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Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes¹

Department of Biomedical Sciences, Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins 80523

	Abstract

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Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 µM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm.

Key Words: Bovine Serum Albumin • Capacitation • Follicular Fluid • Oocytes • Spermatozoa • Stallion

	Introduction

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There have been few advances in equine in vitro fertilization (IVF) since the first reports of sporadic success (Bezard et al., 1989; Palmer et al., 1991; Hinrichs et al., 2002). One reason is the difficulty of capacitating stallion sperm and maturing equine oocytes (Blue et al., 1989; Zhang et al., 1990). The zona-free hamster oocyte penetration test (Brackett et al., 1982; Landim-Alvarenga et al., 2001), homologous equine sperm-hemizona pellucida binding assay (Fazeli et al., 1993), and zona-free equine oocytes (Hochi et al., 1996; Landim-Alvarenga et al., 2001) have been used to assess equine sperm fertilizing capacity. However, these tests are expensive.

Follicular fluid has been used during in vitro maturation (IVM) to improve oocyte maturation, as estimated by IVF and embryonic development, in pigs (Naito et al., 1988; 1989; Vatzias and Hagen, 1999), sheep (Sun et al., 1994), and cattle (Kim et al., 1996; Romero-Arredondo and Seidel 1996). Follicular fluid increased male pronucleus formation or cleavage after IVF (Hinrichs et al., 2002) or intracytoplasmic sperm injection (Dell’Aquila et al., 1997; Schmid et al., 1998) of equine oocytes. Romero-Arredondo and Seidel (1994) reported that cumulus expansion and meiotic maturation were enhanced relative to controls when bovine oocytes were cultured with bovine follicular fluid obtained from follicles 8 or 20 h after the LH surge.

Bovine serum albumin is widely used to supplement capacitation and fertilization media. In part because BSA is contaminated by other serum components (Maurer, 1992; Bavister, 1995), polyvinylalcohol (PVA) has been used as a substitute for BSA during IVF to examine mechanisms of capacitation or fertilization in pigs (Wang et al., 1995), cattle (Tajik et al., 1994; Keskintepe and Brackett, 1996), and mice (Choi and Toyoda, 1998).

The main objective of our studies was to compare effects of BSA and PVA during sperm capacitation and fertilization using various models of equine IVF.

	Materials and Methods

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Experiment 1. Comparison of BSA and PVA in Penetration of Zona-Free Bovine Oocyte by Stallion Sperm
In Vitro Maturation and Preparation of Zona-Free Bovine Oocytes.
Bovine oocytes were aspirated from follicles of slaughterhouse-derived ovaries. Oocytes with compact cumulus cells were cultured in tissue culture medium (TCM)-199 (Sigma Chemical Co., St. Louis, MO, catalog No. M-7528) with Earl’s salts and 25 mM HEPES supplemented with 10% fetal calf serum (FCS), 15 ng/mL of FSH (NIH-oFSH-20), 1 µg/mL of LH (USDA-bLH-B-6), 1 µg/mL of estradiol-17ß and 0.67 mM glutamine for 22 h at 39°C in 5% CO₂ in air.

After maturation, cumulus cells surrounding the oocytes were removed by gentle pipetting in modified Dulbecco’s PBS (Sigma, catalog No. D-6607) containing 300 U/mL of hyaluronidase (Sigma). Oocytes with polar bodies were selected. Zonae pellucidae were dissolved in acid Tyrode’s solution (pH 2.1) for 1 to 2 min, and zona-free oocytes were washed three times in TYH medium (119.37 mM NaCl, 4.78 mM KCl, 1.71 mM CaCl₂•2H₂O, 1.19 mM MgSO₄•7H₂0, 1.19 mM KH₂PO₄, 25.07 mM NaHCO₃, 5.56 mM glucose, 0.5 mM sodium pyruvate, 75 µg/mL potassium penicillin G, 50 µg/mL streptomycin sulfate, 2 µg/mL of phenol red; Toyoda et al., 1971) supplemented with PVA (1 mg/mL, cold water soluble, Sigma) or BSA (4 mg/mL, Fraction V, Amersham Biosciences, Cleveland, Ohio).

Preparation of Stallion Sperm and IVF.
Stallion semen was collected using an artificial vagina and washed twice by centrifugation (660 x g, 5 min) in either TYH with 0.1% PVA (TYH/PVA) or TYH with 0.4% BSA (TYH/BSA). Washed sperm were incubated at 5 x 10⁷ sperm/mL in 100 µL of TYH/PVA or TYH/BSA media containing 0.5 mM 8-bromoadenosine cyclic monophosphate (8BrcAMP; Sigma) for 3.5 h. Sperm in the 8BrcAMP + ionomycin (Sigma) treatments were then treated with 0.1 µM ionomycin for 15 min by adding 1 µL of a 7.47-µg/mL solution to the 8BrcAMP-containing aliquot.

Bovine zona-free oocytes (5 to 10) were placed in a 100-µL droplet of TYH/PVA or TYH/BSA. Stallion sperm from the corresponding macromolecule treatment were added to give 1 x 10⁶ sperm/mL, and the sperm and oocytes were incubated at 39°C for 18 to 20 h. Afterward, oocytes were fixed in ethanol:acetic acid (3:1, vol/vol), stained with 1% orcein in 45% acetic acid, and examined at 400x magnification. Oocytes containing enlarged sperm heads or a male pronucleus were considered penetrated.

Experiment 2. Viability and Acrosome Reaction of Stallion Sperm
The viability and acrosome reaction rates of live sperm collected from four stallions and cultured in media including either PVA or BSA were assessed by staining with fluorescent dyes. One ejaculate was collected from each of four stallions of known good fertility and was washed twice by centrifugation (660 x g, 5 min) in TYH/BSA or TYH/PVA media. Sperm were resuspended to 5 x 10⁷ sperm/mL in 100 µL of the corresponding medium and treated for capacitation with 8BrcAMP for 3.5 h, and then with ionomycin for 15 min at 39°C as described above. After this time, sperm were added to a 100-µL droplet of the corresponding BSA- or PVA-containing medium to give 1 x 10⁶ sperm/mL, and incubated at 39°C. Acrosome reaction rates in live stallion sperm before capacitation treatment and after 0.5, 4, or 16 h of incubation in the droplet were studied by incubation of sperm with fluorescein isothiocyanate-labeled Pisum sativum agglutinin (75 µg/mL) and propidium iodide (1.2 µg/mL) in either TYH/BSA or TYH/PVA for 5 min. Samples were examined with epifluorescence microscopy at 400 to 1,000x. Live, acrosome-intact sperm did not stain (no color); live, acrosome-reacted sperm showed patchy green fluorescence over the rostral portion of the head and equatorial segment; and dead sperm, regardless of acrosome reaction, showed red fluorescence over the head. This experiment was replicated three times; 200 sperm were counted at each time in each subclass.

Experiment 3. Effects of BSA and PVA on Penetration of Partially Zona-Removed Equine Oocytes
In Vitro Maturation and Manipulation of Equine Oocytes.
Equine oocytes were collected by the method described by Choi et al. (1993) with a slight modification. Briefly, ovaries were collected at a slaughterhouse over a 3- to 4-h period, sliced at 5-mm intervals, and then washed in PBS supplemented with 0.5% FCS. Pooled solution was sedimented for 10 to 20 min, and the sediment was searched for oocytes. Oocytes with compact cumulus cells were selected, put into 15-mL conical tubes filled with TCM-199 plus 10% FCS, and then transported to the laboratory within 4 h. They were matured at 39°C in 5% CO₂ in air in TCM-199 supplemented with 10% FCS, 15 ng/mL of FSH, 1 µg/mL of LH, 1 µg/mL of E₂, and 0.67 mM glutamine at 39°C in 5% CO₂ in air. After 28 to 30 h of IVM, cumulus cells were removed in HEPES-buffered TCM-199 containing hyaluronidase, and oocytes with a first polar body were used for manipulation. Approximately one-third of zona pellucida was removed with a fragment of a razor blade by micromanipulation in protein-free PBS supplemented with 0.3 M sucrose.

In Vitro Fertilization
. Based on the results of Experiment 1, stallion sperm were collected and washed as described above and capacitated with 0.5 mM 8BrcAMP for 3.5 h and 100 nM ionomycin for 15 min in either TYH/BSA or TYH/PVA media at 39°C. Partially zona-removed (PZR) equine oocytes (Choi et al., 1994) were placed into 98 µL of TYH/BSA or TYH/PVA media, and 2 µL of sperm suspension was added to give 1 x 10⁶ sperm/mL. After 18 to 20 h, oocytes were fixed and stained as described above.

Experiment 4. Effects of Follicular Fluid on Zona-Free Bovine Oocyte Penetration by Stallion Sperm
Preparation of Follicular Fluid
. Human chorionic gonadotropin (2,500 U, i.v.) was administered to an estrous mare when the dominant follicle exceeded 35 mm in diameter. After 27 h, follicular fluid was aspirated by transvaginal ultrasound-guided puncture of the follicle. The follicular fluid was immediately centrifuged (4,000 x g, 10 min) at 4°C. The supernatant was kept at -80°C until use.

In Vitro Maturation and Preparation of Zona-Free Bovine Oocytes.
Bovine oocytes were collected from slaughterhouse-derived oocytes as described above and were matured in TCM-199 supplemented with 0, 20, 50, or 100% follicular fluid and 1 IU/mL of eCG (Sigma, catalog No. G-4877) at 39°C in 5% CO₂ in air. After 22 h of IVM, the oocytes were denuded of cumulus cells and oocytes with a polar body were treated with acid Tyrode’s solution for removal of the zona pellucida.

In Vitro Fertilization
. Semen from three stallions was used. Sperm preparation and insemination were conducted as described in Experiment 3, using both BSA- and PVA-based media and capacitation with 8BrcAMP plus ionomycin. Oocyte fixation and evaluation were performed as described in Experiment 1.

Experiment 5. Effects of Follicular Fluid on IVF of Bovine Oocytes
To evaluate whether follicular fluid affects standard bovine IVM and IVF, we treated bovine oocytes with 0 to 100% follicular fluid as described in Experiment 4 during IVM. After 22 h of culture, cumulus cell expansion was estimated by scoring from 0 to 4: 0 = no expansion; 1 = slight expansion of peripheral cumulus mass; 2 = moderate expansion of outer layers of cumulus mass; 3 = outer layers of cumulus mass expanded except corona radiata; 4 = fully expanded cumulus mass. A proportion of oocytes were denuded, fixed, and stained to evaluate nuclear maturation as described above.

Preparation of Bovine Sperm
. Frozen bovine semen was thawed and washed (660 x g, 8 min) in Sp-TALP (Parrish et al., 1988) containing 5 U/mL heparin (Sigma) and 20% BSA. The sperm pellet was resuspended in fresh Sp-TALP containing heparin and 6 mg/mL BSA and centrifuged (660 x g, 5 min). Sperm were resuspended to 10⁷ sperm/mL and preincubated for capacitation at 39°C for 2 to 2.5 h in 5% CO₂ in air.

In Vitro Fertilization.
After 22 h of maturation, bovine cumulus–oocyte complexes were washed twice in Fert-TALP (Parrish et al., 1988) supplemented with 6 mg/mL BSA and transferred to 200 µL of the same medium. Bovine sperm (50 µL) from the preparation described above was added to Fert-TALP to give a final sperm concentration of 2 x 10⁶ sperm/mL. After 18 to 20 h of incubation, oocytes were fixed, stained, and evaluated as described in Experiment 1. Oocytes not exposed to sperm with a first polar body and metaphase II chromosomes were considered matured.

Experiment 6. Comparison of BSA and PVA for Bovine IVF
To determine whether BSA and PVA affect bovine IVF in a manner similar to equine IVF, we used zona-intact and zona-free bovine oocytes; sperm were capacitated and fertilized in either TYH/BSA or TYH/PVA instead of Fert-TALP. Bovine oocytes were matured as described in Exp. 1 using conventional media. For zona-free bovine oocytes, zona pellucidae were dissolved using acid Tyrode’s solution as described in Experiment 1.

Preparation of Bovine Sperm.
Bovine sperm were centrifuged through a Percoll gradient (45/90%) at 330 x g for 20 min. The sperm pellet was then washed in either TYH/BSA or TYH/PVA by centrifugation at 330 x g for 5 min. Sperm were incubated for 2 h at 5 x 10⁷ sperm/mL in each medium containing 5 U/mL heparin for capacitation.

In Vitro Fertilization
. For insemination, zona-intact bovine oocytes were placed into 96 or 98 µL of either TYH/BSA or TYH/PVA media, and then 2 or 4 µL of preincubated bovine sperm suspension in the corresponding medium was added to give a final sperm concentration of 1 or 2 x 10⁶ sperm/mL. For zona-free bovine oocytes, sperm were diluted 10 times, and 4 or 10 µL of preincubated sperm suspension was added to either TYH/BSA or TYH/PVA to give a final sperm concentration of 0.2 or 0.5 x 10⁶ sperm/mL. After 18 to 20 h insemination, oocytes were fixed, stained, and evaluated as described above.

Statistical Analysis
. Each experiment was done in three to nine replicates. Data from Exp. 1 and 3 were analyzed by single-df ² analysis using the Fisher-Yates correction. Experiment 2 was analyzed by repeated-measures ANOVA with sources of variation that included stallions, macromolecules, hours, replicates, and their interactions. Experiment 4 was analyzed by ANOVA as a complete factorial, with percentage of follicular fluid, macromolecules, and stallions as sources of variation. The highest order interaction was used as the error term. The cumulus expansion response in Exp. 5 was analyzed as a two-factor factorial (equivalent to a randomized complete block), with percentage of follicular fluid and replicates as factors. The interaction term was used as the error term. The other responses for Experiment 5 were analyzed by ². Experiment 6 was analyzed as two complete factorial analyses, one for zona-intact and one for zona-free, with sources of variation that included macromolecules, sperm concentration, and bulls, plus all interactions. Replicates within bulls constituted the error terms. For all ANOVA, the arc sin transformation was used for all percentage data. Untransformed least squares means are presented.

	Results

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Experiment 1. Comparison of BSA and PVA in Penetration of Zona-Free Bovine Oocyte by Stallion Sperm
The penetration rate of sperm treated with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (Table 1).

	Discussion

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The reason that PVA was superior to BSA as a macromolecule for capacitation and fertilization in stallion sperm is not clear; this macromolecule was inferior to BSA for bovine IVF of both zona-free and zona-intact oocytes. However, the capacitation method used for bovine sperm (heparin) and that used for stallion sperm (8BrcAMP and ionomycin) differ. It is possible that in the stallion sperm, BSA partially quenched the action of ionophore and reduced its efficiency in causing capacitation-like changes. In contrast, high BSA levels increase capacitation of bovine sperm, and other workers have also shown that BSA is superior to PVA for capacitation for bovine IVF (Parrish et al., 1989). Perhaps the explanation is simply that we observed much more agglutination of stallion sperm in BSA than PVA treatments, and this was much less pronounced with bull sperm.

Brackett et al. (1982) first evaluated the ability of in vitro-capacitated stallion sperm to penetrate zona-free hamster oocytes (83% penetration rate) by preincubation in Biggers, Whitten, Whittingham medium + 1% BSA for 18 to 24 h. Penetration rates of zona-free hamster oocytes by fresh (30 to 78%; Graham et al., 1987; Okolski et al., 1987; Landim-Alvarenga et al., 2001) and cooled stallion sperm (75 to 80%; Padilla et al., 1991) have also been reported after capacitation of sperm using long preincubation (4 to 10 h), and calcium ionophore, lysophosphatidyl-serine, dilauroylphosphatidyl-choline (PC12), or heparin. Landim-Alvarenga et al. (2001) reported that stallion sperm treated with either PC12 or calcium ionophore A23187 could penetrate zona-free bovine oocytes; a 26% penetration rate was obtained in that study. In the present study, we achieved a 47% penetration rate of zona-free bovine oocytes by sperm treated with PVA/8BrcAMP/ionomycin. This is the first study to evaluate penetration of zona-free bovine oocytes by stallion sperm in relation to the macromolecule used in capacitating medium. Direct injection of stallion sperm into bovine oocytes also has been reported (Choi et al., 2002; Li et al., 2003); up to 80% of bovine oocytes injected with stallion spermatozoa formed a male pronucleus. Therefore, bovine oocytes may be useful for evaluation of both cytoplasmic penetration and fertilizing (activation) capacity of stallion sperm in vitro.

In the present study, penetration rates of bovine zona-free oocytes by stallion sperm were improved when the percentage of preovulatory equine follicular fluid was increased in IVM media for bovine oocytes and reached a peak at 100% (45% penetration vs. 18% for 0% follicular fluid). In contrast, addition of equine follicular fluid during maturation of zona-intact bovine oocytes resulted in no benefit in penetration rates (55 to 68%) by bovine sperm, although cumulus expansion was significantly increased. Romero-Arredondo and Seidel (1996) observed positive effects of 20 to 40% preovulatory bovine follicular fluid during bovine oocyte maturation; cumulus expansion, cleavage rates, and rates of blastocyst formation of bovine oocytes improved. An earlier study (Romero-Arredondo et al., 1994) demonstrated that preovulatory follicular fluid speeded up the maturation process; that may have occurred in the current study, resulting in oocytes that could be penetrated sooner by sperm that rapidly were becoming less viable (Table 2). In the horse, 20% equine preovulatory follicular fluid added to in vitro maturation media increased male pronucleus formation rates after intracytoplasmic sperm injection (Dell’Aquila et al., 1997). Hinrichs et al. (2002) obtained significantly improved IVF rates when equine oocytes were matured in 100% preovulatory follicular fluid compared with control medium (10% serum) or 20% follicular fluid (13 to 22% vs. 0 to 12%).

Equine preovulatory follicular fluid is plentiful and contains substances implicated in oocyte maturation and fertilization competence. Gerard et al. (1998) found a high molecular weight preovulatory stage-related protein in equine preovulatory follicular fluid. Gerard and Monget (1998) also observed an increase in intrafollicular IGF-binding proteins after exogenous LH injection. The types of IGF-binding proteins present in the preovulatory follicle are assumed to affect the quality of the follicle-enclosed oocyte (Funston et al., 1996). In the present study, expansion of the cumulus complex was significantly greater for follicular fluid treatments than for the control, and penetration of oocytes by equine sperm was enhanced. Therefore, equine preovulatory follicular fluid (after hCG stimulation) may contain substances that support in vitro maturation of both bovine and equine oocytes.

In conclusion, PVA was superior to BSA for capacitation and fertilization by stallion sperm in the systems used in our study. This chemically defined medium would simplify investigation of detailed mechanisms of capacitation of stallion spermatozoa. Further, equine preovulatory follicular fluid improved the penetration rate of oocytes with stallion sperm when it was used as in vitro maturation medium. Bovine zona-free oocytes proved to be a useful assay for in vitro fertilizing ability of stallion sperm.

	Implications

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Most media for capacitation and fertilization of stallion spermatozoa contain serum albumin, and thus vary in composition from batch to batch. We conducted studies to examine whether bovine serum albumin could be replaced by polyvinylalcohol using several approaches. Surprisingly, polyvinylalcohol was superior to bovine serum albumin for capacitating stallion sperm in each system tested. An additional benefit is that polyvinylalcohol is chemically defined; however, this benefit did not occur in bovine in vitro fertilization using zona-intact or zona-free oocytes and bovine sperm. These findings will enable more meaningful studies of species differences on capacitation and fertilization and should lead to better understanding of equine sperm capacitation that has been difficult to induce in vitro.

	Footnotes

 
¹ We thank Y. Toyoda for his valuable suggestions, K. Hinrichs for critically reviewing the manuscript, J. K. Graham for his help with the acrosome reaction assay, E. Carnevale for collecting equine follicular fluid, Z. Brink for assistance with bovine ovaries, A. DeLille for statistical analysis, and graduate students at the Equine Reproduction Unit for collection of stallion semen. This experiment was supported by the Preservation of Equine Genetics Program and the Lucy Whittier Foundation. The gonadotropins were obtained through the National Hormone and Pituitary Program, the National Institute of Diabetes and Digestive and Kidney Diseases, and A. F. Parlow.

² Present address: Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station 77843-4466.

³ Present address: Department of Animal Reproduction and Veterinary Radiology, FMVZ-UNESP, Botucatu, SP 18618.000, Brazil.

⁴ Correspondence: ARBL Bldg., Foothills Campus (phone: 970-491-8409; fax: 970-491-3557; E-mail: esquires{at}colostate.edu).

Received for publication September 9, 2002. Accepted for publication March 28, 2003.

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