Impact of endocrine-active compounds on adrenal androgen production in pigs during neonatal period.

This study investigated the impact of neonatal exposure to endocrine-active compounds (EACs): flutamide (antiandrogen), 4-tert-octylphenol (an estrogenic compound), and methoxychlor (an organochlorine insecticide exhibiting estrogenic, antiestrogenic and antiandrogenic activities) on androgen production within porcine adrenal glands. The expression of genes related to androgen synthesis and the level of androgen production were analyzed (i) in the adrenal glands of piglets exposed to EACs during the first 10 days of life (in vivo study), and (ii) in adrenal explants from sow-fed or formula-fed 10-day-old piglets incubated with EACs (ex vivo study). EACs affected the expression of genes linked to adrenal androgen biosynthesis. The prominent effect of methoxychlor on downregulation of StAR, CYP11A1 and HSD3B and upregulation of CYP17A1 and SULT2A1 were demonstrated. Furthermore, our study revealed divergent response to EACs between sow-fed and formula-fed piglets, suggesting that natural feeding may provide protection against adverse EACs effects, particularly those interfering with estrogens action.

Atretic preovulatory follicles could be precursors of ovarian lutein cysts in the pig.

Ovarian cysts contribute to reduced reproductive performance in pigs. Unfortunately, the mechanism of lutein cysts formation remains unknown. Here, we compared the endocrine and molecular milieus of intact, healthy preovulatory follicles (PF), gonadotropin (eCG/hCG)-induced healthy and atretic-like PF, as well as gonadotropin-provoked and spontaneous ovarian cysts in gilts. Several endocrine and molecular indicators and microRNA were compared in walls of PF and cysts. Intact and healthy PF, showed high estradiol/androstendione and low progesterone levels associated with CYP17A1, HSD17B1, and CYP19A1 elevation and reduced StAR/HSD3B1 protein expression. In contrast, low estradiol/androstendione and high progesterone concentrations, accompanied by decreased CYP17A1, HSD17B1, CYP19A1 and increased HSD3B1 protein abundance, appeared in atretic-like PF, gonadotropin-induced and spontaneous cysts. High progesterone receptor (PGR) protein abundance was maintained in intact and healthy PF, while it dropped in atretic-like PF, gonadotropins-induced and spontaneous cysts. The atretic PF showed high level of TNFα compared to healthy PF. In conclusion, follicular lutein cysts could be recruited from atretic-like PF with lost estrogenic milieu and inability to ovulate. Ovulatory cascade was presumably disrupted by a low PGR and high TNFα levels associated with earlier luteinization of follicular walls. These results suggest a novel mechanism of lutein ovarian cysts development in pigs and, perhaps, other species.

The expression and localization of selected matrix metalloproteinases (MMP-2, -7 and -9) and their tissue inhibitors (TIMP-2 and -3) in follicular cysts of sows.

Matrix metalloproteinases (MMPs) are a family of enzymes that degrade extracellular matrix (ECM) molecules, playing a vital role in tissue remodeling under physiological and pathological conditions. Their expression and/or activity are regulated by specific tissue inhibitors of MMPs named TIMPs. Recently, an imbalance in the MMP/TIMP system has been found in human and bovine ovarian cysts, but its role in porcine cyst pathogenesis is unknown. This study examined mRNA expression, protein abundance and localization for selected members of the MMP/TIMP system in follicular cysts of sows. Based on histological analysis, we have assessed follicular (FC) and follicular lutein (FLC) cysts with preovulatory follicles (PF) used as a control. Regarding the pattern of MMP expression, increased MMP2, MMP7 and MMP9 mRNA levels were observed in FLC. Furthermore, both pro- and active forms of MMP-2 and MMP-9 proteins were more abundant in FLC. In FC, the abundance of latent and active forms of MMP-9 and the active form of MMP-2 were greater when compared with PF. In relation to TIMPs, TIMP-2 mRNA and protein expression were increased in FLC, whereas TIMP-3 was up-regulated in both FC and FLC only at the protein level. Using immunofluorescence, MMP-2, MMP-7, TIMP-2 and TIMP-3 were detected in granulosa and theca compartments of FC and within the entire luteinized wall of FLC. Notably, MMP-9 occurred weakly in the granulosa layer of FC, but abundantly in the theca compartment of FC and in the luteinized FLC. Taken together, our findings indicate altered expression of the MMP/TIMP system, suggestive of increased ECM degradation, in sow follicular cysts. These components may be involved in the pathogenesis of porcine ovarian cysts through the ECM remodeling.

Vitamin D3 Metabolic Enzymes in the Porcine Uterus: Expression, Localization and Autoregulation by 1,25(OH)2D3 In Vitro.

The role of vitamin D3 has been confirmed in female reproductive organs. This study aimed to examine vitamin D3 metabolic enzymes, i.e., CYP27B1 and CYP24A1, mRNA transcript and protein abundance, and protein localization in the uterus of pigs on days 2-5, 10-12, 15-16 and 18-20 of the estrous cycle. Additionally, we determined 1,25(OH)2D3 concentration in uterine flushings and the effect of 1,25(OH)2D3 (10, 50 and 100 ng/mL) in vitro on CYP27B1 and CYP24A1 mRNA transcript abundance in endometrial and myometrial slices. In the endometrium, a greater CYP27B1 mRNA transcript abundance was noted on days 10-12 and 18-20 than on days 15-16, whereas encoded protein abundance was greater on days 18-20 when compared to days 15-16. Endometrial CYP24A1 mRNA transcript abundance was greater on days 18-20 than on days 10-12 and 15-16. In the myometrium, CYP27B1 mRNA transcript abundance was greater on days 18-20 than on days 2-5 and 15-16, while protein abundance was larger in slices collected on days 18-20 than on days 15-16. Neither CYP24A1 mRNA transcript nor encoded protein abundance were detected in the myometrium. The highest 1,25(OH)2D3 concentration in uterine flushings was observed on days 18-20. Furthermore, the 1,25(OH)2D3 increased the abundance of the CYP24A1 mRNA transcript in endometrial slices. Overall, our results suggest that porcine uterus is an extra-renal site of vitamin D3 metabolism. Both the endometrium and the myometrium possess the ability to synthesize vitamin D3, while only the endometrium contributes to its catabolism.

Long-Term Changes in Ovarian Follicles of Gilts Exposed Neonatally to Methoxychlor: Effects on Oocyte-Derived Factors, Anti-Müllerian Hormone, Follicle-Stimulating Hormone, and Cognate Receptors.

In this paper, we investigated the effects of neonatal exposure to methoxychlor (MXC), a synthetic organochlorine used as an insecticide with estrogenic, antiestrogenic, and antiandrogenic activities on ovarian follicles of adult pigs. Piglets were injected with MXC (20 µg/kg body weight) or corn oil (controls) from postnatal Day 1 to Day 10 (n = 5 per group). Then, mRNA expression, protein abundance and immunolocalization of growth and differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), anti-Müllerian hormone (AMH) and cognate receptors (ACVR1, BMPR1A, BMPR1B, TGFBR1, BMPR2, and AMHR2), as well as FSH receptor (FSHR) were examined in preantral and small antral ovarian follicles of sexually mature gilts. The plasma AMH and FSH levels were also assessed. In preantral follicles, neonatal exposure to MXC increased GDF9, BMPR1B, TGFBR1, and BMPR2 mRNAs, while the levels of AMH and BMP15 mRNAs decreased. In addition, MXC also decreased BMP15 and BMPR1B protein abundance. Regarding small antral follicles, neonatal exposure to MXC upregulated mRNAs for BMPR1B, BMPR2, and AMHR2 and downregulated mRNAs for AMH, BMPR1A, and FSHR. MXC decreased the protein abundance of AMH, and all examined receptors in small antral follicles. GDF9 and BMP15 were immunolocalized in oocytes and granulosa cells of preantral follicles of control and treated ovaries. All analyzed receptors were detected in the oocytes and granulosa cells of preantral follicles, and in the granulosa and theca cells of small antral follicles. The exception, however, was FSHR, which was detected only in the granulosa cells of small antral follicles. In addition, MXC decreased the plasma AMH and FSH concentrations. In conclusion, the present study may indicate long-term effects of neonatal MXC exposure on GDF9, BMP15, AMH, and FSH signaling in ovaries of adult pigs. However, the MXC effects varied at different stages of follicular development. It seems that neonatal MXC exposure may result in accelerated initial recruitment of ovarian follicles and impaired cyclic recruitment of antral follicles.

Effects of neonatal methoxychlor exposure on the ovarian transcriptome in piglets.

Methoxychlor (MXC) is a man-made organochlorine insecticide capable of disrupting endocrine functions due to its mixed steroidal properties (estrogenic, anti-estrogenic and/or anti-androgenic). Retarded follicle development was recently reported in neonatal pigs treated with MXC. The goal of the current study was to better understand the mechanism of MXC action in the ovary of newborn piglets. By employing RNA-Seq we studied the expression of protein coding (mRNA) and long non-coding RNA (lncRNA) transcripts in the ovary of the MXC-treated piglets. Piglets were injected (sc) daily with MXC (100 mg/kg body weight) or corn oil (controls) between postnatal Days 1 and 10 (n = 3 piglets/group). The ovaries excised from 11-day-old piglets were processed for total RNA isolation and subsequent RNA sequencing. Four hundred sixty differentially expressed genes (DEGs) and 143 differentially expressed lncRNAs (DELs) were identified in the ovaries of MXC-treated piglets (P-adjusted < 0.05; abs(log2FC) > 1). Functional enrichment analysis showed that MXC altered the expression of genes associated with intracellular and membrane transport, intra-ovarian signaling as well as cell-cell junction and communication. Moreover, positive and negative correlations determined between the identified DEGs and DELs suggest that some lncRNAs may mediate the MXC action in the ovary. The results support the hypothesis that MXC-induced changes in the expression of genes involved in neonatal ovarian folliculogenesis increase the risk of fertility problems in adults.

Endocrine and molecular milieus of ovarian follicles are diversely affected by human chorionic gonadotropin and gonadotropin-releasing hormone in prepubertal and mature gilts.

Different strategies are used to meet optimal reproductive performance or manage reproductive health. Although exogenous human chorionic gonadotropin (hCG) and gonadotropin-releasing hormone (GnRH) agonists (A) are commonly used to trigger ovulation in estrous cycle synchronization, little is known about their effect on the ovarian follicle. Here, we explored whether hCG- and GnRH-A-induced native luteinizing hormone (LH) can affect the endocrine and molecular milieus of ovarian preovulatory follicles in pigs at different stages of sexual development. We collected ovaries 30 h after hCG/GnRH-A administration from altrenogest and pregnant mare serum gonadotropin (eCG)-primed prepubertal and sexually mature gilts. Several endocrine and molecular alternations were indicated, including broad hormonal trigger-induced changes in follicular fluid steroid hormones and prostaglandin levels. However, sexual maturity affected only estradiol levels. Trigger- and/or maturity-dependent changes in the abundance of hormone receptors (FSHR and LHCGR) and proteins associated with lipid metabolism and steroidogenesis (e.g., STAR, HSD3B1, and CYP11A1), prostaglandin synthesis (PTGS2 and PTGFS), extracellular matrix remodeling (MMP1 and TIMP1), protein folding (HSPs), molecular transport (TF), and cell function and survival (e.g., VIM) were observed. These data revealed different endocrine properties of exogenous and endogenous gonadotropins, with a potent progestational/androgenic role of hCG and estrogenic/pro-developmental function of LH.

Effects of neonatal exposure to methoxychlor on corpus luteum in gilts: A transcriptomic analysis.

This study investigated the effects of neonatal exposure to methoxychlor (MXC), a synthetic organochlorine used as an insecticide with estrogenic, antiestrogenic, and antiandrogenic activities, on luteal function in pigs. Piglets were injected subcutaneously with MXC (20 µg/kg body weight) or corn oil (control) between postnatal Days 1 and 10 (N = 5/group). Corpora lutea from sexually mature gilts were examined for luteal steroid and prostaglandin concentrations and processed for total RNA isolation and subsequent RNA sequencing. Intra-luteal concentrations of androstenedione and prostaglandin E2 were greater, while that of estrone was lower when compared to control. Fifty-three differentially expressed (DE) microRNAS (miRNAs) (p-adjusted <.05 and log2(fold change) ≥.5) and 359 DE genes (p-adjusted <.05 and log2(fold change) ≥1) were identified in luteal tissue in response to neonatal MXC treatment. MXC was found to affect the expression of genes related to lipogenesis, steroidogenesis, membrane transport, immune response, cell signaling and adhesion. These results suggest an earlier onset of structural luteolysis in pigs caused by MXC actions in neonates. Since negative correlation analysis showed the potential interactions of miRNAs with specific messenger RNAs, we propose that these miRNAs are potential mediators of the long-term MXC effect on the CL function in pigs.

Effect of neonatal exposure to endocrine-active compounds on epigenetic regulation of gene expression in corpus luteum of gilts.

Recently, we have demonstrated that neonatal exposure to environmental endocrine-active compounds (EACs) with androgenic/antiandrogenic and estrogenic/antiestrogenic activities led to morphological and functional changes in the porcine corpus luteum (CL). To gain insight into the regulatory mechanisms of the long-term effects of EACs, we analyzed the impact of neonatal exposure of such compounds on global DNA methylation and the expression of miRNA biogenesis components in the porcine CL. Piglets were injected subcutaneously with testosterone propionate (TP, an androgen), flutamide (FLU, an antiandrogen), 4-tert-octylphenol (OP, an estrogenic compound), ICI 182,780 (ICI, an antiestrogen), methoxychlor (MXC, a compound with mixed activities) or corn oil (control) between postnatal days 1 and 10 (n = 5/group). The CLs from sexually mature gilts were examined for global DNA methylation and for the abundance of proteins related to DNA methylation (DNMT1, DNMT3A and DNMT3B) and miRNA biogenesis (DROSHA, XPO5, DICER1, AGO2) using an enzyme-linked immunosorbent assay, Western blotting and immunohistochemical staining. ICI and MXC increased the global DNA methylation levels and DNMT1 protein abundance in the luteal tissue. OP treatment led to a lower DROSHA protein abundance, while ICI treatment resulted in a greater DROSHA protein abundance. Both FLU and ICI increased DICER1 protein abundance in the luteal tissue. In addition, XPO5 showed immunolocalization exclusively in small luteal cells in the OP-treated pigs, in contrast to localization in both small and large luteal cells in the controls. In conclusion, the changes in DNA methylation, as well as the altered miRNA biogenesis components, seem to be a part of the regulatory network that mediates the long-term effects of EACs on CL function in pigs.

Vitamin D3 in ovarian antral follicles of mature gilts: Expression of its receptors and metabolic enzymes, concentration in follicular fluid and effect on steroid secretion in vitro.

Recent studies have reported that vitamin D3 (VD) regulates ovarian function under physiological and pathological conditions. Due to a lack of information concerning the expression of VD-related molecules (receptors: VDR, PDIA3, and metabolic enzymes: CYP27B1, CYP24A1) in the porcine ovary, this research aimed to determine the mRNA expression, protein abundance and localization of VDR, PDIA3, CYP27B1 and CYP24A1 in small (SFs), medium (MFs) and large (LFs) antral follicles of sexually mature gilts. We also examined the concentration of active VD in the follicular fluid of SFs, MFs and LFs, and the effect of 1,25(OH)2D3 on their steroidogenic activity in vitro. In the present study, we have demonstrated for the first time the patterns of VDR, PDIA3, CYP27B1 and CYP24A1 immunolocalization in porcine antral follicles of different sizes. Furthermore, the expression of VD-related molecules was influenced by follicle developmental stage. VDR and PDIA3 mRNA expression and protein abundance decreased with the follicle size: they were the greatest in SFs, and the lowest in LFs. CYP27B1 mRNA expression was the highest in MFs and differed from that in SFs, whereas protein abundance was greater in MFs and SFs than in LFs. The expression of mRNA for CYP24A1 was higher in MFs than in SFs and LFs, while protein abundance did not differ between follicle classes. We have also described changes in the concentration of 1,25(OH)2D3 in the follicular fluid of antral follicles with its highest level in MFs. These findings show that the porcine antral follicle is a target tissue for direct VD action and is a local site of VD metabolism. Furthermore, we found that 1,25(OH)2D3 increased the secretion of progesterone and estradiol-17ß by SFs and MFs in vitro, implying a crucial role of VD in the regulation of ovarian steroidogenesis in mature gilts. Therefore, VD appears to be an important intraovarian factor that could regulate follicular development and function in pigs.

Altrenogest affects the development and endocrine milieu of ovarian follicles in prepubertal and mature gilts†.

Altrenogest with gonadotropins is commonly used to synchronize the estrous cycle, but it can also lead to follicular cyst formation, especially in prepubertal gilts. Here, we aimed to investigate how maturity and altrenogest treatment affect the development, endocrine milieu, and molecular control of ovarian follicles. Crossbred prepubertal and mature gilts were challenged or not (control) with altrenogest, and ovaries were collected in the morning on the first day of behavioral estrus. In prepubertal gilts, altrenogest decreased the percentage of primordial and atretic small follicles, but increased large antral follicles when compared with controls. In mature gilts, altrenogest reduced the percentage of primary follicles and elevated the total number of antral follicles. Maturity affected the estradiol level in the follicular fluid of preovulatory follicles, luteinizing hormone (LH)-stimulated cyclic adenosine monophosphate (cAMP) generation, and LH receptor messenger RNA (mRNA) expression in granulosa. Moreover, cytochrome P45017A1 (CYP17A1) mRNA levels in the theca layer were affected and correlated with follicular androstendione and estradiol concentration. Altrenogest negatively affected follicular fluid progesterone concentration and decreased levels of prostaglandin (PG) E2 in prepubertal gilts and PGF2alpha metabolite in mature gilts. LH-stimulated cAMP release in granulosa cells of mature gilts as well as human chorionic gonadotropin- and forskolin-induced cAMP were also affected. In addition, altrenogest downregulated CYP17A1 mRNA in the prepubertal theca layer and PGF2alpha synthase expression in the granulosa and theca layer of mature gilts. To the best of our knowledge, this is the first study to report multiple effects of maturity and altrenogest on the endocrine milieu and molecular regulations governing ovarian follicle development in gilts.

Transcriptomic profiles of the ovaries from piglets neonatally exposed to 4-tert-octylphenol.

The environmental pollutants with hormonal activities may influence steroid-mediated processes in neonatal ovaries and increase the incidence of reproductive disorders. The aim of the current study was to examine effects of 4-tert-octylphenol (OP), a non-ionic surfactant widely used in a variety of industrial applications which has been reported to mimic the 17ß-estradiol activity, on the expression of protein-coding (mRNAs) and long non-coding (lncRNAs) transcripts in neonatal ovaries of the pig. By employing RNA-Seq we aimed to gain insights into regulatory networks underlying the OP effects on the follicular development in pigs. Piglets were injected (sc) daily with OP (100 mg/kg bw) or corn oil (controls) between postnatal Days 1 and 10 (n = 3/group). Ovaries were excised from the 11-day-old piglets and total cellular RNA was isolated and sequenced. Two hundred three differentially expressed genes (DEGs; P-adjusted < 0.05 and log2 fold change ≥1.0) and 23 differentially expressed lncRNAs (DELs; P-adjusted < 0.05 and log2 fold change ≥ 1.0) were identified in OP-treated piglet ovaries. The DEGs were assigned to Gene Ontology terms, covering biological processes, molecular functions and cellular components, which linked the DEGs to functions associated with movement of cell or subcellular component, regulation of plasma membrane bounded cell projection assembly as well as hydrolase and endopeptidase activity. In addition, STRING analysis demonstrated the strongest interactions between genes related to negative regulation of endopeptidase activity. Some correlations between DEGs and DELs were also found, revealing that the OP action on the ovary may be partially executed via the changes in the lncRNA expression. These results suggest that neonatal exposure of pigs to OP induces changes in the ovarian transcriptomic profile associated with genes encoding serine protease inhibitors and involved in steroid synthesis as well as genes linked to intracellular and membrane transport. We suggest that the changes in the mRNA and lncRNA expression in the ovaries of OP-treated piglets may disturb ovarian cellular function, including steroidogenesis, proliferation and apoptosis.

A transcriptome approach evaluating effects of neonatal androgen and anti-androgen treatments on regulation of luteal function in sexually mature pigs.

The current study was designed to gain insights into regulatory mechanisms mediating long-term effects of androgen excess or deficiency on corpus luteum function in pigs. Piglets were injected subcutaneously with testosterone propionate (TP, an androgen), flutamide (FLU, an anti-androgen) or corn oil (control) between postnatal Days 1 and 10. Corpora lutea from sexually mature gilts were examined for luteal steroid concentrations and processed for total RNA isolation and subsequent RNA sequencing to determine abundances of mRNA transcripts and microRNAs (miRNAs). Potential miRNA-mRNA interactions were explored in silico. Androstenedione, testosterone and estrone concentrations in corpora lutea were altered due to the disrupted androgen action in neonates. The luteal tissue had 465 and 353 genes for which there were differential mRNA abundances as compared with the control group (P-adjusted < 0.05; log2FC ≥ 1.0) in response to neonatal TP and FLU piglet treatments, respectively. Disruption of androgen signalling in neonates affected mRNA transcript abundance, as compared with the control group, for genes associated with apoptosis, angiogenesis and immune functions in the corpora lutea. Furthermore, there was a differential abundance of a group of miRNAs in the treatment groups compared with the control group. These results indicate the neonatal androgenic milieu affects the onset of luteolysis when these animals are sexually mature, although mechanisms for responses to TP or FLU likely differ. It is proposed that changes in specific miRNAs and mRNAs may, in part, account for long-term effects of androgen excess or androgen deficiency on corpus luteum function in pigs.

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

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

Effect of estrogen-related receptor silencing on miRNA protein machinery expression, global methylation, and deacetylation in bank vole (Myodes glareolus) and mouse tumor Leydig cells.

The function of estrogen-related receptor (ERR) in testicular cells is at the beginning of exploration. Our previous findings showed that expression pattern of estrogen-related receptor (ERR) in mouse Leydig cell depends on physiological status of the cell. Exogenous hormones/hormonally active chemicals affect ERR expression. In Leydig cells in vitro, ERRα and ERRγ show opposing regulatory properties. The aim of this study was to examine the role of ERR in epigenetic processes in cells with altered level of secreted estrogens; mouse tumor Leydig cells and bank vole Leydig cells, respectively. In Leydig cells, ERRα and ERRγ were silenced via siRNA. mRNA and protein expression and protein localization of molecules required for miRNA biogenesis and function (Exportin 5, Dicer, Drosha and Argonaute 2; Ago2) were studied with the use of qRT-PCR, Western blotting, and immunohistochemistry. Global DNA methylation and histone deacetylation status together with estradiol secretion were determined with fluorometric, and immunoenzymatic assays. Regardless of ERR type knockdown in tumor Leydig cells, downregulation (P < 0.05; P < 0.01; P < 0.001) of Exportin5, Dicer, Drosha but not Ago2 was revealed while at protein level only Drosha was downregulated (P < 0.01) by both ERRα and ERRγ. Oppositely, Exportin5, Dicer and Ago2 showed ERR type-dependent regulation (downregulation; P < 0.01 by ERRα and upregulation; P < 0.01; P < 0.001 by ERRγ). In ERR-silenced vole Leydig cells, expression of Exportin5, endonucleases and Ago2 was not changed. Immunolocalization of Dicer and Ago2 was independent of the cell origin in contrast to localization of Exportin5 and Drosha which was dependent on the cell origin and ERR type knockdown. Absence of ERR effected on cell methylation status (ERRα increased it; P < 0.01 while ERRγ decreased it; P < 0.01, P < 0.001) but it not changed histone deacetylates activity. ERRα and ERRγ silencing decreased (P < 0.01, P < 0.001) estradiol secretion in both tumor and vole Leydig cells. In mouse and bank vole Leydig cell, Exportin5, Dicer, Drosha and Ago2 expression as well as methylation status are regulated by ERR in a manner related to receptor type, molecule type, cell origin and level of secreted estrogen.

Neonatal exposure to agonists and antagonists of sex steroid receptors induces changes in the expression of oocyte-derived growth factors and their receptors in ovarian follicles in gilts.

The objective of the present study was to examine the effects of neonatal exposure to either agonists or antagonists of androgen and estrogen receptors on the expression of growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) and their cognate receptors (TGFBR1, BMPR1B, and BMPR2) in ovarian follicles of adult pigs. Piglets were injected subcutaneously with testosterone propionate (TP, an androgen, at 20 mg/kg bw), flutamide (FLU, an antiandrogen, at 50 mg/kg bw), 4-tert-octylphenol (OP, an estrogenic compound, 100 mg/kg bw), ICI 182,780 (ICI, an antiestrogen, 400 µg/kg bw), or corn oil (control) between postnatal Days 1 and 10 (n = 5/group). Ovarian follicles were excised from adult pigs on Days 8-11 of the estrous cycle. The expression of GDF9, BMP15, TGFBR1, BMPR1B and BMPR2 were examined in the population of preantral and small antral ovarian follicles using real-time PCR, Western blot and immunohistochemistry. In preantral follicles, the upregulation of GDF9 mRNA and protein expression was found in pigs that were neonatally exposed to TP or FLU, while administration of TP or ICI resulted in upregulation of BMP15. TGFBR1 and BMPR2 mRNA and protein expression were upregulated in preantral follicles of adult pigs that were neonatally exposed to TP or FLU, while administration of TP or ICI resulted in upregulation of BMPR1B. In small antral follicles, the mRNA and protein for TGFBR1 and BMPR2 were upregulated, while BMPR1B was downregulated in response to neonatal OP treatment. In addition, treatment with FLU upregulated BMPR1B and BMPR2 mRNA and protein expression, while downregulated the expression of TGFBR1. Moreover, GDF9 and BMP15 were immunolocalized in oocytes and granulosa cells of preantral follicles obtained from both control and treated ovaries. TGFBR1, BMPR1B and BMPR2 receptors were observed in the oocytes and granulosa cells of preantral follicles as well as in granulosa and theca cells of small antral follicles. In conclusion, the present study demonstrated neonatal exposure to either agonists or antagonists of androgen and estrogen receptors affected GDF9 and BMP15 signalling in ovaries of adult pigs. It seems that neonatal androgen excess or deficiency may lead to the acceleration of initial follicle recruitment, while neonatal exposure to compounds with antiandrogenic and estrogenic activity may disturb small antral follicles fate. Therefore, it confirms that neonatal window is critical for programming of ovarian function in pigs.

Flutamide-induced alterations in transcriptional profiling of neonatal porcine ovaries.

BACKGROUND: Androgens are involved in the regulation of ovarian development during fetal/neonatal life. Environmental chemicals displaying anti-androgenic activities may affect multiple signal transduction pathways by blocking endogenous androgen action. The aim of the current study was to examine effects of the anti-androgen flutamide on the expression of coding transcripts and long non-coding RNAs (lncRNAs) in neonatal porcine ovaries. By employing RNA-Seq technology we aimed to extend our understanding of the role of androgens in neonatal folliculogenesis and examine the impact of the anti-androgen flutamide on ovarian function. METHOD: Piglets were subcutaneously injected with flutamide (50 mg/kg BW) or corn oil (controls) between postnatal days 1 and 10 (n = 3/group). Ovaries were excised from the 11-day-old piglets and total cellular RNAs were isolated and sequenced. RESULTS: Flutamide-treated piglet ovaries showed 280 differentially expressed genes (DEGs; P-adjusted < 0.05 and log2 fold change ≥1.0) and 98 differentially expressed lncRNAs (DELs; P-adjusted < 0.05 and log2FC ≥ 1.0). The DEGs were assigned to GO term, covering biological processes, molecular functions and cellular components, which linked the DEGs to functions associated with cellular transport, cell divisions and cytoskeleton. In addition, STRING software demonstrated strongest interactions between genes related to cell proliferation. Correlations between DEGs and DELs were also found, revealing that a majority of the genes targeted by the flutamide-affected lncRNAs were associated with intracellular transport and cell division. CONCLUSIONS: Our results suggest that neonatal exposure of pigs to flutamide alters the expression of genes involved in ovarian cell proliferation, ovarian steroidogenesis and oocyte fertilization, which in turn may affect female reproduction in adult life.

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.

The impact of antiandrogen flutamide on the hypoxia inducible factor 1a and vascular endothelial growth factor A gene and protein expression in the pig placenta during late pregnancy.

INTRODUCTION: In contrast to estradiol action, little is known about androgen signaling in placental development. The purpose of this study was to evaluate the impact of diminished androgen action on hypoxia inducible factor 1a (HIF-1a) and vascular endothelial growth factor A (VEGFA) protein expression as well as their mRNAs in the structures of fetal and maternal parts of porcine placenta during late pregnancy. MATERIAL AND METHODS: Pregnant pigs were injected daily with antiandrogen flutamide, at a dose of 50 mg/kg body weight at different stages of pregnancy: between gestational days 83-89 (90 dpc) and 101-107 (108 dpc). Control groups (90 dpc or 108 dpc) were treated with vehicle (corn oil). One day after the last injection animals were sacrificed and tissues were collected. Tissue samples were frozen for mRNA isolation or fixed for immu-nohistochemistry (IHC). The expression of HIF-1a and VEGFA were investigated by real-time PCR and IHC. RESULTS: Flutamide treatment caused changes in both HIF-1a and VEGFA mRNA levels only in the placentas of the 90 dpc group. Relative optical density analysis showed decreased HIF-1a and increased VEGFA protein expression in the placentas obtained from flutamide-treated 108 dpc group while no differences were observed in the 90 dpc group. CONCLUSIONS: Experimentally induced androgen deficiency in pigs deregulates the expression of some genes important for placental blood circulation. We suggest that androgens are involved in the control of expression of HIF-1a and VEGFA in porcine placenta during late pregnancy.

Effect of gestational antiandrogen treatment on Dicer1 expression in the porcine fetal gonads.

Recently, we have demonstrated that flutamide-induced androgen deficiency during fetal life led to changes in gene expression that affected both testicular functions and follicular formation. It is known that microRNA-mediated genes regulation is essential for gonadal development and function. Thus, the aim of the present study was to examine whether prenatal flutamide exposure influences expression of Dicer1, an enzyme involved in microRNA maturation, in gonads of porcine fetuses during mid- and late gestation. Pregnant gilts were injected with flutamide (50mg/day/kg b.w.) or corn oil (controls) between days 43-49, 83-89 or 101-107 of gestation. The fetal gonads were obtained on gestational day 50 (GD50), 90 (GD90) or 108 (GD108). To assess Dicer1 mRNA expression real-time PCR were performed. Furthermore, immunohistochemical Dicer1 localization was conducted. In testes from flutamide treated fetuses, increased Dicer1 mRNA expression was observed in the GD50 and GD108 groups, but decreased in the GD90 group. Dicer1 was immunolocalized in the fetal Leydig cells in both control and flutamide-treated groups. In fetal ovaries, antiandrogen treatment increased Dicer1 mRNA level in the GD50 and GD90 groups. In control and flutamide-exposed groups, Dicer1 was localized in the germ cells within oogonia/oocyte nests as well as in the granulosa cells and oocytes of forming follicles. Concluding, diminished androgen action during gestation induces changes in Dicer1 mRNA expression, which may affect post-transcriptional gene regulation via miRNAs in porcine fetal gonads. However, it seems that androgens exert diverse biological effects depending on the gestational period.