Effect of electrofusion pulse in either electrolyte or nonelectrolyte fusion medium on subsequent murine embryonic development.

This study was designed to determine what effect electropulse parameters would have on rate of fusion, lysis, and embryo viability when embryos were subjected to electrofusion treatment in nonelectrolyte or electrolyte pulse media. Previous experiments have shown electrolyte medium (i.e., phosphate-buffered saline; PBS) to have a positive effect on electric pulse-induced murine oocyte activation. In addition, these results also indicated that pulse media containing 0.9 mM Ca2+ induced a dramatic increase in the rate of murine oocyte activation compared with oocytes pulsed in media containing 0.0 or 0.05 mM Ca2+. Pronuclear or two-cell-stage embryos were obtained from superovulated prepubertal randomly bred Swiss (albino) female mice. Embryos were randomly assigned to three nonelectrolyte and three electrolyte treatment media. Nonelectrolyte media consisted of 0.3 M mannitol (T1), 0.3 M mannitol + 0.05 mM CaCl2 (T2), and 0.3 M mannitol + 0.9 mM CaCl2 (T3). Electrolyte media consisted of Ca(2+)-free PBS (T4), PBS containing 0.05 mM CaCl2 (T5), and PBS containing 0.9 mM CaCl2 (T6). Three experiments were carried out; the objective of the first was to determine the rate of fusion and rate of lysis in murine two-cell embryos placed in the two types of (0.3 M mannitol, T1-T3; and PBS, T4-T6) fusion media and subjected to a fusion procedure (3 V, 5 sec AC alignment pulse, followed by a 1.56 kV.cm-1, 99 microsec DC fusion pulse). Control two-cell embryos were placed in T1 for 2 min and did not receive a fusion pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrically induced calcium elevation,activation, and parthenogenetic development of bovine oocytes

The influence of electrical stimulation on the level of intracellular Ca²⁺ in bovine oocytes, as well as activation and extent of parthenogenetic development, was investigated. Mature oocytes were electrically stimulated at 29 hr of maturation, and intracellular Ca²⁺ concentration was determined with the Ca²⁺ indicator fura-2 dextran (fura-2 D). The Ca²⁺ response of oocytes to a given electrical pulse was variable. Oocytes responded with either no Ca²⁺ rise from baseline (≈? 12 nM), a short-duration Ca²⁺ rise (from 12 nM to 300 nM) that returned to baseline within 2 min of the pulse, or a long-duration Ca²⁺ rise (from 12 nM to 1,000–2,000 nM) that never returned to baseline during the 8 min period over which the oocytes were monitored. In these oocytes, Ca²⁺ level returned to baseline when oocytes were removed from 0.30 M mannitol and placed in an ionic medium. Increasing field strength or pulse duration tended to increase the proportion of oocytes displaying a Ca²⁺ rise, and at 1.0 kVcm^?1 for 40 μsec, all oocytes displayed a long-duration Ca²⁺ elevation. Direct transfer of oocytes from culture medium to mannitol also triggered a Ca²⁺ rise. Multiple stimulations, either electrical or by transferring to mannitol, produced multiple Ca²⁺ rises. This mannitol-induced Ca²⁺ rise could be inhibited by first washing the oocytes in medium containing equal parts of 0.30 M mannitol and phosphate buffered saline (PBS). The level of Ca²⁺ stimulation affected activation and development of oocytes. Insufficient, or, conversely, excessive Ca²⁺ stimulation impaired development. Optimum development was obtained with (1) three pulses of 0.2 kVcm^?1 for 20 μsec, each pulse 22 min apart, after direct transfer of oocytes from culture medium to mannitol (22% blastocysts) or (2) three pulses of 1.0 kVcm^?1 for 20 μsec after transfer of oocytes from culture medium to medium containing equal parts mannitol and PBS, then to mannitol (24% blastocysts). This procedure avoided induction of a Ca²⁺ rise prior to the pulse. The results indicate that the level of Ca²⁺ stimulation can be regulated by incubation conditions prior to the pulse and, to some extent, by field strength and pulse duration. The level of electrical stimulation influenced oocyte Ca²⁺ response, activation, and parthenogenetic development. © 1993 Wiley-Liss, Inc.     

Calcium concentration in vitrification medium affects the developmental competence of in vitro matured ovine oocytes

The present study was designed to determine whether different calcium concentrations in the vitrification solutions could improve the developmental competence of in vitro matured ovine oocytes after cryopreservation. In vitro matured oocytes were vitrified with 16.5% ethylene glycol (EG) + 16.5% dimethylsulfoxide (DMSO) vitrification media. The base media contain different calcium concentrations, so that five experimental groups were obtained: TCM/FCS (TCM 199 + 20% fetal calf serum (FCS), [Ca²⁺] 9.9 mg/dl); PBS/FCS (Dulbecco Phosphate Buffered Saline (PBS) + 20% FCS, [Ca²⁺] 4.4 mg/dl); PBS^{CaMg free}/FCS (PBS without Ca²⁺ and Mg²⁺ + 20% FCS [Ca²⁺] 2.2 mg/dl); PBS/BSA (PBS + 0.4% bovine serum albumin (BSA), [Ca²⁺] 3.2 mg/dl) and PBS^{CaMg free}/BSA (PBS without Ca²⁺ and Mg²⁺ +0.4% BSA, [Ca²⁺] 0.4 mg/dl). After warming, the oocytes from the five experimental groups were assessed for survival, spontaneous parthenogenetic activation and developmental capacity via in vitro fertilization. Oocyte survival after vitrification procedures was better preserved in group PBS^{CaMg free}/FCS compared to the others (P < 0.05). In addition, a positive correlation was found between calcium concentration in vitrification solutions and spontaneous parthenogenetic activation (correlation index 0,82; P < 0.001). Development of vitrified oocytes was significantly affected by vitrification media composition (P < 0.01). In particular, oocytes from group PBS^{CaMg free}/FCS led to higher cleavage rates and blastocyst rate compared to the others. Our data showed that lowering calcium concentration in the vitrification medium improves the blastocyst rate of vitrified ovine oocytes, probably reducing the effect of EG + DMSO during vitrification. On the contrary, the replacement of FCS with BSA dramatically reduces the developmental potential of these oocytes.     

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.     

Optimization of parthenogenetic activation protocol in porcine

The effects of the electrical field strengths, number of pulses, and post-activation media on chromatin conformation and parthenogenetic development were studied to optimize the activation protocol for porcine nuclear transfer. In experiment 1, electrical field strengths were examined. Oocytes were subjected to square direct current pulses at output voltages of 1.2, 1.7, 2.2, and 2.7 kV/cm for 1 x 30 microsec. The voltage resulting from experiment 1 was 2.2 kV/cm, in which 50.0% of activated oocytes developed to blastocysts in vitro. In experiment 2, the influence of 1, 2, and 3 pulses on blastocyst development was tested using field strengths and post-activation medium described in experiment 1. Oocytes activated by a single 30 microsec pulse of 2.2 kV/cm DC yielded a higher blastocyst rate (56.3%) than oocytes activated by 2 or 3 pulses (<42.5%). In experiment 3 and 4, we investigated the effects of cytochalasin B (CB), cycloheximide (CH), and CB + CH on nuclear development stages and parthenogenetic development following a single 30 microsec pulse of 2.2 kV/cm DC. The percentage of activated oocytes was not different among CB (93.3%), CB + CH (98.3%), control (80.0%), and CH (80.0%) groups 12 hr after activation. Treatment with CB (57.5%) or CB + CH (53.8%) enhanced the blastocyst rate compared with other groups, CH (23.8%) treated- and control group (18.8%). The results demonstrated that a single 30 microsec pulse of 2.2 kV/cm DC followed by culturing in post-activation medium with CB for 5 hr were effective parameters for parthenogenetic activation and blastocyst formation of in vitro matured porcine oocytes which suggests that a single calcium rise is sufficient to activate pig oocytes and to achieve high rate of blastocyst development.     

Development of pronuclei in pig oocytes activated by a single electric pulse.

Pig oocytes were matured in vitro in a modified M-199 medium for 44 h, subjected to electrical stimulation and scored for activation 6 h later. Sham pulsed oocytes, exposed to electroporation medium and an a.c. field, did not develop the female pronucleus any more frequently than occurs spontaneously (8.3% within 50 h of culture). However, a single d.c. pulse proved extremely efficient in activating pig oocytes. Pulses of 0.75-1.65 kV cm-1 lasting 30 or 100 microseconds activated at least 90% of matured oocytes. The developmental pathway taken by the activated oocytes depended on the parameters of the pulse. The lowest effective stimulation (0.45 and 0.60 kV cm-1 for 30 microseconds) frequently produced oocytes that remained in pre-pronuclear stages of activation (29.4 and 42.3%, respectively). Extrusion of the second polar body and creation of one pronucleus was the most frequent type of activation (in up to 88.2% among the activated oocytes). The strongest stimulations used (1.05-1.65 kV cm-1 for 100 microseconds) often yielded oocytes that failed to extrude the second polar body and formed two or more pronuclei (up to 56.3%). Under optimal stimulation (0.75 kV cm-1), the activated oocytes proceed synchronously to interphase of the first mitotic division. Anaphase II is reached within 30 min and telophase Ii at 1 h after application of the pulse. The second polar body is extruded about 2 h after activation. Well-defined swelling pronuclei were found in oocytes 5-6 h after activation. The relationship between the stage of oocyte maturation and susceptibility to activation was investigated. The period of culture in which the oocytes develop the activation competence (32-36 h of culture) overlapped with the period in which the oocytes complete meiosis (28-38 h). This suggests that ageing in meiotic arrest is not essential for pig oocytes to become activated by electric pulses. Activation of pig oocytes was accompanied by release of cortical granules. In sections of control (metaphase II) oocytes, an average of 7.3 intact cortical granules per 10 microns of overlying cytoplasmic membrane was found. This number dropped to 1.5 in 10 microns within 30 min after the pulse.     

Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor

This study examined the ability of epidermal growth factor (EGF) to improve the developmental competence of pig oocytes matured in a protein-free (PF) in vitro maturation (IVM) system. Oocyte maturation was done in one of three media: 1. PF-TCM: tissue culture medium (TCM) 199 + 0.1% polyvinylalcohol (PVA); 2. PF-TCM+EGF: PF-TCM + 10 ng/ml EGF; and 3. +ve CONT: North Carolina State University (NCSU) 23 medium + 10% porcine follicular fluid. All media contained 0.57 mM cysteine. Hormonal supplements, 0.5 microg/mL LH and 0.5 microg/mL FSH, were present only for the first half (20 to 22 h) of the culture period. After maturation, oocytes were co-incubated with frozen-thawed spermatozoa for 5 to 6 h and transferred to embryo culture medium, NCSU 23 containing 0.4% BSA, for 144 h. In Experiment 1, differences in cumulus expansion were observed for oocytes matured in +ve CONT (Category 4), PF-TCM (Category 2) and PF-TCM+EGF (Category 3). However, no significant differences in nuclear maturation to metaphase II stage were observed. In Experiment 2, no differences in fertilization parameters were observed. Significant (P < 0.01) differences in cleavage rates were observed among the three media for a proportion of the oocytes matured (52, 60 and 69% in PF-TCM, PF-TCM+EGF, and +ve CONT, respectively). Oocytes matured in PF-TCM showed the lowest (P < 0.01) blastocyst development (22%). However, the same rate of blastocyst development was obtained for +ve CONT (37%) and PF-TCM+EGF (37%). Blastocyst cell numbers were significantly higher when oocytes were matured in the presence of EGF (26 vs. 37 to 41). In Experiment 3, oocytes matured in PF-TCM+EGF had a significantly (P < 0.05) higher intracellular glutathione (GSH) concentration (5.9 vs. 11.4 pmol/oocyte) compared with PF-TCM. Twenty-two of 25 embryo transfer recipients became pregnant (Experiment 4). Four animals returned to estrus in within 60 days. Six pregnant animals slaughtered at 26 to 45 days had 43 fetuses (range: 4 to 12) and the remaining 12 animals farrowed 82 piglets (range: 3 to 12). These results indicate that EGF enhances the developmental competence of pig oocytes matured in a protein-free culture medium which is correlated with higher GSH level in oocytes. Birth of piglets indicate that embryos derived from oocytes matured in the presence of EGF are viable.     

A long-lasting potentiation of calmodulin-mediated chloride channel activity without a mediation of protein kinase C in Xenopus oocytes injected with rat brain mRNA.

When Xenopus oocytes injected with rat brain poly(A)+RNA were voltage-clamped in a recording solution containing Ca2+, a depolarization pulse induced a transient current, ICl(Ca), which reflects calmodulin-mediated opening of endogenous Cl- channels in response to a Ca2+ influx through Ca2+ channels of brain origin. ICl(Ca) could be repetitively observed with a steady amplitude over 1 h, whereas the response was greatly potentiated for more than 30 min after a brief stimulation of muscarinic or other Ca2(+)-mobilizing receptors. The enhancement of ICl(Ca) was mimicked by an injection of inositol-1,4,5-trisphosphate or by a treatment with A23187, but not affected by treatments that stimulate or inhibit protein kinase C activity. Isolated Ba2+ current flowing through voltage-sensitive Ca2+ channels was not augmented during the facilitation of ICl(Ca). These observations indicate that the endogenous calmodulin/Cl- channel system may memorize an over-threshold increase in the intracellular Ca2+ concentration and potentiate the Ca2(+)-sensitiveness of the Cl- channel. A long-lasting autoregulation of Ca2(+)-dependent ion channel activity is suggested.     

Stimulation of the T3-T cell receptor complex induces a membrane-potential-sensitive calcium influx

Three monoclonal antibodies selected for their recognition of parts of the T3-T cell receptor complex on human T lymphocytes were found to induce an increase in cytoplasmic free Ca2+ (Ca2+i) in the T cell leukemia line HPB-ALL as measured by Quin2 fluorescence. These reagents are directed against T3 (OKT3), a nonvariable T3-associated structure (WT-31) and the variable region of the T3-associated antigen receptor (T40/25) of this cell line. The rise in Ca2+i was dependent on the presence of extracellular Ca2+, occurred within 30 sec of stimulation, and was sustained for at least 10 min. Fab fragments of OKT3 also caused a rapid increase in Ca2+i, indicating that cross-linking is not necessary to induce a Ca2+ response. Alterations in plasma membrane potential and La3+ blocked the Ca2+ influx induced by OKT3 and T40/25. These data suggest that the T3-T cell receptor complex of human T lymphocytes may be an antigen-regulated Ca2+ channel.     

Uptake and release of calcium by canine gastric corpus smooth muscle plasma membrane enriched fraction

Calcium movements across plasma membrane enriched vesicles isolated from canine gastric corpus smooth muscle were investigated. The ATP-dependent Ca2+ uptake increased with time up to 10 min. The uptake for the initial 2-min period was approximately linear with time. The apparent initial velocity of the ATP-dependent Ca2+ uptake increased monotonically with free Ca2+ concentration from 0.1 to 2 microM, and further increases in free Ca2+ concentration did not increase the Ca2+ uptake. The free Ca2+ dependence curve could be described with a Hill coefficient of approximately 1.0 and Km of 0.85 +/- 0.01 microM for free Ca2+ concentration. Passive Ca2+ uptake (reaction time = 1 h) also increased with increasing free Ca2+ concentrations from 0.02 to 4.0 mM. Dilution of loaded vesicles in isotonic media containing EGTA led to initial rapid loss (less than 1 min) followed by a slower release which showed simple exponential decay. The t 1/2 values of the slower Ca2+ loss from these vesicles were 16.1 +/- 0.9 min (actively loaded n = 5) and 18.4 +/- 0.9 min (passively loaded n = 3), respectively. Dilution in isotonic medium containing both EGTA and A23187 released all the sequestered Ca2+ from these loaded vesicles.     

Factors affecting electrical activation of porcine oocyte matured in vitro

This work was undertaken to improve conditions for in vitro maturation and activation of porcine oocytes. Experiments were designed to compare: (i) electrical pulse frequency, (ii) methods of oocyte preparation, (iii) maturation conditions, and (iv) electrical poration medium on development. Oocytes were harvested by follicle dissection or aspiration, co-cultured with follicle shells in M199 based medium with or without media changes at 38.5°C in 5% CO₂ under non-static conditions for 48 h and electroactivated using single or multiple pulses (current strength 1.0 kV/cm for 50 μs in 0.28 M inositol or mannitol based media with 10 mM histidine) at different time intervals. The results showed: (i) neither the pulse frequency nor the pulse interval influenced rates of pronuclear formation but multiple pulse activation (3 pulses at 5 min intervals) induced a higher incidence of development and progression through the 4-cell block in contrast to one pulse activation; (ii) both the rate of nuclear maturation (88.6% vs. 77.6%) and post-activation cleavage (89.8% vs. 67.4%) were higher (P < 0.05) when oocytes were collected by follicle dissection rather than by aspiration; (iii) while changing to a hormone-free medium at 24 h was without effect on maturation (91.9% vs. 91.7%), rate of cleavage (81.6% vs. 72.3%, P < 0.05) at 24 h was enhanced by the medium change; and (iv) oocytes activated with 3 pulses 5 min apart in mannitol based medium at 48–49 h and at 53–54 h formed pronuclei at a comparable rate but subsequent parthenogenetic development was higher in the older eggs. By contrast, inositol-based medium supported development of young and old eggs equally well. Calcium and magnesium ions are, however, necessary in both mannitol and inositol media for activation of porcine oocytes matured in vitro. The present results suggest that optimal parthenogenetic activation and early development of IVM pig oocytes could be obtained if oocytes are harvested by dissection, cultured for 24 h in hormone-containing medium before being placed in hormone free medium and activated at 48 h in inositol based medium using a three pulse activation system.     

Activation of pig oocytes by intracytoplasmic injection of strontium and barium

Ovulated mouse oocytes are activated by exposure to culture medium containing Sr2+ or Ba2+ or by intracytoplasmic injection of the divalent cations. It is known that in vitro matured pig oocytes are activated by the intracytoplasmic injection of Ca2+. In this study, we examined the effect of exposure and of intracytoplasmic injection of Sr2+ or Ba2+ on in vitro matured pig oocytes (MII-oocytes). When MII-oocytes were exposed to the medium containing divalent cations, no oocytes were activated. However, in the case of oocytes that were injected with Sr2+, Ba2+ and Ca2+, at 6 h after injection, 64%, 71% and 86% of the oocytes had been released from MII-arrest, and 51%, 67% and 84% formed female pronuclei, respectively. The initial transient in intracellular Ca2+ concentration ([Ca2+]i) was measured by the Ca2+ indicator dye fluo-4 dextran. Microinjection of Sr2+, Ba2+ or Ca2+ induced a rapid elevation of [Ca2+]i. The exocytosis of cortical granules was examined by staining with fluorescein isothiocyanate (FITC)-labelled peanut agglutinin. After an injection of divalent cations, a release of cortical granules was observed in the oocytes. Maturation promoting factor (MPF) activity declined to a low level after 6 h in all the oocytes injected with divalent cations. To test their developmental ability, injected oocytes were treated with cytochalasin B and then cultured for 168 h in NCSU23 medium. After 168 h, 29% (Sr2+), 29% (Ba2+) and 51% (Ca2+) of the oocytes had developed to the blastocyst stage. These results indicate that intracytoplasmic injection of Sr2+ and Ba2+, like that of Ca2+, induces in vitro matured pig oocytes to be released from MII-arrest and leads them into a series of events related to oocyte activation.     

A new infrared spectroscopoic marker for cochleate phases in phosphatidylserine-containing model membranes.

Fourier transform-infrared (IR) spectroscopic and electron microscopic studies are reported for 1,2-dimyristoylphosphatidylserine (DMPS) and for DMPS/1,2-dimyristoylphosphatidylcholine mixtures in the presence and absence of Ca2+ ion. The frequency of the methyl symmetric deformation mode near 1,378 cm-1, previously assumed insensitive to changes in lipid morphology, has been found to respond to cochleate phase formation by undergoing an approximately 8 cm-1 increase. The new IR spectroscopic marker at 1,386 cm-1 has been used to identify and verify structures suggested from the phase diagram of J. R. Silvius and J. Gagné (1984. Biochemistry. 23:3241-3247) for this system. In addition, the ability of Mg2+ ion to induce cochleate formation has been demonstrated. Higher Mg2+ than Ca2+ levels are required for this process. Finally, IR spectroscopy has been used to monitor dehydration of the lipid surface through changes in the asymmetric PO2- stretching mode. Dehydration precedes cochleate phase formation (i.e., occurs at a lower Ca2+/phosphatidylserine level).     

Conditions for in vitro maturation and artificial activation of ferret oocytes

The ferret represents an attractive species for animal modeling of lung diseases because of the similarity between ferret and human lung biology and its relatively small size and short gestation time. In an effort to establish experimental protocols necessary for cloning ferrets, optimized conditions for in vitro maturation and artificial activation of ferret oocytes were examined. Cumulus-oocyte complexes were harvested from ovaries of superovulated ferrets, and in vitro maturation was evaluated in three different culture media: medium 1 (TCM-199 + 10% FBS), medium 2 (TCM-199 + 10% FBS with eCG [10 IU/ml] and hCG [5 IU/ml]), or medium 3 (TCM-199 + 10% FBS with eCG, hCG, and 17beta-estradiol [2 microg/ml]). After 24 h of maturation in vitro, the maturation rate of oocytes cultured in medium 2 (70%, n = 79) was significantly greater (P < 0.01) than those of oocytes cultured in the other two media (27%-36%, n = 67-73). At 48 h, similar maturation rates (56%-69%, n = 76-87) were observed for all three types of media. For activation experiments, oocytes cultured in medium 2 were stimulated with electrical and chemical stimuli either individually or in combination. Treatment with cycloheximide and 6-dimethylaminopurine (6-DMAP) following electrical stimulation resulted in 43% (n = 58) of the oocytes developing to the blastocyst stage. Such an activation rate represented a significant improvement over those obtainable under other tested conditions, including individual treatment with electrical pulses (10%, n = 41), cycloheximide (3%, n = 58), or 6-DMAP (5%, n = 59). Blastocysts derived from in vitro activation appeared to be normal morphologically and were composed of an appropriate number of both inner cell mass (mean +/- SEM, 10.3 +/- 1.1; n = 11) and trophectoderm (60.8 +/- 2.9, n = 11) cells. These results have begun to elucidate parameters important for animal modeling and cloning with ferrets.     

In vitro spontaneous parthenogenetic activation of golden hamster oocytes

Parthenogenetic activation is a major hurdle to be cleared for the examination of the human sperm chromosome after intracytoplasmic injection (ICSI) into golden hamster oocytes. Various factors that affect spontaneous activation of hamster oocytes were, therefore, investigated in this study. We collected cumulus-oocyte complexes (COC) from the oviducts of superovulated females and washed them thoroughly with Ca2+-containing or Ca2+-free TALP-HEPES medium (handling media). We cultured oocytes with intact cumulus or those without cumulus (removed by previous hyaluronidase treatment) in Ca2+-containing or -free m-TALP-3 for 6 or 12 h before examining for their activation. Among the oocytes recovered 17 h post-hCG, 92-94% were parthenogenetically activated by 6 h of in vitro culture. Activation rate in the oocytes collected at 13.5 h post-hCG (53%) was significantly (P < 0.05) lower than that in the oocytes collected 17 h post-hCG (92%), indicating that the spontaneous activation rate increased as the oocytes became older. Both cumulus-intact and cumulus-free oocytes had similar (P > 0.05) activation rates when cultured in vitro, suggesting that hyaluronidase treatment had no effect on the rate of oocyte activation. Omission of Ca2+ from the handling medium also had no effect on the activation of the oocytes. The rate of spontaneous activation of the oocytes cultured in calcium-free medium for 6 (9%) and 12 h (16%) was significantly (P < 0.01) lower than that (94%) of the control oocytes cultured in Ca2+-containing medium, implying a positive influence of Ca2+ on in vitro activation of hamster oocytes. When we cultured the oocytes first in calcium-free medium for 6 h, and then in calcium-containing medium for 6 h, 94% were activated, which is comparable to the rate for oocytes continuously cultured in Ca2+-containing medium. This indicates that the inhibition of hamster oocyte activation in Ca2+-free medium is reversible and can be used to control spontaneous activation of golden hamster oocytes.     

Brief exposure to cycloheximide prior to electrical activation improves in vitro blastocyst development of porcine parthenogenetic and reconstructed embryos

To investigate the effects of cycloheximide exposure before electrical activation of in vitro-matured porcine oocytes on the subsequent development of parthenogenetic embryos, cumulus-free mature oocytes were exposed to NCSU-23 medium containing cycloheximide (10 microg/mL) for 0, 5, 10, 20, 30 and 60 min, activated by electrical pulse treatment (1.5 kV/cm, 100 micros) and then cultured in PZM-3 for 7 days. To evaluate the effects of cycloheximide on the activation of nuclear transfer embryos, reconstructed embryos were electrically activated by two DC pulses (1.2 kV/cm, 30 micros) before or after exposure to cycloheximide. The reconstructed embryos were allocated into four groups: electrical pulse treatment alone (Ele); exposure to cycloheximide for 10 min followed by electrical activation (CHX+Ele); electrical activation followed by exposure to cycloheximide for 6h (Ele+CHX); exposure to cycloheximide for 10 min, followed by electrical activation and a further exposure to cycloheximide for 6h (CHX+Ele+CHX). The activated reconstructed embryos were cultured in PZM-3 for 6 days. Oocytes treated with 10 min exposure to cycloheximide followed by electrical activation had a significantly higher percentage of blastocyst formation compared to control oocytes and oocytes exposed for > or =30 min. In the reconstructed embryos, the blastocyst development rates of embryos exposed to cycloheximide (CHX+Ele, Ele+CHX and CHX+Ele+CHX) were significantly higher than those of the control group (Ele). Among the cycloheximide-treated groups, the CHX+Ele group had increased development rate and total blastocyst cell number, though these values were not significantly different from those observed in the other cycloheximide-treated groups. To evaluate the quality of NT embryos treated with cycloheximide, apoptosis in blastocysts was analyzed by TUNEL assay. The 10 min exposure to cycloheximide prior to electrical activation significantly reduced cell death compared with longer exposure to cycloheximide after electrical fusion. In conclusion, brief exposure to cycloheximide prior to electrical activation may increase the subsequent blastocyst development rates in porcine parthenogenetic and reconstructed embryos.     

Intracellular Ca2+ response of rabbit oocytes to electrical stimulation.

Electrical stimulation is known to cause activation in mammalian oocytes, possibly by eliciting an elevation in intracellular calcium (Ca2+). This study reports intracellular Ca2+ concentrations in mature rabbit oocytes using the Ca2+ indicator fura-2. Calcium levels were determined prior to, during, and after the administration of an electrical pulse (3.6 kV/cm for 60 microseconds). Baseline Ca2+ levels ranged from 30 to 90 nM. The intracellular Ca2+ transient evoked by a pulse, peaked at 11 sec, was highly variable in amplitude (40-300 nM) and returned to prepulse levels within 300 sec. Electrically stimulated oocytes did not exhibit repetitive Ca2+ transients. The size of the cytoplasmic Ca2+ rise was influenced by the duration of the pulse, the field strength and the concentrations of external Ca2+ rise was influenced by the duration of the pulse, the field strength and the concentrations of external Ca2+ (P less than 0.05). Oocytes electrically stimulated in the presence of 100 microM CaCl2, which evoked Ca2+ transients with a mean magnitude of 120 nM, activated at a higher rate (P less than 0.05) than oocytes stimulated in the presence of either higher or lower levels of external Ca2+. Although oocytes electrically shocked at 16-18 hr after administration of human chorionic gonadotropin (hphCG) activated at a lower rate than oocytes stimulated at 22-24 hphCG (P less than 0.05), their intracellular Ca2+ response to the pulse was similar (P less than 0.05). These results indicate that electrical pulse parameters and extracellular Ca2+ concentrations can be used to modulate intracellular Ca2+ levels and optimize oocyte activation rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effects of calcium channel blockers on thyroid hormone uptake in neonatal rat cardiomyocytes

The effects of the Ca2+ channel blockers verapamil, nifedipine, and diltiazem on triiodothyronine (T3) and thyroxine (T4) uptake were tested in cultured cardiomyocytes from 2-day-old rats. Experiments were performed at 37 degrees C in medium with 0.5% BSA for [125I]T3 (100 pM) or 0.1% BSA for [125I]T4 (350 pM). The 15-min uptake of [125I]T3 was 0.124 +/- 0.013 fmol/pM free T3 (n = 6); [125I]T4 uptake was 0.032 +/- 0.003 fmol/pM free T4 (n = 12). Neither T3 nor T4 uptake was affected by 1% DMSO (diluent for nifedipine and verapamil). Uptake of [125I]T3 but not of [125I]T4 was dose dependently reduced by incubation with 1-100 microM verapamil (49-87%, P < 0.05) or nifedipine (53-81%, P < 0.05). The relative decline in [125I]T3 uptake after 4 h of incubation with 10 microM verapamil or nifedipine was less than after 15 min or 1 h, indicating that the major inhibitory effect of the Ca2+ channel blockers occurred at the level of the plasma membrane. The reduction of nuclear [125I]T3 binding by 10 microM verapamil or nifedipine was proportional to the reduction of cellular [125I]T3 uptake. Diltiazem (1-100 microM) had no dose-dependent effect on [125I]T3 uptake but reduced [125I]T4 uptake by 45% (P < 0.05) at each concentration tested. Neither the presence of 20 mM K+ nor the presence of low Ca2+ in the medium affected [125I]T3 uptake. In conclusion, the inhibitory effects of Ca2+ channel blockers on T3 uptake in cardiomyocytes are not secondary to their effects on Ca2+ influx but, rather, reflect interference with the putative T3 carrier in the plasma membrane.     

Methods for collecting and maturing equine oocytes in vitro

The objective of our study was to develop an effective method for collecting and maturing equine oocytes. In Experiments 1 and 2, oocytes were collected from excised ovaries obtained via colpotomy. In Experiment 3, oocytes were collected from ovaries obtained after slaughter. Follicles were aspirated and flushed with various treatments to recover the oocytes, which were then cultured and stained to observe the stage of meiosis. In Experiment 1, the aspiration treatments consisted of 0.5 ml of modified Dulbecco's PBS with 0, 100 or 500 lU/ml hyaluronidase. There was no increase (P>0.05) in oocyte recovery with the addition of hyaluronidase. The oocytes were cultured in either TCM-199 or Ham's F-10 medium containing 0.5 ug/ml FSH, 1 ug/ml LH, 1 ug/ml estradiol 17β, 250 uM Na-pyruvate and 10% estrual mare serum for 0, 24, 36 or 48 h. Maturation rates were higher (P<0.05) at 36 h for oocytes cultured in TCM-199 (79%) than for those in Ham's F-10 (21%). There was no difference (P>0.05) in the percentage of maturation of oocytes between the 2 media at 48 h of culture. In Experiment 2, a single aspiration was performed with no flushing medium (dry aspiration) in 0.5 ml of PBS or in PBS with 1000 IU/ml hyaluronidase. The oocytes were then cultured in TCM-199 for 24, 30 or 36 h. There was an increase (P<0.05) in oocyte recovery when follicles were flushed with PBS, with or without hyaluronidase. There was also a difference (P<0.05) in the percentage of maturation of oocytes between 30 and 24 h (86 vs 48%), but no further increase was seen by 36 h (84%). In Experiment 3, follicles were aspirated with PBS 5 to 6, 6 to 7 or 7 to 8 h after slaughter. The oocytes were cultured for 30 h in TCM-199 either with or without 100 IU/ml eCG. There was no effect of eCG or time from slaughter on oocyte maturation or cumulus expansion (P>0.05).     

Rabbit cloning: improved fusion rates using cytochalasin B in the fusion buffer.

This study investigated the possibility of producing rabbits from embryos obtained by transfer of somatic cell nuclei; Muscle biopsies were taken from the upper part of the hind limb of fetuses at 24 days of gestation. Fetal fibroblasts were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% FCS at 38 degrees C in 5% CO(2) in air. Cells were passaged at least three times; for 5 days prior to nuclear transfer, cells were cultured in 0.5% FCS medium to induce them into G(0). These cells then were placed into the perivitelline space of enucleated rabbit MII stage oocytes. Fusion media were Ca/Mg-containing 0.3 M mannitol (Buffer 1) or 0.3 M mannitol plus 7.5 microg/ml cytochalasin B (CB) (Buffer 2). Fusion was studied with 6 protocols: culture of donor cell-cytoplast complexes (CCCs) in TCM199 for 0, 30, or 60 min after micromaniupulation and electrical stimulation in Buffer 1 or Buffer 2. Fusion rates were 57.4, 42.9 and 52.8%, respectively, in groups 0 + B1, 30 + B1, and 60 + B1, significantly lower than the rates in groups 0 + B2, 30 + B2 and 60 + B2 (75.9, 75.7, and 76.4%, respectively) (P < 0.05). Thus, CB in electrical fusion buffer improved the fusion rate. The percentages of blastocyst formation were 40% in 0 + B2 and 37.1% in 0 + B1 groups, significantly higher (P < 0.05) than those in 30 + B1 (20%), 30 + B2 (15.6%), 60 + B1 (13%) and 60 + B2 (8.3%). A total of 653 cloned embryos were at 1-cell, 2-4-cell, or morula/blastocyst stages were transferred to 44 mated or non-mated synchronized recipients. No cloned embryos developed to term.