Oocyte development takes place in ovarian follicles. During folliculogenesis, oocytes grow and reach metaphase of meiosis II (MII). To be developmentally competent, it is very important that the cytoplasm matures at the same time when oocyte’s meiosis resumed. This include accumulation of proteins and energy substrates, organelles reallocation and changes in the structure of chromatin etc. If the meiosis resumed precociously before cytoplasmic maturation, the developmental competence of oocyte, e.g. the ability to be fertilized and the ability to develop to blastocyst, will be negatively affected.
The importance of cytoplasmic maturation was confirmed by a test reported by Cheng et al. When the cytoplast at GV stage was used as recipient and karyoplast at metaphase in meiosis I (MI) or MII stage as donor, the constructed oocyte extruded a polar body after electrofusion and culture. While both the cytoplasm and the polar body had a metaphase spindle in the MI-GV pair, only a clutch of condensed chromatin was observed in the cytoplasm and polar body of the MII-GV pair. When MI cytoplast was used as recipient and GV or MII karyoplast as donor, the reconstructed oocyte also extruded a polar body. Each had one spindle and a group of metaphase chromosomes in the cytoplasm and polar body, respectively. When MII cytoplast was used as recipient and GV or MI karyoplast as donor, the reconstructed oocytes were activated, became parthenogenetic embryos and even developed to hatching blastocysts after electrofusion. Immunoblotting showed that mitogen-activated protein kinase (MAPK) activity was high in MI and MII cytoplasts but not detected in GV cytoplast. The results demonstrated that the cytoplsmic environment determines the behavior of asynchronous donors .
In ovary, follicular cells communicate via paracrine and juxtacrine mechanism. CNP is encoded by natriuretic peptide precursor C (Nppc) gene expressed mainly in mural granulosa cells. It stimulates natriuretic peptide receptor B (NPRB) on the membrane of cumulus cells to produce cyclic guanosine monophosphate (cGMP). cGMP of cumulus origin diffuses into oocytes to suppress phosphadiesterase 3 (PDE3) activity, leading to elevation of cyclic adenosine 3’,5’-monophosphate (cAMP) in oocyte. cAMP binds to protein kinase A (PKA) which in turn activates WEE 1 homolog 2 (WEE1B) and myelin transcription factor 1 (MYT1) kinase. WEE1B and MYT1 are known to block cyclin-dependent kinase 1 (CDK1). Thus, cAMP-dependent activation of PKA results in CDK1 inhibition leading to meiotic arrest in oocyte [2,3,4]. This effect of CNP was confirmed in mouse[5,6], procine ,bovine [8,], cat , goat  and sheep  etc.
In most Graffian follicles of Nppc or Npr2 mutant mice, meiotic arrest was not sustained and meiosis resumed precociously. In in vitro condition, the physiological condition including the cAMP level within oocyte is changed. This will probably result in spontaneous resumption of meiosis. As the maturation of cytoplasm does not catch up with that of nuclear, the developmental competence of such oocytes might decrease. Zhang et al reported that approxiamately 50%-70% of fertilized oocytes cultured in vitro fail to develop into preimplantation embryos[12,13].
To solve this issue, CNP was supplemented in the in vitro culture system to retard nuclear maturation, which in turn allows time for ooplasmic maturation to catch up and results in better synchronization of nuclear and cytoplasmic compartments [3,6,7,8,9,10,11,13]. But, it was reported that although CNP alone could enhance the concentration of cGMP during in vitro culture of COC from porcine antral follicle, follicle stimulating hormone (FSH) suppressed the effect if it was added simultaneously with CNP. However, if CNP was added into culture medium 1 h before FSH supplementation, the high level of cGMP induced by pretreatment of CNP could be maintained for up to 8-12 hours. As the oocytes for in vitro culture were isolated from follicles close to ovulation, if the pretreatment of CNP lasted longer, the oocytes cultured in vitro demonstrated a significant decrease in meiotic arrest . So, to prevent premature meiotic resumption, some scholars pretreated COC from antral follicle with CNP for only several hours before conventional in vitro maturation (IVM) [11,12,13]. This protocol was called two-step in vitro culture method.
Besides maintaining meiotic arrest, CNP could also stimulate ovarian follicle growth and increase ovarian follicle viability. In mice, Nppc and Npr2 begin expression in early preantral follicles and increase during early to late preantral follicle development. In cultured somatic cells from infantile ovaries and granulosa cells from prepubertal animals, CNP stimulated cGMP production. Also, treatment of cultured preantral follicles with CNP stimulated follicle growth, whereas treatment of cultured ovarian explants from infantile mice with CNP promoted the development of primary and early secondary follicles to the late secondary stage. In vivo studies indicated that in infantile mice, daily injection of CNP for 4 d promoted ovarian growth and the follicles ovulated after ovulation induction. In prepubertal mice, CNP treatment alone also promoted early antral follicle growth to the preovulatory stage resulting in efficient ovulation by gonadotropin. Mature oocytes retrieved after CNP treatment could be fertilized in vitro and developed into blastocysts. After embryo transfer (ET), viable offspring was delivered [15,16]. It was reported that CNP increased the expression of paracrine or autocrine factors such as Wingless-type mouse mammary tumor integration site family 2b (Wnt2b), Wnt5a, cytochrome P 450 11 a 1 (Cyp11a1) and repressed the expression of estrogen metabolic enzymes cytochrome P 1a1 (Cyp1a1) resulting in follicle growth . This beneficial effect of CNP is more prominent in small follicles compared with large follicles in in vitro culture .
As FSH receptor was expressed in preantral follicle onwards, the development of preantral ovarian follicles were jointly regulated by CNP and FSH physiologically. To mimic the in vivo condition, the present study cultured murine late secondary follicles with supplementation of CNP and FSH during the whole period of IVG. Follicle development, oocyte’s maturity and fertilization competency were evaluated. The objective is to investigate if supplementation of CNP in combination with FSH during the whole IVG period could stimulate preantral follicle growth and at the same time effectively maintain oocyte meiotic arrest.