"生存还是毁灭",发育还是阻滞--小鼠早胚体外发育2-细胞阻滞产生机制初析

在体外培养条件下,小鼠受精卵往往经一次分裂后就停滞在２－细胞,不能完成到达囊胚的后续发育过程,称作２－细胞阻滞。氧自由基伤害、培养液成分不平衡等外界因素都能引起阻滞。小鼠的２－细胞阻滞现象受细胞质内母型物质制约,具有品系依赖性。在阻滞品系小鼠胚胎的细胞质内可能缺乏某些重要的蛋白因子,在无外源信号的培养体系内不能继续分裂。发生阻滞的胚胎细胞内ＭＰＦ前体物虽然储备充足,但因无法去磷酸化激活而最终导致发     

Analysis of specific factors generating 2-cell block in AKR mouse embryos

The phenomenon of the developmental arrest at the 2-cell stage of 1-cell embryos from some mouse strains during in vitro culture is known as the 2-cell block. We investigated the specific factors involved in the 2-cell block of AKR embryos by means of a modified culture system, the production of reconstructed embryos by pronuclear exchange and a cross experiment. In a culture medium with phosphate, 94.6% of 1-cell embryos from the C57BL mouse strain developed to the blastocyst stage, but 95.7% of embryos from the AKR mouse strain showed 2-cell block. Phosphate-free culture medium rescued the 2-cell block of AKR embryos and accelerated the first cell cycle of the embryos. Co-culture with BRL cells and a BRL-conditioned medium fractionated below 30 kDa also rescued the 2-cell block of AKR embryos. Examinations of in vitro development of reconstructed embryos and of embryos from F1 females between AKR and C57BL strains clearly demonstrated that the AKR cytoplast caused the 2-cell block. In the backcrossed female progeny between (AKR x C57BL) F1 males and AKR females, about three-quarters of the embryos were of the 2-cell blocking phenotype and about one-quarter were of the non-blocking phenotype. These results suggest that two genes are responsible for the 2-cell block of AKR embryos.     

A deficiency in the mechanism for p34cdc2 protein kinase activation in mouse embryos arrested at 2-cell stage.

Mouse embryos of the ddY strain fertilized in vitro undergo the first cleavage to the 2-cell stage but not the second cleavage even 45 hr after insemination (2-cell block). We examined the phosphorylation state of p34cdc2 and histone H1 kinase activity in mouse 2-cell embryos to investigate the relationship of p34cdc2 with 2-cell block. In the first mitotic cell cycle, the amount of phosphorylated forms of p34cdc2, which were detected as the bands of retarded mobility on SDS-PAGE followed by immunoblotting with anti-p34cdc2 antibody, increased during interphase and abruptly decreased at M phase. Concomitant with this dephosphorylation, histone H1 kinase activity was increased. After the embryos cleaved to the 2-cell stage, the amounts of phosphorylated forms of p34cdc2 increased up to 33 hr after insemination. However, the activation of histone H1 kinase did not occur and the states of phosphorylation of p34cdc2 did not show any significant changes until 45 hr. In contrast, 2-cell embryos of B6C3F1 mice, which do not show a 2-cell block and develop normally to blastocysts in vitro, exhibit the dephosphorylation of p34cdc2 and an increase in histone H1 kinase activity between 31 and 45 hr after insemination. When the ddY mouse embryos arrested at the 2-cell stage were treated with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, the dephosphorylation of p34cdc2 occurred and histone H1 kinase activity increased. The chromosomes of these embryos stained with 4',6'-diamidino-2-phenylindole revealed the initiation of condensation. These results suggest that 2-cell-blocked embryos contain enough p34cdc2 to induce mitotic events but the protein remains in a latent form.     

Delay of ZGA initiation occurred in 2-cell blocked mouse embryos

One-cell mouse embryos from KM strain and B6C3F1 strain were cultured in M16 medium, in which2-cell block generally occurs. Embryos of KM strain exhibited 2-cell block, whereas B6C3F1 embryos,which are regarded as a nonblocking strain, proceeded to the 4-cell stage in our culture condition. It is oftenassumed that the block of early development is due to the failure of zygotic gene activation (ZGA) in culturedembryos. In this study we examined protein synthesis patterns by two-dimensional gel electrophoresis of[35^S] methionine radiolabeled 2-cell embryos. Embryos from the blocking strain and the nonblocking strainwere compared in their development both in vitro and in vivo. The detection of TRC expression, a markerof ZGA, at 42 h post hCG in KM embryos developed in vitro suggested that ZGA was also initiated even inthe 2-cell arrested embryos. Nevertheless, a significant delay of ZGA was observed in KM strain as comparedwith normally developed B6C3F1 embryos. At the very beginning of major ZGA as early as 36 h post hCG,TRC has already been expressed in B6C3F1 embryos developed in vitro and KM embryos developed in vivo.But for 2-cell blocked KM embryos, TRC was still not detectable even at 38 h post hCG. These evidences suggest that 2-cell-blocked embryos do initiate ZGA, and that 2-cell block phenomenon is due not to the disability in initiating ZGA, but to a delay of ZGA.     

Cyclin B1 levels in the porcine 4-cell stage embryo

The relative quantity of cyclin B1 was determined during the development of in vitro and in vivo derived porcine 4-cell embryos by western blotting and immunolocalised during the 4-cell stage. After cleavage to the 4-cell stage cyclin B1 localised to the cytoplasm at the 5, 10, 18 and 25 time points and localised to the nucleus 33 h post 4-cell cleavage (P4CC). The relative abundance of cyclin B1 was not significantly different in in vivo or in vitro derived 4-cell stage embryos cultured in the absence of the RNA polymerase inhibitor alpha-amanitin. Cyclin B1 protein was not detectable in embryos cultured in medium without alpha-amanitin for 5, 10, 18 or 25 h P4CC followed by culture in medium with alpha-amanitin to 33 P4CC. These results suggest that the maternal to zygotic transition of mRNA production that occurs at the 4-cell stage of the pig embryo does not result in an increase in cyclin B1 production. In addition, cyclin B1 protein levels remained constant in the absence of embryonic genome activation at the 4-cell stage.     

Cell-cycle-dependent subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse

M phase or maturation promoting factor (MPF), a kinase complex composed of the regulatory cyclin B and the catalytic p34cdc2 kinase, plays important roles in meiosis and mitosis. This study was designed to detect and compare the subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse. We found that all these proteins were concentrated in the germinal vesicle of oocytes. Shortly after germinal vesicle breakdown, all these proteins were accumulated around the condensed chromosomes. With spindle formation at metaphase I, cyclin B1 and phosphorylated cyclin B1 were localized around the condensed chromosomes and concentrated at the spindle poles, while p34cdc2 was localized in the spindle region. At the anaphase/telophase transition, phosphorylated cyclin B1 was accumulated in the midbody between the separating chromosomes/chromatids, while p34cdc2 was accumulated in the entire spindle except for the midbody region. At metaphase II, both cyclin B1 and p34cdc2 were horizontally localized in the region with the aligned chromosomes and the two poles of the spindle, while phosphorylated cyclin B1 was localized in the two poles of spindle and the chromosomes. We could not detect a particular distribution of cyclin B1 in fertilized eggs when the pronuclei were initially formed, but in late pronuclei cyclin B1 was accumulated in the pronuclei. p34cdc2 and phosphorylated cyclin B1 were always concentrated in one pronucleus after parthenogenetic activation or in two pronuclei after fertilization. At metaphase of 1-cell embryos, cyclin B1 was accumulated around the condensed chromosomes. Cyclin B1 was accumulated in the nucleus of late 2-cell embryos but not in early 2-cell embryos. Furthermore, we also detected the accumulation of p34cdc2 in the nucleus of 2- and 4-cell embryos. All these results show that cyclin B1, phosphorylated cyclin B1 and p34cdc2 have similar distributions at some stages but different localizations at other stages during oocyte meiotic maturation and fertilization, suggesting that they may play a common role in some events but different roles in other events during oocyte maturation and fertilization.     

Inactivation of M-phase promoting factor at exit from first embryonic mitosis in the rat is independent of cyclin B1 degradation

Exit from M-phase and completion of cell division requires inactivation of M-phase promoting factor (MPF), a heterodimer composed of the regulatory cyclin B1 and the catalytic p34cdc2 kinase. Inactivation of MPF is associated with cyclin B1 degradation that is brought about by the ubiquitin-proteasome pathway. Our study examined the role of the proteasome in the first mitosis of rat embryos and its participation in the regulation of cyclin B1 degradation and MPF inactivation. We show that in the early zygote the proteasome is evenly distributed in the ooplasm and the nucleus, whereas during mitosis it accumulates on the spindle apparatus. We further demonstrate that inhibition of proteasomal catalytic activity prevents 1-cell embryos from undergoing mitosis. This mitotic arrest is associated with the presence of relatively high amounts of cyclin B1, which unexpectedly does not result in elevated MPF activity. Our findings strongly imply that completion of the first embryonic division depends on proteasomal degradation and that cyclin B1 is included among its target proteins. They also provide the first evidence that MPF inactivation at this stage of development is not solely dependent upon cyclin B1 degradation and is insufficient to allow the formation of the 2-cell embryo.     

Effects of protein kinase C on M-phase promoting factor in early development of fertilized mouse eggs

The mechanism of development of mouse fertilized eggs from the one-cell stage to the two-cell stage remains unclear to date. In the present study, we have evaluated protein kinase C (PKC) and M-phase promoting factor (MPF) kinase activity in fertilized mouse eggs treated with a PKC modulator. PKC and MPF activity have similar activity. The two subunits of MPF, p34(cdc2) and cyclin B, were shown to be included in the substrates phosphorylated by PKC in fertilized mouse eggs, while PKC modulator affected the electrophoretic mobility shift of cdc2 and cdc25C by dephosphorylation and phosphorylation. These results clearly indicate that PKC may affect the progression of the cell cycle through post-translational modification of MPF activity.     

Acquisition of meiotic competence in mouse oocytes: absolute amounts of p34(cdc2), cyclin B1, cdc25C, and wee1 in meiotically incompetent and competent oocytes

M-Phase promoting factor (MPF) is a complex of p34(cdc2) and cyclin B. Results of previous studies in which relative mass amounts of these cell cycle regulators were determined suggested that the accumulation of p34(cdc2), rather than cyclin B, could be a limiting factor in the acquisition of meiotic competence in mouse oocytes. Nevertheless, in the absence of measurements of the absolute amount of these components of MPF, it is possible that the molar amount of p34(cdc2) is in excess to that of cyclin B, i.e., the accumulation of p34(cdc2) is not a limiting factor. We report measurements of the absolute mass of p34(cdc2) and cyclin B1, as well as the two proximal regulators of MPF, namely cdc25C and wee1, in meiotically incompetent and competent mouse oocytes. We find that the numbers of molecules of p34(cdc2), cyclin B1, cdc25C, and wee1 in meiotically incompetent oocytes are 1.4 x 10(6), 11.3 x 10(6), 24.6 x 10(6), 15. 6 x 10(6), respectively, and in meiotically competent oocytes the numbers are 14.3 x 10(6), 95.5 x 10(6), 80.0 x 10(6), 40.1 x 10(6), respectively. Thus, the concentration of cyclin B1 is always in excess to that of p34(cdc2), and this is consistent with the hypothesis that the accumulation of p34(cdc2) plays a role in the acquisition of meiotic competence. Last, the concentration of cdc25C is greater than that of wee1 and the concentration of each is greater than that of p34(cdc2) in both meiotically incompetent and competent oocytes.     

Phosphorylation and activation of human cdc25-C by cdc2--cyclin B and its involvement in the self-amplification of MPF at mitosis.

We have investigated the mechanisms responsible for the sudden activation of the cdc2-cyclin B protein kinase before mitosis. It has been found previously that cdc25 is the tyrosine phosphatase responsible for dephosphorylating and activating cdc2-cyclin B. In Xenopus eggs and early embryos a cdc25 homologue undergoes periodic phosphorylation and activation. Here we show that the catalytic activity of human cdc25-C phosphatase is also activated directly by phosphorylation in mitotic cells. Phosphorylation of cdc25-C in mitotic HeLa extracts or by cdc2-cyclin B increases its catalytic activity. cdc25-C is not a substrate of the cyclin A-associated kinases. cdc25-C is able to activate cdc2-cyclin B1 in Xenopus egg extracts and to induce Xenopus oocyte maturation, but only after stable thiophosphorylation. This demonstrates that phosphorylation of cdc25-C is required for the activation of cdc2-cyclin B and entry into M-phase. Together, these studies offer a plausible explanation for the rapid activation of cdc2-cyclin B at the onset of mitosis and the self-amplification of MPF observed in vivo.     

The Mos pathway regulates cytoplasmic polyadenylation in Xenopus oocytes.

Cytoplasmic polyadenylation controls the translation of several maternal mRNAs during Xenopus oocyte maturation and requires two sequences in the 3' untranslated region (UTR), the U-rich cytoplasmic polyadenylation element (CPE), and the hexanucleotide AAUAAA. c-mos mRNA is polyadenylated and translated soon after the induction of maturation, and this protein kinase is necessary for a kinase cascade culminating in cdc2 kinase (MPF) activation. Other mRNAs are polyadenylated later, around the time of cdc2 kinase activation. To determine whether there is a hierarchy in the cytoplasmic polyadenylation of maternal mRNAs, we ablated c-mos mRNA with an antisense oligonucleotide. This prevented histone B4 and cyclin A1 and B1 mRNA polyadenylation, indicating that the polyadenylation of these mRNAs is Mos dependent. To investigate a possible role of cdc2 kinase in this process, cyclin B was injected into oocytes lacking c-mos mRNA. cdc2 kinase was activated, but mitogen-activated protein kinase was not. However, polyadenylation of cyclin B1 and histone B4 mRNA was still observed. This demonstrates that cdc2 kinase can induce cytoplasmic polyadenylation in the absence of Mos. Our data further indicate that although phosphorylation of the CPE binding protein may be involved in the induction of Mos-dependent polyadenylation, it is not required for Mos-independent polyadenylation. We characterized the elements conferring Mos dependence (Mos response elements) in the histone B4 and cyclin B1 mRNAs by mutational analysis. For histone B4 mRNA, the Mos response elements were in the coding region or 5' UTR. For cyclin B1 mRNA, the main Mos response element was a CPE that overlaps with the AAUAAA hexanucleotide. This indicates that the position of the CPE can have a profound influence on the timing of cytoplasmic polyadenylation.     

多能性转录因子OCT4和SOX2在体外发育的小鼠2-细胞胚胎的表达与定位

为探讨多能性转录因子OCT4和SOX2在昆明小鼠(Mus musculus)2-细胞胚胎发育过程中与2-细胞胚胎阻滞发生的相关性,本研究应用实时荧光定量PCR技术检测了小鼠卵母细胞及在M16培养液中培养的不同发育阶段体外受精胚Oct4和Sox2基因的表达,并利用实时荧光定量PCR和免疫荧光技术比较了2-细胞胚、2-细胞阻滞胚和4-细胞胚的OCT4和SOX2的表达与定位。采用ANOVA对实验所得的数据进行分析,P0.05被认为是具有显著性差异。研究结果显示,2-细胞胚只有24.8%发育成4-细胞胚,75.2%的2-细胞胚发生了阻滞。Sox2和Oct4的m RNA在MⅡ期卵母细胞、原核胚、2-细胞胚、4-细胞胚、桑椹胚和囊胚中都有表达。Oct4 m RNA的表达水平在4-细胞胚显著高于2-细胞胚和2-细胞阻滞胚(P0.05),Sox2 m RNA的表达水平在2-细胞胚显著高于2-细胞阻滞胚和4-细胞胚(P0.05),而后两者之间没有差异(P0.05)。OCT4蛋白在2-细胞胚和4-细胞胚中与核共定位,但在2-细胞阻滞胚中弥散存在于胞质中。SOX2蛋白在以上3类胚胎中始终定位于细胞核。上述结果提示,转录因子OCT4和SOX2的表达和定位与小鼠2-细胞胚胎发育阻滞相关,母源性SOX2表达的维持对胚胎合子基因组激活(ZGA)的发生具有重要作用,母源性OCT4的异常定位可能影响了合子基因组激活相关基因的激活,而合子中Oct4的表达影响合子基因组激活后胚胎的发育。     

An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro

One-cell CF-1 x B6SJLF1/J embryos, which usually exhibit a 2-cell block to development in vitro, have been cultured to the blastocyst stage using CZB medium and a glucose washing procedure. CZB medium is a further modification of modified BMOC-2 containing an increased lactate/pyruvate ratio of 116, 1 mM-glutamine and 0.1 mM-EDTA but lacking glucose. Continuous culture of one-cell embryos in CZB medium allowed 83% of embryos to develop beyond the 2-cell stage of which 63% were morulae at 72 h of culture, but blastocysts did not develop. However, washing embryos into CZB medium containing glucose after 48 h of culture (3-4-cell stage) was sufficient to allow development to proceed, with 48% of embryos reaching the blastocyst stage by 96 h of culture. Exposure of embryos to glucose was only necessary from the 3-4-cell stage through the early morula stage since washing back into medium CZB without glucose at 72 h of culture still promoted the development of 50% of embryos to the blastocyst stage. The presence of glucose in this medium for the first 48 h of culture (1-cell to 4-cell stage) was detrimental to embryo development. Glutamine, however, exerted a beneficial effect on embryo development from the 1-cell to the 4-cell stage although its presence was not required for development to proceed during the final 48 h of culture. Blastocysts which developed under optimum conditions contained an average of 33.7 total cells. The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.     

Cdc2 protein kinase is complexed with both cyclin A and B: evidence for proteolytic inactivation of MPF

In the clam, Spisula, two previously described proteins known as cyclin A and B display the unusual property of selective proteolytic degradation at the end of each mitosis. We show here that clam oocytes and embryos contain a cdc2 protein kinase. This protein kinase is a component of the M phase promoting factor (MPF) in frog eggs and the M phase-specific histone H1 kinase in starfish. Clam cdc2 is found in association with both cyclin A and B, probably not as a trimolecular association, but as separate cdc2/cyclin A and cdc2/cyclin B complexes. Clam cdc2 and the associated cyclins bind to p13suc1-Sepharose. The p13-bound complex, and also anti-cyclin A or B immunoprecipitates, each display cell cycle-dependent histone H1 kinase activity. We suggest that in addition to the cdc2 protein kinase, the cyclins are further components of the M phase promoting factor and that cyclin proteolysis provides the mechanism of MPF inactivation and thus exit from mitosis.     

Initial activation of cyclin-B1-cdc2 kinase requires phosphorylation of cyclin B1

At the G₂/M transition of the cell cycle, the cdc25c phosphatase dephosphorylates inhibitory residues of cdc2, and cyclin-B–cdc2 kinase (MPF) is activated. Phosphorylation of cyclin B1 induces its nuclear accumulation, and, since cdc25c is also believed to accumulate and activate shortly before G₂/M in the nucleus, it has been proposed that this induces cyclin-B1–cdc2 kinase activation. We demonstrate that cyclin B1 phosphorylation has another essential function in vivo: it is required for cdc25c and MPF activation, which does not require nuclear accumulation of cyclin B1, and occurs in the cytoplasm.     

Involvement of protein kinase B/AKT in early development of mouse fertilized eggs

The activation of AKT (also called protein kinase B) is thought to be a critical step in the phosphoinositide 3-kinase pathway that regulates cell growth and differentiation. In this report, we investigated the role of AKT in the regulation of mouse early embryo development. Injection of mRNA coding for a constitutively active myristoylated AKT (myr-Akt1) into one-cell stage fertilized eggs induced cell division more effectively than injection of wild-type AKT (Akt1-WT) mRNA, whereas microinjection of mRNA of kinase-deficient AKT (Akt1-KD) delayed the first mitotic division. Meanwhile, microinjection of different kinds of mRNA of AKT affected the phosphorylation status of CDC2A-Tyr15 and the activation of M-phase promoting factor (MPF). To investigate the intermediate factor between AKT and MPF, we then injected one-cell stage eggs first with Akt1-WT mRNA or myr-Akt1 mRNA and then with mRNA encoding either wild-type CDC25B (Cdc25b-WT) or a AKT-nonphosphorylatable Ser351 to Ala CDC25B mutant (Cdc25b-S351A). Cdc25b-S351A strongly inhibited the effect of AKT. Therefore, AKT causes the activation of MPF and strongly promotes the development of one-cell stage mouse fertilized eggs by inducing AKT-dependent phosphorylation of CDC25B, a member of the CDC25 phosphatase family. Our finding that CDC25B acts as a potential target of AKT provides new insight into the effect of AKT in the regulation of early development of mouse embryos.     

Cyclin B in fish oocytes: its cDNA and amino acid sequences, appearance during maturation, and induction of p34cdc2 activation.

Under the influence of maturation-inducing hormone (MIH) secreted from follicle cells, oocyte maturation is finally triggered by maturation-promoting factor (MPF), which consists of a homolog of the cdc2+ gene product of fission yeast (p34cdc2) and cyclin B. Two species of cyclin B clones were isolated from a cDNA library constructed from mature goldfish oocytes. Sequence comparisons revealed that these two clones are highly homologous (95%) and were found to be similar to Xenopus cyclin B1. Using monoclonal antibodies against Escherichia coli-produced goldfish cyclin B and the PSTAIR sequence of p34cdc2, we examined the levels of cyclin B and p34cdc2 proteins during goldfish oocyte maturation induced in vitro by 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-DP), a natural MIH in fish. Protein p34cdc2 was found in immature oocyte extracts and did not remarkably change during oocyte maturation. Cyclin B was not detected in immature oocyte extracts and appeared when oocytes underwent germinal vesicle breakdown. Cyclin B that appeared during oocyte maturation was labelled with [35S]methionine, indicating its de novo synthesis. Introduction of E. coli-produced cyclin B into immature oocyte extracts induced p34cdc2 (MPF) activation. Although the possibility that immature goldfish oocytes contain an insoluble cyclin B is not completely excluded, these results strongly suggest that 17 alpha, 20 beta-DP induces oocytes to synthesize cyclin B, which in turn activates preexisting p34cdc2, forming active MPF.     

Quantification of cyclin B1 and p34(cdc2) in bovine cumulus-oocyte complexes and expression mapping of genes involved in the cell cycle by complementary DNA macroarrays

Although high amounts of cyclin B1 mRNA are present in bovine oocytes arrested at the germinal vesicle (GV) stage, the protein is not detectable. Furthermore, there is a depletion of the stored cyclin B1 mRNA in the oocyte as follicular growth progresses. To assess the effect of follicular growth on the accumulation of M-phase promoting factor (MPF) components, mRNA and protein levels of cyclin B1 and p34(cdc2) were measured in GV oocytes collected from diverse follicle size groups (<2 mm, 3-5 mm, and >6 mm). Because oocytes collected from very small follicles have high levels of cyclin B1 mRNA, the onset of its accumulation in the oocytes was evaluated by in situ hybridization of fetal ovaries. Also, a comparative expression map of cell cycle-related genes expressed in the oocyte and cumulus cells was established using nylon-based cDNA arrays, which allowed the detection of 35 different genes transcribed mostly in oocytes. Both components of the pre-MPF complex were expressed at the mRNA level in GV oocytes, whereas p34(cdc2) was the only pre-MPF protein detected at that stage, thus indicating that meiosis resumption in bovine oocytes is differentially regulated as compared with other mammals, and meiosis resumption seems to be regulated by the translation of cyclin B1 mRNA.