Effect Of SIRT1 Deacetylase Enzyme Soak Sow Ovary Development

The porcine follicular development is a complex physiological processes regulated by endocrine, paracrine, and gene expression. Studies on growth and regulation mechanism of follicle have important theoretical and practical significance to better understand reproductive mechanism of sows. SIR2(silent information regulator2)-related enzyme1(SIRT1) is a kind of dependent NAD+deacetylase. The current studies found that SIRT1deacetylase used histone and non-histone as substrates involved in a variety of cell biological functions such as gene transcription silence, cell cycle regulation, energy metabolism, insulin secretion, angiogenesis, neuro-protection, cellular senescence and so on. In recent years, studies have shown that SIRT1mediated reproductive hormone receptor signaling pathways, involved in the regulation of animal breeding performance. However, studies of SIRT1gene in the regulation of porcine reproductive has not been reported. To further investigate follicular mechanism of sows, we used porcine ovarian tissue as the main materials, designed experiments to explore the role of SIRT1gene in follicular development of sows. The results were as follows:1. Expression of SIRT1gene has tissue specificityqRT-PCR and Western blot were used to detect expression levels of SIRT1mRNA and protein in porcine brain, liver, pancreas, adipose, ovary, kidney, heart, spleen tissue. The results showed that SIRT1gene was expressed in each tissue, but expression levels among tissues was very significant difference. Expression levels of SIRT1mRNA in various tissues:ovary> brain> spleen> fat> kidney> liver> heart> pancreas. And expression levels of SIRT1protein:ovary> brain> fat> liver> kidney> spleen> heart> pancreas. Expression levels of SIRT1mRNA and protein in ovary, brain, adipose tissue were significantly higher than those in other tissues.2. Expression of SIRT1gene in porcine ovarian tissueImmunohistochemistry was used to detect expression and localization of SIRT1gene in ovary tissue. qRT-PCR and Western blot were used to detect expression levels of SIRT1mRNA and protein in different developmental stages of follicles and different atresia degree of follicles. The results showed that SIRT1gene was detected in porcine oocytes, granulosa cells, membrane cells, luteal cells. Expression levels of SIRT1gene in granulosa cells were significantly higher than that in membrane cells and luteal cells, expression levels of SIRT1was not detected in stromal cells, and subcellular localization of SIRT1gene was in nuclei. Expression of SIRT1gene in different developmental stages of follicles and different atresia degree of follicles had regularity. Expression levels of SIRT1gene in different developmental stages of follicles:diameter1.5-3.Omm> diameter≤1.5mm>diameter3.0-5.0mm>diameter>5.0mm. Expression levels of SIRT1gene in different atresia degree of follicles:atretic follicles>early atretic follicle>healthy follicles. It was suggested that SIRT1gene plays an important role in follicle development.3. Effect of SIRT1gene on ovarian granulosa cell apoptosis of sowsActivator resveratrol (RES) and inhibitor nicotinamide (NAM) of SIRT1gene were added to cultured porcine ovarian granulosa cells in vitro, qRT-PCR was used to detect expression levels of SIRT1mRNA in the different treatment groups. Flow cytometry was used to detect apoptosis rates of granulosa cell in the different treatment groups. MTT was used to assay proliferation of granulosa cells in different treatment groups. The results showed that RES and NAM dose-dependent activated and inhibited SIRT1expression respectively. Granulosa cell apoptosis in RES group was significantly increased, the rate of cell proliferation declined. Granulosa cell apoptosis rate in NAM group reduced, the rate of cell proliferation increased. Expression levels of apoptosis inhibitor BcL-2declined in both RES and NAM treatment group. Expression levels of pro-apoptotic factors Bax mRNA was significantly increased in RES treatment group, but had not change significantly in NAM group. Expression levels of caspase-3in the RES group increased, and significantly decreased in the NAM group. In conclusion, RES and NAM caused the change of SIRT1expression. Granulosa cells were more susceptible to apoptosis in RES group, while granulosa cells apoptosis were inhibited in NAM group. The above results indicated that expression of SIRT1gene plays an important role in the process of porcine ovarian granulosa cell apoptosis.4. Effects of SIRT1gene on estrogen secretion of porcine granulosa cells and variation rules with expression levels of related receptorPorcine estrogen enzyme-linked immunosorbent assay kits was used to detect the concentration of estrogen in the culture medium, it was found that with the increase of resveratrol concentration, expression levels of SIRT1mRNA and estrogen concentrations in the culture medium increased. On the contrary, with the increase of nicotinamide concentration, expression levels of SIRT1mRNA and estrogen concentration in culture medium decreased, the correlation coefficient was0.9668.qRT-PCR was used to detect expression of FSHR, LHR, ER2mRNA in porcine ovarian granulosa cells cultured by resveratrol and nicotinamide, it was found that expression levels of FSHR and ER2were increased in RES group. Expression levels of FSHR and ER2mRNA in nicotinamide group has decreased, and had significant difference (p<0.05). Expression levels of LHR mRNA were decreased in both resveratrol and nicotinamide group, and the difference was significant (P<0.05).This study found that SIRT1gene was widely expressed in porcine ovarian tissue, its overexpression was associated with granulosa cell apoptosis, and its expressional changes affected reproductive hormone receptor expression and estrogen secretion of granulosa cells. These results suggest that SIRT1plays an important role in the regulation of porcine ovarian follicular development.