Long non-coding RNAs (lncRNAs) may regulate gene expression in numerous biological processes including cellular response to xenobiotics. The exposure of living organisms to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, results in reproductive defects in many species including pigs. The aims of the study were to identify and characterize lncRNAs in porcine granulosa cells as well as to examine the effects of TCDD on the lncRNA expression profile in the cells. One thousand six hundred sixty-six lncRNAs were identified and characterized in porcine granulosa cells. The identified lncRNAs were found to be shorter than mRNAs. In addition, the number of exons was lower in lncRNAs than in mRNAs and their exons were longer. TCDD affected the expression of 22 lncRNAs (differentially expressed lncRNAs [DELs]; log₂ fold change ≥ 1, P-adjusted < 0.05) in the examined cells. Potential functions of DELs were indirectly predicted via searching their target cis- and trans-regulated protein-coding genes. The co-expression analysis revealed that DELs may influence the expression of numerous genes, including those involved in cellular response to xenobiotics, dioxin metabolism, endoplasmic reticulum stress and cell proliferation. Aryl hydrocarbon receptor (AhR) and cytochrome P450 1A1 (CYP1A1) were found among the trans-regulated genes. These findings indicate that the identified lncRNAs may constitute a part of the regulatory mechanism of TCDD action in granulosa cells. To our knowledge, this is the first study describing lncRNAs in porcine granulosa cells as well as TCDD effects on the lncRNA expression profile. These results may trigger new research directions leading to better understanding of molecular processes induced by xenobiotics in the ovary.

Low doses of endocrine disrupting chemicals (EDCs) used in combination may act in a manner different from that of individual compounds. The objective of the study was to examine in vitro effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 pM) and genistein (500 nM) on: 1) progesterone (P4) and estradiol (E2) secretion (48 h); 2) dynamic changes in aryl hydrocarbon receptor (AhR) mRNA and protein expression (1, 3, 6, 24 and 48 h); 3) dynamic changes in estrogen receptor β (ERβ) mRNA and protein expression (1, 3, 6, 24 and 48 h); and 4) induction of apoptosis in porcine granulosa cells derived from medium follicles (3, 6 and 24 h). TCDD had no effect on P4 or E2 production, but potentiated the inhibitory effect of genistein on P4 production. In contrast to the individual treatments which did not produce any effects, TCDD and genistein administered together decreased ERβ and AhR protein expression in granulosa cells. Moreover, the inhibitory effect of TCDD on AhR mRNA expression was abolished by genistein. The treatments did not induce apoptosis in the cells. In summary, combined effects of low concentrations of TCDD and genistein on follicular function of pigs differed from that of individual compounds. The results presented in the current paper clearly indicate that effects exerted by low doses of EDCs applied in combination must be taken into consideration when studying potential risk effects of EDCs on biological processes.

Commercially available, but not yet characterized, the AVG-16 granulosa cell line was established from granulosa cells of medium porcine follicles. To examine the suitability of the AVG-16 cell line for studying the molecular mechanism of action of various environmental oestrogens, we investigated: 1/ cell morphology (by standard haematoxylin and eosin (HE) staining); 2/ basal and follicle-stimulating hormone (FSH) or luteinizing hormone (LH) -stimulated steroid hormone (progesterone; P ₄ and 17β-oestradiol; E ₂ ) secretion (by radioimmunoassay) and 3/ expression of receptors involved in the regulation of granulosa cell function: FSH receptor (FSHR), LH receptor (LHR), oestrogen receptor α (ERα), oestrogen receptor β (ERβ) and aryl hydrocarbon receptor (AhR). mRNA and protein expression was determined by RT-PCR and fluorescence immunocytochemistry, respectively. The secretion of P ₄ and E ₂ by AVG-16 cells was in the range of steroid hormone secretion by porcine cultured primary granulosa cells. Neither FSH (100 ng/ml) nor LH (100 ng/ml) affected P ₄ and E ₂ secretion by AVG-16 cells. The presence of FSHR and LHR at both mRNA and protein level was not demonstrated in the cells. However, AVG-16 cells were found to express mRNA and protein of ERα, ERβ and AhR. The results of our study showed that AVG-16 cells possess the capability of steroid hormone production, and both oestrogen receptors and AhR are present in these cells. Therefore, AVG-16 cells may serve as an unlimited source of homogenous porcine granulosa cells useful for studying the effects of environmental oestrogens on ovarian physiology.