Sperm Selection by a Climbing-over-a-Wall IVF Method Reduces the Incidence of Polyspermic Penetration of Porcine Oocytes

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1 Journal of Reproduction and Development, Vol. 46, No. 5, 2000 Original Sperm Selection by a Climbing-over-a-Wall IVF Method Reduces the Incidence of Polyspermic Penetration of Porcine Oocytes Hiroaki FUNAHASHI 1) and Takashi NAGAI 2) 1) Faculty of Agriculture, Okayama University, Okayama , 2) Tohoku National Agricultural Experiment Station, Morioka , Japan Abstract. Effect of sperm selection according to the degree of motility after insemination on in-vitro penetration was examined by using a new in-vitro fertilization system designated as a climbing-overa-wall (COW) IVF method. When the sperm penetration rate in the COW-IVF method was compared with a standard method at the same sperm concentration ( cells/ml), the rates (95.1 ± 1.9 and 98.2 ± 1.0%, respectively) were similar, but the incidence of monospermic penetration was higher in the COW-IVF (25.5 ± 4.5%) than the standard method (10.4 ± 2.5%). When sperm concentration was changed from to cells/ml in the COW-IVF method, sperm penetration rate was higher at a higher concentration, whereas monospermic penetration rate was increased at a lower concentration. The proportion of monospermic oocytes in matured oocytes was similar among sperm concentrations, to cells/ml, at fertilization in the COW method. These results demonstrate that the COW-IVF method, selection according to the degree of sperm motility after insemination, can increase the normal penetration of frozen-thawed boar spermatozoa into IVM oocytes without any reduction in the sperm penetration rate. Key words: Caffeine, In vitro fertilization, Polyspermy, Porcine oocytes. (J. Reprod. Dev. 46: , 2000) ince a high sperm penetration rate was achieved in a workable porcine IVF system [1], an abnormally high incidence of polyspermic penetration has been repeatedly observed, to be a persistent problem in almost all of porcine IVF systems (see recent reviews [2 5]). In these systems, after washing by centrifugation a relatively high number of spermatozoa have been introduced into the medium containing caffeine and oocytes. In the current IVF system, simultaneous sperm penetration appears to be one of the main reasons for the increased incidence of polyspermy [6]. In physiological conditions, spermatozoa are capacitated during transport Accepted for publication: June 28, 2000 Correspondence: H. Funahashi through the female reproductive tract, and only a limited number of capacitated and/or partially acrosome-reacted spermatozoa reach the surface of oocytes, zona pellucida, and initiate the penetration [7 9]. Therefore, polyspermic penetration may decrease without a reduction in the proportion of sperm penetration if the number of spermatozoa that contact the oocytes is limited after the induction of capacitation and/or partial acrosomereaction. In the present study, effect of selection of spermatozoa according to the degree of motility after insemination on monospermic fertilization was examined in the presence of caffeine by using a new in-vitro fertilization system, designated as a climbing-over-a-wall (COW) IVF method. Here we show that the COW-IVF method can reduce the

2 320 FUNAHASHI and NAGAI incidence of polyspermic penetration of frozenthawed boar spermatozoa without reducing the penetration rate. Materials and Methods Culture media The medium used for the collection of oocytecumulus complexes and washing was modified TL- HEPES-PVA medium composed of 114 mm NaCl, 3.2 mm KCl, 2 mm NaHCO 3, 0.34 mm KH 2PO 4, 10 mm Na-lactate, 0.5 mm MgCl 2 6H 2O, 2 mm CaCl 2 2H 2O, 10 mm HEPES, 0.2 mm Na-pyruvate, 12 mm sorbitol, 0.1% (w/v) polyvinylalcohol, 25 µg/ml gentamicin and 65 µg/ml potassium penicillin G. The basic maturation medium (OMM37) used was BSA-free North Carolina State University 37 medium [10] supplemented with 0.6 mm cysteine, 5 µg/ml insulin and 10% (v/v) porcine follicular fluid [11]. The basic medium used for in vitro fertilization was modified BO solution (mbo; [12]), composed of mm NaCl, 4.02 mm KCl, 37.0 mm NaHCO 3, 0.83 mm NaH 2PO 4 2H 2O, 1.25 mm Na-pyruvate, 0.50 mm MgCl 2 6H 2O, 7.50 mm CaCl 2 2H 2O, 50 µg/ml gentamicin, 6 mg/ml BSA (Sigma, A6003), 1 mg/ ml casein phospho peptide (Meiji Seika Inc., Tokyo) and 1.0 mm caffeine [13, 14]. All media without modified TL-HEPES-PVA were equilibrated at 38.5 C in an atmosphere of 5% CO 2 in air overnight prior to incubation with oocytes. Porcine follicular fluid was prepared from antral follicles (3 to 6 mm in diameter) as described previously [15]. Preparation and culture of cumulus-oocyte complexes Ovaries were collected from prepubertal gilts at a local abattoir. Transportation of ovaries to the laboratory was carried out at C. Cumulusoocyte complexes were aspirated through an 18- gauge needle into a disposable 10-ml syringe from antral follicles (3 to 6 mm in diameter) on the surface of ovaries, washed three times with modified TL-HEPES-PVA medium, and then collected in 3 ml of fresh modified TL-HEPES-PVA medium. Fifty cumulus-oocyte complexes with uniform ooplasm and a compact cumulus cell mass were washed three times with OMM37 supplemented with 1mM dibutyryl camp, 10 iu/ ml ecg and 10 iu/ml hcg, and subsequently cultured in 500 µl of the same medium covered with paraffin oil for 20 h at 38.5 C in an atmosphere of 5% CO 2 in air [11]. The complexes were then transferred to 500 µl of OMM37 (without dibutyryl camp, ecg and hcg) after washing three times with the same medium. The complexes were cultured for an additional 24 h [11, 15, 16]. After culture, oocytes were stripped of cumulus cells by pipetting with 0.1% (w/v) hyaluronidase and washed carefully three times with mbo. Preparation of frozen-thawed boar spermatozoa According to Prof. T. Niwa [17], frozen boar semen packed in 0.25-ml straws was produced from sperm-rich fractions of the ejaculated semen that were collected from three boars. After thawing the semen at 38.5 C, spermatozoa were resuspended in 10 ml of Dulbecco's PBS supplemented with 0.1% BSA (Sigma A8022) and then washed three times with the same medium by centrifugation at 1000 g for 4 min each time. The pellets containing spermatozoa were resuspended at cells/ml in mbo medium and used for in vitro fertilization. In vitro fertilization and assessment of sperm penetration With COW-IVF method, thirty denuded oocytes were placed in the inner plate of the COW chamber (the cap of round bottom tube, Falcon #2058) containing 495 µl of mbo (Fig. 1). Five microliters of diluted sperm suspension was added to a part of the outer ditch of the chamber (Fig. 1) for final sperm concentrations of , , , cells/ml. As controls (Standard IVF Method), fifty microliters of sperm suspension diluted to cells/ml in mbo was added to a 50-µl droplet of the same medium containing 30 denuded oocytes for a final sperm concentration of cells/ml. Oocytes were co-cultured with spermatozoa for 9 h Fig. 1. Perspective drawing and cross-sectional view of the COW-IVF method. The chamber of the method was the cap of round bottom tube (Falcon #2058).

3 A NEW IVF METHOD FOR PORCINE OOCYTES 321 at 38.5 C in an atmosphere of 5% CO 2 in air and fixed for observation. At 9 h after insemination, the cultured eggs were fixed, stained with 1% (w/v) orcein, and examined at 200 and 400 magnification. Oocytes were designated as penetrated when they had at least one sperm head, a decondensed sperm nucleus, or a male pronucleus and corresponding sperm tail in the vitellus. Only oocytes containing male and female pronuclei with intact nuclear membrane were considered to have formed pronuclei. at the same sperm concentration ( cells/ml) for both the standard and COW-IVF methods (Table 1). At the same sperm concentration, however, the incidence of monospermic penetration was higher in the COW method (25.5 ± 4.5%) than controls (10.4 ± 2.5%). With the COW-IVF method, spermatozoa that were inseminated did not attach to the surface of the chamber. When sperm concentration was Statistical analysis Statistical analyses of data from four replicate trials were made by analysis of variance (ANOVA) and Fisher's protected least significant difference test using the STATVIEW (Abacus Concepts, Inc., Berkeley, CA, USA) program to determine treatment differences. All percentage data were subjected to arc sine transformation before statistical analysis. Data were expressed as means ± SEM. Probability of p<0.05 was considered to be statistically significant. Results When 5 µl of medium was collected from the bottom of the inner plate at 1 h after insemination, sperm concentration in the medium was cells/ml (data from 2 replications) and had not increased 3 and 5 h after insemination ( cells/ml). The penetration rate in IVM oocytes was similar Fig. 2. The incidence of monospermy in matured oocytes 9 h after insemination with the COW-IVF method. Oocytes were co-cultured with frozen-thawed boar spermatozoa at various concentrations in mbo containing 1 mm caffeine. Data obtained by coculturing oocytes with spermatozoa at a concentration of cells/ml in the standard method are also shown as a control. All data are presented as mean % ± SEM of monospermic penetration of matured oocytes. Different letters above the bars denote statistically significant differences (P<0.05). Table 1. Sperm penetration in different fertilization systems in which modified BO medium containing 1 mm caffeine was used as a fertilization medium In-vitro Sperm No. of % (mean ± SEM) 2 of oocytes No. (mean ± SEM) 1 fertilization concentration oocytes of sperm in systems 1 ( 10 5 cells/ml) examined Matured Penetrated 3 Monospermy 4 PN-formed 4,5 penetrated eggs Standard ± 0.9 a 98.2 ± 1.0 a 10.4 ± 2.5 a 99.0 ± ± 0.31 a COW ± 3.0 b 33.7 ± 12.1 b 81.7 ± 7.3 b 92.6 ± ± 0.09 b COW ± 1.1 ab 61.9 ± 4.9 c 49.5 ± 6.7 c 97.2 ± ± 0.14 c COW ± 6.3 a 95.1 ± 1.9 a 25.5 ± 4.5 d 97.1 ± ± 0.25 d COW ± 0.0 a 100 ± 0.0 a 2.6 ± 1.6 a 96.5 ± ± 0.15 e 1 COW: Climbing-Over-a-Wall IVF method. 2 Data from 4 replicated experiments. 3 Percentage of oocytes matured. 4 Percentage of oocytes penetrated. 5 Oocytes with both male and female pronuclei formed. a-e Values with different superscripts within each column are significantly different (P<0.05).

4 322 FUNAHASHI and NAGAI changed from to cells/ml, the rate of sperm penetration and the number of spermatozoa in a penetrated oocyte were higher at a higher concentration. In contrast, monospermic penetration rate was increased at a lower concentration (Table 1). The proportion of monospermic oocytes in matured oocytes did not differ at to cells/ml in the COW-IVF method, and the proportions at all the concentrations were higher than controls (standard method at cells/ml, Fig. 2). Discussion Although polyspermic porcine zygotes have been shown to develop to the blastocyst and later stages [18, 19], polyploid embryos fail to develop to full term. Therefore, polyspermic penetration is still one of the main causes of reduced efficiency in producing piglets from zygotes produced in vitro. In the present study we demonstrated that the incidence of monospermic penetration was lower in the COW-IVF method than standard IVF method without any reduction in the sperm penetration rate. It was reported [20] that when boar spermatozoa with a high motility were selected by Percoll gradient centrifugation before insemination in vitro, both sperm penetration and polyspermy were significantly increased as compared with nonselected spermatozoa at the same concentration. The swim-up method has also been used to select viable spermatozoa. In bovine spermatozoa, sperm penetration has been reported to be higher with swim-up than after treatment with Percoll gradients, whereas the incidence of polyspermic penetration was not different [21]. In our study with the COW-IVF method, it was also expected that, of spermatozoa inseminated into a part of the outer ditch of the chamber, only spermatozoa with a high motility after insemination could gradually traverse the wall, to enter the inner plate containing oocytes, and penetrate the oocytes resulting in a high rate of monospermic fertilization. In contrast in the standard IVF Method, capacitated and/or partially acrosome-reacted spermatozoa were expected to reach the surface of oocytes, regardless of the motility. The current results suggest that spermatozoa with even a low motility contribute to the penetration into oocytes, and that rejection of these cells in the COW-IVF method can reduce the polyspermic penetration rate without any reduction in the incidence of sperm penetration. One possibility to explain this phenomenon may be that capacitated and/or partially acrosome-reacted cells even with a low motility can penetrate into IVM oocytes in vitro. Alternatively, the presence of decorative spermatozoa that can not traverse the wall in the COW-IVF method may increase the population of penetrable cells in the current IVF conditions. In the physiological conditions of pigs, the majority of spermatozoa ejaculated do not pass through the uterotubal junction (UTJ) [22]. The limited number of spermatozoa was permitted to reach the oviduct, and capacitation of the spermatozoa was induced during the transportation from the UTJ to the ampulla region of the oviduct [23, 24]. Then acrosome reaction occurs during passage through the cumuli [25] or the surface of the zona pellucida [26, 27]. Limitations in the number of spermatozoa that can initiate penetration may be achieved in vivo not only at the UTJ but also during capacitation and/or acrosome reaction. A similar limitation procedure, when only capacitated spermatozoa can advance to the place of fertilization and can meet oocytes, may be required in the IVF system to overcome the persistent problem, polyspermic penetration in vitro. In many IVF systems, an increased number of spermatozoa per oocyte is parallel with a higher incidence of polyspermic penetration [28, 29]. In the present study we could reduce the incidence of polyspermic penetration by using the COW-IVF method, but we observed higher rates of sperm penetration and a larger number of spermatozoa in a penetrated oocyte at a higher sperm concentration. In contrast a higher monospermic penetration rate was obtained at a lower concentration. Therefore, spermatozoa still penetrate with a similar manner as controls even in the COW-method. Caffeine has been used to induce sperm capacitation in a majority of current porcine IVF systems [1, 30], especially with frozenthawed boar spermatozoa [31 34]. In the COW-IVF method, we also supplemented fertilization medium with caffeine. Recently, CTC fluorescence assay has demonstrated that caffeine induces both capacitation and acrosome reaction of boar spermatozoa [35]. The sticky matrix, proacrosine, has been considered to be a secondary ligand molecule that binds acrosome-reacted spermatozoa

5 A NEW IVF METHOD FOR PORCINE OOCYTES 323 tightly to the zona pellucida [36]. Not only capacitated cells but also just or partially acrosomereacted ones may be able to bind tightly to the zona, resulting in successful penetration. For further improvement of a low incidence of monospermic penetration in porcine IVF systems, consideration of other chemicals to induce capacitation may be required. In conclusion, our present study demonstrates that polyspermy was decreased in the COW-IVF method by using frozen-thawed boar spermatozoa selected according to the degree of sperm motility after insemination without any reduction in the incidence of sperm penetration. These evidences indicate that even capacitated and/or partially acrosome-reacted boar spermatozoa with a low motility contribute to the penetration, and the presence of those spermatozoa around oocytes may be one of factors to increase the incidence of polyspermic penetration in vitro. Acknowledgments This work was supported in part by a visiting grant to HF from the Japanese Agriculture, Forestry and Fisheries Research Council, a Grant-in-Aid for Scientific Research (B ) to HF from the Ministry of Education, Science, Sports and Culture of Japan, and a Grant-in-Aid (Bio Design Program) to TN from the Ministry of Agriculture, Forestry and Fisheries (BDP-00-III-1). We thank Prof. B. N. Day and Ms. B. Nichols of University of Missouri- Columbia for critical reading and editorial assistance, Ms. N. Nishimoto for technical assistance, and Prof. H. Masuda of the Iwate University for preparation of frozen boar semen. References 1. Cheng WTK, Polge C, Moor RM. In vitro fertilization of pig and sheep oocytes. Theriogenology 1986; 25: 146 (abstr.). 2. Niwa K. Effectiveness of in vitro maturation and in vitro fertilization techniques in the pig. J Reprod Fertil Suppl 1993; 48: Day BN, Funahashi H. In vitro maturation and fertilization of pig oocytes. In: Day BN, Funahashi H (eds.), Beltsville Symposia in Agricultural Research XX. Biotechnology's role in the genetic improvement of farm animals. Savoy, IL, USA. American Society of Animal Science; 1996: Nagai T. In vitro maturation and fertilization of pig oocytes. Anim Reprod Sci 1996; 42: Funahashi H, Day BN. Advances in in-vitro production of porcine embryos. J Reprod Fertil Suppl 1997; 52: Wang WH, Machaty Z, Abeydeera LR, Prather RS, Day BN. Time course of cortical and zona reaction of pig oocytes upon intracellular calcium increase induced by thimerosal. Zygote 1999; 7: Hunter RHF. Interrelationships between spermatozoa, the female reproductive tract, and the egg investments. In: Hunter RHF (ed.), Control of Pig Reproduction. London. Butterworths; 1982: Hunter RHF. Oviduct function in pigs, with particular reference to the pathological condition of polyspermy. Mol Reprod Dev 1991; 29: Mburu JN, Einarsson S, Lundeheim N, Rodriguez- Martinez H. Distribution, number and membrane integrity of spermatozoa in the pig oviduct in relation to spontaneous ovulation. Anim Reprod Sci 1996; 45: Petters RM, Wells KD. Culture of pig embryos. J Reprod Fertil Suppl 1993; 48: Funahashi H, Cantley TC, Day BN. Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic AMP improves developmental competence following in vitro fertilization. Biol Reprod 1997; 57: Brackett BG, Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biol Reprod 1975; 12: Nagai T, Takenaka A, Mori T, Hirayama M. Effects of caffeine and casein phosphopeptides on fertilization in vitro of pig oocytes. Mol Reprod Dev 1994; 37: Mori T, Hirayama M, Suzuki K, Shimizu H, Nagai T. Effect of casein phospho peptides and Ca 2+ on penetration of boar spermatozoa into pig oocytes matured in vitro. Biol Reprod 1996; 55: Funahashi H, Cantley TC, Day BN. Different hormonal requirement of porcine oocyte-complexes during maturation in vitro. J Reprod Fertil 1994; 101: Funahashi H, Day BN. Effects of the duration of exposure to supplemental hormones on cytoplasmic maturation of pig oocytes in vitro. J Reprod Fertil 1993; 98: Niwa T. Artificial Insemination (in Japanese). In: Niwa T (ed.), Handbook for Swine Production. Tokyo. Yokendo Ltd.; 1994:

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