Comparative analysis between endometrial proteomes of pregnant and non-pregnant ewes during the peri-implantation period

Background： Early pregnancy failure has a profound impact on both human reproductive health and animal production. 2/3 pregnancy failures occur during the peri-implantation period; however, the underlying mechanism（s） remains unclear. Well-organized modification of the endometrium to a receptive state is critical to establish pregnancy Aberrant endometrial modification during implantation is thought to be largely responsible for early pregnancy loss. Result： In this study, using well-managed recipient ewes that received embryo transfer as model, we compared the endometrial proteome between pregnant and non-pregnant ewes during implantation period. After embryo transfer, recipients were assigned as pregnant or non-pregnant ewes according to the presence or absence of an elongated conceptus at Day 17 of pregnancy. By comparing the endometrial proteomic profiles between pregnant and non-pregnant ewes, we identified 94 and 257 differentially expressed proteins （DEPs） in the endometrial caruncular and intercaruncular areas, respectively. Functional analysis showed that the DEPs were mainly associated with immune response, nutrient transport and utilization, as well as proteasome-mediated proteolysis. Conclusion： These analysis imply that dysfunction of these biological processes or pathways of DEP in the endometrium is highly associated with early pregnancy loss. In addition, many proteins that are essential for the establishment of pregnancy showed dysregulation in the endometrium of non-pregnant ewes. These proteins, as potential candidates, may contribute to early pregnancy loss.

Modified Bfat-1 gene and its biological verification in mice by hydrodynamic tail vein injection

Omega-3 polyunsaturated fatty acids （w-3 PUFAs） are essential components required for normal cellular function and have been shown to have important therapeutic and nutritional benefits in humans. But humans or mammals cannot naturally produce w-3 PUFAs, due to the lack of the co-3 fatty acid desaturase gene （fat-1 gene）. Previously, fat-1 gene has been cloned from Caenorhabditis elegans and transferred into mice, pigs and sheep, but not yet into beef cattle. We attempt to transfer it into beef cattle. The object of this paper is to edit the fat-1 gene from C. elegans to express more efficiently in beef cattle and verify its biological function in mice model. As a result, the fat-1 gene from C. elegans was modified by synonymous codon usage and named it Bfat-l. We have demonstrated that degree of codon bias of Brat-1 gene was in- creased in beef cattle. Moreover, Bfat-1 gene could be transiently expressed in mouse liver and muscle, the w-6/co-3 PUFAs ratio of 18 and 20 carbon was decreased significantly in liver （P〈0.05）, and the ratio of 20 carbon decreased significantly in muscle 24 and 72 h after injection （P〈0.05）. This confirms that the Bfat-1 gene modification was successful, and the protein encoded was able to catalyze the conversion of w-6 PUFAs to e0-3 PUFAs.

Transgenic Pigs Carrying a Synthesized Fatty Acid Desaturase Gene Yield High Level of ω-3 PUFAs

Polyunsaturated fatty acids （PUFAs） are essential for normal growth in mammals, especially the ω-3 PUFAs, which play important roles in preventing several life-threatening diseases, such as coronary heart disease and diabetes. In this study, we aimed to investigate whether the sFat-1 gene from Caenorhabditis briggsae could be functionally expressed in transgenic pigs, and whether the transgenic could synthesize high quality ω-3 PUFAs endogenously. In this study, a gene construct consisting of CMV promoter and 1.9 kb cDNA of ω-3 fatty acid desaturase gene （sFat-1） from C. briggsae was injected into the male pronucleus of pig embryos by microinjection. The piglets were screened for the transgene by PCR, Southern blot and reverse transcription-PCR analysis. Pigs that give positive results were mated with wild-type pigs to produce the next generation and the transmission of transgene was examined by PCR analysis. Fatty acids compositions of various tissues in the transgenic pigs were then analyzed by gas chromatograph. A total of 878 embryos were transferred into 42 recipients, among which 29 successfully got pregnant and gave birth to a total of 162 piglets, and 8 of them were identified to be transgenic. Fatty acid compositions in the transgenic pigs were altered, and the levels of ω-6：ω-3 ratios were decreased from 14.53 in the control to 2.62 in Fat-1 transgenic pigs. A number of primary sFat-1-transgenic pigs were bred in this study, which lays the foundation for cultivation of new varieties of transgenic pigs.

Production of Transgenic Mice by Type-A Spermatogonia-Mediated Gene Transfer

Type-A spermatogonia first appear at between 3-7 d postnatally in mice and are the only immortalized diploid cells that reproduce in adulthood in these animals. In our current study, we explored the feasibility of producing stable transgenic mice using these cells. Enhanced pEGFP-N1 plasmids were suspended in ExGen500 transfection reagent and injected at different angles into the testes of 7-d-old male ICR mice. The resulting type-A spermatogonia-mediated gene transfer （TASMGT） mice were then mated with normal females at different stages of sexual maturity （6, 12, and 24 wk）. The integration and expression of the introduced EGFP gene was evaluated in the F1 transgenic offspring by PCR and Southern blotting analysis. The foreign gene integration rates for a low-dose group （15 μL gene suspension injected into each testis） and a high-dose group （30 μL suspensions injected） at the three stages of female sexual maturity tested were 11.76% （2/17）, 14.29% （3/21）, and 11.11% （2/18）, and 5% （1/20）, 5.56% （1/18）, and 0 （0/17）, respectively. The average integration rates for these two dose groups were 12.5% （7/56） and 3.64% （2/55）, respectively, which was a significant difference （P0.05）. Semi-quantitative RT-PCR analysis further showed that the introduced GFP gene was expressed in 3/9 integration mice. In addition, GFP expression was observed in the sperm cells from the TASMGT mice, and also in the embryos and F2 pups from the F1 generation transgenic mice. Hence, although the foreign gene integration rate for TASMGT is not high and the transgenic offspring show as yet unexplained defects, our results indicate that this method is a potentially feasible and reproducible new approach to creating transgenic mice.