爪蟾p21活化激酶2参与卵母细胞胞质分裂过程不依赖于Cdc42

研究p21活化激酶2(p21-activated kinase2,PAK2)在卵母细胞成熟过程中的作用.以爪蟾卵母细胞为模型,分别向爪蟾卵母细胞显微注射PAK2-N端(PAK2-N-terminal,PAK2-NT)和PAK2-N端突变体(PAK2-N-terminal mutation,PAK2-NTm)mRNA,荧光显微镜下观察胚泡破裂发生.采用共聚焦显微镜,时间延迟摄影法观察正常卵母细胞、PAK2-NTmRNA注射组和PAK2-NTm mRNA注射组卵母细胞胞质分裂、极体形成及与Cdc42活性的关系.结果表明,PAK2-NTmRNA和PAK2-NTm mRNA注射组的卵母细胞与正常卵母细胞胚泡破裂发生相似,但PAK2-NTmRNA和PAK2-NTm mRNA注射组未见胞质分裂和极体形成.结果提示,PAK2参与卵母细胞胞质分裂和极体形成可能不依赖于Cdc42的调节过程.     

p21活化蛋白激酶2在卵母细胞成熟中的作用

研究p21活化蛋白激酶2（p21-activated kinase 2,PAK2）在爪蟾卵母细胞成熟中的作用。利用特异性抑制PAK2活性的PAK2-N端（PAK2-N terminal,PAK2-NT）片段显微注射爪蟾卵母细胞。荧光显微镜下比较PAK2-NT mRNA注射组和未注射对照组卵母细胞胚泡破裂发生。共聚焦显微镜下,时间延迟摄影法观察两组卵母细胞胞质分裂过程中肌动蛋白和纺锤体的变化。与未注射PAK2-N端mRNA的对照组卵母细胞相比,注射组卵母细胞胚泡破裂发生无异常,但未见胞质分裂发生和极体形成。结果提示PAK2可能参与爪蟾卵母细胞胞质分裂过程。     

Cdc42和球形肌动蛋白在卵母细胞胞质分裂中的定位分析

研究活性Cdc42与球形肌动蛋白(G-actin)在爪蟾卵母细胞胞质分裂中的定位关系。分别用GFP-wGBDmRNA与罗丹明-594-微管蛋白、Alexa-488-球形肌动蛋白与罗丹明-594-微管蛋白、GFP-wGBDmRNA与Alexa-594-球形肌动蛋白共同显微注射爪蟾卵母细胞。利用共聚焦显微镜,时间延迟摄影方法,分别观察活体卵母细胞中活性Cdc42、球形肌动蛋白在胞质分裂过程中的定位,以及活性Cdc42与球形肌动蛋白在胞质分裂中的定位关系。在卵母细胞胞质分裂中,活性Cdc42与球形肌动蛋白存在空间上共定位现象,并且在时相上具有一致性。结果提示活性Cdc42和球形肌动蛋白在卵母细胞胞质分裂过程中密切相关。     

牛卵母细胞玻璃化冷冻对基因mRNA表达量的影响

目的 将处于生发泡期(GV期)和体外成熟期(IVM)的牛卵母细胞进行玻璃化冷冻.解冻,对其卵裂率和囊胚率以及一些与发育相关基因的mRNA表达量进行评价.方法 玻璃化冷冻GV期(n=224)和IVM期(n=235)牛卵母细胞,解冻后对其进行体外培养并采用quantitative real time-PCR技术对冷冻.解冻...     

猪卵母细胞c-mos基因表达以及RNA干扰

c-mos基因在动物卵母细胞减数分裂调控中起作用,但其作用机制目前仍不清楚。本实验通过RT-PCR、免疫荧光激光共聚焦检测方法检测了猪卵母细胞在体外成熟培养过程中c-mos基因在转录水平、翻译水平上的表达以及蛋白的分布,并应用注射小干扰RNA(siRNA)方法对其进行了RNA干扰(RNAi)研究。结果显示,猪卵母细胞在体外成熟培养过程中c-mos基因mRNA量逐渐增高,电激活后6h接近完全降解;MOS(c-mos基因蛋白产物)在GV卵母细胞生发泡中有一定量的表达,生发泡破裂(GVBD)前表达量增加且开始向卵母细胞胞质弥散,成熟培养44h未成熟卵母细胞中的MOS表达量要高于成熟卵母细胞,激活后6h核区MOS明显减少,但仍然有少量MOS分布于胞质中;成熟培养前干扰c-mos基因,所用三个siRNA都能成功敲低mRNA量,分别是同时期对照组mRNA量的0.08±0.03,0.11±0.06和0.20±0.06倍,干扰后虽然没有完全剔除MOS,但MOS量比同期卵母细胞有明显下降,仍可以引发成熟卵母细胞染色体解凝集。研究结果揭示了猪卵母细胞体外成熟及发育进程中c-mos基因在转录和翻译水平上的动态表达规律,建立了猪卵母细胞c-mos基因RNAi体系,为MOS在猪卵母细胞发育过程中的功能研究建立了重要的基础。     

小鼠Cdc25B核输出序列

为探讨小鼠卵母细胞中Cdc25B(cell division cycle 25 homolog B)核输出序列在卵母细胞G2/M转换过程中的调控机制,应用显微注射方法将Cdc25B的野生型、N末端缺失1～51位氨基酸片段(Cdc25B-Δ51)、1～65位氨基酸片段(Cdc25B-Δ65)突变体的mRNA和pEGFP-Cdc25B-WT、pEGFP-Cdc25B-Δ51、pEGFP-Cdc25B-Δ65的融合质粒显微注射到含有完整生发泡的小鼠卵母细胞中,观察不同注射组小鼠卵母细胞发生生发泡破裂的情况及蛋白质亚细胞定位。结果显示Cdc25B-Δ51及Cdc25B-Δ65都丧失了诱导小鼠卵母细胞减数分裂的能力;同时亚细胞定位研究表明在G2期野生型Cdc25B主要分布在细胞浆中,Cdc25B-Δ51在核浆均有分布,Cdc25B-Δ65则主要分布于细胞核中。研究结果表明Cdc25B在52～65位氨基酸之间存在核输出序列(nuclear export sequence,NES),NES参与的核转运机制作为一种重要的调控机制控制着细胞的生理进程;N末端的氨基酸对减数分裂的重启动起促进作用。     

Cdc25B蛋白过表达可使小鼠胚胎突破2-细胞期阻滞

目的：探讨Cdc25B蛋白过表达对小鼠2-细胞期胚胎发育的影响。方法：利用体外转录试剂盒将Cdc25B转录成mRNA,将mRNA显微注射入小鼠2-细胞期胚胎中,观察胚胎发育情况和卵裂率。用蛋白激酶活性测定方法和Western印迹分别检测Cdc25B蛋白过表达小鼠胚胎MPF的活性及Cdc2-Tyr15的磷酸化状态。结果：hCG后48 h,mRNA注射组有超过40%的2-细胞期胚胎分裂到4-细胞期而对照组仍停留在2-细胞期;激酶活性测定显示注射Cdc25B mRNA后,MPF的活性显著升高;Cdc2-Tyr15的磷酸化状态变化与激酶活性测定结果一致。结论：Cdc25B蛋白过表达可以激活有丝分裂促进因子（MPF）,从而使小鼠2-细胞期胚胎突破2-细胞期阻滞,发育到4-细胞期。     

原癌基因c-erbB2和c-myb经丝裂原活化蛋白激酶和成熟促进因子途径调节卵母细胞的成熟

本研究探讨了原癌基因c-erbB:和c-myb对小鼠卵母细胞成熟的影响及其在调控卵母细胞成熟中与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和成熟促进因子(mamration promoting factor,MPF)的上下游关系.c-erbB2反义寡脱氧核苷酸(antisense oligodeoxynucleotide,ASODN)和c.myb ASODN均呈剂量依赖方式抑制卵母细胞的生发泡破裂(germinalvesicle breakdown,GVBD)率和第一极体(first polar body,PBl)排放率,并显著延迟其成熟时间.小鼠卵母细胞显微注射重组人c-erbB2蛋白和c-myb蛋白后,培养6 h其GVBD率分别比对照组上升了23.1%(P<0.05)和32.2%(P<0.05),.培养12 h其PBl排放率分别比对照组上升了17.3%(P<0.05)和23.5%(P<0.05).RT-PCR结果显示,小鼠卵母细胞中存在c-erbB2mRNA和c-myb mRNA表达;c-erbB2ASODN能明显抑制卵母细胞中c-erbB2mRNA和c-myb mRNA的表达,c-myb ASODN能明显抑制卵母细胞中c-myb mRNA的表达,对c-erbB2 mRNA无明显影响;MAPK抑制剂PD98059以及MPF抑制剂roscovitine在抑制卵母细胞成熟的同时,均能阻断显微注射重组人c-erbB:蛋白和重组人c-myb蛋白对卵母细胞成熟的促进作用,但对卵母细胞中c-erbB2mRNA和c-myb mRNA表达无明显影响.Western blot结果显示,c-erbB2ASODN、c-mybASODN、PD98059、roscovitine均使卵母细胞中MAPK磷酸化水平和cyclinB 1含量下降.结果提示,原癌基因c-erbB2、c-myb在卵母细胞成熟中起重要作用,可能是调控卵母细胞成熟中关键蛋白激酶如MAPK、MPF的上游激活物.     

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.     

利用斑马鱼胚胎快速鉴定真核质粒中目的基因表达的研究

目的建立利用斑马鱼胚胎快速鉴定真核质粒中目的基因表达的实验体系。方法选20枚斑马鱼受精卵,在显微镜下每隔1h记录胚胎的发育情况。另选250枚单细胞期斑马鱼胚胎,平均分成5组,一组胚胎作为对照,剩余4组分别向胚胎的单细胞内注射pEGFP-N1(真核表达质粒)、pCMV-DsRed-Express2(真核表达质粒)、pET28-GFP(原核表达质粒)、pET28-RFP(原核表达质粒)质粒,在不同时间点连续观察绿色荧光及红色荧光的表达情况。另选600枚单细胞期斑马鱼胚胎,平均分成3组,一组胚胎作为对照,一组向胚胎单细胞内注射pEGFP-N1质粒,另外一组向胚胎单细胞内注射pEGFP-N1-MUC1外源基因融合重组质粒,注射4h后在荧光显微镜下观察绿色荧光的表达情况,并用RT-PCR的方法检测目的基因MUC1mRNA的转录情况。结果注射pEGFP-N1、pCMV-DsRed-Express2真核表达质粒的胚胎,注射4h后分别观察到很强的绿色荧光及红色荧光;注射pET28-GFP、pET28-RFP原核表达质粒的胚胎,10h内都未观察到绿色荧光及红色荧光;注射pEGFP-N1-MUC1外源基因融合质粒,注射4h后同样...     

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.     

Nicotinamide Impairs Entry into and Exit from Meiosis I in Mouse Oocytes

Following exit from meiosis I, mammalian oocytes immediately enter meiosis II without an intervening interphase, accompanied by rapid reassembly of a bipolar spindle that maintains condensed chromosomes in a metaphase configuration (metaphase II arrest). Here we study the effect of nicotinamide (NAM), a non-competitive pan-sirtuin inhibitor, during meiotic maturation in mouse oocytes. Sirtuins are a family of seven NAD⁺-dependent deacetylases (Sirt1-7), which are involved in multiple cellular processes and are emerging as important regulators in oocytes and embryos. We found that NAM significantly delayed entry into meiosis I associated with delayed accumulation of the Cdk1 co-activator, cyclin B1. GVBD was also inhibited by the Sirt2-specific inhibitor, AGK2, and in a very similar pattern to NAM, supporting the notion that as in somatic cells, NAM inhibits sirtuins in oocytes. NAM did not affect subsequent spindle assembly, chromosome alignment or the timing of first polar body extrusion (PBE). Unexpectedly, however, in the majority of oocytes with a polar body, chromatin was decondensed and a nuclear structure was present. An identical phenotype was observed when flavopiridol was used to induce Cdk1 inactivation during late meiosis I prior to PBE, but not if Cdk1 was inactivated after PBE when metaphase II arrest was already established, altogether indicating that NAM impaired establishment rather than maintenance of metaphase II arrest. During meiosis I exit in NAM-treated medium, we found that cyclin B1 levels were lower and inhibitory Cdk1 phosphorylation was increased compared with controls. Although activation of the anaphase-promoting complex-Cdc20 (APC-Cdc20) occurred on-time in NAM-treated oocytes, Cdc20 levels were higher in very late meiosis I, pointing to exaggerated APC-Cdc20-mediated proteolysis as a reason for lower cyclin B1 levels. Collectively, therefore, our data indicate that by disrupting Cdk1 regulation, NAM impairs entry into meiosis I and the establishment of metaphase II arrest.     

Germ cell–specific expression of Venus by Tol2-mediated transgenesis in endangered endemic cyprinid Honmoroko (Gnathopogon caerulescens)

Fishes expressing a fluorescent protein in germ cells are useful to perform germ cell transfer experiments for conservation study. Nonetheless, no such fish has been generated in endangered endemic fishes. In this study, we tried to produce a fish expressing Venus fluorescent protein in germ cells using Honmoroko (Gnathopogon caerulescens), which is one of the threatened small cyprinid endemic to the ancient Lake Biwa in Japan. To achieve germ cell-specific expression of Venus, we used piwil1 (formally known as ziwi) promoter and Tol2 transposon system. Following the co-injection of the piwil1-Venus expression vector and the Tol2 transposase mRNA into fertilized eggs, presumptive transgenic fish were reared. At 7 months of post-fertilization, about 19% (10/52) of the examined larvae showed Venus fluorescence in their gonad specifically. Immunohistological staining and in vitro spermatogenesis using gonads of the juvenile founder fish revealed that Venus expression was detected in spermatogonia and spermatocyte in male, and oogonia and stage I and II oocytes in female. These results indicate that the Tol2 transposon and zebrafish piwil1 promoter enabled gene transfer and germ cell-specific expression of Venus in G. caerulescens. In addition, in vitro culture of juvenile spermatogonia enables the rapid validation of temporal expression of transgene during spermatogenesis.     

Improving in vitro development of cloned bovine embryos with hybrid (Holstein-Chinese Yellow) recipient oocytes recovered by ovum pick up

In the present study, oocytes from F1 hybrid cattle, as well as their parental lines, were recovered by ovum pick up (OPU) and used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Four F1 hybrid (Holstein dam x Chinese Yellow sire), 10 Holstein and four Chinese Yellow cattle were subjected to OPU once weekly. There were no significant differences among breeds for number of recovered oocytes per session (overall average, 7.8+/-0.5; mean+/-S.E.M.), quality of the recovered oocytes, or oocyte maturation rate (72-73%). Matured oocytes were all used as recipient cytoplasm (without selection) and a single batch of cumulus cells collected from a Holstein cow were used as donor cells. Although reconstructed embryos initiated cleavage sooner when the recipient cytoplasm was from hybrid cattle versus the two parental breeds, the overall cleavage rate was indistinguishable among breeds. At Day 8, the blastocyst rate from the cleaved embryos (51% versus 37% and 27%), the total number of cells per blastocyst (135+/-4.1 versus 116+/-3.6 and 101+/-4.2), and the percentage of Grade-A (excellent quality) blastocysts (54% versus 42% and 29%) in the hybrid group were all higher than that of Holstein and Yellow groups. Furthermore, the proportion of blastocysts obtained at Day 7 (as a percentage of the total number of blastocysts) was greater in the hybrid group than in Holstein and Yellow groups (89% versus 71% and 63%). In conclusion, the use of F1 hybrid oocytes as recipient cytoplasm significantly improved in vitro development of cloned bovine embryos relative to oocytes derived from the parental lines.     

Differential regulation of cyclin B1 degradation between the first and second meiotic divisions of bovine oocytes

During mammalian oocyte maturation, two consecutive meiotic divisions are required to form a haploid gamete. For each meiotic division, oocytes must transfer from metaphase to anaphase, but maturation promoting factor (cyclin-dependent kinase 1/cyclin B1) activity would keep the oocytes at metaphase. Therefore, inactivation of maturation promoting factor is needed to finish the transition and complete both these divisions; this is provided through anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. The objective of this study was to examine meiotic divisions in bovine oocytes after expression of a full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion, coupled with the fluorochrome Venus, by microinjecting their complementary RNA (cRNA). Overexpression of full-length cyclin B1-Venus inhibited homologue disjunction and first polar body formation in maturing oocytes (control 70% vs. overexpression 16%; P < 0.05). However at the same levels of expression, it did not block second meiotic metaphase and cleavage of eggs after parthenogenetic activation (control: 82% pronuclei and 79% cleaved; overexpression: 91% pronuclei and 89% cleaved). The full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion caused metaphase arrest in both meiotic divisions, whereas degradation of securin was unaffected. Roscovitine, a potent cyclin-dependent kinase 1 (CDK1) inhibitor, overcame this metaphase arrest in maturing oocytes at 140 μM, but higher doses (200 μM) were needed to overcome arrest in eggs. In conclusion, because metaphase I (MI) blocked by nondegradable cyclin B1 was distinct from metaphase II (MII) in their different sensitivities to trigger CDK1 inactivation, we concluded that mechanisms of MI arrest differed from MII arrest.     

Wee1B,Myt1, and Cdc25 function in distinct compartments of the mouse oocyte to control meiotic resumption

After a long period of quiescence at dictyate prophase I, termed the germinal vesicle (GV) stage, mammalian oocytes reenter meiosis by activating the Cdc2–cyclin B complex (maturation-promoting factor [MPF]). The activity of MPF is regulated by Wee1/Myt1 kinases and Cdc25 phosphatases. In this study, we demonstrate that the sequestration of components that regulate MPF activity in distinct subcellular compartments is essential for their function during meiosis. Down-regulation of either Wee1B or Myt1 causes partial meiotic resumption, and oocytes reenter the cell cycle only when both proteins are down-regulated. Shortly before GV breakdown (GVBD), Cdc25B is translocated from the cytoplasm to the nucleus, whereas Wee1B is exported from the nucleus to the cytoplasm. These movements are regulated by PKA inactivation and MPF activation, respectively. Mislocalized Wee1B or Myt1 is not able to maintain meiotic arrest. Thus, cooperation of Wee1B, Myt1, and Cdc25 is required to maintain meiotic arrest and relocation of these components before GVBD is necessary for meiotic reentry.     

In vitro maturation and fertilization of bovine oocytes and in vitro culture of presumptive zygotes in the presence of bovine pestivirus

A pathogen which has been shown to commonly contaminate in vitro bovine embryo production system is bovine pestivirus (bovine viral diarrhea virus). Three experiments were designed to evaluate the in vitro maturation (experiment I), fertilization (experiment II) and embryo development (experiment III) of immature oocytes, inseminated oocytes and presumptive zygotes in the presence of a bovine pestivirus (non-cytopathic, nCP type 1). The virus inoculum used was derived from a persistently infected cow. In experiment I, follicular oocytes (n=1257) recovered from slaughterhouse derived ovaries were randomly assigned to either a control group (n=578) which did not become exposed to bovine pestivirus and a treatment group (n=679) which was inoculated with bovine pestivirus (2.20-3.69 log(10) TCID(50)/50 microl) at the time of commencement of in vitro maturation. Overall, there was no significant difference between the control and pestivirus inoculated oocytes in either the cumulus cell expansion rate (79+/-7.5% versus 74+/-10.7%) or the nuclear maturation rate (89+/-4.8% versus 85+/-7.4%), respectively. In experiment II, in vitro matured oocytes (n=607) were inseminated either in the absence (control; n=301) or the presence of bovine pestivirus (4-4.6 log(10) TCID(50)/50 microl; n=306). A significant (P<0.01) reduction in the overall number of fertilized oocytes with two well formed male and female pronuclei was observed in the treatment group compared to the control group (58.5+/-5.8% versus 73.3+/-3.6%, respectively). In experiment III, after in vitro maturation and fertilization, presumptive zygotes were randomly assigned to either a control group (n=139) which was not exposed to bovine pestivirus or a treatment group which was inoculated with bovine pestivirus (2.97-4.47 log(10) TCID(50)/30 microl; n=139). The zygotes were then cultured under mineral oil in an atmosphere of 88% N(2), 7% O(2) and 5% CO(2) at 39 degrees C. The morphologic appearance of the embryos was assessed 48 h after the commencement of culture, and then every 48 h up to days 7-8 after insemination. The 22% (31/139) and 3.6% (5/139) of the presumptive zygotes developed to the morula or blastocyst stage in the control and the bovine pestivirus inoculated groups, respectively (P<0.001). This study demonstrates that bovine pestivirus has a significant detrimental effect on in vitro fertilization and early in vitro embryo development.     

mTOR is required for asymmetric division through small GTPases in mouse oocytes

Mammalian target of rapamycin (mTOR) is central to the control of cell proliferation, growth, and survival in mammalian cells. Prolonged treatment with rapamycin inhibits mTOR complex 2 (mTORC2) activity, and both the mTORC1-mediated S6K1 and 4E-BP1/eIF4E pathways are essential for TORC2-mediated RhoA, Cdc42, and Rac1 expression during cell motility and F-actin reorganization. The functions of mTOR in the mouse oocyte remain unclear, however. The present study shows that rapamycin affects mTOR expression and cytoskeleton reorganization during meiotic maturation of mouse oocytes. mTOR mRNA was expressed in germinal vesicles (GV) until metaphase I (MI), and increased during metaphase II (MII). Immunostaining showed that mTOR localized around the spindle and in the cytoplasm of oocytes. Treatment of oocytes with rapamycin decreased mTOR at the RNA and protein level, and altered asymmetric division. Formation of the actin cap and the cortical granule-free domain were also disrupted after rapamycin treatment, indicating the failure of spindle migration. Injection of an anti-mTOR antibody yielded results consistent with those obtained for rapamycin treatment, further confirming the involvement of mTOR in oocyte polarity. Furthermore, rapamycin treatment reduced the mRNA expression of small GTPases (RhoA, Cdc42, and Rac1), which are crucial regulatory factors for cytoskeleton reorganization. Taken together, these results suggest that rapamycin inhibits spindle migration and asymmetric division during mouse oocyte maturation via mTOR-mediated small GTPase signaling pathways.     

Speriolin is a novel spermatogenic cell-specific centrosomal protein associated with the seventh WD motif of Cdc20

The fundamental mechanisms of mitosis are conserved throughout evolution in eukaryotes, including ubiquitin-mediated proteolysis of cell cycle regulators by the anaphase-promoting complex/cyclosome. The spindle checkpoint protein Cdc20 activates the anaphase-promoting complex/cyclosome in a substrate-specific manner. It is present in the cytoplasm and concentrated in the centrosomes throughout the cell cycle, accumulates at the kinetochores in metaphase, and is no longer detected following anaphase. However, it is unknown whether Cdc20 has the same activities and distribution during meiosis in male germ cells. We found that in mice, Cdc20 accumulates in the cytoplasm of pachytene spermatocytes during meiosis I, is distributed throughout spermatocytes undergoing meiotic division, and is present in the cytoplasm of postmeiotic spermatids. Several proteins bind to and regulate the function of Cdc20 during mitosis. We identified speriolin and determined that it is a novel spermatogenic cell-specific Cdc20-binding protein, is present in the cytoplasm, and is concentrated at the centrosomes of spermatocytes and spermatids and that a leucine zipper domain is required to target speriolin to the centrosome. The seven tandem WD motifs of Cdc20 probably fold into a seven-blade beta-propeller structure, and we determined that they are required for speriolin binding and for localization of Cdc20 to the centrosomes and nucleus, suggesting that speriolin might regulate or stabilize the folding of Cdc20 during meiosis in spermatogenic cells.