Effects Of Ghrelin On Bovine Cumulus Proliferation And Oocyte Maturation In Vitro

Ghrelin is the orexigenic gut hormone that has been put forward to regulate thebalance between energy and reproduction in mammals, and is the endogenous ligandof growth hormone secretion receptor (GHS-R), distribution in endocrine cells ofgastric tissue. In recent years, Ghrelin has been demonstrated that it plays essentialroles in reproductive system because of the high expression of Ghrelin in thereproductive gonads. Oocyte maturation and development is a prerequisite forfertilization and the subsequent embryo development in mammals, however, theprocess of oocyte maturation is affected by multiple factors. Ghrelin is a new type ofhormone affected the reproductive system. Therefore, it has an important theoreticaland practical significance for promoting fertilization in vitro and embryo engineeringtechnology development and application to explore the potential role of ghrelin inoocyte maturation process.It has been demonstrated that Ghrelin and GHS-R mainly expressed in the lutealcells and interstitial cells with ovarian steroid secretion activity, and Ghrelin andghrelin receptor expressed in oocyte as well. Moreover, Ghrelin stimulated granulosacell proliferation and inhibited apoptosis in vitro, and delayed oocyte maturation.Whether Ghrelin would be regulated bovine oocyte maturation and cumulus cellproliferation has not yet been reported. In this study, the technologies ofimmunofluorescence staining, real-time PCR, proliferation assay and WesternBlotting were adopted to research the expression of Ghrelin and GHS-R1a in bovineoocytes and cumulus cell and the effects of Ghrelin and [D-Lys~3]-GHRP-6on bovineoocyte maturation and cumulus cell proliferation in vitro. The results are as follows:1. It has been confirmed that Ghrelin protein expressed in the cytoplasm ofbovine oocytes, GHS-R protein specifically located in the oocyte cell surface andGhrelin and GHS-R protein also expressed in cumulus cell.2. In the course of maturation in vitro, Ghrelin and GHS-R1a mRNA relativeexpression level in oocyte decreased significantly with mature process; ghrelin mRNAexpression levels in cumulus cells showed prominently increased at8h and decreasedat16h, while GHS-R1a mRNA expression gradually increased with the maturationtime.3. It has been detected that FSH inhibited Ghrelin and GHS-R1a mRNA expression in oocyte, but it promoted Ghrelin and GHS-R1a mRNA expression incumulus cells. However, FSHBI removed the inhibition effect from FSH on Ghrelinand GHS-R1a mRNA expression in oocyte and the promotion effcet on Ghrelin andGHS-R1a mRNA in cumulus cell induced by FSH.4. Ghrelin and FSH significantly promoted the expression of PCNA and BCL-2mRNA in cumulus cell, but this promotion was removed after addition of[D-Lys~3]-GHRP-6(P<0.05). BAX mRNA expression was not influenced by FSH and[D-Lys~3]-GHRP-6while Ghrelin prominently inhibited BAX mRNA expression and[D-Lys~3]-GHRP-6could reverse this inhibition (P<0.05).5. Ghrelin promoted PCNA protein expression, and [D-Lys~3]-GHRP-6reversethe effect of Ghrelin on stimulating PCNA expression.6. Ghrelin and FSH significantly promoted cumulus cell proliferation activity,while [D-Lys~3]-GHRP-6inhibited the increase of cumulus cell activity (P<0.05).Moreover,[D-Lys~3]-GHRP-6could be removed stimulated effect of Ghrelin and FSHon the increase of proliferation activity in cumulus cell.7. Ghrelin inhibited BMP15and GDF9mRNA expression in oocyte, as well asGVBD occurs, and this effect was removed by [D-Lys~3]-GHRP-6.