Study Of The Correlation Between Main Components Of Hippo Pathway And The Size Of Mouse Primordial Follicle Pool

Background and objective:Clarifying the molecular mechanisms by which the initiation of primordialfollicles is regulated is crucial for the prevention and treatment of female infertilityand premature ovarian failure. Recent reports have shown that changes of ovariancortical density could trigger the onset of the primordial follicle. On the other hand,Hippo signaling pathway which consists of several oncogenes（Mst/Lats/Yap）couldsense the changes of cell density in Drosophila ovary. To further verify the functionand regulative mechanism of ovary, we explored the expression changes of the corecomponents of Hippo pathway (MST, LATS, YAP) before and after primordialfollicle activation and its spatio-temporal correlation with the size of primordialfollicle pool in mice.Methods:1. To analyze the spatio-temporal correlation between the core component ofHippo pathway and the size of primordial follicle pool, Western blot, Real-time PCRand immunohistochemistry were used, and the expression and localization of mst, latsand yap mRNA and protein were examined in3d,1m,5m, and16m postnatal miceovary.2. The culture model of mice primordial follicle in vitro was established, and theexpression change and localization of mst, lat and yap mRNA and protein weredetected by using Western blot, Real-time PCR and immunohistochemistry beforeand after follicle activation.3. Using western blot, changes of P-YAP expression were examined andP-YAP/YAP were analyzed during follicle development. Results:1. The results of western blot showed that: The results of western blot showedthat: The expression of MST was decreased significantly as mouse age,(p <0.05),but there was no difference among1m,5m and16m groups; the expression ofLATS was also declined significantly (p <0.05, p <0.01, p<0.05), but increasedsignificantly in16m compared with5m (p <0.05). The expression of YAP showedthe opposite trend compared with MST. The significant expression of P-YAP wasincreased before1m, after that, no significant change was obeserved. Moreover, theratio of P-YAP/YAP was declined significantly, while no significant differencebetween5m and16m (p>0.05).2. The mRNA expression of mst decreased significantly with age (p <0.05, p <0.01, p<0.01), while increased significantly in16m compared with5m (p<0.05).The mRNA expression of lats also decreased significantly (p <0.05, p <0.01, p<0.01), but increased significantly in16m compared with5m (p <0.05). yap mRNAincreased remarkably (p <0.01, p <0.001, p<0.01), but decreased significantly in16m (p <0.05).3. The immunohistochemical analyses showed that: The expression of MST,LATS and YAP in primordial follicles was distributed mainly in the cytoplasm ofoocytes. From primary to mature follicles, MST, LATS and YAP were localized inthe cytoplasm of oocytes, envelope and granulosa cells, with higher expression insecondary follicles compared to granulsa cells, but a low or almost no experssion inatresia follicles. In addition, P-YAP also expressed in ovarian follicles. It expressed incytoplasm of oocytes in primordial follicles, and principally localized in thecytoplasm of oocytes, envelope and granulosa cells in primary, secondary and maturefollicles, while mainly expressed in granulosa cells in atresia follicles.4. After cultured8days in vitro, the results of HE showed that plenty ofprimordial follicles in3d postnatal mice ovary were activated and developed into primary follicles, and the results of Western blot and immunohistochemical showedthat PCNA increased significantly (p <0.05), which suggested that the waymouthculture system of mice primordial follicle be estabilished successfully.5. Based on the culture system, we used Western blot and Real-time PCR todetect the expression change of MST, LATS and YAP before and after primordialfollicle activation respectively. All the results showed that: the mRNA and proteinexpression of MST decreased significantly (p<0.05); the mRNA and proteinexpression of LATS decreased significantly (p<0.05); the expression of YAPincreased significantly (p<0.05, p<0.01); the ratio of P-YAP/YAP decreasedsignificantly (p <0.05).Conclusions:1. The core components of Hippo pathway (MST, LATS, YAP) express in miceovary and have spatio-temporal correlation with the size of primordial follicle pool.2. The expression of the core components of Hippo pathway (MST, LATS, YAP)changes during mouse follicular development especially before and after primordialfollicle activation.3. The primordial follicle activation may be related to the significant decrease ofthe ratio of P-YAP/YAP.