Parthenogenetic Development and Protein Patterns of Newly Matured Bovine Oocytes After Chemical Activation

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1 MOLECULAR REPRODUCTION AND DEVELOPMENT 49: (1998) Parthenogenetic Development and Protein Patterns of Newly Matured Bovine Oocytes After Chemical Activation LIN LIU, JYH-CHERNG JU, AND XIANGZHONG YANG* Department of Animal Science, University of Connecticut, Storrs, Connecticut ABSTRACT Development of an effective activation protocol is of great importance for studying oocyte competence and embryo cloning. Experiments were designed to examine effects of intracellular calcium elevating agents such as calcium ionophore A23187 (CaA) and ethanol, or protein synthesis and phosphorylation inhibitors such as cycloheximide (CH) and 6-dimethylaminopurine (6-DMAP), or a sequential combination of these agents on both parthenogenetic development and protein patterns of newly matured bovine oocytes. Oocytes were matured for 24 hr in M-199 supplemented with follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol at 39 Cin humidified air. They were then activated by various treatments and cultured in KSOM. Protein patterns at 15 hr after treatment were determined on 8 15% gradient SDS-PAGE and silver stained. Results demonstrated that none of the chemical agents CaA, ethanol, 6-DMAP, or cycloheximide could effectively induce parthenogenetic development of young bovine oocytes. When compared with the single treatments, sequentially combined treatments of CaA with 6-DMAP or with cycloheximide plus cytochalasin D (CD) significantly increased the rates of cleavage (78 82% versus 3 13%) and blastocyst development (31 40% versus 0%), which were comparable with those of IVF group (80% and 35%, respectively; P 0.05). Supplementation with CD to the combined CaA and CH treatment improved rates of cleavage and blastocyst development versus without CD supplementation (31% versus 7%; P 0.05). Fluorescent microscopy revealed that 95% (n 40) of oocytes treated with CaA plus 6-DMAP had one pronucleus (PN) and one polar body (PB), while 88% (n 40) in the CaA plus cycloheximide treated group had one PN and two PBs and 85% (n 40) in CaA plus cycloheximide and CD group had two PNs and one PB. Treatment by CaA alone resulted in 73% of oocytes (n 40) arrested at a metaphase stage with two PBs (named as metaphase III or MIII). Protein patterns were similar for chemically activated and in vitro fertilized (IVF) oocytes in that the 138- and 133- kda proteins, whose functions are not yet known, were present in the metaphase-stage (MII 24 hr, MII 40 hr, and MIII) oocytes but were absent in PN-stage oocytes regardless of treatment. Therefore, these proteins seem to be metaphase-associated proteins. Taken 1998 WILEY-LISS, INC. together, we conclude that optimal parthenogenetic development of newly matured bovine oocytes can be obtained by calcium ionophore treatment followed by incubation in either 6-DMAP or cycloheximide plus cytochalasin D and that the reduction of the 138- and 133-kDa proteins might be necessary for the full activation of bovine oocytes. Mol. Reprod. Dev. 49: , Wiley-Liss, Inc. Key Words: maturation; activation; oocyte; protein profile; MIII; cattle INTRODUCTION Matured oocytes of most mammalian species generally are arrested at the second metaphase until fertilization or parthenogenetic activation occurs. In bovine oocytes, parthenogenetic activation has been studied extensively during the past decade (Nagai, 1987; Presicce and Yang, 1994a, 1994b; Stice et al., 1994; Susko- Parrish et al., 1994; Ware et al., 1989). Ethanol or calcium ionophore A23187 (CaA) or reduced-temperature culture has been shown to induce activation of aged bovine oocytes but not young ones (Nagai, 1987; Stice et al., 1994; Ware et al., 1989). Ethanol, calcium ionophore, or electric pulse sequentially combined with cycloheximide (CH), a protein synthesis inhibitor, has been reported to be effective in activating newly matured bovine oocytes (Presicce and Yang, 1994a; Shi et al., 1993; Yang et al., 1994). Additionally, protein kinase inhibitors have been shown to induce or accelerate the activation of metaphase II oocytes (Rickords et al., 1992). A protein serine/threonine kinase (or phosphorylation) inhibitor, 6-dimethylaminopurine (6-DMAP) (Meijer and Pondaven, 1988; Néant and Guérrier, 1988), has been shown to enhance the activation stimulus and to accelerate pronuclear formation and parthenogenetic development in the young mouse and bovine oocytes (Moses and Masui, 1994; Moses et al., 1995; Susko-Parrish et al., 1994; Szöllösi et al., 1993; Takahashi et al., 1996). Furthermore, young bovine oocytes *Correspondence to: Dr. X. Yang, Department of Animal Science, 3636 Horsebarn Road, Ext U-40, University of Connecticut, Storrs, CT xyang@ansc1.cag.uconn.edu Received 5 June 1997; Accepted 6 August 1997

2 CHEMICAL ACTIVATION OF BOVINE OOCYTES 299 have been activated and developed to the blastocyst stage by treatment with either chemicals or electrostimulation followed by protein synthesis or phosphorylation inhibitors (Du et al., 1995; Presicce and Yang, 1994b; Stojkovic et al., 1997; Susko-Parrish et al., 1994). Despite many reports on bovine oocyte activation, limited information is available on directly comparing the activation efficiency of various agents mentioned above. Additionally, a direct comparison on parthenogenetic development and development of in vitro fertilized (IVF) oocytes should be conducted because the sperm is the natural activator of oocytes. Furthermore, the mechanisms involved with parthenogenetic development stimulated by different sequential combination treatments remain unclear. Bovine oocytes matured for 24 hr (called newly matured or young oocytes) are used routinely for in vitro fertilization in laboratories worldwide. Moreover, young oocytes generally were considered to have higher developmental potential than aged oocytes in IVF and cloning experiments (Lavoir et al., 1997; Susko-Parrish et al., 1991). In the present study, experiments were undertaken with the following objectives: (1) to determine the treatment effects on parthenogenetic development of young bovine oocytes by different agents that stimulate calcium increase as well as inhibitors that prevent protein synthesis or phosphorylation (these agents or inhibitors were applied either singly or in combination and were compared with our standard IVF protocol for activating oocytes), (2) to examine nuclear behavior of oocytes after activation treatments, and (3) to analyze and compare protein profiles of the activation-treated, IVF, and metaphase control oocytes. The results demonstrated that the combined activation procedures of CaA DMAP, and CaA CH CD were effective for the activation and development of young bovine oocytes. Moreover, the 138- and 133-kDa proteins were present in metaphasearrested bovine oocytes and became undetectable following activation or fertilization. MATERIALS AND METHODS Collection and Maturation of Oocytes Bovine ovaries were transported from a slaughterhouse to the laboratory in a thermocontainer at C. Collection and culture of oocytes were as described previously (Presicce and Yang, 1994a, 1994b; Yang et al., 1993). Briefly, oocytes were collected in a 50-ml conical tube by aspiration of antral follicles (2 8 mm in diameter) using an 18-gauge needle and a syringe. After being washed three times in Dulbecco s phosphate-buffered saline (DPBS-PVA), cumulus-enclosed oocytes were selected and washed three times in maturation medium. For maturation culture, cumulusenclosed oocytes were cultured in 100-µl droplets of maturation medium (20 25 oocytes per droplet) covered with mineral oil (Sigma, St. Louis, MO) at 39 C in 5% CO 2 and humidified air. The maturation medium was bicarbonate medium M-199 with Earle s salts, 25 mm HEPES, and 7.5% fetal calf serum (Gibco, Grand Island, NY, lot ) supplemented with 0.5 µg/ml ovine follicle-stimulating hormone (ofsh), 5.0 µg/ml ovine luteinizing hormone (olh), 1.0 µg/ml estradiol, and 0.25 mm pyruvate (Sigma). Chemical Activation and In Vitro Culture After maturation culture for 24 hr, oocytes were stripped of their cumulus cells by vortexing. Denuded oocytes with a polar body were selected and then assigned to the following treatments: exposure to ethanol (Eth, 7% for 7 min), CaA (Sigma, 5 µm for 5 min), 6-DMAP (Sigma, 2.5 mm for 3.5 hr), or cycloheximide (Sigma, CH, 10 µg/ml for 6 hr) either alone or Eth or CaA followed by incubation with 6-DMAP, CH, or CH CD (Sigma, 2.5 µg/ml for 6 hr). The CaA and CD stocks were prepared in dimethyl sulfoxide and 6-DMAP and CH stocks in sterile distilled water. All the activation chemicals were diluted to the desired concentration in KSOM (Liu and Foote, 1995; Liu et al., 1996) supplemented with 0.1% bovine serum albumin (BSA, Sigma, A-9647) before use. Following a treatment or combination of treatments, oocytes were washed and then cultured in 100-µl drops of KSOM containing 0.1% BSA under mineral oil (Sigma, embryo tested) at 39 C in an atmosphere of 5% CO 2 in humidified air for the first 4 days and in KSOM containing 1% BSA for the remaining days, with the medium changed every 2 days. IVF of Bovine Oocytes IVF procedure was described previously (Yang et al., 1993). Briefly, frozen semen was thawed in a 37 C water bath. The sperm were washed twice by centrifugation with washing medium, which was based on Brackett s defined medium (Brackett and Oliphant, 1975) and then capacitated with heparin and coincubated for 6 hr with cumulus-enclosed oocytes in the Brackett s fertilization medium (Yang et al., 1993). Afterwards, the fertilized oocytes were denuded of cumulus cells as in the activation experiment, washed, and then cultured in KSOM medium as described above. Assessment of Activation and Developmental Ability In Vitro After hr of culture, treated oocytes were stained with 10 µg/ml Hoechst for 10 min and checked by epifluorescence microscopy. In the control group, denuded oocytes were cultured for the same period of time. Oocytes exited from the MII stage were considered to have been activated. To assess cleavage and developmental ability, embryos were maintained in culture for 10 days. Statistical significance of the differences between treatments was analyzed with the SAS package chisquare program.

3 300 L. LIU ET AL. Fig. 1. Cleavage of bovine oocytes matured for 24 hr treated by different chemicals alone or in sequential combination. Two to four replicates per treatment. The pooled number of oocytes used is indicated in parentheses. A versus B indicates significant difference (P 0.05); a versus b versus c indicates significant difference (P 0.05). CaA (5 µm calcium ionophore A23187 for 5 min); Eth (7% ethanol, 7 min); CH (10 µg/ml cycloheximide, 6 hr); DMAP (2.5 mm 6-dimethylaminopurine, 3.5 hr); CD (2.5 µg/ml cytochalasin D, 6 hr). Cleavage at 24 hr in IVF group is not included. Gel Electrophoresis and Staining The same number of oocytes (45 or 50) from each group were washed in DPBS-PVA and collected in Eppendorf tubes at hr after treatment. To each tube, 30 µl of sample buffer (Laemmli, 1970) was added, heated for 2 min, and then stored frozen. The samples were loaded and electrophoresed on a standard 8 15% linear gradient SDS-polyacrylamide slab gel as described by Moor et al. (1981) and revealed by silver stain (Sigma kit). Immunofluorescence Microscopy of Tubulin and Chromatin Denuded oocytes were fixed and extracted for 30 min at 37 C in a microtubule-stabilizing buffer (Albertini and Clark, 1981; Allworth and Albertini, 1993; Ju et al., 1998a, 1998b). The oocytes were washed extensively and blocked overnight in the wash medium, which is composed of PBS supplemented with 0.02% NaN 3, 0.01% Triton X-100, 0.2% nonfat dry milk, 2% goat serum, 2% BSA, and 0.1 M glycine. Afterwards, oocytes were incubated with - and -tubulin mouse monoclonal antibody (1:200; Sigma), washed, and then incubated with fluorescein isothiocyanate (FITC) conjugated antimouse IgG (1:200; Cappel) at 37 C for 2 hr. After washing, oocytes were stained for DNA with Hoechst (10 µg/ml) in mounting medium containing PBS and glycerol (1:1) and finally mounted onto slides. The samples were observed under an Olympus Axiophot epifluorescein microscope. RESULTS Effects of Different Activation Agents Alone or in Combination on Cleavage and Development of Newly Matured Bovine Oocytes To compare parthenogenetic cleavage and development, oocytes were subjected to ethanol, CaA, 6-DMAP, or cycloheximide (CH) treatments either alone or in combination. Figure 1 shows that the rate of cleavage at 24 hr was very low (0 10%; n 62 66) when oocytes were treated by CaA, ethanol, 6-DMAP, or cycloheximide alone. Treatment of oocytes by CaA or ethanol followed by incubation with 6-DMAP or cycloheximide supplemented with CD significantly increased the cleav-

4 CHEMICAL ACTIVATION OF BOVINE OOCYTES 301 Fig. 2. Blastocyst development of bovine oocytes matured for 24 hr treated by different chemicals alone or in sequential combination. Two to four replicates per treatment. The pooled number of oocytes used is indicated in parentheses in Fig. 1. The percentage of blastocysts was based on the cleaved oocytes at day 2. A versus B indicates significant difference (P 0.05); a versus b indicates significant difference (P 0.05). CaA (5 µm calcium ionophore A23187 for 5 min); Eth (7% ethanol, 7 min); CH (10 µg/ml cycloheximide, 6 hr); DMAP (2.5 mm 6-dimethylaminopurine, 3.5 hr); CD (2.5 µg/ml cytochalasin D, 6 hr). No blastocyst development from A23187 group was obtained. age rate (63 73%; n 105 and 111; P 0.01). The cleavage rate at 24 hr in IVF oocytes was low; however, it increased to about 70% at 30 hr after sperm-oocyte coincubation (data not shown in Fig. 1). The cleavage rate of oocytes was low (14%; n 100) in the CaA CH group in the absence of cytochalasin D (CD). At day 2, the rates of cleavage among the three combination treatment groups of CaA DMAP, CaA CH CD, and Eth DMAP were very similar (78 82%; P 0.05) to that of IVF oocytes and significantly higher than that in the CaA CH group (27%; P 0.001). Furthermore, there was no blastocyst formation in oocytes treated singly with any of these agents (Fig. 2; only CaA group listed). The effectiveness of sequential combination treatments also was evident in the rate of blastocyst development (21 29%) from three of the combined treatment groups (CaA DMAP, CaA CH CD, or Eth DMAP) after 8 days of culture in vitro, which was again similar to that of IVF controls (29%; n 172; P 0.05; Figs. 2 and 3A). The rate of development to the blastocyst stage was significantly lower (4%; P 0.001) in CaA CH treated oocytes. Similar trends of blastocyst development (31 40%) were observed at day 10 except that hatched blastocysts (see Fig. 3B) were observed only in CaA DMAP and IVF groups. Another phenomenon we noticed was that blastocyst formation was delayed in the Eth DMAP group, in which 21% of blastocyst development was obtained by day 8 compared with 35% at day 10. However, overall, it appeared that developmental competence of oocytes treated with CaA DMAP or CaA CH CD was quite similar to that obtained from IVF oocytes. Activation Evaluation of Newly Matured Bovine Oocytes The following five treatment groups were compared for oocyte nuclear progression at hr after activation treatment: CaA, CaA DMAP, CaA CH CD, CaA CD, and controls. Denuded oocytes matured for 24 hr without activation treatment were further cultured in the same condition for hr as controls. All these oocytes (n 40) remained arrested at the MII stage, but in some oocytes the chromosomes migrated further away from the first polar body (Table 1 and Fig. 4A). All treated oocytes (100%; n 40) in the groups treated with CaA DMAP, CaA CH, and CaA CH CD were found activated and progressed to the pronuclear stage. However, the patterns of pronuclear formation were very different among these treatment groups. Ninety-five percent of activated oocytes (n 40)

5 302 L. LIU ET AL. Changes of Protein Patterns in Activated Bovine Oocytes The protein pattern of treated oocytes was directly compared with that of the controls following treatments, including MII 24 hr, CaA, CaA DMAP, CaA CH CD, CaA CH, MII 40 hr, and IVF at 16 hr. Basically, the protein profiles of these oocytes were very similar. However, proteins of 138 and 133 kda were found only in metaphase-stage oocytes (MII 24 hr, MIII, and MII 40 hr oocytes), while they disappeared in pronuclear-stage eggs (CaA DMAP, CaA CH CD, CaA CH, and IVF oocytes) (Fig. 5). The fact that these proteins found at metaphase stage were not detectable at pronuclear stage suggests that they are metaphase-related proteins that seemed to disappear at pronuclear stage. Fig. 3. Blastocysts developed from bovine oocytes activated by calcium ionophore followed by 6-DMAP. (A) Blastocysts developed at day 8 of in vitro culture. (B) Hatched blastocysts at day 10 of in vitro culture. ( 200.) in the CaA DMAP treatment group displayed one pronucleus and one polar body (see Fig. 4B). In CaA CH treatment group, most of the oocytes (88%; n 40) had one pronucleus accompanied by two polar bodies (see Fig. 4C). In contrast, oocytes with two pronuclei and one polar body were found as the predominant pattern (85%; n 40) in the CaA CH CD group (see Fig. 4D). Interestingly, the majority of the treated oocytes (73%; n 40) in the CaA treatment group manifested a partial activation with the release of a second polar body, but the remaining chromosomes arrested at the metaphase stage (MIII) (see Fig. 4E). Microtubular Changes in Activated Bovine Oocytes In order to correlate the changes of protein patterns with spindle structures in activated bovine oocytes, microtubular organization and nuclear changes were examined by double staining and fluorescent microscopy. As shown in Fig. 6, intact spindles existed in matured oocytes with condensed chromosomes attached on the spindle. In CaA-treated oocytes, a second polar body extruded and an MIII spindle formed, with reduced number of separated chromosomes. The MIII spindle differed from the MII spindle in that a longer spindle and more scattered chromosomes were seen at the MIII stage, similar to an early anaphase structure. In CaA DMAP treated oocytes, spindle destruction occurred, and one pronucleus with a microtubular network formed in the cytoplasm. A slightly elongated spindle with a little spread of chromosomes was found in aged oocytes. Fertilization induces similar changes in the microtubules to those in pronuclear formed oocytes caused by parthenogenetic activation. These observations also confirmed our parallel studies on extensive analyses of the nuclear and cytoskeleton dynamics following single or combined activation treatments (Ju et al., 1998a, 1998b). DISCUSSION This work demonstrated that optimal parthenogenetic development of newly matured bovine oocytes can be obtained by calcium ionophore followed by incubation in 6-DMAP or cycloheximide plus cytochalasin D. The cleavage and developmental competence is comparable with that of IVF oocytes. This study also confirmed previous findings that agents that increase intracellular calcium (CaA or ethanol) or inhibitors that prevent protein synthesis/phosphorylation each alone resulted in low cleavage and development rates. Combined treatments of these agents with the inhibitors were more efficient in inducing activation and development of young bovine oocytes than any single treatment alone (Presicce and Yang, 1994a, 1994b; Stojkovic et al., 1997; Takahashi et al., 1996). Our data are also in

6 Treatment TABLE 1. Chemical Activation of Bovine Oocytes Matured In Vitro for 24 hr* No. of oocytes No. MII CHEMICAL ACTIVATION OF BOVINE OOCYTES 303 No. (%) activated Total 1PN 1PB 1PN 2PB 2PN 1PB Others MII 40 hr (5) 2 (control) CaA DMAP (100) 38 (95) 2 2-cell CaA CH (100) 35 (88) 2 2 Tel, 1 MIII CaA CH CD (100) 34 (85) 6 3PN 1PB CaA (83) (73) MIII *MII 40 hr (control) IVM oocytes at 40 hr of maturation; CaA DMAP calcium ionophore treatment for 5 min and then treated with 6-DMAP for 3.5 hr; CaA CH calcium ionophore treatment for 5 min and then treated with cycloheximide for 6 hr; CaA CH CD calcium ionophore treatment for 5 min and then treated with cycloheximide plus cytochalasin D for 6 hr; PN pronucleus; PB polar body; MIII metaphase III; Tel telophase II. Fig. 4. Fluorescence microscopy of nuclear status of bovine oocytes by chemical activation treatments after staining with Hoechst (A) Newly matured bovine oocytes cultured for another 16 hr as control, with visible chromosomes. (B) CaA DMAP, with one PN and one PB. (C) CaA CH, with one PN and two PB. (D) CaA CH CD, with two PN and one PB. (E) CaA only, with metaphase III chromosomes and two PB. ( 300.)

7 304 L. LIU ET AL. Fig. 5. Protein profiles in bovine oocytes revealed by 8 15% gradient SDS-PAGE and silver stain. Molecular weight standards from Sigma wide range marker are on the right (lane 8). Lane 1: MII 24 hr; lane 2: CaA; lane 3: CaA DMAP; lane 4: CaA CH CD; lane 5: CaA CH; lane 6: MII 40 hr; and lane 7: IVF control. agreement with the previous results that cytochalasins inhibit the polar body release and significantly improve blastocyst development (Presicce and Yang, 1994a, 1994b; Stojkovic et al., 1997), suggesting a significant role of ploidy in the development of embryos. Evaluation of nuclear status revealed that young bovine oocytes can be efficiently activated by exposure to calcium ionophore followed by either 6-DMAP or cycloheximide treatment. The rate of pronuclear formation by treatment with CaA followed by cycloheximide or 6-DMAP was very high ( 90%). Nonetheless, spontaneous activation is quite low during the aging process of bovine oocytes with the time period studied (Presicce and Yang, 1994a; our unpublished observations). The treatment and incubation medium might be an important factor in the activation process. KSOM medium was used throughout the present experiment; in contrast, in a previous report, only 66% of oocytes had pronuclear development after exposure to 50 µm CaA in PBS followed by 10 µg/ml cycloheximide for 10 hr in M-199 medium (Goto et al., 1994). Calcium ionophore was reportedly able to induce an increase in cytoplasmic calcium, which was the initial stage of activation, resulting in the inactivation of maturation-promoting factor (MPF). It is likely that a single intracellular calcium rise induced by the CaA stimulation would inactivate the existing MPF and thus allow young bovine oocytes to resume meiosis and emit the second polar body. However, active protein synthesis and phosphorylation activities in the young oocytes would quickly restore the MPF activity (Collas et al., 1993) and thus force the nuclear materials to enter a new metaphase arrest (metaphase III), which has been reported previously in mice (Kubiak, 1989; Vincent et al., 1992). A similar phenomenon was observed when ionomycin alone was used in activating young bovine oocytes (Susko-Parrish et al., 1994). Further incubation with protein synthesis or phosphorylation inhibitor is necessary for the development of a pronucleus. The present study also elucidated different patterns of pronuclear formation following different activation treatments. Only one pronucleus and one polar body appeared in the CaA DMAP group, while two pronuclei and one polar body were observed after CaA CH CD treatment and one pronucleus and two polar bodies were seen in the CaA CH group. It was reported previously that ionomycin sequentially combined with 6-DMAP led to one pronuclear formation in bovine oocytes (Susko-Parrish et al., 1994). These data show that 6-DMAP treatment induced diploid activation by preventing chromosomal separation and extrusion of the second polar body, while cycloheximide did not prevent chromosomal segregation and second polar body extrusion, resulting in haploid development. However, when cytochalasin D was supplemented in the medium, chromosomal segregation manifested, but cytokinesis (second polar body extrusion) was prohibited in cycloheximide-treated oocytes, leading to diploid development with two pronuclei in activated oocytes. In normal IVF oocytes, two pronuclei and two polar bodies were observed. Therefore, different mechanisms were involved in the very early stage of parthenogenetic and normal embryonic development. However, the common pathway is through the inactivation of MPF, resulting in pronuclear formation and subsequent development. Two distinct proteins were found different after activation of oocytes. These proteins, which disappeared in activated and fertilized oocytes, may serve as marker proteins for activation in fertilized or chemically activated oocytes. Most other proteins are consistently observed in oocytes following various combination procedures or IVF treatment, suggesting that the protein pattern is highly conserved, even though initial activation may be triggered by diverse pathways that merge into a final stage. The 138- and 133-kDa proteins were present only in oocytes at the metaphase stage when a spindle was present (MII 24 hr, MII 40 hr, and MIII induced by CaA treatment alone). These two proteins were nondetectable in oocytes at the pronuclear stage (artificially activated and IVF oocytes), in which the spindle disappeared and microtubular network formed. Similarly, the two proteins were not found in GV-stage oocytes (data not shown). These observations suggest that these two proteins might be microtubule-associated proteins that are crucial for spindle organization or stabilization. The disappearance of these proteins might be a prerequisite for pronuclear formation. These proteins may be involved in the inactivation of MPF, but the cause-effect relationship requires further investigation.

8 CHEMICAL ACTIVATION OF BOVINE OOCYTES 305 Fig. 6. Microtubular and nuclear staining and fluorescent microscopic examination of in vitro matured bovine oocytes following activation treatments. (A) Matured oocytes at 24 hr, with intact spindle. (B) MIII spindles in oocytes treated by CaA alone at 15 hr after treatment. (C) Disappearance of spindle and formation of microtubular network in the cytoplasm of oocytes treated by CaA DMAP at 15 hr after treatment. (D) Aged oocytes at 40 hr. Blue: stained for chromatin; Green: stained for spindle or microtubules. S, spindle; M, microtubule; CH, chromosome; PB, polar body; PN, pronucleus. Oocytes can be partially activated by agents that induce a rise in the intracellular concentration of calcium, either by enabling the entrance of this cation from the external medium (ethanol or electrical stimulus) or by the action of specific ionophore that liberate calcium from intracellular stores. A single calcium increase can induce early activation events, which are characterized by resumption of meiosis and MIII arrest, exocytosis of cortical granules (cortical reaction), and the modifications in the glycoproteins of the zona pellucida, but cannot induce late events, such as mrna recruitment, pronuclear formation, and DNA synthesis and cleavage (Schultz and Kopf, 1995; Soloy et al., 1997; Susko-Parrish et al., 1994). The use of IP 3 as an activator elicits repetitive calcium oscillations, as occurred in normal fertilization and results in activation in bovine oocytes (White and Yue, 1996). Following fertilization or parthenogenetic activation of mammalian oocytes, H1 kinase activity, presumably MPF activity, is inactivated and maintained at basal levels in all species studied (Choi et al., 1991; Collas et al., 1993; Kikuchi et al., 1995). However, a rebound of H1 kinase activity occurs within 2 4 hr after a single calcium stimulation in bovine oocytes, (Collas et al., 1993; Liu et al., 1998; Wu et al., 1998), while multiple electrical stimuli (Collas et al., 1993) or a combined treatment inhibited the reactivation of H1 kinase activity (Liu et al., 1998; Wu et al., 1998). The combined treatments described in this study that resulted in the full activation of oocytes are effective likely through preventing reactivation of MPF, as hypothesized previously (Presicce and Yang, 1994a, 1994b; Yang et al., 1994) and confirmed by our more recent studies (Liu et al., 1998; Wu et al., 1998). It is noteworthy that different initial activation mechanisms were involved in 6-DMAP and cycloheximide treatments. Unlike sperm or cycloheximide treatment, 6-DMAP seems to bypass the normal chromosome segregation and second polar body extrusion process and instead induces instant chromosome condensation, decondensation, and pronuclear formation. This phenomenon also was observed previously in mouse and bovine oocytes (Moses and Masui, 1994; Susko-Parrish et al., 1994; Szöllösi et al., 1993). Cycloheximide inhibits the synthesis of proteins, including cytostatic factors (CSF), which are believed to stabilize cyclin B (Moos et al., 1996) and thus to prevent MPF reactivation (Presicce and Yang, 1994a; Yang et al., 1994). It has been shown that 6-DMAP has no effect on protein synthesis (Neant and Guerrier, 1988). However, it inhibits phosphorylation of specific proteins, which results in the inactivation of MPF. Moreover, modifications in protein patterns were observed in oocytes

9 306 L. LIU ET AL. treated either with calcium ionophore followed by either 6-DMAP or cycloheximide or with IVF. Two proteins of 138 and 133 kda that were found in metaphasestage oocytes became nondetectable in pronuclearstage oocytes. Microtubule analysis of spindles suggests that these two proteins might be accompanied by spindles. However, further work is needed to identify the role of the 138- and 133-kDa proteins in the activation process of bovine oocytes. Finally, it should be noted that blastocyst development was delayed in bovine oocytes treated by ethanol, compared with ionophore, followed by DMAP. In addition, hatched blastocysts were observed only in the CaA DMAP and IVF groups in the present experiment, although the rate was not high. More experiments are needed to investigate the poor hatching rate for most activation treatments reported in this study. The low rate of blastocyst hatching may have been caused by a deficiency in the serum-free KSOM medium used in this experiment. Blastocyst hatching may require more nutritionally complex medium and serum supplementation. In conclusion, both sequential combination activation procedures of CaA DMAP, and CaA CH CD were effective for the activation and development of young bovine oocytes. Moreover, the disappearance of the 138- and 133-kDa proteins might be prerequisite for the full activation of bovine oocytes. This study confirms earlier findings that sequential combined incubation in CaA with 6-DMAP or CH CD induces sustained inactivation of MPF and thus leads to efficient activation of young bovine oocytes (Susko-Parrish et al., 1994). In addition, inhibition of the second polar body extrusion caused by these treatments, resulting in diploid development, also may contribute to the high rate of activation development obtained in this study. 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