Jerzy Galas

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 3β-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.

Expression of estrogen receptor α (ERα) and estrogen receptor β (ERβ) 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.

The expression of FSH receptor (FSHR) in the neonatal porcine ovary and its regulation by flutamide.

This study was designed to reveal the FSHR mRNA and protein expression in the neonatal porcine ovary and to determine whether maternal administration of antiandrogen flutamide may affect FSHR expression in the ovary of newborn piglets using real-time PCR, immunohistochemistry and Western blot analysis. Pregnant sows were injected with flutamide at a dose of 50 mg/kg body weight, given five times, every second day, starting at day 20 post-coitum (p.c.) or day 80 p.c., and ovaries were obtained from neonatal pigs. The FSHR mRNA expression was significantly decreased after flutamide administration. Furthermore, higher down-regulation was observed following exposure to antiandrogen at day 20 than at day 80 p.c. Immunohistochemistry showed the positive immunostaining for FSHR in the oocytes, granulosa cells of primary follicles and the surface epithelium of the ovaries from both control and flutamide-treated pigs. However, oocytes and granulosa cells of primary follicles in the ovaries exposed in utero to flutamide were weakly immunostained when compared to those in the control ones. The presence of FSHR protein in all investigated ovaries was confirmed by Western blot analysis. Based on our findings, we suggest that FSHR may be involved in the early follicle formation in pigs, which begins during prenatal life. Furthermore, the regulation of FSHR mRNA and protein expression in neonatal porcine ovaries after maternal exposure to flutamide confirms that androgens play a crucial role in porcine folliculogenesis at the early stages.

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.

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

Immunoexpression of 3β-hydroxysteroid dehydrogenase (3β-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, 3β-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 3β-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 3β-HSD over P450c17, suggesting more intensive progesterone than androgen synthesis in these structures.

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.

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.

Primary and tumor mouse Leydig cells exposed to polychlorinated naphthalenes mixture: Effect on estrogen related-receptors expression, intracellular calcium level and sex hormones secretion.

We report the effects of polychlorinated napthalanes (PCNs) on the mRNA expression of estrogen-related receptors (ERRs) α, β and γ, calcium (Ca2+) concentration, and sex steroid secretion in mouse primary and tumor Leydig cells. The cells were exposed to a mixture of PCNs (10nM) alone or in combination with one of sex steroid receptor antagonists; 182,780 (ICI; 10μM); hydroxyflutamide (HF; 10(-4)M) and G-coupled estrogen receptor antagonist (G15; 10nM) respectively. The expression of mRNAs and protein for ERRα, β, and γ was detected in primary and tumor Leydig cells. The expression of ERRs was always lower in primary Leydig cells. Exposure of Leydig cells to PCNs significantly increased the expression of ERRs mRNA irrespective of the cell type. Concomitantly, an increased concentration of Ca2+ and sex steroids was revealed in exposed cells. After ICI, HF or G15 was added no changes in expression of ERRs was found. In Leydig cells changes in ERRs expression at mRNA level are clearly linked to changes in Ca2+ level and steroid secretion. Estrogen and androgen receptors are not involved in PCNs action in Leydig cells. The effect of PCNs on mouse Leydig cells is independent on the cell of origin (primary or tumor).

Expression of androgen receptor and 3β-hydroxysteroid dehydrogenase in corpora lutea during pregnancy in the rat

Immunoexpression of androgen receptor (AR) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) was investigated in three generations of corpora lutea (CLs), found in the ovaries of rats on Days 1, 2, 5, 9, 14 and 20 of pregnancy. The youngest generation of CLs functioned during the whole pregnancy, whereas the older and the oldest generations underwent earlier regression. The newly formed CLs exhibited weak cytoplasmic 3beta-HSD expression. During subsequent days, a gradual increase in 3beta-HSD immunolabelling was observed, followed by a decrease on Day 20. In the older and the oldest CLs, surviving luteal cells demonstrated strong, although in the oldest CLs mostly perinuclear, 3beta-HSD immunoreaction. The newly formed CLs showed weak nuclear AR immunolabelling, which became stronger during the following days. On Day 20, luteal cells demonstrated a weaker nuclear immunoreaction. The older and oldest generations of CLs exhibited weaker and almost negative AR immunolabelling, respectively. Of special interest was the richly vascularised apical region of young CLs. Here luteal cells with more intensive 3beta-HSD staining predominated, whereas cytoplasmic AR immunoreaction was accompanied by positive or negative nuclear AR immunoexpression. The present studies showed differences in AR and 3beta-HSD distribution within various generations of CLs and within particular regions of the same young CL.

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(Ydel) 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(Ydel) 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 Ydel males that poorly fertilize B10.BR(Ydel) oocytes demonstrates positive staining of membrane progesterone receptors. This proportion is significantly higher for sperm of control males that fertilize B10.BR(Ydel) and B10.BR oocytes with the same efficiency.