Regulations And Mechanisms Of Protein Kinase MAP3K8 On Progesterone Synthesis In Mouse Corpus Luteum

The corpus luteum (CL) is a transient endocrine gland developed from the ovulated follicles. The most important function of CL is to synthesize and secrete progesterone (P4), a key hormone to maintain normal pregnancy and estrus cycle in most mammals. Prolactin (PRL), luteinizing hormone (LH), estradiol (E2) and prostaglandin F2a (PGF2 a) are key hormones regulating progesterone synthesis in CL. However, in rodents PRL and E2 are the major hormones to stimulate progesterone synthesis. It is known that estrogen has a vital role in stimulating P4 synthesis in CL, but it still remains unclear about the mechanism of E2 regulating P4 production in CL.In the present study, we firstly located Mitogen-activated Protein Kinase 8 (MAP3K8) expression in the ovary by Immunohistochemistry (IHC). The results showed that MAP3K8 was stained in all of the luteal cells, and no MAP3K8 signal was observed in the follicular and stromal cells. Using Real-Time quantitative PCR (RT-qPCR) and Western blot (WB), we found that MAP3K8 level was much higher at mid-stage than at early and late stages during CL development.The further functional studies showed that the inhibition of endogenous MAP3K8 by MAP3K8-siRNA and MAP3K8 signaling inhibitor (MAP3K8i) in the luteal cells significantly blocked P4 synthesis. The results in vivo showed that after blocking the signal of MAP3K8, the level of P4 in the serum and CL also decreased that was accordant with the cells experiments. In order to find the effect of the altered P4 levels after MAP3K8 inhibition, the implantation sites were visualized after MAP3K8i treatment. The results showed that the MAP3K8i treatment significantly decreased the number of implantation sites. These data confirmed that the altered P4 levels after MAP3K8 inhibition have significant influence on the related physiological functions of P4.To understand the mechanism of MAP3K8 influencing the P4 synthesis, we found that only E2 but not PRL or LH up-regulated MAP3K8 expression in vitro or vivo. The forced inhibition of endogenous MAP3K8 by MAP3K8-siRNA or MAP3K8i omitted the enhancing effect of E2 on P4 production in the CL. These data suggest that MAP3K8 participates the passway of E2 stimulating P4 synthesis in CL.It is generally thought that the actions of E2 are ascribed to classical nuclear receptors, including estrogen receptor alpha (ERa) and and beta (ERβ). Our above results showed that E2 rapidly up-regulated the MAP3K8 expression at 3h after treatment. On these basis, we hypothesized that E2 up-regulated the MAP3K8 expression through G protein-coupled receptor 30 (GPR30) rather than nuclear receptors. Then, we found that GPR30 was expressed in the mouse CL, follicular and stromal cells. We used ICI 182,780 (ICI) and G15 to inhibit E2 from binding with ERa or ERβ and GPR30. Our results showed that ICI blocked the nuclear receptors but MAP3K8 mRNA remained up-regulated by E2. On the other hand, when G15 blocked E2 binding with GPR30, the effect of E2 up-regulating MAP3K8 mRNA level disappeard. These data suggest that MAP3K8 is involved in mediating the GPR30 signaling of E2 affecting P4 synthesis. Meanwhile, MAP3K8 acts as a mediating molecule in the effect of E2 on production of P4 in the granulosa-luteinized cells and CL through ERK1/2.In conclusion, our results showed that MAP3K8 was highly expressed in mouse CL and involved in regulating P4 synthesis in mouse CL, and the signaling of E2 stimulating P4 synthesis is through GPR30 and MAP3K8 which subsequently enhanced ERK1/2 phosphorylation and the expressions of cyplla and 3β-HSD. In light of the results of our study, MAP3K8 plays a critical role in regulating CL hormone synthesis and the reported signal molecules of E2 affecting P4 synthesis are potential for our understanding the related mechanisms of ovary physiology and the pharmacological intervention.