Sertoli cell ectoplasmic specializations in the seminiferous epithelium of the testosterone-suppressed adult rat

The Sertoli cell ectoplasmic specialization is a unique junctional structure involved in the interaction between elongating spermatids and Sertoli cells. We have previously shown that suppression of testicular testosterone in adult rats by low-dose testosterone and estradiol (TE) treatment causes the premature detachment of step 8 round spermatids from the Sertoli cell. Because these detaching round spermatids would normally associate with the Sertoli cell via the ectoplasmic specialization, we hypothesized that ectoplasmic specializations would be absent in the seminiferous epithelium of TE-treated rats, and the lack of this junction would cause round spermatids to detach. In this study, we investigated Sertoli cell ectoplasmic specializations in normal and TE-treated rat testis using electron microscopy and localization of known ectoplasmic specialization-associated proteins (espin, actin, and vinculin) by immunocytochemistry and confocal microscopy. In TE-treated rats where round spermatid detachment was occurring, ectoplasmic specializations of normal morphology were observed opposite the remaining step 8 spermatids in the epithelium and, importantly, in the adluminal Sertoli cell cytoplasm during and after round spermatid detachment. When higher doses of testosterone were administered to promote the reattachment of all step 8 round spermatids, newly elongating spermatids associated with ectoplasmic specialization proteins within 2 days. We concluded that the Sertoli cell ectoplasmic specialization structure is qualitatively normal in TE-treated rats, and thus the absence of this structure is unlikely to be the cause of round spermatid detachment. We suggest that defects in adhesion molecules between round spermatids and Sertoli cells are likely to be involved in the testosterone-dependent detachment of round spermatids from the seminiferous epithelium.     

Effects of flutamide on testicular involution induced by an antagonist of gonadotrophin-releasing hormone and on stimulation of spermatogenesis by follicle-stimulating hormone in rats.

The effects of combined treatment with an antagonist of gonadotrophin-releasing hormone (ANT) and the antiandrogen flutamide (FL) on spermatogenesis were studied in the presence and absence of exogenous follicle-stimulating hormone (FSH). After treatment for 2 weeks, the combination of ANT (RS 68439, 450-500 micrograms/kg per day, s.c.) with 10, 20 or 40 mg FL/day, s.c. was as effective as ANT plus the Leydig cell toxin ethane dimethane sulphonate (75 mg/kg per week, i.p.) in terms of reduction in weight of testes, epididymides and seminal vesicles. Thus, a daily dose of 10 mg FL/kg was sufficient to block the androgen action in the testes of ANT-treated rats. In a second experiment, rats received ANT and ANT+FL (10 mg/kg) alone or in combination with a highly purified human FSH preparation (5 or 10 iu, twice a day) for 2 weeks. FSH did not affect testosterone concentration or weight of epididymides and seminal vesicles, but ANT+FL markedly enhanced the ANT-induced reduction of testis weight, seminiferous tubule diameter and numbers of germ cells, as revealed by qualitative and quantitative analysis of testis histology. In the absence of FL, testis size and numbers of germ cells, including elongated spermatids, were increased by FSH. In the presence of FL, the effects of FSH were less pronounced with respect to the germ cells, in terms of both numbers of cells and the effective dose of FSH. Irrespective of treatment with FL, exogenous FSH increased the inhibin concentrations in serum, indicating that Sertoli cells remained responsive to FSH. From the present study it is concluded that (i) FL accelerates ANT-induced testicular involution, (ii) FSH has a role in adult spermatogenesis and (iii) the effects of FSH on advanced germ cells are influenced by androgens.     

Gonadal dysgenesis induced by prenatal exposure to ethinyl estradiol in mice

Daily oral administration of ethinyl estradiol (0.02, 0.2, or 2.0 mg/kg of body weight) to pregnant Jc1:ICR mice resulted in ovotestis and intra-abdominal testis with persistent Müllerian duct and Wolffian duct in male fetuses and ovarian hypoplasia in female fetuses when it was given from day 11 through day 17 of gestation (before gonadal differentiation in the fetus). The ovotestis consisted of testicular and ovarian portions. In the testicular portion, a few solid seminiferous tubules containing spermatogonia, some with pachytene nuclei with Sertoli cells and compact interstitial tissue including Leydig cells, were seen. In the ovarian portion, pachytene nuclei were seen. The intra-abdominal testis was smaller and contained more spermatogonia per tubule in cross section than the control testis. These findings suggest that in male fetuses ethinyl estradiol affects Sertoli cell differentiation resulting in suppression of Müllerian inhibiting factor. On the other hand, in the ovarian hypoplasia, the primordial follicles and follicular cells in a primordial follicle were significantly decreased in number, and the number of the degenerated primordial follicles was significantly increased. It seems likely that ethinyl estradiol affects the intimate contact between follicular cells and oocytes to cause degeneration of primordial follicles.     

The pesticide methoxychlor given orally during the perinatal/juvenile period,reduced the spermatogenic potential of males as adults by reducing their Sertoli cell number

Perinatal and juvenile oral treatment of rats with the insecticide, methoxychlor (MXC), reduced testicular size and other reproductive indices including the number of epididymal spermatozoa in those animals as adults 161. The objective was to determine if these males exposed during development had fewer Sertoli cells which might explain these testicular effects. Rat dams were gavaged with MXC at 0, 5, 50, or 150 mg x kg(-1) x day(-1) for the week before and after they gave birth. Resulting male pups (15/group) then were dosed directly from postnatal day 7 to 42. Testes were fixed in Bouin's and in OsO4, embedded in Epon and sectioned at 0.5 microm, stained with toluidine blue, and evaluated stereologically or cut at 20 microm to measure Sertoli cell nuclei with Nomarski optics. Sertoli cell number was calculated as the volume density of the nucleus times the parenchymal weight (90% of testicular weight) divided by the volume of a single Sertoli cell nucleus. Across dose groups, there were no changes in the nuclear volume density, the volume of a single nucleus, or the number of Sertoli cells per g parenchyma. There were highly significant dose-related changes in the volume of Sertoli cell nuclei per testis and the number of Sertoli cells per testis. Reduced testicular weight (r = 0.94) and reduced numbers of epididymal spermatozoa (r = 0.43) were significantly (p < 0.01) correlated to reduced number of Sertoli cells per testis. Hence, perinatal and juvenile oral exposure to MXC can reduce spermatogenic potential of males as adults by reducing their number of Sertoli cells.     

Distribution and levels of cellular retinol- and cellular retinoic acid-binding protein in various types of rat testis cells

The distribution and levels of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) were measured in rat testicular peritubular and Sertoli cells and in isolated rat pachytene spermatocytes and spermatids. Two Sertoli cell preparations, one containing some germ cells and another that had been osmotically shocked to destroy germ cells, were examined. CRBP and CRABP levels were measured by specific and sensitive radioimmunoassays. Testicular peritubular cell cytosol preparations were found to contain high levels of CRBP (1.48 +/- 0.87 microgram CRBP/mg protein) but CRABP could not be detected. The mean CRBP level in Sertoli cell preparations that contained some germ cells was 0.93 +/- 0.24 microgram CRBP/mg protein; this value was similar to the level of 1.11 +/- 0.20 microgram CRBP/mg protein measured for Sertoli cells free of germ cells. The level of CRABP found in Sertoli cell preparations containing germ cells (0.81 +/- 0.32 microgram CRABP/mg protein) was approximately five times greater than was observed in Sertoli cells free of germ cells (0.16 +/- 0.03 microgram CRABP/mg protein). CRBP and CRABP levels in cultured Sertoli cells were not affected by time in culture for up to five days of culture. Pachytene spermatocytes and spermatids were very enriched in CRABP (0.72 +/- 0.26 microgram CRABP/mg protein for spermatocytes and 0.65 +/- 0.21 microgram CRABP/ml protein for spermatids). A search for a high molecular weight retinol-binding protein did not demonstrate the existence of such a protein in Sertoli cell-conditioned medium. In summary, these studies provide quantitative information about the distribution of the cellular retinoid-binding proteins in the cell types that compose the rat testis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testicular Histomorphometry and Ultrastructure of Rats Treated with Cadmium and Ginkgo biloba

The aim of this study is to investigate the association of a single low dose of Cd and daily doses of Ginkgo biloba extract (GbE) on the testis and accessory glands of rats. The animals were treated with a single dose of 3 µmol/kg body weight of cadmium chloride (CdCl₂) and/or 100 mg/kg body weight of GbE. The plasma testosterone levels; corporal, testicular, and accessory glands weight; gonadosomatic index, volumetric proportion; and absolute volume of testicular components did not change after the treatments. CdCl₂ caused significant reduction in Leydig cells volume and altered Leydig cell morphology, as well as vacuolated Sertoli cells cytoplasm, irregular chromatin condensation of late spermatids, and modified acrosome formation. However, animals that received GbE did not show these alterations. The reversal of Cd-induced alterations by the extract is a strong indication that G. biloba is helpful in diminishing the effect of Cd toxicity.     

Effect of styrene on testicular enzymes of growing rat.

Effect of styrene (100 or 200 mg/kg body wt/day) for 60 days was observed on testicular enzymes of postnatally maturing rats. A significant decrease in epididymal spermatozoa count was observed only at 200 mg/kg body weight dose. Activities of testicular sorbitol dehydrogenase and acid phosphatase decreased while activities of lactate dehydrogenase, beta-glucuronidase, glucose-6-phosphate dehydrogenase, and gamma-glutamyl transpeptidase significantly increased only in animals exposed to styrene at a dose of 200 mg/kg body weight. The results suggest that exposure to high dose of styrene during developmental period alters the activities of enzymes associated with specific cell type of testis.     

Age-related and seasonal variation in the Sertoli cell population, daily sperm production and serum concentrations of follicle-stimulating hormone, luteinizing hormone and testosterone in stallions

Testes and blood samples were obtained from 201 stallions aged 6 months to 20 years in either December-January (nonbreeding season) or June-July (breeding season) to study the effect of age and season on reproductive parameters. Seasonal differences in the Sertoli cell population of adult (4-20 years old) horses were characterized by a 36% larger number of Sertoli cells in the breeding season than in the nonbreeding season. Seasonal elevation in the Sertoli cell population was associated with an increase in testicular weight and daily sperm production per testis (DSP/testis). Concentrations of luteinizing hormone (LH) and testosterone in serum varied with season. Although follicle-stimulating hormone (FSH) concentrations also tended to be higher in the breeding season, this trend was not statistically significant (P less than 0.08). Sertoli cell numbers averaged over both seasons, like testicular weights, increased with age until 4-5 years of age, but were stabilized thereafter. This age-related difference was also associated with increased concentrations of FSH, LH and testosterone, and with increased DSP/testis. The Sertoli cell population was capable of increasing in the adult horse by fluctuating its size with season. The number of elongated spermatids per Sertoli cell over both seasons increased with age up to 4-5 years of age and was stabilized thereafter. Thus, seasonal and/or age-related differences in DSP/testis were associated with significant elevations in serum concentrations of FSH, LH and testosterone, testicular weights, numbers of elongated spermatids per Sertoli cell and elevation of the Sertoli cell population.     

Effect of gossypol on pituitary reproductive axis: ultrastructural and biochemical studies

Oral administration of gossypol induced sterility in male rats by 10 weeks, at a dose of 15 mg/kg body weight/day. The pituitary FSH gonadotroph cells showed dilated endoplasmic reticulum and accumulation of secretory granules in the cytoplasm. LH cells were degranulated. The Leydig cells showed enhanced synthetic activity. There was no change in testis weight and testicular RNA, lipids and cholesterol in the treated group while significant increase was observed in DNA content. Testicular sialic acid content decreased significantly over controls. The Sertoli cells, spermatogonia, spermatocytes and early spermatids were not affected after the treatment. The weights of prostate, seminal vesicle were recorded normal and there were no ultrastructural variations. The levels of acid and alkaline phosphatase and RNA in prostatic tissue were insignificant as compared with controls. However, DNA content of prostate gland showed a significant increase. Sialic acid of seminal vesicle + coagulating gland were within the control range. A marked reduction in fructose values from the same organ was noted.     

Application of morphometric techniques to postnatal rat testes in organ culture: insights into testis growth

The extent of Sertoli cell proliferation during fetal and neonatal development determines the final adult testis size and potential for sperm output. To gain further knowledge of the factors that regulate Sertoli cell proliferation, the present study used a new approach to analyse changes in morphology and proliferation in the postnatal testis by combining organ culture with morphometric analysis. Fragments of rat testes from days 0 to 10 postpartum were cultured in contact with DMEM for 6 h or 72 h and fixed. The effects of ovine follicle-stimulating hormone (FSH) and activin were studied in an additional 72-h organ culture experiment using day 9 testes. Bromodeoxyuridine (BrdU) was added for the last 6 h of culture to mark proliferating cells. Two-microm sections of the fragments were analysed for morphological changes of the seminiferous cords, and the proportion of BrdU-labelled Sertoli and germ cells was determined. Assessment of 6-h samples revealed growth characteristics consistent with those observed in vivo during days 1-10 of postnatal development. From day 2 onwards, the volume fraction of seminiferous cords began to increase, while significant growth in cross-sectional area of the cords occurred only after day 6. In these culture conditions, germ cell proliferation and testicular architecture was consistent with that expected for the age of the tissue at time of explant. The proportion of dividing Sertoli cells declined from 15-20% at days 0-4 postpartum to below % at day 10 postpartum in the 6-h culture, and it was low or abolished in the 3-day culture at all time points. Activin and FSH together, but not singly, stimulated Sertoli cell proliferation in the 72-h culture. This paper presents a new approach to analysis of in vitro testis development. The combination of fragment culture and stereological analysis permits rigorous and detailed assessment of developmental changes in the postnatal testis.     

Male reproductive toxic effects of carbendazim: hitherto unreported targets in testis

Carbendazim (MBC), a widely used fungicide, is toxic to male reproductive mechanisms. Various cellular targets in the testis for MBC action are being understood only recently and still more targets have been conceived. The present study was aimed at finding such newer targets. Male rats were administered through oral route a single dose of carbendazim (400 mg/kg) and the testis was studied adopting routine histological technique. It has been observed that pachytene spermatocytes could also be targets for MBC action in the testis. The study also reports selective loss of step 14 spermatids, asynchrony of the stages in the spermatogenic cycle and development of Sertoli cell fibrosis of the seminiferous tubules of carbendazim-treated rats. From the different kinds of responses seen in the seminiferous tubules in the same testis to MBC, particularly in the Sertoli cell, MBC action in the testis appears dependent on the stage in the spermatogenic cycle at first exposure.     

Effects of various doses of testosterone propionate on intratesticular and plasma testosterone levels and maintenance of spermatogenesis in adult hypophysectomized rats.

Adult hypophysectomized rats were maintained on different regimens of testosterone propionate (TP) treatment for 27 days (0.2, 0.4, 0.6 and 1 mg/day) and autopsied 16 hours after the last injection. Blood samples were taken, sex organs were weighed and one testis from each animal was fixed in Bouins fluid for histologic analysis. The other testis and blood were used for testosterone (T) determinations. Both testicular and plasma T were below detectable levels in hypophysectomized control rats. The plasma T level showed a dose response relationship with increasing dose of TP but such was not the case for intratesticular T concentrations. Qualitative and quantitative evaluation of testis sections showed that spermatogenesis was incomplete in rats receiving 0.2 mg TP/day characterized by the absence of step 15 to 19 spermatids, degeneration of some pachytene spermatocytes and a significantly lower yield of B type spermatogonia. Analysis of testis sections from animals treated with 0.4 to 1 mg TP/day showed complete maintenance and maturation of pachytene spermatocytes, meiosis and spermiogenesis. However, even with the highest dose of TP (1 mg/day) the total yield of B type spermatogonia was only about 58% of the intact controls. It is concluded that at least 0.4 mg/day of exogenous TP is essential for qualitative maintenance of spermatogenesis in hypophysectomized rats with an intratesticular T concentration of 17 to 18 ng/gm testis.     

Immunofluorescence localization of vinculin in ectoplasmic ("junctional") specializations of rat Sertoli cells

We have investigated, using indirect immunofluorescence techniques, the possibility that vinculin is a component of Sertoli cell ectoplasmic specializations. Affinity-purified polyclonal antibodies produced against human platelet vinculin were used to probe fixed frozen sections of rat testis. Specific fluorescence occurs in Sertoli cell regions adjacent to spermatids and to basally situated junctional complexes, sites at which ectoplasmic specializations are known to occur. Staining also occurs in Sertoli cell regions associated with tubulobulbar complexes. The antibody also labels focal contacts in cultured human dermal fibroblasts, apical junctional sites of rat epididymal epithelium, and dense plaques of smooth muscle. Our results are consistent with the prediction that vinculin is likely a component of ectoplasmic specializations and are also consistent with the hypothesis that these structures are a form of actin-associated adhesion complex.     

Effect of cyproterone acetate on testis of albino rats: ultrastructural and biochemical approach

The animals were treated with cyproterone acetate (1 mg/100 g body weight) for 60 days after which the testicular tissue was studied at the ultrastructural level to determine the stage at which the effect took place and to study the magnitude of the effect. The testis showed changes in Sertoli cells, Leydig cells, germ cells and in the spermatids. To correlate these changes, important marker enzymes and other biochemical parameters essential for spermatogenesis were studied after 30 days and 60 days of the treatment. They registered a decrease in their levels as compared with the controls.     

Ethyl methanesulphonate induced changes in the differentiation, structure and functions of spermatozoa of the house rat,Rattus rattus L.

Intraperitoneal administration of 500 mg/kg and 625 mg/kg doses of the germ cell mutagen, ethyl methanesulphonate (EMS) in 5 consecutive days to the house rat,Rattus rattus caused a dose-dependent reduction in its body weight, cauda epididymides weight, concentration, motility and percentage of live spermatozoa with simultaneous increase in the percentage of their abnormal forms. Compared to 0·65% spermatozoa with abnormal heads in the cauda epididymidis of untreated control rats, 24·86% and 65·72% such spermatozoa were observed in rats on day 14 post treatment with 500 mg/kg and 625 mg/kg doses of EMS respectively. On day 28 post treatment corresponding values for abnormal spermatozoa were 16·21% and 14·32%. Similarly, spermatozoa with abnormal flagella increased from 0.78% in control rats to 9·25% and 5·75% on day 14 post treatment of 500 and 625 mg/kg doses of EMS respectively and declined to 2·91% and 2·40% on day 28 post treatment. Abnormality in the sperm head was mainly due to acrosomelessness and in the flagellum due to bending at proximal region. However, the main effect of EMS was the development of spermatozoa without or deformed acrosomes which may impair the fertility of rats. Analysis of various stages of differentiation of spermatozoa inthe testis revealed that population of preleptotene and pachytene spermatocytes and of round spermatids showed a gradual decline which became significantly less than controls on day 28 of EMS treatment. Occurrence of abnormal heads of testicular spermatids indicated that the sperm head abnormalities originated in the testis during late spermiogenesis.     

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.     

Effect of an acute exposure of rat testes to gamma rays on germ cells and on Sertoli and Leydig cell functions.

Germ cells and Sertoli and Leydig cell functions were studied from 7 to 180 days after an acute exposure of 2-month-old rat testes to 9 Gy of gamma rays. Body weight, testis and epididymal weights were recorded. Sertoli cell parameters (androgen-binding protein, ABP, in caput epididymis and plasma follicle stimulating hormone, FSH) and Leydig cell parameters (plasma luteinizing hormone, LH, testosterone and prostate and seminal vesicle weights) were determined together with the number of germ cells and Sertoli cells. Irradiation did not affect body weight but significantly reduced testicular and epididymal weights from day 7 and day 15 post-irradiation respectively. The cells killed by irradiation were mainly spermatogonia and preleptotene spermatocytes engaged in replicating their DNA at the time of exposure, but all spermatocytes seemed damaged as they gave abnormal descendent cells. By day 34, only elongated spermatids remained in a few tubules and thereafter very little regeneration of the seminiferous epithelium occurred, except for one rat which showed a better regeneration. Levels of ABP decreased by day 15 when the germ cell depletion had reached the pachytene spermatocytes, whereas FSH and LH levels rose when the number of elongated spermatids decreased. Levels of testosterone and the weight of the seminal vesicles did not change; occasionally, the prostate weight was slightly reduced. These results support our hypothesis that pachytene spermatocytes and elongated spermatids are involved in influencing some aspects of Sertoli cell function in the adult rat.     

Dehydrodolichyl diphosphate synthase in Sertoli and spermatogenic cells of prepuberal rats

The specific activity of 2,3-dehydrodolichyl diphosphate synthase in homogenates of protease-treated seminiferous tubules, enriched spermatogenic cells, and Sertoli cells changed as a function of the age of prepuberal rats. The highest enzymatic activity occurred in each case in 23-day-old rats. Homogenates of pachytene spermatocytes, spermatids, or Sertoli cells had higher synthase activity than a whole testicular homogenate prepared by protease treatment of tubules. Enzymatic activity in pachytene spermatocytes expressed per mg of protein was about 1.7-fold higher than in spermatids, 5.3-fold higher than in spermatogonia, and about 8.3-fold higher than in spermatozoa. Therefore, the increase in spermatogenic cell synthase before day 23 can be accounted for by the appearance of the pachytene spermatocytes. Enzymatic activity decreased remarkably after the differentiation of spermatids into spermatozoa. Synthase activity in enriched Sertoli cell preparations was 1.5-2.3-fold higher than in spermatogenic cell preparations between days 15 and 30. Therefore, both spermatogenic cells and Sertoli cells contribute to changes in the enzymatic activity in seminiferous tubules during development. These changes may be important in regulating the availability of dolichyl phosphate for glycoprotein synthesis during early stages of differentiation.     

Localization of transferrin and transferrin receptors in rat testes

One of the major proteins secreted by rat Sertoli cells in culture is a transferrin-like protein (Skinner and Griswold, 1980). The purpose of this study was to quantitate the amount of testicular transferrin in fluids isolated from the testis by the use of a radioimmunoassay and to determine the location of transferrin and transferrin receptors in the testis by indirect immunofluorescence. Seminiferous tubule fluid, rete testis fluid, and testicular lymph were collected from rat testes and were found to contain 141 micrograms, 47 micrograms and 3.7 mg transferrin per ml of fluid, respectively. Serum was found to contain 3.7 mg/ml transferrin. Paraffin sections of rat testis were incubated with rabbit anti-rat transferrin, biotinylated goat anti-rabbit and fluorescein-conjugated avidin. Immunoreactive transferrin was thus localized on the proacrosome and nuclear cap of developing spermatids. Late spermatids showed transferrin over the entire region of the head but mature testicular spermatozoa exhibited little fluorescence. The interstitial tissue between seminiferous tubules fluoresced brightly, indicating a large amount of transferrin in this area. By pretreating sections with rat transferrin, the receptor for the protein was localized on and in spermatocytes and early round spermatids. Dividing germ cells were brightly fluorescent.