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'1".51"1' '1'v1'1e'1'531L' * ‘1." ‘ " .«1‘111111'..'11:;‘1f1111j1111'11-1..11.1'1"«11111‘-11""11“" ""1111”; "" LW“”[’M W H afiWWN 51H“ «1 .. « 1., :::.- 11111111111 11.1111 1“ 115‘“ . .1 - . , «..;11'1':'.‘«. ‘: 1:11:11 111' 1: "1"" 1'1'.,11 ::1_111 "Wm“; . 11'1" " " 1 11.11 1" '11. In 1‘. I| ‘ T . ,. .1" ". 119'. (111'? 1 "I :1 h, '11 “1'11"” ,1 1 1 ..1 lI11I 11. '111 .11 11'1'"' 1' ,',‘ '1 . “11 . . 1 .11 ' " :1". 11111111,: .. ‘1 1'1 I II , 1 "'1. 1 '1 ,1 .1...' 111M.1':""nhu1n1m1|h'L'Ii m11""1"1'1'111' "'"1 111 11"1 '1'1'1'1151 111.}:11I 1 '111 111111 111111'11'11111'1'11'1 « " Hfg" Illllll IIIHII llllllll "Till" This is to certify that the thesis entitled EFFECTS OF THE GENEyfl ON REPRODUCTION IN THE SYRIAN HAMSTER MESOCRICETUS AURATUS presentedby Susan C. James has been accepted towards fulfillment of the requirements for _Masmns__ degree in .ZQQngJL._ W/%/ 4. / Major professor Date—WE EFFECTS OF THE GENE EH ON REPRODUCTION IN THE SYRIAN HAMSTER, MESOCRICETUS AURATUS By Susan C. James A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1979 ABSTRACT EFFECTS OF THE GENE E§_ON REPRODUCTION IN THE SYRIAN HAMSTER, MESOCRICETUS AURATUS By Susan C. James The mutant gene Eh, Anophthalmdc white, in the Syrian hamster, Mesocricetus auratus, is a highly pleitropic gene causing aplasia of the eye, lack of coat skin and eye pigmentation and infertility in male animals homozygous for this gene. Since the cause of infertility was unknown, the anatomy and histology of the testis was investigated. At the gross anatomical level, testis were severely atrOphic. Light and electron microscopic investigations revealed that the testis were aspermic and possessed abnormalities in the Leydig cells, Sertoli cells and deve10ping germ cells. It was concluded that the infertility in eyeless homozygotes may be due to: (l) a defect at the level of the testis, (2) a defect at the level of the pituitary, or (3) lack of eyes and non-function of the visual pathway. Since infertility may be due to (2), light and electron microscOpic examination of the pituitary was also undertaken. Mutant glands contained a 40% reduction in cell number, were highly vacuolated and contained numerous specialized junctional complexes and atypical cilia. These abnoumalities may represent abnormalities in embyronic cellular differentiation caused by Eh. I dedicate this thesis to Mom and Dad. 11 ACKNOWLEDGMENTS I would like to express my sincere appreciation and gratitude to Dr. James H. Asher, Jr. for chairing my graduate committee. His en- couragement and guidance were invaluable. I would like to thank Dr. Gary H00per, Dr. Harold Hafs and Dr. John Shaver for serving on my committee. A special thanks to Dr. Hooper for his patience and expert advice in electron microscopy and to Dr. Hafs for his thoughtful criticism of this thesis. Special thanks are due to Dr. Shirley Siew for her encouragement, instruction and unending interest in all of my undertakings. I wish to thank Linda Chaney and Karen Baker for their friendship and extend a special acknowledgment to Linda for her enthusiasm and continuing encouragement, critical evaluations and stimulating discus- sions which have given me better insight into the problems discussed in this thesis. Thanks to my friends Bruce, Anna and Bob for their unending patience and hours of entertainment throughout the course of this en- deavor. Last, but certainly not least, a very special thanks to my parents, Richard and Patricia James, for their encouragement and material as- sistance. Their interest and advice will always be deeply appreciated. iii TABLE OF CONTENTS EXAMINATION MUTANT ANOPHTHALMIC LIST OF TABLES . . . . . . . . . . . . LIST OF FIGURES O O O O O O O O 0 O O 0 CHAPTER 1. THE MAMMALIAN TESTIS AND PITUITARY CONTROL OF FUNCTION 0 O O O O O O O O A. General Morphology and Function of the Testis . . B. General Morphology of the Pituitary Gland . . . . C. Hypophyseal Control of Testis Function . . . . . . D. Genes Causing Sterility Including Eh . . . . . . . CHAPTER 2. _ HAMSTER MESOCRICETUS AURATUS A. Introduction . . . . . . . . . B. Methods and Materials . . . . C. ReSUItS O I O O O O O O O O O D. Discussion . . . . . . . . . . E. Plates 0 I O O O O O O I O O 0 CHAPTER 3. AN ELECTRON MICROSCOPIC GLAND OF THE SYRIAN HAMSTER (HE) C o o o o o o o o o o A. Introduction . . . . . . . . . B. Methods and Materials . . . . C. Results . . . . . . . . . . . D. Discussion . . . . . . . . . . E. Plates . . . . . . . . . . . . SWY O O O O O O O O O O O O O O O A. PrOblem O O O O O O O O I O O B. MethOdS O O O O O O O O O O O C. Results .,. . . . . . . . . . D. Conclusions . . . . . . . . . LITERATURE CITED . . . . . . . iv EFFECTS OF THE GENE Wh ON REPRODUCTION IN THE SYRIAN OF THE PITUITARY WHITE 10 13 15 15 17 18 32 4O 52 52 53 54 65 72 84 84 84 85 85 87 LIST OF TABLES CHAPTER 2 Table 1. Measurements of Hamster Body Weight (g), Testis Weight (g) and Tubule Diameter (mm) from 10 Normal (+/+), 10 Heterozy- gous (+/-) and 10 Mutant (-/-) Animals in the AN/Asfiflh strain 0 O O O O O O O O O O O O O O O O O O O O O O O O I 0 2. Mean Measurements of Hamster Body Weight, Testis Weight and Tubule Diameters from Table l . . . . . . . . . . . . . . . . 3. Computations for the Analysis of Variance on Adult Body Weight Measurements from Table l . . . . . . . . . . . . . . 4. Computations for the Analysis of Variance on Testis Weight Measurements from Table I . . . . . . . . . . . . . . . . . . 5. Computations for the Analysis of Variance on Testis Weight as Percent of Adult Body Weight Measurements from Table 1 . . 6. Computations for the Analysis of Variance on Tubule Diameter MeasurementSfromTableloo000.000.000.000. 7. F-Test of the Hypothesis that Population Means are Identical for Average Weights of Various Organs . . . . . . . . . . . . 8. Hartleys Fmax-test for Homogeneous Variance . . . . . . . . . 9. Modified f'-statistic of Brown and Forsythe to Compare Variances for Population Means which are Heterogeneous in Nature 0 O O O O O O O O O C O O O O O O O O C O O O O O O O 10. Results from Tukey's Honestly Significant Difference (HSD) Test Comparing Means Summarized in Table 2 . . . . . . . . . LIST OF TABLES CHAPTER 3 Table Page 1. Measurements of the Number of Nuclei per 10 x 7 cm Plot from the Ventromedial Zone of the Pars Distalis and Testis Weight as Percent Body Weight from 5 Normal (+/+), 5 Heterozygous (+/-) and 9 Mutant (-/-) Hamsters from the AN/Asfiflh Strain . . . . . . . . . . . . . . . . . . . . . . . 59 Computations for the Analysis of Variance on the Number of Nuclei per 10 x 7 cm Plot from Table 1 . . . . . . . . . . . 60 Mean Measurements of Cellular Densities from Table 2 . . . . 61 Analysis of Variance Comparing Data from Tables 1 thru 3 . . 62 A Linear Regression Analysis Between Genotypeso and Within the Mutant Genotyped where the Number of Nuclei per Plot was Regressed Against the Gram Percent Body Weight of the Testis for 5 Normal (+/+), 5 Heterozygous (+/-) and 9 Mutant (-/-) Hamsters in the AN/Asfflh Strain. Number of Nuclei per Plot; Gram Percent Body Weight of the Testis . . . . . . . . . . . 63 vi LIST OF FIGURES CHAPTER 2 Figure l. Homozygous normal hamster of the genotype whwhee . . . . . 2. Heterozygous hamster of the genotype thhee . . . . . . . . 3. Homozygous mutant hamster of the genotype WhWhee . . . . . 4. Testis and epididymis from a homozygous normal hamster . . 5. Testis and epididymis from a heterozygous hamster . . . . . 6. Testis and epididymis from a homozygous mutant hamster . . 7. Light micrograph of normal/heterozygous seminiferous tubules . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Light micrograph of normal/heterozygous germinal epithelium from a field comparable to that in the box in Fig. 7 . . . 9. Light micrograph of mutant seminiferous tubules . . . . . . 10. Light micrograph of mutant germinal epithelium in a field comparable to that in the box in Fig. 9 . . . . . . . . . . 11. Electron micrograph of a normal/heterozygous Leydig cell . 12. Electron micrograph of normal/heterozygous Leydig cytOplasm 13. Electron micrograph of mutant Leydig cells . . . . . . . . 14. Electron micrograph of mutant Leydig cytoplasm . . . . . . 15. Electron micrograph of a normal/heterozygous Sertoli cell . 16. Electron micrograph of normal/heterozygous Sertoli cell cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . 17. Electron micrograph of mutant Sertoli cells . . . . . . . . 18. Electron micrograph of mutant Sertoli cell cytoplasm . . . vii 41 41 . 41 41 43 43 43 43 45 45 45 45 47 47 47 47 Figure 19. Electron micrograph of an early spermatid from normal/ heterozygous testicular tissue . . . . . . . . . . . . . 20. Electron micrograph of an early spermatid from mutant tes tiCUIar tissue 0 O O O O O O O C O O O O O O I I O O O 21. Electron micrograph of normal/heterozygous germinal epithelium towards the tubular lumen . . . . . . . . . . 22. Electron micrograph of mutant germinal epithelium towards the tUbUlar lumen O O O O O O O O O O O O O O O O O O O 0 viii Page 49 49 51 51 LIST OF FIGURES CHAPTER 3 Figure 1. A schematic representation of the areas of the hyp0physis processed for light and electron microscoPe . . . . . . . 2. A schematic representation of the hyp0physis of male hamsters from the AN/As-W_h_ strain 0 o o o o o o o o o o o 3. Hypophysis from a homozygous normal hamster (whwhee) . . 4. Hypophysis from a heterozygous hamster (thhee) . . . . . 5. HypOphysis from a homozygous mutant hamster (WhWhee) . . 6. Light micrograph of the ventromedial section of the pars distalis representative of normal and heterozygous tissue 7. Light micrograph of the ventromedial section of the pars distalis representative of mutant tissue . . . . . . . . 8. Electron micrograph of gonadotropes from the ventromedial zone of normal/heterozygous tissue . . . . . . . . . . . 9. Electron micrograph of the most prevalent cell type found in the ventromedial zone of mutant tissue . . . . . . . . 10. Electron micrograph of cellular membranes of adjacent cells representative of normal and heterozygous tissue . . . . 11. Electron micrograph of cellular membranes of mutant tissue containing SpeCialized junCtions o o o o o o o o o o o c 12. Electron micrograph of an isolated cilium, occasionally found in the ventromedial zone of the pars distalis in normal and heterozygous tissue . . . . . . . . . . . . . . . . . . . . . 13. Electron micrograph of a portion of a cell cord in mutant tissue, containing one cell which is representative of a lactotropic cell and one highly ciliated secretory cell . . . 14. Electron micrograph of a field comparable to that in the box in Fig. 13 O O O O O O O O O O O O O O C O O O O O 0 ix 73 73 73 73 75 75 77 77 79 79 81 81 83 Figure Page 15. Electron micrograph of the box in Fig. 14 . . . . . . . . . . 33 CHAPTER 1 THE MAMMALIAN TESTIS AND PITUITARY CONTROL OF TESTIS FUNCTION A. General Morphology and Function of the Testis The mammalian testis as described by Bloom and Fawcett ('75) is a compound tubular gland which is enclosed in a tough fibrous coat referred to as the tunica albuginea. Fibrous septa divide the organ into about 250 pyramidial compartments each called a lobule testis. Each lobule testis consists of one to four highly convoluted seminiferous tubules. At the apex of the lobule testis, its seminiferous tubule or tubules pass directly into the first segment of the system of excretory ducts each referred to as a tubule recti. The recti are confluent with the rete testis, a plexiform system of epithelium lined spaces in the connec- tive tissue. The seminiferous tubules are cylindrical, and when stacked together create a series of three-sided spaces. A loose connective tis- sue containing fibroblasts, mast cells, macrophages and perivascular mesenchymal cells, extends inward from the tunica to fill the inter- sticies among the tubules. In addition, the spaces contain epithelioid cells, or interstitial cells of Leydig. Each testis is susPended in the scrotom at the end of a long vascular pedicle, the spermatic cord, which consist of the excretory ducts of the testis, blood vessels, and nerves supplying the testis on that side. The epididymis, an elongated organ closely applied to the surface of the testis, is made of the convoluted l 2 proximal part of the excretory duct system and is the site of accumula- tion and storage of spermatozoa (Bloom and Fawcett, '75). The testis is known to serve two functions. The first function in- volves the synthesis and secretion of testosterone by the endocrine come ponent of the testis or interstitial cells of Leydig. The second in- volves the development and maturation of germ cells in the exocrine portion of the testis or epithelium of the seminiferous tubules. Interstitial Tissue The endocrine component of the testis, or interstitial cells, was first described by Leydig (1850) as modified connective tissue. Their function in producing male sex hormones was suggested as early as 1903 by Bouin and Ancel. It is now generally accepted that these cells pro- duce the majority of the steriod hormone testosterone (Means, '76). The interstitial tissue of the rodent testis is sparse while the bulk of the intersticies is filled with large lymphatic sinusiods. Leydig cells are seen clustered around blood capillaries. The ultrastructure of Leydig cells is consonant with their function of steroid synthesis (Christensen, '65; Setchell, '78a). Leydig cells are irregularly poly- hedral in shape, measuring 14-20 um in diameter, and contain a large spherical nucleus with a small amount of peripherally disposed hetero~ chromatin and one or two prominent nucleoli. Leydig cytoplasm contains a well developed Golgi complex, abundant mitochondria, lipid draplets and vacuoles (Christensen, '65). In common with other steroid secreting endocrine cells, Leydig cells possess an extensive system of branching and anastomosing smooth end0p1asmic reticulum, which is known to contain enzymes necessary for several steps in the synthesis of androgenic steroids (Christensen, '65; Setchell, '78s). 3 Seminiferous Tubules The exocrine portion of the testis, or seminiferous tubules, are lined with a complex stratified epithelium composed of two categories of cells: (I) a fixed population of non-proliferating supporting cells or Sertoli cells, and (2) a proliferating and differentiating population of germ cells or spermatogenic cells. Sertoli cells, as described by Sertoli (1865), are columnar epithe- lial cells which are fixed to the basement membrane or basal lamina of the seminiferous tubules and perform a supportive and nutritive function during the maturation of spermatogenic cells. A number of specific functions have now been ascribed to Sertoli cells including secretion of fluid, phagocytosis, aiding the maturation and release of spermatozoa and synthesis of intratubular androgen-binding protein (Setchell, '78b). These cells form an elaborate system of thin processes that extend to- wards the lumen of the tubule surrounding the spermatogenic cells and filling the intersticies among them. Sertoli cytoplasm contains numer- ous slender elongate mitochondria, numerous lipid droplets, primary and secondary lysosomes, a small Golgi zone, free polysomes and a well develOped agranular endoplasmic reticulum. The Sertoli nucleus is large and ovid in shape with one or more deep infoldings or lobulations in its surface and consists of homogenous nucleoplasm. Sertoli nuclei also contain a large and highly characteristic nucleolar complex, con- sisting of a central mass and two laterally associated baSOphyllic bodies (Dym, '73). Spermatogenic cells are of several morphologically distinguishable types including spermatogonia, spermatocytes, spermatids, and spermatozoa. These are not ontogenetically distinct cells types, but are clearly 4 distinguishable successive stages in the continuous process of differen- tiation of male germ cells. Continual proliferation of the seminiferous epithelium is confined to spermatogonia and spermatocytes near the basal lamina. Neogenesis of succeeding generations of cells in this region displaces the more mature forms towards the lumen of the tubule as they differentiate. Spermatogonia arise from gonocytes of the fetal testis. They are large diploid cells which lie against the boundary tissue of the tubule and divide mitotically (Setchell, '78c). Studies by Rowley £5 31., '71 have revealed four types of human spermatogonia consisting of dark type A (AD), pale type A (AP), type B (B), and a new type A (AL). Subcellular criteria used in distinguishing these four cells types include: (1) shape of the nucleus, (2) density of nucleoplasm, (3) type and placement of nucleoleus, (4) structure of mitochondrial cristae, (5) association of endoplasmic reticulum with the mitochondria, (6) amount of glycogen, and (7) presence of filamentous structures in the cytoplasm (Rowley 25 al., '71). As diploid spermatogonia proliferate, some daughter cells are retained as future progenitors (stem cells), while others deve10p into primary spermatocytes. With the last mitotic division of spermato- gonia (Type B), pre-leptotene spermatocytes are produced (Clermont, '72; Steinberger and Steinberger, '75). Primary spermatocytes initially resemble spermatogbnia, but as they move from the basal lamina towards the luman, they accumulate cytoplasm and become larger (Setchell, '78c). Immediately after their formation, they enter prophase of the first meiotic division, characterized by the formation of filaments (synaptonemal complexes) within the nuclear chroma- tin (Bloom and Fawcett, '75). The reticulum of the nucleus of the primary 5 spermatocyte is reorganized into the diploid number of very long slender threads characteristic of the leptotene stage of meiosis. Homologous threads undergo pairing and thickening during the zygotene stage, then gather in a boquet-like arrangement around the synaptonemal complex. Paired threads shorten and thicken to form four obvious chromosomal strands or a tetrad, typical of the pachytene stage. Duplicated chromo- somes in pachytene can be identified as dyads held together by centro- meres. During diplotene and diakinesis, chromosomes complete their pro— cess of shortening, and the synaptonemal complexes disappear. At the end of prophase the nuclear envelop disappears and the tetrads rearrange themselves at the equatorial plane in metaphase I. At anaphase I, centro- meres move to Opposite poles of the spermatocyte taking both dyads along with them. Anaphase I and telophase I are quickly completed resulting in the formation of secondary spermatocytes carrying the haploid number of chromosomes. Secondary spermatocytes quickly complete the second phase of meiotic division resulting in spermatids which have a haploid set of chromosomes (Bloom and Fawcett, '75). Spermiogenesis is the term which describes the developmental events leading to the transformation of spermatids into spermatozoa. Each of the relatively small spherical or polygonal spermatids contain a small Golgi apparatus in the juxtanuclear cytoplasm. Several small granules, proacrosomal granules, appear within the developing Golgi complex. Each proacrosomal granule is found to be enclosed within a membrane limited vesicle of the Golgi apparatus. As maturation of the spermatid proceeds, separate granules in the Golgi region fuse together into a single large globule or acrosomal granule which is contained within a membrane bound acrosomal vesicle. The granule and vesicle become adherent to the 6 outer aspect of the nuclear envelop. The point of adherence marks the future anterior tip of the sperm nucleus. The Golgi apparatus remains closely associated with the surface of the acrosomal capsule, continuing to form small vesicles which feed into the membrane of the acrosomal vesicle and contribute to its enlargement. The limiting membrane of the acromomal vesicle increases its area of adherence and forms a thin fold, initially covering the anterior one-third of the nucleus. The bulk of the acrosome remains localized at the anterior pole of the nucleus, but during Spermiogenesis its substance gradually spreads in a thin layer into the fold. Condensation of nuclear chromatin causes the spermatid nucleus to transform into a homogeneous dense mass devoid of structure, at which point the acrosome assumes a position covering the anterior two-thirds of the nucleus. Once the acrosome is formed, the remaining Golgi complex migrates posteriorally and is included as part of the residual cytOplasm. While the early stages of acrosome formation are in progress, cen- trioles migrate to the opposite end of the spermatid. The distal cen- triole becomes oriented perpendicular to the cell surface, giving rise to a slender flagellum. Along with a marked elongation of the spermatid, cytoplasmic microtubules arise and become laterally associated to form a manchette (Bloom and Fawcett, '75). The flagellum consists only of the axial filament complex, or axoneme. It contains two central fibrils and nine peripheral doublets, which are continuous with the wall of the distal centriole. During development of the tail, the proximal centriole gives rise to a thin sheet of dense material which arches over its upper surface and eventually develops into the articular surface of the connect- ing piece, which joins the tail to the nucleus. As spermeogenesis 7 proceeds, the manchette disappears and mitochondria aggregate around the segment of flagellum between the annulus and the nucleus. Simultane- ously, a succession of circumferentially oriented ribs are deposited around the tail, distal to the annulus, to form the fibrous sheath of the principal piece. As differentiation proceeds, most of the residual cytoplasm is cast off as the residual body. Only a thin layer of cyto- plasm remains to cover the nucleus, middle piece and tail piece. In summary, the testis is a compound tubular gland which serves two functions, one hormonal, the second reproductive. Interstitial cells of Leydig comprise the endocrine portion of the testis and are responsible for the synthesis and secretion of the steroid hormone testo- sterone. Seminiferous tubules comprise the exocrine portion of the testis and are lined with a complex stratified epithelium containing Sertoli cells (supporting cells), and spermatogenic cells. Proper division and maturation of spermatogenic cells results in spermatozoa which are ex- truded from the tubules and stored in the epididymis until ejaculated. B. General Morphology of the Pituitary Gland The pituitary gland, or hyp0physis cerebrei, is composed partly of nervous tissue, the neurohypOphysis, and partly of epithelial tissue, the adenohypophysis. The gland is suspended from the floor of the third ventricle of the brain. The neurophypophysis as described by Rhodin ('74) is composed of the eminentia medealis (median eminence) of the tuber cinereum, the infundibular stem and the infundibular process. It arises as an evagina- tion from the floor of the diencephalon. It is composed of large numr bers of non-myelinated nerve fibers and some neuroglial cells, or 8 pituicytes. The nerve fibers are derived from the cells of the supra— optic and paraventricular nuclei of the hypothalmus. The nerve terminals end in close apposition to the capillaries of the posterior lobe. The hormones secreted by the neurohypoPhysis include oxytocin and vasopressin. These hormones are produced in the cell bodies of the hypothalmic nuclei, but are stored and liberated by the neurohypophysis (Rhodin, '74). The epithelial portion of the gland, or adenohypophysis, is com- posed of the pars distalis, pars tuberalis, and pars intermedia (Rhodin, '74). It develops from an invagination of the oral ectoderm (Rathke's pouch). The pouch loses contact with the oral cavity and forms a vesicle which later becomes a solid cell mass. The anterior lobe, pars distalis and pars tuberalis, consists of five cellular types, which were initially characterized by differential staining affinities of the cytoplasmic granules (Herlant, '64). Each cell type is responsible for the secre- tion of a characteristic hormone such as somatotropin (GH), prolactin (PRL), adrenocorticotropin (ACTH), thyrotropin (TSH), follicle stimulat- ing hormone (FSH), or lutinizing hormone (LH). Research into the cellular origins of the anterior pituitary hor- mones has resulted in the elucidation of five classes of cells charac- terized by their reactivity to physiological change. At the light micro- scopic level, Herlant and colleagues demonstrated that the anterior lobe contained two varieties of PAS negative cells (acidophils): (1) GH— secreting alpha cells and (2) PRL-secreting epsilon cells. In addition, three varities of PAS positive cells (basophils) were identified: (1) TSH-secreting delta cells, (2) FSH-secreting beta cells and (3) LR- secreting gamma cells (Herlant, '56; Herlant, '59; Herlant and Canivenc, '60; Herlant and Racodot, '57; Peyre and Herlant, '61). There is yet little evidence as to the cellular origin of ACTH. 9 Since many cellular structures of interest are beyond the limits of resolution of the light microscope, electron microsc0py has proved to be a powerful tool for the investigation of pituitary interactions in complex endocrine states, or in particular, of pituitary influences in certain pathologic conditions. In the normal condition, GH-secreting acidophils are medium sized cells containing a large round nucleus and secretory granules of approximately 3000 to 3500 X in diameter. LTH- secreting acidophils are large cells with electron lucent cyt0plasm and contain a large indented nucleus and secretory granules of approximately 6000 to 9000 A. TSH—secreting basophils are fairly large cells of poly- gonal shape, containing a large nucleus and secretory granules of approxi- mately 1000 to 1600 A in diameter. Electron microscopic studies have identified gonadotropes (FSH and LH producing cells) in the normal and castrated rat (Farquhar and Rinehart, '54; Yoshimura and Harumiya, '65; Costoff, '73), mouse (Barnes, '62) and hamster (Giroud and Dubois, '65; Dekker, '67). The controversy over cellular autonomy of FSH and LH continues, although the use of immuno- cytochemical techniques have indicated that one gonadotropic cell con- tains both FSH and LH (Baker and Gross, '78). Gonadotropes are numerous and distributed throughout the pars distalis, although they form a dense aggregation in the cephalic ventromedial "sex zone." Also, gonadotropes are thought to be more numerous in the male than in the female gland (Baker and Cross, '78). Gonadotropic cells are large and polyhedral, measuring 10 to 15 pm in diameter and contain an eccentrically positioned nucleus. The nucleus is irregular in contour and possesses finely scattered and peripherally clumped heterochromatin and one or two nucleoli. The secretory granules are electron dense and range in size 10 from 750 to 2350 A. When these cells are in an inactive state, the Golgi and endoplasmic reticulum are rather inconspicuous and poorly developed, whereas in actively secreting cells the Golgi is extensive and contains areas with granules in different stages of formation. The endoplasmic reticulum is usually irregular, consisting of dialated sacs and scattered vesicular areas. The membranes are intermittantly dotted with ribosomes, although some ribosomes are free in the cytOplasm. Mitochondria are generally elongate but, short, rod-like and round mitochondria are also present. Most gonadotrOpes are found in associa- tion with vascular spaces (Farquhar and Rinehart, '54; Dekker, '67; Costoff, '73). Summarizing, the pituitary gland consists of a neural lobe (neuro- hypophysis), and an epithelial lobe (adenohypOphysis). The anterior lobe of the adenohypophysis, or pars distalis and pars tuberalis, con- tains five cellular types, determined at the light microscopic level, by differential staining affinities of characteristic cytoplasmic granules. Electron microscopy has elucidated the gross cellular morphology of these cell types on the basis of their response to changes in endocrine states. C. Hypophyseal Control of Testis Function The importance of the hyp0physis in maintaining gonadal function was demonstrated as early as 1930 by Smith using hypophysectomized rats. After hypophysectomy the gonads atrOphied, but were restored to normal appearance and function when pituitary extracts were injected or implants of the gland were made. After Smith's discovery, many attempted to separate the gonadotrOpins from the other hormones derived from the anterior pituitary. Fevold ggual. ('31) were the first to accomplish 11 a partial separation of FSH and LH. They accompanied the above with an intensive study of the biological effects of each hormone, concluding that the action of these two hormones was to promote growth of ovarian folliCles and affect the germinal epithelium of the testis. Greep.g£.§l. ('41) and Fevold ('43) reported that the hormone LH stimulated luteini- zation of the ovaries and maintained interstitial cells of the testis. In the male mammal, follicle stimulating hormone was tentatively identified as being responsible for the maintenance of the germinal epithelium within the seminiferous tubules of the testis. Presently, FSH appears to play an important role in the initiation of the spermato- genic process in immature mammals and for the maintenance of testicular function in the adult. The precise mechanisms by which FSH exerts its effects are still unknown, although considerable evidence has appeared, as of late, to identify and sequence the biochemical events initiated by the interaction of FSH with membrane receptors on the Sertoli cell (Means, '77). The Sertoli cell has been shown to be the primary target for FSH action in the testis, since it is the only cell to possess FSH-specific receptors and responds to FSH stimulation by increasing its level of endogenous cyclic AMP (Steinberger and Steinberger, '77). Specific receptors present on the plasma membrane of Sertoli cells recognize and bind FSH. This binding activates membrane bound adenylate cyclase. Cyclic AMP, formed in response to this stimulus, promotes DNA-dependent RNA synthesis and the formation of proteins, including the androgen binding protein (ABP) (Means, '76). The ABP is transported into the intracellular spaces where it binds androgens. This ABP-androgen com- plex then interacts with the membrane of a developing germ cell and 12 transfers the androgen to a hypothetical cyt0plasmic androgen receptor. The mechanism of action of androgens subsequent to this step are unknown. After delivery of the androgen to the germ cell, ABP is free to bind an- other androgen, and the process thus is repeated. ABP—androgen com- plexes are eventually either secreted into the lumen of the seminifer- ous tubule of broken down by proteolytic enzymes within the germinal epi- thelium (Steinberger and Steinberger, '77). It is known that the primary role of LH in the testis is to regu- late testosterone secretion from testicular interstitial tissue (Leydig cells). LH most likely affects spermatogenesis by controlling produc- tion of the androgen testosterone. Testosterone is required for spermatogenesis, although the precise mechanism by which it exerts its effects is not well understood (Lostroh, '76). Binding of LH to Leydig cells was demonstrated in rats using labelled hormone both in gixg (deKretsler g£_§l,, '69; '71) and EB ‘zi££2_with testis homogenates (Catt §£_§1,, '72. Apart from.hCG, no other hormones bind to these sites (Setchell, '78a). Stimulation by LH enhances adenylate cyclase activity and the formation of cyclic AMP. A protein kinase frees cholesterol from lipid stores (lipid droplets) and catalyses the formation of a protein which then activates the con- version of cholesterol to pregenenolone. This conversion occurs by splitting off of the long side chain attached to the carbon-17 atom of cholesterol. The reaction requires NADPH and 02 and occurs in the mitochondria (Setchell, '78a). Pregenenolone is converted to other steroid hormones in the smooth endoplasmic reticulum (Christensen, '65). In experiments concerned with the restoration of spermatogenesis in testes which have regressed following hypophysectomy, germ cell 13 deve10pment proceeded as far as the early spermatid stage in animals treated with testosterone, but FSH was necessary for the completion of Spermiogenesis (Steinberger, '71). Spermatogonia persist after hypo- physectomy, and do not require exogenous hormone to develop to the pachytene stage of the primary spermatocyte (Lostroh, '76). Testosterone, however, is necessary in the transformation of the prophase to metaphase primary spermatocyte. Thus, with the presence of testosterone alone, spermatocytes complete meiosis to form spermatids (Lostroh, '76). Modest levels of FSH and LH are subsequently necessary for spermatid development while substantial levels of LH, FSH and testosterone are necessary for the transformation of spermatids to spermatozoa (Lostroh, '76). Thus, testosterone, LH and FSH control gametogenesis in the post- pubertal male from the prophase of meiotic reduction division through final maturation of spermatozoa. D. Genes Causing Sterility Including Eh Setchell ('78d) points out that the normal function of the testis can be directly interrupted by illness, heat, chemical agents such as cadmium acetate or lead salts or drugs such as cyproterone acetate. Likewise, normal function may be secondarily affected by depressing the function of the pituitary with drugs such as l9-norspiroxenone (Setchell, '78d). In addition to these environmental agents, mutant genes may also interrupt the normal function of the testis, rendering the mutant indi- vidual infertile. These mutant genes may act, for example, to inhibit Wolffian duct deve10pment (testicular feminization), causing an absence of germ cells in many species including mice (Lyon and Hawkes, '70; 14 Blackburn 33 31., '73) and rats (Stanley and Gumbreck, '64; Stanley 53%., '73).. Mutant genes may directly affect Leydig cell deve10pment in the rat as in vet-rat (Stanley 25 al., '73) or seminiferous tubule deve10pment as in Sertoli-cell only syndrome (deKrestsler gt §l°» '72). Mutant genes may affect spermiogenesis in the mouse as in“: (Olds, '71), hop:sterile (Johnson and Hunt, '71), p-sterile (Hunt and Johnson, '71) and guaking (Bennett ggugl., '71), or in the bull as in dag-defect (Blom, '66) and pseudodroplet (Blom, '68). Hypothetically, genes might secondarily affect testis function by acting to alter pituitary function. At least three genes in the Syrian hamster, Mesocrecetus auratus, are known to cause infertility: (l) ruby-eyed mutants (52) are known to be sterile due to an inadequate sperm count (Robinson, '68), (2) piebald mutants (g) are known to possess urogenital abnormalities and reproductive tract aplasia (Robinson, '68), and (3) Anophthalmic white mutants (Eh) do not reproduce but the cause of the infertility is unknown. Hamsters homozygous for the mutant gene Eh are known to possess three distinct characteristics: (1) a complete suppression of pigment, (2) an0phthalmia or severe microphthalmia and (3) an interruption in the reproductive system causing infertility (Robinson, '62; '64; Asher, '68). Since infertility exists in anophthalmic males and females without ex- planation, the question as to what causes infertility may be proposed. Thus, the purpose of this research is to determine why male mutants are sterile. Similarly, since genes are known to affect testis function either directly or indirectly, the purpose of this research is also to determine whether the gene affects testis function alone or whether the pituitary is involved. CHAPTER 2 EFFECTS OF THE GENE EH 0N REPRODUCTION IN THE SYRIAN HAMSTER, MESOCRICETUS AURATUS A. Introduction One of the many mutants occurring in the Syrian hamster, Mesocricetus auratus, is Anophthalmic white. The mutant gene was first described by Knapp and Polivanov ('58) as an autosomal recessive gene (an), inherited independently of the albino gene 29. Studies by Behr and Behr ('59) distinguished heterozygous animals from homozygous wild-type (golden hamsters) and suggested that the gene acted as a partial dominant. With this in mind, they proposed the symbol Eh for the mutant gene. AdOpting the designation Eh, heterozygous animals (Whyh) were found to be agouti but possessed white venter fur as opposed to the pale cream venter fur of the wild-type. Later studies by Robinson ('62;'64) described Eh_as incompletely dominant and indicated that ani- mals homozygous for Eh exhibited a complete absence of coat and skin pigmentation while aplasia of the eyes resulted in extreme microphthal- mia or anophthalmia. Hughes and Geerarts ('62) reported that the development of the eye proceeded normally until the last days of fetal development, at which time the action of the gene Eh_became apparent. Studies by Berman ('64) revealed that the choroid fissure of Ehflh_embryos remained open and the lack of intra-occular pressure was prOposed to be the cause of the 15 l6 extreme microphthalmia. Finally, studies by Yoon ('73; '75) suggested that the gene Eh caused extreme optic degeneration along with deafness. Studies of the mutant Eh_in the AN/Asfiflh strain have been in pro- gress for the last seventeen years (Asher, '68). As a result of low breeding-capacity and a lack of resistance to environmental stress, this mutant hamster proved to be exceptionally hard to maintain. Throughout the development of the strain (AN/Asjflh), a strong inter— action was noticed between Eh and cream (c). Since the expression of .Eh_was enhanced in the presence of g_and since homozygous cream (fig) hamsters appeared to be more vigorous than the contrasting wild-type golden hamsters, hamsters homozygous for the cream background were selected. Thus, animals described in this study exhibited the follow- ing phenotypes: (1) Normal hamsters (whwhee) were cream colored with white Spotted bellies and black eyes (Fig. l); '(2) Heterozygous hamsters (thhee) were white with black eyes and possessed less ear, eye and skin pigmentation than normal (Fig. 2); (3) Homozygous mutant hamsters (WhWhee) were white, lacked eyes and possessed no ear or skin pigmentation (Fig. 3). Earlier studies by Asher ('68) indicated that all Ehflh_individuals in the AN/Asfiflh strain appeared to: be anophthalmic, lack pigmentation, be comatose when sleeping, have a different fur texture, exhibit extreme nervousness, exhibit general growth retardation, have small adrenal glands, and lack proper sexual development. It was first noted by Knapp and Polivanov ('58) that the anaphthal- rue male was not able to reproduce. Breeding data from the strain l7 AN/Asjflh also indicated a lack of reproductive ability of both male and female Ehflh_individuals. Preliminary histological examination by Asher ('68) suggested that the seminiferous tubules from mutant testes con- tained no sperm. Therefore, the present study was undertaken to deter- mine whether testicular abnormalities existed and contributed to the infertility of male animals homozygous for the mutant gene Eh. B. Methods and Materials The strain used in this study was designated AN/Asjflh by Asher ('68), and is maintained by a system of full-sibling mating where at least one parent is heterozygous for Eh, Hamsters were housed in poly- carbonate cages with galvanized or stainless steel tops, cleaned weekly and provided with pine shavings for bedding. Nayne Laboratory and Breeder Chow and water were provided 2d libidum. Lighting of the animal room was on a regime of 13 hours of light and 11 hours of darkness. Ten sets of matched-siblings at approximately 135 days of age were employed in this investigation. Activity patterns for each animal were determined on running wheels over a one week period prior to killing. Four hours prior to the calculated onset of running activity, hamsters were weighed, anesthetized with ether, and perfused through the left ventricle with Ringer's solution followed by 0.5 percent cacodylate buffered glutaraldehyde at pH 7.3 (Glauert, '75s). After perfusion, testicular tissue was collected and further processed for both light and electron microscopy. Light Microscopy After perfusion, each testis prepared for light microscopic exami- nation was fixed for two hours in Bouin's solution, weighed, cut into 18 small pieces and fixed in Bouin's solution for at least 3 days. Small tissue pieces were dehydrated through alcohols, embedded in paraffin, cut into 5 um sections and stained with Harris' Hematoxylin and Eosin. Five random sections from each testis were photographed and the photo- graphs were contact printed. Tubule diameter measurements were deter- mined from the contact prints at a magnification of 64X. Electron Microscopy Each testis prepared for electron microscopic examination was imme- diately cut into small pieces and fixed in 0.5 percent cacodylate (0.2 M) buffered glutaraldehyde at pH 7.3 (Glauert, '75a). The tissue was post- fixed in 2.0 percent cacodylate buffered osmium tetroxide (Glauert, '75b) and embedded in Epon-Araldite (Mollenhauer, '64). Thick (l um) epoxy sections, taken for light microscopy, were stained in a 1.0 percent toluidine blue 0 solution. Ultra-thin sections were stained in uranyl acetate (watson, '58) and lead citrate (Reynolds, '63) and examined in the Phillips 300 transmission electron microscope at 60 kV. C. Results Homozygous mutant hamsters in this strain were small in comparison to normal and heterozygous siblings, as revealed by mean body weight measurements (Table 2). Along with a general reduction in body size, homozygous mutant individuals possessed testes and epididymi which were reduced in size (Figs. 4-6). Although all mutant testes were hypoplastic, the severity of reduction was not uniform within the genotype as indi- cated by the large standard deviation in testes weights exhibited in Table 2. Six out of 10 mutant hamsters possessed testes which were severely reduced in size while four possessed testes which approached 19 the normal phenotype. When normalized to body weight (Table 2), abnormal testes were more than proportionately reduced in size suggesting that the general growth retardation was not responsible for the severe reduc- tion seen in testes weight. Light Microscopy It was evident from light microscopic examinations of testes from ten normal and ten heterozygous hamsters, that these testes were identi- cal in composition and possessed large seminiferous tubules containing proliferating and differentiating germ cells. Spermatogonia were quite prevalent, lying adjacent to the basal lamina. A myriad of spermatozoa tails were seen filling the lumen of each tubule (Figs. 7-8). By con- trast, the most severely affected mutant tissue, found in testes which were severely reduced in size, contained small seminiferous tubules in which spermiogenesis was arrested in the early spermatid stage (Figs. 9-10). Less affected mutant tissue, found in testes which approached the normal phenotype, contained seminiferous tubules with identical composition to normal and heterozygous tubules. Seminiferous tubules from mutant animals were reduced in size as revealed by mean diameter measurements (Table 2). In order to determine the magnitude of the effect that the gene ‘Wh_had on body weight, testis weight and tubule diameter measurements (Tables 1 and 2), an analysis of variance was used (Tables 3 through 7). Since the variances for testis weight, testis weight normalized to body weight and tubule diameter were heterogeneous (Table 8) and since vio- lations of the mathematical assumption of homogeneous variance will re- sult in an underestimate of the true difference between genotypes, the modified f'-statistic of Brown and Forsythe (Gill, '78s) was employed 20 (Table 9). The Honestly Significant Difference (HSD) test of Tukey (Gill, '78b) was used to determine whether the heterozygous as well as the homozygous mutant individual contributed to the large differences observed in population means (Table 10). Electron Microscopy The ultrastructure of normal and heterozygous Leydig cells were identical in morphology and exhibited a classial appearance as described by Christensen ('65). The cells contained a large spherical nucleus with peripherally-disposed heterochromatin, and cytoplasm with a well developed Golgi complex, abundant mitochondria, polysomes, lipid droplets and vacuoles (Fig. 11). In common with other steriod-secreting endocrine cells (Christensen, '65), both normal and heterozygous Leydig cells possessed an extensive system of smooth endoplasmic reticulum (Fig. 12). Ultrastructural examination of all Leydig cells from animals severely affected by Eh suggested that these cells were in a state of degeneration. The nuclei contained clumped masses of peripherally- and centrally-dis- posed heterochromatin while the cytoplasm contained highly vesiculated agranular endoplasmic reticulum (AER), some mitochondria and lipid droplets (Figs. l3-14). The ultrastructure of normal and heterozygous Sertoli cells also exhibited an identical and classial appearance as described by Dym ('73). These cells were found next to the basal lamina and contained numerous mitochondria, lipid droplets, a small Golgi zone, free polysomes and well developed agranular endoplasmic reticulum. Developing germ cells assumed their close relationship to Sertoli cells and were enveloped by the extension of the folds of cytoplasm. The lobulated nucleus was large and oval in shape (Figs. 15-16). Sertoli cells from all of the 21 Table 1. -- Measurements of Hamster Body Weight (g), Testis Weight (g), and Tubule Diameter (mm) from 10 Normal (+/+), 10 Heterozy- hous (+/-) and 10 Mutant (-/-) Animals in the AN/ASjWh Strain. Animal Body Testis Tubule Number Generation Age Weight Weight Diameter (0') (F) Genotype (dayS) (g) (3) (mm) 1766 3 178 101.20 2.18 17.5 1771 3 135 122.80 2.47 18.0 1750 3 219 116.10 2.40 17.5 1821 4 135 102.50 2.27 17.5 1822 4 whwhee 136 99.90 2.42 18.0 1847 5 (+/+) 138 85.60 1.85 18.0 1867 5 136 94.30 2.32 23.5 1874 5 137 89.60 2.41 18.5 1889 6 141 95.30 2.50 16.5 1890 6 140 90.10 2.06 20.5 1763 3 177 108.80 2.27 17.5 1825 3 136 95.50 2.27 18.0 1818 3 136 97.40 2.41 19.5 1797 3 188 106.10 2.22 20.5 1783 3 thhee 137 106.20 2.48 19.5 1858 5 (+/-) 136 88.20 1.68 21.0 1861 5 137 89.60 2.41 23.0 1866 5 137 90.50 2.35 18.5 1879 5 149 101.80 2.49 20.5 1883 4 137 92.60 2.34 23.0 1736 3 239 95.00 0.18 08.5 1811 3 135 91.90 1.53 18.0 1812 3 136 82.10 0.42 14.0 1816 3 136 84.20 1.59 17.5 1803 4 WhWhee 135 83.40 1.53 19.0 1329 4 (-/-) 137 98.90 0.43 08.0 1782 3 135 91.90 0.19 07.0 1844 5 136 81.10 1.42 17.0 1857 5 135 96.40 0.49 08.0 1860 5 136 78.20 0.25 06.5 22 come mums muouoEMHm mannnu moouowfiowaom .:0fiumowmwowma x so am museum uomuaoo scum ofiOflHmH>0fi CHNUGGUQ H GNQE mm GQ>HW 0H03 mUGOEOHQENQE HH< AH.H H mm.~H ee.o H mm.o Ham.o H mam.o sm.o H Hm.mw AoHV amaze: mm.H H OH.oN mm.o H om.~ HNN.o H ~a~.~ NH.A H Ho.ea AoHV amaze: No.~ H mm.wH m~.o H Hm.~ om~.o H AmH.~ o~.HH H s~.mm AoHV amaze: Aaav Houseman ANV mwom\moumofi Amy moumoe va zoom Aav mmhuoaou saunas .H canny aoum muouoamfio masons mum Dawes: saunas .unwfloz moon Hmumfimm MO mafimfiwkfimmwz saw: u. .H «Hana 23 Table 3. -- Computations for the Analysis of Variance on Adult Body Weight Measurements from Table l. (yi.) S.E. 2 Si Sum of Squared Observations within A Group +/+ 101.20 122.80 116.10 102.50 99.90 85.60 94.30 89.60 95.30 90.10 997.40 10 99.74 111.20 125.42 100734.86 +/— 108.80 95.50 97.40 106.10 106.20 88.20 89.60 90.50 101.80 92.60 976.70 10 97.67 i7.22 52.14 95915.75 Correction Factor for Each Group 99480.68 2999.34 741.81 2257.53 370.90 83.61 95394.29 _/_ 95.00 91.90 82.10 84.20 83.40 98.90 91.90 81.10 96.40 78.20 883.10 10 88.31 :6.94 48.18 78468.45 77986.56 272119.72 [A] 30 275119.06 [B] 272861.53 [C] 24 Table 4. -— Computations for the Analysis of Variance on Testis Weight Measurements from Table l. +/+ +/- -/- 2.18 2.27 0.18 2.47 2.27 1.53 2.40 2.41 0.42 2.27 2.22 1.59 2.45 2.48 1.53 1.85 1.68 0.43 2.32 2.41 0.19 2.41 2.35 1.42 2.50 2.49 0.49 2.06 2.34 0.25 (yi ) 22.87 22.92 8.03 96.55 [A] ri 10 10 10 30 (§i 2.287 2.292 0.803 S.E. :0.236 :0.221 $0.592 8: 0.0556 0.0487 0.3502 Sum of Squared Observations within a Group 52.86 53.02 9.95 115.83 [B] Correction Factor for Each Group 52.30 52.53 6.45 111.28 [C] SS 19.28 y SST 14.73 SSE 4.55 MST 7.37 MS 0.169 25 Table 5. -- Computations for the Analysis of Variance on Testis Weight as Percent of Adult Body Weight Measurements from Table 1. S.E. 82 1 Sum of Squared Observations within a Group +/+ NNNNNNNNNN 00.... NO‘O‘J-‘l-‘bNOOI" \DNOO‘O‘NNNHUI 23.09 10 2.31 .23 .05 53.79 Correction Factor for SS Y SST 53.31 19.14 13.14 5.97 6.57 .22 +/— 2.09 2.38 2.48 2.09 2.34 1.91 2.69 2.60 2.45 2.53 23.56 10 2.36 .25 .06 56.08 Each Group 55.51 I \ l OOHOOHHOHO o o WU'INmemU'IO‘H NHUHWUQHO‘O 9.29 10 0.93 0.74 0.55 13.58 8.63 104.31 [A] 30 123.45 [B] 117.45 [C] 26 Table 6. - Computations for the Analysis of Variance on Tubule Diameter Measurements from Table 1. +/+ +/- -/- 17.5 17.5 8.5 18.0 18.0 18.0 17.5 19.5 14.0 17.5 20.5 17.5 18.0 19.5 19.0 18.0 21.0 8.0 23.5 23.0 7.0 18.5 18.5 17.0 16.5 20.5 8.0 20.5 23.5 6.5 (yi ) 185.50 201.00 123.50 8670 [A] ri 10 10 10 30 (T1 ) 18.55 20.10 12.35 S.E. $2.02 il.89 15.19 s: 4.08 3.60 26.95 Sum of Squared Observations within a Group 3477.75 4072.50 1767.75 9318 [B] Correction Factor for Each Group 3441.03 4040.10 1525.23 9006.35 [C] SS 648.00 Y SST 336.35 SSE 311.65 MST 168.18 MS 11.54 27 Table 7. - F- Test of the Hypothesis that Population Means are Identi- cal for Average Weights of Various Organs. Mean Square Mean Square between classes df within classes df F Body Weight 370.90 2 83.610 27 4.44* Testes weight 7.37 2 0.169 27 43.58* Testes/Body 6.57 2 0.220 27 29.86* Tubule Diameter 168.18 2 11.540 27 14.57* *Indicates significance at the five percent level where F = 3.35. 2,7 28 Table 8. - Hartleys Fmax-test for Homogeneous Variance. +/+ vs +/- +/+ vs -/- +/- vs -/- Body weight 2.40 2.60 1.08 Testes weight 1.14 6.30* 7.19* Testes/Body 1.20 11.00* 9.17* Tubule Diameter 1.13 6.61* 7.49* *Indicates significance at the five percent level where F max,9,9, = 3.18. 29 Table 9. -- Modified f'-statistic of Brown and Forsythe to Compare Variances for Papulation Means which are Heterogeneous in Nature. SST d f v Testes weight 14.73 0.3045 48.37*(a) 14.51 Testes/Body 13.14 0.44 29.86*(b) 12.57 Tubule Diameter 336.35 23.20 14.50*(c) 14.27 *Indicates significance at the five percent level where: (a)f2,15 = 3.68 (b)f2,13 = 3.41 (c)f 2.14 = 3.74 30 Table 10. - Results from Tukey's Honestly Significant Difference (HSD) Test Comparing Means Summarized in Table 2. +/+ vs +/- +/+ vs -/- +/- vs -/- Body weight 0.716 3.96* 3.24 Testes weight 0.385 11.42* 11.45* Testes/Body 0.338 9.32* 9.66* Tubule Diameter 1.450 5.79* 7.25* *Indicates significance at the five percent level where q3 27 = 3.509. 9 31 severely affected mutant testicular tissue again exhibited an appearance suggestive of a degenetative condition. These cells assumed a position further towards the lumen of the tubule due to reduplication and invagi- nation of the basal lamina, and contained some mitochondria, Golgi and endoplasmic reticulum. In addition, these cells contained numerous large osmiophilic and non-osmiophilic lipid droplets, while the absence of more mature germ cells was associated with total vacuolation towards the tubular lumen (Figs. 17-18). Since light microscopic examination of the germinal epithelium from all animals severely affected by Wh_indicated that spermiogenesis was arrested at the early spermatid stage, an electron microscopic examina- tion of germ cell development was undertaken. In the normal condition, early spermatids contained a Golgi apparatus which developed in the juxtanuclear cytoplasm (Fig. 19). Several proacrosomal granules, en- closed within a membrane limited vesicle of the Golgi, fused into a single large acrosomal granule which was contained within a membrane- bound acrosomal vesicle. The acrosomal vesicle, containing the granule, became adherent to the outer aspect of the nuclear envelope, while the Golgi apparatus continued to form small vesicles feeding into the mem- brane of the acrosomal vesicle. The limiting membrane of the acrosomal vesicle increased its area of adherence, forming a thin fold that ini- tially spread over the anterior one-third of the nucleus. The bulk of the acrosomal granule remained localized at the anterior pole, gradually spreading as a thin layer into the fold (Fig. 19), giving rise to the acrosome. As differentiation proceeded within the Sertoli cytoplasm, condensation of nuclear chromatin along with development and attachment of the tail followed the normal progression of events leading to the formation of spermatozoa (Fig. 21). 32 Early spermatids from all of the severely affected abnormal tissue lacked the well-defined Golgi apparatus, since the Golgi seemed to de- generate promptly after the formation of the granule at the anterior pole of the nucleus. Commensurate with the normal condition, the limit- ing membrane of the acrosomal vesicle spread over the anterior one-third of the nucleus. However, spreading of the acrosomal contents did not occur (Fig. 20). The cytoplasm, irrespective of the Golgi, also exhibi- ted major degenerative changes exemplified by the overabundance of large lipid inclusions and lysosomal vacuoles. The marked degeneration seen in the developing germ cells resulted in tubules which were completely devoid of spermatozoa of spermatozoal tails and consisted mostly of cellu- lar debris (Fig. 22). D. Discussion Past breeding data indicated that most hamsters homozygous for the gene Wh_in the AN/Asjflh strain were sterile. In this investigation, testes from six mutant hamsters were severely affected by the gene Eh. These testes exhibited a severe reduction in both size and weight. Testes from four mutant hamsters were only slightly affected by the gene. These testes were atrophic, but morphologically and histologically re- sembled those of their normal matched-siblings. Thus, the expression of the gene Wh_among individuals varied considerably from almost complete atrophy of the testes to almost normal testes. Since the hamsters used in this study were from generations F3 to F5, they should only be identi- cal for 6 to 29 percent of their genome (Falconer, '60). Because of the low percentage of inbreeding, animals could differ by numerous genes, including genes near the E3 locus. Matched-siblings were employed for 33 comparisons between genotypes in order to eliminate possible problems caused by this difference in genetic background, although great vari— ability existed in comparisons made within a genotype. Since the pur- pose of this investigation was to explore the question of infertility in anophthalmic homozygotes, the remaining discussion will focus on those mutant animals which were most severely affected by the gene Eh. At the anatomical level, hamsters severely affected by the gene .Wh_possessed atrophic testes. Seminiferous tubules from atrophic testes were reduced in size, were aspermic and contained germinal cells arrested in the early spermatid stage of spermiogenesis. A highly significant genotypic difference existed in mean testicular weights and tubule dia- meters (Tables 2 and 7-9). Comparing data for all doses of the gene, the large difference in mean testicular weights and tubule diameters was due to animals which were homozygous for Wh_(Table 10). At the ultrastructural level, major morphological differences in Leydig cells, Sertoli cells and developing germ cells were observed in normal and heterozygous testicular tissue in comparison to mutant tissue. Studies by Christensen ('65) revealed that the characteristic agranular endoplasmic reticulum (AER) of the steroid-secreting Leydig cell was difficult to preserve for electron microscopy. According to his study, the tendency toward vesiculation of the AER was minimal in tissue fixed with glutaraldehyde without perfusion and was virtually absent in material perfused with glutaraldehyde. The appearance of normal and heterozygous Leydig cells in this study was consistent with those in previous reports on guinea pig (Christensen, '65) and mouse (Christensen and Fawcett, '66). Since abnormal tissue was processed in exactly the same manner, it was concluded that the degenerative condition 34 of this cell type was indicative of abnormal tissue and not an artifact of fixation. The appearance of normal and heterozygous Sertoli cells in this study was consistent with those in previous reports of monkey (Dym, '73) and human (Schultze, '74). Sertoli cells from abnormal testicular tissue were highly vacuolated and contained numerous osmiophilic and non- osmiophilic lipid droplets. Spermatogenesis and spermiogenesis proceeded properly in normal and heterozygous tubules, whereas spermiogenesis arrested at the early spermatid stage in mutant tubules. The Golgi phase (Sa) of early sperma- tid formation was consistent with studies by deKretsler ('69). All Sa spermatids possessed an oval nucleus and a prominent Golgi apparatus which was perinuclear and showed evidence of formation of membrane-bound pro- acrosomal granules. Normal apposition of the acrosomal granule and vesicle to the nuclear membrane took place. The cap phase of the Sb1 stage (deKretsler, '69) included spreading of the acrosomal vesicle over the anterior pole of the nucleus, forming the head cap. During the cap phase, the acrosomal granule remained anterior and centrally positioned. It is evident that the arrest of spermatid formation occurred in the acrosomal phase of the Sb stage (deKretsler, '69), exemplified by l the failure of the acrosomal granule to spread and fill the head cap (Fig. 20). The Golgi apparatus showed signs of degeneration, suggesting that premature failure of the Golgi, which was no longer capable of feeding vesicles into the head cap, was the cause of infertility in this mutant. This further suggested that continued function of the Golgi apparatus contributed to the acrosomal phase. The significance of this contribution has not been objectively dealt with in the past, 35 since it was commonly thought that the Golgi functions solely in the production of the acrosomal granule. However, the present study suggests that extension of the acrosome is dependent on continued function of the Golgi apparatus throughout the acrosomal phase and the Golgi begins its posterior migration only after proper acrosomal formation has occurred. Similarly, the nucleus of the normal and heterozygous Sb spermatid had 1 started to elongate (Fig. 19), while the nucleus of the mutant Sbl spermatid was still oval (Fig. 20). Pedersen and Rebbe ('74) and Holstein .g£.§1. ('73) reported that the absence of the acrosome in infertile men resulted in a high percentage of round-headed sperm. The results of these studies, along with the present study, also suggest that changes in nuclear shape from oval (Sb spermatids) to pear-shaped (sz sperma- 1 tids) may be dependent on normal acrosome development. Therefore, the present study has confirmed that the primary cause of male infertility in anophthalmic hamsters was the failure of mutant testes to produce spermatozoa. The data showed that abnormalities existed in Leydig cells, Sertoli cells, and in the deve10ping germ cells from the testes of homozygous mutant hamsters. Since the single primary action of the gene is unknown, several hypotheses can be formulated to try to explain the effects of this gene on reproduction in the Syrian hamster. Speculation as to the primary cause of the aforementioned abnormalities might include: (1) a defect at the level of the testes along, (2) a defect at the level of the pituitary (or high regulatory centers) or, (3) a phenomenon of eyelessness. Studies by Dym ('73) suggested that the production of spermatozoa was largely dependent on the normal function of the Sertoli cell. If this gene acted primarily to alter membrane function in the testes, 36 Sertoli cells might not respond to hormonal stimulation by FSH to trigger the enzymatic or biochemical processes necessary for prOper germ cell deve10pment. On the other hand, studies by Christensen ('65) proposed that the membrane of the AER in the Leydig cell was the location of ens zymes essential to steroid biosynthesis. Vesiculation of the AER along with the virtual absence of lipid droplets and mitochondria, containing villous cristae and an electron dense matrix, might suggest an altera- tion of the biosynthetic activity in the Leydig cells of severely affected mutant tissue. It is not impossible, but highly improbable, that a pleio- tropic gene, affecting pigmentation, eye development and reproduction, exerts its primagy effect on the testes alone. Smith ('27) first reported that the pituitary gland was responsible for the maintenance of gonad function. Studies by Smith ('30) and Greep and Fevold ('36; '37) first indicated that pituitary hormones were essential in spermiogenesis from the spermatocyte stage through the spermatozoa, but not for the earlier stages. Lostroh ('69) reported that spermatogonia persisted after hypophysectomy and did not require exogenous hormone to evolve to the pachytene stage of the primary sperma- tocyte. Thus, no hormone requirement existed in germ cell development from the spermatogonium to prophase of the primary spermatocyte. Testo- sterone was found to be necessary to transform the prophase spermatocyte to the metaphase spermatocyte (Lostroh, '76). Modest levels of FSH and LH were necessary for spermatid deve10pment while substantial levels of FSH, LH and testosterone were required for maturation of spermatids to spermatozoa (Lostroh, '76). Since germ cell development arrested at the spermatid stage in.mutant tubules, one prudent argument might be made for the suppression of gonadotropin secretion from the pituitary 37 gland. This hypothesis is consistent with studies by Steinberger and Dunkett ('65) who indicated that suppression of gonadotropin secretion resulted in incomplete spermatid formation in rat testes. It is also well known that testosterone alone does not promote normal germ cell deve10pment in gonadotropin-deficient mammals (Steinberger and Dunkett, '65; Lostroh, '69; utuly and Cutuly, '40). The most advanced germ cell development in hypOphysectomized and testosterone treated rats included spermatids, which were randomly distributed in the tubules and appeared abnormal (Lostroh, '69). Commensurate with the observation that mutant Sertoli cells contain numerous lipid draplets were studies by Lynch and Scott ('51) who demonstrated that hypophysectomized animals showed a dramatic increase in lipid accumulation. Therefore, if the gene causes a defect in the maintenance of gonad function, the pituitary gland, or higher regulatory centers may be involved. This possibility is currently under investigation. A third proposal for the primary effect of this gene on reproduc- tion is that the abnormalities presented were due solely to the lack of vision, due to the failure of prOper eye deve10pment (Asher, '68) and the absence of the visual pathway (unpublished results). It is well known that light, perceived by the eyes, plays a major role in mammalian production. The reproductive organs of male and female golden hamsters are particularly sensitive to darkness as seen by the rapid involution of these organs when animals were experimentally blinded (Reiter and Hester, '66; Reiter, Hoffman and Hester, '66; Reiter, '68a,b) or exposed to short photoperiods (Hoffman and Reiter, '65; '66). The reduction in testicular size and weight caused by the gene Wh_(Tables 2, 7 through 10) was consistent with the aforementioned studies by Reiter and Hester ('66), 38 Reiter, Hoffman and Hester ('66) and Reiter ('68a,b), who first reported a severe reduction in testicular size and weight in experimentally blinded hamsters. Similarly, involution of the seminiferous epithelium, accom- panied by a concomitant decrease in tubular size and a thickening of the basement membrane was observed in experimentally blinded hamsters (Reiter, '67) as well as in the genetically blinded (Whflh) hamsters re- ported here. Histologically, the atrophic testes of experimentally blinded ham- sters exhibited a complete loss of spermatogenesis (Reiter, '67) whereas testes from genetically anOphthalmic hamsters exhibited an arrest at the early spermatid stage of psermiogenesis. This result may be due to strain differences, although the cream gene has no apparent effect on reproductive ability in the Syrian hamster. It is also plausible that the technique of electron microscopy has made it possible to more clearly differentiate the developing germ cells in testicular tubules, since normal hamsters in the AN/Asfflh strain which have been experimentally blinded possess tubules identical in composition to those from anophthal- mic individuals (unpublished results). The exact mechanisms whereby light deprivation alters gonal function remains unknown. It has been suggested by Reiter ahd Hester ('66) that the pineal gland, activated by the absence of light, alters the central control of either the synthesis or release of pituitary gonadotropins. Reiter ('68) suggested that the pineal influences reproduction by modi- fying the neruoendocrine axis, rather than a direct effect on the target organs. Recent studies by Quay ('74) suggest that the pineal gland exerts a retardant effect on hypothalmic-pituitary activity in general. 39 Since the effects of the gene Wh_in the Syrian hamster closely parallel the effects of experimental blinding, the infertility observed in anOphthalmic homozygotes (Whflh) is probably caused by the absence of eyes. Much of the evidence presented in this investigation also points to a defect in endocrine control of testes function, which may or may not be related to anophthalmia. Thus, further embryological, anatomical, and physiological examination of this mutant will provide useful informa- tion as to the effects of anOphthalmia on reproduction, and may further reveal the primary action of the gene Wh_in the Syrian hamster. Acrosomal contents Agranular endoplasmic reticulum Basal lamina Cellular debris Epididymis Free polysomes Golgi Developing germ cells Germinal epithelium Heterochromatin Osmiophylic lipid Non-osmiophilic lipid Lipofuchsin pigment Mitochondria 40 ABBREIVATIONS PLATE 1 Nucleue Outer limiting membrane of the acrosomal vesicle Primary spermatocyte Early spermatid Late spermatid Sertoli cytoplasm Smooth endoplasmic reticulum Spermatogonia Spermatozoal head Sertoli nucleus Spermatozoal tail Testis Vacuole EXPLANATION OF FIGURES Homozygous normal hamster of the genotype whwhee. Heterozygous hamster of the genotype thhee. Homozygous of eyes Testis (T) Testis (T) Testis (T) mutant hamster of the genotype WhWhee. and pigmentation. Note the reduction in testis size. Note the absence and epididymis (e) from a homozygous normal hamster. and epididymis (e) from a heterozygous hamster. and epididymis (e) from a homozygous mutant hamster. 41 u : l»...— :E: 3 vi....l :52 N 8:... p 3“]...- =_::: 2:2: 42 PLATE 2 EXPLANATION OF FIGURES Figures 7 and 9 are 5 pm paraffin sections stained with Hemotoxylin and Eosin. For increased resolution, Figures 8 and 10 are 1 pm Epon sections stained with Toluidine Blue 0. 7. Light micrograph of normal/heterozygous seminiferous tubules. The germinal epithelium (GE) proliferates and differentiates within each tubule to form spermatozoa which are seen as heads (SH) and tails (ST) filling the tubular lumen. x 256. (Paraffin, H and E) 8. Light micrograph of normal/heterozygous germinal epithelium from a field comparable to that in the box in Figure 7. Note spermato- gonia (SG) and Sertoli nuclei (SN) lying adjacent to the basal lamina (BL), along with maturation of the germinal epithelium to the late spermatid stage (32). x 640. (Epon, Toluidine Blue) 9. Light micrograph of mutant seminiferous tubules. Note the reduction in tubular diameter along with the lack of properly differentiated germinal epithelium (GE). x 256. (Paraffin, H and E) 10. Light micrograph of mutant germinal epithelium in a field comparable to that in the box in Figure 9. Note the thickened basal lamina (BL) and the arrest of spermiogenesis in the early spermatid stage (81). x 640. (Epon, Toluidine Blue) 43 11. 12. 13. 14. 44 PLATE 3 EXPLANATION OF FIGURES Electron micrograph of a normal/heterozygous Leydig cell. Note the abundance of mitochondria (M) and lipid (L1). x 6,900. Electron micrograph of normal/heterozygous Leydig cytoplasm. Note the abundance of smooth endoplasmic reticulum (SER). x 25,000. Electron micrograph of mutant Leydig cells. Note the degenerative condition of these cells which is exemplified by the clumped nuclear heterochromatin (H) and lack of defined cytoplasmic organelles. x 4,800. Electron micrograph of mutant Leydig cell cytoplasm. Note the vacuolated state of the smooth endOplasmic reticulum (SER) along with the lack of mitochondria and lipid. x 19,750. 45 15. 16. 17. 18. 46 PLATE 4 EXPLANATION OF FIGURES \ Electron micrograph of a normal/heterozygous Sertoli cell. The large lobulated Sertoli nucleus (SN) assumes a position next to the basal lamina (BL). Developing germ cells (GC) are found embedded within the Sertoli cytoplasm (SC). x 5,850. Electron micrograph of normal/heterozygous Sertoli cell cytoplasm. x 18,750. Electron micrograph of mutant Sertoli cells. Note the position of the nucleus (SN) next to the basal lamina (BL) which has re- duplicated and invaginated (arrow) toward the tubular lumen. Sertoli cell cytOplasm (SC) does not envelope developing germ cells and hence appears vacuolated (V) towards the lumen. x 5,850. Electron micrograph of mutant Sertoli cell cytoplasm. Note the abundance of osmiophilic (L1) and non-osmiOphilic (L2) lipids within the cytoplasm. x 18,750. 47 48 PLATE 5 EXPLANATION OF FIGURES 19. Electron micrograph of an early spermatid from normal/heterozygous testicular tissue. The Golgi apparatus (G) in the juxtanuclear cytoplasm produces material which feeds into the acrosomal vesicle (arrow). x 21,875. 20. Electron micrograph of an early spermatid from mutant testicular tissue. Note the degenerative appearance of the Golgi apparatus (G), vacuolation of the cytoplasm (V) and restriction of the acrosomal contents (A) to the anterior pole of the nucleus. x 21,875. 50 PLATE 6 EXPLANATION OF FIGURES 21. Electron micrograph of normal/heterozygous germinal epithelium towards the tubular lumen. Note spermatozoal head (SH) and tail (ST) enveloped by the Sertoli cytoplasm (SC). x 5,600. 22. Electron micrograph of mutant germinal epithelium towards the tubular lumen. Note that the absence of germ cells was accompanied by tubules filled with cellular debris (CD). x 7,200. 51 CHAPTER 3 AN ELECTRON MICROSCOPIC EXAMINATION OF THE PITUITARY GLAND OF THE SYRIAN HAMSTER MUTANT ANOPHTHALMIC WHITE (Eh) A. Introduction Previous studies of the gene Eh, Anophthalmic white, in the Syrian hamster revealed that animals homozygous for this gene were anophthalmic (Knapp and Polivanov, '58; Robinson, '62; '64; Asher, '68; Yoon, '73; '75), exhibited a complete absence of skin and coat pigmentation (Robinson, '62; '64; Asher, '68), exhibited severe growth retardation (Asher, '68; Chapter 2), and lacked reproductive ability (Knapp and Polivanov, '58; Asher, '68; Chapter 2). Along with these obvious de- fects, earlier studies by Asher ('68) suggested that growth retarda- tion, sexual infantilism and adrenal insufficiency were the result of an improper hormone balance due to improper pituitary function, caused by ammonia toxification during development. The study in Chapter 2 revealed that most male hamsters homozygous for the gene Eh in the AN/Asfiflh strain possessed atroPhic testes. These atrophic testes contained small seminiferous tubules, abnormal Leydig and Sertoli cells and possessed germ cells arrested in the early sperma- tid stage of spermiogenesis. This investigation also suggested that the primary cause of infertility in eyeless homozygotes might be correlated with a defective hypophysis, since it is known that the pituitary is 52 53 responsible for the maintenance of gonad function (Smith, '27; '30) and that the pituitary hormones FSH and LH are essential to spermiogene- sis (Smith, '30; Greep and Fevold, '36; Greep, Fevold, and Hisaw, '37; Lostroh, '76). Therefore, the present study was undertaken to determine whether abnormalities existed in hypophyses of male hamsters homozygous for the mutant gene Eh and whether a correlation existed between the hyp0physis and the testicular abnormalities described in Chapter 2. Since the pur- pose of this investigation was to correlate pituitary and gonal activity, only the "sex zone" (Baker and Cross, '78) of the pars distalis was examined. B. Materials and Methods The strain used in this investigation was designated AN/Asjflh_by Asher ('68) and is maintained by a system of full-sibling mating where at least one parent is heterozygous for Eh. Hamsters were housed in poly- carbonate 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 3d libidum. Lighting of the animal room was on a regime of 13 hours of light and 11 hours of darkness. The same sets of ten matched-siblings at approximately 135 days of age were used in this investigation as were used in the testicular study described in Chapter 2. Activity patterns for each animal were determined on running wheels over a one week period prior to killing. Four hours prior to the calculated onset of running activity, hamsters were weighed, anesthetized with ether, and perfused through the left ventricle with Ringer's solution followed by 0.5 percent cacodylate buffered glutaraldehyde 54 at pH 7.3 (Glauert, '75a). The time from anesthetization to perfusion with fixative took approximately 3 minutes. After perfusion and removal of the superior part of the cranium, hypophyses were excised and immersed in 0.5 percent cacodylate buffered glutaraldehyde at pH 7.3 (Glauert, '75a). Some of the glands were photo- graphed. With the aid of a dissecting microscope, the pars intermedia and the infundibular processes were carefully separated from the pars distalis and discarded. A mid-sagital cut was made in the pars distalis (B of Fig. 1), along with two cross cuts (A of Fig. 1), to divide the gland into four pieces of tissue (1 through 4 of Fig. 1). Each piece of tissue, which was approximately one millimeter in width, was post-fixed in 2.0 percent cacodylate buffered osmium tetroxide (Glauert, '75b), and embedded in Epon-Araldite (Mollenhauer, '64). Random think and thin mid-sagital sections, found ventromedial to the hypophyseal cleft (shadded boxes in B-B of Fig. 1), were examined. Thick Epon-embedded sections (1 pm) were stained with a 1.0 percent solution of toluidine blue 0, examined and photographed with a Zeiss light microscope. All nuclei were counted and cellular densities were calculated from replicate, 10 x 7 cm plots, taken from photographs printed at 680K magnification. Ultra-thin sections were stained in uranyl acetate (Watson, '58), and lead citrate (Reynolds, '63) and examined in the Phillips 300 electron microscope at 60 kV. C. Results In this investigation, no gross anatomical differences existed in the hypophyses of male animals in the AN/AsfiWh strain. All glands possessed a pars distalis, pars tuberalis, pars intermedia, infundibular 55 Fig. l. A schematic representation of the areas of the hyp0physis processed for light and electron microscopy. After removal of the pars intermedia and infundibular process, a mid—sagital cut (B) was made in the pars distalis, along with two cross cuts (A), to divide the gland into four pieces of tissue (1 through 4). Sections were examined from the medial zone, ventral to the hyp0physeal cleft (shaded boxes in B-B). P, peripheral zone; L, lateral zone; HC hypophyseal cleft. 56 57 process and infundibular stem (Figs. 2-5). All pars intermedia and infundibular processes were in close apposition to one another and were separated from the pars distalis by the hypophyseal cleft. Unfortunately, no weight measurements were taken to determine if weight differences existed between genotypes. At the light microscopic level, ventromedial sections of the pars distalis from ten normal and ten heterozygous tissues were identical in composition and contained closely packed cells which were arranged in irregular anastomosing cords, separated by capillary channels. In cross section, these cords were seen as nests or rosettes made up of 8 to 9 cells. AcidOphils, basophils, and chromophobes were present in a single cord or rosette. Each cell contained a large centrally or eccentrically positioned nucleus, while acidophilic and basophilic cells possessed darkly stained secretory granules (Fig. 6). Ventromedial sections of the pars distalis from 9 mutant tissues contained some cords or rosettes of cell which approached the normal phenotype. However, many of the cells in this region contained darkly stained nuclei, which were oriented to one side near the cell membrane, and one large cytoplasmic vacuole. These vacuolated structures were greatly enlarged and at the light micro- scopic level seemed to be completely devoid of cell cytoplasm, secretory granules and cellular organelles (Fig. 7). Since hyp0physes of mutant tissue appeared to contain fewer cells per plot, a comparison of cellular densities was made between genotypes. Mean measurements of cellular densities (Tables 1 through 3) demonstrated that the glands from anophthalmic individuals contained approximately 40 percent fewer cells per plot than did the glands from normal and heterozygous individuals. An analysis of variance (Gill, '78s), comparing 58 means from Table 3, proved that a highly significant reduction in cell number existed in mutant tissue (Table 4). A linear regression analysis (Gill, '78c) was performed to determine whether the reduction in pitui- tary cell density was correlated with differences in testis size found in homozygous mutant individuals (Chapter 2). When the number of nuclei per plot was regressed against the gram percent body weight of the testis, a significant correlation was observed between the reduction in cell densities of the hyp0physis and the severity of testicular involution caused by the gene (Table 5). At the electron microscopic level, the fine structure of gonadotropes from 10 normal and 10 heterozygous tissues were identical in composition. These cells were large and polyhedral, measuring 10 to 15 pm in diameter, and each contained an eccentrically positioned nucleus. The nucleus was of irregular contour and possessed finely scattered and peripherally clumped chromatin. The secretory granules were uniformly electron-dense, measuring 200 um maximally, and were randomly distributed throughout the cell. Mitochondria were generally elongate and the Golgi complex was not prominent. Free and attached ribosomes were present and the endoplasmic reticulum was vesiculated, resembling a filagreed pattern throughout the cell. Most of these cells were found in association with vascular spaces (Fig. 8). By contrast, 9 mutant tissues from this region possessed very few cells resembling those from normal and heterozygous tissue. Most of the cells in this region were greatly hypertOphied and contained very irregular and indented nuclei, which were oriented to one side of the cell membrane. Electron dense cellular cytoplasm was located next to the nucleus and immediately adjacent to the periphery of the cell membrane, and contained a few lysosomes and mitochondria. Mbst of the 59 Table l. -- Measurements of the Number of Nuclei per 10 x 7 cm Plot from the Ventromedial Zone of the Pars Distalis and Testis Weight as Percent Body Weight from 5 Normal (+/+), 5 Het- erozygous (+/-) and 9 Mutant (-/-) Hamsters from the AN/AS- .Wh_Strain. Animal Generation Genotype Age Nuclei per Testis as Number (F) (days) 10 x 7 cm Plot Z Body Weight 1821 4 135 79.0 2.22 1867 5 136 83.0 2.46 1874 5 whwhee 137 79.5 2.69 1889 6 (+/+) 141 77.5 2.62 1890 6 140 79.5 2.29 1825 3 136 86.5 2.38 1818 3 136 78.0 2.48 1797 3 thhee 188 75.5 2.09 1861 5 (-/+) 137 75.5 2.69 1883 4 137 75.0 2.53 1736 3 239 41.5 0.19 1811 3 135 59.5 1.66 1812 3 136 55.0 0.51 1816 3 WhWhee 136 52.0 1.88 1803 4 (-/-) 135 42.5 1.83 1829 4 137 68.5 0.43 1782 3 135 56.0 0.21 1857 5 135 31.0 0.51 1860 5 136 41.0 0.32 60 Table 2. -- Computations for the Analysis of Variance on the Number of Nuclei per 10 x 7 cm Plot from Table l. +/+ +/— -/- 79.0 86.5 41.5 83.0 78.0 59.5 79.5 75.5 55.0 77.5 75.5 52.0 79.5 75.0 42.5 68.5 56.0 31.0 41.0 (Y, ) 398.50 391.00 447.0 80470.12 [A] r, 5 5 9 19.0 1 (Ti ) 79.70 78.20 49.67 S.E. :2.02 :1.97 14.76 s: 4.08 3.89 22.65 Sum of Squared Observations Within a Group 31776.75 30591.75 23268.00 85636.50 [B] Correction Factor for each Group 31760.45 30576.20 23086.78 85423.43 [C] SSY 5166.38 SST 4953.31 SSE 213.07 MST 2476.66 MS 13.32 61 Table 3. - Mean Measurements of Cellular Densities from Table 2*. GENOTYPE whwhee thhee WhWhee 51 79.70 78.20 49.67 in 5 5 9 S.E. :2.02 :1.97 :4.76 *All measurements were calculated from 10 x 7 cm plots taken from photographs at 680K magnification. 62 Table 4. -- Analysis of Variance Comparing Data from Tables 1 thru 3. Parameter examined MSBetween df MSWithin df F Cellular density 2476.66 2 13.32 16 185.94** **Denotes significance at the one percent level where F2 = 6.23. ,16 63 Table 5. - A Linear Regression Analysis Between GenotypesO and Within the Mutant Genotype6 where the Number of Nuclei per Plot was Regressed 5 Heterozygous (+/-) and 9 Mutant (-/-) Hamsters in the AN/Asfiflh Strain. Number of Nuclei per Plot; Gram Percent Body Weight of the Testis. +/+ +/— -/- 79.0; 2.22 86.5, 2.38 41.5; 0.19 83.0; 2.46 78.0, 2.48 59.5; 1.66 79.5; 2.69 75.5; 2.09 55.0; 0.51 77.5; 2.62 75.5, 2.69 52.0; 1.88 79.5; 2.29 75.0, 2.53 42.5; 1.83 68.5; 0.43 56.0; 0.21 31.0; 0.51 41.0; 0.32 OSy.x = 0.64; 4 = 0.77**. **Indicates significance at the one percent level where r = 0.573. 6Sy.x = 0.56; r = 0.09. 17 64 cells were occupied by huge lumens, containing a colloid-like substance. No secretory granules, Golgi or endoplasmic reticulum were present (Fig. 9). At the cell boundaries, cellular membranes of adjacent cells in all normal and heterozygous tissue were parallel and separated by an inter- cellular space of approximately 200 A in width. This uniform spacing of Opposed membranes was interrupted occasionally by some intercellular canaliculi and small desmosomes (Fig. 10). Each adjacent cord of cells was separated from the next by an intercordal or vascular space. Most cell boundaries between adjacent cells in mutant tissue were identical in composition to those of the normal phenotype. However, all nine mutants possessed some boundaries which contained specialized junctions, including tight junctions (zonulae occludens), intermediate junctions (zonulae adherens), and desmosomes (maculae adherens) as described by Fawcett ('66). These specialized junctions were occasionally found between granulated cells in this region (Fig. 11). Probably the most interesting observation from hypophyses was the presence of many highly ciliated secretory cells within the pars distalis of all homozygous mutant individuals. The ventromedial zone from normal and heterozygous glands in 3 out of 20 cases examined contained cells with a single cilium, basal body, and an associated centriole lying free in the cytoplasm proximal to the base of the cilium. These isolated cilium were located close to the cell membrane and projected into an intercellular space. In each of these comparatively rare cases, the single cilium was bound by an extension of the plasma membrane of the cell, which invaginated along the shaft to the level of the junction between the cilium and the basal body (Fig. 12). The tubules or fibrils 65 in the center of the cilium had a 9 + 0 configuration which continued into the basal body (Fig. 12, inset). Mutant tissue contained some cells with a single cilium which were identical to the ciliated cells of the 3 cases described above. How- ever, all mutant tissue possessed numerous ciliated cells which contained numerous ciliary shafts and basal bodies cut in cross section and longi- tudinal section. Ciliated cells were found within cell cords and con- tained a highly indented, bizarre-looking nucleus. Along with cilia, these cells still possessed mitochondria, endOplasmic reticulum and secretory granules (Fig. 13). Commensurate with the normal condition, cilia in mutant cells con- sisted of a cilary shaft and basal body, although no centrioles were observed. Each single cilium was bound by an extension of the plasma membrane of the cell and apparently projected into an intercellular space. Each basal body possessed many rootlets, giving it a "hairy" appearance (Fig. 14). The tubules or fibrils in the center of the cilium had a 9 + 2 configuration which continued into the basal body. The peripheral doublets consisted of three microtubules. The outer two, commonly referred to as subfibre A and subfibre B (Fawcett, '61), were joined by a third microtubule located towards the central core (Fig. 15). D. Discussion It is evident from this investigation that the gene Eh did not cause gross anatomical differences in hypophyses of the three genotypes. All glands were of similar size and shape and possessed a pars distalis, pars tuberalis, pars intermedia, infundibular process and infundibular 66 stem. All pars intermedia and infundibular processes were in close apposition to one another and were separated from the pars distalis by the hypophyseal cleft. Since the purpose of this investigation was to determine whether the gene Eh altered the hypophysis of homozygous hamsters in addition to causing infertility, light and electron microscopy of the ventromedial "sex zone" of the pars distalis (Purves and Griesbach, '55; Costoff, '73; Baker and Cross, '78) was undertaken. Light microscopic examination of the "sex zone" of hypophyses from 10 normal and 10 heterozygous hamsters were consistent with previous observations on the cytology of the anterior pituitary as reviewed by Herlant ('64). Acidophils, basophils and chromophobes possessed a centrally or eccentrally positioned nucleus and the acid0phils and baso- phils contained darkly stained secretory granules. Mutant tissue from this zone contained some cords or rosettes of cells which approached the normal phenotype. However, most of the cells in this region con- tained darkly stained nuclei, which were oriented to one side near the cell membrane, and one large cytoplasmic vacuole. These vacuolated cells were greatly enlarged and, at the light microscopic level, seemed to be devoid of cytoplasm, secretory granules and cellular organelles. In addition to the enlargement of cells in this region, mutant tissue appeared to contain far less cells per plot than did normal and heterozygous tissue. Since the later genotypes were absolutely identi- cal in composition, five random plots from these normals and heterozygotes were compared to plots from all of the mutant tissues. Mean measure- ments of cellular densities indicated that, when homozygous, the gene ‘Eh caused a 40 percent reduction in pituitary cell number (Tables 1 and 67 2). When cell density between genotypes was regressed against the gram percent body weight of the testis, a significant correlation was observed between the severity of testicular involution caused by the gene and the reduction in cell density of the hypophysis (Table 3). Thus, hamsters who possessed pituitaries containing cells which were vacuolated also possessed atrophic testes. Since the cellular structures of interest were beyond the resolu- tion of the light microscope, electron microscopy has proven to be a power- ful tool for the investigation of changes in pituitary ultrastructure correlated with changes in endocrine states and to study pituitary in- fluences in certain pathologic conditions (Barnes, '62). The ultrastruc- ture of gonadotropes and the changes they undergo following castration have been reported in the rat (Farquhar and Rinehart, '54; Yoshimura and Harumiya, '65; Costoff, '73), mouse (Yamada and Sano, '60; Barnes, '62), and hamster (Girod and Dubois, '65; Dekker, '67). The appearance of gonadotropes from the 10 normal and 10 heterozygous glands reported here were consistent with the previous studies of gonadotropes from intact hamster glands reported by Girod and Dubois ('65) and Dekker ('67). Schleidt ('14) observed vacuolation of basophils after castration and designated these castration cells as "signet ring cells." Studies by Dekker ('67) revealed that ten days after castration, hamster gona- dotropes began to enlarge. By one month after castration of the rat, the majority of cells were transformed into signet ring cells (Costoff, '73). Studies by Yoshimura and Harumiya ('65) suggested that the de- velopment of signet ring cells occurred through the dialation of fusion of the endoplasmic reticulum within the cell, resulting in the gradual formation of a large vacuole. 68 Electron microscopic examination has revealed considerable differ- ences among cells which with light microscopy were known as signet ring cells (Farquhar and Rinehart, '54). Some cells with one large vacuole had large quantities of cytoplasm containing many mitochondria, granules and smaller Golgi vesicles. Cells of this description resembled more a signet ring. However, some cells consisted of one large vesicle con- taining uniformly light staining material which entirely replaced the cytoplasm of the cell so that the only other elements remaining were a few short rod-like mitochondria and a few granules concentrated in the Golgi region. These larger vesicles were surrounded by only a few deli- cate strands of cytoplasm and possessed an indented and darkly stained nucleus (Farquhar and Rinehart, '54). The exaggerated signet ring cells described above by Farquhar and Rinehart ('54) were the long-term response to castration (35 to 75 days post castration) in the rat. All of the other studies, described above, also attribute exaggerated signet ring formation to long term effects of castration. Since all hamsters homozygous for the gene Wh_in the AN/Asfiflh strain possessed cells identical in appearance to these exag- gerated signet ring cells, the mutant glands not only resembled glands from castrated hamsters, but resembled glands with long term effects of castration. Thus, anophthalmic hamsters must have possessed atrophic testes for quite some time, possibly since the onset of puberty. Since vacuolation and enlargement of cells in the "sex zone" closely parallel the long term effects of castration, it is possible that ab- normalities in this region of the hypophysis were only caused by an interruption in the pituitary-gonadal axis. However, Dekker ('67) and Yoshimura and Harumiya ('65) both reported that all basophilic cells 69 enlarged gpg_concurrently proliferated as the long term response to castration. These results were inconsistent with the present finding that the cells in the ventromedial zone were reduced by approximately 40 percent in anOphthalmic individuals. Furthermore, if the infertility caused by Wh_was analogous to castration, a strong correlation within the genotype (WhWhee) would be expected between the severity of testicu- lar involution and the severity of vacuolation, enlargement and reduction in cell number in the hypophysis. This was not the case (Table 5). Therefore, it is possible that the morphologic abnormalities of pituitary cells represent a morphologic manifestation of the primary action of this gene in addition to a response to castration. Since cell membranes and cell surfaces are highly specialized, depending on the cell's functional demands and relationships to neighboring cells, the appearance of small desmosomes, pinocytotic vesi- cles and cilia in normal and heterozygous tissue was not surprising. Among normal and heterozygous cells, the presence of small desmosomes, located at the boundary of adjacent cell membranes, must represent points of cellular adhesion, offering an area of anchorage for converging cytoplasmic filaments. The presence of many pinocytotic vesicles along these cellular membranes, functioning in either cellular endocytosis or exocytosis, suggested that fluid, possibly cerebro-spinal fluid, must flow between anterior pituitary cells and thatxthe presence of small desmosomes did not interrupt this flow. The quantity of vesicles found along the membrane further suggested that endocytosis and exocytosis were regularly occurring events within anterior pituitary cells and it would seem that normal function of these cells would depend on an intact inter- action between the vesicles and the cellular membrane. 70 Mutant tissue contained many cells with typically opposed membranes, small desmosomes and pinocytotic vesicles as described above. However, the presence of many occluding junctions, intermediate junctions and large desmosomes was unexpected, since these junctions isolate cells from any membrane interaction with their external environment. Special- ized junctions also seemed to be associated with cells which were less granulated and possessed extensive areas of free ribosomes. Throughout the course of general study of the fine structure of the adenohypoPhysis, ciliated cleft cells (Costoff, '73), ciliated follicle cells (Farquhar, '57; Rennels, '64; Yoshida, '66; Costoff, '73) and ciliated secretory cells (Barnes, '61; Millhouse, '67; Wheatly, '67; Dubois and Girod, '70; Dingemans, '70) have been observed. Barnes ('61) reported that ciliated secretory cells were occasionally found in normal glands but seemed to be more frequent in castrated and lactating animals. These rare ciliated secretory cells, like those few from the normal and heterozygous hamsters reported here, possessed a cilium with a basal body and proximal centriole and possessed a 9 + 0 fibril configuration. In order to understand the effect on pituitary ultrastructure caused by the gene Eh, the development of the pituitary, beginning with the embyronic epithelium of Rathke's pouch, should be undertaken. It is known that embyronic epithelium, along with typical differentiated epithelium, possess two prevalent features: (1) tight, intermediate and gap junctions and (2) many cilia or microvilli (Fawcett, '65). Barnes ('61) suggested that the presence of cilia in the adult pituitary was not surprising, as long as this glandular tissue retained its embyronic capacity to form cilia. Also, Campbell and Campbell ('71) pointed out that despite the general permance of cell attachments, there 71 are specific and important developmental and pathologic circumstances which lead to their disappearance, especially when epithelial cells differentiate into nonepithelial tissues. Thus, junctional complexes, found in all mutant tissue, were either retained from the embyronic con- dition or were a result of de-differentiation in the adult condition. Similarly, the abnormal cilia may either represent an initial abnormal differentiation of embyronic ciliated epithelium which is retained in the adult mutant or may represent an abnormal de-differentiation from the adult condition. This suggests that the pituitary may be one site or primary action of the gene Wh_which caused abnormal cellular differ- entiation, exemplified by the formation of abnormal membrane structures. Similarly, eyelessness, caused by the failure of closure of the chroid fissure (Asher, '68), may also be caused by an abnormal membrane differ- entiation. Cells which possessed junctional complexes in embyronic epi- thelium may not allow proper cell-cell interactions and the closure of this fissure might thus be obstructed. Furthermore, vacuolation and en— largement in adult hypophyses could be the result of junctional complexes which isolate cells from their external environment. Thus, it is possi- ble that the gene Eh causes a retention of cell junctions and cilia from the embyronic state. This lack of differentiation in the sex zone may have caused, secondarily, a disruption in the pituitary-gonadal axis which resulted in malfunction of the testis. Further anatomical, embyrological and biochemical examinations of this mutant will provide useful information as to the effects of the gene Eh on pituitary morpho- logy and will ultimately reveal the primary action of this gene in the Syrian hamster. ISP, LTH, ABBREVIATIONS Blood capillary in longitudinal section Blood capillary in cross section Basal body Centriole Cell membrane Colloid-like substance Desmosome Endoplasmic reticulum Nucleus of a gonadotrope Intercellular canaliculi Infundibular stem Intercellular space Lysosome Nucleus of a lactropic cell EXPLANATION OF FIGURES 72 PLATE 1 Mitochondria Nucleus Pars distalis Pars intermedia Plasma membrane Pars tuberalis Pinocytotic vesicle Rootlets in logitudinal section Rootlets in cross section Ciliary shaft Secretory granules Cytoplasmic vacuole Vascular space Zonulae adherens Zonulae occludens A schematic representation of the hypophysis of male hamsters from the AN/Asjflh strain. Each gland consisted of a para distalis (PD), pars intermedia (PI), pars tuberalis (PT), infundibular process (IP) and infundibular stem (IS). Hypophysis from a homozygous normal hamster (whwhee). x 10. Hypophysis from a heterozygous hamster (thhee). x 10. Hypophysis from a homozygous mutant hamster (WhWhee). x 10. 74 PLATE 2 EXPLANATION OF FIGURES Figures 6 and 7 are light micrographs of 1 pm thick Epon—embedded sections stained with Toluidine Blue. 6. Ventromedial section of the pars distalis representative of normal and heterozygous tissue. Note the closely packed arrangement of cells in cords or rosette patterns. Each cell contained a large nucleus (N) and most cells contained secretory granules (SG). x 1,127. (Light, Toluidine Blue). 7. Ventromedial section of the pars distalis representative of mutant tissue. Note that many of these cells were greatly enlarged and possessed a darkly stained nucleus (N) and one large cytoplasmic vacuole (V). x 1,127. (Light, Toluidine Blue). 76 PLATE 3 EXPLANATION OF FIGURES 8. Electron micrograph of gonadotropes from the ventromedial zone of normal/heterozygous tissue. Note the large nucleus of irregular contour (GN), containing finely scattered and peripherally clumped chromatin. Secretory granules (SG) were of uniform density and randomly distributed throughout. Mitochondria (M) were generally elongate and the endoplasmic reticulum (ER) was vesiculated. Most gonadotropes were found in association with vascular spaces (VS). x 5,600. 9. Electron micrograph of the most prevalent cell type found in the ventromedial zone of mutant tissue. Note the very irregular and indented nucleus (N) oriented to one side of the cell meme brane (CM). Cell cytoplasm was oriented next to the nucleus and immediately adjacent to the periphery of the cell membrane and contained a few lysosomes (L) and mitochondria (M). Most of the cell was occupied by a huge lumen, containing a colloid- like substance (CS). x 5,600. 77 . .1 .4 V1.7. 78 PLATE 4 EXPLANATION OF FIGURES 10. Electron micrograph of cellular membranes (CM) of adjacent cells representative of normal and heterozygous tissue. Note that the cell boundaries were parallel and separated by an intercellular space of approximately 200A in width. The uniform spacing of opposed membranes was interrupted by some intercellular canalicule (IC), small desmosomes (D) and pinocytotic vesicles (PV). x 56,000. 11. Electron micrograph of cellular membranes (CM) of mutant tissue con- taining specialized junctions. Note the presence of zonulae occludens (Z0), zonulae adherens (ZA), and desmosomes (D). x 56,000. 79 8O PLATE 5 EXPLANATION OF FIGURES 12. Electron micrograph of an isolated cilium, occasionally found in the ventromedial zone of the pars distalis in normal and heterozygous tissue. These cilium consisted of a shaft (S), basal body (BB), and associated centriole (C), and were bound by an extension of the plasma membrane (PM) of the cell, which invaginated to the junction between the shaft and the basal body (arrow)x 32,000. Inset: The tubules or fibrils in the center of the cilium had a 9 + 0 configuration which continued into the basal body (BB). x 96,000. 13. Electron micrograph of a portion of a cell cord in mutant tissue, containing one cell which is representative of a lactotropic cell (LTH) and one highly ciliated cell (arrow). Note the bizarre appearance of the nucleus (N) and cilium, cut in cross section and longitudinal section (Box). Highly ciliated cells contained secretory granules (SG), mitochondria (M), and endoplasmic reticulum (ER). x 9,750. 81 82 PLATE 6 EXPLANATION OF FIGURES 14. Electron micrograph of a field comparable to that in the box in Fig. 13. Cilia in these secretory cells consisted of a ciliary shaft (8), which possessed a 9 + 2 microtubular arrangement (Box), and a basal body (BB). Each basal body contained many rootlets, cut in longitudinal section (R1) and cross section (R2), giving them a "hairy" appearance. Each single cilium was bound by an extension of the plasma membrane of the cell (PM), which invaginated along the shaft to the junction between the cilium and the basal body (arrow). x 32,000. 15. Electron micrograph of the box in Fig. 14. The tubules or fibrils in the center of the cilium had a 9 + 2 configuration. Note that the peripheral doublets were modified into triplets with an extra tubule located towards the central core (arrow). x 104,000. 83 SUMMARY A. Problem The gene Wh_causing anophthalmia in the Syrian hamster, Mesocricetus auratus, is a highly pleitropic gene which has profound effects upon eye deve10pment, pigmentation and reproduction. Since male hamsters homozy- gous for this gene are usually sterile, the objective of this study was to determine: (1) whether testicular abnormalities existed and contri- buted to the infertility of male hamsters, and (2) to determine whether abnormalities existed in hypophyses of male hamsters. Ultimately, it was hoped that the primary action of the gene Eh might be identified. B. Methods In order to determine whether anatomical differences existed in the testes and hypophyses of 10 normal, 10 heterozygous and 10 mutant matched-siblings at approximately 135 days of age, the following para- meters were measured: (1) adult body weight, (2) testis weight, (3) tubular diameter and (4) pituitary cells densities. Gross anatomical observations were made upon adult hypophyses. Histological examinations were made at the light and electron microscopic level of both testicular tissue, including seminiferous epithelium and interstitial tissue, and the rostral ventromedial zone of the pars distalis of the hypophysis. 84 85 C. Results Testes from most homozygous mutant hamsters were hypoplastic 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 cells arrested in the early spermatid stage of spermiogenesis, possibly due to premature failure of the Golgi apparatus to feed vesicles into the acrosomal vesicle and a subsequent dysgenesis of the acrosome. Hypophyses from mutant hamsters were of similar size and shape to those of the normal phenotype. Rostral ventromedial sections from the "sex zone" of the gland contained a 40 percent reduction in cell density. Abnormalities in cellular membranes were observed including the unexpected appearance of specialized junctional complexes and atypical cilia. D. Conclusions Since the primary action of the gene is unknown, it was postulated that the gene either acts directly to alter pituitary function, via abnormal cellular differentiation, or that the abnormalities were due to a failure of eye development and subsequent lack of the visual pathway. 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