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Gamete Biology |
Department of Animal Sciences,4 University of Missouri, Columbia, Missouri 65211
Department of Obstetrics and Gynecology,5 College of Medicine, University of Florida, Gainesville, Florida 32610
Reproductive Genomic Program,6 Monsanto, St. Louis, Missouri 63017
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, in vitro fertilization, oviduct, ovum, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In benchmark experiments in the rabbit, Oliphant et al. [3] identified an estrogen-dependent protein synthesized de novo by nonciliated oviductal epitheilial cells that was secreted into the oviduct lumen, where it bound to the egg coat. Analyses of de novo synthesized and secreted oviductal proteins in the pig has led to the identification and characterization of 10 proteins. The major secretory product, a high-molecular-weight oviduct-specific, estrogen-dependent glycoprotein (OGP; see [4] for review) has been identified in a variety of species, including the mouse [5, 6], hamster [7, 8], rabbit [3], cow [9, 10], pig [11, 12], baboon [13, 14], rhesus monkey [15], and human [16, 17]. The nucleotide and amino acid sequence of OGP is highly conserved among mammalian species [4, 6, 8, 10, 12, 14, 15, 17], and several structurally related molecular-weight variants and isoforms exist within species [4]. This OGP family belongs to an emerging mammalian family of chitinase-like proteins that lack chitinase enzyme activity. In the pig, three major isoforms of OGP (previously called OSP) were identified as porcine (p) OGP-E1, -E2, and -E3 [2]. Although OGP displays regional and temporal differences in expression in the pig oviduct, electron microscopy/immunogold localization studies demonstrated that pOGP binds to the zona pellucida, perivitelline space, and plasma membrane of ovulated oocytes and oviductal embryos [18]. These interactions suggest that pOGP plays a functional role regulating fertilization and/or early embryonic development.
The efficiency of in vitro production (IVP) of porcine embryos through in vitro fertilization (IVF) of in vitro-matured (IVM) oocytes has improved dramatically in recent years; however, deficiencies in IVP of pig embryos are indicated by both a high incidence of polyspermic penetration and low success rates for embryonic development. Polyspermy remains a major obstacle to successful production of large-scale numbers of viable IVP embryos (see [19] for review). Various attempts to control polyspermy have included preincubation of oocytes with oviductal fluid [20, 21] and coculture of oocytes with oviductal epithelial cells and/or conditioned medium [22–24]. Treatment of spermatozoa with oviductal epithelial cells reduced polyspermy and improved embryonic development [25]. Although such studies demonstrated that compounds of oviductal origin could affect IVF, significantly reduce the incidence of polyspermy, and increase numbers of embryos, they were not well suited to address the identity of important mediators of fertilization and development in vitro. In contrast, the development of in vitro maturation and IVF under defined serum-free culture conditions in the pig [26] provides a powerful tool for functional evaluation of specific purified oviduct proteins that may be involved at the level of both fertilization and development. Kouba et al. [27] demonstrated that pOGP present during IVF reduced the incidence of polyspermy, reduced sperm-zona pellucida binding, and increased the percentage of pig embryos that developed to the blastocyst stage. Whether the observed effects of pOGP were mediated through the oocyte or through changes in spermatozoa, or both, was not determined. To gain insight into the mechanism(s) of action of pOGP, the objectives of this study were to determine whether 1) exposure of oocytes and/or spermatozoa to pOGP prior to IVF reduces the rate of polyspermy and improves embryo development, 2) spermatozoa function (e.g., capacitation and zona binding) is altered by pOGP, and 3) pOGP has embryotrophic effects. Information gained from such studies may prove useful for improving the efficiency of embryo production in vitro and understanding the role of oviduct proteins in fertilization and development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unless otherwise stated, all chemicals used in this study were purchased from Sigma Chemical Company (St. Louis, MO). The medium used for oocyte maturation was BSA-free tissue culture medium 199 (Gibco, Grand Island, NY) supplemented with 0.57 mM cysteine, 10 ng/ml epidermal growth factor, 0.5 µg/ml FSH, 0.5 µg/ml LH, and 0.1% (w/v) polyvinyl alcohol (PVA). Detailed procedures for oocyte collection and in vitro maturation in this protein-free medium have been described previously [26, 28].
The basic medium used for IVF was modified Tris-buffered medium (mTBM) consisting of 113.1 mM NaCl, 3 mM KCl, 10 mM CaCl2, 20 mM Tris, 11 mM glucose, 5 mM sodium pyruvate, 1 mM caffeine, and 0.1% (w/v) fatty acid-free BSA (A-7888) [29, 30]. The embryo culture medium was North Carolina State University medium (NCSU) 23 containing 0.4% (w/v) BSA. All gamete incubations were performed in microdrops under paraffin oil (Fisher Scientific, Fair Lawn, NJ) equilibrated at 39°C in an atmosphere of 5% CO2 in air.
Porcine OGP Preparation
Porcine OGP used in this study was a highly purified preparation isolated from oviduct culture medium by heparin-agarose affinity chromatography that was immunoreactive to a polyclonal anti-pOGP IgG. This preparation contained four major isoforms of OGP, E1–E4. The purification and characterization of pOGP was described previously [11, 27]. Lyophilized pOGP (5 mg) was resuspended in BSA-free mTBM (for IVF experiments) or NCSU 23 (for in vitro culture experiments), insoluble particulates were removed by centrifugation (12 000 x g), and the total protein content was determined by bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL) using a BSA standard. Aliquots were stored at -20°C until used, and all experimental replicates were performed using pOGP from the same batch.
In Vitro Fertilization
After 44 h of in vitro maturation, cumulus cells were removed with 0.1% (w/v) hyaluronidase in Hepes-buffered Tyrode lactate (TL-Hepes) medium containing 0.1% (w/v) PVA. Nude oocytes were initially washed three times in 500 µl of TL-Hepes medium and subsequently in mTBM. Thirty to 35 oocytes were transferred into preequilibrated 50-µl drops of mTBM under paraffin oil. Cryopreserved semen [30] was thawed, and spermatozoa were washed two times by centrifugation (1000 x g for 4 min) in Dulbecco PBS (DPBS; Gibco) supplemented with 1 mg/ml BSA. Spermatozoa were resuspended in mTBM, and 50 µl of this suspension was added to the fertilization drop containing IVM oocytes to give a final concentration of 3 x 105 spermatozoa/ml. At 6 h postinsemination, oocytes were washed three times and cultured in NCSU 23. Oocytes were fixed at 12 h postinsemination in 25% (v/v) acetic acid in ethanol and stained with 1% (w/v) orcein in 45% acetic acid to assess meiotic stages, spermatozoa penetration rate, and the incidence of polyspermy, as described previously by Hunter and Polge [31]. Oocytes were considered penetrated when one or more decondensed spermatozoa heads and/or male pronuclei and corresponding spermatozoa tails were present. The rate of polyspermy was determined from the number of oocytes penetrated. To evaluate the effect of treatment on embryo development, putative zygotes were incubated for 144 h in NCSU 23, at which time the number of embryos reaching the blastocyst stage of development was determined. Efficiency of fertilization was determined as the number of embryos with two pronuclei divided by the total number of oocytes inseminated. Blastocyst formation rate was calculated as a percentage of the total number of oocytes inseminated.
Fertilization of Oocytes Pretreated with pOGP
To test the hypothesis that exposure of oocytes to pOGP after maturation but prior to fertilization reduces the incidence of polyspermy in vitro, cumulus-free IVM oocytes were exposed to 0, 1, 10, 20, or 40 µg/ml pOGP in preequilibrated mTBM for 3 h, washed three x in pOGP-free mTBM, and transferred to a pOGP-free fertilization drop. Untreated spermatozoa were then added to fertilization drops containing pretreated eggs, and the gametes were cocultured for 6 h. Following coincubation, putative zygotes were washed three x in NCSU 23, incubated 6–12 h in NCSU 23, fixed and examined as described above, or cultured for 144 h to determine the number of embryos reaching the blastocyst stage of development.
Fertilization of Untreated Oocytes by Spermatozoa Pretreated With pOGP
To study the effect of pOGP on the fertilizing ability of spermatozoa, washed spermatozoa (107) were resuspended in 100 µl mTBM containing 0, 1, 10, 20, or 40 µg/ml OGP and incubated for 1 h at 39°C under an atmosphere of 5% CO2 in air. Preincubated spermatozoa were washed three x in pOGP-free mTBM, diluted in the same medium to yield a final concentration of 3 x 105 spermatozoa/ml, and coincubated with untreated IVM oocytes for 6 h at 39°C under an atmosphere of 5% CO2 in air. After sperm-oocyte coincubation, putative zygotes were washed three x in NCSU 23, incubated for 6–12 h, fixed, and examined as described above.
Effect of Preincubation of Both Oocytes and Spermatozoa Prior to IVF
Independent dose-response experiments indicated that the efficiency of IVF was optimized when oocytes or spermatozoa were preincubated with 10 µg/ml pOGP. Therefore, 10 µg/ml was the concentration chosen to evaluate whether a synergistic effect of preincubation of both oocytes and spermatozoa could be observed on polyspermy. Oocytes were preincubated with 0 or 10 µg/ml pOGP for 3 h and washed prior to insemination as described above. Spermatozoa were preincubated with 0 or 10 µg/ml pOGP for 1 h and washed as described above. IVF was performed by coincubating 1) control spermatozoa with pretreated oocytes, 2) pretreated spermatozoa with control oocytes, 3) pretreated spermatozoa and pretreated oocytes, and 4) untreated spermatozoa and oocytes (control). After fertilization, putative zygotes were washed three x in NCSU 23 and fixed as described above or were incubated for 144 h at which time the number of embryos reaching the blastocyst stage of development was determined. Four replicates with a total of 109–127 oocytes per treatment were evaluated for penetration and polyspermy. A total of 122–133 embryos per treatment from four replicates were scored for blastocyst development.
Evaluation of Sperm Capacitation
Semen was collected from fertile boars by the gloved hand method [32]. The sperm-rich fraction was centrifuged (800 x g for 5 min), and the pellet was resuspended in DPBS and centrifuged two more times. Prior to the last wash, the diluted semen was split into two equal portions, and the resulting pellet was resuspended in capacitating medium (CM; 4.8 mM KCl, 1.2 mM KH2PO4, 95 mM NaCl, 5.55 mM glucose, 25 mM NaHCO3, 2 mM CaCl2, 2 mM pyruvic acid, and 0.4% BSA, pH 7.4) or noncapacitating medium (NCM; modified CM devoid of calcium, BSA, and NaHCO3) as described by Tardif et al. [33]. Spermatozoa were diluted to 1–2 x 107/ml, pOGP was added at 0 or 10 µg/ml, and the spermatozoa suspensions were incubated in a final volume of 1 ml at 39°C in a humidified 5% CO2 atmosphere for 4 h. Sperm capacitation was assessed by the ability of spermatozoa to undergo the calcium ionophore A23187-induced acrosome reaction. After 4 h of incubation, spermatozoa suspensions were divided into 500-µl aliquots to which 10 µM A23187 (Cambridge Isotope Laboratories, Andover, MA) in dimethyl sulfoxide (DMSO) or DMSO alone (control) was added, and spermatozoa were incubated for an additional 15 min. Calcium (2 mM) was added to the NCM immediately prior to addition of A23187 or DMSO to ensure availability of sufficient calcium to allow spermatozoa to undergo the acrosome reaction. The percentage of acrosome-reacted spermatozoa was determined by Coomassie-blue staining, as described by Larson and Miller [34]. Following exposure to A23187 or DMSO, spermatozoa were fixed in an equal volume of 4% paraformaldehyde for 15 min and washed twice in 100 mM ammonium acetate (pH 9). Fifteen microliters of the spermatozoa suspension was smeared onto a slide and air dried. Slides were incubated in 0.22% Coomassie blue G-250 diluted in 50% methanol, 10% acetic acid for 5 min, rinsed with distilled H2O, and air dried, and coverslips were mounted with Permount mounting medium (Fisher). Three hundred spermatozoa were evaluated and classified as acrosome intact or acrosome reacted.
Sperm-Zona Binding
IVF of oocytes pretreated with OGP was performed as described above. After fertilization, oocytes were transferred into one well of a nine-well Pyrex glass plate filled with 400 µl of 37°C PBS, and 100 µl of 10% formaldehyde was slowly added to bring the final concentration of formaldehyde to 2%. After 40 min of fixation at room temperature, oocytes were washed in two wells of PBS, and the DNA fluorochrome 4',6'-diamidino-2-phenylindole (2.5 µg/ml) was added to the solution. Samples were mounted on microscope slides in VectaShield (Vector Laboratories, Burlingame, CA) mounting medium and sealed with clear nail polish. Images was recorded with an Eclipse 800 microscope, CoolSnap camera, and Metamorph software (Nikon, Tokyo, Japan). Images of three focal planes were acquired, each image was pseudo-colored (blue: metaphase II chromatin; red and green: upper and lower distinct planes of spermatozoa, respectively), and a composite image was generated to depict the entire depth of the zona pellucida in a single focal plane. Data were archived on CD-Rs, edited with Adobe Photoshop 5.5 (Adobe Systems, San Jose, CA), and printed on an Epson Stylus photo printer (Epson America, Long Beach, CA).
Embryotrophic Effects of OGP
Putative zygotes, produced in vitro under standard fertilization conditions without pOGP, were washed and randomly assigned to in vitro culture (NCSU 23 supplemented with 0.4% BSA) drops (n = 35/drop; 2 drops/treatment for each replicate) containing 0 (control) or 10 µg/ml pOGP (continuous) and cultured for 144 h. A second treatment group was cultured in pOGP-supplemented medium for 48 h, washed, and cultured in control medium without pOGP for an additional 96 h to examine the effect of transient exposure to pOGP. Cleavage rate and blastocyst formation were determined at 48 and 144 h after insemination, respectively. The experiment was replicated four times with 276–284 embryos examined per treatment. Developmental effects of pOGP were evaluated by determining the total number of cells present in control and pOGP-treated embryos. Blastocyts (Day 6) were fixed in 4% glutaraldehyde, washed, and placed in TL-Hepes-PVA containing Hoechst 33342 (5 µg/ml) for 10 min. Embryos were mounted under a coverslip, and the number of cells in each blastocyst was determined by counting under ultraviolet illumination the number of nuclei stained with Hoechst 33342. For cell number determination, 30 embryos from three replicates were analyzed for each treatment.
Statistical Analysis
All data analysis was performed using SAS/STAT software [35]. Dependent variables were analyzed for normality using the Wilk-Shapiro test. Percentage data not normally distributed were square root or log transformed. After transformation, data were analyzed by the general linear models procedure of SAS. When a significant treatment effect was detected, a Duncan multiple-range test was used to determine treatment differences. Means are reported as least squares ± SEM unless noted otherwise. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When oocytes were preincubated with various concentrations of pOGP for 3 h and washed prior to IVF, penetration rate did not differ regardless of treatment; however, the incidence of polyspermy and the number of penetrated spermatozoa per oocyte was reduced (P < 0.01) by preincubation with 10 µg/ml OGP (27.6 ± 4.4 and 1.32 ± 0.04, respectively) or 20 µg/ml OGP (25.0 ± 3.7 and 1.33 ± 0.06, respectively) compared with the control (50.5 ± 6.4 and 1.65 ± 0.13, respectively; Table 1). A trend (P = 0.07) for reduced polyspermy was apparent when the highest (40 µg/ml) concentration of pOGP was used. A common observation in porcine IVF is the tightly linked relationship between penetration rate and polyspermy. Those two parameters generally rise and fall concomitantly. Most important in this study, fertilization was not inhibited by preincubation of oocytes with pOGP, even though polyspermy was significantly reduced. The addition of pOGP maximized efficiency of fertilization (number of oocytes with two pronuclei/total number of oocytes inseminated x 100) to 47.2% when oocytes were preincubated with 10 µg/ml pOGP compared with 29.6% in the control (Table 1). No difference was observed in the percentage of embryos that formed blastocysts (range, 28.2–34.7%) regardless of the concentration of pOGP used (Table 2).
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Pretreatment of Oocytes, Spermatozoa, or Both Prior to IVF
To determine whether pOGP differentially modulates oocyte and spermatozoa function or whether exposure of both spermatozoa and oocyte to pOGP elicits a synergistic effect on fertilization parameters, the effect on fertilization rate of preincubation of oocytes, spermatozoa, or both gametes with pOGP and the incidence of polyspermy and blastocyst formation were assessed. The optimal concentration of pOGP (10 µg/ml as determined above and in separate experiments where spermatozoa were pretreated) was used in this experiment. Preincubation of oocytes or spermatozoa with pOGP significantly reduced (P < 0.01) polyspermy (oocytes: 19.8 ± 2.0; spermatozoa: 23.1 ± 3.1) compared with the control (35.6 ± 1.8). However, no synergistic effect was observed when both spermatozoa and oocytes were preincubated with pOGP (Fig. 1). Similar to the previous experiment, no difference was observed in the percentage of embryos that formed blastocysts (range, 27.6–30.5%) regardless of treatment.
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Sperm Capacitation and Sperm-Zona Binding
Spermatozoa were incubated in CM or NCM in the presence or absence of pOGP and induced to undergo the acrosome reaction with calcium ionophore A23187 to determine whether OGP treatment modulated sperm capacitation. No difference was observed between control and pOGP-treated groups in the number of spermatozoa that underwent the acrosome reaction in response to A23187, indicating that under the conditions used in this experiment (fresh spermatozoa exposed to low concentrations of pOGP, 10 µg/ml), pOGP did not affect capacitation (Table 3).
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To determine whether the observed reduction in polyspermic fertilization resulting from the addition of exogenous pOGP was due to altered sperm-zona binding, pretreated oocytes were fixed immediately after sperm-oocyte coincubation, and the number of spermatozoa bound per oocyte was determined by immunofluorescence analysis. Significantly fewer spermatozoa were bound to pOGP-treated oocytes (n = 92) compared with control oocytes (n = 93; 43.5 ± 6.2 vs. 79.2 ± 7.1, P < 0.01; Fig. 2). These data suggest that the observed reduction in polyspermic penetration caused by pOGP is at least partially explained by a reduction in the number of spermatozoa interacting with the zona pellucida and not by modulation of sperm capacitation. The acrosomal status of zona-bound spermatozoa was examined by immunofluorescence (using an acrosomal marker protein c-yes or fluorescein isothiocyanate-PNA) in pOGP-pretreated and control oocytes. No differences in the percentage of acrosome-reacted spermatozoa were apparent; however, results were not quantified. Therefore, the possibility that pOGP affects the physiological zona pellucida-induced acrosome reaction cannot be discounted.
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Embryotrophic Effects of pOGP
In a previous study, pOGP improved in vitro development of fertilized oocytes to blastocysts [27]. However, in that study, both oocytes and spermatozoa were exposed to pOGP during in vitro maturation and IVF, which also resulted in a reduced incidence of polyspermic penetration, confounding interpretation of those results. In the present study, porcine embryos were exposed to pOGP, which was absent during in vitro maturation and IVF, to evaluate effects on cleavage and blastocyst formation. The addition of 10 µg/ml pOGP, either transiently or continuously, significantly increased (P < 0.01) the percentage of embryos that underwent cleavage (57.0% ± 3.1% and 57.7% ± 3.3%, respectively) and the percentage of embryos that formed blastocysts (35.6% ± 1.8% and 36.6% ± 1.8%, respectively) compared with the control (42.3% ± 1.8% and 28.5% ± 1.3%, respectively; Fig. 3). There was no difference in the number of cells per blastocyst (34.9 ± 2.7 vs. 31.5 ± 1.7) for control embryos and pOGP continuously treated embryos, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two areas that are likely deficient in porcine IVF systems and are therefore receiving much attention are 1) the regulation of sperm capacitation and acrosome stability and 2) zona pellucida composition. Simultaneous penetration may be the underlying cause of polyspermy and may be related to the adverse effect of caffeine present in porcine IVF medium on spermatozoa function [39]. Sperm capacitation, measured by the ability of spermatozoa to undergo the calcium ionophore-induced acrosome reaction, was not enhanced by exposure to pOGP in vitro in the current study. Because of the reduced viability of frozen-thawed spermatozoa and the associated spontaneous acrosomal shedding that occurs during the incubation period in IVF medium (data not shown), fresh spermatozoa were used to examine capacitative effects of pOGP, and the assay was conducted with a sperm capacitation medium that differs from the caffeine-containing IVF medium. In a previous study using frozen-thawed spermatozoa, we found that preincubation of spermatozoa with concentrations of pOGP similar to those used in the current study increased penetration rate compared with controls [40]. However, the incidence of polyspermy tended to increase concomitantly with penetration rate. The results of the present study demonstrated that transient exposure of spermatozoa to low concentrations (10 µg/ml) of pOGP decreased the incidence of polyspermy without affecting penetration rates. These results suggest that the reduction in polyspermy after spermatozoa treatment with pOGP was not due to stimulation (or inhibition) of capacitation. Whether pOGP binds to porcine spermatozoa is unclear; reports of OGP binding to spermatozoa in other species are contradictory [41–44]. Recently, specific binding of an enriched preparation of human OGP to bonnet monkey spermatozoa was demonstrated [45]. Consequences of OGP binding to spermatozoa and its potential effects on spermatozoa function remain to be determined. Because of concerns with background staining observed with preimmune sera, immunofluorescence analysis of pOGP-spermatozoa binding was not conducted in the current study.
The addition of pOGP to oocytes consistently improved the percentage of oocytes that underwent normal (monospermic) fertilization in repeated experiments. One explanation for the reduction in polyspermy is that pOGP binds to the zona pellucida and forms a physical barrier to spermatozoa binding or occupies/modifies spermatozoa binding sites on the zona pellucida (number of zona-bound spermatozoa was significantly reduced by exposing oocytes to pOGP). Although a major effect of pOGP treatment of oocytes was observed during fertilization in vitro, including a reduction of spermatozoa binding to the zona pellucida, direct effects on spermatozoa function cannot be ruled out, because spermatozoa treatment alone reduced polyspermy in this study.
Inclusion of pOGP in embryo culture medium, without exposure to oocytes or spermatozoa before or during fertilization, resulted in embryotrophic effects. A previous study showed that pOGP improved in vitro development of fertilized oocytes to the blastocyst stage [27]. However, in that study, the effect on embryo development coincided with exposure of both oocytes and spermatozoa to pOSP during in vitro maturation and IVF, which also resulted in a reduced incidence of polyspermic penetration, and no additional effect of OGP was observed during in vitro culture. In vivo, embryos are exposed to pOGP only during early cleavage-stage divisions (one- to four-cell stage); therefore, possible beneficial effects of OGP should be exerted at that time. Stimulatory effects of pOGP on blastocyst formation of IVF embryos have been described in other studies [46]. In the present study, embryos were exposed to pOGP for 48 h (at which time porcine embryos are at the two- to four-cell stage) to mimic the expression/exposure pattern of pOGP and embryos in vivo. The short-term exposure to pOGP enhanced cleavage and subsequent blastocyst development, suggesting that pOGP exerts some developmental (i.e., metabolic) stimulus in addition to reducing polyspermy. Continuous exposure to pOGP did not provide additional embryotropic effects, suggesting a narrow window in which developmental effects may be manifested.
Although polyspermy occurs in other species [47–49], the extent of polyspermic fertilization is much higher in the pig. Therefore, the problem of polyspermic penetration is largely species specific and is confined to porcine IVF [30, 36]. Abnormal fertilization is relatively rare in pig oocytes fertilized in vivo if insemination occurs in a timely manner with respect to ovulation [50, 51]. In vivo, polyspermic fertilization is controlled by at least two major mechanisms: 1) the reproductive tract restricts the number of capacitated spermatozoa that reach the site of fertilization, and 2) spermatozoon penetration triggers cortical granule exocytosis and the subsequent cortical reaction at the surface of the zona pellucida and/or plasma membrane. The environment in vitro drastically increases the number of spermatozoa that reach the surface of the oocyte and bind to the zona pellucida; however, the reason this is more problematic for pig oocytes than for oocytes of other species is unknown. The distribution of cortical granules, their migration to the cortex of the oocyte during in vitro maturation, and the ability of IVM oocytes to undergo exocytosis [52] appears to be similar in oocytes matured in vivo. Those data, along with the fact that induction of cortical granule exocytosis by ionophore treatment prevents spermatozoa penetration of the zona pellucida [53], suggest that polyspermic penetration is due to an incomplete or delayed zona reaction [54] and not due to abnormal cortical granule exocytosis. The role that OGP may play in the cortical reaction or overcoming an incomplete or delayed zona reaction is unknown. However, preliminary unpublished studies with OGP and a cortical granule lectin suggest that an interaction may occur.
A phenomenon described as incomplete maturation of the zona pellucida has been observed at the ultrastructural level in porcine oocytes matured in vitro [55, 56]. It has been hypothesized that these morphological differences in the zona pellucida of IVM compared with ovulated oocytes reflect failure in the final maturation of the zona pellucida during in vitro maturation and may be responsible for a delayed and/or incomplete zona reaction in response to cortical exocytosis [56]. Thus, OGP probably is a key component involved in reducing the number of spermatozoa that bind, without affecting penetration, and possibly may have a role in the cortical reaction; the absence of OGP probably contributes to the problem of polyspermic penetration. OGP displays an expression pattern that is temporally associated with fertilization and early cleavage-stage development, and it associates with the zona pellucida and perivitelline space/vitelline membrane of ovulated oocytes and oviductal embryos. Exogenous OGP successfully reduces the incidence of polyspermy to levels approaching 20%. Macromolecules such as OGP, which are added to the zona pellucida in vivo as the oocyte transits the oviduct, may be necessary in vitro to confer full functionality on the zona pellucida, but OGP alone is not sufficient to eliminate polyspermic fertilization. It remains to be determined whether the lack of exposure of oocytes to OGP in IVF systems is manifest by incomplete maturation of the zona pellucida, creating oocytes that are compromised in their ability to mount a rapid and effective zona block.
Although the results of this study indicate that the biological effect on spermatozoa binding is elicited through an OGP interaction with the oocyte and spermatozoa prior to fertilization, the embryotrophic effect appears to occur in a narrow window of time following fertilization. Future studies using highly purified preparations of OGP isoforms, OGP-E1, -E2, and -E3, are needed to determine whether a specific isoform may be responsible for a specific function and by what mechanism(s) that activity occurs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Billy N. Day, Department of Animal Sciences, 159 Animal Science Research Center, University of Missouri, Columbia, Missouri 65211. FAX: 573 884 7827; dayb{at}missouri.edu
3 Current address: TMI Laboratories, 850 N. Kolb Rd., Tucson, Arizona 85710
Received: 20 February 2003.
First decision: 17 March 2003.
Accepted: 30 April 2003.
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D. S. McBride, C. Boisvert, G. Bleau, and F. W.K. Kan Evidence for the Regulation of Glycosylation of Golden Hamster (Mesocricetus auratus) Oviductin During the Estrous Cycle Biol Reprod, January 1, 2004; 70(1): 198 - 203. [Abstract] [Full Text] [PDF]
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