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Research Article |
Division of Animal Sciences,4
Comparative Medicine Center,5
and Departments of Biology6
and Microbiology and Immunology,7 University of Missouri-Columbia, Columbia, Missouri 65211
Monsanto Company,8 St. Louis, Missouri 63167
ABSTRACT
This study was designed to determine the role of osteopontin (SPP1) in in vitro fertilization (IVF) in swine. The initial objective was to evaluate the effect of various concentrations of SPP1 (0, 0.001, 0.01, 0.1 and 1 µg/ml) on spermatozoa and oocytes during IVF. The results demonstrate that SPP1 reduced the rate of polyspermy in a dose-dependent manner (P < 0.05). SPP1 also reduced both the number of sperm in oocytes as compared to the control and the number of spermatozoa bound to the zona pellucida (ZP) (P < 0.05). High doses of SPP1 (1 µg/ml) reduced penetration and male pronucleus formation as compared to the control (P < 0.05). Interestingly, compared to the control group, medium doses of SPP1 increased fertilization efficiency (42.6% and 44.6% vs. 31.6%; P < 0.05), representing a 41% improvement for 0.1 µg/ml SPP1). The ZP of 0.1 µg/ml SPP1-treated oocytes was more difficult to digest than control oocytes (P < 0.05). The percentage of acrosome-reacted spermato zoa bound to the ZP during IVF increased after 4 h of 1.0 µg/ml SPP1 treatment compared to 0 or 0.1 µg/ml SPP1. SPP1 did not have an effect on sperm motility, progressive motility, and sperm viability. To confirm that the reduction of polyspermy was specific to SPP1, a mixture of pregnancy-associated glycoproteins was included in the IVF protocol and shown to have no effect on polyspermy. Furthermore, Western blotting demonstrated that a 50-kDa SPP1 form was present in the oviducts on Days 0, 3, and 5 in pregnant and nonpregnant gilts, and the concentration of SPP1 on Day 0 was higher than on Days 3 and 5. The current study represents the first report to demonstrate that SPP1 plays an important role in the regulation of pig polyspermic fertilization; it decreases polyspermy and increases fertilization efficiency during IVF.
fertilization, in vitro fertilization, oocyte, osteopontin, oviduct, pig, polyspermy, sperm, spermatozoa
INTRODUCTION
Although porcine embryos produced by in vitro maturation (IVM) and in vitro fertilization (IVF) develop to the blastocyst stage, the high incidence (often exceeding 50%) of polyspermy remains a major impediment [1]. Polypronuclei can participate in karyosyngamy and the resulting polyploid eggs can develop into diploid, triploid, or mosaic fetuses [2, 3] that may have difficulty completing gestation. The development of efficient systems for in vitro production of porcine embryos has been hampered by a high incidence of polyspermic fertilization. Polyspermic fertilization occurs more frequently in the pig than in other species, even for in vivo fertilization under diverse experimental conditions [4–6]. Different approaches have been taken to solve this problem, such as coculture of spermatozoa with oviduct cells [7], follicle cells [8], oviductal fluid [9], follicular fluid [10], and other substances [11]. Reducing sperm number during IVF decreases polyspermic penetration, but it also reduces overall sperm penetration rates [12] and results in an overall reduction in the efficiency of fertilization. In addition, undefined biologicals (such as coculture with oviduct cells, or addition of follicular fluid or oviductal fluid) are unstable factors, causing results to be less readily repeatable [13]. A recently reported method, using embryo cryopreservation straws instead of microdrops to mimic the process of spermatozoa movement, binding, and fertilization of oocytes in the oviducts, can reduce polyspermic penetration by 25% [13]. However, a satisfactory IVF system in pigs has not been established. To overcome the current problems of polyspermy, future efforts might focus on controlling boar sperm-zona binding to achieve normal maturation associated with normal zona pellucida (ZP) modifications of porcine oocytes at fertilization [14]. Osteopontin (SPP1) is an extracellular matrix (ECM) protein first described in the bone matrix. It is an acidic single chain phosphorylated glycoprotein, ranging in length from 264 to 301 amino acids. It undergoes extensive posttranslational modification, resulting in molecular mass variants ranging from 25 to 75 kDa [15]. In pig, both 70-kDa and 45-kDa forms of SPP1 were detected in cyclic and pregnant endometrium [16]. So far, SPP1 has been found not only on epithelial cells and in secretions of the gastrointestinal tract, kidney, thyroid, and breast, but also in reproductive tissues, such as oviduct, uterus, placenta, and testes. Expression of SPP1 by various cells in the uterus has been demonstrated in sheep, pigs, cows, mice, baboons, and humans [15]. The Arg-Gly-Asp sequence of SPP1 interacts with cell surface receptors, including integrins. SPP1 has been reported to stimulate cell-cell adhesion, increase cell-ECM communication, promote migration of immune cells, osteocytes, and tumor cells, decrease cell death by reducing reactive oxygen species and nitric oxide production by injured tissues, stimulate immunoglobulin production by B cells, induce changes in the phosphorylation state of focal adhesion kinase and paxillin, stimulate phosphatidylinositol 3'-kinase activity, alter intracellular calcium levels, and affect tissue mineralization and promote calcium phosphate deposition in bone. SPP1 also appears to play multiple key roles in the mammalian male and female reproductive systems [15].
We found SPP1 messages to be relatively abundant in a cDNA library derived from the oviducts of a gilt exhibiting estrus (Day 0) (unpublished results). Randomly selected clones, from the Day 0 library and another oviduct library generated from a gilt on Day 3, were sequenced and clustered into groups of sequence similarity. Twenty of 1808 (1.1%) cDNAs from the estrous oviduct and one of 1772 (0.06%) cDNAs from the Day 3 oviduct represented SPP1 message (http://genome.rnet.missouri.edu/Swine/). Similarly, results from a preliminary microarray study suggest that SPP1 and oviductal glycoprotein 1 both decreased 8-fold from Day 0 to Day 3 (unpublished results). Since there is a polyspermy problem in pig IVF and since SPP1 has many functions that relate to cell-cell interactions, we decided to determine the effect of SPP1 on porcine fertilization. This study evaluated the effect of various concentrations of SPP1 on IVF and polyspermy in the pig.
MATERIALS AND METHODS
SPP1
Recombinant rat SPP1 (0.76 mg/ml, in PBS; lot r8/10.03.02, 90–94% pure [17]) was donated by Robert Burghardt (Texas A&M University, College Station, TX). SPP1 was diluted to 0.001, 0.01, 0.1, or 1.0 µg/ml in a modified Tris-buffered medium (mTBM) that contained 2 mg/ml BSA and 2 mM caffeine [12]. A negative control (no SPP1) was also included.
Pregnancy-Associated Glycoproteins
A mixture of affinity purified bovine pregnancy-associated glycoproteins (0.56 mg/ml in PBS) was donated by Jon A. Green, University of Missouri-Columbia, Columbia, MO; see Fig. 1A in [18]). Pregnancy-associated glycoproteins were diluted to 0.1 and 1.0 µg/ml in mTBM, and compared to 0.1 µg/ml SPP1. A negative control (no SPP1 or pregnancy-associated glycoproteins) was also included.
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Media
Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). The oocyte maturation medium was TCM 199 (Gibco BRL) supplemented with 0.1% (w/v) polyvinyl alcohol (PVA), 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 75 µg/ml penicillin G, and 50 µg/ml streptomycin. The following were added fresh before use: 0.57 mM cysteine, 0.5 µg/ml luteinizing hormone, 0.5 µg/ml follicle stimulating hormone, and 10 ng/ml epidermal growth factor. The medium used for IVF was a mTBM of the same formulation as described above. Sperm washing medium was Dulbecco PBS (dPBS; Gibco) supplemented with 1 mg/ml BSA (pH 7.3). The embryo culture medium was Porcine Zygote Medium-3 (pH 7.3) supplemented with 3 mg/ml BSA [19, 20].
Collection of Oocytes and IVM
Ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in 0.9% sodium chloride (NaCl) solution at 30–35°C. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles (3–6 mm in diameter) with an 18-gauge needle fixed to a 10-ml disposable syringe. COCs with uniform cytoplasm and several layers of cumulus cells were selected and rinsed three times in TL-Hepes medium [21] containing 0.1% (w/v) PVA. Approximately 50–70 COCs were transferred into 500 µl IVM medium. The medium had been covered with mineral oil in a four-well Nunclon dish (Nunc, Roskilde, Denmark). Oocytes were matured for 40–44 h at 38.5°C, in a humidified atmosphere of 5% carbon dioxide (CO2) in air [22].
IVF and Embryo Culture
Cumulus-free oocytes were derived after vortexing and were then washed three times in IVF medium. Approximately 30–35 oocytes were transferred into 50-µl droplets of IVF medium covered with mineral oil that had been equilibrated for 40 h at 38.5°C in 5% CO2 in air. Dishes were kept in a CO2 incubator until sperm were added for insemination. For IVF, one 0.1-ml frozen semen pellet was thawed at 39°C in 10 ml sperm washing medium [12]. After washing twice by centrifugation (1900 x g, 4 min), cryopreserved ejaculated spermatozoa were resuspended with fertilization medium to a concentration of 2 x 106 cells/ml. Fifty microliters of the sperm sample was added to the fertilization droplets containing the oocytes, giving a final sperm concentration of 1 x 106 cells/ml. Oocytes were co-incubated with sperm for 6 h. At 6 -h post-insemination, oocytes were washed three times and cultured in 500 µl embryo culture medium in 4-well Nunclon dishes (Nunc) at 38.5°C in 5% CO2 in air.
At 18 h after the onset of IVF, oocytes were transferred into a 1-ml centrifuge tube, and spermatozoa were removed by vortexing for 1 min. After washing three times, oocytes were transferred to a glass microscope slide, covered with a cover slip, and fixed with freshly prepared fixation medium (25% [v/v] acetic acid in ethanol) for 72 h at room temperature. Oocytes were stained with 1% (w/v) orcein in 45% (v/v) acetic acid for 10 min at room temperature, and then washed with 20% glycerol and 20% acetic acid in water. Nuclear status (pronuclear, sperm head, sperm tail, metaphase II chromosome, polar body [PB]1, PB2) was then observed by phase contrast microscopy (Nikon) at 400x magnification.
Western Blotting
Ten gilts that exhibited at least two normal estrous cycles (18–21 days) were assigned randomly to a cyclic status. Fluid from oviducts was collected from two gilts on Day 0 of the cycle and from two bred gilts and two open gilts on Day 3 and 5 of the estrous cycle. Oviducts were removed from killed gilts and a capillary tube was used to aspirate oviductal fluid. The cellular debris in the fluid was removed by centrifugation (3000 x g, 5 min), and the fluid was stored at –80°C until analysis.
Proteins from oviductal fluids (20 µg/sample) were denatured in Laemmli buffer (Bio-Rad, Hercules, CA) by boiling for 8 min, electrophoresed through a 10% Tris-HCl Ready Gel (Bio-Rad) at 100 V for 1 h, and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA) using a semidry transfer cell (Bio-Rad). After blocking with 5% nonfat milk in 20 mM Tris pH 7.4, 150 mM NaCl, 0.02% Tween 20 (TBST) overnight at 4°C, the membrane was incubated with goat anti-human SPP1 polyclonal antibody (1:1000; R&D Systems, Minneapolis, MN; catalog number AF1433) or normal goat serum (5 µg/ml) as control, for 1 h at room temperature. The membrane was washed three times with TBST and incubated with rabbit anti-goat IgG (ZYMED; catalog number 81–1620) conjugated to horseradish peroxidase in blocking solution for 1 h at room temperature. Standards (lot r8/10.03.02., Robert Burghardt, Texas A&M University) containing SPP1 at 30 µg/ml, 20 µg/ml, 10 µg/ml, 5 µg/ml, and 1 µg/ml were analyzed by Western blotting. Immunodetection of SPP1 was performed using the Enhanced Chemiluminescence System (Amersham, Piscataway, NJ; RPN 2106/8/9). Intensity of SPP1 bands was determined using Kodak 1D Image Analysis Software and the Kodak Electrophoresis Documentation and Analysis System 290 (EDAS 290).
Immunohistochemical Localization of SPP1 in Porcine Oviducts
Oviductal tissue was collected from gilts and embedded in Tissue-Tek Optimal Cutting Temperature Compound (Miles Inc., Oneonta, NY) and frozen in liquid nitrogen vapor for 5 min. The tissue was wrapped in aluminum foil and stored at –80°C.
Frozen sections (4–8 µm) of oviductal tissues were sectioned with a cryotome and fixed in –20°C methanol for 10 min. Sections were permeabilized with 0.3% Tween 20 in 0.02 M PBS for 1 h at room temperature and blocked in antibody dilution buffer (5% BSA in 0.3% Tween 20, pH 8.0) for 1 h at room temperature. Sections were rinsed in PBS three times (10 min each) at room temperature and incubated with 10 µl goat anti-human SPP1 polyclonal antibody (1:200; R&D Systems) or normal goat serum (5 µg/ml) as control overnight at 4°C. After washing three times in PBS, sections were incubated with 5 µl Texas Red-conjugated rabbit anti-goat IgG (1:100; Gene Tex, San Antonio, TX; catalog number GTX27130) for 1 h at room temperature and again washed in PBS containing 4',6'-diamidino-2-phenylindole (DAPI) to stain nuclei. Sections were then overlaid with a coverslip and ProLong Antifade Mounting Reagent (Molecular Probes, Eugene, OR), sealed with nail polish, and stored at 4°C in the dark. Samples were viewed at 400x magnification with an epifluorescence microscope (Nikon, Tokyo, Japan) and images were acquired with a digital camera.
Sperm Function Assays
Thawed sperm were incubated in mTBM containing 0, 0.1, or 1 µg/ml SPP1 for 2, 4, or 6 h at 38.5°C in 5% CO2 in air. The time immediately after thawing served as the 0 h group for all experiments. Porcine sperm motility and progressive motility of samples were analyzed at 0, 2, 4, and 6 h on a computer-aided semen analyzer (IVOS v 12.2c; Hamilton Thorne, Beverly, MA). Motility was defined as the percentage of spermatozoa that exhibited any movement of the sperm head. Progressive motility was defined as the percentage of spermatozoa that had a linear velocity of at least 45 µm/sec with a straightness of 45% or more.
Sperm viability was assessed in a fluorometric assay after staining with propidium iodide (PI; Sigma). Thawed semen samples were transferred to 50 µl IVF medium containing various concentrations of SPP1 (0, 0.1, or 1 µg/ml) and incubated for 0, 2, 4, or 6 h. At different timepoints, PI (10 µg/ml) was added for 30 min in an incubator with 5% CO2 in the dark. At the end of incubation, 10-µl sperm samples were transferred onto a glass slide, smeared, mounted with an antifade reagent (ProLong, Molecular Probes), covered with a glass cover slip, and sealed. Spermatozoa were examined by epifluorescence microscopy (Nikon) at 400x magnification; at least 200 cells were evaluated per sample. Spermatozoa stained with PI were considered to have damaged membranes. Spermatozoa that blocked uptake of PI were considered to be viable. Each experiment was replicated six times.
The sperm acrosome reaction was investigated by lectin-PNA/DAPI staining according to the procedure described by Sutovsky et al. [23] and Katayama et al. [24], with slight modifications: At 4 h or 6 h after IVF, oocytes were washed three times in 400 µl dPBS-polyvinylpyrrolidone (dPBS-PVP) medium in a prewarmed glass plate with 4-well dish on a slide warmer set to 37°C, and pipetted 10 times to remove loosely bound spermatozoa. Oocytes were fixed in 400 µl 2% formaldehyde in dPBS for 40 min at room temperature. After fixation, oocytes were washed twice in dPBS-PVP, and then permeabilized in 0.1% Triton X-100 in dPBS for 40 min at room temperature. Oocytes were then incubated in 0.4 µg/ml Alexa-Fluor 488-PNA (1:500; Molecular Probes, catalog number L-21409) in 0.1% Triton X-100 in dPBS for 40 min in the dark, and then transferred into 0.1% Triton X-100 in dPBS for 5 min. Oocytes were transferred to a standard microscopy slide, in 8 µl mounting medium with DAPI (Vectashield, Vector Laboratories, Burlingame, CA; H-1200) and covered with a cover slip that was then sealed with nail polish. Fl uorescence was determined by epifluorescence microscopy (Nikon). Sperm were observed at 1000x magnification, and 10 oocytes were evaluated per sample. Spermatozoa around the ZP were counted according to Alexa-PNA and DAPI staining: spermatozoa were considered to be acrosome intact as determined by a green-colored acrosome at its top and a blue-colored nucleus. Sperm in which the acrosome was not stained with Alexa- PNA were considered to be acrosome reacted [25]. The percentage of acrosome-intact and acrosome-reacted spermatozoa around the ZP was determined. Each experiment was replicated three times.
Effect of SPP1 on Oocyte Function
Zona pellucida solubility or hardness was measured after exposure to 0.1% pronase. Cumulus-free oocytes matured in vitro were transferred to 50 µl mTBM containing various concentrations of SPP1 (0, 0.1, or 1.0 µg/ml) and incubated 6 h with or without spermatozoa at 39°C, 5% CO2 in air. Groups of 10 oocytes were used for the experiment without SPP1 (0 µg/ml control) or with SPP1 (0.1 and 1.0 µg/ml). Oocytes were washed three times in PBS, and then transferred into 100 µl 0.1% (w/v) pronase in dPBS. ZP were continuously observed for dissolution under an inverted microscope (Nikon Diaphot) equipped with a warm plate at 37°C. The ZP dissolution time for each oocyte was registered as the interval of time between placement of the samples in pronase solution and disappearance of the ZP observed at 200x magnification. Each experiment was replicated six times.
Sperm binding to the ZP was examined as described by Kouba et al. [26], with slight modification: Cumulus-free oocytes matured in vitro were transferred to 50 µl mTBM containing SPP1 (0, 0.1, or 1.0 µg/ml) and co-incubated with spermatozoa for 4 h or 6 h. After fertilization, oocytes were washed three times in 500 µl mTBM and pipetted 10 times to remove loosely bound sperm. Oocytes were then placed into 50-µl drops of mTBM containing Hoechst 33342 (bis-benzamide; 1.3 mg/ml) and incubated for 30 min at 39°C with 5% CO2 in air in the dark. Oocytes were then washed twice in 300 µl TL-Hepes-PVA, mounted, and the number of tightly bound sperm per zygote was counted with an epifluorescence microscope at 400x magnification (Nikon). Each treatment was replicated six times, with 10 oocytes counted from each replicate.
Experiment 1: Effect of SPP1 on In Vitro Fertilization
The effect of different concentrations of SPP1 in the fertilization medium on penetration rate (number of oocytes penetrated by sperm/total number of oocytes inseminated), polyspermic fertilization rate (number of oocytes with >1 sperm/total number of oocytes penetrated), male pronuclear formation rate (number of oocytes with >1 male pronucleus/total number of oocytes penetrated), normal fertilization efficiency (number of oocytes with one male and one female pronucleus/total number of oocytes inseminated), and number of sperm penetrated per oocyte were determined. SPP1 was added to the fertilization medium prior to addition of sperm or oocytes.
Experiment 2: Effect of SPP1 on Sperm Function
The second set of experiments assessed the effect of SPP1 on sperm motility, progressive motility, viability, and acrosome reaction.
Experiment 3: Effect of SPP1 on Oocyte Function: ZP Solubility and Sperm Binding to the ZP
Zona pellucida solubility or hardness was measured after exposure to pronase, and sperm binding to the ZP was examined.
Experiment 4: Effect of SPP1 on Polyspermy with Sperm from Different Boars and Pregnancy-Associated Glycoproteins During IVF
The fourth experiment was designed to investigate whether there are differences in the effect of SPP1 on polyspermy among three different boars (number 110–7, 110–9, and 139–5), as well as to confirm that the effect of SPP1 was not as an unspecific glycoprotein. Affinity purified pregnancy-associated glycoproteins were added at 0.1 µg/ml. The thawing method for the sperm pellet was the same as Experiment 1. We chose a sperm concentration of 2 x 107/ml for IVF with SPP1 (0.1 µg/ml). Eighteen hours after onset of IVF, oocytes were fixed and fertilization parameters were assessed as in Experiment 1.
Experiment 5: SPP1 in the Oviduct
The fifth experiment was designed to determine if SPP1 was detectable in the oviduct. The relative amount of oviductal SPP1 was determined on Days 0, 3, and 5 of the estrous cycle.
Statistical Analysis
All dependent variables were analyzed for normality using the Wilk-Shapiro test (SAS Institute, Cary, NC). Penetration rate and polyspermic fertilization rate were Arcsine transformed. Sperm number penetrated in oocytes was logarithmically transformed to approach a normal distribution. Data for the function variables were analyzed using the general linear model and Duncan multiple range tests of SAS. Culture wells were the experimental units used to test SPP1 treatment effects. Data are expressed as a least-squares (LS) mean ± SEM.
RESULTS
Experiment 1: Effect of SPP1 on In Vitro Fertilization
As shown in Table 1, SPP1 treatment reduced the polyspermic fertilization rate in a dose-dependent manner. SPP1 at 0.01–1.0 µg/ml significantly decreased the polyspermy rate compared to the control. As a result, the monospermy rate was higher in the three experiments with the highest concentration of SPP1 (0.01, 0.1, and 1.0 µg/ml) than in the control. SPP1 also reduced the number of sperm per oocyte as compared to the control, an effect which was most pronounced at the highest SPP1 concentration. However, the highest concentration of SPP1 (1.0 µg/ml) decreased the percentage of penetration and male pronucleus formation compared to the control (Table 1). The most relevant measure of these endpoints is the normal fertilization efficiency (number of oocytes with one male and one female pronucleus per total number of oocytes inseminated), and it was highest in experiments with the medium doses of SPP1 as compared to the control.
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Experiment 2: Effect of SPP1 on Sperm Function During IVF
To examine whether or not the decreased polyspermy observed in vitro resulted from changes in sperm function, the effect of SPP1 on sperm motility, progressive motility, viability, and acrosome reaction were investigated. The motility of cryopreserved spermatozoa was low immediately after thawing. There was no significant increase in sperm motility (P > 0.47), or progressive motility (P > 0.67) as SPP1 dose increased; sperm viability tended to decrease in response to SPP1 (P = 0.052) (data not shown). The acrosome reaction was observed using fluorescence microscopy (Fig. 1). At 4 h after IVF, the percentage of acrosome-reacted spermatozoa bound to the ZP of 1.0 µg/ml SPP1-treated oocytes was higher than the control. At 6 h after IVF, 0.1 µg/ml SPP1 resulted in the lowest level of acrosome reaction (Table 2).
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Experiment 3: Effect of SPP1 on ZP and Oocyte Function During IVF
To test whether the decreased polyspermy observed in vitro resulted from changes in the oocytes, the number of spermatozoa binding to the ZP and ZP solubility were examined. The number of sperm bound to the ZP of oocytes decreased as the concentration of SPP1 increased, but this was only significant (P < 0.05) at 6 h after IVF (Table 2). The solubility of the ZP was examined by enzymatic digestion. The time in seconds for the ZP to be digested in the 0.1 µg/ml SPP1 group was longer than the control group (P < 0.05) after 6 h incubation (Table 3).
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Experiment 4: The Effect of SPP1 and Pregnancy-Associated Glycoproteins on Polyspermy with Sperm from Different Boars During IVF
To investigate whether the effect of SPP1 on polyspermy is boar-specific, sperm from three different boars were used for IVF. In this experiment, pregnancy-associated glycoproteins were also added to the fertilization system to determine if the effect of SPP1 was specific or simply due to the addition of a glycoprotein. Whereas SPP1 decreased the rate of polyspermy without reducing penetration, pregnancy-associated glycoproteins had no effect on polyspermy (Table 4). Treatment with SPP1 also resulted in a fertilization efficiency increase of approximately 24%, while pregnancy-associated glycoproteins actually decreased fertilization efficiency. The polyspermy rate of these three boars was lower (25%) than the boar used in Experiment 1 (39%).
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Experiment 5: SPP1 in the Oviduct
To demonstrate the presence of SPP1 in porcine oviducts, we conducted Western blotting and immunohistochemical staining experiments. Western blotting analysis indicated that an 50-kDa SPP1 band was present on Days 0, 3, and 5 in oviductal fluid in both pregnant and cyclic gilts (Fig. 2). The concentration of SPP1 in Day 0 oviductal fluid was higher than that of Day 3 and Day 5 oviductal fluid (Fig. 2). The two gels were run and analyzed at the same time and they showed an identical pattern of changes in SPP1. Figure 3 indicates that SPP1 was present in the luminal epithelium (LE) of the isthmus of the oviduct, but not in the ampulla area.
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DISCUSSION
In the pig, the rate of polyspermy not only depends on the quality of semen at the time of fertilization [9, 23, 24], but also on the quality of oocytes. This study demonstrates that SPP1 can help decrease the incidence of polyspermy in pig IVF and result in an overall increase in the efficiency of standard IVF protocols by up to 41%. SPP1 achieved a reduction in polyspermy and an increase in overall fertilization efficiency without altering the penetration rate. This study documents further that SPP1 caused decreased polyspermy in vitro by reducing the number of spermatozoa bound to the ZP or by stabilizing the ZP. These data also showed that SPP1 is present in cells in the isthmus of the oviduct and in the secretions of the oviduct. It is known that SPP1 is produced in some tissues of the male reproductive tract and in some species binds to sperm [27, 28]; since SPP1 is also located in the oviduct, there may be additional binding as the sperm traverses the isthmus prior to fertilization. It is also important to note that while SPP1 expression appears to be predominantly in the isthmus, there is retrograde movement of oviductal fluid [5] that could transport SPP1 to the ampullary region for availability at the time of fertilization.
SPP1 Reduced the Polyspermy Rate During IVF
Polyspermic fertilization occurs less frequently in vivo than in vitro in the pig. The incidence of polyspermy in pigs in vivo is less than 5% [6]. Pig oocytes flushed from the oviduct and subsequently fertilized in vitro had a much lower incidence of polyspermy (28%) than oocytes matured and fertilized in vitro (62%) [29]. In addition, peri-ovulatory oviduct-conditioned media [30], oviduct fluid [9, 31], follicular fluid [10, 30], and co-incubation of boar spermatozoa or pig oocytes with oviductal epithelial cells [32, 33] significantly reduced polyspermic fertilization in vitro. These reports suggest that an unknown factor of oviductal origin associates with either oocytes or spermatozoa and effectively decreases the incidence of polyspermy. In vitro fertilization medium plays a significant role in any successful IVF program. One of the components important for maintaining sperm motility, capacitation, and the acrosome reaction is protein supplementation. Supplementation of IVF medium with fetal calf serum (FCS) can significantly increase polyspermy in pig oocytes, but supplementation of BSA does not [30]. It has been found that FCS reduces the effective concentration of calcium in the medium; thus, supplementation of FCS inhibits cortical granule (CG) content dispersion after exocytosis and delays their reaction with the ZP [34]. FCS also inhibits the zona reaction in oocytes of some mammals [35, 36]. It was found that fetuin in FCS regulates the effect of CGs on the ZP [37]. Another protein family, porcine oviduct-specific glycoprotein reduces the incidence of polyspermy in pig oocytes. Furthermore, it reduces the number of sperm bound to the ZP and increases postcleavage development to the blastocyst stage [26].
The effect of SPP1 on polyspermy has not been previously reported, but it was known that SPP1 does affect reproductive functions. In a cDNA library made from oviducts of gilts in estrus, SPP1 represented 1.3% of the total population of transcripts, but by Day 3 of the cycle it represented only 0.08% of the total transcripts (unpublished results). Moreover, three different SPP1 isoforms (55 kDa, 48 kDa, and 25 kDa) are present in the oviductal fluid of cattle [38], while we found one dominate form at 50 kDa. Taken together, we hypothesize that SPP1 is involved in the fertilization process and might reduce polyspermic fertilization in the pig.
In the male, previous studies reported that SPP1 could play a role in adhesion of early germ cells to the basement membrane and/or epididymal maturation of sperm [28, 39]. Nonciliated cells of the efferent ducts express SPP1, and a cell- and region-specific distribution of SPP1 is observed in the epididymis. The presence of SPP1 in the apical region of cells corresponding to microvilli, small endocytic vesicles, and endosomes has been suggested to indicate that SPP1 serves to remove calcium from the epididymal lumen and thus prevent mineral accumulation and subsequent decrease in sperm fertility [38]. In higher-fertility Holstein bulls, a higher level of seminal plasma SPP1 was found in comparison to lower-fertility bulls [27, 40]. The present study found that a 50-kDa SPP1 form was present in the fluids of porcine oviducts on Days 0, 3, and 5, and the concentration of SPP1 in oviductal fluid on Day 0 was higher than on Days 3 and 5. It is possible that SPP1 in the oviduct may act to affect sperm or oocyte function, possibly contributing to the decreased rates of polyspermy in vivo.
SPP1 Affected the Zona Reaction and Acrosome Reaction, but had less Affect on Sperm Motility and Viability During IVF
The fusion of sperm and egg and the release of CG contents during the cortical reaction lead to changes in the ZP, oolemma, and perivitelline space. It is thought that these changes facilitate the block to polyspermy. Depending on the species, the polyspermic block resides either at the ZP, the egg plasma membrane, or both. For example, polyspermy is primarily blocked by ZP changes in hamster, goat, ovine, and bovine oocytes, by oolemma changes in rabbit oocytes, and by both in mouse, rat, guinea pig, and cat oocytes [35].
In the pig, the in vivo block to polyspermy is thought to result from a restriction of the number of spermatozoa that can reach an egg and the ZP block to penetration [5]. In vivo polyspermy is prevented by modification of two primary oocyte structures, the plasma membrane and ZP. The relative importance of the ZP vs. plasma membrane block to polyspermy varies among species [41–43]. Two events are associated with the zona block to polyspermy: the cortical reaction and the zona reaction. After CG exocytosis, CG exudates act on the ZP, causing biochemical and structural changes that cause the ZP to both lose the ability to be penetrated by already-bound spermatozoa and decrease its ability to bind additional sperm. This study found that SPP1 treatment decreased both the number of sperm bound to the ZP and the percentage of acrosome-reacted sperm on the ZP, but increased the time required for ZP digestion. The mammalian oocyte ZP consists of three glycoproteins, ZP1, ZP2, and ZP3. It is thought that ZP2 and ZP3 are present as heterodimers and act as the primary and secondary sperm receptors, respectively, in mouse oocytes. Acrosome-intact sperm bind to the O-linked oligosaccharides attached to ZP3 and undergo the acrosome reaction. Acrosome-reacted sperm then bind by their inner acrosomal membranes to ZP2. Short after fertilization, CG exudates modify ZP2 and ZP3 to ZP2f and ZP3f, which thus prevents both additional sperm from binding the ZP and migration through the ZP by sperm that are already bound [42–44]. An uneven distribution of different sugar residues in the rat ZP and postfertilization changes in the distribution of galactose are thought to be correlated with the polyspermy block [45].
This study suggests that SPP1 might assist in modification of ZP sperm receptors immediately after fertilization in a process known as "zona hardening." The changes in the ZP are permanent, as further exposure of fertilized oocytes to capacitated sperm did not increase sperm penetration. SPP1 changed the ability of the ZP to bind sperm and induced the sperm acrosome reaction, further preventing sperm penetration.
Our current results show that treatment with a low concentration of SPP1 increased ZP hardness but a much higher concentration did not. In the control group, ZP hardness was only caused by the CG reaction after sperm penetration into the oocyte. In the 0.1 µg/ml SPP1 group, ZP hardness was not only caused by the CG reaction, but might have also been modulated by SPP1. In the 1.0 µg/ml SPP1 group, ZP hardness might have been altered by SPP1 resulting in less of a cortical reaction. Clearly this speculation needs to be experimentally verified.
In conclusion, our study demonstrates that SPP1 significantly decreased the incidence of polyspermy during IVF. The effect of SPP1 on polyspermy was similar among sperm from different boars during IVF, and SPP1 increased the efficiency of the fertilization process by up to 41%. The increase in efficiency was highest in boars with a high incidence of polyspermy and lower in boars with lower incidences of polyspermy. In contrast, a non-xoviductal family of glycoproteins, pregnancy-associated glycoproteins, had no affect on polyspermy. The SPP1-mediated decrease in polyspermy may have decreased the percentage of sperm undergoing the acrosome reaction. These data suggest that SPP1 might play an important role in the in vivo fertilization process and its inclusion in IVF can improve fertilization in vitro.
ACKNOWLEDGMENTS
We thank Robert Burghardt of Texas A&M University for providing SPP1 protein, Peter Sutovsky and Lisa Overman-Martin for help with immunocytochemistry, and Mary Sakla, Boh Yang, Hongsheng Men, Rongfeng Li, Kristin M. Whitworth, Melissa S. Samuel, David Wax, Lee Spate, Zhisheng Zhong, Clifton Murphy, August Rieke, Mika Katayama, Lonnie Dowell, and Aaron Bonk for assistance in this study.
FOOTNOTES
2 Correspondence: Randall S. Prather, Division of Animal Sciences, University of Missouri-Columbia, E125D ASRC, 920 East Campus Drive, Columbia, MO 65211. FAX: 573 884 7827; PratherR{at}Missouri.edu 
3 Visiting scientist from Life Science and Biotechnology Research Center, Northeastern Agricultural University, Harbin, People's Republic of China.
1 Supported by the Monsanto Company and Food for the 21st Century.
Received: 20 March 2006.
First decision: 4 April 2006.
Accepted: 17 July 2006.
REFERENCES
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