Orexin A Influence Gran?Lar Cell Secretion Of Progesterone In Ovary Via Action Of PKA-CREB Signaling Pathway

Orexin A is a neuropeptide secreted by the hypothalamus in recent years.Its receptor is G protein coupled receptor,which is involved in reproductive,neuroendocrine,energy metabolism,feeding and visceral activities.At present,It has been proved that the addition of Orexin A can affect the secretion of progesterone by sheep ovarian luteinized granulosa cells,and this effect is achieved by up-regulating the concentration of intracellular cAMP,but it has not been reported whether it is through the PKA-CREB signaling pathway.Therefore,in this study,ELISA,Western blot and immunohistochemical methods were used to study whether the effect of Orexin A on progesterone secretion by sheep ovarian luteinized gran?Losa cells was mediated by PKA-CREB signaling pathway,In order to provide theoretical reference for Orexin A to regulate the reproductive function of sheep.Referring to the sheep ovarian gran?Losa cell c?Lture system established in our laboratory,the cells were identified by Gimsa staining and follicp?Lar stim?Lating hormone(FSHR)immunofluorescence.Determination and Localization of CREB by Immunohistochemical staining with specific Antibody to CREB;The granulosa cells were luteinzied and identified by adding follic?Lar stimulating hormone(FSH),luteinizing hormone(LH)and estradiol(E2).The progesterone secretion of luteinzied granulosa cells and luteinized granulosa cells added with Orexin A were measured by ELISA after treated with different concentrations(100 nmol/L,1?mol/L,10?Lmol/L)of CREB inhibitor(666-15),at the same time,the corresponding control group was set up.After treatment with PKA inhibitor(H89),CREB inhibitor(666-15),Orexin A receptor agonist and Orexin A receptor inhibitor(Almorexant),progesterone secretion was measured by ELISA and compared with the corresponding control group.After the cell proteins were collected,the expression of CREB,CREB active form p-CREB and progesterone synthesis key protein StAR in each group were analyzed by Western blot.The results show that:CREB was found in sheep ovarian granulosa cells and mainly expressed in the nucleus.The addition of 666-15 cop-Ld reduce the progesterone secretion of luteinized granulosa cells,and the inhibitory effect was significant when the concentration of 666-15 was 10 ?mol/L,and the inhibitory effect was also significant when Orexin A was added.In the luteinized gran?Losa cell group supplemented with Orexin A,the secretion of progesterone was significantly decreased by adding H89,and the secretion of progesterone was affected by the addition of Orexin Receptor Agonist and Almorexant.The results of Western blot and ELISA confirmed each other,and the expression of StAR,CREB and p-CREB were related to the inhibitory effect,and their expression was also affected by Orexin Receptor Agonist and Almorexant.It is suggested that Orexin A plays a role in affecting the secretion of progesterone in sheep ovarian luteinized gran?Losa cells by binding to receptors via cAMP-PKA-CREB signaling pathway.