| Many events of modern pig production management procedures may be stressors for animals,such as high stocking densities,poor feeding conditions,long transportation periods,rough or intense interaction patterns between humans and animals,heat stress,etc.Hypothalamic pituitary adrenal（HPA）axis was activated during stress.The main central regulator of HPA axis is Corticotropin releasing hormone（CRH）,which acts on the anterior pituitary gland to promote ACTH secretion.Subsequently,ACTH acts on adrenal cortex to promote adrenal synthesis and release of Glucocorticoid（GCs）.HPA in turn mediated the function of the Hypothalamic pituitary gonadal axis（HPG）,which was responsible for the maturation of reproductive organs and the reproductive capacity of organisms.CRH inhibits Gonadotropin releasing hormone（Gn RH）secretion,while GCs inhibits Luteinizing hormone（LH）and Estrogen（E2）and Progesterone（P）in the ovary produced,which made the target tissues or organs resistant to E2.HPA axis hormones have multi-level adverse effects on female reproduction at different levels.It has been reported that heat stress can inhibit the early embryo implantation in pigs and damage the pre-implantation embryo development.Sows are more sensitive to heat stress during the first 15 days after fertilization than during the next 15-30 days.In the early stages of pregnancy,food deprived stress within 48 h after ovulation or repeated injection of ACTH 6 h after ovulation significantly reduced embryo cleavage rate and blastocyst development rate,and serum Cortisol（CORT）concentration was significantly increased.In pigs,heat stress significantly reduced the proportion of normal oocytes fertilized.Studies have shown that fertilization and pre-implantation stage are sensitive to stress during pregnancy.However,the effects of HPA-axis hormones CRH,ACTH and CORT on preimplantation embryo development in pigs are still unclear,and the direct or indirect mechanism of their damage to pig embryos remains unclear.Before the oocytes fertilization and embryo implantation development process,the sperm and oocytes or embryos in the free state in the fallopian tube,and the mother did not establish direct contact of blood,combined with the complex environment of oviductal lumen,therefore,it is difficult to use a particular stress model in the research of mechanism of specific HPA axis hormones affect the pig oocytes fertilization and preimplantation embryo development.The test was carried out to investigate the relationship between the HPA axis hormone concentration and Oviductal epithelial cells（OECs）and the development of Oviductal cells and preimplantation embryos using an in vitro model.CRH 1×10-7 M,1×10-6 M,1×10-5 M or ACTH1×10-10 M,1×10-9 M,1×10-8 M or CORT 1×10-7 M,1×10-6 M,1×10-5 M were added directly during the development of pig parthenogenetic preimplantation embryos in vitro.The effects of different concentrations of three hormones on embryo development were observed.In the presence of CRH,ACTH or CORT,parthenogenetic embryos were co-cultured with pig primary OECs in vitro to observe the effect of embryo development.Conditional Medium-in vitro fertilization medium（CM-m TBM）or Conditional medium-pig zygotes medium（CM-PZM）prepared by pretreatment of OECs using CRH,ACTH or CORT for in vitro fertilization of oocytes or the development of pig preimplantation embryos and to observe the fertilization and development of embryos;Apoptosis of OECs cells was observed by adding CRH,ACTH or CORT with different concentrations in vitro culture.The results show that:（1）Directly add CRH 1×10-7 M,1×10-6 M,1×10-5 M or ACTH1×10-10 M,1×10-9 M,1×10-8 M or CORT 1×10-7 M,1×10-6 M,1×10-5 M to in vitro parthenogenetic preimplantation embryos.There’s no effects on the blastocyst rate and blastocyst cell number of parthenogenetic preimplantation embryos（P<0.05）;（2）Porcine primary OECs cells cultured in vitro and added CRH 1×10-7 M,1×10-6 M,1×10-5 M or ACTH1×10-10 M,1×10-9 M,1×10-8 M or CORT 1×10-7 M,1×10-6 M,1×10-5 M,respectively.Hoechst33342 nuclear staining showed that apoptosis of OECs cells was induced on 2×10-6 M CRH or10-6 M CORT（P<0.05）;Flow cytometry further detected OECs cultured with 2×10-6 M CRH or 1×10-6 M CORT,and the results showed that the proportion of early and late apoptotic cells was significantly higher than that of the group without hormone（P<0.05）,but the proportion of early and late apoptosis of OECs cultured with 1×10-8 M ACTH did not increase significantly（P<0.05）;Western Blot analysis showed that the expression of activated caspase-3 protein in OECs cultured with 2×10-6 M CRH or 1×10-6 M CORT increased significantly（P<0.05）;（3）In the presence of 2×10-6 M CRH or 1×10-6 M CORT,preimplantation parthenogenetic embryos were co-cultured with primary OECs.The results showed that the blastocyst rate of embryos compared with the no-hormone group was significantly reduced（P<0.05）,the number of blastocyst cells decreased significantly（P<0.05）;When CM-m TBM or CM-PZM pretreated with 2×10-6 M CRH or 1×10-6 M CORT were fertilized and cultured in vitro,it was found that the rate of polyspermy was significantly increased in the hormone group（P<0.05）.The embryo blastocyst rate was significantly lower than that of the control group（P<0.05）and the number of blastocyst cells decreased significantly compared with the control group（P<0.05）;（4）The m RNA relative expression level of apoptotic molecule Bcl-2/Bax was detected by RT-QPCR in CM cultured blastocysts supplemented with hormone CRH or CORT.The results showed that the m RNA ratio of Bcl-2/Bax in CM cultured blastocysts supplemented with hormone CRH or CORT was significantly lower than that in the control group（P<0.05）;Immunofluorescence staining analysis of activated caspase-3 in blastocysts showed that the expression of activated caspase-3 in blastocysts in hormone group was significantly higher than that in control group（P<0.05）;（5）The m RNA relative expression levels of growth factors BDNF,IGF1,TGF-β1 and OVGP1 in OECs cultured with 2×10-6 M CRH or 1×10-6 M CORT were detected by RT-QPCR.The m RNA relative expression levels of 4 growth factors in OECs in hormone supplemented group were significantly lower than those in hormone free control group（P<0.05）;（6）ELISA was used to detect the concentration of Fas L or TNF-αin the supernatant of OECs cultured with 2×10-6 M CRH or 1×10-6 M CORT.The results showed that the concentration of soluble Fas L or TNF-αin the supernatant of OECs in the hormone supplemental group was significantly higher than that in the control group（P<0.05）;（7）Western Blot was used to detect the expression of Fas or TNFR in OECs cultured with 2×10-6M CRH or 1×10-6 M CORT.The results showed that the expression of Fas or TNFR in OECs in the CRH or CORT group was significantly higher than that in the control group（P<0.05）;Western Blot analysis showed that the expression of CRHR in OECs in 2×10-6 M CRH group was significantly higher than that in control group.The GR expression of OECs cultured with1×10-6 M CORT was significantly lower than that of OECs cultured without CORT（P<0.05）;（8）Liposome transfection of Fas L or TNF-αsi RNA was used to knock down the apoptotic ligand Fas L or TNF-αin OECs,and then 2×10-6 M CRH or 1×10-6 M CORT was added for OECs culture.Flow cytometry was used to detect cell apoptosis.The early and late apoptosis rates of OECs cells transfected with Fas L or TNF-αsi RNA were significantly lower than those transfected with random si RNA（P<0.05）;（9）Fas,TNFR,CRHR and GR were detected by immunofluorescence staining at 2-cell stage,4-cell stage and blastocyst stage of preimplantation parthenogenetic embryos.Fas,TNFR and GR were all expressed in each stage,and Fas and TNFR were mainly located in the cytoplasmic membrane,while GR was mainly located in the cytoplasm.CRHR was not expressed in 2-cell stage and 4-cell stage,but was only expressed in cytoplasmic membrane of blastocyst stage.（10）RT-q PCR was used to detect the relative expression of 11β-HSD1 and 11β-HSD2 in OECs or parthenogenetic embryos at 2.4-cell stage.The results showed that the relative expression of 11β-HSD1 in OECs was 561 times that of 11β-HSD2.The relative expression of 11β-HSD2 in embryos was 43 times that of 11β-HSD2（P<0.05）.In this study,although ACTH has no effects on pig embryo development,CRH and cortisol indirectly impair preimplantation embryo development and increase polyspermy by inducing apoptosis of tubal epithelial cells.Therefore,after treatment by CRH or CORT,the expression level of GR decreased,and the expression level of CRHR,Fas and TNFR increased significantly,leading to cell apoptosis.Apoptotic OECs produced more Fas L and TNF-α,but less growth factor and OVGP1.The interaction of Fas L and TNF-αwith Fas and TNFR on embryos,respectively,coupled with a significant reduction of growth factors,induced apoptosis in embryos.The decrease of OVGP1 and growth factor increased the rate of polyspermia.Both apoptosis and polyspermy result in the decrease of embryonic development potential.Porcine preimplantation embryos did not express CRHR but expressed CRHBP and were insensitive to CRH.The expression of 11β-HSD2 is higher than 11β-HSD1,which converts cortisol into inactive cortisone and thus has no damage from cortisol.These data are important for us to understand the mechanism by which adverse injury affects reproduction and to take measures to ameliorate the harmful effects of adverse injury on fertilization and embryo development. |
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