Effects of duration, concentration, and timing of ionomycin and 6-dimethylaminopurine (6-DMAP) treatment on activation of goat oocytes

The protocol of ionomycin followed by 6-dimethylaminopurine (6-DMAP) is commonly used for activation of oocytes and reconstituted embryos. Since numerous abnormalities and impaired development were observed when oocytes were activated with 6-DMAP, this protocol needs optimization. Effects of concentration and treatment duration of both drugs on activation and development of goat oocytes were examined in this study. The best oocyte activation (87-95%), assessed by pronuclear formation, was obtained when oocytes matured in vitro for 27 hr were treated with 0.625-20 microM ionomycin for 1 min before 6-hr incubation in 2 mM 6-DMAP. Progressional reduction of time for 6-DMAP-exposure showed that the duration of 6-DMAP treatment can be reduced to 1 hr from the second up to the fourth hour after ionomycin, to produce activation rates greater than 85%. Activation rates of oocytes in vitro matured for 27, 30, and 33 hr were higher (P < 0.05) than that of oocytes matured for 24 hr when treated with ionomycin plus 1-hr (the third hour) 6-DMAP, but a 4-hr incubation in 6-DMAP enhanced activation of the 24-hr oocytes. Goat activated oocytes began pronuclear formation at 3 hr and completed it by 5-hr post ionomycin. An extended incubation in 6-DMAP (a) impaired the development of goat parthenotes, (b) quickened both the release from metaphase arrest and the pronuclear formation, and (c) inhibited the chromosome movement at anaphase II (A-II) and telophase II (T-II), leading to the formation of one pronucleus without extrusion of PB2. In conclusion, duration, concentration, and timing of ionomycin and 6-DMAP treatment had marked effects on goat oocyte activation, and to obtain better activation and development, goat oocytes matured in vitro for 27 hr should be activated by 1 min exposure to 2.5 microM ionomycin followed by 2 mM 6-DMAP treatment for the third hour.     

6—DMAP对小鼠卵母细胞减数分裂启动及孤雌发育作用

小鼠卵泡卵母细胞体外培养过程中加入２ｍｍｏｌ／Ｌ６－ＤＭＡＰ可抑制卵母细胞自发的染色持浓缩和生发泡破裂（ＧＶＢＤ）。源自超排的ＭⅡ期卵母细胞则能为６－ＤＭＡＰ所激活。ｈＣＧ注射后１８－１９ｈ的卵母细胞置于２ｍｍｏｌ／Ｌ６－ＤＭＡＰ的ＣＺＢ溶液中培养０．５ｈ、１ｈ、２ｈ、３ｈ,卵母细胞的激活率分别为２６．１％、７５．２％、７５．８％、７７．３％、卵裂率分别为８８．２％、７３．２％、６７．０％、５８．     

Development of mouse oocytes superovulated at different ages.

The development of oocytes superovulated at 25, 50, or 90 days in four mouse strains (C57BL/6N, DBA/2N, ICR, and B6D2F1) was examined using the techniques of in vitro fertilization, culture, and transfer of two-cell embryos to pseudopregnant recipients. The highest number of ova were obtained from superovulated 25-day-old mice in all strains. Approximately 80% of oocytes developed to the two-cell stage after in vitro fertilization. Of these living oocytes, 60% developed to weanling stage through the recipient. These results suggested that donor age among 25, 50, or 90-day-old mice has no influence on the viability of superovulated oocytes. Consequently, we conclude that superovulated 25-day-old mice offer an economical and efficient source of viable oocytes for the production of transgenic mice.     

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

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

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.     

不同活化方法对小鼠卵母细胞孤雌发育的影响（简报）

In order to study effects of electro-fusion and strontium chloride (SrCl2) activation in nuclear transfer experiment on activation and development of mouse oocytes, concentration and treatment duration of SrCl2, electro-pulse and electro-pulse combining SrCl2 were used to activate mouse oocytes which were obtained after hCG 17h. Activated oocytes were cultured in vitro in CZB medium. The results were as follows: 82.4% activation percentage was obtained when the oocytes were treated with 10mmol/L SrCl2 for 6h, it was significantly (P>0.05) higher than those obtained from that treated with the 5mmol/L or 10mmol/L SrCl2 for 4h. The activation percentage was not significantly different between 5mmol/L and 10mmol/L SrCl2 for 6h, but the percentage of morula and blastocyst in 10mmol/L SrCl2 6h group was significantly (P > 0.05) higher than those in 5mmol/L SrCl2 6h group. In the groups of treatment with electro-pulse, the best activation percentage (70.9%) was obtained when the oocytes were treated with 1.0kv/cm, 320micros, 3 pulses, but M + B percentage (7.9%) was low. In the groups of treatment with electro-pulse combining with SrCl2, the best result was acquired (activation and M + B percentage were 75.0% and 57.3% separately) when the oocytes were treated in 10mmol/L SrCl2 for 6h interval 1h after treated with 1.8kv/cm, 10s, 1pulse. These results show that the treatment with electro-pulse combining SrCl2 is a better way to mouse parthenogenesis.     

Effect of 6-dimethylaminopurine on germinal vesicle breakdown of bovine oocytes

The effect of 6-dimethylaminopurine (6-DMAP) on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%, 120/124) in control medium, presence of 6-DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3% GV, 349/355) or absence (97.1% GV, 165/170) of cumulus cells. When lower concentrations of 6-DMAP were used (100-500 microM), GVBD was observed in 87.9% of oocytes, but their maturation was arrested at late diakinesis-metaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%). To assess whether the action of 6-DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6-DMAP, respectively. Whereas in cycloheximide-supplemented medium approximately 80% of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6-DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6-DMAP supplemented medium, the level of the maturation-promoting factor (MPF) is decreased. These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes.     

A combined treatment with ethanol and 6-dimethylaminopurine is effective for the activation and further embryonic development of oocytes from Sprague-Dawley and Wistar rats

In nuclear-transferred or round spermatid-injected oocytes, artificial activation is required for further development in mammals. Although strontium chloride is widely used as the reagent for inducing oocyte activation in mice, the optimal method for oocyte activation remains controversial in rats because ovulated rat oocytes are spontaneously activated in vitro before artificial activation is applied. In our previous study, we found that cytostatic factor activity, which is indispensable for arrest at the MII stage, is potentially low in rats and that this activity differs greatly between two outbred rats (Slc: Sprague-Dawley (SD) and Crj: Wistar). Therefore, it is necessary to establish an optimal protocol for oocyte activation independent of strains. Given that comparative studies of the in vitro development of oocytes activated by different activation protocols are very limited, we compared four different protocols for oocyte activation (ethanol, ionomycin, strontium and electrical pulses) in two different SD and Wistar rats. Our results show that oocytes derived from SD rats have significantly higher cleavage and blastocyst formation than those from Wistar rats independent of activation regimes. In both types of rat, ethanol treatment provided significantly higher developmental ability at cleavage and blastocyst formation compared to the other activation protocols. However, the initial culture in a fertilization medium (high osmolarity mR1ECM) for 24 h showed a detrimental effect on the further in vitro development of parthenogenetic rat oocytes. Taken together, our results show that ethanol treatment is the optimal protocol for the activation of rat oocytes in SD and Wistar outbred rats. Our data also suggest that high-osmolarity media are inadequate for the in vitro development of parthenogenetically activated oocytes compared with fertilized oocytes.     

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

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

Chromatin behaviour under influence of puromycin and 6-DMAP at different stages of mouse oocyte maturation

Preovulatory mouse oocytes were cultured in vitro up to each subsequent stages of maturation: germinal vesicle (GV), germinal vesicle breakdown (GVBD), groups of not yet individualized bivalents, circular bivalents, late prometaphase I, metaphase I, anaphase I and telophase I. The stages were identified in living oocytes by fluorescence microscopy using Hoechst 33342 as a specific vital dye. Oocytes from each stage of development developed in vitro and ovulated metaphase II oocytes were subsequently cultured in the presence of puromycin or 6-dimethylaminopurine (6-DMAP), an inhibitor of protein phosphorylation. The effects on chromatin of these drugs were studied during and at the end of culture by fluorescence and electron microscopy. We found that puromycin and 6-DMAP stop meiosis when applied at all stages of oocyte maturation, except for metaphase II. Oocytes at this stage are activated by puromycin. Reaction of the oocytes to the two drugs is different at GV and at metaphase II. All of the other stages react to the drugs by chromatin compaction, which can be followed by chromatin decondensation to form a nucleus. Our results suggest that late prophase chromatin condensation, bivalent individualization and retention of their individuality, as well as individualization of monovalents from telophase and retention of their individuality at metaphase II, are dependent on protein phosphorylation. The events occurring between metaphase I and telophase I are independent of protein synthesis and phosphorylation. The events occurring between metaphase II and formation of the nucleus are independent of protein synthesis.by U. Scheer     

Parthenogenetic activation of bovine oocytes using single and combined strontium, ionomycin and 6-dimethylaminopurine treatments

In vitro-matured (IVM) bovine oocytes were activated with single and combined treatments of strontium (S), ionomycin (I) and 6-DMAP (D). Using oocytes IVM for 26 h, we observed that activation altered cell cycle kinetics (faster progression, MIII arrest, or direct transition from MII to pronuclear stage) when compared to in vitro fertilization. The effect of oocyte age on early parthenogenesis was assessed in oocytes IVM for 22, 26 and 30 h. Better results in pronuclear development were obtained in treatments ISD (81.7%) at 22 h; D (66.7%), IS (63.3%), ID (73.3%) and ISD (76.7%) at 26 h; and D (86.7%), IS (85.0%) and ID (78.3%) at 30 h. Higher cleavage occurred on ISD (80.0%) at 22 h; ID (83.3%) and ISD (91.7%) at 26 h; and I (86.7%), IS (90.0%), ID (85.0%) and ISD (95.0%) at 30 h. More blastocysts were achieved in ID (25.0%) and ISD (18.3%) at 22 h; and in ID at 26 h (45.0%) and 30 h (50.0%). We also observed that IS allowed higher haploid (77.4%) embryonic development, whilst ID was better for diploid (89.1%) development. It was concluded that association of S and D without I was not effective for blastocyst development; treatments using S were less influenced by oocyte age, but when S was associated with D there was a detrimental effect on aged oocytes; treatment ISD promoted higher activation and cleavage rates in young oocytes and ID protocol was the best for producing blastocysts.     

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

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

Activation of Xenopus laevis eggs in the absence of intracellular Ca activity by the protein phosphorylation inhibitor, 6-dimethylaminopurine (6-DMAP).

Xenopus laevis eggs pricked or microinjected with water or saline in medium containing a limited quantity of free Ca (1.0 to 2.0 microM) remain unactivated for at least 6 hr, even after transfer to oocyte medium containing Ca at higher concentrations (0.5-1.0 mM). These injected eggs, when later pricked in oocyte medium or exposed to A23187 or urethane are fully capable of activation. This confirms the observations of Wangh ('89). However, eggs injected in this Ca-limited medium (CaLM) with 6-DMAP as well as those simply exposed to this drug undergo changes characteristic of activation, including cortical contraction, cortical granule breakdown, a loss of MPF and CSF activities, and pronuclear formation. The time required for 6-DMAP to induce egg activation is inversely correlated to its concentration. Interestingly, eggs that have been injected with EGTA, and thus are unable to respond to activation stimuli such as pricking and A23187 or urethane treatment, can also be activated by exposure to 6-DMAP. In contrast, eggs exposed to or injected with a 6-DMAP analogue (6-aminopurine or puromycin) or a protein synthesis inhibitor (cycloheximide or emetine or puromycin) are not activated. As well, eggs injected in CaLM with 6-DMAP simultaneously with a phosphatase inhibitor (NaF or ammonium molybdate) fail to become activated. Although 6-DMAP-activated eggs remain at the pronucleus stage so long as 6-DMAP is present, they resume cell cycle activities after the drug is withdrawn. They form cleavage furrows, disassemble pronuclear envelopes, and recondense chromosomes. Also, MPF activity reappears and cycles at least twice, peaking each time shortly before cleavage furrow formation. These results suggest that activation of Xenopus eggs arrested at metaphase II by inhibition of protein phosphorylation does not require intracellular Ca release and that maintenance of the egg at metaphase II depends upon continuous protein phosphorylation.     

Differential effects of 6-DMAP, olomoucine and roscovitine on Xenopus oocytes and eggs

The effects of the new cyclin-dependent kinase inhibitors, roscovitine and olomoucine, on oocytes and eggs of Xenopus laevis were investigated and compared with those of 6-dimethylamino purine (6-DMAP). The inhibitory properties of 6-DMAP, olomoucine and roscovitine towards p34cdc2-cyclin B isolated from Xenopus eggs revealed K-IC50 values of 300, 40 and 10 microM respectively. The three compounds inhibited progesterone-induced maturation with M-IC50 values of 200, 100 and 20 microM. These values were consistent with the K-IC50 values but the ratio M-IC50/K-IC50 was higher for roscovitine and olomoucine than for 6-DMAP. The disappearance of spindle and condensed chromosomes without pronucleus formation was observed when 1 mM 6-DMAP was applied for 4 h at germinal vesicle breakdown or at metaphase II, whereas no effect was observed using 1 mM olomoucine or 50 microM roscovitine. Changes in the electrophoretic mobility of p34cdc2 and erk2 were observed only in homogenates of matured oocytes or eggs exposed for 4 h to 1 mM 6-DMAP. When the drugs were microinjected into matured oocytes, olomoucine (100 microM) and roscovitine (50 microM) induced pronucleus formation more efficiently than did 6-DMAP (100 microM). Taken together, these results demonstrate that Xenopus oocytes possess a lower permeability to olomoucine and roscovitine and that these new compounds are suitable for in vivo studies after germinal vesicle breakdown provided they are microinjected.     

6—DMAP和胚胎干细胞代数对异种重构卵体外发育的影响（简报）

The development of reconstructed oocytes and the survival rate of cloned animal were affected by many factors during nuclear transfer. The genetic constitution and the genetic state of donor nucleus were proposed to be primary factors, which affected the survival rate of cloned animal. In addition, the survival rate of cloned animal might be influenced by nuclear transfer technique itself and passages of donor cells as well as the activation methods of oocytes. We reconstructed oocytes with outbreeding Kunming albino mouse ES cells and enucleated rabbit oocytes, and analyzed the effects of the passages of ES cells and 6-DMAP on the development of interspecific reconstructed oocytes. The interspecific reconstructed ES-rabbit oocytes were activated either by combined two set electric pulses and 6-DMAP or by two set electric pulses alone. The rate of cleavage was significantly higher for the group (86.2%) treated with 6-DMAP than the group (64.2%, P < 0.05) treated with electric pulses only, and the rate of blastocysts was 17.0% and 13.4% respectively, which were not significantly different between two groups. When ES cells that had been passed for 24 and 14 generations were used as donors, the cleavage rates of the reconstructed oocytes were 88.5% and 82.1%, respectively (P > 0.05), and the rates of blastolation were 16.7% and 15.4%, respectively (P > 0.05). The results show that 6-DMAP increases the cleavage rate of reconstructed oocytes derived from ES cells, and affects slightly the developmental rate of blastocysts. There are no differences when high passage and low passage ES cells are used as nuclear donors.     

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.     

Effects of chronic exercise on the kidneys at different ages

In order to determine the effects of chronic exercise on the kidneys at different ages, young, adult, and old rats swam 1 hour either daily or twice a week for 10 weeks and then were killed along with unexercised controls. The kidneys were removed and sections were prepared for histometric analysis including planimetric measurements on camera lucida drawings of renal components and line sampling. With both degrees of exercise young rats showed lower kidney weight, fewer glomeruli and less medullary tissue than unexercised controls. In the adult group no significant differences were noted between exercised and unexercised rats. In old rats both degrees of exercise resulted in a loss of kidney weight and medullary and cortical mass, and a decrease in size of glomeruli while total number of glomeruli remained unchanged. Thus the effects of chronic exercise on the kidneys varied with age. Retarded kidney development occurred in young animals; a loss of renal tissue in old animals; and no change in adult animals.     

c-Mos proteolysis is independent of the CA(2+) rise induced by 6-DMAP in Xenopus oocytes

In Xenopus oocytes, metaphase II arrest is due to a cytostatic factor (CSF) that involves c-Mos, maintaining a high MPF (cdk1/cyclin B) activity in the cell. At fertilization, a rise in intracellular calcium triggers the proteolysis of both cyclin B and c-Mos. The kinase inhibitor 6-dimethylaminopurine (6-DMAP) is also able to release matured Xenopus oocytes from metaphase II block. This is characterized by c-Mos proteolysis without degradation of cyclin B. We hypothesized that 6-DMAP induced an increase in intracellular calcium. Using the calcium-sensitive fluorescent dye Fura-2, we observed a systematic increase in intracellular calcium following 6-DMAP application. In matured oocytes previously microinjected with the calcium chelator BAPTA, no calcium changes occurred after 6-DMAP addition; however, c-Mos was still proteolysed. In oocytes at the GVBD stage, c-Mos proteolysis occurred in response to 6-DMAP but not to calcium ionophore treatment. We suggest that c-Mos proteolysis is rather controlled by a phosphorylation-dependent process.     

Effects of different activation treatments on fertilization of horse oocytes by intracytoplasmic sperm injection

The effects of four reagents on the activation and subsequent fertilization of equine oocytes, and the development of these after intracytoplasmic sperm injection, were investigated. Cumulus-oocyte complexes collected from equine ovaries obtained from an abattoir were matured in vitro for 40-44 h in TCM199 medium before being injected, when in metaphase II, with an immobilized stallion spermatozoon. The cumulus-oocyte complexes were then subjected to one of five activation treatments: (a) 10 micromol ionomycin l(-1) for 10 min; (b) 7% (v/v) ethanol for 10 min; (c) 100 micromol thimerosal l(-1) for 10 min; (d) 250 micromol inositol 1,4, 5-triphosphate l(-1) injection; and (e) no treatment (control). After 18-20 h further culture, the cumulus-oocyte complexes were assessed for activation by observing whether they had progressed through second anaphase-telophase and had formed a female pronucleus. The proportions of oocytes activated after each treatment were: 16/27 (59%) for ionomycin; 14/25 (56%) for ethanol; 22/28 (79%) for thimerosal; 15/27 (56%) for inositol 1,4,5-triphosphate; and 0/20 (0%) for the untreated controls. Thus, significantly more oocytes (P < 0.05) were activated by treatment with thimerosal than by the other four treatments. The proportions of oocytes that cleaved to the two-cell stage at 24-30 h after sperm injection in the groups treated with ionomycin, ethanol and thimerosal were 7/20 (35%), 5/19 (26%) and 11/23 (48%), respectively. No cleavage was observed in any of the control oocytes or those treated with inositol 1,4, 5-triphosphate. Furthermore, evidence of normal fertilization was observed in 2/7 (29%), 2/5 (40%) and 7/11 (64%) of the oocytes treated with ionomycin, ethanol and thimerosal, respectively. These results demonstrated that: (a) it is possible to activate equine oocytes with the chemical stimulants, ionomycin, ethanol, thimerosal and inositol 1,4,5-triphosphate; (b) thimerosal is more effective than the other three reagents in facilitating both meiotic activation and normal fertilization of equine oocytes; and (c) chemical activation may also stimulate parthenogenetic cleavage of oocytes without concurrent changes in the head of the spermatozoon.     

Effect of cycloheximide, 6-DMAP,roscovitine and butyrolactone I on resumption of meiosis in porcine oocytes

Improvement of the ability to maintain germinal vesicle stage oocytes in vitro is important for the acquisition of developmental competence. Maintaining oocytes at this stage without damaging their quality would allow synchronization of maturation and homogenization of the oocytes population. More investigations are needed to better understand how the oocyte cell cycle is blocked without consequences to future developmental competence. This study tested the efficacy of pharmacological inhibitors of the G2/M cell cycle transition in keeping porcine oocytes at the germinal vesicle (GV) stage and the reversibility of this inhibition. Porcine cumulus-oocyte complexes (COCs) were thus incubated without any hormones for 24 h in the presence or absence of tested inhibitors: 6-DMAP (protein kinase inhibitor, 2 mM), cycloheximide (protein synthesis inhibitor, 2 microg/ml), roscovitine (cyclin-dependent kinase inhibitor, 50 microM) and butyrolactone I (cyclin-dependent kinase inhibitor, 50 microM). Cumulus-oocyte complexes cultured with any of the inhibitors were significantly blocked at the GV stage. The inhibitory effect varied according to the products, with cycloheximide being the most efficient. Reversibility of the pharmacological inhibitors was assessed by culturing COCs an additional 24 h in inhibitor-free culture medium. Examination of oocytes revealed that the inhibitory effect was fully reversible. This study suggests that 6-DMAP, cycloheximide, roscovitine and butyrolactone I can be use to block meiotic resumption in porcine oocytes in NCSU culture medium.