Constitutive proteins of the oocytes and early embryos of rats]

5 protein fractions were identified and their relative mobility was determined in the rat oocytes and cleaving embryos by means of vertical capillary microdisc-electrophoresis in 7.5% polyacrilamide gel (PAA-gel). The same fractions were identified in the cleaving embryos devoid of zona pellucida. A conclusion was drawn that these proteins were present in the oocyte cytoplasm and kept in the cleaving embryos until the stage of implantation. 4 groups of proteins with different anodic mobility were identified in the isolated zona pellucida by means of microdisc-electrophoresis in 7.5% PAA-gel added with 1% sodium dodecylsulfate (SDS). The molecular weight of low molecular weight proteins of oocytes and preimplantation embryos was determined by means of disc-electrophoresis in 14% PAA-gel with 1% SDS. The zona pellucida of one embryo contained, according to the data of capillary spectrophotometry, 5 ng of protein.     

The role of autophagy during the oocyte-to-embryo transition

After fertilization, the maternal proteins stored in oocytes are degraded and new proteins encoded by the zygotic genome are synthesized. Although several proteins are degraded by the ubiquitin-proteasome system, the mechanism underlying the dynamic protein turnover during this process remains largely unknown. We recently reported that autophagy plays a critical role during preimplantation embryonic development. We found that the level of autophagy was low in unfertilized oocytes; however, autophagy was activated shortly after fertilization. The function of autophagy was further analyzed using oocyte-specific Atg5 (autophagy-related 5) knockout mice. Atg5-null oocytes could develop if they were fertilized with wild-type sperm, but could not develop beyond the four- and eight-cell stages if they were fertilized with Atg5-null sperm. Furthermore, protein synthesis rates were reduced in the autophagy-deficient embryos. We have previously reported that Atg5-null oocytes derived from Atg5+/- mice, which should contain maternally inherited Atg5 protein in the oocyte, were able to produce Atg5-/- neonates, emphasizing the specific importance of autophagy during very early embryogenesis. Thus, the degradation of maternal factors by autophagy is essential for preimplantation development in mammals.

Addendum to: Tsukamoto S, Kuma A, Murakami M, Kishi C, Yamamoto A, Mizushima N. Autophagy is essential for preimplantation development of mouse embryos. Science 2008; 321:117-20.     

Microelectrophoretic analysis of changes in protein expression patterns in mouse oocytes and preimplantation embryos.

One- and two-dimensional polyacrylamide microslab gel electrophoresis followed by silver staining was devised to visualize picogram to nanogram levels of proteins and was applied to the analysis of 1-20 mouse oocytes and embryos (approximately 16.5-330 ng of protein) during preimplantation development. Compared with values in embryos, more bands in the higher molecular weight range were found only for unfertilized oocytes in one-dimensional microelectrophoresis. A marked decrease in the number of protein spots occurred after fertilization in two-dimensional microelectrophoresis. Both findings indicate a decrease in maternal proteins caused by fertilization. Silver-staining densities were almost invariable for 8 major spots, but increased, decreased, or varied for 32 minor spots in developing embryos from the 1-cell to the morula stage, signifying spot-specific changes in the expression of zygotic proteins during development. The protein patterns in cumulus cells and blastocysts were different from those in oocytes and embryos. Even in a single 1-cell embryo, major spots and some minor spots were detectable by our two-dimensional microelectrophoretic technique, but many more minor spots were visualized in five 1-cell embryos, exemplifying the limit of our microelectrophoretic technique. As a preliminary result, a two-dimensional immunoblot pattern is shown for glucose transporter 1 expressed in morulae.     

Determination of protein concentration in cleaving mammalian ova by the capillary spectrophotometry method]

A micromodification of the Lowry's method is described which allows to measure reliably the protein content in 5-20 embryos of white rats and CBA mice. Differences in the protein content in rat embryos at the stages of 2 blastomeres and blastocyst were shown to be statistically unreliable (20.2 +/- 1.4 and 18.9 +/- 1.3 ng, resp.). The protein content in the mouse embryos at the same two stages differs reliably (19.1 +/- 1.1 and 22.0 +/- +/- 1.7 ng, resp.). The protein content in zona pellucida does not differ reliably from those in rat embryos both at the stages of 2 blastomeres (4.5 ng) and blastocyst (2.3 ng). The protein content in embryos devoid of zona pellucida decreased after 3 hours incubation in the medium 199 at 37 degrees at the stages of morula and blastocyst by 17-20% in rats and by 50% in mice. Addition of 1% serum albumin to the incubation medium did not prevent the partial "loss" of protein by the embryos. The protein content in the rat and mouse embryos at the stage of 2 blastomeres suffered no changes under long-term incubation in the medium 199.     

Cloned mice derived from embryonic stem cell karyoplasts and activated cytoplasts prepared by induced enucleation

Our objective was to induce enucleation (IE) of activated mouse oocytes to yield cytoplasts capable of supporting development following nuclear transfer. Fluorescence microscopy for microtubules, microfilaments, and DNA was used to evaluate meiotic resumption after ethanol activation and the effect of subsequent transient treatments with 0.4 micro g/ml of demecolcine. Using oocytes from B6D2F1 (C57BL/6 x DBA/2) donors, the success of IE of chromatin into polar bodies (PBs) was dependent on the duration of demecolcine treatment and the time that such treatment was initiated after activation. Similarly, variations in demecolcine treatment altered the proportions of oocytes exhibiting a reversible compartmentalization of chromatin into PBs. Treatment for 15 min begun immediately after activation yielded an optimized IE rate of 21% (n = 80) when oocytes were evaluated after overnight recovery in culture. With this protocol, 30-50% of oocytes were routinely scored as compartmentalized when assessed 90 min postactivation. No oocytes could be scored as such following overnight recovery, with 66% of treated oocytes cleaving to the 2-cell stage (n = 80). Activated cytoplasts were prepared by mechanical removal of PBs from oocytes whose chromatin had undergone IE or compartmentalization. These cytoplasts were compared with mechanically enucleated, metaphase (M) II cytoplasts whose activation was delayed in nuclear transfer experiments using HM-1 embryonic stem cells. Using oocytes from either B6D2F1 or B6CBAF1 (C57BL/6 x CBA) donors, the in vitro development of cloned embryos using activated cytoplasts was consistently inferior to that observed using MII cytoplasts. Live offspring were derived from both oocyte strains using the latter, whereas a single living mouse was cloned from activated B6CBAF1 cytoplasts.     

Precocious initiation of meiosis by male germ cells of the mouse

12 1/2-15 1/2 day embryonic mouse testes of 129/terSv and CBA/T6T6 strains were transplanted under the kidney capsule of adult hosts. After 3-5 days in 41% of CBA/T6T6 transplants and in 82% of 129/terSv transplants a limit number of germ cells began meiosis. The percentage of meiotic germ cells was inversely related to the total number of gonocytes and the organization of seminiferous cords. The presented evidence indicates that the ability of the germ cells to begin meiosis precociously depends on: 1) genotype of donor embryos; 2) age of transplanted testis, and 3) using whole of half of gonad for transplantation. After 10-15 days in two out of 46 129/terSv testes (4%) growing oocytes were observed.     

Stability of liver nuclear proteins in inbred strains of mice

1. A remarkable similarity in the gel patterns of liver nuclear proteins between four inbred strains of mice (A.CA, B10.A, CBA and DBA/2) was observed. 2. Only a very few quantitative differences were detected in the protein spot patterns of nucleoplasmic (spot of about 41 kDa) and chromatin (spot of about 37 kDa) non-histone proteins between those strains of mice. 3. Comparison of two-dimensional gel patterns of non-histone proteins from males and females revealed a few sex-linked spots. Nucleoplasmic protein with molecular weight of about 59 kDa and chromatin proteins with molecular weights of approximately 47 and 57 kDa were more abundant in liver nuclei of male mouse.     

Cdc42 protein acts upstream of IQGAP1 and regulates cytokinesis in mouse oocytes and embryos

Cdc42 and Rac1 Rho family GTPases, and their interacting protein IQGAP1 are the key regulators of cell polarity. We examined the role of Cdc42 and IQGAP1 in establishing the polarity of mouse oocyte and regulation of meiotic and mitotic divisions. We showed that Cdc42 was localized on the microtubules of meiotic and mitotic spindle and in the cortex of mouse oocytes and cleaving embryos. IQGAP1 was present in the cytoplasm and cortex of growing and fully-grown oocytes. During maturation it disappeared from the cortex and during meiotic and mitotic cytokinesis it concentrated in the contractile ring. Toxin B inhibition of the binding activity of Cdc42 changed the localization of IQGAP1, inhibited emission of the first polar body, and caused disappearance of the cortical actin without affecting the migration of meiotic spindle. This indicates, that in maturing oocytes accumulation of cortical actin is not indispensable for spindle migration. In zygotes treated with toxin B actin cytoskeleton was rearranged and the first and/or subsequent cytokinesis were inhibited. Our results indicate that Cdc42 acts upstream of IQGAP1 and is involved in regulation of cytokinesis in mouse oocytes and cleaving embryos, rather than in establishing the polarity of the oocyte.     

The development of diploid parthenogenetic mouse embryos of the inbred C57BL and CBA strains]

We studied preimplantation development in vitro and postimplantation development in vivo of diploid parthenogenetic mouse embryos of C57BL/6 and CBA strains, as well as of (CBA x C57BL/6)F1 hybrids. Development to blastocyst stage of diploid eggs obtained from C57BL/6, CBA, and hybrid mice was observed in 90, 15, and 73% cases, respectively. After implantation, C57BL/6 embryos did not develop to somite stages, while CBA and hybrid embryos reached various stages of somite formation in 45 and 30% cases, respectively. Cultivation of embryos beginning from one-cell stage in the medium containing 2% newborn calf serum increased the yield of blastocysts from 15 to 59% in CBA embryos and from 73 to 90% in hybrids; However, such effect was not observed with C57BL/6 embryos. The latest stages of development observed in CBA and hybrid diploid parthenogenetic embryos were 33-35 somites and 25-30 somites, respectively. Imprinting patterns in chromosomes of CBA and C57BL/6 gametes are discussed.     

The role of autophagy during the oocyte-to-embryo transition

After fertilization, the maternal proteins stored in oocytes are degraded and new proteins encoded by the zygotic genome are synthesized. Although several proteins are degraded by the ubiquitin-proteasome system, the mechanism underlying the dynamic protein turnover during this process remains largely unknown. We recently reported that autophagy plays a critical role during preimplantation embryonic development. We found that the level of autophagy was low in unfertilized oocytes; however, autophagy was activated shortly after fertilization. The function of autophagy was further analyzed using oocyte-specific Atg5 (autophagy-related 5) knockout mice. Atg5-null oocytes could develop if they were fertilized with wild-type sperm, but could not develop beyond the four- and eight-cell stages if they were fertilized with Atg5-null sperm. Furthermore, protein synthesis rates were reduced in the autophagy-deficient embryos. We have previously reported that Atg5-null oocytes derived from Atg5(+/-) mice, which should contain maternally inherited Atg5 protein in the oocyte, were able to produce Atg5(-/-) neonates, emphasizing the specific importance of autophagy during very early embryogenesis. Thus, the degradation of maternal factors by autophagy is essential for preimplantation development in mammals.     

Expression of mRNAs of the aquaporin family in mouse oocytes and embryos

The molecular basis of water and cryoprotectant permeability in mammalian oocytes and embryos is poorly understood. Therefore, we investigated the expression of mRNAs of water channel proteins (aquaporins) in mouse oocytes and embryos by RT-PCR. The total RNA of mouse oocytes at metaphase II and embryos at the 4-cell, morula, and blastocyst stages was isolated, reverse-transcribed, and subjected to nested PCR amplification. Aquaporins were expressed in both oocytes and embryos, but the types were different among the developmental stages: aquaporins 3 and 7 were expressed in oocytes and embryos at all stages examined, but aquaporins 8 and 9 were expressed only in blastocysts. On the other hand, aquaporins 1, 2, 4, 5, and 6 were not detected in any of the stages examined. The present study shows for the first time that aquaporins are expressed in mammalian oocytes and embryos. These aquaporins may play a role in water transport and conceivably also in cryoprotectant transport across the plasma membrane in these cells.     

Sex ratio in hybrid mice CBA X C57BL

Sex ratio and postimplantation mortality were studied in (CBAXC57BL)F1, CBA and C57BL mouse embryos. It has been shown that female fetuses are predominant in the progeny of hybrid mice. In CBA and C57BL mice the sex distribution was 1 : 1. The disorder in the balanced sex ratio in the progeny of hybrid mice confirms a conclusion on the effect of the mouse genetic features on the sex distribution in embryos. Equal sex ratio in CBA and C57BL mice indicates the absence of selective mortality in the embryos of either sex during embryogenesis.     

Injection of somatic cell cytoplasm into oocytes before intracytoplasmic sperm injection impairs full-term development and increases placental weight in mice

This study investigated the effects on fertilized embryo development of somatic cytoplasm after its injection into intact mouse oocytes. Mature oocytes collected from female B6D2F1 mice were injected with cumulus cell cytoplasm of different volumes and from different mouse strains (B6D2F1, ICR, and C57BL/6), or with embryonic cytoplasm. After culture for 1 h, B6D2F1 sperm were injected into those oocytes by intracytoplasmic sperm injection (ICSI). The oocytes were examined for pre- and postimplantation developmental competence. Increases in the volume of the somatic cytoplasm from onefold to fourfold resulted in an impairment of blastocyst development and full-term development (28% and 7%, respectively, vs. 96% and 63%, respectively, in the control group; P < 0.01). An increase in the volume of somatic cytoplasm reduced the expression of POU5F1 (more commonly known as OCT4) in expanded blastocysts. The frequency of embryos that developed to the blastocyst stage did not differ when B6D2F1 or ICR somatic cytoplasm was injected, but injection of C57BL/6 somatic cytoplasm induced a two-cell block in embryo development. Injection of the cytoplasm from fertilized embryos did not reduce the frequency of embryos attaining full-term development. Interestingly, somatic cytoplasm significantly increased the placental weight of ICSI embryos, even the injection of onefold cytoplasm (0.20 +/- 0.02 [n = 32] vs. 0.12 +/- 0.02 in the control group [n = 87]; P < 0.01). These findings indicate that the injection of somatic cytoplasm into oocytes before ICSI causes a decrease in preimplantation development, clearly impairs full-term development, and causes placental overgrowth in fertilized embryos. To our knowledge, placental overgrowth phenotypes are only caused by interspecies hybridization and cloning, and in genetically modified mice. Here, we report for the first time that somatic cytoplasm causes abnormal placentas in fertilized embryos. This study suggests that somatic cell cytoplasmic material is one cause of the low rate of full-term development in cloned mammals.     

Effects of recombinant human follicle-stimulating hormone on embryo development in mice

We have developed a protocol using recombinant human follicle-stimulating hormone (rhFSH) to induce ovarian stimulation in the mouse to investigate its impact on preimplantation embryo development. Embryos were collected from adult female C57Bl/6 x CBA F1 mice treated with rhFSH (0, 2.5, 5.0, 10.0, or 20.0 IU) or 5 IU equine chorionic gonadotropin (eCG). Embryos were also recovered from nontreated control mice. Embryos were cultured in vitro for 88 h, and the stage of development was morphologically assessed. The allocation of cells to the inner cell mass or trophectoderm of blastocysts was determined by differential nuclear staining. The expression of insulin-like growth factor 2 (IGF-II), the insulin-like growth factor receptor (IGF-II receptor), and vascular endothelial growth factor (VEGF) in blastocysts was measured by real-time RT-PCR. Blastocyst development was reduced in the 10 (72.3 +/- 5.1%) and 20 (77.3 +/- 5.6%) IU rhFSH groups compared with control embryos (96.7 +/- 1.0%). The number of inner cell mass cells was reduced (P < 0.001) in the 5, 10, and 20 IU rhFSH groups and the eCG group compared with control embryos. We did not find any effect of rhFSH treatment on IGF-II, IGF-II receptor, or VEGF expression in blastocysts compared with the control group. eCG treatment, however, significantly increased the expression of IGF-II in blastocysts. These results indicate that ovarian stimulation with rhFSH impairs the in vitro development of preimplantation mouse embryos, and these results may have potential implications for clinical ovarian stimulation during infertility treatment and subsequent embryo quality.     

不同品系小鼠胚胎玻璃化冷冻保存的比较研究

目的　研究甘油作为冷冻保护剂、不同基因型小鼠对胚胎玻璃化冷冻的影响。方法　采用 6 5mol L的甘油作为冷冻保护剂 ,采用二步法对CBA、NOD、C57BL 6J、ICR及CD1小鼠 3 5d的胚胎进行玻璃化冷冻 ,并比较了不同品系小鼠胚胎的复苏率及移植受孕率。结果和结论　CBA、NOD、C57BL 6J,ICR及CD1的复苏率分别为 5 7 6 %、4 8%、31 3%、86 5 %及 88% ,移植受孕率为 2 1%、2 3 5 %、11%、38%和 35 5 % ,封闭群小鼠的胚胎复苏率、移植受孕率均显著高于近交系小鼠。这提示胚胎的复苏率及移植受孕率可能与小鼠的不同基因型有关。五个品系中 ,桑椹胚及早期囊胚的体外复苏率均显著高于扩张囊胚。这说明不同基因型及胚胎的不同发育阶段对胚胎玻璃化冷冻效果有影响     

Different response of maturing oocytes from two inbred strains of mice to sperm penetration

Oocytes from two inbred strains of mice, CBA and KE, were inseminated at prometaphase of the first meiotic division. Pronuclei were formed in 21-36% of inseminated oocytes from the CBA strain. In the KE strain the formation of pronuclei occurred in 2-5% of oocytes and was at the same level as in the non-inseminated control. These results show that in oocytes of the CBA strain maturity of the cytoplasm is acquired earlier than in those of the KE or other so far studied strains of mice. This fact is discussed in the context of different maturation rates of oocytes from different strains. In oocytes which remained non-activated after penetration, transformation of sperm heads into separate chromosomes was observed. An interstrain difference in efficiency of this process was also found.     

Development of enucleated mouse oocytes reconstituted with embryonic nuclei

The chromosomes of mouse oocytes at telophase of the first meiotic division were removed using micromanipulation and differential interference microscopy. The enucleated oocytes were used as recipients for nuclear transplantation, after culture for 4-6 h. The newly synthesized proteins of the enucleated oocytes showed the same pattern as those of secondary oocytes matured in vivo. When the enucleated oocytes received a nucleus from late 2- and 8-cell embryos, or a cell from the inner cell mass (ICM) of blastocysts, 23, 4 and 10%, respectively, of reconstituted embryos developed to blastocysts. After transfer to recipient females, live young were produced from the reconstituted eggs that received a nucleus from late 2-cell embryos.