Interplay of maturation-promoting factor and mitogen-activated protein kinase inactivation during metaphase-to-interphase transition of activated bovine oocytes.

The objective of the present study was to examine the activity changes in histone H1 kinase (also known as maturation-promoting factor [MPF]) and mitogen-activated protein kinase (MAPK) and their constituent proteins in in vitro-matured bovine oocytes after in vitro fertilization (IVF) or after parthenogenetic activation induced by calcium ionophore A23187 alone or by the ionophore followed by either 6-dimethylaminopurine (6-DMAP) or cycloheximide (CHX). Inactivation of both H1 kinase and MAPK occurred after both A23187+6-DMAP treatment and IVF; inactivation of H1 kinase preceded inactivation of MAPK. However, MAPK was inactivated much earlier in 6-DMAP-treated oocytes. Further analysis of constituent cell cycle proteins of these kinases by Western blot showed that A23187 alone could not induce changes in cdc2, cdc25, or ERK2 but induced reduction of cyclin B1. IVF and A23187+CHX induced similar changes: cyclin B1 was destroyed shortly after activation followed by accumulation of cyclin B1, phosphorylation of cdc2, and dephosphorylation of ERK2 at pronuclear formation 15 h after activation. No change in cdc25 was observed at this time. In contrast, A23187+6-DMAP treatment resulted in earlier phosphorylation of cdc2 and dephosphorylation of ERK2 at 4 h after treatment when the pronucleus formed. Moreover, accumulation of both cdc25 and cyclin B1 was detected at 15 h. Microinjection of ERK2 antibody into A23187-treated oocytes resulted in pronuclear formation. In conclusion, activation of bovine oocytes with 6-DMAP led to earlier inactivation of MAPK, while CHX induced inactivation of MAPK parallel to that following sperm-induced oocyte activation. Destruction of cyclin B is responsible for inactivation of MPF, while phosphorylation of cdc2 is likely responsible for maintaining its low activity. Inactivation of MAPK is closely associated with pronuclear development regardless of the activation protocol used.     

Surface alterations of the bovine oocyte and its investments during and after maturation and fertilization in vitro

Surface characteristics of the bovine oocyte and its investments before, during, and after maturation, and fertilization in vitro were evaluated by scanning electron microscopy (SEM). Oocyte diameters were also measured during SEM analysis of the oocyte. The cumulus cells manifested a compact structure with minimal intercellular spaces among them in the immature oocytes. These became fully expanded with increased intercellular spaces after maturation in vitro, but contracted again after fertilization. The zona pellucida (ZP) showed a fibrous, open mesh-like structure in the maturing and matured oocytes. The size and number of meshes on the ZP decreased dramatically after fertilization. The vitelline surface of immature oocytes was characterized by distribution of tongue-shaped protrusions (TSPs) varying in density. After 10 and 22 hr of maturation incubation, oocyte surface microvilli (MV) increased to become the predominant surface structure, and TSPs decreased substantially. The vitelline surface of fertilized oocytes (at 6 and 20 hr) was similar to that of the matured oocytes, but unfertilized oocytes had less dense MV than did fertilized oocytes (at 20 hr). The diameter of the oocytes decreased from 99 to 80 μm during maturation and increased to 106 μm after insemination (P < 0.05). Membrane maturation was characterized by surface changes from a TSP-predominant pattern to a MV-predominant pattern. Thus, the bovine oocyte maturation process was found to involve the expansion of cumulus cells and the maturation of the ZP, which changes dramatically upon fertilization. Also, volumetric changes occurred in ooplasm processed for SEM following oocyte maturation and insemination. © 1994 Wiley-Liss, Inc.     

Structural aspects of the zona pellucida of in vitro-produced bovine embryos: a scanning electron and confocal laser scanning microscopic study

Structural aspects of the bovine zona pellucida (ZP) of in vitro-matured (IVM) oocytes and in vitro-produced (IVP) embryos were studied in two experiments to find a tentative explanation for the zona's barrier function against viral infection. In Experiment 1, the ultrastructure of the outer ZP surface was studied. The diameter (nm) and the number of the outer pores within an area of 5000 microm(2) of 10 IVM oocytes, 10 zygotes, 10 8-cell-stage embryos, and 10 morulae were evaluated by scanning electron microscopy. In oocytes and morulae, the ZP surface showed a rough and spongy appearance with numerous pores. In zygotes, the ZP surface was found to have a smooth, melted appearance with only a few pores. In 8-cell-stage embryos, both surface patterns were found. The mean number (per 5000 microm(2)) and the mean diameter of the outer pores were different between the four stages of development (P < 0.001): 1511 pores in oocytes, 1187 in zygotes, 1658 in 8-cell-stage embryos, and 3259 in morulae, with mean diameters of 182, 223, 203, and 155 nm, respectively. In Experiment 2, the continuity of the meshes (network of pores) towards the embryonic cells was examined by confocal laser scanning microscopy. Therefore, the passage through and the location in the ZP of fluorescent microspheres, with similar dimensions as bovine viral diarrhea virus (BVDV, 40-50 nm) and bovine herpesvirus-1 (BHV-1; 180-200 nm), were evaluated. For all stages, the smallest beads were detected halfway through the thickness of the ZP, whereas the beads with a size of 200 nm were found only within the outer-fourth part of the ZP. It can be concluded that the intact ZP of bovine IVM oocytes and IVP embryos are constructed in such a way that BVDV and BHV-1 should not be able to traverse the ZP and reach the embryonic cells. However, the risk exists that viral particles can be trapped in the outer layers of the ZP.     

Effects of different activation protocols on preimplantation development, apoptosis and ploidy of bovine parthenogenetic embryos

The objective of this study was to optimize the protocols for bovine oocytes activation through comparing the effectiveness of different treatments on the activation and subsequent development of oocytes and examining the effects of two combined activation treatments on the blastocyst apoptosis and ploidy. Cumulus-oocyte complexes (COCs) were recovered from abattoir-derived ovaries and matured in vitro. After maturation, cumulus-free oocytes were activated according to the experiment designs. Activated oocytes were cultured in vitro in modified synthetic oviductal fluid (mSOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage. In Experiment 1, the matured oocytes were treated with single activation agents, including ionomycin (5 microM for 5 min), ethanol (7% for 7 min), calcium ionophore A23187 (5 microM for 5 min) or strontium (10mM for 5h). The pronuclear formation and cleavage rate were higher significantly in ionomycin (39.0 and 30.7%) and ethanol (41.5 and 28.1%) treatment alone compared to other treatments (9.7-25.2 and 11.3-23.7%, respectively, P<0.05). Very low blastocyst rates (3.9-5.3%) resulted which were not significantly different among treatments (P>0.05). For the combined activation treatment (Experiment 2), the same concentrations of ionomycin and ethanol as in Experiment 1 were used in combination with either 6-dimethylaminopurine (6-DMAP, 2.0 mM for 3 h) or cycloheximide (CHX)+cytochalasin B (CB, 10 microg/ml for 3 h). The pronuclear formation, cleavage rate, blastocyst rate and cell number of blastocyst were higher significantly (P<0.05) in ionomycin+6-DMAP treatment (67.1, 69.2, 28.0 and 91.3%, respectively) and ethanol+CHX+CB treatment (68.9, 70.2, 25.5 and 89.3%, respectively) compared to other treatments (11.7-58.1, 10.2-47.1, 1.5-24.2 and 34.2-62.7%, respectively). In Experiment 3, the parthenogenetic blastocysts produced by activation with ionomycin+6-DAMP and ethanol+CHX+CB and in vitro fertilized blastocysts (control group) were examined for apoptosis using a terminal deoxynucleotidyl transferase mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The ethanol+CHX+CB treatment (7.0%) showed significantly lower blastocyst apoptosis index compared to ionomycin+6-DAMP treatment (9.1%, P<0.05). Furthermore, the chromosomal composition in the parthenotes embryos differed (P<0.05) among treatments. The percentage of haploid parthenotes was higher in ionomycin+6-DMAP treatment than ethanol+CHX+CB treatment. These results suggested that ethanol+CHX+CB treatment was more favorable protocol for parthenogenesis of bovine oocytes.     

Developmental capacity of bovine oocytes following inhibition of meiotic resumption by cycloheximide or 6-dimethylaminopurine

This study was conducted to assess the fertilizability and developmental capacity of bovine oocytes which had been maintained in meiotic arrest by either a protein synthesis inhibitor, cycloheximide (CHX), or an inhibitor of serine/threonine protein kinases, 6-dimethylaminopurine (6-DMAP). Both CHX and 6-DMAP reversibly prevented nuclear maturation of nearly all oocytes for 24 h. After the reversal of arrest, CHX-treated oocytes could be successfully matured and fertilized. They developed to the blastocyst stage at slightly lower rates than oocytes cultured without inhibition for 22 h prior to sperm addition but at higher rates than those incubated in a medium containing no inhibitors for 46 h prior to fertilization. Oocytes inhibited by CHX for 48 h matured and fertilized normally but failed to develop into blastocysts. Even though 6-DMAP-treated oocytes completed meiosis I after removal from the drug, the rates of fertilization and blastocyst formation were lower than for untreated oocytes or CHX-treated oocytes. Effects of adding FSH and/or estradiol-17 beta (E(2)) during CHX-inhibition for 24 h were also examined. Embryos from oocytes treated with CHX and E(2) or with CHX and FSH + E(2) developed into blastocysts at similar rates as the controls. Further development of inhibited oocytes was examined by transferring blastocysts derived from oocytes inhibited by CHX with FSH and E(2) for 24 h to recipient heifers. Two calves were obtained following transfer. These results indicate that CHX-inhibited oocytes retain developmental competence, while 6-DMAP-inhibited oocytes after the reversal of arrest have reduced capacities for fertilization and further development. The addition of FSH and E(2) during CHX-inhibition improves development to the blastocyst stage of the oocytes that are capable of initiating and maintaining pregnancy after embryo transfer to recipient animals.     

Parthenogenetic development of porcine oocytes treated by ethanol, cycloheximide, cytochalasin B and 6-dimethylaminopurine

This study was carried out to investigate the various concentrations and exposure times of ethanol, one of many intracellular calcium elevating agents, and a sequential combination of ethanol (8%), cycloheximide (CHX, 10 microg/ml), cytochalasin B (CCB, 7.5 microg/ml) and 6-dimethylaminopurine (6-DMAP, 2 mM) to improve parthenogenetic activation and development of in vitro matured porcine oocytes. Cumulus-oocyte complexes (COCs) were matured in tissue culture medium (TCM) 199 for 44 h at 38.5 degrees C, 5% CO2 in air. Cumulus-free oocytes showing first polar body were activated by concentrations of 0, 5, 6, 7, 8, 9 and 10% ethanol for 10 min and exposure times of 0, 5, 8, 10, 12 and 15 min with 8% ethanol in HEPES buffered (25 mM) NCSU-23 medium. Also, oocytes were activated with the NCSU-23 medium containing 8% ethanol for 10 min. After that, oocytes were incubated in the NCSU-23 medium supplemented with CHX, CCB, 6-DMAP, CHX + CCB, CHX + 6-DMAP, CCB + 6-DMAP and CHX + CCB + 6-DMAP for 3h, respectively. Following activation, oocytes were transferred into the NCSU-23 medium containing 0.4% BSA for further culture of 20 and 144 h at 38.5 degrees C, 5% CO2 in air. The activation rates of oocytes were higher in 6, 7 and 8% ethanol concentrations compared with 0, 5, 9 and 10% ethanol concentrations. Significantly, more oocytes (29.3-33.7%) were activated in the exposure for 8, 10, 12 and 15 min than those in the exposure for 0 and 5 min, but there was no difference due to exposure to 8% ethanol for 8-15 min. Oocytes treated by chemical agents (40.5-70.5%) after exposure to ethanol significantly improved the rate of oocyte activation compared with ethanol alone (31.2%). The percentage of cleaved oocytes was higher in the ethanol+CHX+CCB+6-DMAP treatment (66.4%) than in other treatments (24.9-57.6%). Also, the rate of blastocyst formation was higher in the ethanol+CHX+CCB+6-DMAP treatment (25.0%) than in other treatments (0.0-19.3%). In conclusion, the optimal activation treatment of ethanol exposure alone for the in vitro matured porcine oocytes was 8% ethanol for 8-15 min. Oocytes activated by 8% ethanol for 10 min and incubated in the NCSU-23 medium supplemented with CHX, CCB and 6-DMAP for 3 h were more efficient for parthenogenetic development of in vitro matured porcine oocytes.     

In vitro and in vivo developmental competence of dromedary (Camelus dromedarius) oocytes following in vitro fertilization or parthenogenetic activation

Parthenogenetic activation of the oocyte represents an important step in the somatic cloning. The aim of the present study was to evaluate the effectiveness (in term of in vitro development) of different methods of parthenogenetic activation of dromedary oocytes. Selected cumulus-oocytes-complexes (n=1264) collected by follicular aspiration from ovaries obtained postmortem were matured in vitro (IVM) for 30 h then divided randomly into seven groups and submitted to artificial activation. Two groups were preactivated with 25 microM of calcium ionophore (CaI) for 20 min then incubated for 4h with either 2mM 6-dimethylaminopurine (6-DMAP) (group 1, n=202) or with 10 microg/mL cycloheximide (CHX) (group 2, n=194). Group 3 (n=172) and group 4 (n=184), oocytes were pretreated with 5 microM ionomycin (Iono) for 5 min then incubated with either 2mM 6-DMAP or 10 microg/mL cycloheximide for 4h, respectively. Group 5 (n=161) and group 6 (n=155) oocytes were preactivated with electrical stimulation (ES) then activated with either 2mM 6-DMAP or 10 microg/mL cycloheximide for 4h, respectively. Group 7 (n=196) oocytes were submitted to in vitro fertilization (IVF) and served as a control. All groups containing oocytes were cultured in vitro following activation or IVF, at 38.5 degrees C under 5% CO(2) in air with >95% humidity. The in vitro development rates of dromedary oocytes exposed to 6-DMAP after CaI (61%), ES (74%) and the IVF group (71%) were similar and significantly greater (P<0.05) than other treatments (10% for group 2, 47% for group 3, 27% for group 4 and 41% for group 6). The blastocyst developmental rate was better (P<0.05) in parthenotes following activation with Iono/6-DMAP (21%) compared to activation with Iono/CHX (12%). However, all were less than that achieved in the IVF group (35%). We conclude that parthenogenetic activation of camel oocytes with 6-DMAP is more effective than activation with CHX for all pre-treatments tested (CaI, Iono or ES). The viability of activated (n=15) or IVF (n=10) hatched-dromedary embryos was examined by transfer to synchronized recipients. An embryonic vesicle was seen by ultrasonography at 15 days post transfer in four females (CaI/6-DMAP: 1/5; 20%, IVF: 3/10; 30%). The only pseudopregnancy obtained with an activated embryo resorbed at 25 days. One of the females receiving the IVF produced embryos aborted at 2 months and the other two females carried to term and gave birth to healthy calves (one female and one male). This study shows that artificial activation of dromedary oocytes with CaI/6-DMAP or ES/6-DMAP is more effective than other treatments in terms of in vitro embryo development. This provides efficient activation conditions which may lead to the development of the somatic cell nuclear transfer procedure in dromedary.     

Zona reaction in porcine oocytes fertilized in vivo and in vitro as seen with scanning electron microscopy

Morphological changes in zona pellucidae (ZP) isolated from in vitro-matured (IVM) and ovulated porcine oocytes were compared before or after fertilization in vitro and in vivo, respectively, by using scanning electron microscopy (SEM). The ZP of some ovulated or IVM oocytes and in vivo- or in vitro-fertilized (IVF) zygotes were equally split into two halves while immersed in an enzyme-inhibitor solution, using a surgical blade. After washing, intact and ZP halves were fixed in 1% glutaraldehyde solution in 0.1 M cacodylate buffer, processed, and examined using SEM. The outer surface of ZP in ovulated oocytes had a mesh-like structure. The outer morphology in IVM oocytes was more smooth although the mesh-like structure was still visible at high magnification. In in vivo zygotes and IVM-IVF zygotes, this lysed, mesh-like structure was more obvious. The inner surface of ZP had some small depressions (orifices). The mean number of orifices per 100 micrometer(2) of ZP surface was larger in IVM oocytes as compared to ovulated ones. The number of orifices per 100 micrometer(2) decreased in IVM-IVF zygotes as compared to IVM oocytes; whereas, in vivo zygotes did not differ from ovulated oocytes. The mean diameter of intact ZP as well as their mean thickness was greater in ovulated oocytes than IVM oocytes. The mean thickness of the ZP was larger in ovulated oocytes than IVM ones. The ZP thickness was larger in zygotes than in in vivo oocytes, whereas that of IVM-IVF zygotes did not differ from that of IVM oocytes. These results indicate that the morphology of ZP and the ZP reaction at sperm penetration appears to be much different between IVM oocytes and ovulated ones.     

Improved parthenogenetic development of vitrified-warmed bovine oocytes activated with 9% ethanol plus 6-DMAP

The objective was to compare various activation protocols on developmental potential of vitrified bovine oocytes. Bovine oocytes matured in vitro for 23 h were vitrified with EDFSF30 in open pulled straws. After warming, they were cultured in vitro for 1 h, followed by parthenogenetic activation. Vitrified-warmed oocytes had a morphologically normal rate similar to that of controls (nonvitrified oocytes cultured in vitro for 24 h; 98.6% vs. 100%, P > 0.05). When vitrified-warmed oocytes were first activated with 7% ethanol for 5 min and then incubated in 6-dimethylaminopurin (6-DMAP) for 4 h, cleavage and blastocyst rates were 41.2% and 23.2%, respectively, which were lower than those of controls (77.5% and 42.0%, P < 0.05). Subsequently, we varied the ethanol concentration to increase the effectiveness of parthenogenetic activation. When either 5%, 6%, 7%, 8%, 9%, 10%, or 11% ethanol alone (for 5 min) or in combination with 6-DMAP (4 h) was used to activate vitrified-warmed oocytes, cleavage rates ranged from 22.3% to 61.1% and blastocyst rates ranged from 1.1% to 30.6%. These rates were optimized when oocytes were treated with 9% ethanol plus 6-DMAP; this was verified in experiments evaluating other activation protocols with 9% ethanol, calcium ionophore A23187, or ionomycin alone, or in combination with DMAP or cycloheximide (CHX). In conclusion, the oocyte activation protocol affected developmental capacity of vitrified bovine oocytes; 9% ethanol (5 min) followed by 6-DMAP (4 h) promoted optimal parthenogenetic activation.     

Maturation conditions and boar affect timing of cortical reaction in porcine oocytes

The cortical reaction induces changes at the egg's Zona pellucida (ZP), perivitelline space and/or oolemma level, blocking polyspermic fertilization. We studied the timing of sperm penetration and cortical reaction in pig oocytes matured under different conditions and inseminated with different boars. Immature (germinal vesicle stage) and in vitro matured (IVM) (metaphase II stage) oocytes were inseminated and results assessed at different hours post insemination. Penetrability and polyspermy rates increased with gamete coincubation time and were higher in IVM oocytes. A strong boar effect was observed in IVF results. Cortical reaction (assessed as area occupied by cortical granules) and galactose-β(1-3)-Nacetylgalactosamine residues on ZP (area labeled by peanut agglutinin lectin, PNA) were assessed in IVM and in vivo matured (IVV) oocytes at different hours post insemination. After maturation, IVM and IVV oocytes displayed similar area occupied by cortical granules and it decreased in fertilized oocytes compared to unfertilized ones. Cortical reaction was influenced by boar and was faster in polyspermic than in monospermic oocytes, and in IVM than in IVV oocytes. The outer ZP of inseminated oocytes appeared stained by PNA and the labeled area increased along with gamete coculture time. This labeling was also observed after insemination of isolated ZP, indicating that this modification in ZP carbohydrates is not induced by cortical reaction. The steady and maintained cortical reaction observed at 4 to 5 h post insemination in IVV monospermic oocytes might reflect the physiological time course of this important event in pigs. Both maturation conditions and boar affect cortical granules release.     

Somatic cell cloning in Buffalo (Bubalus bubalis): effects of interspecies cytoplasmic recipients and activation procedures

Successful nuclear transfer (NT) of somatic cell nuclei from various mammalian species to enucleated bovine oocytes provides a universal cytoplast for NT in endangered or extinct species. Buffalo fetal fibroblasts were isolated from a day 40 fetus and were synchronized in presumptive G(0) by serum deprivation. Buffalo and bovine oocytes from abattoir ovaries were matured in vitro and enucleated at 22 h. In the first experiment, we compared the ability of buffalo and bovine oocyte cytoplasm to support in vitro development of NT embryos produced by buffalo fetal fibroblasts as donor nuclei. There were no significant differences (p > 0.05) between the NT embryos derived from buffalo and bovine oocytes, in fusion (74% versus 71%) and cleavage (77% versus 75%) rates, respectively. No significant differences were also observed in blastocyst development (39% versus 33%) and the mean cell numbers of day 7 cloned blastocysts (88.5 +/- 25.7 versus 51.7 +/- 5.4). In the second experiment, we evaluated the effects of activation with calcium ionophore A23187 on development of NT embryos after electrical fusion. A significantly higher (p < 0.05) percentage of blastocyst development was observed in the NT embryos activated by calcium ionophore and 6-DMAP when compared with 6-DMAP alone (33% versus 17%). The results indicate that the somatic nuclei from buffalo can be reprogrammed after transfer to enucleated bovine oocytes, resulting in the production of cloned buffalo blastocysts similar to those transferred into buffalo oocytes. Calcium ionophore used in conjunction with 6-DMAP effectively induces NT embryo development.     

The effect of 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and nuclear transfer embryos

The effects of activation by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and SCNT embryos were investigated. The results revealed that the blastocyst development of parthenogenetic embryos was significantly higher (P < 0.05) in 6-DMAP activated oocytes, compared to those activated with CHX (21.0 +/- 0.9 vs. 14.9 +/- 0.5, respectively). In contrast, the blastocyst frequencies did not significantly differ (P > 0.05) between the two activation treatment groups for SCNT embryos. The 6-DMAP or CHX treatment did not result in any significant difference in the blastocyst total cell number in either parthenote or SCNT embryos. The chromosomal analysis revealed that all the parthenogenetic embryos (100.0%) derived from 6-DMAP treatment, were chromosomally abnormal whereas in CHX-treated embryos, it was significantly lowered (93.6%, P < 0.05). Conversely, the proportions of chromosomally abnormal SCNT embryos did not significantly differ (P > 0.05) among the 6-DMAP and CHX- treated embryo groups (60.0% vs. 56.2%, respectively). This study demonstrated that oocyte activation agents such as DMAP and CHX have differing effects on meiotic or mitotic nuclei. The study also highlighted the feasibility of using bovine X and Y chromosome specific painting probes in sheep embryos.     

Effect of different parthenogenetic activation methods on the developmental competence of in vitro matured porcine oocytes

The aim of this study was to investigate the effect of electrical pulse, ethanol, and ionomycin combined with cycloheximide (CHX), cytochalasin B (CB), and 6-dimethylaminopurine (6-DMAP) on parthenogenetic developmental competence of in vitro matured porcine oocytes. In experiment 1, oocytes were treated with direct current electrical pulse (DC pulse) and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX, and CB + 6-DMAP for 6 h, respectively. The rate of blastocyst development in DC pulse + CB + 6-DMAP group was significantly higher than those in other groups (42.4% vs 23.9% approximately 35.8%; P < 0.05); however, there were no differences in both of the cleavage rate and the cell number of blastocysts among four groups. In experiment 2, oocytes were treated with NCSU-23 medium containing 20 muM ionomycin for 40 min and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX and CB + 6-DMAP for 6 h, respectively. The rates of cleavage and blastocyst development in ionomycin + 6-DMAP group were higher than those obtained in other groups (66.2% vs 46.3% approximately 57.3%; 22.3% vs 7.4% approximately 16.1%; P < 0.05). In experiment 3, the activation effects of ethanol combined with 6-DMAP, CHX, CB + 6-DMAP and CB + CHX were investigated. The rates of cleavage and blastocyst development in ethanol + CB + 6-DMAP group were significantly higher than those in other groups (55.5% vs 42% approximately 46.2%; 18.0% vs 7.1% approximately 11.9%; P < 0.05). In experiment 4, the optimal activation protocols in each group plus DC pulse + ionomycin + 6-DMAP were compared. The results showed the rates of cleavage in DC pulse + CB + 6-DMAP group and ionomycin + 6-DMAP were higher than those in ethanol + CB + 6-DMAP and DC pulse + ionomycin + 6-DMAP (73.8-74.4% vs 56.5-57.5%; P < 0.05), but the blastocyst development only in DC pulse + CB + 6-DMAP group was significantly higher than that in other groups (34.1% vs 13.4% approximately 22.3%; P < 0.05). Total cell number of blastocysts in the group of DC pulse + ionomycin + 6-DMAP was higher than that in other groups (34.1 vs 25.3-27.2; P < 0.05). In conclusion, DC pulse, ethanol, CB, and 6-DMAP all affected the parthenogenesis of porcine oocytes matured in vitro, but their combination of DC pulse + CB + 6-DMAP showed the best result in both of cleavage and blastocyst development.     

Effects of monensin and forage:concentrate ratio on feed intake, endocrine, and ovarian function in beef heifers

Several reports have suggested that a treatment before in vitro maturation might improve oocyte competence and increase its developmental potential. Therefore, the objectives of the present study were to establish the kinetics of IVM in Zebu oocytes, to assess the effect of 6-dimethylaminopurine (6-DMAP), a phosphorylation inhibitor, on meiotic resumption, and to verify the developmental potential of the blocked oocytes after removal of the inhibitory conditions. To establish the kinetics of in vitro maturation 1422 oocytes were obtained from Nellore cows ovaries and matured in presence and absence of gonadotropins. Samples of oocytes were taken from culture at 0, 6, 9, 12, 15, 18, 21 and 24h, and the oocytes were fixed, stained and evaluated for nuclear morphology. Germinal vesicle break down (GVBD) occurred between 6 and 12h of culture in both groups. By 21h the majority of the oocytes had reached metaphase II in presence (71%) and absence (62%) of gonadotropins. In order to examine the inhibitory effect of 6-DMAP, 585 oocytes were cultured for 12, 18 and 24h in the presence or absence of 2mM of 6-DMAP. At each time point the oocytes were evaluated for nuclear morphology. To test the reversibility of meiotic inhibition 366 oocytes were incubated for 0, 12, 18 and 24h in the presence of 6-DMAP and then were transferred to the maturation medium and cultured for further 24h. A total of 429 oocytes were used to evaluate the developmental potential after meiotic inhibition. The oocytes were cultured in the presence of 6-DMAP for 0, 12, 18 and 24h, and then were matured, fertilized and cultured in vitro. Culture of bovine oocytes in the presence of 6-DMAP up to 24h completely blocked GVBD with more than 90% of the oocytes at GV stage. The inhibitory effect of 6-DMAP was fully reversible since maturation rates were similar (P>0.05) among all treatment groups. The evaluation of embryo development after various periods of meiotic blockage showed that inhibition, regardless the time period, had no effect (P>0.05) on penetration and cleavage rates. However, the proportion of embryos at blastocyst stage was reduced after inhibition for 12 (20.2%), 18 (20.1%) and 24h (19.0%) compared with the control group (35.6%). 6-DMAP has a reversible effect on maintenance of meiotic arrest, but reduced further embryo development.     

Parthenogenetic development and protein patterns of newly matured bovine oocytes after chemical activation

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°C in 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 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. 49:298–307, 1998. © 1998 Wiley-Liss, Inc.     

Germinal vesicle chromatin configuration and meiotic competence is related to the oocyte source in canine

This study investigated the effect of deriving oocytes from different stages of the estrous cycle on oocyte diameter, germinal vesicle (GV) chromatin configuration, and in vitro meiotic competence in canine oocytes. Cumulus oocyte complexes (COCs) were recovered from both ovaries during anestrous, follicular, and luteal phases and in vivo ovulated oocytes. The diameter of canine oocyte was compared with or without the zona pellucida (ZP) before in vitro maturation (IVM). Also, GV chromatin configuration was evaluated before (0 h) or 72 h after IVM by fixation with 3.7% formaldehyde supplemented with 10 microg/ml Hoechst 33342 for 30 min. COCs were matured in TCM199 supplemented with 10% fetal bovine serum (FBS), 0.6 mM cysteine, 0.2 mM pyruvic acid, 50 microg/ml gentamycin sulfate, and 20 microg/ml 17beta-estradiol (E(2)) at 39 degrees C and 5% CO(2) in air for 72 h. The diameter of in vivo ovulated oocytes with the ZP (167.5+/-12.7 microm) or without ZP (133.9+/-5.3 microm) was significantly greater (p<0.05) than those of anestrous, follicular, and luteal oocytes (with ZP, 151.2+/-7.4, 153.1+/-8.8 and 152.8+/-5.4 microm, respectively; without ZP, 115.3+/-7.6, 122.1+/-4.9 and 114.3+/-6.6 microm, respectively). At 0 h, the GV-II configuration was more prevalent in oocytes from anestrual ovaries than from follicular or luteal ovaries or in vivo ovulated oocytes (63.6% versus 14.8%, 33.0%, and 0.0%; p<0.05), whereas the proportion of oocytes with the GV-V configuration was higher in follicular phase and ovulated oocytes than in oocytes from anestrus and luteal phase (57.4% and 100% versus 2.0% and 22.7%; p<0.05). However, oocytes in luteal phase exhibited diverse GV configurations (10.3%, 33.0%, 16.5%, 13.4%, and 22.7% in GV-I, GV-II, GV-III, GV-IV, and GV-V, respectively). After 72 h post-IVM, a greater percentage of in vivo ovulated oocytes progressed to MII than those oocytes collected during anestrous, follicular, and luteal phases (50.0% versus 5.5%, 11.5%, and 9.1%; p<0.05). In conclusion, the oocyte diameter, GV chromatin configuration, and meiotic maturation of canine COCs are related to the oocyte source. These results indicated that the oocyte source could be critical to nuclear progression to MII stage in canines.     

Effect of 6-dimethylaminopurine on electrically activated in vitro matured porcine oocytes

The effect of the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), on the maturation promoting factor (MPF) activity, pronuclear formation, and parthenogenetic development of electrically activated in vitro matured (IVM) porcine oocytes was investigated. Oocytes were activated by exposure to two DC pulses, each of 1.5 kV/cm field strength and 60 microsec duration, applied 1 sec apart. In the first experiment, subsequent incubation with 2 or 5 mM 6-DMAP for 3 hr increased the incidence of blastocyst formation compared with no treatment, whereas incubation with 2 or 5 mM 6-DMAP for 5 hr did not. In the proceeding experiments, oocytes exposed to 6-DMAP were incubated with 2 mM of the reagent for 3 hr. Assaying histone H1 kinase activity in the second experiment revealed that the levels of active MPF in electrically activated oocytes treated with 6-DMAP were depleted more rapidly and remained depleted for longer compared with electrical activation alone. The kinetics of MPF activity following 6-DMAP treatment were similar to that found in inseminated oocytes in the third experiment. The effect of 6-DMAP was correlated with an increased incidence of parthenogenetic blastocyst formation. A fourth experiment was undertaken to examine the diploidizing effect of 6-DMAP. Electrically activated oocytes treated with 6-DMAP and cytochalasin B, either alone or in combination, displayed a higher incidence of second polar body retention compared with those that were untreated or treated with cycloheximide alone. After 6 days of culture in vitro, parthenotes exposed to 6-DMAP, either alone or in combination with cytochalasin B, formed blastocysts at a greater rate compared with those exposed to cytochalasin B alone, cycloheximide alone or no treatment. The combined 6-DMAP and cytochalasin B treatment induced the highest rate of blastocyst formation (47%), but the numbers of trophectoderm and total cells in these blastocysts were lower compared with those obtained following exposure to 6-DMAP alone. These results suggest that the increased developmental potential of 6-DMAP-treated parthenotes may be attributable to the MPF-inactivating effect of 6-DMAP, rather than the diploidizing effect of 6-DMAP.     

Embryo development,oocyte morphology,and kinetics of meiotic maturation in bovine oocytes exposed to 6-dimethylaminopurine prior to in vitro maturation

The developmental competence of bovine follicular oocytes that had been meiotically arrested with the phosphokinase inhibitor 6-dimethylaminopurine (6-DMAP) was studied. After 24 h in vitro culture with 2 mM 6-DMAP, 85 ± 12% of the oocytes were at the germinal vesicle stage compared to 97 ± 3% at the start of culture (P > 0.05). After release of the 6-DMAP inhibition, followed by 24 h IVM, 82 ± 18% were at MII stage, compared with 93 ± 7% in the control group (P > 0.05). The 6-DMAP oocytes displayed a much higher frequency of abnormal MII configurations than the control oocytes (67% vs 23%; P < 0.0001). In addition spontaneous oocyte activation was more frequent than among control oocytes (5% vs 0.3%; P 0.0006). After IVF of 6-DMAP oocytes, normal fertilization was lower (76 ± 8% vs 89 ± 7%; P < 0.01), oocyte activation higher (11 ± 5% vs 2 ± 2%; P < 0.01), and polyspermy slightly but not significantly higher (8 ± 7% vs 4 ± 4%; P > 0.05), compared with the control group. Cleavage was lower (61 ± 13% vs 81 ± 6%; P < 0.001), as well as day 8 blastocyst formation (17 ± 7% vs 36 ± 8%; P < 0.001). The MII kinetics was different for 6-DMAP and control oocytes. Maximum MII levels were reached at 22 h IVM in both groups, but 50% MII was reached at 17 h in 6-DMAP oocytes, compared to 20 h in control oocytes. Ultrastructure of MII oocytes was similar in the two groups, but in 6-DMAP oocytes the ooplasmic vesicle pattern at GV was at a more advanced stage than in control oocytes. In conclusion, 6-DMAP exposure of GV oocytes prior to IVM induce asynchronous cytoplasmic maturation, leading to aberrant MII kinetics. Thus, at the time of insemination a smaller cohort of oocytes will be at the optimal stage for normal fertilization and subsequent blastocyst development. Mol. Reprod. Dev. 50:334–344, 1998. © 1998 Wiley-Liss, Inc.     

Cytoplasmic activation of starfish oocytes by sperm and divalent ionophore A-23187

The relationship between onset of the early cytoplasmic stages of oocyte activation (vitelline membrane separation and elevation) and nuclear meiotic maturation was investigated in starfish oocytes after their exposure to divalent ionophore (A-23187) or sperm. Meiotically mature oocytes, isolated in calcium-free seawater, underwent activation in response to sperm or ionophore as previously reported. Large, immature starfish oocytes, arrested in prophase I of meiosis (germinal vesicle stage), underwent vitelline membrane elevation when treated with divalent ionophore A-23187 or starfish sperm. Histological studies demonstrated that cortical granule breakdown in the oocyte cortex was associated with vitelline membrane elevation after these treatments. Activation of oocytes by sperm occurred only in response to starfish sperm. Sea urchin, sand dollar, surf clam, or marine worm sperm did not induce vitelline membrane elevation of either immature or mature starfish oocytes. Sperm- or ionophore-activated immature oocytes underwent nuclear maturation after addition of the meiosis-inducing hormone, l-methyladenine; however, parthenogenetic development did not occur and embryonic development was markedly inhibited. In contrast to previous studies, the present results indicate that cytoplasmic activation can be initiated before and without hormone induction of the nuclear maturation process. Differentiation of the oocyte cell surface or cortex reactivity therefore appears to occur during oogenesis rather than as a consequence of maturation. The data further support the view that divalent ions mediate certain of the early activation responses initiated by sperm at the time of fertilization and that synchronization of fertilization to the meiotic process in the oocyte is important for the occurrence of normal development.     

Time-dependent effects of heat shock on the zona pellucida ultrastructure and in vitro developmental competence of bovine oocytes

The aim of this study was to determine the effects of different exposure lenght to heat shock (HS) during in vitro maturation (IVM) on zona pellucida (ZP) ultrastructure and developmental competence of bovine oocytes. Cumulus-oocyte complexes (COCs) were matured in vitro (IVM) at 38.5 °C for 24 h (control group, CG), or incubated at 41 °C (HS) for 6 h (HS-6h), 12 h (HS-12h), 18 h (HS-18h), and 22h (HS-22h) followed by incubation at 38.5 °C to complete a full 24-h period of maturation. After IVM, oocytes were subjected to scanning electron microscopy (SEM) or in vitro fertilization and culture until the blastocyst stage. For heat-shocked oocytes, with exception of those in the HS-6h group, SEM examinations revealed that ZP surfaces were rough and characterized by a presence of spongy network. Oocytes from the HS-22h group displayed an increase in the number of pores, as well as a higher proportion of oocytes with amorphous ZPs. The proportion of oocytes that reached metaphase II (MII) stage decreased in all HS groups, regardless of the duration of exposure to 41 °C. These results provide evidence that HS during IVM for 12–22 h reduces the developmental competence of bovine oocytes, increasing the percentage of oocytes with abnormal chromosomal organization, and reducing fertilization and blastocysts formation rate. The effects of HS were more pronounced for the 22-h exposure group. The damage induced by HS on oocyte function clearly increased upon exposure to elevated temperature.