NPR2 protein is the primary receptor for C-type natriuretic peptide (CNP), which upon ligand binding exhibits greatly increased guanylyl cyclase activity.////////CNP is likely the Oocyte Maturation Inhibitor (OMI) proposed by Alex Tsafriri. Regulation of the development of meiotic competence and of the resumption of oocyte maturation in the rat. Tsafriri A et al. The first meiotic maturation division of mammalian oocytes is initiated in the embryo or during the early postnatal period. However, when the germ cells reach the diplotene stage the meiotic process is arrested. Meiosis is normally kept in abeyance up to a short period prior to ovulation, when the process is resumed in preovulatory follicles. Resumption of meiosis is studied in mammalian oocytes mainly in two dissimilar in vitro models, isolated oocytes maturing spontaneously in culture and hormone-induced maturation of follicle-enclosed oocytes. A third approach, namely, co-culture of oocytes with follicular constituents was adopted in order to test the role of follicular components in the control of meiosis. Such studies demonstrated an inhibitory action of granulosa cells, granulosa-cell conditioned medium and of follicular fluid (FF1) upon the spontaneous maturation of co-cultured oocytes. By contrast, theca tissue was without effect on meiosis. Addition of luteinizing hormone (LH) to co-cultures of rat granulosa cells and rat oocytes induced resumption of meiosis, as it does in vivo or in vitro in follicle-enclosed oocytes. It is therefore suggested that within antral follicles meiosis is held in abeyance by a granulosa cell product, the inhibitor of oocyte maturation (OMI). Further studies led to the conclusion that OMI is not species specific, that its production by granulosa cells is enhanced by follicle stimulating hormone (FSH) and that its concentration in FF1 is dependent upon the development of the follicle and not the stage of the oestrous cycle. OMI appears to be a peptide of less than 2000 Da. Its action on the oocyte appears to be mediated, at least partially, by cumulus cells and is potentiated by cyclic AMP. Since OMI activity has been demonstrated only in antral follicles, we examined the development of the ability of rat oocytes to undergo spontaneous maturation during their growth phase in preantral follicles. We have found that the ability of rat oocytes to resume maturation ('meiotic competence') is acquired between days 20-26 post partum. By the use of hypophysectomy on day 15 of life and by treatment with hormones and inhibitors we demonstrated that the acquisition of meiotic competence is dependent upon FSH stimulation and that it is mediated, at least partially, by ovarian oestrogen production. The findings that oocytes from preantral follicles are meiotically incompetent suggests that the physiological role of follicular OMI is limited only to antral follicles i.e. when the oocytes acquire meiotic competence.(ABSTRACT TRUNCATED AT 400 WORDS)
Oocyte maturation inhibitor. Tsafriri A et al. The preovulatory surge of gonadotropins induces within the mature Graafian follicle a series of changes culminating in the release of a fertilizable ovum. These include resumption of the meiotic division, a process held in abeyance from a short time after birth, and the progression of the oocyte from the dictyate stage to the metaphase of the second meiotic division. Here the role of a follicular factor, oocyte maturation inhibitor (OMI), in preventing resumption of meiosis by ova of antral follicles prior to the surge of gonadotropins has been reviewed. The suggested involvement of OMI in regulation of meiosis is based on the following observations: (1) fully grown mammalian oocytes explanted from their follicles undergo meiotic maturation spontaneously, whereas follicle-enclosed ova remain immature until stimulated; (2) co-culture of oocytes isolated from their follicles with follicular granulosa cells, granulosa cell extract and follicular fluid inhibits the spontaneous maturation; (3) the inhibition of oocyte maturation by OMI is reversible and in several of the models employed can be removed by the addition of the physiological trigger of meiosis, luteinizing hormone (LH). The current state of OMI characterization and purification has been described and the involvement of additional factors, such as cyclic AMP, in the regulation of meiosis discussed.
Huang et al. (1996) isolated, mapped, and expressed the gene encoding mouse c-type natriuretic peptide. Natriuretic peptides comprise a family of 3 structurally related molecules: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (NPPC). ANP and BNP act mainly as cardiac hormones, produced primarily by the atrium and ventricle, respectively. C-type natriuretic peptide occurs in a wide variety of tissues, where it acts as a local regulator. Natriuretic peptides can influence body fluid homeostasis and blood pressure control through the activation of 2 guanylyl cyclase-coupled natriuretic peptide receptor subtypes--GC-A and GC-B. ///////// Intraovarian control of early folliculogenesis. Hsueh AJ 2014 et al.
Although hormonal regulation of ovarian follicle development has been extensively investigated, most studies concentrate on the development of early antral follicles to the preovulatory stage, leading to the successful use of exogenous FSH for infertility treatment. Accumulating data indicate that preantral follicles are under stringent regulation by FSH and local intra-ovarian factors, thus providing the possibility to develop new therapeutic approaches. Granulosa cell-derived C-type natriuretic factor (CNP) not only suppresses the final maturation of oocytes to undergo germinal vesicle breakdown before ovulation but also promotes preantral and antral follicle growth. In addition, several oocyte-and granulosa cell-derived factors stimulate preantral follicle growth by acting through WNT, receptor tyrosine kinase, receptor serine kinase, and other signaling pathways. In contrast, the ovarian Hippo signaling pathway constrains follicle growth and disruption of Hippo signaling promotes the secretion of downstream CCN growth factors capable of promoting follicle growth. Although the exact hormonal factors involved in primordial follicle activation has yet to be elucidated, the AKT and mTOR signaling pathways are important for the activation of dormant primordial follicles. Hippo signaling disruption following ovarian fragmentation, combined with treating ovarian fragments with PTEN inhibitors and phosphoinositide-3-kinase stimulators to augment AKT signaling, promote the growth of preantral follicles in patients with primary ovarian insufficiency (POI), leading to a new infertility intervention for such patients. Elucidation of intraovarian mechanisms underlying early folliculogenesis may allow the development of novel therapeutic strategies for patients diagnosed with POI, polycystic ovary syndrome (PCOS), and poor ovarian response to FSH stimulation, as well as for infertile women of advanced reproductive age.
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NCBI Summary:
This gene encodes a preproprotein that is proteolytically processed to generate multiple protein products. These products include the cardiac natriuretic peptides CNP-53, CNP-29 and CNP-22, which belong to the natriuretic family of peptides. The encoded peptides exhibit vasorelaxation activity in laboratory animals and elevated levels of CNP-22 have been observed in the plasma of chronic heart failure patients. [provided by RefSeq, Oct 2015]
General function
Ligand, Hormone
Comment
Prophase I Arrest of Mouse Oocytes Mediated by Natriuretic Peptide Precursor C Requires GJA1 (connexin-43) and GJA4 (connexin-37) Gap Junctions in the Antral Follicle and Cumulus-Oocyte Complex. Richard S 2014 et al.
Fully grown germinal vesicle stage mouse oocytes remain arrested in meiotic prophase I until ovulation. This arrest is maintained by cGMP produced in cumulus granulosa cells surrounding the oocyte. Recently, it was found that cGMP production in cumulus cells depends on NPR2 guanylate cyclase activated by its ligand natriuretic peptide precursor C (NPPC). It is assumed that cGMP reaches the oocyte through gap junctions that couple cumulus granulosa cells to each other and to the oocyte. Previous work identified two main types of gap junctions in the follicle, connexin-43 gap junctions (GJA1 protein) between granulosa cells and connexin-37 gap junctions (GJA4) between cumulus cells and the oocyte. However, it had not been established that both types are required for meiotic arrest mediated by NPPC/NPR2 signaling. To investigate this, we used connexin mimetic peptides (CMP) that specifically disrupt gap junction isoforms within cumulus-oocyte complexes (COCs) and isolated antral follicles in culture. We furthermore developed a punctured antral follicle preparation to permit CMP access to the antral cavity in an otherwise intact follicle. CMP directed against connexin-43 (Cx43 CMP) overcame NPPC-mediated meiotic arrest in both isolated COCs and antral follicles. Cx37 CMP, in contrast, had no effect when present in the medium, but released oocyte arrest in the presence of NPPC when microinjected into the perivitelline space near the oocyte surface in COCs. This is consistent with both connexin isoforms being required for meiotic arrest, and with the reported localization of connexin-43 throughout the cumulus cells and connexin-37 at the oocyte surface.
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Cellular localization
Secreted
Comment
Correlation analysis between C natriuretic peptide and pregnancy outcome. Guo L et al. (2020) C-type natriuretic peptide (CNP) plays a key role in female reproduction and is related to oocyte quality. This study analyzed the relationship of CNP with pregnancy outcome to provide a new indicator of pregnancy outcome. Follicular fluids were collected from 158 patients undergoing the IVF/ICSI procedure at the Center for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology. CNP and cGMP levels in human follicular fluids were tested by ELISA. Then, the distribution patterns of CNP and NPR-B from GV oocytes to blastocysts in mice were tested by confocal microcopy. Finally, CNP was added to the fertility or embryo development medium to observe fertility rate and the development of the embryo. CNP levels in follicular fluids from nonpregnant women were significantly higher than those in follicular fluids from pregnant women. A strong positive correlation between CNP and cGMP concentrations in human follicular fluids was observed. Both CNP and NPR-B were expressed in the plasma of cells at different stages from GV to blastocyst. CNP could increase the 2-cell rate of embryos and the rate of blastocysts when added to either fertility culture medium or embryo culture medium. In a word, CNP in human follicular fluid could predict the pregnancy outcome of IVF patients, and the mechanism of CNP in follicular fluid is related to the quality of oocyte or embryo competence and could promote the development of embryos.//////////////////
An improved IVM method for cumulus-oocyte complexes from small follicles in polycystic ovary syndrome patients enhances oocyte competence and embryo yield. Sánchez F et al. (2017) Are meiotic and developmental competence of human oocytes from small (2-8 mm) antral follicles improved by applying an optimized IVM method involving a prematuration step in presence of C-Type Natriuretic Peptide (CNP) followed by a maturation step in presence of FSH and Amphiregulin (AREG)? A strategy involving prematuration culture (PMC) in the presence of CNP followed by IVM using FSH + AREG increases oocyte maturation potential leading to a higher availability of Day 3 embryos and good-quality blastocysts for single embryo transfer. IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for the patients, but the approach is not widely used because of an efficiency gap compared to conventional ART. In vitro systems that enhance synchronization of nuclear and cytoplasmic maturation before the meiotic trigger are crucial to optimize human IVM systems. However, previous PMC attempts have failed in sustaining cumulus-oocyte connections throughout the culture period, which prohibited a normal cumulus-oocyte communication and precluded an adequate response by the cumulus-oocyte complex (COC) to the meiotic trigger. A first prospective study involved sibling oocytes from a group of 15 patients with polycystic ovary syndrome (PCOS) to evaluate effects of a new IVM culture method on oocyte nuclear maturation and their downstream developmental competence. A second prospective study in an additional series of 15 women with polycystic ovaries characterized and fine-tuned the culture conditions. Fifteen women with PCOS (according to Rotterdam criteria) underwent IVM treatment after 3-5 days of highly purified human menopausal gonadotropin (HP-hMG) stimulation and no human chorionic gonadotropin (hCG) trigger before oocyte retrieval. A first study was designed with sibling oocytes to prospectively evaluate the impact of an IVM culture method: 24 h PMC with CNP + 30 h IVM with FSH and AREG, on embryo yield, in comparison to the standard (30 h) IVM clinical protocol (Group I, n = 15). A second prospective study was performed in 15 women with polycystic ovaries, to characterize and optimize the PMC conditions (Group II, n = 15). The latter study involved the evaluation of oocyte meiotic arrest, the preservation of cumulus-oocyte transzonal projections (TZPs), the patterns of oocyte chromatin configuration and cumulus cells apoptosis following the 24 and 46 h PMC. Furthermore, oocyte developmental potential following PMC (24 and 46 h) + IVM was also evaluated. The first 20 good-quality blastocysts from PMC followed by IVM were analysed by next generation sequencing to evaluate their aneuploidy rate. PMC in presence of CNP followed by IVM using FSH and AREG increased the meiotic maturation rate per COC to 70%, which is significantly higher than routine standard IVM (49%; P ≤ 0.001). Hence, with the new system the proportion of COCs yielding transferable Day 3 embryos and good-quality blastocysts increased compared to routine standard IVM (from 23 to 43%; P ≤ 0.001 and from 8 to 18%; P ≤ 0.01, respectively). CNP was able to prevent meiosis resumption for up to 46 h. After PMC, COCs had preserved cumulus-oocyte TZPs. The blastocysts obtained after PMC + IVM did not show increased aneuploidy rates as compared to blastocysts from conventional ART. The novel IVM approach in PCOS patients was tested in oocytes derived from small antral follicles which have an intrinsically low developmental potential. Validation of the system would be required for COCs from different (larger) follicular sizes, which may involve further adjustment of PMC conditions. Furthermore, considering that this is a novel strategy in human IVM treatment, its global efficiency needs to be confirmed in large prospective randomized controlled trials. The further application in infertile patients without PCOS, e.g. cancer patients, remains to be evaluated. The findings of this pilot study suggest that the efficiency gap between IVM and conventional IVF can be reduced by fine-tuning of the culture methods. This novel strategy opens new perspectives for safe and patient-friendly ART in patients with PCOS. IVM research at the Vrije Universiteit Brussel has been supported by grants from: the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680); the Fund for Research Flanders (Fonds Wetenschappelijk Onderzoek-Vlaanderen-FWO, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69). The authors have no conflicts of interest.//////////////////
Live births after oocyte in vitro maturation with a prematuration step in women with polycystic ovary syndrome. Vuong LN et al. (2020) Standard oocyte in vitro maturation (IVM) usually results in lower pregnancy rates than in vitro fertilization (IVF). IVM preceded by a prematuration step improves the acquisition of oocyte developmental competence and can enhance embryo quality (EQ). This study evaluated the effectiveness of a biphasic culture system incorporating prematuration and IVM steps (CAPA-IVM) versus standard IVM in women with polycystic ovarian morphology (PCOM). Eighty women (age < 38 years, ≥ 25 follicles of 2-9 mm in both ovaries, no major uterine abnormalities) were randomized to undergo CAPA-IVM (n = 40) or standard IVM (n = 40). CAPA-IVM uses two steps: a 24-h prematuration step with C-type natriuretic peptide-supplemented medium, then 30 h of culture in IVM media supplemented with follicle-stimulating hormone and amphiregulin. Standard IVM was performed using routine protocols. A significantly higher proportion of oocytes reached metaphase II at 30 h after CAPA-IVM versus standard IVM (63.6 vs 49.0; p < 0.001) and the number of good quality embryos per cumulus-oocyte complex tended to be higher (18.9 vs 12.7; p = 0.11). Clinical pregnancy rate per embryo transfer was 63.2% in the CAPA-IVM versus 38.5% in the standard IVM group (p = 0.04). Live birth rate per embryo transfer was not statistically different between the CAPA-IVM and standard IVM groups (50.0 vs 33.3% p = 0.17]). No malformations were reported and birth weight was similar in the two treatment groups. Use of the CAPA-IVM system significantly improved maturation and clinical pregnancy rates versus standard IVM in patients with PCOM. Furthermore, live births after CAPA-IVM are reported for the first time.//////////////////
Pre-IVM treatment with C-type natriuretic peptide in the presence of cysteamine enhances bovine oocytes antioxidant defense ability and developmental competence in vitro. [Zhenwei J et al. (2019) The use of C-type natriuretic peptide (CNP) combined with cysteamine during pre-in vitro maturation (IVM) can help establish an effective pre-IVM system. This study was designed to investigate the effects of CNP combined with cysteamine on meiotic maturation and developmental competence of bovine oocytes in vitro. Oocytes were cultured in pre-IVM medium supplemented with 100 nM CNP for 6 h in the absence or presence of 100 μM cysteamine, followed by standard IVM for 24 h. Oocytes matured by standard IVM for 24 h (no pre-IVM) were used as the control. The percentage of oocytes arrested at germinal vesicle (GV) stage in each treatment group was examined at 0 h of IVM, and the nuclear maturation rate was assessed by evaluating the percentages of oocytes that reached metaphase II (M II) stage at 24 h of IVM. After 24 h of IVM, oocytes were subjected to in vitro fertilization (IVF) and in vitro embryo culture (IVC). Cleavage rates were assessed 48 h post-insemination. Blastocyst production rates were recorded on day 8 after IVF. In addition, intra-oocyte glutathione (GSH) and reactive oxygen species (ROS) content for each treatment were tested at 0 h and 24 h of IVM. No differences were observed between the CNP pre-IVM treatment and control groups in the rate of bovine oocytes maturing to the M II stage, cleavage and blastocysts production rates after IVF, and intra-oocyte GSH levels. Notably, the presence of cysteamine during pre-IVM culture with CNP significantly improved the rate of embryos developed to the blastocyst stage after in vitro maturation and fertilization, moreover, it increased the levels of GSH and reduced the levels of ROS in bovine oocytes. The improvement to IVM bovine oocyte developmental competence through pre-IVM with CNP combined with cysteamine may be associated with an increased antioxidant defense. Therefore, such an approach may be a good option for establishing a pre-IVM system.//////////////////
C-type natriuretic peptide enhances mouse preantral follicle growth. Xi G et al. (2019) Compared to ovarian antral follicle development, the mechanism underlying preantral follicle growth has not been well documented. Although C-type natriuretic peptide (CNP) involvement in preantral folliculogenesis has been explored, its detailed role has not been fully defined. Here, we used mouse preantral follicles and granulosa cells (GCs) as a model for investigating the dynamic expression of CNP and natriuretic peptide receptor 2 (NPR2) during preantral folliculogenesis, the regulatory role of oocyte-derived growth factors (ODGFs) in natriuretic peptide type C (Nppc) and Npr2 expression, and the effect of CNP on preantral GC viability. Both mRNA and protein levels of Nppc and Npr2 were gradually activated during preantral folliculogenesis. CNP supplementation in culture medium significantly promoted the growth of in vitro cultured preantral follicles and enhanced the viability of cultured GCs in a follicle-stimulating hormone (FSH)-independent manner. Using adult and prepubertal mice as an in vivo model, CNP pre-treatment via intraperitoneal injection before conventional superovulation also had a beneficial effect on promoting the ovulation rate. Furthermore, ODGFs enhanced Nppc and Npr2 expression in the in vitro cultured preantral follicles and GCs. Mechanistic study demonstrated that the regulation of WNT signaling and estrogen synthesis may be implicated in the promoting role of CNP in preantral folliculogenesis. This study not only proves that CNP is a critical regulator of preantral follicle growth, but also provides new insight in understanding the crosstalk between oocytes and somatic cells during early folliculogenesis.//////////////////
Effect of pre-maturation with C-type natriuretic peptide and 3-isobutyl-1-methylxanthine on cumulus-oocyte communication and oocyte developmental competence in cattle. Soto-Heras S et al. (2019) In vitro embryo production depends on oocyte competence, which is acquired during folliculogenesis, involving cytoplasmic and nuclear processes. In vitro maturation (IVM) induces spontaneous resumption of meiosis, preventing full competence acquisition. The incorporation of a pre-IVM phase with supplementation with C-type natriuretic peptide (CNP) and 3-Isobutyl-1-methylxanthine (IBMX) was used with the aim of improving developmental competence of cattle oocytes. In a preliminary experiment, COCs were cultured with increasing CNP concentrations and nuclear stage assessment was performed. Supplementation with both 100 and 200 nM CNP resulted in more germinal vesicle (GV) arrest at 6 h of culture than those in the control group (79.3%, 76.4% and 59.2%, respectively). In a second experiment, use of 100 nM CNP plus 500 μM IBMX resulted in retention of more oocytes in the GV stage (92.0%) at 6 h of culture compared to supplementation with either CNP or IBMX alone (74.8% and 86.7%, respectively). A subsequent assessment of the effect of the pre-IVM system (6-h of culture with CNP plus IBMX), followed by 20-h of IVM, with comparison to the control at 24-h of IVM was performed. Blastocyst development rate was greater after the pre-IVM phase (45.1% compared with 34.5%). The inclusion of the pre-IVM phase also resulted in an enhanced mitochondrial activity in matured oocytes and sustained integrity of transzonal projections for longer after IVM. In conclusion, CNP and IBMX function synergistically to arrest meiosis in cattle oocytes during a pre-IVM phase, which improves cumulus-oocyte communication and embryo development.//////////////////
Effects of pre-incubation with C-type natriuretic peptide on nuclear maturation, mitochondrial behavior, and developmental competence of sheep oocytes. Zhang T et al. (2018) In vitro produced mammalian embryos suffer from developmental failure, with a large proportion showing embryonic retardation, degradation, or apoptosis. This failure is due, in part, to incomplete oocyte cytoplasmic maturation. C-type natriuretic peptide (CNP) has been reported to act as a meiotic inhibitor. Here we explored the potential effects of CNP pre-treatment sheep oocytes on nuclear maturation, changes in mitochondrial behavior and developmental competence of in vitro fertilized embryos. Sheep cumulus-oocyte complexes (COCs) were aspirated from abattoir-derived ovaries. Nuclear progression was assessed using DAPI chromatin staining, the expression of natriuretic peptide receptor 2 (NPR2) was evaluated by RT-qPCR, active mitochondria localization was assessed with a confocal laser scanning microscopy using MitoTracker Red, and the developmental competence of sheep oocytes subjected to one-step IVM or two-step IVM with or without CNP pretreatment was also investigated. Our results showed that 200 nM CNP could effectively maintain meiotic arrest of sheep COCs in vitro within 4 h. Furthermore, NPR2 mRNA was mainly expressed in cumulus cells. COCs pre-treated with 200 nM CNP for 4 h followed by 24 h IVM showed significantly higher (P < 0.05) cleavage rate and blastocyst rate after in vitro fertilization (IVF), and significantly lower (P < 0.05) proportion of DNA-fragmented nuclei in blastocysts when compared to the conventional 24 h IVM (standard IVM). Non-matured oocytes mainly displayed brilliant circumferential and fine diffuse distribution of mitochondria throughout the cytoplasm. By comparison, 200 nM CNP pre-treated COCs for 4 h led to cytoplasmic mitochondrial granule localization to the peripheral and perinuclear regions. Moreover, oocytes pre-treated with 200 nM CNP for 4 h followed by 24 h IVM, showed mitochondrial organization were similar to those of conventional 24 h matured oocytes in which mitochondria were aggregated more toward the cortical regions of the oocytes, but with larger clumps of stained mitochondria. These results indicate that CNP pre-treatment improves the quality and developmental competence of sheep oocytes and has great potential for facilitating in vitro embryo production.//////////////////
Steroid hormones interact with natriuretic peptide C to delay nuclear maturation, to maintain oocyte?cumulus communication and to improve the quality of in vitro-produced embryos in cattle. Soares AC et al. (2017) In vivo, oocyte maturation is triggered by the ovulatory LH surge, whereas in vitro it is precociously induced when the cumulus-oocyte complex is removed from the follicle. Natriuretic peptide C (NPPC) delays germinal vesicle breakdown (GVBD) while increasing oocyte-cumulus communication during in vitro maturation (IVM) in cattle. In the present study we first tested the hypothesis that steroids secreted by the follicle (17β-oestradiol, progesterone and androstenedione) interact with NPPC to delay GVBD and to maintain oocyte-cumulus communication as assessed by transfer of a dye (Lucifer Yellow) from the oocyte to cumulus cells. Then, we assessed the effects of steroid hormones and NPPC, alone and in combination in a pre-IVM culture, on embryo production. The combination of NPPC with steroids delayed GVDB, increased natriuretic peptide receptor 2 (NPR2) mRNA abundance in cumulus cells during culture, and maintained oocyte-cumulus communication at levels not different from non-cultured controls. The addition of steroids and/or NPPC to a pre-IVM culture did not alter blastocyst rates after IVF, but supplementation with steroids increased blastocyst total cell number. The present study provides evidence, for the first time in cattle, that steroids interact with NPPC to regulate oocyte nuclear maturation and oocyte-cumulus communication, and improve oocyte developmental competence.//////////////////
C-Type Natriuretic Peptide Stimulates Ovarian Follicle Development. Sato Y et al. C-type natriuretic peptide (CNP) encoded by the NPPC (Natriuretic Peptide Precursor C) gene expressed in ovarian granulosa cells inhibits oocyte maturation by activating the natriuretic peptide receptor (NPR)B (NPRB) in cumulus cells. RT-PCR analyses indicated increased NPPC and NPRB expression during ovarian development and follicle growth, associated with increases in ovarian CNP peptides in mice. In cultured somatic cells from infantile ovaries and granulosa cells from prepubertal animals, treatment with 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, similar to FSH, increased ovarian weight gain that was associated with the development of primary and early secondary follicles to the late secondary stage. Of interest, treatment with FSH increased levels of NPPC, but not NPRB, transcripts in ovarian explants. In vivo studies further indicated that daily injections of infantile mice with CNP for 4 d promoted ovarian growth, allowing successful ovulation induction by gonadotropins. In prepubertal mice, CNP treatment alone also promoted early antral follicle growth to the preovulatory stage, leading to efficient ovulation induction by LH/human chorionic gonadotropin. Mature oocytes retrieved after CNP treatment could be fertilized in vitro and developed into blastocysts, allowing the delivery of viable offspring. Thus, CNP secreted by growing follicles is capable of stimulating preantral and antral follicle growth. In place of FSH, CNP treatment could provide an alternative therapy for female infertility.///////
Immature Oocytes from Unprimed Juvenile Mice Become a Valuable Source for Embryo Production When Using C-Type Natriuretic Peptide as Essential Component of Culture Medium. Romero S et al. (2016) C-type natriuretic peptide (CNP) and its receptor Natriuretic peptide receptor 2 (NPR2) play a paramount role in the maintenance of oocyte meiotic arrest in antral follicles via the regulation of the intra-oocyte levels of cyclic guanosine monophosphate and cyclic adenosine monophosphate. We investigated the potential of CNP 1) to maintain oocyte meiotic arrest during a prolonged prematuration culture, and 2) to sustain acquisition of developmental competence of immature cumulus-oocyte complexes (COCs). Compact COCs were collected from small antral follicles of pre-pubertal unprimed mice and placed in prematuration culture under different CNP-supplemented media conditions. A preliminary analysis showed a dose-dependent effect of CNP on the maintenance of meiotic arrest. A dose of 25nM maintained oocytes under meiotic arrest for 24 h, and this period was extended to 48 h in presence of Estradiol. Analysis of transzonal projections of COCs cultured with CNP indicated that oocyte-cumulus connections were well preserved after the prolonged prematuration culture. Furthermore, CNP medium supplemented with FSH and GDF9 promoted oocyte growth and induced a shift in oocyte chromatin configuration from a predominant disperse- to a condensed configuration. Following IVM, oocytes cultured under CNP were capable to extrude the first polar body at a high rate (around 80%). Blastocyst formation was significantly improved when oocytes were cultured under CNP-supplemented medium containing FSH and GDF9. This study reports for the first time a prolonged prematuration culture system, having CNP as pivotal factor, which can efficiently maintain oocytes retrieved from unprimed prepubertal mice under meiotic arrest while promoting their acquisition of developmental competence.//////////////////
C-type natriuretic peptide improved vitrified-warmed mouse cumulus oocyte complexes developmental competence. Yang L et al. (2016) Cryopreservation of cumulus oocyte complexes (COCs) is important for reproductive medicine. However, the vitrified-warmed COCs have lower maturation rate and subsequent developmental competence compared with fresh COCs. The present study was aimed to evaluate the effects of supplementation of the maturation medium with C-type natriuretic peptide (CNP) on the developmental competence of vitrified-warmed mouse COCs. Addition of CNP to the maturation medium improved the maturation rate and enhanced the developmental competence of vitrified-warmed mouse COCs. The reason may be that vitrified COCs led to a decline in cyclic guanosine monophosphate (cGMP) levels. Furthermore, addition of CNP to the maturation medium elevated cGMP levels of the vitrified-warmed COCs. In conclusion, cryopreservation-associated lower maturation rate and developmental competence of COCs may be ameliorated by CNP during maturation culture after warming.//////////////////
Effect of C-Type Natriuretic Peptide on Maturation and Developmental Competence of Goat Oocytes Matured In Vitro. Zhang J et al. (2015) The developmental competence of oocytes during in vitro maturation (IVM) is compromised due to asynchronous nuclear and cytoplasmic maturation. To improve IVM efficiency, a pre-maturation culture or two-step maturation strategy has been established, involving meiosis arrest induced by pharmacological agents to provide oocytes with sufficient time to synchronize the maturation of the nucleus and cytoplasm. C-type natriuretic peptide (CNP), which has been demonstrated to function as an oocyte maturation inhibitor (OMI) in many species, provides a new alternative to improve the developmental capacity of oocytes matured in vitro. However, the effect of CNP on meiosis arrest and the maturation of goat oocytes remains unclear. In the present study, CNP was shown to function as an OMI in goat oocytes. CNP could temporarily maintain the meiotic arrest of goat oocytes cultured in vitro for 4 hours. This transient effect was partly due to the reduction of natriuretic peptide receptor 2 (Npr2). Estradiol could delay the decrease in Npr2 expression and prolong the duration of meiosis arrest up to 6 hours. Based on the above results, a two-step method was established for goat oocyte maturation, in which the oocyte maturation rate was significantly increased. After parthenogenetic activation, the cleavage rate, blastocyst rate and total cell number of blastocysts were significantly improved. Our results suggested that CNP can be used to delay meiotic resumption and enhance the developmental competence of goat oocytes matured in vitro.//////////////////
/Natriuretic Peptide Precursor C Delays Meiotic Resumption and Sustains Gap Junction Mediated Communication in Bovine Cumulus Enclosed Oocytes. Franciosi F 2014 et al.
Oocyte in vitro maturation (IVM) has become a valuable technology for animal breeding and cloning and the treatment of human infertility, since it does not require the administration of exogenous gonadotropin to obtain fertilizable oocytes. However, embryo development after IVM is lower compared to in vivo maturation, most likely because oocytes collected for IVM are heterogeneous with respect to their developmental competencies. Attempts to improve IVM outcome have relied upon either pre-maturation culture (PMC) or two-step maturation strategies in the hope of normalizing variations in developmental competence. Such culture systems invoke the pharmacological arrest of meiosis, in theory providing oocytes sufficient time to complete the acquisition of developmental competence after cumulus-enclosed oocytes isolation from the follicle. The present study was designed to test the efficiency of natriuretic peptide precursor C (NPPC) as a non-pharmacologic meiosis-arresting agent during IVM in a mono-ovulatory species. That NPPC maintains meiotic arrest in vivo and in vitro has been shown in mice and pigs, however, the use of this molecule for PMC has yet to have been explored. Towards this end, meiotic cell cycle re-entry, gap-junction functionality and chromatin configuration changes were investigated in bovine cumulus enclosed oocytes cultured in presence of NPPC. Moreover, oocyte developmental competence was investigated after IVM, IVF and embryo culture and compared to standard IVM-IVF protocol without PMC. Our results suggest that NPPC can be used to delay meiotic resumption and increase the developmental competence of bovine oocytes when used in PMC protocols.
/////////////////////////C-Type natriuretic peptide maintains domestic cat oocytes in meiotic arrest. Zhong Y et al. (2015) Recent studies have shown that C-type natriuretic peptide (CNP; encoded by the natriuretic peptide C (NPPC) gene) plays an essential role in maintaining meiotic arrest of mouse and porcine oocytes. However, whether CNP inhibits feline meiotic resumption is not known. In the present study we used a domestic cat model to explore the role played by CNP in feline oocyte meiotic resumption. We determined mRNA expression of genes encoding CNP and its cognate receptor natriuretic peptide receptor 2 (NPR2) in antral follicles. NPPC mRNA was primarily expressed in mural granulosa cells, whereas NPR2 mRNA was predominantly expressed in cumulus cells. Following in vitro culture for 24h, 100nM CNP increased cGMP levels, and maintained meiotic arrest of oocytes associated with cumulus cells. When the duration of in vitro culture increased from 24h to 36h, the ability of CNP to maintain meiotic arrest decreased, and this was accompanied by a decrease in the steady state levels of NPR2 mRNA in cumulus cells. In addition, CNP decreased the rate of degeneration of oocytes. These results indicate that CNP is required to maintain meiotic arrest and prevent degeneration in domestic cat oocytes.//////////////////
Porcine natriuretic peptide type B (pNPPB) maintains mouse oocyte meiotic arrest via natriuretic peptide receptor 2 (NPR2) in cumulus cells. Zhang Y 2014 et al.
In mouse ovarian follicles, the oocyte is maintained in meiotic prophase arrest by natriuretic peptide type C (NPPC) acting via its cognate receptor, natriuretic peptide receptor 2 (NPR2). As there is a marked species difference in the receptor selectivity of the natriuretic peptide family, this study examined the functional effect of other natriuretic peptides, type A (NPPA) and type B (NPPB), acting via NPR2 on mouse-oocyte meiotic arrest.. The results by quantitative, reverse-transcriptase PCR showed that Npr2 was the predominant natriuretic peptide receptor transcript, and that Npr1 and Npr3 mRNA levels were negligible in cumulus cells isolated from equine chorionic gonadotropin-primed, immature female mice. While NPPA and NPPB from human and rat had no effect on oocyte maturation, porcine NPPB (pNPPB) maintained oocyte meiotic arrest in a dose-dependent manner. Furthermore, pNPPB-mediated meiotic arrest and cGMP production could be completely blocked by the NPR2 inhibitor sphingosine-1-phosphate (S1P). Neither the NPR1 antagonist anantin or Npr1 knockout, however, had an effect on pNPPB-mediated meiotic arrest. Thus, pNPPB has the functional effect of maintaining mouse-oocyte meiotic arrest by the receptor NPR2 of cumulus cells. These findings demonstrate that pNPPB may be used as a probe to identify the essential amino acid sequences for activation of NPR2. Mol. Reprod. Dev. ? 2014 Wiley Periodicals, Inc.
/////////////////////////Effect of C-type natriuretic peptide on maturation and developmental competence of immature mouse oocytes in vitro. Wei Q et al. (2015) In vitro maturation (IVM) of oocytes is an important assisted reproductive technology for infertility treatment and livestock breeding programs. Because of asynchronous nuclear and cytoplasmic maturation, the developmental competence of oocytes matured in vitro is compromised. C-Type natriuretic peptide (CNP), which has been proved to be an inhibitor of oocyte maturation, provides a new alternative to optimise synchronisation of nuclear and cytoplasmic maturation and improve developmental capacity of immature oocytes in vitro. To investigate the effect of temporary meiotic arrest mediated by CNP on maturation and subsequent development of immature oocytes, immature mouse oocytes from small antral follicles were temporarily arrested in meiosis by CNP (0, 5, 10 and 50 nM) for 24 h and then matured for 16 h. CNP treatment significantly increased the oocyte maturation rate from less than half to above 80%. After IVF, temporary meiotic arrest mediated by 10 and 50 nM CNP significantly improved fertilisation and blastocyst rate of oocytes matured in vitro up to approximately 55% and 30% respectively. Moreover, this positive effect of CNP was attributed, in part, to an increase in the number of mature oocytes with aligned chromosomes and a normal spindle. The present findings indicate the potential to use CNP to improve the efficiency of oocyte IVM.//////////////////
CNP/NPR2 signaling maintains oocyte meiotic arrest in early antral follicles and is suppressed by EGFR-mediated signaling in preovulatory follicles. Tsuji T et al. Oocyte meiosis is arrested at prophase I by factors secreted from surrounding somatic cells after oocytes acquire meiotic competence at an early antral stage, and meiosis resumes in preovulatory follicles as a result of the luteinizing hormone (LH) surge. Recently, signaling by C-type natriuretic peptide (CNP) through its receptor, natriuretic peptide receptor 2 (NPR2), was found to be essential for meiotic arrest at the late antral stage. Whether or not CNP/NPR2 signaling maintains oocyte meiotic arrest in earlier follicular stages and how it is associated with meiotic resumption induced by the LH surge is unclear. In this study, we examined the expression of Nppc and Npr2, respectively encoding CNP and NPR2, in the ovaries of immature mice. Nppc and Npr2 mRNA were specifically expressed in the outer and inner granulosa cell layers, respectively, in early antral follicles. Histological analysis of mice with a mutation in Npr2 revealed precocious resumption of oocyte meiosis in early antral follicles. Ovaries of mice treated with excess human chorionic gonadotropin (hCG) exhibited markedly decreased Nppc mRNA levels in granulosa cells of preovulatory follicles. Moreover, we found that amphiregulin, a mediator of LH/hCG activity through epidermal growth factor receptor (EGFR), suppressed Nppc mRNA levels in cultured granulosa cells. These results suggest that CNP/NPR2 signaling is essential for oocyte meiotic arrest in early antral follicles and that activated LH/amphiregulin/EGFR signaling pathway suppresses this signal by downregulating Nppc expression. Mol. Reprod. Dev. ? 2012 Wiley Periodicals, Inc.
Since C-type natriuretic peptide (CNP) and the guanylyl cyclase receptors A and B (GC-A and GC-B) showed coordinate estrous cycle-dependent variation with maximal expression at proestrus, it was concluded that the natriuretic peptides may play an important role in either the development of ovulatory follicles or in the ovulatory process (Jankowski et al., 1997) . Furthermore, Gutkowska et al.(1999) studied that the hormonal regulation of natriuretic peptide system during induced ovarian follicular development showed a coordinated hormonal stimulation of the NPs and their receptors in the rat ovary. CNP in the ovary is most likely due to local synthesis since CNP is not detected or circulates in very low concentrations. Together with the presence of guanylyl cyclase B receptor subtypes in the ovary and the responsiveness to hormonal regulation, CNP and GC-B receptors emerge as specific regulators of ovarian functions.
CNP has been found in, and could be released from, endothelial cells (Stingo et al., 1992). Thus, ovarian CNP could be involved in the alteration of cellular permeability that leads to antrum formation, which is essentially a buildup of fluid along with accumulation of albumin in the follicles that are destined for ovulation (Gutkowska et al., 1999).
NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation during follicular development in the mouse ovary. Kiyosu C et al. Natriuretic peptide type C (NPPC) and its high affinity receptor, natriuretic peptide receptor 2 (NPR2), have been assumed to be involved in female reproduction, and have recently been shown to play an essential role in maintaining meiotic arrest of oocytes. However, the overall role of NPPC/NPR2 signaling in female reproduction and ovarian function is still less clear. Here we report the defects observed in oocytes and follicles of mice homozygous for Nppclbab or Npr2cn, mutant alleles of Nppc or Npr2, respectively, to clarify the exact consequences of a lack of NPPC/NPR2 signaling in female reproductive systems. We found that (1) Npr2cn/Npr2cn female mice ovulated a comparable number of oocytes as normal mice but never produced a litter; (2) all ovulated oocytes of Npr2cn/Npr2cn and Nppclbab/Nppclbab mice exhibited abnormalities, such as fragmented or degenerated ooplasm and never developed to the two-cell stage after fertilization; (3) histological examination of the ovaries of Npr2cn/Npr2cn and Nppclbab/Nppclbab mice showed that oocytes in antral follicles prematurely resumed meiosis and that immediately before ovulation, oocytes showed disorganized chromosomes or fragmented ooplasm; and (4) ovulated oocytes and oocytes in the periovulatory follicles of the mutant mice were devoid of cumulus cells. These findings demonstrate that NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation, which affects female fertility through the production of oocytes with developmental capacity.
C-type natriuretic peptide inhibits porcine oocyte meiotic resumption. Hiradate Y et al. Summary C-type natriuretic peptide (CNP) is a recently identified meiotic inhibitor in mice. However, it has not been investigated in porcine oocytes to date. This study aimed to demonstrate the inhibitory effect of CNP against germinal vesicle breakdown (GVBD) in porcine oocyte meiotic resumption. Immunohistochemical analysis revealed intense natriuretic peptide receptor 2 (NPR2) immunoreactivity in the oocyte surrounded cumulus cells in the follicles. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) analysis showed the expression of npr2 mRNA only in cumulus cells but not in oocytes, suggesting that cumulus cells are the targets of CNP. When cumulus-oocyte complexes (COCs) or denuded oocytes (DOs) were cultured with various concentrations of CNP (10, 50, 100, 500, and 1,000 nM), inhibitory effect was observed in the COC group, but not in the DO group, confirming that CNP indirectly inhibits GVBD via cumulus cells. This evidence is the first indication that the CNP-NPR2 pathway is involved in meiotic arrest in porcine oocytes. Furthermore, we investigated the effect of oocyte-derived paracrine factor (ODPF) on npr2 mRNA expression level in cumulus cells by evaluating changes in mRNA expression in oocytectomised COCs (OXCs) by real-time PCR. A significant decrease in npr2 mRNA expression level was observed in OXCs, whereas mRNA expression level was restored in OXCs with DOs, indicating that ODPF participates in the regulation of npr2 expression in porcine cumulus cells.///////
The mRNA-destabilizing protein Tristetraprolin targets "meiosis arrester" Nppc mRNA in mammalian preovulatory follicles. Xi G et al. (2021) C-natriuretic peptide (CNP) and its receptor guanylyl cyclase, natriuretic peptide receptor 2 (NPR2), are key regulators of cyclic guanosine monophosphate (cGMP) homeostasis. The CNP-NPR2-cGMP signaling cascade plays an important role in the progression of oocyte meiosis, which is essential for fertility in female mammals. In preovulatory ovarian follicles, the luteinizing hormone (LH)-induced decrease in CNP and its encoding messenger RNA (mRNA) natriuretic peptide precursor C (Nppc) are a prerequisite for oocyte meiotic resumption. However, it has never been determined how LH decreases CNP/Nppc In the present study, we identified that tristetraprolin (TTP), also known as zinc finger protein 36 (ZFP36), a ubiquitously expressed mRNA-destabilizing protein, is the critical mechanism that underlies the LH-induced decrease in Nppc mRNA. Zfp36 mRNA was transiently up-regulated in mural granulosa cells (MGCs) in response to the LH surge. Loss- and gain-of-function analyses indicated that TTP is required for Nppc mRNA degradation in preovulatory MGCs by targeting the rare noncanonical AU-rich element harbored in the Nppc 3' UTR. Moreover, MGC-specific knockout of Zfp36, as well as lentivirus-mediated knockdown in vivo, impaired the LH/hCG-induced Nppc mRNA decline and oocyte meiotic resumption. Furthermore, we found that LH/hCG activates Zfp36/TTP expression through the EGFR-ERK1/2-dependent pathway. Our findings reveal a functional role of TTP-induced mRNA degradation, a global posttranscriptional regulation mechanism, in orchestrating the progression of oocyte meiosis. We also provided a mechanism for understanding CNP-dependent cGMP homeostasis in diverse cellular processes.////////////////// Global analysis of FSH-regulated gene expression and histone modification in mouse granulosa cells. Madogwe E et al. (2020) Follicle-stimulating hormone (FSH) regulates ovarian follicular development through a specific gene expression program. We analyzed FSH-regulated transcriptome and histone modification in granulosa cells during follicular development. We used super-stimulated immature mice and collected granulosa cells before and 48 h after stimulation with equine chorionic gonadotropin (eCG). We profiled the transcriptome using RNA-sequencing (N = 3/time-point) and genome-wide trimethylation of lysine 4 of histone H3 (H3K4me3; an active transcription marker) using chromatin immunoprecipitation and sequencing (ChIP-Seq; N = 2/time-point). Across the mouse genome, 14,583 genes had an associated H3K4me3 peak and 63-66% of these peaks were observed within ≤1 kb promoter region. There were 72 genes with differential H3K4me3 modification at 48 h eCG (absolute log fold change > 1; false discovery rate FDR] < 0.05) relative to 0 h eCG. Transcriptome data analysis showed 1463 differentially expressed genes at 48 h eCG (absolute log fold change > 1; FDR < 0.05). Among the 20 genes with differential expression and altered H3K4me3 modification, Lhcgr had higher H3K4me3 abundance and expression, while Nrip2 had lower H3K4me3 abundance and expression. Using ChIP-qPCR, we showed that FSH-regulated expression of Lhcgr, Cyp19a1, Nppc, and Nrip2 through regulation of H3K4me3 at their respective promoters. Transcript isoform analysis using Kallisto-Sleuth tool revealed 875 differentially expressed transcripts at 48 h eCG (b > 1; FDR < 0.05). Pathway analysis of RNA-seq data demonstrated that TGF-β signaling and steroidogenic pathways were regulated at 48 h eCG. Thus, FSH regulates gene expression in granulosa cells through multiple mechanisms namely altered H3K4me3 modification and inducing specific transcripts. These data form the basis for further studies investigating how these specific mechanisms regulate granulosa cell functions.////////////////// Transforming growth factor-β is involved in maintaining oocyte meiotic arrest by promoting natriuretic peptide type C expression in mouse granulosa cells. [Yang J et al. (2019) Natriuretic peptide type C (NPPC) secreted by mural granulosa cells (MGCs) maintains oocyte meiotic arrest via the activation of guanylyl cyclase-linked natriuretic peptide receptor 2 (NPR2). Here, we investigated the effect of transforming growth factor (TGF)-β on NPPC expression in MGCs and oocyte maturation. TGF-β ligands (TGFB1 and TGFB3, but not TGFB2) and receptors (TGFBR1 and TGFBR2) were predominantly expressed in MGCs. The activation of the follicle-stimulating hormone (FSH) receptor by FSH/equine chorionic gonadotropin (eCG) increased the levels of TGFB1, TGFBR2, and TGF-β downstream SMAD proteins in MGCs, which were decreased following the activation of the luteinizing hormone (LH) receptor by human chorionic gonadotropin (hCG). TGF-β significantly increased the gene and protein levels of NPPC in cultured MGCs through SMAD3 binding to Nppc promoter regions. In the presence of FSH, TGF-β further increased NPPC levels and inhibited oocyte meiotic resumption of cumulus-oocyte complexes (COCs). Moreover, Tgfbr2-specific depletion in granulosa cells using Fshr-Cre mice reduced NPPC mRNA and protein levels, resulting in the weak maintenance of oocyte meiotic arrest within large antral follicles. Tgfbr2 depletion also impaired follicle development, ovulation, and female fertility. Taken together, TGF-β-promoted NPPC in MGCs is involved in maintaining oocyte meiotic arrest. FSH and LH could regulate NPPC levels in MGCs via TGF-β and then control the process of oocyte meiosis.//////////////////Impacts of macrophage colony-stimulating factor (M-CSF) on the expression of natriuretic peptide precursor type C (NPPC) and regulation of meiotic resumption. Liu C et al. (2019) In mammalian follicles, oocytes are arrested at the diplotene stage of prophase I until meiotic resumption following the LH surge. C-type natriuretic peptide (CNP), encoded by natriuretic peptide precursor type C (NPPC), was found to be reduced by the LH surge in the follicle, and then lead to meiotic resumption by decreasing the level of cAMP in the oocyte. As a wide-spread cytokine, macrophage colony-stimulating factor (M-CSF) takes part in the oocyte development to maturation and ovulation. Our study describes the expression curve of M-CSF and its receptor and investigates the impact on the levels of CNP/NPPC to explore the possible mechanism for meiotic resumption in both vivo and vitro. The result shows after the LH/HCG surge, the expressions of M-CSF and its receptors decline significantly inside ovarian follicles, thus leading to transduction of a range of signals. Consequently, the expression of CNP reaches the peak at 2 h and immediately declines to a relatively low level.//////////////////
KITL regulates meiosis and expression of NPPC in bovine cumulus-oocyte complexes. de Lima PF et al. (2016) In vitro maturation (IVM) of oocytes in cattle is inefficient, and there is great interest in the development of approaches to improve maturation and fertilization rates. Intraovarian signaling molecules are being explored as potential additives to IVM media. One such factor is kit ligand (KITL), which stimulates the growth of oocytes. We determined if KITL enhances oocyte maturation in cattle. The two main isoforms of KITL (KITL1 and KITL2) were expressed in bovine cumulus-oocyte complexes (COC), and levels of mRNA increased during FSH-stimulated IVM. The addition of KITL to the culture medium increased the percentage of oocytes that reached meiosis II but did not affect cumulus expansion after 22 hours of IVM. Addition of KITL reduced levels of mRNA encoding natriuretic peptide precursor C (NPPC), a protein that holds oocytes in meiotic arrest, and increased levels of mRNA encoding YBX2, an oocyte-specific factor involved in meiosis. Removal of the oocyte from the COC resulted in increased KITL mRNA levels and decreased NPPC mRNA levels in cumulus cells, and addition of denuded oocytes reversed these effects. Taken together, our results suggest that KITL enhances bovine oocyte nuclear maturation through a mechanism that involves NPPC, and that the oocyte regulates cumulus expression of KITL mRNA.//////////////////
MAPK3/1 is conducive to LH-mediated CNP decrease in bovine granulosa cells. Yang L et al. (2015) C-type natriuretic peptide (CNP) plays a role as an oocyte maturation inhibitor (OMI) in many species, including the bovine. However, the effects of luteinizing hormone (LH) on CNP expression and its potential mechanisms have not reported in the bovine. In the present study, we aimed to study the effects of LH on CNP expression and to illuminate the potential molecular mechanism in this process. Our results showed that LH induced epidermal growth factor receptor (EGFR) phosphorylation, mitogen-activated protein kinase3/1 (MAPK3/1) activation and CNP mRNA decrease in cultured bovine granulosa cells. Further study revealed that LH suppressed CNP expression via the MAPK3/1 signaling pathway, which was activated by the EGFR pathway. In conclusion, our research suggested that MAPK3/1 is involved in LH-mediated decrease of CNP and that this process is related to the EGFR and MAPK3/1 signal pathways.//////////////////
Identification and gene expression analyses of natriuretic peptide system in the ovary of goat (Capra hircus). Peng JY et al. (2013) Natriuretic peptides (NPs) are involved in maintaining cardiovascular and fluid homeostasis, regulating reproductive processes and bone growth, and other numerous functions. To better understand the role of NPs in goat (Capra hircus), in the present study, full-length cDNAs of goat Nppa (natriuretic peptide precursor A), Nppb (natriuretic peptide precursor B) and Nppc (natriuretic peptide precursor C), respectively encoding ANP, BNP and CNP, were cloned from adult goat heart and ovary. The putative prepropeptide ANP (prepro-ANP) and prepro-CNP share a high amino acid sequence identity with other species. Real-time PCR showed that Nppa, Nppb and Nppc were widely expressed in adult goat tissues. The mRNA expression of Nppa and Nppb in the heart was extremely higher compared with other tissues. Nppc mRNA expression in the lung and uterus was also higher than in other tissues. The expression of Nppa, Nppb and Nppc genes was examined at different ovarian follicle stages using RT-PCR. The mRNAs of Nppa and Nppb were detected in secondary follicles as well as in COCs (cumulus-oocyte-complexes) and granulosa cells of antral follicles. However, the mRNA expression of Nppc was observed throughout ovarian follicle development, and it was especially higher in granulosa cells of antral follicles. In vitro, stimulating goat granulosa cells with FSH led to an increase in the expression of Nppc by dose- and time-dependent manners and a rapid decline was induced by LH stimulation, but the expression of Nppa and Nppb did not change after FSH or LH treatment. These results suggest that Nppc is a gonadotropin-induced gene in granulosa cells of goat ovary and CNP may be involved in the regulation of ovarian follicle development and oocyte maturation.//////////////////
Dephosphorylation and inactivation of NPR2 guanylyl cyclase in granulosa cells contributes to the LH-induced decrease in cGMP that causes resumption of meiosis in rat oocytes. Egbert JR 2014 et al.
In mammals, the meiotic cell cycle of oocytes starts during embryogenesis and then pauses. Much later, in preparation for fertilization, oocytes within preovulatory follicles resume meiosis in response to luteinizing hormone (LH). Before LH stimulation, the arrest is maintained by diffusion of cyclic (c)GMP into the oocyte from the surrounding granulosa cells, where it is produced by the guanylyl cyclase natriuretic peptide receptor 2 (NPR2). LH rapidly reduces the production of cGMP, but how this occurs is unknown. Here, using rat follicles, we show that within 10 min, LH signaling causes dephosphorylation and inactivation of NPR2 through a process that requires the activity of phosphoprotein phosphatase (PPP)-family members. The rapid dephosphorylation of NPR2 is accompanied by a rapid phosphorylation of the cGMP phosphodiesterase PDE5, an enzyme whose activity is increased upon phosphorylation. Later, levels of the NPR2 agonist C-type natriuretic peptide decrease in the follicle, and these sequential events contribute to the decrease in cGMP that causes meiosis to resume in the oocyte.
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Multiple Pathways Mediate Luteinizing Hormone Regulation of cGMP Signaling in the Mouse Ovarian Follicle. Liu X 2014 et al.
LH regulation of the EGF network is critical for oocyte maturation and the ovulatory process. Recent studies have indicated that C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR2) play an important role in the control of meiotic arrest. Here, we investigated the involvement of the EGF network in the LH-dependent regulation of the CNP/NPR2 axis and cGMP accumulation. LH/hCG treatment causes a major decrease in both cGMP and the CNP precursor (natriuretic peptide precursor C, Nppc) mRNA accumulation in vivo and in vitro. However, the cGMP downregulation precedes the decrease in Nppc mRNA by more than 1h. Amphiregulin, an EGF-like factor, suppresses Nppc mRNA levels in cultured follicles to the same extent as LH, and this effect is completely prevented by the EGFR kinase inhibitor AG1478. However, the LH-dependent suppression of Nppc is insensitive to AG1478. Similarly, Nppc suppression by LH occurs in follicles from EGFR null mice. These findings document that EGFR signaling is sufficient to downregulate CNP but is not necessary for LH action. When cGMP concentration in the follicle is measured, the short- but not long-term LH effects on cGMP are prevented by AG1478, suggesting that ligand availability may be responsible for the late response. hCG decreases the CNP-dependent cGMP synthesis in wild type and EGFR knockdown cumulus-oocyte complexes. These findings demonstrate that redundant pathways are involved in the regulation of cGMP. EGFR-dependent events are involved in the short-term regulation of cGMP, whereas the long-term effects may involve regulation of the CNP.
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Identification and gene expression analyses of natriuretic peptide system in the ovary of goat (Capra hircus). Peng JY 2013 et al.
Natriuretic peptides (NPs) are involved in maintaining cardiovascular and fluid homeostasis, regulating reproductive processes and bone growth, and other numerous functions. To better understand the role of NPs in goat (Capra hircus), in the present study, full-length cDNAs of goat Nppa (natriuretic peptide precursor A), Nppb (natriuretic peptide precursor B) and Nppc (natriuretic peptide precursor C), respectively encoding ANP, BNP and CNP, were cloned from adult goat heart and ovary. The putative prepropeptide ANP (prepro-ANP) and prepro-CNP share a high amino acid sequence identity with other species. Real-time PCR showed that Nppa, Nppb and Nppc were widely expressed in adult goat tissues. The mRNA expression of Nppa and Nppb in the heart was extremely higher compared with other tissues. Nppc mRNA expression in the lung and uterus was also higher than in other tissues. The expression of Nppa, Nppb and Nppc genes was examined at different ovarian follicle stages using RT-PCR. The mRNAs of Nppa and Nppb were detected in secondary follicles as well as in COCs (cumulus-oocyte-complexes) and granulosa cells of antral follicles. However, the mRNA expression of Nppc was observed throughout ovarian follicle development, and it was especially higher in granulosa cells of antral follicles. In vitro, stimulating goat granulosa cells with FSH led to an increase in the expression of Nppc by dose- and time-dependent manners and a rapid decline was induced by LH stimulation, but the expression of Nppa and Nppb did not change after FSH or LH treatment. These results suggest that Nppc is a gonadotropin-induced gene in granulosa cells of goat ovary and CNP may be involved in the regulation of ovarian follicle development and oocyte maturation.
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Hormonal Coordination of Natriuretic Peptide Type C and Natriuretic Peptide Receptor 3 Expression in Mouse Granulosa Cells. Lee KB et al. Natriuretic peptide type C (NPPC) and its receptor natriuretic peptide receptor 2 (NPR2) regulate cGMP in ovarian follicles and participate in maintaining oocyte meiotic arrest. We investigated the regulation of Nppc expression in mouse granulosa cells in vivo and in vitro. In mural granulosa cells (MGCs) in vivo, eCG caused an increase in Nppc mRNA and subsequent hCG-treatment caused a decrease. A culture system was established for MGCs isolated from follicles not stimulated with eCG to further define mechanisms controlling Nppc expression. In this system, expression of Nppc mRNA was increased by E2, with augmentation by FSH, but FSH or LH alone had no effect. Thus estrogens are important for regulating Nppc expression, probably by feedback mechanisms enhancing the action of gonadotropins. In MGCs treated with E2 plus FSH in vitro, subsequent treatment with EGF, but not LH, decreased Nppc mRNA. MGCs express higher levels of both Nppc and Lhcgr mRNAs than cumulus cells. Oocyte-derived paracrine factors suppressed cumulus cell Lhcgr but not Nppc expression. Thus, higher Nppc expression by MGCs is not the result of oocyte suppression of expression in cumulus cells. Another possible regulator of the LH-induced NPPC decrease is NPR3, an NPPC clearance receptor. hCG increased Npr3 expression in vivo and LH increased Npr3 mRNA in cultured MGCs, independently of EGF-receptor activation. Interestingly, despite the increase in Npr3 mRNA, the hCG-induced decrease in ovarian NPPC occurred normally in an Npr3 mutant (lgj), thus NPR3 probably does not participate in regulation of ovarian NPPC levels or oocyte development.
Pre-ovulatory LH/hCG surge decreases C-type natriuretic peptide secretion by ovarian granulosa cells to promote meiotic resumption of pre-ovulatory oocytes. Kawamura K et al. BACKGROUND In mammalian follicles, oocytes are arrested at the diplotene stage of prophase I until meiotic resumption following the LH surge. Recently, C-type natriuretic peptide (CNP), encoded by natriuretic peptide precursor type C (NPPC) was found to suppress mouse oocyte maturation by promoting cyclic guanosine 5'-monophospate (cGMP) production in cumulus cells. However, regulation of NPPC/CNP expression during the pre-ovulatory period and their regulation by the LH surge have not been investigated. METHODS AND RESULTS Based on genome-wide analysis of DNA microarray data sets using samples from periovulatory ovaries, we found increases in NPPC transcripts in granulosa cells during pre-ovulatory follicle growth in mice and a rapid decline induced by the pre-ovulatory LH/hCG stimulation. Treatment of pre-ovulatory animals with hCG decreased ovarian CNP content. In isolated ovarian cells, NPPC mRNA was predominantly expressed in mural granulosa cells exhibiting similar regulation following gonadotrophin treatment. In cultured mouse pre-ovulatory follicles, meiosis resumption in oocytes by hCG treatment was accompanied by decreases in NPPC transcript levels. In cultured mouse cumulus cell-oocyte complexes, CNP treatment inhibited the resumption of meiosis with increases in cGMP levels in both cumulus cells and oocytes. In human ovaries, CNP levels in ovarian follicular fluid were also decreased following treatment of patients with an ovulatory dose of hCG. CONCLUSIONS Our findings demonstrate gonadotrophins regulation of NPPC/CNP expression in mouse and human ovaries and confirm the role of CNP as a potent paracrine oocyte maturation inhibitor.
Gutkowska et. al. (1999) studied the hormonal regulation of the NP system during follicular growth and ovulation induced by gonadotropins eCG and hCG in the rat. After treatment, the expression level of both receptors (GC-A and GC-B) increased, and ovarian ANP and C-type natriuretic peptide (CNP) gene expression was also activated.
Ovarian localization
Cumulus, Granulosa
Comment
C-type natriuretic peptide signaling in human follicular environment and its relation with oocyte maturation. Casalechi M et al. (2019) Studies in mice have shown that C-type natriuretic peptide (CNP) is produced by granulosa cells and contributes to ovarian follicle growth and oocyte meiotic arrest until the preovulatory LH surge. In humans, the relationship between intraovarian CNP levels and oocyte meiotic resumption is unknown. The aim of this study was to investigate whether CNP and its receptor NPR2 are expressed in human ovarian follicles and if their levels change according to the meiotic phase of oocytes. We collected follicular fluid (FF) and luteinized granulosa cells (LGC) from follicle pools (n = 47), and FF, LGC and cumulus cells (CC) from individual follicles (n = 96) during oocyte pickup for in vitro fertilization. There was a positive linear correlation between CNP levels in FF pools and basal antral follicle counting (rs = 0.458; p = 0.002), number of preovulatory follicles >16 mm (rs = 0.361; p = 0.016) and number of oocytes retrieved (rs = 0,378; p = 0.011) and a negative correlation between CNP levels in FF pools and the percentage of mature (MII) oocytes retrieved (rs = -0.39; p = 0.033). FF CNP levels in follicles containing MII oocytes were significantly lower than in follicles containing immature (MI) oocytes (median = 0.44 vs. 0.57 ng/ml, p < 0.05). Accordingly, the CNP precursor gene NPPC was 50% less expressed in LGC from follicles containing MII oocytes than in follicles containing MI oocytes (p < 0.01). In addition, NPR2 mRNA was down-regulated in CC surrounding MII oocytes (60% reduction, p < 0.01). CNP signaling is downregulated in human ovarian follicles containing mature oocytes. Further studies should clarify whether CNP signaling is essential to keep oocyte meiotic arrest in humans.//////////////////
Follicle stages
Secondary, Antral, Preovulatory
Comment
CNP has been found in, and could be released from, endothelial cells (Stingo et al., 1992). Thus, ovarian CNP could be involved in the alteration of cellular permeability that leads to antrum formation, which is essentially a buildup of fluid along with accumulation of albumin in the follicles that are destined for ovulation (Gutkowska et al., 1999).
Phenotypes
Mutations
2 mutations
Species: mouse
Mutation name: None
type: null mutation fertility: subfertile Comment: Granulosa cell ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes. Zhang M et al. Granulosa cells of mammalian Graafian follicles maintain oocytes in meiotic arrest, which prevents their precocious maturation. We show that mouse mural granulosa cells, which line the follicle wall, express natriuretic peptide precursor type C (Nppc) messenger RNA (mRNA), whereas cumulus cells surrounding oocytes express mRNA of the NPPC receptor NPR2, a guanylyl cyclase. NPPC increased cGMP levels in cumulus cells and oocytes and inhibited meiotic resumption in vitro. Meiotic arrest was not sustained in most Graafian follicles of Nppc or Npr2 mutant mice, and meiosis resumed precociously. Oocyte-derived paracrine factors promoted cumulus cell expression of Npr2 mRNA. Therefore, the granulosa cell ligand NPPC and its receptor NPR2 in cumulus cells prevent precocious meiotic maturation, which is critical for maturation and ovulation synchrony and for normal female fertility.
Species: mouse
Mutation name: None
type: null mutation fertility: None Comment: Dwarfism and early death in mice lacking C-type natriuretic peptide. Chusho H et al. Longitudinal bone growth is determined by endochondral ossification that occurs as chondrocytes in the cartilaginous growth plate undergo proliferation, hypertrophy, cell death, and osteoblastic replacement. The natriuretic peptide family consists of three structurally related endogenous ligands, atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP), and is thought to be involved in a variety of homeostatic processes. To investigate the physiological significance of CNP in vivo, we generated mice with targeted disruption of CNP (Nppc(-/-) mice). The Nppc(-/-) mice show severe dwarfism as a result of impaired endochondral ossification. They are all viable perinatally, but less than half can survive during postnatal development. The skeletal phenotypes are histologically similar to those seen in patients with achondroplasia, the most common genetic form of human dwarfism. Targeted expression of CNP in the growth plate chondrocytes can rescue the skeletal defect of Nppc(-/-) mice and allow their prolonged survival. This study demonstrates that CNP acts locally as a positive regulator of endochondral ossification in vivo and suggests its pathophysiological and therapeutic implication in some forms of skeletal dysplasia.