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.     

Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos.

The objective of this study was to examine the effects of different culture media used for maturation of bovine oocytes on in vitro embryo development following in vitro fertilization. Oocytes were aspirated from 2-5 mm follicles of ovaries collected at a local abattoir. The oocyte-cumulus complexes (OCCs) were cultured for 23-25 h in one of seven commercially available media supplemented with 6 mg/ml bovine serum albumin (BSA), 0.25 mM pyruvate, 10 micrograms/ml luteinizing hormone (LH), 0.5 microgram/ml follicle-stimulating hormone (FSH), and 1 microgram/ml estradiol. After maturation for 23-25 h, all eggs were subjected to the same in vitro fertilization protocol using modified TALP medium and subsequently cultured in the same serum-free embryo culture medium (HECM-1/BSA) for 8 days, after which embryo development was assessed. Five media (SFRE, MEM alpha, TCM199, MEM alpha/+, RPMI:MEM alpha) better supported normal oocyte maturation as determined by embryo development to the two-cell (76-82%), morula/blastocyst (25-32%), and blastocyst (12-19%) stages. Oocytes that were matured in Waymouth's medium MB 752/l or Ham's F-12 had a significantly reduced incidence of cleavage to the two-cell stage (52% and 37%, respectively), which was not attributed to failure of fertilization. Of the eggs that did cleave to the two-cell stage in these two media, 27% and 9% developed to morulae/blastocysts but only 6% and 3%, respectively, developed into blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of protein synthesis in morphologically classified bovine follicular oocytes before and after maturation in vitro

Bovine cumulus oocyte complexes (COCs) were isolated from antral ovarian follicles (4-8 mm). Immature COCs were classified into four categories, based on the homogeneity and clearness of the ooplasm and the transparency and compactness of the cumulus investment. In this study, the incorporation of TCA-precipitable 35S-methionine and the protein synthesis patterns of oocytes of these four categories were examined. Before maturation in vitro, similar incorporation rates and identical protein synthesis patterns were observed between oocytes of categories 1-3. Immature oocytes of category 4 showed reduced incorporation rates and exhibited aberrant protein synthesis patterns. After maturation in vitro, the patterns of category 4 oocytes were identical with the patterns of those in categories 1-3. The incorporation of 35S-methionine into in vitro matured oocytes was lower (P less than .001) in all categories. Based on these results, it is concluded that the initial classification of oocytes into four categories can be reduced to two categories.     

Parthenogenetic development of activated in vitro matured bovine oocytes

Bovine oocytes matured in vitro for 26 hours were electrically stimulated 1) by a single pulse (Treatment A); 2) by 3 pulses 30 minutes apart (Treatment B); 3) by a single pulse followed by 5 minutes of incubation in the stimulation medium (Treatment C); or 4) by a single pulse at 27 hours of maturation (Treatment D). The oocytes were then cultured for up to 8 days to assess parthenogenetic activation and development. Each electrical stimulation consisted of a 60-mus square wave pulse of 2.5 or 3.6 kV/cm. Treatment A was less effective than the other treatments (P<0.05), activating 47 or 59% of oocytes at 2.5 or 3.6 kV/cm, respectively. However, there were no differences due to voltage nor among the other treatments, which activated 64 to 78% of the oocytes. The cleavage rate, 28 to 38%, was not affected by the activation treatment, but development to the 8-cell stage or beyond was greater after activation with the higher voltage. While the numbers of morulae or blastocysts resulting from any given treatment were too small to support meaningful statistical comparison, the results indicate that bovine parthenogenotes produced in vitro are capable of development to the blastocyst stage.     

Parthenogenetic development of bovine oocytes matured in vitro for 24 hr and activated by ethanol and cycloheximide

This research was undertaken to improve development of parthenogenetic embryos following various combined treatments of ethanol and cycloheximide. In Experiment 1 in vitro matured oocytes (IVM, 24 hr) were treated with 7% ethanol for 5 min followed by incubation in 10 μg/ml cycloheximide in Medium 199 for 0 (control), 5, 10, and 20 hr. Development to 2–8 cells following culture for 3 days was similar among treated groups (32–41%; P > 0.05), which was higher than that of controls (6%; P < 0.05). Experiment 2 compared pre-ethanol exposures for 0, 1, 2.5, and 5 min, followed by 5 hr cycloheximide treatment on activation development. One- to 5-min groups resulted in 42–44% cleavage contrasted to 1–12% for controls (P < 0.05). Experiment 3 examined the effect on oocyte development of ethanol and different concentrations of cycloheximide (0, 1, 5, and 10 μg/ml). Cleavage to 2–8 cells was similar among the 5 and 10 μg/ml cycloheximide groups (36% and 42%, P > 0.05) but lower (P < 0.05) for the 1 μg/ml group (24%) and the controls (2–13%). When 5 μg/ml cycloheximide was used (Experiment 4), pre-exposure to ethanol (1, 2.5, and 5 min) resulted in more oocytes cleaved (38–41%) than in the cycloheximide alone group (0%) or the control (0%, P < 0.05). Experiment 5 tested blastocyst development of the activated oocytes with or without cytochalasin B treatment. Oocytes developed to blastocyts were 0%, 14%, 3%, and 3% (P < 0.05), respectively, for control, treatment with ethanol and cycloheximide in the presence, or absence of cytochalasin B, or electrical pulse plus cycloheximide. In conclusion, the combined ethanol and cycloheximide treatment supported high rates of parthenogenetic development using 24 hr IVM bovine oocytes. Blastocyst rate was significantly higher when cytochalasin B was added to the combined activation regimen. © 1994 Wiley-Liss, Inc.     

Parthenogenetic development of in vitro matured porcine oocytes treated with chemical agents

Parthenogenetic activation is a possible way to produce homogeneous embryos with the same ploidy. These embryos could develop to the blastocyst stage during the cultivation. Probably such embryos could be used in other areas of biotechnology. The objectives of the present study were first to assess the ability of strontium-chloride to induce activation and parthenogenetic development in porcine oocytes in comparison with cycloheximide and 6-dimethylaminopurine; second to verify whether the combination of the two treatments improved activation and parthenogenetic development rates. At first, the effects of cycloheximide, 6-dimethylaminopurine and strontium-chloride on oocyte activation and embryonic development were compared. Oocytes from slaughterhouse ovaries were matured for 42h in tissue culture medium (TCM) 199 at 38.5 degrees C, 5% CO(2) in air. Matured oocytes were activated with 10mM strontium-chloride (S), 0.04mM cycloheximide (CX), 2mM 6-dimethylaminopurine (D) for 5h. The activation rate was judged by pronuclear formation of oocytes. Following the activation, oocytes were incubated in NCSU 37 medium for 6 days and in all groups more than 45% of oocytes activated. The activation rate for CX treatment was significantly higher (P<0.05) than for D (57.37+/-4.21% and 48.09+/-3.43%, respectively). In a second experiment in vitro matured porcine oocytes were activated using a combined treatment of strontium-chloride with cycloheximide (SCX) and strontium-chloride combined with 6-dimethylaminopurine (SD). In S and SCX groups more than 50% of oocytes were activated (53.29+/-5.39% and 54.3+/-7.29%, respectively). However a large portion of embryos stopped their development at the two- or four-cell stage. Significantly higher numbers of embryos could reach the eight-cell stage in SD and SCX than for S (7.8+/-1.0%, 7.2+/-4.0% and 3.9+/-3.1%, respectively). Blastocyst formation was only observed in S, CX and SCX. These results show that porcine in vitro matured oocytes can be artificially activated by cycloheximide, 6-dimethylaminopurine and strontium-chloride.     

Parthenogenetic activation of cattle follicular oocytes in vitro with ethanol

Cattle follicular oocytes cultured in vitro for 24–33 h were treated with ethanol to induce artificial activation. When oocytes were cultured for 27–33 h before ethanol treatment, 60–68% of oocytes were activated and were found to have a female pronucleus(ei). In contrast, maturation culture of oocytes for 24–26 h resulted in low activation rates (25–38%). The female pronucleus was formed in the activated oocytes within 8–10 h of incubation after ethanol treatment. And it became visible under interference-contrast microscope by centrifugation for 3 min at 15,000g and 10 min at 20,000g. These results indicate that ethanol treatment is effective for activation of cattle follicular oocytes and that the pronucleus formed in the activated oocyte can be visualized by centrifugation.     

Control of in vitro maturation of bovine cumulus-oocyte complex by preovulatory granulosa cells

The present study was designed to investigate (1) the influence of the secretions of follicular cells on the in vitro maturation of bovine cumulus-oocyte complexes (COCs) and (2) the origin of the factors controlling the metabolic function of cumulus cells during the preovulatory period. Preovulatory granulosa cells were collected from synchronized heifers either before or 7–9 hr after the luteinizing hormone (LH) surge, and their secretions were recovered after a 3 hr incubation. Follicular fluids (FFs) originating from the same follicles and sera from the same animals were also collected. The effects of FFs, sera, and secretions of granulosa cells on COC metabolism were compared during 24 hr of culture. FF stimulated cumulus expansion, progesterone secretion, and overall protein synthesis by COCs but decreased the amount of a major protein of 28 kDa. The time at which FF was collected influenced both cumulus expansion and protein synthesis by COCs. The effects of FF on COC metabolism were detected at the lowest protein concentration studied (0.073 mg/ml) and could be mimicked with serum, but only at a protein concentration 100-fold higher. The inhibitory effect of FF and serum on the amount of the 28 kDa protein was reproduced with the secretions of granulosa cells, acting at protein concentrations five- and 500-fold lower, respectively. However, the secretions of granulosa cells enhanced slightly cumulus expansion and had no effect on progesterone secretion and overall protein synthesis by COCs. These results suggest that COC metabolism is influenced both by endocrine and by local factors secreted by granulosa cells in response to gonadotropins. The paracrine control of COC metabolism by preovulatory granulosa cells could be exerted not only via intercellular contacts but also via substances secreted in FF. © 1993 Wiley-Liss, Inc.     

Parthenogenetic activation of bovine oocytes maturing in vitro]

The effect of hyaluronidase (0.3%) and killed bull spermatozoa on the parthenogenetic activation of cow oocytes matured in vitro until metaphase II was studied. It is shown that hyaluronidase, killed spermatozoa, and both agents in combination activate 3.4, 15.0 and 29.6% oocytes, respectively.     

Parthenogenetic activation and subsequent development of rat oocytes in vitro.

Studies were undertaken to determine whether electrical stimulation, or ethanol treatment alone or in combination with 6-dimethylaminopurine (6-DMAP) influenced the rate of parthenogenetic activation of rat oocytes. The percentages of activated oocytes with pronuclei (89-91%) and those developed to the two-cell stage (68-72%) were significantly higher after electrical stimulation with direct current (DC) at 100 V/mm, 99 microsec once or twice, than when other DC voltages (75, 150, and 200) were applied or when ethanol or 6-DMAP treatment was given alone. However, none of the activated oocytes developed beyond the four-cell stage. The percentages of activated oocytes with pronuclei (100%) that developed to the two-cell (100%), eight-cell (89%) and blastocyst stages (50%) were significantly higher when electrical stimulation was followed by treatment with 2 mM 6-DMAP for 4 hr than when other combined procedures were applied. In conclusion, the results of the present study clearly showed that combined treatment of electrical stimulation or ethanol with 6-DMAP induces parthenogenetic activation and subsequent development of rat oocytes in vitro.     

Embryo development after vitrification of immature and in vitro-matured equine oocytes

Effects of meiotic stage and cumulus status on development of equine oocytes after vitrification was evaluated. Immature oocytes with corona radiata (IMM); in vitro-matured oocytes with corona radiata (MAT CR+); and in vitro-matured oocytes denuded of cumulus (MAT CR-) were vitrified using the Cryotech® method. Warming medium was equilibrated either in 5% CO₂ or Air. IMM oocytes underwent in vitro maturation after warming. Recovery, survival, and maturation rates, and cleavage and blastocyst rates after ICSI, were evaluated. Recovery was higher for oocytes warmed in CO₂- than Air-equilibrated medium (86 ± 3 vs. 76.9 ± 4%, respectively). Maturation for all vitrified-warmed oocyte treatments (37 ± 6.5 to 45.9 ± 5.8%) was not different from control (50 ± 4.1%), except for MAT CR- CO₂ (20.3 ± 4.6%). Cleavage for MAT CR- CO₂ and Air groups was similar to control (67.7 ± 12.1, 71.4 ± 8.1, and 78 ± 5.3%, respectively). One blastocyst was produced (MAT CR + CO₂), representing the first equine blastocyst reported after vitrification of an in vitro-matured oocyte.     

Thyroid stimulating hormone enhancement of bovine oocyte maturation in vitro.

Efforts to improve bovine embryonic development in vitro involved study of effects of thyroid stimulating hormone (TSH) alone or in combination with LH on bovine oocyte maturation (IVM). Putative effects were assessed by observing cumulus expansion (CE), fertilization (IVF), and development to morulae/blastocysts (M/B). Effects of prolactin (PRL) were also investigated. Variables for the 24-hr IVM interval were no hormone (control), TSH (0.1, 0.5, or 1.0 micrograms/ml) or PRL (10, 100, or 1000 micrograms/ml), luteinizing hormone (LH) (0, 10, or 100 micrograms/ml) + TSH (0.1 or 0.5 micrograms/ml), and serum (20%, v/v) + 0.5 micrograms TSH/ml; data were from 4-5 trials for each IVM treatment. Higher proportions of oocytes exhibited complete CE with hormones or serum than without (P less than 0.05). All oocytes (with and without CE) were inseminated with heparin-capacitated sperm. A higher proportion of inseminated oocytes cleaved after IVM with 0.5 micrograms TSH/ml (53.4%) than for other TSH treatments (P less than 0.05). The combination of TSH (0.1 and 0.5 micrograms/ml) with 10 micrograms LH/ml for IVM enabled higher proportions (P less than 0.05) of ova to fertilize (67.4 and 69.2%) than did medium alone (28.3%), LH (10 micrograms/ml) alone (54.1%) or serum + 0.5 micrograms TSH/ml (55.6%). No improvement in proportions undergoing fertilization was seen after addition of TSH to 100 micrograms LH/ml for IVM. Frequency of CE and cleavage did not differ among PRL treatments. More M/B developed from cleaved ova after IVM with LH or TSH than with PRL or no hormone (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Nuclear dynamics of parthenogenesis of bovine oocytes matured in vitro for 20 and 40 hours and activated with combined ethanol and cycloheximide treatment

Bovine oocytes matured in vitro (IVM) for 20 hr vs. 40 hr were treated for activation with 7% ethanol in Dulbecco's phosphate-buffered saline for 5 min followed by incubation in M199 + 7.5% fetal calf serum containing cycloheximide (10 μg/ml). TreatedIVM oocytes and the controls (no ethanol and cycloheximide exposures) were fixed after 0, 1, 2, 3, 4, 5, 7, 10, and 20 hr of incubation and stained 24 hr later with 1% acetoorcein to examine nuclear events. Different stages of nuclear development of the activated oocytes were identified on the basis of nuclear and chromosomal morphology. Pronuclear development was classified into four stages (PN I, II, III, and IV) according to pronuclear progression in chromatin decondensation, nucleoplasm appearance, and nuclear size. The results demonstrated that the combined activation treatment effectively drove the IVM oocytes, both young (20 hr) and aging (40 hr), out of metaphase arrest. The activation rates for young oocytes examined immediately after 0, 1, 2, 3, 4, 5, 7, 10, and 20 hr of incubation with cycloheximide were, respectively, 7%, 24%, 77%, 96%, 92%, 97%, 98%, 93%, and 98%. For aging oocytes (40 hr) the corresponding activation values at the same time intervals were 6%, 84%, 100%, 100%, 100%, 100%, 98%, 100%, and 100%, respectively. These values were significantly higher than those for the corresponding controls. The activated aging oocytes achieved peak activation response more rapidly than did young oocytes. In addition, nuclear events in aging oocytes proceeded faster than those in young ones. Spontaneous activation rates of the aging oocytes were also higher (6–57%) than those of the young ones (0–14%). © 1994 Wiley-Liss, Inc.     

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 bovine oocyte maturation in vitro.

Bovine cumulus-oocyte complexes (COCs) were collected from 4-8 mm follicles and graded into four categories on their morphological characteristics. These four categories were matured in vitro and processed for transmission electron microscopy at 24 h after the onset of culture. The morphology of the four groups of oocytes was analysed and compared with that of oocytes that had matured in vivo and were collected 20-23 h after the preovulatory luteinizing hormone peak. After in vivo maturation, oocytes formed a homogeneous group with respect to their morphological characteristics. After in vitro maturation, the oocytes formed a heterogeneous group with respect to their morphology between as well as within the four categories of oocytes. Oocytes from the first three categories showed the same morphology after in vitro maturation. The fourth category showed some specific characteristics: 1) vacuolization, 2) flattening of cumulus cells, and 3) almost complete lack of cortical granules in some category 4 oocytes. These characteristics are interpreted as signs of degeneration. Besides these aspects of degeneration, other deviations from normal development were seen: 1) retraction of cumulus cell process endings from the oocyte without the breaking down of these processes, 2) retardation of some aspects of the cytoplasmic maturation, and 3) incomplete cumulus expansion. It is concluded that oocytes capable of development in vitro show a large morphological variability before the onset of culture. In vitro maturation systems can support normal development, but many oocytes show signs of degeneration and deviant development after in vitro maturation.     

Parthenogenetic activation by electric stimulus of bovine oocytes matured in vitro

The purpose of this study was to determine the optimal conditions for parthenogenetic activation of in vitro-matured bovine oocytes by electric stimulus in vitro. Oocytes were assigned to a factorial treatment structure with direct current ranging from 0.5 to 1 KW/cm for 25 to 100musec and single or double pulses. The optimal conditions for activation were found to be direct current pulses of 1 KV for 25 musec x 2, under which 84% of stimulated oocytes formed one (70%), two (13%) or three (2%) pronuclei. When the stimulated oocytes were incubated in a culture medium containing cytochalasin B, 80% of the oocytes formed two pronuclei. A proportion of the parthenogenetic oocytes developed to the two-cell stage or higher (27%, 83 312 ) in vitro; however, this was significantly (P<0.001) lower than that of the oocytes fertilized in vitro (46%, 736 1608 ).     

Involvement of steroid hormones in bovine oocytes maturation in vitro.

Influences of steroid hormone additions or of their binding by specific antisera on nuclear maturation and subsequent fertilization and cleavage of bovine oocytes were studied in vitro. It was found that progesterone in doses of 50 ng/ml, 250 ng/ml, 1 μg/ml or 5 μg/ml stimulates reinitiation and in doses of 1 or 5 μg/ml stimulates further development of meiosis. Antiserum to progesterone had opposite effects on nuclear maturation, but has no influence on the ability of matured oocytes to subsequent fertilization and cleavage. Testosterone additions (10 ng, 100 ng, 1 μg or 5 μg/ml) did not influence nuclear maturation, but antiserum to this hormone inhibited both meiosis reinitiation and completion, as well as lowered the rate of oocytes fertilized and embryos obtained. Estradiol (5, 50, 100 or 500 ng or 5 μg/ml) treatment stimulated reinitiation, but not nuclear maturation. Antiserum to estradiol activated both reinitiation, development and completion of meiosis, but the cells matured by estradiol deficit were as a rule uncapable of fertilization and further cleavage. Estradiol addition (1 μg/ml) to maturation medium together with FSH (10 μg/ml) (but not of FSH alone) lead to a significantly higher rate of fertilization and cleavage of matured cells.

Results obtained suggest (1) relative independence of reinitiation, further development of nuclear maturation and cytoplasmic maturation regulation in bovine oocytes as well as (2) the involvement of steroid hormones in these three processes.     

Holding bovine oocytes in the absence of maturation inhibitors: kinetics of in vitro maturation and effect on blastocyst development after in vitro fertilization

Holding immature oocytes before maturation simplifies the transport of oocytes and aids in scheduling later manipulations. We examined the effect of holding bovine oocytes in the absence of meiotic inhibitors on their subsequent meiotic and developmental competence. Oocytes were matured immediately after recovery (control) or were held in a mixture of 40% TCM 199 with Earle's salts, 40% TCM 199 with Hanks' salts, and 20% FBS, at room temperature for 16 to 18h (EH-held) and then matured. Chromatin status was determined at 0, 10, 14, 18, and 22h of maturation culture. Oocytes were fertilized in vitro after either 18 or 22-24h maturation. The EH treatment maintained oocytes at the germinal vesicle stage (79.3%, vs. 87.7% for control oocytes at 0h; P>0.05). Upon culture, held oocytes matured more quickly than did control oocytes. The proportions of mature oocytes were not significantly different between groups at 18h (EH-held, 80.6% and control, 79.3%); however, after 22h significantly more EH-held than control oocytes had degenerated (24.1% vs. 4.5%, P<0.0001). Blastocyst development was similar between groups for oocytes fertilized after 18h maturation (EH-held, 29.6% and control, 27.8%). When oocytes were fertilized after 22-24h maturation, EH-held oocytes yielded lower blastocyst development than did control oocytes (16.5% vs. 29.3%, P<0.05). In conclusion, bovine oocytes may be effectively held in the EH treatment before maturation without adversely affecting meiotic or developmental competence. However, holding affects the kinetics of maturation and this must be taken into account when subsequent manipulations are performed.