Spontaneous Oocyte Maturation Research Articles

Dual roles of PDE9a in meiotic maturation of zebrafish oocytes

The essential role of cyclic guanosine monophosphate (cGMP) signaling in regulating the oocyte meiotic cell cycle has been established. However, control of the level of cGMP in ovarian follicles is unclear. The cGMP-hydrolyzing phosphodiesterases (PDEs) are important in regulating the cellular cGMP level. We used zebrafish as a model to study the role of a cGMP-hydrolyzing phosphodiesterase-9a (PDE9a) in meiotic maturation of oocytes. Three PDE9a coding genes (PDE9aa, PDE9ab, and PDE9ac) were identified in zebrafish. Both pde9aa and pde9ac are expressed in most adult tissues including the ovary, but pde9ab is only expressed in the ovary, kidney, pituitary, and brain. All three pde9as mRNA exhibited different expression profiles during folliculogenesis. All of them are highly expressed in the oocyte but not in the follicular cell. The expression of both pde9aa and pde9ab, but not pde9ac, in ovarian follicles increases during oocyte maturation either in natural ovulatory cycle or induced by administration of hCG in vivo. We overexpressed pde9aa by injection of capped pde9aa mRNA into the oocytes. The cGMP level was decreased, and oocyte maturation was stimulated. When the activity of PDE9a was blocked by a specific inhibitor, Bay736691, the oocyte maturation was also stimulated. The stimulatory effect could be blocked by a gap junction blocker. However, the spontaneous oocyte maturation of denuded oocytes was not largely affected after treatment with Bay736691. All of the mature oocytes obtained by either treatment of Bay736691 or injection of pde9aa mRNA, could be fertilized in vitro. These results demonstrate the dual roles of PDE9a in oocyte maturation. The basal level of PDE9a is responsible for maintaining the meiotic arrest, and the increased level of PDE9a induced by LH signaling is helpful for stimulating meiotic maturation by hydrolyzing cGMP in oocytes.

The Phosphodiesterase Inhibitor, Isobutyl-1-Methylxanthine Prevents the Sudden Drop in Cyclic Adenosine Monophosphate Concentration and Modulates Glucose Metabolism of Equine Cumulus–Oocyte Complexes Matured in Vitro

Spontaneous nuclear maturation of mammalian oocytes can occur when physically removed from the ovarian follicle during in vitro oocyte maturation (IVM), largely because of a decrease in cyclic adenosine monophosphate (cAMP) concentration. Modulation of oocyte cAMP during IVM by using phosphodiesterase inhibitors has been shown to maintain elevated oocyte cAMP concentrations and control meiotic resumption of bovine and ovine oocytes. This study determined the effect of inclusion of isobutyl-1-methylxanthine (IBMX) during collection and the first 12 hours of incubation of equine oocytes on cAMP concentration and glucose metabolism of cumulus–oocyte complexes (COCs). Abattoir-derived COCs were collected in aspiration medium with (Asp-IBMX) or without (Asp) IBMX. Cumulus–oocyte complexes were then incubated for 12 hours in IVM medium with (Mat-IBMX) or without (Mat) IBMX, followed by additional 24 hours in Mat medium. The cAMP concentration, glucose consumption, lactate production, and metaphase II rates of the COCs were assessed. Cumulus–oocyte complexes aspirated into Asp-IBMX (62.2 ± 2.6 fmol per COC) medium had higher cAMP concentration than Asp (31.8 ± 2.8 fmol per COC) control group (P < .05). Likewise, at 12 hours of IVM, Mat-IBMX group (33.2 ± 2.1 fmol per COC) had higher cAMP concentration than the Mat group (7.68 ± 0.5 fmol per COC; P < .05). Glucose consumption and lactate production were lower during the first 12 hours of incubation in COCs cultured in Mat-IBMX (P < .05). Isobutyl-1-methylxanthine prevented the rapid drop in cAMP concentration and altered metabolism of glucose by the COC. Preventing the sudden drop in cAMP prevents the premature nuclear maturation of in vitro–matured oocytes causing poor developmental competence.

C-C motif chemokine receptor 2 as a novel intermediate in the ovulatory cascade.

Expression of immune function genes within follicle cells has been reported in ovaries from many species. Recent work from our laboratory showed a direct effect of the monocyte chemoattractant protein 1/C-C motif chemokine receptor 2 system within the feline cumulus oocyte complex, by increasing the mRNA levels of key genes involved in the ovulatory cascade in vitro. Studies were designed to evaluate if C-C motif chemokine receptor 2 acts as a novel mediator of the ovulatory cascade in vitro. Therefore, feline cumulus oocyte complexes were cultured in the presence or absence of a highly selective C-C motif chemokine receptor 2 antagonist together with known inducers of cumulus-oocyte expansion and/or oocyte maturation to assess mRNA expression of key genes related to periovulatory events in other species as well as oocyte maturation. Also, the effects of recombinant monocyte chemoattractant protein 1 on spontaneous or gonadotrophin-induced oocyte maturation were assessed. This is an in vitro system using isolated cumulus oocyte complexes from feline ovaries. The present study reveals the modulation of several key ovulatory genes by a highly selective C-C motif chemokine receptor 2 antagonist. However, this antagonist was not enough to block the oocyte maturation induced by gonadotropins or amphiregulin. Nonetheless, recombinant monocyte chemoattractant protein 1 had a significant effect on spontaneous oocyte maturation, increasing the percentage of metaphase II stage oocytes in comparison to the control. This is the first study in any species to establish C-C motif chemokine receptor 2 as a mediator of some actions of the mid-cycle gonadotrophin surge.

High cGMP and low PDE3A activity are associated with oocyte meiotic incompetence

ABSTRACT Resumption of meiosis in mammalian oocytes, defined as oocyte maturation, is stimulated by luteinizing hormone (LH). Fully grown oocytes can also mature spontaneously, upon their release from the ovarian follicle. However, growing oocytes fail to resume meiosis in vitro and the mechanism underlying their meiotic incompetence is unknown. It is commonly accepted that a drop in intraoocyte cyclic guanosine monophosphate (cGMP) resulting in the elevated activity of the oocyte-specific PDE3A leads to a decrease in cAMP content, essential for reinitiation of meiosis. We explored the regulation of these cyclic nucleotides and their degrading PDE3A in growing oocytes. Our research addressed the LH-induced rather than spontaneous oocyte maturation. We examined 16–21 as compared to 25-day-old, PMSG-primed rats, treated with the LH analog, hCG. The effect of LH was also examined ex vivo, in isolated ovarian follicles. We found that hCG failed to induce oocyte maturation and ovulation in the younger animals and that ovulation-associated genes were not upregulated in response to this gonadotropin. Furthemore, the drop of intraoocyte cGMP and cAMP observed in fully grown oocytes upon exposure of the ovary to LH, was not detected in growing oocytes. Interestingly, whereas the global expression of PDE3A in growing and fully grown oocytes is similar, a significantly lower activity of this enzyme was determined in growing oocytes. Our findings show that meiotic incompetence is associated with a relatively high oocyte cGMP concentration and a low activity of PDE3A, which in follicle-enclosed oocytes may represent the failure of the somatic follicle cells to respond to LH.

The role of PKG in oocyte maturation of zebrafish

Recently the importance of cyclic guanosine monophosphate (cGMP) signaling pathway in oocyte maturation has been well demonstrated in several species. However, as the primary downstream effector of the cGMP signaling pathway, little is known on the role of cGMP-dependent protein kinase (PKG) in oocyte maturation. In the present study, the expression, regulation and function of PKG in oocyte maturation was investigated in zebrafish. We identified four distinct PKG coding genes (named Prkg1a, Prkg1b, Prkg2, and Prkg3) in zebrafish. All prkgs are expressed in the ovary, and both prkg1a and prkg1b could be regulated by human chronic gonadotropin in follicular cells during oocyte maturation. We found that a cGMP analogue, 8-Br-cGMP, could stimulate oocyte maturation in a dose- and time-dependent manner. Such stimulatory effects of cGMP could be totally blocked by a PKG specific inhibitor, KT-5823. Intriguingly, we further found KT5823 could significantly attenuate spontaneous oocyte maturation in intact follicles but not in the denuded oocytes, suggesting that the activity of PKG in follicular cells is important for oocyte maturation. All of these results clearly demonstrate that PKG is involved in oocyte maturation in zebrafish.

Xenopus laevis oocyte maturation is affected by metal chlorides

Few studies have been conducted using Xenopus laevis germ cells as oocytes, though these cells offer many advantages allowing both electrophysiological studies and morphological examination.Our aim was to investigate the effects of metal (cadmium, lead, cobalt and zinc) exposures using cell biology approaches. First, cell survival was evaluated with both phenotypical and electrophysiological approaches. Secondly, the effect of metals on oocyte maturation was assessed with morphological observations and electrophysiological recordings.From survival experiments, our results showed that metal chlorides did not affect cell morphology but strongly depolarized X. laevis oocyte resting potential. In addition, cadmium chloride was able to inhibit progesterone-induced oocyte maturation. By contrast, zinc, but also to a lesser extent cadmium, cobalt and lead, were able to enhance spontaneous oocyte maturation in the absence of progesterone stimulation. Finally, electrophysiological recordings revealed that some metal chlorides (lead, cadmium) exposures could disturb calcium signaling in X. laevis oocyte by modifying calcium-activated chloride currents. Our results demonstrated the high sensitivity of X. laevis oocytes toward exogenous metals such as lead and cadmium. In addition, the cellular events recorded might have a predictive value of effects occurring later on the ability of oocytes to be fertilized. Together, these results suggest a potential use of this cellular lab model as a tool for ecotoxicological assessment of contaminated fresh waters.

G-protein coupled estrogen receptor (GPER) inhibits final oocyte maturation in common carp, Cyprinus carpio

GPR-30, now named as GPER (G protein-coupled estrogen receptor) was first identified as an orphan receptor and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. Later studies demonstrated that GPER has the characteristics of a high affinity estrogen membrane receptor on Atlantic croaker and zebra fish oocytes and mediates estrogen inhibition of oocyte maturation in these two distantly related teleost. To determine the broad application of these findings to other teleost, expression of GPER mRNA and its involvement in 17β-estradiol mediated inhibition of oocyte maturation in other cyprinid, Cyprinus carpio was investigated. Carp oocytes at pre-vitellogenic, late-vitellogenic and post-vitellogenic stages of development contained GPER mRNA and its transcribed protein with a maximum at late-vitellogenic oocytes. Ovarian follicular cells did not express GPER mRNA. Carp oocytes GPER mRNA was essentially identical to that found in other perciformes and cyprinid fish oocytes. Both spontaneous and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)-induced oocyte maturation in carp was significantly decreased when they were incubated with either E2, or GPER agonist G-1. On the other hand spontaneous oocyte maturation was significantly increased when carp ovarian follicles were incubated with an aromatase inhibitor, fadrozole, GPER antagonist, G-15 and enzymatic removal of the ovarian follicle cell layers. This increase in oocyte maturation was partially reversed by co-treatment with E2. Consistent with previous findings with human and fish GPR30, E2 treatment in carp oocytes caused increase in cAMP production and simultaneously decrease in oocyte maturation, which was inhibited by the addition of 17,20β-P. The results suggest that E2 and GPER play a critical role in regulating re-entry in to meiotic cell cycle in carp oocytes.

Effects of different oocyte retrieval andin vitromaturation systems on bovine embryo development and quality

Cyclic adenosine monophosphate (cAMP) modulators have been used to avoid spontaneous oocyte maturation and concomitantly improve oocyte developmental competence. The current work evaluated the effects of the addition of cAMP modulators forskolin, 3-isobutyl-1-methylxanthine (IBMX) and cilostamide during in vitro maturation on the quality and yields of blastocysts. The following experimental groups were evaluated: (i) slicing or (ii) aspiration and maturation in tissue culture medium (TCM)199 for 24 h (TCM24slicing and TCM24aspiration, respectively), (iii) aspiration and maturation in the presence of cAMP modulators for 30 h (cAMP30aspiration) and in vivo-produced blastocysts. In vitro-matured oocytes were fertilized and presumptive zygotes were cultured in vitro to assess embryo development. Cleavage, blastocyst formation, blastocyst cell number, mRNA abundance of selected genes and global methylation profiles were evaluated. Blastocyst rate/zygotes for the TCM24aspiration protocol was improved (32.2 ± 2.1%) compared with TCM24slicing and cAMP30aspiration (23.4 ± 1.2% and 23.3 ± 2.0%, respectively, P 0.05), while those from the other groups were significantly elevated. It is concluded that retrieval, collection systems and addition of cAMP modulators can affect oocyte developmental competence, which is reflected not only in blastocyst rates but also in global DNA methylation and gene expression patterns.

Bi-directional communication with the cumulus cells is involved in the deficiency of XY oocytes in the components essential for proper second meiotic spindle assembly

The oocyte becomes competent for embryonic development by involving mutual communication with cumulus cells (CCs) during folliculogenesis. How this communication takes place under physiological conditions is not fully understood. Current study examined oocyte–CCs communication in the XY sex-revered female mouse. We have previously found that the XY oocyte is defective in its cytoplasm, causing abnormal MII-spindle assembly and a failure in embryonic development. Our present study showed that transcript levels of Pfkp, Pkm2 and Ldh1 involved in glycolysis were lower in the CCs surrounding XY oocytes than in those surrounding XX oocytes. ATP contents in XY oocytes were also lower than those in XX oocytes, suggesting that lower glycolytic gene expression in CCs resulted in lower ATP contents in the enclosed oocyte. Co-culture of oocytectomized CC-oocyte complexes (COCs) with denuded oocytes showed that XY oocytes were less efficient than XX oocytes in promoting glycolytic gene expression in CCs. Furthermore, both glycolytic gene expression levels in CCs and ATP contents in oocytes of XY COCs increased to similar levels to those of XX COCs after culture for 20h in the presence of milrinone (=preincubation), which prevented spontaneous oocyte maturation. By increasing ATP levels in XY oocytes by either COC preincubation or ATP microinjection into oocytes prior to in vitro maturation, an improvement in MII-spindle assembly was observed. We conclude that the XY oocyte produces lesser amounts of paracrine factors that affect its companion CCs, which in turn make the ooplasm deficient in its components, including ATP, essential for MII-spindle assembly.

Reversible meiotic arrest in feline oocytes

Increasing intracellular concentrations of cyclic adenosine monophosphate (cAMP) within the cumulus-oocyte complex (COC) inhibits or delays spontaneous oocyte maturation and improves the developmental competence of the oocyte in many species, but information for carnivores is limited. The objectives of the present study were to describe the effects of isobutyl methylxanthine (IBMX), which decreases cAMP degradation, and forskolin, which increases cAMP production, on spontaneous and induced maturation (by equine chorionic gonadotrophin (eCG) and epidermal growth factor (EGF)) of feline oocytes and to evaluate the reversibility of IBMX-induced arrest by measuring the resumption of meiosis and embryonic development following IVF. IBMX decreased (P<0.05) the incidence of spontaneous (6.7% vs 42.0%, metaphase II (MII)) and induced (5.6% vs 66.1% MII) maturation after 24 h of culture. In contrast, forskolin stimulated meiosis (81.7% MII; P<0.05). Following 12 h of culture with IBMX and an additional 24h with eCG and EGF in the absence of IBMX, the proportions of oocytes reaching MII (66.1%), cleaving (79.9%) and developing to the blastocyst stage (15.3%) were similar (P>0.05) to oocytes cultured continuously with eCG and EGF (70.2%, 83.0% and 18.1%, respectively). These results demonstrate that IBMX reversibly inhibits both spontaneous and eCG+EGF-induced meiosis in feline oocytes without compromising the oocyte's developmental competence.

268 CYCLIC GUANOSINE MONOPHOSPHATE INHIBITS PHOSPHODIESTERASE 3 ACTIVITY IN PORCINE OOCYTES

The level of cyclic adenosine monophosphate (cAMP) within oocytes has been shown to play a critical role in maintaining meiotic arrest. High levels of intracellular cAMP prevent spontaneous oocyte maturation in vitro, whereas a decrease in oocyte cAMP is associated with the resumption of meiosis. Another cyclic nucleotide that also was recently proposed as being involved in meiotic resumption is cyclic guanosine monophosphate (cGMP), which could be regulating phosphodiesterase (PDE) 3 activity. The aim of this study was to determine whether cGMP inhibits cAMP-PDE activity in porcine oocytes. With the method described previously by Sasseville et al. (2006 BMC Dev. Biol. 6, 47), PDE activity was measured in groups of 10 oocytes cultured in the absence (control) or presence of different concentrations of cGMP (1, 3, 10, 30, 100, 300, 1000, and 3000 nM) or with the PDE3 inhibitor cilostamide (10 µM). Before assaying PDE activity, the cumulus–oocyte complexes (COC) were matured in vitro for 24 h in the presence of pregnant mare serum gonadotropin (5 IU) and hCG (5 IU) at 38.5°C in 5% CO2. The COC were homogenised in a hypotonic buffer. Data were analysed using one-way ANOVA followed by Duncan’s post-hoc test. Differences with P &lt; 0.05 were considered significant. Results showed that 300, 1000, and 3000 nM cGMP inhibited PDE3 activity (7.9, 5.1, and 4.1 fmol min–1 per COC; P &lt; 0.05) at levels below the controls (13.2 fmol min–1 per COC) and were similar to the activity observed in the presence of (2.4 fmol min–1 per COC; P &gt; 0.05). The other concentrations tested were similar to activity levels seen in the control (1 to 100 nM; 12.2, 11.3, 10.8, 11.5, and 10.4; P &gt; 0.05). In conclusion, the results support the concept that increasing concentrations of cGMP inhibit PDE activity, suggesting the inhibition of the predominant form of cAMP-PDE present in porcine oocytes, PDE3. These results support the hypothesis that cGMP inhibits PDE activity in porcine oocytes. Further work is needed to determine the role this plays in maintaining high cAMP levels and inhibiting oocyte nuclear maturation. Financial support from FGZ FAPESP 2010/20188-6 and 2010/18023-9 is acknowledged.

Spatiotemporal Expression of Bone Morphogenetic Protein Family Ligands and Receptors in the Zebrafish Ovary: A Potential Paracrine-Signaling Mechanism for Oocyte-Follicle Cell Communication1

The bone morphogenetic proteins (BMPs), originally identified by their abilities to induce bone and/or cartilage formation, have been reported to be involved in various growth and differentiation processes, including reproduction. Although mammalian models are more frequently used to study the BMP system in reproduction, we have extended the study to the zebrafish, an excellent model for studying female reproduction in teleosts. Reverse transcription-PCR analysis revealed the expression of the Bmp ligands (bmp2a, bmp2b, bmp4, bmp6, and bmp7a) and the type II Bmp receptors (bmpr2a and bmpr2b) in various tissues, including the ovary. Spatiotemporal distribution of these Bmp ligands and receptors in the ovary was then investigated in this study. Reverse transcription-PCR on isolated follicle layers and denuded oocytes demonstrated that all Bmp ligands examined were exclusively or abundantly expressed in the oocyte, whereas the two receptors were expressed exclusively in the follicle layers, strongly suggesting a potential paracrine signaling from the oocyte towards the follicle layer by various Bmp ligands. This supports the current view that instead of being passively controlled and nurtured by the follicle layer for its growth and development, the oocyte may play an active role by releasing various growth differentiation factors to regulate follicle layer function. Quantitative analysis of temporal expression profiles during folliculogenesis revealed an increased expression of bmp2a, bmp2b, bmp4, and bmp6 from primary growth (stage I) to previtellogenic (stage II) stages, followed by steady declines toward the end of folliculogenesis when the follicles became fully grown. On the contrast, the BMP receptors (bmpr2a and bmpr2b) consistently showed an increase in expression during folliculogenesis, with the peak levels reached at the full-grown stage prior to final oocyte maturation. The spatiotemporal expression patterns of the Bmp family in the zebrafish follicles provide important insights into potential roles for Bmps during follicle development as oocyte-derived factors. Further experiments using recombinant zebrafish Bmp4 showed that Bmp4 had an inhibitory effect on spontaneous oocyte maturation in vitro, but not 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP)-induced oocyte maturation in vitro.

Involvement of Epidermal Growth Factor Receptor Signaling in Estrogen Inhibition of Oocyte Maturation Mediated Through the G Protein-Coupled Estrogen Receptor (Gper) in Zebrafish (Danio rerio)1

Oocyte maturation (OM) in teleosts is under precise hormonal control by progestins and estrogens. We show here that estrogens activate an epidermal growth factor receptor (Egfr) signaling pathway in fully grown, denuded zebrafish (Danio rerio) oocytes through the G protein-coupled estrogen receptor (Gper; also known as GPR30) to maintain oocyte meiotic arrest in a germinal vesicle breakdown (GVBD) bioassay. A GPER-specific antagonist, G-15, increased spontaneous OM, indicating that the inhibitory estrogen actions on OM are mediated through Gper. Estradiol-17beta-bovine serum albumin, which cannot enter oocytes, decreased GVBD, whereas treatment with actinomycin D did not block estrogen's inhibitory effects, suggesting that estrogens act at the cell surface via a nongenomic mechanism to prevent OM. The intracellular tyrosine kinase (Src) inhibitor, PP2, blocked estrogen inhibition of OM. Expression of egfr mRNA and Egfr protein were detected in denuded zebrafish oocytes. The matrix metalloproteinase (MMP) inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor, increased spontaneous OM, whereas the MMP activator, interleukin-1alpha, decreased spontaneous OM. Moreover, inhibitors of EGFR (ErbB1) and extracellular-related kinase 1 and 2 (Erk1/2; official symbol Mapk3/1) increased spontaneous OM. In addition, estradiol-17beta and the GPER agonist, G-1, increased phosphorylation of Erk, and this was abrogated by simultaneous treatment with the EGFR inhibitor. Taken together, these results suggest that estrogens act through Gper to maintain meiotic arrest via an Src kinase-dependent G-protein betagamma subunit signaling pathway involving transactivation of egfr and phosphorylation of Mapk3/1. To our knowledge, this is the first evidence that EGFR signaling in vertebrate oocytes can prevent meiotic progression.

262 ISOBUTYLMETHYLXANTHINE REVERSIBLY SUPPRESSES SPONTANEOUS AND EQUINE CHORIONIC GONADOTROPIN/EPIDERMAL GROWTH FACTOR-STIMULATED MEIOSIS IN FELINE OOCYTES

Recent studies have shown that short-term exposure to phosphodiesterase inhibitors (decreased cAMP degradation) or adenylate cyclase stimulators (increased cAMP synthesis) inhibits spontaneous oocyte maturation and improves the developmental competence of oocytes after gonadotropin-induced maturation. A similar approach may improve the developmental competence of in vitro matured feline oocytes. The objectives of this study were to 1) determine if a nonspecific phosphodiesterase inhibitor [isobutylmethylxanthine (IBMX); 100 μM] or an adenylate cyclase stimulator (forskolin; 100 μM) would suppress spontaneous or eCG (1 IU mL–1)/epidermal growth factor (EGF; 25 ng mL–1)-induced maturation of feline oocytes in vitro; 2) evaluate the reversibility of chemically induced meiotic arrest; and 3) assess the developmental competence of meiotically arrested oocytes. Cumulus–oocyte complexes were cultured in modified feline optimized culture medium [6 mM glucose, 0.1 mM cysteamine, 0.6 mM cysteine, 0.5× minimal essential medium (MEM) essential amino acids, 1× MEM vitamins, and 1× insulin-transferrin-selenium; Herrick et al. 2010 Biol. Reprod. 82, 552–562] supplemented with (+) or without (–) IBMX, forskolin, or eCG/EGF (eE). The IBMX decreased (P &lt; 0.05; mixed model ANOVA) the incidence of spontaneous maturation (–eE) after 24 h of culture (84.6%, germinal vesicle, GV; 6.7% metaphase II, MII) compared with control (no supplements) oocytes (18.8% GV, 42.0% MII). Forskolin (–eE) stimulated (P &lt; 0.05) meiosis (6.1% GV, 81.7% MII), so it was not tested in subsequent experiments. The IBMX also inhibited (P &lt; 0.05) eE-induced meiosis compared with control (–IBMX +eE) oocytes after 18 h (87.6 v. 27.1% GV, 2.5 v. 11.0% MII), 24 h (68.0 v. 11.9% GV, 5.6 v. 66.1% MII), and 30 h (58.1 v. 9.4% GV, 14.6 v. 64.4% MII) of culture. To evaluate the reversibility of IBMX, oocytes were cultured +IBMX –eE for 12 h and then transferred to medium –IBMX +eE. After 12 h of culture –IBMX +eE (24 h total), fewer (P &lt; 0.05) IBMX-exposed oocytes were MII (6.6%) compared with control oocytes cultured for 24 h +eE –IBMX (70.2%). The proportion of IBMX-exposed oocytes reaching MII increased (P &lt; 0.05) following 18 h of culture –IBMX +eE (30 h total; 49.4% MII), and by 24 h of culture –IBMX +eE (36 h total; 66.1% MII) was similar (P &gt; 0.05) to the proportion of MII oocytes (78.3%) that had been cultured continuously for 36 h –IBMX +eE. Finally, oocytes were cultured for 0 h or 12 h +IBMX –eE followed by 24 h of culture –IBMX +eE, coincubated with frozen–thawed spermatozoa (5 × 105 sperm mL–1, 22 h), and the resulting embryos cultured (6% CO2, 5% O2) until day 7 post-insemination (Herrick et al. 2007 Biol. Reprod. 76, 858–870). The proportion of oocytes cleaving (83.0 v. 79.9%) and the proportions of oocytes (20.8 v. 18.7%) or embryos (25.2 v. 23.6%) developing to the blastocyst stage were not affected (P &gt; 0.05) by 12 h of IBMX exposure during oocyte maturation. These results demonstrate that IBMX reversibly inhibits both spontaneous and eCG/EGF-induced meiosis in feline oocytes without compromising the oocyte’s developmental competence. This work was supported by the College of Veterinary Medicine’s Companion Animal Memorial Fund at the University of Illinois.

Role of G protein-coupled estrogen receptor 1, GPER, in inhibition of oocyte maturation by endogenous estrogens in zebrafish

Estrogen inhibition of oocyte maturation (OM) and the role of GPER (formerly known as GPR30) were investigated in zebrafish. Estradiol-17β (E2) and G-1, a GPER-selective agonist, bound to zebrafish oocyte membranes suggesting the presence of GPER which was confirmed by immunocytochemistry using a specific GPER antibody. Incubation of follicle-enclosed oocytes with an aromatase inhibitor, ATD, and enzymatic and manual removal of the ovarian follicle cell layers significantly increased spontaneous OM which was partially reversed by co-treatment with either 100 nM E2 or G-1. Incubation of denuded oocytes with the GPER antibody blocked the inhibitory effects of estrogens on OM, whereas microinjection of estrogen receptor alpha (ERα) antisense oligonucleotides into the oocytes was ineffective. The results suggest that endogenous estrogens produced by the follicle cells inhibit or delay spontaneous maturation of zebrafish oocytes and that this estrogen action is mediated through GPER. Treatment with E2 and G-1 also attenuated the stimulatory effect of the teleost maturation-inducing steroid, 17,20β-dihyroxy-4-pregnen-3-one (DHP), on OM. Moreover, E2 and G-1 down-regulated the expression of membrane progestin receptor alpha (mPRα), the intermediary in DHP induction of OM. Conversely DHP treatment caused a > 50% decline in GPER mRNA levels. The results suggest that estrogens and GPER are critical components of the endocrine system controlling the onset of OM in zebrafish. A model is proposed for the dual control of the onset of oocyte maturation in teleosts by estrogens and progestins acting through GPER and mPRα, respectively, at different stages of oocyte development.

Involvement of estradiol-17β and its membrane receptor, G protein coupled receptor 30 (GPR30) in regulation of oocyte maturation in zebrafish, Danio rario

The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20β-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17β, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes.

Estrogen Signaling Characteristics of Atlantic Croaker G Protein-Coupled Receptor 30 (GPR30) and Evidence It Is Involved in Maintenance of Oocyte Meiotic Arrest

Human G protein-coupled receptor 30 (GPR30) mediates estradiol-17beta (E2) activation of adenylyl cyclase in breast cancer cells and displays E2 binding typical of membrane estrogen receptors (mERs). We identified a mER in Atlantic croaker ovaries with characteristics similar to those of human GPR30. To confirm the proposed role of GPR30 as a mER in this distantly related vertebrate group, we cloned GPR30 from croaker ovaries and examined its distribution, steroid binding, and signaling characteristics. Western blot analysis showed the GPR30 protein (approximately 40 kDa) is expressed on the plasma membranes of croaker oocytes and HEK293 cells stably transfected with GPR30 cDNA. Plasma membranes prepared from croaker GPR30-transfected cells displayed high-affinity, limited-capacity, and displaceable binding specific for estrogens, characteristic of mERs. Consistent with previous findings with human GPR30, estrogen treatment of plasma membranes from both croaker ovaries and GPR30-transfected cells caused activation of a stimulatory G protein (Gs) resulting in increased cAMP production. Treatment with E2 as well as G-1, a specific GPR30 ligand, significantly reduced both spontaneous and progestin-induced maturation of both croaker and zebrafish oocytes in vitro, suggesting a possible involvement of GPR30 in maintaining oocyte meiotic arrest in these species. Injection of antisense oligonucleotides to GPR30 into zebrafish oocytes blocked the inhibitory effects of estrogen on oocyte maturation, confirming a role for GPR30 in the control of meiotic arrest. These findings further support our previous suggestion that GPR30 is a vertebrate mER. In addition, the results suggest GRP30 may play a critical role in regulating reentry into the meiotic cell cycle in fish oocytes.

A role for GPRx, a novel GPR3/6/12-related G-protein coupled receptor, in the maintenance of meiotic arrest in Xenopus laevis oocytes

Progesterone-induced Xenopus laevis oocyte maturation is mediated via a plasma membrane-bound receptor and does not require gene transcription. Evidence from several species suggests that the relevant progesterone receptor is a G-protein coupled receptor (GPCR) and that a second receptor–GPR3 and/or GPR12 in mammals–tonically opposes the progesterone receptor. We have cloned a novel X. laevis GPCR, GPRx, which may play a similar role to GPR3/GPR12 in amphibians and fishes. GPRx is related to but distinct from GPR3, GPR6, and GPR12; GPRx orthologs are present in Xenopus tropicalis and Danio rerio, but apparently not in birds or mammals. X. laevis GPRx is mainly expressed in brain, ovary, and testis. The GPRx mRNA increases during oogenesis, persists during oocyte maturation and early embryogenesis, and then falls after the midblastula transition. Microinjection of GPRx mRNA increases the concentration of cAMP in oocytes and causes the oocytes to fail to respond to progesterone, and this block is reversed by co-injecting GPRx with morpholino oligonucleotides. Morpholino injections did not cause spontaneous maturation of oocytes, but did accelerate progesterone-induced maturation. Thus, GPRx contributes to the maintenance of G2-arrest in immature X. laevis oocytes.

The influence of cadmium on Prussian carp oocyte maturation, development of eggs and hatching

The influence of cadmium (Cd) on the last stage of Prussian carp (&lt;I&gt;Carassius auratus gibelio&lt;/I&gt; B.) oocyte maturation and embryonic development was investigated. The (postvitellogenic) oocytes were incubated with carp pituitary homogenate (Chh), 17&amp;alpha;,20&amp;beta;-dihydroxy-4-pregnen-3-one (17&amp;alpha;,20&amp;beta;OH-P) and Cd at the concentrations of 20, 100 and 200&amp;mu;M. Cd was found to stimulate spontaneous oocyte maturation, however it inhibited Chh-stimulated maturation. Cd had no influence on the effects of 17&amp;alpha;,20&amp;beta;OH-P (MIS – maturation inducing steroid). It seems that the action of Cd on the last stage of oocyte maturation takes place before the MIS production. In an &lt;I&gt;in vivo&lt;/I&gt; study Prussian carp eggs were incubated in the water containing Cd at the concentrations of 0, 0.01, 0.1, 1 or 10 mg/l. There were no differences between the final numbers of hatched larvae in the respective groups, however Cd at the highest concentration accelerated hatching and increased the number of deformed larvae. These results suggest that Prussian carp eggs are very resistant to waterborne Cd.

PDE5 modulates oocyte spontaneous maturation via cGMP-cAMP but not cGMP-PKG signaling

Phosphodiesterase type 5 (PDE5), a cGMP specific, cGMP binding phosphodiesterase, specifically hydrolyzes cGMP to 5'-GMP. Here, we examine the distribution of PDE5 in mouse ovary and its effects on spontaneous maturation of mouse oocytes. PDE5 is present in oocytes and cumulus cells of big, antral follicles. Inhibition of activity of PDE5 significantly and reversibly inhibits spontaneous maturation of cumulus-oocyte complexes (COCs). Suppressive effect of PDE5 on spontaneous maturation of COCs is not blocked by the inhibitor of cGMP-dependent protein kinase (PKG). While Sildenafil, an inhibitor of PDE5, has a poor effect on cGMP levels, it significantly increases cAMP levels. These results suggest that the activity of PDE5 plays a role in regulating spontaneous maturation of mouse oocytes and imply that an interaction between cGMP and cAMP signal is involved in this process.