The aim of this study was to show whether the connexin43 (Cx43) expression in gonads is affected by an anti-androgen action. To test this, 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 that was given in five doses, every second day and its effect was observed in prepubertal gilts and boars. Morphology and expression of Cx43 was investigated in testes and ovaries by means of routine histology, immunohistochemistry, Western blotting, and RT-PCR, respectively. Qualitative analysis of immunohistochemical staining for Cx43 was confirmed by quantitative image analysis in which the staining intensity was expressed as relative optical density of diaminobenzidine deposits. There were statistically significant differences in Cx43 signal intensity between interstitial tissue of control and GD20 pigs (**p less than 0.01), between seminiferous tubules of control and PD2 boars (**p less than 0.01), between granulosa cells of preantral follicles of control and GD20 and PD2 pigs (**p less than 0.01 and *p less than 0.05, respectively), and between theca cells of control and GD80 and PD2 gilts (**p less than 0.01). In Western blotting Cx43 appeared as a band of 43 kDa, whereas the size of the PCR-amplified product was 232 bp in all gonad tissue samples. Since we demonstrated changes in gonad morphology and in the expression of Cx43 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 Cx43 gene expression in pig gonads.

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. 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. 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. 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 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.