Journal of

Brain Natriuretic Peptide and C-Type Natriuretic Peptide Maintain Porcine Oocyte Meiotic Arrest

Cellular Physiology

WENQIANG ZHANG, YE YANG, WEI LIU, QIAN CHEN, HUARONG WANG, XIAO WANG, YANHAO ZHANG, MEIJIA ZHANG, AND GUOLIANG XIA* State Key Laboratory of Agro-biotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China Recent studies have shown that C-type natriuretic peptide (CNP) serves as a key control system during mouse oocyte maturation. We used pig models (in vitro and in vivo) to explore the role played by the natriuretic peptide family in porcine oocyte maturation. We reported the expression and location of natriuretic peptide system in different stages of porcine antral follicles. Atrial natriuretic peptide (ANP) and CNP were expressed primarily in granulosa cells, whereas brain natriuretic peptide (BNP) and natriuretic peptide receptor-B (NPRB) receptor were expressed in granulosa cells (both cumulus and mural granulosa cells) and thecal internal cells, and the natriuretic peptide receptor-A (NPRA) receptor predominantly in thecal cells. Upon in vitro culture, BNP and CNP maintained meiotic arrest of oocytes associated with cumulus cells. The expression levels of BNP, CNP, and the NPRB receptor increased upon treatment of prepubertal gilts with pregnant mare's serum gonadotropin and decreased upon subsequent human chorionic gonadotropin injection. Such dynamic changes in the expression of natriuretic peptides and their receptor paralleled the proportions of oocytes exhibiting nuclear maturation in vivo. These data indicated that BNP and CNP co-contributed to maintaining porcine meiotic arrest under physiological condition and lutenizing hormone (LH) relieved this inhibitory effect by decreasing the expression levels of BNP and CNP in vivo. Our present work, combined with previous data, improved the understanding of the oocyte meiotic arrest mechanisms and further revealed that natriuretic peptides serve as oocyte maturation inhibitor (OMI) to inhibit oocyte maturation in mammals. J. Cell. Physiol. 230: 71–81, 2015. © 2014 Wiley Periodicals, Inc.

In mammals, 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. Germinal vesicle breakdown (GVBD) is the first change observed as meiosis recommences. This obvious morphological feature is commonly used to assess the resumption of meiosis (Pincus and Enzmann, 1935). Maintenance of meiotic arrest at the germinal vesicle (GV) stage is associated with sustained high levels of adenosine-30 ,50 monophosphate (cAMP), which may diffuse from somatic cells into oocytes (Cho et al., 1974; Magnusson and Hillensjo, 1977; Eppig, 1989). However, in mouse, knockout of the adenylyl cyclase (ADCY)-encoding gene in oocytes, the expression of which is controlled by constitutive action of the G-proteincoupled receptors GPR3 and GPR12 via the intermediacy of Gs proteins, causes precocious GVBD in vivo. This indicates that oocytes can constitutively generate cAMP levels sufficient to sustain meiotic arrest (Conti et al., 1998; Horner et al., 2003; Mehlmann et al., 2004; Hinckley et al., 2005; Vaccari et al., 2008). In vitro, oocyte cAMP levels decrease after oocytes exit follicles, and in vivo, the catalytic activity of an oocyte-specific phosphodiesterase (phosphodiesterase 3 A, PDE3A) increases following the LH surge. This enzyme degrades oocyte cAMP, thereby indirectly activating cell cycle-promoting proteins to initiate meiotic resumption. However, activation of Gs proteins by GPR3 is not affected by enzyme action, indicating that somatic cells contribute to the maintenance of elevated oocyte cAMP levels. 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, thus preventing the decrease in oocyte cAMP levels and inhibiting development of GVBD. In response to LH stimulation, the levels of cGMP in follicle-enclosed oocytes © 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .

decreases to levels that stimulate PDE3A activity, thus relieving the inhibitory effect of cAMP on PDE3A hydrolysis (LaPolt et al., 2003; Norris et al., 2009, 2010; Vaccari et al., 2009). In a previous study, we showed that natriuretic peptide system plays an important role in cGMP production (Zhang et al., 2010, 2011; Zhang and Xia, 2012). The natriuretic peptide forms a family of three structurally related polypeptide hormones: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and c-type natriuretic peptide (CNP). ANP and BNP activate the same transmembrane guanylyl cyclase (natriuretic peptide receptor-A [NPRA]), whereas CNP activates the related cyclase natriuretic peptide receptor-B (NPRB) (Potter et al., 2009). Recent studies have shown that CNP and NPRB are expressed in mouse ovaries and the expression of which is The authors have no conflict of interest to declare. Contract grant sponsor: National Basic Research Program of China; Contract grant numbers: 2014CB138503, 2013CB945500, 2012CB944701. Contract grant sponsor: Chinese Universities Scientific Fund; Contract grant number: 2013YJ002. [Correction added on 7 October 2014, after first online publication 29 September 2014: The citation year has been corrected to 2015.] *Correspondence to: Guoliang Xia, State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, People's Republic of China. E-mail: [email protected] Manuscript Received: 6 March 2014 Manuscript Accepted: 21 May 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 9 June 2014. DOI: 10.1002/jcp.24682




regulated by gonadotropins. Injection of immature mice with pregnant mare's serum gonadotropin (PMSG) increases the expression levels of mRNAs encoding CNP and NPRB, and subsequent injection with human chorionic gonadotropin (hCG) reduces those levels. More importantly, resumption of precocious meiosis is evident in Graafian (late antral) follicles from ovaries lacking CNP or NPRB expression, and CNP sustains inhibition of the spontaneous maturation of cumulus cell-enclosed oocytes in vitro (Zhang et al., 2010; Kawamura et al., 2011; Tsuji et al., 2012). Previous studies found that cumulus cell–oocyte complexes (COCs) from a variety of mammalian species, cultured without hormonal stimulation after removal from ovarian follicles, spontaneously resume meiosis (Pincus and Enzmann, 1935; Edwards, 1965). The material inhibiting meiosis in porcine follicular fluid is likely a peptide of molecular weight less than 2,000 Da, and not species-specific in terms of inhibition of oocyte maturation; the action thereof is reversed by the addition of LH (Tsafriri and Channing, 1975a,b; Leibfried and First, 1980; Tsafriri et al., 1976, 1982). These results, together with evidence that the 22amino-acid forms of CNP are identical in human, dog, pig, horse (Rose and Giles, 2008; Potter et al., 2009), suggest that the material is probably CNP. Indeed, the CNP level in human ovarian follicular fluid decreases after treatment with a dose of hCG sufficient to induce ovulation (Kawamura et al., 2011). Therefore, CNP addition may enhance the maturation of cultured oocytes during clinical in vitro maturation/in vitro fertilization (IVM/IVF). An interesting question arises: Do CNP and the CNP receptor play such a role in all mammals, particularly large mammals including humans? Human oocyte maturation is known to be affected by the CNP level in follicular fluid, but no evidence has supported the role for CNP in this context. This may be because collecting adequate amounts of human oocyte-derived materials in vitro is difficult. In the present study, we showed that in vitro both BNP and CNP, acting with their cognate receptor NPRB, stimulated the production of sufficient cGMP to maintain meiotic arrest. Furthermore, we used in vivo pig models to demonstrate that BNP, CNP, and NPRB expression levels reflected the nuclear status when pigs were injected with PMSG and later with hCG. Materials and Methods Animals and ovaries Ovaries obtained from 6- to 12-month-old gilts at a local slaughterhouse were transported to our laboratory in phosphatebuffered saline supplemented with 100 U/ml penicillin (Huabei Medical, Jizhou, China) and 50 mg/ml streptomycin sulfate (Sigma, St. Louis, MO) at 37°C, and cells used in vitro were derived from these tissues. For in vivo work, triple crossbred prepubertal pigs (male Landrace  female Large Yorkshire, followed by mating with a male Duroc) were reared on Anyang Farm. Forty prepubertal gilts aged 130–150 days were allocated to five treatment groups (Morbeck et al., 1992; Soede et al., 2011): control (n ¼ 8) (slaughtered without treatment); group P48 (slaughtered 48 h after intravenous injection of PMSG from ear vein; 1,000 IU); group P72 (slaughtered 72 h after treatment with PMSG); group h18 (slaughtered 90 h after PMSG treatment and injection of 500 IU hCG at 72 h); and group h36 (slaughtered 108 h after PMSG treatment and injection of 500 IU hCG at 72 h) (Torner et al., 1998). Porcine cumulus–oocyte complex (COC) culture and assessment of nuclear maturation Porcine ovaries were collected from prepubertal gilts slaughtered at a local abattoir and delivered to our laboratory within 1.5–2 h of slaughter in sterile saline maintained at 35–38°C. COCs were aspirated from healthy follicles 3–6 mm in diameter, as described JOURNAL OF CELLULAR PHYSIOLOGY

previously (Tsafriri and Channing, 1975a). Groups of 60 COCs or denuded oocytes (DOs) were transferred into individual wells of 24-well culture dishes (Nunclon; Nunc, Roskilde, Denmark) containing 0.5-ml amounts of TCM199 medium supplemented with 0.23 mmol/l sodium pyruvate, 2 mmol/l glutamine, 3 mg/ml lyophilized crystalline BSA, 100 IU/ml penicillin G, and 25 mg/ml streptomycin sulfate. When COCs were cultured in FSH-induced maturation medium, all culture groups were supplied with 0.05 U/ ml FSH. Gonadotropin was not included in the medium when COCs were cultured using the spontaneous maturation model. After culture, oocytes were harvested, fixed in acetic acid/ethanol (1/3 v/v) for 48 h, and stained with 1% (w/v) orcein prior to phasecontrast microscopic examination (200) for evaluation of chromatin configuration. Immunohistochemistry Follicles were fixed in Bouin's solution at room temperature overnight. After fixation, the follicles were dehydrated in alcohol baths, cleared in xylene, and embedded in paraffin. Each process mentioned above for small antral follicles (1–3 mm) and large antral follicles (3–6 mm) lasted for 4 and 8 h, respectively. The slides were dried for at least 48 h at 42°C, and then deparaffined with xylene and rehydrated in graded ethanol before being washed in water. Sections (8 mm) were heated (three times for 5 min) in sodiumcitrate buffer (0.01 M, pH 6.0) in a microwave oven to inhibit antigenicity. To block endogenous peroxidase activity, the sections were incubated for 20 min in 0.5% hydrogen peroxide. Nonspecific staining was blocked with 10% normal goat serum (for natriuretic peptides) or 15% normal goat serum (for NPRA). Finally, the sections were incubated at 4°C overnight with polyclonal antibodies against ANP (1:500, Phoenix Pharmaceuticals, Inc., Burlingame, CA; catalog #H-005-06), BNP (1:300, #H-011-08), CNP (1:600, #H-012-03), or monoclonal antibody against NPRA (1:200, Thermo Scientific, Waltham, MA; catalog #PA5-15390), followed by biotinylated goat anti-rabbit IgG (1:500) or goat antimouse IgG (1:200) and streptavidin–horseradish peroxidase complex (ABC/HRP; 1:200). The color reaction was developed in TBS buffer pH 7.4 containing 0.01% H2O2, 0.05% diaminobenzidine, and 0.1% imidazole. The primary antibodies were replaced with normal rabbit serum, or 5 mg/ml mouse IgG for control sections. After washing with distilled water, sections were counterstained with hematoxylin. Subsequently, sections were washed with tap water, acetic acid and redistilled water, dehydrated in ethanol and toluene and mounted in DPX. In each independent experiment, we used porcine follicles got from one same ovary for IHC and ISH detection. Ovaries from different pigs are used in three independent experiments. In situ hybridization To generate probes, DNA regions containing sequences unique to various genes were amplified using primers (Table 1) and subcloned into the pGEM-T Easy Vector (Promega, Madison, WI). Complementary antisense or sense cRNA probes were prepared using DIG RNA Labeling Kits (Roche Diagnostics, Indianapolis, MN). Follicles were embedded in optimal cutting temperature (OCT) compound and frozen at –80°C prior to analysis. Frozen sections (15 mm) were prepared using a CM 1950 cryostat microtome (Leica, Wetzlar, Germany) and mounted on SuperFrost Plus slides (Thermo Scientific). Sections were pre-hybridized at 55°C with hybridization buffer (50% formamide, 5  SSC, 5  Denhardts, 1.5% [w/v] polyvinylpyrrolidone, 200 mg/ml yeast tRNA [Roche], and 500 mg/ml herring sperm DNA [Roche]) for 5 h in a hybridization oven with 5  SSC. After pre-hybridization, sections were hybridized with a hybridization mixture for 18 h. The hybridization mixture was prepared by adding 400 ng/ml DIG-cRNAs to hybridization buffer, followed by heating for 10 min at 95°C to denature probes and chilled on ice. Hybridization was



TABLE 1. Primers used for ISH and quantitative real-time PCR Gene ISH ANP BNP CNP NPRA NPRB QRT-PCR BNP CNP NPRB GAPDH


PCR cycles

Annealing temp (°C)

Product length (bp)






























performed overnight at 55°C in a 5  SSC hybridization oven. Slides were then washed in 5  SSC at 55°C for 30 min, 2  SSC at 55°C for 30 min, 1  SSC at 55°C for 30 min, 0.2  SSC at 55°C for 15 min and at room temperature in 0.2  SSC for 15 min. Immunological detection was performed as described previously (Wandji et al., 2000). Eukaryotic expression and binding assays with intact cells Total RNA was isolated from porcine granulosa cells and fulllength NPRB cDNA was prepared using polymerase chain reaction (PCR). NPRB-specific primers containing restriction sites (to allow subsequent cloning) were as follows: forward, 50 -CTTAAGATGGCACTGCCATCACTCCTG-30 and reverse, 50 -TCTAGATTATAGGAGGCCAGCAGGTCCT-30 . Purified cDNA fragments were ligated into the pMD18-T vector (Takara, Kyoto, Japan). The recombinant plasmid was appropriately digested and the cloned DNA purified and inserted into the eukaryotic expression vector pcDNA3.1 (þ) (Invitrogen, Carlsbad, CA). COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 100 IU/ml penicillin G, 100 mg/ml streptomycin, and 10% (v/v) fetal bovine serum. After 24 h, cells were transfected with the recombinant plasmid pcDNA3.1-NPRBCDS using the Lipo2000 protocol (Invitrogen). Binding assays with COS-7 cells mentioned above were performed in 24-well plates. Cells (106) were incubated at 4°C for 2 h with 125I-pBNP-32 (Phoenix Biotech, Burlingame, CA; BNP-32, porcine, T-011-08) and various concentrations of unlabeled peptides in 200-ml Hank's Balanced Salt Solution containing 10 mg/ml pepstatin. After incubation, cells were washed five times in Hank's Balanced Salt Solution and then solubilized with 1 ml of 0.5 N NaOH for detection. RNA extraction and quantitative real-time PCR Total RNA was isolated using TRIzol (Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. Quantification and quality analysis of total RNA isolated from the above samples were determined using Nanodrop (Thermo Im Heiligen Feld 17, Germany). Total RNA (1 mg) from each sample was incubated for 20 min at 25°C with 0.5 U DNase I (Invitrogen) before reverse transcription to eliminate genomic DNA contamination. Firststrand cDNA was created by RT (Promega Reverse Transcription System) from 1 mg of total RNA. Reverse transcription proceeded for 1 h at 42°C. DNA was amplified by an initial incubation at 94°C for 5 min followed by 24– 33 cycles of denaturation at 94°C for 30 sec, annealing at different temperatures showed in Table 1 for 30 sec, and extension at 72°C JOURNAL OF CELLULAR PHYSIOLOGY

for 30 sec, and a final extension at 72°C for 5 min. b-Actin was used as the internal standard. Amplified products were sequenced (Invitrogen, Beijing, China) to confirm specificity. Quantitative real-time PCR was performed in 96-well plates (Applied Biosystems, Foster City, CA) in reaction volumes of 25 ml containing 12.5-ml SYBR Green PCR Master Mix (Applied Biosystems), 15-ng cDNA, appropriate primers, and nuclease-free water. PCR was performed on an ABI 7500 Sequence Detection System (Applied Biosystems) using the following parameters: 10 min at 95°C followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. The primer pairs used are shown in Table 1. All test data were normalized to levels of GAPDH transcription. A melt curve was generated after PCR at temperature increments of 0.5°C every 2 cycles (62 cycles total) starting at 65°C, with fluorescence acquisition after each step. The relative level of target gene expression in each sample was calculated using the 2–DDCt formula, as described previously (Livak and Schmittgen, 2001). Radioimmunoassay (RIA) for assay of BNP-32, and CNP-22 in porcine follicular fluid and plasma Pig follicular fluid (FF) was centrifuged at 10,000g at 4°C for 15 min and supernatants were collected in centrifuge tubes containing aprotinin (Phoenix Biotech; catalog #RK-APRO; 0.6 TIU/ml) and stored at –80°C prior to the assay. Blood samples were collected into centrifuge tubes containing EDTA and gently rocked several times immediately after collection to inhibit coagulation. These samples were then transferred to fresh centrifuge tubes containing aprotinin (0.6 TIU/ml) and gently rocked several times to inhibit proteinase action. All samples were then centrifuged at 1,600g for 15 min at 4°C and stored at –80°C prior to assay. Natriuretic peptide levels were measured with the aid of commercial RIA kits: porcine BNP-32 (#T-011-10); CNP (Human, Rat, Mouse, Porcine; #T-012–03). These RIA kits showed no cross reactivity with each other. The sensitivity of the BNP assay is 47.1 pg/ml and the CNP assay is 17.9 pg/ml. In our experiments, BNP was measured with an intra-assay variation of less than 4.3% and an inter-assay variation of less than 8.7%. CNP was measured with an intra-assay variation of 5% and an inter-assay variation of 12.2%. Measurement of cGMP by RIA cGMP levels were measured with the aid of commercial RIA kits (Institute of Isotopes Ltd., Budapest, Hungary). The manufacturer states that the working range of the assay is 2–128 fmol/tube using an acetylation protocol, or 50–6,400 fmol/tube using a non-acetylation protocol. This kit has no cross reactivity with



cAMP, AMP, ADP, ATP, GMP, GDP, or GTP. After culture of COCs as described above, groups of 250 COCs were mechanically dissociated via pipetting with a small-bore glass pipette in TCM199 medium (Invitrogen) containing 0.2 mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma) to separate cumulus cells from oocytes. After three washes in phosphate-buffered saline, all samples were solubilized in 100 ml of 0.1 M hydrochloric acid on ice for 20 min, snap-frozen in liquid nitrogen, and stored at –80°C prior to assay. The in vivo samples were prepared following the same procedure mentioned above. The sensitivity of the cGMP assay was 0.5 fmol, the intra-assay coefficient was less than 3.8%, and the inter-assay coefficient was less than 9.2%. Chemicals All chemicals were purchased from Sigma, unless otherwise noted. PMSG and hCG were the products of Sansheng Pharmaceutical Co. Ltd. (Ningbo, China). Human ANP-28 (Sigma, catalog # A1663) and CNP-22 (Sigma, catalog # N8768) were used in the present study because the biologically active 28-amino acid form of ANP and the 22-amino acid form of CNP are identical in the human, dog, pig, horse, and sheep. Porcine BNP-32 (catalog # B6651) was purchased from Sigma. Results Immunohistochemical localization of natriuretic peptides and their receptors in antral follicles of different sizes

We immunohistochemically assessed the expression levels of natriuretic peptides and their receptors in antral follicles of the

pig. In small antral follicles (1–3 mm) (Fig. 1A2), ANP was mainly found in mural granulosa, which was stronger than in cumulus cells and thecal internal cells. BNP (Fig. 1B2) was detected in granulosa cells (both cumulus cells and mural granulosa cells) and thecal internal cells. CNP (Fig. 1C2) was principally stained in both cumulus cells and mural granulosa cells. The positive signal of CNP in thecal cells was weaker than in those cell types. The volume of a large porcine follicle (diameter 3–6 mm) is 27,000– 64,000 times as large as a COC (diameter 100–150 mm). It is too difficult to find a COC in a large antral porcine follicle. Therefore, we were not able to detect the expression of each natriuretic peptide and respective receptors in COCs. The peptides of ANP and CNP (Figs. 1A3 and C3) were predominantly present in mural granulosa cells and the expression levels of them were weak in thecal internal cells. BNP (Fig. 1B3) was localized in both mural granulosa cells and thecal internal cells. NPRA (Figs. 1D2 and D3) was detected only in thecal internal cells at all follicular stages. No positive staining was detected in the control groups (Figs.1A1,B1,C1 and D1). We could not record the expression pattern of NPRB because an antibody recognizing this receptor is not available. Based on this reason, we further explored the locations of natriuretic peptides and their receptors in the pig ovary by in situ hybridization. In situ localization of mRNAs encoding natriuretic peptides and their receptors in antral follicles of different sizes

Our result indicated that mRNAs encoding ANP and CNP (Figs. 2A1,A2,C1 and C2) were mainly expressed in mural granulosa cells in both small and large antral follicle. In small

Fig. 1. Immunohistochemical detection of ANP, BNP, CNP, and NPRA in small antral follicle (1–3 mm) and large antral follicle (3–6 mm). A1–C1: Sections were immunohistochemically stained with normal rabbit serum as a control for ANP, BNP, and CNP, respectively. D1: Sections were immunohistochemically stained with mouse IgG as a control for NPRA. A2,A3: Follicles of different sizes stained with ANP antibody. B2,B3: Immunohistochemical detection of BNP expression in different-sized follicles. C2,C3: Immunohistochemical detection of CNP expression in different-sized follicles. D2,D3: Follicles of different sizes stained with anti-NPRA antibody. Control sections exhibited background staining only. Different color arrowheads indicate different cell types. Purple arrowhead for oocytes, blue for cumulus cell, green for granulosa cell, red for thecal cell. Bars ¼ 100 mm.



Fig. 2. Expression and location of mRNAs encoding natriuretic peptides and their receptors in small antral follicle (1–3 mm) and large antral follicle (3–6 mm). Frozen sections (15 mm) of pig follicles were hybridized with DIG-labeled antisense probes detecting ANP, BNP, CNP, NPRA, and NPRB mRNAs (left and middle panels), and sense probes detecting control groups (right panels). A1–E1: Sections from small antral follicles. A2–E2: Sections from large antral follicles. A1,A2: Sections hybridized with ANP antisense probe. B1,B2: Sections hybridized with BNP antisense probe. C1,C2: Sections hybridized with CNP antisense probe. D1,D2: Sections hybridized with NPRA antisense probe. E1, E2: Sections hybridized with NPRB antisense probe. A3–E3: Sense probes yielded only background staining. Different color arrowheads indicate different cell types. Purple arrowhead for oocytes, blue for cumulus cell, green for granulosa cell, red for thecal cell. Bars ¼ 200 mm.

antral follicles, the expression levels of ANP and CNP in cumulus cells and internal cells were much lower than in mural granulosa cells. mRNA encoding NPRA appeared to be expressed predominantly in thecal internal cells (Figs. 2D1 and D2). mRNAs JOURNAL OF CELLULAR PHYSIOLOGY

encoding BNP and NPRB were expressed in both granulosa cells (cumulus cells and mural granulosa cells) and thecal cells (Figs. 2B1,B2,E1 and E2). None of these mRNAs were expressed in oocytes.


Fig. 3. The effects of peptides (ANP, BNP, and CNP) on the meiotic arrest of oocytes cultured in FSH-induced maturation medium and spontaneous maturation medium. A: Pig oocyte nuclear morphological features. GV, germinal vesicle; GVBD, germinal vesicle breakdown; PB1, first polar body. B: Pig denuded oocytes (DOs) were cultured in TCM199 medium for different times at natriuretic peptide concentrations of 100 nM. C: Pig cumulus–oocyte complexes (COCs) were cultured in TCM199 medium supplemented with 0.05 U/ml folliclestimulating hormone (FSH) and different natriuretic peptides for 24 h. D: Pig COCs were cultured in different maturation media in the presence of 1,000 nM ANP for 44 h. E,F: Pig COCs were cultured in TCM199 medium with 100 nM BNP, with or without FSH, for 44 h. G,H: The kinetics of porcine oocyte maturation in COCs cultured in TCM199 with 100 nM CNP, with or without FSH. The concentration of estradiol (E2) in the culture medium was 100 nM. The means  SEMs of data from three independent experiments assaying 60–80 COCs at each time point are shown. Bars with different letters are significantly different (P < 0.05).



BNP and CNP maintain oocyte meiotic arrest

None of the three tested peptides (ANP, BNP, and CNP) had any effect on DO meiotic maturation (Fig. 3B). It indicated that none of natriuretic peptide receptors were expressed in oocyte. However, both BNP and CNP (100 nM) significantly inhibited GVBD of porcine COCs cultured in FSH-containing maturation medium for 24 h. ANP, even at 1,000 nM, showed no obvious inhibitory effect on meiotic resumption after either 24 or 44 h of culture in both FSH-induced maturation medium and spontaneous maturation medium (Figs. 3C and D). We further tested the abilities of BNP and CNP to inhibit spontaneous and FSH-induced resumption of meiosis by cultured COCs for 44 h (Figs. 3E–H). Both BNP and CNP maintained meiotic arrest, even when porcine oocytes cultured for 44 h. The inhibitory effect of CNP gradually decreased as the duration of COC culture increased (Figs. 3G and H). Addition of E2 prolonged CNP-mediated meiotic arrest of mouse oocytes for at least 24 h (Zhang et al., 2011). We found that E2 (100 nM) stabilized both BNP- and CNPmediated porcine oocyte meiotic arrest for at least 44 h (Fig. 3D,E,G,H). However, E2 alone exerted no such effect. Porcine BNP and CNP bind to NPRB with high affinity

Several previous reports demonstrated that the rank order of binding of NPRB ligands stimulating cGMP production was CNP >> ANP  BNP (Suga et al., 1992; Potter et al., 2009). However, early reports found that porcine BNP bound to human and rat NPRB with high affinity (Chang et al., 1989; Koller et al., 1991; Suga et al., 1992). Whether pBNP activates porcine NPRB (pNPRB) is not known. We found that BNP or CNP binds to recombinant pNPRB with an affinity much greater than that of ANP (Fig. 4A). This showed that in pig, both CNP and BNP could activate NPRB. We next measured cGMP production in COS-7 cells incubated with various peptides. CNP and pBNP significantly increased cGMP accumulation in such cells (Fig. 4B). ANP was notably less effective in this context. Thus, pBNP and CNP activated NPRB to a similar extent.

BNP and CNP sustain cGMP production by binding to NPRB and thus maintain meiotic arrest

A decrease in cGMP levels in somatic cells surrounding the oocyte lowers the cGMP level in the latter cell type, thus triggering meiotic resumption (Norris et al., 2009; Vaccari et al., 2009). CNP binding to NPRB of mouse cumulus cells increased cGMP levels in both cumulus cells and oocytes (Zhang et al., 2010, 2011). The cGMP levels of control porcine oocytes of COCs cultured for 44 h decreased rapidly (Fig. 5). However, the cGMP levels in both oocytes and somatic cells were sustained at relatively high levels when the COCs were incubated in medium containing 100 nM BNP or CNP with or without FSH. Addition of E2 enhanced this effect, although the cGMP levels were somewhat lower than those of oocytes and cumulus cells freshly isolated from follicles. Effect of hormonal treatments on the relative levels of mRNA encoding NPRB in cumulus cells cultured in vitro

In vitro, the levels of cGMP synthesized by CNP- or BNPactivated NPRB in oocytes and cumulus cells were closely associated with the proportions of oocytes maturation. E2 seemed to enhance this process when added together with CNP or BNP. We explored whether E2 alone affected NPRB activity. Figure 6 shows that the level of cumulus cell mRNA encoding NPRB decreased gradually as COC culture time increased. E2 inhibited this decrease of the NPRB mRNA level to some extent. As E2 also enhanced the inhibition of meiotic arrest by BNP or CNP added to FSH-containing medium, we next explored whether FSH alone or FSH-plus-E2 sustained the expression of NPRB mRNA. In the FSH group, the level of mRNA encoding NPRB decreased in COCs after 24 h of culture and was sustained at this reduced level during culture for a further 12 or 24 h. However, in the FSH-plus-E2 group, the level of gene expression was greater than in freshly isolated cells. These results may interpret why E2 or FSH well sustain meiotic arrest for a long time when COCs cultured with BNP or CNP.

Fig. 4. The binding affinities of peptides (ANP, BNP, and CNP) to the natriuretic peptide receptor-B (NPRB). A: Forty-eight hours after transfection, COS-7 cells (106) were incubated at 4°C for 2 h with 50 pmol 125I-pBNP-32 and various concentrations of unlabeled peptides. B: To measure the stimulation of cGMP synthesis in transfected cells by different peptides, cells were incubated with varying concentrations of peptides (ANP-28; porcine BNP-32; CNP-22; sigma) at 37°C for 15 min and then measured by RIA. The bars show the means  SEMs of data from three independent experiments. Bars with different letters are significantly different (P < 0.05).





Fig. 5. cGMP levels in cumulus cells and oocytes derived from freshly isolated COCs or COCs cultured in different maturation media with BNP (100 nM), CNP (100 nM), estradiol (E2), E2 plus BNP, or E2 plus CNP. A,B: cGMP levels in oocytes and cumulus cells in the treatment groups upon culture of COCs in spontaneous maturation medium. C,D: cGMP levels in oocytes and cumulus cells in the treatment groups upon culture of COCs in FSH-induced maturation medium. The concentration of estradiol (E2) in the culture medium was 100 nM. cGMP levels were measured by RIA (means  SEMs are shown) and are shown as fmol cGMP/cumulus cells surrounding an oocyte (cumulus cells) or fmol cGMP/oocyte. Three independent samples of 250 COCs were analyzed at each time point. Bars with different letters are significantly different (P < 0.05).

Oocyte maturation in vivo is closely linked to the expression levels of natriuretic peptides and their receptor

Fig. 6. In vitro effects of FSH and estradiol on the levels of mRNA encoding NPRB in cumulus cells of cultured COCs. COCs divided into four groups (Control, E2, FSH, FSH plus E2) and cultured for 24, 36, or 44 h, after which the NPRB mRNA levels were measured. Three independent experiments were conducted using 50 COCs at each time point. Bars show the means  SEMs of data derived from three independent samples. Bars with different letters are significantly different (P < 0.05).


To further explore the roles played by natriuretic peptides in oocyte maturation, we obtained ovaries from triple-crossbred prepubertal pigs. As shown in Figure 7A, 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 (Fig . 7B). 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 NPRB gene (Figs. 7E and F). Furthermore, the expression of both BNP and CNP increased at both the mRNA and protein levels after PMSG treatment and decreased following hCG injection (Figs. 7C–F). As both BNP and CNP are circulatory hormones, we examined the plasma concentrations of these peptides (Fig. 7D). The concentration of BNP in FF increased to 86 nM and next decreased to 14 nM. The CNP concentration increased from 27 to 50 nM and next decreased to 7 nM. However, the plasma concentration of BNP was 20 fmol, and that of CNP 10 fmol, which were considerably lower than those of FF (Figs. 7C and D). These results indicated that BNP and CNP originate from follicles, not plasma, and the expression thereof is regulated by gonadotrophic paracrine factors of the follicle.


Fig. 7. Relationships between the extent of oocyte maturation in vivo, and the levels of expression of natriuretic peptides and their receptors. Triple crossbred prepubertal pigs were allocated to five treatment groups as described in the Materials and Methods Section. A: Oocytes were collected for assessment of the extent of nuclear maturation. C,D: Peptide concentrations were assayed in plasma and follicular fluid. RIA kits exhibited no cross reactivity with each other. B,E,F: Cumulus and granulosa cells were prepared for the examination of cGMP production and expression of genes of interest. At least three follicles were examined at each time point for each experiment. Bars show the means  SEMs of data from three independent experiments. Bars with different letters are significantly different (P < 0.05).


Under physiological conditions, oocytes from primordial follicles arrest at meiotic prophase until there is a surge in ovulatory gonadotropin levels. Previous studies in mouse showed that cGMP was transferred from cumulus cells to oocytes and involved in the maintenance of meiotic arrest by inhibiting PDE3A activity (Norris et al., 2009; Vaccari et al., 2009; Conti et al., 2012). Recent reports indicated that CNP and NPRB maintain meiotic arrest in mouse ovaries. Further reports showed that that the CNP concentration in follicle fluid JOURNAL OF CELLULAR PHYSIOLOGY

decreased in females treated with LH/HCG injections. Here, we used both in vitro and in vivo porcine models to show that CNP and BNP maintained oocyte meiotic arrest by binding to the NPRB receptor. An inhibitory effect of CNP on porcine oocyte maturation had been described (Hiradate et al., 2013). In our report, we showed the expression and location of natriuretic peptide system in different stages of porcine antral follicles. We further found that CNP was insufficient to inhibit porcine oocyte maturation in vivo because only some of the porcine oocytes were maintained at meiotic arrest at a concentration of 20 nM



ZHANG ET AL. in vitro (Hiradate et al., 2013). In porcine follicle fluid, PMSG injection increased CNP concentrations from 27 to 50 nM and LH injection decreased CNP concentrations from 50 to 5 nM. In human follicle fluid, CNP concentrations decreased from 100 to 5 nM after LH injection (Sato et al., 2012). However, porcine oocytes were well-maintained at the GV stage in the presence of 100 nM CNP (Hiradate et al., 2013). This indicated that ANP or BNP in large antral follicles would be involved in the maintenance of porcine oocyte maturation. When porcine COCs were cultured in vitro, porcine BNP showed similar inhibitory effects compared to CNP, whereas ANP had no effect. No effect on GVBD was observed when mouse oocytes were cultured for 24 h in medium containing ANP (Zhang et al., 2010). We reported previously that ANP exerted a limited inhibitory effect on porcine oocyte maturation when COCs were cultured in NCSU37 medium with 10% (v/v) porcine FF (Zhang et al., 2005). This low-level inhibition was likely caused by BNP and CNP present in porcine FF. These results suggested that ANP could not sustain meiotic arrest during the oocyte maturation process. Human or rat BNP had no effect on maintaining mouse oocyte meiotic arrest (Zhang et al., 2010, 2011) because NPRA, the specific receptor for ANP and BNP, was not expressed in cumulus cells. Although numerous studies have indicated that NPRA is the principal receptor for ANP and BNP, whereas NPRB is unique for CNP (Schmitt et al., 2004; Rose and Giles, 2008; Potter et al., 2009), porcine BNP is known to bind with high affinity to human, rat, and mouse NPRB (Chang et al., 1989; Koller et al., 1991; Suga et al., 1992). We further showed that pBNP has a high affinity for porcine NPRB. The concentration of BNP in porcine follicle fluid before PMSG injection was 60 nM. The addition of 100 nM BNP or CNP in the medium, which is similar with the summation of 20–30 nM CNP and 60–70 nM BNP under physiological condition, can arrest porcine oocyte meiosis. This indicated that BNP and CNP both contributed to maintaining porcine meiotic arrest in vivo. The nuclear status of porcine oocytes in vivo, which is under gonadotrophic control, is strongly associated with the expression levels of natriuretic peptides system. This was consistent with previous studies, in which PMSG increased CNP and NPRB expression, inducing the production of cGMP to maintain meiotic arrest (Zhang et al., 2010; Tsuji et al., 2012), while hCG decreased expression levels of CNP and NPRB, triggering oocyte maturation (Zhang et al., 2010). The decrease in NPRB expression levels in vivo after the LH surge is likely regulated by ovulatory LH stimulation and reduced E2 signaling, since E2 stabilizes CNP-mediated mouse oocyte meiotic arrest for at least 24 h and porcine oocytes for at least 44 h by promoting and maintaining the expression of mRNA encoding NPRB in cumulus cells (Zhang et al., 2010, 2011). Moreover, injection of immature rats with the synthetic estrogen diethylstilbestrol (DES) increased the levels of mRNA encoding NPRB (Noubani et al., 2000). However, the LH surge rapidly changed the concentration of E2 from 20 to 1 pg/ml in vivo (Soede et al., 2011), which may decrease NPRB expression. Further studies are required as these signaling networks remain uncharacterized. Previous studies showed that the addition of LH rapidly decreased NPRB catalytic activity without a corresponding decrease in the NPRB protein level (Robinson et al., 2012). However, since no antibody to porcine NPRB is available, further studies in this area are required using porcine follicles. An oocyte maturation inhibitor (OMI) had been isolated from porcine FF and was shown to be a polypeptide with a molecular weight of 2,000 Da (Tsafriri and Channing, 1975a,b; Tsafriri et al., 1976; Leibfried and First, 1980; Tsafriri et al., 1982). OMI required that cumulus cells surround the oocytes, and OMI action could be overcome by the addition of LH. OMI acted similarly in all tested species. BNP and CNP were JOURNAL OF CELLULAR PHYSIOLOGY

expressed predominantly by mural granulosa cells, and previous studies showed that porcine granulosa cells completely inhibited meiosis by secreting inhibitory material into follicle fluid (Tsafriri and Channing, 1975b). Since CNP is identical in human, pig, cow, mouse, and rat, and because pBNP activates NPRB of the mouse, rat, cow (Potter et al., 2009), and pig, we hypothesized that pBNP and/or CNP might be the inhibitory material designated as OMI. Indeed, our research revealed that BNP and CNP both contributed to maintaining porcine meiotic arrest under physiological condition and LH triggered meiotic resumption by decreasing the expression levels of BNP and CNP in vivo. Therefore, we confirmed that porcine BNP and CNP was probably the OMI. This report, combined with previous data, improved our understanding of the cellular mechanisms underlying meiosis resumption of oocytes and further proved that natriuretic peptide serves as OMI to maintain oocyte meiotic arrest in mammals. Since optimal fertility requires synchrony in the regulation of oocyte meiotic resumption (Mehlmann et al., 2004; Hinckley et al., 2005; Zhang et al., 2011), elucidation of the potentially regulating mechanisms underlying physiological condition could provide a better understanding to improve the quality of cultured oocytes during IVM/IVF practice. Acknowledgments

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Brain natriuretic peptide and C-type natriuretic peptide maintain porcine oocyte meiotic arrest.

Recent studies have shown that C-type natriuretic peptide (CNP) serves as a key control system during mouse oocyte maturation. We used pig models (in ...
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