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Biology of Reproduction 62, 1702-1709 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Ability of Integrins to Mediate Fertilization, Intracellular Calcium Release, and Parthenogenetic Development in Bovine Oocytes1

Kenneth D. Campbella, William A. Reeda, and Kenneth L. White2,a

a Department of Animal, Dairy and Veterinary Sciences, Center for Developmental and Molecular Biology, Biotechnology Center, Utah State University, Logan, Utah 84322-4815

ABSTRACT

The ability of arginine-glycine-aspartic acid (RGD; a sequence recognized by integrins) or non-RGD-containing peptides to block fertilization, induce intracellular Ca2+ oscillations, and initiate parthenogenetic development in bovine oocytes was investigated. Addition of a soluble RGD peptide during fertilization at concentrations ranging from 10 to 1000 µg/ml significantly decreased (P < 0.05) fertilization as compared to the in vitro-fertilized controls. The addition of non-RGD peptide had no effect on fertilization. Two intracellular Ca2+ transients 21.5 ± 1.9 min apart were observed in 56 of 60 oocytes incubated in RGD peptide concentrations ranging from 20 to 1000 µg/ml. No intracellular Ca2+ transients were observed in medium alone, non-RGD treatment groups or in the RGD peptide at 10 µg/ml. The percentage of oocytes activated with ionomycin and 6-dimethylaminopurine (63% cleavage and 34% blastocyst development) was significantly higher (P < 0.05) than those activated with the RGD peptide and 6-dimethylaminopurine (35% cleavage and 19% blastocyst development). These groups were significantly higher (P < 0.05) than either peptide alone, 6-dimethylaminopurine alone, or the non-RGD peptide and 6-dimethylaminopurine treatment groups. These data provide evidence that ligation of an integrin on bovine oocytes with a soluble RGD peptide is capable of blocking fertilization, inducing intracellular Ca2+ transients, and initiating parthenogenetic development.

INTRODUCTION

The adhesion of sperm to an oocyte and resumption of meiosis is a complex process that involves mutual recognition, membrane fusion, signal transduction, and the reorganization of the oocyte cytoskeleton. At present, the components and mechanisms of this process in mammals are not well defined.

Cell-cell adhesion is mediated by adhesion molecules and their counterreceptors on opposing membranes. Integrin molecules are cell surface adhesion receptors that form a family of transmembrane glycoproteins with heterodimeric structure ({alpha}-chain and ß-chain; [1]). Many integrins have been shown to recognize the tripeptide sequence arginine-glycine-aspartic acid (RGD; [1]). Integrins facilitate attachment of the cell to the extracellular matrix (ECM), facilitate cell migration, mediate cell–cell adhesion, link the ECM with the cellular cytoskeleton, and act as two-way signaling molecules [1, 2]. Integrins have been shown to be involved in the process of fertilization [3–5]. In 1990, Bronson and Fusi reported that coincubation of RGD-containing peptides in a heterologous system (human sperm and zona-free hamster eggs) or a homologous system (hamster sperm and zona-free hamster eggs) resulted in a significant decrease in the number of adherent sperm, egg penetration, and fertilization. In addition, the presence of a molecule on the oocyte capable of binding the RGD sequence was demonstrated by using immunobeads coupled with an RGD-containing peptide that adhered to numerous points on the egg surface. In 1995, Almeida et al. [5] concluded that the integrin {alpha}6ß1 serves as a murine sperm receptor. A number of integrins and their ligands have been described on human oocytes and sperm [6–8].

At fertilization, sperm binding and/or penetration produces transient but periodic increases in intracellular Ca2+ ([Ca2+]i) that are essential for resumption of meiosis and embryogenesis [9–11]. These calcium transients or oscillations involve the periodic release and uptake of calcium from intracellular stores. The biological significance of the changes in [Ca2+]i concentration ([Ca2+]i) is not fully understood, however, calcium ions are known to be involved in cortical granule release that leads to a block in polyspermy and in the control of cell cycle progression [9–11].

Mammalian eggs analyzed thus far express integrins on their surface [3–5, 12]. Prior studies have addressed the role of particular integrins in sperm–egg binding and have concluded that RGD-containing peptides inhibit the binding of spermatozoa to oocytes [5]. Because oocytes from all mammalian species analyzed express integrins on their surface it seems plausible that the binding between oocytes and sperm as mediated by integrins is a general mechanism for interaction among mammalian gametes [5]. It has been shown that interaction between integrins and their ligands is capable of producing calcium spikes or transients in a variety of cell types including platelets, neutrophils, monocytes, lymphocytes, fibroblasts, endothelial cells, osteoclasts, epithelial cells, pancreatic acinar cells, and hepatocytes [2]. Previous research on Xenopus eggs indicated the ability of RGD peptides to induce intracellular calcium transients [13]. Based on these observations the role of integrins in bovine oocyte fertilization, activation, and subsequent development was studied. The ability of an RGD peptide to inhibit fertilization, to generate intracellular Ca2+ transients, and to initiate parthenogenetic development were investigated.

MATERIALS AND METHODS

Reagents

All reagents were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise stated.

Oocyte Collection and In Vitro Maturation

All procedures were performed according to published methods routinely used in this laboratory [14–18]. Briefly, bovine ovaries were collected from a local abattoir (E. A. Miller, Hyrum, UT). Oocytes from follicles between 3 and 8 mm were aspirated into 50-ml centrifuge tubes using an 18-gauge needle connected to a vacuum pump. Oocytes with intact layers of cumulus cells and evenly shaded cytoplasm were selected and washed with HEPES-buffered Tyrode's medium (TL-HEPES; [19]) supplemented with 3 mg/ml BSA. Oocytes were then transferred into 250 µl of maturation medium; M199 containing 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT), 0.5 µg/ml FSH (Sioux Biochemicals, Sioux City, IA), 5 µg/ml LH (Sioux Biochemicals), 100 U/ml penicillin (Life Technologies, Grand Island, NY), and 100 µg/ml streptomycin (Life Technologies) into four-well culture dishes (Nunc, Milwaukee, WI) and cultured at 39°C in a humidified atmosphere of 5% CO2 and air for 24 h. At 24 h after the initiation of maturation, oocytes were vortexed in 1 ml TL-HEPES containing 10 mg/ml hyaluronidase to completely remove cumulus cells. Denuded oocytes having an extruded first polar body were selected for use in all experiments except those dealing with in vitro fertilization.

Peptide Synthesis and Coupling of Peptides to Beads

Peptides with the sequence CQDSETRTFY (non-RGD; [6, 20]) and GRGDSPK (RGD; [21]) were synthesized at the USU Biotechnology Center. The non-RGD sequence corresponds to amino acids 470–479 and is contained within the nonadhesive domain 1F7, a fibronectin type 1 repeat [22–24]. The RGD sequence corresponds to amino acids 1492–1498 and is contained within the RGD cell attachment domain 3F11, a fibronectin type 3 repeat [22–24]. The peptides were bound to 6-µm-diameter fluorescein isothiocyanate (FITC)-labeled carboxylated latex microparticles (Polysciences, Inc., Warrington, PA) following the manufacturer's protocol and binding kit (Polysciences, Inc.). Immediately before use, the beads were washed two times with PB1+ (PBS [Life Technologies], containing 0.32 mM sodium pyruvate, 5.55 mM glucose, and 3 mg/ml BSA; [25]).

Binding of RGD Peptide-Coated Beads to Zona-Free Bovine Oocytes

Twenty-four hour in vitro-matured (IVM) bovine oocytes were incubated in 0.05% pronase to remove zonae pellucidae (ZP). As soon as the ZP swelled and began to thin, the oocytes were transferred to PB1+ medium and worked up and down through a drawn glass pipette. The oocytes were then washed extensively in PB1+ medium. Exposure time to pronase was kept as short as possible in order to maintain the integrity and composition of the plasma membrane. These zona-free IVM oocytes were allowed to recover after removal of the ZP for 8 h in PB1+ at 39°C in 5% CO2. Zona-free oocytes were then incubated for 10 h at 39°C in 5% CO2 in a 30-µl drop of PB1+ containing 6-µm FITC-labeled latex beads alone or coupled to either the non-RGD or RGD peptide. After incubation, oocytes were washed through six 50-µl drops of PBS containing 5 mg/ml BSA. Oocyte/bead complexes were visualized by fluorescence, phase-contrast, or confocal microscopy (Bio-Rad Laboratories, Inc., Hercules, CA).

Effect of an RGD-Containing Peptide on In Vitro Fertilization

Oocytes were collected and matured according to methods previously described. Twenty-four-hour IVM cumulus cell-intact bovine oocytes were subjected to our standard in vitro fertilization protocol [15]. Cryopreserved bovine semen (Hoffman AI, Logan, UT) was thawed and live sperm separated by centrifugation on a 45%/95% layered Percoll gradient [26]. Motile spermatozoa obtained by this method were diluted in fert-TALP [15] to a final concentration of 1.0 x 106 per ml. Capacitation occurred in fert-TALP containing 10 µg/ml heparin. Treatment groups included no peptide and either the non-RGD peptide or the RGD peptide at 10, 100, and 1000 µg/ml. In vitro-matured oocytes were randomly separated into the seven treatment groups and fertilized in vitro for 18–24 h in the presence of the RGD or non-RGD peptide at 39°C in 5% CO2 and air. After the fertilization period, oocytes were vortexed in a 15-ml conical centrifuge tube containing TL-HEPES for 4 min to remove cumulus cells. A random sample of 30 oocytes were selected from all treatment groups and incubated for 30 min with 10 µg/ml Hoechst 3342. Sperm-oocyte fusion was assessed by visualization of sperm heads or pronuclei under UV light and a Chroma Technology (Brattleboro, VT) Hoechst filter set (exciter D360, emitter D460). Embryos were cultured with buffalo rat liver (BRL) cells in CR2 medium plus 10% FBS [15] at 39°C in 5% CO2 and cleavage was determined 48 h after removal of sperm. Data were analyzed with a chi-square analysis for independence in an r x 2 contingency table, where r equals the number of treatments.

Calcium Indicator Fura-2 AM Loading

Twenty-four-hour cumulus cell-free oocytes were loaded with Ca2+ indicator by incubation in 2 µM Fura-2 AM ester (Molecular Probes Inc., Eugene, OR) and 0.02% Pluronic F-127 (Molecular Probes Inc.) in PB1- solution (Ca2+- and Mg2+-free PBS [Hyclone Laboratories] containing 0.32 mM sodium pyruvate, 5.55 mM glucose, 3 mg/ml BSA, and 100 µM EGTA) at 39°C in darkness for 40–50 min. After loading Fura-2 AM, oocytes were washed extensively in M2- medium, pH 7.4 (Ca2+- and Mg2+-free M2+ with 100 µM EGTA; [27]).

Intracellular Calcium Monitoring and Calibration

Immediately prior to calcium measurements, the Fura-2 loaded oocytes were transferred to a 30-µl drop of M2- medium containing either 1 mg/ml of non-RGD or 1 mg/ml of RGD peptide and covered with warmed mineral oil. A duplicate set of experiments was carried out in M2+ medium, pH 7.4 that contained both Ca2+ and Mg2+ [27]. Three additional controls were included with each replication: electroporation with inositol (1,4,5)-triphosphate (IP3; Molecular Probes Inc.), electroporation with caffeine, and electroporation with medium (PBS minus Ca2+ and Mg2+ plus 100 µm EGTA) alone. These controls assured that the oocytes are of good quality and are capable of intracellular Ca2+ transients. Intracellular Ca2+ monitoring as well as electroporation conditions including concentrations of IP3 and caffeine and electroporation pulse were according to published methods [17, 18].

Effect of an RGD Peptide on Parthenogenetic Development

Oocytes were collected, matured in vitro, and stripped of cumulus cells according to the methods presented above. Oocytes were randomly assigned to one of the following seven treatment groups: 1, fertilized control; 2, 5 µM ionomycin for 4 min immediately followed by 0.2 mM 6-dimethylaminopurine (6-DMAP) for 4 h [28]; 3, 1 mg/ml non-RGD peptide for 15 min immediately followed by 0.2 mM 6-DMAP for 4 h; 4, 1 mg/ml RGD peptide for 15 min immediately followed by 0.2 mM 6-DMAP for 4 h; 5, 1 mg/ml non-RGD peptide for 15 min; 6, 1 mg/ml RGD peptide for 15 min; 7, 0.2 mM 6-DMAP for 4 h. After the various treatments were completed, the oocytes were extensively washed in CR2 medium plus 10% FBS and cultured in the CR2-BRL coculture system [15]. Cleavage was recorded on Day 2 and blastocyst development was recorded on Day 8. These treatment groups were compared to in vitro-fertilized oocytes according to our standard laboratory procedures [15, 16]. Data were analyzed with a chi-square analysis for independence in an r x 2 contingency table, where r equals the number of treatments.

RESULTS

Ability of an RGD-Containing Peptide to Bind to the Plasma Membrane of Bovine Oocytes

After removal of the ZP, bovine oocytes were able to recognize an RGD-containing peptide in that fluorescent immunobeads coated with GRGDSPK (RGD-containing peptide) bound to the plasma membrane. In contrast, bead matrix alone or immunobeads coupled to CQDSETRTFY (non-RGD-containing peptide) did not bind to the surface of the bovine oocyte. The results for all oocytes (four repetitions) are summarized in Table 1, and representative photographs are shown in Figure 1. Although the data are not shown, when the peptide/bead complex is incubated with the zona-free oocytes in PB1-, no beads were observed binding to the oocyte in any of the treatment groups.


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  		TABLE 1. Binding of RGD peptide–coated beads to zona-free bovine oocytes



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  		FIG. 1. Binding of peptide-coated beads to zona-free bovine oocytes. Six-micrometer beads coated with either non-RGD peptide (A) or RGD peptide (B–D) were allowed to adhere to zona-free bovine oocytes. Oocyte/bead complexes were then washed and subjected to either fluorescent microscopy (A–C) or confocal microscopy (D). Panels A and B are combination phase-contrast and fluorescence. Panels C and D are fluorescent images only. Scale bar = 50 µm, which applies to panels A–D.

Ability of an RGD-Containing Peptide to Block Fertilization

In four repetitions, GRGDSPK impaired the ability of spermatozoa to bind to or penetrate the surface of the bovine oocyte in a dose-dependent manner as indicated by the decrease in cleavage. The GRGDSPK peptide at a concentration of 10 µg/ml interfered with sperm binding or penetration thereby reducing fertilization, cleavage, and subsequent development of bovine oocytes as compared to the control (P < 0.05; Table 2). The percentage of oocytes that cleaved decreased in a dose-dependent manner with a concentration of 1000 µg/ml effectively blocking the cleavage resulting from fertilization (Table 2). In contrast, the non-RGD-containing peptide had no effect on fertilization regardless of the concentration as compared to the standard in vitro fertilization treatment group (Table 2). Exposure to the RGD- or non-RGD-containing peptides at all concentrations had no adverse effect on the morphology of the oocyte or the motility, life span, or morphology of the spermatozoa. The random samples of oocytes indicated there were no sperm heads or pronuclei seen in 29 of 29 oocytes when treated with RGD peptide. Sperm heads or pronuclei were observed in 25 of 30 oocytes in the fertilized control group and in 26 of 31 oocytes in the non-RGD treatment group.


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  		TABLE 2. Effect of an RGD peptide on fertilization rate

Ability of an RGD-Containing Peptide to Induce Intracellular Calcium Transients

To establish whether an RGD-containing peptide could induce intracellular Ca2+ transients similar to those observed at fertilization, MII bovine oocytes were incubated in the presence of the RGD- or non-RGD-containing peptides. Immediately prior to calcium measurements, the Fura-2-loaded oocytes were transferred to M2- medium containing either 1 mg/ml of non-RGD or 1 mg/ml of RGD peptide (Fig. 2A). A duplicate set of experiments was carried out in M2+ medium (Fig. 2B). Oocytes incubated with the RGD-containing peptide in M2- medium showed no detectable calcium transients in the absence of divalent cations (representative graphs are shown in Fig. 2A). When oocytes were incubated in medium that contained the divalent cations Ca2+ and Mg2+, two intracellular Ca2+ transients were observed (representative graphs are shown in Fig. 2B). Oocytes incubated in medium alone or in the presence of the non-RGD-containing peptide failed to generate any intracellular Ca2+ oscillations regardless of the presence or absence of divalent cations in the extracellular medium.



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  		FIG. 2. Effect of a non-RGD or RGD peptide at 1 mg/ml on [Ca2+]i in Ca2+- and Mg2+-free medium (A) and Ca2+- and Mg2+-containing medium (B). IVM, cumulus-free, Fura-2-loaded oocytes were incubated in the presence of peptide in medium. [Ca2+]i was determined by measuring ratio changes of fluorescence intensity at 510 nm wavelength emission when excited at 340 nm and 380 nm. The system is calibrated at 39°C using Calibration Buffer Kit I with magnesium according to the protocol provided by Molecular Probes. Fura-2 potassium salt is used as the Ca2+ indicator with a Kd value of 224 nM. However, we present our data in a ratio with a value of 3 approximately equal to 717 nM free [Ca2+]i. Representative tracings of all treatment groups are shown.

To investigate further the ability of the peptides to induce intracellular Ca2+ oscillations, oocytes were incubated in various concentrations of the RGD- and non-RGD-containing peptides (ranging from 10 to 1000 µg/ml in M2+ medium). All measurements were carried out in M2+ medium (Tables 3 and 4). In a total of 10 replicates, intracellular Ca2+ transients were observed in 20 of 20 oocytes incubated in 1000 µg/ml RGD peptide, in 19 of 20 oocytes in 100 µg/ml RGD peptide and in 17 of 20 oocytes in 20 µg/ml RGD peptide. No intracellular Ca2+ transients were observed in any of the oocytes in medium alone, non-RGD treatment groups, or in the RGD peptide at 10 µg/ml. In all the responding oocytes, two intracellular Ca2+ transients 21.5 ± 1.9 min apart were observed. Although not shown, all batches of oocytes used for these treatments failed to respond to electroporation in medium alone, while electroporation with either IP3 or caffeine produced intracellular Ca2+ transients in agreement with previous reports [14, 17, 18, 29].


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  		TABLE 3. Number of oocytes responding and height of intracellular Ca2+ peak

Ability of an RGD-Containing Peptide to Induce Parthenogenentic Development

The ability of a receptor to initiate parthenogenetic development in bovine oocytes was also investigated. The percentage of oocytes treated with 1 mg/ml RGD peptide alone that cleaved (3%) and reached the blastocyst stage (0%) was statistically no different than those activated with 1 mg/ml non-RGD peptide and 6-DMAP (5% cleavage and 0% blastocyst development) or those activated with non-RGD peptide alone (3% cleavage and 0% blastocyst development). The percentage of oocytes treated with ionomycin and 6-DMAP that cleaved (63%) and reached the blastocyst stage (34%) was statistically higher (P < 0.05) than those activated with 1 mg/ml RGD peptide and 6-DMAP (35% cleavage and 19% blastocyst development). In a total of eight replicates, these two treatments were statistically different (P < 0.05) as compared to all other treatment groups mentioned above (Table 5).


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  		TABLE 5. Effect of an RGD peptide on parthenogenetic development

DISCUSSION

In this study, we reveal a potential role for integrins in sperm-egg interactions by investigating the effects of an RGD-containing peptide on in vitro fertilization, generation of intracellular Ca2+ transients, and parthenogenetic activation and subsequent development of bovine oocytes. In the experiments presented, we showed that the RGD ligand will bind to the bovine oocyte. The binding of this tripeptide appears to be specific in that the bead matrix alone or bead matrix incubated with the non-RGD peptide failed to adhere to the surface of bovine oocytes. To our knowledge this is the first report identifying the potential role of integrins and their ligands in the process of bovine fertilization. The results indicate that the sperm-egg interaction may be mediated through the cell surface receptors that contain RGD recognition sequences. The inhibitory effects of the RGD-containing peptide were not associated with changes in the sperm motility or morphology or with oocyte morphology. These results indicate that sperm-oocyte interactions in cows are mediated by RGD-dependent pathways via an integrin on the surface of bovine oocytes. These results are in agreement with reports that RGD-containing peptides can competitively inhibit sperm-oocyte adhesion and subsequent egg penetration [3, 4]. Using peptide-coated beads, these authors also showed that the RGD-binding molecule localized on the oocyte. In 1992, Fusi et al. [6] also reported, using immunobeads coated with RGD-containing peptides, that the oolemma of human oocytes is capable of recognizing the RGD sequence. In 1995, Almeida et al. [5] demonstrated the presence of integrins and suggested their possible role as a sperm receptor in murine oocytes. In 1995, Evans et al. [30] demonstrated that fertilization in the mouse could be partially inhibited with various RGD peptides that caused a decrease in sperm-egg fusion by 30 to 60%. They also indicated that peptides containing the tripeptide QDE decreased the binding and fusion of murine sperm and oocytes by approximately 70%. These results suggest that in the mouse sperm-egg binding and fusion may utilize multiple molecules and/or multiple sites on molecules.

Although an RGD-containing peptide accompanied [Ca2+]i release in Xenopus [13], this is the first published report in mammalian species that demonstrates the ability of an RGD-containing peptide to initiate intracellular Ca2+ transients similar to those observed at fertilization [31]. The ability of integrins and their ligands to mediate calcium transients or spikes is well documented in a variety of cell types [2]. The ability of an RGD peptide to initiate calcium transients would seem to support our hypothesis that the initiation of these transients is a receptor-mediated process and that no soluble factor from the sperm is necessary to initiate calcium transients similar to those seen at fertilization. The apparent requirement for divalent cations for bead–RGD binding and for the initiation of the sperm-oocyte binding, the initiation of intracellular Ca2+ transients is in agreement with published work on integrin structure and function [32]. Data presented in this paper indicate peptide binding to integrin or integrin-like molecules on bovine oocytes depends on the presence of extracellular Ca2+ and/or Mg2+. The nature of the cations can affect both the affinity and specificity of the receptor for its ligand [1]. The importance of receptor/ligand binding and the nature and concentration of divalent cations on the processes of fertilization and oocyte activation is well established [1, 2, 5, 13].

Three different hypotheses have been put forth to explain how sperm initiate [Ca2+]i oscillations in mammalian eggs [33, 34]. One hypothesis is that spermatozoa interact with a receptor located in the plasma membrane of the oocyte. This receptor is postulated to be coupled to a G-protein or to have tyrosine kinase activity and to be able to activate phospholipase C (PLC) that, in turn, stimulates the production of diacylglycerol (DAG) and IP3, a common Ca2+ releasing agent, produced from phosphatidyl inositol (4,5)-bisphosphate (PIP2). In support of this hypothesis, serotonin [35], acetylcholine [36], or injection of GTP{gamma}[S] [37] have been shown to elicit [Ca2+]i oscillations. In addition, recent studies have shown that activation of pig oocytes via an exogenously introduced rat M1 muscarinic receptor resulted in events associated with oocyte activation: calcium release, cortical granule release, a decrease in histone H1 kinase activity, and progression through the cell cycle [38, 39]. These results provide evidence indicating porcine oocytes possess a G-protein-coupled signal transduction pathway that leads to a series of intracellular changes associated with oocyte activation. The participation of a tyrosine kinase pathway in mammalian fertilization remains under investigation, although some components of this pathway have been detected in mouse eggs [40]. The importance of tyrosine kinases has also been investigated in Xenopus oocytes [41, 42].

The second hypothesis proposes that a factor from the sperm is released into the egg and, by interacting with unknown cytosolic targets, generates [Ca2+]i oscillations. Several lines of evidence support this theory. First, injection of a sperm preparation into hamster and mouse eggs triggered [Ca2+]i oscillations similar to those observed during fertilization [43, 44], and in one study, egg activation was demonstrated [45]. Secondly, in most species, sperm-egg fusion appears to precede the initiation of oscillations [33]. Thirdly, injection of intact sperm into the cytoplasm of human eggs has been shown to initiate [Ca2+]i oscillations and embryonic development [46].

The third hypothesis proposes that the sperm acts as a channel or pore to allow extracellular Ca2+ to enter the egg causing intracellular Ca2+ release [47]. Alternatively, in 1983 Jaffe [48] suggested that the sperm may be the source of Ca2+; favored by an increase in mammalian sperm Ca2+ during capacitation and acrosome reaction.

Data presented in this paper would seem to support the hypothesis that [Ca2+]i oscillations in bovine eggs similiar to those observed at fertilization is a receptor-mediated process not requiring a cytoplasmic factor delivered by spermatozoa.

One of two or a combination of both Ca2+ receptors or channels are likely to mediate Ca2+ release in mammalian oocytes. As [Ca2+]i channels, at least three isoforms of both ryanodine receptors (RyR) and IP3 receptors (IP3R) have been identified, and the existence of both receptors and different isoforms have been observed in excitable and nonexcitable cells [17, 49–54]. Whether these receptors/channels act together or act as two separate components remains to be elucidated. The IP3R is clearly active, as injection or electroporation of IP3 elicits [Ca2+]i transients [17, 29, 35]. Inhibition of IP3R-mediated Ca2+-release, either by injecting GDPß[S], an inhibitor of G-proteins [55] or by competitively inhibiting IP3R with a blocking antibody [56] or heparin [17], inhibits fertilization-induced [Ca2+]i oscillations and egg activation. The second mechanism for Ca2+ mobilization is a Ca2+-induced release (CICR) that is believed to be mediated by the RyR [57]. This pathway appears to be totally distinct from the IP3R pathway [17]. In bovine eggs, injection of ryanodine, caffeine, and cADPR (all RyR agonists) elicited a Ca2+ response [17]. Sun et al. [11] reported that preinjection of the IP3 antagonist heparin into bovine oocytes did not inhibit characteristic calcium changes following subsequent fertilization. In addition, Nakada et al. [58] reported that preinjection of a monoclonal antibody directed against the mouse brain IP3R failed to inhibit fertilization-induced calcium oscillations in two thirds of fertilized bovine oocytes examined. Yue et al. [18] and Machaty et al. [38] evaluated the ability of IP3 and ryanodine agonists to induce intracellular calcium release in bovine and porcine oocytes, respectively, at various times during maturation. At all concentrations of ryanodine evaluated, the germinal vesicle (GV) oocytes either failed to respond or exhibited a reduced intracellular calcium response as compared to metaphase II (MII) oocytes. Similar results were reported by Yue et al. [18] when comparing the intracellular calcium response of GV, metaphase I (MI), and MII staged bovine oocytes to either IP3 or ryanodine. The results from our laboratory [18] as well as others [38, 59] indicating changes in both biological activity and location/presence of RyR and IP3R during oocyte maturation potentially have important implications relative to normal development. The role of the IP3R and/or RyR pathway in the generation of Ca2+i oscillations remains to be elucidated.

The results relative to parthenogenetic development point to several interesting possibilities about the action of this peptide and the mechanism and/or factors involved in oocyte activation. Despite the growing evidence supporting the principal role of the RGD domain in determining the functional characteristics of the adhesive protein, it is becoming clear that the secondary contact sites, distinct from the RGD tripeptide, contribute to the binding and functional properties of RGD-containing peptides [4, 13, 60]. Even though the linear RGD-containing peptide used in this study was able to block fertilization and induce intracellular Ca2+ transients, a cyclic RGD peptide or modifying the amino acid environment proximal to the tripeptide RGD may enhance the biological activity of this peptide and may negate the observed requirement for 6-DMAP in the parthenogenetic activation protocol. The requirement for 6-DMAP in the activation protocol may suggest a possible mechanism for sperm–egg binding and subsequent activation. The ability of the peptide to initiate intracellular Ca2+ transients but not full activation as evidenced by parthenogenetic development may suggest the need for two factors to produce complete oocyte activation. It is possible that intracellular calcium transients induced by the ligand–receptor pair may work in conjunction with a soluble factor produced by and released from the sperm into the oocyte cytoplasm upon fusion with the oocyte. This soluble factor is postulated to mimic the effects of 6-DMAP, which inhibits protein phosphorylation [61]. These results may indicate that both a receptor-mediated pathway and a soluble factor supplied by the spermatozoa are required for complete activation and subsequent development of bovine oocytes. It is also possible that the need for 6-DMAP represents two receptor-signaling pathways that are required for complete activation. The ligation of this receptor with the RGD peptide may only activate one part of the complete pathway that is needed for activation. It seems possible that a similarity with the mouse could be drawn in that multiple sites or receptors may be required for the complete activation of the bovine oocyte.

A 1999 report by Perry et al. [62] further supports the data presented here and the two-component activation system hypothesis proposed above. Using the mouse model, it was reported that complete activation was not obtained without both a cytosolic factor and a factor referred to as a submembrane fraction. In their system, both components were required by the oocyte before complete activation and subsequent development would occur. The submembrane factor or spermborne oocyte-activating factor(s) (SOAF) was proteinaceous in nature and appears obligate for activation but not sufficient for it, requiring at least one additional sperm component that is heat stable. An attractive hypothesis is that the RGD ligand stimulates the SOAF-induced pathway and the 6-DMAP, used herein, induces the heat-stable companion pathway for complete activation. Further, it seems logical that, at least in cattle, one or both of these pathways may use either the IP3 or ryanodine calcium receptors/channels to mediate their response.

Research on mammalian spermatozoa has shown the presence of a detergent-insoluble complex that is specifically adherent to the luminal surface of the outer acrosomal membrane in cows [63–66] and hamsters [65, 67–69]. In 1996, NagDas et al. [66] termed this detergent-insoluble continuous sheet attached to the bovine sperm head at the anterior margin of the equatorial segment the outer acrosomal membrane–associated matrix complex (OMC). In addition, it has been reported that hamster spermatozoa are comprised of two ultrastructurally distinct matrix domains containing a detergent-insoluble membrane skeleton of the outer acrosomal membrane termed the acrosomal lamina–matrix complex (ALM) [69]. The acrosome contents of hamster spermatozoa exhibit differences in structural appearance [64, 65, 67, 70] and elements of the acrosomal matrix remain intact and associated with the membrane complex after the acrosome reaction [70–72]. The structural stability of these acrosomal matrix elements in cattle and the hamster is emphasized by their resistance to solubilization using various extraction regimens [64, 65, 68]. The stability is further emphasized knowing that complete removal of the OMC and ALM from the sperm heads required detergent extraction followed by homogenization [66, 69].

Evidence for the presence of integrin or integrin-like molecules on the surface of oocytes has been found in many species including hamsters [3, 4], humans [6], Xenopus [73], mouse [30], and pigs [74]. We hypothesize that there is a ligand for integrins that contains the RGD recognition sequence associated with the stable acrosomal matrix elements of bovine spermatozoa. The nature or mechanisms that establish this association are not known; it probably represents a specific protein-protein interaction between matrix peptides or polypeptides and a peptide or protein localized to the outer acrosomal membrane. The results in this study strongly imply that a sperm protein containing the RGD sequence induces egg activation through interaction with a receptor molecule on the surface of the oocyte plasma membrane. This report also implicates the involvement of integrins in the receptor-mediated induction of signal transduction pathways capable of generating intracellular Ca2+ transients. The isolation, identification, and sequence of this RGD-containing ligand and the contribution of the IP3 and/or ryanodine pathway(s) remains to be studied. Altogether the data strongly indicate that integrins have a role in fertilization and activation of bovine oocytes.


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  		TABLE 4. Time to peak Ca2+ concentration and duration of peaks.a

ACKNOWLEDGMENTS

We thank E. A. Millers in Hyrum, Utah for their generous contribution of the bovine ovaries used in these experiments. We also thank Dr. Ilka Nemere for her critical review of this manuscript and Dr. Kurt Albertine and the staff of the University of Utah Microscopy Laboratory for use of the Edge Real-Time 3D microscope model number R4000.

FOOTNOTES

First decision: 14 January 2000.

1 This work was supported in part by a grant from the United States Department of Agriculture National Research Initiative Competitive Grants program 19992247. Utah State University Agricultural Experiment Station Publication No. 7210. Back

2 Correspondence. FAX: 435 797 2118; kwhite{at}cc.usu.edu Back

Accepted: February 3, 2000.

Received: December 10, 1999.

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