Anabolic Steroids Activate the NF-κB Pathway in Porcine Ovarian Putative Stem Cells Independently of the ZIP-9 Receptor.

Boldenone (Bdn) and nandrolone (Ndn) are anabolic androgenic steroids (AASs) that, as our previous studies have shown, may increase the risk of neoplastic transformation of porcine ovarian putative stem cells (poPSCs). The NF-κB pathway may be important in the processes of carcinogenesis and tumour progression. Therefore, in this work, we decided to test the hypothesis of whether Bdn and Ndn can activate the NF-κB pathway by acting through the membrane androgen receptor ZIP-9. For this purpose, the expression profiles of both genes involved in the NF-κB pathway and the gene coding for the ZIP-9 receptor were checked. The expression and localization of proteins of this pathway in poPSCs were also examined. Additionally, the expression of the ZIP-9 receptor and the concentration of the NF-κB1 and 2 protein complex were determined. Activation of the NF-κB pathway was primarily confirmed by an increase in the relative abundances of phosphorylated forms of RelA protein and IκBα inhibitor. Reduced quantitative profiles pinpointed not only for genes representing this pathway but also for unphosphorylated proteins, and, simultaneously, decreased concentration of the NF-κB1 and 2 complex may indicate post-activation silencing by negative feedback. However, the remarkably and sustainably diminished expression levels noticed for the SLC39A9 gene and ZIP-9 protein suggest that this receptor does not play an important role in the regulation of the NF-κB pathway.

The Molecular Quality and Mitochondrial Activity of Porcine Cumulus-Oocyte Complexes Are Affected by Their Exposure to Three Endocrine-Active Compounds under 3D In Vitro Maturation Conditions.

Thus far, the potential short- and long-term detrimental effects of a variety of environmental chemicals designated as endocrine-active compounds (EACs) have been found to interfere with histo- and anatomo-physiological functions of the reproductive system in humans and wildlife species. For those reasons, this study sought to examine whether selected EACs, which encompass the fungicide vinclozolin (Vnz), the androgenic anabolic steroid nandrolone (Ndn) and the immunosuppressant cyclosporin A (CsA), affect the developmental competence and molecular quality (MQ) of porcine cumulus-oocyte complexes (COCs) subjected to in vitro maturation (IVM) under 3D culture conditions. The COCs underwent 3D-IVM in the presence of Vnz, Ndn or CsA for 48 h. To explore whether the selected EACs induce internucleosomal DNA fragmentation in cumulus cells (CCs), TUNEL-assisted detection of late apoptotic cells was performed. Additionally, for the detailed evaluation of pro- and antiapoptotic pathways in COCs, apoptosis proteome profiler arrays were used. To determine changes in intracellular metabolism in COCs, comprehensive assessments of mitochondrial ultrastructure and activity were carried out. Moreover, the relative abundances (RAs) of mRNAs transcribed from genes that are involved in scavenging reactive oxygen species (ROS), such as SIRT3 and FOXO3, and intramitochondrial bioenergetic balance, such as ATP synthase subunit (ATP5A1), were ascertained. Finally, to investigate the extent of progression of oocyte maturation, the intraooplasmic levels of cAMP and the RAs of mRNA transcripts encoding regulatory and biocatalytic subunits of a heterodimeric meiosis-promoting factor, termed cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDC2), were also estimated. The obtained results provide, for the first time, strong evidence that both Vnz and Ndn decrease the developmental competence of oocytes and stimulate apoptosis processes in CCs. The present study is also the first to highlight that Vnz accelerates the maturation process in immature oocytes due to both increased ROS production and the augmented RA of the CCNB1 gene. Furthermore, Vnz was proven to trigger proapoptotic events in CCs by prompting the activity of the FOXO3 transcription factor, which regulates the mitochondrial apoptosis pathway. In turn, Ndn was shown to inhibit oocyte maturation by inducing molecular events that ultimately lead to an increase in the intraooplasmic cAMP concentration. However, due to the simultaneous enhancement of the expression of TNF-ß and HSP27 proteins in CCs, Ndn might be responsible for the onset of their neoplastic transformation. Finally, our current investigation is the first to clearly demonstrate that although CsA did not interfere with the nuclear and cytoplasmic maturation of oocytes, by inducing mitophagy in CCs, it disrupted oocyte metabolism, consequently attenuating the parameters related to the MQ of COCs. Summing up, Vnz, Ndn and CsA reduced not only the processes of growth and IVM but also the MQ of porcine COCs, which might make them unsuitable for assisted reproductive technologies (ARTs) such as in vitro fertilization by either gamete co-incubation or intracytoplasmic sperm injection (ICSI) and cloning by somatic cell nuclear transfer (SCNT).

Anabolic Steroids-Driven Regulation of Porcine Ovarian Putative Stem Cells Favors the Onset of Their Neoplastic Transformation.

Nandrolone (Ndn) and boldenone (Bdn), the synthetic testosterone analogues with strong anabolic effects, despite being recognized as potentially carcinogenic compounds, are commonly abused by athletes and bodybuilders, which includes women, worldwide. This study tested the hypothesis that different doses of Ndn and Bdn can initiate neoplastic transformation of porcine ovarian putative stem cells (poPSCs). Immunomagnetically isolated poPSCs were expanded ex vivo in the presence of Ndn or Bdn, for 7 and 14 days. Results show that pharmacological doses of both Ndn and Bdn, already after 7 days of poPSCs culture, caused a significant increase of selected, stemness-related markers of cancer cells: CD44 and CD133. Notably, Ndn also negatively affected poPSCs growth not only by suppressing their proliferation and mitochondrial respiration but also by inducing apoptosis. This observation shows, for the first time, that chronic exposure to Ndn or Bdn represents a precondition that might enhance risk of poPSCs neoplastic transformation. These studies carried out to accomplish detailed molecular characterization of the ex vivo expanded poPSCs and their potentially cancerous derivatives (PCDs) might be helpful to determine their suitability as nuclear donor cells (NDCs) for further investigations focused on cloning by somatic cell nuclear transfer (SCNT). Such investigations might also be indispensable to estimate the capabilities of nuclear genomes inherited from poPSCs and their PCDs to be epigenetically reprogrammed (dedifferentiated) in cloned pig embryos generated by SCNT. This might open up new possibilities for biomedical research aimed at more comprehensively recognizing genetic and epigenetic mechanisms underlying not only tumorigenesis but also reversal/retardation of pro-tumorigenic intracellular events.

A Simplified Method for Three-Dimensional (3D) Porcine Preantral Follicles Culture Utilizing Hydrophobic Microbioreactors.

The technological revolution in reproductive biology that started with artificial insemination procedures and embryo transfer led to the development of assisted reproduction techniques such as in vitro fertilization or even cloning of domestic animals by nuclear transfer from somatic cells. Currently, procedures of isolated immature ovarian follicles in vitro culture are becoming the prominent technology aimed to preserve or restore fertility especially of young oncological patients or those at risk of premature ovarian failure.Here, we describe a protocol that can be applied for in vitro growth of porcine, preantral ovarian follicles in three-dimensional (3D) culture conditions. After enzymatic isolation from the ovarian cortex, preantral follicles are suspended in a drop of medium and enclosed with fluorinated ethylene propylene (FEP) powder particles (microbioreactors). Such microbioreactors maintain the 3D structure of the follicles during the whole process of in vitro growth what is crucial to ensure proper folliculogenesis progression and their ability to survive.

Neonatal exposure to agonists and antagonists of sex steroid receptors induces changes in the expression of oocyte-derived growth factors and their receptors in ovarian follicles in gilts.

The objective of the present study was to examine the effects of neonatal exposure to either agonists or antagonists of androgen and estrogen receptors on the expression of growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) and their cognate receptors (TGFBR1, BMPR1B, and BMPR2) in ovarian follicles of adult pigs. Piglets were injected subcutaneously with testosterone propionate (TP, an androgen, at 20 mg/kg bw), flutamide (FLU, an antiandrogen, at 50 mg/kg bw), 4-tert-octylphenol (OP, an estrogenic compound, 100 mg/kg bw), ICI 182,780 (ICI, an antiestrogen, 400 µg/kg bw), or corn oil (control) between postnatal Days 1 and 10 (n = 5/group). Ovarian follicles were excised from adult pigs on Days 8-11 of the estrous cycle. The expression of GDF9, BMP15, TGFBR1, BMPR1B and BMPR2 were examined in the population of preantral and small antral ovarian follicles using real-time PCR, Western blot and immunohistochemistry. In preantral follicles, the upregulation of GDF9 mRNA and protein expression was found in pigs that were neonatally exposed to TP or FLU, while administration of TP or ICI resulted in upregulation of BMP15. TGFBR1 and BMPR2 mRNA and protein expression were upregulated in preantral follicles of adult pigs that were neonatally exposed to TP or FLU, while administration of TP or ICI resulted in upregulation of BMPR1B. In small antral follicles, the mRNA and protein for TGFBR1 and BMPR2 were upregulated, while BMPR1B was downregulated in response to neonatal OP treatment. In addition, treatment with FLU upregulated BMPR1B and BMPR2 mRNA and protein expression, while downregulated the expression of TGFBR1. Moreover, GDF9 and BMP15 were immunolocalized in oocytes and granulosa cells of preantral follicles obtained from both control and treated ovaries. TGFBR1, BMPR1B and BMPR2 receptors were observed in the oocytes and granulosa cells of preantral follicles as well as in granulosa and theca cells of small antral follicles. In conclusion, the present study demonstrated neonatal exposure to either agonists or antagonists of androgen and estrogen receptors affected GDF9 and BMP15 signalling in ovaries of adult pigs. It seems that neonatal androgen excess or deficiency may lead to the acceleration of initial follicle recruitment, while neonatal exposure to compounds with antiandrogenic and estrogenic activity may disturb small antral follicles fate. Therefore, it confirms that neonatal window is critical for programming of ovarian function in pigs.

Stress differentially affects the systemic and leukocyte estrogen network in common carp.

Both systemic and locally released steroid hormones, such as cortisol and estrogens, show immunomodulatory actions. This research gives evidence that circulating and leukocyte-derived estrogens can be involved in the regulation of the immune response in common carp, during homeostasis and upon restraining stress. It was found that stress reduced level of blood 17ß-estradiol (E2) and down-regulated the gene expression of components of the "classical" estrogen system: the nuclear estrogen receptors and the aromatase CYP19, in the hypothalamus, the pituitary and in the ovaries. In contrast, higher gene expression of the nuclear estrogen receptors and cyp19a was found in the head kidney of stressed animals. Moreover, stress induced changes in the E2 level and in the estrogen sensitivity at local/leukocyte level. For the first time in fish, we showed the presence of physiologically relevant amounts of E2 and the substrates for its conversion (estrone - E1 and testosterone - T) in head kidney monocytes/macrophages and found that its production is modulated upon stress. Moreover, stress reduced the sensitivity of leukocytes towards estrogens, by down-regulation the expression of the erb and cyp19 genes in carp phagocytes. In contrast, era expression was up-regulated in the head kidney monocytes/macrophages and in PBLs derived from stressed animals. We hypothesize that, the increased expression of ERα, that was observed during stress, can be important for the regulation of leukocyte differentiation, maturation and migration. In conclusion, these results indicate that, in fish, the estrogen network can be actively involved in the regulation of the systemic and local stress response and the immune response.

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.

On the trail of the glycan codes stored in cancer-related cell adhesion proteins.

Changes in the profile of protein glycosylation are a hallmark of ongoing neoplastic transformation. A unique set of tumor-associated carbohydrate antigens expressed on the surface of malignant cells may serve as powerful diagnostic and therapeutic targets. Cell-surface proteins with altered glycosylation affect the growth, proliferation and survival of those cells, and contribute to their acquisition of the ability to migrate and invade. They may also facilitate tumor-induced immunosuppression and the formation of distant metastases. Deciphering the information encoded in these particular glycan portions of glycoconjugates may shed light on the mechanisms of cancer progression and metastasis. A majority of the related review papers have focused on overall changes in the patterns of cell-surface glycans in various cancers, without pinpointing the molecular carriers of these glycan structures. The present review highlights the ways in which particular tumor-associated glycan(s) coupled with a given membrane-bound protein influence neoplastic cell behavior during the development and progression of cancer. We focus on altered glycosylated cell-adhesion molecules belonging to the cadherin, integrin and immunoglobulin-like superfamilies, examined in the context of molecular interactions.

Androgen receptor-mediated non-genomic effects of vinclozolin on porcine ovarian follicles and isolated granulosa cells: Vinclozolin and non-genomic effects in porcine ovarian follicles.

The present study investigated the influence of the androgen receptor (AR) agonists testosterone (T) and dihydrotestosterone (DHT), and vinclozolin (Vnz), a fungicide with antiandrogenic activity, on non-genomic signal transduction within ovarian follicles. Porcine granulosa cells (GCs) isolated from mature follicles were cultured for 48h. For the last 24h of culture, they were exposed to T (10(-7)M), DHT (10(-7)M), Vnz (1.4×10(-5)M), T and Vnz (T+Vnz), or DHT and Vnz (DHT+Vnz) at the same concentrations. To better imitate in vivo conditions, whole follicles (4-6mm in diameter) were incubated (24h) in an organ culture system with the same factors. Expression of AR mRNA and protein was determined by real-time PCR and western blot analyses. To demonstrate AR localization in cultured GCs and whole follicles, immunocytochemistry and immunohistochemistry were performed, respectively. To elucidate the possible non-genomic action of Vnz in GCs, protein expression and the activity of ERK1/2 and Akt kinases were determined by western blot and ELISA analyses. The immunocytochemistry and immunohistochemistry results showed that exposure of GCs and follicles to Vnz resulted in cytoplasmic and perinuclear AR localization. Real-time PCR and western blot analysis showed that AR mRNA and protein expression increased (P≤0.001) in GC cultures after combined treatment with an androgen and Vnz. In whole follicles, such treatment also increased AR mRNA with a decrease in the respective protein expression (P≤0.001). Moreover, addition of T or DHT with Vnz increased the activity of ERK1/2 and Akt kinases in cultured GCs (P≤0.001). The results suggest a novel mechanism for Vnz action in porcine ovarian follicles on both AR mRNA and protein levels. Thus, this environmental antiandrogen activates non-genomic signaling pathways, as indicated by the increased activity of both investigated kinases observed within minutes of Vnz addition. Given the widespread presence of Vnz in the environment, elucidation of its non-genomic action should be the subject of studies on female fertility.

The impact of antiandrogen 2-hydroxyflutamide on the expression of steroidogenic enzymes in cultured porcine ovarian follicles.

We used our model system for agonism and antagonism of the androgen receptor (AR), in which the porcine ovarian follicles were exposed on the excessive concentration of an AR agonist- testosterone (T) or an AR antagonist- 2-hydroxyflutamide (2-Hf) to: (1) analyze the spatiotemporal expression of ovarian 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cytochrome P450 17α-hydroxylase/c17,20-lyase (P450c17) and cytochrome P450 aromatase (P450arom); (2) to determine the contribution of AR-mediated action during steroidogenesis and (3) to establish some correlations between the onset and expression pattern of the investigated proteins. Whole follicles (6-8 mm in diameter) isolated from mature porcine ovaries have been incubated (for 24 h) in an organ culture system in the presence of T (10(-7 )M), 2-Hf (1.7 × 10(-4) M) or both T and 2-hydroxyflutamide (T+2-Hf, at the same concentrations as when added separately). Thereafter, sections obtained from cultured follicles were processed for main steroidogenic enzymes detection by immunohistochemistry. Moreover, expression of their mRNA and protein was determined by real-time PCR and Western blot analysis. Progesterone, androgens and estradiol concentrations in the culture media were measured by radioimmunoassays (RIA). Our results demonstrated that 2-Hf can influence the steroidogenic activity of porcine follicles in vitro through the blockade of AR. It was shown that follicular 2-Hf treatment brought about dramatic decline in the production of the investigated steroids. What is more the addition of 2-Hf separately caused a negative effect on 3ß-HSD and P450c17 mRNA and protein expression by ovarian follicles, while it was without effect on P450arom mRNA level. Quite opposite effect was observed in case of the simultaneous addition of 2-Hf and T. It caused high increase, in both P450arom mRNA and its protein. What was interesting, addition T+2-Hf evoked 3ß-HSD and P450c17 increase on mRNA level, but decreased their protein expression. This was against our expectations but the reason for that finding remains undiscovered, intriguing and worth reporting. These results suggest that alike, steroidogenic enzymes activity and their expression is associated with the presence of androgens and AR in the porcine ovary.

Effect of flutamide on folliculogenesis in the fetal porcine ovary--regulation by Kit ligand/c-Kit and IGF1/IGF1R systems.

In pigs, primordial to primary follicle transition occur in the late pregnancy. The interactions between Kit ligand (KL) and its receptor (c-Kit), as well as insulin-like growth factor 1 (IGF1) and cognate receptor (IGF1R) are crucial for the primordial follicle activation. It is well established that hormonal disruption induces abnormalities in the developing reproductive system. Hence, this study investigated the influence of antiandrogen, flutamide, on genes involved in the primordial to primary follicle transition. Pregnant gilts were injected with flutamide (50mg/kg bw, seven times, every day) or corn oil (control groups) starting on gestation days 83 (GD90) or 101 (GD108). Fetal ovaries were excised on days 90 and 108 of gestation. The proportion of primordial and primary follicles was determined, and immunohistochemistry for c-Kit and IGF1R was conducted. To assess KL, c-Kit, IGF1 and IGF1R mRNA expression real-time PCR was performed. Ovaries from both GD90 and GD108 animals exhibited a greater proportion of primordial to primary follicles when compared to respective control groups. C-Kit and IGF1R were immunolocalized in the oocytes of primordial and primary follicles. Both c-Kit mRNA and protein levels and KL mRNA expression were diminished in GD90 group. IGF1R expression decreased at mRNA and protein levels, whereas IGF1 mRNA expression was increased in GD90 and GD108 groups. In summary, our findings may indicate that the interactions between KL and c-Kit as well as IGF1 and IGF1R are relevant to the initiation of follicular transition from primordial into primary follicles and can be affected by AR signaling.

Does 2-hydroxyflutamide inhibit apoptosis in porcine granulosa cells? - An in vitro study.

In mammalian ovaries, the majority of follicles are lost before ovulation by atresia. This degenerative process is initiated or caused by granulosa cell apoptosis. To reveal the androgen-dependent mechanism of selective follicular atresia, the culture model system for agonism and antagonism of the androgen receptor has been established. We examined the influence of an androgen receptor antagonist, 2-hydroxyflutamide (2-Hf), on the incidence of apoptosis in cultured porcine granulosa cells. They were incubated (6 and 12-h) in the presence of testosterone (T, 10⁻7M), 2-Hf (1.7×10⁻4 M) or both T and 2-Hf (T+2-Hf), and then analyzed by flow cytometry with fluorescein labelled annexin V. To better imitate in vivo conditions, the intact porcine follicles (6-8 mm in diameter) have been incubated in an organ culture system with the addition of the same factors. Sections obtained from follicles fixed after culture were stained with hematoxylin and eosin, and the presence of apoptosis-related DNA strand breaks was evaluated by the TUNEL method. Estradiol and progesterone concentrations in the culture media were measured by radioimmunoassays. The addition of T or 2-Hf to the culture media caused an increase in the number of apoptotic granulosa cells, while treatment with T+2-Hf decreased it in both in vitro and organotypic models. Follicles cultured with the addition of T or 2-Hf exhibited morphological changes indicating follicular atresia. Granulosal estradiol secretion was considerably stimulated by T+2-Hf. The highest increase in follicular estradiol secretion was observed after the anti-androgen addition. In both granulosal and follicular cultures, the production of progesterone declined in the presence of T or 2-Hf but increased after their simultaneous addition. In conclusion, androgen receptor antagonist 2-Hf attenuates induction of granulosa cell apoptosis in the presence of a high T level. The nature of this protective mechanism as yet is unknown and requires further research.

Luteal macrophage conditioned medium affects steroidogenesis in porcine granulosa cells.

The purpose of the study was to examine the effect of luteal macrophage conditioned medium (LMCM) on progesterone and estradiol production by cultured granulosa cells. Porcine granulosa cells were cultured for 48 h with or without LMCM in the absence or presence of 100 ng/ml LH, FSH or prolactin. Progesterone and estradiol concentrations were measured by radioimmunoassay. Granulosa cells were analyzed histochemically and immunocytochemically for the activity and presence of Δ5, 3ß-hydroxysteroid dehydrogenase (3ß-HSD), respectively. LMCM stimulated basal and LH-, FSH- or prolactin-induced progesterone secretion. Similarly, LMCM augmented basal and stimulated activity of 3ß-HSD in the examined cells. In contrast, LMCM decreased LH- and prolactin-induced estradiol secretion but increased FSH-induced estradiol secretion. These data demonstrate the clear stimulatory effect of LMCM on granulosal progesterone production. It is concluded that substances secreted by macrophages modulate gonadotropin effect on follicular progesterone secretion in a paracrine manner via 3ß-HSD activity.

Octylphenol affects morphology and steroidogenesis in mouse tumor Leydig cells.

Exposure to xenoestrogens has been shown to cause adverse effects on reproductive function of various animal species. However, direct effects of xenoestrogens on Leydig cell function remain scarcely known. The aim of our study was to demonstrate the effects of 4-tert-Octylphenol (OP) on the expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and androgen receptor (AR) in MA-10 Leydig cells. Cells were treated for 3 and 12h with OP (10(-4)-10(-8)M). In cell cultures treated with high OP concentrations for 3 and 12h morphological alterations were observed. Oil Red O staining revealed differences in lipid droplet size and distribution when compared to controls. Immunoreactive 3ß-HSD was found in the cytoplasm, whereas immunoreactive AR was detected in the nuclei of both control and OP-exposed Leydig cells. A dose-related decrease in the expression of both proteins in OP-exposed cells was found. Qualitative results by immunocytochemistry and Western blot were confirmed by further quantitative analyses. Radioimmunological analysis revealed time-dependent secretion of progesterone (P(4)) by control and OP-exposed Leydig cells. In 3 and 12h OP-treated Leydig cells a significant decrease in P(4) level was found. Our study demonstrated a dose- and time-dependent action of OP on both morphology and steroidogenic function of MA-10 cells. It seems likely that OP besides acting via ER may mediate its action through AR modulating Leydig cell function.

Immunolocalisation of oestrogen receptors alpha (ERalpha) and beta (ERbeta) in porcine embryos and fetuses at different stages of gestation.

The sites of oestrogen action can be shown by the localisation of their receptors in the target tissues. The aim of the present study was to show the localisation of oestrogen receptors in porcine embryos and fetuses obtained on days 18, 22, 32, 40, 50, 60, 71 and 90 post coitum (p.c.). The visualisation of proteins was conducted in embryos and various fetal organs such as gonads, uterus, lung, kidney, intestine and adrenal gland. Both ERs were observed in the blastocysts on day 18 p.c. In the male, ERbeta was detected in the testis and epididymis, whereas ERalpha was present in the efferent ductules. In the female, ERbeta was detected in the ovarian stromal cells investing the oocyte nests, while ERalpha protein was detected in the surface epithelium. In the uterus, ERs were present in the stromal cells, while ERbeta was present in the luminal epithelium. In the non-reproductive fetal porcine tissues ERbeta was localised in the lungs, kidneys, adrenal glands and in the umbilical cords. Both ERs were observed in the intestine. It is possible that ERbeta may play important roles in the development of the adrenal gland, testis, kidney and lungs, while both ERs are involved in the development of the ovary, uterus, epididymis and intestine of the porcine fetus.

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.

Effects of transferrin on aromatase activity in porcine granulosa cells in vitro.

Proliferating cells have an absolute requirement for iron, which is delivered by transferrin with subsequent intracellular transport via the transferrin receptor. Recent studies have reported that transferrin plays a crucial role in the local regulation of ovarian function, apart from its iron-binding characteristic. Therefore, the present study was undertaken to explore the possible role of transferrin in porcine granulosa cells function by examining its influence on aromatase activity, the most important indicator of follicular cell differentiation. In the first series of studies, pig granulosa cells isolated from small, immature follicles were cultured in the presence of transferrin alone (10 microg/ml or 100 microg/ml) or with the addition of FSH (100ng/ml). The second series of studies was undertaken to determine transferrin-stimulated granulosa cells ability to aromatize exogenous testosterone (1x10(-7)M). One hour after the establishment of cultures an aromatase inhibitor CGS16949A was added to test its influence on estradiol production. After 48 hours, cultures were terminated and cells were processed for immunocytochemical staining of aromatase. Media were frozen for further estradiol level analysis. Positive immunostaining for aromatase was found in all granulosa cell cultures. The intensity of immunostaining was always stronger in cultures supplemented with FSH whereas the addition of transferrin had no effect. Granulosa cells in vitro synthesized the highest amount of estradiol after the addition of FSH and exogenous testosterone as measured radioimmunologically. Concomitant treatment with FSH and transferrin caused an inhibition of FSH-stimulated aromatase activity. The production of estradiol also declined in the presence of FSH, testosterone and transferrin. This study demonstrates that transferrin had a dose-dependent inhibitory effect on FSH-stimulated aromatase activity, which was confirmed by radioimmunoassay. Our results indicate that transferrin may be an important factor in the regulation of granulosa cell diferentiation.

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.

Androgens and FSH affect androgen receptor and aromatase distribution in the porcine ovary.

In the present study the authors investigated whether androgens could interact with FSH to induce aromatase and androgen receptor expression in porcine granulosa cells. Dissected whole porcine follicles (small, medium, and large) were incubated for 8 hours in M199 medium supplemented with testosterone (10(-7) M), FSH (100 ng/ml) or both those hormones. After incubation, the follicles were fixed and immunostained to visualise androgen receptor and aromatase. In cultures of granulosa cells isolated from small and large follicles, oestrogen secretion was measured by appropriate RIA. Incubation of follicles with testosterone and FSH increased aromatase immunoreactivity in preantral and early antral (i.e. small) follicles. The immunostaining for androgen receptor was slightly higher in medium follicles, while such hormonal stimulation had no effect on small and large follicles. Moreover, granulosa cells isolated from small follicles cultured with both testosterone and FSH produced more estradiol than control cultures (40 pg vs. 100 pg/10(5) cells). The level was relatively close to that obtained in the culture of control granulosa cells isolated from large preovulatory follicles (105 pg/10(5) cells). These results indicate that testosterone acts synergistically with FSH to increase aromatase expression in the small porcine follicles.