Study On The Mechanisms Of Heat Stress Affecting Rat Functional Luteal Regression

Heat stress affects a variety of reproductive functions in female species,such as follicular development,oocyte maturation and early embryonic development.Studies have shown that heat stress regulates reproductive functions by interfering with the reproductive endocrine.Meanwhile,heat shock response depends on the regulation of cell signaling pathways.Thus,pseudopregnant rats were used as experimental subjects to establish the heat stress model in this study.Techniques such as ELISA,Western blot and IHC were used to analyze the expression pattern of proteins associated with progesterone synthesis and catabolism and the potential pathways,therefore to reveal the mechanism of heat stress effecting luteal regression which provides a theoretical basis for the further study of heat stress on female reproductive function.The main contents are as follows:1 Effects of heat stress on the expression of proteins associated with rat luteal progesterone synthesis and catabolismTo investigate the effect of heat stress on the expression of proteins associated with the progesterone synthesis and catabolism,a total of 70(28 days old)rats were induced pseudopregnancy by sequential PMSG/hCG(DO)treatment paradigm and then randomly distributed into non-heat stress(NHS)group and heat stress(HS)group.The rats in HS group were exposed to 40? at the same time every day for 2 h.The water intake,food intake and the rectal temperature before and after heat stress were determined every day.On day 3,after 2 h post-stress recovery,serum and ovaries of two groups were collected(n=5).On day 7,after 2 h post-stress recovery,rats were killed direcrtly or killed after treatment with PGF for 0 h,1 h,2 h,8 h or 24 h,and then the serum and ovaries were collected(n=5).Serum were collected to determine progesterone concentrations by ELISA,and ovaries were collected to determine the proteins associated with the progesterone synthesis and catabolism by Western blot and IHC.ELISA results showed that heat stress alone had no effect on progesterone concentrations;Western blot results revealed that heat stress treatment alone didn't affect StAR,P450scc and 3?HSD expressions during luteal formation,but phospho-PKA substrates expression on day 3 was higher than those on day 7(P<0.05).PGF decreased progesterone concentrations in both HS and NHS groups,and heat stress blunted PGF-induced decline in the progesterone concentrations in luteal regression(P<0.05);PGF or heat stress treatment alone didn't alter the expressions of phospho-PKA substrates,StAR,P450scc,3?HSD and NR4A1-F,but significantly increased NR4A1-S expression(P<0.05).Furthermore,heat stress reduced NR4A1-S expression in response to PGF(P<0.05).The immunostaining density for the phospho-PKA substrates were expressed mainly in the nuclei of luteal cells,while StAR,P450scc,3?HSD and NR4A1-F were uniformly expressed in the cytoplasm of luteal cells.These results indicate that heat stress could reduce NR4A1-S expressions during PGF-induced luteal regression in pseudopregnant rats,which might be correlated with regulation of the progesterone catabolism during luteal regression.2 Effects of heat stress on the ERK 1/2 phosphorylation,HSP70 and ATF3 expression during rat luteal regressionTo investigate the effect of heat stress on the expressions of HSP70,MAPK/ATF3 and PKB signaling during PGF-induced luteal regression,pseudopregnant rats were used as experimental subjects to establish the heat stress model(described as in experiment 1).On day 7,after 2 h post-stress recovery,rats were treated with PGF for 0 h,1 h,2 h,8 h or 24 h and then were killed.Ovaries were collected to determine the expressions of HSP70 as well as activation of MAPK/ATF3 and PKB by Western blot and IHC.Western blot results showed that heat stress didn't change the basal levels of HSP70 and p-ERK-1/2 compared with the NHS group.However,heat stress promoted the PGF-induced HSP70 and p-ERK-1/2 expression(P<0.05).Heat stress significantly elevated the basal levels of phosphorylation of p38 MAPK(P<0.05).PGF treatment increased the expression of ATF3 at 2 h which was inhibited by heat stress(P<0.05).For the PKB signaling,the p-Akt(Thr308/Ser473)were reduced 8 h and 24 h(P<0.05)post PGF treatment,while no significant differences were found at all time points between HS and NHS,The results indicated that heat stress promote HSP70 and p-ERK-1/2 expression,inhibted ATF3 expression during PGF-induced luteal regression in pseudopregnant rats which may help to reduce the decline in PGF-induced progesterone to maintain the luteal function.