Development of Pig Oocytes Activated by Stimulation of an Exogenous G Protein-Coupled Receptor¹

^a Department of Animal Sciences, University of Missouri-Columbia, Missouri 65211

	ABSTRACT

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This study determined whether stimulation of a G protein-coupled receptor could initiate the events that occur at fertilization in pig oocytes and, if so, whether the activated oocytes were competent to form blastocysts. After maturation for 30 h, oocytes received microinjections of mRNA encoding the rat M1 muscarinic receptor, a G protein-coupled acetylcholine (ACh) receptor. Oocytes were then incubated for an additional 15 h to complete maturation of oocytes and translation of microinjected mRNA, and they were subsequently cultured in the presence of ACh. ACh treatment of these oocytes triggered pronuclear formation (50.4%) as well as cortical granule exocytosis. SDS-PAGE showed that mRNA-microinjected oocytes treated with ACh were activated (61.1%), as characterized by the appearance of the 22-kDa polypeptide derived from dephosphorylation of the 25-kDa precursor. Furthermore, after being cultured in a ligated pig oviduct for 6 days, 17.4% of treated oocytes developed to the compact morula or blastocyst stage. Transmission electron microscopy revealed that blastocysts recovered from ligated oviducts contained reticulated nucleoli with fibrillar cores surrounded by fibrillar and granular components. In addition, mitochondria in the blastocysts were dispersed throughout the cytoplasm and contained numerous transverse cristae. These results show that pig oocyte activation mediated by a G protein-coupled signal transduction system can signal a series of intracellular changes that lead to activation events associated with fertilization. Furthermore, oocytes activated through this pathway showed preimplantation development consistent with normal development.

	INTRODUCTION

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Oocyte activation refers to global changes in the oocyte after the interaction of sperm and oocyte plasma membranes. Oocyte activation can be classified into early and late events [1]. Early events include repetitive calcium transients and cortical granule exocytosis. Late events include recruitment of maternal mRNAs, pronuclear formation, and initiation of DNA synthesis.

There are two major theories concerning the mechanism by which sperm activate oocytes. One hypothesis states that a certain diffusible substance that sperm carry in their cytoplasm activates oocytes [2, 3]. Microinjection of a fraction prepared from sperm induced repetitive calcium waves, cortical granule exocytosis, and pronuclear formation. Recently, Parrington et al. [4] characterized a soluble sperm protein, oscillin in hamster sperm, that triggers calcium oscillations. Oscillin is located at the equatorial segment of the sperm head, and its cDNA has high homology with a hexose phosphate isomerase found in prokaryotes.

The other hypothesis states that oocyte activation is mediated by a ligand-receptor-effector system. It proposes that sperm bind the receptor, which is coupled to either a G protein or a tyrosine kinase-coupled signal transduction pathway. To test the hypothesis that a G protein is involved in the stimulatory pathway leading to oocyte activation, oocytes received microinjections of GTP--S, a hydrolysis-resistant analogue of GTP that activates G proteins, or with GDP-ß-S, which acts as a competitive inhibitor of GTP [5–8]. Oocytes receiving microinjections of GTP--S released calcium and underwent cortical granule exocytosis, whereas sperm-induced activation was inhibited in GDP-ß-S-microinjected oocytes. In support of the G protein-coupled signal transduction pathway, frog [9], mouse [10, 11], and pig [12] oocytes have received microinjections of mRNA for rat M1 muscarinic receptors, which are acetylcholine (ACh) receptors, to express receptor in the oocyte plasma membrane. This receptor acts by way of a G protein to stimulate phosphatidylinositol metabolism [13]. After ACh application, the oocytes showed several responses that normally occur at fertilization. This suggested that receptor-mediated activation of a G protein may be sufficient to mimic activation events.

Recently, we have shown that the activation of pig oocytes via an exogenously introduced rat M1 muscarinic receptor resulted in some of the events associated with oocyte activation: calcium release, cortical granule exocytosis, decrease in histone H1 kinase activity, and progression to first interphase [12]. In the investigation reported here, these studies were carried a step further with an evaluation of the changes in the patterns of protein synthesis, developmental competence, and morphological changes in nucleoli and mitochondria.

	MATERIALS AND METHODS

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In Vitro Maturation

Porcine oocytes were aspirated from pig ovaries collected at a slaughterhouse. Cumulus oocyte complexes (COC) with uniform ooplasm and a compact cumulus cell mass were prepared in Hepes-buffered Tyrode's medium [14] containing 0.1% polyvinylalcohol (HbTP). After COCs were washed in HbTP medium, they were matured in BSA-free Whitten's medium for 30 h at 39°C in a humidified atmosphere of 5% CO₂ in air. During the first 20 h of maturation, the medium contained 10 IU/ml eCG, 10 IU/ml hCG, and 10% porcine follicular fluid; during the last 10 h of maturation, the same medium without hormonal supplementation was used. After maturation, oocytes were denuded by vigorous pipetting in 0.3 M mannitol solution supplemented with 0.3 mg/ml hyaluronidase (Sigma Chemical Co., St. Louis, MO). After a rinse in HbTP, the oocytes with homogenous cytoplasm were used for experiments.

In Vitro Transcription of Rat M1 Muscarinic Receptor cDNA

In vitro transcription of cDNA and identification of RM1 mRNA were carried out as previously described [12]. Briefly, cDNA, a gift from Dr. Tom I. Bonner (National Institutes of Health) [13], was transcribed in vitro with T7 RNA polymerase using the RiboMAX Large Scale RNA Production System (Promega Corp., Madison, WI) according to the manufacturer's instructions. The reaction was performed in the presence of m⁷G(5')ppp(5')G (Boehringer-Mannheim, Indianapolis, IN) to produce capped RNA transcripts. The RNA transcripts were dissolved in diethyl pyrocarbonate-treated water to a final concentration of 1.4 µg/µl. The samples were stored in 3-µl aliquots at -80°C.

The identity of the RNA transcripts was verified by Northern blotting. The integrity of the RNA was tested by in vitro translation in the Rabbit Reticulocyte Lysate System (Promega), and its size was consistent with the size of the core receptor protein (data not shown).

Microinjection of Rat M1 Muscarinic Receptor mRNA and ACh Treatment

After maturation for 30 h, each oocyte received a microinjection at the center of the cytoplasm of approximately 56 pg mRNA encoding the rat M1 muscarinic ACh receptor; a microinjector (Narishige Co. Ltd., Tokyo, Japan) was used. Microinjections were performed in calcium-free HbTP to prevent calcium-induced activation. After microinjection, the oocytes were cultured for 15 h in Whitten's medium without hormonal supplementation. This period is required for completion of maturation, translation of microinjected mRNA, and incorporation of the receptor into the plasma membrane [12]. Subsequently, mRNA-microinjected and noninjected oocytes were cultured in HbTP medium in the presence of 5 µM ACh for 6 h or 4 h to evaluate cortical granule exocytosis.

Radiolabeling of Oocytes and SDS-PAGE

Protein profiles were evaluated by L-[³⁵S]methionine (Amersham Corp., Arlington Heights, IL) incorporation followed by one-dimensional SDS-PAGE as previously described [8]. Briefly, the labeled oocytes were placed individually into siliconized 0.5-ml centrifuge tubes containing reducing SDS lysis buffer, and the tubes were then stored at -80°C. Before analysis, samples were thawed and boiled, and 12.5% SDS-PAGE was performed with prestained molecular weight markers in parallel (Rainbow Protein Molecular Weight Markers; Amersham). After electrophoresis, the gels were fixed, impregnated, and precipitated. Gels were dried under a vacuum and exposed to Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY) at -80°C for 3 days.

As a positive control for protein profiles, fully matured electrostimulated oocytes were used. COC were matured in BSA-free Whitten's medium supplemented with 10 IU/ml eCG, 10 IU/ml hCG, and 10% porcine follicular fluid for 20 h, and cultured in the same medium without hormonal supplementation for 24 h. Denuded oocytes were placed in electrostimulation medium containing 0.3 M mannitol, 1 mg/ml BSA, and 5% Hepes-buffered Tyrode's medium (pH 7.0) for 5 min and pulsed for 10 sec with 3 V AC and then by five 30-µsec pulses at 40 V DC (BTX 200; BTX Inc., San Diego, CA). After electrostimulation, oocytes were rinsed with HbTP.

Evaluation of Pronuclei Formation

Oocytes were fixed on slides for 48 h in ethanol:glacial acetic acid (3:1). After being stained with 1% (w:v) aceto-orcein, the oocytes were evaluated for the presence of pronuclei using Hoffman modulation contrast optics (Hoffman Modulation Optics, Inc., Greenvale, NY).

Oocyte Transfer and Embryo Recovery

To evaluate the developmental potential of mRNA-injected oocytes treated with ACh, treated oocytes were transferred surgically into ligated oviducts of gilts 6-9 days postestrus. Experiments were conducted according to institutional ACUC guidelines. By Day 6, ovulated oocytes had passed into the uterus and did not confound the data [15]. As a negative control, mRNA-injected oocytes without ACh treatment were transferred into the contralateral oviduct. Gilts were anesthetized by i.v. injection of 5% pentothal (23 ml) into an ear and then given 5% halothane/oxygen through a closed-circuit system. A midventral laparotomy was performed to transfer the oocytes to the ligated oviducts, using a Tomcat catheter (Sherwood Medical, St. Louis, MO) attached to a 1-cc syringe. After 6 days, the oviducts were removed and flushed with HbTP, and the stages of embryos were determined. After embryo recovery, blastocysts were used for examination of nucleolar and mitochondrial morphology.

Transmission Electron Microscopy

Oocytes and blastocysts were fixed in 2% glutaraldehyde/2% paraformaldehyde with 0.1 M sodium cacodylate buffer (pH 7.3) for 4 h at 4°C. After fixation, all samples were washed with the buffer and postfixed with a 1% OsO₄ in distilled water for 1 h, stained in 1% aqueous uranyl acetate for 1 h, dehydrated in a graded series of ethanol, and embedded in a mixture of Epon/Araldite (Electron Microscopy Sciences, Fort Washington, PA). Serial ultrathin sections were cut with a diamond knife and stained with uranyl acetate for 20 min and lead citrate for 3 min. Sections were examined under a transmission electron microscope (Hitachi, Ltd., Tokyo, Japan).

Statistical Analysis

The data for pronuclear formation was analyzed by ANOVA. Means were compared by Duncan's multiple-range procedure. Developmental rates for compact morulae or blastocysts were compared by Student's t-test [16].

	RESULTS

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Cortical Granule Exocytosis

As shown in Figure 1 (A–C), cortical granules containing specialized enzymes and glycoproteins were observed within the cortex and identified by their electron-dense cores. After oocyte activation, cortical granules were no longer present in the oocyte cortex (Fig. 1D). Five of six mRNA-microinjected oocytes treated with ACh released their cortical granule contents into perivitelline space, while there was no occurrence of exocytosis in ACh-treated or mRNA-injected oocytes. In addition, only one of six nontreated oocytes released cortical granules.

View larger version (150K): [in this window] [in a new window]   FIG. 1. Transmission electron micrographs of cortical granule exocytosis. A) Nontreated oocyte; B) ACh-treated oocyte; C) ACh receptor mRNA-injected oocyte; D) ACh receptor mRNA-injected and ACh-treated oocyte. Arrows indicate cortical granules. x5520.

Changes in Protein Profiles

In addition to cortical granule exocytosis, activation causes a number of fertilization-associated changes in the patterns of protein synthesis. We compared protein profiles in each group in order to determine the posttranslational changes in mRNA-injected oocytes treated with ACh (Fig. 2). SDS-PAGE (4 replications) showed, by the disappearance of the 25-kDa and the appearance of the 22-kDa polypeptide (lanes 5-8), that 61.1% (11 of 18) of the mRNA-injected oocytes cultured with ACh were activated. As a positive control, electrostimulated oocytes (4 of 4; lane 3) revealed the same pattern as mRNA-injected oocytes treated with ACh. There were no posttranslational changes in ACh-treated (0 of 4) or mRNA-injected oocytes (0 of 3); however, one of the nontreated oocytes (1 of 4) showed both the 22-kDa and 25-kDa band.

View larger version (123K): [in this window] [in a new window]   FIG. 2. Protein profile of pig oocytes. Lane 1, ACh-treated oocyte; lane 2, nontreated oocyte; lane 3, electrostimulated oocyte; lane 4, mRNA-injected oocyte; lanes 5-8, mRNA-injected and ACh-treated oocytes. Activated oocytes underwent posttranslational changes in the protein profile that resulted in the disappearance of the 25-kDa polypeptide and the appearance of a 22-kDa polypeptide.

Pronuclear Formation and Development of Activated Oocytes

Pronuclear formation and developmental competence were also evaluated. ACh treatment of mRNA-microinjected oocytes induced resumption of meiosis and progression to first interphase. Fifty percent of these oocytes formed one or two pronuclei (Table 1). This is significantly higher than the formation rate in ACh-treated (0.7%), mRNA-injected (12.1%), or nontreated (3.5%) oocytes. To test the developmental potential of mRNA-injected oocytes treated with ACh, the oocytes (treated with ACh or nontreated) were transferred surgically into ligated oviducts of gilts. After 6 days, 17.4% of the oocytes treated with ACh developed to compact morulae or blastocysts, while none of the oocytes that were not treated with ACh showed development to even the compact morula stage (Table 2).

Morphology of Nucleolus and Mitochondria

In order to determine whether mRNA-microinjected oocytes treated with ACh undergo morphological changes similar to those in normal embryonic development, the morphology of the nucleolus and mitochondria was examined in blastocysts recovered six days after the oocytes were transferred to ligated gilt oviducts. The blastocysts contained reticulated nucleoli composed of fibrillar centers, a fibrillar component, and a granular component (Fig. 3). Fibrillar centers with loose fibrillar material were surrounded by a dense fibrillar component. The granular component contained small particles similar to cytoplasmic ribosomes.

View larger version (160K): [in this window] [in a new window]   FIG. 3. Transmission electron micrograph of nucleolus in blastocyst derived from ACh receptor mRNA-injected oocyte treated with ACh. The fibrillar center (FC) is surrounded by the dense fibrillar component (DFC) scattered in the granular component (GC) region. x22 400.

The majority of mitochondria in the blastocysts were elongated and dispersed throughout the cytoplasm, and they had many transverse cristae, as do mature somatic forms (Fig. 4, A and B). Some of them appeared to be in a transition state from immature to mature morphology; they contained areas of low electron density including a filamentous substance, vacuoles, and an electron-dense matrix. The mitochondria in matured oocytes were markedly different from those in mature blastocysts: they were spherical and contained an electron-dense matrix and only a few transverse cristae (Fig. 4C).

View larger version (83K): [in this window] [in a new window]   FIG. 4. Transmission electron micrographs of (A) mitochondria in a blastocyst derived from ACh receptor mRNA-injected oocyte treated with ACh (x10 752); (B) typical matured elongated mitochondria in a blastocyst embryo (x30 800); and (C) matured oocyte (x30 240).

	DISCUSSION

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G proteins play important roles in signal transduction in response to extracellular signals [17]. They interact not only with cell surface receptors but also with various intracellular effector enzymes, which eventually leads to physiological responses. Muscarinic ACh receptors transduce their intracellular signals by coupling with G proteins [18]. The receptors are composed of a single polypeptide containing seven hydrophobic transmembrane helics, an extracellular NH₂-terminal domain, and a cytoplasmic COOH-terminal, which have the same structural features as the G protein-coupled receptors. The principal signaling pathway through the G protein-coupled receptor, or the M1 muscarinic ACh receptor, activates phospholipase C-ß, which produces the second messengers inositol 1,4,5-trisphosphate and diacylglycerol [19, 20]. Inositol trisphosphate acts on the receptors in endoplasmic reticulum to cause transient calcium mobilization and diacylglycerol acts to activate protein kinase C.

In a previous study [12], we reported that a G protein-coupled signal transduction pathway might be involved in pig oocyte activation. We have shown that agonist treatment induced various activation-related changes such as calcium release, cortical granule exocytosis, decrease in H1 kinase activity, and pronuclear formation. The present study carries these results a step further. Here it is shown that stimulation of exogenously introduced M1 muscarinic ACh receptor triggered additional oocyte activation events: cortical granule exocytosis, change in protein profile, pronuclear formation, and development to the blastocyst stage. In addition, the activated oocytes underwent the ultrastructural changes in nucleoli and mitochondria similar to the changes in normal embryos.

During oocyte activation, an increase in intracellular calcium concentration occurs as an early event. It is well accepted that calcium release from intracellular stores is essential for cortical granule exocytosis and resumption of meiosis [21]. This calcium release is induced by inositol 1,4,5-trisphosphate derived from the phosphatidylinositol 4,5-bisphosphate with stimulated phospholipase C. Initially, to confirm the effect of G protein-coupled signal pathway on the pig oocyte activation, we examined the results in our previous report [12] that ACh treatment of the pig oocytes microinjected with mRNA encoding M1 muscarinic ACh receptor triggered cortical granule exocytosis and pronuclear formation (58.8%). In the present study, 50.4% of mRNA-microinjected oocytes treated with ACh formed pronuclei, and some oocytes contained two pronuclei without second polar body extrusion, suggesting that the second pronucleus might be derived from the second polar body. In addition, ACh treatment caused the release of cortical granules in microinjected oocytes. These results are similar to those obtained in our previous study. Furthermore, when the oocytes were transferred into ligated oviducts, 17.4% of them developed to the compact morula or blastocyst stage. While we observed some degree of pronuclear formation in only mRNA-injected oocytes, the rate of pronuclear formation in mRNA-injected oocytes was significantly lower than in mRNA-injected oocytes treated with ACh, but it was notably higher than in only ACh-treated or nontreated oocytes. The injection process apparently caused some oocyte activation. Some reports have shown that oocytes were activated by pricking with a fine needle [22].

In addition to pronuclear formation, activation causes a number of fertilization-associated changes in the patterns of protein synthesis [23]. After fertilization in pig oocytes, one of the most characteristic posttranslational changes in the protein profile is the disappearance of a 25-kDa polypeptide and the appearance of a dominant 22-kDa polypeptide [24]. The positively charged 22-kDa polypeptide is derived from the 25-kDa precursor by dephosphorylation. This modification coincides with the time of pronuclear formation. Several treatments have been reported to trigger the posttranslational modification associated with fertilization in pig oocytes. The treatments include electrical stimulation [25], GTP--S microinjection [8], CaCl₂ microinjection [26], treatment with protein kinase inhibitors [27], and now, ACh treatment of oocytes microinjected with the M1 muscarinic ACh receptor [present study].

During embryogenesis, nucleoli and mitochondria undergo morphological and functional changes associated with differentiation processes. During early stages of mammalian embryonic development, embryos depend on proteins and mRNAs previously stored in the oocytes, because of the absence of their own functional nucleoli. The embryos are under control of maternal messages. Early embryos have a compact nucleolus with fine fibrils in a spherical center surrounded by an agranular core and are unable to synthesize ribosomal RNAs, which are required for protein synthesis. At the specific time of transition from the maternal to embryonic control of development, the four-cell stage in the pig embryo [28], the nucleoli begin to reticulate, have fibrillar cores surrounded by a fibrillar component, and are capable of synthesizing ribosomal RNA [29]. The mitochondria in early embryos also undergo a change in morphology. Mammalian oocytes and early embryos have an electron-dense matrix and a few peripherally arranged and concentrated cristae, while morula- or blastocyst-stage embryos contain greater numbers of traverse cristae [30]. It has been recognized that morphological changes in mitochondria have a temporal relationship to changes in respiration rate and glucose metabolism [31, 32]. Mitochondria in pig [33] oocytes and early embryos tend to occur in small groups or aggregates. They have an electron-dense matrix, few cristae, and vacuole-like inclusions as in other mammals. In contrast, mitochondrial aggregates are not found in blastocysts. The mitochondria in pig blastocysts are more dispersed throughout the cytoplasm, and they have a less electron-dense matrix. In addition, like the somatic type, they contain numerous transverse cristae. In the present study, morphological changes in nucleoli and mitochondria were examined in M1 muscarinic ACh receptor mRNA-injected oocytes treated with ACh and were found to be consistent with normal embryo development.

In conclusion, these results suggest that pig oocyte activation might be mediated by a G protein signal transduction pathway. Stimulation of a G protein-coupled receptor does lead to both early and late events of pig oocyte activation. Furthermore, these oocytes showed preimplantation developmental competence as measured by blastocoele formation as well as morphological changes similar to those in normal embryo development, i.e., reticulation of nucleoli and appearance of somatic-type mitochondria.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. T.I. Bonner at the National Institutes of Health for the gift of rat M1 muscarinic receptor cDNA.

	FOOTNOTES

 
¹ This material is based upon work supported by Food for the 21st Century and the Cooperative State Research, Education and Extension Service, U.S. Department of Agriculture, under agreement No. 95-37203-2073. The manuscript is a contribution from the Missouri Agriculture Experiment Station Journal Series No. 12,727.

² Correspondence: Randall S. Prather, 162 Animal Science Research Center, University of Missouri-Columbia, Columbia, MO 65211. FAX: (573) 882-6827; randall_prather{at}muccmail.missouri.edu

Accepted: April 28, 1998.

Received: January 29, 1998.

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