproductive capacity (weeks 1-4), 65.6% of the matings led to litters with viable implants. Once fertility was established (weeks 5—12), 93.4% of the 320 mat- ings of the 40 males were successful. During the onset of repro— ductive capacity, the mean value of total implants per litter pro- duced each week increased from 9.0 in the first week to 14.5 in the third week, and stayed between 13.4 and 14.2 thereafter. The average number of resorptions per litter was one, at the most, for the whole mating study. In addition to the 404 matings leading to viable implants, one litter found in week 2 and one found in week 6, contained resorptions only. The mean number of viable implants per litter increased from 9.0 in week 1 to 12.7 in week 2. The weekly mean values of viable implants per litter were between 12.9 and 14.0 thereafter. The two control groups were analyzed statistically and they were not comparable. In particular, Control 2 had higher ferti- lity, more implantations per litter and higher fetal mortality than Control 1. Therefore, treated animals were always compared to their corresponding control group. ay0\ 1.2 ULLLTHNRNEHH M83 - J 1..» :(l 095.3 #00 mucoHQEH momma: mHQMflxw 94. HMS fig 0HQMH> 0H9MH> wCOHunmnowwm wCOHUnmuowmwm wucanwnH H308 H308 @505er 5.43 ”“6003 wahmvm “MANN—fig N HoyNUHHOO CH: 38 \finwwnfli.” h u 55 0 0 0.00 0000 00.0 000 0.00 0000 00. 00 0.0 H 0.00 000 00. H 0m. 00 0.0 H 0.00 000 00. H 0m. 00 0.0 H 0.00 000 00. H 00.0 00 0.0 H 0.00 000 00. 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Clinical Signs No overt clinical signs of systemic toxicity were noted in any animal at any time. 2. Gross Necropsy During Phase II, of the animals treated at six days of age, one male died spontaneously during week 6, one during week 7, and two during week 8 of the serial mating study. Gross necropsy of these animals revealed peritoneal anad pleural effusions. None of the animals treated at six days of age died during Phase I. Of those treated at 16 days, no animals died during the first phase of the study but during the second phase, one animal died during week 5 and two animals during week 6; the necropsy findings were similar to those reported above. None of the animals treated at 24 or 45 days of age died during the study. In animals surviving to sacrifice points, gross pathologic change was minimal with the exception of decreased size and weight of testes and epididymides of rats treated during Phase II at 6 and 16 days of age and sacrificed after 5 and 12 weeks of serial mating. However, during Phase I, two animals treated at 6 days of age and euthanatized 14 days later had a small amount of clear ascitic fluid in the peritoneal cavity. Also, one animal treated at 45 days of age and sacrificed at 129 days of age had an en— larged pale rounded liver. 57 3. Body Weight: Testicular and Epididymal Weights {Tables 8I 9 and 10) a. Treatment at 6 Days of Age No significant changes in body weight were observed during Phase I of the study. However, the body weights of the animals sacrificed at the age of 80 days (after 5 weeks of mating) and 129 days (after 12 weeks of mating) during Phase II were signifi— cantly lower than those of the controls (72.6% and 77.1%, respec— tively, of the control values). Testicular and epididymal weights were also not affected dur- ing the first phase of the study but they were significantly de- creased during the second phase at both 80 days (16.2% and 16.1%, respectively, of control values) and 129 days of age (12.9% and 20.3%, respectively). b. Treatment at 16 Days of Age No decreases in body weight or the testicular and epididymal weights were noted during Phase I, but during Phase II the body weights of the l29-day-old animals were significantly lower than those of the controls (82.3%). Testicular and epididymal weights were significantly decreased at both 80 and 129 days of age (testes: 41.4% and 38.7% of the control values; epididymides: 43.5% and 39.8%). c. Treatment at 24 Da 5 of A e Significant changes in body weights were found only at 7 and 14 days after treatment during Phase I (76.1% and 74.1% of con- trol, respectively). 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Moo #:0050008 pm 090 . m B 0500sz 0900000030000 :0 Ago: 00 000030000008 mo ubmmmm .00 0.000. e) 61 during the second phase of the study and no changes in epididymal or testicular weights during the first phase. Testicular and epi- didymal weights, however, were significantly lower than controls (65.5% for testis, 76.3% for epididymis) at 80 days of age as well as at 129 days of age (68.8% for testis, 89.6% for epididymis) during Phase II. d. Ireatment at 45 Days of Age No significant changes in body weights were found at any ob— servation point. The testicular and epididymal weights were sig- nificantly lower from the controls only at 3 days post treatment during Phase I (83.5% for testis and 63.7% for epididymis) and not at all during Phase II. 4. Morphologic Evaluation a. Treatment at 6 Days of Age: Phase I Acute cytotoxic damage to the seminiferous epithelium of the testis was evident 3 days after exposure; however, few changes were seen at 7 or 14 days after treatment. Of the germ cells, the spermatogonia were the most obviously affected with evidence of degeneration, necrosis and mitotic arrest at 3 days after treatment but no such alterations were seen 7 days after treat- ment. A few tubules near the periphery of testes 14 days post exposure had moderate to complete germinal hypoplasia, reflect- ing irreversible damage to the stem cell population. However, more than 95% of the tubules contained a full complement of ger- minal cells that appeared morphologically normal. Sertoli cells had mild acute cytotoxicity only at three days post exposure 62 evidenced by slightly vacuolated cytoplasm. Testicular morphology was normal 7 days post treatment, but after 14 days post exposure, the seminiferous tubular lumina were effected. At an age where lumen formation is normally prominent (20 days of age), lumina were occasionally smaller than normal or nonexistent. No visible morphologic lesions occurred in any of the other, organs examined (kidney, liver, epididymis, seminal vesicles, prostate) at any observation period with the exception of a mild mixed peribronchial inflammatory cell infiltrate in the lungs of all animals after 7 days of exposure. This was not considered to be drug related. b. Treatment at 6 Days of age: Phase II All tubules of all animals had marked pathologic alteration after 5 weeks of serial mating (80 days of age). Germinal cell hypoplasia was severe. Only one to a few spermatogonia were found per tubule with complete absence of all more advanced germ cell types. The spermatogonia were slightly larger and more round than usual and had decreased contact with the basement membrane. No other germ cell types were present. All tubules contained nu— merous Sertoli cells, all of which were morphologically abnormal. These cells were shrunken, possessed pale cytoplasm, were markedly vacuolated and the nuclei were small, pale and extremely angular. Long fingerlike cytoplasmic extensions occluded most lumens. In the interstitium, cellular density of the Leydig cells was in— creased with cells often forming solid sheets. Leydig cell size was often increased, occasionally tw0 to four times that of the we 61. 63 controls. The cells were usually stellate or fusiform shaped ra- ther than round to oval, with abundant dark basophilic cytoplasm. Nuclei were enlarged with prominent nucleoli and heterochromatin. Some Leydig cells were multinucleate with 2-4 nuclei/cell (Figure 9). Capsular walls were thickened with a relative increase of both smooth muscle and connective tissue and contained a mild diffuse and a few dense multifocal areas of calcification. Mast cells with abundant metachromatic granules were occasionally seen in the perivascular areas. After 12 weeks of serial mating (129 days of age), most tu- bules of all animals were morphologically similar to those after 5 weeks of serial mating. However, some tubules possessed more advanced germ cell types than those seen after 5 weeks of mating. In these tubules spermatogonial cell numbers were almost normal and spermatocytes and Spermatids could occasionally be identified. However, all these cell types were frequently degenerative or ne- crotic with resorption or sloughing of the spermatocytes and sper— matids. In addition, there was marked architectural disorganiza- tion of the Spermatid cell layer with disruption of cell-cell contact and loss of mitochrondrial alignment along cell borders of early Spermatids and a few binucleate cells. Acrosomal forma- tion was often abnormal; many were small, indistinct and degenera— tive. The step 6 Spermatid was the most mature cell type that could be clearly identified. In later stages, Spermatid heads were abnormally shaped, usually involving failure of the head to elongate or the lack of nuclear chromatin condensation. During Figu 64 Figure 9. Testicular Tissue of Rat Treated with Doxorubicin at 6 Days of Age and Sacrificed at 80 Days of Age. All tu— bules were atrophied with severe germinal cell hypoplasia. Tubular lumens had not formed and Leydig cell density were not increased (Magnification: 180x). 65 stages 2-4, late Spermatids should be embedded deep within the layers of the seminiferous epithelium. Only a few Spermatid heads could be identified and these were either abnormally shaped or phagocytized within Sertoli cell cytoplasm. Thus germinal devel- opment was arrested in late Spermatid stages. The nuclei of Ser- toli cells in these partially functioning tubules were usually smaller, more angular than the controls with nucleoplasm that was slightly granular. The luminal diameters of several tubules were decreased (Figure 10) as compared to those of a control animal (Figure 11). Though Leydig cell size, shape and cellular density were si— milar to that described at the 5 week observation point (80 days of age), the degree of change was not as marked. Capsular mor- phologic alterations were consistent with those at five weeks after treatment. No visible morphologic lesions were detected in any other organ at either 80 or 129 days of age (5 or 12 weeks of mating) with the exception of the epididymis. At both 5 and 12 weeks of mating, there was complete absence of spermatozoa in the tubular lumen and the tubular epithelium was atrophic. c. Treatment at 16 Days of Age: Phase I Acute cytotoxic alterations were obvious at 3 days after ex- posure. The spermatocytes appeared to be the cell type most se- verely affected. Change included evidence of degeneration, ne- crosis and a few multinucleate cells in many tubules. Though spermatogonia did not show morphologic damage at this time, stem 66 Figure 10. Testicular Tissue of Rat Treated with Doxorubicin at 6 Days of Age and Sacrificed at 129 Days of Age. Seminiferous tubules were atrophic, germinal epithelium hypoplastic, Sertoli cell cytoplasm occluded some tubular lumens and Leydig cell size and density was increased (Magnification: 180x). FigL Figure 11. Testicular Tissue of Control Rat Sacrificed at 129 Days of Age. A Stage VIII tubule from a control animal illustrates normal testicular morphology (Magnification: 180x). FigUre 67 :orubicin at of Age- .nal epithelium LdEd some iensity W35 . Ced at 129 Control phology Figure 11. ——————"7—7 68 cells had been affected in a few tubules since, by 7 and 14 days after treatment, tubules in the subcapsular area contained only one to a few spermatogonia and no other, more advanced germinal cell types. In these tubules, pathologic changes of the Sertoli cells were similar to those in animals treated at 6 days of age. However, the majority of the tubules at these two time points appeared morphologically normal. No visible morphological lesions were present in any other organ examined. d. Treatment at 16 Days of Age: Phase II After 5 weeks of serial mating (at 80 days of age) all tu- bules of all animals had germinal cell hypoplasia and usually spermatogenic arrest. Though spermatogonia usually appeared nor- mal, one animal had a few seminiferous tubules with many degenera- tive, basophilic or necrotic spermatogonia with decreased contact to the basement membrane. Damage to spermatocytes was extensive in all animals. Spermatocytes through the zygotene stage were least affected and were normal in many tubules. Pachytene sper- matocytes, however, were frequently decreased in number and ex- isting cells were often degenerative, swollen and pale or necrotic with loss of cell—cell contact. Many tubules had moderate to severe hypoplasia of early Spermatids (Figure 12). Remaining cells were usually degenerative, occasionally multinucleate and often necrotic. Architectural disorganization was extensive and acrosomal abnormalities were common (Figure 13). Spermatogenic cells above Step 8 usually had abnormally shaped heads involving Figure 12. Figure 13. 69 Testicular Tissue of Rat Treated with Doxorubicin at 16 Days of Age and Sacrificed at 80 Days of Age. Germinal cell hypoplasia was severe with spermatogenic arrest in most tubules; early Spermatids were frequently the most mature cell type (Magnification: 180x). FL Testicular Tissue of Rat Treated with Doxorubicin at 16 Days of Age and Sacrificed at 80 Days of Age. Architectural disorganization was extensive, many germ cells were degenerative or necrotic (Magnifica— tion: 360x). Figu .xorubicin at : of Age. 1 spermatogenic '. were frequently 1: 180x). oxorubici“ at s of Age. Siva: man}: c (Magnifica— Figure 13. 70 71 failure to elongate and absence of nuclear chromatin condensa- tion. Late Spermatids were absent in many tubules and there was a moderate to severe decrease in cell numbers in most others. Thus, spermatogenesis was arrested in most tubules and those few sperma- tids that continued to develop were morphologically abnormal and usually prematurely released with the cytoplasmic droplet still attached or phagocytized within Sertoli cells. In tubules with severe germinal epithelial hypoplasia, Sertoli cells were atro- phic. Though morphologic characteristics of the germinal epithe— lium varied between animals after 12 weeks of serial mating (129 days of age), there was spermatogenic arrest in most tubules. Most tubules had a prominent population of germinal epithelial cells and pathologic alterations were generally consistent. Early Spermatids were most severely damaged and late Spermatids were often absent. Spermatogonia and preleptotene spermatocytes were usually abundant and morphologically normal except in an occa— sional severely damaged tubule where they were slightly enlarged and rounded with decreased contact to the basement membrane. Later spermatocytes, especially pachytenes, were moderately hypo- plastic and many existing cells were degenerative or necrotic. Architecture of spermatocytes and Spermatids was disrupted: cel- lular arrangement was disorganized and an occasional pachytene spermatocyte was noted at the adluminal border. A few meiotic cells were arrested and necrotic. Early Spermatids were often hypoplastic and only a few late Spermatids could be identified. Many early Spermatids through Step 11 were abnormal: cells were often in varying stages of degeneration, necrosis and premature 72 release; acrosomes were often malformed. A few abnormally shaped Step 11 Spermatids with no Step 12 or 13 in Stage XIII tubules was evidence of asynchronous development with failure of nuclear chro- matin condensation. Spermatogenic arrest was apparent in many tu- bules in Stages I-VII where there was complete absence of or only a few rare late Spermatids (Figure 14). Sertoli cell morphology ranged from relatively normal in tu— bules with prominent germinal epithelial populations to slightly rounded in moderately affected tubules to extremely small pale cells with angular nuclei in the few tubules with severe germinal epithelial hypoplasia. Though cellular density of Leydig cells appeared slightly increased, this was likely due to testicular atrophy since cell size and shape approximated those of the con— trols. The macrophage population of the interstitium was slightly increased and many of these cells were laden with large clear vacuoles. Tubular morphology of one animal differed markedly. Approximately 98% of the tubules had severely dilated lumens with only a few epithelial cells. Sertoli cells were small and pale with angular nuclei and their cytoplasm formed a thin rim around the tubule. Spermatogonia were rare and spermatocytes were ab- sent. A few early spermatids rested on the adluminal border of a All of these cells were abnormal, often binucleate few tubules. and degenerative. These cells had obviously been released from elsewhere in the tubule and were passing through the lumen. On glycol methacrylate sections, 100 tubules were examined. Of these, all but two were as described above and the remaining appeared to have a complete, normal complement of germinal epithe- Figure 14. Testicular Tissue of Rat Treated with Doxorubicin at 16 Days of Age and Sacrificed at 129 Days of Age. Architectural disruption and spermatogenic arrest of the seminiferous epithelium was evidenced by almost total absence of the spermatocyte and early Spermatid layers and no late Spermatids. (Magnification: 180x). 74 lium. One tubule was a Stage VIII and mature spermatozoa were be— ing released. Leydig cells were slightly hypertrophic with abun— dant basophilic cytoplasm and stellate shape. Interstitial macro- phages were similar to other animals of the group. After both 5 and 12 weeks of serial mating, epididymal tu- bules of all animals contained mild to moderate numbers of de- generative germinal epithelial cells, most of which appeared to be spermatids. Spermatozoa were absent or severely decreased in number. Occasionally, there were small foci of inflammatory cells, primarily lymphocytes, in the peritubular areas of the in- terstitium. One animal after 5 weeks of serial mating and all after 12 weeks had mild hepatic change, including sinusoidal disruption, swollen hepatocytes and dilated central veins. These alterations may have been secondary to cardiotoxicity which is often asso- ciated with doxorubicin. Another animal at 5 weeks of serial mating had severe unilateral hydronephrosis. e. Treatment at 24 Days of Age: Phases I and II Three days post exposure (Phase I), occasional spermatocytes were degenerative or necrotic. Spermatogonia appeared unaffected. At 7 and 14 days after treatment, spermatogenic cells were morpho— logically normal. At 80 days of age (Phase II, 5 weeks of mat- ing), one animal had a few tubules in the subcapsular area (glycol methacrylate section) that were atrophic due primarily to hypo- plasia of spermatocytes and spermatids. When present in affected tubules, these cell types were often degenerative or necrotic. 75 This pathologic change, however, was the only alteration observed in any of the animals at 5 weeks and 12 weeks of mating (Figure 15). Therefore, when exposed at 24 days of age, there appeared to have been only mild acute cytotoxicity affecting primarily sperma- tocytes. No visible morphologic lesions were observed in any organs at 3, 7 or 14 days post exposure. During the second phase, at 80 days of age, the lumen of epididymides possessed spermatozoa and a small population of degenerative germinal epithelial cells. At 129 days of age, only one animal had evidence of sloughed degen- erative germinal epithelial cells in the epididymal lumen and the sperm populations approximated those of the controls. All animals after 5 weeks of mating (80 days of age) and one after 12 weeks of mating (129 days of age) had liver changes simi- lar to those described for the 16-day treatment animals and one rat in the 5 week mating group had unilateral hydronephrosis. f. Treatment at 45 Days of Age: Phases I and II An occasional tubule of one animal had mild to moderate de- generative changes in spermatocytes and spermatids 3 days after treatment (Phase I). At 7 and 14 days post treatment, all evi— dence of acute cytotoxicity had disappeared and testicular mor- phology was normal in all animals. In Phase II, at 5 and 12 weeks of mating (80 and 129 days of age), all testicular morphology was normal with the exception of one animal. In this animal, 20% of 100 tubules examined were altered, and spermatocytes were most severely affected. Pachytene spermatocytes were often hypoplastic 76 Figure 15. Testicular Tissue of Rat Treated with Doxorubicin at 24 Days of Age and Sacrificed at 129 Days of Age. TestiCular morphology was normal (Magnification: 180x). Fig Figure 16. Testicular Tissue of Rat Treated with Doxorubicin at 45 Days of Age and Sacrificed at 129 Days of Age. Testicular morphology was normal (Magnification: 180x). FiSUr 77 t icin a Doxorub Days of Age. _ification ) VA 0 00 1 a o Figure 15. Figure 16. ———r————_" 78 and existing cells were frequently swollen and degenerative. Oc— casionally, there was mild architectural disorganization of the spermatid cell layer. A few Sertoli cell nuclei appeared slightly enlarged and rounded. Morphologic alterations in ani- mals exposed at 45 days of age were extremely minimal (Figure 16). No visible lesions were observed in other organs at 3, 7 or 14 days post treatment in Phase I. In Phase II, there was very mild hepatic congestion in all animals at 80 and 129 days of age. Because the degree of change was slight, it was probably not of clinical significance. 5. Serial Mating Data a. Treatment at 6 Da 5 of A e and 19) Fertility data of the animals treated with doxorubicin at Table 11 Fi ures 17 18 6 days of age showed that these animals were sterile, with the exception of one male who produced two viable implants in week 5. b. Treatment at 16 Da 5 of A e Table 12 Fi ures l7 l8 and 192 The onset of reproductive capacity was delayed for about one week, and fertility was 30% for this group during week 2 (Table 12, Figure 17). Both findings were statistically different from those of the controls. The percentages of fertile males were lower than control values during the entire mating study. The differences were significant in weeks 5 to 7, and 11 to 12. For the period of onset of reproductive capacity (weeks 1 to 4), overall fertility was only 40.0%. During the period of — ‘ 79 established fertility (weeks 5 to 12), 57.4% of the matings of males treated with doxorubicin resulted in viable implants. Dur— ing the first three weeks of the mating study and in weeks 8 and 11, the total number of implants and the number of viable im— plants were reduced significantly compared to the control values. Overall, doxorubicin treated males produced significantly smaller numbers of implants (11.8 total implants per female) and a smaller number of viable implants per female (11.0) than the con- trol animals (13.8 total implants per female and 13.4 viable im- plants per female). The mean number of resorptions (0.88 per female) was slightly higher than that of the controls (0.47 per litter)(Table 12, Figure 19). c. Treatment at 24 and 45 Days of Age (Tables 13 and l4I Figures l7. l8 and 19). Serial mating data of males treated on day 24 and day 45 of age were not significantly different from the control values, with the exception of an increased resorption rate in week 2 of the mating study with the animals treated at 45 days of age. Taking into account the large number of comparisons made, this one marginal effect was not regarded as treatment related. 6. Functional and Biochemical Data (Table 15, Figures 17 and 20) a. Treatment at 6 Days of Age Spermatids were absent in testicular homogenates of 80 day old animals (after 5 weeks of mating), as were sperm heads in epididymal homogenates. At the end of the trial (129 days of age; 12 weeks of mating), however, a few (0.5% of control values) 80 spermatids and sperm heads were found in the respective tissue homogenates (Table 15, Figure 17). ABP, measured as 3H-DHT bound to a cytosolic preparation, was absent in caput epididymides in the doxorubicin—treated ani— mals sacrificed at 80 days of age. A slight increase in ABP (28.7% of control value) occurred in epididymal cytosols of the drug—treated animals at the end of the study (Figure 20). b. Treatment at 16 Days of Age Spermatid reserves in the testes and sperm counts in the epididymides performed in 10 animals per group were signifi- cantly decreased at 80 days of age (14.4% and 5.4% of the control values) and particularly at 129 days of age (2.6% and 0.5% of the control values)(Table 15, Figure 17). ABP measurements in epididymal cytosols did not reveal any differences between the treated animals and the controls (Figure 20). c. Treatment at 24 Days of Age The number of spermatid heads in the testes and the sperm counts in the epididymides were significantly decreased at 80 days of age only (16.8% and 27.4% of control values, respec- tively)(Table 15, Figure 17). A decrease in ABP in the epididymal homogenates to 57.3% of the control values in the animals at 80 days of age was followed by a significant increase to 176.9% of the control value at the end of the study (129 days of age)(Table 15, Figure 20). 11“! (I...:\...(Ar.l§(\l£ FLT fig ENHHHUHNMRH. uH-H: wflfirfi M0.0 VAM 01 M33 #93 nmflo wmeE 05. AU #83 pmfi no? mama mac 3 OH H Egon H3238 you H8» mom 3 II o In In I: o o m\o NH II o I: II I: o o m\o d I: 0 II In I: o o m\o 3 II o I: I: I: o o m\o m ul 0 ..l I: I: o o no? m 010. I: o ul ul II o o nmxo s ..I o I: ul ul 0 o o“<0 6 am an I: 0 am an *3 S) m I: o .l I- I: o o 33 a II o In nl I. o o 23 .m I: o In I: .l o o Sxo m I. o I. I: II o o 33 H mm + x mm + x nappflx mm H x umfiflx E 8%.: x83 384”an 383cm uflpfl\ mommies $5.“an 8?: 8§wuaflsfi gums 8303 £83 mcoflmuommm maoflmuommm Eon H305 338m 20353 @8qu HEwm £932 8 mos mo mama e um voyage manna Bug 5035969 5 $8 sfiflfiwm u: wanna Fertility Data in Doxorubicin heated Animals Treated at 16 Days of Age (3 mg/kg)a Table 12 Viable Implants Viable Implants ter Lit /_ Resorptions tions Total Reso Total Implants Implants Males Litters w/Viable Fertile Inplants/Males erl Ma S x+SE /Litter Xi-SE ter x+SE Lit / Mated (9.) Week 0/10 17 5.7 i- 2.9* 0 .14 i .14 17 5.7 i- 2.9* 30* 3/10 72 10.3 i- 1.9* .14 i- .14 10.4 i- 1.9* 73 70 7/10 .33 i .21 75 12.5 i- 0.8 26 77 60 6/10 49 16.3 i- 2.4 16 5.33 i 5.33 33 11.0 1- 3.1 30* 3/10 68 11.3 i- 2.0 .33 j .21 11.7 i 2.0 70 60* 6/10 82 81 13.5 i 1.4 .83 i- .54 86 14.3 i- 1.5 60* 6/10 61 8.7 i- 1.4* .29 i .18 9.0 i 1.5* 63 70 7/10 14.4 i- 0.8 101 .29 i- .29 14.7 i- 0.8 103 70 7/10 70 91 13.0 i- 1.9 7 1.00 i .53 84 12.0 i 1.8 7/10 10 5/10 50* 66 9.4 -_i-_ 2.4* 14 2.00 i 1.23* 52 10.4 i 2.9* 11 57 11.4 i- 2.5 1.00 i- 1.00 5 62 50* 5/10 See text for experimental detail N=10 a) * p < 0.05 . ‘ .wlnllnué (A... “1+5 BHHERN um.” mHfiH 83 +| +l +l +l +1 +l +l +1 +| +l +l NMH bHH va hmH ovH hm bNH ONH mm +| mm. H 0%. H 00. +| om. + on. +1 +1 +l I O c> O m r1 m H \9 1n +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +l mm 11. x mummies flfiasfi “wound anmH> wcoHuQHomwm msoHugnomwm OH minnow Egg you tap OVH HNH owH me mvH hm OmH mNH mm om OOH OOH OOH OOH om OOH OOH OOH om ow O E flfiaafi 3:305 wows 38. SR 33H 33H OQOH 33H oa\m oQoa 33H SSH 38 Saw oqo 8pm: megmpcmHmE. gums wflflmm 3833. @8qu fiflmm magma 8 was no g8 am pm Bug 39:5. 6388. 530.30on 5 8.8 bags mod Va a do NH HH OH Mama "MH anmH Doxorubicin Treated Animals Treated at 45 Days of age (3 mg/kg)a Fertility Data in Table 14 : Viable Inplants x+SE Viable /;.itter Implants XiSE Resorptions Resorptions Total (95) 10 Males Implants w/Viable Fertile es ants Mated Litters eri Mating Impl Week S 14.0 14 0/10 11.0 i- 1.2 105 11.7 i 1.3 6* .67 i- .33 99 90 9/10 12.5 _-|_- 1.9 125 .20 i- .13 127 12.7 i 1.9 100 10/10 142 14.2 i- 0.5 16 .10 i- .10 141 14.1 i- 0.5 100 10/10 14.3 -_I- 1.7 114 .56 121 13.4 i- 1.2 76 .78 i 80 8/10 11.7 i- 1.8 117 .70 i- .33 124 12.4 + 1.6 100 10/10 84 14.2 i- 0.4 142 .40 i .30 14.6 i- 0.4 146 100 10/10 13.1 i 0.8 131 .27 .50 i- 136 13.6 i 0.9 100 10/10 121 13.4 i 0.8 .67 i- .24 14.1 i 0.8 127 90 9/10 124 12.4 + 1.3 .90 i- .35 10/10 100 133 13.3 i 1.1 10 14.3 i- 1.1 129 .56 i- .44 9/10 90 134 14.9 i 1.1 11 13.2 i- 1.6 119 .33 i .17 13.6 i 1.4 122 90 9/10 tal deta' s experimen See text for N=10 * p<0.05 a) 85 m00.0 v a 4 was mo when $4 8. monommwhfioo 9.4384 HMHwa Ho 0483 fimH 9.0 mHflficm no 08804404 40 mm H 8548 594 3 .80 mo 948 00 8 8884.48 gums 48.48 Ho 48.3 5m 0m 40$ 0.0 H «.04 and 0mm H 445 34V 00.4 H 40.4 34V 00. H 40.4 4 I I I mHofiEoo 1| 33 m8 + 84 ad 8. + 8.4 34V mm. + 404 m 84081094 84v to H 0.04 5 0mm H 0044 03 mm. H 004 3; mm. H 004 m4 111 5 mam H 0004 E 04. H .444 5 mm. H 04.4.. m m4 >8 84V 5.0 H m.04 E 4004 H 84 43 0.4. H 40.4 E 40. H E4 4 111 5 Re H 4404 5 40. H 40.0 5 «mm. H 40.0 m «N >8 84V 3.4 H 4.0 E 004 H 40m 5 .440. H 40.0 E .48. H m0.0 44 1| 5 000 H «404 5 to H «4.0 5 48. H mmd m 04 m8 84V *0. H 4.0 5 .84. H 404 E .440. H 40.0 3 +8. H 40.0 nm4 111 E .0 E *0 05 *0 am 0 >8 A 04 5 044 04 as a N4 04 33439.5 0: 004>§e mmamgflom @4me 04. 0483 um 80.8.44. mm 8834 40 181mm 885 04 0588 840.8 04 Ag? 8 89052 $90,492 mmd $9400 044me 3444844.QO nooflHHomm 443.438.3048 654484 44.4.4.8 Ho 043m 8&2 9483 NH .40 m boom 14.408 .08 00 £48 04 448 4m .04 .0 p0 8488. mg . Ems 5.88.4 94.62444 58.4484 so 80 wHEmanHmm 5 3.4800 482.4 04.8% .mHummH. CH wmzwmmm 6384.4me :0 494504 8 5034440089 Ho ”48.4.4.4 "m4 mHoBH FERTILITY p-O-q lawn-T200 100+ pr-O-o—o‘wp-ZOO d I O 1 4-- fl 4 04> I» nm 0 LIVE IMPLANTS AS PERCENTAGE OF CONTROL :00 . . 24 C I O o I I 1 1 4 8 12 ( 4 m— _a N WEEK OF SERIAL MATING Age in days at treatment ‘ Fertile males control, N = 10 Fertile males doxorubicin N = 10 Sperm counts in cauda epididymidis, N = 3 p < 0.05 ** p < 0.01 Figure 17. Fertility and Sperm Counts of Animals Treated with Doxorubicin *‘DOKB SPERM COUNT AS PERCENTAGE OF CONTROL 87 TOTAL IMPLANTS 20[ ZOP 9' b'o‘oodp'o‘O-o IO— II IO 55 ,’ E III———1Hr a (Z) 0 V I/\ I i 0 <2, 0 4 e 12 o E z 20- 20 5' o. F3 0 IO' IO 243 453 C t l 1 1 C 1 1 I O 4 8 l2 0 4 8 I2 WEEK OF SERIAL MATING a Age in days at treatment 0 Control; N = 10 ‘ Doxorubicin; N = 10 * p <0.05 ** p < 0.01 Figure 18. Total Implants of Animals Treated with Doxorubicin LIVE IMPLANTS/LITTER 20’» IO‘ a O A k * F I [F————tt-————————¥66 5 O- O LIVE IMPLANTS AND RESORPTIONS 20" fetch-adpofl‘o I _ RESORPTIONS/LITTER WEEK OF SERIAL MATING Age in da 3 of treatment Control; 8 = 10 Doxorubicin; N = 10 p < 0.05 * p < 0.01 igure 19. Live Implants and Resorptions of Animals Treated with Doxorubicin PERCENTAGE OF CONTROL ANDROGEN BINDING PROTEIN 200” 200[ IOO IOO_——- _..._._._..._ ‘ i n: .1. 63. I63 0 T O 1 b 0 5b 12b 0 5 12b .1. 1328 t 1 200[- I 200i- IOO i IOO 1 243 l 45a 0 T C 1 1 0 5b I2b 0 5b [29 WEEK OF SERIAL MATING a) Age in days of treatment b) N = 3 * p < 0.05 ** p < 0.01 Figure 20. Androgen Binding Protein of Animals Treated with Doxorubicin 90 d. Treatment at 45 Days of Age The parameters measured in the males treated at 45 days of age showed no statistically significant differences from the controls. With the exception of the ABP levels at the end of the study, all values fell between 90% and 120% of the control values. The high level of ABP measured in 129 day old animals (181.2% of the control value) was associated with a large vari- ability in individual values and was therefore not significantly different from the control (Table 15, Figures 17 and 20). Procarbazine 1. Clinical signs During Phase I, partial alopecia occurred in most animals between 7 and 14 days post treatment. 2. Gross necropsy At sacrifice 14 days after exposure (Phase I), most animals had partial hair loss. No other gross changes were observed and no deaths occurred during either one of the phases. 3. Body Weight: Testicular and Epididymal Weights (Tables l6. l7 and 18) a. Treatment at 6 Days of age The body, testicular and epididymal weights were not signi— ficantly different from control values at any time point during either Phase I or II. c. Treatment at 16 Days of Age Body weights of males treated at 16 days of age and sacri- ficed at 14 days after exposure showed a significant increase in 91 body weight (121.8%) as compared to the controls. No differences from the controls were seen in any other group of animals. Testicular weights were significantly decreased from con— trols at the 14 day post treatment period (44%) during Phase I and at the age of 129 days (66.5%) during Phase II. Epididymal weights were also decreased at 129 days of age (64.2% from con- trols). c. Treatment at 24 Days of Age Body weights were significantly higher than controls at 14 days after exposure (122.1%, Phase I). No other differences from the controls were noted at any other observation points in either Phase I or II. No statistical differences in testicular or epididymal weights were seen during Phase I. In Phase II, testicular weights were only significantly decreased at 129 days of age (67.2% of controls) and the epididymal weights only at 80 days of age (59.4% of controls). d. Treatment at 45 Days of Age In Phase I of the study, body weights were significantly lower than controls at 3 days post exposure (86.6%) and higher at 7 days post exposure (109.8%). No changes in body weight occurred during the second phase of the study. Both testicular and epididymal weights were significantly decreased from controls at 3 days post exposure (Phase I), and at 80 and 129 days of age (Phase II; testis: 79.3%, 59.4%, and 92 40.0 v a t. 00.0 v a an am H 0548 :85 0 00 won no mama 0N4 B mgommmunoo 0483 fiNH .04 u z «o 0008 0000 00888888483430 .012 3 0 u z H0 0.04 H 0.000. 0.04 H 0.000 0.0 H 0.000 30.0 H 0.4.00 0.0 H 4.44.4 0044084808 0.00 H 0.4.00 0.00 H 0.004 0.44 H 0.000 4.4 H 0.000 0.04 H 0.4.04 4940.08 04. 0.00 H 0.040 4.04 H 0.000 0.0 H 0.044 400.0 H 0.404 0.0 H 0.00 84.00.880.404 0.00 H 0.000 0.0 H H.000 0.0 H 0.444 0.4 H 0.00 0.0 H 0.04. 40.4028 40 0.00 H 0.000 0.0 H 0.000 +4.0 H 0.00 0.0 H 4.4.4 0.0 H 0.00 68030088 04 H 0.000 0.04 H 4.400 0.4 H 0.00 0.4 H 0.00 0.4 H 0.04. 40528 04 0.04 H 0.000 0.00 H 0.400 0.0 H 0.04. 40.4 H 0.00 0.4 H 0.04 09.408.480.404 0.04 H 0.040 0.0 H 0.000 0.4 H 4.44. 4.0 H 0.44 4004 H 0.04 408.28 0 004 n0 004 as 00 90.4440: 48me Ho Moo: 0.85848 .4802 >8 0558.48 0.0 0mm 4448.33 >408 so 80R? 008 $50? no 003mm "0H anmH. 93 HO.o v m .34 00.0 v m 4 4.40 H 0548 480440 02 8 000 00 008 004 8 08808.48 483 £04 .04 1 z 8 000 no 0.48 00 8. 08880.48 0483 6.0 .0 u z 3 0 u z 10 30.000 H 0.000 30.004 H 0.000 0.044 H 0.4404 0.40 H 0.0044 0.00 H 0.000 84084088 0.00 H 0.0004 0.00 H 0.0004 0.400 H 0.0044 0.04 H 0.0044 0.00 H 0.4004 49.508 04 3.0.04 H 0.0044 0.00 H 0.0044 0.04 H 0.000 0.00 H 0.004 0.00 H 0.400 0040094808 0.400 H 0.4040 0.40 H 0.0004 0.00 H 0.000 0.0 H 0.440 0.40 H 0.000 40.40.80 40 3.0.004 H 0.4004 0.000 H 0.0044 40.04 H 0.004 0.0 H 0.00 0.0 H 0.44 840848940 0.40 H 0.0004 0.04 H 0.0004 0.04 H 0.000 0.0 H 0.044 0.4 H 0.00 405:8 04 0.404 H 0.0004 0.00 H 0.0004 0.0 H 0.00 0.4 H 0.00 0.0 H 0.04 840840840 0.00 H 0004 0.044 H 0.0004 0.0 H 0.00 0.4 H 0.00 00.0 H 0.44 40.488 0 004 n40 4.44 400 00 94.4.8.4 4.04400 .40 0483 pgmeH. 34 mg 05:58.04. pm $3 0.443003 404503.009 no 493004 oomv 05000041400004 mo poommm "AH GHQMB HO.O V m .404 00.0 v d .4 4.40 H 0540> 44er 0“ M400 04 1 0.000 m6 044040 0048m§400400H40044003fiNH . u n 0008044040 00308098048350 mum M0 iii IIIIIIIIII 44.40 H 0.044 40.00 H 0.000 4.00 H 0.040 4.00 H 0.000 440.0 H 0.004 044400004008 0.40 H 0.000 0.00 H 0.000 0.00 H 0.400 0.04 H 0.004 0.04 H 0.400 494.048 04 IIIIIIIII 0.4 H 0.004 44000 H 0.040 4.44 H 0.004 0.4 H 0.00 I1 0040084088 1 1 1 1 0 4 0.0 + 0.000 0.00 + 0.000 0.04 + 0.00 0.04 + 0.00 I 40.40.08 4 9 $40.00 H 0.000 0.04 H 0.400 111 111 111 0040000440098 1 1 1 . c H 0.40 + 0.000 4.4 + 0.004 I 0.0 + 0 04 I1 494» 8 0 I I III N 0.00 + 0.400 0.00 + 0.000 I1 I 004 840080 1 1 111 coo 0 0.00 + 0.000 00.04 + 0.004 111 I 40.40. 004 0.0 044 00 00 0 094 0443044 404.400 .16 04003 0005008 000040 .08 00050000 0. 0.00403 4800404040 :0 80402 0000 004080088 no H0800 "04 0400.4 95 56.7%, respectively; epididymis: 64.0%, 77%, 73.4%, respec- tively). 4. Morphologic Evaluation a. Treatment at 6 Days of Age In the first phase of the study, acute cytotoxic damage to the seminiferous epithelium of the testis was not evident 3 days after exposure. The integrity and population of the cells ap- peared normal and mitotic figures in both gonocytes and Sertoli cells were evident. At 7 days post exposure, large clumps of Leydig cells were present at an age (13 days) when this cell type should be relatively rare. Necrotic spermatogonia and primary spermatocytes could occasionally be identified. Mild to moderate damage to the germinal epithelium was still obvious in occasional tubules. Binucleate spermatocytes could occasionally be identi- fied near tubular lumens and there were a few necrotic spermato- gonia and primary spermatocytes near the basement membrane. In the second phase of the study, after 5 weeks of serial mating (80 days of age), although most tubules appeared morpho— logically normal, occasional tubules were severely damaged. In the affected tubules, the architecture was disrupted with loss of most of the spermatogonia and primary spermatocyte cell layers. Sertoli cell cytoplasm was vacuolated and filled with debris, much of which was likely necrotic cellular debris (Figure 21). At 129 days of age, almost all tubules of all animals appeared morphologically normal with the exception of a few necrotic sper~ matogonia Figure 21. 96 .. 1.0 ~ 7" .‘A r Testicular Tissue of Rat Treated with Procarbazine at 6 Days of Age and Sacrificed at 80 Days of Age. Severe architectural disruption was present with loss of most of the spermatogonial and spermatocyte layers. Sertoli cell cytoplasm was vacuolated and filled with debris (Magnification: 360x). 97 and/or early spermatocytes along the basement membrane. There— fore, by the end of Phase II (Figures 22 and 23), there was no longer evidence of the mild Leydig cell hyperplasia or germinal cell damage that was observed at earlier time points. b. Treatment at 16 Days of Age Three days following exposure in Phase I (19 days of age) there was marked retardation of tubular lumen formation. Lumen formation is temporally associated with formation of the blood- testis barrier. Necrotic spermatocytes could occasionally be identified but the spermatogonia did not appear to be damaged. By 7 days after exposure (21 days of age), tubular lumen forma- tion was still retarded. Of those present, diameters were still smaller than normal. At this time point, the germinal epithelium was also affected. Decreased numbers of spermatogonia and sper— matocytes were present in many tubules. If present, the sperma- tocytes were often degenerative or necrotic. Large clumps of Leydig cells could occasionally be identified. By 14 days after exposure (30 days of age), there was a severe loss of germinal epithelium in many tubules; remaining cells were usually degener— ative or necrotic. Spermatocytes and spermatids were most se- verely affected. Sertoli cells were atrophic, with pale vacuo- lated cytoplasm in damaged tubules. Leydig cell density appeared increased, but this was likely due to seminiferous tubular atro- phy. At 80 days of age (Phase II, 5 weeks of mating), most tu- bules appeared morphologically normal and there was frequent Figure 22. Figure 23. 98 Testicular Tissue of Rat Treated with Procarbazine at 6 Days of Age and Sacrificed at 129 Days of Age. Testicular morphology was similar to that of control animals (Figure 23), indicating recovery of the toxic effects of procarbazine on the germinal epithelium (Magnification: 180x). Testicular Tissue of Control Rat Sacrificed at 129 Days of Age. Testicular tissue from a control animal With normal morphology (Magnification: 180x). 99 11th Procarbazine at :9 Days of Age. to that of control :covery of the toxic minal epithelium Figure 22. 1N acrificed at ‘ an1mal on a control ion: 180x)- Figure 23. 100 evidence of mature spermatozoa ready for release. In occasional tubules, however, there was severe germinal epithelial hypoplasia with usually only a few spermatogonia and primary spermatocytes remaining. These affected tubules had marked architectural dis- ruption with decreased tubular diameters, atrophic Sertoli cells with pale vacuolated cytoplasm and the remaining germinal epithe— lium was usually degenerative (Figure 24). At 129 days of age (12 weeks of mating), there was a marked variation in severity of tubular damage between individual animals. Though some appeared morphologically normal, others had severe disruption and hypopla- sia of the germinal epithelium in most tubules (Figures 25 and 26). In these affected animals, the most mature germinal epithe- lial cells were most severely damaged. Often, only spermatogonia and spermatocytes remained and these were degenerative. Sertoli cells were atrophic, pale and vacuolated. Some tubules had no lumen. Leydig cell density appeared increased in most animals. During Phase I, most animals had moderately acute damage in many tubules. However, by the end of the serial mating (at 129 days of age), only a few tubules were seen that were permanently damaged. c. Treatment at 24 Days of Age Three days after exposure during Phase I, there was no evi- dence of acute toxic damage. Cells appeared morphologically nor- mal and there were abundant mitotic and meiotic figures in the germinal epithelial cells. By 7 days post exposure, the germinal epithelium showed evidence of moderate to severe degeneration and Figure 24. Testicular Tissue of Rat Treated with Procarbazine at 16 Days of Age and Sacrificed at 80 Days of Age. Severe germinal hypoplasia was evidenced by the presence of only a few spermatogonia and spermatocytes. These remaining germinal cells were degenerative (Magnification: 180x). 102 Figure 25. Testicular Tissue of Rat Treated with Procarbazine at 16 Days of Age and Sacrificed at 129 Days of Age. Testicular tissue was morphologically normal (Magnification: 180x). Figure 26. Testicular Tissue of Rat Treated with Procarbazine at 16 Days of Age and Sacrificed at 129 Days of Age. Most tubules were atrophic, with severe germinal cell hypoplasia and spermatogenic arrest (Magnification: 180x). 103 ith Procarbazine at 29 Days of Age. lly normal Figure 25_ ith Procarbazine at 29 Days of Age. evere ermina t (Magnifi Figure 26. — V 104 necrosis in many tubules; the spermatocytes were most severely affected. At 14 days post exposure, only mild degeneration and necrosis of the germinal epithelium was seen in a few tubules. In Phase II of the study, at 80 days of age (after 5 weeks of serial mating), most tubules appeared to be morphologically normal with a slight decrease in spermatocytes and spermatids in only a few tubules. At 129 days of age, most tubules were mor— phologically normal. A few were severely damaged with almost no germinal epithelial cells remaining and those present were de- generative and necrotic (Figure 27). d. Treatment at 45 Days of Age An early loss of spermatogonia along the basement membrane was apparent by 3 days after exposure (48 days of age) during Phase I. Acute and severe damage was obvious in many tubules by 7 days after exposure. There was marked degeneration and necro- sis of all germinal epithelial cell types. Large multinucleate cells in tubular lumens were evidence that spermatids had sloughed. Spermatogonia and spermatocytes were often degenera- tive or necrotic. Loss of spermatogonia and spermatocytes was common at 14 days after exposure. Many necrotic spermatogonia and spermatocytes were located near the basement membrane. To further emphasize the evidence of spermatogonia and spermato- cyte loss, Stage II spermatids could frequently be identified near the basement membrane. In Phase II, by 80 days of age (after 5 weeks of mating), there was a marked loss of germ cells (especially spermatocytes 105 and spermatids) in many tubules of all animals. Leydig cells appeared more densely populated, but again this was probably due to decreased testicular weight (Figure 28). At the final sacri- fice point, 129 days of age, many tubules remained permanently damaged, often with essentially a "Sertoli cell only" pattern and an occasional spermatogonium. These tubules were atrophic and there was total spermatogenic arrest (Figures 29 and 30). 5. Serial Mating Data a. Treatment at 6 Days of Age (Table 19, Figures 31, 32 and 33) The onset of reproductive capacity was delayed for about 1 week and fertility was 40% in week 2 (Table 19, Figure 31). Though this is decreased from the control, it is not statistic- ally significant. By week 3, normal reproductive capacity was attained and remained so throughout the duration of the study. All other serial mating data were not significantly different from the control values. b. Treatment at 16 Da 3 of A e Table 20 F1 ures 31 32 and 33) The onset of reproductive capacity was delayed 2 weeks. During the third week, only 30% of the males were fertile. Both findings were statistically different from those of the controls (Table 20, Figure 31). During the third week, both total im- plants per female and viable implants per female were also sta- tistically lower than control values (Table 20, Figures 32 and 33). Due to these early effects on fertility, the average number of litters produced per male in 12 weeks (Table 23) was significantly less than control values (73.5% of control). 106 Figure 27. Testicular Tissue of Rat Treated with Procarbazine at 24 Days of Age and Sacrificed at 129 Days of Age. Although most tubules were morphologically normal, a few tubules were severely damaged with almost total absence of germinal epithelial cells. Early spermatids were most mature cell type present. (Magnification: 180x) . Figure 28. Testicular Tissue of Rat Treated with Procarbazine at 45 Days of Age and Sacrificed at 80 Days of Age. Severe germinal hypoplasia involved primarily spermatocytes and early spermatids with a complete absence of late spermatids (Magnification: 360x). ith Procarbazine at 29 Days of Age. ogically normal, 2 with almost total ls. Early spermatids (Magnification: at - h Procarbazine 1‘ Severe 0 Days of Age. Figure 28. Figure 29. Figure 30. 108 Testicular Tissue of Rat Treated with Procarbazine at 45 Days of Age and Sacrificed at 129 Days of Age. Many tubules remained severely damaged, often with essentially a 'Sertoli—cell' only pattern with occasional spermatogonia. These tubules were atro- phic with spermatogenic arrest (Magnification: 180x). Testicular Tissue of Rat Treated with Procarbazine at 45 Days of Age and Sacrificed at 129 Days of Age. Many seminiferous tubules had marked depletion of spermatid cell layer. Remaining spermatocytes were often swollen, degenerative and located near the adluminal border (Magnification: 360x). 109 :arbazine ays of Age. Eten with wifl1 vere atro— :ion: 180x). Figure 29. arbazine at Of Age, Many of spermatid aften swollen: 1 border Figure 30.\ 110 By week 4, all remaining serial mating data were not sta- tistically different from control values with the exception of an increased resorption rate in week 12. Taking into considera- tion the number of comparisons made, this marginal effect was not regarded as treatment related. c. Treatment at 24 Days of Age (Table 21, Figures 31, 32 and 33) The onset of reproductive capacity was delayed for about 1 week and fertility was 20% in week 2 (Table 21, Figure 31). The percentages of fertile males were lower than those of the con- trols during the first 6 weeks of the mating study. The differ- ences were significant in weeks 2, 4, and 6. The number of total implants per female and viable implants per female were statis- tically lower than control values in weeks 3 and 4 (total im- plants: 71.0% and 59%, respectively; viable implants: 68.4% and 57.6%, respectively)(Tab1e 21, Figures 32 and 33). The number of resorptions was significantly increased in weeks 6 and 12 (Table 21, Figure 33). Over the 12 weeks of serial mating, the average number of litters produced per male was 86.3% of control values, which was significantly decreased (Table 23). d. Treatment at 45 Da 5 of A e Table 22 Fi ures 31 32 and 33) Fertility of animals treated at 45 days of age was severely affected. Though there was no delay in the onset of reproductive capacity, the percentage of fertile males was lower than control values in weeks 3-12 and differences were statistically signifi- cant in weeks 3, 5, 6, 7, 8, 9 and 12 (57.1%, 31.6%, 73.1%, 111 64.9%, 31.6%, 21% and 61.5%, respectively)(Table 22, Figure 31). Total implants and viable implants were significantly decreased in weeks 2, 3, 4, 7, 8 and 10 (total implants: 56.0%, 77.2%, 40.8%, 59.3%, 42.5% and 55.3%, respectively; viable implants: 52.8%, 67.6%, 39.6%, 56.6%, 38.8% and 56.5%, respectively)(Table 22, Figures 32 and 33). Overall, procarbazine treated males pro— duced significantly fewer litters (49 litters/ 120 matings), smaller litters (8.8 total implants/female) and a smaller number of viable implants per female (8.2) than did control animals (13.8 total implants per female and 13.4 viable implants per female). The mean number of resorptions (0.49 per female) was approximately the same as the control values. These severe de- creases in fertility caused the average number of litters pro- duced per male (Table 23) to be significantly lower than control values (58.8% of control). 6. Functional and Biochemical Data (Table 23, Figures 31 and 331 aw Spermatids in testicular homogenates and sperm heads in epi- didymal homogenates approximated control values at both 80 and 129 days of age. ABP, measured as 3H—DHT bound to an epididy- mal cytosolic preparation, was also within the normal range set by control animals. b. Treatment at 16 Days of Age Testicular spermatid reserves and epididymal sperm counts were both significantly decreased as compared to controls in ani- mals at 80 days of age (60.9% and 25.1% of control, 112 respectively). At 129 days of age (after 12 weeks of mating), the testicular spermatid reserves were within control values, but the epididymal count remained decreased (35.6% of control). Cytosolic ABP measurement did not differ from control. c. Treatment at 24 Days of Age Spermatid reserves in the testes and the sperm counts in the epididymides performed in 10 animals per group were significantly decreased at 80 days of age (50.0% and 20.1% of control values, respectively), but both testicular and epididymal counts were within control range at 129 days of age. ABP measurements in epididymal cytosols did not reveal any differences between the treated animals and controls. 0W Testicular spermatid reserve values were significantly de— creased from controls only at 80 days of age (17.8% of control). This value was within normal range at 129 days. There were no significant differences in sperm counts in epididymides or ABP at either time point. Cyclophosphamide 1. Clinical Signs Animals in all treatment groups showed evidence of systemic toxicity. Growth rate was retarded, animals were thin and had varying amounts of alopecia. During Phase II, skulls of ani- mals treated at 6 days of age did not develop normally. Both the maxilla and mandible were shortened and there was significant loss of teeth. (For this reason, a special pulverized food was fed to these animals.) OH H z 03000.0 344.054.4080 140“ 8400 00m 40 0.0 H 0.04 004 0.0 H 0.0 0 0.0 H 4.44 444 004 04\04 04 0.0 H 4.44 444 4.4 H 0.0 0 0.0 H 0.44 044 004 04\04 44 0.0 H 0.44 044 0.0 H 4.0 4 0.0 H 0.44 044 004 04\04 04 0.0 H 0.44 044 0 H 0 0 0.0 H 0.44 044 004 04\04 0 0.0 H 0.04 404 4.0 H 0.0 0 4.0 H 0.44 404 00 04\0 0 m 4.0 H 0.44 044 0.0 H 4.0 4 4.0 H 4.44 444 004 04\04 0 0.0 H 0.04 404 4.0 H 0.0 0 0.0 H 0.44 004 00 30 0 0.4 H 0.04 004 0.0 H 4.0 4 0.4 H 0.04 004 004 04\04 0 4.4 H 4.44 004 0.0 H 4.0 4 4.4 H 0.44 004 00 30 4 0.4 H 0.04 004 0 H 0 0 0.4 H 0.04 004 00 04\0 0 0.0 H 0.04 00 0.0 H 00.0 4 0.0 H 0.04 40 04 04\4 0 III III III III III O O OH\O H mm + m .40 + x WWW? 00.004944 WWW “04ng 000040.44 00404.“: 8%....”048 0540“: 044404.5 0H040H> 02039400004 wcoHéommm H309 H309 0.431400 0.30sz 0.00053 H0HH0m 0804004 008 03 Ho 04400. 0 00 00000.49 mHOEHc< 400000.49 0500044009424 00.4 0909 434.430.40.04. "04 0.30.4. OH H 2 0309040 34.484.41.090 08.14 0040» 00m 40 0.4 H 0.04 004 0.4 H 4.4 444 4.4 H 4.44 044 00 04\0 04 0.4 H 0.44 004 0.0 H 4.0 4 0.0 H 0.44 004 00 30 44 0.0 H 0.44 044 0.0 H 00.0 0 0.0 H 0.44 044 00 04\0 04 0.0 H 4.44 044 0.0 H 4.0 0 0.0 H 0.44 044 00 040 0 0.4 H 0.44 004 0.0 H 0.0 0 0.4 H 4.04 004 00 04\0 0 w... 0.0 H 0.04 004 0.4 H 0.0 0 0.0 H 0.04 004 00 04\0 0 0.0 H 0.04 044 0.0 H 4.0 4 0.0 H 0.04 004 00 30 0 0.0 H 4.0 40 0.0 H 4.4 0 0.0 H 0.04 00 00 04\0 0 0.0 H 0.04 00 0.4 H 0.0 4 0.0 H 0.44 00 00 040 4 .304 H 0.0 04 0.4 H 0.0 0 40.4 H 0.4 04 3.00 04\0 0 I1 0 I 11 I 0 .20 04\0 0 I1 0 I1 I I1 0 0 04\0 4 mm + x 034% 1 00 + m . s MMMwNH Megan WWW WWW? 8004944 0%“: 840qu0m04efi 0.0m“: . 38> 00040000000 0840.880 408.4. 400.94. 0440.400 038?3 0.400044 404000 084\0044 0000 094 no 0448 04 #0 4000.00.04. 040594 400000.49 024000040008 CH 38 5430.400 "00 04040.4. Viable Implants /Li X + SE 6.0 i- 4.0 Viable /L.itter Implants XiSE Resorptions Resorptions 'Ibtal Implants /Li x + SE 6.0 + 4.0 Total 12 (9°) 20** Males Implants Mated 0/10 2/10 Implants/Males Litters w/Viable Fertile erJ. Week Table 21: Fertility Data in Procarbazine Treated Animals Treated at 24 Days of Age (200 mg/kg)a Mating S 115 a: a: -x N m on 1-1 0 cu m to as 01 N N H 1-1 0 o 6 <5 ‘H 'H +I 'H +l 'H ‘H 'H 'H 'H 1v: 0 N m o ox 4 \0 <1- as oo o m 1-1 1*; ° 9} 1-1 14 1-1 91 7—‘1‘ H :1) tn 0 oo \o 0 xx m N \o <- xo <1- ox 1\ m m 1-1 1-1 9} 31' 21 1—1 m m to <11 <1- 4- m xx 1-1 0 o o o o +| 'H “H ‘H 'H 'H “H "H 'H "H 1-1 <11 g [x N N N N o 4- o - o o o o N o o E a: a: l\ N N m N N N N 3 <11 4: 4: N m m N ox as «1 <11 xo 0) N N 1—1 1—1 0 o -1 o o o 'H 'H 'H ‘H +I 'H +I +l 'H ‘H m <11 10 \o N 1—1 1\ \o «3 0:2 ’ ' ' 1—7 16 N <1. m m 3 °° 3 1—1 1-1 1—1 53 1-1 1-1 1-1 N N o .-1 N ox 1x \9 \o oo 0 <11 «1 l‘ " .91 °° m 9} 14 1'91 1-1 m H '5 a) o 3 o '5 o o o o o o '6 o o o o [x lo 00 1‘ S 0‘ ,4 1—1 r-i H a o o o o o o o O O E < q q q < q q q q *a o o o o ‘H l‘ to co xx 3 0‘ 1-1 ._1 .4 H m :5 o o o . . .—1 oo 33 II V V {Dz 949-1 «1 <11 10 \o b 00 0‘ O N 7'7 _x a: Viable Implants /Li x + SE 5.5 + 4.5 6.7 i' 2.0* 11 47 Implants 0.4 /Litter xifi 0.5 + 0.7 0.1 i- Resorptions Viable tions 1 Total Reso Inplants /Li 6.0 + 4.0 6.9 i- 2.0* x+SE Total Males Implants 12 48 (%) 20 7o es Mated 2/10 7/10 Fertility Data in Procarbazine Treated Animals Treated at 45 Days of Age (200 mg/kg)a Litters w/Viable Fertile ing Implants Table 22 Week Serial Mat 1- 2.4** 9.2 46 +| 10 11.2 i 1.9* 56 50* 5/10 5.5 i 2.0** 10.0 i- 3.8 10.6 i- 2.1 7.7 + 2.6 * 5.0 i 3.5* 32 30 74 46 0.5 + 0.5 1.3 + 2.3 0.1 + 0.4 0.7 + 1.6 0.7 + 1.2 3 4 2 5.8 i 1.9* 11.3 i- 2.6 8.3 i- 2.7* 5.7 i- 3.3* 10.7 + 2.2 35 34 75 50 17 60 30** 70** 60* 30** 6/10 3/10 7/10 6/10 3/10 15 +| 15 20** 2/10 7.4 i- 2.0* 11.7 i- 2.1 ll.5 + 1.7 37 92 70 0.5 + 1.0 0.5 + 0.8 1.0 + 2.0 2 7.8 i 1.9* ubils 12.7 + 1.8 39 96 76 S 50 80 60** mental de 4/10 8/10 6/10 12 See text for exper N= 10 10 11 a) 117 mo.o v Q 0. 000 me 0000 004 8. 0000000008 04.4000 404000 me 4003 0004 so 04004.45 .00 .4085: AU 00 H 000405 0000 3 000 no 04400 ow 00 044.409.000.400 040.4003 404.400 Mo 0003 :00 A0 84V 0.0 H 0.04 4040 000 H 440 4040 00.4 H 40.4 4040 40.0 H 40.4 04 1 1 400008 111 A040 000 H 0004 4040 00.0 + 00.0 0400.0 + 00.0 0 00480 000 840 44.0 H 0.0 5 000 H 000 5 00. H 00. 5 00. H 00. 04 111 5 0000 H 0404 5 00. H 00.4 5 400. H 00. 0 04 000 840 404.4 H 0.0 5 004 H 040 05 00. H 4.0.4 5 40. H 00.0 04 111 5 040 H 04.04 5 44.4. H 04. 5 40.0 H 40.4 0 40 000 840 400.0 H 0.0 5 400 H 0004 5 400. H 00. 5 00 H 00.4 04 111 5 0040 H 4000 5 4 40. H 00. 5 400 H 00.4 0 04 0400 840 00. H 0.04 5 000 H 000 5 04. H 00.4 5 00 H 40.0 0404 111 5 004. H 0044 5 400 H 00.4 05 44 H 00.4 00 0 000 01004 H 00 04004 H 00 W00: 04 04 0340080 0.4 00404000 0000040494 04000.4 04 0003 00 00000.44. 4020000044 00 40010 45000 04 03.0000 94.400: 04 40400 0000 0400052 0.00.0020 mm< 004500 44ng Uflugwmm 000404.400m 0500044000044 .300: 404.400 .00 0.4000 400.50 9003 04 020 m 00 000004.400m .090 00 04400 04 0:0 40 .04 .0 00 00000.44 0400454 . 40040 040080 04440040 00000000 so 0:0 04000404040. 5 004500 400004 2.0090 4040.009 441.4 0050094 040% so 804504 83 04440004408404 00 00000.4... "00 040405 ‘- _lJ LIVE IMPLANTS AS PERCENTAGE OF CONTROL 118 FERTILITY >f. X- *‘DOm WEEK OF SERIAL MATING Age in days at treatment Fertile males control, N = l Fertile males procarbazine, N . 0 Sperm counts in cauda‘epidldymldis, N = 3 p < 0.05 p < 0.01 Figure 31. Fertility and Sperm Counts of Animals Treated with Procarbazine SPERM COUNT AS PERCENTAGE OF CONTROL TOTAL IMPLANTS/LITTER 20- 119 TOTAL IM PLANTS 20 WEEK OF SERIAL MATING a Age in days at treatment 0 Control, N = 10 A Procarbazine, N = 10 * p < 0.05 ** p < 0.01 Figure 32. Total Implants of Animals Treated with Procarbazine g LIVE IMPLANTS/LITTER LIVE IMPLANTS AND RESORPTIONS 120 WEEK OF SERIAL MATING 8 Age in days at treatment 2 Control, N = 10 Procarbazine, N = 10 * p < 0.05 ** p < 0.01 Figure 33. Live Implants and Resorptions of Animals Treated with Procarbazine RESORPTIONSILITTER 121 ANDROGEN BINDING PROTEIN ‘ J . 2004 200 1% ------------ 1w---- ---- .n 63 16a V ) 5b 12b _I O I p. Z c 8 o 5 b 12b LL 0 LU 2 . . 5 zooj 2°°J W U 0: Lu 0. 1Mq ----------- 100.- ——————————— 24a 45a 9 c 0 5b 12b 0 5b 12b WEEK OF SERIAL MATING a) Age in days at treatment b) N=3 * p < 0.05 Figure 34. Androgen Binding Protein of Animals Treated . with Procarbazine 122 KM): During Phase I, no gross lesions were observed in animals of any age. Animals treated at 6 days of age and sacrificed after 12 weeks of serial mating (129 days of age) had malformations of the skull as described above. These animals were small and thin with marked alopecia. Other than poor body condition and alope- cia, there were no gross abnormalities in animals treated at 16, 24 or 45 days of age. 3. Body Weight: Testicular and Epididymal Weights (Tables 24, 25 and 26) a. Treatment at 6 Days of Age Body weights were significantly lower than control values at 14 days post exposure (40.8%) in Phase I treated animals. The animals treated during the first part of the serial mating study (Phase II), showed severe signs of systemic toxicity and died during the study. A second group of animals were treated and survived Phase II of serial matings. Because of concern for ani- mal loss, no animals were sacrificed after 5 weeks of serial mat- ing. At the end of Phase II, body, testicular and epididymal weights were significantly less than control values (59.5%, 70.1% and 52.7%, respectively). kw Body weights were significantly decreased at 3 and 7 days post exposure (64.5% and 72.1% of the control) in Phase I and at both the 5 and 12 week serial mating sacrifice (80 and 129 days of age; 56.5% and 38.7% of the control, respectively). Although 123 testicular weights were less than controls only at the 129 days of age(80.6%), epididymal weights were decreased at both 80 and 129 days of age (69.0% and 70.6% of the control). c. Treatment at 24 Days of Age Body weights were lower than controls at all sacrifice points in both Phases I and II of the study (all p < 0.01). No changes in testicular and epididymal weights were observed during Phase I, but the testicular weights were significantly decreased at 129 days of age (76.1% of control) and the epididymal weights at both 80 and 129 days of age (70.2% and 73.3% of control). d. Treatment at 45 Days of Age Body weights were decreased at three observation periods, 3 and 7 days post exposure (Phase I) and at 129 days of age dur- ing Phase II (87.2%, 91.7% and 74.8%, respectively). Testis weight was statistically different from the control value at 14 days post exposure when it was increased to 115.2% and epididy- mal weight also varied only at one time point (3 days post expo- sure: 60.2% of control). No changes in testis or epididymal weight were observed during Phase II. 4. Morphological Evaluations a. Treatment at 6 Days of Age Mild cytotoxic damage in testis was observed at 3 days post exposure (Phase I). Spermatogonia were often rounded with loss of contact to the basement membrane and cytoplasm was more baso- philic than normal. Spermatocytes were enlarged with loss of 124 H .o v m is mod v d 1. am H 93> :82 m 8 man no mama m3 8 €88.38 #83 fifl .3 u z 8 wow no man omeEommmuHwawzfim .muz 3 m u z 3 35m H tag as H s48 s." H «$2 .335 H odmfl in: H o.mh mvHemfimofioHoso was H cams men H 0.9m mAH H 0.8m s4 H teen 3: H MKS Hoficoo me 1.3.2 H odmm 53.3 H meow is; H mama 3mg H 0.8 scam H 0.8 muggmmofioaoso 92 H in? 6.3 H fimmm 5m H s43 m4 H 78 m.m H Q? 3380 am 30K H méom 15.9% H «zomm m4. H «2% $4 H tam 1&5 H 0.8 magnumofioflomo 6.: H team 92 H 908 «A H mém m4 H 9mm «4 H mdw H0380 ma $8.8 H mama In 3.0.0 I 0.2 «4 H 93 «A H mad moHaanmofioHomo «an H Qmwm ma H Ewen «A H ES To H oz: emé H WE H9328 m 02 am «.3 as on 95a: 338 no x83 ufifig g son #5385 um mod ufisz seom so Ago: 03 meflsdmonmoaoso co Booms u vN wHQMH. H0.0 V Q anew m0.0 v m 1. am H 93> :88 ms 8 own no when man” on mucomwmuuoo M83 $3 .3” u 2 A0 emu mo meme 0e 8 mEommeHHB Mews fie _e u z 3 m u z 3 06s H meeea p.05 H 0.33 6.9 H Emofl eem H 733 0.0m H p.83 meHsEdmosmoHomo ede H toeea 0.ee H flea? eémm H Emmi .053 H 905.3 0.2 H mémma Hoficoo me 3.de H 0.8g e.ee H 0.83 e.me H Dim 0.em H been edfl H m.mmm megsmmofioaomo 5 n 0&3 H 0.83 den H QSeH 0.0m H n.0se ms H 5va ease H 50% Hoficoo em $0.3 H tween” «63 H 583 e60. 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I e we 5 + p.03 I 35.80 e IIIIIIIIIIIIIIIIIIIIIIIII III III 03” IL e3 es en 05%: . define . m mo Mews unmauemga Hopwm.»mo unmaummha He mom new magma £0sz HQSGHHAH no Amine o8 moHfimfimonmoHoxo mo ”63mm 127 cell-cell contact. Testicular morphology was normal at all other observation points. b. Treatment at 16 Days of Age Mild cytotoxic change was observable in spermatocytes at 3 days post exposure as evidenced by swollen cytoplasm and loss of cell—cell contact. Morphology was normal at 7 and 14 days post exposure (Phase I). There were no visible morphologic lesions at either observation point during Phase II. c. Treatment at 24 and 45 Days of Age Testicular morphology was normal at all observation points. Spermatogenesis was complete with release of mature spermatozoa. Other tissues examined showed no significant pathologic changes in animals of any age group. Serial Mating Data a. Treatment at 6 Da s of A e Table 27 Onset of reproductive capacity was severely delayed and fer— tility was markedly decreased. No animal reached reproductive capacity until week 5. From weeks 5 to 12, normal fertility le— vels were not achieved. However, due to the small group size (six animals), meaningful statistical analyses were not pos- sible. Throughout the entire mating study, the group had only 18.1 i 10.2% fertility as compared to the controls. There was a significant decrease in the average number of litters produced per male in 12 weeks (Table 31). Again, because of the small group size, statistical analyses of total implants, resorptions and viable implants was not reliable. Overall, the number of 128 total and viable fetuses was low compared to the controls, but the resorption rate did not appear to be effected. b. Treatment at 16 Da 5 of A e Table 28 Onset of reproductive capacity was delayed by 1 week. Dur- ing weeks 2 and 3, only 10% of the males were fertile. After week 3, the percent of fertile males approached (but did not reach) control values. Overall, there was a significant decrease in the average number of litters produced per male after 12 weeks of mating. The number of resorptions showed a significant in- crease above control levels in week 5 (154% of control) and vi- able implants were significantly decreased in week 8 (75% of con- trol values). c. Treatment at 24 and 45 Days of Age (Tables 29 and 302 Animals treated at these ages had minimal effects on repro- ductive abilities. There was a one week delay in the onset of reproductive capacity in animals treated at 24 days of age and only 30% of the males were fertile in week 2, but after that, fertility values quickly reached and stayed at control levels. Animals treated at 45 days had an increased resorption rate in week 2 (1600% of control). All other end points approximated control values. 6. Functional and Biochemical Data Table 31 Spermatid reserves in the testis were only significantly de- creased in animals treated at 24 days and sacrificed at 129 days (12 weeks of mating). Sperm counts in the epididymides were de- creased in the animals treated at 16 and 24 days of age at the 80 m0.0 vm I Enhance gamma How :96 00» 33 39wa 3 e H z .33me Egg How 889 00m Aw 129 no.0 H 0.3 S 0.0 + 0.H H noéH sH fieH e\H NH o“0.0 H 0.3 3 e 0 noeH eH fieH e\H HH O_00 H 0.2 «N 0 0 O_0.0 H 0.~H em mam exm 0H 30.3 «H o o no.3 «H 0.3 e\H m «Ha H as mm 0.0 H mm. H 00 H es on be e} e n06 H oé e to H e.H m nee H me HH 9mm e\m s no.0 H 0.mH H 0 0 n0.eH 3 DH e\H e OH0.0 H 00 0 0 0 no.0 e fieH e\H e 0 0 0 0 0 0 0 e\0 a 0 0 0 0 0 0 0 e\0 m 0 0 0 o 0 0 0 e\0 m 0 0 0 0 0 0 0 e\0 H Mm + x mm + X 38mg mm H x 3H3 8 Bee: x83 BfiHsfi 3535 umfiafl 3:205 fifiHoen 8?: 8§B§H§H gums fined, $083 meoflmnomem mcoflmuowmm H82. 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H.H H m.NH mHH om eH\m NH N m.o H H.NH HmH 00H 0H\6H HH m m.o H m.NH mNH 00H OH\dH oH o m.o H m.HH mHH 00H OH\oH m N m.o H H.NH wHH om OH\N w o H.o H m.mH mmH OOH OH\OH H H m.H H w.HH oHH om oH\m m N H.H H H.NH NOH om oH\m m .ON o.H H o.NH NOH om 0H\m H o m.H H H.0H mm om Ome m Hm.H H H.N mH m.¢ H N.m *HoH H.H H N.m Hm om Ome N 0.0 H o.H H 0.0 H o.v H o.m m 0H OH\H H mm + x mm + x 3.33 mm H x umpflfl 8 Runs :83 BfiHgfi BfiHQfi HBHHQ BfiHgfi BfiHgfi 8?: mmgwaHgfi gum: 2%.? Sang mcoflfiommm mcoflfiomwm H53 H38 238m 2803} £833 HwHHmm mflmxxma omv mom yo mama m¢ um noummya mHmchm Hopmmue mvHamnamozmaHoxo cH mama muHHHuuwm "om mHan 133 H0.0 V Q *% .0 wk mod v Q H w m. no no mNH op mgommmufioo Eng HmHme mo M83 £3 8 mg no .Hwoanfi Au mm + m8? acme 0.0m. mo mmmv ow B mgommmnnoo Eng .53me mo M003 5m 3 3 8i m6 H N.oH ANHV mmm H HHH ANHV oo.H H H.NH ANHV 3.0 H 3H NH In ANHV mam H mmNH N: 8.0 H NN.N ANHV mmd H No.N m fiwamfimmw SHV mm. H om.m 5 Nom H HHNH E 3H.H H NH.N 5 Hm. H wo.N NH In E NSH H HMS 5 mm. H SH 5 «a. H 8N m 3 >8 SHV mN.H H 86 5 HwH H $8 5 Hm. H 9.3 5 *Ho. H 2H NH In E 5 H 08 5 3H. H 9H 5 NH. H «H.N m N m8 8d *HNH H 0H.» 5 *mmNH H mva E 9.. H SN 5 8. H om.N NH I... 5 NS H movH 5 Km. H mN.H 5 H.H H mmH m mH >8 6V Boa H H.N ANINHHN H mmwN 5 8. H SH 05 i. H 3H HUNH III .in II. III mm m >8 0H 5 Hang? 93%??? am? fig 3%? £33 $952 momma?“ m mpfifio.§&m .0399QO vooflHHomm mUHEEQmoHHQOHoa 69m .mxflpmz HMHHmm mo gm 3 @603 NH Em m pm 6003308 98 mo mwmd mv 98 «N 6H .0 pm 638.3. mg . Ema 539E gm 5% no 98 mHSHUWWw "Hm mHonB 5 fiasco 68m :9me £389 2H 858m Eng no Ag? 08 mUHfimfimosmoHomo mo Pow —: . 134 day observation point (5 weeks of mating) and in the animals treated at 45 days of age at the 129 day observation point (12 weeks of mating). ABP was statistically different from control values in ani- males treated at both 6 and 16 days at 129 days of age. In both cases, the ABP levels were markedly elevated (371% and 400% of control, respectively). H. Vincristine 1. Clinical Signs No evidence of systemic or clinical effects in any age group was detected during the study. 2. Gross Necropsy No gross abnormalities were detected in any age group at any of the observation periods. 3. Body Weight: Testicular and Epididymal Weights Tables 32 33 and 34 , a. Treatment at 6 Days of Age No significant differences occurred in either body weight or testicular and epididymal organ weights at any sacrifice point in animals treated at 6 days of age. b. Treatment at 16 Days of Age Body weights were decreased during Phase I at 7 days after exposure and during Phase II at 129 days of age (63.6% and 86.1% of control values, respectively). Epididymal weights were de- creased at 80 days of age (after 5 weeks of serial mating) (74.4% ——7—7‘” 135 of control) and testicular weights were decreased only at 129 days of age (77.7% of control). c. Treatment at 24 Days of Age Body weights were significantly decreased at all three ob- servation points during Phase I (62.3%, 74.6% and 69.4% of control, respectively, at 3, 7 and 14 days post exposure). Body weights were within normal range at 80 and 129 days of age, the two sacrifice points during Phase II. Testicular weight was de- creased only at 14 days post exposure and epididymal weights showed no significant change from control at any time point. d. Treatment at 45 Days of Age Though body weights were not statistically different from controls at any sacrifice point, both testicular and epididymal weights showed marked decreases from control values at 80 days of age during Phase II (49.0% for testis and 53.8% for epididy- mis). At 129 days of age, however, there was no difference in either testis or epididymal weights from control values. 4. Morphologic Evaluation «it-We Three days following exposure, there was extremely mild cy- totoxicity of the germinal epithelium as evidenced by occasional swollen or degenerative cells. At 7 and 14 days post exposure, testicular morphology was normal. At both the 5 and 12 week ob- servation points during Phase II, germinal epithelial cells occa- sionally were degenerative or necrotic. 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Houpcoo o ofi gm «.3 an on 3%: $38 no x83 “gamma g >8 €93me um mom ”59¢: Bicéam no Agog Ye wfiumflong no pomfim 3% game 139 b. Treatment at 16 Days of Age All animals of this group had occasional tubules with only spermatogonia or Sertoli cells at 3 days post exposure. This could have been due to either loss of spermatocytes or the de- creased formation of these cells due to mitotic failure. Tubular lumen formation was retarded in one animal. Though these changes were relatively mild, they were thought to be treatment related. Changes were likely not prevalent enough to alter testicular function. By 7 days post exposure, the animals of this group had similar changes, but they varied in severity. A mild to moderate number of tubules, most often located near the periphery of the testis, had decreased numbers of spermatocytes sometimes with decreased or no spermatogenesis. This alteration in populations of spermatogonia and spermatocytes produced a decreased number of spermatids also. Sertoli cells appeared normal in morphology and number. At the 14 day observation point, one animal showed no significant morphologic change, but the other two had severe da- mage in most tubules near the periphery and occasionally in the tubules near the center of the cross-section. In the affected tubules, there was a marked loss of spermatids and spermatocytes, and a moderate decrease in spermatogonia. During Phase II, there was only an extremely mild morphologic change in testicular tis- sue. At the 5 week observation period, there was a slight loss of cell contact between spermatids and Sertoli cells. A mild decrease in spermatid numbers also occurred in a few tubules in- dicating that this loss of cell contact may have resulted in pre- mature release of spermatids. After 12 weeks of mating, no mor- phological changes in the testis were observed. 140 c. Treatment at 24 Days of Age Three days post exposure, one animal had severe disruption of several tubules in the subcapsular area. The main cells affected were spermatocytes and spermatids. The other two animals showed no significant morphologic change. At 7 and 14 days post treatment, the pattern was similar. One animal in each group showed marked loss of spermatocytes and spermatids in sev- eral tubules, especially in the subcapsular area. Testicular morphology of the remaining animals was normal. There did not seem to be a gradient of damage. In Phase II after 5 weeks of mating, only mild damage to the testis was noted; however, it was present in all animals. The change involved disruption of cell-cell contact between Sertoli cells and spermatids in occa- sional tubules. Vacuolation in the cytoplasm of some Sertoli cells also occurred. A few tubules had what appeared to be large holes which was most likely due to focal areas of cell loss. After 12 weeks of mating, no significant changes were seen in any of the animals. d-Wm Mild cytotoxic change was observed in between 10% and 20% of tubules in all animals during Phase I. Three days after expo- sure, spermatocytes and spermatids were most commonly affected and change involved degenerative signs and the presence of occa- sional necrotic cells. By 7 and 14 days after exposure, only rare tubules had a few degenerative cells showing any damage. During Phase II after 5 and 12 weeks of mating, the severity of 141 damage varied. After 5 weeks, two out of three animals had sig- nificant damage to several tubules and the third animal appeared morphologically normal. In affected tubules, Sertoli cells were damaged. The cytoplasm was vacuolated and there was loss of cell-cell contact between Sertoli cells and germinal epithelial cells. Late spermatocytes and spermatids were the cells of the germinal epithelium most affected. Spermatogonia and early sper- matocytes appeared normal in both number and morphology. Later spermatocytes and spermatids were decreased in number or absent, therefore arresting spermatogenesis. After 12 weeks of mating, two out of three animals had no significant morphologic changes. In the third animal, about 50% of tubules were seriously damaged with almost complete loss of germinal epithelium. This may have been caused by Sertoli cell damage or mitotic arrest. No significant morphologic lesions were observed in other organs of any age group. 5. Serial Mating Data a. Treatment at 6 Da 3 of A e Table 35 The onset of reproductive capacity was delayed for about two weeks. In the second week, only one male was fertile, which is significantly decreased from the control. By week 3 of mating, reproductive capacity for the group was similar to control values and remained so throughout the rest of the serial mating. The total number of resorptions was significantly different from the control during weeks 5, 7 and 8 of mating (146%, 200% and 150%, respectively). All other serial mating data were similar to N 142 control values with the exception of viable implants/litter in week 12. However, overall, there was a significant decrease in the number of litters produced per male after 12 weeks of mating (Table 39). b. Treatment at 16 Da 5 of A e Table 36 The onset of reproductive capacity was significantly de— layed, with no evidence of fertility until week 3 and then only 40% of the males were fertile. After week 3, fertility was not statistically decreased from the control, but it remained at a lower level through most of the study. Though the other weekly serial mating data were similar to the control, an overall signi- ficant decrease in the average number of litters produced per male was noted after 12 weeks of mating (Table 39). c. Treatment at 24 Da 5 of A e Table 37 The onset of reproductive capacity was delayed by one week, only 20% of the males were fertile in week 2 and 30% (statistic- ally significant at p < 0.01) in week 3. Though fertility rose after week 3, it remained lower than that of the control through- out most of the 12 weeks of mating, resulting in an overall fer- tility of 60.8 i 7.2% which was significantly lower than the con- trol fertility. Also, a significant decrease in the average num- ber of litters produced per male occurred during the 12 weeks of mating (Table 39). d. Treatment at 45 Da 5 of A e Table 38 Onset of reproductive capacity was delayed and the level of fertility never reached that of the controls. Onset was delayed 143 by one week and, in weeks 2 and 3, only 10% and 50% of the males were fertile, both values being statistically different from con- trol values. The overall percentage of fertile males for the en- tire 12 weeks was 55.8 : 9.0%. Total implants were significantly decreased in week 12 (61.6% of control) and viable implants in weeks 8 and 12 (68.0% and 60.0% of control, respectively). The average number of litters produced per male during the 12 weeks was statistically lower than that of the controls (Table 39). 6. Functional and Biochemical Data (Table 39) Sperm counts in the epididymides were decreased in ani- mals treated at 45 days of age after 5 weeks of serial mating, but were similar to control values after 12 weeks of mating. ABP measurements in epididymal cytosols did not reveal any differ- ences between the animals of any age group and the controls at either the 5 or the 12 week sacrifice point during Phase II. Cytosine Arabinoside 1. Clinical Signs No evidence of systemic or clinical effects in any age group during the study. 2. Gross Necropsy No gross abnormalities were observed in any age group at any observation point. a Treated Animals Treated at 6 Days of Age (0.6 mg/kg) stine incri DatainV Table 35: Fertility /;.'1tter x + SE Viable Viable Implants Implants xisa Resorptions /;.itter Total Resorptions Implants /L.itter X + SE Total Males Inplants (96) es /V1able Fertile Implants Mated a1 littersw er1 Mating Week S 0/10 10* 1/10 11.0 i- 2.0 74 12.0 i 1.8 84 70 7/10 77 78 90 9/10 82 101 11.2 i 1.8 19* 90 9/10 144 H ox H o +I +l \o m H N H H to H H H H H [x ox o H H H \o H o H a: a: so 0 H H I\ H o H H N <- N m H H N H m N H o o o a: H o o "‘ i 3 m H 10 xx 11.4 i 1.2 144 120 12.2 i 1.2 6** 100 10/10 14.3 i 0.9 143 147 14.7 i 0.7 100 10/10 13.2 i 1.7 132 10/10 100 133 13.3 i 0.7 10 10.2 + 1.7 102 117 11.7 i 1.1 15 100 10/10 111 11.1 3: 1.2* 0.2 i 0.4 113 11.3 i 1.1 100 10/10 S See text for exper' d N=10 a) 145 H m 06va .3. o.ovm .. OHHZ 33% Egg Hoe Bap 8m A... 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Morphologic Evaluation 6I l6I 24 and 45 Day Treatment Groups Treatment related morphologic changes in the testis did not appear to be significant in any age group at any observation per- iod. Some evidence of acute cytotoxicity occurred at 3 days post exposure in animals treated at 6 days of age; however, morphology was normal at 7 days after exposure and at all subsequent time points. No significant Visible morphologic lesions were observed in other organs of any age group. 5. Serial Mating Data (Tables 43, 44, 45 and 462 Only very few alterations from the control could be detected in the serial mating studies. The 6 day group had fewer total implants in week 3 than the control (80%). Animals treated at 16 days were one week delayed in reaching reproductive capacity and only 40% of the males were fertile in week 2. There were statistically greater resorptions than in the controls at week 3 (150%) and fewer viable implants per litter in week 12 (84.4%). Animals treated at 24 days had fewer viable implants at week 12 150 (76.9% of control). The 45 day treatment group had statistic- ally more resorptions (175% of control) and fewer viable im- plants per litter (73.1% of control) only during week 8. All other serial mating data were within the control range. 6. Biochemical and Functional Data During Serial Mating (Table 472 Sperm heads in epididymal homogenates were significantly less than in the controls in the animals treated at 6 days and sacrificed after 5 weeks of mating and also in animals treated at 24 days and sacrificed after 12 weeks of mating. 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The highly irritating qualities of doxorubicin (Pratt and Ruddon, 1979) which had been injected IP was the likely cause of ascites in two animals during Phase I. The chronic systemic toxic ef- fects of pleural and peritoneal effusions, the likely cause of death in four animals during Phase II, was probably secondary to the well documented cardiotoxic properties of doxorubicin (Pratt and Rudon, 1979). General systemic toxicity was evidenced by long-term growth retardation. Decreased body weight values remained relatively consistent at the two sacrifice points dur- ing Phase II (73% and 77%, respectively, of control values.) There was, therefore, little evidence of recovery. Testicular and epididymal weights showed few or no acute ef— fects, but long-term chronic change was usually severe. This finding was consistent with testicular morphology where semini- ferous tubules were atrophic. Epididymal changes were most likely secondary to decreased testicular fluid and sperm produc- tion. Also, epididymal weight change may have resulted from an- drogen deficiency and/or disturbance of androgen action. Nei- ther the testicular nor the epididymal weights had recovered by the end of the study, therefore indicating long term or possibly permanent damage. 160 Acute morphologic change was observed in the testis dur- ing Phase I. The spermatogonia were the cell type most affec- ted. The effect was generally reversible within 14 days after exposure. However, a few tubules in all animals remained se- verely hypoplastic, indicating irreversible injury and death of stem cells. Lu and Meistrich (1979) reported similar findings involving spermatogonial and stem cell death in mice following administration of doxorubicin. Sertoli cells appeared normal until 2 weeks post treatment when there was evidence of disrup- tion of Sertoli cell function. At that time, the tubular lumina were occasionally smaller than normal or even nonexistent. 0n chronic observation, severe morphologic alterations were prominent. All three major cell types of the testis were af- fected at 80 days of age. Spermatogonia were usually the only germinal cell type present. Therefore, doxorubicin damage to the germinal epithelium must have been severe and long-lasting, since there was essentially no recovery over time sufficient for an en- tire cycle of the seminiferous epithelium. The lack of spermato— genesis was confirmed by the total absence of spermatids in tes- ticular homogenates and of sperm in epididymides. Sertoli cells were also severely damaged and appeared abnormally shaped. The tubular lumina were often small or nonexistent, reflecting de- creased fluid production by the Sertoli cells. The ABP levels in epididymal cytosols were zero, indicating a lack of Sertoli cell activity. Leydig cells were occasionally hyperplastic and multi~ nucleate. This change was likely a secondary response to the toxic damage in the seminiferous tubules rather than a direct 161 toxic injury of the Leydig cells by doxorubicin. Leydig cells produce testosterone in response to stimulation by LH through the hypothalamo-pituitary-gonadal axis. Since the function of both the germinal epithelium and Sertoli cells were disrupted, the in- crease in Leydig cell number and shape may have been a compensa- tory response to loss of negative feedback. At the end of the serial mating study, severe morphologic damage remained after the duration of approximately two full seminiferous cycles. There was not total stem cell death, how— ever, since occasional tubules possessed more advanced germ cells through the early spermatid stages. Indeed, spermatids and sperm heads were detectable in testis and epididymis. The numbers were small, however, about 0.5% of controls. Although the Sertoli cells were still often morphologically abnormal and lumens of se- veral tubules were still decreased in size, the ABP levels im- proved somewhat (from 0% to 18.7% of the controls) indicating im- proved function. Leydig cell size and shape also indicated re- turn to normal at the end of serial mating. The severe toxic effects of doxorubicin on the testis were confirmed by the mating studies. All animals were sterile with the exception of one which produced two implants in week 5. 2. Treatment at 16 Days of Age As with the treatment at 6 days of age, administration of doxorubicin to animals at 16 days of age caused both severe re- productive impairment and systemic toxicity (as evidenced by decreased weight gain and the deaths of two animals). Usually, 162 little or no evidence of acute toxicity was present, but long— term chronic changes were marked. Testicular and epididymal weights were significantly decreased only during Phase II and there was no evidence of recovery. The epididymal weight changes were most likely secondary to decreased testicular function, es- pecially sperm and fluid production. The chronic decrease in these organ weights was less than that seen when the animals were treated at 6 days of age and it appears that, at 16 days of age, the rats are less susceptible to the toxic effects of doxorubi- cin, at least in regard to testicular and epididymal develop- ment. Acute morphologic change in the testis was similar to that in animals treated at 6 days of age, but fewer tubules were af- fected. Spermatogonia and spermatocytes were the germ cells most commonly affected and Sertoli cell alterations were similar to those described in the previous section. Though chronic damage to the germ cell epithelium was severe, no changes in the Leydig cells were observed. Germinal cell hypoplasia and spermatogenic arrest were evident in most tubules. However, unlike animals treated at 6 days of age, the germ cells developed into the sper— matid stages. It was these later cell types, pachytene sperma- tocytes through late spermatids, that were most severely damaged. The few spermatids that did develop were morphologically abnormal and usually prematurely released. Also, severe alterations in spermatid and sperm head counts were observed. These observa- tions imply that the seminiferous tubules were unable to support 163 spermatogenesis, either by a defect in the germinal cells them— selves and/or a functional or structural defect in the Sertoli cells. In support of the latter is the fact that the Sertoli cells showed morphologic alterations and that animals were treated at 16 days of age, the time period during which the Ser— toli cells are forming the tight occluding junctions which con— stitute the blood-testis barrier. Although the ABP levels were normal, the production of ABP does not reflect the integrity of the blood—testis barrier and the maintenance of the germ cell milieu by the Sertoli cells which, if altered, could lead to ab— normal Spermiogenesis. Reproductive performance as indicated by the onset of repro- ductive capacity, fertility and litter size was significantly de— creased from the controls, confirming the morphological observa— tions. It was notable, however, that many of the animals still did father offspring, showing that animals can reproduce with se- verely decreased sperm counts. 3. Treatment at 24 Days of Age In contrast to the previous two groups, animals treated at 24 days of age had only minimal systemic or reproductive effects. Growth retardation was only acutely evident, but, chronically, animal weights and appearance were similar to control animals. Testicular and epididymal weights had the reverse trend. Acutely, no changes occurred, but chronically, both organs showed significant decreases in weight, although not nearly as severely as in the animals treated at 6 or 16 days of age. How- ever, morphologic changes in the testis were minimal. Also, no 164 changes in spermatid and sperm head count, or in the ABP levels were observed. Thus, animals treated at 24 days of age appeared much less susceptible to the toxic effects than animals treated at 6 or 16 days of age, even though time since treatment, and, therefore, time available for recovery, was less. Consistent with the above findings, there were no significant effects on the reproductive performance of animals treated at 24 days of age. 4. Animals Treated at 45 Days of Age Similar to the immature animals treated at 24 days of age, rats treated at the start of puberty (45 days of age) showed very minimal effects. Body weights were normal at all observation periods and the testicular and epididymal organ weights were only acutely decreased at 3 days post exposure, but not thereafter. Acute morphologic lesions were observed in the testis at 3 days after exposure, but cytotoxicity was minimal and the morpho- logy returned to normal thereafter. Normal levels of spermatids and sperm head counts verified the morphologic findings. Neither the onset of reproductive capacity or mating performance of these animals was altered. The high ABP level was associated with a large variability in individual animals and was not thought to be treatment—related, i.e., there is no basis for concluding change in Sertoli cell function, particularly since they appeared nor- mal morphologically and spermatogenesis was not altered. Summary Differential susceptibility of the mature testis to treat- ment with doxorubicin was clearly demonstrated. Animals treated at 6 days of age were most severely affected in all end—points 165 measured. Spermatogonia and stem cells were the germ cell type most affected, and there was only minimal recovery 123 days after treatment. As a result, there was essentially no sperm produc- tion and animals were sterile. Sertoli cell function was damaged as evidenced by decreased ability to produce fluid and ABP. Ani- mals treated at 16 days of age had severe reproductive damage. Spermatogonia and stem cells were the germ cells most severely affected, but over time there was evidence of recovery and gener- ation of more advanced germ cell types. Incomplete spermatogene- sis was likely due either to a defect in the germ cell line or damage to the Sertoli cell. At the time of exposure, Sertoli cells were forming the blood-testis barrier. Disruption of this, and therefore permanent alteration of the adluminal enviroment, would explain failure of cells to continue development after entering the adluminal compartment. Minimal effects in animals treated at 24 and 45 days of age may be credited to the presence of mature, fully functioning Sertoli cells and blood-testis bar- rier. Biochemically, the severe effects of doxorubicin on the ra— pidly dividing germ cells of the testis is not unexpected. Doxo- rubicin binds tightly to DNA and its cytotoxicity appears to be a result of this binding (see Introduction). The findings of others regarding the reproductive effects of doxorubicin (Parvi— nen and Parvinen, 1978; Lu and Meistrich, 1979; Au and Hsu, 1980; Meistrich gt al., 1985; Hacher-Klom g; 1., 1986; see Introduc- tion) in mature animals are consistent with the findings of this study. However, these reports offer no explanation for the 166 dramatic results reported here. In the current study, a clear inverse relationship between severity of reproductive effects and age at exposure was seen. The reasons for these findings may either be associated with the properties of the compound or the differential structure and function of the male reproductive sys~ tem at the time of exposure. Doxorubicin is primarily metabol— ized by the liver (Takanashi and Bachur, 1976). Immature animals treated at 6 or 16 days of age may not be fully competent in he- patic metabolism, therefore producing a higher physiologic dose than animals treated at older ages. Also, since doxorubicin is retained in tissues for a long period of time, this may act to further increase tissue dose levels. Whether the differential susceptibility was due solely to stage of testicular maturation at exposure, or to the metabolism of the compound or to some com- bination of the two requires further study. Procarbazine 1. Treatment at 6 Days of Age Administration of procarbazine (200 mg/kg) to animals at 6 days of age produced only mild systemic and reproductive effects. Generalized toxicity was evidenced only by slight alopecia soon after treatment. Mild alterations of testicular morphology in— volving spermatogonia and spermatocytes during the early parts of the study were the only indication of reproductive target organ effects. Testicular function appeared unchanged following expo~ sure to procarbazine since there were no alterations in the re- sults of serial matings, the spermatid and sperm head counts, and 167 the ABP levels were essentially identical to those of the con- trols. From these results, it appears that morphology is the most sensitive indicator of the toxic effects of procarbazine on the reproductive system. Additionally, it is clear that mild and transient morphologic alterations can occur in the testis without changing testicular function or the capacity to reproduce. 2. Treatment at 16 Days of Age The only evidence of systemic toxicity in the animals treated with procarbazine at 16 days of age was acute partial alopecia. Although alterations in testicular and epididymal weights were generally only seen long after treatment (113 days), testicular morphology showed early pathologic changes. Tubular lumen formation was markedly retarded and the germinal epithelium showed degenerative and necrotic changes that were most prominent in the spermatocytes and spermatids. Since lumen formation oc- curs concurrently with fluid production by the Sertoli cells (Ritzen g; al., 1981), it is likely that this impairment of Ser— toli cell function was the cause of the retardation of tubular lumen formation. Toxic effects by procarbazine on the germinal epithelium, especially on spermatocytes and spermatids, have pre— viously been noted by Meierhofer (1973) and Parvinen (1979). The damaging effects of procarbazine on the cells of the testis were not as dramatic on chronic observation. Thus, it appeared that most tubules were able to recover from the acute effects. Repro— ductive function was impaired during the early part of serial mating, but recovered considerably by the end of the study. 168 Sexual maturation was delayed and the normal fertility levels were not achieved until the sixth week of serial mating. Even though both testicular and epididymal sperm counts were signifi- cantly reduced during Phase II, fertility was similar to control during the second half of the serial mating study. These results show the variable sensitivity of the different endpoints mea- sured. As described by Aafjees g; g1. (1980), sperm counts can be decreased by approximately 80% before reproduction is altered. 3. Treatment at 24 Days of Age As with both the 6 and 16 day old treated animals, the only evidence of systemic toxicity was acute partial alopecia. Evi- dence of reproductive toxicity was present throughout the study. Although neither testicular or epididymal weights were altered in Phase I, both were decreased during Phase II indicating chro- nic changes. The testicular morphology showed marked alterations 7 days after treatment; spermatocytes were the most severely af- fected. During subsequent observation periods, morphological changes were seen in only a few tubules. Thus, although acute damage to the germinal epithelium was severe, long-lasting or permanent damage was rare. The onset of reproductive capacity was delayed only one week, but fertility did not reach that of the control animals until the seventh week of serial mating. Even though spermatid reserves and sperm head counts were markedly decreased throughout the study, fertility was normal during the second half of serial mating. This finding confirms the observation with the 16 day treatment group that severe decreases in sperm production may 169 occur without altering fertility. ABP levels were significantly higher than those of the controls during Phase II. This observa- tion, combined with the decrease in epididymal weights, may in- dicate an effect on the hypothalamo~pituitary-gonadal axis and/or on androgen action. Increased fetal mortality during weeks 6 and 11 may have been due to genetic effects, but one would expect to have seen similar changes during the other weeks of mating as well. 4. Treatment at 45 Days of Age Acute partial alopecia was again the only evidence of sys- temic toxicity. Alterations in reproductive endpoints indicated significant target organ effects. Both testicular and epididymal weights were significantly lower than those of the controls dur- ing Phase II. Reduced epididymal weights may reflect an effect on Leydig cells and/or a defect in androgen action. Dramatic pathologic alterations were seen in the germinal epithelium acutely. The spermatogonia and spermatocytes were most severely damaged. Prominent germinal epithelial damage persisted through- out the serial mating study with marked germinal hypoplasia in many tubules. However, no change in the ABP levels was observed, indicating that Sertoli cell function was intact. Even though onset of reproductive capacity was unaffected, fertility was reduced during the entire mating study with signs of recovery only appearing from weeks 10 to 12. Average litter size was reduced from weeks 1 to 10. The fertility pattern sug- gested that the late spermatogonia, early spermatocytes and sper- matids were most severely affected, confirming the morphological 170 observations. Decreased litter size occurred without a signifi— cant reduction in sperm counts, possibly suggesting a genetic mechanism of toxicity. 5. Summary The present studies confirm the adverse affects of procar- bazine on spermatogenesis and reproductive tract function as pre- viously reported by several investigators. Such adverse effects were reported in mice by Lee and Dixon (l972c) and Ehling (1974), in rats by Hilscher and Reichett (1968) and Russell g; g1. (1983), in rhesus monkeys by Sieber g; _1. (1978), and in humans by Sherins and deVita (1973). Procarbazine is known to have a variety of biological effects (see Introduction). In the present study, pathologic effects on all the end- points measured were most severe in animals treated at 45 days of age and were progressively less in animals treated at 24, 16 and 6 days of age. Throughout the study, spermatogonia and early spermatocytes were the cell types most severely altered. Sertoli cell function appeared to be changed in animals treated at 16 days of age, but recovery was complete in these animals during Phase II. Procarbazine has a short biological half-life. Approxi— mately 70% of the drug is eliminated from the body within one day 1., 1967). Therefore, it is likely that the pri— (Schwartz gg mary action of procarbazine occurred shortly after administra- tion. Cell structure and function eventually returned to normal unless stem cell death occurred in a given tubule. The spermato- genic process offers unique stages of susceptibility to toxic 171 chemicals: replication of spermatogonia requires intensive DNA, RNA and protein synthesis, all of which are affected by procar- bazine as described in the Introduction. Considerable DNA syn- thesis also takes place in early spermatocytes in preparation for meiosis (Burgin g; al., 1979). Protein synthesis is high in preleptotene and pachytene primary spermatocytes as well as in elongated spermatids (Courot g2 g1., 1970). RNA synthesis is particularly active in late primary spermatocytes, secondary spermatocytes and early spermatids (Courot g; al., 1970). The response of animals treated at 45 days of age confirm these find- ings. However, the progressive decrease in severity of effects noted in animals treated at younger ages is not consistent with these previous observations. This discrepancy may partially be explained by the length of time between treatment and the begin- ning of the serial mating procedures. Since this time period in- creased as the age of treatment decreased, progressively longer recovery periods were present when the animals were treated at an earlier age. Cyclophosphamide 1. Treatment at 6 Days of Age Cyclophosphamide (80 mg/kg) caused severe systemic effects when administered to animals at 6 days of age as indicated by alopecia, growth retardation, skeletal deformities and death. Body, testicular and epididymal weights were all significantly decreased from control animals. However, since values were pro- portionately decreased, there was not a clear target organ 172 effect. Both testicular morphology and spermatid and sperm head counts had no apparent alterations from control animals. How- ever, there were significant effects on reproductive function throughout the serial mating study. The onset of reproductive capacity was delayed until week 5 and animals were subfertile throughout the remainder of the study. ABP was markedly ele- vated; 371% of control. Therefore, although testicular morpho- logy and sperm numbers appeared normal, the reproductive capabi- lities of these animals were severely impaired. There are two major possibilities which could explain this. First, due to sys- temic toxicity, these animals may not have been able, or had the energy, to copulate. This could account for animals that pro- duced no implants. However, oftentimes, there were viable im- plants present, but in decreased numbers. Secondly, a biochemi- cal or morphological defect in sperm would explain the decreased fertility. The markedly elevated level of ABP may indicate a defect in the hormonal and/or biochemical mechanisms necessary to produce sperm capable of maturation, capacitation and/or fertili— zation. Sperm did not appear to have genetic defects since there was not a significant difference in resorption rate from control animals. 2. Treatment at 16 Days of Age Systemic effects of cyclophosphamide administration to ani- mals at 16 days of age caused growth retardation and alopecia. Testicular and epididymal organ weights were significantly de- creased from control values, but, like animals treated at 6 days of age, decreased organ weights were proportional to decreased 173 body weights so there did not appear to be target organ effects. Also, as in animals treated at 6 days of age, testicular morpho- logy and spermatid and sperm head counts were normal. Reproduc- tive function was adversely affected, but not as severely as in the younger treatment group. Maturation was delayed for one week and fertility was subnormal during the early part of the serial mating study. As in the animals treated at 6 days, ABP levels were markedly elevated. The general response of animals treated at 16 days of age was therefore similar to, but not as severe as, animals treated at 6 days of age. 3. Treatment at 24 Days of Age Administration of cyclophosphamide to animals 24 days of age dramatically decreased body weights throughout the study. The systemic effects of cyclophosphamide on growth was more consis- tent in this group of animals who were higher on the growth curve and more metabolically competent than those in the two younger age groups. Testicular and epididymal weights were altered, but, as in the other two age groups, this did not appear to be a tar— get organ effect. Though testicular morphology appeared normal at all observation points, spermatid reserves were decreased at 80 days of age and sperm head counts at 129 days of age. Despite the decreased number of sperm, reproductive performance was not significantly altered. 4. Treatment at 45 Days of Age Treatment of animals at 45 days of age did not have severe effects on either body weights, or testicular or epididymal weights. Although significant differences from control occurred 174 at various time points for all three values, the consistency and severity of toxic effects was much less than in animals treated at younger ages. Testicular morphology was normal and reproduc— tive performance was similar to controls except for an increased resorption rate in only week 2. With the exception of sperm counts at 129 days of age, spermatid reserves, sperm head counts and ABP levels were not altered by cyclophosphamide. Summary Cyclophosphamide is a derivative of nitrogen mustard and de- pends on i_ vivo hepatic activation to form reactive metabolities with alkylating and cytotoxic capabilities (IARC, 1975). The 4 toxicity of cyclophosphamide has been reviewed by Gershwin g; a1. (1974). The reproductive effects of long-term chronic exposure in the human, treated both as children and adults, are well docu- mented (see Introduction). Several reports describe the toxic effects of cyclophosphamide on the reproductive system of adult laboratory animals (Sotomayor and Cumming, 1975; Sram, 1976; Vigil and Bustos-Obregon, 1985; Moreland g; al., 1981; Adams g; al., 1981; Trasler g; al., 1985; Trasler gt al., 1986; Auroux and Dulioust, 1985). The effects of either single or chronic admin- istration of cyclophosphamide in the immature rodent has pre- viously been unknown. The single dose used in this study, 80 mg/kg/bw, is consistent with the studies in adult males described above where doses ranged from 50-100 mg/kg/bw. In the present study, toxic effects of cyclophosphamide on the reproductive sys- tem were not as great as those described in the references cited. There were only minor changes in testicular morphology and sperm 175 production. Decreased reproductive capacity in animals treated at 6 or 16 days of age was most likely associated with a biochem— ical or hormonal effect on sperm function or secondary to sys- temic toxicity. The lack of reproductive alterations in animals treated at 24 or 45 days of age was not consistent with many of the reports cited above. In this study, the systemic toxic ef— fects of cyclophosphamide were greater than reproductive disrup- tion. Decreased reproductive outcomes with normal testicular morphology and sperm count is consistent with genetic defects in sperm, but absence of increased resorption rates suggests that genetic alterations in sperm were not responsible for the repro- ductive changes in this study. Vincristine 1. Treatment at 6 Days of Age Vincristine, administered to animals at 6 days of age (0.6 mg/kg), caused alterations in only a few of the many endpoints measured. Although the epididymal sperm counts were signifi- cantly decreased throughout the serial mating study, there were only minimal differences in the mating results between treated and control animals. Overall, the animals treated at 6 days of age appeared to be resistant to the toxic effects of vincristine. Even though vincristine is known to be a microtubule disrupting agent, there was no observable effect on mitotic activity of Sertoli cells which were actively dividing at the time of treat— ment. 176 2. Treatment at 16 Days of Age Administration of vincristine to animals at 16 days of age produced alterations in testicular morphology and mating results. Although morphologic change was observed during the early parts of the study, testicular morphology was normal by the end of ser- ial mating. With time, the germinal epithelium was apparently capable of recovering from the toxic effects of vincristine. The duration of spermatogenesis in the Sprague-Dawley rat is approxi- mately 52 days and transit time through the epididymis is one to two weeks. Even if vincristine caused acute death of all germ cell types, including spermatogonia, sufficient time between treatment to observation may allow repopulation of affected tu- bules. Indeed, although the serial mating data reflected dam- age to reproductive function during the early part of Phase II, recovery was seen during the latter weeks of mating. Maturation was delayed for 2 weeks and, during the third week, only 40% of the males were fertile. However, fertility improved thereafter and was not significantly different from control animals for the remainder of the study. 3. Treatment at 24 Days of Age Administration of vincristine to animals at 24 days of age caused primarily only mild acute changes; body and testicular weights were decreased only during the early phase of the study. The onset of reproductive capacity was delayed and fertility was decreased through the third week of mating. Thereafter, ferti- lity levels approximated those of control. It, therefore, appears that animals were able to fully recover from the acute systemic and reproductive toxic effects of vincristine. 177 3. Treatment at 45 Days of Age Vincristine produced no evidence of systemic toxicity in the animals treated at 45 days of age. Damage to the reproduc- tive system, however, was evidenced by decreased testicular and epididymal weights and decreased sperm counts during the early part of the serial mating study, and changes in testicular mor- phology and mating results. Morphologically, minimal acute cytotoxic damage involved primarily spermatocytes and spermatids. The sensitivity of indi- vidual animals to long—lasting damage by vincristine varied, 2/3 of the animals were affected at the 5 week sacrifice and only 1/3 at the end of serial mating. Changes in both the germinal epi- thelium and the Sertoli cells were noted. The serial mating data reflected disruption of reproductive function, especially during the first three weeks of the mating study. Maturation was de- layed, fertility was decreased and the litter size was less than that of the controls. However, normal or near normal fertility was maintained thereafter, indicating recovery from the toxic ef— fects of vincristine on the reproductive system. 4. Summary Vincristine is a microtubule disrupting agent. Microtubules are important elements in many different cell types and are in- dispensable for normal cell division. Microtubules also play an important role in the dynamics of spermatogenesis. Microtubules are a major component of the cytoskeleton of the Sertoli cell and the Sertoli cells are responsible for the movement of the germ cells within the seminiferous epithelium from the basal to the 178 adluminal compartments and for the release Of spermatozoa into the tubular lumen (Russell g; al., 1981). For the above reasons, treatment of animals with vincristine can cause mitotic and meiotic arrest as well as damage to the ul- trastructure of the Sertoli cell (Russell g; al., 1981; Parvinen _t _1., 1978), and could explain the effects on spermatogenesis and fertility seen during the present study. These effects were variable among animals, indicating that not all rats are equally susceptible to vincristine treatment. It was particularly no- ticeable that the age of treatment had a major influence on the alterations that were noted. This is probably due to the fact that the changes were not permanent, i.e., recovery occurs over time. Thus, the longer the time period between treatment and ob- servation, the more chance there is for a return to normal func- tional activity, possibly independent of the age at which the animals were treated. Cytosine Arabinoside Even though the endpoints measured in animals treated at 6, 16, 24 and 45 days with cytosine arabinoside occasionally dif- fered significantly from control values, no clear reproductive effects were seen in any age group. Cytosine arabinoside is an antimetabolite and pyrimidine analog. This compound specifically disrupts the S-phase of cell division, the time of DNA synthesis (Pratt and Ruddon, 1979). Indeed, Lee and Dixon (1972d) reported that treatment of adult male rats with cytosine arabinoside only caused damage to the spermatogonia in the S-phase. 179 The reason for the lack of effect seen in the present study is most likely associated with the metabolism of cytosine arabi— noside. 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F., Gambetta, R.A., and diMarco, A.: The inhibition in vitro of DNA polymerase and RNA polymerase by daunomycin and adriamycin. Biochem. Pharmacol., 24:309-311, 1975. VITA The author was born in Midland, Michigan on January 27, 1947. She received her primary and secondary education in the public school system of Midland, Michigan. In 1969, she earned a Bachelor of Arts Degree in psychology from the University of Michigan, Ann Arbor, Michigan. She received the Degree of Doctor of Veterinary Medicine from the School of Veterinary Medicine, Michigan State University, East Lansing, Michigan in 1977. The author was admitted into the Department of Pathology as a doctoral candidate in 1980 on a Dow Special Fellowship. From 1984 until the present, she has been working as a science advisor to the Assistant Administrator for Research and Development, Environmental Protection Agency, Washington, D.C. 193 STATE UNIVE Willi/WNW“ H I ”111111111111“ 3 62 1316 1293 030