Orexin A Influence Granular Cell Secretion Of Progesteronein Ovary Via Action Of CAMP-PKA Signaling Pathway

The aim of this experiment is to clarify the effects of orexin A on the progesterone secretion from granular cell and observe the possible mechanism of action.The Sheep ovary granulosa(SOG)cells was cultured in vitro,HE and Immunohistofluorescence staining of FSHR were used for ovarian granulose cell identification.The SOG cells has been pre-incubated with luteinizing hormone(LH)(2.5 IU/ml),follicle-stimulating hormone(FSH)(2.5 IU/ml),estrogen(E2)(1 ug/ml).Then the SOG cells were treated with different concentrations(1nM,10 n M,50 nM,100nM,and 150 n M)of Orexin A for different time(0 h,24 h,48 h,and 72 h).The progesterone P4 have been determined.The cell supernatant was treated with 10 nmol/L and 50 nmol/L orexin A for 24 h,and the progesterone concentration in the supernatant of ovine granulosa cells was detected by ELISA.The cells were collected and ELISA was used to detect c AMP in ovarian granulosa cells.concentration.Different concentrations of Almorexant(10 nmol/L,100 nmol/L,1 ?mol/L)were added for 2 h,and different concentrations(10 nmol/L,50 nmol/L)of orexin A were added to incubate for 24 h to obtain cell supernatant.The ELISA method was used to detect the change of cAMP concentration in the cell lysate.Western blot was used to detect the expression of St AR and PKA protein in the cells.Alorexant,Orexin Receptor Agonist,and PKA inhibitor(H-89)were added to detect changes in the expression of StAR and PKA in cells and the concentration changes of cAMP in cells.The results showed that granulose cells form was complete,the edge was clear,like triangle cone or irregular star.The cytoplasm was pink,the nucleus were dyed blue.The positive rate was more than 90% compared with control group.Luteinized granulosa cells in sheep's ovary were supplemented with orexin A.At the same time,the secretion of progesterone gradually increased and then decreased with the increase of orexin concentration.When the concentration of progesterone was added at 10 nmol/L,the secretion of progesterone reached the highest;At the concentration of orexin A,the secretion of progesterone gradually increased and then decreased with the prolongation of the action time,and the secretion reached the maximum at 24 h.The concentration of cAMP in Almorexant group was significantly lower than the control group(P<0.01).By examining the effects of Almorexant,Orexin Receptor Agonist,and H-89 on the expression of PKA and St AR protein in ovarian granulosa cells,Compared with the control group,the expression of St AR and PKA protein in orexin A group was significantly increased(P<0.05);orexin A was added after adding orexin Receptor Agonist as an orexin receptor activator,and the St AR in the luteinized sheep granulosa cells.The expression of PKA protein was significantly increased(P<0.001);the expression of St AR and PKA protein in luteinized sheep granule cells was significantly decreased in the inhibitor-treated group;and the expression of St AR and PKA protein was significantly decreased in H-89 group added granulosa cells(P<0.05).These results reveal that orexin A can promote the secretion of progesterone from ovine ovary granulosa cells through its cAMP-PKA signaling pathway through its receptor binding.