Aquaporin 9 expression in the developing rat epididymis is modulated by steroid hormones.

Fluid and solute transport across the epithelium of the male excurrent duct is important for sperm maturation and storage. Aquaporin 9 (AQP9), which allows permeation of water and neutral solutes, is abundant throughout the male reproductive tract, where it is expressed at the apical membrane of rat epididymal principal cells as early as at 1 week of age. We evaluated the effect of neonatal exposure to: 1) a GNRH antagonist (GNRHa); 2) diethylstilbestrol (DES); 3) ethinyl estradiol (EE); 4) DES plus testosterone (DES+TE); and 5) the anti-androgen flutamide on AQP9 expression in the epididymis of peripubertal rats. Control groups received the vehicle alone. In 25-day-old rats, quantification of the mean pixel intensity of immunofluorescence-stained sections showed a significant decrease in AQP9 staining in the apical membrane of epididymal principal cells after treatments with GNRHa, DES, or flutamide, compared to controls. These results were confirmed by western blotting. While EE induced a marked decrease in AQP9 levels by western blotting, the decrease in AQP9-associated fluorescence was not significant compared to controls. DES+TE-treated rats showed levels of AQP9 protein similar to controls, indicating maintenance of AQP9 expression by testosterone treatment in the presence of DES. Our data show that expression of AQP9 in the developing rat epididymis is downregulated by neonatal DES, GNRHa, EE, and flutamide, and that the effects mediated by estrogens can be prevented by testosterone administration.

Cellular origins of testicular dysgenesis in rats exposed in utero to di(n-butyl) phthalate.

Foetal exposure of male rats to di(n-butyl) phthalate (DBP) induces testicular changes similar to testicular dysgenesis syndrome in humans, including the formation of focal 'dysgenetic areas' within post-natal testes, surrounded by otherwise normal tubules exhibiting complete spermatogenesis. We hypothesize that these dysgenetic areas form when Sertoli (and other) cells are 'trapped' during the abnormal formation of large Leydig cell (LC) clusters in foetal life and by post-natal day (d) 4 these groups of intermingled cells attempt to form seminiferous tubules. It is likely that the malformed tubules resulting correspond to the dysgenetic areas evident in later life. This also provides a plausible explanation for the occurrence of LCs within seminiferous cords/tubules in or bordering the dysgenetic areas. In our previous studies intratubular LCs (ITLCs) were identified by immunostaining for 3beta-hydroxysteroid dehydrogenase (3beta-HSD), the definitive LC cytoplasmic marker. However, the possibility remained that the 'presumptive' ITLCs were in fact Sertoli cells that had aberrantly gained the ability to express 3beta-HSD. Therefore, the aim of the present study was to fully characterize the ITLCs induced by in utero DBP exposure in d25 rats using a number of LC- (3beta-HSD, P450 side-chain cleavage enzyme, insulin-like factor 3, oestrogen receptor alpha) and Sertoli cell- (vimentin, Wilm's tumour-1) specific markers. Our results show that ITLCs express all four LC-specific markers but do not express either of the Sertoli cell markers. It is therefore concluded that the ITLCs are bona fide LCs that are abnormally located within the seminiferous tubules of DBP-exposed rats in post-natal life.

Evidence that androgens and oestrogens, as well as follicle-stimulating hormone, can alter Sertoli cell number in the neonatal rat.

Neonatal treatment of male rats with diethylstilboestrol (DES) or a gonadotrophin-releasing hormone antagonist (GnRHa) reduces final Sertoli cell number, an effect presumed to occur via suppression of follicle-stimulating hormone (FSH). As both treatments also suppress androgen action, we investigated whether androgens and oestrogens might affect Sertoli cell nuclear volume/number in the rat using single or combined treatments that differentially affected FSH, testosterone and oestrogen (DES) levels. Neonatal treatment with flutamide (50 mg/ kg) significantly reduced Sertoli cell nuclear volume/ number per testis and blood inhibin-B levels at day 18, despite elevating FSH levels; this treatment also exacerbated the reduction in Sertoli cell nuclear volume per testis induced by treatment with 0.1 mug DES without affecting FSH levels. Treatment with 0.1 mug DES on its own also reduced Sertoli cell nuclear volume per testis without affecting FSH/testosterone levels, but co-administration of 0.1 mug DES+GnRHa, which suppressed FSH and testosterone levels, resulted in a markedly greater effect. Treatment with GnRHa alone or 10 mug DES alone grossly suppressed FSH and testosterone levels and reduced Sertoli cell nuclear volume/number per testis by approximately 60%, but co-administration of the two treatments had no greater effect than either alone. Co-administration of testosterone esters with 10 mug DES partially prevented the 10 mug DES-induced reduction in Sertoli cell nuclear volume per testis, and normalized FSH levels. In all treatment groups, plasma levels of inhibin-B paralleled changes in Sertoli cell nuclear volume/number per testis, but treatments that suppressed FSH levels (GnRHa, 10 mug DES) caused a proportionately greater reduction (approximately 90%) in inhibin-B levels than in Sertoli cell nuclear volume/number (50-60%). Germ cell volume per unit Sertoli cell was reduced in animals treated with 10 mug DES alone or in those treated with 0.1 mug DES plus either flutmaide or GnRHa, but otherwise remained relatively constant between treatment groups. It is concluded that, in the neonatal rat, (1) endogenous androgens, as well as FSH, play a physiological role in increasing Sertoli cell number, (2) exogenous oestrogen exposure can decrease Sertoli cell number without altering FSH levels, (3) these effects probably share a common pathway and (4) blood inhibin-B provides a robust indicator of change in Sertoli cell number.

Abnormal Leydig Cell aggregation in the fetal testis of rats exposed to di (n-butyl) phthalate and its possible role in testicular dysgenesis.

Fetal exposure of male rats to di (n-butyl) phthalate (DBP) induces testicular changes remarkably similar to testicular dysgenesis syndrome in humans; these include induction of focal areas of dysgenetic tubules in otherwise normal testes. In searching for the fetal origins of the latter, we used image analysis to show that exposure to 500 mg/kg DBP [embryonic day (E)13.5-20.5)] caused abnormal aggregation of Leydig cells centrally in the fetal testis. This aggregation was not due to increase in Leydig cell number, and Leydig cell size was significantly reduced in DBP-exposed animals, as were testosterone levels and immunoexpression of P450 side-chain cleavage enzyme. The Leydig cell aggregates did not exhibit evidence of focal proliferation at E17.5-19.5. Using confocal microscopy and Leydig (3beta-hydroxysteroid dehydrogenase) and Sertoli (anti-Mullerian hormone) cell-specific markers, we show that fetal Leydig cell aggregates in DBP-exposed animals trap isolated Sertoli cells within them at E21.5. These areas of intermingled cells are still apparent on postnatal d 4, after cessation of DBP treatment, when they may form misshapen seminiferous cords that trap (intratubular) Leydig cells within them. These centrally located dysgenetic tubules contain germ cells in early puberty, but by adulthood they are Sertoli cell only, implying that presence of intratubular Leydig cells interferes with spermatogenesis. It is concluded that DBP-induced fetal Leydig cell aggregation may be a key event in formation of focal dysgenetic areas in the testis, and identification of the mechanisms underlying these events may give new insights into the fetal origins of testicular dysgenesis syndrome disorders in the human.

Are all EDC effects mediated via steroid hormone receptors?

Despite the surge of interest in the endocrine disruption field, there is still no globally accepted definition of the term. There is a great political will to test chemicals to determine whether they have endocrine disrupting potential. This is a huge task and the US Environmental Protection Agency has taken the lead by setting up an Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC), which with international co-operation, will ultimately deliver a validated testing strategy to detect endocrine disrupting chemicals (EDCs) in humans and wildlife. One of the major developments will be the use of high throughput pre-screening (HTPS) methods that will detect binding to steroid receptor hormones, although many other testing methods will be employed. This paper describes two mechanisms of EDC action that are not mediated via steroid receptors; firstly, the suppression of fetal testosterone synthesis in rodents by in utero exposure to phthalates; and secondly, the ability of several chemicals to interfere with steroid metabolism by inhibiting estrogen sulfotransferases. These examples will be discussed with reference to pertinent human disorders, which have been associated with exposure to EDCs. Issues and questions about how scientists and regulators can deal with these types of chemicals or potential mechanisms in a risk assessment paradigm are raised.

Environmental anti-androgens and male reproductive health: focus on phthalates and testicular dysgenesis syndrome.

The amount of research into endocrine disruption has exploded over the past decade and a re-evaluation of the state of research in this area is timely. There are debates about whether human male reproductive health is really declining and whether endocrine disrupting chemicals play any role in the perceived decline. Most data currently conclude that there are wide geographical variations in semen quality and in the incidence of testicular cancer, cryptorchidism and hypospadias. This review aims to give a brief overview of the issues surrounding the perceived decline in human male reproductive health and the importance of the hormonal environment for the development of the testis and reproductive tract. The consequences for the male reproductive tract of abnormal androgen levels or action are discussed with reference to environmental anti-androgenic compounds. The in vivo data on several anti-androgenic compounds that have been administered to pregnant rodents during the period of male reproductive tract development are assessed with attention to the effects on the male offspring. Finally, the data on in utero phthalate administration are discussed in detail to illustrate the similarities between the effects of some phthalate esters and the human male reproductive tract disorders which comprise testicular dysgenesis syndrome (TDS).

Human 'testicular dysgenesis syndrome': a possible model using in-utero exposure of the rat to dibutyl phthalate.

BACKGROUND: The disorders comprising human 'testicular dysgenesis syndrome' (TDS) may be increasing in incidence. TDS originates in fetal life but the mechanisms are not known, and discerning them requires an animal model. METHODS AND RESULTS: The study investigated whether male rats exposed in utero to dibutyl phthalate [DBP; 500 mg/kg on gestational days (GD) 13-21] would provide a suitable model for human TDS. DBP induced a high rate (>60%) of cryptorchidism (mainly unilateral), hypospadias, infertility and testis abnormalities, similar to those in human TDS. Cell-specific immunohistochemistry and confocal microscopy were used to track development of Sertoli [anti-Müllerian hormone (AMH), Wilm's tumour (WT-1) protein, p27(kip)], Leydig [3beta-hydroxysteroid dehydrogenase (3beta-HSD)], germ (DAZL protein) and peritubular myoid (smooth muscle actin) cells from fetal life to adulthood. In scrotal and cryptorchid testes of DBP-exposed males, areas of focal dysgenesis were found that contained Sertoli and Leydig cells, and gonocytes and partially formed testicular cords; these dysgenetic areas were associated with Leydig cell hyperplasia at all ages. Suppression ( approximately 90%) of testicular testosterone levels on GD 19 in DBP-exposed males, coincident with delayed peritubular myoid cell differentiation, may have contributed to the dysgenesis. Double immunohistochemistry using WT-1 (expressed in all Sertoli cells) and p27(kip) (expressed only in mature Sertoli cells) revealed immature Sertoli cells in dysgenetic areas. DBP-exposed animals also exhibited Sertoli cell-only (SCO) tubules, sporadically in scrotal and predominantly in cryptorchid, testes, or foci of SCO within normal tubules in scrotal testes. In all SCO areas the Sertoli cells were immature. Intratubular Leydig cells were evident in DBP-exposed animals and, where these occurred, Sertoli cells were immature and spermatogenesis was absent. Abnormal Sertoli cell-gonocyte interaction was evident at GD 19 in DBP-exposed rats coincident with appearance of multinucleated gonocytes, although these disappeared by postnatal day 10 during widespread loss of germ cells. CONCLUSIONS: Abnormal development of Sertoli cells, leading to abnormalities in other cell types, is our hypothesized explanation for the abnormal changes in DBP-exposed animals. As the testicular and other changes in DBP-exposed rats have all been reported in human TDS, DBP exposure in utero may provide a useful model for defining the cellular pathways in TDS.

Neonatal coadministration of testosterone with diethylstilbestrol prevents diethylstilbestrol induction of most reproductive tract abnormalities in male rats.

The primary purpose of this study was to evaluate whether the coadministration of testosterone (TE; 200 micro g) with 10 micro g of diethylstilbestrol (DES) between days 2 and 12 postnatally could prevent the adverse gross reproductive tract changes and associated loss of androgen receptor (AR) expression induced by DES treatment alone. Various endpoints (rete testis area, efferent duct lumen area, epithelial cell height of efferent ducts, and vas deferens) were quantified to check for the abnormal changes that have been shown to occur after neonatal treatment with a high dose (10 micro g) of DES. Additionally, DES induction of an aberrant pattern of estrogen receptor alpha (ER-alpha) immunoexpression in the vas deferens and seminal vesicles was evaluated. The coadministration of DES with TE prevented the induction of all but one of the abnormalities induced by DES treatment on its own, coincident with the restoration of normal/supranormal TE levels and normal immunoexpression of the AR and ER-alpha in the tissues studied. The exception was DES-induced lumenal distension of the efferent ducts, which was only partially prevented by the coadministration of DES with TE. These evaluations were made on day 18, but the described abnormalities were already somewhat evident by day 8 in DES-treated animals. It was therefore tested whether a delay of TE replacement until days 8-12 was still able to reverse the abnormalities already induced by DES treatment alone. A delayed treatment with TE reversed the adverse changes in epithelial cell height and in ER-alpha and AR immunoexpression in the same tissues by day 18; however, rete testis overgrowth was only partially prevented, and efferent duct distension was not prevented at all. These results provide further evidence that DES-induced disorders of reproductive tract development in the male result from a disturbance of the androgen-estrogen balance rather than from estrogen action alone.

Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood.

Disorders of testicular function may have their origins in fetal or early life as a result of abnormal development or proliferation of Sertoli cells. Failure of Sertoli cells to mature, with consequent inability to express functions capable of supporting spermatogenesis, is a prime example. In a similar way, failure of Sertoli cells to proliferate normally at the appropriate period in life will result in reduced production of spermatozoa in adulthood. This review focuses on the control of proliferation of Sertoli cells and functional maturation, and is motivated by concerns about 'testicular dysgenesis syndrome' in humans, a collection of common disorders (testicular germ-cell cancer, cryptorchidism, hypospadias and low sperm counts) which are hypothesized to have a common origin in fetal life and to reflect abnormal function of Sertoli (and Leydig) cells. The timing of proliferation of Sertoli cells in different species is reviewed, and the factors that govern the conversion of an immature, proliferating Sertoli cell to a mature, non-proliferating cell are discussed. Protein markers of maturity and immaturity of Sertoli cells in various species are reviewed and their usefulness in studies of human testicular pathology are discussed. These markers include anti-Mullerian hormone, aromatase, cytokeratin-18, GATA-1, laminin alpha5, M2A antigen, p27(kip1), sulphated glycoprotein 2, androgen receptor and Wilms' tumour gene. A scheme is presented for characterization of Sertoli-cell only tubules in the adult testis according to whether or not there is inherent failure of maturation of Sertoli cells or in which the Sertoli cells have matured but there is absence, or acquired loss, of germ cells. Functional 'de-differentiation' of Sertoli cells is considered. It is concluded that there is considerable evidence to indicate that disorders of maturation of Sertoli cells may be a common underlying cause of human male reproductive disorders that manifest at various life stages. This recognition emphasizes the important role that animal models must play to enable identification of the mechanisms via which failure of proliferation and maturation of Sertoli cells can arise, as this failure probably occurs in fetal life.

Effect of neonatal treatment of rats with potent or weak (environmental) oestrogens, or with a GnRH antagonist, on Leydig cell development and function through puberty into adulthood.

This study addressed whether reduced Sertoli cell number or manipulation of the neonatal hormone environment has an influence on final Leydig cell number per testis in the rat, by applying neonatal treatments known to affect these parameters, namely administration of a GnRH antagonist (GnRHa) or diethylstilboestrol (DES, in doses of 10, 1 or 0.1 microg per injection). The effect of treatment with either of two 'environmental oestrogens', bisphenol-A (Bis-A) or octylphenol (OP), was also evaluated. Leydig (3beta-hydroxysteroid dehydrogenase immunopositive) cell development and function (plasma testosterone levels) were studied through puberty into adulthood. Treatment with GnRHa impaired testis growth, Leydig cell (nuclear) volume per testis and testosterone levels during puberty, when compared with controls, but final Leydig cell volume/number in adulthood was comparable with controls. As adult testis weight was reduced by 45% in GnRHa-treated rats, the percentage Leydig cell volume per testis was approximately double (p < 0.01) that in controls, and also at day 35. Testosterone levels in adulthood in GnRHa-treated rats were lower (p < 0.01) than in controls but were within the lower end of the normal range. Treatment with DES caused largely dose-dependent suppression of testis growth, Leydig cell (nuclear) volume per testis and testosterone levels up to day 35. Although by adulthood, Leydig cell volume/number per testis was comparable with controls in DES-treated rats, testosterone levels remained grossly subnormal. Neonatal treatment with either Bis-A or OP had little consistent effect on any of the parameters studied except that both treatments significantly elevated testosterone levels on day 18, as did treatment with DES-0.1 microg. The present findings are interpreted in the context of what is known about the hormonal regulation of Leydig cell development. These lead to the conclusion that final Leydig cell number per testis is not determined by the number of Sertoli cells per testis and appears not to be influenced in any major way by gonadotrophins, androgens or oestrogens in the first 2 weeks of postnatal life. This implies that adult Leydig cell number may be determined prior to birth.

Induction of reproductive tract developmental abnormalities in the male rat by lowering androgen production or action in combination with a low dose of diethylstilbestrol: evidence for importance of the androgen-estrogen balance.

This study tested the hypothesis that testis/reproductive tract abnormalities induced in the rat by neonatal treatment with diethylstilbestrol (DES) result from disturbance of the androgen-estrogen balance. Male rats were treated neonatally with a dose of DES (0.1 micro g) that induced either no or small effects on its own or with a dose (10 micro g) that induced major reproductive tract abnormalities. To allow quantification, the abnormalities chosen for study were distension of the rete testis and efferent ducts and reduction in epithelial cell height in the efferent ducts and vas deferens. To alter the androgen-estrogen balance, other rats were treated with DES (0.1 micro g) in combination with a treatment to suppress either androgen production [GnRH antagonist (GnRHa)] or androgen action (flutamide); other rats were treated with GnRHa or flutamide alone. Testosterone levels were measured to verify the effects of treatment. Combined administration of DES (0.1 micro g) plus GnRHa or flutamide induced significantly greater distension/overgrowth of the rete testis and efferent ducts (ED) and a reduction in epithelial cell height of the ED than did DES (0.1 micro g) administered alone. Neither GnRHa nor flutamide affected rete or ED distension when administered alone, but both significantly reduced ED epithelial cell height. Neonatal treatment with bisphenol-A (100 micro g) with or without GnRHa had no significant effect on any of these parameters. In contrast to the ED, a reduction in cell height of the vas deferens was induced to an equal extent by DES (10 micro g), DES (0.1 micro g) with GnRHa, and GnRHa alone, suggesting greater sensitivity of this tissue to both androgen and estrogen action. The induction of major abnormalities in rats treated with DES (10 micro g) was coincident with loss of androgen receptor immunoexpression in affected tissues. Reduced androgen receptor immunoexpression was also induced by combined treatment with DES (0.1 micro g) plus GnRHa or flutamide, whereas treatment with any of these compounds alone had no or only minor effects. These findings suggest that reduced androgen action sensitizes the reproductive tract to estrogens, demonstrating that the balance in action between androgens and estrogens, rather than their absolute levels, may be of fundamental importance in determining normal or abnormal development of some regions of the male reproductive tract.

Modulation of the onset of postnatal development of H(+)-ATPase-rich cells by steroid hormones in rat epididymis.

Vacuolar type H(+)-ATPase is involved in lumenal acidification of the epididymis. This protein is highly expressed in narrow and clear cells where it is located in the apical pole, and it contributes to proton secretion into the lumen. We have previously shown that in rats, epididymal cells rich in H(+)ATPase appear during postnatal development and reach maximal numbers at 3-4 wk of age. The factors that regulate the appearance of these cells have not been investigated, but androgens, estrogens, or both may be involved. This study examined whether neonatal administration of estrogens (diethylstilbestrol [DES] or ethinyl estradiol) or an antiandrogen (flutamide), or the suppression of androgen production via administration of a GnRH antagonist (GnRHa), was able to alter the appearance of cells rich in H(+)-ATPase in the rat epididymis when assessed at age 25 days. Surprisingly, all of these treatments were able to significantly reduce the number of H(+)-ATPase positive cells; this was determined by immunofluorescence and confirmed by Western blotting. In contrast, neonatal coadministration of DES and testosterone maintained the expression of H(+)-ATPase in the epididymis at Day 25 despite the high level of concomitant estrogen exposure. These findings indicate that androgens, acting via the androgen receptor, are essential for the normal development of epididymal cells rich in H(+)-ATPase, and that treatments that interfere directly or indirectly with androgen production (GnRHa, DES) or action (flutamide, DES) will result in reduced expression of H(+)-ATPase. Our findings do not exclude the possibility that estrogens can directly suppress the postnatal development of cells in the epididymis that are rich in H(+)-ATPase, but if this is the case, this suppression can be prevented by testosterone administration.