Proteomic changes in rat spermatogenesis in response to in vivo androgen manipulation; impact on meiotic cells

The production of mature sperm is reliant on androgen action within the testis, and it is well established that androgens act on receptors within the somatic Sertoli cells to stimulate male germ cell development. Mice lacking Sertoli cell androgen receptors (AR) show late meiotic germ cell arrest, suggesting Sertoli cells transduce the androgenic stimulus co-ordinating this essential step in spermatogenesis. This study aimed to identify germ cell proteins responsive to changes in testicular androgen levels and thereby elucidate mechanisms by which androgens regulate meiosis. Testicular androgen levels were suppressed for 9 weeks using testosterone and estradiol-filled silastic implants, followed by a short period of either further androgen suppression (via an AR antagonist) or the restoration of intratesticular testosterone levels. Comparative proteomics were performed on protein extracts from enriched meiotic cell preparations from adult rats undergoing androgen deprivation and replacement in vivo. Loss of androgenic stimulus caused changes in proteins with known roles in meiosis (including Nasp and Hsp70-2), apoptosis (including Diablo), cell signalling (including 14-3-3 isoforms), oxidative stress, DNA repair, and RNA processing. Immunostaining for oxidised DNA adducts confirmed spermatocytes undergo oxidative stress-induced DNA damage during androgen suppression. An increase in PCNA and an associated ubiquitin-conjugating enzyme (Ubc13) suggested a role for PCNA-mediated regulation of DNA repair pathways in spermatocytes. Changes in cytoplasmic SUMO1 localisation in spermatocytes were paralleled by changes in the levels of free SUMO1 and of a subunit of its activating complex, suggesting sumoylation in spermatocytes is modified by androgen action on Sertoli cells. We conclude that Sertoli cells, in response to androgens, modulate protein translation and post-translational events in spermatocytes that impact on their metabolism, survival, and completion of meiosis.     

Development of Fetal Testicular Cells in Androgen Receptor Deficient Mice

During mammalian development, androgens produced by the fetal testis are the most important hormones controlling the masculinization of the reproductive tract and the genitalia. New findings show that the male germ line is the most sensitive to anti-androgenic endocrine disruptors during the embryonic period. In a recent study, we reported that endogenous androgens physiologically control germ cell growth in the male mouse fetus during early fetal life. In the present study, we extended this result by showing the presence of a functional androgen receptor in the gonocytes in the latter part of the fetal life. We also studied the effect of androgens on the development of the somatic testicular cells using the Tfm mice which carry a naturally inactivating mutation of the androgen receptor. Fetal Leydig cell are largely independent of endogenous androgens during fetal development whereas fetal Sertoli cell number is decreased following a default of peritubular myoid cells differenciation. They also point to the gonocyte as a special target for androgens during the embryonic period and indicate a novel mechanism of androgen action on gonocytes. Elucidation of this new pathway in the fetal testis will clarify not only fetal testis physiology but also the effects of environmental anti-androgens that act during fetal life and open new perspectives for future investigations into the sensitivity of fetal germ cell to androgens.     

Sertoli cell-specific deletion of the androgen receptor compromises testicular immune privilege in mice

In the mammalian testis, meiotic and postmeiotic germ cell antigens are granted immune privilege. Both local immune suppression and specialized intercellular junctions between somatic Sertoli cells have been proposed to contribute to a highly restricted and effective blood-testis barrier (BTB) that helps maintain tolerance to germ cell antigens. Several studies have suggested that androgens play a role in immune suppression, although direct evidence for this is lacking. We previously reported that Sertoli cell-specific ablation of the androgen receptor (Ar) decreases expression of Cldn3, an androgen-regulated gene and component of Sertoli cell tight junctions, and increases the permeability of the BTB to biotin, a small-molecular-weight tracer. The physiological consequences of Sertoli cell-specific Ar (S-Ar) ablation on immune privilege are unknown. Here we show that in the testes of S-Ar mutant mice, the ultrastructure of Sertoli cell tight junctions is defective and testicular IgG levels are elevated. The interstitium of S-Ar mutant testes becomes populated with macrophages, neutrophils, plasma cells, and eosinophils, and serum samples of mutant mice contain antibodies against germ cell antigens. Together, these results suggest that Sertoli cell-specific deletion of the androgen receptor results in loss of testicular immune privilege. Suppressed levels of androgen signaling may be a contributing factor in idiopathic male infertility.     

Immunolocalization of androgen receptors in testicular cells of prepubertal and pubertal pigs

In the testis, androgen receptors are known to mediate autocrine and paracrine effects of androgens on Leydig cell function and spermatogenesis. The pig presents some unusual features with regard to the synthesis of testosterone and estrogens in the male gonads. In testes from prepubertal males, testosterone level was lower than in testes from adult boars, while estrogen secretion was relatively high and comparable to that of mature porcine gonad. Immunolocalization of androgen receptors and intensity of immunohistochemical staining was age-dependent. In testis sections from adult boars, androgen receptors were found in nuclei of all somatic cells such as Leydig cells, Sertoli cells, and peritubular-myoid cells, whereas in sections from immature pigs only in the Leydig cell cytoplasm showed positive immunoreaction for androgen receptors. In control tissue sections incubated with omission of the primary antibody, no positive staining was observed. Detection of the androgen receptors in testicular cells of the pig is important for understanding of their central role in mediating androgen action.     

Effect of testosterone on testicular steroidogenesis in the hypogonadal (hpg) mouse

Previous studies have shown that androgens have direct inhibitory effects on steroidogenesis in active Leydig cells. It is not clear what effect androgens have on inactive Leydig cell either through direct action on the cell itself or indirectly through stimulation of Sertoli cell activity. The hpg mouse has undetectable levels of circulating gonadotrophins and the gonads fail to develop post-natally. The effect of androgen treatment on testicular steroidogenesis and morphology was examined in these animals. Treatment with testosterone propionate for two weeks significantly increased testicular and seminal vesicle weight. Seminiferous tubules showed marked development in androgen-treated animals, indicating increased Sertoli cell activity, but the abnormal Leydig cell morphology of the hpg testis was unchanged. Androgen production per testis in vitro was low in control hpg animals and remained unaffected by treatment with androgen. Similarly, the pattern of [3H]pregnenolone metabolism was not significantly affected by androgen treatment. The androgen content of the testis was higher in androgen-treated animals but this could be accounted for by uptake of administered steroid from the circulation. It is concluded that androgens have no direct trophic effect on Leydig cells and that stimulation of Sertoli cell activity is not, in itself, sufficient to affect Leydig cell function.     

Testosterone immunoreactivity in the seminiferous epithelium of rat testis: effect of treatment with ethane dimethanesulfonate

Androgens drive spermatogenesis by processes that are largely unknown. Direct effects on germ cells and indirect effects mediated via testicular somatic elements are currently under consideration, and specific localization of androgens in seminiferous tubules may provide information as regards this. Adult male rats were injected with ethane dimethanesulfonate (EDS; 75 mg/kg body weight) or vehicle. Testes were fixed and paraffin-embedded for localization of testosterone immunoreactivity 1 and 2 weeks after treatment, using the unlabeled antibody (PAP) technique. Plasma testosterone dropped from a pre-treatment level of 2.3 ng/ml to below 0.2 ng/ml 3 days after EDS injection and remained at low levels until the end of observation, accompanied by a progressive decrease in testicular weight. In the seminiferous tubules of vehicle-injected males, testosterone immunoreactivity was found in nuclei of spermatocytes and spermatids and in nuclei and the cytoplasm of Sertoli cells, and showed typical variations according to the stage of spermatogenesis. One week after EDS treatment, immunoreactivity had disappeared from the seminiferous epithelium. Two weeks after treatment, staining of germ cells was detected in two out of four males. The disappearance and reappearance of immunoreactivity coincided with the time course of EDS effects on rat Leydig cells, and we conclude that it corresponds to androgen specifically localized in fixed, paraffin-embedded tissue. Because staining of germ cell nuclei varied with the stage of spermatogenesis, the technique may detect a physiologically relevant androgen fraction; its location suggests that androgens may also directly affect certain germ cell stages.     

Hormonal induction of all stages of spermatogenesis in germ-somatic cell coculture from immature Japanese eel testis

In cultivated male eel, spermatogonia are the only germ cells present in testis. Our previous studies using an organ culture system have shown that gonadotropin and 11-ketotestosterone (11-KT, a potent androgen in teleost fishes) can induce all stages of spermatogenesis in vitro. for detailed investigation of the control mechanisms of spermatogenesis, especially of the interaction between germ cells and testicular somatic cells during 11-KT-induced spermatogenesis in vitro, we have established a new culture system in which germ cells and somatic cells are cocultured after they are aggregated into pellets by centrifugation. Germ cells (spermatogonia) and somatic cells (mainly Sertoli cells) were isolated from immature eel testis. Coculture of the isolated germ cells and somatic cells without forming aggregation did not induce spermatogenesis, even in the presence of 11-KT. In contrast, when isolated germ cells and somatic cells were formed into pellets by centrifugation and were then cultured with 11-KT for 30 days, the entire process of spermatogenesis from premitotic spermatogonia to spermatozoa was induced. However, in the absence of 11-KT in the culture medium spermatogenesis was not induced, even when germ cell and somatic cells were aggregated. These results demonstrate that physical contact of germ cells to Sertoli cells is required for inducing spermatogenesis in response to 11-KT.     

Heat-induced testicular apoptosis occurs independently of hormonal depletion

Previous studies have demonstrated that testicular germ cell apoptosis can be induced both by heat stress and by withdrawal of androgens and gonadotrophins. To investigate whether heat-induced germ cell apoptosis occurs independently of the altered levels of hormones that occur with heat exposure, mouse testicular apoptosis was studied using an in vitro system with controlled levels of testosterone, FSH and LH. It was observed that cells underwent apoptosis sooner in the absence of hormones at the same temperature. Apoptosis also occurred earlier at abdominal temperature compared to scrotal temperature with the same hormonal levels. No somatic tissues studied underwent apoptosis at 37°C under the same culture conditions. These results suggest that heat stress may independently activate an apoptotic pathway in the testis, and that hormone deprivation may induce apoptosis via a separate mechanism.     

Testicular development in mice lacking receptors for follicle stimulating hormone and androgen

Post-natal testicular development is dependent on gonadotrophin and androgen stimulation. Follicle stimulating hormone (FSH) acts through receptors (FSHR) on the Sertoli cell to stimulate spermatogenesis while androgens promote testis growth through receptors (AR) on the Sertoli cells, Leydig cells and peritubular myoid cells. In this study we have examined the effects on testis development of ablating FSHRs (FSHRKO mice) and/or ARs ubiquitously (ARKO mice) or specifically on the Sertoli cells (SCARKO mice). Cell numbers were measured using stereological methods. In ARKO mice Sertoli cell numbers were reduced at all ages from birth until adulthood. FSHR ablation also caused small reductions in Sertoli cell numbers up to day 20 with more marked effects seen in the adult. Germ cell numbers were unaffected by FSHR and/or AR ablation at birth. By day 20 ubiquitous AR or FSHR ablation caused a marked reduction in germ cell numbers with a synergistic effect of losing both receptors (germ cell numbers in FSHRKO.ARKO mice were 3% of control). Germ cell numbers in SCARKO mice were less affected. By adulthood, in contrast, clear synergistic control of germ cell numbers had become established between the actions of FSH and androgen through the Sertoli cells. Leydig cell numbers were normal on day 1 and day 5 in all groups. By day 20 and in adult animals total AR or FSHR ablation significantly reduced Leydig cell numbers but Sertoli cell specific AR ablation had no effect. Results show that, prior to puberty, development of most testicular parameters is more dependent on FSH action than androgen action mediated through the Sertoli cells although androgen action through other cells types is crucial. Post-pubertally, germ cell numbers and spermatogenesis are dependent on FSH and androgen action through the Sertoli cells.     

The effect of a sertoli cell-selective knockout of the androgen receptor on testicular gene expression in prepubertal mice

To unravel the molecular mechanisms mediating the effects of androgens on spermatogenesis, testicular gene expression was compared in mice with Sertoli cell-selective androgen receptor knockout (SCARKO) and littermate controls on postnatal d 10. Microarray analysis identified 692 genes with significant differences in expression. Of these, 28 appeared to be down-regulated and 12 up-regulated at least 2-fold in SCARKOs compared with controls. For nine of the more than 2-fold down-regulated genes, androgen regulation was confirmed by treatment of wild-type mice with an antiandrogen (flutamide). Some of them were previously described to be androgen regulated or essential for spermatogenesis. Serine-type protease inhibitors were markedly overrepresented in this down-regulated subgroup. A time study (d 8-20), followed by cluster analysis, allowed identification of distinct expression patterns of differentially expressed genes. Three genes with a pattern closely resembling that of Pem, a prototypical androgen-regulated gene expressed in Sertoli cells, were selected for confirmation by quantitative RT-PCR and additional analysis. The data confirm that the SCARKO model allows identification of novel androgen-regulated genes in the testis. Moreover, they suggest that protease inhibitors and other proteins related to tubular restructuring and cell junction dynamics may be controlled in part by androgens.     

Regulation of gonadal androgen secretion

The various mechanisms regulating testicular and ovarian androgen secretion are reviewed. Testicular androgen secretion is controlled by luteinizing hormone (LH) and follicle stimulating hormone (FSH), which influence the Leydig cell response to the LH. The contribution of prolactin, growth hormone and thyroid hormones to the Leydig cell function is discussed. The ovarian androgen secretion is regulated in a very similar fashion as the Leydig cell of testis. Prolactin, however, has an inhibitory effect on androgen secretion in the ovary. The intratesticular action of androgens is linked to spermatogenesis. Sertoli cells, by producing the androgen-binding protein, contribute to the intratubular androgen concentration. Inhibin production of the Sertoli cell is stimulated by androgens. In the ovary, androgens produced by the theca interna are used as precursors for the aromatization of estradiol, which stimulates together with FSH the mitosis of granulosa cells. The feedback control of androgen secretion is complicated, as the direct feedback mechanisms are joined by indirect feedback regulations like the peptide inhibin, which can be stimulated by androgens. Intragonadal mechanisms regulating androgen production are the cybernins for testicles and ovaries. In the testicle, estrogens from the Sertoli cells regulate the Leydig cell testosterone biosynthesis. In the ovary, nonaromatizable androgens are potent inhibitors of the aromatization activity in the granulosa cell. A peptide with a FSH receptor binding inhibiting activity is found in male and female gonads. Finally, LH-RH-like peptides have been found in the testicle, which are capable of inhibiting steroidogenesis. These gonadocrinins are similarly produced in granulosa cells of the ovary.