The uterusmasculinus of the Eurasian beaver (Castor fever L.) - The appraisal of fast hormone regulation by membrane androgen and estrogen receptors involvement.

The phenomenon of remaining paramesonephric ducts (uterus masculinus) in males of some animal species concerning its role is still an unresolved issue. Now it is well-recognized that sex hormonal regulation of reproductive physiology involves also fast nongenomic control of cellular processes through noncanonical signaling. Herein, in the uterus masculinus of Eurasian beaver membrane androgen receptor (metal ion transporter Zrt- and Irt-like protein 9; ZIP9) and membrane estrogen receptor (G protein-coupled estrogen receptor; GPER) were studied. Scanning electron microscopy together with anatomical analysis revealed that Eurasian male beavers possess one double uterus (uterus duplex). Two odd parts open into the vagina but do not form a common lumen. The length of the horns is the most differential feature of this organ in studied animals. Uterus masculinus is not a tightly closed tubular structure. Histological analysis showed an analogy to the female uterus structure however no glands but gland-like structures were observed. The presence and abundant localization of ZIP9 and GPER proteins in cells of uterus masculinus was confirmed by immunohistochemistry while their expression was measured by western blotting. GPER expression in remnants was lower (P < 0.001) than those in the female uterus. Parallelly, the concentration of progesterone and estradiol but not testosterone was lower (P < 0.05 and P < 0.01, respectively) in comparison to the female uterus. Our study, for the first time, reports the involvement of fast hormonal regulation in the uterus masculinus of Eurasian beavers reflecting the participation of this organ in the creation local hormonal environment. Moreover, the uterus masculinus seems to be a useful research model for understanding and resolving urgent biological problems such as gender identities and having children by women with a lack of uterus or anatomical barriers on this level.

Extracellular vesicles in follicular fluid of sexually mature gilts' ovarian antral follicles - identification and proteomic analysis.

Ovarian follicular growth and development involves extensive bidirectional cell-to-cell communication between somatic cells and the oocyte. Recently it has been found that extracellular vesicles (EVs) represent a new mechanism of the communication inside the ovarian follicle. The present research shows for the first time the presence of EVs in follicular fluid of small (SFs), medium (MFs) and large (LFs) antral follicles of sexually mature gilts using nanoparticle tracking analysis and Western blot. The highest (P = 0.0338) concentration of EVs was found in follicular fluid of MFs, as compared to LFs and SFs. Furthermore, nanoparticle tracking analysis showed the existence of the population of particles in follicular fluid of all analyzed follicles, which resembles exosomes. That was confirmed by the abundance of exosomal markers, CD9 and CD63, in those samples. The proteomic analysis of EVs from MFs using the nano-liquid chromatography-matrix-assisted laser deposition/ionization time-of-flight mass spectrometry allows to identify 249 proteins that predominantly indicated binding function and catalytic activity. Most of them belong also to the group of cytoskeleton and extracellular matrix proteins (ECM) suggesting their role in the building of cell components. Functional annotation predicted association of identified proteins with processes crucial for follicle development and function, as well as ovulation and corpus luteum formation. Therefore, EVs through their protein cargo might regulate follicle development in sexually mature gilts.

Effect of neonatal exposure to endocrine-active compounds on plasma lipid and steroid concentrations, and morphology of luteal tissue in the adult pig.

To study the long-term impact of neonatal exposure to endocrine-active compounds (EACs) on plasma lipid profiles, steroid concentrations, and morphology of porcine luteal tissue, piglets were injected with testosterone propionate (TP), flutamide (FLU), 4-tert-octylphenol, ICI 182,780 (ICI), methoxychlor, or corn oil (controls) between postnatal days 1 and 10 (n = 5/group). Blood samples and corpora lutea were obtained from sexually mature gilts. The investigated compounds differentially affected plasma lipid and steroid concentrations. Moreover, we demonstrated hypertrophy of luteal cells after neonatal EAC administration. In addition, a predominant abundance of lipid droplets was found in luteal cells of TP-, FLU-, and ICI-treated animals. It seems that the pathways leading to changes in the plasma lipid profile may contribute to the development of long-term alterations that have the potential to affect luteal steroidogenic capability in pigs.

Do G-protein coupled estrogen receptor and bisphenol A analogs influence on Leydig cell epigenetic regulation in immature boar testis ex vivo?

Organotypic culture of testicular fragments from 7-day-old male pigs (Polish White Large) was used. Tissues were treated with an antagonist of G-protein coupled estrogen receptor (GPER) (G-15; 10 nM), and bisphenol A (BPA), and its analogs (TBBPA, TCBPA; 10 nM) alone or in combination and analyzed using electron and light (stainings for collagen fibers, lipid droplet and autophagy markers) microscopes. In addition, mRNA and protein abundances and localization of molecules required for miRNA biogenesis and function (Drosha, Exportin 5; EXPO5, Dicer, and Argonaute 2; AGO2) were assessed together with calcium ion (Ca2+) and estradiol concentrations. Regardless of GPER blockade and/or treatment with BPA, TBBPA and TCBPA, there were no changes in Leydig cell morphology. Also, there were no changes in lipid droplet content and distribution but there were changes in lipid and autophagy protein abundance. In the interstitial tissue, there was an increase of collagen content, especially after treatment with BPA analogs and G-15 + BPA. Independent of the treatment, there was downregulation of EXPO5 and Dicer genes but the Drosha and AGO2 genes were markedly upregulated as a result of treatment with G-15 + BPA and TCBPA, respectively. There was always a lesser abundance of EXPO5 and AGO2 proteins regardless of treatment. There was markedly greater abundances of Drosha after G-15 + BPA treatment, and this also occurred for Dicer after treatment with G-15 + TCBPA. Immunolocalization of miRNA proteins indicated there was a cytoplasmic-nuclear pattern in control and treated cells. There was an increase of Ca2+ concentrations after treatment with G-15 and BPA analogs. Estradiol secretion decreased after antagonist and chemical treatments when these were administered alone, however, there was an increase in estradiol secretion after treatment with combinations of these compounds.

The expression of autophagy-related proteins within the corpus luteum lifespan in pigs.

Autophagy is a cellular process that involves the degradation of intracellular components. Recent studies suggested a role for autophagy in corpus luteum (CL) regression; however, a complete understanding of its contribution to CL function remains unclear. The present research using porcine CLs obtained from gilts at the early (CL1, n = 5), middle (CL2, n = 5), and late (CL3, n = 5) luteal phase of the estrous cycle aimed to assess the incidence of autophagy during CL development. The stages of collected CLs were verified through morphological analysis and intraluteal progesterone concentration. The presence of autophagosomes was assessed using transmission electron microscopy, and the expression of autophagic markers was examined at mRNA (BECN1 and Lamp1) and protein (Beclin 1, LC3-II, and Lamp 1) levels. Lamp 1 immunolocalization was also performed in luteal tissue. Double-membrane autophagosomes and autophagy-related proteins were found in all examined CLs. Interestingly, there was a greater expression of Beclin 1 (P = 0.005 and P = 0.025) and Lamp 1 (P = 0.009 and P = 0.032) protein in CL3 as compared with CL1 and CL2. In addition, the presence of autolysosomes in CL3 indicated advanced autophagy at that developmental stage. Overall, the occurrence of autophagy throughout CL development and regression suggests it has a role in the regulation of CL lifespan in pigs. In the early and mature CL, autophagy is proposed to promote luteal formation and function, whereas in the late CL, it may participate in luteal regression.

Expression of apelin and apelin receptor (APJ) in porcine ovarian follicles and in vitro effect of apelin on steroidogenesis and proliferation through APJ activation and different signaling pathways.

Apelin was thought to be an adipocyte-specific hormone, but recent studies indicate a link between apelin and female reproductive function. Using real-time PCR, immunoblotting, immunohistochemistry and ELISA, we demonstrated expression of apelin and its receptor (APJ) in ovarian follicles of different sizes from mature pigs. Apelin concentration in the follicular fluid, and expression of both apelin and APJ, increased with follicular growth; greatest values were found in large follicles. Immunohistochemistry revealed the positive staining for apelin and APJ in membranes of granulosa, than theca cells. Furthermore, we observed strong expression of apelin in oocytes and APJ in the zona pellucida. The effect of apelin (0.02, 0.2, 2 and 20 ng/ml) on basal and IGF1- and FSH-induced steroid hormone (progesterone [P4], and estradiol [E2]) secretion, steroidogenic enzyme (3ßHSD and CYP19A1) expression and cell proliferation (Alamar blue) was determined. Apelin was found to increase basal steroid secretion, but decrease IGF1- and FSH-induced steroid secretion, and 3ßHSD and CYP19 expression. Apelin also increased cell proliferation and the phosphorylation level of 5'-monophosphate-activated protein kinase (AMPK), phosphatidyl inositol 3' kinase/Akt (Akt) and extracellular signal-regulated kinases (ERK1/2). AMPKα was involved in the action of apelin in P4 production, and MAPK/ERK and Akt/PI3 mediated the proliferative effect of apelin. However, these effects on steroid secretion and cell proliferation were abolished when cultured in the presence of ML221, an APJ antagonist. In conclusion, apelin appears to regulate ovarian follicular functions such as steroidogenesis and proliferation via APJ activation and different signaling pathways.

The impact of flutamide on prostaglandin F2α synthase and prostaglandin F2α receptor expression, and prostaglandin F2α concentration in the porcine corpus luteum of pregnancy.

Recently, we have indicated that flutamide-induced androgen deficiency diminished progesterone production in the porcine corpus luteum (CL) during late pregnancy and before parturition, as a sign of functional luteolysis. In pigs, the main luteolytic factor is prostaglandin F2α (PGF2α), which acts via specific receptors (PTGFRs), and its biosynthesis is catalyzed by prostaglandin F2α synthase (PGFS). The present study investigated the impact of flutamide on luteal PGFS and PTGFR expression, as well as intraluteal PGF2α content during pregnancy in pigs. Flutamide (50 mg/kg BW per day, for 7 d) or corn oil (control groups) were administered subcutaneously into pregnant gilts (n = 3 per group) between 83 and 89 (GD90) or 101-107 (GD108) days of gestation (GD). On GD90 and GD108 ovaries were collected and CLs were obtained. Real-time PCR and Western blot analyses were conducted to quantify PGFS and PTGFR mRNA and protein expression, respectively. In addition, immunohistochemical localization of both proteins was performed and the concentration of PGF2α was analyzed by enzyme immunoassay method. Flutamide caused upregulation of PGFS mRNA and protein in GD90F (P = 0.008; P = 0.008, respectively) and GD108F (P = 0.041; P = 0.009, respectively) groups. The level of PTGFR mRNA increased only in the GD90F (P = 0.007) group, whereas PTGFR protein expression was greater in both gestational periods (P = 0.035; P = 0.038, respectively). On GD90 PGFS was immunolocalized in the cytoplasm of large luteal cells only, whereas on GD108, sparse small luteal cells also displayed positive staining. PTGFR showed membranous localization within large luteal cells on both days of pregnancy. In luteal tissue, PGF2α concentration was greater after flutamide exposure on both days (P = 0.041; P = 0.038, respectively), when compared with control groups. Overall, the enhanced luteal PGF2α content due to increased PGFS expression after flutamide administration might contribute to premature CL regression. Moreover, higher PTGFR protein levels indicate enhanced sensitivity of luteal cells to PGF2α under androgen deficiency.

Induction of autophagy in the porcine corpus luteum of pregnancy following anti-androgen treatment.

Experimentally induced androgen deficiency during late pregnancy leads to decreased progesterone synthesis in the porcine corpus luteum (CL), which suggested an onset of functional luteolysis. It was shown that luteal regression in mammals involves not only apoptosis but also autophagy. Therefore, this study aimed to examine whether anti-androgen flutamide treatment during late pregnancy in pigs induces apoptosis and/or autophagy within luteal cells. Flutamide (50 mg/kg b.w.) was administered into pregnant gilts between 83 - 89 (GD90F) or 101 - 107 (GD108F) gestational days (GD). CLs were retrieved on day 90 or 108 of pregnancy (n = 3/each group). Detection of apoptosis was performed by TUNEL assay and assessment of cleaved caspase 3 level. Both assays revealed that luteal rate of apoptosis was unaffected by flutamide treatment either in the GD90F or GD108F groups. Moreover, pro-apoptotic protein Bax was downregulated on GD108. The autophagy was examined by expression of two markers, LC3-II and Lamp1. Flutamide led to greater expression of LC3-II protein form in the GD90F and GD108F groups. Likewise, the mRNA and protein levels of Lamp1 were elevated in both flutamide-treated groups. The activation of autophagy is regulated by Beclin 1 and the increased Beclin 1 mRNA and protein expression was found in the GD90F and GD108F groups. Beclin 1 is a Bcl-2-binding protein, thus Beclin1/Bcl-2 interactions were examined using immunoprecipitation. Beclin 1/Bcl-2 complexes were less abundant following flutamide treatment in both flutamide-exposed groups. In summary, we concluded that androgen deficiency induced autophagy by disrupting Beclin 1/Bcl-2 interplay in the porcine CL during pregnancy. The role of autophagy in luteal regression in pigs requires further evaluation.

Effect of Prenatal and Neonatal Anti-Androgen Flutamide Treatment on Aquaporin 5 Expression in the Adult Porcine Ovary.

The growth of ovarian follicles is accompanied by fluid-filled antrum formation. Water movement within the follicular wall is predominantly transcellular via membranous water channels named aquaporins (AQPs). Androgens are important regulators of mammalian folliculogenesis, and their prenatal and/or neonatal deficiency affects female fertility in adulthood. Therefore, this study was performed to determine whether gestational or neonatal exposure to the anti-androgen flutamide influences androgen-dependent AQP5 expression in pre-antral and large antral follicles of adult pigs. Flutamide was injected into pregnant gilts between days 80 and 88 of gestation and into female piglets between days 2 and 10 post-natally. The ovaries were collected from flutamide-treated and non-treated (control) sexually mature pigs. In pre-antral follicles, AQP5 mRNA and protein levels were both downregulated following maternal (p < 0.01 and p < 0.01, respectively) and neonatal (p < 0.01 and p < 0.01, respectively) flutamide exposure. Likewise, the expression of mRNA (p < 0.01 and p < 0.001, respectively) and protein (p < 0.05 and p < 0.01, respectively) for AQP5 were diminished in large antral follicles in both groups. Immunohistochemistry showed decreased intensity of AQP5 immunoreaction in pre-antral (p < 0.01) and large antral (p < 0.001) follicles following flutamide treatment. Moreover, radioimmunological analysis revealed that changes observed in AQP5 expression corresponded with diminished follicular androgens production after both maternal (p < 0.05 and p < 0.05, respectively) and neonatal (p < 0.05 and p < 0.01, respectively) flutamide administration. Therefore, AQP5 appears to be a potential regulator of follicular fluid accumulation, under androgen control, and may be a key factor in antral follicle growth.

Altered expression of 3ß-HSD, CYP17 and 17ß-HSD in the foetal porcine gonads in response to anti-androgen flutamide exposure.

We investigated whether the limited access to androgens during late prenatal period alters expression of steroidogenic enzymes involved in androgen production: 3ß-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3ß-HSD), cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17) and 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) or type 3 (17ß-HSD3) in the foetal porcine gonads. Pregnant gilts were injected with anti-androgen flutamide (for seven days, 50 mg/day/kg bw) or corn oil (control) starting at 83 (GD90) or 101 (GD108) gestational day. To assess 3ß-HSD, CYP17 and 17ß-HSD1 or 17ß-HSD3 expression, real-time PCR and immunohistochemistry were performed. In testes from flutamide-treated foetuses, increased 3ß-HSD and CYP17 mRNA expression was observed in the GD90 group, while decreased 3ß-HSD and 17ß-HSD3 mRNA expression and increased CYP17 mRNA expression were found in the GD108 group. CYP17 and 17ß-HSD3 were localized in Leydig cells. Following flutamide administration, the intensity of CYP17 immunostaining was higher in both treated groups, while 17ß-HSD3 intensity was lower in the GD108 group. In ovaries from flutamide-treated foetuses in the GD90 group, mRNA level for 3ß-HSD was elevated, but it was diminished for CYP17 and 17ß-HSD1. In the GD108 group, flutamide treatment led to lower mRNA level for 3ß-HSD but higher for CYP17. 3ß-HSD was found in granulosa cells, while CYP17 was localized within egg nests and oocytes of forming follicles. Following flutamide treatment, the intensity of 3ß-HSD and CYP17 immunostaining was higher in the GD90 and GD108 groups, respectively. Immunohistochemical staining for 3ß-HSD was restricted to the ovary. Concluding, diminished androgen action in the porcine foetal gonads during late gestation induces changes in steroidogenic enzymes expression, which may led to changes in gonadal function. However, it seems that androgens exert diverse biological effects depending on the gestational period.

Flutamide influences placental aldo-keto reductase family 1 member C1 (AKR1C1) expression in pigs.

Aldo-keto reductase family 1 member C1 (AKR1C1) catalyses the conversion of progesterone into inactive 20α-dihydroxyprogesterone. It is suggested that AKR1C1 expression in the placenta prevents from the cytotoxic effect of progesterone on foetuses during late pregnancy. The aim of the study was to determine whether the anti-androgen flutamide administered during late pregnancy (83-89 days of gestation) or before parturition (101-107 days of gestation) influences AKR1C1 expression in the porcine placenta. AKR1C1 mRNA and protein levels were measured using real-time PCR and western blotting, respectively. Immunolocalization of AKR1C1 within placentas was also performed. Flutamide significantly increased AKR1C1 mRNA (p = 0.008) and protein (p = 0.019) expression only during the pre-parturient period in pigs. AKR1C1 protein was immunolocalized in the epithelial and stromal cells of foetal and maternal part of placenta at both stages of gestation. Following flutamide treatment, the intensity of staining was higher (p = 0.045) on day 108 of gestation. In conclusion, porcine placental AKR1C1 expression seems to be regulated by an androgen signalling pathway and may be involved in foetal survival by preventing the detrimental effect of progesterone.

In utero exposure to the anti-androgen flutamide influences connexin 43 and ß-catenin expression in porcine fetal gonads.

Recent reports have indicated a role of cell-to-cell interactions during gonadal development and functions. Numerous reports indicate that fetal hormonal disruption induces abnormalities in the developing reproductive system and, therefore, may interfere with reproductive functions later in adult life. Hence, this study investigated the effect of androgen deficiency during late prenatal periods on the gap junction-associated connexin 43 (Cx43) and the adherens junction-associated ß-catenin expression in the fetal porcine gonads. Thus, pregnant gilts were injected with anti-androgen flutamide (for 7 d, 50 mg/kg BW per day) or corn oil (control groups) starting at 83 (GD90) or 101 (GD108) gestational day. On GD90 and GD108 the fetuses were excised and fetal gonads were obtained. To assess Cx43 and ß-catenin expression real-time PCR and immunohistochemistry were performed. In fetal testes, Cx43 was localized between Leydig cells, whereas ß-catenin was observed mainly within the seminiferous tubules. In fetal ovaries, Cx43 was detected between interstitial cells and between granulosa cells of forming follicles, whereas ß-catenin was found within egg nests, in oocytes' membrane, and in granulosa cells of forming follicles. Immunohistochemistry showed decreased Cx43 and ß-catenin expression in fetal gonads from flutamide-treated pigs compared with respective controls. However, the ovaries from animals treated with flutamide on GD108 showed increased Cx43 expression. The changes of Cx43 and ß-catenin expression after prenatal flutamide treatment were confirmed at the mRNA level. These findings suggest that androgen deficiency during late gestation may lead to disturbed intercellular interactions in fetal porcine testes affecting testicular functions, as well as impaired follicular formation in fetal ovaries. Our results further signify the role of androgens in the regulation of cell-to-cell interactions within fetal porcine gonads.

Expression of connexin 43 in the porcine foetal gonads during development.

This study was designed to reveal connexin 43 (Cx43) mRNA and protein expression in porcine foetal gonads using RT-PCR, immunohistochemistry and Western blot analysis. Expression of Cx43 was investigated in porcine foetal ovaries and testes on days 50, 70 and 90 post coitum (p.c.). RT-PCR results indicated that Cx43 mRNA was expressed in both foetal ovaries and testes at all gestational ages examined. Cx43 protein was found in the foetal ovary but its distribution varied across ovarian compartments and changed during development. In foetal ovaries, Cx43 was localized between the interstitial cells surrounding egg nests on all investigated days of prenatal period. Moreover, Cx43 expression was observed between germ cells on day 50 p.c. as well as between pre-granulosa and granulosa cells of primordial and primary follicles on days 70 and 90 p.c. In the foetal testes, Cx43 protein was detected between neighbouring Leydig cells on all examined days of prenatal period and between adjacent Sertoli cells exclusively on day 90 p.c. The presence of Cx43 protein in all investigated foetal gonads was confirmed by Western blot analysis. Cx43 protein detection between pre-granulosa cells of primordial follicles suggests its role in regulation of the initial stages of follicle development. The Cx43 immunoexpression between neighbouring Leydig and between Sertoli cells indicates its involvement in controlling their functions. We propose that Cx43-mediated gap junctional communication is involved in the regulation of porcine foetal gonadal development.

The influence of the antiandrogen 2-hydroxyflutamide on the androgen receptor expression in the porcine ovarian follicles - an in vitro study.

Androgens are one of the most important agents influencing ovarian follicles growth and development. The biological action of androgens is primarily exerted through transcriptional regulation by the androgen receptor (AR), a member of the steroid hormone receptor superfamily. The purpose of this study was to test the role of androgen receptor agonist testosterone (T) or antagonist 2-hydroxyflutamide (2-Hf) and in combination on AR expression in cultured porcine granulosa cells (GC) or whole follicles. Granulosa cells isolated from mature pig follicles were cultured for 48 h. During the last 12 and 24 h of culture, they were incubated in the presence of T (10(-7)  m/ml), 2-Hf (1.7 × 10(-4)  m) or both T and 2-Hf (T + 2-Hf, at the same concentrations as when added separately). To better imitate in vivo conditions, whole follicles (6-8 mm in diameter) isolated from porcine ovaries have been incubated (for 12 and 24 h) in an organ culture system with the addition of the same factors. Thereafter, cells or sections obtained from cultured follicles were processed for AR detection by immunocytochemistry or immunohistochemistry. Moreover, expression of AR mRNA and protein was determined by real-time PCR and Western blot analysis. It was shown that the addition of 2-Hf in the presence of T had a positive effect on AR mRNA and protein expression in porcine GC and ovarian follicles. Moreover, the addition of 2-Hf influenced AR distribution in GC cultures which is seen as change of its localization from nuclear to perinuclear. Our results suggest that androgens acting through AR could be involved in the control of AR expression in porcine GC in vitro and in vivo.

Elevated level of 17ß-estradiol is associated with overexpression of FSHR, CYP19A1, and CTNNB1 genes in porcine ovarian follicles after prenatal and neonatal flutamide exposure.

Recent studies suggest that disturbed androgen action during gestational and neonatal periods leads to reprogramming of the trajectory of ovarian development, manifested by altered follicular functioning in adulthood. In this study, we tested whether prenatal and neonatal exposure to antiandrogen flutamide affected ovarian 17ß-estradiol (E(2)) synthesis and the associated gene expression in large antral follicles of adult pigs. Flutamide was injected into pregnant gilts between Days 80 and 88 of gestation and into female piglets between Days 2 and 10 postnatally. After animals reached sexual maturity, the ovaries were collected from treated and nontreated (control) pigs. The analysis of E(2) concentration in follicular tissues, as well as FSH and LH levels in plasma of control and flutamide-treated animals were conducted. In addition, the expression of mRNAs and proteins for FSH receptor (FSHR), cytochrome P450 aromatase (CYP19A1) and ß-catenin (CTNNB1) was examined in large antral follicles of adult pigs. The E(2) concentration was greater in response to flutamide administered prenatally (P < 0.05) and neonatally (P < 0.01), whereas there was no changes in plasma gonadotropin concentration. Real-time polymerase chain reaction analysis revealed significant upregulation of FSHR, CYP19A1, and CTNNB1 at the mRNA level after maternal (P < 0.001, P < 0.01, P < 0.05, respectively) and neonatal (P < 0.001, P < 0.001, P < 0.01, respectively) flutamide exposure. The expression of FSHR protein was higher (P < 0.01) only after neonatal exposure to flutamide, whereas CYP19A1 and CTNNB1 proteins were upregulated in response to both prenatal (P < 0.01) and neonatal (P < 0.001) flutamide administration. Furthermore, membranous CTNNB1 immunolocalization indicates that it is not involved in regulation of FSH-mediated CYP19A1 activity as a transcription factor, but rather contributes to the intercellular adhesion. Concluding, it appears that the higher E(2) level in response to flutamide treatments is a result of the intensified aromatization and local E(2) action at the ovary level. The observed changes might influence the normal follicle development and pig fertility as a consequence.

Differential expression of connexin 43 in adult pig testes during normal spermatogenic cycle and after flutamide treatment.

Evidence is mounting that the foetal and neonatal period of reproductive tract development is highly sensitive to hormonal disruption induced by various endocrine active compounds. Thus, we asked whether androgen withdrawal caused by prenatal (GD20, GD80) or neonatal (PD2) exposure to an anti-androgen flutamide alters Cx43 gene expression and may induce delayed effects on morphology and function of adult pig testes. Flutamide was given in five doses (50 mg/kg bw). Our histological analysis and TUNEL staining revealed varying degrees of seminiferous tubules abnormalities in all experimental pigs. Testes of pigs exposed to flutamide in utero exhibited moderate alterations of the spermatogenic process, whereas those of exposed neonatally were severely impaired. The most striking effects were spermatogenic arrest, germ cell detachment and a statistically significant increase in the frequency of germ cell apoptosis (p<0.01). Moreover, all pigs exposed to flutamide displayed Leydig cell hyperplasia. Because the network of cell-cell communication provided by gap junction channels plays an essential role in the regulation and maintenance of spermatogenesis, the physiological significance of Cx43-based gap junctions with regards to the gonadal impairment was evaluated by analysis of its expression using immunohistochemical, Western blot and qRT-PCR approaches. Significantly, lower Cx43 expression was found when flutamide was administered neonatally, which has coincided with severe disruption of spermatogenesis. Our data suggest that neonatal exposure to flutamide induces long-term effects on the spermatogenic capacity of the pig testis through alterations of Cx43-mediated intercellular communication and permanent alteration of both Sertoli and Leydig cell functions.

Prenatal and neonatal exposure to the antiandrogen flutamide alters connexin 43 gene expression in adult porcine ovary.

Connexin 43 (Cx43) is the predominant gap junction protein within porcine ovary and is required for proper follicle and corpus luteum (CL) development. Recent research suggests maternally or neonatally mediated effects of antiandrogens on reproductive function during adulthood, notably those dependent on gap junctional communication. The current study was conducted to determine whether late gestational or neonatal exposure to the antiandrogen flutamide influences Cx43 gene expression in the adult porcine ovary. Flutamide was injected into pregnant gilts between days 80 and 88 of gestation and into female piglets between days 2 and 10 posnatally. After animals reached sexual maturity, the ovaries were collected from treated and nontreated (control) pigs. Expression of Cx43 mRNA and protein was determined for preantral and antral follicles and for CLs. In addition, 3ß-hydroxysteroid dehydrogenase (3ß-HSD) expression and progesterone concentration were determined for luteal tissues. In preantral follicles, Cx43 mRNA was down-regulated (P < 0.01) following maternal and neonatal flutamide exposure. In large antral follicles, Cx43 mRNA was up-regulated (P < 0.01) after neonatal flutamide administration. Immunofluorescence showed that Cx43 expression decreased (P < 0.001) in preantral follicles and increased (P < 0.001) in large antral follicles following flutamide exposure. In luteal tissues, Cx43 and 3ß-HSD expression and progesterone concentration decreased (P < 0.01) after postnatal flutamide treatment. Overall, these results suggest the involvement of androgens in the regulation of Cx43 expression in pig ovary. Moreover, alteration of Cx43 expression by the administration of flutamide during particular prenatal and neonatal time periods may affect porcine follicle development, as well as CL formation and function.

Expression of oestrogen receptor α and oestrogen receptor ß in the uterus of the pregnant swine.

The uterus is a well-known target of endocrine, paracrine and autocrine acting molecules among which steroid hormones are of special importance. The objective of our work was to localize oestrogen receptors (ERα and ERß) mRNA and protein in the pig uterus throughout pregnancy (10, 18, 32, 50, 71, 90 days post coitum) using RT-PCR, Western-blot and immunohistochemistry. The present study is the first one to demonstrate the presence of ERs protein in the porcine uterus not only at the beginning but also at mid- and late pregnancy. In the pregnant swine, ERα was immunolocalized in the luminal epithelium (LE) and glandular epithelium (GE) and the myometrium of the uterus with differences in the intensity of staining at different stages of pregnancy studied. The LE and GE of pregnant swine stained for ERß regardless of the day of pregnancy examined, whereas only a few cells within the myometrium showed a weak immunoreactivity. Western blot analysis confirmed the presence of ERα and ERß proteins on all investigated days of gestation. The expression of ERα and ERß mRNA was detected by RT-PCR in all examined samples corresponding to each of the consecutive stages of pregnancy. The obtained results show that ERα is more abundant in comparison to ERß within the porcine pregnant uterus. The presence of ERα and ERß in all compartments of the pig uterus during pregnancy may indicate direct action of oestrogens on proliferation and differentiation of these cells.

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.