RESEARCH ARTILCE Molecular Reproduction & Development 81:1030–1041 (2014)

Epidermal Growth Factor-Network Signaling Mediates Luteinizing Hormone Regulation of BNP and CNP and Their Receptor NPR2 During Porcine Oocyte Meiotic Resumption WENQIANG ZHANG, QIAN CHEN, YE YANG, WEI LIU, MEIJIA ZHANG, GUOLIANG XIA,

AND

CHAO WANG*

State Key Laboratory of Agro-biotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, People’s Republic of China

SUMMARY The epidermal growth factor (EGF) network, induced by luteinizing hormone (LH), plays an essential role in the regulation of oocyte maturation, cumulus expansion, and ovulation. Binding of brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) to natriuretic receptor 2 (NPR2) generates cyclic guanosine monophosphate (cGMP), a key inhibitor that sustains porcine oocyte meiotic arrest. This correlation suggests that LH interacts with natriuretic-peptide signaling, possibly via the EGF network, to promote porcine meiotic resumption. In testing this hypothesis, we found that the majority of porcine oocytes remain arrested in the germinal-vesicle stage after 44 h of co-culturing cumulus-oocyte complexes with 107 granulosa cells, which secreted active BNP and CNP; these natriuretic peptides associate with NPR2 on cumulus cells, thereby inhibiting porcine oocyte maturation. This inhibitory effect of BNP and CNP was relieved by EGF-like growth factors, whose expression naturally increases in granulosa cells 18 h after human chorionic gonadotropin injection. LH and the EGF-like peptide amphiregulin (AREG) decreased BNP and CNP production in granulosa cells and down-regulated NPR2 expression in cumulus cells, which together decreased oocyte cGMP to levels that permit meiotic resumption. The effects of AREG on the gene expression of natriuretic-peptide signaling components and on oocyte maturation were completely blocked by the EGF receptor kinase inhibitor AG1478; the effect of LH, however, was only partially reversed by AG1478. Based on these results, LH regulates natriuretic-peptide signaling, although other pathways also cooperate with the EGF network to induce porcine oocyte maturation. Mol. Reprod. Dev. 81: 10301041, 2014. ß 2014 Wiley Periodicals, Inc. Received 17 June 2014; Accepted 7 September 2014



Corresponding author: State Key Laboratory of Agrobiotechnology College of Biological Sciences China Agricultural University Beijing 100193, China. E-mail: [email protected]

Grant sponsor: National Basic Research Program of China; Grant numbers: 2014CB138503, 2013CB945500 Grant sponsor: Chinese Universities Scientific Fund; Grant number: 2013YJ002

Published online 27 October 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22424

INTRODUCTION In mammalian oocytes, meiosis is initiated during fetal life and subsequently arrested at the diplotene stage of prophase I. In preantral follicles, maintenance of this arrest is due to the absence of key components that are required for cell-cycle progression (Kanatsu-Shinohara et al., 2000). After formation of a fluid-filled antrum within the follicle, oocytes reach their full size and acquire the competence to

ß 2014 WILEY PERIODICALS, INC.

Abbreviations: AREG, amphiregulin; BNP, brain natriuretic peptide; BTC, betacellulin; cA/GMP, cyclic adenosine/guanosine-30 ,50 -monophosphate; CNP, c-type natriuretic peptide; COC, cumulus-oocyte complex; EGF, epidermal growth factor; EREG, epiregulin; GV[BD], germinal vesicle [breakdown]; hCG, human chorionic gonadotropin; LH, luteinizing hormone; NPPB/C, gene encoding natriuretic peptide B/c-type; NPR2, natriuretic peptide receptor 2; PDE3A, phosphodiesterase 3A; PMSG, pregnant mare serum gonadotropin.

EGF SIGNAL DECREASES BNP

resume meiosis  although an oocyte may remain arrested by factors derived from mural granulosa cells. This constraint is released during the pre-ovulatory stage, when a surge of luteinizing hormone (LH) is secreted by the pituitary gland. Germinal-vesicle breakdown (GVBD) is often used to assess meiotic resumption (Pincus and Enzmann, 1935; Edwards, 1965). A high intra-oocyte concentration of cyclic adenosine30 ,50 -monophosphate (cAMP) also maintains meiotic arrest at the germinal-vesicle (GV) stage (Cho et al., 1974; Dekel and Beers, 1978; Mehlmann et al., 2004; Hinckley et al., 2005). An increase in the catalytic activity of phosphodiesterase 3A (PDE3A), an oocyte-specific enzyme that degrades cAMP, follows the LH surge, thereby activating cell cycle-promoting factors that help resume meiosis (Richard et al., 2001; Shitsukawa et al., 2001). Cyclic guanosine monophosphate (cGMP) from granulosa cells was recently reported to inhibit PDE3A activity, thus preventing the hydrolysis of cAMP and sustaining meiotic arrest (Norris et al., 2009). Prior to the LH surge, direct transfer of cGMP from granulosa cells to oocytes occurs; following LH stimulation, this shuttling is blocked, allowing cGMP in follicle-enclosed oocytes to decrease to levels that stimulate PDE3A activity, thus promoting meiotic resumption (Norris et al., 2009; Vaccari et al., 2009). In mural granulosa cells, the epidermal growth factor (EGF)-like growth factors amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC) are rapidly induced by LH in preovulatory follicles (Park et al., 2004; Panigone et al., 2008). Activation of EGF signaling by these growth factors is essential for mediating many of the effects of LH, including oocyte meiotic resumption, cumulus expansion, and gap-junction closure (Park et al., 2004; Norris et al., 2008; Norris et al., 2010; Hsieh et al., 2011). In response to LH, EGF-like growth factors also induce a rapid decrease in follicle and oocyte cGMP levels (Vaccari et al., 2009; Norris et al., 2010; Liu et al., 2014). Previous studies demonstrated that natriuretic peptides play an important role in cGMP production and oocyte maturation (Zhang et al., 2010, 2014; Kawamura et al., 2011; Liu et al., 2014; Santiquet et al., 2014). The natriuretic peptides include three structurally related polypeptide hormones: atrial natriuretic peptide (ANP, from gene NPPA), brain natriuretic peptide (BNP, from gene NPPB), and C-type natriuretic peptide (CNP, from gene NPPC) (Potter et al., 2009). At least three different subtypes of natriuretic peptide receptors (NPRs) have been identified: NPR1, NPR2, and the clearance receptor NPR3. NPR1 and NPR2 are transmembrane guanylyl cyclases that, when activated, catalyze the synthesis of the intracellular signaling molecule cGMP (Potter and Hunter, 1998; Rose and Giles, 2008). Following human chorionic gonadotropin (hCG) administration, murine Nppc, and Npr2 mRNA expression decrease in the ovary (Zhang et al., 2010; Kawamura et al., 2011; Tsuji et al., 2012; Liu et al., 2014) and porcine NPPB and NPPC mRNA levels as well as BNP and CNP protein production also decrease (Zhang et al., 2014). Further, almost all porcine oocytes remain in the GV stage after injection of pregnant-mare

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serum gonadotropin (PMSG), with some oocytes undergoing GVBD 18 hr following hCG injection. The proportion of oocytes that resume meiosis increases between 18 and 36 hr following hCG administration, which is closely timed with decreasing cGMP levels in follicle cells from hCGprimed pigs (Zhang et al., 2014). Thus, LH likely induces a decrease in the natriuretic peptide system in the ovaries of mice and pigs, thereby decreasing cGMP accumulation (Zhang et al., 2010, 2014; Kawamura et al., 2011; Liu et al., 2014). Here, we link the processes by demonstrating that the EGF network activated by LH stimulates porcine oocyte maturation by regulating BNP/NPR2 and CNP/ NPR2 signaling.

RESULTS Effect of In Vivo Gonadotropin Treatment on Relative Levels of EGF-Like Growth Factor mRNA in Porcine Granulosa Cells Porcine LH receptors (LHRs) include four isoforms, three of which are loss-of-function (Loosfelt et al., 1989). We used reverse-transcriptase PCR to determine the mRNA abundance of each isoform in different antral follicle cell types. LHR mRNA was not detectable in oocytes or cumulus cells, but was present in antral follicle mural granulosa cells and thecal cells (Fig. 1A). Consistent with these expression data, LH did not promote oocyte maturation when cumulusoocyte complexes (COCs) were cultured in vitro (Fig. 1B) despite its ability to induce oocyte maturation in vivo. The EGF network is essential to efficiently propagate and amplify LH signals. In mice, the LH surge rapidly promotes Areg, Ereg, and Btc transcription in granulosa cells, resulting in EGFR kinase activation in cumulus cells (Park et al., 2004). mRNA levels of EGF-like growth factors (EREG, BTC, and particularly AREG) also significantly increased in porcine granulosa cells 18 h after hCG injection (Fig. 1C). Although expression levels decreased by 36 h, mRNA abundance for each growth factor remained higher than those in the control and PMSG-treated groups. Our in situ hybridization results further showed that hCG injection strongly induced AREG mRNA expression in vivo (Fig. 1D).

Effect of AREG on BNP- and CNP-Mediated Meiotic Arrest To further assess the role of AREG-dependent EGF signaling during porcine oocyte maturation, COCs were cultured with various concentrations of AREG for 24 or 44 hr. AREG significantly enhanced oocyte maturation (Fig. 2B and C). Since BNP and CNP both contribute to the maintenance of porcine meiotic arrest under physiological conditions (Zhang et al., 2014), the effect of AREG on meiotic resumption was determined in COCs cultured with BNP, CNP, or both peptides together. Despite the presence of these inhibitors or the synergizer b-estradiol, AREG induced oocyte maturation under all conditions (Fig. 2DF). But, AREG failed to induce oocyte maturation

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Figure 1. Effect of LH on porcine oocyte maturation in vitro and on the mRNA expression of EGF-like growth factors AREG, EREG, and BTC in vivo.

A: The expression of LH receptor mRNA (1,121 bp), and its three splice variants (855, 411, and 185 bp) without putative transmembrane domains, in different antral-follicle cell types. CC, cumulus cell; GC, granulosa cell; TC, theca cell. B: Porcine COCs were cultured in TCM199 supplemented with different LH concentrations for 44 hr. After culturing, oocytes were harvested, fixed, and then stained to evaluate chromatin configuration. C: AREG, EREG, and BTC mRNA in granulosa cells from pigs injected with and hCG. At least three follicles were examined at each time point for each experiment. Bars show the means  standard errors of data from three independent experiments. Bars represented by different letters were significantly different (P < 0.05). D: Frozen sections (15 mm) from pig follicles were hybridized with digoxigenin-labeled antisense probes and sense probes detecting AREG mRNA. Different-colored arrowheads indicate different cell types: purple arrowhead for granulosa cells, red for thecal cells. Scale bars, 200 mm. In all cases, follicles were from triple-crossbred, prepubertal pigs.

when EGFR kinase activity was blocked by its specific inhibitor, AG1478 (Fig. 2DF). It was shown previously that lowering cGMP in the somatic cells surrounding the oocyte reduces cGMP levels in the oocyte, thus triggering meiotic resumption (Norris et al., 2009). In our hands, BNP and CNP together triggered cGMP synthesis specifically in cumulus cells (Fig. 2G-H), thereby inhibiting oocyte maturation (Fig. 2F). AREG reduced cGMP levels in cumulus cells and oocytes, thus facilitating oocyte maturation; again, AG1478 completely blocked this effect (Fig. 2G-H).

Effect of the EGF Network on NPR2 mRNA Expression in Porcine Cumulus Cells Levels of NPR2 mRNA in cumulus cells of COCs decreased gradually with increasing culture time (Fig. 3).

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AREG accelerated this decrease within the first 12 h of culture, but caused no additional decrease over the next 12 h (Fig. 3 A-B). Although b-estradiol sustained NPR2 mRNA levels in the control group, the addition of AREG still promoted a decrease in NPR2 mRNA whereas AG1478 completely blocked this effect (Fig. 3B).

LH and AREG Induce Oocyte Meiotic Resumption in COCs Co-Cultured With Granulosa Cells COCs from a variety of mammalian species, cultured without hormonal stimulation after removal from ovarian follicles, resume meiosis spontaneously (Pincus and Enzmann, 1935; Edwards, 1965). One study indicated that meiotic progression in porcine COCs is completely inhibited when co-cultured with 107 granulosa cells (Tsafriri and Channing, 1975b). The agent responsible for inhibiting

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Figure 2. Effect of AREG on BNP- and CNP-mediated meiotic arrest. A: Morphological features of porcine oocyte nuclei. After culturing, oocytes were harvested, fixed in acetic acid/ethanol (1/3 v/v) for 48 hr, and stained with 1% (w/v) orcein prior to assessment of maturation by scoring for GV (meiotic arrest) or GVBD (meiotic resumption). Non-matured oocytes (GV stage) exhibited intact nuclear envelopes (green arrow) and nucleoli (red arrow) with chromatin in a loose conformation. Matured oocytes (GVBD) exhibited condensed chromosomes (blue arrow). B: Porcine COCs were cultured in TCM199 medium supplemented with different concentrations of AREG (AR) for 24 hr. C: Porcine COCs were cultured in TCM199 supplemented with 100 ng/mL AREG or 1 mM AG1478 (AG) for 44 hr. DF: Porcine COCs were cultured in TCM199 medium in the presence of 100 nM BNP, 100 nM CNP, or 50 nM BNP and 50 nM CNP, supplemented with either 100 ng AREG or 100 ng AREG plus 1 mM AG1478 for 44 hr. G and H: Effects of AREG on cGMP levels in cumulus cells and oocytes. Porcine COCs were cultured in TCM199 in the presence of 50 nM BNP and 50 nM CNP, supplemented with either 100 ng AREG or 100 ng AREG plus 1 mM AG1478 for 44 hr. cGMP levels were measured by radioimmunoassay, and expressed in fmol cGMP/cumulus cells surrounding an oocyte or fmol cGMP/oocyte. Bars represent the means  standard errors of data from three independent experiments. Bars with different letters are significantly different (P < 0.05).

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Figure 3. Effects of AREG on the relative NPR2 mRNA levels in cumulus cells cultured in vitro Porcine COCs were cultured in TCM199 supplemented with either 100 ng AREG (AR) or 100 ng AREG plus 1 mM AG1478 (AG) for 12 and 24 hr, respectively. A: Porcine COCs cultured in TCM199 without b-estradiol (E2); B: Porcine COCs cultured in TCM199 with 100 nM b-estradiol. The means  standard errors of data from three independent experiments that assayed 100 COCs at each time point are shown. Bars with different letters are significantly different (P < 0.05).

meiosis was hypothesized to be a peptide of approximately 2,000 Da that is secreted from granulosa cells (Tsafriri et al., 1976; Hillensjo et al., 1979)  quite similar to the molecular weights of BNP and CNP. We therefore used this co-culture system to test whether LH or AREG could decrease BNP and/or CNP expression, thereby promoting oocyte maturation. Indeed, LH and AREG reversed granulosa-cell-dependent arrested meiosis when COCs were co-cultured with 107 granulosa cells (Fig. 4). AG1478, however, completely blocked the effect of AREG on oocyte maturation in this culture system, while partially reversing the effect of LH (Fig. 4).

Effect of LH and AREG on the Expression of Natriuretic Peptides and Their Receptors and on cGMP Levels in COCs co-Cultured With Granulosa Cells Since AREG is the most abundant EGF-like factor induced by LH in vivo, we explored AREG expression in granulosa cells cultured in vitro. LH significantly elevated AREG transcript levels within 12 hr, although its abundance decreased over the next 32 h (Fig. 5A). In vivo, LH caused a decrease in BNP and CNP in granulosa cells and in their receptor, NPR2, in cumulus cells, allowing meiosis to resume (Zhang et al., 2014). Similarly in vitro, NPPB (encoding BNP) and NPPC (encoding CNP) transcription in granulosa cells, BNP and CNP secretion into culture medium, and NPR2 expression in cumulus cells decreased with LH and AREG treatment (Fig. 5BD). We further examined the effects of LH and AREG on cGMP to evaluate their roles in regulating the natriuretic-peptide system; both significantly decreased cGMP levels in oocyte and cumulus cells (Fig. 5EF). AG1478, on the other hand, completely reversed the effect of AREG and partially reversed the effect of LH (Fig. 5BF).

DISCUSSION

Figure 4. Effect of LH and AREG on oocyte meiotic arrest in COCs co-

cultured with granulosa cells. COCs cultured with or without 107 granulosa cells in the presence of 1 mg/mL LH, 100 ng/mL AREG (AR), and/or 1 mM AG1478 (AG) for 44 hr. Bars represent the means  standard errors of data from three independent experiments. Bars with different letters are significantly different (P < 0.05).

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In this study, EREG, BTC, and especially AREG transcript levels increased significantly in porcine granulosa cells 18 h after hCG induction. When co-cultured with granulosa cells for 44 h, however, the majority of oocytes in COCs remain arrested in the GV stage; this inhibitory effect likely results from BNP and CNP signaling during porcine oocyte meiosis. In contrast, LH and AREG downregulate NPR2 expression in cumulus cells and decrease BNP and CNP production in granulosa cells, thereby decreasing cGMP levels and allowing oocyte meiosis to resume. Although the influence of LH was only partially reversed by AG1478, the effects of AREG on natriuretic

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EGF SIGNAL DECREASES BNP

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Figure 5. Effect of LH and AREG on natriuretic peptides and their receptor expression, and on cGMP levels in COCs co-cultured with granulosa cells. A: Effect of LH on AREG mRNA in granulosa cells. COCs cultured with 107 granulosa cells treated with 1 mg/mL LH for 12, 24, or 44 hr. B and C: Effect of LH and AREG on NPR2 mRNA in cumulus cells and NPPB (encoding BNP) and NPPC (encoding CNP) mRNA in granulosa cells. COCs cultured with 107 granulosa cells in the presence of 100 nM b-estradiol (E2), 1 mg/mL LH, 100 ng/mL AREG (AR), and/or 1 mM AG1478 (AG) for 44 hr. D: Effects of LH and AREG on BNP and CNP concentrations in the culture medium. E and F: cGMP levels in cumulus cells and oocytes in different treatment groups. cGMP levels were measured by radioimmunoassay, and are expressed in fmol cGMP/cumulus cells surrounding an oocyte or fmol cGMP/oocyte. The bars represent the means  standard errors of data from three independent experiments. Bars with different letters are significantly different (P < 0.05).

peptide expression and oocyte maturation were blocked completely by AG1478. Therefore, we conclude that the expression of LH-induced EGF-like growth factors (especially AREG) decreases BNP and CNP protein production in granulosa cells and down-regulates NPR2 mRNA in cumulus cells, resulting in the stimulation of oocyte maturation. Although LH induces the meiotic resumption of oocytes, cumulus cells and oocytes express very low levels of the LH receptor (LHR/LHCGR) and are insensitive to direct LH stimulation (van Tol et al., 1996; Shimada et al., 2003).

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Instead, LH-activated EGF signaling is believed to be a key pathway involved in the induction of oocyte maturation, cumulus cell expansion, and ovulation (Park et al., 2004; Panigone et al., 2008). In mice, the LH surge rapidly promotes Areg, Ereg and Btc mRNA expression in granulosa cells, resulting in EGFR kinase activation in cumulus cells (Park et al., 2004) as well as decreased cGMP production, which helps release oocytes from meiotic arrest (Norris et al., 2009; Vaccari et al., 2009). LH also induces a decrease in ovarian natriuretic peptide synthesis, which halts cGMP accumulation in mice (Zhang et al., 2010) by

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possibly activating EGF signaling (Norris et al., 2010). BNP and CNP also inhibit porcine oocyte maturation in vivo, which is closely and inversely linked to cGMP levels and follicular expression of the natriuretic peptides BNP and CNP and their receptor NPR2 (Zhang et al., 2014). Despite the existence of interspecies differences, it is therefore reasonable to believe that the natriuretic peptide system plays an essential role during meiotic arrest in mammals. Steady-state levels of NPR2 in cumulus cells are essential for the maintenance of meiotic arrest in mouse COCs cultured with CNP for 24 h (Zhang et al., 2011) or in porcine COCs cultured with BNP and CNP for 44 h (Zhang et al., 2014). Given that exogenous BNP and/or CNP supplementation can also maintain meiotic arrest, the dominant effect of AREG in inducing COC maturation in the presence of BNP and/or CNP may be to down-regulate NPR2 expression and to decrease cGMP production. Indeed, NPR2 mRNA levels were lower when porcine COCs were cultured for 24 or 44 h in the presence of AREG. On the other hand, EGF receptor signaling was reported to trigger meiotic resumption in murine COCs by modifying NPR2 protein activity (Wang et al., 2013). After hCG injection, NPR2 mRNA levels in cumulus cells decreased gradually over 4 h in mice and 36 h in pigs, and oocytes showed signs of nuclear maturation (Wang et al., 2013; Zhang et al., 2014). Therefore, NPR2 protein activity is likely important for mouse oocyte maturation in vivo, whereas the longer period utilized by porcine oocytes suggests that the abundance of NPR2 mRNA also influences the timing of maturation in this animal. Synthetic estrogen diethylstilbestrol and b-estradiol, on the other hand, increase and sustain NPR2 mRNA levels (Noubani et al., 2000; Zhang et al., 2011; Zhang and Xia, 2012; Zhang et al., 2014). The concentration of b-estradiol in porcine plasma rapidly declines from 20 to 1 pg/mL during an LH surge, which can be mimicked by hCG administration (Soede et al., 2011). Considering these results together with our present work, it is possible to speculate that b-estradiol and EGF signaling impact the in vivo steady-state NPR2 mRNA levels between the control and hCG-administered groups. Thus, coordinated activation of EGFR signaling and inactivation of b-estradiol signaling induced by LH together reduce NPR2 mRNA expression in cumulus cells, leading to a subsequent decrease in cGMP levels in the oocyte. After LH injection, the CNP concentration in human follicular fluid decreases from approximately 100 nM to 5 nM (Kawamura et al., 2011). Similarly, the BNP and CNP concentrations in porcine follicular fluid decrease from 86 nM to 14 nM and 50 nM to 5 nM, respectively (Zhang et al., 2014). In this study, the addition of 50 nM BNP and 50 nM CNP to the medium, which is similar to the physiological conditions of 2030 nM CNP and 6070 nM BNP, can maintain meiotic arrest in porcine oocytes. Therefore, BNP and CNP both contribute towards the maintenance of porcine meiotic arrest in vivo. Co-culture of porcine oocytes with 1  107 granulosa cells results in complete inhibition of meiosis (Tsafriri and Channing, 1975b). Using this system, we found that

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co-culture of granulosa cells with COCs also maintained meiotic arrest, suggesting that the BNP and CNP originating from granulosa cells may be secreted into the culture medium, binding to NPR2 on cumulus cells and thereby maintaining meiotic arrest in the oocyte. When AREG and LH were added to this co-culture system, porcine NPPB and NPPC mRNA levels as well as BNP and CNP protein production decreased, which promoted oocyte maturation. As the biologically active C-terminal 26-amino acids BNP are identical among the porcine, goat, and bovine orthologs, it would be useful to assess how conserved this BNP-LH balance is among livestock species. The EGFR kinase inhibitor AG1478 was able to completely reverse the effect of AREG but only partially reverse the effect of LH in porcine oocytes, which is in agreement with previous data indicating that EGF signaling negatively regulates CNP expression in mice (Tsuji et al., 2012; Lee et al., 2013; Wang et al., 2013; Liu et al., 2014). AREG strongly suppressed Nppc expression in cultured follicle and granulosa cells (Tsuji et al., 2012; Liu et al., 2014), yet similar to wild-type mice, Nppc mRNA levels in mutant mice (Areg/ and Areg/Egfrwa2/wa2) and in granulosa-cell-specific EGF receptor (EGFR) knockout mice (conditional allele mice Egfrfl/fl Cyp19-Cre) decreased after recombinant LH administration (Liu et al., 2014). Thus, EGFR activation is sufficient, but not required, to mediate LH regulation of NPPB and NPPC expression. Protein kinase C (PKC) signaling also mediates the effects of LH in oocyte maturation (Morris and Richards, 1993; Shimamoto et al., 1993; Morris and Richards, 1995; Kawashima et al., 2014). Although PKC has been reported to regulate BNP and CNP signaling in cell lines (Hanford and Glembotski, 1996; Mendonca et al., 2006; Chu et al., 2008; Mendonca et al., 2009; Mendonca et al., 2012), further study is required to establish if and how PKC interacts with EGF signaling downstream of LH to regulate natriuretic peptide signaling within the ovarian follicle. In summary, the maintenance of mammalian oocyte meiotic arrest at the GV stage is associated with sustained, high levels of cAMP. These concentrations of cAMP are established by the activity of oocyte adenylyl cyclase, which synthesizes cAMP in response to the constitutive activity of GPR3 and GPR12 via Gs, and by the inhibition of oocyte PDE3A, which degrade the cyclic nucleotides, via cGMP (Fig. 6). cGMP is produced in cumulus cells upon activation of NPR2 by CNP in mice and by BNP and CNP in pigs, and it is transferred to the oocyte via gap junctions from granulosa-associated cumulus cells. Inhibition of PDE3A is the primary mechanism that maintains oocyte cAMP levels, thereby blocking GVBD. On the other hand, LH promotes the secretion of EGF-like growth factors and thus activates EGF signaling in mural granulosa and cumulus cells. Increasing LH concentrations also lower follicular-fluid b-estradiol concentration. Activation of EGF signaling and deactivation of b-estradiol signaling act together to decrease NPR2 activity in cumulus cells, weakening the effects of granulosa-secreted natriuretic peptides. At the same time, activated EGF signaling plus undetermined signal(s) reduce BNP and CNP secretion by granulosa

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Agricultural University, according to the guidelines of Animal Care Committee of China Agricultural University, and all efforts were made to minimize suffering. These prepubertal pigs, 130150 days old, were allocated to five treatment groups (Zhang et al., 2014): control (n ¼ 8) (slaughtered after saline injection); group P48 (slaughtered 48 hr after intravenous injection of PMSG into the ear vein; 1,000 IU); group P72 (slaughtered 72 hr after PMSG treatment); group h18 (slaughtered 90 hr after PMSG treatment plus injection of 500 IU hCG at 72 hr); and group h36 (slaughtered 108 hr after PMSG treatment plus injection of 500 IU hCG at 72 hr).

Porcine COC Culture and Assessment of Nuclear Maturation

Figure 6. Model depicting the signaling pathways that mediate LH regulation of BNP and CNP and their receptor, NPR2, during porcine oocyte meiotic resumption. See Discussion for details. PDE3A, phosphodiesterase 3A; ADCY, adenylyl cyclase.

cells. The resulting reduction in oocytic cGMP accumulation relieves the inhibitory effect on PDE3A, resulting in a decrease in cAMP levels and subsequent resumption of meiosis.

MATERIALS AND METHODS Chemicals All chemicals were purchased from Sigma, St. Louis, MO, unless otherwise noted. PMSG and hCG were purchased from Sansheng Pharmaceutical Co., Ltd. (Ningbo, China). Human CNP-22 (Sigma, catalog # N8768) was used in the present study because the biologically active 22-amino acid CNP is identical in human, dog, pig, horse, and sheep. Human LH (catalog # L5259), AREG (catalog # A7080), b-Estradiol (catalog # E2758), and porcine BNP-32 (catalog # B6651) were purchased from Sigma.

Animals and Ovaries Ovaries obtained from 12-month-old pigs at a local slaughterhouse were transported to our laboratory in phosphate-buffered saline (PBS) supplemented with 100 U/mL penicillin (Huabei Medical, Jizhou, China) and 50 mg/mL streptomycin sulfate (Sigma) at 378C. COCs and granulosa cells used for in vitro experiments were derived from these tissues. To detect transcript abundance of EGF-like growth factors in vivo, we used samples collected in our previous studies (Zhang et al., 2014). A total of 40 triple-crossbred, prepubertal pigs (male Landrace  female Large Yorkshire, followed by mating with a male Duroc) were reared at the Anyang Farm. All animals were kept in the facility at China

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Porcine ovaries were collected from pigs slaughtered at a local abattoir, and delivered to our laboratory within 1.52 hr of slaughter in sterile saline maintained at 35388C. COCs were aspirated from healthy follicles 36 mm in diameter, as described previously (Tsafriri and Channing, 1975a). Only COCs with a uniform ooplasm and compact cumulus were selected. These COCs were then washed in Tyrode’s lactate-HEPES medium with 0.1% polyvinyl alcohol. Each group, consisting of 150 COCs (unless otherwise stated), were transferred into individual wells of 24-well culture dishes (Nunclon; Nunc, Roskilde, Denmark) containing 500 mL of tissue-culture medium (TCM)199 supplemented with 0.23 mM sodium pyruvate, 2 mM glutamine, 100 nM b-estradiol, 3 mg/mL lyophilized crystalline bovine serum albumin, 100 IU/mL penicillin G, and 25 mg/mL streptomycin sulfate. The culture medium was equilibrated for more than 6 hr before culture. Cultures were maintained in an atmosphere of 5% CO2 in air at 38.58C. After culturing, oocytes were harvested, fixed in acetic acid/ethanol (1/3 v/v) for 48 hr, and stained with 1% (w/v) orcein prior to phase-contrast microscopic examination (200) to evaluate the chromatin configuration via visualization of a GV (intact nuclear envelope and nucleolus with loose chromatin mass) or GVBD (condensed chromosome).

RNA Extraction and Quantitative Real-Time PCR Total RNA was isolated using TRIzol (Life Technologies, Carlsbad, CA), according to the manufacturer’s protocol. Total RNA (1 mg) from each sample was incubated for 20 min at 258C with 0.5 U DNase I (Invitrogen, Carlsbad, CA) before reverse transcription to eliminate genomic DNA contamination. First-strand cDNA was synthesized by reverse transcription (Promega Reverse Transcription System, Madison, WI) of 1 mg total RNA. Reverse transcription was performed for 1 hr at 428C. Quantitative real-time PCR was performed in 96-well plates (Applied Biosystems, Foster City, CA) in 25-mL reaction volumes containing 12.5 mL SYBR Green PCR Master Mix (Applied Biosystems), 15 ng cDNA, the appropriate primers, and nuclease-free water. PCR was performed on an ABI 7500 Sequence Detection System (Applied Biosystems) using

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TABLE 1. Primers Used for ISH, PCR, and Quantitative Real-Time PCR Gene

PCR cycle

Annealing temp (8)

Product length (bp)

50 -CCAATCTCCTAGATGCCACATTGAC 50 -GCTCAGCAACAGAAAGAAATCCC

28

55

1,121/855 411/185

AREG

50 -CACCAGAACAAAAAGGTTCTGTC 50 -AAGTCCATGAAGACTCACACCAT

30

57.5

330

ACTIN

50 -GGGAGATCGTGCGGGACATC 50 -CCGCCAGACAGCACCGTGTT

24

57.5

298

50 -TGCTCCTGTTCTTGCACCTGTTG 50 -GCTCCTGTATCCCTGGCAGTTCT

40

60

93

NPPC

50 -AGGCAACAAGAAGGGTTTGTC 50 -ACTAACATCCCAGGCCGCT

40

60

83

NPR2

50 -CTACTCAGGAGCCGAGAAGCAG 50 -CGCCACAATCGCCAGAGTTGAA

40

60

151

AREG

50 -CCGTGGTGCTGTCACTCTTGATC 50 -CGAGGACATCTCACTTCTGGAGGT

40

60

149

EREG

50 -ACCACACCACCGAAGCGAAGA 50 -GCCACCACGAAGCGACATCTCCGTCCAA 50 -CGAGCACTTCTTCTTAA 50 -GGAACCAGCAACTATGATGAGGAAC 50 -AGCAATGCCTCCTGCACCACCA 50 -TGAGTCCCTCCACGATGCCGAA

40

60

100

40

60

92

40

60

82

ISH and PCR LHR

qRT-PCR NPPB

BTC GAPDH

Primers

the following parameters: 10 min at 958C, followed by 40 cycles of 15 s at 958C and 1 min at 608C. The primer pairs used are shown in Table 1. All expression levels were normalized to those of GAPDH. A melting curve was generated after the PCR was complete, using temperature increments of 0.58C every two cycles (62 cycles total) starting at 658C, with fluorescence acquired after each step. The relative level of target gene expression in each sample was calculated using the 2DDCt formula, as described previously (Livak and Schmittgen, 2001).

hybridization, sections were incubated with hybridization solution at 558C for 18 hr. The hybridization solution was prepared by adding 400 ng/mL DIG-cRNAs to hybridization buffer, followed by heating for 10 min at 958C to denature probes, and immediate chilling on ice. Slides were then washed in 5 SSC at 558C for 30 min, 2 SSC at 558C for 30 min, 1 SSC at 558C for 30 min, 0.2 SSC at 558C for 15 min, and 0.2 SSC at room temperature for 15 min. Immunological detection was performed as described previously (Wandji et al., 2000).

In Situ Hybridization

Radioimmunoassay for BNP-32 and CNP-22 in Cultured Medium

To generate AREG probes, DNA regions containing sequences unique to AREG were amplified using specific primers (Table 1) and subcloned into the pGEM-T Easy Vector (Promega, Madison, WI). Complementary antisense or sense cRNA probes were prepared using digoxigenin (DIG) RNA Labeling Kits (Roche Diagnostics, Indianapolis, IN). Follicles from treated gilts were embedded in optimal cutting temperature (OCT) compound and frozen at 808C prior to analysis. Frozen sections (15 mm thick) were prepared using a CM 1950 cryostat microtome (Leica, Wetzlar, Germany), and mounted on Super-Frost Plus slides (Thermo Scientific, Schwerte, Germany). Sections were pre-hybridized at 558C with hybridization buffer [50% formamide, 5 SSC, 5 Denhardts, 1.5% (w/v) polyvinylpyrrolidone, 200 mg/mL yeast tRNA (Roche, Germany), and 500 mg/mL herring sperm DNA (Roche)] for 5 hr in a hybridization oven with 5 SSC. After pre-

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Medium was harvested at the end of the culture period, and centrifuged at 10,000g at 48C for 15 min. The supernatants were collected in centrifuge tubes containing aprotinin (Phoenix Biotech, Burlingame, CA; catalog #RK-APRO; 0.6 TIU/mL) and stored at 808C prior to the assay. The assay for BNP and CNP in culture medium was performed based on our previous radioimmunoassay (Zhang et al., 2014). BNP and CNP levels were measured using commercial kits that exhibited no cross reactivity [porcine BNP-32 (Phoenix Biotech; catalog #T-011-10) and CNP (human, rat, mouse, pig; #T-012-03)]. The sensitivity of the BNP assay was 47.1 pg/mL and the CNP assay 17.9 pg/mL. BNP was measured using an intra-assay variation of less than 4.3% and an inter-assay variation of less than 8.7%. CNP was measured using an intra-assay variation of 5% and an inter-assay variation of 12.2%.

Mol. Reprod. Dev. 81:1030–1041 (2014)

EGF SIGNAL DECREASES BNP

COC Co-Culture With Granulosa Cells Since serum-free systems established for granulosa cells do not support adequate attachment of porcine granulosa cells, we developed COC and granulosa-cell coculture medium as previously reported (Tsafriri and Channing, 1975b): Medium 199 with 25 nM HEPES buffer supplemented with 15% charcoal-stripped special fetal bovine serum (FBS) (GIBCO, catalog # 12676-029), 1 mM L-glutamine, 0.03 nM sodium pyruvate, 5 mg/mL insulin, 100 nM b-estradiol, 100 IU/mL penicillin G, and 25 mg/mL streptomycin sulfate. We used charcoal-stripped FBS instead of normal FBS in our culture system to minimize the effects that various residual hormones in untreated FBS can exert on oocyte maturation. Granulosa cells (107) from healthy follicles (36 mm) were harvested as described previously (Tsafriri and Channing, 1975b), cultured in the abovementioned medium for 12 hr until the cells attached, and then transferred into fresh medium (400 mL) for another 12 hr. COCs were then co-cultured with these granulosa cells for 44 hr. Prior to culture, we determine the presence of residual BNP and CNP in the culture medium powder and in the charcoal-stripped special FBS; minimal amounts of BNP and CNP were detected.

Measurement of cGMP by Radioimmunoassay cGMP levels were measured using commercial radioimmunoassay kits (Institute of Isotopes Ltd., Budapest, Hungary), as in our previous work (Zhang et al., 2014). The working range of the assay, according to the manufacturer, is 2128 fmol per tube using an acetylation protocol or 506,400 fmol per tube using a non-acetylation protocol. This kit showed no cross-reactivity with cAMP, AMP, ADP, ATP, GMP, GDP, or GTP. After COCs were cultured as described above, groups of 250 COCs were mechanically dissociated via pipetting using a small-bore glass pipette in TCM-199 medium (Invitrogen) containing 0.2 mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma) to separate cumulus cells from oocytes. After three washes in PBS, all samples were solubilized in 100 mL 0.1 M hydrochloric acid on ice for 20 min, snap-frozen in liquid nitrogen, and stored at 808C until needed. 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%.

ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (No. 2014CB138503, 2013CB945500), Chinese Universities Scientific Fund (2013YJ002).

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Epidermal growth factor-network signaling mediates luteinizing hormone regulation of BNP and CNP and their receptor NPR2 during porcine oocyte meiotic resumption.

The epidermal growth factor (EGF) network, induced by luteinizing hormone (LH), plays an essential role during the regulation of oocyte maturation, cu...
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