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Maturation in Porcine Oocytes K Fluctuation of histone H1 kinase activity during meiotic maturation in porcine oocytes K. Naito and Y. Toyoda Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan Summary. Porcine oocytes cultured in follicular fluid for various periods of up to 48 h were stained with Hoechst-33342 and classified according to maturation before assay- ing. Histone H1 kinase activity at metaphase I was \m=~\10 times that at the germinal vesicle stage. An abrupt reduction in activity was observed in oocytes emitting the first polar body; then the activity increased again to the same level as at metaphase I. This pattern is similar to those reported in non-mammalian species and supports the con- cepts that histone H1 kinase is ubiquitous in eukaryotes and controls the meiotic cell cycle in mammals. Keywords: histone H1 kinase; oocyte; fluorochrome; pig Introduction Histone Hl kinase was first identified in Chinese hamster cells as an enzyme whose activity in¬ creases during metaphase and that phosphorylates histone HI in a cAMP-independent manner (Lake & Salzman, 1972). Recently, histone HI kinase has been purified and suggested to be homologous with maturation-promoting factor (MPF) (Arion et ai, 1988; Labbe et ai, 1988a), which promotes meiosis in immature oocytes arrested at the germinal vesicle stage (Masui & Markert, 1971; Kishimoto & Kanatani, 1976). Furthermore, p34cdc2, first identified in Schizo- saccharomyces pombe as a gene product controlling the cell cycle (Nurse & Bissett, 1981), and cyclin, first identified in marine organisms as a protein that varies in concentration during the cell cycle (Evans et ai, 1983), have been reported as components of histone HI kinase/MPF (Gautier et ai, 1988, 1990). The high activity of histone H1 kinase during metaphase has been suggested to cause nuclear lamina disassembly (Peter et ai, 1990b), nucleolar disassembly (Peter et ai, 1990a), chromosome condensation (Moreno & Nurse, 1990), transcription regulation (Cisek & Corden, 1989), translation regulation (Belle et ai, 1989) microfilament rearrangement (Morgan et ai, 1989), and reorganization of the intermediate filament network (Chou et ai, 1990). Numerous studies have been reported on the changes of histone H1 kinase activity during oocyte meiotic maturation (Mailer et ai, 1977; Dorée et ai, 1983; Picard et ai, 1985; Karsenti et ai, 1987; Labbe et ai, 1988a, b) and its characteristics (Sano, 1985; Pelech et ai, 1987; Erikson & Mailer, 1989) in Xenopus and marine organism oocytes. In mammalian oocytes, however, changes of histone HI kinase activity during meiotic maturation have not been reported, although there are a few reports on the change in activity of MPF and HI kinase at different times during maturation in mouse (Hashimoto & Kishimoto, 1988; Rime & Ozon, 1990) and porcine oocytes (Prochazka et ai, 1989). In this study, we established an assay system for histone HI kinase activity in porcine oocytes that matured in vitro, and investigated changes in activity during meiotic maturation. Downloaded from Bioscientifica.com at 09/29/2021 10:41:49PM via free access Materials and Methods Oocyte preparation. Gilt ovaries were obtained from a slaughterhouse and carried to the laboratory within 40 min in saline solution at 37-39°C. Follicular oocytes were collected from medium-sized follicles (2-5 mm in diameter) as described by Naito et ai (1988). Each of the 10 to 15 oocyte-cumulus complexes was cultured in 01 ml porcine follicular fluid (pFF) supplemented with 1-Oiu pregnant mares' serum gonadotrophin/ml (Peamex, Sankyo Co., Japan) for various periods of up to 48 h at 37°C in 5% C02 in air; pFF was collected from gilt ovaries and stored as described by Naito et ai (1988). After culturing, the oocytes were treated with 150 U hyaluronidase/ml (Sigma, Type IV-S) in a modified Krebs-Ringer bicarbonate solution (TYH; Toyoda et ai, 1971) for a few minutes at room temperature. Thereafter, the surrounding cumulus cells were removed by repeated pipetting with a fine-bore pipette. The denuded oocytes were then centrifuged at 15 000 g for 5 min, to localize the lipid granules, then stained in TYH containing 10 µg Hoechst-33342 (H-33342: Calbiochem Co., German Federal Republic)/ml for 15-60 min at 37°C in 5% C02 in air. H-33342 was dissolved in TYH at 1 mg/ml and stored in the dark at 70°C until use. The stained oocytes were examined under a fluorescent microscope (Nikon DIAPHOT-TMD microscope— equipped with a 100-W mercury lamp and Nikon DM400 filter) and classified according to meiotic maturation stages (Fig. 1). Photographs were taken of the oocytes (Nikon FE2 camera with Fujichrom DX 400D film) through the fluorescent microscope at different times during culture. Preparation of oocyte extracts. Oocytes at different stages were picked up under the fluorescent microscope and collected in ice-cooled TYH for assaying the histone HI kinase. Oocytes in which the maturation stage could not be determined under the fluorescent microscope were not used for assaying, but were mounted on a glass slide for determination of the stage after fixing and staining with 0-75% acetic orcein. The fresh oocytes of determined stages were washed 3 times in buffer A (60 mmol ß-glycerol phosphate/1, 30 mmol p-nitro-phenyl phosphate/1, 25 mmol Mops/1 (pH 7-2), 15 mmol EGTA/1, 15 mmol MgCl2/l and 0-1 mmol sodium vanadate/1) and put into plastic tubes at 2 oocytes to 1 µ buffer A; they were then frozen to 70°C to break the oocyte membrane. Repeated freezing and thawing treatment in a preliminary experiment had been— shown to reduce the enzyme activity and so all oocytes were subjected to frozen-thaw treatment only once. After thawing, the oocyte suspension was centrifuged at 15 000 g for 5 min at 4°C and mixed well; it was then centrifuged again for 20 min under the same conditions. The supernatants were used immediately for the kinase assay. Kinase assay. Histone HI kinase was assayed according to the method of Pelech et ai (1987), but with several modifications. Unless stated otherwise, all kinase assays contained the following in a final volume of 25 µ buffer: 500 nmol protein kinase inhibitor (TTYADFIASGRTGRRNAIHD: Sigma)/1, 50 µ histone 1/1 (Sigma type III- S), 1 mmol dithiothreitol/1 (Wako Pure Chemical Ind., Japan), 20pmol [ -32 ] /1 (3-10c.p.m./fmol: Amersham) and 5 µ oocyte extract. Kinase reactions commenced upon the addition of [ -32 ] and were usually completed within 60 min at 36°C. Assays were terminated by 0-4 ml of 20% trichloroacetic acid solution (TCA); 0-1 ml of 1% bovine serum albumin (BSA-FR.V.: Wako Pure Chemical Ind.) was added as a carrier protein for precipitation. The assay suspensions were centrifuged at 15 000 g for 5 min and supernatants were discarded. The precipitates were washed once with 0-4 ml of 20% TCA and dissolved in 0-4 ml of lM-NaOH. The solution was then transferred into vials containing 5 ml of scintillation fluid (ACS II: Amersham) and radioactivity was measured using a liquid scintillation counter (Aloka, LSC-1000). The value for blank tubes which contained all materials except for a cytosol preparation was subtracted from each experimental value to obtain the kinase activity. Statistical analysis. Student's t test was used for evaluation of the results. Probability of < 005 was considered to be statistically significant. Results The maturation stages of most oocytes were distinguishable by fluorescent microscopy after stain¬ ing with H-33342 (Fig. 1: b-h). The germinal vesicles were also visible by light microscopy after centrifugation (Fig. la). The maturation stages of some oocytes whose chromosomes were within the located lipid granules could not be determined without fixing and staining. All oocytes remained at the germinal vesicle (GV) stage after 4 h in culture (Table 1); 24 h after the start of culturing, 39 and 29% of oocytes were at first prometaphase (PMI) and first metaphase (MI), respectively. The proportion of MI oocytes increased until 33 h after the start of culturing. Oocytes emitting the first polar body appeared after 24 h of culturing, but the incidence of these oocytes at first anaphase and telophase (ATI) remained <20% even at 33-37 h in culture. Most of the others were either at MI or at second metaphase (Mil). Matured Mil oocytes were observed mainly after 35 h in culture and increased gradually to reach 85% of the total at 45^f8 h in culture. Downloaded from Bioscientifica.com at 09/29/2021 10:41:49PM via free access Fig. 1. Porcine follicular oocytes at different stages of maturation stained with Hoechst-33342. The photographs were taken with a light microscope (a) or a fluorescent microscope (b-h), 200; (a) and (b) germinal vesicle after 4 h in culture, (c) prometaphase I at 31 h, (d) metaphase I at 31 h, (e) anaphase I (early stage) at 31 h, (f) anaphase I (late stage) at 33 h, (g) telophase I at 33 h and (h) metaphase II at 45 h. Table 1. Temporal relationship of the maturation stages in porcine follicular oocytes cultured in vitro No. (%) of oocytes at the stage of Culture Total no. - period of ova Germinal Prometaphase Metaphase Anaphase and Metaphase (h) examined vesicle I I telophase I II 2^1 119 119(100) 0 0 0 0 24-28 148 42 (28) 58(39) 43(29) 3 (2) 2 (1) 31 157 3 (2) 58(37) 80(51) 11 (7) 5 (3) 33 101 4 (4) 13(13) 60(60) 16(16) 8 (8) 35 156 6 (4) 2 (1) 73(47) 30(19) 45(29) 37 152 5 (3) 8 (5) 51(34) 22(14) 66(43) 45-48 91 2 (2) 0 12(13) 0 77(85) Data are means from 3 experiments.
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