Malgorzata Grzesiak

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.

Elevated level of 17β-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 17β-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 β-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.

Androgen Deficiency During Mid- and Late Pregnancy Alters Progesterone Production and Metabolism in the Porcine Corpus Luteum

We determined whether androgen deficiency induced by flutamide treatment during mid- and late pregnancy affects the functions of the porcine corpus luteum (CL). Pregnant gilts were injected with flutamide between days 43 and 49 (gestation day [GD] 50F), days 83 and 89 (GD90F), or days 101 and 107 (GD108F) of gestation. Antiandrogen treatment increased the luteal progesterone concentration in the GD50F group and decreased progesterone content in the GD90F and GD108F groups. Luteal levels of side-chain cleavage cytochrome P450 (CYP11A1) mRNA and protein were significantly downregulated in the GD90F and GD108F groups as compared with the respective controls. The 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (HSD3B) mRNA and protein expression were significantly reduced only in the GD108F group as compared with the control. Decreased luteal 20α-hydroxysteroid dehydrogenase (AKR1C1) mRNA and protein levels were observed in the GD50F group. Thus, androgen deficiency during pregnancy in pigs led to CL dysfunction that is marked by decreased progesterone production. Furthermore, exposure to flutamide during late pregnancy downregulated steroidogenic enzymes (CYP11A1 and HSD3B) in pigs. We conclude that androgens are important regulators of CL function during pregnancy.

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

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

Alterations in luteal production of androstenedione, testosterone, and estrone, but not estradiol, during mid- and late pregnancy in pigs: Effects of androgen deficiency

Recently, we have found that flutamide-induced androgen deficiency altered progesterone production in the porcine corpus luteum (CL) during mid- and late pregnancy. Herein, we tested whether flutamide administration subsequently influences androgen and estrogen metabolism in the CL of pregnancy. Pregnant gilts were treated with flutamide between Days 43 and 49 (GD50F), 83 and 89 (GD90F), or 101 and 107 (GD108F) of gestation. Corpora lutea (CLs) were collected from treated and nontreated (control) pigs. The concentrations of androstenedione (A4), testosterone (T), estrone (E1), and estradiol (E2) together with the levels of expression of mRNAs and proteins for cytochrome P450 17α-hydroxylase/c17-20 lyase (CYP17A1), 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), cytochrome P450 aromatase (CYP19A1), and 17β-hydroxysteroid dehydrogenase type 7 (17β-HSD7) were measured in the CL of control and flutamide-treated animals. Steroidogenic enzymes were also immunolocalized in luteal tissues. The luteal concentrations of A4 and T were higher in the GD50F (P = 0.006, P = 0.03) and GD108F (P = 0.005, P = 0.035) groups, but lower in the GD90F (P = 0.004, P = 0.014) group. The E1 level was greater only in the GD90F (P = 0.03) and GD108F (P = 0.035) groups, whereas E2 concentration was not affected by flutamide treatment. Increased luteal CYP17A1 mRNA and protein expression was found in the GD50F (P = 0.002, P = 0.03) and GD108F (P = 0.0026, P = 0.03) groups, but reduced in the GD90F (P = 0.002, P = 0.03) group. mRNA of 17β-HSD1 was upregulated in the GD50F (P = 0.0005) group, but downregulated in the GD90F (P = 0.002) and GD108F (P = 0.0005) groups. In contrast, 17β-HSD1 protein expression was higher in the GD50F and GD108F (P = 0.03) groups, but lower in the GD90F (P = 0.03) group. Both CYP19A1 mRNA and protein levels were greater in the GD90F (P = 0.001, P = 0.028) and GD108F (P = 0.005, P = 0.03) groups. Neither 17β-HSD7 mRNA nor protein level were affected by flutamide exposure. Both CYP17A1 and 17β-HSD1 were immunolocalized exclusively in small luteal cells, whereas CYP19A1 and 17β-HSD7 were found in large luteal cells of control and flutamide-treated CLs. Overall, flutamide administration led to the alterations in A4, T, and E1, but not in E2, production in the CL of pregnancy in pigs, probably because of disrupted steroidogenic enzymes expression. These changes suggest that androgens are important modulators of luteal function during pregnancy in pigs.

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.

Flutamide alters β-catenin expression and distribution, and its interactions with E-cadherin in the porcine corpus luteum of mid- and late pregnancy.

This study examined whether flutamide-induced androgen deficiency during mid- and late pregnancy in pigs affected luteal expression of adherens junction protein, β-catenin, and its interactions with E-cadherin. Flutamide (50 mg/kg body weight) was administered into pregnant gilts between days 43-49 (GD50F), 83-89 (GD90F) or 101-107 (GD108F) of gestation. Corpora lutea (CLs) were obtained on day 50, 90 or 108 of pregnancy (n=8-11 per each group). Total β-catenin and E-cadherin expression was examined at mRNA (real-time PCR) and protein (Western blot) level. Moreover, subcellular β-catenin fractions were extracted and immunoblotted. Immunohistochemistry was used for β-catenin localization. To determine whether flutamide disturbs β-catenin/E-cadherin mutual interactions, coimmunoprecipitation using anti-β-catenin antibody was performed. Furthermore, phosphorylation of E-cadherin was assessed. Flutamide exposure led to decreased β-catenin mRNA expression in all examined groups (p<0.001 or p<0.01), but protein level was lower only in the GD90F and GD108F groups (p<0.05). E-cadherin mRNA (p<0.05 or p<0.01) and protein (p<0.05) levels were up-regulated in all flutamide-treated groups when compared to controls. β-catenin was predominantly found in membranes of luteal cells with no significant changes after antiandrogen treatment. β-catenin/E-cadherin complexes were more abundant in the GD90F (p<0.05) and GD108F (p<0.01) groups than in controls due to enhanced E-cadherin phosphorylation at serine 838/840 in those animals (p<0.05). Overall, although androgen deficiency affected β-catenin expression in the CL of pregnancy in pigs, a compensatory mechanism by enhanced interactions with E-cadherin is possible. Thus, androgen signaling via androgen receptors appears to be crucial in the regulation of luteal cells cross-talk.

The impact of sex steroid agonists and antagonists on folliculogenesis in the neonatal porcine ovary via cell proliferation and apoptosis

The objective of the study was to examine the effects of androgen and estrogen agonists or antagonists on the follicle formation, ovarian cell proliferation and apoptosis as well as plasma steroid concentration in neonatal pigs. Piglets were injected with testosterone propionate (TP, 20 mg/kg bw), flutamide (FLU, 50 mg/kg bw), 4-tert-octylphenol (OP, 100 mg/kg bw), ICI 182,780 (ICI, 400 μg/kg bw), methoxychlor (MXC, 100 mg/kg bw) or corn oil (CTR, controls) between postnatal Days 1 and 10 (n = 4/group). Heart blood was collected and ovaries were excised from the 11-day-old piglets. The lower percentage of oocytes within an egg nest and higher ovarian expression of active caspase 3 were found in TP (androgen excess) piglets compared to controls. FLU-induced androgen deficiency decreased the percentage of primordial follicles, increased that of early primary follicles and diminished ovarian cell proliferation. OP-induced estrogen action increased the percentage of primordial and developing follicles as well as cell proliferation. ICI-induced estrogen deficiency decreased the percentage of transitional follicles and ovarian cell proliferation, while increased the percentage of primordial follicles and the abundance of active caspase 3. Treatment with MXC, exhibiting estrogenic, antiestrogenic, and antiandrogenic activities, declined the percentage of developing follicles and cell proliferation. Moreover, the investigated compounds differentially affected plasma steroid level. In conclusion, the present study demonstrated clear effects of TP and FLU during the earliest stages of folliculogenesis in pigs (nest breakdown and follicle assembly), whereas OP and ICI influenced also the subsequent stages of follicle initial recruitment and growth. Therefore, the androgen and estrogen seems to be important for the follicle assembly and follicle growth in neonatal porcine ovaries.

Selection of reference genes for quantitative real-time PCR analysis in chicken ovary following silver nanoparticle treatment

Reliable results of quantitative real time PCR (qPCR) analysis require normalization of target gene expression level using reference genes (RGs). However housekeeping genes expression may vary under experimental conditions, so selection of the proper RGs is a crucial step in a qPCR analysis. Several algorithms have been developed to address this problem: geNorm, NormFinder and BestKeeper. In this study, we have used these three tools to evaluate the stability of RGs in the ovarian tissues of hens treated with silver nanoparticles. Eight genes were selected for the validation: HPRT, HMBS, VIM, SDHA, TBP, RPL13, GAPDH and 18S rRNA. According to geNorm the best combination of reference genes is SDHA and TPP. NormFinder also selected SDHA as the most suitable gene, but in combination with RPL13. Analysis in BestKeeper showed that SDHA, RPL13 might be the best choice in gene expression studies using the chicken ovary. In conclusion, the results obtained depend on the algorithm used and it arises from the diverse calculation strategies used in these programs. The outcome from the NormFinder is considered to be the most trustworthy and used in further qPCR analysis.

Retrograde and destination transfer of sex steroid hormones in the spermatic cord vessels of the mature boar (Sus scrofa) in short-daylight and long-daylight periods, as well as vernal and autumnal equinox.

The aim of this study was to examine the seasonal changes in concentration of steroid hormones in the spermatic cord vessels of the mature boar. Cytochrome P450 aromatase (P450arom) was also localized in the arteries and veins of the spermatic cord. Arterial blood was collected from the common carotid artery and from two branches of the testicular artery supplying the testis and epididymis to determine progesterone (P4), androstenedione (A2), testosterone (T2) and estradiol (E2) plasma concentrations. The greatest concentration of P4 was found in testicular artery during December (P<0.001), when compared with other periods and vessels. In contrast, the greatest A2 concentration was observed in the epididymal artery during the same season (P<0.001). Greater T2 concentrations were found in both testis and epididymal arteries than in common artery in March (P<0.001, P<0.001; respectively) and in September (P<0.01, P<0.001; respectively). The E2 concentration was weakly affected by seasonal periods, but greater E2 concentrations were found within vessels in the testis and epididymis than in the common artery. The P450arom was immunolocalized in all layers of the arteries and veins of the testicular spermatic cord. The intensity of P450arom staining was greater in December than in June (P<0.001). There were greater steroid concentrations in arterial vessels during December in comparison to June and this may explain the summer infertility in boars and may be related to the local retrograde and destination transfer into the spermatic cord area. The P450arom gene expression in this area seems to be involved in the conversion of T2 into E2 to enrich the testes and epididymis.