Malgorzata Duda

Connexin 43 gene expression in male and female gonads of porcine offspring following in utero exposure to an anti-androgen, flutamide.

The aim of this study was to show the effect of maternal exposure to flutamide on connexin 43 (Cx43) gene expression in testes and ovaries of 2-day-old piglets. Additionally, anogenital distance (AGD) was measured both in male and female offspring. Immunohistochemistry, Western blotting, and RT-PCR were performed to assess the immunoreactivity and the presence of Cx43 protein and its mRNA, respectively. Following flutamide exposure strong immunostaining for Cx43 was observed between testicular Leydig cells, between granulosa cells of primary follicles, and between interstitial cells surrounding clusters of oocyte nests in the ovarian cortex as in the respective controls. Differences between the flutamide-treated groups and the controls obtained by qualitative immunohistochemistry were confirmed by quantitative image analysis (*P<0.05; **P<0.01). In Western blotting, Cx43 appeared as a band of 43kDa, whereas electrophoresis revealed PCR products of the predicted sizes. Screening for Cx43 expression revealed the presence of a transcript, both in control and in flutamide-treated pigs. The AGD values differed significantly from the control (*P<0.05). Overall, since no obvious changes in gonad morphology were observed and the Cx43 signal was present in all the examined tissues, it seems likely that androgens acting through ARs are not involved in the control of Cx43 gene expression in neonatal pig gonads.

Effects of pre- and postnatal exposure to flutamide on connexin 43 expression in testes and ovaries of prepubertal pigs

The aim of this study was to show whether connexin43 (C×43) expression in gonads is affected by an anti-androgen action. To perform this test, pigs were prenatally (on gestational days 20–28 and 80–88; GD20, GD80) and postnatally (on days 2–10 after birth; PD2) exposed to flutamide, which was given in five doses every second day and its effect was observed in prepubertal gilts and boars. Morphology and expression of C×43 was investigated in testes and ovaries by means of routine histology, immunohistochemistry, Western blotting, and RT-PCR. In boars exposed to flutamide varying degrees of seminiferous tubule abnormality, including reduced number of Sertoli cells, tubules with severely dilated lumina and multinucleated germ cells were observed, whereas in gilts, the administration of flutamide at GD20 resulted in delayed folliculogenesis. Only follicles at the preantral stage were observed. Qualitative analysis of immunohistochemical staining for C×43 was confirmed by quantitative image analysis, where the staining intensity was expressed as relative optical density of diaminobenzidine deposits. After flutamide exposure, statistically significant increase in C×43 signal intensity was observed between interstitial tissue of GD20 and control pigs (**P<0.01), between seminiferous tubules of PD2 and control boars (**P<0.01) and between theca cells of GD80, of PD2 and control gilts (**P<0.01). In contrast, statistically significant decrease in C×43 signal intensity was found between granulosa cells of GD20, of PD2 and control gilts (**P<0.01 and *P<0.05, respectively) and between theca cells of GD20 and control gilts (**P<0.01). Since we demonstrated changes in gonad morphology and in the expression of C×43 at the level of protein of prepubertal boars and gilts, it seems possible that flutamide, through blocking androgen action, causes delayed gonadal maturation in later postnatal life and, among other factors, may be involved in the regulation of C×43 gene expression in pig gonads.

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, 3β-hydroxysteroid dehydrogenase (3β-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 3β-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.

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.

Expression of estrogen receptor α (ERα) and estrogen receptor β (ERβ) in the ovarian follicles and corpora lutea of pregnant swine

The objective of the study was to demonstrate the presence of estrogen receptor alpha (ERalpha) and beta (ERbeta) protein and corresponding mRNA in porcine ovarian follicles and corpora lutea obtained on day 10, 18, 32, 50, 71 and 90 post coitum (p.c.) using immunohistochemistry, Western blot, and RT-PCR analysis. Immunohistochemistry showed that ERalpha protein was located in the granulosa cells of ovarian follicles and the strongest immunoreaction was observed on days 32 and 50 p.c. The ERbeta protein was found mainly in theca cells of follicles as well as in luteal cells. The most intense immunoreaction was observed on day 18 p.c. within theca cells, while in the corpus luteum (CL) the intensity of ERbeta staining gradually increased and remained elevated at mid and late pregnancy. In CL by day 50 p.c. immunoreaction for ERbeta was present only in small luteal cells, but starting from day 71 to 90 p.c. it was observed in both small and large luteal cells. Western blot analysis was performed and validated data obtained from immunohistochemistry. RT-PCR results indicated that ERalpha mRNA was expressed only in ovarian follicles of the pregnant swine, while that of ERbeta in both follicles and CL. The results suggest an autocrine/paracrine role of estrogens acting via both ERalpha and ERbeta in the regulation of the ovarian function during pregnancy and for the process of successful reproduction.

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.

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.

Expression of ghrelin and the ghrelin receptor in different stages of porcine corpus luteum development and the inhibitory effects of ghrelin on progesterone secretion, 3β-hydroxysteroid dehydrogenase (3β-honestly significant difference (HSD)) activity and protein expression

Recent studies have suggested that ghrelin plays a direct role in controlling female reproduction. The aim of the present study was to investigate the mRNA and protein expression of ghrelin and its receptor (via real time PCR, Western blot and immunohistochemistry analysis, respectively) in porcine corpora lutea (CL) collected during early (CL1: 1-2 days after ovulation), middle (CL2: 7-10 after ovulation), and late luteal phase (CL3: 13-15 after ovulation). Ghrelin expression and concentration of both acylated and unacylated forms of ghrelin significantly increased during CL development. Immunohistochemistry analysis shown localization of ghrelin protein in the cytoplasm of large luteal cells. No changes in the expression of the ghrelin receptor were observed. Direct in vitro effects of ghrelin on progesterone (P4) secretion and 3-beta-hydroxysteroid dehydrogenase (3β-honestly significant difference (HSD)) activity, which were measured by the conversion of pregnenolone (P5) to P4, and 3β-HSD protein expression were then analyzed. To assess 3β-HSD activities, mature luteal cells were first cultured for 24 h with ghrelin at 100, 250, 500 and 1000 pg/mL with P5, or with aminoglutethimide (AMG). AMG is an inhibitor of CYP11A1-mediated hydroxylation; an addition of AMG and P5 enabled P4 production to serve as an index of 3β-HSD activity. Inhibitory effects of ghrelin on P4 secretion, 3β-HSD activity and protein expression were observed. In conclusion, the presence of ghrelin and its receptor in porcine corpora lutea and the direct inhibitory effects of ghrelin on luteal P4 secretion and 3β-HSD suggest potential auto/paracrine regulation by ghrelin in the luteal phase of ovary function.

Octylphenol affects morphology and steroidogenesis in mouse tumor Leydig cells

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

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.