Identification And Characterization Of Key Factors To Porcine Fertilization And Embryonic Development

Assisted reproductive technology（ART） has attracted significant public interest since the birth of the first in vitro fertilization（IVF） baby,but its efficiency is still low. Therefore, elucidation of the mechanism during the fertilization and preimplantation embryo development is extremely important. Pigs represent an ideal model for reproduction and biomedical applications owing to their morphological and functional similarities with humans, thus we expect this study could expand our knowledge of fertilization and preimplantation embryo development. A large amount of proteins and transcripts play critical roles in reproductive processes, such as oocyte maturation, fertilization, zygotic gene activation（ZGA） and embryonic development. Transcriptomic and proteomic are currently underway to gain further insights into the significance of specific factors, however, the key factors to the fertilization and preimplantation embryo development were needed to be explored. Long non-coding RNAs（lncRNAs） have emerged as a new aspect of biology, playing vital roles in a wide variety of normal biological processes, and disease development as well. However, their functional involvement in pig early embryo development has not been elucidated. Therefore, we explore the key factors on both protein and lncRNA levels. Previous research showed low transcription active from germinal vesical breakdown（GVBD） to ZGA. Therefore, we investigate the core proteins in fertilization, and lncRNA for embryonic development. The main results were as following:（1）In general, porcine oocytes with the first polar body at 42 h of in vitro maturation（42O） are used for IVF studies, but we found previously that most of the oocytes maturated in vitro for 33 h（33O） had completed nuclear maturation. In the current study, we demonstrated that 33 O sustain IVF with higher sperm decondensation and pronuclear formation rates and support in vitrodevelopment with higher cleavage and blastocyst rates, compared with 42O（ P < 0.05）. Proteomic compared between 33 O and 42 O led to the identification of 18 differentially expressed proteins, which 11 proteins expressed higher in 42 O while the other 7 proteins expressed higher in 33 O. Bioinformatics analysis found that the functions of the differentially expressed proteins mainly concentrated in oocyte maturation, chromotine remolding, epigenetics and embryo development et al. The results showed the tight connection between the higher IVF competence of 33 O and the its specific protein components, therefore we we focused on the proteins abundantly expressed in 33 O.（2）To examine the role of PDIA3 in sperm nucleus decondensation, its expression was inhibited or enhanced by injection of anti-PDIA3 antibody or PDIA3 mRNA, respectively, into MII oocytes at 40 h of IVM at 2 h before IVF. Inhibition of maternal PDIA3 disrupted sperm decondensation; conversely, overexpression of PDIA3 in improved sperm decondensation. In addition, sperm decondensation failure in PDIA3 antibody-injected oocytes was rescued by dithiothreitol. Our results collectively report that maternal PDIA3 plays a crucial role in sperm decondensation by reducing protamine disulfide bonds in porcine oocytes.（3）Due to the limited sequence annotation, porcine lncRNA have not yet been characterized and it is unknown if they are associated with early embryonic development. Here, we identified 4776 lnc RNA genes derived from RNA sequence（RNA-seq） data sets of 30 samples through Coding-Non-Coding Index（CNCI） and Coding Potential Assessment Tool（CPAT）, which are two tools for lncRNA identification independent of known annotations. Weighted co-expression network analysis suggested that each developmental stage can be delineated concisely by a small number of functional modules of co-expressed genes. We predicted the function of lncRNA through gene ontology analysis for the known gene in the same moudle. This study provides the first comprehensive analysis of pig lincRNAs and gives a revealing insight into the gene regulatory mechanism responsible for early embryonic development in pig.（4）Previous research suggested the ZGA happened from 4- to 8-cell stage, thus we focused on two lncRNAs which are abundantly transcripted started from 4-cell. The two transcripts were identified through PCR and sequencing; The direction of transcripts were confirmed by SSRT; We found two alternative splicing of TCONS₀0166370 by specific primer; Using q PCR, we found the two lncRNAs were 4-cell to morula, and showed the top level in 8-cell stage; Depletion of the two lncRNA by injection of the specific locked nucleic acid caused the embryonic developmental arrest at 4-cell stage, especially, one LNA-injection group of TCONS₀0166370 even could not enter the 2-cell stage.