Effect of exogenous hormones on the ovulation of primary and secondary oocytes in LT/Sv strain mice

LT/Sv strain mice ovulate both primary and secondary oocytes. These are fertilizable and give rise to digynic triploid and normal diploid conceptuses, respectively. A previous study [Kaufman and Speirs, 1987] had indicated that just over 20% of embryos recovered on the 10th day of gestation from spontaneously ovulating females had a triploid chromosome constitution. This value was considerably lower than might have been expected by extrapolation from earlier studies in which LT/Sv mice had been given exogenous gonadotrophins. In the present study, therefore, cytogenetic analysis of fertilized eggs was performed at the first cleavage mitosis in (1) spontaneously ovulating females mated to F1 hybrid males, and (2) superovulated females mated to similar males. Additional females from group (1) were autopsied on the 10th day of gestation, and the ploidy of embryos isolated at this stage of gestation was determined. Exposure to exogenous gonadotrophins significantly increased the proportion of eggs that were ovulated as primary oocytes (34.4%), compared to the situation observed following spontaneous ovulation (24.4%). All the triploids encountered in both series were of the digynic type and characteristically (for LT/Sv mice) had an oocyte-derived set with 40 chromosomes present, and a sperm-derived set containing 20 chromosomes. Similar numbers of eggs were recovered from spontaneously ovulating females on the 1st and 10th days of gestation, and the incidence of triploidy observed on the 10th day was 22.1%. The influence of exogenous hormones in increasing the “spontaneous” level of triploidy in LT/Sv and in other strains of mice is briefly reviewed.     

Spontaneous and sperm-induced activation of oocytes in LT/Sv strain mice

The oocytes of LT/Sv strain mice are unique in that a high proportion of them (∼40% in this study) are ovulated before reaching metaphase of the second meiotic division (metaphase II). The remaining oocytes of LT/Sv mice are ovulated at metaphase II, as in other strains of mice. When recently ovulated oocytes were cultured in vitro for 11–12 h, those ovulated at metaphase II remained at this stage, whereas those ovulated at metaphase of the first meiotic division (metaphase I) commonly resumed meiosis during in vitro aging. These oocytes extrude the polar body and form a diploid pronucleus. This oocyte activation is not coupled with cortical granule exocytosis. The oocytes ovulated at metaphase II are fully capable of normal fertilization, whereas those ovulated at metaphase I are not. Approximately 50% of metaphase I oocytes penetrated by spermatozoa remain at this stage, and sperm nuclei frequently undergo premature chromosome condensation. Only 13% of spermpenetrated metaphase I oocytes formed a diploid female pronucleus and a haploid male pronucleus by 4 h after insemination. These results demonstrate that the two types of ovulated LT/Sv oocytes have different potentials to undergo either spontaneous or sperm-induced activation.     

Ovulation and fertilization of primary and secondary oocytes in LT/Sv strain mice

In this study, the chromosome constitution of both unfertilized oocytes and fertilized eggs isolated from the oviducts of LT/Sv strain mice were analyzed. Air-dried chromosome preparations from unfertilized oocytes revealed that about one-third of those examined were ovulated as primary oocytes. These were arrested at metaphase of the first meiotic division and exhibited the characteristic “tetrad” chromosome configuration. The remaining two-thirds of the unfertilized oocytes were ovulated at metaphase of the second meiotic division. The fertilized eggs were isolated from the oviducts of LT/Sv females previously mated to (C57BL × CBA) F1 hybrid males. Analysis of the fertilized eggs at metaphase of their first cleavage mitosis revealed that about one-third of the eggs examined were digynic triploids, whereas the remaining two-thirds had the normal diploid chromsome constitution. In the triploids, the 40 female chromosomes present (mouse, n = 20) were derived from a single diploid pronucleus formed after the extrusion of a first polar body, and following the monospermic fertilization of primary oocytes. The female pronuclear-derived chromosomes invariably exhibited “homologous pairing,” and these were associated at their centromeres. The ovulation, penetration, and subsequent fertilization of primary oocytes is an extremely unusual phenomenon in mammals and only appears to occur on a regular basis in LT/Sv mice. The premature “cytoplasmic maturation” of these oocytes is of interest, as they clearly have the same developmental capacity as secondary oocytes. The significance of these observations in relation to folliculogenesis and litter size in LT/Sv mice is discussed.     

Cytostatic Activity Develops during Meiosis I in Oocytes of LT/Sv Mice

Oocytes of wild-type mice are ovulated as the secondary oocytes arrested at metaphase of the second meiotic division. Their fertilization or parthenogenetic activation triggers the completion of the second meiotic division followed by the first embryonic interphase. Oocytes of the LT/Sv strain of mice are ovulated either at the first meiotic metaphase (M I) as primary oocytes or in the second meiotic metaphase (M II) as secondary oocytes. We show here that duringin vitromaturation a high proportion of LT/Sv oocytes progresses normally only until metaphase I. In these oocytes MAP kinase activates shortly after histone H1 kinase (MPF) activation and germinal vesicle breakdown. However, MAP kinase activation is slightly earlier than in oocytes from wild-type F1 (CBA/H × C57Bl/10) mice. The first meiotic spindle of these oocytes forms similarly to wild-type oocytes. During aging, however, it increases in size and finally degenerates. In those oocytes which do not remain in metaphase I the extrusion of first polar bodies is highly delayed and starts about 15 h after germinal vesicle breakdown. Most of the oocytes enter interphase directly after first polar body extrusion. Fusion between metaphase I LT/Sv oocytes and wild-type mitotic one-cell embryos results in prolonged M-phase arrest of hybrids in a proportion similar to control LT/Sv oocytes and control hybrids made by fusion of two M I LT/Sv oocytes. This indicates that LT/Sv oocytes develop cytostatic factor during metaphase I. Eventually, anaphase occurs spontaneously and the hybrids extrude the polar body and form pronuclei in a proportion similar as in controls. In hybrids between LT/Sv metaphase I oocytes and wild-type metaphase II oocytes (which contain cytostatic factor) anaphase I proceeds at the time observed in control LT/Sv oocytes and hybrids between two M I LT/Sv oocytes, and is followed by the parthenogenetic activation and formation of interphase nuclei. Also the great majority of hybrids between M I and M II wild-type oocytes undergoes the anaphase but further arrests in a subsequent M-phase. These observations suggest that an internally triggered anaphase I occurs despite the presence of the cytostatic activity both in LT/Sv and wild-type M I oocytes. Anaphase I triggering mechanism must therefore either inactivate or override the CSF activity. The comparison between spontaneous and induced activation of metaphase I LT/Sv oocytes shows that mechanisms involved in anaphase I triggering are altered in these oocytes. Thus, the prolongation of metaphase I in LT/Sv oocytes seems to be determined by delayed anaphase I triggering and not provoked directly by the cytostatic activity.     

Exposure to light at night accelerates aging and spontaneous uterine carcinogenesis in female 129/Sv mice

The effect of the constant illumination on the development of spontaneous tumors in female 129/Sv mice was investigated. Forty-six female 129/Sv mice starting from the age of 2 mo were kept under standard light/dark regimen [12 h light (70 lx):12hr dark; LD, control group], and 46 of 129/Sv mice were kept under constant illumination (24 h a day, 2,500 lx, LL) from the age of 5 mo until to natural death. The exposure to the LL regimen significantly accelerated body weight gain, increased body temperature as well as acceleration of age-related disturbances in estrous function, followed by significant acceleration of the development of the spontaneous uterine tumors in female 129/Sv mice. Total tumor incidence as well as a total number of total or malignant tumors was similar in LL and LD group (p > 0.05). The mice from the LL groups survived less than those from the LD group (χ² = 8.5; p = 0.00351, log-rank test). According to the estimated parameters of the Cox’s regression model, constant light regimen increased the relative risk of death in female mice compared with the control (LD) group (p = 0.0041). The data demonstrate in the first time that the exposure to constant illumination was followed by the acceleration of aging and spontaneous uterine tumorigenesis in female 129/Sv mice.     

The postimplantation development of spontaneous digynic triploid embryos in LT/Sv strain mice

When spontaneously ovulating LT/Sv female mice are mated with fertile males, between one third and one half of the zygotes analyzed at the first cleavage mitosis are found to be triploid. This is due to the fact that LT/Sv females ovulate both primary and secondary oocytes, all of which are capable of being fertilized. Fertilization of the former group results in the production of digynic triploid conceptuses, while their diploid littermates result from the fertilization of normal secondary oocytes. The present study was therefore carried out in order to investigate the 'spontaneous' level of triploidy in these mice, and to provide insight into the developmental fate of the LT/Sv triploid embryos, as previous studies had indicated that in this species triploids invariably fail to develop beyond the early postimplantation period. This study revealed that when autopsies were carried out on the 7th and 8th days of gestation, it was generally difficult to distinguish between the karyologically normal diploids and the digynic triploid conceptuses when only morphological criteria were used. However, by the 10th day of gestation, the triploid conceptuses could usually be readily distinguished from their diploid littermates by their smaller size and (occasionally) by their disorganized or abnormal morphological appearance.     

Kinetics of primary and secondary infections with Strongyloides ratti in mice

Dawkins H. J. S. and Grove D. I. 1981 Kinetics of primary and secondary infections with Strongyloides ratti in mice. International journal for Parasitology11: 89–96. The kinetics of infection with S. ratti were quantitated in normal and previously exposed C57B1 /6 mice. In primary infections, larvae penetrated the skin rapidly and were seen in peak numbers 12 h after infection. By 24 h after infection, larval numbers had declined appreciably and there was a slow decrease in numbers thereafter. Larvae were first observed in the lungs at 24 h and maximal recovery occurred at 48 h. It is thought that larval migration through the lungs is rapid. Worms were first seen in the intestines two days after infection. Maximum numbers were seen on the fifth day and worm expulsion was complete by day 10. Two moults took place in the small intestine during days 3 and 4 after infection. Rhabditiform larvae were first noted on the fourth day after infection. Mice exposed to S. ratti four weeks previously had significantly less larvae in the skin 4 and 12 h after infection but by 24 h there was no difference when compared with mice with primary infections. Peak recovery of larvae from the lungs occurred 24 h after infection; significantly less larvae were recovered on days 2 and 3 when compared with normal mice. There was a marked reduction in the adult worm burden in the gut; the number of worms recovered was less than one fifth of that seen in primary infections. Those worms which did mature were less fecund and were expelled from the intestines within 7 days of infection. It is suggested that in previously exposed animals, the migration of larvae from the skin is hastened, many of these larvae are destroyed in the lungs and that expulsion of worms which do mature in the intestines is accelerated.     

Defective calcium release during in vitro fertilization of maturing oocytes of LT/Sv mice

Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in contrast to the oocytes of wild-type mice, a significant fraction of LT/Sv oocytes remains arrested at the metaphase of the first meiotic division and is unable to undergo sperm-induced activation when fertilized 15 hours after the resumption of meiosis. We also show that LT/Sv oocytes experimentally induced to resume meiosis and to reach metaphase II are unable to undergo activation in response to sperm penetration. However, the ability for sperm-induced activation developed during prolonged in vitro culture. Both types of LT/Sv oocytes, i.e. metaphase I and those that were experimentally induced to reach metaphase II, underwent activation when they were fertilized 21 hours after germinal vesicle breakdown (GVBD). Thus, the ability of LT/Sv oocytes to become activated by sperm depends on cytoplasmic maturation rather than on nuclear maturation i.e. on the progression of meiotic division. We also show that sperm penetration induces fewer Ca(2+) transients in LT/Sv oocytes than in control wild-type oocytes. In addition, we found that the levels of mRNA encoding different isoforms of protein kinase C (alpha, delta and zeta), that are involved in meiotic maturation and signal transduction during fertilization, differed between metaphase I LT/Sv oocytes which cannot be activated by sperm, and those which are able to undergo activation after fertilization. However, no significant differences between these oocytes were found at the level of mRNA encoding IP(3) receptors which participate in calcium release during oocyte fertilization.     

Histochemical identification of primordial germ cells in diandric and digynic triploid mouse embryos

Diandric and digynic triploid mouse embryos were isolated in the morning on day 10 of gestation. The embryos were separated from their extraembryonic membranes, and the latter were analysed cytogenetically by G-banding to establish the ploidy and sex chromosome constitution of these embryos. The diandric triploid embryos were produced by the technique of nuclear micromanipulation. Females were mated with male mice with a morphologically distinguishable "marker" chromosome to confirm the diandric status of these embryos. Digynic triploid and normal diploid embryos were isolated from LT/Sv strain females. These females spontaneously ovulate both primary and secondary oocytes, which are fertilisable and give rise to digynic triploid and normal diploid embryos, respectively. All the embryos were serially sectioned and processed in order to demonstrate the presence of alkaline phosphatase enzyme activity. This histochemical technique allowed primordial germ cells to be readily recognised, due to their characteristic location, cellular morphology, and staining appearance. Primordial germ cells were found in all the embryos studied, being located within the visceral yolk sac, at the base of the allantois, and/or in association with the wall or mesentery of the hindgut. The total number of germ cells present was established in nine diandric triploids and in five digynic triploids. The findings presented here represent the first demonstration that primordial germ cells can differentiate in either diandric or digynic triploid mammalian embryos.     

Developmental potential of LT/Sv parthenotes derived from oocytes matured in vivo and in vitro

Release of oocytes of LT/Sv mice from the meiosis-inhibiting influence of antral follicles promotes parthenogenetic activation and development to early cleavage stages of 14% of the eggs. However, to attain the potential to develop to blastocysts under the culture conditions used, the oocytes must mature within follicles for 8–9 hr after human chorionic gonadotropin (HCG) administration. The results suggest that some positive influence, which does not occur during spontaneous oocyte maturation under defined conditions in vitro, occurs within preovulatory follicles and imparts developmental competence to the maturing oocytes.     

Membrane potential,pH and the activation of surf clam oocytes

Unfertilized oocytes of the surf clam, Spisula solidissima, have resting membrane potentials of ?18 ± 7 mV (n = 20). Within five seconds of sperm addition, an electrophysiologically detectable response was apparent, which was characterized by a rapid and prolonged depolarrization depolarization followed four to five minutes post-insemination by the beginning of the beginning of a steady hyperpolarization to approximatelv ?70 mV. This final hyperpolarization was completed within ten minutes of sperm addition. The initial rapid depolarization following insemination may result from a transient increase in sodium conductance, and it may be crucial in preventing polyspermy, since the degree of polyspermy in Spisula oocytes was sensitive to external sodium ion concentrations. Evidence was obtained that changes in intracellular pH are essential for oocvte activation. Using germinal vesical breakdown (GVB) as a marker for activation, it was shown that agents that raise intracellular pH (ammonia and procaine) induced GVB, whereas agents that lower intracellular pH pH (Na-acetate or Na-propionate seawater) inhibited GVB.     

Histopathological appearances in primary and secondary infections with Strongyloides ratti in mice

Dawkins H. J. S., Muir G. M. & Grove D. I. 1981. Histopathological appearances in primary and secondary infections with Strongyloides ratti in mice. International Journal for Parasitology11: 97–103. The histological appearances of the skin, lungs and small intestines of mice with primary and secondary infections with S. ratti are described. When the skins of mice with a primary infection were examined, larvae were seen scattered throughout the dermis. An inflammatory reaction of neutrophils and eosinophils was first noted around larvae 12 h after infection. By 48 h, mononuclear cells were prominent. The intensity of the inflammatory reaction gradually increased to a maximum on the fifth day and the larvae were destroyed. Very few larvae were seen in the lungs; those observed were located in the alveolar spaces and were not surrounded by an inflammatory infiltrate. Worms in the small intestines were found mostly in the crypts of Leiberkuhn, and were probably located within the epithelial layer; there was no significant villous atrophy or cellular infiltration. Marked differences were found in the tissues of mice with secondary infections. In the skin, oedema and neutrophils and eosinophils were seen around worms as early as 2 h after infection. By 24 h after infection, there was a mixed inflammatory infiltrate and worms were undergoing disintegration. Larvae in the lungs were surrounded by polymorphonuclear and mononuclear cells 48 h and 72 h after infection and the engulfed larvae were undergoing lysis. Only a few worms were seen in the intestines of mice with a secondary infection; the histological appearances were similar to that found in animals with primary infections. It is suggested that the rapid development of an oedematous reaction in the skins of immune mice may facilitate the entry of larvae into the bloodstream and that inflammatory cells destroy many larvae in the lungs of immune mice.     

Parthenogenetic activation and subsequent development of rat oocytes in vitro.

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

Course of heavy primary infections and earlier immunologically mediated rejection of secondary infections of Hymenolepis diminuta in mice

Roepstorff A. and Andreassen J. 1982. Course of heavy primary infections and earlier immunologically mediated rejection of secondary infections of Hymenolepis diminuta in mice. International Journal for Parasitology12: 23–28. The worms of heavy (50–100 worms) primary Hymenolepis diminuta infections in inbred C57-mice were 1–2 mm long when growth ceased about day 4. Thereafter the mean length decreased by shrinkage and/or ‘decollation’, the worms moved backwards in the small intestine and were rejected from day 6 to day 10. Heavy secondary infections given 14 days after a heavy primary infection were severely stunted (0.2–0.3 mm) but normally situated in the intestine on day 2 and nearly all were rejected by day 4. Even when the time between the primary and secondary infections was increased to 21 or 42 days, therecovery, position and length of the secondary worms were significantly different from primary infections. These results show that an immunologically mediated memory was involved, and that functional antigens can be released from the scolex and/or the neck alone.     

Association of MPF, MAPK, and nuclear progression dynamics during activation of young and aged bovine oocytes

We have previously shown that bovine oocytes parthenogenetically activated after 40 hours (hr) of in vitro maturation proceed through the cell cycle faster than those after 20 hr of maturation. In the present study, we used this model of different speed of nuclear progression to investigate the correlation of two hallmarks of nuclear events, exit of metaphase arrest and pronuclear formation, with dynamics of MPF and MAPK. Bovine oocytes were matured in vitro for 20 hr (young) or 40 hr (aged) and activated in 7% ethanol followed by incubation in cycloheximide for 0, 0.5, 1, 3, 5, or 7 hr. Activity of MPF and MAPK was lower in aged than young oocytes. The responses to oocyte activation by both the two kinases and nuclear progression were faster in aged than in young oocytes. The activity of MPF declined to undetectable levels (P < 0.05) as early as 0.5 hr after activation in aged oocytes, while this did not happen in young oocytes until 3 hr after activation. The inactivation of MAPK occurred approximately 2 hr earlier in aged oocytes (5 hr post-activation) than in young oocytes (7 hr post-activation). Furthermore, the decline in MPF activity preceded that of MAPK in both young and aged oocytes by about 2 hr. The decrease in activity of MPF and MAPK corresponded with the exit from meiosis and pronuclei formation regardless of the speed of nuclear progression. Despite dramatic changes in activity of MPF and MAPK, the levels of Cdc2 and Erk2 proteins were unchanged (P > 0.05) during the first 7 hr of activation. These observations suggest that inactivation of MPF and MAPK are pre-requisite for the release from metaphase arrest and formation of pronuclei in bovine oocytes.     

The pattern of X-chromosome inactivation in the embryonic and extra-embryonic tissues of post-implantation digynic triploid LT/Sv strain mouse embryos

Spontaneously cycling LT/Sv strain female mice were mated to hemizygous Rb(X.2)2Ad males in order to facilitate the distinction of the paternal X chromosome, and the pregnant females were autopsied at about midday on the tenth day of gestation. Out of a total of 222 analysable embryos recovered, 165 (74.3%) were diploid and 57 (25.7%) were triploid. Of the triploids, 26 had an XXY and 31 an XXX sex chromosome constitution. Both embryonic and extra-embryonic tissue samples from the triploids were analysed cytogenetically by G-banding and by the Kanda technique to investigate their X-inactivation pattern. The yolk sac samples were separated enzymatically into their endodermally-derived and mesodermally-derived components, and these were similarly analysed, as were similar samples from a selection of control XmXp diploid embryos. In the case of the XmXmY digynic triploid embryos, a single darkly-staining Xm chromosome was observed in 485 (82.9%) out of 585, 304 (73.3%) out of 415, and 165 (44.7%) out of 369 metaphases from the embryonic, yolk sac mesodermally-derived and yolk sac endodermally-derived tissues, respectively. The absence of a darkly staining X-chromosome in the other metaphase spreads could either indicate that both X-chromosomes present were active, or that the Kanda technique had failed to differentially stain the inactive X-chromosome(s) present. In the case of the XmXmXp digynic triploid embryos, virtually all of the tissues analysed comprised two distinct cell lineages, namely those with two darkly-staining X-chromosomes, and those with a single darkly staining X-chromosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamate receptor subunit expression in primary neuronal and secondary glial cultures

We report on the expression of ionotropic glutamate receptor subunits in primary neuronal cultures from rat cortex, hippocampus and cerebellum and of metabotropic glutamate (mGlu) receptor subtypes in these neuronal cultures as well as in cortical astroglial cultures. We found that the NMDA receptor (NR) subunits NR1, NR2A and NR2B were expressed in all three cultures. Each of the three cultures showed also expression of the four AMPA receptor subunits. Although RT-PCR detected mRNA of all kainate (KA) subunits in the three cultures, western blot showed only expression of Glu6 and KA2 receptor subunits. The expression analysis of mGlu receptors indicated the presence of all mGlu receptor subtype mRNAs in the three neuronal cultures, except for mGlu2 receptor mRNA, which was not detected in the cortical and cerebellar culture. mGlu1a/alpha, -2/3 and -5 receptor proteins were present in all three cultures, whereas mGlu4a and mGlu8a receptor proteins were not detected. Astroglial cultures were grown in either serum-containing or chemically defined medium. Only mGlu5 receptor protein was found in astroglial cultures grown in serum-containing medium. When astrocytes were cultured in chemically defined medium, mGlu3, -5 and -8 receptor mRNAs were detected, but at the protein level, still only mGlu5 receptor was found.     

Versatile inducible activation system of Akt/PKB signaling pathway in mice

We report a transgenic mouse line in which Akt/protein kinase B (PKB) pathway can be activated in an inducible manner in defined cell types. In this transgenic mouse line, Cre expression allows the expression of a tamoxifen-activatable form of Akt/PKB in a defined cell type. Subsequent injection of tamoxifen triggers the transient activation of Akt/PKB in mice. Thus, this transgenic line allows the transient activation of Akt/PKB pathway in a predefined cell type. We expect that this transgenic system will provide a unique tool to study the roles of Akt/PKB pathway in mice.     

Nuclear transfer in sheep embryos: The effect of cell-cycle coordination between nucleus and cytoplasm and the use of in vitro matured oocytes

The developmental ability of nuclear transplant sheep embryos derived from in vitro matured oocytes was studied by controlling cell-cycle coordination of donor embryonic nuclei and recipient cytoplasts. Oocytes were recovered from nonatretic antral follicles of adult sheep ovaries and cocultured with follicle shells in M199-based medium supplemented with gonadotrophins in a nonstatic system. Effective activation of IVM oocytes was obtained by applying two pulses of 1.0 kv/cm 22 min apart in inositol-based electroporation medium to oocytes matured in vitro for 27 hr. Synthesis of DNA (S-phase) was assessed by BrdU incorporation and was found to initiate around 5 hpa (hours postactivation) and to persist until 18 hpa. Mitotic blastomeres were induced by treating embryos with 6.6 μM nocodazole for 14–17 hr. Three types of transfers were compared directly: “S → S,” early embryonic nuclei (mostly in S-phase) were transferred to presumptive S-phase cytoplasts; “M → MII,” nocodazole-treated embryonic nuclei (most in M-phase) were transferred to MII-phase cytoplasts; and control (S → MII), conventional nuclear transfer of fusion and activation simultaneously. The results showed that fusion and recovery rates did not differ among the three groups. However, after 6 days of in vivo culture, the morula and blastocyst formation rate was significantly higher for the M → MII combination than for the control (28.3% vs. 8.1%, P < 0.05), while no significant differences in developmental rate were observed between S → S and M → MII, and between S → S and control, though developmental rate was also increased for S → S compared to control (20.9% vs. 8.1%, P > 0.05). Transfer of blastocysts derived from M → MII or S → S nuclear cytoplasmic reconstitution to synchronized recipient ewes resulted in the birth of lambs. These data suggest that in vitro matured oocytes can support full-term development of nuclear transplant sheep embryos when the cell cycle of nucleus and cytoplasm is coordinated, and that M → MII nuclear transfer might be an efficient and simple way to improve the developmental competence of the reconstituted embryos. Mol. Reprod. Dev. 47:255–264, 1997. © 1997 Wiley-Liss, Inc.     

Effects of trehalose vitrification and artificial oocyte activation on the development competence of human immature oocytes

Sucrose and trehalose are conventional cryoprotectant additives for oocytes and embryos. Ethanol can artificially enhance activation of inseminated mature oocytes. This study aims to investigate whether artificial oocyte activation (AOA) with ethanol can promote the development competence of in vitro matured oocytes. A total of 810 human immature oocytes, obtained from 325 patients undergoing normal stimulated oocyte retrieval cycles, were in vitro maturated (IVM) either immediately after collection (Fresh group n = 291)) or after being vitrified as immature oocytes (Vitrified group n = 519). These groups were arbitrarily assigned. All fresh and vitrified oocytes which matured after a period of IVM then underwent intra-cytoplasmic sperm injection (ICSI). Half an hour following ICSI, they were either activated by 7% ethanol (AOA group) or left untreated (Non-AOA group). Fertilization, cleavage rate, blastocyst quality and aneuploidy rate were then evaluated. High-quality blastocysts were only obtained in both the fresh and vitrified groups which had undergone AOA after ICSI. Trehalose vitrification slightly, but not significantly, increased the formation rates of high-quality embryos (21.7% VS 15.4%, P > 0.05) and blastocysts (15.7% VS 7.69%, P > 0.05)) when compared with sucrose vitrification. Aneuploidy was observed in 12 of 24 (50%) of the AOA derived high quality blastocysts. High-quality blastocysts only developed from fresh or vitrified immature oocytes if the ICSI was followed by AOA. This information may be important for human immature oocytes commonly retrieved in normal stimulation cycles and may be particularly important for certain patient groups, such as cancer patients. AOA with an appropriate concentration of ethanol can enhance the developmental competence of embryos.