Effect And Mechanism Of Inhibin Gene Transfection Mediated By Liposome On The Development Of Bovine Follicular Cells And Embryos

Follicular inhibin is one of the important regulators of ovarian function.In monotocous mammalian,passive or active immunization against inhibin or pEGISI gene could be used to induce superovulation and twinning.Furthermore,inhibin is able to be used as one of best marker of ovarian cancer.However,little is known that inhibin gene plays a role on oocyte development in vitro.In this trial,effect of overexpression of inhibinα(1-32) fragment on bovine granulosa cell proliferation, apoptosis,steroidogenesis,oocyte maturation and embryo development were investigated respectively,by transfection with inhibin recombinant plasmid pEGISI. Techniques such as cell culture,plasmid extraction,RT-PCR,transfection,TUNEL, radioimmunoassay,and flow cytometry were used.The plasmid pEGISI was constructed previously in our laboratory.1.Effect of overexpression of inhibinα(1-32) fragment on bovine granulosa cell proliferation,apoptosis,steroidogenesis,and development of co-cultured oocytes.The objective of the present trial was to determine the effects of an inhibinα(1-32) fragment gene on proliferation,apoptosis,and steroidogenesis of bovine granulosa cells(GC) isolated from medium and small follicles(diameter＞4 to 8 mm and 1 to 4 mm,respectively),and the effect of GC,previously transfected with pEGISI,on oocyte maturation and in vitro embryo development.To enhance expression of the inhibinα(1-32) fragment,GC was transfected with pEGISI.Our results showed that transfection with pEGISI inhibited GC proliferation(88.8±2.1%; mean±SEM) compared to control and EGFP group(100%,97.5±2.1%) from medium follicles(P＜0.05),with no significant effect on GC from small follicles. Apoptosis was higher(P＜0.01) in transfected GC than in controls,transfection with pEGISI increased(P＜0.05) estradiol synthesis from both medium and small follicles(0.57±0.13 and 0.86±0.13 pg/mL vs.0.19±0.05 and 0.35±0.09 pg/mL in controls) after culturing for 48 h,with suppression(P＜0.05) in transfected GC after 96 h.Transfection reduced(P＜0.05) progesterone synthesis in GC from both medium and small follicles(24.5±3.4 and 75.4±4.6 ng/mL vs.45.42±5.33 and 117.32±11.99 ng/mL in the control) after culture for 48 h,with no significant difference after 96 h.Maturation rate of oocytes co-cultured with transfected GC from medium follicles was decreased relative to control(61.5±6.8%vs.71.2±5.7%,P＜0.05),with no significant effect on embryo development.In conclusion, overexpression of inhibinα(1-32) fragment regulated GC development;effects on subsequent oocyte maturation were both time-and stage-dependent.2.Effect of overexpression of inhibinα(1-32) fragment on oocyte maturation and embryo development by transfectionIn this trial,denuded oocytes were transfected with pEGISI.The transfection efficiency,oocyte maturation,embryo development were investigated.The results showed that pEGISI could be transfected into oocyte through the zona pellucida with liposome and overexpressed in high efficiency(53%),which was observed under the fluorescence microscope.Transfection was not harmful to oocyte structure, maturation and fertilizability.The overexpression of pEGISI has no effect on oocyte maturation and embryo development.3.Effect of overexpression of inhibinα(1-32) fragment on bovine cumulus-oocyte complex maturation embryo development in vitroThe aim of this trial was to examine the effect of inhibin on the oocyte maturation and hormone secretion in vitro.For this purpose,we carried out the transfection of cumulus-oocyte complex(COC) with recombinant plasmid pEGISI,enhanced inhibin peptide fragment expression.The cumulus expansion,apoptosis,hormone secretion and oocyte maturation were investigated.The apoptosis of COC was assessed using TUNEL,and the release of progesterone,estradiol was evaluated using RIA technique.The transfection of pEGISI significantly decreased cumulus expansion and nuclear maturation in medium follicles(87.5%vs.69.8%,77.4%vs.57.9%),on the contrary,increased the cumulus expansion and nuclear maturation in the small follicles(55.9%vs.58.1%,50.3%vs.54.9%).The cumulus apoptosis was significantly increased in the pEGISI treatment group compared with the controls both in MFs and SFs.It was observed that transfection of COC with pEGISI increased the synthesis of progesterone,estradiol of COCs.The estradiol and progesterone synthesis in transfected cumulus cells of COCs were 6.9pg/mL and 0.47ng/mL, respectively,both of which were higher(P＜0.05) than that observed in the control group.These results suggested that inhibin is one of key regulator of COC development,effect of transfection with pEGISI on the COCs changed with different stage of follicles during in vitro maturation.