The Leptin Regulates Granulosa Cell Proliferation, Apoptosis, Lipid Metabolism And Steriod Hormone Synthesis

The development of poultry follicles is accompanied with the proliferation and apoptosis of granulosa cells, the increasing lipid content and the changes of steroid hormone content within the follicle. Many studies showed, leptin plays an important regulatory role in the developing process of follicles, but its regulation roles have been less consistent. For granulosa cells, leptin may have three roles, promoting proliferation and suppressing apoptosis, or promoting apoptosis and suppressing proliferation, or no obvious effect; for the steroid hormones and fatty acids within different cells, leptin may have a promotion or a suppression role. In addition, it is generally agreed that the ovarian follicle of birds has no ability to synthesize lipids, and the lipids in follicle are almost entirely from liver. However, such views remain to be verified. In order to study the role and mechanism of leptin on regulating follicular development, in this experiment, goose was taken as research material, the relationship between leptin and granulosa cell proliferation and apoptosis, follicles steroids contents change, and lipid metabolism within the follicle was explored, from the levels of tissue, cells, genes and hormone levels, respectively. The main contents include:(1) the geese granulosa cells were cultured with leptin and PI3K/Akt/mTOR signaling pathway inhibitors, the relationship among leptin-PI3K/Ak/mTOR signaling pathway-granulosa cell proliferation and apoptosis was analyzed using the experimental methods such as MTT staining, BrdU staining and real-time quantitative analysis; (2) the geese granulosa cells were cultured with leptin and PI3K/Akt/mTOR signaling pathway inhibitors, the relationship among leptin-PI3K/Ak/mTOR signaling pathway-follicular lipids metabolism was analyzed using the experimental methods such as oil red O staining and real-time quantitative analysis; (3) the geese granulosa cells were cultured with leptin and PI3K/Akt/mTOR signaling pathway inhibitors, the relationship among leptin-PI3K/Ak/mTOR signaling pathway-follicular steroid hormone levels was analyzed using the experimental methods such as ELISA and real-time quantitative analysis.The main results are as follows:(1) The mRNA of leptin-R (leptin receptor) was detected in granulosa cells of different size of geese follicles.(2) Leptin regulated leptin-R, and the genes involved in proliferation, apoptosis and PI3K/Akt/mTOR signal in vitro cultured geese granulosa cells, but it had a dose-dependent effect. The mRNA levels of leptin-R, CD1, CD2, CD3, Bcl-2 and genes involved in PI3K/Akt/mTOR signaling pathway in the low concentrations of leptin (1 or 10 ng/mL) treated group, were significantly higher than control group (p< 0.05); the mRNA levels of these genes in high concentration of leptin (100 ng/mL) treated group was significantly lower than the low concentration of leptin (1 or 10 ng /mL) treated group (p< 0.05).(3) PI3K/Akt/mTOR signaling pathway inhibited the proliferation of cultured goose granulosa cells, inhibited the transcriptional expression of CD 1, CD2, CD3, and Bcl-2. The mRNA levels of these genes in LY294002 and Rapamycin treated group were significantly lower than control group (p< 0.05).(4) In vitro serum-free cultured goose granulosa cells have the ability to synthesize lipids, lipid droplets content increased with the prolonged incubation time (lipid droplets was little when 0 h after the adherent culture, and there was an increase of lipid droplets after 24 h, and it mainly gathered in the cell membrane, the lipid droplets filled with whole cell after 48 h); number of cells reduced with the prolonged incubation time.(5) The expression of genes involved in lipid metabolism is unbalanced in vitro serum-free cultured goose granulosa cells. After 48 h of the adherent culture, the expression levels of the key genes related with de novo synthesis of fatty acids(ACC, FAS), the rate-limiting enzyme genes of TG synthesis(DGAT1, DGAT2), the genes involved in lipid deposition regulation (PPARa, PPARy) were higher in goose granulosa cells; the expression level of fatty acid β oxidation rate-limiting enzyme gene (CPT1) was lower; the expression levels of lipid transporter genes(MTP, apoB) were almost undetectable.(6) Leptin regulated the mRNA expression of genes involved in lipid metabolism in cultured goose granulosa cells, but it had a dose-dependent effect. the mRNA levels of ACC, FAS, DGAT1, DGAT2, PPARa and PPARy in the low concentrations of leptin (1 or 10 ng/mL) treated group, were significantly higher than control group (p< 0.05); the expression of CPT1 was significantly lower than control group (p< 0.05); the mRNA levels of ACC, FAS, DGAT1, DGAT2, PPARa and PPARy in high concentration of leptin (100 ng/mL) treated group was significantly higher than the low concentration of leptin 1 or 10 ng/mL) treated group (p< 0.05), and the expression of CPTl was significantly higher than low concentration of leptin (1 or 10 ng/mL) treated group (p< 0.05);(7) PI3K/Akt/mTOR signaling pathway inhibited the transcriptional expression of ACC, FAS, DGAT1, DGAT2, PPARa and PPARy, but promoted the transcriptional expression of CPT1.(8) Leptin promoted the secretion of E2 and P4, but inhibited the secretion of T in cultured goose granulosa cells.(9) Leptin regulated the mRNA expression of steroid synthesis genes (SREBP-1, HMGCS, CYP51, DHCR24), steroid transporter gene (StAR, CYP11) and steroid hormone synthesis gene (17β-HSD, CYP17, CYP19,17β-HSD) in cultured goose granulosa cells, but it had a dose-dependent effect. The mRNA levels of SREBP-1, HMGCS, CYP51, DHCR24, StAR, CYP11,17β-HSD, CYP17, CYP19,3β-HSD in the low concentrations of leptin (1 or 10 ng/mL) treated group, were significantly higher than control group; the mRNA levels of these genes in high concentration of leptin (100 ng/mL) treated group were significantly lower than the low concentration of leptin (1 or 10 ng/mL) treated group (p< 0.05).(10) PI3K/Akt/mTOR sigmal pathway inhibited the secretion of E2, P4 and T in cultured goose granulosa cells, and the expression of StAR, CYP11, CYP19 and 3β-HSD.