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Spermatogenesis is absolutely dependent on FSH and androgens; suppression of these hormones inhibits germ cell development and thus sperm production. The final release of spermatids by the Sertoli cell, a process known as spermiation, is particularly sensitive to hormone suppression. To define the molecular mechanisms that mediate FSH and androgen effects in the Sertoli cell, we investigated the expression and regulation of micro-RNAs (miRNAs), small noncoding RNAs that regulate protein translation and modify cellular responses. By array analysis, we identified 23 miRNAs up-regulated more than 2-fold after hormone suppression in vivo and in vitro in primary Sertoli cell cultures. The regulation of four of these miRNAs (miR-23b, -30c, -30d, and -690) was confirmed by quantitative RT-PCR. Bioinformatic analysis of potential targets of hormonally regulated miRNAs identified genes important for focal adhesion and regulation of the actin cytoskeleton, processes known to be intimately associated with adhesion of spermatids to Sertoli cells. Two of the identified genes, Pten, an intracellular phosphatase, and Eps15, a mediator of endocytosis, were down-regulated by the withdrawal of hormones in vivo and possess miR-23b target sites in their 3'-untranslated regions. Overexpression of miR-23b in vitro resulted in decreased translation of PTEN and EPS15 protein as assessed by Western blot and luciferase analysis. We conclude that FSH and androgens act on Sertoli cells in stage VIII to control the expression of miRNAs that operate in a coordinated manner to regulate cell adhesion pathways and male fertility and that miRNA transcription is a new paradigm in the hormone dependence of spermiation.

Spermatogenesis occurs within the highly complex seminiferous epithelium. This cyclic process is accompanied by dynamic stage-specific transcriptional changes and is driven by androgens and FSH by mechanisms that are unclear. Here we report the impact of acute androgen and FSH suppression on the transcriptional dynamics of the seminiferous epithelium. We used transcriptional profiling to compare the most hormone-sensitive seminiferous epithelial stages (VII and VIII) from control and hormone-suppressed adult rats, together with publicly available datasets to delineate stage- and cell-specific transcriptional changes. The analyses reveal that, in these stages, there was a hormone-responsive down-regulation of spermatogonial and Sertoli cell transcripts maximally expressed in the earlier spermatogenic stages (I–VI). Transcripts expressed in Sertoli cells from stage VII and beyond were both up- and down-regulated by hormone suppression, with lysosome function, immune system-related genes, and lipid metabolism predicted to be hormone responsive. Hormone-responsive genes with putative roles in integrin-mediated cell adhesion were also identified. In pachytene spermatocytes, there was an initiation of transcription likely important for the completion of meiosis. A transcriptional switch in round spermatids was observed, from a hormone-responsive down-regulation of transcripts expressed in steps 1–7 spermatids to a hormone-independent up-regulation of transcripts expressed in steps 8–11 and likely involved in spermatid differentiation and DNA compaction. This study points to the existence of hormone-responsive global transcriptional repressors in Sertoli cells, spermatogonia, and spermatids and reveals novel and diverse cell-specific responses of the seminiferous epithelium to hormone suppression.

Spermiation is the process by which mature sperm are released from the Sertoli cell into the lumen of the seminiferous tubule. Previous studies have shown that FSH and LH/testosterone suppression causes a significant increase in the degeneration of mature elongated spermatids. The purpose of this study was to investigate the extent to which spermiation failure contributes to the overall failure of spermatogenesis during hormone suppression. We used in vivo models to selectively suppress either FSH, by passive immunization, and or testosterone, by administration of SILASTIC brand (Dow Corning) testosterone and estradiol implants to suppress LH and testicular testosterone production. Stereological quantitation of the number of step 17-18 spermatids before spermiation and the number of step 19 spermatids retained within the epithelium after spermiation showed that 2% of spermatids failed to spermiate in control animals, and 11% and 14% of spermatids failed to spermiate after 1 week of FSH inhibition or testosterone suppression, respectively. After 1 week of combined FSH and testosterone withdrawal, 50% of the spermatids in the testis failed to be released. A time course of testosterone suppression showed that after 4-5 weeks over 90% of spermatids failed to spermiate. We conclude that spermiation is highly sensitive to hormone suppression, with T and FSH acting synergistically to support spermiation, and that spermiation inhibition is a potential target for contraception.

Testosterone (T) is required for spermatogenesis, particularly in the conversion of round spermatids between stages VII and VIII of spermatogenesis. T is generally thought to be the major androgen involved in adult spermatogenesis due to the high local concentration within the testis, whereas its more potent 5alpha-reduced metabolite dihydrotestosterone (DHT) is thought to be the active androgen in peripheral tissues. The current study investigated whether 5alpha-reduction of T to DHT is involved in the restoration of spermiogenesis in vivo in a setting in which testicular T levels were markedly reduced. Adult male rats were given 3-cm T plus 0.4-cm estradiol implants for 9 weeks to suppress serum LH and testicular T levels and thereby inhibit spermatogenesis. Increasing doses of T (3-, 6-, 10-, and 24-cm implants) were then given for 4 days to restore spermatogenesis in the presence or absence of a 5alpha-reductase inhibitor (L685,273). The hourly production rates of round spermatids in stages I-III, IV-VI, VII, and VIII were assessed using stereological techniques, and the conversion of round spermatids between stages VII and VIII was then assessed as an index of androgen action on spermiogenesis. Testicular androgen levels were measured by HPLC and RIA. The 5alpha-reductase inhibitor significantly suppressed (P < 0.05) the hourly production rate of round spermatids at the 3- and 6-cm T doses, but not at the 10- and 24-cm doses. The conversion of round spermatids between stages VII and VIII was suppressed (P < 0.05) by the inhibitor only at the 3- and 6-cm doses. The 5alpha-reductase inhibitor had no effect on testicular T levels, but suppressed (P < 0.05) DHT levels at the 6-, 10-, and 24-cm doses. We conclude that the 5alpha-reduction of T is involved in the restoration of spermiogenesis at the lower administered doses of T and that these data are the first description of a role for 5alpha-reduced androgens in adult spermatogenesis.

The procession of round spermatids through stages VII and VIII of the rat spermatogenic cycle is critically dependent on testosterone (T). When intratesticular T levels are reduced, round spermatids appear to slough from the seminiferous epithelium, resulting in the disappearance of elongated spermatids. We hypothesized that T-dependent cell adhesion molecules are involved in Sertoli cell-round spermatid interactions. This study examined the hormonal regulation of one candidate cell adhesion molecule, N-cadherin, in vitro and its participation in Sertoli cell-round spermatid adhesion in coculture. Sertoli cells were isolated from 20-day-old Sprague-Dawley rats; treated with FSH and T, alone or in combination; and incubated for 48 h before determination of N-cadherin concentrations in Sertoli cell extracts by RIA. Both FSH and T significantly increased the cellular content of N-cadherin (3.7-fold), whereas FSH or T alone had no effect. Round spermatids were isolated from adult rats, and their adhesion to Sertoli cells was assessed in a 48-h coculture in the presence of FSH, T, or FSH plus T. Adherent round spermatids were quantitated by histological evaluation after staining with the periodic acid-Schiff reaction. A dose-dependent increase in round spermatid density (number of round spermatids bound per 10,000-microns2 Sertoli cell culture surface area) was observed with increasing T doses (7-28 ng/ml) in the presence of FSH (1 microgram/ml), whereas FSH and T alone at these doses produced no effect. T also increased the N-cadherin content of the cocultures in a dose-dependent manner in the presence of FSH. Addition of an N-cadherin antiserum to the Sertoli cell-round spermatid coculture in the presence of FSH and T significantly (P0.0001) reduced round spermatid density by 65%. It is concluded that both the production of N-cadherin by Sertoli cells and the binding of round spermatids to Sertoli cells are stimulated in a synergistic manner by T and FSH. Furthermore, the immunoneutralization data suggest the active involvement of N-cadherin in round spermatid-Sertoli cell adhesion in vitro. N-Cadherin may be one of the factors that subserve the androgen-dependent process of round to elongated spermatid maturation.

The role of FSH in spermatogenesis is unclear as testosterone alone has been reported to be sufficient in the gonadotropin-deficient rat. This study examined the effects of recombinant FSH on the restoration of spermatogenesis after gonadotropin withdrawal by GnRH immunization. Adult Sprague-Dawley rats received GnRH immunogen (100 micrograms, sc, every 4 weeks) to induce gonadotropin deficiency, with severe spermatogenic regression occurring by 12 weeks. Recombinant human FSH was then given (10 or 50 IU/kg, sc, daily) for 7, 14, and 21 days, with data from both dosages combined in the analyses. Testes were perfusion fixed, and germ cell numbers were quantified by the optical disector technique. After 7 days of FSH, testis weight significantly increased by 43% (P0.01), with no further increases at 14 and 21 days. GnRH immunization severely reduced germ cell numbers, which were then significantly (P0.05) restored in all cell types, except elongated spermatids, by 7 days of FSH; type A spermatogonia (45%--61% of control), type B spermatogonia/preleptotene spermatocytes (46%--65%), leptotene/zygotene spermatocytes (39%--55%), pachytene spermatocytes in stages I-VIII (11%--30% control) and IX-XIV (4.3%--22% control), and round spermatids in stages I-VIII (1.4%--4.4% control). Prolonged FSH treatment did not further increase type A spermatogonial or pachytene spermatocyte number, whereas round spermatids increased to a peak of 12.8% of the control value. At no stage did FSH increase elongated spermatid numbers above 1% of the control level. The incorporation of bromode-oxyuridine into spermatogonial and early spermatocyte nuclei did not change after GnRH immunization or FSH treatment. Sertoli cell number was not altered by any treatment; however, Sertoli cell nuclear volume was significantly decreased from the control value by GnRH immunization (142 +/- 9 vs. 455 +/- 22 microns 3; P0.01) and increased after 7 and 14 days of FSH treatment to 212 +/- 10 and 259 +/- 24 microns 3, respectively. FSH treatment restored serum inhibin levels to normal, but did not increase serum or testicular androgen levels. We conclude that recombinant FSH partially restores spermatogenesis in the gonadotropin-deficient rat by increasing the number of spermatogonia and promoting subsequent maturational steps up to the round spermatid stage. Spermatid elongation was not restored by FSH, indicating the need for an additional factor(s), most likely testosterone.

Several spermatogenic cell types have been reported to be responsive to testosterone (T) in vivo. We have proposed that the principal action of T is to facilitate the maturation of round to elongated spermatids during spermiogenesis. To identify T-dependent cell types during spermiogenesis, round spermatid populations were counted using stereological techniques in adult rats after T withdrawal and replacement. The number of round spermatids per testis in stages I-III, IV-VI, VII, and VIII of the spermatogenic cycle were determined and, based on the known duration of each stage, the hourly production rates of round spermatid populations calculated. Sprague-Dawley rats received 3 cm T and 0.4 cm estradiol implants (TE treatment) for 11 weeks to suppress LH, testicular T levels, and spermatogenesis. To restore sperm production, high-dose T (24 cm) implants were then given, and animals were perfused 0, 2, 4, and 7 days later. Total testicular T levels were suppressed to 2.9% of control levels by TE treatment and significantly increased (P < 0.05) after 2 days of high-dose T, to remain between 7-12% of control. The hourly production rates of round spermatids between stages I and VII were suppressed to 29-35% of control by TE treatment and remained unchanged by high-dose T administration. Stage VIII round spermatid hourly production rates however, were markedly reduced to 5% of control by TE treatment and increased significantly (P < 0.05) to 27% of control after 4 days of high-dose T. The efficiency of conversion of spermatids through spermiogenesis was estimated from the ratios of the hourly production rates of successive spermatid groups. The conversion of spermatids between stages I-VII of the cycle did not differ from control regardless of the treatment. However, the conversion of round spermatids between stages VII and VIII was markedly suppressed to 16% of control by TE treatment and was then normalized after 4 days of high-dose T administration. We conclude that the conversion of round spermatids between stages VII and VIII is a highly T-dependent step during spermiogenesis.

Activin-A is a homodimer of the beta A inhibin subunit that stimulates FSH secretion by pituitary cells in vitro; however, the physiological relevance of this effect is unknown. We have examined whether recombinant human activin-A (activin-A; 80 micrograms/kg/day iv infusion for 50.5 h) has in vivo bioactivity in the adult male macaque (n = 5). Serum FSH and LH bioactivity and serum testosterone (T) levels were measured on 2 control days and after 24 and 48 h of activin-A administration. Basal FSH levels increased significantly (p less than 0.05) by 17% at 24 h and 82% at 48 h during activin-A administration. No changes in basal LH or T levels were seen. The FSH and LH responses to GnRH (5 micrograms/kg, iv bolus) increased significantly (p less than 0.05) by 117% and 55% after 48 h of activin-A, respectively. A small (16%), but statistically significant (p less than 0.05), increase in the T response to the GnRH challenge was also noted. These data are preliminary evidence in support of a physiological role for activin-A in the control of gonadotropin secretion in the male primate.