| Arsenic(As) is a kind of trace element in animals, with both benefit and toxicity, and it has posed a serious threat to livestock’s living environment and health. At present, effects of As on livestock reproductive mainly focus on male reproduction, and there’s little research done in the female livestock. pGCs is important on ovarian function, and play an important role in reproduction activity. In this study pGCs were used as study modle to choose appropriate-diameter antral follicles for subsequent reproductive biology research, and tried to explore the effects of NaAsO2 on pGCs’ proliferative activity.In this study, follicles was divided into three grades by diameter, then pGCs was got by mechanical way, the cell survival rate was calculated by trypan blue staining, cell morphology were observed under inverted microscope, pGCs growth curve cultured in vitro determined by 3-(4,4-dimethylthiazoly-2)-2,5-diphenylttrazoliu mbromide(MTT), a real-time quantitative reverse transcript polymerase chain reaction(qRT-PCR) was used to detect the follicle-stimulating hormone receptor(FSHR) and luteinizing hormone receptor(LHR) gene expression for further identification of GCs.Then pGCs from appropriate follicle diameter range were used for following experimental object, the effect of NaAsO2 on the cell survival was tested by CCK-8 method, mRNA expression levels of FSHR, LHR, CYP-19, ER1 and ER2 were detected via qRT-PCR, Flow cytometry(FCM) method for apoptosis detection, and enzyme-linked immunoassay(ELISA) method for hormone secretion test.Following results were got from our experimental research:1. The optimum culture system in vitro of pGCs was determined finally: DMEM/F12, FBS 3%, BSA 0.3%, Insulin 50ng/mL, FSH 0.1 IU/mL, penicillin-streptomycin 1%.2. Viabilities and purities of pGCs, extracted from three antral follicles of different diameters(Small Follicles(SFs):< 2mm, Middle Follicles(MFs):2~5mm, Large Follicles(LFs): ≥5mm), were 68%, 75%, 72% and 97%, 95%, 90%, respectively; growth behavior of pGCs from the MFs was best; FSHR gene expression from SFs was highest, and there were no significant difference between MFs and LFs(P>0.05); LHR gene expression was highest in pGCs from SFs and higher significantly than those from MFs and LFs(P<0.05). After the comprehensive analysis, MFs were selected for flowing study.3. Growth curve detected by CCK-8 assay showed, a certain concentration of NaAsO2 can affect the survival rate and growth status of pGCs, high-concentration NaAsO2(50 μM) resulted in death of pGCs, low-concentration NaAsO2(0.08μM) had almost no effect, there were no significant differences between middle-concentrations(0.4μM, 2μM, 10μM)(P>0.05), and an increase after 24 h appeared in 10μM NaAsO2 group.4. FITC-AnnexinV/PI double-staining method showed, pGCs treated with different-concentration NaAsO2(0, 0.08μM, 0.4μM, 2μM, 10μM, 50μM), in addition to 10μM NaAsO2 group(easing the apoptosis phenomenon), other groups’ apoptosis rate increased as the increasing concentration of NaAsO2, which was dose-dependence; The pGCs cultured in vitro were treated with 10 μM Na AsO2 over 24 h, Bcl-2 gene expression was up-regulated, while Caspase-3 was down-regulated, which were consistent with previous FITC-AnnexinV/PI method result.5. The pGCs cultured in vitro were treated with 10 μM NaAsO2 concentration and different duration(4 h, 8 h, 12 h, 24 h), qRT-PCR results showed that LHR, CYP19, ER1 and ER2 gene expression in pGCs were down-regulated at 4h,8h,12 h, however, there was an up-regulation tendency at 24h; The pGCs cultured in vitro were treated with different concentration NaAsO2 during same period of time, qRT- PCR results showed that FSHR, LHR, CYP19, ER1 and ER2 gene expression quantity were rised as increasing NaAsO2 concentration, which showed dose-dependence.6. ELISA assay result showed, progesterone and estradiol levels were statistically insignificant between NaAsO2 treatment groups and control group, as well as among the treatment groups(P>0.05)。... |
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