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RESEARCH REPORT
Regulator of G-protein signaling 2 (RGS2) suppresses premature calcium release in mouse eggs
Miranda L. Bernhardt, Katie M. Lowther, Elizabeth Padilla-Banks, Caitlin E. McDonough, Katherine N. Lee, Alexei V. Evsikov, Tracy F. Uliasz, Peter Chidiac, Carmen J. Williams, Lisa M. Mehlmann
Development 2015 142: 2633-2640; doi: 10.1242/dev.121707

Abstract

During oocyte maturation, capacity and sensitivity of Ca2+ signaling machinery increases dramatically, preparing the metaphase II (MII)-arrested egg for fertilization. Upon sperm-egg fusion, Ca2+ release from IP3-sensitive endoplasmic reticulum stores results in cytoplasmic Ca2+ oscillations that drive egg activation and initiate early embryo development. Premature Ca2+ release can cause parthenogenetic activation prior to fertilization; thus, preventing inappropriate Ca2+ signaling is crucial for ensuring robust MII arrest. Here, we show that regulator of G-protein signaling 2 (RGS2) suppresses Ca2+ release in MII eggs. Rgs2 mRNA was recruited for translation during oocyte maturation, resulting in ∼20-fold more RGS2 protein in MII eggs than in fully grown immature oocytes. Rgs2-siRNA-injected oocytes matured to MII; however, they had increased sensitivity to low pH and acetylcholine (ACh), which caused inappropriate Ca2+ release and premature egg activation. When matured in vitro, RGS2-depleted eggs underwent spontaneous Ca2+ increases that were sufficient to cause premature zona pellucida conversion. Rgs2−/− females had reduced litter sizes, and their eggs had increased sensitivity to low pH and ACh. Rgs2−/− eggs also underwent premature zona pellucida conversion in vivo. These findings indicate that RGS2 functions as a brake to suppress premature Ca2+ release in eggs that are poised on the brink of development.

INTRODUCTION

Fully grown mammalian oocytes are arrested in meiotic prophase until a mid-cycle release of luteinizing hormone from the pituitary stimulates resumption of meiosis to the metaphase II (MII) stage, referred to herein as eggs. Fusion of the egg and sperm at fertilization introduces into the egg cytoplasm a sperm-specific phospholipase C (PLCζ) (Kashir et al., 2014), which initiates inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from intracellular stores. An initial, prolonged rise in cytoplasmic Ca2+ is followed by several hours of repetitive, low-frequency Ca2+ oscillations (Runft et al., 2002). These Ca2+ oscillations drive the conversion of the egg to an early embryo by causing a series of downstream responses, including resumption of meiosis, cortical granule exocytosis, which prevents polyspermy, and recruitment of maternal mRNAs needed for successful embryonic development (Ducibella and Fissore, 2008).

During meiotic maturation, oocytes undergo several cytoplasmic changes that dramatically increase the ability of mature eggs to release Ca2+ in response to sperm or exogenous signals (Fujiwara et al., 1993; Jones et al., 1995; Mehlmann and Kline, 1994). These changes include an approximately fourfold increase in Ca2+ stores (Tombes et al., 1992), an increase in IP3 receptor protein levels by 1.5- to twofold (Fissore et al., 1999; Mehlmann et al., 1996) and reorganization of the endoplasmic reticulum (ER) towards the plasma membrane (FitzHarris et al., 2007; Mehlmann et al., 1995), which places the Ca2+ stores proximal to exogenous signals. Priming of the egg for Ca2+ release, although needed for proper Ca2+ signaling after fertilization, is also associated with the risk of parthenogenetic activation should Ca2+ signals occur prior to sperm-egg fusion. Indeed, activation of overexpressed Gq-protein-coupled muscarinic receptors in mouse eggs by exogenous ligands in the absence of sperm causes IP3-mediated Ca2+ release, Ca2+ oscillations and parthenogenetic egg activation (Moore et al., 1993; Williams et al., 1998, 1992). Ca2+ signaling prevents subsequent fertilization by inducing cortical granule release, which modifies the zona pellucida (ZP), a proteoglycan-rich extracellular matrix that surrounds the egg, to cause the ‘block to polyspermy’.

Because the mature egg is exceedingly sensitive to stimuli that can activate IP3-mediated Ca2+ release via G-protein-coupled receptors, we reasoned that a mechanism was in place to suppress this response prior to sperm-egg fusion. Regulator of G-protein signaling (RGS) proteins are a ubiquitous family of proteins that downregulate G-protein-coupled receptor signaling by inhibiting the activity of G-proteins (Heximer and Blumer, 2007). In general, RGS proteins stimulate the hydrolysis of GTP bound to activated Gα subunits, leading to signal termination (Willars, 2006). Here, we tested the hypothesis that RGS2, which inhibits both Gq and Gs signaling (Ingi et al., 1998; Roy et al., 2006; Wang et al., 2004), suppresses Ca2+ release in mature mouse eggs. We report that RGS2 translation during meiotic maturation serves as a safety mechanism to prevent parthenogenetic egg activation prior to fertilization.

RESULTS AND DISCUSSION

RGS2 protein levels increase during oocyte maturation

To determine which RGS proteins are expressed in mouse oocytes, we searched an oocyte gene expression database (Evsikov et al., 2006). Among RGS isoforms, RGS2 had the highest expression levels, ∼500 transcripts per million (Blake et al., 2014). Rgs2 mRNA was highly expressed in germinal vesicle (GV)-stage oocytes, decreased during oocyte maturation and was greatly reduced at the 2-cell stage (Fig. 1A). By contrast, RGS2 protein was minimally detected in GV oocytes but increased ∼20-fold during maturation to the MII stage (Fig. 1B,C). These findings indicate that RGS2 is developmentally regulated and suggest that it functions during oocyte maturation or beyond.

Fig. 1.
Fig. 1.

RGS2 expression in oocytes, eggs and early embryos. (A) Rgs2 mRNA levels; all stages expressed relative to GV oocytes. N=3; graph shows mean±s.e.m. (B) Immunoblot of RGS2 protein in oocytes and eggs. Blot representative of 4 independent replicates; 50 oocytes or eggs/lane. (C) Quantitation of RGS2 immunoblot signal. N=4; graph shows mean±s.e.m. *P<0.05, Mann–Whitney test. GV, GV-stage oocytes; MII, MII eggs; 1C, 1-cell embryos; 2C, 2-cell embryos.

Exposure to acidic pH causes spontaneous activation in eggs lacking RGS2

To test functional roles of RGS2, we used both overexpression and knockdown approaches. Because Gs activity is crucial for the maintenance of prophase arrest prior to oocyte maturation (Mehlmann et al., 2002, 2004), and RGS2 can inhibit the activity of Gs (Roy et al., 2006, 2003; Sinnarajah et al., 2001), we first tested whether altering RGS2 levels affected maturation success. Overexpressing RGS2 in GV oocytes did not stimulate meiotic resumption, and depleting RGS2 in oocytes using RNA interference did not affect the progression of meiosis or meiotic spindle formation (Fig. 2A,B; see supplementary material Table S1 and Fig. S1). These results suggest that RGS2 is not required during oocyte maturation and that these approaches could be used to examine its function in MII eggs.

Fig. 2.
Fig. 2.

Acid-induced Ca2+ release causes resumption of meiosis in eggs lacking RGS2 protein. (A) Immunoblot of GV oocytes following microinjection with cRNA encoding HA-tagged RGS2, probed with monoclonal anti-RGS2 antibody. Arrow, full-length HA-RGS2 band. 20 oocytes/lane. (B) Immunoblot of RGS2 in control MII eggs or eggs matured to MII following microinjection at the GV stage with Rgs2 siRNA. 20 eggs/lane. Arrow, RGS2 band. (C) Appearance of siRNA-injected eggs following ZP removal with acid Tyrode's solution. Eggs in different groups are separated by dashed line. Arrowheads indicate second polar bodies. (D) Average percentage of eggs with second polar body (PB2) emitted by 4.5 h after the indicated treatment. N=5 independent replicates with 8-27 cells per group/replicate; graph shows mean±s.e.m. *P<0.05, ANOVA with Bonferroni's multiple comparison test. (E) Relative level of intracellular Ca2+ in response to lowering pH in control eggs or eggs lacking RGS2. Color indicates approximate pH at each time point. Eight representative tracings shown/group. (F) Percentage of siRNA-injected eggs with a rise in intracellular Ca2+ beginning at the indicated pH. (G) Percentage of morpholino (MO)-injected eggs with a rise in intracellular Ca2+ beginning at the indicated pH. Control MO, scrambled MO; Rgs2 MO, Rgs2-targeted MO. Graphs in F and G indicate cumulative percentage of 3-8 cells/group from n=4 or n=2 experiments, respectively.

RGS2 potently suppresses Gq signaling (Ingi et al., 1998; Wang et al., 2004), and, in mouse eggs, activation of Gq in the absence of sperm leads to Ca2+-mediated resumption of meiosis and complete egg activation (Moore et al., 1993; Williams et al., 1998). Sperm do not appear to utilize this pathway (Williams et al., 1998) but instead stimulate IP3 generation directly by introducing PLCζ (Saunders et al., 2002; Knott et al. 2005). However, Gq activation triggers the same downstream IP3 receptor-mediated Ca2+ release that is essential for fertilization. To determine whether RGS2 activity could impact Ca2+ signals at fertilization, we tested the effect of RGS2 depletion on Ca2+ oscillatory patterns during in vitro fertilization (IVF). We found that RGS2-depleted eggs exhibited normal Ca2+ oscillations, with the exception that the duration of the first Ca2+ transient was slightly, but consistently, shorter (see supplementary material Fig. S2A-D). This finding suggests that the Ca2+ stores were depleted prior to fertilization, which could be explained by either impaired Ca2+ store accumulation during maturation or by premature Ca2+ release. To distinguish between these possibilities, we analyzed Ca2+ stores in control and RGS2-depleted MII eggs by measuring thapsigargin-mediated ER Ca2+ release. There was no difference in Ca2+ stores between control and RGS2-depleted MII eggs when the ZPs were intact (see supplementary material Fig. S2E-G).

As is typical for IVF experiments, the ZP was removed prior to insemination to promote rapid synchronous fertilization during Ca2+ imaging. We noticed that many ZP-free, RGS2-depleted eggs began emitting second polar bodies before sperm addition (Fig. 2C), indicating spontaneous activation. Spontaneous activation was only observed in ZP-free RGS2-depleted eggs, not in ZP-intact eggs (see supplementary material Fig. S3A). Our standard protocol for ZP removal is a brief treatment with acid Tyrode's medium. We therefore tested whether absence of the ZP or the acid exposure was causing the spontaneous activation by using pronase treatment as an alternative method for ZP removal. Pronase-treated eggs did not activate, whereas eggs exposed to acid had high rates of second polar body emission, and most of the activated eggs went on to form pronuclei or to cleave (Fig. 2D; see supplementary material Fig. S3A-D). These findings are consistent with previous observations of acid induction of parthenogenetic activation in mouse and human eggs (Johnson et al., 1990). In addition, spontaneous Ca2+ changes were observed in 25% (4/16) of the siRNA-injected cells prior to addition of sperm, but never (0/15) in controls (see supplementary material Fig. S3E). These Ca2+ changes could explain the shortened first transients of eggs lacking RGS2, because premature Ca2+ release would result in Ca2+ store depletion prior to fertilization. To test this idea, we analyzed Ca2+ stores in ZP-free control and RGS2-depleted MII eggs. Ca2+ stores were significantly lower in RGS2-depleted eggs when the ZPs were removed using acid Tyrode's medium, but not different when the ZPs were removed by manual microdissection (see supplementary material Fig. S3F-H). Taken together, these findings indicate that lack of RGS2 during oocyte maturation does not affect Ca2+ accumulation into ER stores, and suggest that acid-induced premature Ca2+ release in RGS2-depleted eggs causes a reduction in Ca2+ stores and, as a consequence, shortened first Ca2+ transients following fertilization.

Acidic pH induces a rise in intracellular Ca2+ in eggs lacking RGS2 but not in control eggs

To characterize the acid sensitivity of RGS2-depleted eggs, we examined the effect of gradually lowering pH on Ca2+ release. The majority of control eggs treated with acid did not exhibit increases in Ca2+ even at a pH as low as 5 (Fig. 2E,F). However, most RGS2-depleted eggs showed marked increases in Ca2+ starting between pH 6.2 and 6.9, suggesting that RGS2 inhibits acid-induced Ca2+ release. Similar results were obtained using an Rgs2-targeted morpholino oligonucleotide (Fig. 2G), indicating that this response was not due to a non-specific effect of siRNA.

We also examined the Ca2+ response to increased acid in GV-stage oocytes, which have low levels of RGS2 protein, and throughout oocyte maturation. We found that GV- and GVBD-stage oocytes (maturing oocytes that have undergone nuclear envelope breakdown) were very sensitive to lower pH, with virtually all oocytes displaying Ca2+ release starting at pH 6.4-6.6 (Fig. 3A,B). Acid sensitivity decreased during maturation such that fewer metaphase I oocytes and MII-stage eggs responded to lower pH with Ca2+ release (Fig. 3A,B). This decrease in sensitivity during maturation correlated with increased translation of Rgs2. To examine directly the role of RGS2 in inhibiting acid-induced Ca2+ release, we overexpressed RGS2 protein in GV-stage oocytes and tested their response to acid treatment. Control oocytes released Ca2+ in response to lowering pH beginning at pH 6.4-6.6. By contrast, RGS2-overexpressing oocytes had a greatly reduced Ca2+ response, with fewer cells responding, and the total amount of Ca2+ released being much lower when Ca2+ release occurred at all (Fig. 3C,D).

Fig. 3.
Fig. 3.

RGS2 mediates loss of acid-induced Ca2+ response during oocyte maturation. (A) Relative level of intracellular Ca2+ in response to lowering pH in maturing oocytes. Graphs show 4-5 representative tracings/group. (B) Percentage of oocytes with a rise in intracellular Ca2+ beginning at the indicated pH. Graph indicates cumulative percentage of 2-5 cells/group from n=5 independent replicates. (C) Relative level of intracellular Ca2+ in response to lowering pH in control GV oocytes or GV oocytes overexpressing RGS2 (Rgs2 cRNA). Six representative tracings/group. (D) Percentage of oocytes with a rise in intracellular Ca2+ beginning at the indicated pH levels. Graph indicates cumulative percentage of 3-8 cells/group from n=4 independent replicates. (E) Gpr68 mRNA level; all stages expressed relative to GV oocytes. N=3; graph shows mean±s.e.m. GV, GV oocyte; GVBD, oocytes immediately following GV breakdown; MI, metaphase I stage; MII, MII eggs; 1C, 1-cell embryos; 2C, 2-cell embryos.

In somatic cells, RGS2 inhibits acid-induced responses, such as those induced by alterations in pH that result from inflammatory conditions including asthma (Liu et al., 2013). Studies in airway epithelial cells suggest that lower pH activates the Gq-coupled proton sensor GPR68 (also called OGR1) to release Ca2+, which stimulates the production of MUC5AC (Liu et al., 2013; Ludwig et al., 2003; Saxena et al., 2012). RGS2 overexpression prevents acid-induced secretion of MUC5AC, and RGS2 depletion increases this response (Liu et al., 2013). These results indicate that RGS2 acts as an inhibitory regulator of acid-induced cellular responses by binding to Gq. GPR68 or a similar receptor could act as a proton sensor linked to Ca2+ signaling by activating Gq in oocytes. Indeed, GV-stage oocytes have significant levels of Gpr68 transcripts compared with those in MII eggs and early embryos (Fig. 3E). It is unclear whether mouse eggs are exposed to low pH under physiological conditions within the ovarian follicle or oviduct, as direct measurements have not been reported. In larger species, follicular fluid and oviduct pH is generally in the range of 7-8 [reviewed by Edwards (1974); Stone and Hamner (1975)], but pH has been measured as low as 6.8 in bovine follicles and pig oviducts (Smiljaković et al., 2008; Zachariae and Jensen, 1958). In addition, follicle or oviductal pH could drop during an inflammatory process as has been observed in inflamed airways (Liu et al., 2013).

Acetylcholine causes Ca2+ release in oocytes and RGS2-depleted eggs

In addition to an acidic environment, eggs could be exposed to other stimuli that activate Gq in vivo. One such stimulus is acetylcholine (ACh). Choline acetyltransferase, which synthesizes ACh, is expressed in granulosa cells from large antral follicles of human, monkey and rat ovaries (Fritz et al., 1999, 2001; Mayerhofer et al., 2006). Moreover, ACh has been measured in cultured human and rat granulosa cells (Fritz et al., 2001) and in adult rat ovaries (Mayerhofer et al., 2006). Interestingly, the amount of ACh in rat granulosa cells is significantly increased by follicle stimulating hormone (Mayerhofer et al., 2006), suggesting that maturing oocytes are exposed to ACh in vivo. ACh treatment stimulates Ca2+ release via Gq-coupled muscarinic receptors, which are expressed in oocytes of several species including mouse (Caratsch et al., 1984; Eusebi et al., 1979, 1984). In addition, stimulation of the muscarinic receptor induces Ca2+ release in immature growing mouse oocytes (Carroll et al., 1994) but not in MII eggs (Williams et al., 1998).

We examined the ability of ACh to stimulate Ca2+ release in oocytes, eggs and RGS2-depleted eggs. All of the GV-stage oocytes and RGS2-depleted eggs released Ca2+ in response to addition of ACh, whereas the majority of control eggs that contained RGS2 showed no response (Fig. 4A,B). Treatment of GV-stage oocytes with the muscarinic receptor antagonist atropine almost completely suppressed ACh-induced Ca2+ release (Fig. 4C), but did not affect acid-induced Ca2+ release (data not shown). These findings indicate that RGS2 effectively inhibits Gq-mediated Ca2+ release in response to ACh, and suggest that one function of the maturation-associated accumulation of RGS2 protein is to suppress this physiological response.

Fig. 4.
Fig. 4.

RGS2 inhibits acetylcholine (ACh)-induced Ca2+ release and premature ZP2 cleavage. (A) Relative level of intracellular Ca2+ in response to the indicated ACh concentrations. One representative tracing is shown per group, along with the proportion of cells displaying a similar pattern. GV, GV oocytes; Control MII, in vitro-matured MII eggs; Rgs2 siRNA MII, MII eggs matured in vitro following microinjection at the GV stage with Rgs2 siRNA. (B) Percentage of cells with a rise in intracellular Ca2+ beginning at the indicated ACh concentrations. Graph indicates cumulative percentage of 4-6 cells/group from n=4 independent replicates. (C) Effect of atropine on ACh-induced Ca2+ response in GV oocytes. Graph indicates cumulative percentage of 25 cells/group from n=3 independent replicates. (D) Immunoblot of ZP2 protein. Oocytes were microinjected with scrambled siRNA (control) or Rgs2 siRNA, then matured in vitro to MII. Blot represents 3 independent replicates; 12 eggs per lane. ZP2, full-length ZP2 protein; ZP2f, cleaved form of ZP2. (E) Quantitation of ZP2-to-ZP2f conversion. Graph shows mean±s.e.m. of 3 independent replicates. *P<0.05; t-test. (F) Relative level of intracellular Ca2+ in response to 2 µM ACh. Representative tracings are shown along with the proportion of cells displaying a similar pattern. Control, Rgs2+/+ eggs; Rgs2 KO, Rgs2−/− eggs. (G) Relative area under the curve (AUC) of ACh response. N=25-26 total eggs in 5 independent experiments. Graph shows mean±s.e.m. *P<0.05, t-test. (H) Number of eggs with a rise in intracellular Ca2+ beginning at the indicated pH. (I) Relative AUC of acid response. N=21-23 total eggs in 4 independent experiments. Graph shows mean±s.e.m. *P<0.05, t-test. (J) Average litter size for the indicated genotype. N=31-33 litters; *P<0.05, t-test. (K) Immunoblot of ZP2 protein from Rgs2+/+ (control) and Rgs2−/− (Rgs2 KO) eggs. Blot shows 2 of 3 replicates; 10 eggs per lane. (L) Quantitation of ZP2-to-ZP2f conversion in the indicated groups. Graph shows mean±s.e.m. of 3 replicates. *P<0.05; t-test. (M) Schematic summarizing RGS2 function after oocyte maturation in suppressing Ca2+ signaling mediated by Gq prior to fertilization.

RGS2-depleted eggs undergo premature ZP conversion

Ca2+ release at fertilization triggers cortical granule exocytosis and release of the protease ovastacin, which cleaves the ZP protein ZP2 to ZP2f (Burkart et al., 2012). This cleavage event is responsible for the ZP block to polyspermy. Because cortical granule release in eggs is easily triggered in response to rises in cytoplasmic Ca2+ (Ducibella et al., 2002), ZP2 cleavage can be used as a proxy for Ca2+ release over time. To test whether RGS2 regulates Ca2+ release during oocyte maturation, we microinjected GV-stage oocytes with Rgs2 siRNA, matured them and then measured the percentage of ZP2-to-ZP2f conversion. RGS2-depleted eggs consistently had elevated levels of ZP2 conversion compared with controls (Fig. 4D,E). These findings demonstrate that during in vitro maturation of RGS2-depleted oocytes, Ca2+ increases occurred that were sufficient to cause cortical granule release and promote ZP2 conversion.

A full RGS2 knockout mouse has been developed and this mouse has at least two defects due to inappropriate Ca2+ regulation. RGS2 knockout pancreatic acinar cells have significantly higher IP3-mediated Ca2+ release in response to muscarinic receptor activation (Wang et al., 2004). In addition, these knockout mice have high blood pressure due to increased Ca2+ release in response to vasoconstrictors, which act through Gq-coupled receptors. To determine whether loss of RGS2 in vivo resulted in abnormal responses to G-protein-coupled receptor agonists, we tested the effects of ACh and acidic pH on MII eggs from Rgs2+/+ and Rgs2−/− females. Consistent with our findings in RGS2-depleted eggs, 100% of the Rgs2−/− eggs responded to ACh by releasing Ca2+ (Fig. 4F). Only 12/26 Rgs2+/+ eggs showed some degree of response to ACh, but far less Ca2+ was released than in Rgs2−/− eggs (Fig. 4G). Similarly, Rgs2−/− eggs responded at a higher pH and released more Ca2+ than Rgs2+/+ eggs (Fig. 4H,I). Of note, control eggs in these experiments, which were from C57BL/6J females, were more sensitive to both ACh and acid when compared with the CF-1 eggs used in previous experiments, as demonstrated by the greater proportion showing responses. These findings draw attention to mouse strain differences that probably underlie conflicting reports regarding responses of MII eggs to ACh (Kang et al., 2003; McGuinness et al., 1996; Williams et al., 1992). In fact, the MF-1 mouse strain, which produces eggs that have a high incidence of activation following acid-mediated ZP removal (Johnson et al., 1990), was the subject of a quantitative trait locus analysis that identified the Rgs2 gene locus as a modulator of anxiety (Yalcin et al., 2004). These findings suggest that differences in RGS2 expression or function contribute to the increased sensitivity of MF-1 eggs to ACh and acid activation.

RGS2 knockout mice are viable and fertile, but no formal breeding study has been reported. We analyzed litter sizes from ongoing production of Rgs2+/+ and Rgs2−/− mice over a 32-month period. The average litter size of Rgs2−/− females was significantly lower than that of Rgs2+/+ females (Fig. 4J). This finding was due to subfertility of the Rgs2−/− females, because litters of Rgs2+/+ females mated to Rgs2−/− males were not smaller than those of wild-type pairs (data not shown). A possible explanation for the reduced litter size was premature cleavage of ZP2 similar to that observed in RGS2-depleted eggs, which could result in impaired fertilization. To test this idea, we collected MII eggs from Rgs2+/+ and Rgs2−/− females 16 h after hCG administration and quantified ZP2-to-ZP2f conversion. Indeed, Rgs2−/− eggs had increased levels of ZP2 conversion compared with those from Rgs2+/+ females (Fig. 4K,L). These findings indicate that RGS2 suppresses Ca2+ release in vivo. The finding that the mice were not completely infertile suggests that, in addition to RGS2, other mechanisms are in place to help prevent premature Ca2+ release.

In conclusion, a dramatic rise in intracellular Ca2+ is of paramount importance for successful egg activation and embryo development in all animals (Kashir et al., 2013). Mammalian oocytes do not develop the ability to efficiently release large amounts of Ca2+ until immediately before ovulation, thereby preventing premature Ca2+ release that could preclude successful fertilization. As the oocyte becomes fertilization-competent, it is exquisitely sensitive to signals that release Ca2+ (Fig. 4M). Our findings suggest that RGS2 functions to inhibit premature G-protein-mediated Ca2+ release in eggs that are poised for activation by PLCζ from the fertilizing sperm.

MATERIALS AND METHODS

Gamete collection and oocyte microinjection

All mice used (see details in the supplementary Materials and Methods) were maintained under approved protocols and complied with the Institute of Laboratory Animal Research Guide for the Care and Use of Laboratory Animals. Oocytes, eggs, embryos and sperm were collected and oocytes were microinjected with 5-10 pl volume, essentially as previously described (Bernhardt et al., 2011; Jefferson et al., 2009). Pipette concentrations of microinjected reagents were as follows: Rgs2 siRNA (Ambion) and control nontargeting siRNA (Santa Cruz), 2 μM; Rgs2 and control morpholinos (GeneTools), 2 mM; and HA-RGS2 cRNA, 1 µg/µl. siRNA- and morpholino-injected oocytes were cultured in 10 µM milrinone for 5-8 h to allow protein turnover and then matured. cRNA-injected oocytes were incubated in milrinone-containing medium for 18-20 h prior to imaging to allow for protein overexpression. For some experiments, ZPs were removed by brief exposure to acidic Tyrode's solution, pH 1.6, by incubation for 4-8 min in 5 mg/ml pronase, or by piezoelectric-actuated drilling of a 50-µm slit in the ZP, followed by gentle pipetting using a 70-μm inner diameter capillary. Culture media used are detailed in the supplementary Materials and Methods.

Calcium imaging

Ca2+ imaging was performed as previously described (Miao et al., 2012). For fertilization experiments, ZP-free eggs were adhered to Cell-Tak-coated dishes in BSA-free medium under mineral oil. Capacitated sperm were added to a final concentration of 105 sperm/ml. For acid addition and ACh experiments, ZP-intact cells were used. HCl (1 N) was diluted 1:100, and 150 μl of this solution was added to 2 ml medium several minutes apart during imaging. pH measurements were made using parallel additions of the dilute HCl to the same medium volume. ACh was prepared as a 5 mM stock in water and added to L-15 medium during imaging to achieve final concentrations of 2 µM, 200 µM and 1 mM.

Immunofluorescence and immunoblotting

For spindle staining, eggs were fixed, extracted and blocked as previously described (Bernhardt et al., 2012). Oocytes, eggs or embryos were lysed in sample buffer, separated under reducing conditions and immunoblotted as previously described (Jefferson et al., 2013). Details regarding antibodies are provided in the supplementary Materials and Methods.

Real-time RT-PCR

Total RNA was isolated from 50 oocytes, eggs or embryos using an Arcturus Pico Pure kit (Life Technologies). EGFP cRNA was generated as previously described (Miao et al., 2012) and 10 pg was added to each sample prior to RNA isolation as an internal control. Real-time RT-PCR was performed as previously described (Jefferson et al., 2013), using cDNA from two oocytes or embryos per reaction. Primer sequences are provided in the supplementary Materials and Methods. Relative gene expression was calculated by the ΔCt method (Pfaffl, 2001) using EGFP expression for normalization.

Statistical analysis

Data were analyzed using one-way ANOVA, Student's t-test, Mann–Whitney U-test or chi-square test, as indicated in the figure legends. Statistical tests were performed using GraphPad Prism.

Acknowledgements

We thank Yaping Tu for providing Rgs2−/− mice, Dean Betts and Nicole Edwards for obtaining Rgs2−/− eggs, Jurrien Dean for the ZP2 antibody, and Laurinda Jaffe and Bruce White for helpful discussions.

Footnotes

  • Competing interests

    The authors declare no competing or financial interests.

  • Author contributions

    M.L.B., K.M.L., P.C., C.J.W. and L.M.M. designed the experiments. M.L.B., K.M.L., E.P.-B., C.E.M., K.N.L., A.V.E., T.F.U. and L.M.M. performed the experiments and analyzed the data. M.L.B., C.J.W. and L.M.M. wrote the manuscript. All authors edited and approved the final manuscript.

  • Funding

    This work was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Environmental Health Sciences [Z01ES102985 to C.J.W.], NIH grant HD056366 and the University of Connecticut Health Center Research Advisory Council (to L.M.M.), and a grant from the Canadian Institutes of Health Research (to P.C.). Deposited in PMC for release after 12 months.

  • Supplementary material

    Supplementary material available online at http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.121707/-/DC1

  • Received January 5, 2015.
  • Accepted June 25, 2015.

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Regulator of G-protein signaling 2 (RGS2) suppresses premature calcium release in mouse eggs
Miranda L. Bernhardt, Katie M. Lowther, Elizabeth Padilla-Banks, Caitlin E. McDonough, Katherine N. Lee, Alexei V. Evsikov, Tracy F. Uliasz, Peter Chidiac, Carmen J. Williams, Lisa M. Mehlmann
Development 2015 142: 2633-2640; doi: 10.1242/dev.121707
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