Laminin ultrastructural immunolocalization in rat testis during ontogenesis

Summary The synthesis of one of the main glycoproteins of the basement membrane, the laminin, was demonstrated by ultrastructural immunolocalization during rat foetal (16th day to 20th day of gestation) and postnatal development of the testis. The lamina densa, part of seminiferous tubular basement membrane, is labeled uniformly at all studied stages. The lamina lucida is not well defined before the postnatal stages, at which times discrete immunostaining extends from the lamina densa to the adjacent seminiferous epithelial cells (spermatogonia and Sertoli cells). The extracellular matrix around the peritubular cells is not labeled before birth. Intracellular immunostaining was detected as early as the 16th day of gestation in both Sertoli cells and cells around the seminiferous tubules which will transform later into peritubular cells. It was located in rough endoplasmic reticulum (RER) cisternae and secretory vesicles. After 18–20 days of postnatal life, the immunostaining faints progressively. Some positive material is seen in the RER of the gonocytes at all studied stages.Sertoli cells and peritubular cells are the main producing cells of laminin after the 16th of gestation. The laminin secreted by gonocytes may play an important role in adhesion of gonocytes to the lamina densa and adjacent Sertoli cells before their transition from basal compartment to adluminal compartment.     

Immunolocalization of type IV collagen and laminin during rat gonadal morphogenesis and postnatal development of the testis and epididymis

Summary The distribution of type IV collagen and laminin was studied by immunocytochemistry during rat gonadal morphogenesis and postnatal development of the testis and epididymis. Immunostaining appeared as early as the 12th day of gestation along the basement membranes of the mesonephric-gonadal complex. The connection between some mesonephric tubules and coelomic epithelium was seen between the 12th and 13th day of gestation. Discontinuous immunostained basement membranes delineated the differentiating sexual cords in 13-day-old fetuses; this process probably began in the inner part of the gonadal ridge. The seminiferous cords surrounded by a continuous immunoreactive basement membrane are separated from the coelomic epithelium by the differentiating tunica albuginea in 14-day-old fetuses. During the postnatal maturation of epididymis and testis, the differentiation of peritubular cells is accompanied by a progressive organisation of the extracellular matrix into a continuous basement membrane. This change is associated with a gradual condensation of peritubular cells inducing an increase of immunostaining. In adult animals, the tubular wall of epididymis is thicker than the lamina propria of seminiferous tubules. Both type IV collagen and laminin immunostaining paralleled during ontogenesis at the light-microscope level.     

Immunolocalization of type IV collagen and laminin during rat gonadal morphogenesis and postnatal development of the testis and epididymis

The distribution of type IV collagen and laminin was studied by immunocytochemistry during rat gonadal morphogenesis and postnatal development of the testis and epididymis. Immunostaining appeared as early as the 12th day of gestation along the basement membranes of the mesonephric-gonadal complex. The connection between some mesonephric tubules and coelomic epithelium was seen between the 12th and 13th day of gestation. Discontinuous immunostained basement membranes delineated the differentiating sexual cords in 13-day-old fetuses; this process probably began in the inner part of the gonadal ridge. The seminiferous cords surrounded by a continuous immunoreactive basement membrane are separated from the coelomic epithelium by the differentiating tunica albuginea in 14-day-old fetuses. During the postnatal maturation of epididymis and testis, the differentiation of peritubular cells is accompanied by a progressive organisation of the extracellular matrix into a continuous basement membrane. This change is associated with a gradual condensation of peritubular cells inducing an increase of immunostaining. In adult animals, the tubular wall of epididymis is thicker than the lamina propria of seminiferous tubules. Both type IV collagen and laminin immunostaining paralleled during ontogenesis at the light-microscope level.     

Immunocytochemistry of extracellular matrix in the lamina propria of the rat testis: electron microscopic localization

The distribution of laminin, type IV collagen, heparan sulfate proteoglycan, and fibronectin was investigated in the rat testicular lamina propria by electron microscopic immunocytochemistry. Distinct patterns were observed for each antigen within the extracellular matrix (ECM) layers of the lamina propria. Laminin, type IV collagen, and heparan sulfate proteoglycan all localized to the seminiferous tubule basement membrane. Type IV collagen and heparan sulfate proteoglycan, but not laminin, localized to the seminiferous tubule side of the peritubular myoid cells. All four of the antigens were localized between the peritubular and lymphatic endothelial cells. Failure to localize fibronectin in the ECM layer between the Sertoli and peritubular myoid cells tends to support the concept that adult Sertoli cells do not produce this protein in vivo. Intracellular immunostaining was insufficient to allow unambiguous identification of the cellular source of any of the ECM molecules.     

Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV–VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.     

Differential expression of extracellular matrix components in rat Sertoli cells.

We studied expression of laminin, fibronectin, and Type IV collagen in the testis by means of immunofluorescence and immunoblot analysis and also examined gene expression of fibronectin using the ribonuclease protection assay. By immunofluorescence on sections from 20-day-old rats, laminin, fibronectin, and Type IV collagen were found in the basement membrane of the seminiferous tubules and in the interstitial regions of the testis. No localization of any extracellular matrix components was found inside the sectioned cells. However, when Sertoli cells were cultured on glass coverslips, laminin and Type IV collagen were both found inside the cells, suggesting new synthesis. In cultured peritubular cells, Type IV collagen, laminin, and fibronectin were found within the cells. When examined by immunoblot analysis, freshly isolated Sertoli and peritubular cells from 20-day-old rats did not demonstrate production of laminin or fibronectin. After 5 days in culture, peritubular cells produced both laminin and fibronectin, whereas cultured Sertoli cells produced only laminin. In contrast, freshly isolated and cultured Sertoli and peritubular cells all produced Type IV collagen. Moreover, the ribonuclease protection assay indicated that the bulk of fibronectin gene expression occurs within the first 10 days of postnatal development, with lower maintenance levels occurring thereafter. These results indicate that in the testis the highest levels of expression of laminin and fibronectin occur during development and in primary cell culture, whereas expression of Type IV collagen is higher at later stages.     

Regulation of P450 oxidoreductase by gonadotropins in rat ovary and its effect on estrogen production

Cimetidine, referred as antiandrogenic agent, has caused alterations in the seminiferous tubules, including alterations in the peritubular tissue and death of myoid cells by apoptosis. Regarding the structural and functional importance of the peritubular tissue for the maintenance of Sertoli cells (SC), we purpose to investigate the SC-basement membrane interface, focusing the morphological features of SC and their interaction with the basement membrane in the affected tubules by cimetidine. Ten animals were distributed into two groups, control (CG) and cimetidine (CmG) which received saline solution and 50 mg of cimetidine per kg of body weight, respectively, for 52 days. The testes were fixed, dehydrated and embedded for analyses under light and transmission electron microscopy. Paraffin sections were submitted to the TUNEL method; sections of testes embedded in glycol methacrylate were submitted to PAS method and stained by H&E for morphological and quantitative analyses of Sertoli Cells. In the CmG, the SC nuclei were positive to the TUNEL method and showed typical morphological alterations of cell death by apoptosis (from early to advanced stages). A significant reduction in the number of Sertoli Cells was probably due to death of these cells by apoptosis. A close relationship between SC nuclear alterations (including a high frequency of dislocated nuclei from the basal portion) and damage in the peritubular tissue was observed. The ultrastructural analysis showed a parallelism between the gradual advancement of apoptotic process in SC and detachment of the anchoring sites (hemidesmosomes) of SC plasma membrane from the lamina densa. The presence of portions of lamina densa underlying the detached hemidesmosomes indicates a continuous deposition of lamina densa, resulting in the thickening of the basal lamina. The results indicate a possible disarrangement of the SC cytoskeleton, including the focal adhesion structure. These alterations are related to SC apoptosis and probably result from disturbs induced by cimetidine on the peritubular tissue.     

Anionic sites of the basement membrane of rat seminiferous tubules during ontogenesis

The anionic sites of the basement membrane of rat seminiferous tubules were demonstrated ultrastructurally in the lamina densa by using cationic polyethyleneimine (PEI). The sites were largely digested out after incubation with heparitinase, indicating a large proportion of heparan sulfates. The anionic sites were present as early as day 16 of gestation on the interstitial side of the lamina densa, and after gestation day 20 they were symmetrically organized on both sides of the lamina densa. The number of sites is not modified postnatally. They appear more irregular in density with advancing age. Experimental conditions as cryptorchidism, fetal irradiation, and ligation of the ductuli efferents lead to unspecific alterations in the distribution of the anionic sites that are parallel to the modifications in the basement membrane.     

Fine structure of the glomerular basement membrane and immunolocalization of five basement membrane components to the lamina densa (basal lamina) and its extensions in both glomeruli and tubules of the rat kidney

Electron microscopic immunostaining was used to examine the localization of type IV collagen, laminin, entactin , heparan sulfate proteoglycan, and fibronectin within the basement membranes of the rat kidney. In preliminary experiments, various methods of processing formaldehyde-fixed kidney were compared using antilaminin antiserum and the indirect immunoperoxidase method. Little or no laminin immunostaining of the glomerular basement membrane was present in sections unless they had been frozen-thawed; and even in this case, the immunostaining was light in comparison to that of basement membranes in adjacent tubules. However, when frozen-thawed sections were treated with 0.5% sodium borohydride, immunostaining was then as strong in glomerular as in tubular basement membranes. Accordingly, this treatment was applied to frozen-thawed sections before immunostaining for any of the substances under study. Immunostaining of the glomerular basement membrane for each of the five substances was fairly uniform throughout the lamina densa (also called basal lamina), but uneven in the lamina lucida interna and externa (also called lamina rara interna and externa) in which stained bands extended from the lamina densa. Similarly in the basement membranes of tubules, immunostaining for the five substances was localized to the lamina densa and bands extending into the lamina lucida. When the ultrastructure of the glomerular basement membrane was examined, three structures were found: (1) a network of 4-nm-thick "cords," which seems to be the main component; the cords are closely packed in the lamina densa and more loosely arranged in the lamina lucida interna and externa; (2) straight, hollow 7-10-nm-thick structures referred to as " basotubules "; and (3) 3.5-nm elements composed of minute paired rods, referred to as "double pegs." The distribution of the cords, but not that of the other two structures, was related to the immunostaining pattern. It is concluded that (1) to fully reveal the antigenicity of the glomerular basement membrane, frozen-thawed sections must be treated with sodium borohydride prior to immunostaining, possibly because this basement membrane is more compact than the others; and (2) in both glomerular and tubular basement membranes, type IV collagen, laminin, entactin , heparan sulfate proteoglycan and fibronectin are colocalized in the lamina densa and its extensions to the laminae lucidae . Since the distribution of the cords corresponds to that of immunostaining, it is likely that the five substances are present within the cords.     

Methodological dependence in the ultrastructural immunolocalization of laminin in tubular basement membranes of the mouse kidney

Summary The ultrastructural localization of the basement membrane glycoprotein laminin was investigated in basement membranes of proximal tubules of the mouse kidney. The localization of laminin was determined using two different immunoperoxidase and one immunogold preembedding technique and one immunogold postembedding technique on unfixed and formaldehyde fixed tissue. Strong differences in the immunolocalization for laminin were found in the lamina densa of the tubular basement membrane using different techniques.After preembedding immunostaining for laminin using JgG-PO as secondary antibody, a positive reaction for the lamina densa was found in the formaldehyde fixed as well as in the unfixed kidney. After preembedding immunostaining for laminin using Protein-A-PO, staining of the l. densa was seen in the unfixed, but not in the fixed kidney. It was striking that no clear immunoreaction in the l. densa of the tubular basement membrane was seen in either the fixed or unfixed tissue after preembedding immunostaining for laminin using protein A-gold. With a direct postembedding immunogold technique laminin was localized only in the l. fibroreticularis and the l. rara but not in the l. densa of basement membranes of proximal tubules of the unfixed and the fixed kidney.     

High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.     

Involution of seminiferous tubules in aged hamsters: an ultrastructural, immunohistochemical and quantitative morphological study

In this study, we examined the age-related changes on morphometric parameters and ultrastructure of seminiferous tubules, and on the expression of extracellular matrix proteins in lamina propria of Syrian hamsters. A significant decrease in the percentage of normal tubules and an increase in the percentage of hypospermatogenic and arrested maturation tubules was observed with aging. Aged animals showed a decrease in tubular diameter, tubular lumen, seminiferous epithelium volume and total tubular volume. However, the total length of seminiferous tubules was significantly increased with aging. The most important ultrastructural changes with aging were the thickening of the lamina propria, the presence of diverse abnormalities in the spermiogenesis process, degeneration of germ cells, and vacuolization and flattening of Sertoli cells showing abundant lipofucsin droplets and residual bodies. Laminin immunoreactivity was found along the lamina propria of seminiferous tubules both in young and aged animals. Fibronectin immunoreactivity was found along the lamina propria and blood vessels. Both laminin and fibronectin total volume of immunostaining per testis was increased in aged hamsters. In conclusion, the age-related changes in seminiferous tubules of hamster include: a decrease in tubular width and an increase in tubular length; widening of the lamina propria caused by a more extensive connective matrix between the peritubular cells and the basal membrane; and a strong disarrangement of the seminiferous epithelium, including germ cell degeneration and important alterations in both spermiogenesis and Sertoli cell structure.     

Methodological dependence in the ultrastructural immunolocalization of laminin in tubular basement membranes of the mouse kidney

The ultrastructural localization of the basement membrane glycoprotein laminin was investigated in basement membranes of proximal tubules of the mouse kidney. The localization of laminin was determined using two different immunoperoxidase and one immunogold preembedding technique and one immunogold postembedding technique on unfixed and formaldehyde fixed tissue. Strong differences in the immunolocalization for laminin were found in the lamina densa of the tubular basement membrane using different techniques. After preembedding immunostaining for laminin using IgG--PO as secondary antibody, a positive reaction for the lamina densa was found in the formaldehyde fixed as well as in the unfixed kidney. After preembedding immunostaining for laminin using Protein-A--PO, staining of the 1. densa was seen in the unfixed, but not in the fixed kidney. It was striking that no clear immunoreaction in the 1. densa of the tubular basement membrane was seen in either the fixed or unfixed tissue after preembedding immunostaining for laminin using protein A-gold. With a direct postembedding immunogold technique laminin was localized only in the 1. fibroreticularis and the 1. rara but not in the 1. densa of basement membranes of proximal tubules of the unfixed and the fixed kidney.     

Localization and synthesis of entactin in seminiferous tubules of the mouse.

Localization and synthesis of entactin in seminiferous tubules of mouse testis was studied by immunocytochemistry. Frozen sections from adult mice testes were subjected to anti-entactin and anti-laminin immunofluorescence. Both entactin and laminin were localized within the seminiferous tubule basement membrane and intertubular region of the testis. The addition of excess amount of entactin (but not fibronectin), premixed with anti-entactin antiserum, abolished the immunostain. Western blotting showed that a protein extract from a seminiferous tubule basement membrane preparation was recognized by anti-entactin anti-serum and comigrated with recombinant entactin. Enriched fractions of isolated primary Sertoli cells and peritubular myoid cells cultured for 6 days on a glass coverslip were able to synthesize and secrete entactin as detected by immunofluorescence microscopy. Entactin was also produced by TM3 (Leydig-like) and TM4 (Sertoli-like) cell lines as detected by both immunofluorescence and Western blotting. The distribution of entactin vs. laminin within both the cultured primary cells and the TM3 and TM4 cell lines differed. Entactin appeared mainly localized extracellularly. In contrast, laminin was mainly localized intracellularly. The above findings suggested that 1) entactin existed in the seminiferous tubule basement membrane and intertubular region of adult mice testis, co-localized with laminin; 2) entactin was synthesized by the cultured primary Sertoli cells and peritubular myoid cells and the TM3 and TM4 cell lines; 3) entactin was exocytosed with little intracellular accumulation, in contrast to an intracellular accumulation of laminin.     

Hypoplastic basement membrane of the lens anlage in the inheritable lens aplastic mouse (lap mouse)

Adult homozygous lap mice show various eye abnormalities such as aphakia, retinal disorganization, and dysplasia of the cornea and anterior chamber. In the fetal eye of a homozygous lap mouse, the lens placode appears to develop normally. However, the lens vesicle develops abnormally to form a mass of cells without a cavity, and the mass vanishes soon afterward. Apoptotic cell death is associated with the disappearance of the lens anlage. We examined the basement membranes of the lens anlage of this mutant by immunohistochemical methods under light microscopy using antibodies against basement membrane components of the lens anlage, type IV collagen, fibronectin, laminin, heparan sulfate proteoglycan, and entactin and by transmission electron microscopy. Immunohistochemistry showed the distribution and intensity of antibody binding to the lens anlage to be almost the same for each these antibodies regardless of the stage of gestation or whether the anlagen were from normal BALB/c or lap mice. Thus, positive continuous reactions were observed around the exterior region of the lens anlage from day 10 of gestation for type IV collagen, fibronectin, laminin, heparan sulfate proteoglycan antibodies, and at least from day 11of gestation for entactin antibody. The basement membrane lamina densa of both normal and lap mice was shown by electron microscopy to be discontinuous at days 10 and 10.5 of gestation. However, by day 11 the lamina densa was continuous in the lens anlagen of normal mice but still discontinuous in the lap mice. By day 12 of gestation, the lamina densa had thickened markedly in normal mice, whereas in lap mice it remained discontinuous and its thinness indicated hypoplasia. These results indicate that, while all basement components examined are produced and deposited in the normal region of the lens anlage in the lap mouse, the basement membrane is, for some reason, imperfectly formed. The time at which hypoplasia of the basement membrane was observed in this mutant coincided with the stage during which apoptosis in the lens anlage occurred. This result may indicate a possibility of the relationship between the basement membrane and apoptosis in this mutant.     

Extracellular matrix components and testicular peritubular cells influence the rate and pattern of Sertoli cell migration in vitro

We report the patterns of migration of Sertoli cells plated on specific substrata, and the influences of testicular peritubular cells on these processes. Data presented indicate that while peritubular cells readily spread when explanted onto Type I collagen, Sertoli cells do not. A delay of 4 to 6 days occurs after Sertoli cells are plated before they begin to migrate randomly to form plaque-like monolayers on Type I collagen. These processes are dependent upon the synthesis and subsequent deposition of laminin and/or Type IV collagen by Sertoli cells, and are independent of fibronectin. A different behavior occurs when reconstituted mixtures of purified Sertoli cells and pertiubular cells are sparsely plated onto Type I collagen. Peritubular cells rapidly spread to form chains of cells between Sertoli cell aggregates. Sertoli cells then migrate on the surfaces of the peritubular cells, culminating in the formation of cable-like structures between aggregates. Evidence is presented that the Sertoli cell migration to form "cables" under these conditions is dependent upon fibronectin synthesized by peritubular cells, and is independent of the presence of laminin or Type IV collagen. We discuss the possible relevance of these data to the role which precursors of peritubular cells may play in determining the behavior of Sertoli cell precursors in vivo during tubulogenesis, or in the remodelling of the seminiferous tubule which occurs during different stages of the cycle of the seminiferous epithelium in spermatogenesis.     

Desert hedgehog (Dhh) gene is required in the mouse testis for formation of adult-type Leydig cells and normal development of peritubular cells and seminiferous tubules

Testes from adult and prepubertal mice lacking the Desert hedgehog (DHH:) gene were examined in order to describe further the role of Dhh in spermatogenesis because, in a previous report, DHH:-null male mice were shown to be sterile. Dhh is a signaling molecule expressed by Sertoli cells. Its receptor, patched (Ptc), has been previously localized to Leydig cells and is herein described as being localized also to peritubular cells. Two phenotypes of the mice were observed: masculinized (7.5% of DHH:-null males) and feminized (92.5%), both of which displayed abnormal peritubular tissue and severely restricted spermatogenesis. Testes from adult feminized animals lacked adult-type Leydig cells and displayed numerous undifferentiated fibroblastic cells in the interstitium that produced abundant collagen. The basal lamina, normally present between the myoid cells and Sertoli cells, was focally absent. We speculate that the abnormal basal lamina contributed to other characteristics, such as extracordal gonocytes, apolar Sertoli cells, and anastomotic seminiferous tubules. The two DHH:-null phenotypes described have common peritubular cell defects that may be indicative of the essential role of peritubular cells in development of tubular morphology, the differentiation of Leydig cells, and the ultimate support of spermatogenesis.     

Immunoexpression of Tyro 3 family receptors--Tyro 3, Axl, and Mer--and their ligand Gas6 in postnatal developing mouse testis.

Tyro 3 family receptors contain three members-Tyro 3, Axl, and Mer-that are essential regulators of mammalian spermatogenesis. However, their exact expression patterns in testis are unclear. In this study, we examined the localizations of Tyro 3, Axl, Mer, and their ligand Gas6 in postnatal mouse testes by immunohistochemistry. All three members and their ligand were continuously expressed in different testicular cells during postnatal development. Tyro 3 was expressed only in Sertoli cells with a varied distribution during testis development. At day 3 postnatal, Tyro 3 was distributed in overall cytoplasmic membrane and cytoplasm of Sertoli cells. From day 14 to day 35 postnatal, Tyro 3 appeared on Sertoli cell processes toward the adlumenal compartment of seminiferous tubules. A stage-dependent Tyro 3 immunoexpression in Sertoli cells was shown by adulthood testis at day 56 postnatal with higher expression at stages I-VII and lower level at stages IX-XII. Axl showed a similar expression pattern to Tyro 3, except for some immunopositive Leydig cells detected in mature testis. In contrast, immunostaining of Mer was detected mainly in primitive spermatogonia and Leydig cells, whereas a relative weak signal was found in Sertoli cells. Gas6 was strongly expressed in Leydig cells, and a relative weak staining signal was seen in primitive spermatogonia and Sertoli cells. These immunoexpression patterns of Tyro 3 family receptors and ligand in testis provide a basis to further study their functions and mechanisms in regulating mammalian spermatogenesis.     

Effect of ethinyl estradiol on the differentiation of mouse fetal testis

In an evaluation of the effect of ethinyl estradiol (EE) on the differentiation of fetal mouse testes, the ratio of the seminiferous tubular region to the testicular tissue region, the ratio of Sertoli cells to gonocytes in tubule cross sections, and the size of Leydig cells were determined by the Texture Analyse System (T.A.S., Leitz) in histological preparations of the testes. The testes were those of fetuses taken from dams given orally 0, 0.02, 0.2 or 2.0 mg/kg of body weight of EE in olive oil from day 11 through day 17 of gestation and killed at term. From experimental and the control testes, five sections were taken at 40-micron intervals. The areas of the seminiferous tubular region and the testicular region were determined and the Sertoli cells and gonocytes in tubule cross section were counted in each of the five sections. The diameters of 100 Leydig cells selected at random were averaged. These data were analyzed by Student's t test. The seminiferous tubular region was significantly increased in the testes treated with 0.02 mg/kg of EE and significantly decreased in those treated with 0.2 mg/kg of EE. The number of gonocytes per tubule cross section was significantly increased in the testes treated with 0.02 or 2.0 mg/kg of EE. The number of Sertoli cells per tubule cross section and the number of Sertoli cells per gonocyte were significantly decreased in the experimental testes. The size of the Leydig cells was significantly decreased in the testes treated with 0.2 mg/kg of EE. These findings suggest that prenatal exposure to EE before testicular differentiation affects tubular formation, the proliferation of fetal Sertoli cells, and Leydig cell differentiation, resulting in disturbances of spermatogenesis.     

Dysplastic development of seminiferous tubules and interstitial tissue in rat hypogonadic (hgn/hgn) testes

The hypogonadic rat is characterized by male sterility, reduced female fertility, and renal hypoplasia controlled by a single recessive allele (hgn) on chromosome 10. Plasma testosterone is low and levels of gonadotropins are high in adult male hgn/hgn rats, indicating that the cause of hypogonadism lies within the testis itself. We found that the postnatal growth of the seminiferous tubules was severely affected. Here we describe the details of postnatal testicular pathogenesis of the hgn/ hgn rats. In these rats, gonadal sex determination and initial differentiation of each type of testicular cell occur, but proliferation, differentiation, and maturation of these cells during postnatal testicular development is severely affected. Postnatal pathological changes include reduced proliferation and apoptotic cell death of Sertoli cells, abnormal mitosis and cell death of gonocytes, reduced deposition of extracellular matrix proteins into the basal lamina, lack of the formation of an outer basal lamina, formation of multiple layers of undifferentiated peritubular cells, and the delayed appearance and islet conformation of adult-type Leydig cells. Apoptotic cell death of Sertoli cells and disappearance of FSH receptor mRNA expression indicate that this mutant rat is a useful model for Sertoli cell dysfunction. The abnormalities listed above might be caused by defective interactions between Sertoli cells and other types of testicular cells. Because the results presented here strongly indicate that a normal allele for hgn encodes a factor playing a critical role in testicular development, the determination of the gene responsible for hgn and the analysis of early alterations of gene expression caused by mutations in this gene would provide important information on the mechanisms of testicular development.