Expression of aquaporin 4 in the chicken ovary in relation to follicle development.

In the mammalian ovary, aquaporins (AQPs) are thought to be involved in the regulation of fluid transport within the follicular wall and antrum formation. Data concerning the AQPs in the avian ovary is very limited. Therefore, the present study was designed to examine whether the AQP4 is present in the chicken ovary, and if so, what is its distribution in the ovarian compartment of the laying hen. Localization of AQP4 in the ovarian follicles at different stage of development was also investigated. After decapitation of hens the stroma with primordial follicles and white (1-4 mm), yellowish (4-8 mm), small yellow and the three largest yellow pre-ovulatory follicles F3-F1 (F3 < F2 < F1; 20-36 mm) were isolated from the ovary. The granulosa and theca layers were separated from the pre-ovulatory follicles. The AQP4 mRNA and protein were detected in all examined ovarian compartments by the real-time PCR and Western blot analyses, respectively. The relative expression of AQP4 was depended on follicular size and the layer of follicular wall. It was the lowest in the granulosa layer of pre-ovulatory follicles and the highest in the ovarian stroma as well as white and yellowish follicles. Along with approaching of the largest follicle to ovulation the gradual decrease in AQP4 protein level in the granulosa layer was observed. Immunoreactivity for AQP4 was present in the granulosa and theca cells (theca interna ≥ theca externa > granulosa). The obtained results suggest that AQP4 may take part in the regulation of water transport required for follicle development in the chicken ovary.

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.

Periovarian Adipose Tissue - an Impact on Ovarian Functions.

Periovarian adipose tissue (POAT) is a type of gonadal white adipose tissue that surrounds the ovary. POAT is a source of various bioactive molecules, such as adipokines, cytokines, chemokines, growth factors and hormones. Thereby it could influence crucial ovarian functions. Recent findings showed that removal of POAT affects folliculogenesis and steroidogenesis in the ovary. Furthermore, changes in the morphology and function of POAT were observed in women during menopause or polycystic ovary syndrome. Although the relationship between the body's energy status and fertility in females is generally well known, the contribution of POAT remains still elusive. Therefore, the objective of this review is summarizing the actual state of knowledge about POAT function in physiological and pathological processes within the ovary.

Testosterone influences water transport in porcine granulosa cells.

The development of antral ovarian follicles entails fluid accumulation, but the mechanisms regulating water flux are unknown. Aquaporins are small, integral membrane proteins facilitating passive movement of water, some of which are known to be regulated by steroid hormones. The aim of this study was to determine whether testosterone (T) influences water transport in porcine granulosa cells. To assess water movement, the swelling of granulosa cells when moved from isotonic (319 mOsm) to hypotonic (95 mOsm) medium was measured after 12-hour pre-incubation in the presence of either testosterone (T), the antiandrogen 2-hydroxyflutamide (HF) or HF and T together. Pre-incubation with T increased the swelling of granulosa cells (p < 0.01) and this was abolished by HF (p < 0.001). Neither T nor HF affected cells in isotonic medium (319 mOsm). The results indicate that T acting via intracellular androgen receptors increases water permeability of porcine granulosa cells, probably through the regulation of aquaporin activity.

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.

The relationship between vitamin D3 and insulin in polycystic ovary syndrome - a critical review.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder affecting women of reproductive age. It is characterized by hormonal, reproductive and metabolic disturbances, including hyperandrogenism, altered gonadotropin level, ovarian cysts and ovulatory dysfunction as well as insulin resistance, hyperinsulinemia and dyslipidemia. It was shown that increased insulin concentration is a plausible factor in the pathogenesis of PCOS. Insulin leads to overstimulation of ovarian theca cells to androgen biosynthesis and contributes to insulin resistance in tissues such as muscle, liver, adipose tissue and ovary of PCOS patients. Noteworthy, recent studies suggested that supplementation with vitamin D3 may be an alternative therapy increasing insulin sensitivity and thereby improving reproductive parameters in PCOS women. Indeed, various action of vitamin D3 on the ovarian, hormonal and metabolic features observed in PCOS were presented. Many studies reported therapeutic effects of vitamin D3, but some research found a lack of influence or contradicted these findings. Therefore, the aim of this review was to summarize the available evidence about vitamin D3 and insulin interaction in PCOS, and discusses the potential usefulness of VD3 in PCOS treatment.

The effect of Roundup on embryonic development, early foxr1 and hsp70 gene expression and hatching of common carp (Cyprinus carpio L.).

The effects of herbicide Roundup (based on glyphosate) on the embryonic development, survival and hatching of common carp (Cyprinus carpio L.) larvae and alteration in foxr1 and hsp70 gene expression were determined. The eggs (obtained from 6 females) were fertilised and incubated in water containing 0; 1 or 10 µl L-1 of Roundup formulation. During early embryonic development (24 and 48 h post-fertilisation - hpf), Roundup caused a statistically important decrease in the embryonic survival rate of common carp. Moreover, retardation of the hatching rate was observed in the group treated with the higher concentration of Roundup at 81 to 99 hpf. At the end of the experiment (99 hpf), an important increase in number of deformed larvae was observed in both groups treated with Roundup in comparison to the control group (52.06; 16.02 and 5.08%, respectively). Significant differences in transcript of the gene foxr1 were found in Roundup-intoxicated groups in comparison to the controls. In the case of hsp70 transcripts, no important changes in exposed groups were observed. These results showed that even small, environmentally relevant amount of Roundup present in the aquatic environment is able to affect the early life stages of common carp and change the transcripts of foxr1, which may have an adverse effect on the later proper development of the reproductive system.

Vitamin D3 action within the ovary - an updated review.

Vitamin D3 is well-known as a major regulator of calcium and phosphorus homeostasis. A growing body of evidence highlights its crucial role in the regulation of reproductive processes in females. The role of vitamin D3 in the female reproductive tract has been extensively investigated because its receptor is abundant in reproductive organs, including ovary. Importantly, besides expression of vitamin D3 receptor, the ovary is an extrarenal site of vitamin D3 metabolism. The influence of vitamin D3 on follicular development and ovarian steroidogenesis has been investigated. Furthermore, vitamin D3 deficiency has also been associated with polycystic ovary syndrome, premature ovarian failure and ovarian cancer. The objective of this review is to summarize our knowledge about the contribution of vitamin D3 to physiological and pathological processes within the ovary.

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.

Correction: C-Peptide and leptin system in dichorionic, small and appropriate for gestational age twins-possible link to metabolic programming?

The original version of this Article was updated shortly after publication to correct two mistakes.Under Figure 2, "The results were recalculated accordingly: nanograms [ng] of studied protein per 100 µ" should read "The results were recalculated accordingly: picograms [pg] of studied protein per 100 µg".

C-Peptide and leptin system in dichorionic, small and appropriate for gestational age twins-possible link to metabolic programming?

Children born small for gestational age (SGA) are at increased risk of future glucose intolerance and type 2 diabetes, possibly after due intrauterine metabolic programming. Soluble leptin receptor (SLR) limits leptin access to signal-transducing membrane receptors. The present study examines whether SGA and appropriate for gestational age (AGA) twins differ with regard to their C-peptide, glucose and leptin systems. The markers C-peptide, glucose, fetal leptin, and SLR in cord blood were assessed in children from dichorionic twin pregnancies at delivery. In 32 cases, weight differed by >15% between twins: one demonstrated Intrauterine Growth Retardation (IUGR) (<10th percentile-SGA), while the other did not (AGAI). The other 67 pairs presented appropriate weight for gestational age (AGAII). Placental leptin and placental leptin receptor content were also assessed. Despite the same concentrations of glucose, the SGA twins maintained a higher level of C-peptide [44.48 pmol/l vs. 20.91 pmol/l, p < 0.05] than the AGAI co-twins, higher HOMA index, calculated as [C-peptide] x [Glucose] (p = 0.045), in cord blood, and a higher level of SLR [SGA vs AGAI-mean: 28.63 ng/ml vs. 19.91 ng/ml, p < 0.01], without any differences in total leptin (p = 0.37). However, SGA placentas demonstrated higher leptin level [130.1 pg/100 g total protein vs 83.8 pg/100 g total protein, p = 0.03], without differences in placental leptin receptor (p = 0.66). SGA/IUGR twins demonstrate relative insulin resistance accompanied by decreased fetal and increased placental leptin signaling. We speculate that relative insulin resistance and changes in the leptin system might be the first evidence of processes promoting deleterious metabolic programming for post-natal life.

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.

Nitrophenols suppress steroidogenesis in prehierarchical chicken ovarian follicles by targeting STAR, HSD3B1, and CYP19A1 and downregulating LH and estrogen receptor expression.

To assess the effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on steroidogenesis in the chicken ovary, white (WF, 1-4 mm) and yellowish (YF, 4-8 mm) prehierarchical follicles were incubated in a medium supplemented with PNP or PNMC (10-8-10-4 M), ovine LH (oLH; 10 ng/mL), and combinations of oLH with PNP or PNMC (10-6 M). Testosterone (T) and estradiol (E2) concentrations in media and mRNA expression for steroidogenic proteins (STAR, HSD3B1, and CYP19A1), and LH receptors (LHR), estrogen receptor α (ESR1) and ß (ESR2) in follicles were determined by RIA and real-time qPCR, respectively. PNP and PNMC decreased T and E2 secretion by the WF and YF, and oLH-stimulated T secretion from these follicles. PNP decreased basal STAR and HSD3B1 mRNA levels both in the WF and YF, and CYP19A1 mRNAs in the WF. PNP reduced oLH-affected mRNA expression of these genes in the YF. PNMC inhibited basal STAR, HSD3B1, and CYP19A1 mRNA expression in the WF, but not in the YF. PNMC reduced oLH-stimulated STAR and CYP19A1 expression in the YF and WF, respectively. PNP decreased basal mRNA expression of LHR, ESR1, and ESR2 in the WF, but it increased ESR1 and ESR2 mRNA levels in the YF. PNMC reduced both basal and oLH-affected LHR, ESR1, and ESR2 mRNA expression in the WF; however, it did not influence expression of these genes in the YF. We suggest that nitrophenols by influencing sex steroid synthesis and transcription of LH and estrogen receptors in prehierarchical ovarian follicles may impair their development and selection to the preovulatory hierarchy.

The influence of testosterone on the expression and function of vitamin D(3) receptor (VDR) protein in the porcine ovarian follicle.

Recently it has been shown that vitamin D(3) acting via its cognate receptor (VDR) regulates the growth, differentiation and function of female reproductive tissues including ovary. The aim of the study was to examine the effect of testosterone (T) and its antagonist 2-hydroxyflutamide (HF) on VDR protein expression and function in porcine ovarian follicles. Medium size antral follicles expressing great amount of androgen receptors and represent high steroidogenic activity were used in this research. After 6 h incubation of whole follicles with T, HF or T+HF, immunohistochemical analysis of VDR revealed its nuclear localization in granulosa and theca interna cells in control and experimental groups. The expression of VDR protein was shown as a band of 48 kDa. There were no significant differences between either experimental group and the control. T influenced the function of VDR through decreased formation of VDR/RXR (retinoid X receptor) complexes (P<0.05) in both granulosa and theca interna cells, but HF abolished this effect only in granulosa cells (P<0.05). These results suggest that androgens regulate the response of follicular cells to vitamin D3 in pigs ovary via regulation of VDR transcriptional activity.

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.

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.