The Role Of Natriuretic Peptide System In Porcine Oocyte Meiotic Arrest

In mammals,the maturation of oocyte is an important prerequisite for subsequent fertilization and embryo development.Oocytes in antral follicles are held under meiotic arrest for a prolonged period at the diplotene stage of prophase I.This constraint is released at the preovulatory stage.At this time,a surge of luteinizing hormone(LH)secreted by the pituitary gland triggers the resumption of meiosis.Maintenance of meiotic arrest at the germinal vesicle(GV)stage is associated with sustained high levels of adenosine-3’,5’-monophosphate(cAMP).The adenylyl cyclase(ADCY)in oocyte can constitutively generate cAMP levels sufficient to sustain meiotic arrest.In vivo,the catalytic activity of an oocyte-specific phosphodiesterase(phosphodiesterase 3A,PDE3A)increases following the LH surge.This enzyme degrades oocyte cAMP,thereby indirectly activating cell cycle-promoting proteins to initiate meiotic resumption.Recently,cyclic guanosine monophosphate(cGMP)produced in granulosa cells and transferred via gap junctions from granulosa-associated cumulus cells to oocytes has been shown to inhibit PDE3A activity,thus preventing the decrease intraoocyte cAMP levels and inhibiting development of germinal vesicle breakdown(GVBD).In response to LH stimulation,the levels of cGMP in follicle-enclosed oocytes decrease to levels that stimulate PDE3A activity,thus relieving the inhibitory effect of cAMP on PDE3 A hydrolysis.In a previous study,we showed that natriuretic peptide plays an important role in cGMP production in mice.To further explore whether the mechanism of natriuretic peptide system on oocyte maturation found in mouse is versatile or different in mammals,we used porcine oocyte as a model in our present study.We firstly explored the location and expression levels of natriuretic peptides and their receptors in the porcine ovary by measuring levels of the mRNAs encoding these proteins.The mRNAs of natriuretic peptides were predominantly expressed in granulosa cells.mRNA encoding NPR1 appeared to be expressed only in thecal interna cells.mRNA encoding NPR2 was expressed in both granulosa cells(cumulus cells and mural granulosa cells)and thecal cells.Our immunohistochemical results further confirmed the localization of natriuretic peptides in follicle.Based on abovementioned results,we tested the ability of these three peptides in porcine oocyte maturation in vitro.Both BNP and CNP significantly inhibited GVBD of porcine COCs while ANP had no such inhibitory effcet.In vitro,the cGMP levels of oocytes and cumulus cells were closely associated with the proportions of oocytes demonstrating nuclear maturation.The CNP-or BNP-activated NPR2 sustained the cGMP concentration at a high level.We further showed that porcine BNP has a high affinity for porcine NPR2.This indicated that BNP contributed to maintaining porcine meiotic arrest by activating NPR2.Since mammal oocyte maturation is under the regulation of various hormones,we further explored whether hormones regulate natriuretic peptides in meiotic resumption.FSH and E2 increased the expression levels of NPR2 while LH decreased the expression via EGFR network.Meanwhile,EGFR network mediated the function of LH in the regulation of BNP and CNP.To further explore the physiological role played by natriuretic peptides in porcine oocyte maturation,we obtained ovaries from triple-crossbred prepubertal pigs.All oocytes remained in meiotic arrest after injection of PMSG By 18 h after the administration of hCG,some oocytes showed nuclear maturation and the proportion of meiotically mature oocytes increased from 18 to 36 h after hCG injection.This process was closely associated with the cGMP levels of follicle cells.cGMP synthesis initially increased after PMSG administration and next decreased to a level lower than that of control ovaries.This change was paralleled by a change in expression level of the NPR2 gene.Furthermore,the expression of both BNP and CNP increased(at both the mRNA and protein levels)after PMSG treatment and decreased following hCG injection.In summary,we used both in vitro and in vivo porcine models to show that BNP and CNP maintained oocyte meiotic arrest by binding to the NPR2 receptor.LH decreased natriuretic peptide system expression levels to induce meiosis resumption.Our results further elucidate the role of natriuretic peptide system in mammal oocyte maturation and provide a better understanding to improve the quality of cultured oocytes during IVM/IVF practice.