Effect Of Lipopolysaccharide On Expression Of Toll-like Receptors And Steroidogenesis In Granular Layer And Thecal Layer Of Goose Ovarian Follicles

To study the role of avian Toll-like receptors(TLRs) in waterfowl reproductive immunology and its relationship with reproductive endocrine, to elucidate the regulation of reproductive infection and reproductive endocrine function, and to find effective treatment methods and new immune targets, Yang Zhou goose was choosen as experimental animals. After 8h of laying, mature follicles and blood samples were collected from physiological geese and LPS treated geese, LPS treated at 0h, 6h, 12 h, 24 h, 36 h respectively. The study includes three parts: Various expression levels of Toll-like receptors(TLRs) in granulosa and theca layers of laying geese, changes in the expression of TLRs during follicular growth and in response to lipopolysaccharide(LPS), concentration of P4 and E2 in blood and the expression of genes involved in sex steroids synthesis, including St AR and CYP11A1 in granulosa layer(GL), CYP17A1 and CYP19A1 in thecal layer(TL). All the research was completed by q RT-PCR and Radioimmunoassay techniques respectively.The results showed that all ten members of poultry TLRs genes were expressed in the GL and TL of laying geese. Among them, only TLR2-2 in GL almost showed no expression.In laying geese, the genes expression level of four members of TLRs family in GL was significantly higher than that of TL(P<0.01), while three members of the TLRs expression level in TL was significantly higher than that of GL(P<0.01). During follicular growth, the expression levels of all TLRs were relatively stable, only the expression level of TLR15 in GL and TL was significantly enhanced(P<0.05), and TLR2-1 in TL significantly increased(P<0.05).There was no visual pathological changes in follicles within 12 hours after intravenous injection of LPS. After 24 h, partial geese follicles showed irregular shape of jelly-like lesions, and after 36 h, all experimental goose follicles appeared jelly-like lesions.In GL, during the period of LPS treatment, the expression level of eight members of TLRs genes increased significantly(P<0.01), but the expression level of TLR3 decreased. All TLRs showed significance in different time. Unlike in GL, the expression level of all TLRs family genes that increased extremely significant in response to LPS in TL(P<0.01). After LPS treatment, concentration of P4 and E2 decreased significantly during 12 h to 36h(P<0.01),the expression level of St AR and CYP11A1 was reduced significantly at 6h each, CYP17A1 and CYP19A1 was also reduced significantly at 12 h and 6h respectively(P<0.01). The expression level of St AR was first increased and then decreased too.In summary, the expression profiling of TLRs in geese follicles indicate the existence of innate immune response mediated by TLRs. Different expression levels in GL and TL showed their different sensitivity and potential ability to various pathogenic microorganisms. With the growth and development of follicles, the pathogen recognition function of GL and TL was enhanced, and both of them maintained an efficient immune status. LPS harmed follicular development and ovulation, and all TLRs family genes showed changes in their expression levels in response to LPS. These TLRs can be divided into early, middle and late response genes. Follicular damage occurred slightly in early stage, so it is the best time for treatment of LPS invasion. In addition, LPS also reduced the concentration of P4, E2, and genes expression level involved in sex steroids synthesis. It also harmed goose reproductive endocrinology.As LPS up-regulation the expression level of TLRs and down-regulation the concentration of P4, E2 and the genes involved in their synthesis, we speculated that synthesis of P4 and E2 is mediated by TLRs after LPS invasion, and this process may exist time-dependent. This study would be of great significance for the environment control of waterfowl industry, development of endotoxin inhibitors, new attenuated vaccines and adjuvants. This study also lay a foundation for the further study of the relationship between reproductive immune and reproductive endocrine.