Essential Role Of C-type Natriuretic Peptide(CNP)in The Development Of Mouse Preantral Follicle

The ovarian follicle is a unique structure that consists of an oocyte,as well as surrounding granulosa cells and theca cells.Follicles are the function unit for supporting oocyte growth.Based on their morphology and structure,ovarian follicles,from primordial to mature follicles,can be categorized into preantral and antral follicles.Unlike the well-documented role of FSH and LH in regulating the development of ovarian antral follicles,the mechanism underlying preantral follicular growth is largely elusive.Pre-experiments indicated that C-type natriuretic peptide(CNP)may be implicated in the growth of preantral follicles,and the expression of Nppc,the gene encoding precursor of CNP,was gradually activated during development of preantral follicles.Based on these observations,we hypothesize that CNP may plays important role in the preantral folliculogenesis.The test this,following experiments were contracted:(1)The expression patterns of Nppc and Npr2 in preantral and antral follicles were detected,and we found that a significant increase in expression levels of Nppc and Npr2,both which are located in granulosa cells,during development from early to late preantral follicles.(2)Using in vitro cultured preantral follicles as model,we detected the effect of CNP at different concentrations(0.1,1,10,100,500nM),and found in the medium without FSH supplementation,CNP treatment alone over lOnM prompt preantral follicle growth in a dose-dependent manner.(3)To further confirm the prompting-effect of CNP on preantral follicle growth,we tested the effect of different concentration of CNP(0nM,0.1 nM,lOnM,100nM,and 500nM)on the proliferation of in vitro culture granulosa cells(GCs)isolated from the preantral follicles.Under this culture condition supplemented a low level FBS,showed that CNP treatment at 10nM,100nM and 500nM,significant increased proliferation of preantral GCs.(4)Next,by pretreating adult(8 weeks of age)and infantile(13 day of age)mice with intra-peritoneal CNP injection,we detect the function of CNP to stimulate preantral follicle development in vivo.Results showed that CNP promote the development of preantral follicles to the early antral stage,and thus significantly enhancing the outcome of superovulation in both adult and infantile mice.(5)To understand the mechanism responsible for the regulation of Nppc and Npr2 expression in preantral follicles,we tested the effects of oocyte-derived growth factors(ODGFs),e.g.,BMP15,GDF9,and FGF8,on the Nppc and Npr2 expression in cultured preantral follicles and GCs,and found both Nppc and Npr2 expression was synergistically regulated by ODGFs.(6)To further elucidate molecular mechanisms underlying the prompting-effect of CNP on the development of preantral follicles,selected a set of key ovarian genes that are important for the growth of preantral follicle or granulosa cell and steroidogenesis,and detected the effect of CNP treatment on the expression of these genes in cultured preantral follicles,and results demonstrated that CNP probably promoted preantral follicular development through facilitating the Wnt/?-catenin signaling and enhancing the estrogen synthesis.This study,not only present that CNP is a critical regulator in the preantral follicle growth,but also provides a new insight in understanding the crosstalk between oocytes and somatic cells during folliculogenesis.