Effect Of Nesfatin-1 On In Vitro Maturation Of Porcine Oocytes

As an essential part of in vitro embryo production,in vitro maturation(IVM)of oocytes and in vitro culture(IVC)of embryos are of great importance to animal husbandry,human assisted reproduction,and developmental basic research.Mining and interpretation of factors regulating development of oocytes and embryos are beneficial to modify culture media and increase embryo production rate.Nesfatin-1,a polypeptide discovered recently,can regulate reproductive activities of rodents,fish and other animals.But whether Nesfatin-1 also participates in regulating livestock reproductive activities,impacting the development of oocytes and embryos has not yet been reported.The study was designed to investigate the effect of Nesfatin-1 on in vitro development of oocytes and embryos,to provide evidence for optimizing the pig IVM,IVC media formulations.The specific test and results are as follows:Experiment I aimed to analyze the Nesfatin-1 gene of the pig.The search results indicate that pig Nesfatin-1 gene(pNesfatin-1)of 151.8Kb is located in the second autosome,consists of 15 exons and 14 introns.As for amino acid sequence,pNesfatin-1showed 92% and 83% homology to human and rat.As for nucleotide sequence pNesfatin-1dispayed 93% and 96% similiarity to human and rat homologous.The evolutionary tree clustering showed that pig's Nesfatin-1 have the most homology with rat Nesfatin-1 gene.Experiment II was designed to explore the expression and distribution of Nesfatin-1on pig ovarian.Immunofluorescence staining of ovarian tissue slice showed that Nesfatin-1is mainly distributed in the cumulus granule cells,parietal granule cells and thecal cells.Nesfatin-1 stably expressed in primary cultured granule cells and thecal cells and the expression amount of Nesfatin-1 in both cumulus oocyte complexes and granule cells showed a significant downward trend as the follicle diameter increases.Experiment III aimed to explore the role of Nesfatin-1 in pig oocytes which were randomly selected for IVM.Nesfatin-1 at different concentrations(control group 0ng/mL;the experimental group 0.1,1,10,100 ng/mL)was added into the IVM medium.No significant differences in maturation rate,cleavage rate and blastocyst rate were observed.Experiment ? was to investigate the effect of Nesfatin-1 in pig oocytes with different degrees of granule cells for IVM.Nesfatin-1 at different concentrations(control group0ng/mL;the experimental group 1,10,100,1000ng/mL)was added into the IVM medium.No obvious differences in maturation rate and blastocyst rate was found in both encapsulated granule cell layer wrap >5 and granule cell layer wrap <2.Experiment ? was to investigate whether Nesfatin-1 play roles in oocytes maturation by energy metabolism.Nesfatin-1 at different concentrations(control group 0ng/mL;the experimental group 0.1,1,10,100 ng/mL)was added into IVM medium removal of pyruvate.It was noted that adding 1ng/mL,10ng/mL,100ng/mL Nesfatin-1 could significantly increase the rate of nuclear maturation of pig oocytes.Experiment ? was to investigate the effect of Nesfatin-1 in pig parthenogenetic embryos for IVC.Nesfatin-1 at different concentrations(control group 0ng/mL;the experimental group 0.1,1,10,100 ng/mL)was added into IVC.The results showed that0.1ng/mL Nesfatin-1 significantly promoted blastocyst formation,and 100ng/mL Nesfatin-1 significantly increased the total cell number of per blastocyst.In conclusion,the study revealed the distribution and expression pattern of pig Nesfatin-1 in ovarian tissues and in vitro cultured granulosal and cumulus cells,and established that Nesfatin-1 has effect on oocyte maturation and parthenogenetic embryo development: Nesfatin-1 could promote oocytes development when the medium energy substrates deletion,regardless of the degree of granules cells wrap oocytes;suitable concentration Nesfatin-1 has a positive effect on parthenogenetic embryo development.