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.     

In vitro fertilization analysis of squirrel monkey oocytes produced by various follicular induction regimens and the incidence of triploidy

An in vitro fertilization system utilizing squirrel monkeys was used to compare follicle-stimulating hormone, clomiphene citrate and prostaglandin E₁ as follicular induction regimens, analyze culture medium characteristics, and examine the physiological phenomenon of polyspermy. Induction of follicular growth was poor with clomiphene citrate when compared to the control group and increased the incidence of atretic follicles at all levels tested. When prostaglandin E₁ was administered, larger numbers of mature oocytes were recovered at laparoscopy. There was no difference in fertilization rate between the treatment and control groups. Homologous serum was an adequate protein source in TC-199 fertilization medium for squirrel monkey oocytes. Although the rate of triploidy was increased with in vitro fertilization, there was no relationship between sperm concentration and the incidence of polyspermy. These findings demonstrate that the squirrel monkey is a valuable primate system for studies of in vitro fertilization and preimplantation development. © 1993 Wiley-Liss, Inc.     

Effects of caffeine,Ca++, capacitation time,and strain on in vitro fertilization in mice

Oocytes (N=2922) were collected from superovulated female C57B16/J X DBA2/J (B6D2F₁) mice and distributed among 48 treatments consisting of a 2×3×2×2×2 factorial design. The factors were strain of spermatozoa, B6D2F₁ or SJL/J; caffeine concentration in the fertilization medium, 0,2, or 6 mM; time oocytes were exposed to sperm, 1 or 2 hours; Ca⁺⁺ concentration in the capacitation medium, 0 or 1.8 mM; and capacitation time, 1 or 2 hr. Ova were observed 400 min after they were initially exposed to 10⁵ spermatozoa per ml. Ova with two or more pronuclei and a second polar body were considered fertilized, In vitro embryonic development was monitored for 5 days. B6D2F₁ spermatozoa resulted in consistently higher rates of fertilization than SJL spermatozoa, 77.5% vs 38.7% when averaged over other treatments. Caffeine concentrations of 0,2, and 6 mM resulted in respective mean fertilization rates of 50.1%, 58.8%, and 65.4% (P<0.005) when averaged over other factors. Fertilization rates of ova exposed 1 and 2 hr to sperm were 53.0% and 63.3% (P<0.005). B6D2F₁ spermatozoa capacitated in medium with 1.8 mM Ca⁺⁺ fertilized more ova (P<0.01), 83.1%, than when no Ca⁺⁺ was present, 71.9%; this effect was absent with SJL spermatozoa. The effect of capacitation time depended on strain. Fertilization rates with B6D2F₁ spermatozoa were higher, 80.1%, with a 2-hour capacitation time than with a 1-hour capacitation time, 75.0%. Exactly the opposite was true for the SJL spermatozoa; 43.4% for the 1-hour and 34.1% for 2-hour capacitation (P<.01). Development to the blastocyst stage was significantly greater (P<0.025) for ova fertilized by B6D2F₁ (26.8%) than by SJL spermatozoa (17.7%).     

Morphology and fertilizability of frozen human oocytes

Human oocytes were frozen and thawed by four methods previously used for cryopreser-vation of human embryos. Most of these oocytes were inseminated after thawing to assess their capacity to fertilize and form pronuclear ova. Their morphology was assessed by phase-contrast microscopy used in routine IVF. Twenty-three oocytes were examined by electron microscopy to critically evaluate the effects of cooling and cryopreservation and to confirm fertilization. Morphological survival was observed in more than 60% of the oocytes examined after freeze-thawing. The main features of cryoinjury were cracks in the zona pellucida, disruption of the plasma membrane and extensive disorganization of the ooplasm. Subtle changes in the cytosol of cumulus cells was also observed. Cooling to 0°C or ?6°C had little effect on cytoplasmic structure. Spindles were damaged in two frozen oocytes. Cumulus cell activity, sperm binding to the zona, sperm penetration of the zona seem to be largely unaffected by freeze-thawing. Fertilization was observed in eight oocytes after postthaw insemination and three embryos (8-cell to morula stages) were developed from pronuclear ova on further culture. Both monospermic and polyspermic fertilization were confirmed by electron microscopy and micronuclei were detected in three pronuclear ova. The genetic implications of these nuclear aberrations are discussed. These preliminary studies indicate that oocyte freezing needs to be integrated cautiously with clinical IVF by further assessment of embryos developed from frozen oocytes.     

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.     

Monoclonal antibodies against unfertilized zona-free mouse oocytes: Characterization and effects on fertilization

A panel of anti-oocyte antibodies was raised against unfertilized zona-free mouse oocytes by intrasplenic immunization and checked for their effects on in vitro fertilization. Four antibodies decreased the fertilization rate from about 90% in controls to 8% (B5-2 F7), 12% (A2-2 A7), 13% (4-G1), and 25% (A2-2 F2), when the sperm cell concentration was 1 × 10⁵ to 1 × 10⁶. Antigen localization: All the antibodies labelled components in the cell membrane of zona-free oocytes as demonstrated by indirect immunofluorescence and/or by complement-mediated oocyte lysis. In various patterns, the ooplasm and zona pellucida were also labelled with different intensities. Western blotting: A2-2 A7 and A2-2 F2 recognized a protein with a molecular weight of approximately 65 kDa, while antibody B5-2 F7 bound a 97 kDa protein. Complement activation and complement-mediated oocyte lysis: Systemically injected antibodies, C3 and C4 were detected on zona-free oocytes recovered from the mouse oviduct indicating the activation of C3 and C4 by antigen-antibody complexes. The recovered oocytes were not damaged, suggesting a presence of complement-regulating factors. In vitro, however, a large number of zona-free oocytes preincubated with antibodies were lysed or protruded ooplasma vesicles in complement-active serum. Stage, tissue, and species specificity: None of the antibodies, except A2-2 A7, showed a positive immunolabelling to the pronuclear stage. Antibodies 4-G1 and A2-2 F2 cross-reacted with the ovarian oocytes. No antibodies bound to any of the tissues tested, indicating that the corresponding antigen epitopes are not commonly expressed. A2-2 A7, A2-2 F2, and B5-2 F7 cross-reacted with hamster and human unfertilized oocytes, suggesting the presence of developmentally conserved molecules and the possibility to apply these antibodies in hamster and human in vitro fertilization. It is concluded that the approach used could be a useful strategy in searching for anti-fertilization antibodies for human contraception. © 1996 Wiley-Liss, Inc.     

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.     

Failure of spermatozoa from T/t mice to fertilize in vitro is overcome by zona drilling

Failure of epididymal spermatozoa from T/t mutant mice, but not from t/t individuals, to fertilize oocytes in vitro was partially overcome by opening a small aperture in the zona pellucida with acidified Tyrode's solution to permit direct access of the spermatozoon to the vitellus. This study provides a model system to evaluate requirements for successful zona drilling in the treatment of human infertility and further insights into the effects of the t complex on sperm fertility.     

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.     

Second meiotic nondisjunction is not increased in postovulatory aged murine oocytes fertilized in vitro

Summary We used an in vitro fertilization system to examine the effects of postovulatory oocyte age on nondisjunction at the second meiotic division. After ovulatory-inducing injections of hormone, we recovered mouse oocytes either at the estimated time of ovulation (controls) or 2, 4, 5, 10, or 14 h later. Oocytes were subjected to an in vitro fertilization procedure, and chromosomal preparations were made from first cleavage metaphase eggs. The first cleavage assay reveals morphologically distinguishable paternal and maternal chromosomes. Many of the aged oocytes were activated rather than fertilized by the in vitro procedure, but could still be analyzed for nondisjunction. We foun a tendency toward retention of the second polar body after 10 and 14 h aging. A total of 488 maternal genomes, 290 of which were in the control group, were analyzable for nondisjunction. Seven hyperhaploid genomes (2.4%) were observed in the controls and 6 (3.0%) in the combined aged group. The difference between these two frequencies is not significant (G _adj=0.164,P>0.50). In the aged group, one hyperhaploid genome was in the 2-h population, three in the 5-h population, and two in the 10-h population. We were unable to find any significant increase in the frequency of nondisjunction after postovulatory oocyte aging. This work was supported by National Institutes of Health, Bethesda, MD, grants HD-12035 and HD-19040 to PAM-D.     

Influence of luteinizing hormone and progesterone on maturation and fertilizability of rat oocytes in vitro

The effects of luteinizing hormone (NIH-bovine LH) and progesterone on maturation in vitro of oocyte-cumulus complexes from adult proestrous rats were studied by comparing proportions of oocytes showing germinal vesicle breakdown, mucification of the cumulus oophorus, and fertilizability. Addition of either or both of the hormones to the medium in concentrations between 1.25 and 10 μg/ml during maturation had no discernible effect on germinal vesicle breakdown or on fertilization. Mucification was stimulated by LH and even more by LH plus progesterone. It was concluded that maturation in vivo is the result of concerted action of the two hormones. However, addition of LH + progesterone had no effect on the fertilizability of these oocytes. We attribute this to a relative insensitivity of the system for fertilization in vitro to subtle changes in the oocyte.     

Differences in sperm function in vitro but not in vivo between inbred and random-bred mice

Genetic variation in spermatozoa was used to examine mechanisms important for fertilization in the mouse. A significantly greater proportion of cauda epididymal sperm from C57BL/6 (inbred) males were motile than from random-bred (CFW) males. Random-bred sperm, however, were able to fertilize a significantly greater percentage of eggs in vitro than were inbred sperm. When sperm of these two genotypes were used for insemination in vivo, and the penetrated eggs cultured through the first cleavage, the levels of cleavage were similar, suggesting that neither levels of sperm motility nor sperm penetration in vitro accurately reflect the ability of the same sperm populations to penetrate eggs in vivo.     

小鼠体外受精、胚胎培养及胚胎快速冷冻的研究

目的　为扩大胚胎来源并获取特定胚龄胚胎 ,建立小鼠冷冻胚胎库。方法　运用超数排卵、体外受精与胚胎培养及胚胎冷冻技术系统研究了小鼠受精卵的体内发育与运行规律。卵母细胞的体外成熟与受精、单细胞胚胎培养及胚胎快速冷冻。结果　 (1)注射hCG后 12～ 2 0h受精卵发育至原核期 ,4 2～ 4 8h为 2 细胞期 ,4 8～ 6 0h为 4 细胞期 ,6 0～ 6 8h为 8 细胞期 ,以上各期受精卵均处于输卵管中 ;75～ 78h为桑椹胚 ,78～ 80h为致密桑椹胚 ,90～ 92h为早期囊胚 ,92～ 96h为囊胚 ,以上各期均处于子宫角中。 (2 )培养液中添加促性腺激素 (FSH与hCG) ,能显著提高卵母细胞的体外受精率 ,添加FCS和激素组的体外受精率又显著高于单独添加激素组 ,FCS还能显著提高胚胎发育。 (3)在培养液中添加EDTA ,能有效克服小鼠胚胎的 2 细胞阻断 ,其 2 细胞胚的发育率达 10 0 % ,8 细胞胚发育率达 5 5 %以上 ;牛、羊上皮细胞培养液上清也能有效克服 2 细胞阻断。添加乳酸钠和丙酮酸钠可使 2细胞与 8细胞期胚的发育率显著提高。 (4)以D PBS +甘油 +蔗糖为冷冻液 ,以D PBS +蔗糖为稀释液 ,对小鼠胚胎进行快速冷冻 ,桑椹胚的存活率为 6 9 3% ,早期囊胚的存活率为 6 0 4 %。结论　研究为将生物技术应用于小鼠 ,扩大卵子和胚胎来源     

Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid

The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine (Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (−) FCs (5.2 × 10⁶ cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, −FC/S, +FC/R, and −FC/R) under 5% or 20% O₂. Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O₂ using the −FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both −FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the −FC/S culture system for promoting fertilization.     

Blastocysts derived from in vitro-fertilized cat oocytes after vitrification and dilution with sucrose

Experiments were conducted to find an optimal incubation period in a sucrose solution during dilution of cryoprotectants for obtaining a higher level of survival and development of cat oocytes cryopreserved by vitrification method. In the first experiment, in vitro-matured fresh oocytes were exposed to 0.5M sucrose solution for 1 or 5 min before in vitro fertilization (IVF). The percentage of development to the blastocyst stage significantly decreased in oocytes exposed for 5 min, compared with oocytes exposed for 1 min and control oocytes without exposure to sucrose (P<0.05). In the second experiment, oocytes that had been vitrified in 40% ethylene glycol and 0.3M sucrose were liquefied and then incubated in 0.5M sucrose for 0.5, 1 or 5 min to dilute the cryoprotectant. The percentage of cleavage (>or=2-cell stage) of vitrified-liquefied oocytes incubated for 0.5 min was significantly higher (P<0.05) than that of other groups. Development of vitrified-liquefied oocytes to the morula and blastocyst stages after IVF was observed only in oocytes incubated in sucrose for 0.5 min. The present study indicates that the oocytes have sensitivity to the toxic effect of sucrose and that the incubation period during dilution of the cryoprotectant is of critical importance for developmental competence of vitrified-liquefied cat oocytes.     

Parthenogenetic activation of Chinese hamster oocytes by chemical stimuli and its cytogenetic evaluation

This study was undertaken parthenogenetically to activate Chinese hamster oocytes in vitro by chemical stimuli. Oocytes were exposed to five different chemical agents, ethanol (EtOH), strontium chloride (SrCl₂), cycloheximide (CHX), phorbol ester (PMA), and ionophore A23187 (IA23). No parthenogenetic activation was observed in the oocytes treated with 8% EtOH for 8–11 min, 1.7 mM and 5.0 mM SrCl₂ for 1 hr, 100 μM and 400 μM CHX for 2 hr, and 81 nM and 162 nM PMA for 5 min. In contrast, 89.7% of oocytes parthenogenetically extruded the second polar body in treatment with 3 μM IA23 for 5 min, but only 22.6% of them formed a pronucleus and developed to 2-cell embryos. The remaining ova stopped their cell cycle immediately after completion of the second meiotic division. They had unichromatid chromosomes (monads), which are called MIII chromosomes. Treatment with 5 μM IA23 for 5 min was so deleterious that >90% of oocytes were degenerated. However, oocyte activation was significantly improved when the treatment with 3 μM IA23 for 5 min was followed by treatment with 8% EtOH for 10 min, 100 μM CHX for 2 hr, 81 nM PMA for 5 min or 3 μM IA23 for 5 min: rates of pronuclear formation were 54.4%, 84.3%, 34.2%, and 54.6%, respectively. More than 80% of pronucleate ova successfully developed into 2-cell stage. Additive treatment with 5 mM SrCl₂ for 1 hr had no positive effect on pronuclear formation. Incidences of aneuploidy (4.6%) and structural chromosome aberrations (1.0%) in parthenogenons produced by combined stimuli of IA23 and CHX were not significantly different from those (3.8% and 1.6%, respectively) in female pronuclei of ova fertilized in vitro, showing that combined treatments with IA23 and CHX cause neither nondisjunction at the second meiotic division nor structural aberrations in MII chromosomes. The present technique for parthenogenetic activation of Chinese hamster oocytes may be useful as an assessment system to detect aneugenic and clastogenic effects of mutagens on mammalian oocytes. Mol. Reprod. Dev. 47:72–78, 1997. © 1997 Wiley-Liss, Inc.     

Wheat germ agglutinin treatment of follicle cell-free mouse oocytes inhibits fertilization

Controversy exists whether treatment of follicle cell-free oocytes with wheat germ agglutinin (WGA) prevents fertilization. Lack of inhibition in one case has led to the suggestion that acrosin may not be a zona lysin. To re-examine the effect of the WGA, the zona pellucida of follicle cell-free mouse oocytes was made more resistant to proteinase digestion by treatment with 10 or 50 μg/ml WGA. Such WGA-treated oocytes showed decreased fertilizability when washed to remove excess WGA and incubated with capacitated spermatozoa. Oocyte cleavage was used as an end point, because a large number of spermatozoa adhered to the eggs after WGA treatment, making observation of sperm penetration and pronucleus formation unreliable. Resistance to proteinase digestion increased, and the fertilizability decreased with the higher amount of WGA. The action of WGA was most likely not mediated by a direct effect on sperm motility, sperm acrosin activity, sperm binding to the zona pellucida, or oocyte cleavage. WGA did not affect the acrosome reaction of guinea pig spermatozoa. These data show that WGA treatment of follicle cell-free mouse oocytes results in decreased fertilizability, possibly by rendering the zona pellucida more resistant to sperm proteinase digestion.     

In vitro maturation and artificial activation of donkey oocytes

Three media were evaluated for their ability to support in vitro maturation of donkey (Equus asinus) oocytes and their development after parthenogenetic activation. The basal medium for Medium 1 (M1) and Medium 2 (M2) was M199 and DMEM/F12 respectively, whereas, Medium 3 (M3) consisted of equal parts (v/v) of M199 and DMEM/F12. All three media were supplemented with 10% (v/v) fetal calf serum, 0.01 units/mL porcine FSH, 0.01 units/mL equine LH, 200 ng/mL insulin-like growth factor 1(IGF-I), 10 μl/mL insulin-transferrin-selenium (ITS), 0.1 mg/mL taurine, 0.1 mg/mL L-cysteine, 0.05 mg/mL L-glutamine, 0.11 mg/mL sodium pyruvate, and 25 mg/mL gentamycin. There were no significant differences among the three maturation media for oocyte maturation. Maturation rate of donkey oocytes in M1 was 53% for compact (Cp) cumulus-oocyte complexes and 75% for expanded (Ex) cumulus-oocyte complexes; in M2 these were 55 and 77%, respectively; and in M3, 58 and 75%. The percentage of cleaved parthenotes and 4- or 8-cell embryos were not significantly different for oocytes matured in the various media (61 and 24% for M1; 66 and 32% for M2; and 67 and 33% for M3). Oocytes matured in M3 tended to yield a higher rate of advanced embryo development (morula) than oocytes matured in M1 (22 vs 9%; P = 0.07). In conclusion, donkey oocytes were matured and parthenogenetically activated in vitro, using methods similar to those used in the horse.     

显微受精废弃的人类卵母细胞体外成熟及孤雌激活

以卵胞浆单精注射(intracytoplasmic sperm injection,ICSI)后废弃的未成熟人类卵母细胞(生发泡期卵母细胞(the germinal vesicle,GV)和第一次减数分裂中期卵母细胞(the metaphase,MI))为材料,使用卵母细胞体外成熟培养液培养未成熟的卵母细胞,分别在人类绒毛膜促性腺激素(human chorionic gonadotrophin,hCG)注射后45、60、84 h观察卵母细胞成熟情况.分别使用钙离子载体(calcium ionophore,CI)A23187联合6-二甲基氨基嘌呤(6-DMAP)法或精子提取物卵胞质内注射(sperm extracts intracytoplasmic injection,SEII)法两种不同的激活方法对体外成熟MII的卵母细胞进行孤雌激活,评价其体外发育潜能.MI卵子体外成熟率要显著高于GV(75.2%vs 30.6%)(P<0.01).与CI/6-DMAP法相比使用SEII/6-DMAP法在激活率(87.5%vs 70.2%)上要明显高于CI/6-DMAP法(P<0.05),但在卵裂率(65.7%vs 72.5%)和桑囊率(0%vs 5.0%)上SEII/6-DMAP法要低于CI/6-DMAP法.注射hCG 45 h组的卵母细胞激活率(91.3%vs 57.9%)、卵裂率(85.7%vs 57.9%)及桑囊率(9.5%vs 0%)均显著高于注射hCG 60 h组(P<0.01).56.8%(117/206)的ICSI废弃的未成熟卵母细胞可以在体外发育成熟,激活后具有一定的发育潜能,卵龄对卵母细胞的质量和发育能力影响较大.