DES exposure alters neonatal germ cell differentiation through Nr0b2

The orphan nuclear receptor small heterodimer partner mediates male infertility induced by diethylstilbestrol in mice

2009 Study Abstract

Studies in rodents have shown that male sexual function can be disrupted by fetal or neonatal administration of compounds that alter endocrine homeostasis, such as the synthetic nonsteroidal estrogen diethylstilbestrol (DES). Although the molecular basis for this effect remains unknown, estrogen receptors likely play a critical role in mediating DES-induced infertility. Recently, we showed that the orphan nuclear receptor small heterodimer partner (Nr0b2), which is both a target gene and a transcriptional repressor of estrogen receptors, controls testicular function by regulating germ cell entry into meiosis and testosterone synthesis. We therefore hypothesized that some of the harmful effects of DES on testes could be mediated through Nr0b2. Here, we present data demonstrating that Nr0b2 deficiency protected mice against the negative effects of DES on testis development and function. During postnatal development, Nr0b2-null mice were resistant to DES-mediated inhibition of germ cell differentiation, which may be the result of interference by Nr0b2 with retinoid signals that control meiosis. Adult Nr0b2-null male mice were also protected against the effects of DES; however, we suggest that this phenomenon was due to the removal of the repressive effects of Nr0b2 on steroidogenesis. Together, these data demonstrate that Nr0b2 plays a critical role in the pathophysiological changes induced by DES in the mouse testis.

Discussion

Here, we demonstrated that DES induced abnormalities in the male genital tract upon fetal and/or neonatal exposure in mice. In animal models, neonatal administration of DES decreased fertility in adult male mice as a result of altered morphology of the male genital tract, with decreased relative weights of epididymis, vesicle seminals, and testis. This was associated with a decrease in epididymis sperm count, which was caused by the increased germ cell death we observed in male mice exposed to DES. Interestingly, for all these parameters, deficiency for Nr0b2 protected the males exposed to DES, which suggests that Nr0b2 plays a critical role in the testicular pathophysiology induced by DES. However, we have demonstrated that DES acts via several signaling pathways. Through the use of estradiol and/or of the Er antagonist ICI, we showed that Nr0b2 protected the male mice against both estrogenic and nonestrogenic effects of DES. Even though it is not established in humans, these rodent data are the basis of the hypothesis of a potential link between these environmental factors, including the exposure to EDs, and the fast increase of the incidence of male reproductive disorders. Our data could potentially establish a link between EDs and male reproductive disorders in humans.

High doses of EDs induce a severe decrease in testicular weight caused by germ cell death, which leads to important modifications in the relative cell type proportions. Such changes could lead to false interpretations, because the observed differences in gene expression are the result of changes in testicular cell content rather than of the real regulation of gene expression. Through a dose-effect experiment of DES, we first determined the concentration that did not dramatically affect testicular weight. This allowed us to assume that at the doses used for the molecular analyses, the modifications of gene expression were significant, not the consequences of altered cell content.

Unexpectedly, we observed higher levels of active caspase-3 in Nr0b2L–/L– mice compared with Nr0b2+/+littermates. This increase in basal levels of active caspase-3 in Nr0b2L–/L– mice was significant in P10 animals, which could reflect the earlier entry in meiosis. In adult mice, we also detected a significant difference between active caspase-3 levels in Nr0b2+/+ and Nr0b2L–/L– mice. Interestingly, and in line with our previous studies, there was no difference in the number of TUNEL-positive cells between vehicle-treated Nr0b2+/+ and Nr0b2L–/L– males. The increase in active caspase-3 was not consistent with the unchanged results of the TUNEL assay and requires further study; this finding reflects the complexity of apoptosis, which can be regulated at both activation and inhibition levels.

Even though the present report showed that Nr0b2L–/L– mice were protected against the deleterious effect of DES and EB on the male reproductive tract, basal estrogen metabolism was similar between Nr0b2+/+ and Nr0b2L–/L–males . These data suggested that the role of Nr0b2 on the estrogenic pathway is only revealed under pathophysiological conditions when exposed to estrogenic endocrine disrupters. However, the lack of difference between the genotypes under basal condition was surprising and difficult to explain; it suggests the existence of some yet-unidentified compensatory mechanisms. One could speculate that Nr0b1, which is very close to Nr0b2 at the structural level, could compensate for the lack of Nr0b2.

Nr0b1 and Nr0b2 are closely related nuclear receptors. Moreover, the impact of Nr0b1 on steroidogenesis has been well demonstrated. In our experiments, however, we have not shown a significant effect of DES treatment on Nr0b1 expression in P10 pups or in adult males. These results suggest that Nr0b1 is not involved in the DES-induced phenotypes. Several studies have shown that Nr0b1 KO mice have normal levels of testosterone. The Nr0b1 KO mouse phenotype can mostly be explained by the altered expression of aromatase, the enzyme that synthesizes estrogens, whereas other steroidogenic genes, like Star, were not affected. Together, these observations imply a converging role of both Nr0b members to control full testicular steroid metabolism, in which Nr0b2 would predominantly regulate steroidogenesis up to the level of testosterone synthesis, and Nr0b1 would instead control aromatization.

In adult mice, Nr0b2 was responsible for the DES-induced decrease in testosterone production. The absence of a decrease of steroidogenic genes in Nr0b2L–/L– males after DES exposure is suggestive of a major role for Nr0b2 in the DES-induced repression of steroidogenesis. This hypothesis is further corroborated by the induction of Nr0b2mRNA in response to DES. This effect was also observed with EB, which suggests that it might be driven by Nr3a1/2 signaling. This is consistent with previous studies showing that estrogen can directly regulate steroidogenesis at the Leydig cell levels. The ability of estrogen to decrease steroid synthesis in the mouse Leydig cell line MA-10, highlighted by the decrease in the mRNAs of Star and Cyp11a1, has been described previously. To identify the possible direct effect of Nr0b2 in Leydig cells following DES administration, we also performed experiments on MA-10 cells. Similar to our in vivo results, EB and DES resulted in an increase of Nr0b2 mRNA 12 hours after treatment. This increase in Nr0b2 mRNA was followed at 24 hours by a significant decrease in Star mRNA. The kinetics of the mRNA increase of Nr0b2, followed by the decrease of Star mRNA, is consistent with Nr0b2 being a major inhibitor of steroidogenesis in Leydig cells. This is consistent with our previous work demonstrating that overexpression of Nr0b2 in a Leydig cell line represses Star and Cyp11a1expression and that Nr0b2 binds to the promoter of these 2 genes. Moreover, our conclusion that Nr0b2 controls steroidogenesis directly at the testicular level after DES exposure was confirmed by the finding that plasma LH and FSH levels were both unaltered by DES as well as Nr0b2 genotype.

At the molecular level, we have previously shown that Nr0b2 regulates testicular androgen synthesis through repressed mRNA expression of 2 activators of steroidogenesis, Nr5a2 and Nr5a1, and/or through inhibited transcriptional activities of the same. Here, the repression of steroidogenesis by Nr0b2 at the lower 0.5-μg dose is likely caused by protein-protein interaction, as mRNA expression of both Nr5a1 and Nr5a2 was not affected, whereas Star accumulation was already reduced at this DES concentration, in Nr0b2+/+ mice (data not shown). At the dose of 0.75 μg DES, this effect was further amplified, probably by the decreased mRNA expression of Nr5a2. Even though Nr5a1 was defined as a target gene of Nr0b2, its expression was not affected by DES administration. The absence of any effect of DES was consistent with previous studies. These data demonstrate the complexity of the regulation of Nr5a1, which is known to be controlled by multiple factors; furthermore, it would be too simplistic to assume that the mRNA expression of Nr5a1 is only regulated by Nr0b2.

In contrast to our findings in adult mice, neonatal steroidogenesis seemed not to be controlled by Nr0b2, as Nr0b2L–/L– and Nr0b2+/+ mice showed similar profiles . This difference in Leydig cell response seems to occur in parallel with the cells’ transition from the fetal to the adult population and is in line with the finding that from P20 onward, Nr0b2 becomes expressed only in interstitial cells, where it controls steroidogenesis. Moreover, P10 testis samples showed that Nr0b2 was mainly expressed in the tubular compartment of the testis (data not shown). This suggests that, at P10, the impact of Nr0b2 following DES exposure occurs in the intratubular compartment.

In addition to its role on androgen synthesis in adult mice after neonatal DES exposure, Nr0b2 has other functions during postnatal testicular development. We administered DES during early postnatal development, close to the beginning of germ cell differentiation. Indeed, the first spermatocytes are seen at P5. Neonatal administration of either DES or EB induced modified expression of genes associated with differentiated or undifferentiated cells, such as Nanos3Stra8, and Dmc1. We demonstrated here that DES induced accumulation of specific transcripts of undifferentiated spermatogonia, specifically in treated Nr0b2+/+ testis, and reduced the expression of transcripts involved in germ cell differentiation. These results suggest a change of the relative proportion of undifferentiated versus differentiating spermatogonia after DES exposure. We thus hypothesize that Nr0b2 mediates the effect of DES on germ cell differentiation, which is in line with our previous report that Nr0b2L–/L– mice showed earlier differentiation of germ cells than did control littermates. To understand the alteration of these relative cell proportions, we analyzed both proliferation and apoptotic processes in P10 mouse testes. In contrast to adult mice, this differential effect observed between Nr0b2+/+ and Nr0b2L–/L– males in early postnatal development did not seem to be linked with androgenic status. The increased apoptosis observed in Nr0b2+/+ males is most likely caused by other alterations. DES treatment clearly affected the meiotic cells. In oocytes, DES induced a severe, yet reversible, deterioration of meiotic spindle microtubule organization during maturation. DES reduces viability of Caenorhabditis elegans and its fertility, associated with the production of aberrant gametes, as a result of nuclear abnormalities and loss of synaptonemal complexes. Moreover, recent studies have demonstrated the importance of germ cell–specific epigenetic processes in the initiation and early progression of meiosis. Interestingly, mice with a loss-of-function mutation for H3K9 histone methyltransferases are sterile, with germ cells undergoing apoptosis during the pachytene stage. Several proteins possess H3K9 methyltransferase activity. Suv39h1, Suv39h2, and G9a are able to perform H3K9 dimethylation, whereas only G9a performs H3K9 monomethylation. In testes of DES-treated P10 Nr0b2+/+ mice, we observed decreases in the H3K9me1 and H3K9me2 marks, which suggests that DES might affect G9a expression and/or activity in the testis. Consistent with this hypothesis, G9a mRNA and G9a protein expression was decreased in Nr0b2+/+ mice treated with DES. Because of its histone methyltransferase activity, G9a acts as a repressor of transcription. The decrease of G9a after DES exposure was confirmed in Nr0b2+/+ mice by higher mRNA accumulation of known G9a target genes, such as Akr1c13 and Chst11. We observed no significant effect in the Nr0b2L–/L– mice. It has been recently demonstrated that Nr0b2 physically interacts with G9a to induce repression of gene expression. Here, we demonstrated another form of cross-talk between Nr0b2 and G9a, in which Nr0b2 inhibited the mRNA expression of G9a. It is possible that both coexist and are part of a feedback loop in which Nr0b2 inhibits G9a gene expression to attenuate the repression of their common target genes. The effect of DES on the histone methylation marks was also found when we used EB, highlighting the involvement of the estrogenic part of DES in this pathway.

If the signaling of G9a was decreased in Nr0b2+/+ mice treated with DES, as evidenced by decreased expression of G9a, decreased H3K9 methylation, and increased expression of G9a target genes, we unexpectedly observed an opposite effect of DES in the Nr0b2L–/L– males. Indeed, the expression of some target genes was found more repressed in the Nr0b2L–/L– males exposed to DES compared with vehicle. This effect, observed in Nr0b2L–/L–males, was confirmed by a higher level of methylated histones on the DNA sequences of these G9a target genes. This effect is surprising and to date remains unexplained. However, this is in line with our conclusion that Nr0b2 participates to control G9a signaling. The observed data in Nr0b2L–/L– mice treated with DES suggest that the lack of Nr0b2 induced an increase in the G9a pathway, or a compensatory pathway through other histone methyltransferases. The exact molecular mechanisms involved are not established yet and will require further studies.

The molecular mechanisms triggering the initiation of germ cell differentiation, in particular the mitotic/meiotic transition, are not completely understood. However, retinoids are key components to induce entry of germ cells in meiosis. Most intriguingly, retinoids have also been shown to induce an increase in H3K9 methylation during differentiation. Based on these reports and our above-described findings, we hypothesized that Nr0b2, via the control of G9a expression, is the link between RA and DES pathways in the control of the meiotic process. This was confirmed by the induction of G9a expression following RA administration in different cell lines. Moreover, we found a specific enrichment of a Rar/Nr0b2 complex on the DNA sequences surrounding the RARE of the G9a promoter. Our results therefore show, for the first time to our knowledge, a potential interaction between the retinoid signaling pathway and the expression of the histone methyltransferase G9a, which could explain, at least in part, the impact of G9a and H3K9 methylation in germ cell differentiation.

Interestingly, this effect of DES on germ cell differentiation seemed to persist in adult mice, as we observed a clear decrease in mRNA accumulation of Stra8 . We have indeed demonstrated that the deregulation of G9a was still observed at the mRNA and protein levels in Nr0b2+/+ adult males treated neonatally with DES . This perpetuation of G9a deregulation in adult testis, combined with the altered testosterone synthesis, might cooperate to decrease germ cell survival and lead to subfertility after DES administration.

It has been previously demonstrated that DES can have estrogenic and nonestrogenic effects. Compared with EB, DES appeared to have a stronger effect. To determine the estrogenic part of DES activity, we used either a pure estrogenic compound, EB, or the Er antagonist ICI. Most of the macroscopic phenotypes observed with DES were also obtained using EB (e.g., organ weight, apoptotic process), and the Nr0b2L–/L– mice were also protected against the deleterious effects of EB. ICI has previously been demonstrated to have deleterious effects on the male genital tract, with loss of germ cells and decrease of fertility. Here, we also observed a significant alteration in sperm count induced by ICI alone. In adult rats and mice, treatment with ICI induced effects similar to those previously observed in the male reproductive tract of Nr3a1 KO mice. These findings suggest that, following ICI treatment, the decrease in sperm concentration in the cauda epididymis could be explained, at least in part, by the fact that Nr3a1 is required for normal fluid reabsorption, as concluded from studies of Nr3a1 KO males. Most interestingly, ICI partially reversed the effect of DES on all the macroscopic testicular abnormalities it induced. Notably, we used only 1 dose of ICI (50×) to compete with DES; perhaps a higher dose would produce even more pronounced competition with DES.

Most of the molecular pathways altered by DES were also found to be affected by EB (e.g., testosterone, retinoid pathway). However, some of our results highlighted differential effects of DES and EB at the dose we used: at P10, the expression of Oct3/4 was changed by DES, not by EB. However, this is an important difference, as Oct3/4plays an important role in the determination of the pluripotency of cells, which could explain why the effect of DES was more potent compared with EB. Indeed, the altered expression of Oct3/4 by DES might more robustly inhibit germ cell differentiation than that by EB. At the molecular level, Nr5a2 has been described to induce Oct3/4expression. At P10, the expression of Nr5a2 was not affected by DES treatment. This result demonstrated that the effect on Oct3/4 expression at P10 age does not seem to be related to the status of Nr5a2. On the one hand, differences in dosing and relative receptor affinity could contribute to the difference in Oct3/4 expression between DES and EB. Indeed, both compounds have a different affinity to either Nr3a1 or Nr3a2, as DES was shown to bind to Ers with a higher affinity than did estradiol . On the other hand, it could be hypothesized that in response to DES, Oct3/4 expression might be regulated by Nr3b1/2/3. DES has previously been demonstrated to activate Nr3b1/2/3, and these receptors positively regulate Oct3/4 expression. Even though we did not determine the underlying molecular mechanisms, the effect of DES on Oct3/4 expression was also inhibited in Nr0b2L–/L– mice; therefore, Nr0b2 deficiency might also protect against the nonestrogenic effects of DES.

In conclusion, our results demonstrated that Nr0b2 plays a major role in the DES signaling pathway, which affects the development and function of the male reproductive system. We showed that Nr0b2L–/L– male mice were protected against the deleterious effects of DES, as they were still able to reproduce even when exposed to high doses of DES. This is caused by the multiple actions of Nr0b2 during testicular development. First, in neonatal animals, Nr0b2 controls germ cell differentiation through inhibition of the retinoid pathway. Nr0b2 regulates the expression of genes involved in the entry and progression of meiosis, such as Stra8 and Nanos3. It also affects meiosis through regulation of the expression of the histone methyltransferase G9a and the subsequent modification of H3K9 methylation marks. These alterations in methylation upon DES exposure induce abnormal chromosomal complexes favoring germ cell apoptosis and could affect the meiosis process. Next to these effects, which appear to be mediated through the estrogenic pathway, DES seems to inhibit germ cell differentiation at P10 through estrogen-independent pathways, as shown by Oct3/4 deregulation specifically in DES-treated mice. Second, in adult animals, the effect of Nr0b2 was dependent on the inhibition of testosterone production, leading to germ cell death. Together, our present data define Nr0b2 as one of the major actors in the molecular events leading to DES-mediated male infertility.

Sources and more information
  • Full study (free access) : The orphan nuclear receptor small heterodimer partner mediates male infertility induced by diethylstilbestrol in mice, Journal of Clinical Investigation, NCBI PubMed PMC2786790, 2009 Dec 1.
  • Featured image (A) Nr0b2 mRNA levels in whole testes of P10 Nr0b2+/+ and Nr0b2L–/L– males exposed to 0.75 μg DES (n = 6), and in whole testes of 10-week-old males neonatally exposed to 0, 0.75, and 5 μg DES (n = 3–5). Values were normalized to β-actin. (B) Each male was bred with 5 C57BL/6J females to analyze the number of pups per litter. (C and D) Spermatozoa count in the tail of epididymis (C) and whole body weight as well as weights of testis, epididymis, seminal vehicles, and liver normalized to body weight (D) of 10-week-old Nr0b2+/+ and Nr0b2L–/L– males neonatally exposed to 0, 0.35, 0.5, 0.75, 1.5, and 5 μg DES (n = 6–14 per group). *P < 0.05 versus vehicle; #P < 0.05 versus Nr0b2+/+ given the same DES dose. credit NCBI PMC2786790/figure/F1.
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