Androgens control spermatogenesis, but germ cells themselves do not express a functional androgen receptor (AR). Androgen regulation is thought to be mediated by Sertoli and peritubular myoid cells, but their relative roles and the mechanisms involved remain largely unknown. Using Cre͞loxP technology, we have generated mice with a ubiquitous knockout of the AR as well as mice with a selective AR knockout in Sertoli cells (SC) only. Mice with a floxed exon 2 of the AR gene were crossed with mice expressing Cre recombinase ubiquitously or selectively in SC (under control of the anti-Mü llerian hormone gene promoter). AR knockout males displayed a complete androgen insensitivity phenotype. Testes were located abdominally, and germ cell development was severely disrupted. In contrast, SC AR knockout males showed normal testis descent and development of the male urogenital tract. Expression of the homeobox gene Pem, which is androgen-regulated in SC, was severely decreased. Testis weight was reduced to 28% of that in WT littermates. Stereological analysis indicated that the number of SC was unchanged, whereas numbers of spermatocytes, round spermatids, and elongated spermatids were reduced to 64%, 3%, and 0% respectively of WT. These changes were associated with increased germ cell apoptosis and grossly reduced expression of genes specific for late spermatocyte or spermatid development. It is concluded that cell-autonomous action of the AR in SC is an absolute requirement for androgen maintenance of complete spermatogenesis, and that spermatocyte͞spermatid development͞ survival critically depends on androgens.

Androgens are essential for normal spermatogenesis and male fertility, but how androgens exert this effect remains uncertain. Androgen receptors (ARs) are expressed in several testicular cell types, but continuing uncertainty exists over which cell type mediates androgen control of spermatogenesis. Androgen signaling via Sertoli cells (SCs) is essential for complete spermatogenesis, but the role for androgen signaling via peritubular myoid (PTM) cells is contentious. To address this controversy, we generated PTM-specific AR-knockout (PTM-ARKO) mice in which gross reproductive development was normal, but all PTM-ARKO males were azoospermic and infertile. Testis weight was reduced beyond puberty, and in adulthood there was an 86% reduction in germ cells, compared with wild-type littermates. These changes were not explained by any deficits in testosterone, luteinizing hormone, or follicle-stimulating hormone concentrations. SC function was impaired in PTM-ARKO males, indicated by reduced seminiferous tubule fluid production and reduced expression of some androgen-dependent SC genes. Androgen action via PTM cells is therefore essential for normal testis function, spermatogenesis, and fertility in males. This study also provides the first direct evidence for the importance of androgen-driven stromal-epithelial interactions underpinning the regulation of spermatogenesis; PTM-ARKO mice will enable identification of the new molecular pathways involved.—Welsh, M., Saunders, P. T. K., Atanassova, N., Sharpe, R. M., Smith, L. B. Androgen action via testicular peritubular myoid cells is essential for male fertility.

The role of androgens in the proliferation and maturation of Sertoli cells (SC) and the development of their capacity to support spermatogenesis remains poorly understood. We evaluated these functions in complete androgen receptor knockout (ARKO) and SC-selective androgen receptor knockout (SCARKO) mice. Compared with controls, ARKO mice exhibited a progressive reduction in SC number/testis, whereas SCARKOs showed minor changes, suggesting that androgen effects on SC number are not mediated via direct action on SCs. Immunoexpression of anti-Mullerian hormone (AMH), p27(kip1), GATA-1, and sulfated glycoprotein-2, which changes according to SC maturational status, occurred normally in ARKOs and SCARKOs. Functional capacity of SCs to support spermatogonia was similar in SCARKOs and controls, whereas ARKOs showed reduced capacity with age. SC capacity to support total germ cells revealed major deficits in ARKO and SCARKO adults, particularly with respect to postmeiotic germ cells. Using quantitative RT-PCR, the expression of SC markers was compared in d 50 testes. In ARKOs, expression of Pem, fatty acid binding protein, platelet-derived growth factor-A, and transferrin were all significantly reduced, whereas FSH receptor and AMH were increased. In SCARKOs, there were modest reductions in expression of cystatin-related gene highly expressed in testis and epididymis (cystatin-TE) and claudin-11, whereas expression of Pem, fatty acid binding protein, and platelet-derived growth factor-A was markedly reduced, highlighting these as potentially androgen-regulated SC genes that merit further study. In conclusion, androgen action is not required for maturation-dependent changes in immunoexpression of the SC markers AMH, p27(kip1), GATA-1, and sulfated glycoprotein-2 but is essential for expression of other SC genes, the attainment of normal SC number, and the support of meiotic and postmeiotic germ cell development.

This study aimed to identify the mechanism(s) for impairment of spermatogenesis in adulthood in rats treated neonatally with estrogens. Rats were treated (days 2-12) with 10, 1, or 0.1 microg diethylstilbestrol (DES), 10 microg ethinyl estradiol (EE), 10 mg/kg of a GnRH antagonist (GnRHa), or vehicle and killed in adulthood. DES/EE caused dose-dependent reductions in testis weight, total germ cell volume per testis, and Sertoli cell volume per testis. Sertoli cell number at 18 days of age in DES-treated rats was reduced dose dependently. GnRHa treatment caused changes in these parameters similar to those in rats treated with 10 microg DES. Plasma FSH levels were elevated (P < 0.001) to similar levels in all treatment groups regardless of differences in Sertoli cell number and levels of inhibin B; the latter reflected Sertoli cell number, but levels were disproportionately reduced in animals treated with high doses of DES/EE. Neonatal estrogen treatment, but not GnRHa, caused dose-dependent reductions (40-80%) in plasma testosterone levels in adulthood, but did not alter LH levels. Preliminary evidence suggests that the decrease in testosterone levels in estrogen-treated rats is not due to reduced Leydig cell volume per testis. GnRHa-treated rats exhibited a significant increase in germ cell volume per Sertoli cell and a reduction in germ cell apoptosis, probably because of the raised FSH levels. Despite similar raised FSH levels, rats treated with DES (10 or 1 microg) or EE (10 microg) had reduced germ cell volume/Sertoli cell and increased germ cell apoptosis, especially when compared with GnRHa-treated animals. The latter changes were associated with an increase in lumen size per testis, indicative of impaired fluid resorption from the efferent ducts, resulting in fluid accumulation in the testis. Rats treated neonatally with 0.1 microg DES showed reduced germ cell apoptosis comparable to that in GnRHa-treated animals. The changes in apoptotic rate among treatment groups occurred across all stages of the spermatogenic cycle. It is concluded that 1) neonatal estrogen treatment results in dose-dependent alterations in Sertoli cell numbers, germ cell volume, efficiency of spermatogenesis, and germ cell apoptosis in adulthood; 2) the relatively poor spermatogenesis in estrogen-treated animals is most likely due to altered testis fluid dynamics and/or altered Sertoli cell function; 3) as indicated by FSH (LH) and testosterone levels, the hypothalamic-pituitary axis and Leydig cells are probably more sensitive than the Sertoli cells to reprogramming by estrogens neonatally; and 4) elevated FSH levels in adulthood may improve the efficiency of spermatogenesis.

This study investigated whether neonatal exposure of male rats to estrogenic compounds altered pubertal spermatogenesis (days 18 and 25) and whether the changes observed resulted in long-term changes in testis size, mating, or fertility (days 90-100). Rats were treated neonatally with a range of doses (0.01-10 microg) of diethylstilbestrol (DES; administered on alternate days from days 2-12), a high dose of octylphenol (OP; 2 mg administered daily from days 2-12) or bisphenol A (Bis-A; 0.5 mg administered daily from days 2-12), or vehicle, while maintained on a standard soy-containing diet. The effect on the same parameters of rearing control animals on a soy-free diet was also assessed as was the effect of administering such animals genistein (4 mg/kg/day daily from days 2-18). Testis weight, seminiferous tubule lumen formation, the germ cell apoptotic index (apoptotic/viable germ cell nuclear volume), and spermatocyte nuclear volume per unit Sertoli cell nuclear volume were used to characterize pubertal spermatogenesis. Compared with (soy-fed) controls, DES administration caused dose-dependent retardation of pubertal spermatogenesis on day 18, as evidenced by decreases in testis weight, lumen formation, and spermatocyte nuclear volume per unit Sertoli cell and elevation of the germ cell apoptotic index. However, the two lowest doses of DES (0.1 and 0.01 microg) significantly increased spermatocyte nuclear volume per unit Sertoli cell. Similarly, treatment with either OP or Bis-A significantly advanced this and some of the other aspects of pubertal spermatogenesis. Maintenance of control animals on a soy-free diet also significantly advanced lumen formation and spermatocyte nuclear volume per unit Sertoli cell compared with controls fed a soy-containing diet. Administration of genistein reversed the stimulatory effects of a soy-free diet and significantly retarded most measures of pubertal spermatogenesis. In general, plasma FSH levels in the treatment groups changed in parallel to the spermatogenic changes (reduced when pubertal spermatogenesis retarded, increased when pubertal spermatogenesis advanced). By day 25, although the changes in FSH levels largely persisted, all of the stimulatory effects on spermatogenesis seen on day 18 in the various treatment groups were no longer evident. In adulthood, testis weight was decreased dose dependently in rats treated neonatally with DES, but only the lowest dose group (0.01 microg) showed evidence of mating (3 of 6) and normal fertility (3 litters). Animals treated neonatally with OP or Bis-A had normal or increased (Bis-A) testis weights and exhibited reasonably normal mating/fertility. Animals fed a soy-free diet had significantly larger testes than controls fed a soy-containing diet, and this difference was confirmed in a much larger study of more than 24 litters, which also showed a significant decrease in plasma FSH levels and a significant increase in body weight in the males kept on a soy-free diet. Neonatal treatment with genistein did not alter adult testis w...

Fetal growth plays a role in programming of adult cardiometabolic disorders, which in men, are associated with lowered testosterone levels. Fetal growth and fetal androgen exposure can also predetermine testosterone levels in men, although how is unknown, because the adult Leydig cells (ALCs) that produce testosterone do not differentiate until puberty. To explain this conundrum, we hypothesized that stem cells for ALCs must be present in the fetal testis and might be susceptible to programming by fetal androgen exposure during masculinization. To address this hypothesis, we used ALC ablation/regeneration to identify that, in rats, ALCs derive from stem/progenitor cells that express chicken ovalbumin upstream promoter transcription factor II. These stem cells are abundant in the fetal testis of humans and rodents, and lineage tracing in mice shows that they develop into ALCs. The stem cells also express androgen receptors (ARs). Reduction in fetal androgen action through AR KO in mice or dibutyl phthalate (DBP) -induced reduction in intratesticular testosterone in rats reduced ALC stem cell number by ∼40% at birth to adulthood and induced compensated ALC failure (low/normal testosterone and elevated luteinizing hormone). In DBP-exposed males, this failure was probably explained by reduced testicular steroidogenic acute regulatory protein expression, which is associated with increased histone methylation (H3K27me3) in the proximal promoter. Accordingly, ALCs and ALC stem cells immunoexpressed increased H3K27me3, a change that was also evident in ALC stem cells in fetal testes. These studies highlight how a key component of male reproductive development can fundamentally reprogram adult hormone production (through an epigenetic change), which might affect lifetime disease risk.adult Leydig stem/progenitor cells | compensated Leydig cell failure | GATA4 | ethane dimethane sulfonate

The effects on reproductive tract development in male rats, of neonatal exposure to potent (reference) oestrogens, diethylstilboestrol (DES) and ethinyl oestradiol (EE), with those of two environmental oestrogens, octylphenol and hisphenol A were systematically compared. Other treatments, such as administration of a gonadotrophin-releasing hormone antagonist (GnRHa) or the anti-oestrogen tamoxifen or the anti-androgen flutamide, were used to aid interpretation of the pathways involved. All treatments were administered in the neonatal period before onset of puberty. The cellular sites of expression of androgen receptors (AR) and of oestrogen receptor-alpha (ERalpha) and ERbeta were also established throughout development of the reproductive system. The main findings were as follows: (i) all cell types that express AR also express one or both ERs at all stages of development; (ii) Sertoli cell expression of ERbeta occurs considerably earlier in development than does expression of AR; (iii) most germ cells, including fetal gonocytes, express ERbeta but not AR; (iv) treatment with high, but not low, doses of potent oestrogens such as DES and EE, induces widespread structural and cellular abnormalities of the testis and reproductive tract before puberty; (v) the latter changes are associated with loss of immunoexpression of AR in all affected tissues and a reduction in Leydig cell volume per testis; (vi) none of the effects in (iv) and (v) can be duplicated by treating with high-dose octylphenol or bisphenol A; (vi) none of the reproductive tract changes in (iv) and (v) can be induced by simply suppressing androgen production (GnRHa treatment) or action (flutamide treatment); and (vii) the adverse changes induced by high-dose DES (iv and v) can be largely prevented by co-administration of testosterone. Thus, it is suggested that many of the adverse changes to the testis and reproductive tract induced by exposure to oestrogens result from a combination of high oestrogen and low androgen action. High oestrogen action or low androgen action on their own are unable to induce the same changes.

Diethylstilbestrol (DES) was administered neonatally (Days 2-12; 10 microg on alternate days) to rats, and developmental changes in Sertoli cell function were evaluated at 18, 25, and 35 days of age and compared to those observed in rats administered a GnRH antagonist (GnRHa; Days 2 and 5; 10 mg/kg) or a vehicle (controls). DES and GnRHa treatments resulted in similar reductions in both Sertoli cell numbers (40% for DES, 48% for GnRHa) and suppression of testicular growth at 18 and 25 days, though by 35 days the suppression was more pronounced (p < 0.001) in DES-treated animals. Plasma FSH levels were suppressed markedly at 18 and 25 days, but not at 35 days, in GnRHa-treated rats, whereas in DES-treated rats the FSH levels were suppressed significantly only at 35 days. Both treatments suppressed plasma levels of inhibin B, though this was more pronounced (p < 0.05) in DES- than in GnRHa-treated rats. In controls, Sertoli cell immunoexpression of inhibin alpha, sulfated glycoprotein-1 (SGP-1), and androgen receptor (AR) increased in intensity and changed to an adult, stage-dependent pattern by 25 days. In GnRHa-treated rats these changes were reduced in intensity but were similar to those in controls at 35 days. In DES-treated rats, the increase in intensity and stage-dependent pattern of immunoexpression of inhibin alpha, SGP-1, and AR were virtually absent at 25 days but were present by 35 days. Germ cell volume per Sertoli cell was reduced in GnRHa- and DES-treated rats compared with controls at 18 and 25 days but was significantly greater (p < 0. 001) in DES- than in GnRHa-treated rats at 35 days. The proportion of apoptotic to viable germ cells was increased (p < 0.01) in GnRHa- and DES-treated rats compared with controls at 18 and 25 days; but at 35 days, values in GnRHa-treated rats had declined to control values whereas those for DES-treated rats remained 10-fold elevated (p < 0.001). In adulthood, testis weight and daily sperm production were reduced by 43% and 44%, respectively, in GnRHa-treated rats, but spermatogenesis was grossly normal. Comparable changes were observed in approximately 25% of DES-treated rats, but the majority exhibited > 60% reduction in testis weight with many Sertoli cell-only tubules and very low daily sperm production. Taken together, these data are interpreted as providing evidence for direct modulation of Sertoli cell (maturational) development by DES.