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I 22” ‘III' III I II, I'III” '2: ' I..I NI I2;II.22,22.I, .2212.” II 21221“ II'IIII2‘I I 332212112 “"111“ "II“I’"'IIIIIIII I "2' JI 2H2 I 22212” I212“ 11.2 2222; 2122III2 2I {II 2222” 2212 I22“ II 1 '22, , 1 1: 222I "1222222221” “JIM/1| 111111.121121 2121” 2222222212 ‘: , 1 1 II I “21112122221122 ’ ' 1 I 21221212” 2II2 I IIi 2222122222I‘2211II 111121|I|I111222I222N1111111111111” I». I II I22 ' 1IIII1II2 l 22222‘2 I 22 2212112122222222IIIII22IIII2I22222122111222222 2222iIIILI2 2222 I III 222II2I222I I2I2I‘222 222222 2 22222 222 22222 It 311} 'III L131?” III. mesifl LIBRARY Michigan State University This is to certify that the dissertation entitled "Effects of the Pineal on Reproduction in the Syrian Hamster Mutant Anophthalmic White (flh)" presented by Susan C. James has been accepted towards fulfillment of the requirements for PhoDo degreein ZOOlogy rMajor professor // Date M MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES .—_ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. EFFECTS OF PINEALECTOMY ON REPRODUCTION IN THE SYRIAN HAMSTER MUTANT ANOPHTHALMIC WHITE (flu) By Susan C. James A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1982 ABSTRACT EFFECTS OF PINEALECTOMY ON REPRODUCTION IN THE SYRIAN HAMSTER MUTANT ANOPHTHALMIC WHITE (Eh) By Susan C. James The gene Eh, causing anophthalmia in the Syrian hamster, flggg; cricetus auratus, is a highly pleiotropic gene affecting eye develop- ment, pigmentation, and reproduction. Since both ED and the pineal organ are known to suppress reproductive function, the objective of this study was threefold: (1) to determine whether Eh, by itself, influences testicular differentiation; (2) to determine whether removal of the pineal gland would restore fertility to both experimentally blinded, genetically normal (wh/wh(8)) and mutant, eyeless (Eh/Eh) hamsters, and (3) to determine if all Nh[!h hamsters possess a pineal organ and if so, are any abnormalities present which might influence its secretory ability. All wh/wh(8) and Nhiflh_hamsters possessed large, mature testes at 60 days. A slight difference was found in testicular weight, favoring mutant individuals, but no differences were observed in body weights or seminiferous tubule diameters when comparing these ,two groups. The gene Eh does not affect initial testicular differ- entiation, since all testes were identical at 60 days and contained normal differentiating germ cells. Testes from all wh/whlB) and most wgryg hamsters at 135 days were hypoplasic and aspermic. Differences were observed in Leydig cells, Sertoli cells, and in the basal lamina and germinal epithelium Susan C. James of the seminiferous tubules. Pinealectomy fully restored adult testi- cular size and morphology in all wh/whlB) and !g[!h_hamsters. Thus, atrophy of the testes in these blinded hamsters was a pineal-mediated phenomenon, and mutant testes appeared to be completely competent to respond to the negative effects of the pineal. The gross morphology of all pineal glands was identical. The superficial pineal rested in the junction of the superior sagittal and transverse sinuses, and was attached to the diencephalon by a long pineal stalk. Histologically, the superficial pineal from all hamsters possessed pinealocytes, blood vessels, and glial cells. Pinealocyte cytology differed between normal and blinded hamsters, and was thought to be related to the differences in functional acti- vity of the gland. The cytoplasm of pinealocytes from all wh/gh(8) and NQLEQ hamsters contained free polyribosomes, increased Golgi vesicles and saccules and decreased rough endoplasmic reticulum. Substantial amounts of smooth endoplasmic reticulum were character- istic of all pinealocytes. ACKNOWLEDGMENTS I would like to express my appreciation to Dr. James H. Asher, Jr. for chairing my graduate committee. Thanks also to Drs. Karen Baker, James Edwards, and John Shaver for serving on my committee. A special thanks to Dr. Karen Baker for the use of all of the equip- ment in the Center for Electron Optics and for providing partial financial support for this project. Thanks to Dr. William Cooper, Chairman of the Department of Zoology, for providing the majority of the electron image film and photographic papers used in this inves- tigation. I would like to thank Dr. Russel J. Reiter, from the University of Texas Health Science Center at San Antonio, for teaching the tech- nique of pinealectomy to me and for his time and stimulating discus- sions while I was in Texas. Special thanks are in order to my husband, Johnny, for his moral and financial assistance, as well as for sharing his expertise in light and electron microscopy with me throughout this endeavor. ii Page LIST OF TABLES ......................... v LIST OF FIGURES ........................ viii CHAPTER 1. POSSIBLE RELATIONSHIPS BETWEEN THE PINEAL GLAND AND INFERTILITY IN THE SYRIAN HAMSTER MUTANT, ANOPHTHAL- MIC WHITE (Wh) ................... l A. Introduction ..................... 1 B. Questions Related to Wh_and Pineal Function ...... 4 CHAPTER 2. A BRIEF OVERVIEW OF THE PINEAL GLAND ........ 6 A. Introduction ..................... 6 B. Comparative Anatomy of the Pineal Gland ........ 8 C. Development of the Pineal in the Syrian Hamster . . . . 12 D. Blood Supply ..................... 15 E. Innervation ...................... 16 F. Cytology ....................... 18 G. Biochemistry ..................... 21 H. Physiology ...................... 23 CHAPTER 3. EFFECTS OF PINEALECTOMY ON REPRODUCTION IN THE SYRIAN HAMSTER MUTANT ANOPHTHALMIC WHITE (Wh). . . . 28 A. Introduction ..................... 28 B. Materials and Methods ................. 30 C. Results ........................ 32 D. Discussion ...................... 42 E. Plates ........................ 47 CHAPTER 4. FINE STRUCTURE OF THE SUPERFICIAL PINEAL FROM THE SYRIAN HAMSTER MUTANT ANOPHTHALMIC WHITE (WE) . . . 65 A. Introduction ..................... 65 B. Materials and Methods ................. 68 C. Results ........................ 69 D. Discussion ...................... 77 E. Plates ........................ 83 TABLE OF CONTENTS iii TABLE OF CONTENTS (Continued) Page SUMMARY ............................ 97 A. Problem ........................ 97 B. Methods ........................ 97 C. Results ........................ 98 D. Conclusions ...................... 99 LITERATURE CITED ........................ 102 APPENDICES ........................... 114 APPENDIX A. Comprehensive Tables Containing All Experimental Data .............. 114 APPENDIX B. Formulations and Procedures ......... 140 iv Table 1A 2A LIST OF TABLES Page CHAPTER 3 Mean Measurements of Hamster Body Weight, Testes Weight Normalized to Body Weight and Tubule Diameters at Approximately 60 Days .................. 37 Results from One-Way Analyses of Variance Comparing Means Summarized in Table 1 ............... 38 Mean Measurements of Hamster Body Weight, Testes Weight, Testes Weight Normalized to Body Weight and Tubule Diameters at Approximately 135 Days ........... 39 Results from One-Way Analyses of Variance Comparing Means Summarized in Table 3 ............... 40 Results from Duncan's Multiple-Range Test Comparing Mean Body Weight (A), Testes Weight (B), Testes Weight Normalized to Body Weight (C), and Tubule Diameter Measurements (D) from Table 3 .............. 41 CHAPTER 4 Mean Measurements of Hamster Body Weight, Pineal Weight, and Pineal Weight Normalized to Body Weight at Approxi- mately 135 Days ..................... 74 Results from One-Way Analyses of Variance Comparing Data Summarized in Table 1 ............... 75 The Resultant Values of Q from Tukey's Honestly Signi- ficant Difference (HSD) Test Comparing Means Summarized in Table 1 ................. . ...... 76 APPENDIX A Data for the Genotype wh/wh(8) (Cream, Blinded) at Approximately 60 Days .................. 114 Data for the Genotype Wh7Wh (Eyeless Mutant) at Approximately 60 Days .................. 115 Table 3A 4A 5A 6A 7A 8A 9A 10A 11A 12A 13A 14A 15A 16A LIST OF TABLES (Continued) Page Computations for the Analysis of Variance on Body Weight Measurements from Hamsters at Approximately 60 Days . . . 116 Computations for the Analysis of Variance on Testes Weight Normalized to Body Weight Measurements from Hamsters at Approximately 60 Days ............ 117 Computations for the Analysis of Variance on Tubule Diameter Measurements from Hamsters at Approximately 60 Days ......................... 118 Data for the Genotype ghflwh (Cream) at Approximately 135 Days ........................ 119 Data for the Genotype yg[!h(8) (Cream, Blinded) at Approximately 135 Days ................. 120 Data for the Genotype wh/wh(B) (Cream, Blinded) at Approximately 135 Days ................. 121 Data for the Genotype Wh/gh (Black-eyed White) at Approximately 135 Days ....... ‘ .......... 122 Data for the Genotype WhXWh (Eyeless Mutant) at Approximately 135 Days ................. 123 Data for the Genotype Wh/Wh (Eyeless Mutant) at Approximately 135 Days ................. 124 Computations for the Analysis of Variance on Body Weight Measurements from Hamsters at Approximately 135 Days . . . . . . . . . . . . . . . . . . . . . . . . 125 Computations for the Analysis of Variance on Testes Weight Measurements from Hamsters at Approximately 135 Days ........................ 127 Computations for the Analysis of Variance on Testes Weight Normalized to Body Weight for Hamsters at Approximately 135 Days ................. 129 Computations for the Analysis of Variance on Tubule Diameter Measurements from Hamsters at Approximately 135 Days ........................ 131 Results from Duncan's Multiple-Range Test Comparing Body Weight Measurements from Hamsters at Approximately 135 Days ........................ 133 LIST OF TABLES (Continued) Table Page 17A Results from Duncan's Multiple-Range Test Comparing Testes Weight Measurements from Hamsters at Approxi- mately 135 Days ..................... 134 18A Results from Duncan's Multiple-Range Test Comparing Testes Weight Normalized to Body Weight from Hamsters at Approximately 135 Days ................ 135 19A Results from Duncan's Multiple-Range Test Comparing Tubule Diameter Measurements from Hamsters at Approximately 135 Days ................. 136 20A Computations for the Analysis of Variance on Body Weight Measurements from Hamsters at Approximately 135 Days ........................ 137 21A Computations for the Analysis of Variance on Pineal Weight Measurements from Hamsters at Approximately 135 Days ........................ 138 22A Computations for the Analysis of Variance on Pineal Weight Normalized to Body Weight Measurements from Hamsters at Approximately 135 Days ........... 139 vii Figure 10 11 12 13 LIST OF FIGURES CHAPTER 3 Light micrograph of seminiferous tubules repre- sentative of testes from !h[!h(8) hamsters at 60 days . . 48 Light micrograph of seminiferous tubules represen- tative of testes from Wh/Wh hamsters at 60 days ..... 48 Light micrograph of seminiferous tubules from testes of wh/gh(8) hamsters at 135 days ............ 50 Light micrograph of seminiferous tubules from testes 0f.!D/!D hamsters at 135 days .............. 50 Light micrograph of seminiferous tubules representa- tive of testes from gh7wh(B)-SPE hamsters at 135 days . . 50 Light micrograph of seminiferous tubules from testes of WhXWh¢SPE hamsters at 135 days ............ 50 Light micrograph of seminiferous tubules representa- tive of testes from wh[!h(B)-PE hamsters at 135 days . . 50 Light micrograph of seminiferous tubules from testes of WhXWthE hamsters at 135 days ............ 50 Electron micrograph of interstitial tissue represen- tative of testes from wh[!h(8) hamsters at 135 days . . . 52 Electron micrograph of interstitial tissue represen- tative of testes from WhXWh hamsters at 135 days . . . . 52 Electron micrograph of interstitial tissue from testes of wh[!h(B)-PE hamsters at 135 days ....... 52 Electron micrograph of interstitial tissue from testes of Wh7thPE hamsters at 135 days ......... 52 Higher magnification electron micrograph of an area representative of interstitial tissue from both ‘wg7wh(B)-PE and Wh/WhrPE hamsters ............ 52 viii Figure 14 15&15 178: 18 19820 21 22 23 24 25 26 27 28 29 30 LIST OF FIGURES (Continued) Higher magnification electron micrograph of the juxtanuclear region in Figure 12 ............ Electron micrographs of the basal lamina of semini- ferous tubules from testes of gh/wh(B) hamsters at 135 days ........................ Electron micrographs of the basal lamina from semini- ferous tubules of testes from WhXWh hamsters at 135 days .......................... Electron micrographs of the basal lamina representa- tive of testes from wh[!h(B)-PE hamsters at 135 days . . Electron micrographs of the basal lamina representa- tive of testes from WhiflthE hamsters at 135 days Electron micrograph of seminiferous epithelium from testes 0f.flfllflfl(3) hamsters at 135 days ........ Electron micrograph of seminiferous epithelium from testes 0f.!h[!h hamsters at 135 days .......... Electron micrograph representative of seminiferous epi- thelium from testes of wh/wh(B)-PE hamsters at 135 days .......................... Electron micrograph of seminiferous epithelium from testes of WhLWhePE hamsters at 135 days ........ Electron micrograph of the most advanced developing germ cells found in testes from gh/gh(8) hamsters at 135 days ........................ Electron micrograph of seminiferous epithelium from testes of Wh7Wh hamsters, at 135 days, near the tubular lumen ..................... Electron micrograph of seminiferous epithelium from testes of wh[!h(B)-PE hamsters, at 135 days ...... Electron micrograph of seminiferous epithelium from testes of Wh/Wh-PE hamsters, at 135 days, toward the tubular lumen ..................... Electron micrograph of early spermatids (S ) from testes of yh/gh(B)-PE hamsters, at 135 day; ...... ix 52 54 54 54 54 56 56 58 6O LIST OF FIGURES (Continued) Figure Page 31 Electron micrograph of a late spermatid ($2) from testes of Wh/WflyPE hamsters, at 135 days ........ 60 32 Electron micrograph of a spermatozoa near the tubular lumen in testes of gh/gg(B)-PE hamsters at 135 days . . 6O 33 Electron micrograph of spermatozoa near the tubular lumen in testes from Wh7WgyPE hamsters at 135 days . . . 6O 34 Electron micrograph of an early spermatid (S ) from Lh/Lh tubules which approached the normal phgnotype, at 135— days ........................ 62 35 Electron micrograph of spermatozoa near the tubular lumen in Lh/Lh tubules which approached the normal phenotype, at —135 days ................. 62 36 Electron micrograph of spermatozoa near the tubular lumen in Lh/Lh tubules which approached the normal phenotype, at _135 days ................. 62 37 Electron micrograph of a Wh/Wh tubule within testes which approached the normET'Efienotype, at 135 days . . . 62 38 Electron micrograph of seminiferous epithelium from the testis of a .WHLWQ hamster at 303 days ....... 62 39 Electron micrograph of seminiferous epithelium from the testis of a EM/Wh hamster at 357 days ...... 62 40 Summary of the relationship between age and testes weight, expressed as gram percent body-weight, in all experimental groups of hamsters from the AN/Asfiflh strain ......................... 64 CHAPTER 4 1 Scanning electron micrograph of a pineal gland repre- sentative of Lh/Lh, Lh/Lh(B), and Lh/Lh hamsters at 135 days ........................ 84 2 Scanning electron micrograph of a pineal gland from a - IWh/Wh_hamster at 135 days ............... 84 3 Low magnification electron micrograph of pineal tissue representative of gh7gh and Wh/gh hamsters at 135 days . 86 4 Electron micrograph of pineal tissue from gh/gg_and .Wh[gh_hamsters at 135 days ............... 86 X Figure 7 & 8 10 11 12 13 14 15 16 17 18 198120 LIST OF FIGURES (Continued) Electron micrograph of a blood vessel (BV) represen- tative of Efllflfl.3"d.flhflflh hamsters at 135 days ..... Electron micrograph of a pinealocyte from pineal tissue representative ongh7gh hamsters at 135 days Electron micrographs of two features characteristic of gh/gh pinealocytes at 135 days ........... Electron micrograph of pineal cell process represen- tative of yfl/gfl tissue at 135 days ........... Cross sections of pineal cell processes from Lh/Lh hamsters showed many microtubules, which were seen as hollows dots within the PCP (arrowheads) ........ Near their terminals, pineal cell processes (PCP) from Lh/Lh and Lh/Lh hamsters contained many granular vesicles (arrows) as well as agranular vesicles (arrow- heads ......................... A survey micrograph of pineal tissue from Lh/Lh(B) and Lh/Lh hamsters at 135 days ............. Electron micrograph of a group of pinealocytes repre- sentative of pineal tissue from Lh/Lh(B) and Lh/Lh hamsters at 135 days .................. Electron micrograph of a blood vessel (BV) represen- tative of yfl/gh(B) and Wh7Wh hamsters at 135 days Electron micrograph of a pinealocyte representative of pineal tissue from gfl7gh(3) and WhLWg hamsters at 135 days ........................ Pinealocytes within the tissues of Lh/Lh(B) and Wh/Wh hamsters possessed an extensive Golgi complex, vmfilfif' was juxtanuclear in position ............... Smooth endoplasmic reticulum (SER) was a prominent feature of pinealocytes from Lh/Lh(B) and Lh/Lh hamsters ........................ In tissues from Lh/wh(B) and Wh/Lh hamsters, pineal cell processes (PCPT—eminated— from the cell body (PCB) and contained many microtubules (MT) .......... Near their terminations, pineal cell processes (PCP) from wh/Lh(B) and Lh/Lh hamsters contained granular and agranETar vesicles_ ................... xi 86 88 88 88 9O 9O 9O 92 92 92 92 92 Figure 21 22 23 24 25 268127 28 LIST OF FIGURES (Continued) Infrequently, the cytoplasm from all tissues con- tained centrioles ............... .. . . . Pinealocytes from all hamsters contained some lipid, but those from Lil/3411(3) and Wthh hamsters contained cells which had an abundance OFTTarge lipid droplets Dense lamellar wholrs (LW) were present and equally as numerous in all hamster pineal glands ....... All pineal tissues possessed glial cells in the form of fibrous astrocytes (GCP) .............. Nerve bundles were frequently seen in all pineal tissues ........................ In tissue representative of both gh/wh hamsters (Fig. 26), as well as Whlflh hamsters—(Fig. 27), nerve fibers were associated with a Schwann cell (SC and SN) and traversed the pineal parenchyma . . . . Unmyelinated nerve fibers (UF) were often seen in bundles (NB) which were closely associated with blood vessels (BV) .................. xii 94 94 94 96 96 96 CHAPTER I POSSIBLE RELATIONSHIPS BETWEEN THE PINEAL GLAND AND INFERTILITY IN THE SYRIAN HAMSTER MUTANT, ANOPHTHALMIC WHITE (WE) A. Introduction Studies of the mutant gene 39, anophthalmic white, in the Syrian hamster, Mesocricetus auratus, have been in progress for many years. The strain AN/Asfiflh was developed by Asher (1968) and consists of hamsters with the following genotypes and phenotypes: (l) Normal hamsters (fly/3g) are cream colored with white spotted bellies and black eyes (James, 1979; James et al., 1980); (2) Heterozygous hamsters (Eh/3h) are white with black eyes and possess less ear, eye and skin pigmentation than normal (James, 1979; James et al., 1980); (3) Homozygous mutant hamsters (Wh7Wh) are white, lack eyes and possess no pigmentation of the ears or skin (James, 1979; James et al., 1980). The gene Wh is highly pleiotropic and causes three major morpho- logical abnormalities as described by Asher (1981): (l) extreme microphthalmia or degenerative anophthalmia, (2) a lack of melanocyte- derived pigmentation, and (3) deafness. In addition, Wh causes a number of secondary problems including sterility, thyroid and adrenal abnormalities, altered sleep patterns, growth retardation, and altered urea cycle amino acid pools (Asher, 1968, 1981; James, 1979; James at al., 1980). One working hypothesis was set forth by Asher (1968) who stated that many of these pleiotropisms may be the result of altered pitui- tary function. In accordance with this hypothesis, Asher (1981) compared ten physiological parameters, in male hamsters, which are controlled by the pituitary-hypothalmic axis (body weight, basal metabolic rate, fasting weight loss at 30°C, body temperature, food and water consumption, urinary volume, respiratory rate and heart rate), and concluded that many aspects of pituitary function are altered by the gene ED: Similarly, James and Asher (1981) reported that pituitary glands from anophthalmic hamsters were histologically different from those of normal hamsters. Pituitary glands from mutant hamsters possessed 33% fewer cells and many of the cells present resembled signet ring cells or folliculo-stellate cells. Cells with numerous cilia, basal bodies and a 9+2 microtubular con- figuration were also found within mutant pituitary tissue (James and Asher, 1981). The presence of many ciliated cells within the pituitary paren- chyma was perplexing and led to the hypothesis that ym_may be involved in the improper differentiation of embryonic ciliated epithelium to glandular epithelium in this tissue (James and Asher, 1981). In addition, we proposed that Wh_may generally influence the conver- sion, or differentiation, of 311 ciliated epithelial cells during early organogenesis, leading to improper development of structures of the diencephalon--pituitary, eyes, hypothalmus and pineal gland (Asher, 1981; Asher and James, 1982). 3 Using the electron microscope, examination of the embryonic eye gave us some positive reinforcement for the hypothesis that '!h affects early organogenesis. By 11.5 days of gestation, the optic cup from mutant hamsters consists of two sensory retinal layers, instead of a sensory layer and a pigmented layer. Differentiation of the pigmented retina was unable to occur because the embryonic cilia, which covers the embryonic epithelial cells, had not been resorbed and the cell-cell interactions necessary for proper induction of the pigmented retina could not take place (Asher and James, 1982). Both the physiological (Asher, 1981) and the morphological (James and Asher, 1981, Asher and James, 1982) data suggested that the primary action of Eh was to alter the development of all structures of the embryonic diencephalon. One of the first questions that I set out to answer concerned the effects 0f.flh on reproduction, since all Wh7Wh hamsters seemed to be infertile. Results of studies using male hamsters demonstrated that some WQLWQ hamsters had normal testes, but most possessed testes which were hypoplasic and aspermic. Using the electron microscope, abnormalities were observed in Leydig cells, Sertoli cells, and in the developing germ cells (James, 1979; James et al., 1980). Seminiferous tubules contained germinal epithelium arrested in the early spermatid stage of spermiogenesis, due to dysgenesis of the acrosome (James, 1979; James et al., 1980). It was postulated that infertility l".!fl[!h hamsters was either (1) the result of altered pituitary function or (2) due to failure of eye development and a subsequent lack of function of the visual pathway acting through the pineal-gonadal axis. While in pursuit of the answers to questions concerning pitui- tary and testicular morphology, the pineal-gonadal relationship was brought to my attention by Dr. Russel J. Reiter. The purpose of Chapter 2 is to familiarize the reader with the many actions of the pineal gland and especially to emphasize the importance of an intact visual pathway for maintenance of reproduction in the Syrian hamster. After completion of the study on pituitary and gonadal morphology (James, 1979), experiments were undertaken to explore the possible interactions between the gonads and the pineal gland in this mutant. B. Questions Related to HQ and Pineal Function Research designed to answer specific questions concerning the involvement of the pineal gland in infertility of these eyeless hamsters LWQLWQ) form the contents of this thesis. These specific questions are as follows: (1) Do testes from mutant, eyeless hamsters (Wh7flh) resemble those from normal, experimentally blinded hamsters (yfl/wh(B)) at puberty (approximately 60 days)--does Wh affect the initial differ- entiation of the testes? (2) Is the morphology of testes from adult WhLWh hamsters similar to the morphology of testes from adult gh[!h(8) hamsters? (3) If the pineal gland generally affects the gonads of blinded hamsters in such a way as to suppress testicular function (small, atrophic, no spermatozoa), does removal of the pineal gland cause wg[!h(8) and Wh7Wh hamsters to maintain normal testicular size and composition? (4) Do all hamsters in the AN/Astflh strain possess a pineal gland, and if so, what are the gross and histological features of the gland? (5) Since some Wh/Wh hamsters are not sterile (they have large testes with spermatozoal-filled seminiferous tubules), does the pineal gland in these few fertile hamsters possess any abnormalities which preclude its function to inhibit reproduction? Answers to this set of questions are found in Chapter 3 (ques- tions 1-3) and 4 (questions 4-5) and will be most informative in the elucidation of inherent pleiotropisms seen in this mutant hamster. The most difficult problem in working with this particular mutant hamster is that animals are non-prolific (approximately 6% of animals born will be Wh/Wfl_male hamsters) and it is almost impossible to do an experiment with numerous replications. If pinealectomy restores fertility to Wh/Wh males, this will greatly increase our abilities to collect information about this mutation. In addition, if pineal- ectomy also restores fertility to female Wh7Wh hamsters, far more mutant progeny can be propagated in a very short period of time. Likewise, the ability to breed homozygous male and female hamsters will greatly aid in the collection of known mutant individuals prior to 10.0 days of gestation for embryological studies on the action 0f.!h- CHAPTER 2 A BRIEF OVERVIEW OF THE PINEAL GLAND A. Introduction The long history of pineal research can be divided into three periods (Kappers, 1981). The first period started about 300 B.C. when Herophilos (300-280 B.C.) first discovered the pineal, and ended in about the middle of the 19th century. Most of this period was characterized by philosophical speculation about the location and composition of the spirit within the brain. One idea was that of Rene Descartes (1596-1650 A.D.) who thought that the pineal gland was the seat of the soul. The second period, lasting until the middle of the 20th century, was characterized by an interest in the comparative anatomy and evolution of the pineal gland. More refined methods of sectioning and staining tissues for histological examination were available and led to the elucidation of many aspects of pineal comparative anatomy, histology, and embryology, which set up the basis for current views on the pineal. The third, or present period, is concerned with the details of pineal function, such as biochemistry, cytochemistry, pharmacology and endocrinology. The discovery of endocrine glands, as hormone producing glands, by Claude Bernard in the middle of the 19th century stimulated interest in the pineal gland (Kappers, 1981). In 1898, Heubner first described a boy showing signs of premature puberty, who was suffering from 6 pinealoma. Marburg (1930) claimed that premature puberty was caused by hypopinealism due to pineal degeneration or to specific tumors where the pineal parenchymal cells were destroyed. He also stated that hyperpinealism would cause an abnormal and incomplete development of the gonads, as well as obesity (Marburg, 1930). By the end of the second period, most authors were of the .Opinion that mamalian pineal gland would exert an antogonadotropic effect by way of the hypothalmo-pituitary-gonadal system, although very little sound proof existed (Kappers, 1981). According to numerous authors, the field of pinealology blossomed from two major contributions. First, Kitay and Altschule published "The Pineal Gland", in 1954, which was a summary of more than 1800 papers, in at least 12 languages, and compiled all of the fragments of pineal research published. The three likeliest conclusions drawn from this literature were as follows: (1) that the pineal is involved in gonadal function, (2) that the pineal is involved in the con- traction of melanophores (causing skin-lightening) in amphibia and (3) that the pineal compounds may be involved in behavioral disorders such as psychoses and schizophrenia (Kitay and Altschule, 1954). This work was most useful in providing a clear outline of all past research and charted new courses for new research. Secondly, Aaron B. Lerner and colleagues (1958) isolated and purified serotonin from over one million bovine pineal glands. Melatonin, the 5-methoxy derivative of serotonin, proved to be 100,000 times more potent than noradrenalin or acetylcholine in invoking the skin-lightening response in frog skin (Lerner et al., 1958). 8 From 1958 until present, numerous publications concerning all aspects of pinealology have emerged. The pineal gland is thought to be a regulator of regulators-~"an endocrine organ of neural origin being of multifunctional significance in modulating the function of endocrine and probably also of nonendocrine regulatory systems synchronizing this function with external and internal conditions" (Reiter, 1980a). Some of the most cursory questions have yet to be solved and are as follows (Kappers, 1981): (l) The true nature of all of the biologically active pineal compounds. (2) The cellular organelles which produce pineal compounds within the mammalian pinealocytes, or within the secretory rudimentary photoreceptor and photoreceptor cells of lower vertebrates. (3) The influence of different physiological conditions upon the synthesis and release of pineal hormones. (4) The mechanisms by which pineal hormones exert their in- fluence within the endocrine and neuroendocrine system. (5) The mechanism of feedback regulation of the pineal. B. Comparative Anatomy of the Pineal Gland The epiphysis cerebri, or pineal gland, is only one portion of the epithalmus, which also includes the frontal organ, parietal organ (parapineal), habenula, posterior commissure and paraphysis (Kappers, 1936). The frontal, parietal and pineal organs all develop eye-like structures in some vertebrates, the former two in other vertebrates, and thelatter evolves into an endocrine organ in higher vertebrates. Ancestral vertebrates had a third eye situated medially and directed upward (Romer, 1970). Median eyes, present in the lampreys, Sphenadon, and some lizards are buried under the skin and function to detect the presence or absence of light (Romer, 1970). It has also been theorized that the parietal eye and pineal body in extinct vertebrates may have functioned as a sensory system.to monitor thermal extremes (Roth and Roth, 1980). With the regression of the photore- ceptive elements through evolution in vertebrates, secretory function of the pineal has assumed a dominant role (Eakin, 1973). The parietal eye was lost in most vertebrates, remaining only in some fishes, in some amphibia, in the iguana-like reptiles and extant lacertilian reptiles. Cyclostomes (lampreys) possess both parapineal and pineal organs, which are both saccular and possess a lumen continuous with the third ventricle (Eakin, 1973; deVlaming and Olcese, 1981). Photo- receptor elements, resembling rods and cones, occur in both the pineal and parapineal organs and the outer segments project into the lumen (Eakin, 1973). Terminals of the sensory cells in cyclo- stomes are associated with ganglion cells (deVlaming and Olcese, 1981). The left habenular nucleus is exceptionally large and accepts nerve tracts from the pineal organ whereas the posterior commissure is small and accepts nerve tracts from the parapineal organ (Kappers et al., 1963; deVlaming and Olcese, 1981). Among adult teleosts, a pineal is present and the parapineal is retained by only a few species (deVlaming and Olcese, 1981). The pineal organ is an expanded, saccular, well-vascularized end vesicle located between the parietal bones (deVlaming and Olcese, 10 1981). A hollow stalk connects the pineal body to the posterio- dorsal portion of the diencephalon. The end vesicle is composed of photosensory cells (similar to the ones of the retina), supporting cells, glial, and sensory nerve cells (bipolar ganglion cells) (de- Vlaming and Olcese, 1981). Outer segments of the sensory cells protrude into the lumen and the basal segments synapse with sensory neurons (deVlaming and Olcese, 1981). The pineal tract projects into the tectal area and the medial longitudinal fasiculus (Kappers et al., 1936). Amphibians have a small epithalmus (Kappers et al., 1936). The pineal complex of the anurans consists of an epiphysis cerebri and a frontal organ (deVlaming and Olcese, 1981). The frontal organ is an extracranial outgrowth of the epiphysis (deVlaming and Olcese, 1981). In urodiles and caecilians the frontal organ is absent (de- Vlaming and Olcese, 1981). Embryologically, the pineal and the parapineal develop, but only the pineal persists (Kappers et al., 1936; Eakin, 1973). The pineal possesses rudimentary photosensory elements which resemble retinal cone cells and bipolar ganglion cells. The outer segments project horizontally into the pineal lumen and the lumen is continuous with the third ventricle (deVlaming and Olcese, 1981). Pineal nerve tracts project to the posterior commissure (Kappers, 1936). The amphibian pineal complex is thought to function as a "dosimeter" for the regulation of body temperature (Ralph et al., 1979a,b). Reptiles possess the most interesting of pineal complexes. In crocodilians the pineal complex is absent (Reiter, 1981). In many lacertilians (lizards) the pineal complex includes bOth an 11 epiphysis and an extracranial parietal eye (the parietal eye is analogous to the frontal organ of amphibians) (Kappers et al., 1936; Eakin, 1973; deVlaming and Olcese, 1981), while in ophidians (snakes) and chelonians (turtles), the parietal eye is absent and the pineal gland has lost all photosensory elements (deVlaming and Olcese, 1981). When present, the pineal gland in reptiles is glandular (Kappers et al., 1936). The parietal eye is present and best represented in the lizards by Sphenadon punctatum and Sceloporus occidentalis and is located beneath a transparent scale at the midline of the skull (parietal foramen) (Eakin, 1973; deVlaming and Olcese, 1981). It has the most highly developed extraocular photoreceptors possessing a cornea, lens, cone-like photosensory cells, and a nerve tract to the habenular commissure and pineal (Eakin, 1973). Embryologically, the parietal eye has been shown to develop from the presumptive pineal tissue by pinching off but migrating to the top of the skull and differentiating independently (Eakin, 1973). Since rudimentary sensory cells in the pineal organ of reptiles are independent of nerve cells and the pineal gland has assumed a secretory function, Collin (1971) argues that lizards represent a phylogenetic turning point in pineal evolution. Accompanying this transformation of the epiphysis into a secretory organ is the presence of pinealopetal (afferent) autonomic innervation. The pineal glands of both turtles and snakes receive this innervation (deVlaming and Olcese, 1981). The epithalmus in Aves (birds) is usually poorly developed, especially in the habenular region (Kappers et al., 1936). Pressure of the enlarged forebrain hemispheres has shifted the pineal posteriorly 12 towards the cerebellum. Embryologically, the pineal and parapineal anlagen are present, but only the pineal develops (Kappers et al., 1936). Even though the morphology of the avian pineal is varied from atrophic (owls, petrels) to large and saccular (penguins, pigeons, chickens, and passerine birds), it is thought to be intermediate in structure (Ralph, 1981). The secretory pinealocytes are rudimen- tary photoreceptor cells and are thought to be evolutionarily derived from the retinal cone-like cells. This secretory pineal gland is thought to function in reproduction (Ralph, 1981). In mammals, the pineal is a gland of internal secretion (Reiter, 1981). It is present in almost all quadrupeds with the exception of some edentates (armidillos, sloths, and ant eaters), sirenians (manatees and dugongs - herbivorous aquatic mammals), cetacae (porposes, and whales), proboscidae (elephants) and perissodactyla (rhinos) (Reiter, 1981). Although no pineal structure exists in these species, there may be scattered cells in the epithalmus that are functionally equivalent (Reiter, 1981). The mammalian pineal is thought to function in the neuroendocrine-reproductive axis. C. Development of the Pineal in the Syrian Hamster The anlage of the mammalian pineal diverticulum is in the dorsal . median area of the neural tube at the level of the neuroaxis, and originates between the posterior and habenular commissures. Even though the pineal is a single median structure, there are two pineal anlagen. Both neural folds possess presumptive pineal cells and when they fuse, a single pineal rudiment is formed. The nature of the double anlagen was discovered by Dalcq (1947), who manipulated 13 the developing hemispheres of early gastrulae of the European toad, Discoglossus pictus, such that the neural folds remained unfused causing the formation of two diverticula and eventually two pineal end vesicles. As the embryonic pineal develops, neuroepithelial cells give rise to pinealoblasts, which differentiate into parenchymal cells or pinealocytes. Glial cells are derived from neural ectoderm and the stroma from mesenchyme (Quay, 1965). Most of what is known about the embryology of the pineal in mammals comes from studies in the rat, where three overlapping develop- mental and maturational phases take place (Vollrath, 1981): (l) the morphogenetic phase--begins at about the 12th embryonic day and proceeds until term, (2) cellular proliferation phase--begins on the 16th embryonic day and terminates several days after birth, and (3) cellular hypertrophy and differentiation phase--commences at birth and extends 9-12 weeks post-partum. Development of the pineal gland in the Syrian hamster is of special interest for two reasons. First, the pineal gland is a complex which is divided into a superficial portion and a deep portion, connected by a long slender stalk. Secondly, most of the knowledge of the endocrine function of the pineal is derived from studies in this species. Clabough (1973) has described the development of the hamster pineal as follows: The epiphyseal evagination starts at 11 days as a thickening of the diencephalic roof. By day 12, the evagination has deepenedand lies rostral to the posterior com- missure. By day 13, the evagination has elongated, dorsocaudally, and lies ventral to the posterior commissure and subcommissural 14 organ. At this stage, the apex is associated with the developing confluence of sinuses and the anterior wall is continuous with the tela choroidae. Day 14 is characterized by profuse cellular proli- feration and secondary evaginations. These factors result in the formation of follicles in the distal end of the organ. On day 15, the folicles arrange themselves randomly or into rosettes. A lumen persists only in the proximal one-third of the pineal organ. At term (16 days), both the follicles and lumen have disappeared. Defini- tive cellular types do not become evident until days 7 through 17 of postnatal life (Clabough, 1973). No neural elements are evident in the fetal stages (Clabough, 1973), but are present by the 11th postnatal day in the rat (Machado et al., 1968). Subdivision of the pineal into a superficial and deep pineal complex begins on the 6th postnatal day (Reiter, 1981). At this point, the gland is rapidly enlarging. A constriction in the pineal becomes more pronounced (Hewing, 1976) and as the two masses separate, the stalk becomes progressively thinner. By 22 days after birth, the stalk consists of a few parenchymal cells, blood vessels, non- myelinated nerves and an evagination of the posterior part of the third ventricle (Sheridan et al., 1969). In the adult, the deep pineal lies in the posteriodorsal diencephalon between the habenular and posterior commissures. The superficial pineal lies under the skull and is adherent to the undersurface of the junction of the superior sagittal and transverse sinuses. 15 D. Blood Supply The major blood supply to the pineal is provided by branches of the posterior choroidal arteries from the posterior cerebral arteries in the dorsolateral mesencephalon (vonBarthold and Moll, 1954). The arteries branch extensively in the pineal capsule, before they penetrate the parenchyma. In most mammals, all areas of this gland possess many capillaries. Hodde (l979) described the constituents of the arterial anatomy after injection of latex into the cephalic arterial system. Scanning electron microscopy of these vascular casts found that the pineal receives two to six arterial branches from the posterior cerebral arteries. The pineal tissue is highly vascularized with the bulk of the vessels being the size of capillaries (Hodde and Veltman, 1979). In rodents, the majority of the pineal tissue protrudes directly into the superior sagittal sinus and the endothelial lining of the sinus forms a portion of the pineal capsule (Reiter, 1981). Venous drainage is via the great cerebral vein (vonBarthold and Moll, 1954). Hodde (1979) has shown that 12 to 16 superficial collecting veins enter the great cerebral vein which drain directly into the superior sagittal sinus. Morphologists have always noticed the perfuse blood supply and those who have examined this have predicted that the pineal is an organ of internal secretion with a high metabolic activity (Reiter, 1981). Goldman and Wurtman (1964) measured the blood flow through the rat pineal and calculated it to be 4 ml/min/g, which is, on a gram percent basis, a greater blood supply than is found 16 in any other gland except the kidney. The blood supply is greater at night than during the day, probably due to the nocturnal rise in indole metabolism (Rollag et al., 1978). In addition, removal of the superior cervical ganglia decreases blood flow by two-thirds (Reiter, 1981). E. Innervation The pineal gland relies on an intact sympathetic innervation for its synthetic (Wurtman et al., 1964a,b) as well as its endocrine (Reiter and Hester, 1966) capabilities. The pineal is relatively unusual among endocrine organs, since sympathetic denervation abolishes its activity (Reiter, 1981). Early physiological (Theiblot et al., 1947) and anatomical data (Kappers, 1960) provided evidence that the nerve processes which terminated within the pineal gland were from postganglionic sympathetic neurons in the superior cervical ganglia. Postganglionic fibers travel from the superior cervical ganglia to the pineal along blood vessels from the internal carotid plexus. In their pathway, these diffuse fibers enter the tentoruum cerebelli and eventually form one or two discrete nerves, the nervi conarii, before entering the pineal gland (Kappers, 1965). The nervi conarii penetrate the pineal capsule, near the apex, and then ramify among the pinealocytes. The nervi terminate in the perivascular spaces or, occassionally, between pinealocyte processes (Romjin, 1975; Matsuhima and Reiter, 1977). Contrary to early thoughts (Wolfe, 1965), nerve terminals generally do not form morphological synapses with pinealoCytes (Matsushima et al., 1981). 17 The neurotransmitter norepinephrine (NE) is found to be asso- ciated with sympathetic nerve endings in the pineal gland where it is presumably packaged in membrane-bound vessels (Matsushima et al., 1981). NE is released during darkness and stimulates indole production within the pineal gland. In the hamster (Morgan et al., 1976) pineal NE levels remain stable throughout the 24 hour lightzdark cycle. In the rat, however, Wurtman and.Axelrod (1966) have shown that concentrations of pineal NE fluctuate to their greatest height during the day (production and storage within the nerve terminal) and depth during the night (maximal release). After NE is released from the nerve terminal, it acts on beta adrenergic receptors (Zatz, 1978; Lipton et al., 1981) to cause an increase in hormone production in the pineal (Axelrod, 1974). Since the mammalian pineal gland is devoid of extra-retinal photoreceptors, it is the action of photoperiodic fluctuations (light and darkness) on the eyes that is responsible for cuing the pineal. Axons of the retinal ganglion cells enter the optic nerve. The optic nerves of the Syrian hamster possess many accessory projections (Pickard and Silverman, 1981) and innervation of the pineal gland is carried out via the suprachiasmatic nucleus (SCN). Saper et al. (1976) demonstrated that the SCN sends projections to the medial tuberal hypothalmus and neurons in this area send axons to the lateral hypothalmus, where they synapse with cells whose fibers descend through the brain stem in the intermediolateral cell column of the upper thoracic cord. The axons of the preganglionic neurons leave the central nervous system in the central roots and ascend in the sympathetic trunk to the superior cervical ganglia (Moore, 1978). 18 The deep pineal of the hamster also receives postganglionic sympathetic fibers from the superior cervical ganglia (Mdller and vanVeen, 1981). Since these fibers are interrupted when the superfi- cial pineal is surgically removed, the axons which innervate the deep pineal either pass in the vicinity or through the superficial pineal on their way to the deep pineal (Reiter and Hedlund, 1976). In addition, the deep pineal is non-functional, with respect to its inhibitory influence on reproduction, when the superficial pineal has been removed (Reiter and Hedlund, 1976). F. Cytology The main unit of the mammalian pineal gland is the chief cell, parenchymal cell or pinealocyte (Reiter, 1981). On morphological grounds, parenchymal cells are subdivided into light and dark pinealo- cytes (Romjin, 1975; Sheridan and Reiter, 1968), which have a nucleus, cell body, and one to several processes. These processes eminate from the cell body, are long, and ramify throughout the gland, usually ending around capillaries (Vollrath, 1981). Besides pinealocytes, the pineal tissue also possesses a varying number of glial cells (usually fibrous astrocytes) and blood vessels (Vollrath, 1981). The nucleus of a mammalian pinealocyte displays the fine struc- tural features of an active interphase nucleus, characterized by electrolucent nucleoplasm (Vollrath, 1981). Nuclei vary in size and shape, but most are highly indented. The nuclear envelop con- sists of an inner and outer membrane, 20 nm in width, which has nuclear pores (Vollrath, 1981). Occasionally diaphragms which close these pores have been described in the hamster (Sheridan and Reiter, 19 1968). Nucleoli are prominent and massive as described by Sheridan and Reiter (1968). The cytoplasm of each pinealocyte contains many organelles, similar to those of any other mammalian cell. Endoplasmic reticulum is rare and is much less abundant than in the protein or steroid secreting endocrine cells, but occasionally substantial amounts are reported (Vollrath, 1981). Rough endoplasmic reticulum has been reported to consist of isolated vesicles, 30 nm to 0.3 pm in size (rat-Bostelman, 1965), isolated flattened sacs (hamster-Sheridan and Reiter, 1968; 1970), or isolated cisternae (rat-Arstila, 1967). Smooth endoplasmic reticulum (SER) consists of randomly oriented vesicular and elongated components which do not interconnect (Leus, 1971). SER has been reported to consist of anastomosing and branching tubular profiles in the pocket gopher (Sheridan and Reiter, 1973), and consists of membranes which are highly concentrated in the hamster (Sheridan and Reiter, 1968). Less typical forms of endoplasmic reticulum are thought to occur after extreme experimental conditions and include transformation of concentric layers into myelin bodies or lamellar whorls (Clabough, 1971; Bucana et al., 1973). The majority of ribosomes are free in the cytoplasm, but some are attached to the endoplasmic reticulum. Golgi is present and juxtanulear. Mitochondria are interesting, because they show a number of different morphological characteristics. Giant mitochondria have a highly packed, electron-dense internum and measure 4-5.8 pm in length in rats or 3-4 um in hamsters (Vollrath, 1981). Small mitochondria are 0.2 to 0.25 um and are nearly devoid of internal structure (Sheridan and Reiter, 1968). Mitochondria are round, oval, 20 elongate, rod-snapped, tortuous, filamentous, branched, club-shaped or cup-shaped (Vollrath, 1981). They are not uniformly distributed, but are found throughout the cell body, cytoplasmic processes, and terminals (Vollrath, 1981). Lysosomes are rare (Vollrath, 1981). Multivescular bodies are found near the Golgi apparatus and are thought to increase in response to blinding (Clabough, 1971; Lin et al., 1973). Centrioles are lacking or rare and often transform into microtubular boquets (Vollrath, 1981). Cilia are rare, but are reported to be quite prevalent in pinealocytes of the bat, mole-rat, and mole. Each cilium is thought to be reminescent of rudimentary outer-segments found in the pineal of lizards and birds and possess axonemes with a 9+0 tubular configuration. Free microtubules are often found at the junction of the cell body and process and run in longitudinal fashion, down the length of the process. Microtubules are thought to function in secretion (Banerjee and Margulis, 1973), in contraction and relaxation of cells, and in changes in cellular size (Vollrath, 1981). Lipid inclusions are present and are thought to be possible morphological correlates of secretory products or storage cites for pineal hormones (Vollrath, 1981). Glycogen is often present within the cytoplasm of normal hamster pinealocytes, but decreases if hamsters are blinded or kept in continuous darkness (Kachi 1971; 1973). Synaptic ribbons are long electron-dense bars studded with synaptic vesicles and are prominent in the guinea pig and rat pinealo- cyte (Vollrath, 1981). Since the relative concentration of synaptic 21 ribbons increases during darkness and decreases during light, they are thought to be involved in intercellular communication (Vollrath, 1981). G. Biochemistry Modern pineal biochemistry began with the identification of N-acetyl-5-methoxytryptamine from bovine pineals (Learner et al., 1958). Due to its lightening effect on melanophores in amphibians, it was named melatonin. It did not take long until the biosynthesis of melatonin from tryptophan, which is taken up by the pinealocytes from the blood, was understood (Giarman and Day, 1958). Axelrod and Weissbach (l960) discovered that the pineal contains enzymes responsible for the conversion of serotonin to melatonin, and soon thereafter, it was established that the light:dark cycle was an important factor in the biochemistry of the gland (Quay, 1963, 1964; Wurtman et al., 1963). After the uptake of tryptophan into the pinealocyte, tryptophan hydroxylase converts it to 5-hydroxytryptophan (Lovenberg et al., 1968). Next, 5-hydroxytryptophan is decarboxylated by l-aromatic amino acid decarboxylase to S-hydroxytraptamine (serotonin) (Quay, 1963). Serotonin is the common precursor of a number of indoTes formed within the pineal gland: (l) Serotonin to melatonin. Serotonin is converted to N-acetyl transferase--NAT (Klein et al., 1971), requiring an acetyl group from acetyl-CoA (Weissbach et al., 1961). N-acetyl-serotonin is converted to melatonin by hydroxyindole-O-methyl-transferase (HIOMT) and requires a methyl group from S-adenosylmethionine (Axelrod and 22 Weissbach, 1960; Axelrod et al., 1965). (2) Deamination of serotonin to S-methoxyindole acetic acid and to S-methoxytryptophol (Klein et al., 1981). Serotonin is con- verted by monoamine oxidase to 5-hydroxyindole acetylaldehyde, which is an unstable intermediate and is either oxidized by aldehydede- hygronase to S-hydroxyindole acetic acid (HIAA) or reduced by alcohol dehydrogenase to S-hydroxytryptophol. Since HIOMT belongs to a family of methyl transferases which transfer methyl groups from the cofactor S-adenosyl methonnine to an acceptor molecule (hydroxy- indole), HIOMT converts both HIAA to methoxyindole acetic acid and hydroxytryptophol to methoxytryptophol. HIOMT enzyme also converts some of the serotonin (hydroxytryptamine) to methoxytryptamine. The conversion of serotonin to melatonin is under control of the light:dark cycle via its action of the peripheral nervous system (Reiter, 1981). Darkness is associated with low serotonin content (Reiter, 1981) whereas periods of light are associated with high pineal serotonin content (Quay, 1963) and low NAT activity (Klein and Weller, 1970; Rudeen et al., 1975). HIOMT was initially thought to exhibit a circadian rhythm (Wurtman et al., 1963), but if the concentration of HIOMT varies from day to night, it is of very low magnitude (Klein et al., 1981). Stimulation of the pineal gland at night results in a very high concentration of melatonin within the gland (Ralph et al., 1967; Lynch, 1971; Goldman, 1981). Unlike the dark-induced rise in pineal melatonin seen in the white-footed deer mouse, Peromyscus leucopus (Petterborg et al., 1981) and Siberian hamster, Phodopus sungorus (Goldman et al., 1981), pineal melatonin levels in the Syrian hamster are amplified only near the end of 23 the dark period (Rollag et al., 1979). At the cellular level, melatonin synthesis and release are under the control of the sympathetic nervous system (Reiter, 1981). The first step in the action of norepinephrine (NE) from the post- ganglionic sympathetic nerve endings is on the membrane surface of pinealocytes. NE binds to B-adrenergic receptors, which results in the stimulation of the enzyme adenylate cyclase (Weiss and Costa, 1968). Adenylate cyclase aids in the formation of cyclic AMP, whose action is involved in protein kinase formation. The pineal gland is rich in cyclic AMP-dependent protein kinase (Zatz, 1968). Protein kinase is involved in the induction of serotonin-NAT. N-acetyl serotonin is made from exogenous tryptophan. HIOMT converts N-acetyl serotonin to melatonin and melatonin is liberated into the blood vascular system (Rollag et al., 1977) or CSF (Hedlund et al., 1977). Following secretion into the blood, melatonin is loosely bound to plasma albumin and is rapidly hydroxylated and conjugated with sulfate (70-80%) or glucuronide (5%) by hepatic microsomes (Reiter, 1981). The metabolites are excreted in the urine as 6-hydroxy- melatonin sulfate which is easily detected (Mathews et al., 1981). H. Physiology Before Quay (1963) and Wurtman et al. (1963) discovered that the light:dark cycle influenced the biosynthesis of methoxyindoles within the pineal gland, the endocrine effects of this gland were poorly understood. Reducing the amount of light to which hamsters are exposed results in total involution of the gonads and accessory sex organs in males (Czyba et al., 1964; Hoffman and Reiter, 1965; 24 Reiter and Hester, 1966) as well as females (Hoffman and Reiter, 1966; Reiter and Hester, 1966). Characteristically, the testes lack spermatogenic activity (Reiter, 1968a,b) and the epithelial, lining of the sex organs appears involuted (Reiter, 1967). In females the ovaries do not produce ova on a regular basis, corpora lutea are rare, the ovarian interstitium is hypertrophic, and the uterei are greatly reduced in weight (Reiter 1968c; 1969). In both sexes, the inhibitory influence of short days is negated by pinealectomy (Reiter, 1981). In males, radioimmunoassayable (RIA) levels of serum leutenizing hormone (LH) and follicle-stimulating hormone (FSH) are usually depressed in intact hamsters maintained under short day conditions (Berndtson and Desjardins, 1974; Tamarkin et al., 1976a; Reiter and Johnson, 1974; Reiter, 1980b). LH and FSH concentrations within the pituitary gland are also depressed (Reiter and Johnson, 1974). Looking at research on the releasing hormones (RH) within the hypo- thalmus yielded many discrepancies. On one hand, Blask et al. (1979) reported depressed levels of LH- and FSH-RH within the hypothalmus in hamsters with pineal induced testicular atrophy and suggested that the pineal gland may impair the synthesis of RH's. 0n the other hand, Pickard and Silverman (l979) found no measurable differ- ences in hypothalmic RH's between animals kept in long days (func- tional testes) or short days (atrophic testes). In females, the pattern of dark-induced hormonal changes is unusual in that circulating gonadotropin (LH and FSH) levels as measured by RIA exhibit a daily afternoon surge (Seegal and Goldman, 1975). When dark-exposed females are exposed to this LH-FSH surge 25 on a daily basis, ovulation does not occur, the ovarian interstitium enlarges and estrous cyclicity is interrupted (Reiter, 1981). Thus, the pineal gland does not depress all of the female reproductive hormones, but causes an imbalance which is incapable of supporting ovarian cyclicity (Reiter, 1981). With these previous factors in mind, the pineal is thought to integrate the reproductive capability of seasonal breeders in the wild to the changes in their environment. These same photo- periodic responses are seen, to a far lesser extent, in the wild rat which is a continual breeder. Besides the Syrian hamster, the gland is linked to seasonal reproduction in the dwarf hamster (Hoff- mann, 1973), white-footed deer mouse (Lynch and Epstein, 1977), grasshopper mouse (Zucker et al., 1980), ferret (Thorpe and Herbert, 1976) and rabbit (Lincoln, 1976). Since the pineal gland has such a strongly antigonadotropic effect, it was postulated that the methoxyindoles (melatonin) may be the active pineal hormone. Many of the early studies of melatonin were unsuccessful until it was discovered that melatonin must be given at precise times during the light:dark cycle in order for it to be effective as an antigonadotropic agent. When hamsters are kept in a light cycle of 14:10, melatonin injections only in the afternoon suppress reproductive function (Tamarkin et al., 1976b). In general, hamsters are sensitive to melatonin between 7-14 hours after lights on (Reiter, 1981). In this case, the degree of repro- ductive atrophy and hormonal changes are identical to those caused by short days. Sackman et al. (1977) reported that this response 26 was specific for melatonin and no other pineal compound will dupli- cate its effects. Several conditions and compounds influence pineal function (from Relkin, 1976; Kappers, 1978). Light, dark, sound, temperature, underfeeding, locomotor activity and sleeping all affect pineal sympathetic innervation, whereas hypothalmic hormone5‘(LH-RH, RSH-RH and TSH-RH) and pituitary hormones (FSH, LH, MSH, and prolactin), sex steroids (androgens, estrogens, and progesterones), and non- pineal indoleamines and catecholamines all affect pineal function via the neurohumoral route. 0n the other hand, many organ systems and organs are known to be influenced by the pineal secretory sub— stances. Locomotor activity, sleep, hypothalmic oxytocin and vaso- pressin as well as hypothalmic LH- and FSH-RH, TSH-RH and ACTH-RH, prolactin-RH and growth hormone-RH melanocyte hormone inhibition factor (MIF) as well as pituitary LH, FSH, TSH, ACTH, prolactin, GH, and MSH are all affected (usually depressed). Pineal indolamines and polypeptides are also known to influence the thyroid, adrenals, growth, pigmentation, parathyroid function and the islet hormones in the pancreas. In addition, Banerji and Quay (1976) suggested a functional significance of the pineal gland on the adrenal medulla. Besides the effects of the pineal on reproduction in the Syrian hamster, Reiter (1981) suggests a more general function. He proposes that the pineal gland is "an intermediary between the external environ- ment and the entire organism". In some vertebrates, the pineal may be important in sensing photoperiodic changes for the purpose of regulating the annual cycle of reproduction. In other vertebrates the same information nay be necessary to regulate body temperature, 27 influence lipid metabolism or to determine behavioral patterns. As described in the introduction, many actions of this multifaceted organ have yet to be uncovered or understood. CHAPTER 3 EFFECTS OF PINEALECTOMY ON REPRODUCTION IN THE SYRIAN HAMSTER MUTANT ANOPHTHALMIC WHITE (WE) A. Introduction The pleiotropic mutation WE (anophthalmic white) in the Syrian hamster, Mesocricetus auratus, causes animals homozygous for this gene to be eyeless (Knapp and Polivanov, 1958; Robinson, 1962; Asher, 1968; Yoon, T973, T975), to lack pigmentation of the coat and skin (Robinson, 1962, T964; Asher, T968) exhibit severe growth retardation (Asher, T968; James at al., T980), and lack reproductive ability (Knapp and Polivanov, T958; Asher, l968; James at al., T980; Jackson, l98l). In addition, Asher (T968, 1981) demonstrated that the gene' causes thyroid and adrenal abnormalities, altered sleep patterns, deafness and extreme nervousness. Knapp and Polivanov (T968), Hughes and Geeraetes (l962), Asher (T968), Yoon (T975) and Jackson (T98T) all discuss the failure of eye development in this mutant, leading to only "raw tissue" beneath the eyelids and complete and progressive degeneration of the retina, retinal derivatives and lens. The eye and its nervous components are completely degenerated in the adult (Asher, l968; Yoon, 1975). These degenerative changes appear to be caused by a failure of the resorption of cilia on cells of the optic stalk and optic cup (Asher and James, T982). 28 29 Most male hamsters homozygous for WE in the An/As1Wh strain have atrophic testes which possess abnormalities in Leydig, Sertoli and developing germ cells (James et al., 1980). Seminiferous tubules contain germinal epithelial cells arrested in the early spermatid stage of spermiogenesis, possibly due to premature failure of the Golgi apparatus and dysgenesis of the acrosome (James et al., 1980). These atrophic testes resemble those from normal hamsters which were experimentally blinded (Reiter and Hester, T966; Reiter, T968a, T968b) or exposed to short photoperiods (Hoffman and Reiter, 1965). The reproductive function of testes and ovaries of light deprived normal hamsters are found to be actively inhibited by the pineal gland (Reiter, 1968a; Reiter and Sorrentino, 1968), which is thought to interfere with normal neuroendocrine function (Reiter, l968a). In general, the hamster pineal is thought to synchronize the annual reproductive cycle of wild hamsters by detecting changes in the photoperiod (Reiter, l98l). When the amount of light to which hamsters are exposed is reduced, as is the case during the winter months of hibernation, the pineal causes a total involution of the gonads and accessory sex organs (Reiter, 1981). On a daily basis, hamsters interpret anything less than 12.5 hours of light as a short day (Elliott, T976). Because of the importance of light to the main- tenance of normal reproductive abilities in the hamster and the negative effects of the pineal on reproduction in the absence of light, it was suggested by James et al. (T980) that Wh causes infer- tility by causing blindness. For this reason, the present study was undertaken to determine whether infertility in anophthalmic 30 white hamsters was a pineal mediated response due to the lack of function of the visual pathway. B. Materials and Methods Animals used in this study exhibited genotypes and phenotypes as described by James et al. (T980) and are as follows: (T) normal hamsters-gwh/gg; (2) experimentally blinded, normal hamsters-- .gh/gfl(8); (3) heterozygous hamsters-fiflh/gh; and (4) homozygous (eye- less) mutant hamsters-1Wfl75h. As a result of low breeding capacity and lack of resistance to environmental stress, these hamsters proved to be exceptionally hard to breed and maintain. Since the expression of ya was enhanced by the presence of cream (9), the strain was made homozygous for g, This portion of the genotype was not included in the genotypic designation since all animals are g/e, All hamsters were from 13 to 16 generations of full-sibling inbreeding. The strain AN/As:Wh_(Asher, T968) is maintained by a system of full-sibling mating where at least one parent is heterozygous for Wh. Hamsters were housed in polycarbonate cages with galvanized or stainless steel tops, cleaned weekly and provided with pine shavings for bedding. Wayne Laboratory or Breeder Chow and water were provided .99 Tibidum. Lighting of the animal room'was on a regime of LD 13:11 with light from 0800 to 2100 hours. At least ten hamsters of each genotype (1 through 4 above) were employed in this investigation. In addition, four to five hamsters from four different treatment groups were used: (l) normal, blinded and sham pinealeCtomized [gh/gh(B)-SPE]; (2) normal, blinded and pinealectomized [fig/gg(B)-PE]; (3) mutant, eyeless sham pinealectomized 31 [LIE-SPE]; and mutant,eyeless pinealectomized [Lh/fl-PE]. Blinding, shampinealectomy [SPE] and pinealectomy [PE] were performed at approxi- mately 30 days. Animals were enucleated by cutting the optic nerve and removing the entire eyeball. The technique for PE was described by Hoffman and Reiter (1965b). Experiments at 60 Days At approximately 60 days, yfl/gg(B) hamsters and Wh7Wh hamsters, who had been SPE or PE at 30 days, were weighed and then unilaterally castrated. These testes were fixed in Bouin's solution for one hour, weighed, cut into small pieces, and fixed in Bouin's solution for at least three days. Small tissue pieces were dehydrated through alcohols, embedded in paraffin, cut into 8 um sections, and stained with Harris' Hematoxylin and Eosin. Six random tubules from each testis were drawn from images made with a Bausch and Lomb microscope slide projector at a magnification of 230x. Tubule diameters were determined by measurement from these drawings. Experiments at 135 Days Testes from all hamsters at approximately 135 days were collected for electron microscopic examination. Hamsters were weighed, anes- thetized with ether and perfused through the left ventricle with saline followed by phosphate buffered Karnovsky's fixative (Glauert, 1975) at pH 7.3. The reason for using this method of perfusion was that the pineal glands were also used for ultrastructural analyses (Chapter 4). One or both testes were then weighed and sliced into thin slivers. These slivers were allowed to fix one hour at 4°C in phosphate buffered Karnovsky's fixative at pH 7.3. Each sliver 32 was cut into small pieces and fixed an additional three hours to 4°C. The tissue was post-fixed for two hours in 1.0% phosphate buffered osmium tetroxide, dehydrated through a graded alcohol series and embedded in Epon-Araldite. Thick (1 pm) epoxy sections were stained with 1.0% toluidine blue. From the thick sections, six random tubules were drawn using images produced by a Bausch and Lamb glass slide projector at a magnification of 230X. Tubule dia- meters were determined by measuring them from these drawings, as above. Ultra-thin sections were stained in uranyl acetate and lead citrate and examined in a Philips 300 transmission electron micro- scope at 80 kV. C. Results The wfl[!h(8) and Wh7Wh_hamsters at approximately 60 days were similar in body weight (Tables 1 and 2). When normalized to body weight, testes from Wh/Wh individuals were slightly larger than those from iii/ENE) individuals (Tables 1 and 2). Morphologically, all testes were identical in composition, containing large semini- ferous tubules and spermatozoa (Figs. 1 and 2). Tubule diameters were similar for both groups (Tables 1 and 2). At approximately 135 days, gg/gh(8) and gh/wfl(B)-SPE hamsters possessed a significantly higher body weight than did all other hamsters (Tables 3 and 4). All gh/wh(8) and wh[!h(B)-SPE hamsters had exceptionally small testes (Table 3). Most Wh7Wh_and Wh/Wh75PE hamsters also had small testes (Tables 3 and 4), although the severity of reduction was not uniform within these groups as indicated by the large standard deviation in testes weights (Table 3). When 33 normalized to body weight (Table 3 and 4), all atrophic testes were more than proportionately reduced in size, suggesting that body weight did not influence the severe reduction in testes weight. Note that the testes of Wh7!h:PE are normal-sized and show normal variation as indicated by the standard deviation (Tables 3 and 4). Light Microscopy Light microscopic morphology of testes from yfl/wh_and.yg[gh hamsters at 135 days were identical to the morphology of testes described by James, Hooper, and Asher (1980). All testes from Ell/1MB) and fl/gh(B)-SPE and most gig/W3 and NEIL-SPE hamsters at 135 days contained small seminiferous tubules in which spermio- genesis was arrested (Figs. 3-6). By contrast, testes from fl]_wfl(B)-PE and thfl-PE hamsters at 135 days were identical to Leg/fl and 111/311 testes and possessed large seminiferous tubules (Figs. 7-8). Semini- ferous tubules from these groups of hamsters contained differentiating germ cells, spermatozoal heads and spermatozoal tails (Figures 7-8). Approximately 30% of the testes from Wh/Wh and WhLWh¢SPE hamsters contained seminiferous tubules identical in composition to testes from Lh/wjl described above. Seminiferous tubules from all wflthjB), gh/wfl(B)-SPE, and 70% 0f.!h[!fl and Wh/WQ:SPE hamsters at 135 days were reduced in size as revealed by mean diameter measurements in Table 3. In order to determine the magnitude of the effect that blinding had on testicular morphology, an analysis of variance was used (Table 4). Since the variances for testis weights from Wh7Wh and Wh7Wh7$PE hamsters at 135 days were heterogeneous, these statistics represent 34 an underestimate of their true differences (Cochran, 1947). Duncan's multiple-range test (Duncan, 1955) was used to determine which indi- viduals contributed to the large differences observed in population means (Table 5). Electron Microscopy The ultrastructure of interstitial tissue from testes of both wh/gh(8) and Wh7Wh hamsters, at 135 days, was identical and was located in the angular interstices between the seminiferous tubules (Figs. 9 and 10). Leydig cells occurred in clusters and were loosely associated with blood vessels (Figs. 9 and 10). Each Leydig cell was small and contained some mitochondria and smooth endoplasmic reticulum. All nuclei were indented‘and possessed, peripherally and centrally, clumped heterochromatin (Figs. 9 and 10). Interstitial tissue from testes ongh/wh(B)-PE and Wh/WthE hamsters, at 135 days, was identical in composition and was found to be loosely asso- ciated with blood vessels (Figs. 11 and 12). Leydig cells contained a large indented nucleus with peripherally disposed heterochromatin, and cytoplasnlwith a well-developed Golgi complex, smooth endoplasmic reticulum, mitochondria, polysomes, and lipid (Figs. 11 and 12-14). Within the testes of PE hamsters, Leydig cells possessed an extensive system of smooth endoplasmic reticulum (Fig. 13) as well as mito- chondria with tubular cristae (Fig. 14). The boundary tissue from seminiferous tubules was very interesting. As previously stated, seminiferous tubules from testes of gh/gh(8) andeh/Wh_hamsters, at 135 days, were atrophic. The boundary tissue, or basal lamina, from these testes appeared highly infolded and 35 reduplicated (Figs. 15-18). The single layer of myoid cells com- prising the basal lamina, met end to end (Figs. 16 and 17) and was greatly thickened (Figs. 16 and 18). Each nucleus was infolded (Fig. 17) or elongate and indented (Figs. 16 and 18). Collagen fibrils were prominent (Figs. 16 and 18), especially where the basal lamina was most thickened (Fig. 15). The basal lamina of testes from wfl/fl(B)-PE and Lh/Lh-PE hamsters, at 135 days, was composed of a single layer of flattened polygonal cells, or myoid cells, which met end to end (Figs. 19 to 21) to form a continuous sheet surrounding the tubule. Each myoid cell consisted of an elongate nucleus, which was smoothly contoured and rounded at the ends, and small bundles of collagen fibrils within the matrix (Figs. 19 and 21). The ultrastructure of Sertoli cells from testes of both gh/gh(8) and WhLWh hamsters, at 135 days, were identical (Figs. 22 and 23). Each Sertoli cell rested on the basal lamina and assumed a position further into the lumen of the tubule due to pronounced infolding of the basal lamina. Sertoli nuclei were compacted or infolded making them appear smaller than normal (Figs. 22 and 23). Sertoli cells from testes of gh/wh(B)-PE and Wh/Wh—PE hamsters, at 135 days, were also identical in composition (Figs. 24 and 25). They were found next to the basal lamina and contained a large, highly lobulated nucleus, which was oval in shape. Developing germ cells assumed a close relationship to Sertoli cells (Figs. 24 and 25). Marked degeneration of developing germ cells within the testes of both yfl/gh(8) and Wh[Wh hamsters, at 135 days, resulted in tubules that were completely devoid of spermatozoa and consisted mainly of cellular debris (Figs. 26 and 27). On the other hand, seminiferous 36 tubules from testes of‘gh/wh(B)-PE and Wh/WhrPE hamsters, at 135 days, were packed with spermatozoal heads and spermatozoal tails (Figs. 28 and 29). Spermatogenesis and spermiogenesis proceeded properly in all testes from gh/wh(B)-PE and Wh/thPE hamsters, at 135 days. Normal apposition of the acrosomal granule and vesicle took place and spread- ing of the acrosomal granule and vesicle formed the acrosomal cap (Figs. 30 and 31). Mitochondria moved to the posterior aspect of the cytoplasm (Fig. 30). Elongation of the nucleus, condensation of nuclear chromatin, manchette formation, and tail assembly also proceeded normally (Fig. 30 and 31), resulting in spermatozoal heads and tails near the tubular lumen (Figs. 32 and 33). As previously mentioned and as described by James, Hooper and Asher (1980), approximately 30% of Wh7Wh hamsters possessed normal ' testes, at 135 days. Within the same testis, three characters were prominent: (l) Normal spermatogenesis and spermiogenesis. Early spermatids developed an acrosomal cap, and tail assembly was normal (Fig. 34). Spermiogenesis proceeded normally, resulting in spermato- zoal heads and spermatozoal tails filling the lumen of the tubule (Fig. 35). (2) Some degeneration of the germinal epithelium resulted in some tubules with far fewer spermatozoal heads and tails and some cellular debris (Fig. 36). (3) Total degeneration of the germinal epithelium in some tubules resulted in aspermic tubules resembling those of wh/wh(8) and infertile Wh7Wh hamsters (Fig. 37). A preliminary investigation of testes from two Lil/ya hamsters at 303 and 357 days of age respectively, showed that these testes contained developing germ cells (Fig. 38) and spermatozoa (Fig. 39). 37 .umucapa appmucmspcmaxm mmumuwv:_ Amv .cmme sue; apuchVLLcm_m m mmumupu:_ sz .xomm Lo coapmuwewcmme um Louumnoca muwpm mmapm 5504 one zumsmm a saw: exact mum: mmpanahm .cowump>mu ucmucmam a came an cm>vm one mucmsmcammme FPPm>wuuoammc .mcmme zap xpucouwe_:m_m co saw; xpucmo_mwcmwm mmumuwucw Any so sz xomm Lo compeuwewcmme m an couumnoca mumpm mmmpm ago; one gumzmm a saw: exact mam: mopsnah N .cowum_>mu vcmvcmum A come we cm>wm use mucwsmcammme FP<~ 85 a o: 8.0 a men 85 a a: m: a £82 E atflhm 3:: a A: :54 a mm; 284 a. $4 2.: a m2: 3 “8.3%. :5: a a: 3:3 8 8.0 384 a 8.0 8.2 a 3.8 :: .mflfi 3.0 a 8.8 Rd A 28 m: 8 8.... 3.2 a 2.5 3: $313. 85 a m: m: a m; :8 a a: 9.2: a 8d: 5 atsvmflfi :32 a an 385 a 35 3:3 8 a: 25.2 a 3;: 3 235.3% 3:: .+. as 335 a 25 385 a 35 $3.3 a 3.9: 8: Efixfi and H. 8.8 8.0 a 25 25 .1. $5 «.5: a 8.8: S: glifi :2 E 3 E E 395 LoamEmwo mpanah xuom\mmummh magma» auom acmEummLh N .mxeo mm“ apmume_xoean< an mcmqum_a mpznap use agape: atom o» ume—mscoz “save: mmummh .wgmmmz mmummh .osmmmz zoom Layman: mo mangmcamam: new: .m apneh 40 Table 4. Results from One-Way Analyses of Variance Comparing Means Summarized in Table 3. Mean Square Mean Square Between Classes df Within Classes df F Body (9) 2686.08 7 221.12 56 12.15** Testes (g) 22.09 7 0.43 56 51.39** Testes per Body (%) 17.67 7 0.46 56 38.87** T“b"‘e 26.90 ’ 7 0.91 55 29.63** Diameter (cm) ‘ v **Indicates significance at the 1% level where F7 56 = 2.99. 41 Table 5. Results from Duncan's Multiple-Range Test Comparing Mean Body Weight (A), Testes Weight (8), Testes Weight Normalized to Body Weight (C), and Tubule Diameter Measurements (D) from Table 3. (A) 8 10 9 6 3 7 4 5 (8) 4 5 8 9 3 7 10 6 (c) 4 5 8 9 7 3 6 10 (0) 5 4 8 9 7 3 6 10 *All means were compared at the 1% level of significance. Numbers indicate the means of the following treatment groups: wh/wh, wh/wh(B), wh/gh(B)-SPE, wh/wh(B)-PE,‘Wh[wh, Wh/Wh, Wh/WE:SPE, and :EE7WthE. Those means underscored by a singTE Tine are considered statistically equivalent. 42 0. Discussion In order to maintain reproductive function, male and female golden hamsters, Mesocricetus auratus, must be exposed to considerable amounts of daily illumination (Reiter, 1969). Blinding hamsters or subjecting them to short daily photoperiods (1-2 hours per day) initiates gonadal regression, such that the testes are hypoplasic and aspermic within 6 to 8 weeks (Hoffman and Reiter, 1965a; Reiter, 1968a,b). Bilateral orbital enucleation of male hamsters, at weanling age, does not delay the onset of puberty (Reiter, 1968b), since testicular maturation, in the golden hamster, is independent of photoperiod (Reiter, 1969). Once mature, the reproductive organs rapidly degenerate and remain so until 150 days of age (Reiter, 1969). The results reported here are consistent with those previously reported for blinded hamsters (above) and are summarized in Figure 40. The scenario for this experiment is as follows: all gh[!h(8) and .Wh7Wh hamsters possessed large, mature testes at 60 days of age (Fig. 40). The gene Wh did not affect testicular differentiation, since all testes were identical at 60 days, and contained normal differen- tiating germ cells. From this point, testes from all gh7wh(8) and most Wh/Wh hamsters degenerated to less than one-tenth of their normal size (Figure 40). The reduction in testicular size and weight caused by the Eh (Table 3) was consistent with the aforementioned studies by Hoffman and Reiter (1965a), Reiter and Hester (1966), and Reiter (1968a,b) who first reported a severe reduction in testicular size and weight in experimentally blinded hamsters. Involution of the seminiferous epithelium, accompanied by a concomitant decrease in tubular diameter 43 and thickening of the basement membrane, was observed in experimentally blinded hamsters (Reiter, 1967) as well as all wfl/wh(8) and most .Whflflh hamsters, reported here. Histologically, the atrophic testes of experimentally blinded hamsters described by Reiter (1967) exhibited a complete loss of spermatogenesis. Testes from Wh/Wh hamsters (James et al., 1980) and from those normal(B) and genetically blinded hamsters in this investigation, exhibited an arrest at the early spermatid stage of spermiogenesis. The difference between our results and those of Reiter (1967) are attributed to the present use of electron microscopy which can better identify those cells present in the two or three layers of cells in atrophic tubules (James et al., 1980). Reproductive atrophy in light-deprived hamsters is negated by a number of surgical manipulations some of which include: pinealectomy, superior cervical ganglionectomy, transection of the nervi conarii and transection of the preganglionic fibers entering the superior cervical ganglia (Reiter, 1972a). These procedures interfere with the ability of the pineal to synthesize its hormonal products. Bi- lateral superior cervical ganglionectomy (Wurtman et al., 1964; Eichler and Moore, 1971; Klein et al., 1971) or interruption of the medial forebrain bundle (Moore et al., 1967), prevent the diurnal fluctuations in N-acetyl transferase and hydroxyindole-o-methyl transferase (HIOMT) which converts serotonin to melatonin. Normally, absence of light exaggerates the concentration of HIOMT in the hamster pineal (Anton- Tay and Wurtman, T968), increasing the concentration of melatonin within the gland. Melatonin is not stored within the gland in any appreciable quantity (Reiter, 1981) but is released into the blood 44 vascular system or directly into the CSF (Reiter, 1981). Melatonin, and other pineal indoles are thought to be antigonadotrophic agents which inhibit the pituitary—gonadal axis and reproductive function. It has been well-documented that the biochemical and physiological parameters of the pineal gland are governed by photic stimuli through the eyes and not by direct penetration through the skull (Reiter, 1969). When blinded hamsters (Anton-Tay and Wurtman, 1968) or rats (Wurtman, 1967) are kept in the light, melatonin is actively synthe- sized. In addition, the reproductive organs of blinded hamsters maintained in 16 hours of illumination per day are severely atrophic (Reiter, 1969). The role that the pineal gland plays in the dark-induced repro- ductive atrophy is well defined, since if hamsters are pinealectomized, neither blinding nor short photoperiods evoke atrophy (Hoffman and Reiter, 1966; Reiter, 1968b). In the investigation described here, reproductive quiescence in genetically blinded, (WhXWh) hamsters is a pineal-mediated phenomenon, since removal of the pineal restores fertility (see Fig. 40). All gh/wh(B)-PE and Wh7WhePE hamsters, at 135 days, had large testes which contained spermatozoal-filled seminiferous tubules. In addition, when two of the HDKEDrPE hamsters were mated to normal, heterozygous and Wh7Wh¢PE female hamsters, over 200 progeny were obtained. After pinealectomy, Wh7!h_females easily became pregnant, but were unable to initiate parturition. According to Reiter (1969), the dark-induced reproductive atrophy ' of seasonal breeders is not permanent. Testes of long-term (25 weeks) light-deprived hamsters eventually re-grow to normal size and function, regardless of the state of the pineal gland (Hoffman, Hester and 45 Towns, 1965). For the seasonal breeder, gonadal atrophy is initiated when day-length falls below 12.5 hours of light. They are sexually infertile during the winter months and, in their natural habitat,- probably hibernate. As spring approaches, the gonads undergo spontan eous or endogenous regeneration, which is a light-independent restora tion of the sexual organs (Reiter, 1975). This ensures that the animals are capable of successful reproduction immediately after their emergence from hibernation (Reiter, 1978). The reproductive organs are capable of full recrudescence in the spring because the pituitary-gonadal axis becomes refractory to the inhibition of pineal hormones (Reiter, 1972b). Reproductive competence and incompetence is repeated on an annual basis (Reiter, 1981). These phenomena might be used to explain two other aspects of altered reproduction in Wh/Wh individuals: (1) the continuum of variation between perfectly normal and very abnormal testes and (2) the large testes of two hamsters at approximately 47 weeks. With regards to (l), all investigators using anophthalmic white (Wh7Wh) hamsters. have complained about their lack of reproductive fitness but have been moderately successful in maintaining the mutation by breeding homozygous mutant males and females. Due to the large variation in testes size, some of these hamsters may be inherently refractive to the action of the pineal, or possess inactive pineals with abnormal structure and function, or maintain some type of extra-retinal control of pineal function. Asher (1968, 1981) has proposed that Wh_may influence the development of all diencephalic derivatives, including the pineal. Also, it has now been established that this mutant gene causes a lack of resorp- tion of cilia from ciliated epithelial cells in early hamster development 46 (James and Asher, 1981; Asher and James, 1981). In conclusion, the effects of the gene HQ in the Syrian hamster, Mesocricetus auratus, closely parallel the effects of experimental blinding, such that the infertility observed in anophthalmic white (Wh7Wh) hamsters is caused by the absence of eyes and lack of a func- tion of the visual pathway. Since the primary ultrastructural defect caused by this gene is related to abnormal ciliary resorption during development, further anatomical studies of the pineal gland are in order. 47 ABBREVIATIONS A Acrosomal contents AC Acrosomal cap BL Basal lamina of the seminiferous tubules BV Blood vessel C Collagen CD Cellular debris 0 Diameter of a seminiferous tubule FP Free and attached polyribosomes G Golgi GE Germinal epithelium H Heterochromatin L Lipid LN Leydig cell nucleus M Mitochondria MA Manchette MN Myoid cell nucleus 81 Early spermatid S Late spermatid S Sertoli cell cytoplasm SER Smooth endoplasmic reticulum SH Spermatozoal head ST Spermatozoal tail SN Sertoli cell nucleus PLATE 1 EXPLANATION OF FIGURES Figures 1 and 2 are 8 pm paraffin sections stained with Hematoxylin and Eosin. For increased resolution, Figures 3-8 are 1 um Epon sections stained with Toluidine Blue 0. Figures 9-39 are ultra- thin sections stained with uranyl acetate and lead citrate. Figure 1 Light micrograph of seminiferous tubules representative of testes from fl/flfl(3) hamsters at 60 days. Note the large tubule size and degree to which the germinal epi- thelium (GE) has differentiated into spermatozoa (SH and ST). X170. (Paraffin, H and E). 2 Light micrograph of seminiferous tubules representative of testes from Wh[Wh hamsters at 60 days. Note the large tubule size and the fact that they are identical in com- position to testes from wh/wh(B) hamsters at 60 days. The germinal epithelium (GET—has proliferated and differ- entiated into spermatoza (SH and ST). X170. (Paraffin, H and E . Figure 49 PLATE 2 EXPLANATION OF FIGURES Light micrograph of seminiferous tubules from testes of Lh/Lh(B) hamsters at 135 days. Note the reduction in tubule diameters, along with the lack of properly differ- entiated germinal epithelium (GE). X130. (Epon, Toluidine Blue Light micrograph of seminiferous tubules from testes of [Eh/Eh hamsters at 135 days. Note the reduction in tubule diameters as well as the lack of properly differentiated germinal epithelium (GE). X130. (Epon, Toluidine Blue). Light micrograph of seminiferous tubules representative of testes from wh/wh(B)- SPE hamsters at 135 days. Note that these tubUTEs_ were identical in composition to those from both wh/wh(B) and Lh/Lh hamsters. X130. (Epon, ToluidineB BTU?) . Light micrograph of seminiferous tubules from testes of .WQLWh:SPE hamsters at 135 days. Note that these tubules were also identical in composition to those from both gh/wh(8) 309.!fllflh hamsters. X130. (Epon, Toluidine Bldg). Light micrograph of seminiferous tubules representative of testes from wh/Lh(B)- PE hamsters at 135 days. The germinal epitheTium (GE) proliferated and differentiated within each tubule to form spermatozoa, which were seen as spermatozoal heads (SH) and spermatozoal tails (ST) filling the tubular lumen. X130. (Epon, Toluidine Blue). Light micrograph of seminiferous tubules from testes of Lh/Lh- PE hamsters at 135 days. As seen in Figure 7, the germinal epithelium (GE) proliferated and differentiated int0)spermatozoa (SH and ST). X130. (Epon, Toluidine Blue . 51 PLATE 3 EXPLANATION OF FIGURES Figure 9 Electron micrograph of interstitial tissue representative of testes from Lh/Lh(B) hamsters at 135 days. Note that the Leydig cells were found to be clumped in the spaces between seminiferous tubules (BL). Each Leydig cell nucleus (LN) possessed clumped nuclear heterochromatin (H) and lacked the well-defined cytoplasmic organelles other than mitochondria (M). X4,536. l0 Electron micrograph of interstitial tissue representative of testes from Lh/Lh hamsters at 135 days. Note that the Leydig cells were small and contained some mitochondria (M), and smooth endoplasmic reticulum (SERL Each Leydig cell nucleus (LN) was indented and possessed clumped hetero- chromatin (H). All interstitial tissue was found within the angular space between seminiferous tubules (BL) and were loosely associated with blood vessels (BV). X4,536. ll Electron micrograph of interstitial tissue from testes of Lh/Lh(B)- PE hamsters at 135 days. Leydig cells were loosely associated with blood vessels (BV) and contained a large indented nucleus (LN) with peripherally disposed heterochromatin (H), and cytoplasm with a well- developed Golgi complex (G), smooth endoplasmic reticulum (SER), abundant mitochondria (M), and lipid (L). X4,536. 12 Electron micrograph of interstitial tissue from testes of Lh/Lh- PE hamsters at 135 days. Leydig cells contained a large, indented nucleus (LN), and cytoplasm with a well- developed Golgi complex (G), smooth endoplasmic reticulum (SER), abundant mitochondria (M) and lipid (L). X4, 536. 13 Higher magnification electron micrograph of an area repre- sentative of interstitial tissue from both Lh/Lh(B)- PE and Lh/Lh- PE hamsters. Leydig cells possessed— an exten- sive system of smooth endoplasmic reticulum. X10,368. 14 Higher magnification electron micrograph of the juxtanu- clear region in Figure 12. Leydig cells possessed mito- chondria (M) with tubular cristae, an extensive Golgi complex (G), and many free and attached polyribosomes (FP). X10,368. 52 Figure 15 & 16 17 G 18 19 & 20 21 S3 PLATE 4 EXPLANATION OF FIGURES Electron micrographs of the basal lamina of seminiferous tubules from testes of'gh[!h(8) hamsters at 135 days. Note the degree to which the basal lamina is reduplicated in Figure 15. Each myoid cell nucleus (MN) was elongated and indented (Fig. 16). Collagen fibrils were prominent (C). X2,592 and X9,072, respectively. Electron micrographs of the basal lamina from seminifer- ous tubules of testes from Wh/Wh hamsters of 135 days. Note the infolding and thiCEEnifig of this boundary tissue. Each myoid cell nucleus (MN) was highly indented or in— folded and the compacted myoid cells met end to end (arrow in Figure 17). Collagen fibrils (C) were prominent. X3,110 and X9,072, respectively. Electron micrographs of the basal lamina representative of testes from yfl/gh(B)-PE hamsters at 135 days. Myoid cells possessed an elongated nucleus, with a smooth con- tour, and small bundles of collagen (C). Each cell met end to end (arrow in Figure 20) to form a continuous sheath. X13,096 and X6,912, respectively. Electron micrograph of the basal lamina representative of testes from Wh[!thE hamsters at 135 days. Myoid cells possessed an elongated nucleus (MN) and small bundles of collagen (C). X13,096. 54 . . ‘I. 1 . ' " “ j? " L" r . “..1-7 ‘ F Figure 22 23 24 25 55 PLATE 5 EXPLANATION OF FIGURES Electron micrograph of seminiferous epithelium from testes of'gfl/wfl(8) hamsters at 135 days. Note that Sertoli cells rest against the basal lamina (BL) and possess compacted nuclei (SN). X4,536. Electron micrograph of seminiferous epithelium from testes of !h[!h hamsters at 135 days. Note that Sertoli cells rest against the basal lamina (BL) and possess a compacted nucleus (SN) identical to those of testes from fl/LYMB) hamsters, above. X4,536. Electron micrograph representative of seminiferous epi- thelium from testes of wh/gfl(B)-PE hamsters at 135 days. Sertoli cells were found next to the basal lamina (BL) and contained a large, lobulated nucleus (SN), which was oval. Developing germ cells (GE) assumed a close rela- tionship to the Sertoli cells. X4,536. Electron micrograph of seminiferous epithelium from testes of flh[flthE hamsters at 135 days. Sertoli cells were found next to the basal lamina (BL) and contained a large, lobulated nucleus (SN). Developing germ cells (GE) assumed a close relationship to Sertoli cells. X4,536. Figure 26 27 28 29 57 PLATE 6 EXPLANATION OF FIGURES Electron micrograph of the most advanced developing germ cells found in testes from Lh/Lh(B) hamsters at 135 days. Early spermatids (S1) possessed a large acrosomal granule (A) at the anterior 1pole of the nucleus. From this point, germ cells degenerated into cellular debris (CD). X3, 629. Electron micrograph of seminiferous epithelium from testes of Lh/Lh hamsters, at 135 days, near the tubular lumen. Note Tthe absence of developing germ cells accompanied by cellular debris (CD). X4, 536. Electron micrograph of seminiferous epithelium from testes of wh/Lh(B)- PE hamsters, at 135 days. The germinal epi- theTTum developed into spermatozoa, seen as spermatozoal heads (SH) and spermatozoal Tails (ST) filling the lumen of the tubule. X2,269. Electron micrograph of seminiferous epithelium from testes of Lh/Lh- PE hamsters, at 135 days, toward the tubular lumen. TGerm cells developed into spermatozoa (SH). Sper- matozoal tails (ST) filled the tubular lumen. X2, 269. Figure 30 31 32 33 59 PLATE 7 EXPLANATION OF FIGURES Electron micrograph of early spermatids (S ) from testes of Lh/Lh(B)- PE hamsters, at 135 days. Not% the acrosomal contents (A) at the anterior pole of the nucleus, the movement of mitochondria (M) to the posterior aspect of the cyt0plasm and the beginning of formation of the spermato- zoal tail (ST). X12,096. Electron micrograph of a late spermatid (S) from testes of Lh/Lh- PE hamsters, at 135 days. Note t6e spreading of the acrosomal contents to form an acrosomal cap (AC), elongation of the nucleus, formation of the manchette microtubules (MA), and formation of the spermatozoal tail (ST). X15,120. Electron micrograph of a spermatozoa near the tubular lumen in testes of Lh/Lh(B)- PE hamsters at 135 days. Each spermatozoal head (SH)— consisted of condensed nuclear chromatin bounded by an acrosomal cap (ACL The typical spermatozoal tail (ST) projected into the tubular lumen. X6,048. Electron micrograph of spermatozoa near the tubular lumen in testes from !h[!h¢PE hamsters at 135 days. X6,048. Figure 34 35 36 37 38 39 61 PLATE 8 EXPLANATION OF FIGURES Electron micrograph of an early spermatid ($1) from Lh/Lh tubules which approached the normal phenotype, at 135 days. Note the spreading of the acrosomal contents (A) and the movement of the mitochondria (M) to the posterior aspect of the cytoplasm towards the spermatozoal tail (ST). X6,048. Electron micrograph of spermatozoa near the tubular lumen in Lh/Lh tubules which approached the normal phenotype, at 135— days. Spermatozoa are seen as spermatozoal heads (SH) and tails (ST) filling the tubular lumen. X2,269. Electron micrograph of spermatozoa near the tubular lumen in Wh/Wh tubules which approached the normal phenotype, at 135—days. These tubules exhibited some degeneration, or regeneration, in the germinal epithelium which resulted in far fewer spermatozoal tails (ST) within the lumen and cellular debris. X3,780. Electron micrograph of a Lh/Lh tubule within testes which approached the normal phenotype, at 135 dayT Total de- generation of the germinal epithelium within this tubule resulted in a totally aspermic tubule, which resembled those from Lil/mm) and infertile Lil/Ln hamsters, as described above. X2,269. Electron micrograph of seminiferous epithelium from the testis of a Wh/Wh hamster at 303 days. Note that the erminal epiTheTTum has differentiated into early spermatids TSI). X5,184. Electron micrograph of seminiferous epithelium from the testis of a Wh/Wh hamster at 357 days. Note that the spermatozoal taTTs (ST) fill the lumen of the tubule. X2, 592. Figure 40 63 PLATE 9 EXPLANATION OF FIGURES Summary of the relationship between age and testes weight, expressed as gram percent body weight, in all experimental groups of hamsters from the AN/As:!h strain. The experi- mental groups are indicated by numbers within brackets. At 60 days, the numbers indicate: [1] gfl/gg(8), and [2] !fl[flh. At 135 days, the numbers indicate: [3] wh/gg, [4] fl/WMB). [5] wh/zh(B)-SPE. [6] wh/wh(B)-PE. [7]— 111%. T8] Lia/ya. 1'91 gym-SPE. and—I151 gym-PE. 64 9.1 n a h 11 7 TW. 6 5 1 .w .IT. 3 0.0 0.40 4. 3 2 .l TIT 2 ..I.l+|..| 0.0.9.0 4 3 . $.83 292$ mete l35doys 60 days CHAPTER 4 FINE STRUCTURE OF THE SUPERFICIAL PINEAL FROM THE SYRIAN HAMSTER MUTANT ANOPHTHALMIC WHITE (HE) A. Introduction The gene yh_in the Syrian hamster, Mesocricetus auratus, causes severe degenerative microphthalmia, or anophthalmia (Knapp and Poli- vanov, 1958; Robinson, 1962; Asher, 1968, 1981; Yoon, 1973, 1975; Jackson, 1981), a lack of reproductive ability (Knapp and Polivanov, 1958; James et al., 1980), and a whole host of other developmental and physiological problems (Asher, 1981). With regard to reproductive ability, James at al. (1980) and Chapter 3 revealed that approximately 30% of male hamsters homozygous for Eh were fertile. Seventy percent of yhfyh_hamsters were sterile and possessed atrophic testes which resembled testes from experimentally blinded, hamsters (Reiter and Hester, 1966; Reiter, 1968a,b; James and Asher, 1982) or hamsters exposed to short daily photoperiods (Hoffman and Reiter, 1965). Since the reproductive organs of light- deprived, normal hamsters are actively inhibited by the pineal gland (Reiter, 1968a; Reiter and Sorrentino, 1970; Chapter 3), it was pro- posed that testicular atrophy caused by Eh was a pineal-mediated phenomenon. Removal of the pineal gland inhibited the action of .yg on the male reproductive system (James and Asher, 1982). The normal testes from !h[!h hamsters were thought to be due to an inherent 65 66 insensitivity of the pituitary-gonadal axis to the action of the pineal or to severely abnormal pineal structure and function. James and Asher (1981) further demonstrated that cells from the pars distalis of 135 day !h[!h hamsters have abnormally ciliated secretory cells while Asher and James (1982) demonstrated that !h causes anophthalmia by preventing the resorption of cilia from embryonic neuroepithelial cells. Finally, Asher (1981) proposed that Eh should alter all structures derived from the embryonic diencephalon, including the posterior lobe of the pituitary and pineal. According to Clabough (1973), development of the hamster pineal gland begins during the last five days of gestation (day 11-16). At days 12 and 13, the epiphyseal evagination elongates in the dorso- caudal direction and forms a thick, tubular structure. The ependymal cells of this thickened tube differentiate into primitive pinealo- blasts which form cellular cords and follicles. By day 15, invading mesenchyme forms connective tissue septae between the parenchymal cords and follicles and the vascular components of the developing gland become detectable. The pineal lumen persists in the proximal one-third of the organ. At term (16 days gestation), the pineal is a dense cellular mass which undergoes rapid growth, cellular proli- feration and compaction. At this point, the lumen and follicular structures are lost (Reiter, 1981; Clabough, 1973). The subdivision of the pineal gland into a superficial and deep pineal begins on the sixth postnatal day (Reiter, 1981). The gland rapidly enlarges and a constriction forms near its center (Hewing, 1976). The constriction becomes more pronounced, and during the next 67 seven days, the pineal stalk becomes thinner. ~By 22 days after birth, and through adulthood, the stalk consists of a few parenchymal cells, blood vessels, non-myelinated nerves, and an evagination of the poster- ior aspect of the third ventricle (Sheridan et al., 1969). The deep pineal lies in the posterodorsal diencephalon and the superficial pineal is adherent to the undersurface of the junction of the superior sagittal sinus and transverse sinuses (Sheridan and Reiter, 1970). With regards to the superficial pineal, cellular hypertrophy and differentiation begins at birth and continues until 9-12 weeks post- partum (Reiter, 1981). Ultrastructural aspects of the adult super- ficial pineal have been described from the normal hamster by Sheridan and Reiter (1968). One interesting aspect of pineal development concerns the transitory surface modifications of the primitive pineal cells which border the pineal lumen or follicles. As long as a lumen or follicles are present (until day 15), apical cytoplasmic bulges possess cilium- like processes containing two or more centrioles and an axoneme exhi- biting a 9+0 tubular configuration (Clabough, 1973). The severe degenerative microphthalmia or anophthalmia in .Hfllflfl is a consequence of abnormal eye development (Hughes and Geeraeters, 1962; Asher, 1968; Jackson, 1981). Asher and James (1982) discovered that EM/!h_embryos were unable to resorb cilia during early organogenesis and consequently the cell-cell interactions neces- sary for proper differentiation could not take place. Since Eh is thought to alter all embryonic derivatives of the diencephalon (Asher, 1981), it was the purpose of this investigation to determine if Eh 68 alters the ultrastructural properties of the adult superficial pineal gland. B. Materials and Methods Animals used in this study exhibited phenotypes and genotypes as illustrated by James et al. (1980) and are as follows: (l) normal hamsters-:whfwh; (2) experimentally blinded normal hamsters-ewh/wh(8); (3) heterozygous hamsters-fighfwh; and (4) homozygous mutant (eyeless) hamsters-1!h[Wh. These animals were exceptionally hard to breed and maintain, due to low breeding capacity and low resistance to environmental stress. Since the action of Wh is enhanced by the presence of cream (g), all hamsters were made homozygous for the gene g, This gene was not included in the above genotypic designations since all animals were homozygous 31;, The strain AN/ASfiEE (Asher, 1968) is maintained by a system of full-sibling mating where at least one parent is heterozygous for Wh. Hamsters were housed in polycarbonate cages with galvanized or stainless steel tops, cleaned weekly and provided with pine shavings for bedding. Wayne Laboratory or Breeder Chow and water were provided 3g libidum. Lighting of the animal room was on a regime of LD 13:11 with light from 0800 to 2100 hours. Tissues were randomly collected between June (1981) and March (1982). The pineal glands from at least ten hamsters of each genotype (1 through 4 above) were collected for electron microscopic examina- tion. Beginning at 0900, hamsters were weighed, anesthetized with ether and perfused through the left ventricle with saline followed by phosphate buffered Karnovsky's fixative (Glauert, 1975) at pH 7.3. 69 All solutions were made less than four hours prior to perfusion. While immersed in fixative, each gland was carefully pulled from the calvaria and dissected from the confluence of sinuses. The entire gland was weighed, and then fixed for four hours in phosphate buffered Karnovsky's fixative, at 4°C. Each pineal gland was post-fixed for two hours in 1.0% phosphate buffered osmium tetroxide and dehydrated through a graded alcohol series. For transmission electron microscopy, all glands were embedded in Epon-Araldite. Thick (1 pm) epoxy sections were stained with 1.0% toluidine blue and used for survey purposes. Ultra-thin sections were stainedin uranyl acetate and lead citrate and examined in a Philips 300 transmission electron microscope at BOkV. For scanning electron microscopy, two additional glands from normal and mutant hamsters were critical point dried, coated with gold/paladium and examined in a JEOL JSM 35 scanning electron micro- scope at 15kV. C. Results In this investigation, no gross structural differences were observed between the pineal glands of male hamsters in the An/Asfiflh strain. Pineals from all groups displayed a high degree of variability, but all possessed a superficial pineal and a long stalk (Figs. 1 and 2). The wh/wh(B) hamsters had significantly increased body weights, while Wh/Wh hamsters had significantly reduced body weights, when compared to both normal and heterozygous hamsters (Table 1). Pineal glands from Wh/Wh_hamsters were approximately one-half the size of those from [ll/Eh, iii/111(8), or gig/1r; hamsters (Table 1). When 70 normalized to body weight, both wh/wh(B) and Whflflh hamsters possessed small pineal glands (Table 1). In order to determine the magnitude of these differences, analyses of variance were employed (Table 2). The Honestly Significant Difference (HSD) test of Tukey was used to determine which genotype or genotypes contributed to the large differences observed in population means (Table 3). These significant differences are indicated by H or L in Table 1. Using the electron microscope, a number of morphological para- meters of the superficial pineal were compared between wig/1h, EVE“), Wh/wh, and lib/fl hamsters. These parameters included: (l) pinealo- cyte morphology, (2) blood vascular relationships, (3) glial components, and (4) nervous components. Within the superficial pineal, no major differences existed in any of these parameters, except that the pinealo- cytes from all light-deprived hamsters contained an excess of lipid and Golgi with vesicles, along with a reduction in the stacks of rough endoplasmic reticulum. The superficial pineal from fl/_w_h_ and Hfl/‘Lh hamsters were identical in composition and contained pinealocytes, blood vessels, and glial cells (Fig. 3). Pinealocytes were the most frequently encountered cells and were of two types, light (PC81) and dark (PCBZ) (Fig. 4). Both light and dark cells had a large nucleus, with a prominent nucleolus (Fig. 4). Numerous capillaries were present and possessed endothelial cells. Capillary fenestrations or pores were absent (Fig. 5). Both light and dark pinealocytes contained all of the expected cytologic features, such as an extensive Golgi complex, lipid, mito- chondria, and microtubules (Figs. 4 and 6). In addition, pinealocytes 71 from normal, and heterozygous hamsters possessed two striking features: (l) numerous stacks of rough endoplasmic reticulum (Figs. 4, 6, and 7), and (2) an abundance of smooth endoplasmic reticulum (Figs. 4, 8, and 10). Pineal cell processes originated from the cell body (Figs. 4, 6, and 9) and ended chiefly in the perivascular spaces (Fig. 6). They possessed many microtubules (Figs. 4, 9 and 10) and showed a regional distribution of organelles such as mitochondria, Golgi, smooth endoplasmic reticulum, and microtubules along their length (Figs. 4, 9, 10 and 11). Near their terminations, pineal cell pro- cesses contained a large number of granular and agranular vesicles (Fig. 11). The superficial pineal from wh/wh(B) and Wh[Wh hamsters were identical in composition and possessed pinealocytes, blood vessels, and glial cells (Fig. 12). Again, pinealocytes predominated and were of two types: light and dark (Fig. 13). Both light and dark cells possessed a large nucleus, with a small nucleolus, and processes which ramified throughout the gland (Fig. 13). Capillaries were numerous and were not fenestrated. Each capillary possessed an endo- thelial cell (Fig. 14). Light and dark pinealocytes contained all of the typical organelles such as an extensive Golgi complex, endo- plasmic reticulum, mitochondria, and microtubules (Figs. 15—17 and 21-22). Within the pinealocytes of'wh/wh(B) and Wh/Wh hamsters, the extensive Golgi complex was juxtanuclear (Fig. 16), and consisted of flattened agranular stacks of membranes. Vesicles and dense granules were often seen at the periphery (Fig. 16). Pinealocytes did not 72 possess numerous stacks of rough endoplasmic reticulum. Infrequently, small stacks were present but most cells exhibited free polyribosomes throughout their cytoplasm (Figs. 15, 21 and 24). Smooth endoplasmic reticulum was also a prominent feature in pinealocytes from whfwh(B) and Wh/Wh hamsters (Figs. 15, 17 and 22). Mitochondria were similar in pinealocytes from wh/wh and Wh/wh hamsters, as well as wh7wh(8) and Wh7Wh hamsters, and varied in shape, size and internal structure (Figs. 15, 17 and 24). In comparison, larger mitochondria (Figs. 19 and 20) had a highly packed internum and were convoluted (Fig. 19), or cup-shaped (Fig. 20). Pineal cell processes of pinealocytes from wh/wh(B) and Wh7Wh hamsters originated from the cell body and possessed many microtubules, both at points of emergence from the cell body (Fig. 13) and along their length (Fig. 18). Each process showed a regional distribution of organelles (Figs. 13, and 18-20). Near their terminations, pineal cell processes contained granular and agranular vesicles (Figs. 19 and 20). Only one structure resembling that of a vesicle-crowned rodlet was found (Fig. 19). Centrioles were infrequently encountered in all pineal tissues (Fig. 21). Lipid droplets were present in light and dark pinealocytes and in all hamster pineal tissues, but were more prominent in the cells of wh7wh(8) and Wh/Wh hamsters (Fig. 22). Multivescular bodies (Fig. 19) and dense lamellar whorls (Fig. 23) were rare and were equally numerous in tissues from wh/_w_h_, W_h/w_h, wh/w_h(B), and gym hamsters. Glial cells were another common feature of all pineal tissues. Processes from fibrous astrocytes ramified throughout the pineal 73 parenchymal cells and were characterized by electron-lucent cytoplasm and fibrils (Figs. 18 and 24). Bundles of nerve fibers were frequently seen in all pineal tissues. Nerve bundles entered through the pineal capsule in all tissues from wh/wh, wh/wh(8), Wh/wh, as well as Wh[Wh, hamsters (Fig. 25). Myelinated and unmyelinated nerve fibers were usually associated with a Schwann cell and traversed the pineal parenchyma tissue (Figs. 26 and 27). Most nerve bundles were closely associated with blood vessels (Figs. 5 and 28). 74 Table 1. Mean Measurements of Hamster Body Weight, Pineal Weight, and Pineal Weigh mately 135 Days. f Normalized to Body Weight at Approxi- Genotype (n) Body (9) Pineal (mg) Pineal/Body gflfwflb (13) 114.23 i 11.73 0.83 i 0.18 0.73 i 0.16 ‘Wfl[gfl(3) (10) 145.45 i 16.42(H) 0.75 i 0.09 0.52 i 0.05(L) Wh_/_v_7h (11) 117.15 i 13.37 0.75 1 0.19 0.63 1 0.15 jflyflfll (11) 95.46 i 15.39(L) 0.38 i 0.14(L) 0.41 i 0.17(L) 1All measurements are given as mean i standard deviation. 2(B) indicates experimentally blinded animals. (H) or (L) indicates significantly high or significantly low means, respectively. 75 Table 2. Results from One-Way Analyses of Variance Comparing Data Summarized in Table 1. Mean Square Mean Square Between Classes df Within Classes df F Body Weight 4430.680 3 221.030 41 20.05** Pineal Weight 0.457 3 0.029 4l 15.76** Pineal/Body 0.230 3 0.022 41 10.45** **Indicates significance at the 1% level where F3 41 = 4.31. 76 Table 3. The Resultant Values of Q from Tukey’s Honestly Significant Difference (HSD) Test Comparing Means Summarized in Table 1. Parameters Compared Pair/Mean Comparisons Body Pineal Pineal (9) (mg) (74) wh/wh_vs‘wh[wh(8) 7.12** 1.59 4.80** Efl/ED.V5.!D/!fl 0.68 1.63 2.34 wh/wh vs Wh/Wh 4.37* 9.15** 7.47** wh/wh(8) vs Wh/wh 6.17** 0.00 2.40 wh/wh(8) vs Wh/Wh 10.89** 7.04** 2.40 .Whflwh vs Wh/Wh 4.84** 7.21** 4.92** *Indicates significance at the 1% level where 04 41 = 4.70. **Indicates significance at the 5% level where 04 41 = 3.79. 77 0. Discussion The morphology of the superficial pineal, reported here, is similar for all hamsters in the AN/Asfiflh strain. All glands possessed a superficial pineal and a long pineal stalk. The deep pineal was not examined. Large variations in the structure of the pineal complex (00 as described by Vollrath, 1981) were expected. In one study using a highly inbred strain of Sprague-Dawley rats, Broeckmann (1980) reported amazing variations in the structure of this complex. Pinealo- cytes, blood vessels, and glial components made up the bulk of the superficial pineal and were identical to those described for the Syrian hamster by Sheridan and Reiter (1968). Several areas of dif- ference in the cytology of the pinealocyte from wh/wh(B) and Wh7Wh hamsters may be related to the physiology of the gland, since the pineal demonstrated marked gonadal inhibitory effects in these hamsters (Chapter 3). Since anophthalmic hamsters were blinded from birth, should this affect the post-natal maturation of the pineal gland? In rabbits kept in constant darkness from birth, a delay in pineal transformation was apparent by six days. Constant darkness reduced the speed of morphologic transformation, as seen in marked differences at 30 days. By 90 days, however, pineal glands from light-deprived rabbits were identical to those from normal animals (Kerenyi and von Westarp, 1971). Itwas concluded from this study, as well as the developmental study by Trakulrungsi and Yeager (1977) using the rat, that light exerts a major effect, upon the development of the pineal but this effect does not appear to be long lasting. 78 Studies by Grunewald and Omura (1975) and Loenstein (1956) found an increase in size, an enlarged lumen and decreased amounts of glycogen in the pineal glands of blind cave fish, Astyanax mexicanus. Roth et al. (1962) and Lin et al. (1975) found that pineal glands were larger in rats and hamsters, respectively, kept in constant darkness and that this weight change was due to an increased volume of parenchymal cells. Kappers (1976) stated that blinding any animal by bilateral optic enucleation caused hypertrophy of the pineal gland. Fiske et al. (1960) found no change in pineal weights of rats kept in con- stant darkness, but when rats were maintained in constant darkness from birth they had smaller pineal glands (Relkin, 1967). On a rela- tive weight basis, all blind animals, reported here, had decreased pineal weights. This decrease was pronounced in animals blinded from birth (homozygous mutant hamsters-:Wh7Wh), but was the direct result of increased body weights of experimentally blinded hamsters (normal(B)--wh[wh(8). On an absolute weight basis, wh/wh(8) hamsters had normal-sized pineals and Wh7Wh hamsters had small pineals. These differences are probably due to the fact that Whfiflh hamsters were blind from birth. The mature pineal gland is a cellular organ which contains one major cell type, the pinealocyte, chief cell, or parenchymal cell (Reiter, 1981). The mammalian pinealocyte is a modified photoreceptor cell (Vollrath, 1981) and usually displays two forms, light and dark. The appearance of this cell is known to change with the time of day, age or stage of development of the animal, reproductive state, expo- sure to light, or season of the year (Ralph, 1978). Some manipulations that may alter the normal cytology of pinealocytes are hypophysectomy, 79 administration of hormones and drugs, stress denervation, and blinding (Ralph, 1978). Blinding has caused variable differences in pinealocyte cytology. The results obtained after constant darkness and blinding are less pronounced and less consistent than those from other forms of mani- pulation (Vollrath, 1981). Quay (1956) reported increased phyloxine- staining granules in pineal nuclei and suggested that protein synthesis increased in mice kept in constant darkness. Freire and Cardinali (1975) claimed that pinealocytes of rats kept in constant darkness, for two weeks, showed changes compatible with the activation of the organ. Stimulation of the pineal was manifested by an increase in free polyribosomes, increased amounts of rough endoplasmic reticulum, procentrioles and microtubules, increased Golgi, prominent nucleolei and increased numbers of annulate lamellae. Leus (1971) also concluded that constant darkness caused pinealocyte activation in guinea pigs. Constant darkness caused an activation of light cells which exhibited increased mitochondria, smooth end0plasmic reticulum, agranular vesicles, vesicle crowned rodlets and cylinders. An increase in dark cell activity was also apparent (Leus, 1971). Romjin (1975) claimed that constant darkness, for 20 days, caused no morphological alterations in rabbit pinealocytes. Both Clabough (1971) and Lin et al. (1975) demonstrated that the pineal gland of the Syrian hamster showed major alterations after blinding. Pineal glands hypertrophied and pineal parenchymal cells contained an increase in smooth endoplasmic reticulum, in Golgi saccules and vesicles, in vesicle-containing terminals, in multivesicular bodies and in membraneous lamellar structures of various sizes. 80 Membraneous whorls were thought to be a consequence of induction of gonadal atrophy in response to light deprivation (Clabough, 1971), but were probably cytoplasmic artifacts (Lin et al., 1975). The cytoplasm of pinealocytes from all light-deprived hamsters, reported here, were rich in ribosomes and showed an increase in lipid, Golgi vesicles and saccules, and a decrease in stacks of rough endo- plasmic reticulum. Large amounts of smooth endoplasmic reticulum were characteristic of all pinealocytes in this study. No size dif- ferences were noted among pineal parenchymal cells. Although the pineal substance responsible for causing gonadal atrophy in blinded hamsters has not been identified, melatonin is believed to be a strong candidate (Wurtman et al., 1968; Reiter et al., 1974; Reiter, 1981). Pineal glands from blinded hamsters show an increase in hydroxyindole-O-methyltransferase (HIOMT), the enzyme responsible for the conversion of serotonin to melatonin (Anton- Tay and Wurtman, 1968). With the assumption that melatonin is the pineal produced which is antagonist to the gonads, Lin et al. (1975) suggested that HIOMT activity resides in the smooth endoplasmic reti- culum (SER) of pinealocytes. As in neurosecretory neurons, the sub- stance made in the cisternae of the SER in pinealocytes is transferred to the Golgi complex, where it is condensed and packaged in the form of membrane-bound, electron-dense granules. These granules are trans- ported down the cell process (or axon in neurosecretory neurons) to its terminal for storage and release (Sheridan and Reiter, 1970). Light-deprivation increases the activity of the pineal (Anton-Tay and Wurtman, 1968; Lin et al., 1975) and the cytology of pinealocytes Treact accordingly (Lin et al., 1975). In this study, all hamsters 81 had significant amounts of SER, but all blinded hamsters showed an increase in Golgi saccules and vesicles indicative of increased glandu- lar activity. Since the SER is thought to be correlated to the syn- thetic activity of this gland (Lin et al., 1975), increased amounts of SER in normal hamsters may be related to either time of year, or the time of day that animals were killed. Since light-deprivation causes pineal rhythms to become free-running (Vollrath, 1981), the appearance of stacks of rough endoplasmic reticulum may also be related to the time of day that the animals were killed. Lipids have been shown to occur in significant amounts in the pineal parenchymal cells of rodents, as well as man (Quay, 1957). Consistent with the study by Lin et al. (1975), all blind hamsters, in this investigation, contained large amounts of lipid and the lipid dr0plets were usually associated with the smooth endoplasmic reticulum. The conclusions drawn from this investigation were both consistent and disconcerting. Since the reproductive atrophy seen in wh/wh(8) and Wh/Wh hamsters was a pineal-mediated phenomenon (Chapter 3), it was expected that the ultrastructure of the pineal gland from these infertile hamsters would be normal, and stimulated. This was the case. Since 30% of Wh7Wh_hamsters are fertile at 135 days of age, we expected these animals to have defective pineals. Contrary to this prediction the superficial pineals from these fertile Wh/Wh hamsters were identical to those from infertile wh/wh(8) hamsters. The pineal gland acts opposite of most endocrine organs in that it is stimulated by the absence of light and then it exerts an antigonado- tropic effect. If abnormalities existed in the function of the gland, 82 the testes of Wh/Wh hamsters would have been normal. The ultrastruc- tural analyses presented here suggest that the pineal glands from all Wh7flh hamsters function normally. Thus, these analyses cannot explain the occurrence of fertile 135 day old thflh hamsters. Since the ultrastructure 0f.!fl[!h pineals appears quite normal in marked contrast to the ultrastructure of their anterpituitaries which are quite abnormal, the analyses presented here suggest that the pineal may develop normally. A complete developmental analysis of the em- bryonic diencephalon is thus required before the fertility of some .Whflflh hamsters can be understood. 83 ABBREVIATIONS BV Blood vessel C Centriole CA Pineal capsule EC Endothelial cell F Filaments in a glial cell process FR Free polyribosomes G Golgi GC Glial cell GCP Glial cell process L Lipid LW Lamellar whorl M Mitochondria MF Myelinated nerve fiber MT Microtubules MVB Multivesicular body N Nucleolus NB Nerve bundle PCBl Pineal cell body--light PCBZ Pineal cell body--dark PN Nucleus of a pinealocyte PS Pineal stalk RER Rough endoplasmic reticulum SC Schwann cell cytoplasm SN Nucleus of a Schwann cell SP Superficial pineal SER Smooth endoplasmic reticulum UF Unmyelinated nerve fiber VCR Vesicle-crowned rodlet PLATE 1 EXPLANATION OF FIGURES Fi ure i Scanning electron micrograph of a pineal gland representa- tive of wh[wh, wh/wh(8), and Wh[wh hamsters at 135 days. The supeFficial pineal (SP) liEk within the undersurface of the junction of the superior sagittal and transverse sinuses. The pineal stalk (PS) projects into the third ventricle. X38.4 2 Scanning electron micrograph of a pineal gland from a Wh[Wh hamster at 135 days. There is a wide variation in the shape of each gland, but each consisted of a super- ficial pineal (SP) and a long pineal stalk (PS). The same spatial relationships existed, as above. X38.4 Figure 85 PLATE 2 EXPLANATION OF FIGURES Low magnification electron micrograph of pineal tissue representative of wh/wh and Wh/wh hamsters at 135 days. Pineal tissue consisted of pineETocytes (PN), blood vessels (BV), and glial cells (GC). X2,160. Electron micrograph of pineal tissue from fl/wh and _W_h]_wh_ hamsters at 135 days. Pinealocytes predominated and were of two types, light (PCB ) and dark (PCBZ). Pinealocytes contained a large, infoléed nucleus (PN), with a prominent nucleolus (N), as well as lipid (L), mitochondria (M), and smooth endoplasmic reticulum (SER). Rough endoplasmic reticulum (RER) and SER were characteristic features of tissue from normal and heterozygous hamsters. Pineal cell processes (PCP) ramified throughout the gland and possessed many microtubules (MT). X3,888. Electron micrograph of a blood vessel (BV) representative of wh7wh and Wh/wh hamsters at 135 days. Capillaries were numerous aha—possessed an endothelial cell (EC). Nerve bundles (NB) were often associated with each capillary. X4,320. 86 Figure 7 & 8 10 ll 87 PLATE 3 EXPLANATION OF FIGURES Electron micrograph of a pinealocyte from pineal tissue representative of wh7wh hamsters at 135 days. Each pinealocyte possessed a lar e nucleus (PN) and many organelles such as Golgi (G), mitochondria (M), and stacks of rough endoplasmic reticulum (RER). Pineal cell pro- cesses (PCP) originated from the pineal cell body, and ramified throughout the gland. Most processes (PCP) terminated in the perivascular spaces associated with blood vessels (BV). X3,888. Electron micrographs of two features characteristic of .wh/wh pinealocytes at 135 days: (7) numerous stacks of rough endoplasmic reticulum (RER); and (8) large areas of smooth endoplasmic reticulum (SER). X15,552. Electron micrograph of pineal cell process representative of wh/wh_tissue at 135 days. Each pineal cell process (PCP) eminated from the cell body (PCB) and contained many microtubules (MT). X7.776. Cross sections of pineal cell processes from whlwh hamsters showed many microtubules, which were seen as FBlTEw dots within the PCP (arrowheads). SER fills one entire section of another PCP. X10,368. Near their terminals, pineal cell processes (PCP) from wh[wh and Wh/wh hamsters contained many granular vesicles 'Tarrows) as «511 as agranular vesicles (arrowheads). Mito- chondria (M) were large and cup-shaped. X10,368. Figure 12 13 14 89 PLATE 4 EXPLANATION OF FIGURES A survey micrograph of pineal tissue from wh]wh(B) and .Wh7Wh hamsters at 135 days. The superficiET pineal gland consisted of pinealocytes (PN), blood vessels (BV), and glial cells (GC). X1,994. Electron micrograph of a group of pinealocytes representa- tive of pineal tissue from ugh/Em) and gym hamsters at 135 days. Pinealocytes were of two types, light (PC81) and dark (P082). They possessed a large nucleus (PN), with a small nucleolus (N). Pineal cell processes (PCP) ramified throughout the gland and contained many micro- tubules (MT). X3,888. Electron micrograph of a blood vessel (BV) representative of wh[wh(8) and Wh/Wh hamsters at 135 days. Each capillary possessed an endothETial cell (EC) and did not have pores or fenestrations. X3,888. 90 S. a 1 .23.». .. Figure 15 16 17 18 198120 91 PLATE 5 EXPLANATION OF FIGURES Electron micrograph of a pinealocyte representative of pineal tissue from Lh/Lh(B) and Wthh hamsters at 135 days. Each pinealoFth possessed —a_Targe nucleus (PN) and organelles such as Golgi (G), mitochondria (M), and smooth endoplasmic reticulum (SER). Small stacks of rough endoplasmic reticulum (RER) were present, but most existed as free polyribosomes (FR) within the cytoplasm. Pineal cell processes (PCP) ramified throughout the paren- chyma. X3, 240. Pinealocytes within the tissues of wh/Lh(B) and WhIWh hamsters possessed an extensive GolET'Fomplex, Which—was juxtanuclear in position. Golgi membranes (G) were flat- tened and possessed vesicles (arrowheads) and dense granules (arrows) at their periphery. X15,552. Smooth endoplasmic reticulum (SER) was a prominent feature of pinealocytes from Lh/Lh(B) and WJ/WJ hamsters. Small mitochondria (M) were present and showed little internal structure. X12, 960. In tissues from Lh/wJ(B) and WJ/WJ hamsters, pineal cell processes (PCP) Fminated from the cell body (PCB) and contained many microtubules (MT). X15, 552. Near their terminations, pineal cell processes (PCP) from Lh/Lh(B) and Wh/Wh hamsters contained granular (arrows) Fnd agranular—(arrowheads) vesicles. Mitochondria (M) were large and convoluted (Fig.19) or cup- -shaped (Fig. 20). Only one structure resembling that of a vesicle- crowned rodlet (VCR) was seen (Fig. 19). x12, 960 and X15, 552, respectively. 92 Figure 21 22 23 24 93 PLATE 6 EXPLANATION OF FIGURES Infrequently, the cytomplasm from all tissues contained centrioles (C). Some mitochondria (M) were small and had little internal structure. Characteristic of all pinealocytes from yfl/gfl(8) and Wh7Wfl hamsters were the small stacks of RER and free poTysomes (FP) throughout the cytoplasm. X10,368. Pinealocytes from all hamsters contained some lipid, but those from wJ/LMB) and WJ/Wh hamsters contained cells which had an abundance of‘Targe lipid droplets (L). X7.776. Dense lamellar whorls (LW) were present and equally as numerous in all hamster pineal glands. X7.776. All pineal tissues possessed glial cells in the form of fibrous astrocytes (GCP). These cells were characterized by their electron-lucent cytoplasm and thin fibrils (F). X10,368. 94 Figure 25 268127 28 95 PLATE 7 EXPLANATION OF FIGURES Nerve bundles were frequently seen in all pineal tissues. Myelinated (MF) and unmyelinated (UF) nerve fibers entered through the pineal capsule (CA). X3,240. In tissue representative of both wh/wh hamsters (Fig. 26), as well as Wh/Wh hamsters (FTgf-27), nerve fibers were associated with—a Schwann cell (SC and SN) and traversed the pineal parenchyma. X3,888. Unmyelinated nerve fibers (UF) were often seen in bundles (NB) which were closely associated with blood vessels (BV). X3,888. 96 SUMMARY A. Problem The gene Eh, causing anophthalmia in the Syrian hamster, Meso- cricetus auratus, is a highly pleiotropic gene which has profound effects upon eye development, pigmentation and reproduction. Since both W5 and the pineal organ are known to suppress reproductive func- tion, the objective of this investigation was to determine: (1) whether HE: by itself, influences testicular differentation, (2) whether removal of the pineal gland will restore fertility to mutant, eyeless hamsters, and (3) to determine if 31].!fllflfl hamsters possess a pineal gland which is normal in terms of its gross and microscopic morphology. Ultimately, it was hoped that the primary effect of the gene Wh upon reproduction might be identified. 8. Methods In order to determine whether anatomical differences existed in the testes of hamsters at 60 days, the following parameters were measured: (1) body weight, (2) testes weight and (3) tubular diameters. Mutant hamsters, as well as normal, blinded hamsters were either pinealectomized or sham pinealectomized and the following parameters were measured at 135 days and compared to the normal conditions: (1) adult body weight, (2) testes weight, and (3) tubular diameters. 97 98 In addition, the pineal gland was extracted from all adult animals for histological examination. Histological examinations were made at the light and electron microscopic level of both testicular tissue, including seminiferous epithelium and interstitial tissue, and tissue from the superficial pineal. C. Results Testes from all normal, experimentally blinded and most mutant, eyeless hamsters were large and mature at 60 days, but degenerated by 135 days and were hypoplasic and aspermic. Abnormalities were observed in Leydig cells, Sertoli cells and in the developing germ cells. Seminiferous tubules were reduced in size and contained germinal epithelium arrested in the early spermatid stage of spermiogenesis. Pinealectomy fully restored adult testicular size and morphology. Approximately 30% of mutant, eyeless hamsters were fertile at 135 days and possessed testes which were similar to thoese of normal hamsters. The gross morphology of all pineal glands were identical and consisted of a superficial pineal with a long pineal stalk. The deep pineal was not examined. The superficial pineal rested in the junction of the superior sagittal and transverse sinuses and was a mass of tissue containing pinealocytes, blood vessels and glial cells. Pinealocyte cytology differed between normal and blinded hamsters, and was thought to be related to the differences in func- tional activity of the gland. 99 D. Conclusions Interactions between the pineal gland and the gonads in ygzyg hamsters have been discussed in this thesis. Chapter 3 answered three specific questions, with regards to the pineal-gonadal axis, and Chapter 4 answered two questions, concerning pineal morphology, as follows: (l) D0 testes from mutant, eyeless hamsters (Wh7Wh) resemble those from normal, experimentally blinded hamsters jwflfwh(8)) at puberty (approximately 60 days)--does Wh affect the initial differ- entiation of the testes? Since all wh/wh(8) and Wh/Wh_hamsters possess large, mature testes at 60 days, it is concluded that Wh does not affect initial differentiation of the testes. (2) Is the morphology of testes from adult yg/yg hamsters similar to the morphology of testes from gh/gfl(8) hamsters? .Wh affects the reproductive system of male hamsters in such a way that hamsters are infertile and possess atrophic testes which are identical in morphology to testes of gh[!g(8) hamsters. (3) If the pineal gland generally affects the gonads of blinded hamsters in such a way as to suppress testicular function, does removal of the pineal gland allow wh/gfl(8) and Wh/Wfl hamsters to maintain normal teSticular size and function? Testes of all adult, male yfljwfl(8) and Wh7flh hamsters, who had been pinealectomized (PE) at 30 days, possessed large, mature testes at 135 days. Histologically, mature testes had developing germ cells and spermatozoa. In addition, two of the Hfl/Eflpr hamsters were fertile and sired over 200 progeny. (4) Do all hamsters in the AN/ASfiyh strain possess a pineal gland, and if so, what are the gross and histological features of 100 the gland? Pineal glands consisted of a superficial pineal, which rested in the junction of the superior sagittal and transverse sinuses and a long pineal stalk. The deep pineal was not examined. The pineal parenchyma from all tissues contained pinealocytes, blood vessels, and glial cells. (5) Since some yg7yg hamsters are not sterile, does the pineal gland in these few fertile hamsters possess abnormalities which preclude its function? Pineal glands from all hamsters, used in this study, were identical in composition. It is proposed that some Wfl/Wh hamsters are fertile because (l) given time, the gonads of blinded hamsters will become refractory to the antigonadotropic compounds liberated by the pineal or (2) hamsters maintain some type of extra-retinal control of pineal function (i.e., through the parasympathetic nervous system or from nerve fibers from the hypothalmus). In summary, it is clear that infertility in this mutant hamster is a pineal-mediated phenomenon, since testes in these animals do not atrophy when the negative influence of the pineal gland is taken away. In addition, it is also clear that the testes of these mutant hamsters are completely competent to respond to the antigonadotrophic effects of the pineal gland. The most interesting aspect of this study is the fact that the pineal-mediated gonadal atrophy occurs in only 70% of mutant hamsters. These results suggested that the pineal gland may be abnormal in 30% of mutant hamsters, allowing normal testicular differentiation to occur. Unfortunately, pineal ultrastructural examinations revealed no differences in any of the pineal glands, including those from fertile, mutant eyeless hamsters. Since the primary ultrastructural defect caused binh is thought 101 to involve the lack of resorption of cilia from epithelial cells within the diencephalon during early organogenesis, it was postulated that the pineal gland would possess numerous ciliated cells which were retained from their embryonic state. This was not the case. At this point it is clear that further embryological, anatomical and biochemical examinations of this mutant will provide useful infor- mation as to the effects of WE. 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APPENDICES APPENDIX A Comprehensive Tables Containing All Experimental Data .ommx eo :owumuwwpcmms a pm souumnoga muwpm mmm_m sec; use sumamm a sum: czmeu mew; mmpanze cowumeummu Pmsmpmpwcam comuwmpuzcm Fmsmumpwmm H em.m HH.m me.H m.¢o~ mm em 8H mHNm mm.m mm.~ eo.H m.mm mm cm «H swam H~.o m~.m mm.H N.Hofi nm mm m“ «can mm.o “H.m m¢.~ m.mm mm mm ea mmHm mm.m -.m mm.~ m.mm mm mm efi HmHm xsouompmmcwa om.m mm.~ eo.H H.mm om om mH mmHm we.m me.~ om.o m.w~ om om mH mmHm mm.m oo.~ mm.o m.~m cm on ma umfim mm.m e~.N no.H “.mm cm on ma mmflm mH.o mm.~ o~.H m.~m cm on ma mmHm xsouumpmmcwa seem :3 E 8: M3 8 mm E 3 gm mauve xuom emu may an em: AmMmuv ma mam soppmemcmm emnsaz mpanzh mmummh p; we: seem mm< Amamnv Fmswc< m_umm» mm< .mxmo om apmumewxoeaa< um humucepm .smmeuv Amvmmxmm maxuocou we» say memo .<_ «_am» 114 116 Table 3A. Computations for the Analysis of Variance on Body Weight Measurements from Hamsters at Approximately 60 Days. LAMB) PE/SPE Lh/L PE/SPE 92.8 92.8 95.7 99.3 92.9 101.1 78.9 94.4 93.1 86.9 83.9 83.9 93.8 84.1 101.7 83.4 89.6 86.4 104.9 91.2 (y, ) 927.30 903.50 167591.43 [A] t‘i 10 10 20 (.371. ) 92.73 90.35 i 17.58 16.46 8% 51.77 37.53 Sum of Squared Observations within a Group 86506.27 82006.49 168512.76 [B] Correction Factor for each Group 85988.53 81631.23 167619.76 [C] 88y 921.33 SST 28.33 SSE 893.00 MST 28.33 MSE 49.61 117 liable 4A. Computations for the Analysis of Variance on Testes Weight Normalized to Body Weight Measurements from Hamsters at Approximately 60 Days. Lil/22(8) PE/SPE WJ/WJ PE/SPE 2.59 2.48 2.24 2.60 2.00 2.55 2.43 3.01 2.23 3.57 3.17 3.62 3.11 3.78 3.31 3.57 2.32 3.06 3.11 2.65 (.y _i ) 26 33 30 89 163.71 [A] Y‘i 10 10 20 (.371. ) 2.63 3 08 15;_i 30.45 30.51 :22 S 1- 0.18 0.23 Sum of Squared Observations within a Group 71.18 97.74 168.92 [B] CO r‘rection Factor for each Group 69.33 95.42 164.75 [C] 88 ‘3’, 5.2l 1:; S-,- 1.04 :5; SE 4.17 IN] 31- 1.04 M SE 0.23 118 Table 5A. Computations for the Analysis of Variance on Tubule Diameter Measurements from Hamsters at Approximately 60 Days. ‘gh[gh(8) PE/SPE ‘2272h PE/SPE 6 15 5.31 5.98 5.77 5 58 5.67 5.48 6.31 5.56 6.29 5.85 6.00 6.29 5.73 6.11 6.21 5.87 5.46 6.34 6.00 (yi ) 59.21 58.75 695.73 [A] ri 10 10 (17,- ) 5.92 5.88 Si 20.31 10.35 s? 0.09 0.11 Sum of Squared Observations within a Group 351.43 346.23 697.66 [B] Correction Factor for each Group 350.58 345.16 695.74 [C] SSy 1.93 SST 0.01 SSE 1.92 MST 0.01 MS 0.107 119 .ommx Lo comumummmcmms m we Lopumwoen mumpm mmmpm 0504 van cumzmm a cup: czmcu mew: mmpznsh m -.o Hw.o fin.o aH.m Hm.m H.mHH mmfi 8H neon om.o ew.o oo.n o~.m oH.¢ H.wNH mmH ma mfiom om.o No.H n¢.n mo.m No.m ~.mHH mmH mH wfiom cm.o mm.o mm.“ ¢¢.m Nn.m H.moH mma mH nfiom mn.o Nw.o mo.~ om.m m~.m m.eo~ mmH ma omom mm.o em.o om.m mm.~ eo.a ~.~mH mm“ «H mmmm eo.o mo.o mH.o Rm.~ Hm.~ w.mo~ mmH m” mmmm mn.o mo.H no.5 on.~ mn.m ~.mmH mm~ 8H oumm Ho.“ NH.H Hm.o nu.~ o~.m «.mHH mm“ «a momm um.o ~o.o m~.~ om.m nm.m m.ooH vmfi NH “emu mm.o mm.o cm.o m~.m mm.m ~.moH mmH Nd Noam mm.o on.o om.“ mm.m mw.m m.mo~ mmH Na comm mm.o om.o -.~ ew.m eo.e m.moH mmH NH menu .4 s: 3.5 23 :3 M53 M3 383 r: 3 uom son ugawmz gm mswwo xuom Log a; em: an em: mm< copumgwcmw gmnsaz _mmcpa Pawnee mpansp mmummh mmummk seem Pmswc< .mxmo mm“ xpmumswxogaa< um flammeuv.mmxmm maxuocmw we» Lou upon . 00 0000000< 000 000 000000000500 .<~0 0000» 6 12 NH.HN~ mm: mo.omo~ hm: m¢.~mm~fi umm mm.~omm~ hmm wo.mmHHm xwm Hog «M.NNNmHm m¢.mmmwm Ho.owmom mm.o¢~oo~ mo.mmmomfl mm.~m¢mo mm.mmnoHH mo.~mmHH~ mo.~mmmc~ gzogu zoom Low gopunu covuumggou mmu ~m.mmfim~m wH.mnowm oH.HmmHm mm.omw~oH Nu.¢mmmmfi mm.ommmo ¢~.~omNHH HH.¢mN¢HN mm.-¢amfi qzogw a cpgupz meowum>gmmao cmgasam we saw II II II II. 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