Chlorinated biphenyls effect on estrogen-related receptor expression, steroid secretion, mitochondria ultrastructure but not on mitochondrial membrane potential in Leydig cells.

To characterize polychlorinated biphenyls (PCBs) action on Leydig cells, PCBs congeners, low-chlorinated (delor 103; d103) and high-chlorinated ones (delor 106; d106) were selected. The cells were treated according to PCBs dose (d103 or d106 0.2 ng/ml in low doses:, or 2 ng/ml in high doses) and type (d103 + d106 in low doses or 103 + 106 in high doses). After 24 h treatment with PCBs, a distinct increase in estrogen-related receptors (ERRs type α, ß and γ) expression was revealed. However, the dose- and type-dependent PCBs effect was mostly exerted on ERRα expression. A similar increase in ERRs expression was demonstrated by estradiol but not testosterone, which was without an effect on ERRs. PCBs caused no decrease in the membrane potential status of Leydig cells (either in dose or type schedule) but had severe effects on the mitochondria number and structure. Moreover, PCBs markedly increased calcium (Ca2+) concentration and sex steroid secretion (both androgens and estrogens were elevated). These findings suggest a similar estrogenic action of PCBs congeners (d103 and d106) on Leydig cell function. We report dose- and type-specific effects of PCBs only on Leydig cell ERRs expression. Both delors showed common effects on the mitochondria ultrastructural and functional status. Based on our results, ERRα seems to be the most sensitive to hormonal modulation. The increases in Ca2+ and sex steroid secretion may be due to the activation of ERRs by PCBs binding and/or direct effect of PCBs on ERRs mRNA/protein expression. Nevertheless, to confirm the existence of possible relationships between ERRs signaling (including PCBs as ligands) and mitochondria function in Leydig cells, further intensive studies are needed.

Effects of testosterone and prolactin on steroidogenesis in post-ovulatory cumuli oophori and on in vitro oocyte fertilisation in the rat.

The main objective of these studies was to determine the in vitro effects of prolactin (PRL) and testosterone (T) on steroidogenic function in post-ovulatory cumuli oophori containing unfertilised (ufCOCs) or fertilised (fCOCs) oocytes and to determine the differences between ufCOCs and fCOCs. In vivo, progesterone (P4) content was distinctly higher in isolated ampullae containing ufCOCs than in those containing fCOCs. Moreover, the expression of androgen (ARs) and prolactin (PRL-Rs) receptors was distinctly higher in ufCOCs than in fCOCs. Also, in vitro P4 profiles were generally higher in incubated ufCOCs, which had very high secretion rates of this steroid, especially after treatment with PRL+T. Testosterone significantly increased P4 levels only in incubated fCOCs, while the anti-androgen dihydroxyflutamide (2-Hf) markedly decreased P4 levels in both ufCOCs and fCOCs. Among post-incubation ufCOCs fertilised in vitro, the highest fertilisation rate was observed for oocytes in ufCOCs exposed to PRL+T, while those incubated with 2-Hf or T+2-Hf were not fertilisable. These studies establish differences in steroidogenic function and expression of ARs and PRL-Rs between post-ovulatory ufCOCs and fCOCs, with higher concentrations of P4 being observed in the microenvironment of ufCOCs. PRL+T stimulated P4 production by ufCOCs and increased in vitro fertilisation rate.

Col-F, a fluorescent probe for ex vivo confocal imaging of collagen and elastin in animal tissues.

A new low-molecular-weight fluorescent probe, Col-F, that exhibits affinity to collagen and elastin, was used successfully in imaging of extracellular matrix in freshly excised animal tissues. Col-F readily penetrates between live cells into tissues and binds to fibers of collagen and elastin by a noncovalent mechanism. Fibers of collagen and elastin have been stained in a variety of tissues, including tendon, skeletal muscle, connective tissue, and arteries. Cells migrating in a Col-F-stained collagenous biomaterial were also imaged. No phototoxic effects were detected when live keratocytes were imaged in the in vitro culture in the presence of Col-F. In conclusion, Col-F provides a simple and convenient tool for fluorescence three-dimensional imaging of intricate collagenous and elastic structures in live and fixed animal tissues, as well as in collagen-containing biomaterials.

Expression of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in the ovarian follicles and corpora lutea of pregnant swine.

The objective of the study was to demonstrate the presence of estrogen receptor alpha (ERalpha) and beta (ERbeta) protein and corresponding mRNA in porcine ovarian follicles and corpora lutea obtained on day 10, 18, 32, 50, 71 and 90 post coitum (p.c.) using immunohistochemistry, Western blot, and RT-PCR analysis. Immunohistochemistry showed that ERalpha protein was located in the granulosa cells of ovarian follicles and the strongest immunoreaction was observed on days 32 and 50 p.c. The ERbeta protein was found mainly in theca cells of follicles as well as in luteal cells. The most intense immunoreaction was observed on day 18 p.c. within theca cells, while in the corpus luteum (CL) the intensity of ERbeta staining gradually increased and remained elevated at mid and late pregnancy. In CL by day 50 p.c. immunoreaction for ERbeta was present only in small luteal cells, but starting from day 71 to 90 p.c. it was observed in both small and large luteal cells. Western blot analysis was performed and validated data obtained from immunohistochemistry. RT-PCR results indicated that ERalpha mRNA was expressed only in ovarian follicles of the pregnant swine, while that of ERbeta in both follicles and CL. The results suggest an autocrine/paracrine role of estrogens acting via both ERalpha and ERbeta in the regulation of the ovarian function during pregnancy and for the process of successful reproduction.

Distribution of androgen receptor and steroid hormone concentrations in ovaries of immature bank voles: effect of photoperiod.

Immunolocalisation of androgen receptor (AR) and steroid contents were analyzed in the ovaries of 7- and 14-day-old bank voles, reared in a long (LD) and short (SD) photoperiod. The strongest AR immunoreaction was found in the stromal cells, especially in the ovaries of 7-day-old animals, and in the granulosa cells of all types of ovarian follicles. Oocytes and the cells of surface epithelium were AR positive. The amount of ovarian androgens was relatively high, whereas the level of estradiol was negligible. This finding, and the presence of numerous ARs in various ovarian compartments, suggest that aromatization was very low during development and the primary function of androgens was hormonal action via a receptor-mediated pathway. Age- and photoperiod-related differences in ovarian progesterone (P4) levels were higher in animals kept in LD than in SD, rising significantly on day 14. Androgen content tended to be lower in LD voles and slightly decreased on day 14. Photoperiod-related differences concerning AR immunolabeling were apparent only in 14-day-old animals. In LD, ovaries already possessed early antral follicles, showing strong AR immunolabeling in the cumulus cells. Immunoreaction of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) showed that the primary interstitial and theca cells were the first to be active in ovarian steroidogenesis. In conclusion, AR is present in juvenile vole ovaries as early as day 7. The influence of the photoperiod on their number is observed beginning on day 14. Differences in steroid contents due to LD conditions occur in 7-day-old, and progresses in 14-day-old animals.

Androgen receptor in the ovary of postnatal bank vole females.

In this experiment, we investigated the influence of the photoperiod upon androgen receptor (AR) distribution and steroid concentrations in ovaries of 21-day-old bank vole females. Sections (6 mum) were assayed immunohistochemically. ARs were localized in the nuclei of granulosa cells. The strongest immunoreaction was observed in primary and early antral follicles, and declined during follicular development. P(4), A, and E(2) contents were measured by RIAs in ovarian homogenates from animals kept in two photoperiods. Ovaries of animals kept in a long photoperiod (18:6 light:dark), which stimulates gonadal activity, contained more P(4) than those kept in a short one (6:18 light:dark). There was no difference in levels of A and E(2).

Increased progesterone production in cumulus-oocyte complexes of female mice sired by males with the Y-chromosome long arm deletion and its potential influence on fertilization efficiency.

It was revealed previously that B10.BR(Y(del)) females sired by males with the Y-chromosome long arm deletion differ from genetically identical B10.BR females sired by males with the intact Y chromosome. This is interpreted as a result of different epigenetic information which females of both groups inherit from their fathers. In the following study, we show that cumulus-oocyte complexes ovulated by B10.BR(Y(del)) females synthesize increased amounts of progesterone, which is important sperm stimulator. Because their extracellular matrix is excessively firm, the increased progesterone secretion belongs presumably to factors that compensate this feature enabling unchanged fertilization ratios. Described compensatory mechanism can act only on sperm of high quality, presenting proper receptors. Indeed, low proportion of sperm of Y(del) males that poorly fertilize B10.BR(Y(del)) oocytes demonstrates positive staining of membrane progesterone receptors. This proportion is significantly higher for sperm of control males that fertilize B10.BR(Y(del)) and B10.BR oocytes with the same efficiency.

Immunohistochemical localization of androgen receptor and aromatase in the ovary of the pregnant pig.

The distribution of androgen receptor (AR) and cytochrome P450 aromatase was investigated in paraffin sections of pregnant pig ovary. Ovarian follicles and corpora lutea were isolated from ovaries obtained on Days 10, 18, 32, 71 and 90 post coitum (p.c.). Androgen receptor was localized in the nuclei of granulosa cells of follicles of various sizes. In addition, some follicles demonstrated staining in the nuclei of theca interna cells. Stroma cells also exhibited a positive immunostaining. At early and mid pregnancy (up to Day 71) AR was expressed in the nuclei of luteal cells. Corpora lutea of Day 71 showed mainly cytoplasmic staining while on Day 90 almost all luteal cells showed staining exclusively in the cytoplasm. Immuno-staining for the presence of cytochrome P450 aromatase was very faint in all investigated ovarian structures. The results could suggest that the process of androgen aromatization plays a negligible role in the ovary of the pregnant pig.

Effect of photoperiod on the distribution patterns of androgen receptors and steroid hormone concentrations in ovaries of bank voles.

Cellular distribution patterns of the androgen receptor in ovaries of female bank voles, born and reared in long (18:6; LD) or short (6:18; SD) photoperiods, have been studied to understand effects of androgens in female gonads. The photoperiod is one of the most important factors in bank voles, which are seasonal breeders, that regulates both morphology and hormonal function of the ovary. Androgen receptors were visualized immunocytochemically, using a specific monoclonal antibody against androgen receptor protein. LD ovaries contained more follicles and showed a different androgen receptor distribution pattern than SD ovaries. In LD ovaries, androgen receptors were strongly expressed in granulosa cells of primordial, primary, and preantral follicles as well as in theca and stromal cells. Positivity was moderate and limited to antral and cumulus regions in large follicles of LD ovaries. In contrast, androgen receptor immunopositivity was intense in the granulosa layer, theca and interstitial cells of large follicles of SD ovaries. A novel observation was the very intense immunostaining of oocyte cytoplasm in primordial and primary unilaminar follicles of LD ovaries. It can be concluded that the androgen receptor is involved in the maturation of oocytes.

TGFß (transforming growth factor ß) superfamily members and their receptors in the fetal porcine ovaries: effect of prenatal flutamide treatment.

INTRODUCTION: We have recently demonstrated that antiandrogen treatment during fetal life resulted in delayed folliculogenesis. The aim of the present study was to investigate the effect of androgen deficiency induced by flutamide on the expression of TGFß superfamily members and their receptors which are involved in follicle formation and its transition to the primary stage. MATERIAL AND METHODS: Pregnant gilts were injected with flutamide (for seven days, 50 mg/day/kg b.w.) or corn oil (controls) starting at 43 (GD50), 83 (GD90) or 101 (GD108) gestational day. The expression in fetal ovaries of selected TGFß superfamily members (AMH, BMP4, GDF9), their receptors (AMHR-II, BMPR-IB, BMPR-II), and Smad1 and Smad3 proteins involved in signal transduction were investigated by real-time PCR and/or immunohistochemistry. RESULTS: Flutamide treatment increased the expression of BMP4 mRNA on GD50 and GD108 and BMPR-IB mRNA on GD50. The expression of BMPR-II was decreased at mRNA level and lower immunostaining intensity was observed after flutamide administration only on GD50. GDF9 and AMHR-II mRNA expression levels were significantly downregulated in both GD90 and GD108 groups. However, AMHR-II was immunolocalized only on GD108 and less positively stained oocytes were found after flutamide treatment. AMH mRNA level was diminished in the GD90 group, while it was elevated in the GD108 group. Moreover, the higher amounts of positively stained oocytes for phosphorylated form of Smad1 were observed following flutamide administration on GD108. CONCLUSIONS: Experimentally-induced androgen deficiency during fetal development deregulates the expression level of some of TGFß superfamily members and their receptors which may affect primordial follicle assembly. Our findings further underline the role of androgens in the early stages of follicle development.

Does 4-tert-octylphenol affect estrogen signaling pathways in bank vole Leydig cells and tumor mouse Leydig cells in vitro?

Primary Leydig cells obtained from bank vole testes and the established tumor Leydig cell line (MA-10) have been used to explore the effects of 4-tert-octylphenol (OP). Leydig cells were treated with two concentrations of OP (10(-4) M, 10(-8) M) alone or concomitantly with anti-estrogen ICI 182,780 (1 µM). In OP-treated bank vole Leydig cells, inhomogeneous staining of estrogen receptor α (ERα) within cell nuclei was found, whereas it was of various intensity among MA-10 Leydig cells. The expression of ERα mRNA and protein decreased in both primary and immortalized Leydig cells independently of OP dose. ICI partially reversed these effects at mRNA level while at protein level abrogation was found only in vole cells. Dissimilar action of OP on cAMP and androgen production was also observed. This study provides further evidence that OP shows estrogenic properties acting on Leydig cells. However, its effect is diverse depending on the cellular origin.

Primary culture of ovarian cells for research on cell interactions in the hormonal control of steroidogenesis.

Ovary is a polymorphic complex structure in which the cells are arranged in two essential endocrine mini glands: the follicle (F) and the corpus luteum (CL). Their secretory function creates an optimal milieu for growth, maturation, and selection of follicles and oocytes competent for ovulation. Monoculture of isolated ovarian cells has identified the secretory potential of the different cell types functioning in this complex gland in vivo. Primary culture of isolated ovarian cells is a good tool for the investigation of cell interactions and its impact on steroidogenesis, dynamics of steroidogenic enzymes, hormone receptors, changes in the cytoskeleton in granulosa cell populations, regulatory mechanisms, and the intracellular pathways of gonadotropin signaling in steroidogenic ovarian cells. Since the granulosa cell number recovered during isolation from the follicle is substantial, this makes primary culture fairly easy and enables many kinds of studies in vitro. Ovarian cells are highly differentiated and express characteristic functional specificity dependent on the dynamics of the sexual cycle. This is important so as not to produce artifacts in vitro.

Steroid concentrations and immunoexpression of steroidogenic enzymes in ovaries of aged bank voles: effect of photoperiod.

The main objective of the present study was to establish morphological and steroidogenic changes occurring in the ovaries of senescent bank voles, with respect to the photoperiod of rearing. Obtained results revealed less pronounced changes in the ovaries of females reared in a long photoperiod (LD). Their gonads still possessed some healthy follicles and old corpora lutea (CLs). Senescence-related changes encompassed the presence of abnormal follicles, large regions containing extra-follicular luteinized granulosa cells and numerous clusters of hypertrophied theca/interstitial cells, exhibiting strong expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and much weaker that of cytochrome P450c17. More pronounced changes were observed in animals reared in short day (SD) conditions and included the presence of only few, usually abnormal follicles and/or remnants of CLs in the surface region, and the isle-like clusters of cells in the ovarian medulla. The clusters were composed of cells generally featuring strong 3ß-HSD and/or P450c17 immunoreaction. Steroid content analysis revealed that progesterone dominated in the ovaries of LD bank voles and androgens in SD animals, while estradiol content was very low in both investigated groups. These studies showed for the first time morphological and steroidogenic changes found in the ovaries of senescent bank voles and indicated an important role of length light conditions in the process of reproductive aging.

Steroid levels and the spatiotemporal expression of steroidogenic enzymes and androgen receptor in developing ovaries of immature rats.

Immunoexpression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cytochrome P450c17 (P450c17), androgen receptor (AR), and steroid contents were studied in the ovaries of immature female Wistar rats killed between postnatal days 1 and 30. During days 1-7, ovarian somatic structures lacked AR, 3ß-HSD and P450c17, except for the surface epithelium, which featured the presence of these three proteins, suggestive of its androgen responsiveness and steroidogenic function. On day 10, AR appeared in many somatic structures, including the granulosa layers, which coincided with the P450c17 immunoexpression in some theca/interstitial cells, and an increase in ovarian androgen concentration. On the following days a further rise in ovarian androgen and progesterone contents paralleled an increase in 3ß-HSD and P450c17 immunoexpression in the theca layer cells and primary interstitial cells. However, the development of the follicles constituting the first follicular wave was aberrant, since they lacked AR expression until the preantral stage and were characterized by a delayed onset and much lower expression of the thecal P450c17. They could not ovulate, since ovarian content of estradiol was too low to evoke the LH surge. The clusters of the secondary interstitial cells found on day 30 exhibited predominant expression of 3ß-HSD over P450c17, suggesting more intensive progesterone than androgen synthesis in these structures.

Effects of 4-tert-octylphenol on the testes and seminal vesicles in adult male bank voles.

The present study was designed to evaluate the effects of 4-tert-octylphenol (OP) on male testes and seminal vesicles of bank vole. Adult males kept under long or short photoperiod were orally administered OP (200mg/kg bw) for 30 or 60 days. Treatment for 30 days had no discernible effect on the parameters examined. Treatment for 60 days adversely influenced weights and histological structure of the testes and seminal vesicles. In these tissues, expression of 3ß-hydroxysteroid dehydrogenase and androgen receptor and testosterone levels were reduced, whereas expression of aromatase and estrogen receptor α and estradiol levels were increased. The alterations were more evident in voles kept in long photoperiod. Taken together, it is suggested that adverse changes in bank vole reproductive tissues induced by long-term OP-exposure result from disturbed androgen and estrogen synthesis and action. Moreover, there might be a subtle difference in the sensitivity to OP between voles kept in different light conditions.

Morphological and functional alterations in testes and efferent ducts of homogametic rainbow trout Oncorhynchus mykiss Walbaum.

In the present study we analyzed the morphology of testes and efferent ducts as well as the localization of androgen receptors (AR) in the tissues of XY, YY, and XX male rainbow trout Oncorhynchus mykiss Walbaum. Testes of the XX trout differed distinctly from those of XY and YY males; the interstitial space was very narrow and lumina of the tubules were densely filled with spermatozoa. There was no efferent duct. The nuclei of Leydig cells and endothelial cells of blood vessels were positively stained for AR, and this was confirmed by quantitative image analysis. Differences in the AR immunoexpression levels indicate the differences in the levels of androgens produced by the Leydig cells, possibly based on the chromosomal constitution of the testes concerned.

Changes in germ cells population and in vitro oocyte maturation in female mice with imperforated vagina.

This study characterized female mice with a congenital defect in their reproductive tract. In females derived from an outbred colony maintained in the Department of Genetics and Evolution, the frequency of the imperforated vaginae was approximately 2%. A consequence of this defect is infertility. Affected animals developed hydrometrocolpos (the uterus and vagina were greatly distended by fluid). Morphology of the ovary and oviduct was normal and similar to that of control mice. In the females with an imperforated vagina numerous corpora lutea were observed. Accordingly, we have found that both spontaneous as well as exogenous gonadotrophin-induced ovulation occurs in such females. Nevertheless, oocytes derived from ovaries of occluded females exhibited a partial block during in vitro maturation. Histological analysis of ovarian tissue revealed an increase in the number of primary follicles (type I follicles) and a decrease in the number of secondary and antral follicles (type IV and type VI follicles) when compared with control mice. The concentration of androgens in the ovarian tissue was higher in the affected females. Our data show that females with an imperforated vagina can be a useful model for studying the mechanism of genetic control of the development of the urogenital tract in mammals.