Effect of in vitro sodium fluoride treatment on CAT, SOD and Nrf mRNA expression and immunolocalisation in chicken (Gallus domesticus) embryonic gonads.

In this study, we examined the effect of sodium fluoride (NaF) on oxidative stress in chicken embryonic gonads. Following exposure to varying concentrations of NaF for 6 h, mRNA expression and immunolocalisation of catalase (CAT), sodium dismutase (SOD1 and SOD2) and nuclear respiratory factors (Nrf1 and Nrf) were analysed in the gonads. In the ovary, a dose-dependent increase in mRNA expression of CAT, Nrf1 and Nrf2 following NaF exposure was found, while the intensity of immunolocalised CAT, SOD2 and Nrf1 was higher in NaF-treated groups. In the testis, no effect of NaF on CAT, SOD1 and Nrf1 mRNA levels was observed; however, NaF (3.5-14.2 mM) elevated Nrf2 mRNA expression. NaF, at a dose of 7.1 mM, increased the immunoreactivity of Nrf1 and SOD2. Further experiments evaluated the ovary and testes when incubated with NaF (7.1 mM), vitamin C (Vitamin C, 4 mM) or NaF + Vitamin C. mRNA expression of all four examined genes in the whole ovary and immunoreactivity of Nrf1 and CAT in the ovarian medulla increased in each experimental group. Similar effects were observed in the testis, where mRNA expression, as well as CAT and Nrf2 immunoreactivity, increased in Vitamin C and NaF + Vitamin C-treated groups. In summary, NaF exposure generated oxidative stress which is manifested by increased expression of free radical scavenging enzymes in chicken embryonic gonads. High doses of Vitamin C did not reverse this effect.

In vitro effects of PNP and PNMC on apoptosis and proliferation in the hen ovarian stroma and prehierarchal follicles.

This study aimed to examine the mRNA expression, activity, and immunolocalisation of apoptosis/proliferation regulating factors following in vitro exposure of the stroma, white (WFs), and yellowish (YFs) follicles of the chicken ovary to 4-nitrophenol (PNP) or 3-methyl-4-nitrophenol (PNMC). PNMC increased the mRNA expression of caspase-3, -8, Apaf-1, and cytochrome c in the ovarian stroma. The activity of caspase-3, -8, and -9 decreased in WFs in both nitrophenol-treated groups. PNP reduced the number of caspase-3-positive cells in the stromal connective tissue (CT) and the theca interna and externa layers of WFs. In the stroma, the proliferating index decreased in the wall of primary follicles in both nitrophenol-treated groups, however, in the CT, the effect of PNMC was opposite. In the theca interna of WFs, PNP diminished the proliferating index. These results suggest that nitrophenols might impact the development of chicken ovarian follicles by affecting cell death and proliferation.

Nitrophenols suppress steroidogenesis in prehierarchical chicken ovarian follicles by targeting STAR, HSD3B1, and CYP19A1 and downregulating LH and estrogen receptor expression.

To assess the effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on steroidogenesis in the chicken ovary, white (WF, 1-4 mm) and yellowish (YF, 4-8 mm) prehierarchical follicles were incubated in a medium supplemented with PNP or PNMC (10-8-10-4 M), ovine LH (oLH; 10 ng/mL), and combinations of oLH with PNP or PNMC (10-6 M). Testosterone (T) and estradiol (E2) concentrations in media and mRNA expression for steroidogenic proteins (STAR, HSD3B1, and CYP19A1), and LH receptors (LHR), estrogen receptor α (ESR1) and ß (ESR2) in follicles were determined by RIA and real-time qPCR, respectively. PNP and PNMC decreased T and E2 secretion by the WF and YF, and oLH-stimulated T secretion from these follicles. PNP decreased basal STAR and HSD3B1 mRNA levels both in the WF and YF, and CYP19A1 mRNAs in the WF. PNP reduced oLH-affected mRNA expression of these genes in the YF. PNMC inhibited basal STAR, HSD3B1, and CYP19A1 mRNA expression in the WF, but not in the YF. PNMC reduced oLH-stimulated STAR and CYP19A1 expression in the YF and WF, respectively. PNP decreased basal mRNA expression of LHR, ESR1, and ESR2 in the WF, but it increased ESR1 and ESR2 mRNA levels in the YF. PNMC reduced both basal and oLH-affected LHR, ESR1, and ESR2 mRNA expression in the WF; however, it did not influence expression of these genes in the YF. We suggest that nitrophenols by influencing sex steroid synthesis and transcription of LH and estrogen receptors in prehierarchical ovarian follicles may impair their development and selection to the preovulatory hierarchy.