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 Relative Abundances of Human Leukocyte Antigen-E, α-Galactosidase A and α-Gal Antigenic Determinants Are Biased by Trichostatin A-Dependent Epigenetic Transformation of Triple-Transgenic Pig-Derived Dermal Fibroblast Cells.

The present study sought to establish the mitotically stable adult cutaneous fibroblast cell (ACFC) lines stemming from hFUT2×hGLA×HLA-E triple-transgenic pigs followed by trichostatin A (TSA)-assisted epigenetically modulating the reprogrammability of the transgenes permanently incorporated into the host genome and subsequent comprehensive analysis of molecular signatures related to proteomically profiling the generated ACFC lines. The results of Western blot and immunofluorescence analyses have proved that the profiles of relative abundance (RA) noticed for both recombinant human α-galactosidase A (rhα-Gal A) and human leukocyte antigen-E (HLA-E) underwent significant upregulations in tri-transgenic (3×TG) ACFCs subjected to TSA-mediated epigenetic transformation as compared to not only their TSA-unexposed counterparts but also TSA-treated and untreated non-transgenic (nTG) cells. The RT-qPCR-based analysis of porcine tri-genetically engineered ACFCs revealed stable expression of mRNA fractions transcribed from hFUT2, hGLA and HLA-E transgenes as compared to a lack of such transcriptional activities in non-transgenic ACFC variants. Furthermore, although TSA-based epigenomic modulation has given rise to a remarkable increase in the expression levels of Galα1→3Gal (α-Gal) epitopes that have been determined by lectin blotting analysis, their semi-quantitative profiles have dwindled profoundly in both TSA-exposed and unexposed 3×TG ACFCs as compared to their nTG counterparts. In conclusion, thoroughly exploring proteomic signatures in such epigenetically modulated ex vivo models devised on hFUT2×hGLA×HLA-E triple-transgenic ACFCs that display augmented reprogrammability of translational activities of two mRNA transcripts coding for rhα-Gal A and HLA-E proteins might provide a completely novel and powerful research tool for the panel of further studies. The objective of these future studies should be to multiply the tri-transgenic pigs with the aid of somatic cell nuclear transfer (SCNT)-based cloning for the purposes of both xenografting the porcine cutaneous bioprostheses and dermoplasty-mediated surgical treatments in human patients.

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.

Porcine ovarian cortex-derived putative stem cells can differentiate into endothelial cells in vitro.

Endothelial cells (ECs), the primary component of the vasculature, play a crucial role in neovascularization. However, the number of endogenous ECs is inadequate for both experimental purposes and clinical applications. Porcine ovarian putative stem cells (poPSCs), although not pluripotent, are characterized by great plasticity. Therefore, this study aimed to investigate whether poPSCs have the potential to differentiate into cells of endothelial lineage. poPSCs were immunomagnetically isolated from postnatal pig ovaries based on the presence of SSEA-4 protein. Expression of mesenchymal stem cells (MSCs) markers after pre-culture, both at the level of mRNA: ITGB1, THY, and ENG and corresponding protein: CD29, CD90, and CD105 were significantly higher compared to the control ovarian cortex cells. To differentiate poPSCs into ECs, inducing medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), ascorbic acid, and heparin was applied. After 14 days, poPSC differentiation into ECs was confirmed by immunofluorescence staining for vascular endothelial cadherin (VECad) and vascular endothelial growth factor receptor-2 (VEGFR-2). Semi-quantitative WB analysis of these proteins confirmed their high abundance. Additionally, qRT-PCR showed that mRNA expression of corresponding marker genes: CDH5, KDR was significantly higher compared with undifferentiated poPSCs. Finally, EC functional status was confirmed by the migration test that revealed that they were capable of positive chemotaxis, while tube formation assay demonstrated their ability to develop capillary networks. In conclusion, our results provided evidence that poPSCs may constitute the MSC population in the ovary and confirmed that they might be a potential source of ECs for tissue engineering.

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.

Efficient generation of neural-like cells from porcine ovarian putative stem cells - morphological characterization and evaluation of their electrophysiological properties.

Until recently, the mammalian ovary was considered to consist of fully differentiated tissues, but evidence for the presence of adult stem cells in this organ appeared. The differentiation potential of these cells, referred to as putative stem cells, is not well defined. Porcine ovarian putative stem cells (poPSCs) were immunomagnetically isolated from postnatal pig ovaries based on the presence of the SSEA-4 surface marker protein. First, they were cultured in the undifferentiated state. After the third passage, a novel 7-day culture method inducing their differentiation into neural-like cells by the addition of forskolin (FSK), retinoic acid (RA) and basic fibroblast growth factor (bFGF) to the culture medium was applied. After 7 days, poPSCs successfully differentiated into neural-like cells, as evidenced by neural morphology and the presence of the neuronal markers nestin, NeuN, and GFAP, as confirmed by immunofluorescence, western blot, and real-time PCR. Electrophysiological analysis of potassium and sodium channel activity (patch clamp) confirmed that they indeed differentiated into neurons. The plasticity of poPSCs offers an excellent opportunity, especially in the field of neuroscience, since they can differentiate into neurons or glial cells. Although poPSCs might not be pluripotent cells, they also escape the rigid classification framework of adult stem cells.

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation.

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Neonatal Exposure to Agonists and Antagonists of Sex Steroid Receptors Affects AMH and FSH Plasma Level and Their Receptors Expression in the Adult Pig Ovary.

In this study piglets were injected with testosterone propionate (TP, an androgen), flutamide (FLU, an antiandrogen), 4-tert-octylphenol (OP, an estrogenic compound), ICI 182,780 (ICI, an antiestrogen) or corn oil (controls) between postnatal days 1 and 10 (N = 5/group). Then plasma anti-Müllerian hormone (AMH) and follicle stimulating hormone (FSH) concentration and the expression of their receptors were examined in the adult pig ovary. TP and FLU decreased plasma AMH and FSH concentration. In preantral follicles, TP resulted in upregulation of AMHR2 and FSHR expression, but decreased AMH protein abundance. FLU upregulated AMHR2 expression, while OP increased FSHR mRNA. In small antral follicles, OP upregulated ACVR1 and BMPR1A expression, while FLU increased BMPR1A mRNA. FLU and ICI resulted in upregulation of AMHR2 expression. TP and FLU upregulated AMH expression, while it was downregulated in response to OP or ICI. Moreover, OP and ICI resulted in downregulation of FSHR expression, while FLU decreased FSHR protein abundance. In conclusion, neonatal exposure to either agonist or antagonist of androgen receptor affected AMH and FSH signalling systems in preantral follicles. In small antral follicles these systems were influenced by compounds with estrogenic, antiestrogenic, and antiandrogenic activity. Consequently, these hormonal agents may cause an accelerated recruitment of primordial follicles and affect the cycling recruitment of small antral follicles in pigs.

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.

Analysis of porcine granulosa cell death signaling pathways induced by vinclozolin.

Recent studies suggest that disturbing androgen-signaling pathways in porcine ovarian follicles may cause granulosa cell (GC) death. For this reason, we investigated which apoptotic pathway is initiated after GC exposure to an environmental antiandrogen, vinclozolin (Vnz), in vitro. Immunocytochemistry, Western blots, and fluorometric assays were used to quantify caspase-3 and -9 expression and activity. To elucidate the specific mechanism of Vnz action and toxicity, GCs were assessed for viability, cytotoxicity, and apoptotic activity using the ApoTox-Glo Triplex Assay. To further determine the mechanism of GC death induced by Vnz, we used the Apoptosis Antibody Array Kit. In response to Vnz stimulus, we found an increased level of caspase-3 protein expression (P ≤ 0.001) and an increase in caspase-3 proteolytic activity (P ≤ 0.001), confirming that Vnz is a potent proapoptotic factor. The strong immunoreaction of caspase-9 after Vnz treatment (P ≤ 0.001) suggests that intrinsic mitochondrial apoptosis pathway was activated during GC death. On the other hand, caspase-8, being a part of the extrinsic receptor pathway, was also activated (P ≤ 0.001). Therefore, it is possible that Vnz induces porcine granulosal apoptosis also through a parallel pathway. Activation of these two pathways was confirmed by the Apoptosis Antibody Array Kit. In conclusion, it is possible that the intrinsic signaling pathway may not act as an initial trigger for GC apoptosis but might contribute to the amplification and propagation of apoptotic cell death in the granulosa layer after treatment with this antiandrogen. Moreover, Vnz disturbs the physiological process of programmed cell death. Consequently, this could explain why atretic follicles are rapidly removed and suggests that normal function of the ovarian follicle may be destroyed.

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.

The effects of the environmental antiandrogen vinclozolin on the induction of granulosa cell apoptosis during follicular atresia in pigs.

The aim of this study was to investigate whether the androgens testosterone and dihydrotestosterone (DHT) and the antiandrogenic fungicide vinclozolin (Vnz) exert proapoptotic effects on porcine granulosa cells (GCs), and to examine the roles of these compounds in follicular atresia. Granulosa cells isolated from pig follicles were cultured for 24 hours, and then exposed to 0.1 µM testosterone, 0.1 µM DHT, 14 µM Vnz, or the equivalent concentrations of testosterone and Vnz or DHT and Vnz for a further 24 hours. Apoptosis and necrosis of the GCs were determined via Hoechst staining and flow cytometry analyses of annexin V-stained cells. Whole porcine follicles were also exposed to the same compounds and combinations of compounds for 24 hours. The sections were stained with hematoxylin and eosin for morphologic assessments, and a Terminal deoxynucleotidyl Transferase Biotyn-dUTP Nick-End Labeling (TUNEL) assay was performed to determine the number of apoptotic cells. The progesterone and estradiol concentrations secreted into the culture media by isolated GCs and follicles were also measured. Exposure to the androgens resulted in an increased number of apoptotic GCs both in vitro and in the organotypic model. Vinclozolin exposure increased and decreased the number of necrotic and apoptotic GCs, respectively. Furthermore, compared with control follicles, those exposed to testosterone, DHT, or Vnz displayed enhanced atresia, and coadministration of Vnz attenuated the promotive effect of these androgens on atresia. Estradiol secretion was stimulated by the combination of testosterone and Vnz, whereas exposure to Vnz alone reduced it. Progesterone production declined after the combined addition of androgens and the antiandrogen. In summary, Vnz caused massive necrosis of GCs in vitro and induced apoptosis of GCs in whole follicles. The androgens testosterone and DHT enhanced these effects. The results presented here suggest that selective destruction of porcine follicles is a serious consequence of exposure to Vnz, and may lead to premature ovarian failure in affected animals.

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.

Effects of testosterone and 2-hydroxyflutamide on progesterone receptor expression in porcine ovarian follicles in vitro.

The purpose of the study was to test the possible role of the androgen receptor (AR) agonist (testosterone; T), an AR antagonist (2-hydroxyflutamide; 2-Hf) or combination of both (T+2-Hf) on progesterone receptor (PGR) expression in cultured porcine granulosa cells (GCs) or whole follicles. GCs isolated from mature pig follicles (6-8mm in diameter) were cultured for 48 h. Experimental cultures were carried out with the addition of T (10(-7)M), 2-Hf (1.7×10(-4)M) or both T and 2-Hf for the last 24 h of culture. To better imitate in vivo conditions, isolated whole porcine follicles (6-8mm in diameter) were cultured for 24 h in an organ culture system, with the addition of the same factors. The cells or sections obtained from cultured follicles were processed for PGR immunocytochemical or immuno-histochemical staining. In addition, expression of PGR protein was determined by Western blot and progesterone (P(4)) concentrations in the culture media were measured by a radioimmunoassay. We found that isoform A of PGR is expressed in both granulosal and follicular cultures. The 2-Hf in the presence of T increased PGR protein expression in porcine GCs and whole follicles. In both granulosal and follicular cultures, 2-Hf or T alone inhibited P(4) secretion, but simultaneous addition of 2-Hf and T increased P(4) secretion. Our results indicate that androgens may be involved in the control of PGR expression in porcine GCs in vitro. Moreover, we suggest a potential auto/paracrine regulation of the follicular function by androgen-dependent signaling pathway.