High potassium concentration and the cumulus corona oocyte complex stimulate the fertilizing capacity of human spermatozoa *

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1 FERTILITY AND STERILITY Copyright'" 1990 The American Fertility Society Printed on acid-free paper in U.S.A. High potassium concentration and the cumulus corona oocyte complex stimulate the fertilizing capacity of human spermatozoa * Luis S. Roblero, D.Sc.t Alejandro Guadarrama, M.T. Maria E. Ortiz, M.S. Emilio Fernandez, M.D. Fernando Zegers-Hochschild, M.D. Departmento de Obstetricia y Ginecologia, Clinica Las Condes, Instituto Chilena de Medicina Reproductiva, Santiago, Chile Progressively motile spermatozoa were incubated for 24 hours in culture media containing 4.7 or 25 mm K, in the presence or absence of hamster cumulus oophorus. The percentage of spermatozoa with progressive motility was significantly higher at 24 hours in the presence of cumulus corona oocyte complexes, irrespective of K concentration. A significant decrease in sperm mortality was observed with the association of 25 mm K and cumulus cells. A higher percentage of acrosome reaction was observed in spermatozoa incubated in 25 mm K when compared with 4.7 mm K, irrespective of time and the presence or absence of cumulus. The percentage of penetrated oocytes at 2 and 5 hours of incubation was higher when sperm had been incubated in 25 mm K than in 4.7 mm K. The presence of cumulus in the culture medium induced an additional significant increase in the percentage of penetrated oocyte. Although at 24 hours of incubation the percentage of acrosome reaction was higher than at 2 and 5 hours, the percentage of penetrated oocytes did not increase proportionally. Fertil Steril54:328, 1990 The acrosome reaction is an essential process experienced by spermatozoa in the acquisition of its fertilizing capacity. This event takes place in the oviduct at a time when the oocyte is surrounded by the zona pellucida and the cumulus oophorus. The factors responsible for the initiation of the acrosome reaction and the site in the genital tract where this event takes place are still matters of controversy. Several studies have shown that the zona pellucida and/or cumulus oophorus can stimulate the acrosome reaction in human spermatozoa cultured in vitro. 1-5 Siiteri et al. 5 were able to detect Received June 29, 1989; revised and accepted April 4, * Supported by the Special Programme of Research and Research Training in Human Reproduction of the World Health Organization, Task Force on the Prevention and Management of Fertility, Geneva, Switzerland. t Reprint requests: Luis S. Roblero. D.Sc., Departamento de Obstetricia y Ginecologia, Clinica Las Condes, Lo Fontecilla 441, Santiago, Chile. 328 Roblero et ai. K + and cumulus effect on human sperm and partially characterize a preovulatory human cumulus oophorus and mural granulosa cell-associated activity capable of initiating the in vitro acrosome reaction in human spermatozoa. On the other hand, we have previously shown that high K concentration, similar to that found in the oviductal fluid,6 significantly increases the acrosome reaction in vitro. 7 Therefore, it is reasonable to believe that in the oviductal microenvironment, where the acrosome reaction takes place, there are at least three constituents that can initiate and stimulate the sperm modifications required for fertilization: zona pellucida, cumulus oophorus, and high K concentration in the oviductal fluid. In this study, we investigated the influence of high K concentration in the presence or absence of the cumulus corona oocyte complexes on the motility, viability, acrosome reaction, and fertilizing capacity of human spermatozoin vitro. The results demonstrate that the interaction between high K

2 concentration and cumulus corona oocyte complexes can increase the percentage of acrosome reaction spermatozoa to significantly higher levels than that induced by K or cumulus corona oocyte complexes alone. It also stimulates the fusion of sperm and oocyte membranes. MATERIALS AND METHODS These experiments were designed to evaluate the in vitro effect of cumulus corona oocyte complexes on the viability, motility, acrosome reaction, and fertilizing capacity, using human tubal fluid culture mediums prepared with 25 mm K, the mean concentration at which K ion is found in the mouse oviductal fluid in the preimplantation period. 9 In each experiment, human tubal fluid medium was prepared with two different K concentrations: 4.7 mm K (control) and 25 mm K. The osmolarity of the resulting media was kept at 275 to 280 mosm/kg, as determined by a vapor pressure osmometer (Wescor Inc., Logan, VT). The media were sterilized by passage through a 0.2 JLm filter membrane and gassed for approximately 12 hours in 5% CO 2 in air and 95% humidity in a Forma scientific incubator (Forma Scientific, Inc., Marietta, OH) at 37 C before each experiment. Preparation of Spermatozoa Semen samples were obtained after at least 3 days of sexual abstinence from two healthy donors of proven fertility. Ten experimental series were run in parallel with semen samples provided by both donors. After liquefaction, progressively motile spermatozoa were obtained in human tubal fluid culture medium prepared with 4.7 mm K and 25 mm K by the swim-up method.lo After swim-up, culture media containing motile spermatozoa were adjusted to concentrations of 5 X 10 6 sperm/ml in an organ culture dish (Falcon Plastics, Oxnard, CA). Preparation of Hamster Cumulus Corona Oocyte Complexes Female hamsters, 8 weeks old, were superovulated by intraperitoneal (IP) injections of 25 IV of pregnant mare serum gonadotropin (Sigma, St. Louis, MO) followed 48 hours later by an IP injection of 25 IV of human chorionic gonadotropin (Sigma). Approximately 15 hours later, the hamsters were killed by cervical dislocation, and the cu- mulus corona oocyte complexes were recovered by flushing the oviducts with a phosphate-buffered saline (PBS) solutiony Zona-free Hamster Oocyte Preparation Harvested cumulus corona oocyte complexes were exposed to hyaluronidase (Sigma), 500 V/mL of PBS, until the cumulus cells were dispersed. Oocytes were collected and washed three times in PBS before exposure to trypsin (Sigma), 300 V /ml, until the zona pellucida dissolved. The denuded ova were immediately removed and washed three times. Aliquots containing 5,000,000 motile spermatozoa were incubated in human tubal fluid culture medium, supplemented with 10% heat inactivated fetal cord serum, under four different conditions: (1) culture medium prepared with 4.7 mm K alone (control condition), (2) culture medium prepared with 4.7 mm K and with 50 cumulus corona oocyte complexes, (3) culture medium prepared with 25 mm K alone, and (4) culture medium prepared with 25 mm K and 50 cumulus corona oocyte complexes. Aliquots of 100 JLL from each condition were obtained at 2, 5, 10, and 24 hours of incubation for the assessment of viability, progressive motility, and acrosome reaction as previously described. 7 Additional aliquots of 100 JLL were obtained at 2, 5, and 24 hours of incubation for reevaluation of acrosome reaction and sperm penetration into the zona-free hamster egg. Incubations were carried out in plastic Petri dishes (Falcon 3037) at 37 C and 5% CO 2 with 90% humidity. Each experiment was repeated 10 times. For the sperm penetration assay, 25 to 40 oocytes were placed under mineral oil with each sperm aliquot and then incubated for three hours. After this time, oocytes were rinsed three times in fresh culture medium and fixed with 5% formalin in PBS. One week later, oocytes were prepared for microscopical examination at high magnification. The criteria used to certify sperm penetration was the presence of a swollen head and sperm tail in the cytoplasm. Statistical Analysis The effects of K concentration, cumulus oophorus, and incubation time on motility, viability, acrosome reaction, and fertilizing capacity were assessed by three-way analysis of variance (ANOVA). Multiple comparisons were made by the least-significant-difference method 12 only if the overall Roblero et al. K + and cumulus effect on human sperm 329

3 100 ::J 6 :::; 80 rn III II: 8 60 g: E i 40 I 20 rn t5 i! mm K + 50 ceo 0 25mMK+50CCO il.s.e TIME OF INCUBATION (Hours) Figure 1 Effect of 25 mm K and hamster cumulus corona oocyte complexes on the motility of human spermatozoa incubated in modified human tubal fluid medium. Each point represents mean ± SE of 10 replicates. ANOVA detected significant influences. The analyses were done using the SAS System version 6.03 (SAS Institute Inc., Cary, NC). RESULTS Complexes on the Motility of Human Spermatozoa An influence of time of incubation (Fig. 1) and a significant interaction between the main factors of incubation time and cumulus corona oocyte complexes were the only significant effects detected by the ANOV A. The percentage of spermatozoa with progressive motility in a long-term culture was significantly higher in the presence of 50 cumulus corona oocyte complexes, irrespective of K concentration. This effect was manifested only after 10 hours of incubation. In the presence of cumulus corona oocyte complexes, nearly 60% of spermatozoa maintained rapid forward displacement, compared with 47% in the absence of cumulus corona oocyte complexes (P < 0.01). Complexes on the Viability of Human Spermatozoa Figure 2 shows the relationship between the percentage of dead spermatozoa (eosin-stained) under the four culture conditions. There were significant influences of time of incubation, K concentration, and cumulus corona oocyte complexes, but the in- cubation time interacted significantly with K concentration and cumulus corona oocyte complexes. When the analysis was repeated excluding results obtained at 24 hours, these interactions disappeared and remained significative of the influence of time of incubation. This means that K concentration and cumulus corona oocyte complexes influenced viability only at 24 hours of incubation (P = 0.005). Multiple comparisons made after the overall ANOV A (i.e., including all the data) for the interactions between time and K and between time and cumulus corona oocyte complexes showed that the percentage of dead spermatozoa was lower after 24 hours of incubation in 25 mm K (9.2%) than in 4.7 mm K (11.3%). After 24 hours of incubation, mortality of spermatozoa was also lower in cultures with cumulus corona oocyte complexes (8.6%) than in cultures without cumulus corona oocyte complexes (11.9%). Complexes on the Acrosome Reaction of Human Spermatozoa There were significant influences of time ofincubation, K concentration, and cumulus corona oocyte complexes, with a slight interaction between time of incubation and K concentration (Fig. 3). The percentage of acrosome reaction increased with time of incubation, and it was larger in the presence than in the absence of cumulus corona oocyte complexes (P < 0.002). Similarly, the percentage of acrosome reaction was larger at 25 mm K <10 8 II: w 3i o 6 u. o 4,. 2 _ o o 4,7 RIM K 25mMK 4,7 mm K 50 ceo 25mMK.50ceo j("s,e. O+O-----r TIME OF INCUBATION (Hours) Figure 2 Effect of 25 mm K and hamster.cumulus corona oocyte complexes on the viability of human spermatozoa incubated in modified human tubal fluid medium. Each point represents mean ± SE of 10 replicates.... (1 330 Roblero et al. K + and cumulus effect on human sperm

4 40 30 i 20 #. 10 o 4.7 mil K 50 ceo o. 215m11K.50ceo XS.E. O-----r------r r TIME OF INCUBATION (HounI) Figure 3 Effect of 25 mm K and hamster cumulus corona oocyte complexes on the acrosome reaction of human spermatozoa incubated in modified human tubal fluid medium. Each point represents mean ± SE of 10 replicates. than at 4.7 mm K (P < ). Multiple comparisons revealed that K concentration influenced acrosome reaction at all incubation times, so that the slight interaction found between these main factors is accounted for by small differences in the magnitude of the effect ofk concentration on acrosome reaction at different times. Complexes on the Zona-free Hamster Oocyte Penetration The interaction between time of incubation, K concentration, and cumulus corona oocyte complexes was highly significant (P = 0.008) (Fig. 4). The presence of cumulus corona oocyte complexes significantly increased the percentage of penetrated oocytes at 2 and 5 hours of incubation with 4.7 mm K (P = ). However, with 25 mm K, the increment produced by the presence of cumulus corona oocyte complexes was significant only at 5 hours of incubation (P < 0.01). Spermatozoa incubated in the presence of 25 mm K alone, reached a high penetration rate after 2 (40%) and 5 (55%) hours of incubation. Paradoxically, although the acrosome reaction continued to increase up to 24 hours, the percentage of penetrated oocytes at this time fell significantly, irrespective of the culture conditions. This fall was even larger in sperm incubated in the presence of 25 mm K and cumulus corona oocyte complexes (P < 0.05). DISCUSSION The results of this study confirm our previous repore that showed a significant increase in the motility, viability, and acrosome reaction of human spermatozoa incubated in capacitating medium containing 25 mm K. In addition, K appears to have a stimulatory effect on the in vitro fertilizing capacity of human spermatozoa, as suggested by the significant increase in the percentage of penetrated zona-free hamster oocytes. The addition of 50 hamster cumulus corona oocyte complexes to the culture medium prepared with 25 mm K further increased the effect on sperm motility, viability, and acrosome reaction. The beneficial effect of hamster cumulus oophorus on the fertilizing capacity was previously reported by Gwatkin et ai., l and recently Siiteri et ai. 5 detected and characterized a factor capable of initiating human sperm acrosome reaction in vitro. This factor appears to be secreted by the cells of mural granulosa and/or by the cumulus oophorus. On the other hand, it had been shown that the K ion has an important role on cell-secreting processesp Both events support the beneficial interaction between cumulus oophorus and high K concentration on sperm physiological and structural changes leading to fertilization. The interaction between cumulus corona oocyte complexes and 25 mm K was also beneficial on sperm penetration into the zona-free hamster oocyte. This observation is in agreement with the fact NC we NC we mmk U:TION 2 HAS NC we NC we NC we NC we ' S...-os Figure 4 Effect of 25 mm K and hamster cumulus corona oocyte complexes on the acrosome reaction and zona-free hamster oocyte penetration. Open and hatched bars represent percentage of acrosome-reacted sperm incubated in the absence (NC) or in the presence (WC) of cumulus corona oocyte complexes. Full bars represent the percentage of penetrated zonafree hamster oocytes. Each bar is the mean ± SE of 10 replicates. Roblero et al. K + and cumulus effect on human sperm 331

5 that acrosome reaction is a requirement for fertilization. The highest percentage of penetrated 00- cytes was observed with spermatozoa incubated in medium with 25 mm K and 50 cumulus corona oocyte complexes. In spite of the fact that at 24 hours of incubation the rate of acrosome reaction was the highest, the percentage of penetrated oocytes fell significantly. This finding suggests that apart from acrosome reaction there are other factors responsible for membrane fusion between sperm and 00- cytes. Since sperm motility and viability were maintained at acceptable rates at 24 hours of in vitro culture, it is probable that a negative effect on the fertilizing capacity of human spermatozoa could occur after a long-term exposure to in vitro conditions. REFERENCES 1. Gwatkin RBL, Andersen OF, Hutchison CF: Capacitation of hamster sperm in vitro: the role of cumulus components. J Reprod Fertil 30:389, Bavister B: Evidence for a role of post-ovulatory cumulus components in supporting ability of hamster spermatozoa. J Androl 3:365, Tesarik J: Comparison of acrosome reaction inducing activities of human cumulus oophorus, follicular fluid and ionophore A23187 in human sperm populations of proven fertilizing ability in vitro. J Reprod Fertil 74:383, Tesarik J, Pilka L, Drahorad J, Cechova D, Deselsky: The role of cumulus cell-secreted proteins in the development of human sperm fertilizing ability: implication in IVF. Hum Reprod3:129, Siiteri JE, Dandekar P, Meizel S: Human sperm acrosome reaction-initiating activity associated with the human cumulus oophorus and mural granulosa cells. J Exp Zool 246: 71, Borland RM, Biggers JD, Lechene CP, Taymor ML: Elemental composition of fluid in the human fallopian tube. J Reprod Fertil 58:479, Roblero L, Guadarrama A, Ortiz ME, Fernimdez E, Zegers Hochschild F: High potassium concentration improves the rate of acrosome reaction in human spermatozoa. Fertil Steril49:676, Quinn P, Warnes GM, Kerin JF, Kirby C: Cultures factors affecting the success rate of in vitro fertilization and embryo transfer. Ann NY Acad Sci 442:195, Roblero L, Biggers JD, Lechene CP: Electron probe microanalysis of the elemental microenvironment of oviductal cleavage stages of the mouse. J Reprod Fertil46:431, Zegers-Hochschild F, Bustos E, Guadarrama A: Morphological evaluation of progressively motile spermatozoa: potential use in fertility evaluation. In Les Colloques de L'INSERM, Edited by A Spira, P Jouannet. Paris, INSERM, 1982, P Whittingham DG, Wales RG: Storage of two-cell mouse embryos in vitro. Aust J BioI Sci 22:1,065, Kleinbaum DG, Kupper LL, Muller KE: Multiple comparison procedure. In Applied Regression Analysis and Other Multivariable Methods, Edited by M Payne. Boston, Kent Publishing Co, 1988, p Mrsny RJ, Meizel S: Potassium ion influx and Na, K ATPase activity are required for the hamster sperm acrosome reaction. J Cell Bioi 91:77, Roblero et al. K + and cumulus effect on human sperm