Investigation On The Biological Characters Of Ovarian Cells Of Xiang Pig And Factors Affect Maturation Of Porcine Granulosa Cells In Vitro

Follicular development is a complex procedure,which includes follicular initiation, recruitment,selection,dominance,ovulation and luteinization.This procedure can be influenced by a lot of factors,such as hormones,growth factors,et al.Only very few follicles will develop into dominant follicles and ovulation and more than 99%turn atresic. Xiang pigs are a world famous breed of the miniature pig mainly distributed in Guizhou and Guangxi provinces of China.Miniature pigs have become a perfect animal model for reproductive and biomedical research.However,knowledge of reproductive physiology in this species is extremely limited.Forkhead transcription factors family O subgroup has 4 members:FOXO1,FOXO3a, FOXO4,and FOXO6,which play different roles in vast physical processes.Recent study showed that FoxO transcription factors regulated cell growth,proliferation,differentiation, and apoptosis through many different signal pathways in ovary cells.Follicle development from primordial follicles to dominant follicles was companied by growth,proliferation, differentiation,and maturation of granulosa ceUs(GCs).Apoptosis of GCs will cause follicular atresia.In this study,apoptosis of follicular cells was firstly detected by TUNEL assay,and cellular localization of FoxOs and several proteins related were studied in porcine ovaries by immunohistochemistry(IHC),then cellular survival rates,[Ca²⁺]i, secretions of E₂,and the expression of FoxO3a protein et al.were detected by FCM,RIA, Western blot et al.to study the mechanisms underlying follicular development and atresia. The conclusions were as follows:1.The apoptosis of follicular cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay.Results indicated that the apoptosis rate of granulosa cells in healthy follicles was lower than that in atretic follicles. Cellular localization of inhibinα-subunit,PKB/Akt,FoxOs,Bax,and Bcl-2 proteins were studied by immunohistochemistry(IHC) in the ovaries of Xiang pigs.The results indicated that inhibinα-subunits were localized in granulosa cells of follicles at all stages but not in the corpora luteal.PKB was localized in granulosa cells of primordial follicles and in the basal layers of granulosa cells of secondary and antral follicles,but not in atretic follicles and corpora luteal.FoxO1 was localized in the cytoplasm of granulosa cells of follicles at all stages but not in corpra luteal cells and apoptotic cells.FoxO3a was localized in granulosa cells of follicles at all stages,granulosa luteal cells and vascular epithelial cells. Both FoxO1 and FoxO3a were localized in oocytes.Bax and Bcl-2 were localized in the cytoplasm and nuclear of granulosa cells of follicles at all stages and the cytoplasm of vascular epithelial cells and corpra lutcal cells.Altogether,the stage- and cell-specific expression pattern of inhibinα-subunit,PKB,FoxO1,FoxO3a,Bax,and Bcl-2 suggested that these proteins might play potential roles in the follicle development,atresia,and luteinization in Xiang pigs.2.To investigate the functions of FoxO6 on matured porcine reproductive system, RNA were extracted from 4 matured mouse brains and 4 matured porcine ovaries.A fragment about 2500 bp was amplified from the mouse RNA,but not yet from porcine ovary RNA.Therefore,FoxO6 may not be expressed in matured porcine ovaries.3.To investigate the factors that influnt the growth and apoptosis of pGCs,GCs were isolated from small(1.5-3 mm),middle(3-5 mm),and large(larger than 5 mm) antral follicles in different diameters.Then,the survival rates of granulosa cells and[Ca²⁺]i were detected by FCM,the expression of FoxO3a was detected by Western blot,and the concentrations of E₂ in pFF were detected.The results showed that the rate of atresic follicles decreased,rate of the healthy follicles increased at the same time;the survival rate of pGC from small follicles was the highest(P＜0.05);[Ca²⁺]i decreased with the growth of follicles,the expression of FoxO3a increased;concentrations of E₂ in porcine follicular fluid increased with the enlargement of antral follicles.These results indicated that FoxO3a transcription factor and[Ca²⁺]i may be involved in the apoptosis of pGC and hence in the atresia of antral follicles.4.Ca²⁺ plays important roles in cell metabolism,synaptic transmission,gene expression,cell cycle control,and intercellular communication et al.The effects of extracelluar Ca²⁺ on granulosa cells were investigated in this study.CaCl₂ or EGTA was supplied in medium of granulosa cells.[Ca²⁺]i,secretion of E₂,and expression of FoxO3a were detected.The main results were as follows:(1)[Ca²⁺]i was not significantly affected by 50 mg/mL CaCl₂ but decreased after 50μg/mL EGTA;(2) The expression of FoxO3a was significantly promoted by CaCl₂ but inhibited by EGTA(P＜0.05);(3) The secretion of E₂ was promoted by CaCl₂ but inhibited by EGTA.It suggested that extracelluar Ca²⁺ may influence the growth,apoptosis,and maturation of granulosa cells through FoxO3a-dependent signal pathways.5.Effects of FSH,EGTA,LY294002,FSH+EGTA,EGTA+LY294002 and FSH+EGT A+LY294002 on the expression of FoxO3a and secretion of E₂ in porcine granulosa cells were investigated in this study.Results showed that There were no significant differences among cell survival rates and secretion of E₂ of pGC after 2 h treatments by FSH,EGTA, and LY294002;The secretion of E₂ decreased after EGTA+LY294002 for 6h(P＜0.05),and expression of FoxO3a is inhibited by EGTA,but promoted by LY294002 for 6h(P＜0.05); Secretion of E₂ were significantly inhibited by LY294002+EGTA and LY294002+EGTA +FSH treatment for 24h,The expression of Bcl-2 was promoted by FSH and LY294002 but inhibited by EGTA.It was implied that Ca²⁺ signal pathway was related with PI3K/PKB signal pathway in granulosa cells of pigs.