Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD14425
C-Type natriuretic peptide maintains domestic cat oocytes in meiotic arrest Yougang Zhong A,B, Jiabao Lin B, Xiaoping Liu B, Jian Hou C, Yong Zhang A and Xingxu Zhao A,D A
College of Veterinary Medicine, Gansu Agricultural University, No.1 Yingmen County, Anning District, Lanzhou 730070, P. R. China. B College of Veterinary Medicine, China Agricultural University, Yuanmingyuan West Road No. 2, Haidian District, Beijing 100193, P. R. China. C State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Yuanmingyuan West Road No. 2, Haidian District, Beijing 100193, P. R. China. D Corresponding author. Email: [email protected]
Abstract. 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 24 h, 100 nM CNP increased cGMP levels, and maintained meiotic arrest of oocytes associated with cumulus cells. When the duration of in vitro culture increased from 24 h to 36 h, 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. Additional keywords: meiosis, natriuretic peptide receptor 2.
Received 5 November 2014, accepted 27 February 2015, published online 15 April 2015
Introduction The domestic cat is a valuable model for the development of assisted reproductive technologies to potentially preserve endangered wild felids, as well as an important mammalian model for investigations into the pathobiology and molecular biology of human diseases (Pope 2000; O’Brien et al. 2002). In this context, improving the quality of in vitro maturation (IVM) of domestic cat oocytes is a crucial step towards maximising the generation of embryos of excellent quality (Go´mez et al. 2006). In mammals, oocytes in Graafian follicles are held in meiotic arrest at the diplotene stage of prophase I until an LH surge before ovulation. Germinal vesicle breakdown (GVBD) is an obvious morphological feature used to assess meiotic resumption of oocytes (Pincus and Enzmann 1935; Edwards 1965). A high level of cAMP in oocytes is considered to be a key ‘controller’ for the maintenance of meiotic arrest at the germinal vesicle (GV) stage (Cho et al. 1974; Magnusson and Hillensjo 1977; Dekel and Beers 1978; Eppig 1989; Mehlmann et al. 2004; Hinckley et al. 2005). Intra-oocyte cAMP levels are controlled by the catalytic activity of phosphodiesterase (PDE) Journal compilation Ó CSIRO 2015
3A, an oocyte-specific PDE (Richard et al. 2001; Shitsukawa et al. 2001). Conversely, cGMP originating from granulosa cells has recently been shown to inhibit PDE3A via cumulus to oocyte transfer, thus preventing the hydrolysis of cAMP and sustaining meiotic arrest (Norris et al. 2009; Vaccari et al. 2009). Upon LH stimulation, cGMP in follicle-enclosed oocytes decreases to levels that stimulate PDE3A activity, thus promoting meiotic resumption (Norris et al. 2010). Recent studies indicate that C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor (NPR) 2 in mouse and pig ovary are responsible for cGMP production (Zhang et al. 2010, 2014, 2015). The natriuretic peptide forms a family of three structurally related polypeptide hormones, namely atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and CNP. ANP and BNP activate NPR1, whereas CNP activates NPR2. NPR1 and NPR2 are transmembrane guanylyl cyclases that, when activated, catalyse the synthesis of the intracellular signalling molecule cGMP (Potter et al. 2009). CNP inhibits the spontaneous maturation of mouse oocytes in vitro, and resumption of precocious meiosis in vivo is evident in late antral www.publish.csiro.au/journals/rfd
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follicles from ovaries lacking CNP or NPR2 expression (Zhang et al. 2010; Kawamura et al. 2011). In addition, CNP inhibits porcine oocyte maturation, and oocyte maturation is closely related to decreased CNP and NPR2 expression and cGMP levels (Hiradate et al. 2014; Zhang et al. 2014, 2015). LH induces a decrease in CNP concentrations and NPR2 activity, and thus decreases cGMP accumulation, resulting the resumption of meiosis (Kawamura et al. 2011; Zhang et al. 2015). Because the circulating 22-amino acid carboxyl terminal form of CNP is identical in human, dog, pig, cat, horse and mouse (Potter et al. 2009) and based on the aforementioned studies, it is reasonable to believe that the natriuretic peptide system plays an essential role in oocyte maturation in mammals. In the present study, we showed that CNP and its receptor NPR2 produce sufficient cGMP to maintain domestic cat oocytes in meiotic arrest. Materials and methods Chemicals Unless noted otherwise, all chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA)). Human CNP-22 (Catalogue no. N8768; Sigma-Aldrich) was used in the present study because the biologically active 22-amino acid CNP is identical in human, dog, pig, cat, horse, and sheep. CNP was dissolved in phosphate-buffered saline (PBS) and kept at 208C until use. Control cultures were performed with the addition of an equal volume of PBS to the culture medium. Animals and ovaries Ovaries were obtained from domestic cats that had undergone surgical sterilisation at the Animal Hospital of China Agricultural University (Beijing, China) and were transported to College of Veterinary Medicine, China Agricultural University, in PBS supplemented with 100 U mL1 penicillin (Huabei Medical, Jizhou, China) and 50 mg mL1 streptomycin sulfate (Sigma) at 378C. The cumulus–oocyte complexes (COCs) and granulosa cells used in vitro were derived from these tissues. In some experiments, cumulus cells were stripped from the oocytes by repeated pipetting using pipettes with a diameter slightly smaller than that of the oocytes. All animals were kept in the facility at China Agricultural University according to the guidelines of the Animal Care Committee of China Agricultural University, and all efforts were made to minimise suffering. Domestic cat COC culture and assessment of nuclear maturation Domestic cat ovaries were collected as described above and delivered to College of Veterinary Medicine, China Agricultural University, within 2–3 h of surgical sterilisation in sterile saline maintained at 35–388C. Only COCs with a uniform ooplasm and compact cumulus were selected. These COCs were washed three times in Tyrode’s lactate–HEPES medium with 0.1% polyvinyl alcohol. Fifty COCs in each group with different doses of CNP (0–1000 nM) were transferred into individual wells of 24-well culture dishes (Nunclon; Nunc, Roskilde, Denmark) containing 500 mL Medium-199 (M199) supplemented with 0.23 mM sodium pyruvate, 2 mM glutamine,
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3 mg mL1 lyophilised crystalline bovine serum albumin (BSA), 100 IU mL1 penicillin G and 25 mg mL1 streptomycin sulfate. The culture medium was equilibrated in an atmosphere of 5% CO2 in air at 38.58C for at least 6 h before culture. After culture for 12, 24 or 36 h, oocytes were harvested, fixed in Bouin’s solution for 48 h and stained with 1% (w/v) orcein before being assessed for maturation by scoring for GVs (meiotic arrest) and GVBD (meiotic resumption). Oocytes reaching MII were classified as being in GVBD. Oocytes at the GV stage exhibited intact nuclear envelopes and nucleoli with chromatin in a loose conformation. Oocytes at the GVBD stage exhibited chromosome condensation. Oocytes with a fragmented cytoplasm or without chromatin were considered to be degenerated. RNA extraction and quantitative real-time PCR Total RNA was isolated using the RNeasy micro-RNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Total RNA (1 mg) from each sample was incubated for 20 min at 258C with 0.5 U DNase I (Life Technologies, Carlsbad, CA, USA) to eliminate genomic DNA contamination. First-strand cDNA was synthesised from 1 mg total RNA using the QuantiTek reverse transcription system (Qiagen). Reverse transcription was performed for 1 h at 428C. Real-time polymerase chain reaction (PCR) was then conducted to quantify steady state mRNA levels using an ABI 7500 realtime PCR instrument (Applied Biosystems, Foster City, CA, USA). Samples were contained in 96-well plates in a 25-mL reaction volume containing 12.5 mL SYBR Green PCR Master Mix (Applied Biosystems), 15 ng cDNA, the appropriate primers and nuclease-free water. The results were first normalised against the expression of ribosomal protein L19 (Rpl19), used as a housekeeping gene. The PCR primer sequences were as follows: natriuretic peptide C (NPPC), 50 -TGAGCGGCTTGG GATGTTAG-30 (forward) and 50 -AGCCACATTGCGTTGGA GAT-30 (reverse); NPR2, 50 -CAGCTCGGAACTAGATG GCG-30 (forward) and 50 -AACAGAAGATCGGGGTCGTG-30 (reverse); and RPL19, 50 -GGCTTGCCTCCAGTGTCCT-30 (forward) and 50 -GATCTGCTGACGGGAGTTGG-30 (reverse). To avoid false-positive signals, a melting curve was generated following real-time RT-PCR at temperature increments of 0.58C every two cycles starting at 658C (62 cycles total) with fluorescence acquisition after each step. The primer efficiencies for NPPC and NPR2 were in the normal range (95–98%). The relative level of target gene expression in each sample was calculated using the 2DDCt method (Livak and Schmittgen 2001). Measurement of cGMP by enzyme immunoassay After 24 h culture of 50–100 COCs in each group with or without 100 nM CNP in an atmosphere of 5% CO2 in air at 38.58C, COCs were collected in M199 (Invitrogen) containing 0.2 mM 3-isobutyl-1-methylxanthine (IBMX). After three washes with PBS, all samples were solubilised in 100 mL of 0.1 M HCl on ice for at least 20 min, snap frozen in liquid nitrogen and then stored at 208C until they were assayed. Concentrations of cGMP were determined using cGMP enzyme immunoassay (EIA) kits obtained from Cayman Chemical (Ann Arbor, MI, USA).
CNP maintains cat oocyte meiotic arrest
Effects of CNP on domestic cat oocyte meiotic resumption To investigate to role of CNP in regulation of domestic cat oocyte meiotic resumption, oocytes were cultured in M199 supplemented with 10, 30, 100 or 1000 nM of CNP. Compared with the control group, 10 and 30 nM CNP had no inhibitory effect on oocyte meiotic resumption. However, 100 nM CNP significantly inhibited oocyte meiotic resumption. Interestingly, although 1000 nM CNP exhibited an inhibitory effect against oocyte meiotic resumption, the proportion of oocytes at the GV stage in the 1000 nM CNP-treated group was much lower than in the group treated with 100 nM CNP (Fig. 2). Kinetics of CNP-inhibited oocyte meiotic resumption Based on the results described above, we further explored the effect of CNP on meiotic arrest after culture for 12, 24 or 36 h. In the control group, the number of matured oocytes gradually increased over a period of 24 h, but there was no further increase in numbers over the following 12 h. 100 nM CNP sustained the majority of cat oocytes at the GV stage over the first 24 h of culture. However, this inhibitory effect gradually decreased as the duration of COC culture increased from 24 to 36 h (Fig. 3). The proportion of oocytes resuming meiosis in the CNP-treated group increased from approximately 16% at 24 h to 37% at 36 h (Fig. 3). Kinetics of NPR2 mRNA expression in cumulus cells Given that the inhibitory effect of exogenous CNP on meiotic resumption decreased when cat oocytes were cultured for 36 h, we examined NPR2 mRNA levels after culture for different periods of time. As shown in Fig. 4, NPR2 mRNA levels in cumulus cells of cultured COCs decreased after 36 h culture. Effects of CNP on cGMP levels in COCs Decreased cGMP levels in cumulus cells decrease cGMP levels in oocytes, thus initiating meiotic resumption (Norris et al. 2009). As shown in Fig. 5, cGMP levels in control COCs were
Mural granulosa cell
Relative mRNA levels
Results Expression of NPPC and NPR2 mRNA in domestic cat antral follicles Quantitative reverse transcription–polymerase chain reaction was used to determine the abundance NPPC (encoding CNP) and NPR2 mRNA in different antral follicle cell types. Expression of NPPC was higher in mural granulosa cells than cumulus cells (Fig. 1). Conversely, NPR2 expression was lower in mural granulosa cells than cumulus cells. Neither of these two mRNAs was detectable in oocytes of antral follicles (Fig. 1).
Fig. 1. Expression of natriuretic peptide C (NPPC) and natriuretic peptide receptor 2 (NPR2) mRNA in domestic cat antral follicles. In this experiment, cumulus cells and mural granulosa cells were harvested from at least three antral follicles for each separate experiment. In all, 200 denuded oocytes were prepared from antral follicles in each experiment. Data are the mean s.e.m. of three independent experiments. Different letters indicate significant differences (P , 0.05). 80
Statistical analysis Unless stated otherwise, each oocyte maturation experiment was conducted at least three times with a minimum of 50 oocytes per group. Results are expressed as the mean s.e.m. All proportional data were subjected to arcsine transformation and were analysed using the least-squares method with SAS software ver. 8.2 (SAS Institute, Cary, NC, USA). Two-tailed P , 0.05 was considered significant.
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Fig. 2. Effects of C-type natriuretic peptide (CNP) on meiotic arrest of oocytes from the domestic cat. Domestic cat cumulus–oocyte complexes were cultured in M199 in the presence of 10, 30, 100 or 1000 nM CNP for 24 h. After culture, oocytes were harvested for the assessment of nuclear morphological features. Data are the mean s.e.m. of three independent experiments. Different letters indicate significant differences (P , 0.05). GVBD, germinal vesicle breakdown.
decreased after in vitro culture for 24 h. However, culture in the presence of 100 nM CNP maintained cGMP concentrations in COCs at relatively high levels. Effects of CNP on oocyte degeneration The proportion of degenerated oocytes (Fig. 6a) in the control group after 12 h culture of COCs was 23.1% (Fig. 6c); this
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100 nM CNP c
f moles cGMP/COC
a 10 0
Culture time (h) Fig. 3. Kinetics of maintenance by C-type natriuretic peptide (CNP) of the meiotic arrest of oocytes from the domestic cat. Domestic cat oocytes were cultured in M199 in the presence of 100 nM CNP for 12, 24 or 36 h. After culture, oocytes were harvested for the assessment of nuclear morphological features. Data are the mean s.e.m. of three independent experiments. Different letters indicate significant differences (P , 0.05). GVBD, germinal vesicle breakdown.
Relative NPR2 mRNA levels
1.0 c 0.8
Culture time (h) Fig. 4. Kinetics of natriuretic peptide receptor 2 (NPR2) mRNA levels in cumulus cells from cumulus–oocyte complexes (COCs) cultured in vitro. Domestic cat COCs were cultured for 12, 24 or 36 h and then cumulus cells were harvested from COCs for real-time quantitative polymerase chain reaction (PCR). Data are the mean s.e.m. of three independent experiments. Different letters indicate significant differences (P , 0.05).
percentage increased further after 24 and 36 h in culture to 36.4% and 56.8%, respectively. Culture of COCs in the presence of 100 nM CNP significantly decreased the proportion of oocytes showing signs of degeneration (Fig. 6c). There was no significant difference in the effects of 10, 100 or 1000 nM CNP on oocyte degeneration after 24 h culture (Fig. 6b).
CNP 24 h
Fig. 5. Effects of C-type natriuretic peptide (CNP) on cGMP levels in oocytes and cumulus cells. Domestic cat cumulus–oocyte complexes (COCs) were cultured in the presence of 100 nM CNP for 24 h. At the end of culture, COCs were harvested for cGMP measurement. Data are the mean s.e.m. of three independent experiments. Different letters indicate significant differences (P , 0.05).
Discussion Recent reports indicate that CNP and NPR2 sustain oocyte meiotic arrest in mouse and pig (Zhang et al. 2010, 2015). In the present study, in domestic cat antral follicles NPPC mRNA was expressed primarily in mural granulosa cells, whereas NPR2 mRNA was expressed mainly in cumulus cells. CNP sustained the majority of oocytes in COCs at the GV stage. However, the proportion of oocytes at the GVBD stage increased gradually when COCs were cultured in vitro for a prolonged period of time. In addition, we confirmed that cGMP levels in cumulus cells and oocytes were closely correlated with the proportion of oocytes at the GV stage. It has been reported previously that 30 nM CNP is sufficient to inhibit mouse oocyte meiotic resumption in vitro (Zhang et al. 2011). In the present study, 100 nM, but not 30 nM, CNP had an inhibitory effect on cat oocyte meiotic resumption, consistent with recent study in pigs showing that .100 nM CNP can significantly inhibit oocyte maturation (Zhang et al. 2015). It has also been found that the CNP concentration in human follicular fluid is approximately 100 nM (Kawamura et al. 2011). At 1000 nM, CNP exhibited a lower inhibitory effect on cat oocyte maturation compared with 100 nM CNP, similar to what has been reported previously (Zhang et al. 2011). The possible reason why the high concentration of CNP decreases NPR2 activity is homologous desensitisation: high concentrations are correlated with receptor dephosphorylation (Potter 1998). The data indicate that different concentrations of CNP are required to maintain meiotic arrest in different species. The present study suggests that expression of CNP and NPR2 in somatic cells may play a physiological role in maintaining the meiotic arrest of cat oocytes during follicular development. It has been reported that oestradiol (E2) enhances the effect of
CNP maintains cat oocyte meiotic arrest
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100 nM CNP 70
40 50 c
40 bc 30
Culture time (h)
Fig. 6. Effects of C-type natriuretic peptide (CNP) on the degeneration of domestic cat oocytes. (a) Morphological features of oocytes from the domestic cat. Oocytes in the germinal vesicle (GV) stage exhibited an intact nuclear envelope and nucleoli with chromatin in a loose conformation. Oocytes in GV breakdown (GVBD) stage exhibited chromosome condensation. Oocytes with fragmented cytoplasm or without chromatin were considered to be degenerated (DE). Scale bars ¼ 30 mm. (b) Domestic cat COCs were cultured in M199 in the presence of 10, 100 or 1000 nM CNP for 24 h. (c) Domestic cat COCs were cultured in M199 in the presence of 100 nM CNP for 12, 24 or 36 h. After culture, oocytes in COCs were harvested to determine the number of degenerated oocytes. Data are the mean s.e.m. of three independent experiments. Different letters indicate significant differences (P , 0.05).
CNP on oocyte meiotic arrest in mice and pigs (Zhang et al. 2011, 2015). However, E2 did not obviously enhance the effects of CNP on oocyte meiotic arrest after 24 h culture of cat COCs (Y. Zhong, J. Lin, X. Liu, J. Hou, Y. Zhang and X. Zhao, unpubl. obs.). A possible explanation for this is the relatively long halflife of NPR2 in the cat or that oocyte-derived paracrine factors are sufficient to maintain NPR2 expression throughout the period of culture. Further experiments are needed to investigate the effects of E2 on the action of CNP in the cat. The inhibitory effect of CNP on meiotic resumption decreased gradually when the duration of culture of cat COCs increased. Because CNP was added exogenously in these experiments, one of the reasons that could explain the increased meiotic resumption in the presence CNP as culture was prolonged is a decrease in NPR2 expression. Expression of NPR2 mRNA in cumulus cells decreased gradually when cat COCs were cultured in vitro. This is consistent with previous results that steady state NPR2 levels in cumulus cells are crucial for inhibition of oocyte meiotic resumption when mouse COCs are cultured with CNP for 24 h (Zhang et al. 2011) or porcine COCs are cultured with CNP for 44 h (Zhang et al. 2014, 2015). It has also been reported that, in vivo, a decrease in NPR2 is correlated with oocyte maturation. After injection of human chorionic gonadotrophin, expression of NPR2 mRNA in cumulus cells
decrease within 4 h in mice (Zhang et al. 2010; Kawamura et al. 2011; Robinson et al. 2012) and within 36 h in pigs (Zhang et al. 2015); at the same time, oocytes in preovulatory follicles exhibit signs of nuclear maturation (Zhang et al. 2015). It has been found that cGMP plays an essential role in maintaining meiotic arrest (Norris et al. 2009). Further studies showed that LH decreases CNP and NPR2 in the mouse ovary, leading to decreased cGMP accumulation (Zhang et al. 2010). In the porcine ovary, the progression of oocyte maturation in vivo is closely and inversely linked to cGMP levels and follicular expression of CNP and its receptor NPR2 (Zhang et al. 2014, 2015). In the present study, cGMP levels in cat COCs paralleled the proportion of oocytes exhibiting meiotic arrest (GV stage). Because oocytes express very low levels of NPR2 mRNA, we speculate that cGMP is produced in cumulus cells upon activation of NPR2 by CNP and transferred via gap junctions from granulosa-associated cumulus cells to oocytes, where it acts to inhibit PDE3A activity and oocyte meiotic resumption in the cat. After follicles form a fluid-filled antrum, mammalian oocytes reach their full size and acquire the ability to complete meiosis. However, they are maintained in meiotic arrest by factors derived from mural granulosa cells. This constraint is released when an LH surge is secreted by the pituitary gland
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during the preovulatory stage (Pincus and Enzmann 1935; Edwards 1965). The majority of oocytes undergo apoptosis during this long period and numerous factors are responsible for this process (Ryle 1973; Anasti 1998; Mehlmann 2005). Interestingly, previous reports indicate that a cGMP analogue prevents apoptosis of preantral follicles (McGee et al. 1997) and that CNP stimulates follicle growth and sustains oocyte survival (Sato et al. 2012). In the present study, we found that CNP significantly decreased the proportion of degenerated oocytes, possibly via NPR2 production through a cGMP pathway. However, the detailed mechanisms involved in this process require further investigation. Because manipulating the kinetics of meiosis during IVM is important to improve subsequent fertilisation and embryonic development, many inhibitors such as hypoxanthine and IBMX are used to promote oocyte cytolasmic maturation in vitro (Eppig and Downs 1987; Hegele-Hartung et al. 1999; Herrick 2014; Huang et al. 2014). However, there is an inverse relationship between the duration of oocyte culture and degeneration (Sirard and First 1988; Ali and Sirard 2002): a long duration of IVM leads to oocyte degradation (Alm et al. 2008). CNP has been reported to be a physiological inhibitor of the process of mouse and porcine oocyte maturation (Zhang et al. 2010, 2015). Herein, we provide the first reported that CNP sustains meiotic arrest and prevents oocyte degeneration in the cat. These results may provide a better understanding of the process and improve the quality of cultured oocytes for IVF or somatic cell nuclear transfer in domestic cats. Acknowledgements The authors thank Dr Meijia Zhang for his helpful suggestions regarding experimental design and manuscript preparation. Domestic cat ovaries were kindly provided by the Animal Hospital of China Agricultural University (Beijing, China) with the dedicated assistance of Min Liu. This work was supported by Chinese Universities Scientific Fund (No. 201105010510305).
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