Maria Szołtys

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.

Immunolocalization of aquaporin 5 during rat ovarian follicle development and expansion of the preovulatory cumulus oophorus

Immunofluorescent localization of aquaporin 5 (AQP5) was investigated in rat ovarian follicles during development and preovulatory cumulus oophorus expansion. Ampullary cumuli oophori complexes (COCs) were examined. Analysis revealed that AQP5 immunostaining appeared in preantral follicles and formed a characteristic ring encircling and touching the oolemma. The staining represented most likely AQP5 functioning at the ends of corona radiata cell projections, anchoring on the oocyte surface. However, several hours after the presumptive preovulatory LH surge, when the process of expansion of COCs started, the AQP5 staining appeared also on the cumulus granulosa cells and in the extracellular matrix. In the postovulatory ampullary COCs the fluorescent ring was not observed, which may be the result of the well-established preovulatory withdrawal of projections from the zona pellucida. At that time-point immunofluorescent staining of AQP5 appeared in most oocytes and was also present in the apical membrane of epithelial cells of the oviduct ampulla. The latter observation suggests that after ovulation AQP5 is involved in the transcellular movement of water in the oviduct ampulla and oocytes in rats.

Hormonal secretion of cultured rat ovarian follicles isolated at various hours of proestrus

SummaryRat ovarian follicles were isolated at nine different times during proestrus and cultured for 3 d. During the period of cultivation, estrogen and progestagen secretion was measured. Other follicles were dissected out at the same time periods and analyzed directly for these hormones. The most intense hormone release was observed during the first 24 h in culture. The concentration of steroids secreted in vitro by follicles previously collected on the morning of proestrus was relatively low. The secreted estrogen concentration increased gradually from follicles previously isolated at later times during proestrus, with a maximum at 1800. The peak secretion of progestagens in culture was from follicles isolated at 2000. There was a marked decrease in secretion of both hormones from follicles isolated past their optimal times. The same secretion pattern of estrogens and progestagens was shown by follicles in vitro at 24 h as with similar follicles in vivo, according to their time of isolation. From these results it is evident that the normal rhythm of steroid secretion, predetermined at the time of follicle excision, was maintained during 24 h in organ culture. After a longer period in culture, the dynamics of hormone synthesis became altered, although follicles appeared morphologically normal up to 48 h.

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.

Immunolocalization of androgen receptor and steroidogenic enzymes in cumuli oophori of pre- and post-ovulatory rats

Immunolocalization of 3β-hydroxysteroid dehydrogense (3β-HSD), cytochrome P450c17 (P450c17) and androgen receptor (AR) were investigated in rat cumuli oophori (COCs) of late pre-ovulatory follicles and in post-ovulatory COCs bearing fertilized oocytes. A gradient of intensity of 3β-HSD immunolabelling was observed in the granulosa layer of pre-ovulatory follicles, with almost negative immunolabelling in COCs and with the strongest immunoreaction in the mural granulosa cells. Post-ovulatory COCs showed strong 3β-HSD immunolabelling in the peripheral regions and weak labelling near the oocyte, suggestive of responsiveness of cumulus cells to an anti-luteinizing effect exerted by the fertilized oocyte. In pre-ovulatory follicles, a weak P450c17 immunopositivity was limited to expanded cumulus granulosa cells and the positive labelling persisted in post-ovulatory COCs. P450c17 immunopositivity was also found in ampullary epithelial cells. A strong AR immunopositivity was confined mainly to the COCs in pre-ovulatory follicles and a similar immunoreaction was present in the granulosa cells of ovulated COCs. Simultaneous AR and cytochrome P450c17 immunolabelling in the pre- and post-ovulatory COCs is suggestive of an intra- and paracrine androgen regulation of the cumulus granulosa cell function.

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.

Steroid hormone content in the submandibular gland of normal and pregnant rats.

Using radioimmunoassay, the levels of steroid hormones were determined in the submandibular gland homogenates from sexually immature, mature male and female and pregnant rats. High progestagen content was established in the whole gland of maturing and mature females and males. The highest level of progestagens was in the last pregnancy trimester. High androgen content was found in maturing and mature males only. Androgens in females and oestrogens in both males and females were present in traces only.

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 3β-hydroxysteroid dehydrogenase (3β-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 3β-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.