Effects Of Factor In The Germline Alpha And Brain-derived Neurotrophic Factor On Development Of Bovine And Porcine Oocytes And Embryos

As one of the emerging reproductive biotechniques, animal cloning is far from mature in theory and practice. For example, the efficiency of cloning is very low ; the cloned animals showed defects in physiology or immunity. However, the knowledge is still lack on the molecular mechanisms underlying the regulation of development of mammanian oocytes and early embryos. Details of signal transduction, caryocinesia, nucleo-cytoplasmic interaction, histone acetylation, DNA methylation, and donor nuclear reprogramming that induced by cell-specific transcription factors during cloning remains unclear. These shortages have become the molecular biological bottleneck for the improvement and industrilization of techniques of animal cloning and oriented differentiation of embryonic stem cells. Therefore, in order to obtain more high quality oocytes and embryos, it is important to explore the molecular mechanism regulating the development process from germ cell to oocyte and then to embryo, particularly focusing on the function of oocyte-specific transcription factors.The factor in the germline alpha (Figla) is a basic helix-loop-helix transcription factor that is expressed exclusively in germ cells and regulates the transcription of zona pellucida (ZP) genes through an E-box motif (CANNTG). Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family which can promote the growth of nerve cells. The recently researches have shown that BDNF is detectable in ovary, indicating that it may be a novel transcription factor controlling the growth of oocyte, follicle and ovary. The main purpose of the present study was to explore the mRNA expression and function of Figla and BDNF in bovine and porcine oocytes and early embryos.Firstly, the complete CDS sequence of bovine Figla gene was cloned from ovarian tissues of Chinese Yellow cows by SMART and RT-PCR. Squencing results showed that the size of figla CDS is 585bp with four exons in the whole open reading frame coding the Figla mature protein consisted of 194 amino acids. Homology, genetic distance and molecular evolution relationship of Figla gene among different species were analyzed by phylogenic NJ tree. The basic characters, secondary structure, tertiary structure and conserved structure domain were predicted by proteomic analysis tools. Results showed that nucleotide sequence homology ranged from 66% to 92% among different species, when bovine sequence shared homology of 90% with those of mouse, anthropoid, and human, indicating that figla is a conserved gene.Secondly, a method for detecting mRNA expression levels in oocytes and embryos was developed by using real time fluorescence quantitative PCR, charaterized by analysing large number of genes with trace amount of total RNA. The bovine SDHA and porcine 18S primers were designed as reference for SYBR Green I real-time fluorescence quantitative PCR, in which the best result was achieved when 0.5μL of primers at 10μM and 2μL of cDNA templates were contained in one reaction system. The results indicated that trace amount of total RNA with high quality can be extracted from small number of oocytes and embryos with QIAGEN RNeasy Micro Kit, and that the designed bovine SDHA and porcine 18S primers are suitable as references for studies on gene expression in oocytes and early embryos.Thirdly, mRNA expression of figla and BDNF genes in porcine oocytes and early parthenogenetic embryos (PA) and bovine oocytes and early embryos derived from in ivtro fertilization (IVF), parthenogenetic activation (PA), and nuclear transfer(NT) were detected. The results showed that: (1) there was no difference in mRNA levels for figla gene between bovine vesicle (GV) oocytes and morulae. While mRNA levels were different in bovine oocytes and IVF embryos at different stages (p<0.01, with the exception of p<0.05 between metaphase II oocytes and 4-cell, 8-cell and blastocysts), with the highest expression in vesicle (GV) oocytes and the lowest expression in 8-cell embryos; mRNA levels for figla were different in porcine oocytes and PA embryos (p<0.01, with the exception of p<0.05 between GV oocytes and blastocysts, metaphase II oocytes and morulae), with the highest expression in 4-cell and the lowest expression in 8-cell embryos; (2) mRNA levels for BDNF gene were different in bovine oocytes and IVF embryos at different stages (p<0.01), with the highest expression in metaphase II oocytes and the lowest expression in 8-cell embryos; there was no difference in mRNA levels for BDNF gene between pocrine 4-cell and 8-cell, morulae and blastocysts, vesicle (GV) oocytes and morulae. While mRNA levels were different in porcine oocytes and PA embryos at different stages (p<0.01); (3) there was no difference in mRNA levels for figla gene among bovine IVF, PA, and NT blastocysts, while the mRNA for BDNF gene was highly expressed in PA and IVF blastocysts compared to the NT blastocysts (p<0.01).In the fourth experiment, effects of BDNF on the development of bovine IVF and PA embryos and porcine PA embryos were studied. Supplementation of culture media with BDNF at the concentration of 40μg/L caused a significant increase in the rates of bovine blastocyst from IVF (31.5% vs 51.2%, p<0.05) and PA oocyte ( 42.0% vs 58.0%, p<0.05). However, the rates of cleavage and hatched blastocyst in the BDNF groups were not significantly different than those in the control (p>0.05). And supplementation of culture media with BDNF at the concentration of 40μg/L caused a significant increase in the rates of porcine blastocyst from PA oocytes ( 20.9% vs 34.1% ,p<0.05). These results indicate that BDNF of 40μg/L in the culture media can promote the development of bovine and porcine early embryos.Effects of NGF on the development of bovine IVF and PA embryos and pocine PA embryos were studied as well. Supplementation of culture media with NGF at the concentration of 10μg/L caused a significant increase in the rates of blastocyst from IVF (35.0% vs 50.0%,p<0.05) and PA oocytes ( 43.9% vs 56.3% ,p<0.05). However, the rates of cleavage and hatched blastocyst in the NGF groups were not significantly different than those in the control (p>0.05). These results indicate that NGF of 10μg/L in the culture media can promote the development of bovine early embryos derived from in vitro fertilization and parthenogenesis. But Supplementation of culture media with NGF did not increase the rates of cleavage, blastocyst and hatched blastocyst from porcine PA oocytes.Finally, effects of NGF and BDNF on proliferation of bovine granulosa cells were studied. Supplementation of DMEM cultured media with NGF at the concentration of 5μg/L or BDNF at the concentration of 20μg/L caused a significant increase in number of granulosa cells after cultrued for 48h, indicating that NGF and BDNF can promote in vitro proliferation of bovine granulosa cells.In conclusion, the full-length cDNA encoding bovine Figla gene was cloned. Oocytes and early embryos from cows and sows were all shown to express mRNAs for Figla and BDNF. Supplementation of culture media with BDNF may promote development of porcine and bovine early embryos. NGF may promote development of bovine early embryo.