| Ovine is a kind of very important herbivorous livestock,which can provide us meat,milk and fur et al.In vitro maturation(IVM),in vitro fertilization(IVF)and embryo transfer(ET)are widely used to improve licstock with good quality and productivity.This technique is initialed with oocytes development and in vitro maturation.Therefore,the quality of oocyte maturation is the key factor to determine survive and the quality of the offsprings.Endogenous gene expression and regulation play key role during oocyte maturation and early embryonic development.In addition,factors in the culture eviorment also participate in the regulation of normal development.One of the most common and direct way of this kind of regulate is paracrine.Some of the paracrine factors such as connective tissue growth factor(CTGF),stromal cell-derived factor-1(SDF-1),nerve growth factor(NGF)and hepatocyte growth factor(Hepatocyte growth factor,HGF)have been found in ovaries.It was found that CTGF can promote ovary follicular development in rodents.SDF-1 has the ability to benifit granulosa cells survival and improve embryos quality.NGF enhances the in vitro maturation of goat primary follicles in a dose-dependent manner.HGF can induce granulosa cell proliferation in cattle.Howere,it has not been well studied if these four paracrine factors have effect on ovine oocyte maturation,fertilization and early embryonic development.Therefore,in this study,we proposed to investigate the effects of CTGF,SDF-1,NGF and HGF on ovine oocyte maturation and early embryonic development.The effects of single or combined supplementation of these fators in the culture medium will be investigated.Effect of different concentrations of CTGF,SDF-1,NGF or HGF in oocyte maturation medium was firstly investigated.oocytes maturation rate was evaluated to determine the optimal concentration of the factors when they were single used.Based on the upper results,effect of combination of these four factors on oocyte maturation and early embryonic development was tested.The ratios of oocyte maturation,parthenogenetic embryo activation and development,cumulus expansion index(CEI),early cell apoptosis as well as the cell number of blastocysts were used as indexes to evaluate the promotional effects of these paracrine factors.Finally,expression of apoptotic related genes such as Bax,Bcl-2,P53 and PTGS2 were tested.The following results were obtained:(1)when the factors were used individually in the culture medium,the optimal concentration to promoted oocyte maturation and early embryonic development is:30.0 ng/mL CTGF,10.0 ng/mL SDF-1,3.0 ng/mL NGF or 100.0 ng/mL HGF;(2)Supplementation of combined paracrine factors(30.0 ng/mL CTGF + 10.0 ng/mL SDF + 3.0ng/mL NGF + 100.0 ng/mL HGF)promotes oocyes maturation rate,pathenogenetic embryo activation and development better than the single factor supplementation group.(3)Supplementation of combined paracrine factors has the effects of promoting CEI,reduce oocytes apoptosis and increase blastocysts cell number.(4)Supplementation of combined paracrine factors has the effects of reducing the expression of apoptotic genes in cumulus cells,oocytes and blastocysts.Our results showed that CTGF,SDF-1,NGF and HGF can promote ovine oocyte maturation.Supplementation of combined paracrine factors in culture medium could promote the maturation of ovine oocyte and the development of early embryos. |
