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 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.     

Optimized combined electrical-chemical parthenogenetic activation for in vitro matured bovine oocytes

Sperm-mediated oocyte activation is a complex procedure, both in steps and duration, not yet been completely mimicked during in vitro studies, e.g., parthenogenesis or somatic cell nuclear transfer. Furthermore, parthenogenetic studies have been recognized as a suitable model for studying activation efficiency for nuclear transfer cloning. This study, therefore, was conducted to develop an optimized artificial activation method, based on bovine cloning. In vitro matured bovine oocytes were initially exposed to electrical pulse, used for cell fusion during cloning, and then treated with 15 temporal sequential combinations of 3 chemical activators [calcium ionophore (CI), strontium (SR) and ethanol (ET)], followed by exposure to a protein kinase inhibitor or used for in vitro fertilization as control group. Treated and naturally fertilized oocytes were further cultured for up to 8 days. Embryo development was scored daily and blastocyst cell counting was carried out using differential staining at day 8 of culture. Among 15 temporal sequential combinations of three chemical activators, the best cleavage rates were associated with double (SR-CI, 84.4%), triple (CI-SR-ET, 79.4%) and single (CI, 73.7%) compounds, respectively, which were not significantly different with each other and with in vitro fertilized (IVF) (85.5%). The highest blastocyst rates were gained with ET-SR (24.5%), SR-CI-ET (20.4%) and CI (24.5%) accordingly which were not significantly different with each other but significantly lower than IVF (47%). Embryo cell counting further confirmed reasonably better quality of blastocysts produced using double, triple and single compounds. Although most of the sequential artificial activation compounds induced high cleavage rate, close to IVF, but this did not assure comparable further embryo development to the blastocyst stage. Nevertheless, the results suggest exposure of in vitro matured bovine oocytes to electrical pulse, followed by exposure to CI-6-dimethylaminopurine (6-DMAP) or ET-SR-6-DMAP could be regarded as the optimal artificial activation protocol for in vitro development of parthenogenic bovine oocytes or as a step for activation protocol in cloning procedure.     

Cytogenetic study of bovine oocytes matured in vitro

Described in the present paper is a cytogenetic study of bovine oocytes matured in vitro. The cumulus-oocyte complexes (COC), punctured from ovaries recovered in a local slaughterhouse, were classified into 3 groups according to follicular diameter 1 to 4mm, 5 to 8mm and 9 to 13 mm. Metaphases available for observation were classified as metaphase I, haploid and diploid metaphase II. High levels of haploid metaphases II (90.6, 86.9 and 94.4 %) among the 3 groups of follicular sizes indicated successful meiotic resumption during in vitro maturation and suggested that cytoplasmic maturation may be responsible for low developmental rate after IVM, IVF and in vitro development (IVD).     

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.     

Artificial activation of porcine oocytes matured in vitro

These studies were undertaken to understand the biological basis of artificially induced activation of meiotic metaphase II oocytes and to develop a source of oocytes as recipients for cloning by nuclear transfer. In vitro matured porcine oocytes were pulsed with various voltages of electricity and evaluated for pronuclear formation. The percentage of eggs that activated was significantly greater for the higher voltages. The effect on activation of the temperature of the ovaries returning from the abattoir was evaluated and it was found that oocytes derived from ovaries returning at 29 degrees C activated at lower rates (45.5%) than those returning at 36 degrees C (78.9%). An experiment was designed to evaluate the pH of electroporation medium (EM) and the duration of exposure to EM on activation. Oocytes were placed in EM at various pHs for 5 minutes, pulsed, and immediately removed to TL-Hepes or allowed an additional 2 minutes in EM prior to rinsing in TL-Hepes. The results indicate an optimum activation rate at a pH of 7.0 and allowing the additional 2 minutes in EM. Additional glucosamine (5 mM) had no affect on development of the oocyte to metaphase but reduced the percent pronuclear formation from 61% and 47%. A final experiment evaluated the developmental competence of oocytes subjected to a optimum combination of the above treatments and illustrated that a significant portion of the activated oocytes can show limited signs of cleavage. Thus in vitro matured pig oocytes can be induced to activate at high rates.     

Diacylglycerol-enhanced electrical activation of porcine oocytes matured in vitro

This study was undertaken to examine the effect of second messengers added to the electroporation medium on electric pulse-induced artificial activation of meiotic Metaphase II porcine oocytes. Six separate experiments evaluated second messengers added to electroporation medium. When added to electroporation medium, neither phospholipase C (PLC: 0 to 2.5 Units/ml), D-myo-inositol triphosphate (IP(3): 0 to 10,000 muM), nor guanosine 5'-O-(3-thiotriphosphate; GTP-gamma-S: 0 to 100 muM) had any effect (P> 0.05) on activation rates. However, addition of 1,2-dioctanoyl-sn-glycerol (DiC(8)) increased activation rates in a dose-dependent response. At a level of 1,000 muM, DiC(8) resulted in a higher activation rate (P< 0,05) than 0.0, 0.1, 1 or 10 muM of DiC(8) with a pulse, and the 1,000 and 10,000 muM of DiC(8) no-pulse control groups. Effects of DiC(8) (1,000 muM) and IP(3) (100 muM) in combination or individually were investigated. At 1,000 muM, DiC(8) caused a higher rate of activation (P< 0.05) than 100 muM IP(3), but the result was not different from DiC(8) + IP(3). In another experiment, no difference (P> 0.05) was observed between DiC(8), GTP-gamma-S and IP(3), but DiC(8) + GTP-gamma-S + IP(3) + PLC yielded a higher (P< 0.05) activation rate than PLC or the rate of the controls. No significant development (blastocyst) was observed after 5 days of culture in any of the experiments. Protein profiles of activated oocytes, determined by 1D SDS-PAGE, were characteristic of pronuclear-stage embryos. These data indicate that the addition of DiC(8) to the electroporation medium synergistically enhances the rate of activation of electrically stimulated in vitro-matured porcine oocytes.     

Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta

Background  

During natural fertilization, sperm fusion with the oocyte induces long lasting intracellular calcium oscillations which in turn are responsible for oocyte activation. PLCZ1 has been identified as the factor that the sperm delivers into the egg to induce such a response. We tested the hypothesis that PLCZ1 cRNA injection can be used to activate bovine oocytes.     

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.     

Development potential of bovine oocytes matured in vitro or in vivo

Bovine oocytes matured in vivo or in vitro were evaluated after sperm-oocyte incubation for frequency of sperm penetration, frequency of male pronuclei formation, and embryonic development. The frequency of sperm penetration was not different for in vitro matured oocytes (216/295, 73%) vs. in vivo matured oocytes (119/176, 70%). However, formation of male pronuclei was reduced (p less than 0.05) for oocytes matured in vitro (149/216, 69%) vs. in vivo (104/119, 88%). Early embryonic development was evaluated 48 h after the onset of sperm-egg incubations. In vitro matured and fertilized oocytes failed to develop to the 2-cell stage (3/88, 3%), whereas oocytes matured in vivo showed normal development (23/56, 40%) to the 2- and 4-cell stage. Development to the blastocyst stage was evaluated after 5 days in ovine oviducts (in vivo). Morulae and blastocysts were obtained only after in vitro fertilization from oocytes that were in vivo-matured (recovered from oviduct, 14/56, 25%; recovered from follicle, 36/80, 45%). Oocytes that were matured in vitro and fertilized in vitro failed to develop to morulae (0/33) in vivo.     

Laparoscopic oviductal transfer of in vitro matured and in vitro fertilized bovine oocytes

The objective of this study was to obtain normal pregnancy following laparoscopic oviductal transfer of in vitro matured and fertilized bovine oocytes. Methods for in vitro maturation and in vitro fertilization were similar to those previously reported (1). Primary oocytes judged to be potentially viable were cultured for 26 h in modified TCM 199 supplemented with heat-treated fetal calf serum (20% v/v), 5mug/ml FSH (USDA-bFSH-B-1), and 1mug/ml estradiol 17-beta. Oocyte cumulus complexes were microscopically evaluated for maturation (first polar body formation) following a brief treatment with hyaluronidase. Mature oocytes were inseminated with heparin-treated spermatozoa and incubated at 39 degrees C under paraffin oil and moist 5% CO(2), 5% O(2), 90% N(2). In this work, 450 oocytes were recovered at slaughter from ovaries of 42 random cows of unknown reproductive status and 336 oocytes (74.7%) with compact cumulus were selected for culture. Of these, 322 (95.4%) matured in vitro. Of 218 inseminated oocytes, 198 (90.8%) were penetrated by sperm and 83 (38.1%) cleaved, with 102 (46.6%) of the embryos reaching four- to eight-cell stages. None of 40 oocytes not exposed to sperm and none of 30 oocytes inseminated with untreated sperm showed signs of activation. In a control experiment with hormones added, 105 of 115 (91.3%) oocytes matured in vitro and 20 of 105 (19.5%) cleaved following in vitro insemination. Laparoscopy was performed on four synchronized recipients under local anesthesia. A catheter containing three embryos in the two to four cell stages was passed through the operating channel of a direct viewing bronchoscope for deposition in the oviduct ipsilateral to the recipients developing corpus luteum while the fimbria and the mesovarium were manipulated with Semm's forceps. A normal term pregnancy confirmed in vitro fertilization and provides feasibility data for use of laparoscopic methodology developed in this work for testing viability of bovine oocytes and embryos. These results are encouraging for the application of in vitro maturation and in vitro fertilization for overcoming infertility in domestic and endangered species.     

In vitro fertilization and cleavage of in vivo matured bovine oocytes

The effect of the interval between onset of estrus and oocyte collection on in vitro fertilization (IVF) rates has been investigated. The oocytes were surgically collected 6-18 h (Group I), 19-24 h (Group II), 25-29 h (Group III) and 30-36 h (Group IV) after the beginning of estrus. Recognizable stages of nuclear maturation were identified in 54.9% of the oocytes used for IVF (5.9% at germinal vesicle, 31.4% at metaphase I, 17.6% at metaphase II); the other 45.1% were degenerate. Considerable between- and within-cow variation in oocyte morphology, oocyte maturation and IVF results was observed. The cverall fertilization and cleavage rates (to four-cell stages) were 26.5 and 6.0%, respectively. The fertilization rate increased as the interval between onset of estrus and collection increased and was optimal 30-36 h after onset. Thus, onset of estrus proved an effective means of timing oocyte collection for IVF.     

Parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro

The present study was undertaken to determine conditions for parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro. The cumulus-free eggs were submitted to square direct current pulses at output voltage of 1.0 to 2.5 kV/cm for 25 to 200 μsec. The best conditions for the activation of mouse eggs were 1.5 kV/cm for 100 μsec, in which 78% of eggs were activated, 32% of which developed to blastocysts in vitro. When the nonelectric solution (0.3M mannitol) was used for electric stimulation, the activation rate was quite low (16%). Optimal conditions for activation of rabbit eggs were 1.5 kV/cm for 200 μsec, in which 77% of eggs were activated, 25% of which developed to blastocysts. Unlike mouse eggs, rabbit eggs frequently had three pronuclei after electric stimulation. It is clearly shown that electric stimulus can induce parthenogenetic activation of the mouse and rabbit eggs in vitro.     

Parthenogenetic activation of porcine oocytes by electric pulse and/or butyrolactone I treatment

The goal of the present research was to study the parthenogenetic activation of porcine oocytes following treatment with the specific cyclin-dependent kinase inhibitor butyrolactone I (BL I). In Experiment I, the effective dose of BL I was determined by the rates of the subsequent pronuclear formation in oocytes after the activation. In Experiment II, BL I was further tested alone or in combination with an electric pulse. The efficiency of the various treatments to induce activation and parthenogenetic development was examined. In Experiment III parthenogenetic development of activated oocytes in two different media was compared. Cleavage and blastocyst developmental rates were examined, and number of cells in the blastocysts was determined. Our results indicate that, in pig, the optimal activation dose for BL I was 150 microM; a combined electrical and BL I treatment resulted in superior cleavage rates compared to an electric pulse, 150 microM of BL I, or 200 microM of BL I alone (74%, 60%, 41%, and 42%, respectively; P < 0.05); and the rate of parthenogenetic development of activated oocytes to the blastocyst stage in mNCSU37 medium was significantly higher than that in Whitten's medium (59% vs. 5%, P < 0.05) and the resulting day-6 blastocysts had higher cell numbers (35.5 +/- 14.1 vs. 19.5 +/- 2.5). This activation protocol might be useful in porcine nuclear transfer experiments and for the generation of parthenogenetic fetuses.     

Effect of the cumulus on in vitro fertilization of bovine matured oocytes

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO₂ in air and heparin.

In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.     

Vitrification of bovine blastocysts obtained by in vitro culture of oocytes matured and fertilized in vitro.

Two experiments were conducted to assess the viability of bovine blastocysts obtained by in vitro fertilization of oocytes matured in vitro (IVM-IVF) and cryopreserved by vitrification. In Expt 1, the optimal concentrations of glycerol and 1,2-propanediol in the basic medium (modified TCM199) for cooling and warming without formation of ice crystals were determined by plunging the solution into liquid nitrogen and then warming it in a water bath at 15 degrees C; when both glycerol and 1,2-propanediol were present in the solution (> 45% v/v), vitrification of the medium was observed. In Expt 2, IVM-IVF blastocysts were equilibrated to the mixture of glycerol and 1,2-propanediol (0% to 45%) at 15 degrees C in a stepwise manner as follows: (i) in one step, for 18 min to the final vitrification solution; (ii) in two steps, for 8 min in the first step and 10 min in the second step; (iii) in four steps, for 4 min in the first three steps and 6 min in the last step; (iv) in eight steps, for 2 min in each step, but 4 min in the last step; and (v) in 16 steps, for 1 min in each step, but 3 min in the last step. After removal of cryoprotectants, the blastocysts were cultured for 24 h in vitro. The survival rates for the embryos equilibrated in 1, 2, 4, 8 and 16 step(s) were 56, 89, 100, 100 and 100%, respectively. The blastocysts equilibrated in 1, 2, 4, 8 and 16 steps were vitrified by plunging the straws containing them into liquid N2, thawed and cultured in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of in vitro matured bovine oocytes after cryopreservation with different cryoprotectants

In vitro matured bovine oocytes at the metaphase-II stage were slowly frozen in phosphate buffered saline (PBS) containing 1.0 M glycerol, 1.0 M dimethylsulfoxide (DMSO) or 1.0 M propylene glycol (PROH). When thawed rapidly, more (P<0.05) oocytes were morphologically normal after being frozen with DMSO (86%) or PROH (83%) than with glycerol (62%). When inseminated in vitro with frozen-thawed bull spermatozoa, higher (P<0.05) penetration rates were observed in DMSO (79%) or PROH (76%) than in glycerol (48%). The percentages of oocytes developing to the 2-cell stage at 48 h postinsemination were also significantly (P<0.05) higher in DMSO (51%) and PROH (54%) than in glycerol (33%). However, a significant increase in the proportions of 8-cell embryos (46 vs 21 to 26%; P<0.05) at 72 h postinsemination and morulae (14 vs. 6 to 8%; P<0.05) was derived from oocytes frozen with PROH than with DMSO or glycerol. In conclusion, the type of cryoprotectant used is one of the critical factors affecting developmental competence of bovine oocytes frozen at the metaphase-II stage. For this stage of oocytes, PROH was the most effective, yielding a large number of 8-cell embryos and morulae than either glycerol or DMSO in a slow freezing method combined with a 3-step thawing protocol.     

In vitro fertilization and development of bovine oocytes matured in serum-free medium

This study was undertaken to investigate the effects of supplementation of serum (fetal calf serum), gonadotropins (LH, FSH, prolactin) and estradiol-17 beta (E2) to culture medium during in vitro maturation of bovine cumulus oocyte complexes on subsequent fertilization and development to the blastocyst stage in vitro. Serum supplementation during bovine oocyte maturation was not required but hormonal supplementation, gonadotropins (LH + FSH) and E2, enhanced the fertilizability and developmental ability of bovine oocytes matured in vitro. The addition of prolactin to maturation medium containing LH, FSH, and E2 did not further enhance frequencies of fertilization and development.     

Activation of bovine oocytes matured in vitro by injection of bovine and human spermatozoa or their cytosolic fractions

The aim of this study was to investigate whether bovine spermatozoa possess so-called sperm factor in the cytosolic fraction (CF) which activates bovine oocytes, and whether bovine oocytes matured in vitro are activated by microinjection of CF extracted from spermatozoa of other species. In the first experiment, bovine and human spermatozoa were microinjected into ooplasm of bovine oocytes matured in vitro. Secondly, CF from bovine and human spermatozoa were injected into bovine oocytes. In the third, CF from human spermatozoa was injected into human unfertilised oocytes obtained 18-20 h after clinical intracytoplasmic sperm injection (ICSI). We found that microinjection of bovine spermatozoa into bovine oocytes induced oocyte activation, as shown by resumption of meiosis and formation of a female pronucleus, at a significantly higher rate than the bovine sham injection (63.0% vs 43.0%; p < 0.05). On the other hand, there was no significant difference in activation rate between the human sperm injection (35.9%) and the human sham injection (22.9%). Furthermore, microinjection of bovine sperm CF into bovine oocytes induced oocyte activation at a significantly higher rate than the human CF injection or sham injection (75.9% vs 14.8%, 20.4%; p < 0.01). Formation of a single female pronucleus and second polar body extrusion was observed in 95.1% of activated oocytes after bovine sperm CF injection. When human sperm CF was injected into human unfertilised oocytes, the activation rate was significantly higher than following sham injection (76.9% vs 44.0%; p < 0.05). These results indicate the presence of sperm factor in bovine sperm CF which activate bovine oocytes, and suggest the possibility that sperm factor has species-specificity at least between bovine and human.