Effects of Microinjection of Glutathione on Male Pronucleus Formation in Porcine Oocytes Matured in Vitro

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1 Journal of Reproduction and Development, Vol. 38, No. 2, 1992 Effects of Microinjection of Glutathione on Male Pronucleus Formation in Porcine Oocytes Matured in Vitro Kunihiko NAITO and Yutaka TOYODA Department of Reproductive and Developmental Biology, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108, Japan Abstract. Porcine follicular oocytes matured in a modified Krebs-Ringer solution were microinjected with glutathione (GSH) and their male pronucleus (mpn) formation ability was examined after in vitro fertilization. While viability of the oocytes injected with 140 pl vehicle solution (91%) was comparable with non-injected control (93%), microinjections of 35 pl and 140 pl of GSH solution (240 mm) decreased oocyte viability dose dependently (76% and 47%, respectively), showing a toxicity of the highly concentrated GSH solution. Male PN formation rate was low in the control (23%) and was not affected by injection of vehicle solution (17%). In contrast, a microinjection of both 35 and 140 pl GSH solution increased the ability significantly (52% and 53%, respectively). These rates were, however, significantly lower than the rate (70%) of those matured in porcine follicular fluid, which have been suggested to be comparable with in vivo matured oocytes. These results suggest that the low mpn formation ability in the porcine oocytes matured in vitro might be attributed in part to their low intracellular GSH concentration. Key words: Glutathione, Microinjection, Male pronucleus, Porcine oocytes, In vitro fertilization. (J. Reprod. Dev. 38: , 1992) I n vitro matured oocytes have been reported to have lower male pronucleus (mpn) formation ability than in vivo matured oocytes in various mammalian species, although the cytoplasmic differences between these oocytes have not been fully explained [1]. The reduction of protamine disulfide bridges in the sperm head is a prerequisite for mpn formation, and glutathione (GSH), the major intracellular free thiol, has been suggested to play a_critical role for the protamine reduction [2-5]. The low and high GSH levels have been reported in immature and mature hamster oocytes, respectively, and the inhibition of GSH synthesis during oocyte maturation caused the decrease in mpn formation ability of the oocytes [3, 5]. The addition of cysteine, a precursor of Accepted for Publication: May 22, 1992 Correspondence to: K. Naito GSH, into the maturation medium of porcine oocytes elevates their mpn formation ability [6]. Therefore, it is conceivable that the GSH level might be related to mpn formation ability in in vitro matured mammalian oocytes and the elevation of their intracellular GSH level might improve the ability. In the present study, to investigate the hypothesis described above, GSH was microinjected into in vitro matured porcine oocytes which have been shown to have very low mpn formation ability [7, 8], and their mpn formation was examined after in vitro fertilization. Materials and Methods Porcine follicular oocytes were collected as described previously [7]. Briefly, gilt ovaries were

2 174 NAITO et al. obtained from a slaughterhouse and carried to the laboratory within 40 min kept in saline at 37-39C. Follicular oocytes were collected from mediumsized follicles (2-5 mm in diameter). Groups of 10 oocytes were matured in 0.1 ml modified Krebs- Ringer bicarbonate solution (mkrb) [9] containing 1.0 IU/ml PMSG (Peamex, Sankyo Co., Japan) ph 7.4, for 44 h at 37C under an atmosphere of 5% CO2 in air. Following the maturation culture, the oocytes were treated with 150 U/ml hyaluronidase (Sigma, Type IV-S) in mkrb for 1 to 3 minutes, then surrounding cumulus cells were removed by repeated pipetting with a fine-bore pipette. The denuded oocytes were transferred to mkrb buffered with 20 mm Hepes (ph 7.4) for microinjection. Microinjection was performed on an inverted microscope (Zeiss) equipped with a micromanipulator (Zeiss) and a microinjector (Eppendorf, 5242) which can control the volume of injecting solution. GSH (Sigma, reduced form, G4251) was dissolved in a vehicle solution (100 mm KC1, 0.5 mm MgCl2, 0.1 mm CaC12) at a concentration of 240 mm, and 35 pl or 140 pl of the GSH solution was microinjected near the center of the ooplasm. The amounts of injected GSH were 8.4 and 31.2 pmol and the concentrations in ooplasm were calculated to be about 10 mm and 40 mm, respectively, on the assumption that GSH was dispersed in cytoplasm homogenously. Some oocytes were not injected or injected with only the vehicle solution (140 pl) without GSH. The oocytes were freed from zona pellucida by the treatment of acidic Ringer's solution [10] for about 10 seconds, then transferred into mkrb containing 2 mm caffeine (Wako Pure Chemical Ind., Japan) at a concentration of about 30 oocytes par 0.2 ml. They were subjected to fertilization in vitro within 2 h after the start of microinjection. Oocytes matured in porcine follicular fluid (pff) containing 1.0 IU/ml PMSG under the same condition were also prepared as described previously [7]. Since such oocytes had a high mpn formation and a high developmental ability comparable to those of in vivo matured oocytes [7, 8, 11], they were not injected but used only for in vitro fertilization after cumulus and zona pellucida removal as a positive control of the mpn formation. Examining the influences of cumulus and zona pellucida removal on mpn formation, intact oocytes matured in mkrb and pff were also fertilized in vitro. Boar spermatozoa were collected by the handglove method from Landraces bred at The Tokyo Metropolitan Livestock Experiment Station. They were diluted with the same volume of Kiev solution [12] at 37C, put in a thermos flask and transported to our laboratory, in a chamber at 5C. The semen was stored at 15C until use. Two days after collection, their capacitation was induced by preincubation in mkrb at a concentration of 4 ~ 108/ml at 37C, 5% CO2 in air for 4 h as described previously [13]. The spermatozoa were diluted to 5 ~ 107/ml with mkrb containing 2 mm caffeine, then added to fertilization medium containing the oocytes 15 min after dilution: concentration of spermatozoa at insemination was 5 ~ 105/ml. Whole-mount preparations of oocytes were made as reported previously [7] about 20 h after insemination. The oocytes were examined using a Nomarski interference-contrast and phasecontrast microscope. Viability of the oocytes was evaluated by morphological characteristics according to Sato et al. [14]. The oocytes which had shrinked cytoplasm, lysis of cytoplasm or torn cytoplasmic membrane were considered as degenerated, and the other oocytes having clear outline and homogenous healthy cytoplasm were considered as viable. The oocytes containing sperm heads with associated sperm tails in the vitellus were considered to be penetrated, and 2 or more PN with second polar body or with detached sperm midpiece in the ooplasm was taken as the criterion of male PN development. The chi-squared test was used for statistical evaluation of the results, as necessary. Differences at a probability of p<0.05 were considered to be significant. Results As shown in Table 1, the maturation rates of the oocytes were 90.0% or more in both maturation media, and mpn formation rate in intact oocytes matured in mkrb (8.3%) was significantly lower than that of oocytes matured in pff (76.6%). These rates were comparable with previous results [7, 8], although penetration rates were relatively low. These low penetration rates might be attributable to stored spermatozoa instead of fresh material [15]. When the oocytes were inseminated after

3 GLUTATHIONE INJECTION INTO PIG OOCYTE 175 cumulus removal and presence of zona pellucida, the oocytes were hardly penetrated (data not shown). After zone removal, however, more than half of the oocytes were penetrated. Male PN formation rates of the zona-free oocytes (14.6 and 77.6% for the oocytes matured in mkrb and pff, respectively) were not significantly different from those in intact oocytes matured in both maturation media. The results of GSH microinjection were shown in Table 2. Viability of vehicle-injected oocytes was high (91%) and not significantly different from those of non-injected oocytes (93-95%). On the other hand, viabilities of the oocytes injected with 35 and 140 pl of 240 mm GSH solution were significantly decreased with dose-dependency (78% and 47%, respectively). The rates of penetrated oocytes were relatively low (42-54%), and were not statistically different among all groups. Seventy-four to 90% of the penetrated oocytes were activated and formed female PN in all experimental groups. Male PN formation ability of oocytes was not affected by the injection of the vehicle solution (17%). In contrast, when GSH was injected into the oocytes matured in mkrb, mpn (Fig. 1) were formed in significantly higher percentages in both 35 pl and 140 pl groups (52% and 53%, respective- Table 1. Male pronucleus (PN) formation of zona intact and zona free porcine oocytes matured in vitro Table 2. Viability and male pronuclei formation of porcine oocytes microinjected with glutathione (GSH)

4 176 Fig. NAITO 1. An oocyte with 35 matured pl of 240 in mm pronucleus (F), a polarbodies (arrows). ~ mkrb GSH male and solusion. pronucleus microinjected It has (M) a female and ly) than non-injected or vehicle injected oocytes. However the percentages did not reach the level of oocytes matured in pff (70.0%). Discussion Male PN formation rates of porcine oocytes matured in mkrb in vitro were low, and agreed well with previous reports [7, 8, 16, 17]. These results support our concept that the porcine oocytes matured in reported culture media in vitro are deficient in some cytoplasmic components involved in mpn formation, although no molecular evidence for the deficiency have been obtained. The low level of disulfide reducing power in the oocytes matured in vitro might be a likely candidate for the deficiency, because the reduction of protamine disulfide bridges in the sperm head has been suggested to be one of the critical processes for mpn formation [2-5]. The high viability of the vehicle-injected oocytes indicates the method of microinjection and composition of the injected solution were not detrimental to the porcine oocytes. An unexpected result is the decrease of the viability in the oocytes injected with 35 pl of GSH solution (240 mm) which was calculated to be about 10 mm in the cytoplasm on the assumption that the solution was dispersed homogenously in the ooplasm. Since the cytosolic GSH concentrations have been reported about 4 mm and 8 mm in immature and mature hamster oocytes, respectively [3, 5], the concentra- et al. tion in the microinjected porcine oocytes was a little higher but might be within physiological range. Therefore, the toxicity might be exhibited by the high concentration of GSH solution before dispersed homogenously in the cytosol. Since GSH functions directly or indirectly in many important biological phenomena, including the synthesis of proteins and DNA, transport, enzyme activity, metabolism, and protection of cells [18], a locally high concentration of GSH in the oocytes would induce an irreversible collapse of homeostasis. The porcine oocytes were fertilized in vitro after zona removal, because the penetration rate of oocytes with zona pellucida was very low. These low rates might be attributable to the removal of cumulus matrix [19, 20], and/or to stored spermatozoa instead of fresh *material [15]. The present result which shows no differences in mpn formation rate between intact and zona-free oocytes indicates that the treatment for zona removal and the absence of zona pellucida at insemination did not affect the mpn formation ability of the porcine oocytes. When the cytosolic reducing power in the porcine oocytes matured in mkrb was elevated by addition of 35 pl GSH solution, mpn formation ability of the oocytes increased significantly. Since the cytosolic GSH concentration of these oocytes seems to be in physiological range as described above, the present result strongly suggests that the low reducing power, possibly low GSH concentration, in the ooplasm contributes, at least in part, to the low mpn formation in the porcine oocytes matured in mkrb. On the other hand, the mpn formation rate of these oocytes did not reach the rate of those matured in pff, which have been suggested to be physiologically normal by their high mpn formation and high developmental abilities [7, 8, 11]. Therefore, we injected higher amount (140 pl) of GSH solution into the oocyte matured in mkrb, but the rate of mpn formation did not increase anymore in the present study. Explanation of these results would be an irreversible damage of the oocytes by the micro injection and/or the presence of other factor(s) involving mpn formation and deficient in the oocytes matured in mkrb. Borsuk [21] has suggested that the high maturation promoting factor activity is necessary for removing sperm nuclear membrane. We have recently found a high activity of this factor in

5 GLUTATHIONE INJECTION INTO PIG OOCYTE 177 porcine oocytes matured in pff [22] and significantly lower activity in those matured in mkrb [23]. Therefore, this activity might be a strong candidate of the factor contributing the low mpn formation in porcine oocytes matured in mkrb. Acknowledgments This research was supported by Grant-in-Aid for Encouragement of Young Scientists (No ) and Grant-in-Aid for scientific research on priority area, "germ-line" (No ) from the Ministry of Education, Science and Culture of Japan. Dr. Y. Itoh, The Tokyo Metropolitan Livestock Experiment Station, is thanked for generous supply of boar spermatozoa. References 1. Moor RM, Crosby IM, Osborn JC. Growth and maturation of mammalian oocytes. In: Crosignani PG, Rubin BL (eds.), In vitro Fertilization and Embryo Transfer. London: Academic Press, 1983, Perreault SD, Naish SJ, Zirkin BR. The timing of hamster sperm nuclear decondensation and male pronucleus formation is related to sperm nuclear disulfide bond content. Biol Reprod 1987; 36: Perreault SD, Barbee RR, Slott VL. Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes. Dev Biol 1988; 125: Perreault SD, Barbee RR, Elstein KH, Zucker RM, Keefer CL. Interspecies differences in the stability of mammalian sperm nuclei assessed in vitro by sperm microinjection and in vitro by flow cytometry. Biol Reprod 1988; 39: Perreault SD. Regulation of sperm nuclear reactivation during fertilization. In: Bavister BD, Cummins J, Roldam ERS (eds.), Fertilization in Mammals. Norwell: Serono Symposia; 1990; Yoshida M, Ishigaki K, Pursel VG. Effect of maturation media on male pronucleus formation in pig oocytes matured in vitro. Mol Reprod Biol 1991; 31: Naito K, Fukuda Y, Toyoda Y. Effects of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in intro. Gamete Res 1988; 21: Naito K, Kosaka M, Fukuda Y, Ishibashi I, Toyoda Y. Analysis of the factor(s) present in follicular fluids promoting male pronucleus formation ability of porcine follicular oocytes.fpn j Anim Reprod 1990; 36: Toyoda Y, Yokoyama H, Hoshi T. Studies on the fertilization of mouse eggs in vitro. I. In vitro fertilization of eggs by fresh epididymal sperm. Jpn J Anim Reprod 1971; 16: (In Japanese). 10. Naito K, Toyoda Y, Yanagimachi R. Production of normal mice from oovytes fertilized and developed without zonae pellucidae. Human Reprod 1992; 7: Naito K, Fukuda Y, Ishibashi I. Developmental ability of porcine ova matured in porcine follicular fluid in vitro and fertilized in vitro. Theriogenology 1989; 31: Plisko NT. A method of prolonging the viability and fertilizing ability for boar spermatozoa. Svinovodstvo 1965; 19: Hamano S, Toyoda Y. In vitro fertilization of pig eggs with ejaculated spermatozoa preincubated at high sperm concentration. Jpn J Anim Reprod 1986; 32: (In Japanese). 14. Sato E, Miyamoto H, Koide SS. Glycosaminoglycans in porcine follicular fluid promoting viability of oocytes in culture. Mol Reprod Dev 1990; 26: Haman S, Toyoda Y, Naito K, Fukuda Y. In vitro fertilization of porcine oocytes with spermatozoa preserved in Kiev extender at 18 C. Jpn J Anim Reprod 1990; 36: (In Japanese). 16. Thibault C. Final stages of mammalian oocyte maturation In: Biggers JD, Schuetz AW (eds.), Oogenesis. Baltimore: University Park Press; 1972, Motlik J, Fulka J. Fertilization of pig follicular oocytes cultivated in vitro. J Reprod Fert 1974; 36: Meister A, Anderson ME. Gultathione. Ann Rev Biochem 1983; 52: Boatman DE, Robbins RS. Detection of a soluble scrosome reaction-inducing factor, different from serum albumin, associated with ovulated eggcumulus complex. Mol Reprod Dev 1991; 30: Bavister BD. A consistently successful procedure for in vitro fertilization of golden hamster eggs. Gamete Res 1989; 23: Borsuk E. Anucleate fragments of parthenogenetic eggs and of maturing oocytes contain com-

6 178 NAITO et al. plementary factors required for development of a male pronucleus. Mol Reprod Dev 1991; 29: Naito K, Toyoda Y. Fluctuation of histone H1 kinase activity during meiotic maturation in porcine oocytes. Reprod Fert 1991; 93: Naito K, Daen FP, Toyoda Y. Comparison of histone H1 kinase activity during meiotic maturation between two types of porcine oocyte matured in different media in vitro. Biol Reprod 1992; (in press).