Androgen suppression-induced stimulation of spermatogonial differentiation in juvenile spermatogonial depletion mice acts by elevating the testicular temperature.

Why both testosterone (T) suppression and cryptorchidism reverse the block in spermatogonial differentiation in adult mice homozygous for the juvenile spermatogonial depletion (jsd) mutation has been a conundrum. To resolve this conundrum, we analyzed interrelations between T suppression, testicular temperature, and spermatogonial differentiation and used in vitro techniques to separate the effects of the two treatments on the spermatogonial differentiation block in jsd mice. Temporal analysis revealed that surgical cryptorchidism rapidly stimulated spermatogonial differentiation whereas androgen ablation treatment produced a delayed and gradual differentiation. The androgen suppression caused scrotal shrinkage, significantly increasing the intrascrotal temperature. When serum T or intratesticular T (ITT) levels were modulated separately in GnRH antagonist-treated mice by exogenous delivery of T or LH, respectively, the inhibition of spermatogonial differentiation correlated with the serum T and not with ITT levels. Thus, the block must be caused by peripheral androgen action. When testicular explants from jsd mice were cultured in vitro at 32.5 C, spermatogonial differentiation was not observed, but at 37 C significant differentiation was evident. In contrast, addition of T to the culture medium did not block the stimulation of spermatogonial differentiation at 37 C, and androgen ablation with aminoglutethimide and hydroxyflutamide did not stimulate differentiation at 32.5 C, suggesting that T had no direct effect on spermatogonial differentiation in jsd mice. These data show that elevation of temperature directly overcomes the spermatogonial differentiation block in adult jsd mice and that T suppression acts indirectly in vivo by causing scrotal regression and thereby elevating the testicular temperature.

Effects of 4-tert-octylphenol on Xenopus tropicalis in a long term exposure.

Endocrine disrupting chemicals that activate the estrogen receptor are routinely detected in the environment and are a concern for the health of both exposed humans and indigenous wildlife. We exposed the western clawed frog (Xenopus tropicalis) to the weak estrogen octylphenol from Nieuwkoop-Faber (NF) stage 46 tadpoles through adulthood in order to document the effects of a weak estrogen on the life history of an amphibian species. Frogs were exposed to 1, 3.3, 11 and 36 µg/L octylphenol in a continuous flow-through water system. Just prior to completion of metamorphosis (NF 65), a random subsample of froglets was collected and assessed, while the remaining frogs received continued exposure through 31 weeks of exposure when the remaining animals were sampled. Significant induction of the female egg yolk protein precursor vitellogenin was observed in the high treatment at the larval subsampling for both males and females, but not at the final sampling for either sex. No significant deviation from the control sex ratio was observed for either sampling period, suggesting minimal to no effect of octylphenol exposure on gonad differentiation. No effects in the adult frogs were observed for mortality, body mass and size, liver somatic index, estradiol and testosterone serum levels, sperm counts, or oocyte counts. The development and growth of oviducts, a female-specific secondary sex characteristic, was observed in males exposed to octylphenol. These results indicate that octylphenol exposure can induce vitellogenin in immature froglets and the development of oviducts in male adult frogs. The lack of effect observed on the developing gonads suggests that in amphibians, secondary sex characteristics are more susceptible to impact from estrogenic compounds than the developing gonads.

Estrogen enhances recovery from radiation-induced spermatogonial arrest in rat testes.

Irradiation of LBNF(1) rat testes induces spermatogonial differentiation arrest, which can be reversed by gonadotropin-releasing hormone (GnRH) antagonist-induced suppression of intratesticular testosterone (ITT) and follicle-stimulating hormone (FSH). Although exogenous estrogen treatment also enhanced spermatogenic recovery, as measured by the tubule differentiation index (TDI), it was not clear whether estrogen stimulated spermatogonial differentiation only by further suppressing ITT or by an additional independent mechanism as well. To resolve this question, we performed the following experiments. At 15 weeks after irradiation, rats were treated with GnRH antagonist; some also received 17beta-estradiol (E2) and were killed 4 weeks later. GnRH antagonist treatment increased the TDI from 0% to 8%, and addition of E2 further increased the TDI to 39%. However, E2 addition further reduced ITT from 7 ng/g testis, observed with GnRH antagonist to 3 ng/g testis, so decreased ITT levels might have contributed to recovery. Next GnRH antagonist-treated rats were given exogenous testosterone and flutamide to stabilize ITT levels and block its action. This increased TDI slightly from 8% to 13%, but the further addition of E2 significantly raised the TDI to 27%, indicating it acted by a mechanism independent of ITT levels. Plots of TDI for all treatment groups compared with ITT, FSH, or a linear combination of ITT and FSH showed that treatments including E2 produced higher TDI values than did treatments without E2. These results indicate that there was an effect of E2 on spermatogonial differentiation because of an additional direct action on the testis that is unrelated to its suppression of testosterone or gonadotropins.

A novel amphibian tier 2 testing protocol: a 30-week exposure of Xenopus tropicalis to the antiandrogen flutamide.

In 1996, the U.S. Congress mandated the development of a screening program for endocrine-disrupting chemicals (EDCs) using validated test systems. Subsequently, the Endocrine Disruptor Screening and Testing Advisory Committee recommended the development of a standardized amphibian assay for tier 2 testing of EDCs. For that reason, a tier 2 testing protocol using Xenopus (Silurana) tropicalis and a 30-week, flow-through exposure to the antiandrogen flutamide from stage 46 tadpoles through sexually mature adult frogs were developed and evaluated in this pilot study. The endpoints for this study included measurements of frog body lengths and weights, liver weights, ovary/egg mass weights, testicular and ovarian histopathology, plasma vitellogenin levels, and notes on any abnormalities observed at necropsy. Increasing exposure concentrations to flutamide caused significant increases in frogs with no recognizable gonadal tissue and increased body and liver weights in male frogs, whereas the body lengths and weights decreased significantly in female frogs. Important issues must be resolved before a tier 2 amphibian assay can be further developed and validated, including the establishment of baseline values in the controls for the parameters under study; the maintenance, measurement, and timing of exposure concentrations; and the development of additional biomolecular markers of effect. This study demonstrated the feasibility of conducting long-term EDC exposure studies using X. tropicalis.

Spermatogonial differentiation in juvenile spermatogonial depletion (jsd) mice with androgen receptor or follicle-stimulating hormone mutations.

The jsd mice experience a single wave of spermatogenesis, followed by an arrest of spermatogenesis because of a block in spermatogonial differentiation. Previous pharmacological and surgical studies have indicated that testosterone (T) and low scrotal temperatures but not FSH block spermatogonial differentiation in jsd mice. We sought to test these observations by genetic approaches by producing male jsd mutant mice with either defective androgen receptor (AR, Tfm mutation) or a deficiency of FSH (fshb(-/-)). In adult jsd-Tfm double-mutant mice, the tubule differentiation index was 95% compared with 14% in jsd littermates, suggesting that general ablation of AR function restored spermatogonial differentiation in jsd mice. The results indicated that this enhancement of differentiation was primarily a result of elevation of temperature caused by the cryptorchid position of the testis in jsd-Tfm double-mutant mice, which resulted from the lack of AR in the gubernaculum. The low levels of T were not a factor in the release of the spermatogonial differentiation block in the jsd-Tfm mice, but we were unable to determine whether inactivation of AR in the adult jsd testis had a direct effect on the restoration of spermatogonial differentiation because the elevated temperature bypassed the T-induced block in spermatogonial differentiation. Although spermatogonia were indeed present in adult jsd-fshb double-mutant mice and were capable of differentiation after androgen deprivation, these mice had a tubule differentiation index of 0%, ruling out the possibility that endogenous FSH inhibited spermatogonial differentiation in jsd mice. The results are consistent in support of the hypothesis that inhibition of spermatogonial differentiation in jsd mice is a result of T acting through the AR only at scrotal temperatures.

Testicular edema is associated with spermatogonial arrest in irradiated rats.

Irradiation of LBNF1 rat testes induces arrest of spermatogonial differentiation, which can be reversed by suppression of testosterone with GnRH antagonist treatment. The cause of the arrest is unknown. We investigated the time course and hormonal effects on radiation-induced arrest and changes in interstitial fluid volume. We postulated that the edema evident in irradiated testes caused the differentiation blockade. Rat testes were irradiated with 3.5 or 6 Gy. Interstitial fluid testosterone (IFT) increased between 2 and 6 wk after irradiation, followed by increased interstitial fluid volume at 6 wk and spermatogonial blockade at 8 wk. Additional rats irradiated with 6 Gy were given GnRH antagonist, alone or with exogenous testosterone, for 8 wk starting at 15 wk after irradiation. In rats treated with GnRH antagonist, IFT started falling within 1 wk of treatment, followed by interstitial fluid volume decreases at wk 2 and 3, with recovery of spermatogenesis starting at wk 4. Addition of exogenous testosterone largely blocked the effects of GnRH antagonist on IFT, interstitial fluid volume, and spermatogenesis. Thus the testicular edema was largely modulated by intratesticular testosterone levels. The time course of changes in the spermatogonial blockade more closely followed that of the testicular edema than of IFT, indicating that testosterone may block spermatogonial differentiation indirectly by producing edema. This conclusion was further supported by an experiment in which irradiated rats were treated with GnRH antagonist plus estrogen; the treatment further reduced IFT and interstitial fluid volume and reduced the time to initiation of recovery of spermatogonial differentiation. These results suggest that studies of the edematous process or composition of the fluid would help elucidate the mechanism of spermatogonial arrest in toxicant-treated rats.

Restoration of spermatogenesis in dibromochloropropane (DBCP)-treated rats by hormone suppression.

The exposure of men to the nematocide dibromochloropropane (DBCP) has caused prolonged oligo- and azoospermia, which occasionally reverses spontaneously. We recently demonstrated that in testes of rats treated with a dose of DBCP sufficient to reduce the percentage of tubules producing differentiating germ cells (tubule differentiation index, TDI) to 20%, the tubules lacking differentiating cells contained type A spermatogonia. To determine whether these type A spermatogonia could be stimulated to differentiate, as had been demonstrated previously in other models of toxicant-induced sterility, we suppressed intratesticular testosterone and serum follicle stimulating hormone (FSH) levels with the GnRH agonist Lupron (leuprolide). When the GnRH agonist was given for 10 weeks starting immediately after DBCP exposure, the TDI was maintained at 94%. Even when GnRH-agonist treatment was stopped at week 10, the TDI remained between 65 and 80% 10 weeks later. Late spermatid counts averaged 10 x 10(6) per testis for the GnRH-agonist-treated rats at week 20 compared with 1.7 x 10(6) per testis in rats treated with only DBCP. To determine whether spermatogonial differentiation could be stimulated after the TDI had declined to below 30%, we initiated GnRH-agonist treatment 6 weeks after DBCP exposure. The GnRH treatment increased the TDI to 53% at week 16. These results indicate that, if the same principles apply to humans, suppression of testosterone may be applied to restore spermatogenesis in men rendered azoospermic by DBCP or other reproductive toxicants.

Dibromochloropropane inhibits spermatogonial development in rats.

Exposure to the nematocide dibromochloropropane (DBCP) has caused prolonged oligo- and azoospermia in men. There are questions regarding the cellular targets resulting in this effect. In this study we characterized an animal model, in which four daily injections of DBCP produced prolonged oligospermia in LBNF(1) rats without any indication of recovery. Between 6 and 20 weeks after DBCP treatment, 70% of seminiferous tubules showed an epithelium with Sertoli cells but no differentiating germ cells. About 20% of tubules contained differentiating germ cells and 10% showed occlusion or major morphologic alterations to Sertoli cells. Since gonadotropin levels and intratesticular testosterone (ITT) concentrations were elevated in the DBCP-treated rats, the failure of spermatogonial development could not have been a result of lack of these hormones. The tubules without differentiating germ cells contained actively proliferating and dividing type A spermatogonia, which underwent apoptosis instead of differentiation. Thus, the target for the damaging effect appears not to be the killing of stem spermatogonia, but the loss of their ability to undergo differentiation. The presence of type A spermatogonia in the atrophic tubules indicates the potential for intervention to restore spermatogenesis.

17beta-estradiol is a hormonal regulator of mirex tumor promotion sensitivity in mice.

Mirex, an organochlorine pesticide, is a potent non-phorbol ester tumor promoter in mouse skin. Previous studies have shown that female mice are 3 times more sensitive to mirex tumor promotion than male mice and that ovariectomized (OVX) female mice are resistant to mirex promotion, suggesting a role for ovarian hormones in mirex promotion. To determine whether the ovarian hormone 17-beta estradiol (E2) is responsible for the sensitivity of female mice to mirex promotion, female mice were initiated with DMBA; 2 weeks later groups of mice were OVX and implants, with or without E2, were surgically implanted subcutaneously. These mice were treated topically twice weekly with mirex for 26 weeks. E2 implanted OVX mice demonstrated high normal physiologic levels of serum E2 throughout the tumor promotion experiment. E2 implants restored by 80% the intact mirex-sensitive phenotype to the OVX mice. Consistent with a role for E2 and ERalpha and ERbeta, treatment of DMBA-initiated female mice with topical ICI 182,780, an estrogen-receptor antagonist, reduced mirex tumor multiplicity by 30%. However, in cells co-transfected with ERalpha or ERbeta and estrogen-responsive promoter reporter, mirex did not stimulate promoter reporter activity, suggesting that the promotion effect of mirex is downstream of ERalpha/beta. Finally, a tumor promotion study was conducted to determine whether E2 implants could increase the sensitivity of male mice to mirex promotion. E2 implants in male mice did increase sensitivity to mirex promotion; however, the implants did not produce the full female sensitivity to mirex tumor promotion. Collectively, these studies indicate that E2 is a major ovarian hormone responsible for mirex tumor promotion sensitivity in female mice.