The Study On Effect Of Kisspeptin On Progesterone Secretion Of Bovine Ovarian Granulosa Cells

In the steroid hormone, progesterone is the activity of the highest hormone progesterone. Inprogesterone secretion process, needs the synergy of some hormones, and kisspeptin and EGFR asan important factor regulation of ovarian function, can strengthen the oocyte maturation, the folliclestimulating process play an important role.Kisspeptin is a peptide hormone, in the hypothalamus, the nerve center, are distributed in areassuch as the placenta, can activate gonadotropin-releasing hormone (GnRH) neurons, adjust the LHand FSH release, at the beginning of promoting animal body plays an important role in the seasonstarted. Roa etc.found that Kisspeptin was expressed in all phases of the estrous rats;Parharetc.found kisspeptin have expressed in tilapia GnRH neurons, and participate in fish KISS-1/GPR54system reproductive regulation; XiaoYunQi etc. found that kisspeptin was expressed in within ovarian granulosa cells of chickens, and it can promote the chicken granular cell proliferation andsecretion of progesterone, whether kisspeptin expression on cattle ovarian granulosa cells, whetherthe regulation of cell proliferation and hormone secretion is unclear. Studies have showed thatEGFR play an important role in granulosa cells progesterone secretion, while kisspeptin is related tothe EGFR signaling pathway about regulation of progesterone secretion of mechanism are needfurther research.This experiment adopts the immunofluorescence technique to detect kisspeptin expressed incattle ovarian granulosa cells; Determined by MTT method and ELISA detection kisspeptin oncattle ovarian granulosa cell proliferation and progesterone secretion;Flow cytometry instrumentmethod to detect kisspeptin’s impact on cattle ovarian granulosa cell apoptosis.In further study,combined with Western blot method preliminarily Kp-10on ovarian granulosa cells and its possiblemechanism of progesterone secretion.This research mainly consists of the following two parts:The first part, Kisspeptin for cattle ovarian granulosa cells progesterone secretion.Select30cow ovaries, suction method of granular cell, training to the F1generation, in fetal bovine serummedium, cultivate24h, respectively choose10nm,100nm,1000nm concentration kisspeptin,observe cells within24h,48h,72h value, combined with ELISA method to detect progesteronecontent in cell supernatant.Experimental results show that by cytology immunofluorescence method,to determine the Kp-10on cattle ovarian granulosa cells express;Kp-10serum free treatment groupcan increase granular cell vigor,100nmol/L can significantly increase the rate of granulosa cells oflive (P<0.05)and promote the secretion of progesterone (P<0.05);Granulosa cell apoptosis rate by PI staining detection, found24h100nmol/L cell apoptosis.Kp-10found expression in ovariangranular cell, can promote cell growth, have influence on progesterone secretion.The second part, select the F1cattle ovarian granulosa cells,5groups, each group has threeparallel group,24h after training in fetal bovine serum, change to serum free medium, culture4h, inaddition to the cell culture, to join the Kp-10serum-free culture medium, respectively after0min,1min,5min and15min,30min time point stimulate cattle ovarian granulosa cell growth, usingWestern blot method preliminarily kisspeptin affects the quantity of p-EGFR proteinexpression.Experimental results show that: in the medium, the highest expression was found inp-EGFR，after treating with100nm Kp-10for5min,.