Effects Of Ghrelin On The Ovine Oocytes During In Vitro Maturation

Ghrelin was discovered by Japanese scientist Kojima in 1999, which is a new endogenous brain-gut peptide made of 28 kinds of amino acid. It is a natural ligard of growth hormone secretagogue receptor. Combined with its receptor it has comprehensive physiological function. So the research progress of Ghrelin is a hot point that people concerned now.With deep research of animal cell project and embryo project, the report of animal oocyte maturation factor is mainly the action principle of some genetic hormone. So, as a natural ligard of growth hormone secretagogue receptor we have not known whether it participated in the maturation of ovine oocyte, whether it exerted action throuth classical MAPK signal passage about cell growth. Because oocyte partly apoptosis in the course of maturation, the expression of anti-apoptosis gene-Bcl-2 and proapoptosis gene-Bax decided whether female reproductive cell was alive. Now we haven't known the relationship between Ghrelin and Bcl-2, Bax in the maturation course of oocyte. Aim at these unknown questions ,my paper esplored the relationship between Ghrelin and MAPK signal, Bcl-2, Bax in the maturation course of ovine oocyte through adding Ghrelin and special antagonist of GHS-R-D-Lys3-GHRP-6 in vitro maturation system of ovine oocye.The results are as follows:1. Expression of GHS-R in the ovine oocye GHS-R was measured in the ovine oocyte through RT-PCR.2. The influence of Ghrelin on the maturation of ovine oocyte(1) Ghrelin has promotion action on the First Polar Body getting–out rate and nucleus mature rate of oocyte, relying on consistency .(2) D-Lys3-GHRP-6 has no effect on the First Polar Body getting–out rate and nucleus mature rate of oocyte.(3) The promotion action of Ghrelin in vitro maturation of oocyte can be obstructed by D-Lys3-GHRP-6. The more complete its antagonism to GHS-R is, the more obvious the promotion action of Ghrelin was obstructed. So it proved that Ghrelin promoted oocyte maturation by combining with GHS-R.3. The effect of Ghrelin on expression of MAPK(ERK1/2), p90~rsk mRNA of ovine oocyte was examined by real-time PCR.(1) Ghrelin has ascending action to the expression of MAPK(ERK1/2), p90~rsk mRNA of oocyte, relying on dosage and time obviously. (2) D-Lys3-GHRP-6 has no effect on expression of MAPK(ERK1/2), p90~rsk mRNA in oocyte vitro maturation.(3)Promotion action of Ghrelin to the expression of MAPK(ERK1/2), p90~rsk mRNA can be obstructed by D-Lys3-GHRP-6, relying on consistency.4. The expression of MAPK(ERK1/2), p90~rsk in ovine oocyte and the influence of Ghrelin on phosphorylated MAPK(ERK1/2), p90~rsk were examined by Western blotting.(1) The expression of MAPK(ERK1/2), p90~rsk protein has no obvious change in vitro maturation.(2) MAPK(ERK1/2), p90~rsk protein were activated at 10h of oocyte vitro maturation, which has high activity untill 24h. So MAPK(ERK1/2), p90~rsk protein were activated after GVBD period.(3) Ghrelin has ascending action to the expression of MAPK(ERK1/2), p90~rsk protein.(4) The ascending action of Ghrelin to the expression of MAPK(ERK1/2), p90~rsk protein can be obstructed by D-Lys3-GHRP-6.(5) Activation of p90~rsk protein was consistent with MAPK(ERK1/2) in vitro maturation.(6) The action of Ghrelin in vitro maturation was exerted through MAPK signal system, and the promotion action of expression after GVBD of oocyte phosphorylated MAPK(ERK1/2), p90~rsk protein was induced by Ghrelin receptor.5. The effect of Ghrelin on expression of oocyte Bcl-2, Bax mRNA was examined by real-time PCR.(1) Ghrelin has ascending action to the expression of Bcl-2 mRNA.(2) Ghrelin has descending action to the expression of Bax mRNA.(3) The ascending action to the expression of Bcl-2 mRNA and the descending action to the expression of Bax mRNA of Ghrelin were obstructed by D-Lys3-GHRP-6, relying on consistency.(4) The effect of Ghrelin on expression of Bcl-2, Bax mRNA in vitro maturation was induced by Ghrelin receptor.