小鼠精子注入兔卵母细胞受精研究

The methods of intracytoplasmic sperm injection (ICSI) and subzonal injection (SUZI) were used to study heterologous fertilization and embryonic development between the mouse and the rabbit. Results were as follows: 1. The mouse sperm nuclei decondensed and formed pronuclei following microinjection into cytoplasm and perivitelline space (PVS) of rabbit oocytes; 2. The hybrid embryos developed to the stage of 8-cell when cultured in vitro; 3. The karyotype analysis showed a normal complement of rabbit oocyte and mouse sperm chromosomes in the 4-cell hybrid embryos; 4. The ultrastructure of 4-cell hybrid embryos was similar to that of normal 4-cell rabbit embryos; 5. The fertilization rate (32.4%) and cleavage rate (22.2%) when 5-10 mouse spermatozoa were injected were higher than those of injection of a single spermatozoon into PVS of the rabbit oocyte, but the difference was not significant (P > 0.05). The fertilization rate (42.3%) and cleavage rate (30.8%) in rabbit oocytes in vitro matured for 11-12 h were higher than those in the oocytes which were in vitro matured for 24-25 h following microinjection of 1-2 mouse spermatozoa into PVS, but the difference was not significant (P > 0.05).     

Enhancement of acrosome reaction and subzonal insemination of a single spermatozoon in mouse eggs.

The acrosome reaction in mouse spermatozoa was induced by various means. These were 1) varying incubation time in T6 medium, 2) incubation in T6 medium with added A23187, 3) incubation in T6 medium with added dbcGMP and imidazole, 4) exposure to an electric field, and 5) a combination of incubation in a medium with dbcGMP and imidazole and electroporation. The mean percentages of acrosome-free spermatozoa obtained by these various methods and assessed on the basis of both Bryan's stain and immunolocalization by FITC-labeled monoclonal antibodies increased by steps from 36% to 67%, 73%, 86%, and 92%. Individual spermatozoa from the various treatments were afterwards microinjected under the zona pellucida of a mouse oocyte. The fertilization rate for eggs microinjected with a spermatozoon treated with A23187, dbcGMP, and imidazole, by electroporation and by a combination of the last two methods also increased by steps from 17% to 34%, 36%, and 70%, respectively. Ninety-five percent of the fertilized oocytes reached the early blastocyst stage, thirty-eight percent of these blastocysts implanted in pseudopregnant mice, and twenty-eight percent developed to term. These results indicated the varying degrees of success of different ways of inducing acrosomal loss in spermatozoa and their subsequent success rates in fertilization and further in vitro and in vivo development.     

Fertilization in vitro with spermatozoa from different mice increased variation in the developmental potential of embryos compared to artificial parthenogenetic activation

Although successful embryo development is dependent upon genetic and epigenetic contributions from both the male and female, the male potential to adversely affect embryo development has been scarcely studied. It is unclear whether the sperm variation among different males would affect the outcome of oocyte evaluation by embryo development following fertilization. In the present study, variation in the developmental potential of mouse embryos was first compared between in vitro fertilization with epididymal spermatozoa from different males and Sr(2+) parthenogenetic activation using oocytes of different qualities, and then the effect of male on fertilization and embryo development was examined using randomly chosen oocytes and spermatozoa from cauda epididymidis, vas deferens or electro-ejaculates. Rates of fertilization and blastocyst formation were significantly higher with spermatozoa from cauda epididymidis or vas deferens than with ejaculated spermatozoa. Rates of embryonic development differed significantly between different males, but not between different ejaculates of the same male. Analysis of standard errors of means and coefficients of variance indicated that as long as multiple males were involved, the variation in oocyte fertilization/activation and blastocyst formation was always higher after fertilization than after Sr(2+) parthenogenetic activation whether spermatozoa were collected from epididymidis, vas deferens or ejaculates and regardless of oocyte qualities. It is concluded that (1) epididymal mouse spermatozoa fertilize more oocytes than ejaculated spermatozoa under identical experimental conditions; (2) like farm animals, the mice also show a remarkable male effect on the developmental potential of in vitro produced embryos although they are supposed to be less genetically diverse; (3) parthenogenetic activation is recommended for assessment of oocyte quality to exclude the effect of male.     

Appearance of the oocyte activation ability of mouse round spermatids cultured in Vitro

This study was undertaken to determine the expression time of fertilization and oocytes activation abilities of spermatids in the mouse. When elongating or elongated spermatids isolated from fresh testes of adult males (B6D2F1) were injected into mature mouse oocytes, both spermatids could activate the mature oocytes and occur fertilization. On the one hand, the round spermatids could not activate mature oocytes, when microinjected into oocytes. In some experiments, recovered round spermatids were cultured under co-culture systems using Sertoli cells as a feeder cell. Under the co-culture system, developed elongating spermatids could stimulate and fertilized mature oocytes. These results indicate that the start of oocyte activation appearance is between the stage of round spermatid and elongating spermatids and the activation ability increases with the advance of spermiogensis. On the other hand, round spermatids isolated from males of ICR strain mouse already have the oocyte activation ability and the fertilizing ability. The result obtained suggests that the expression time of the oocyte activating ability is difficult between the mouse strain.     

小鼠精子注入兔卵母细胞受精研究

利用显微操作仪将小鼠精子注入家兔卵母细胞的胞质内和透明带下,对鼠兔异种精卵互作和异种受精胚胎的发育进行了研究,并对注射精子的数量及卵的体外成熟时间等影响鼠兔异种显微受精的因素进行了探讨,结果如下:(1)将小鼠精子分别注入兔卵胞质内和透明带下,均能激活兔卵母细胞,导致精核解聚和原核形成;(2)小鼠精子注入兔卵胞质内和透明带下受精,杂种胚胎体外培养能发育到8-细胞期;(3)鼠兔异种受精4-细胞胚胎染色体标本制备观察结果表明,它们为正常二倍体;(4)鼠兔异种受精4-细胞胚胎的超微结构观察结果表明,它们极近似兔正常4-细胞胚胎的超微结构;(5)将小鼠精子注入兔卵透明带下,注射5—10个精子组卵的受精率(32.4%)和卵裂率(16.2%)均高于注射单个精子组的,但二组间差异不显著(P>0.05);DM 15%NCS液中体外成熟培养11—12h兔卵透明带下注入1—2个小鼠精子后的受精率(42.3%)和卵裂率(30.8%)均高于体外成熟培养24—25h组的,但二组间差异未达到显著水平(P>0.05)。     

Influence of sperm:oocyte ratio during in vitro fertilization of in vitro matured cumulus-intact pig oocytes on fertilization parameters and embryo development

The present study was conducted to evaluate the influence of sperm:oocyte ratio during in vitro fertilization (IVF) of in vitro matured cumulus-intact oocytes on fertilization parameters and embryo development in pigs. In vitro matured oocytes surrounded by intact cumulus cells (COC) were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000:1, 3000:1, 4000:1, 6000:1, and 8000:1). Denuded oocytes inseminated with 2000 frozen-thawed spermatozoa:oocyte were the control group. A total of 2546 oocytes in five replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture (EC) medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The penetration rate increased in the COC groups with the sperm:oocyte ratio, reaching the highest rates with 8000:1 spermatozoa:oocyte (72.1 +/- 6.5%), similar to the control (73.5 +/- 3.5%). However, the monospermy was highest with the lower spermatozoa:oocyte rates (82.6-94.8%) and decreased drastically (P<0.05) in the COC group fertilized with 8000 sperm:oocyte (36%). The efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) showed no difference among the COC groups (20-30%) but they were significantly lower (P<0.007) than those obtained by the control group (43.7 +/- 2%). Embryo development was highest in the control group (58% for cleavage and 23% for BF) but not significantly different with the 6000 and 8000 sperm:oocyte COC groups (47 and 50% for cleavage and 19 and 17% for BF, respectively). These results indicate that the use of COC for IVF involves a drop in the efficiency of the fertilization and the necessity to increase the frozen-thawed sperm:oocyte ratio three to four times more to obtain similar embryo development to denuded oocytes.     

Fertilizability and chromosomal integrity of frozen-thawed Bryde's whale (Balaenoptera edeni) spermatozoa intracytoplasmically injected into mouse oocytes

Prior to attempting the in vitro production of embryos in the Bryde's whale (Balaenoputera edeni), we investigated whether spermatozoa can retain the capacity for oocyte activation and pronucleus formation as well as chromosomal integrity under cryopreservation by using intracytoplasmic sperm injection (ICSI) into mouse oocytes. Regardless of motility and viability, whale spermatozoa efficiently led to the activation of mouse oocytes (90.3-97.4%), and sperm nuclei successfully transformed into male pronucleus within activated ooplasm (87.2-93.6%). Chromosome analysis at the first cleavage metaphase (M) of the hybrid zygotes revealed that a majority (95.2%) of motile spermatozoa had the normal chromosome complement, while the percentage of chromosomal normality was significantly reduced to 63.5% in immotile spermatozoa and 50.0% in dead spermatozoa due to the increase in structural chromosome aberrations. This is the first report showing that motile Bryde's whale spermatozoa are competent to support embryonic development.     

Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa

Efficient and dependable mouse cryopreservation methods are urgently needed because the production of mice with transgenes and disrupted and mutant genes is now commonplace. Preservation of these unique genomes provides an essential safeguard for future research. Unfortunately, mouse spermatozoa appear more vulnerable to freezing than other species, e.g., bovine and human. In this study, we examined the efficiency of intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) in generating embryos from mouse spermatozoa frozen with 18% raffinose and 3% skim milk for cryoprotection. A comparison was made between the inbred strain C57BL/6J, commonly used in mutagenic and transgenic studies, and a hybrid strain B6D2F1 (C57BL/6J x DBA/2J). C57BL/6J spermatozoa are known to be more sensitive to freezing than B6D2F1. Fertilization of oocytes after IVF was significantly lower with C57BL/6J spermatozoa when compared with B6D2F1 spermatozoa for both fresh and frozen spermatozoa (fresh, 89 vs. 55%; frozen, 56 vs. 9%). Freezing also reduced the fertility of B6D2F1 spermatozoa (89 vs. 56%). Fertilization improved dramatically after ICSI with fresh and frozen C57BL/6J spermatozoa (90 and 85%) and also with frozen B6D2F1 spermatozoa (87%). The development of two-cell embryos to the blastocyst stage was lower for C57BL/6J than B6D2F1 (42-61% and 84-98%) in all treatments but similar for embryos within each strain. The normality of chromosomes from fresh and frozen spermatozoa was assessed in oocytes prior to first cleavage. The majority of oocytes had normal chromosomes after IVF (98-100%) and ICSI (87-95%), indicating that chromosomal abnormalities were not responsible for the poorer development in vitro of C57BL/6J embryos. In conclusion, our data show that ICSI is a more efficient and effective technique than IVF for generating embryos from frozen spermatozoa. More important, ICSI is especially valuable for strains where IVF with fresh spermatozoa produces few or no embryos.     

Mouse offspring after microinjection of heated spermatozoa.

The thermostability of the mammalian sperm genome was previously reported, but no live offspring after conception with heated spermatozoa had yet been obtained. In the present study, mouse spermatozoa were heated at 56 degrees C for 30 min and microinjected into mouse oocytes. Fertilization did not occur unless activation was induced by incubation in a calcium-free medium containing strontium. Under these conditions fertilization and cleavage rates were comparable to those obtained after microinjection of control spermatozoa, but the developmental rate to the blastocyst stage was lower. When transferred to foster mothers, embryos derived from heated sperm developed into phenotypically normal offspring, which grew and reproduced normally. In the mouse, heated spermatozoa can therefore support full embryonic development after microinjection into oocytes.     

Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators

Capacitation of rhesus monkey spermatozoa was assessed by monitoring sperm flagellar beat and trajectory changes during incubation in vitro and by determining sperm penetration into rhesus oocytes and hamster zona-free ova. Rhesus sperm capacitation in vitro depended on the addition to the culture medium of the cyclic nucleotide mediators, caffeine and dibutyryl cyclic AMP. Capacitation was correlated with the development of hyperactivated motility. Spermatozoa treated with the cyclic nucleotide mediators, and showing hyperactivated motility, penetrated 57.4% of all rhesus oocytes and fertilized 88.9% of mature rhesus oocytes that were morphologically normal. Control spermatozoa did not penetrate any of the eggs. Some sperm penetration into hamster ova occurred but was not statistically significant. These data provide a basis for achieving in-vitro fertilization in the rhesus monkey and information on specific sperm motility characteristics associated with fertilizing ability.     

Microinjection of cytoplasm or mitochondria derived from somatic cells affects parthenogenetic development of murine oocytes

Cloned mammals are readily obtained by nuclear transfer using cultured somatic cells; however, the rate of generating live offspring from the reconstructed embryos remains low. In nuclear transfer procedures, varying quantities of donor cell mitochondria are transferred with nuclei into recipient oocytes, and mitochondrial heteroplasmy has been observed. A mouse model was used to examine whether transferred mitochondria affect the development of the reconstructed oocytes. Cytoplasm or purified mitochondria from somatic cells derived from the external ear, skeletal muscle, and testis of Mus spretus mice or cumulus cells of Mus musculus domesticus mice were transferred into M. m. domesticus (B6SJLF1 and B6D2F1) oocytes to observe parthenogenetic development through the morula stage. All B6D2F1 oocytes injected with somatic cytoplasm or mitochondria showed delayed development when compared to oocytes injected with buffer. The developmental rates were not different among injected cell sources, with the exception of testis-derived donor cells injected into B6SJLF1 oocytes (P < 0.01). The developmental rate of B6D2F1 oocytes injected with buffer alone (98.8% survival) was different from those injected with somatic cytoplasm (60.8% survival) or somatic mitochondria (56.5% survival) (P < 0.01). Conversely, injection of ooplasm into B6D2F1 oocytes did not affect parthenogenetic development (100% survival). Our results indicate that injection of somatic cytoplasm or mitochondria affected parthenogenetic development of murine oocytes. These results have further implications for in vitro fertilization protocols employing ooplasmic transfer where primary oocyte failure is not confirmed.     

Activity of a sperm-borne oocyte-activating factor in spermatozoa and spermatogenic cells from cynomolgus monkeys and its localization after oocyte activation

It is widely accepted that mature mammalian oocytes are induced to resume meiosis by a sperm-borne oocyte-activating factor(s) (sperm factor, SF) immediately after normal fertilization or intracytoplasmic sperm injection. The SF is most likely a soluble factor that is localized within the cytoplasm of mature spermatozoa, but the exact stage at which it appears during spermatogenesis and its localization after oocyte activation is not fully understood, except in the mouse. First, we injected mature spermatozoa and spermatogenic cells from cynomolgus monkeys into mouse oocytes to assess their oocyte-activating capacity. More than 90% of mouse oocytes were activated after injection of monkey spermatozoa. Round spermatids and primary spermatocytes (late pachytene to diplotene) also activated oocytes (93% and 79%, respectively). Injection of monkey spermatozoa and spermatids induces intracellular Ca(2+) oscillations in a pattern similar to that seen following normal fertilization. Most spermatocytes did not produce typical intracellular Ca(2+) oscillations. Second, we transferred pronuclei or cytoplasts from mouse oocytes that had been activated by monkey spermatozoa or spermatids into intact mature mouse oocytes by electrofusion in order to examine the localization of the SF after pronuclear formation. Some of the SF was localized within the pronuclei, but some stayed in the ooplasm. This study demonstrated that spermatogenic cells of cynomolgus monkeys acquire oocyte-activating capacity at much earlier stages than those of mice, and that the monkey SF has a pronucleus-directing nature, although to a lesser extent than the mouse SF.     

RNA 3' cleavage and polyadenylation in oocytes and unfertilized eggs of Xenopus laevis

Xenopus laevis histone H4 and H1 genes were transcribed in vitro to generate artificial precursor mRNAs (pre-mRNAs). These pre-mRNAs were microinjected into oocytes, matured oocytes, and unfertilized eggs of Xenopus laevis and their 3' cleavage and polyadenylation were investigated. In the oocyte nucleus both H4 and H1 pre-mRNAs were 3' cleaved but were not detectably polyadenylated. In the oocyte cytoplasm there was neither 3' cleavage nor polyadenylation of these histone pre-mRNAs. When injected into either matured oocytes or unfertilized eggs, the pre-mRNAs underwent 3' cleavage but this was inefficient when compared to the oocyte nucleus. In addition approximately 50% of the remaining uncleaved pre-mRNA was subject to a polyadenylation activity which added A tails of approximately 70 A residues. In contrast, artificial mouse beta-globin pre-mRNAs were not detectably 3' cleaved or polyadenylated in either microinjected oocytes or unfertilized eggs.     

Effect of hyaluronidase, beta-glucuronidase and beta-N-acetylglucosaminidase inhibitors on sperm penetration of the mouse oocyte

The role of hyaluronidase, beta-glucuronidase and beta-N-acetylglucosaminidase in the penetration by mouse spermatozoa through the layers surrounding the oocyte was investigated by in vitro techniques. Myocrisin, fenoprofen, phosphorulated hesperidin and PS53 (a hydroquinone-sulfonic acid-formaldehyde polymer) inhibited fertilization when incubated with capacitated spermatozoa before the treated spermatozoa were mixed with intact oocytes but not when the inhibitor-treated, capacitated spermatozoa were added to oocytes free of follicle cells. The antifertility activity did not appear to be due to an effect on sperm motility or on the oocytes. These 4 compounds are known hyaluronidase inhibitors and, of the acrosomal enzymes tested, only share inhibition of hyaluronidase. Kinetic studies indicated that myocrisin is a reversible inhibitor of mouse sperm hyaluronidase whereas the other three are irreversible inhibitors. Adding saccharolactone, a beta-glucuronidase inhibitor, or N-acetylglucosaminolactone and N-acetylgalactosaminolactone, beta-N-acetylglucosaminidase inhibitors, to capacitated spermatozoa under the same conditions as the hyaluronidase inhibitors did not decrease fertilization. This was the case even though the beta-glucuronidase or beta-N-acetylglucosaminidase activities of the spermatozoa were completely inhibited, at least at the time that the inhibitor-treated, capacitated spermatozoa were mixed with the oocytes. The hyaluronidase activity of mouse spermatozoa remained unaltered during the incubation period required for capacitation; however, prolonged incubation caused a significant decrease in hyaluronidase. Untreated mouse spermatozoa caused hydrolysis of hyaluronic acid more effectively than did sperm extracts obtained by detergent extraction. These results are consistent with the theory of an essential role of hyaluronidase in mouse fertilization. At least in this species, the enzyme appears to be specifically involved in sperm penetration through the follicle cell layer. The data do not support an essential role for beta-glucuronidase and beta-N-acetylglucosaminidase in the penetration by mouse spermatozoa through the oocyte's investments. In contrast to some other species, sperm capacitation in mice does not result in a loss of hyaluronidase although part of the enzyme activity is lost on prolonged incubation. Mouse spermatozoa appear to be able to digest substrate (hyaluronic acid) even though hyaluronidase is not released.     

Mouse-rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species-specific in mammals

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0-14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage.     

Brief coincubation of gametes in porcine in vitro fertilization: role of sperm:oocyte ratio and post-coincubation medium

In this study, a short coincubation time of 10 min was used to determine the effect of different sperm:oocyte ratios during in vitro fertilization (IVF), and different periods of post-coincubation in a medium that is not appropriate for IVF, on fertilization parameters. In the first experiment, a total of 1624 in vitro matured oocytes, from 4 replicates, were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000, 1500, 1000 and 500 sperm:oocyte) and coincubated for 10 min or 6 h. The oocytes from 10 min of coincubation were washed in IVF medium to remove spermatozoa not bound to the zona pellucida and transferred to another droplet of the same medium (containing no spermatozoa) for 6h. The oocytes from the other group remained with the spermatozoa for 6h. Oocytes from both groups were then cultured in embryo culture medium (IVC) for 12h to assess fertilization parameters. In the second experiment, 1872 in vitro matured oocytes, in 3 replicates were inseminated with frozen-thawed spermatozoa using the same sperm:oocyte ratios as in the first experiment. The oocytes were coincubated for 10 min and transferred directly to IVC medium for 18 h (group A), to IVF medium (containing no sperm) only for 2h and then to IVC medium for 16 h (group B), or to IVF medium (containing no sperm) for 6h and then to IVC medium for 12 h (group C or control). There was an effect of sperm:oocyte ratio on all fertilization parameters in experiment 1. The efficiency of IVF (number of monospermic oocytes/total number inseminated) was higher (P<0.05) for oocytes coincubated with spermatozoa for 10 min and inseminated with 1500 and 1000 sperm:oocyte (35.8+/-3 and 37.6+/-2.7%, respectively) and for those coincubated for 6h with 500 spermatozoa per oocyte (37.2+/-3.1%). In experiment 2, the penetration and efficiency rates obtained in group A were poor (between 3 and 15%) irrespective of the sperm:oocyte ratio. However, in group B the fertilization parameters were similar to the controls and were also affected by the sperm:oocyte ratio. These results demonstrate that coincubation time may be reduced to 10 min to increase the efficiency of fertilization depending on the sperm:oocyte ratio, and that the spermatozoa bound to the zona pellucida require a maximum of 2h in an appropriate medium to penetrate the oocytes.     

Effects of Protein Synthesis on the Maturation of Mouse Oocytes in vitro

卵母细胞成熟过程中伴随有多种蛋白质的合成与磷酸化,蛋白质的合成对卵细胞的成熟具有重要作用。本实验较系统地阐述小鼠卵母细胞体外成熟培养的不同阶段蛋白质合成对卵母细胞成熟的影响。放线菌酮是肽链延伸的抑制因子。将生发泡（ＧＶ）期的卵母细胞分别于Ｔ６成熟培养液中培养０、４、６、９小时后,转至含有１０ｍｇ／ｍｌ放线菌酮的Ｔ６成熟培养液继续培养１２１５小时。固定、染色、观察卵母细胞。结果如Ｔａｂｌｅ１。０小时实验组：抑制处理４小时,其生发泡破裂（ＧＶＢＤ）发生率与对照组无明显差异。表明：卵母细胞ＧＶＢＤ所需蛋白质（如：成熟促进因子ＭＰＦ等）是在卵巢的卵泡卵母细胞生长过程中完成的。４、６小时实验组：笫一极体的释放被完全抑制,卵母细胞不能达到ＭＩ期,染色质处于凝集状态（Ｆｉｇ．３＆４）。表明：ＧＶＢＤＭＩ期间所需蛋白质的合成对卵母细胞ＭＩ期中期纺缍体的形成与维持具有重要作用。９小时实验组：可能由于卵母细胞发育速度存在个体间的差异。没有进入ＭＩＩ期的便停滞于ＭＩ期以前。进入ＭＩ期的则能排出笫一极体。因此,笫一极体的释放总体上呈不完全抑制状态,其释放率低于对照组。但是,后者虽然弪过恢复培养至１５小时,可能由于微管蛋白的合成     

Evaluation of boar spermatozoa penetrating capacity using pig oocytes at the germinal vesicle stage

We evaluated the ability of immature pig oocytes (at germinal vesicle stage) to detect differences in the in vitro penetration rates of boar spermatozoa. In Experiment 1, immature and ovulated oocytes (n=303) were exposed to capacitated boar spermatozoa to determine if the penetrability of immature pig oocytes was comparable to that of ovulated oocytes. The percentages of penetrated oocytes and the mean number of spermatozoa per oocyte were similar for immature (88.82 and 7.42+/-0.41) and ovulated oocytes (90.97 and 7.95+/-0.34, respectively). In Experiment 2, immature oocytes (n=1230) were inseminated with semen from 2 boars (A and B) with satisfactory semen characteristics to establish the variability of in vitro penetrating capacity between the boars. Semen was examined for motility, movement quality, acrosome integrity and plasma membrane integrity at various stages of the in vitro procedure. Although the sperm evaluation results were similar between boars, Boar A exhibited a significantly higher (P<0.001) penetration rate (91.49%) and number of spermatozoa penetrated per oocyte (5.90+/-0.25) than Boar B (52.87% and 2.03+/-0.12, respectively). Increasing the sperm concentration at insemination from 1x10(6) to 10x10(6) cells/ml resulted in an increased penetrating capacity for both boars, and the differences in the number of spermatozoa per oocyte between boars also increased. These results indicate that immature pig oocytes can be used in a homologous in vitro fertilization assay, and that despite similarities in semen characteristics a significant boar effect is evident for parameters of in vitro penetration of oocytes.     

CDC20 downregulation impairs spindle morphology and causes reduced first polar body emission during bovine oocyte maturation

The cell division cycle protein 20 (CDC20) is an essential regulator of cell division, encoded by the CDC20 gene. However, the role of CDC20 in bovine oocyte maturation is unknown. In this study, CDC20 morpholino antisense oligonucleotides (MOs) were microinjected into the cytoplasm of bovine oocytes to block the translation of CDC20 mRNA. CDC20 downregulation significantly reduced the rate of first polar body emission (PB1). Further analysis indicated that oocytes treated with CDC20 MO arrested before or at meiotic stage I with abnormal spindles. To further confirm the functions of CDC20 during oocyte meiotic division, CDC20 MOs were microinjected into oocytes together with a supplementary PB1. The results showed that newly synthesized CDC20 was not necessary at the meiosis II-to-anaphase II transition. Our data suggest that CDC20 is required for spindle assembly, chromosomal segregation, and PB1 extrusion during bovine oocyte maturation.     

Cleavage development of bovine oocytes fertilized by sperm injection

Whole in vitro capacitated bovine spermatozoa were microinjected directly into the ooplasm of in vitro matured bovine oocytes in order to determine whether oocytes fertilized by sperm injection could undergo normal pronuclear formation and cleavage development. Immature oocytes recovered from follicles (2-5 mm) of unstimulated ovaries were cultured for 24-25 h in modified TCM 199 medium supplemented with heat-treated day 20 cow serum, luteinizing hormone (LH), and estradiol 17-B. In vitro capacitated, frozen-thawed spermatozoa were injected into the ooplasm, and the injected oocytes were cultured for an additional 24-28 h. Twenty-one percent (21/101) of the sperm-injected oocytes contained a sperm within the ooplasm; however, only 2% (2/101) cleaved. The remaining oocytes either did not contain a sperm or had degenerated. After oocyte activation induced by a 5 min incubation in 1 microM A23187, sperm nuclear decondensation occurred in the A23187-activated, injected oocytes but not in the unactivated, injected controls (37% vs. 0% after 3 h). Those injected, activated oocytes that contained a male pronucleus also exhibited a female pronucleus and second polar body. Furthermore, a significantly higher number (28%, 6/21) of the injected, activated oocytes cleaved to a two- to four-cell stage after 48 h than did the injected, unactivated oocytes (4%). These results indicate that, unlike hamster and rabbit oocytes, bovine oocytes are not sufficiently stimulated by the injection procedure to complete meiosis, but, upon activation by calcium ionophore, they will undergo normal-appearing cleavage development following fertilization by sperm injection.