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Biology of Reproduction 63, 242-250 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular article

Effects of the Porcine Oviduct-Specific Glycoprotein on Fertilization, Polyspermy, and Embryonic Development In Vitro1

Andrew J. Koubaa, Lalantha R. Abeydeerad, Idania M. Alvarezb, Billy N. Dayd, and William C. Buhi2,a,b,c

a Department of Animal Science, b Department of Obstetrics and Gynecology, and c Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610–0294 d Department of Animal Science, University of Missouri-Columbia, Columbia, Missouri 65211

ABSTRACT

This study evaluated the effects of porcine oviduct-specific glycoprotein (pOSP) on in vitro fertilization (IVF), polyspermy, and development to blastocyst. Experiment 1 evaluated the effects of various concentrations (0–100 µg/ml) of purified pOSP on fertilization parameters, including penetration, polyspermy, male pronuclear formation, and mean number of sperm penetrated per oocyte. Experiment 2 examined the ability of an anti-pOSP immunoglobulin G to inhibit the observed effects of pOSP on fertilization parameters. Experiments 3 and 4 examined various concentrations of pOSP (0–100 µg/ml) on zona pellucida solubility and sperm binding, respectively. Lastly, experiment 5 assessed the effects of various concentrations of pOSP (0–100 µg/ml) on the in vitro embryo cleavage rate and development to blastocyst. Pig oocytes matured and fertilized in vitro were used for all experiments. An effect of treatment (P < 0.05) was detected for pOSP on penetration, polyspermy, and mean number of sperm per oocyte. Concentrations for pOSP of 0–50 µg/ml had no effect on sperm penetration rates; however, compared with the control, 100 µg/ml significantly decreased the penetration rate (74% vs. 41%). Addition of 10–100 µg/ml significantly reduced the polyspermy rate compared with the control (61% vs. 24–29%). The decrease in polyspermy achieved by addition of pOSP during preincubation and IVF was blocked with a specific antibody to pOSP. No effect of treatment was observed on zona digestion time relative to the control; however, the number of sperm bound to the zona pellucida was significantly decreased by treatment (P < 0.05). Compared with the control, all concentrations of pOSP examined reduced the number of sperm bound per oocyte (45 vs. 19–34). A treatment effect (P < 0.05) was observed for pOSP on embryo development to blastocyst but not on cleavage rates. Addition of pOSP during preincubation and fertilization significantly increased postcleavage development to blastocyst, but a synergistic stimulation on development was not detected when pOSP was included during in vitro culture. These results indicate that exposure to pOSP before and during fertilization reduces the incidence of polyspermy in pig oocytes, reduces the number of bound sperm, and increases postcleavage development to blastocyst.

fertilization, oviduct

INTRODUCTION

The porcine oviduct, in response to ovarian steroid hormones, synthesizes and releases proteins into the lumen and provides a selective serum transudate [1]. The cumulative synthesis and transport of these proteins into the oviductal lumen during proestrus, estrus, and metestrus creates a microenvironment capable of supporting important reproductive events, which include fertilization and early cleavage-stage embryonic development. Several de novo synthesized proteins of the porcine oviduct have been identified and characterized [2], including the pig oviductal-specific secretory glycoprotein (pOSP) family. This protein is highly conserved across investigated species, which include the human [3], sheep [4], mouse [5], cow [6], hamster [7], baboon [8] and rhesus monkey [9]. Ovariectomy and steroid hormone replacement studies have shown that pOSP mRNA and protein synthesis are estrogen-dependent, and that expression is greatest during the preovulatory period, at ovulation, and during early embryonic development [10]. Recent evidence also suggests that in addition to estrogen, LH may regulate expression of OSP protein by increasing the mRNA half-life [11]. The function of this protein remains unknown. Immunolocalization studies in the pig and other species, however, have revealed that pOSP (and appropriate OSPs) associate with the zona pellucida, perivitelline space and vitelline, and blastomere membranes of ovulated oocytes and oviductal embryos, respectively [2, 12], suggesting a role for this protein during fertilization and early embryonic development.

Embryonic in vitro culture (IVC) systems result in successful in vitro maturation/fertilization (IVM/IVF) and subsequent development of the early cleavage-stage pig embryo to the blastocyst stage [13–16]. However, a high incidence of polyspermy remains a major impediment in porcine IVF and often exceeds 50% [17–20]. Differences in polyspermy rates are much greater with in vitro-matured oocytes (65%) than with ovulated oocytes flushed from the oviduct and then fertilized in vitro (28%) [21]. In addition, coculture of oocytes with oviductal epithelial cells [22] or preincubation of oocytes with oviductal fluid [23] significantly reduced the incidence of polyspermy in pigs. A functional block to polyspermy in vivo has been suggested to result from a factor of oviductal origin, and several investigators have speculated that this activity may result from a specific function of OSP [21, 23–25]. The association of pOSP with ovulated oocytes and early cleavage-stage embryos [12] suggests this glycoprotein may be a likely candidate for such activity. Funahashi and Day [26] have speculated that oviductal proteins in preincubation and/or fertilization media compete with sperm receptors for binding of zona pellucida ligands, stimulate the rate of sperm acrosome reaction, and, thus, reduce the number of capacitated spermatozoa attaching to the surface of pig oocytes.

Specific objectives of this in vitro study were 1) to evaluate the effects of pOSP on fertilization responses, including penetration rate and incidence of polyspermy; 2) to examine the effect of pOSP on zona solubility and sperm binding to the zona; 3) to determine if a polyclonal antibody generated against pOSP could negate specific pOSP effects; and 4) to determine if pOSP can enhance blastocyst development.

MATERIALS AND METHODS

Materials

Acrylamide, N,N'-diallyltartardiamide, urea, Nonidet P-40, and sodium dodecyl sulfate were acquired from Gallard-Schlesinger (Carle Place, NY); X-Omat AR film and photography reagents were products of Eastman Kodak Co. (Rochester, NY). Amino acids and protein standards were purchased from Sigma-Aldrich (St. Louis, MO), and ampholines were from Pharmacia-Biotech (Piscataway, NJ). All other supplies and reagents for gel electrophoresis were procured from either Bio-Rad Laboratories (Richmond, CA) or Fisher Scientific (Orlando, FL), and all medium and culture supplies were obtained from Life Technologies (Grand Island, NY). The L-[4,5-3H]leucine (specific activity, 120 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). Unless otherwise stated, all other chemicals and reagents were acquired from either Sigma or Fisher.

Culture Media

Basic oocyte IVM medium was protein-free Tissue Culture Medium 199 [16] supplemented with 10 ng/ml epidermal growth factor, 0.57 mM cysteine, 0.1% polyvinyl alcohol (PVA), 0.5 µg/ml LH, 0.5 µg/ml FSH, 75 µg/ml potassium penicillin G, and 50 µg/ml streptomycin sulfate and slightly modified by the addition of 3.05 mM D-glucose and 0.91 mM sodium pyruvate. The IVF medium was essentially that described by Wang et al. [19], which was designated as modified Tris-buffered medium (mTBM: pH, 7.2–7.4; 39°C; 5% CO2 [v/v] in air). The mTBM consisted of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2·2H2O, 20 mM Tris (crystallized free base; Fisher), 11 mM glucose, 5 mM sodium pyruvate, and no antibiotics. Embryos were cultured in North Carolina State University (NCSU) 23 medium [27], which was designated as IVC medium supplemented with 4 mg/ml BSA. Media (IVM, IVF, and IVC) were covered with paraffin oil and equilibrated at 39°C, 5% CO2 (v/v) in air, at least 12 h before use.

IVM and IVF

Ovaries from prepubertal gilts were collected from an abattoir and immediately transported to the laboratory at 25–30°C in 0.9% saline containing 75 µg/ml potassium penicillin G and 50 µg/ml streptomycin sulfate. Oocytes were aspirated from follicles (diameter, 3–6 mm) using a 20-guage needle connected to a 10-ml disposable syringe, transferred to a 50-ml conical tube, and allowed to sediment at room temperature (25°C). Supernatant was discarded, and follicular contents were washed with Tyrode's Lactate (TL)-Hepes medium supplemented with 0.01% PVA (TL-Hepes-PVA). Oocytes with an evenly granulated cytoplasm and surrounded by compact cumulus cells were washed twice with TL-Hepes-PVA and three times in IVM medium. Oocytes (n = 50–70) were suspended in 500 µl of IVM medium in a four-well multidish (Nunc, Roskilde, Denmark) and cultured for 42–44 h [20].

On completion of IVM, cumulus cells were removed by treatment with 0.1% (w/v) hyaluronidase in basic IVM medium and vortexed for 1 min. Denuded oocytes were washed three times in 500 µl of IVM medium and then three times in IVF medium containing 1 mM caffeine and 1 mg/ml BSA. Oocytes (n = 35) were placed into 50-µl drops of pre-equilibrated IVF medium and covered with warm paraffin oil in a 35 x 10-mm2 polystyrene culture dish (Becton Dickinson & Co., Lincoln Park, NJ). A frozen semen pellet was thawed and washed three times by centrifugation (1900 x g for 4 min) in Dulbecco's PBS (DPBS; Life Technologies) supplemented with 1 mg/ml BSA, 75 µg/ml potassium penicillin G, and 50 µg/ml streptomycin sulfate (pH 7.2). The sperm pellet was then resuspended in IVF medium supplemented with caffeine (1 mM) and BSA (0.1%, w/v), and 50 µl of the sperm suspension was added to 50-µl drops of IVF medium containing the oocytes. The final sperm concentration was 2.5–3.5 x 105/ml. Spermatozoa and oocytes were coincubated for 6 h at 39°C, 5% CO2 (v/v) in air.

Tissue Collection and Protein Purification

Sexually mature, cross-bred gilts (Yorkshire x Duroc x Hampshire) were observed daily for behavioral estrus for at least two estrous cycles in the presence of an intact boar. The first day of standing estrus was designated as Day 0, and animals were taken to the abattoir for slaughter on Day 0 or 1 of the estrous cycle. After exsanguination, reproductive tracts were collected aseptically and opened longitudinally, and the oviductal tissue was washed in several volumes of (modified) Eagle's minimum essential medium (MEM). Tissue was cut into 1- to 3-mm3 sections, and 500-mg explants were cultured in 15 ml of leucine-deficient MEM containing 100 µCi of [3H]leucine in Petri dishes on a rocking platform at 39°C under a defined atmosphere of 50% N2 (v/v), 47.5% O2 (v/v), and 2.5% CO2 (v/v) [1]. Nonlabeled cultures were generated as described above in complete MEM, without addition of [3H]leucine. After 24 h of culture, media were aspirated and frozen at -20°C until purification.

Molecular cloning and analyses of pOSP cDNA revealed a putative heparin-binding consensus sequence in the protein [10]. Using a heparin-agarose affinity column, highly purified pOSP was obtained (unpublished results). Briefly, culture media from Days 0 and 1 whole-oviduct cultures were thawed, centrifuged (2200 x g for 10 min at 4°C), pooled, diluted (1:3, v/v) with 20 mM Tris-HCl (pH 7.6; 4°C) containing 0.02% (w/v) NaN3, and slowly loaded onto the heparin-agarose column (2.5 x 8.2 cm) at 4°C. Porcine OSP was eluted using stepwise increments of NaCl (0.1–3.0 M), and purification was monitored by two-dimensional SDS-PAGE and fluorography as described elsewhere [1]. Purified preparations from the 0.4 M NaCl elution were used for all experiments. Highly purified pOSP was dialyzed against dH2O (three changes, 4 L each, 24 h each, 4°C), pooled, and measured for total protein content by the Bio-Rad microassay according to manufacturer's instructions and for radioactivity by liquid scintillation spectrophotometry. Pooled samples were then lyophilized and stored at -20°C.

Experiments 1, 2, 3, and 4 used radiolabeled pOSP obtained from whole-oviduct cultures as detailed earlier. Experiment 5, which evaluated pOSP effects on embryonic development to the blastocyst stage, used unlabeled pOSP purified from the ampulla segment of the oviduct. Purification of ampulla-derived pOSP was identical to that of the whole oviduct. Protein (5 mg) from each pOSP preparation was resuspended in 3 ml of either mTBM (39°C) for IVF or NCSU 23 (39°C) for IVC, and total protein content was measured by the Bio-Rad protein assay according to the manufacturer's instructions. Aliquots were stored at -20°C. Oviductal pOSP resuspended in IVF medium was evaluated by two-dimensional SDS-PAGE and fluorography [1].

Immunoglobulin G Purification

Polyclonal anti-pOSP antiserum was prepared according to the method described by Buhi et al. [12]. The immunoglobulin (Ig) G fraction was precipitated from rabbit anti-pOSP serum (5 ml) by ammonium sulfate (saturation, 50%) fractionation and purified by diethylamino ethyl sepharose (DEAE-Sepharose; Pharmacia-Biotech, Piscataway, NJ) ion-exchange chromatography [28]. The isolated IgG fraction was dialyzed against dH2O (two changes, 2 L each, 24 h each, 4°C), and total protein content measured as described earlier. Purity was determined by one-dimensional SDS-PAGE. Aliquots containing 100 µg of IgG were lyophilized, resolubilized in IVF medium, and stored at -20°C. Precipitation of radiolabeled pOSP (10 µg/ml) was evaluated with various concentrations of purified anti-pOSP IgG (10–200 µg/ml). Incubation was allowed to extend overnight at 39°C to mimic conditions established for IVF. Antibody-pOSP complex was precipitated using Protein A-Sepharose (1-h incubation, gentle rotation, at room temperature), washed, and centrifuged (2200 x g for 5 min). Protein A-Sepharose complexes and supernatants were analyzed by one-dimensional SDS-PAGE and fluorography [29]. A protein secreted into explant culture medium that cross-reacted with the pOSP antibody [12] was removed during purification. The pOSP antiserum only cross-reacts with the pOSP radiolabeled proteins shown in Figure 1.



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  		FIG. 1. Representative fluorograph of 3H-labeled proteins (500 µg) from whole-oviduct explant culture media (Day 0/1) subjected to heparin-agarose affinity column chromatography and separated by two-dimensional SDS-PAGE. Lyophilized proteins were resuspended in IVF media before electrophoresis. Arrows mark pOSP (1–4). Molecular weight markers (x103) are indicated, and the pH gradient runs from left (pH 8) to right (pH 4)

Experiment 1: pOSP Effects on IVF

Pig oocytes matured in vitro were exposed to various concentrations of pOSP before and during IVF. Oocytes matured in vitro and devoid of cumulus cells were washed three times in IVF medium and then randomly assigned to each of six treatments. The IVF drops containing pOSP (0–100 µg/ml) were allowed to pre-equilibrate overnight before preincubation and insemination. Oocytes (n = 35/treatment) were placed into 50-µl drops of IVF medium containing 0, 0.1, 1, 10, 50 or 100 µg/ml pOSP and preincubated for 4 h at 39°C, 5% (v/v) CO2 in air. Oocytes were then inseminated with spermatozoa (50 µl), essentially halving the concentration of pOSP used during preincubation. Six hours after insemination, oocytes were washed three times in IVC medium (100 µl) and then incubated in IVC medium (100 µl) for an additional 4 h. After incubation, sperm cells attached to the zona were removed by washing three times in TL-Hepes-PVA (39°C) with a small-bore pipette, mounted, and placed into fixative (25% [v/v] acetic acid in ethanol, room temperature) for 72 h. Next, oocytes were stained with 1% (w/v) orcein in 45% (v/v) acetic acid, examined under a phase-contrast microscope at x200 and x400 magnification, and the meiotic stage assessed according to the method described by Hunter and Polge [30]. Oocytes were considered to be penetrated when one or more sperm heads and/or male pronuclei and corresponding sperm tails were present. The rate of polyspermy, male pronuclei formation, and mean number of sperm (MNS) per oocyte were determined from the oocytes penetrated. Experiment 1 was replicated five times, with multiple observations for each replicate.

Experiment 2: Anti-pOSP IgG Inhibition of Decreased Polyspermy

Preliminary experiments testing various concentrations of pOSP IgG (10, 50, 100, 200, and 400 µg/ml) indicated that 50 µg/ml IgG would inhibit the pOSP effects on polyspermy without affecting the penetration rates. For this experiment, 10 µg/ml pOSP was used. Treatments consisted of the control (no addition), pOSP, pOSP and IgG (50 µg/ml), or antibody alone and were evaluated as a 2 x 2 factorial design. The IVF drops containing pOSP, pOSP and IgG, or IgG alone were allowed to pre-equilibrate overnight before preincubation and insemination. Cumulus-free oocytes were randomly assigned to each of the four treatments (n = 35/treatment), preincubated for 4 hours, and inseminated. The fertilization responses were evaluated as described earlier. Experiment 2 was replicated three times, with multiple observations for each replicate.

Experiment 3: pOSP Effects on Zona Pellucida Solubility

To test zona pellucida solubility, cumulus-free oocytes matured in vitro were washed three times in 500-µl drops of IVF medium and transferred to 50-µl drops of IVF medium (pre-equilibrated with pOSP overnight) containing various concentrations of pOSP (0, 0.1, 1, 10, 50, or 100 µg/ml) for 4 h at 39°C, 5% (v/v) CO2 in air. Oocytes were then washed three times in TL-Hepes-PVA (39°C) and placed (n = 5–15/treatment) into 100 µl of a 0.1% (w/v) pronase solution in PBS. Zona digestion was observed continuously at room temperature (25°C) with an inverted microscope. When the zona pellucida was no longer visible at x200 magnification, the zona pellucida dissolution time was recorded. Experiment 3 was replicated three times.

Experiment 4: Effects of pOSP on Sperm Binding to the Zona Pellucida

To examine pOSP effects on sperm binding, cumulus-free oocytes matured in vitro (n = 35/treatment) were washed and preincubated with various concentrations of pOSP (0, 0.1, 1, 10, 50, or 100 µg/ml, pre-equilibrated in IVF medium overnight) for 4 h at 39°C and coincubated with spermatozoa for 6 h as described earlier. After the fertilization incubation, putative zygotes were washed twice in 500 µl of IVC medium and pipetted in and out (10 times) of a wide-bore pipette to remove loosely bound sperm. Putative zygotes were then placed into 50-µl drops of TL-Hepes-PVA containing Hoescht 33342 (bis-Benzamide; 1.3 mg/ml) and incubated for 30 min at 39°C, 5% (v/v) CO2 in air. Putative zygotes were then washed twice in 300 µl of TL-Hepes-PVA, mounted, and the number of tightly bound sperm/zygote counted under a phase-contrast microscope (Leitz Laborlux D) equipped with ultraviolet illumination (excitation at 330–380 nm, emission at 420 nm). Experiment 4 was replicated three times, with 15 putative zygotes counted from each replicate.

Experiment 5: pOSP Effects on Cleavage Rate and Embryonic Development

Oocytes matured in vitro and devoid of cumulus cells were washed and randomly assigned to each of two treatments: control (0 µg/ml pOSP during preincubation/IVF, n = 35 oocytes/50-µl drop, n = 2 drops), or pOSP (10 µg/ml during preincubation/IVF, n = 35 oocytes/50-µl drop, n = 10 drops). Pre-equilibration, preincubation, and fertilization were performed as described earlier. Six hours after fertilization, control putative zygotes were washed three times in 100-µl drops of IVC medium, transferred to a fresh 100-µl drop (n = 35 oocytes/drop), and incubated at 39°C, 5% (v/v) CO2 in air. Putative zygotes exposed to pOSP during preincubation/IVF were pooled, washed three times in 500 µl of IVC medium, and randomly assigned to 100 µl IVC drops containing 0, 1, 10, 50, or 100 µg/ml of pOSP (n = 35 zygotes/treatment and 2 drops/treatment). At 48 and 144 h after insemination, respectively the cleavage rate and blastocyst formation were evaluated under a stereomicroscope. Experiment 5 was replicated nine times, with multiple observations within each replicate and the percentage blastocyst formation determined from the number of oocytes inseminated.

Statistical Analysis

Data were analyzed by ANOVA using the General Linear Models procedure of the Statistical Analysis System (SAS Institute Inc., Cary, NC). All percentage data were subjected to arc-sine transformation before statistical analysis. Data are expressed as means ± SEM or least-squares means ± SEM. Differences between means were evaluated by the Student-Newman-Kuels t-test. The model for analysis included the main effects of treatment (0–100 µg/ml), replicate, and treatment x replicate. A probability of P < 0.05 was considered to be significant. A 2 x 2 factorial design was used to evaluate the effects of pOSP and anti-pOSP IgG on polyspermy. A set of preplanned, orthogonal contrasts were used to evaluate the effects of pOSP concentrations on embryonic development.

RESULTS

Purification of pOSP

A representative two-dimensional SDS-PAGE fluorograph of fractionated oviductal culture media using a heparin-agarose affinity column resulted in a highly purified preparation of pOSP (Fig. 1). The 0.4 M NaCl elution showed an estimated 80%–85% purification of pOSP protein from oviductal culture media (Alvarez and Buhi, unpublished results). Porcine OSP is the primary protein product that was resuspended in IVF medium. Several minor proteins from the oviduct that contain heparin-binding regions or are complexed together copurify with pOSP. Most of these minor proteins appear not to be de novo synthesized products of the oviduct, however, because they were not labeled with [3H]leucine, suggesting they are from serum transudate. Four fractions of pOSP identified in the purified preparation, pOSP 1–3 (previously identified) and a fourth protein that concentrated during purification, can react with pOSP antibody (unpublished results).

Experiment 1: Effects of pOSP on IVF

To examine the effects of pOSP on IVF, IVM pig oocytes were preincubated with various concentrations of pOSP (0–100 µg/ml) and then fertilized in the presence of pOSP. Effects of pOSP on fertilization parameters are shown in Table 1. A pOSP treatment effect (P < 0.05) was found for sperm penetration. Concentrations of pOSP from 0–50 µg/ml had no effect on penetration, whereas 100 µg/ml significantly decreased sperm penetration of pig oocytes compared with the control (41% vs. 74%; Table 1). A treatment effect (P < 0.05) of pOSP on polyspermy was also determined. Concentrations of pOSP from 10–100 µg/ml significantly decreased polyspermy compared with the control (24%–29% vs. 61%; Table 1). No effect of pOSP was determined on formation of the male pronucleus. A treatment effect (P < 0.05) with pOSP was also determined for the MNS per oocyte. A concentration of 10 µg/ml pOSP was selected for all subsequent experiments, because this concentration significantly decreased polyspermy while maintaining penetration rates similar to that of the control.


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  		TABLE 1. Effect of pOSP on fertilization parameters of pig oocytes matured and fertilized in vitro.*

Experiment 2: Anti-pOSP IgG Inhibition of Decreased Polyspermy

To determine that decreased polyspermy was a specific effect of pOSP and not of an effector that copurified with pOSP, a polyclonal anti-pOSP IgG was included during preincubation/IVF. Experiments testing various concentrations of IgG (10, 50, 100, 200, and 400 µg/ml) indicated that concentrations of 200 µg/ml or greater reduced penetration of oocytes, adversely affecting polyspermy rates (data not shown). However, addition of 50 µg/ml IgG maintained penetration and polyspermy rates similar to those of the control. In addition, and similar to the previous experiment (Table 1), an effect of pOSP (P < 0.05; Fig. 2) was found on polyspermy, and an interaction was detected for pOSP and anti-pOSP IgG (P < 0.05), indicating the effect of pOSP on polyspermy depends on the absence of antibody. This result demonstrates that the pOSP-induced decrease in the polyspermy rate resulted from a specific effect of pOSP and not from that of any other effector molecule. Here, 10 µg/ml pOSP decreased the incidence of polyspermy (23%), whereas addition of anti-pOSP IgG produced a polyspermy rate similar to that of the control (46%; Fig. 2). Compared with the control, no effect was observed for pOSP with IgG or for IgG alone on the penetration rate, male pronuclear formation, or MNS/oocyte.



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  		FIG. 2. Effect of in vitro incubation of pOSP and anti-pOSP IgG on the polyspermy rate of porcine oocytes matured and fertilized in vitro. The concentration was 10 µg/ml for pOSP and 50 µg/ml for the anti-pOSP IgG treatment. A treatment effect (P < 0.05) and an interaction (P < 0.05) was detected for pOSP and anti-pOSP IgG. Bars indicate the least-squares mean ± SEM of three replicates. A total of 210 oocytes are represented for each treatment. Different superscripts indicate significant differences (P < 0.05). The rate of polyspermy was determined from the number of penetrated oocytes

Experiment 3: Effects of pOSP on Zona Pellucida Solubility

Effects of pOSP on zona pellucida solubility were examined to determine if the association of pOSP stabilized or protected the zona pellucida from enzymatic digestion. No treatment effect was observed for the zona digestion time of oocytes when incubated for 4 h in the presence of various concentrations of pOSP (0–100 µg/ml). Most oocytes (62%–80%) lost their zona pellucida within 1–3 min, with 95% lost by 6 min, after exposure to a 0.1% pronase solution.

Experiment 4: Effects of pOSP on Sperm Binding to the Zona Pellucida

To determine if the decreased polyspermy in vitro resulted from a change in the number of spermatozoa attached to the zona pellucida, effects of pOSP on sperm binding were examined. A treatment effect (P < 0.05) of pOSP on sperm binding was demonstrated. The number of sperm bound per zygote was significantly reduced, with a decrease in sperm binding being observed with an increase in the concentrations of pOSP (Fig. 3). The number of sperm bound to each zygote per treatment was evaluated and determined by fluorescent microscopy (Fig. 4) as detailed in Materials and Methods. The decrease in spermatozoa bound to the zona pellucida with an increase in pOSP concentrations begin to reach a plateau with 1 µg/ml pOSP.



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  		FIG. 3. Effect of in vitro incubation of pOSP on sperm binding to porcine putative zygotes. Oocytes were matured in vitro and then preincubated (4 h) and fertilized (6 h) in the presence of increasing concentrations of pOSP (0–100 µg/ml). A treatment effect (P < 0.05) was detected for pOSP on sperm binding. Bars indicate the least-squares mean ± SEM of three replicates. A total of 45 oocytes are represented for each concentration of pOSP. Different superscripts indicate significant differences (P < 0.05)



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  		FIG. 4. Photographs (x200) of sperm binding to putative zygotes after preincubation and fertilization in the presence of 0 µg/ml pOSP (A) or 100 µg/ml pOSP (B). A) Representative photograph of the average sperm bound per zygote in the control (~46 sperm/zygote). B) Photograph of the lower end of the range observed for this treatment (~14 sperm/zygote)

Experiment 5: Effects of pOSP on Embryonic Development

To test whether pOSP had an effect on embryonic development in vitro, pOSP was included during preincubation/IVF alone or during preincubation/IVF with IVC, and both cleavage rate and development to blastocyst were examined. No treatment effects were observed on cleavage rates of oocytes fertilized and cultured in the presence of pOSP (Fig. 5). However, a treatment effect (P < 0.05) of pOSP was found on development of embryos to blastocysts. A significant increase (P < 0.05) in blastocyst number was found when pOSP was included during preincubation/IVF compared with the control (Fig. 6). No additional effect of pOSP at 1, 10, 50, or 100 µg/ml added during IVC could be detected on the number of blastocysts. Higher concentrations of pOSP (50 and 100 µg/ml) added during IVC, however, tended (P = 0.08) to decrease the effect observed on the number of embryos that developed to blastocysts when pOSP was added during preincubation/IVF.



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  		FIG. 5. Effect of in vitro incubation of pOSP on cleavage rate using porcine oocytes matured, fertilized, and cultured in vitro. Oocytes matured in vitro (1) were preincubated and fertilized in the absence (-) or presence of 10 µg/ml pOSP as described in Materials and Methods. Oocytes fertilized in vitro (2) were then cultured in the absence (-) or presence of pOSP (1–100 µg/ml) during early cleavage-stage (two- to four-cell) development. Bars indicate the least-squares mean ± SEM of nine replicates for each treatment. Shown in each bar are the number of oocytes within each treatment. No treatment effect was detected for pOSP on cleavage rates



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  		FIG. 6. Effect of in vitro incubation of pOSP on blastocyst development using porcine oocytes matured, fertilized, and cultured in vitro. Oocytes matured in vitro (1) were preincubated and fertilized in the absence (-) or presence of 10 µg/ml pOSP as described in Materials and Methods. Oocytes fertilized in vitro (2) were then cultured in the absence (-) or presence of pOSP (1–100 µg/ml) during embryonic development to the blastocyst. A treatment effect was detected (P = 0.05) for pOSP on blastocyst development. Bars indicate the least-squares mean ± SEM of nine replicates for each treatment. Shown in each bar is the number of oocytes within each treatment. Differences between treatments were evaluated by orthogonal contrasts, and P values corresponding to these analyses are shown. Blastocyst formation was calculated as a percentage of oocytes inseminated (not cleaved embryos)

DISCUSSION

The present study demonstrated that pOSP administered in vitro decreased the incidence of polyspermy in pig oocytes matured and fertilized in vitro. This antipolyspermic effect of pOSP, which was inhibited by anti-pOSP IgG, indicated the reduction was specific to pOSP and could not be ascribed to an effector molecule that may have copurified with the pOSP. Polyspermy has remained a persistent problem of pig oocytes matured and fertilized in vitro, often reaching levels greater than 50% [13, 17–19]. However, polyspermic fertilization in vivo among pigs mated at the onset of estrus before ovulation is uncommon [31]. Eggs ovulated after luteal phase gonadotrophin treatment show a high incidence of polyspermy (60.6%) [24]. Animals treated with progesterone, either systemically or by local microinjection into the oviduct wall during estrus, also show elevated levels of polyspermic fertilization (40% and 32.3%, respectively) [24]. During the luteal phase, pOSP mRNA and protein expression is at basal levels, and progesterone down-regulates pOSP mRNA and protein synthesis and secretion in ovariectomized gilts [10, 32]. Thus, the in vivo observations [24] may result from the decrease in or an absence of pOSP synthesis by the progestational oviduct during fertilization. A recent study reported that pig oocytes flushed from the oviduct on Day 2 of the estrous cycle and subsequently fertilized in vitro had a much lower incidence of polyspermy (28%) than oocytes matured and fertilized in vitro (62%) [21]. In addition, periovulatory oviduct-conditioned media [33], oviduct fluid [23], and coincubation of boar spermatozoa or pig oocytes with oviductal epithelial cells [22, 25, 34] significantly reduced the in vitro incidence of polyspermy. These reports strongly suggest that an unknown factor of oviductal origin can associate with either oocytes or spermatozoa and effectively decrease the incidence of polyspermy. In the pig, the in vivo block to polyspermy is thought to result from a restriction in the number of spermatozoa that reach an egg and the zona pellucida block to penetration by multiple spermatozoa [35]. However, our findings suggest that a third factor, pOSP, may contribute to the in vivo prevention of polyspermic fertilization. Although the exact concentration of OSPs in the oviduct at fertilization has not been determined, a concentration range of 0.1–100 µg/ml was tested here to determine the physiological response to various fertilization parameters, including polyspermy. Here, pOSP at a concentration of 10 µg/ml or greater decreased the incidence of polyspermy.

Polyspermy also occurs during IVF in humans [36–39] and cows [40–43], although at a much lower rate (approaching 10%). Because polyspermic fertilization is not a significant problem in these species, the reduction in polyspermy (61% vs. 24%–29%) during IVF in pigs may reflect a species-specific difference in the function of pOSP. The exact nature of pOSP in the in vivo prevention of polyspermy at fertilization also becomes more difficult to interpret when one considers the sperm:egg ratio at fertilization. As reviewed by Hunter [44], evidence from both laboratory and farm animals indicates that the sperm:egg ratio at the time of initial penetration of the egg membrane is close to unity, although this number progressively increases with time after ovulation. Therefore, pOSP may be involved in the block to polyspermy by additional spermatozoa attaining the ampulla of the oviduct after penetration and activation of the fertilized oocyte.

The in vitro penetration rate of pig oocytes by sperm was not affected by oocyte exposure to pOSP, except at a high concentration, at which decreased penetration was observed. This high concentration of pOSP and its association with the zona pellucida may lead to a physical block of sperm binding. Previously reported data in other species on the penetration rate vary. In the hamster, partially purified OSP has both increased [45] and decreased [46] the penetration rate, whereas in the cow, the penetration rate has increased with OSP [47]. These observations may result from differences used in scoring penetration rates, species specificity, OSP purity and concentrations, and/or the length of incubation. The increased penetration rate observed in vitro by Martus et al. [47] resulted from a specific effect on the oocyte and not on the spermatozoon; however, in our study, distinctions cannot be made between the gametes.

In the pig, oocytes fertilized in vitro, when compared with those fertilized in vivo, show an incomplete corticol granule exocytosis [23]. We proposed that a factor, such as pOSP, might facilitate a more synchronous exocytosis of corticol granule contents or augment the response of the zona matrix to corticol granule exudate. In addition, pOSP might assist in modification of zona pellucida sperm receptors immediately after fertilization in a process known as "zona hardening." In the mouse, these modified zona receptors appear shortly after fertilization and are cleavage products of ZP2 and ZP3, resulting in the inhibition of sperm binding [48]. Data in this study indicate that added pOSP decreased the number of spermatozoa bound to the zona pellucida during IVF. Because pOSP was incubated with oocytes and sperm, modifications of zona pellucida receptors by co-operative action between pOSP and corticol granule contents may have occurred, as may alterations to the spermatozoa membrane. Results from this study appear to contradict findings observed in the hamster [49, 50] and in humans [51]. Those studies [49–51] found an increased number of spermatozoa bound to the oocyte when exposed to OSP. The differences between our data and these previous findings may result from species-specific differences, purity of OSP, and use of oocytes matured and fertilized in vitro during our study. Although further experiments are required to establish this point, an association of pOSP with the zona pellucida [12] may have a role in the initial recognition and binding of boar spermatozoa by the zona pellucida sperm receptor (ZP1). Porcine OSP, together with ZP1, may assist in a specific selection mechanism of sperm that are capacitated and/or highly motile (i.e., "hyperactivated").

Oviductal-derived oocytes and embryos are more resistant to in vitro proteolytic degradation than either follicular oocytes or embryos recovered from the uterine lumen [52]. Similarly, the zona pellucida of oocytes matured in vitro are more susceptible to protease digestion by pronase than nonfertilized oocytes flushed from the oviduct on Day 2 of the estrous cycle [21]. Therefore, an unknown factor of oviductal origin may be involved in providing zona stability that also contributes to the functional block of polyspermy. Our results, however, indicate that pOSP may not be this factor, because pOSP had no effect on retarding protease digestion of the zona pellucida. Modification in the zona pellucida of oviductal oocytes and embryos that provide protease resistance is likely the result of both known and unknown protease inhibitors of the oviduct [53, 54].

The results of this study further demonstrate that pOSP provided a significant increase in postcleavage development from embryo to blastocyst. This observed increase in blastocyst development might result from decreased polyspermy or other metabolic effects. The pathological condition of polyspermy (i.e., penetration of the vitellus by more than one spermatozoa) is a very early cause of death for the zygote [24, 55, 56]. Addition of pOSP during IVC had no effect on development to blastocyst, and high concentrations of pOSP tended to decrease the observed effect on development. Physiologically, in vivo pig embryos are no longer exposed to oviductal pOSP after the four-cell stage, which coincides with entry into the uterus. Therefore, beneficial effects of pOSP on development to the blastocyst may have accrued during fertilization and early cleavage-stage cell divisions (i.e., one- to four-cell). Bovine OSP was found to increase the number of morula and blastocysts on Day 6 but not on Day 7 of development [57], or it had no effect on blastocyst development [58]. Similarly, ovine OSP had no effect on bovine embryo blastocyst development [59]. Interpreting many of these studies becomes complicated, because the purity of OSP preparations has varied. In this study, as well as those of Schmidt et al. [49, 50] and Martus et al. [47], antibodies specific to that particular species OSP were used to eliminate the possibility of observed effects resulting from copurified or contaminant proteins. To our knowledge, however, no such experiments have been done for other reported studies.

In summary, the results of this study indicates that pOSP significantly reduces the incidence of polyspermy among pig eggs matured and fertilized in vitro. This decrease may result from a reduction in sperm binding to the zona pellucida and not from a protective proteolytic modification of the zona pellucida matrix before fertilization. A postcleavage increase in the number of embryos developing to blastocysts was observed when pOSP was included during preincubation/IVF; however, addition of pOSP during IVC had no synergistic stimulation on development. These data indicate that pOSP may play an important role in vivo in the fertilization process, including a block of polyspermy.

FOOTNOTES

First decision: 4 November 1999.

1 Supported by U.S. Department of Agriculture grant 97-35203-4614. Back

2 Correspondence: William C. Buhi, P.O. Box 100294, Department Obstetrics and Gynecology, University of Florida, Gainesville, FL 32610-0294. FAX: 352 392 2808; buhiwc{at}obgyn.ufl.edu Back

Accepted: February 28, 2000.

Received: October 5, 1999.

REFERENCES

  1. Buhi WC, Alvarez IM, Sudhipong V, Dones-Smith MM. Identification and characterization of de novo synthesized porcine oviductal secretory proteins. Biol Reprod 1990; 43:929–938.[Abstract]
  2. Buhi WC, Alvarez IM, Kouba AJ. Oviductal regulation of fertilization and early embryonic development. J Reprod Fertil 1997; 52:285–300.[CrossRef]
  3. Arias EB, Verhage HG, Jaffe RC. Complementary deoxyribonucleic acid cloning and molecular characterization of an estrogen-dependant human oviductal glycoprotein. Biol Reprod 1994; 51:685–694.[Abstract]
  4. DeSouza MM, Murray MK. An estrogen-dependent secretory protein, which shares identity with chitinases, is expressed in a temporally and regionally specific manner in the sheep oviduct at the time of fertilization and embryo development. Endocrinology 1995; 136:2485–2496.[Abstract]
  5. Sendai Y, Komiya H, Suzuki K, Onuma T, Kikuchi M, Hoshi H, Araki Y. Molecular cloning and characterization of a mouse oviduct-specific glycoprotein. Biol Reprod 1995; 53:285–294.[Abstract]
  6. Sendai Y, Abe H, Kikuchi M, Satoh T, Hoshi H. Purification and molecular cloning of bovine oviduct-specific glycoprotein. Biol Reprod 1994; 50:927–934.[Abstract]
  7. Suzuki K, Sendai Y, Onuma T, Hoshi H, Hiroi M, Araki Y. Molecular characterization of a hamster oviduct-specific glycoprotein. Biol Reprod 1995; 53:345–354.[Abstract]
  8. Donnelly KM, Fazleabas AT, Verhage HG, Mavrogianis PA, Jaffe RC. Cloning of a recombinant complementary DNA to a baboon (Papio anubis) estradiol-dependant oviduct-specific glycoprotein. Mol Endocrinol 1991; 5:356–364.[Abstract/Free Full Text]
  9. Verhage HG, Mavrogianis PA, Boomsma RA, Schmidt A, Brenner RM, Slayden OV, Jaffe RC. Immunologic and molecular characterization of an estrogen-dependent glycoprotein in the rhesus (Macaca mulatta) oviduct. Biol Reprod 1997; 57:525–531.[Abstract]
  10. Buhi WC, Alvarez IM, Choi I, Cleaver BD, Simmen FA. Molecular cloning and characterization of an estrogen-dependant porcine oviductal secretory glycoprotein. Biol Reprod 1996; 55:1305–1314.[Abstract]
  11. Sun T, Lei ZM, Rao ChV. A novel regulation of the oviductal glycoprotein gene expression by luteinizing hormone in bovine tubal epithelial cells. Mol Cell Endocrinol 1997; 131:97–108.[CrossRef][Medline]
  12. Buhi WC, O'Brien B, Alvarez IM, Erdos G, Dubois D. Immunogold localization of porcine oviductal secretory proteins within the zona pellucida, perivitelline space, and plasma membrane of oviductal and uterine oocytes and early embryos. Biol Reprod 1993; 48:1274–1283.[Abstract]
  13. Abeydeera LR, Day BN. Fertilization and subsequent development in vitro of pig oocytes inseminated in a modified Tris-buffered medium with frozen-thawed ejaculated spermatozoa. Biol Reprod 1997; 57:729–734.[Abstract]
  14. Abeydeera LR, Wang WH, Cantley TC, Rieke A, Prather RS, Day BN. Presence of epidermal growth factor during in vitro maturation of pig oocytes and embryo culture can modulate blastocyst development after in vitro fertilization. Mol Reprod Dev 1998b; 51:395–401.
  15. Abeydeera LR, Wang WH, Prather RS, Day BN. Maturation in vitro of pig oocytes in protein-free culture media: Fertilization and subsequent embryo development in vitro. Biol Reprod 1998; 58:1316–1320.[Abstract/Free Full Text]
  16. Abeydeera LR Wang WH, Cantley TC, Rieke A, Prather RS, Day BN. Epidermal growth factor can enhance the developmental competence of pig oocytes matured in vitro under protein-free culture conditions. Theriogenology 1999; 51:365.[CrossRef]
  17. Wang W-H, Okuda K, Niwa K. In vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa. J Reprod Fertil 1991; 93:491–499.[Abstract/Free Full Text]
  18. Wang W-H, Abeydeera LR, Okuda K, Niwa K. Penetration of porcine oocytes during maturation in vitro by cryopreserved, ejaculated spermatozoa. Biol Reprod 1994; 50:510–515.[Abstract]
  19. Wang W-H, Abeydeera LR, Cantley TC, Day BN. Effects of oocyte maturation media on development of pig embryos produced by in vitro fertilization. J Reprod Fertil 1997; 111:101–108.[Abstract/Free Full Text]
  20. Abeydeera LR, Day BN. In vitro penetration of pig oocytes in a modified Tris-buffered medium: effect of BSA, caffeine and calcium. Theriogenology 1997; 48:537–544.
  21. Wang W-H, Abeydeera LR, Prather RS, Day BN. Morphologic comparison of ovulated and in vitro-matured porcine oocytes, with particular reference to polyspermy after in vitro fertilization. Mol Reprod Dev 1998; 49:308–316.[CrossRef][Medline]
  22. Kano K, Miyano T, Kato S. Effect of oviductal epithelial cells on fertilization of pig oocytes in vitro. Theriogenology 1994; 42:1061–1068.
  23. Kim N-H, Funahashi H, Abeydeera LR, Moon SJ, Prather RS, Day BN. Effects of oviductal fluid on sperm penetration and corticol granule exocytosis during fertilization of pig oocytes in vitro. J Reprod Fertil 1996; 107:79–86.[Abstract/Free Full Text]
  24. Hunter RHF. Oviduct function in pigs, with particular reference to the pathological condition of polyspermy. Mol Reprod Dev 1991; 29:385–391.[CrossRef][Medline]
  25. Dubuc A, Sirard M-A. Effect of coculturing spermatozoa with oviductal cells on the incidence of polyspermy in pig in vitro fertilization. Mol Reprod Dev 1995; 41:360–367.[CrossRef][Medline]
  26. Funahashi H, Day BN. Advances in in vitro production of pig embryos. J Reprod Fertil 1997; 52(suppl):271–283.
  27. Petters RM, Wells KD. Culture of pig embryos. J Reprod Fertil 1993; 48(suppl):61–73.
  28. Harlow E, Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor, NY: Cold Springs Harbor Laboratory Press; 1988: 298.
  29. Buhi WC, Vallet JL, Bazer FW. De novo synthesis and release of polypeptides from cyclic and early pregnant porcine oviductal tissue in explant culture. J Exp Zool 1989; 252:79–88.[CrossRef][Medline]
  30. Hunter RHF, Polge C. Maturation of follicular oocytes in the pig after injection of human chorionic gonadotrophin. J Reprod Fertil 1966; 12:525–531.[Abstract/Free Full Text]
  31. Hunter RHF. The effects of delayed insemination on fertilization and cleavage in the pig. J Reprod Fertil 1967; 13:133–147.[Abstract/Free Full Text]
  32. Buhi WC, Ashworth CJ, Bazer FW, Alvarez IM. In vitro synthesis of oviductal secretory proteins by estrogen-treated ovariectomized gilts. J Exp Zool 1992; 262:426–435.[CrossRef][Medline]
  33. Vatzias G, Hagen DR. Effects of porcine follicular fluid and oviduct-conditioned media on maturation and fertilization of porcine oocytes in vitro. Biol Reprod 1999; 60:42–48.[Abstract/Free Full Text]
  34. Nagai T, Moor RM. Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilized in vitro. Mol Reprod Dev 1990; 26:377–382.[CrossRef][Medline]
  35. Hunter RHF. Fertilization of pig eggs in vitro and in vivo. J Reprod Fertil 1990; 40(suppl):211–226.
  36. Rudak E, Dor J, Mashiach S, Nebel L, Goldman B. Chromosome analysis of multipronuclear human oocytes fertilized in vitro. Fertil Steril 1984; 41:538–545.[Medline]
  37. Kola I, Trounson A, Dawson G, Rogers P. Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos. Biol Reprod 1987; 37:395–401.[Abstract]
  38. Plachot M, Crozet N. Fertilization abnormalities in human in vitro fertilization. Hum Reprod 1992; 7(suppl):89–94.
  39. Yie SM, Collins JA, Daya S, Hughes E, Sagle M, Younglai EV. Polyploidy and failed fertilization in in-vitro fertilization are related to patient's age and gamete quality. Hum Reprod 1996; 11:614–617.
  40. Iwasaki S, Shioya Y, Masuda H, Hanada A, Nakahara T. Incidence of chromosomal anomalies in early bovine embryos derived from in vitro fertilization. Gamete Res 1989; 22:83–91.[CrossRef][Medline]
  41. Iwasaki S, Hamano S, Kuwayama M, Yamashita M, Ushijima H, Nagaoka S, Nakahara T. Developmental changes in the incidence of chromosome anomalies of bovine embryos fertilized in vitro. J Exp Zool 1992; 261:70–85.
  42. Iwasaki S, Nakahara T. Incidence of embryos with chromosomal anomalies in the inner cell mass among bovine blastocysts fertilized in vitro. Theriogenology 1990; 34:683–690.
  43. Kawarsky SJ, Basrur PK, Stubbings RB, Hansen PJ, King WA. Chromosomal abnormalities in bovine embryos and their influence on development. Biol Reprod 1996; 54:53–59.[Abstract]
  44. Hunter RHF. Sperm:egg ratios and putative molecular signals to modulate gamete interactions in polytocous mammals. Mol Reprod Dev 1993; 35:324–327.[CrossRef][Medline]
  45. Boatman DE, Magnoni GE. Identification of a sperm penetration factor in the oviduct of the golden hamster. Biol Reprod 1995; 52:199–207.[Abstract]
  46. Kimura H, Matsuda J, Ogura A, Asano T, Naiki M. Affinity binding of hamster oviductin to spermatozoa and its influence on in vitro fertilization. Mol Reprod Dev 1994; 39:322–327.[CrossRef][Medline]
  47. Martus NS, Verhage HG, Mavrogianis PA, Thibodeaux JK. Enhancement of bovine oocyte fertilization in vitro with a bovine oviductal specific glycoprotein. J Reprod Fertil 1998; 113:323–329.[Abstract/Free Full Text]
  48. Hinsch E, Hagele W, Schill W-B, Hinsch K-D. The zona pellucida "receptors." In: Ivell R, Holstein (eds.), The Fate of the Male Germ Cell. New York: Plenum Press; 1997: 313–328.
  49. Schmidt A, Mavrogianis PA, O'Day-Bowman MB, Jaffe RC, Verhage HG. Characterization of antibodies generated against a conserved portion of oviductal glycoprotein (OGP) and endogenous hamster OGP and their ability to decrease sperm binding to the zona pellucida in vitro. Am J Reprod Immunol 1997; 38:377–383.
  50. Schmidt A, Mavrogianis PA, O'Day-Bowman MB, Verhage HG. Species-specific effect of oviductal glycoproteins on hamster sperm binding to hamster oocytes. Mol Reprod Dev 1997; 46:201–207.[CrossRef][Medline]
  51. O'Day-Bowman MB, Mavrogianis PA, Reuter LM, Johnson DE, Fazleabas AT, Verhage HG. Association of oviduct-specific glycoproteins with human and baboon (Papio anubis) ovarian oocytes and enhancement of human sperm binding to human hemizonae following in vitro incubation. Biol Reprod 1996; 54:60–69.[Abstract]
  52. Broermann DM, Xie S, Nephew KP, Pope WF. Effects of the oviduct and wheat germ agglutinin on enzymatic digestion of porcine zona pellucidae. J Anim Sci 1989; 67:1324–1329.
  53. Buhi WC, Alvarez IM, Pickard AR, McIntush EW, Kouba AJ, Ashworth CJ, Smith MF. Expression of tissue inhibitor of metalloproteinase (TIMP)-1 protein and mRNA by the oviduct of cyclic, pregnant, and ovariectomized steroid-treated gilts. Biol Reprod 1996b; 57:7–15.
  54. Kouba AJ, Alvarez IM, Buhi WC. Identification and localization of plasminogen activator inhibitor (PAI)-1 in the porcine oviduct. Biol Reprod 2000; 62:501–510.[Abstract/Free Full Text]
  55. Beatty RA. Parthenogenesis and Polyploidy in Mammalian Development. Cambridge: Cambridge University Press; 1957.
  56. Bomsel-Helmreich O. Heteroploidy and embryonic death. In: Wolstenholme GEW, O'Connor M (eds.), Preimplantation Stages of Pregnancy. Ciba Foundation Symposium. London: Churchill; 246–267.
  57. Martus NS, Verhage HG, Mavrogianis PA, Thibodeaux JK. Enhanced in vitro development of bovine embryos in the presence of a bovine oviductal specific glycoprotein. Theriogenology 1997; 47(suppl 1):334 (abstract).
  58. Van Steenbrugge A, Van Langendonckt A, Massip A, Dessy F. Effect of estrus-associated glycoprotein and tissue inhibitor of metalloproteinase-1 secreted by oviduct cells on in vitro bovine embryo development. Mol Reprod Dev 1997; 46:527–534.[CrossRef][Medline]
  59. Hill JL, Wade MG, Nancarrow CD, Kelleher DL, Boland MP. Influence of ovine oviducal amino acid concentrations and an ovine oestrus-associated glycoprotein on development and viability of bovine embryos. Mol Reprod Dev 1997; 47:164–169.[CrossRef][Medline]



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