Xiaomin Fang

Roles of SIRT1 in granulosa cell apoptosis during the process of follicular atresia in porcine ovary.

Ovarian follicular atresia is characterized by granulosa cell apoptosis, however, the exact mechanism is still unclear. Sirtuin 1 (SIRT1) is a NAD(+)-dependent deacetylase, which is associated with apoptosis in several of cell types, but its exact role in ovarian granulosa cell apoptosis is not clearly defined. In present study, we identified the involvement of SIRT1 in the process of follicle degeneration, which is known as follicular atresia, both from in vivo models and cell culture data. The results of immunohistochemistry showed that SIRT1 was widely detected in non-apoptotic granulosa cells of follicles, but significantly decreased during the process of granulosa cell apoptosis. Quantitative real-time PCR and Western blot analysis showed that the expression levels of SIRT1 mRNA and protein were increased (P<0.05) during follicular atresia. In order to provide more evidences elucidating the roles of SIRT1 during the process of follicular atresia, granulosa cells were cultured in vitro with resveratrol which acts as a potent activator of SIRT1. Results showed that resveratrol caused a dose-dependent increase in both SIRT1 mRNA and protein levels. Meanwhile, apoptotic rate of granulosa cell was increased (P<0.01) in a dose-dependent manner. Additionally, resveratrol significantly increased the expression levels of Caspase-3 (P<0.01) and Bax mRNA (P<0.01), while Bcl-2 mRNA level was significantly decreased (P<0.01). Thus, our results suggest that SIRT1 may play important roles in the regulation of granulosa cell apoptosis during follicular atresia in porcine ovary.

Breed-linked polymorphisms of porcine toll-like receptor 2 (TLR2) and TLR4 and the primary investigation on their relationship with prevention against Mycoplasma pneumoniae and bacterial LPS challenge

Toll-like receptors (TLRs) play a crucial role in innate immunity, serving as pattern-recognition receptors and the first barrier in host defense against microbial infections. Genetic variations of TLR2 and TLR4 are closely associated with a variety of infectious diseases, particularly lung diseases. In this study, we detected six and four single nucleotide polymorphisms (SNPs) in the coding sequences of porcine TLR2 and TLR4 genes, respectively. Only SNP 1027C>A of TLR4 was shown to be markedly biased in Western and Oriental pig populations. Hence, the susceptibility of pigs with different genotype at position 1027C>A to Mycoplasma hyopneumoniae (Mhp) infection was investigated, and changes to the expression of TLR2, TLR4, TNF-α and IL-1β were monitored. The results showed that there was no significant difference in susceptibility to Mhp infection between AA and CC individuals despite expression levels for all detected genes of the challenge groups being significantly higher than the corresponding control groups. Furthermore, porcine alveolar macrophages of different genotype were collected and stimulated by lipopolysaccharide. We found that the expression of TLR2, TLR4, TNF-α and IL-1β genes were enhanced to different levels by lipopolysaccharide stimulation. TLR2 and TLR4 gene expressions and their rates of increase of 1027CC pigs were significantly higher than for 1027AC pigs (Pu2009<u20090.01), while TNF-α and IL-1β expressions were significantly lower than for 1027AC pigs (Pu2009<u20090.01). We predict that allele C at position 1027 of the TLR4 gene contributes to the pigs immune response to gram-negative bacterial infections.

Long form leptin receptor and SNP effect on reproductive traits during embryo attachment in Suzhong sows

To ascertain whether the long form leptin receptor (LEPR) affects the regulation of embryo attachment and whether there are LEPR Single Nucleotide Polymorphisms (SNPs) associated with reproductive traits in pigs, Real-time qPCR was used to detect relative abundance of LEPR mRNA pattern in different tissues of Suzhong sows during the embryo attachment period (pregnancy day 13, 18 and 24) to the uterus, and PCR-RFLP as well as PCR-sequencing were used to investigate the coding sequence for SNPs of LEPR in a population of 512 Suzhong sows. Real-time qPCR results indicated that LEPR mRNA was present in all 22 tissues of pigs with differences in relative abundance of the LEPR mRNA (P<0.05). Among these tissues, the greatest relative abundance occurred at the endometrial attachment site (P<0.01), followed by the hypothalamus and most reproductive tissues (P<0.05), and there was a lesser relative abundance of the LEPR mRNA in the pituitary. During different embryo attachment periods, LEPR mRNA was greatest on Day 18 (attachment; P<0.05), followed by Day 24 (post-attachment), and relative abundance was least on Day 13 (pre-attachment). The prevalence of the LEPR mRNA in pregnant sows was greater than in non-pregnant sows (P<0.05). At the c.2856C>T locus of LEPR, Chi-square test results demonstrated that allele and genotype frequencies were in Hardy-Weinberg disequilibrium at this locus, PCR-RFLP results revealed that Genotype TT was greater than Genotype CC (P<0.05) for reproductive traits of TNB (Total Number Born) and NBA (Number Born Alive), which suggested that T allele at c.2856C>T locus has advantageous effects on litter size and litter weight in Suzhong pigs. In conclusion, the expression of the LEPR gene might be involved in the regulation of embryo attachment mechanisms in pigs, and the LEPR SNP c.2856C>T could be a molecular marker for improving litter size and litter weight in pig breeding.

CYP1A1 mediates the suppression of major inflammatory cytokines in pulmonary alveolar macrophage (PAM) cell lines caused by Mycoplasma hyponeumoniae

Mycoplasmal pneumonia is a lung infection disease caused by Mycoplasma hyopneumoniae in swine. We previously reported that Cytochrome P450 1A1 (CYP1A1) expression was significantly downregulated in pigs infected with M. hyopneumoniae compared to the healthy controls. In this study, pulmonary alveolar macrophage (PAM) cell lines with CYP1A1 overexpression or siRNA-mediated CYP1A1 silencing were used to explore the biological function and regulatory mechanism of CYP1A1 gene expression changed on the inflammatory response of pigs infected with M. hyopneumoniae. The results showed that the cells overexpressing CYP1A1 infected with M. hyopneumoniae led to a rapid increase in PPAR-γ expression, which resulted in decreasing the levels of several inflammatory cytokines including IL-1β, IL-6, IL-8 and TNF-α. On the contrary, this effect was just opposite in CYP1A1-RNAi cells infected with M. hyopneumoniae. We suggest that CYP1A1 suppress the inflammatory response caused by M. hyopneumoniae infection, via PPAR-γ signaling pathway in pigs.

Investigation of Eph-ephrin A1 in the regulation of embryo implantation in sows

Eph A1 and ephrin A1 (Eph-ephrin A1) is a key receptor-ligand pair of Eph-ephrin system, which plays important roles in the migration and adhesion of cells, tissue morphogenesis and vasculogenesis in mammals. In order to investigate the regulation of Eph-ephrin A1 during porcine embryo implantation, the expressions of mRNA and protein of Eph-ephrin A1 were detected in different reproductive tissues from twelve sows during embryo implantation period on pregnancy day 13, 18 and 24, respectively. Functions of Eph-ephrin A1 on the migration and adhesion of porcine endometrial epithelial cells were analysed by RNA interference (RNAi), transwell migration assays and MTT assays. Results showed that mRNA levels of Eph-ephrin A1 were highly expressed in endometrial attachment site when compared to other reproductive tissues (pxa0<xa00.05) and were peaked on pregnancy day 18 during embryo implantation (pxa0<xa00.05). Protein levels of Eph-ephrin A1 were highly expressed in endometrial attachment site and were peaked on pregnancy day 18 (pxa0<xa00.05). Eph-ephrin A1 proteins were located in endometrial luminal epithelium, stroma of attachment site and inter-attachment site during embryo implantation, and the protein levels were higher during implantation compared to pre-implantation or post-implantation. Furthermore, silencing ephrin A1 gene significantly reduced the migration and adhesion capacity of porcine endometrial epithelial cells. These findings suggest that the Eph-ephrin A1 protein likely targets endometrial attachment site to enhance the migration and adhesion of porcine endometrial epithelial cells around pregnancy day 18 during pregnancy in sows.