Modification of Sertoli cell functions in vitamin A-deficient rats

FSH binding and cAMP responses to FSH in Sertoli cell-enriched testes were not affected by the vitamin A (retinol) status of the animals. These results indicate that changes in Sertoli cell functions during vitamin A deficiency are independent of FSH-Sertoli cell interactions. Concentrations of serum androgen binding protein (ABP) in vitamin A-deficient rats were consistently higher than those of control animals throughout the study period. The accumulation of testicular fluid after efferent duct ligation, an indication of Sertoli cell secretory function, was normal in vitamin A-deficient rats at least until 70 days of age, but declined thereafter. ABP concentrations in seminiferous tubular fluid of vitamin A-deficient rats increased transitorily during the 70-80-day age period but returned to normal by 90 days. The increment of ABP in seminiferous tubular fluid after efferent duct ligation, and ABP concentrations in interstitial fluid were consistently lower in vitamin A-deficient rats. The higher serum ABP in vitamin A-deficient rats therefore cannot be explained by an increase in the permeability of Sertoli-cell tight junctions or basement membrane.     

Intratesticular androgen levels, androgen receptor localization, and androgen receptor expression in adult rat Sertoli cells

In the rat, quantitatively normal spermatogenesis is maintained only when intratesticular testosterone (ITT) levels greatly exceed the peripheral T concentration. When ITT concentrations fall below a threshold, germ cells are lost at specific stages of the seminiferous cycle. Germ cells can be restored by high doses of T that binds to androgen receptors (AR) in Sertoli cells. However, the relationships between germ cell dynamics, AR-mediated molecular events, and ITT concentrations are not established. ITT levels may regulate germ cell life and death through an effect on AR localization and AR mRNA or protein levels within Sertoli cells at specific stages of the cycle. We determined AR localization and mRNA and protein expression in adult rat Sertoli cells in relation to reduced and then restored ITT concentrations in vivo. ITT levels were reduced by implanting rats with T- and estradiol (E)-filled capsules for 7-28 days and subsequently restored with large T-filled capsules. AR is normally localized within Sertoli cell nuclei at stages VII-VIII of the seminiferous epithelium. After T/E treatment, AR immunostaining in Sertoli cell nuclei became nondetectable by 14-28 days but was restored 6 h following T restoration. The loss of Sertoli cell nuclear AR localization correlated with increasing numbers of apoptotic germ cells. AR mRNA levels in isolated Sertoli cells did not change through 14 days of T/E treatment, increased significantly by Day 28, and remained elevated 24 h after T restoration. AR mRNA levels in microdissected tubules at stages II-IV, VI-VIII, and IX-XII did not decrease through 14 days of T/E treatment. In contrast, AR protein levels were reduced in seminiferous tubules by Day 14 and in testes at Day 28 post-T/E treatment but were restored within 24 h by T repletion. Therefore, the reduction of ITT concentration results in a time-dependent redistribution of AR and reduced AR protein but not AR mRNA levels in Sertoli cells. Repletion of T restored AR protein and it relocated to Sertoli cell nuclei. By an unknown mechanism, T regulates AR localization within Sertoli cells to determine germ cell life or death.     

The effect of cryptorchidism and subsequent orchidopexy on testicular function in adult rats

Cryptorchidism for 28 or 10 days resulted in a severe disruption of spermatogenesis (assessed histologically or by fertility tests), Sertoli cell function (assessed by seminiferous tubule fluid production after efferent duct ligation, ABP levels, binding of 125I-labelled FSH to testis homogenates and serum FSH levels) and Leydig cell function (assessed by serum LH and testosterone levels, in-vitro testosterone production, binding of 125I-labelled hCG). Orchidopexy after 28 days of cryptorchidism resulted in a poor recovery of spermatogenesis since the majority of tubules were lined by Sertoli cells and a few spermatogonia. No recovery occurred in the indicators of Sertoli and Leydig cell function. Orchidopexy after 10 days of cryptorchidism also resulted in a poor recovery of spermatogenesis, with a few animals showing partial recovery after 6 months. No recovery occurred in seminiferous tubule fluid production but partial recovery occurred in ABP content and production rate. Serum FSH, LH levels and in-vitro testosterone production by the testis remained elevated and did not change from the values found during cryptorchidism. Fertility testing at 6 months revealed a small number of rats in which fertility was restored although the number of embryos was lower than in controls. In this group of animals there was a significant improvement in a number of indicators of Sertoli cell and Leydig cell function. These data provide further evidence to link the changes in Sertoli cell and Leydig cell function to the germ cell complement present in the testis.     

Nociceptin is upregulated by FSH signaling in Sertoli cells in murine testes

In postnatal testes, follicle-stimulating hormone (FSH) acts on somatic Sertoli cells to activate gene expression directly via an intracellular signaling pathway composed of cAMP, cAMP-dependent protein kinase (PKA), and cAMP-response element-binding protein (CREB), and promotes germ cell development indirectly. Yet, the paracrine factors mediating the FSH effects to germ cells remained elusive. Here we show that nociceptin, known as a neuropeptide, is upregulated by FSH through cAMP/PKA/CREB pathway in Sertoli cells in murine testes. Chromatin immunoprecipitation from Sertoli cells shows that CREB phosphorylated at Ser133 associates with prepronociceptin gene encoding nociceptin. Analyses with Sertoli cells and testes demonstrates that both prepronociceptin mRNA and the nociceptin peptide are induced after FSH signaling is activated. In addition, the nociceptin peptide is induced in testes after 9days post partum following FSH surge. Thus, our findings may identify nociceptin as a novel paracrine mediator of the FSH effects in the regulation of spermatogenesis.     

Structural and functional modifications of sertoli cells in the testis of adult follicle-stimulating hormone receptor knockout mice

Follicle stimulating hormone (FSH) plays important roles during testicular development and in the maintenance of spermatogenesis in the adult. However, the cellular events or pathways that FSH regulates to achieve these effects in Sertoli cells, where the FSH receptors (FSH-R) are located, is still not fully elucidated. The development of FSH-R knockout (FORKO) mice provides a model to examine alterations in testicular structure and function in its absence. To this end, light (LM) and electron microscopic (EM) analyses of perfusion-fixed testes of wild-type and FORKO mice of different ages were performed. Under the LM, a significant reduction was noted in the profile area of seminiferous tubules of FORKO mice compared with their wild-type counterparts at different ages. In addition, FORKO testes revealed large irregularly shaped spaces within the seminiferous epithelium, extending from the base to the lumen. Such spaces were often separated by anastomotic cords of spherical germ cells or completely surrounded elongating spermatids. This phenotype was restricted to half or less of the circumference of only some tubules, but was seen at all stages. EM analyses revealed that the spaces corresponded to an apparent accumulation of fluid in the Sertoli cell cytoplasm, coincident with an absence of the fine flocculent ground substance seen in wild-type mice. However, the Sertoli organelles, while less prominent, appeared intact and to be floating in the enlarged fluid-filled cytoplasm. Functionally, androgen-binding protein (ABP), a major secretory protein of Sertoli cells, was dramatically reduced in FORKO mice. These results suggest that FSH-R signaling normally maintains water balance in Sertoli cells in addition to regulating ABP production.     

Regulation by FSH and dibutyryl cyclic AMP of the formation of androgen-binding protein in Sertoli cell-enriched cultures.

Sertoli cell-enriched preparations from testes of 20-day-old rats were cultured in a defined medium in the presence and absence of FSH or dibutyryl cyclic AMP (dcAMP). Androgen-binding activity was assayed in the culture medium, and related to testicular androgen-binding protein (ABP). The production and secretion of ABP by the Sertoli cell-enriched preparation was increased after FSH or dcAMP treatment of the primary culture. It is concluded that ABP is produced by Sertoli cells. The possibility of involvement of other cell types in the testis in ABP production is discussed.     

Isolation and culture of FSH responsive Sertoli cells.

Methods for the isolation and culture of enriched populations of Sertoli cells from 20-60 day old rats are described. The identity of the Sertoli cells was verified by bright light and electron microscopy. Freshly isolated Sertoli cells specifically bound follicle stimulating hormone (FSH) but not luteinizing hormone (LH) and responded to FSH stimulation with dramatic increase in cyclic AMP level. Isolated Sertoli cells, maintained in culture for 11 days, showed no evidence of proliferation but retained their characteristic ultrastructural features and FSH binding ability. Incubation of cultured cells with FSH resulted in a significant stimulation of cyclic AMP and androgen binding protein (ABP). Since the freshly isolated or cultured cells were predominantly (greater than 80%) Sertoli cells, these results provide direct evidence that the Sertoli cells represent a primary target site for FSH activity in the testes. The culture method also provides a valuable in vitro model for the study of chronic effects of various agents on the Sertoli cell.     

Effects of the induction of experimental cryptorchidism and subsequent orchidopexy on testicular function in immature rats

Cryptorchidism surgically induced in 14-day-old rats, was allowed to persist until 35 days when one group was killed to assess testicular function. In a second group the cryptorchid testis was returned to the scrotum surgically (orchidopexy) and subsequently killed at 130 days. A third group remained persistently cryptorchid to 130 days, while in a fourth group two sham operations were performed at 14 and 35 days. At 35 days, cryptorchidism resulted in a significant decline in testis weight due to suppressed spermatogenesis. Sertoli cell function as measured by seminiferous tubule fluid (TF) production after unilateral efferent duct ligation and androgen-binding protein (ABP) production was significantly depressed in the cryptorchid group. Serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were significantly elevated with cryptorchidism but serum testosterone levels were unchanged. Although morphometric measurements showed no change in Leydig cells cross-sectioned area, in vitro human chorionic gonadotropin (hCG)-stimulated testosterone production was significantly increased in the cryptorchid group at higher hCG doses. Similar changes were found in cryptorchid testes at 130 days except that Leydig cell cross-sectional area was now significantly increased. Orchidopexy at 35 days restored spermatogenesis and fertility during test mating was not impaired. TF production, ABP accumulation and serum FSH levels returned to normal following orchidopexy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of germ cell and Sertoli cell development by activin, follistatin, and FSH

We have demonstrated a role for activin A, follistatin, and FSH in male germ cell differentiation at the time when spermatogonial stem cells and committed spermatogonia first appear in the developing testis. Testis fragments from 3-day-old rats were cultured for 1 or 3 days with various combinations of these factors, incubated with bromodeoxyuridine (BrdU) to label proliferating cells, and then processed for stereological analysis and detection of BrdU incorporation. Gonocyte numbers were significantly elevated in cultures treated with activin, while the combination of FSH and the activin antagonist, follistatin, increased the proportion of spermatogonia in the germ cell population after 3 days. All fragment groups treated with FSH contained a significantly higher proportion of proliferating Sertoli cells, while activin and follistatin each reduced Sertoli cell division. In situ hybridization and immunohistochemistry on normal rat testes demonstrated that gonocytes, but not spermatogonia, contain the activin beta(A) subunit mRNA and protein. In contrast, gonocytes first expressed follistatin mRNA and protein at 3 days after birth, concordant with the transition of gonocytes to spermatogonia. Collectively, these data demonstrate that germ cells have the potential to regulate their own maturation through production of endogenous activin A and follistatin. Sertoli cells were observed to produce the activin/inhibin beta(A) subunit, the inhibin alpha subunit, and follistatin, demonstrating that these cells have the potential to regulate germ cell maturation as well as their own development. These findings indicate that local regulation of activin bioactivity may underpin the coordinated development of germ cells and somatic cells at the onset of spermatogenesis.     

Characterization of rat transferrin receptor cDNA: the regulation of transferrin receptor mRNA in testes and in Sertoli cells in culture

A 3.4 kilobase cDNA complementary to rat transferrin receptor mRNA has been isolated from an adult rat testis cDNA library. The rat transferrin receptor nucleotide sequence was shown to be 82% similar to the human transferrin receptor sequence over the amino acid coding region and over 90% similar in the sequences known to be responsible for iron regulation in the human mRNA. The mRNA was shown by Northern blot analysis to be regulated by iron levels in Sertoli cells in culture. Iron depletion resulted in at least a 5-fold increase in receptor message in Sertoli cells, as well as in an actively growing testicular cell line (S10-7). The level of transferrin receptor mRNA in cultured Sertoli cells was not influenced by hormones; however, chronic administration of testosterone or FSH to hypophysectomized rats resulted in increased transferrin receptor mRNA levels in the testis. Northern blot analysis of mRNAs from testes of rats synchronized at various stages of the cycle of the seminiferous epithelium showed that transferrin receptor mRNA was differentially regulated throughout the cycle. Northern blots of mRNA from germinal cell populations derived from synchronized tests showed that the message was regulated in the nongerminal cell components of the tubule, most likely the Sertoli cell. The comparison of transferrin receptor mRNA levels in normal testes and testes from hypophysectomized rats, as well as in isolated germinal cells and cultured Sertoli cells, suggested that transferrin receptor mRNA levels were considerably higher in Sertoli cells than in other cell types of the seminiferous tubules.     

Regulation of testicular and Sertoli cell gamma-glutamyl transpeptidase by follicle-stimulating hormone

Hormonal deprivation achieved by hypophysectomy or gonadotropin-releasing hormone (GnRH)-antagonist treatment of immature rats resulted in markedly lower testicular gamma-glutamyl transpeptidase (GGT) activity than in the testes of age-matched controls. When begun 15 days after hypophysectomy, follicle-stimulating hormone (FSH) treatment significantly increased testicular GGT above that in testes from hypophysectomized controls in a time- and dose-dependent manner. In contrast, testosterone propionate had only a small effect. Testicular GGT was higher in adult hypophysectomized rats treated with FSH from the time of surgery than in untreated hypophysectomized rats; testosterone propionate treatment had no effect. GGT activity in Sertoli cells isolated from GnRH antagonist-treated or hypophysectomized immature rats was also lower than in cells from control rats. FSH treatment from the day of hypophysectomy resulted in Sertoli cell GGT values equivalent to those from intact controls. These data indicate that FSH regulates GGT activity in rat testis and Sertoli cells.     

Hormone interactions in the Sertoli cells.

The effects of follicle-stimulating hormone (FSH) and testosterone in rat Sertoli cells were investigated in vitro by means of isolated cell populations. The Sertoli cells selectively bind FSH, and respond to FSH stimulation with increased accumulation of endogenous cyclic AMP and secretion of androgen-binding protein (ABP). FSH binding and cyclic AMP response in the Sertoli cells change dramatically during sexual maturation. Cyclic AMP response decreases despite an increase in FSH-binding receptors per cell. Evidence has been provided for the existence of cytoplasmic and nuclear androgen receptors and chromatin acceptor-sites that specifically bind the androgen-receptor complex in the Sertoli cells. A model has been proposed for the hormonal interactions in the seminiferous tubule and the possible role of Sertoli cells in mediating the hormonal effects on spermatogenesis.     

Disruption of spermatogenesis and Sertoli cell structure and function by the indenopyridine CDB-4022 in rats.

The present studies were undertaken to determine the testicular cell type(s) affected by the antispermatogenic indenopyridine CDB-4022. At the oral threshold dose (2.5 mg/kg), CDB-4022 induced infertility in all males. CDB-4022 did not alter (P > 0.05) Leydig cell function as assessed by circulating testosterone, seminal vesicle, and ventral prostate weights or body weight gain compared to controls. Conversely, CDB-4022 reduced (P < 0.05) testicular weight, spermatid head counts, and percentage of seminiferous tubules undergoing spermatogenesis. In a second study, adult male rats received a maximally effective oral dose of CDB-4022 (12.5 mg/kg), dipentylphthalate (DPP; 2200 mg/kg; a Sertoli cell toxicant), or vehicle and were necropsied 3, 6, or 12 h after dosing to determine acute effects. Serum inhibin B levels were suppressed (P < 0.05) by 6 h after CDB-4022 or DPP treatment, but epididymal androgen-binding protein (ABP) levels were not altered (P > 0.05), compared to controls. CDB-4022 and DPP increased (P < 0.05) the percentage of tubules with apoptotic germ cells, particularly differentiating spermatogonia and spermatocytes, by 12 h after dosing. Microscopic examination of the testis indicated a greater degree of vacuolation in Sertoli cells and initial signs of apical germ cell sloughing/shedding by 3 or 12 h after CDB-4022 or DPP treatment, respectively. In a third study, prepubertal male rats were treated with vehicle, 12.5 mg/kg of CDB-4022, or 2200 mg/kg of DPP, and the efferent ducts of the right testis were ligated 23 h before necropsy. Seminiferous tubule fluid secretion (difference in weight of testes), serum inhibin B levels, and ABP levels in the unligated epididymis were reduced (P < 0.05) at 24 and 48 h after dosing in CDB-4022- and DPP-treated rats compared to controls. Collectively, these data suggest that CDB-4022 disrupts spermatogenesis by inducing apoptosis in early stage germ cells via a direct action on the Sertoli cell.     

Expression of the enkephalin precursor gene in rat Sertoli cells. Regulation by follicle-stimulating hormone

Searching for somatic cells expressing the preproenkephalin (A) gene in the testis, we have isolated Sertoli cells from the testes of 20-day-old rats. Cultured Sertoli cells contained a single species (about 1.5 kb) of preproenkephalin mRNA, and follicle-stimulating hormone (FSH) transiently increased the mRNA abundance to a maximum (about 30 molecules per cell) at 12 h. Various compounds that activate the cyclic AMP system in Sertoli cells similarly increased the abundance of preproenkephalin mRNA. Moreover, FSH increased intracellular Met-enkephalin immunoreactive peptides in Sertoli cells. Thus, the preproenkephalin gene expression in Sertoli cells is positively regulated by FSH through the cyclic AMP system.