Effects of oxygen toxicity on early development of mouse embryos.

To examine the effects of oxygen toxicity on embryonic development, mouse pronuclear embryos were cultured under low oxygen conditions with or without superoxide dismutase (SOD), and the blastulation rate was compared with that of embryos cultured under standard conditions. The blastulation rate of mouse pronuclear embryos cultured under standard conditions was only 1.5% (2/131). This rate was increased significantly, to 28.5% (43/151), when the embryos were cultured under low oxygen conditions; and to 31.0% (35/113) when SOD (500 micrograms/ml) was added to the medium under standard conditions; the rate was increased to 75.2% (115/153) when the embryos were cultured under low oxygen conditions in the presence of SOD. The minimum effective concentration of SOD in the culture medium was 50 micrograms/ml under conditions of 5% O2. The blastulation rate was significantly decreased after 1-hr exposure of pronuclear embryos to room atmospheric oxygen concentration (20% O2), and subsequent culture under 5% O2 with SOD did not result in an improved blastulation rate. Culture with SOD under 5% O2 promoted the development of two-cell stage embryos to the blastocyst stage. When two-cell stage embryos were collected 48 hr after hCG and cultured for 66 hr, their blastulation rate was similar to that of embryos collected from mice 114 hr after hCG. These results suggested that embryonic development in vitro is greatly affected by atmospheric oxygen throughout the early embryonic stages and that this harmful effect can be prevented by culturing embryos under low oxygen conditions and in the presence of SOD.     

A genetic quality testing system for early stage embryos in the mouse.

We have established a genetic quality testing system for early stage embryos of the mouse. A method of preparation of template DNA for PCR was established using the lysis buffer (1 x PCR reaction buffer supplemented with proteinase K at a concentration of 40 microg/ml) developed by the authors. We demonstrated that two 8-cell embryos of an inbred strain provide sufficient volumes of template DNA for PCR to identify the strain of embryos using four microsatellite markers (D3Mit54, D5Mit18, D6Mit15 and D8Mit50) differentiating 13 inbred strains of mice. This system will be useful in embryo banks that have recently been established worldwide for demonstrating the genetic accuracy of a given strain prior to recovery of live animals.     

The early expression of immunoreactivity for calmodulin in the nervous system of mouse embryos

Summary Calmodulin (CaM) is a major calcium-binding protein in the brain, where its immunoreactivity is mainly localized in the neurons. In this study, ontogenical changes in the distribution of CaM in the nervous system of mouse embryos were investigated immunohistochemically using a specific antibody against CaM and an indirect immunoenzyme method. Immunoreactive staining was first observed in the marginal layer of the cranial neural tube after 9.5 days of gestation; thereafter, the amount of stained structures increased rapidly. Particularly intense staining was observed in the long neuronal processes extending from or into the brain and spinal cord primordia. Intense immunostaining was also observed in the optic nerve layer of early retinae from 12.5 days of gestation. The appearance of CaM immunoreactivity is thus an early event during neuronal differentiation, apparently concominant with the initiation of axon extension and the appearance of neurofilament proteins.     

自分泌生长因子对早期胚胎体外发育的影响(简报)

生长因子(Growth Factors,GFs)对于调节哺乳动物胚胎的早期发育和分化起着重要作用,这些GFs存在于雌性生殖道,也可由胚胎自身合成。正是由于这种自分泌GFs的存在,使我们可以通过改变胚胎培养密度或添加外源性GFs来定性或定量研究GFs对胚胎发育和分化的影响,对于了解GFs在分子水平上的作用途径和机制有着重要的意义。     

The early expression of immunoreactivity for calmodulin in the nervous system of mouse embryos

Calmodulin (CaM) is a major calcium-binding protein in the brain, where its immunoreactivity is mainly localized in the neurons. In this study, ontogenical changes in the distribution of CaM in the nervous system of mouse embryos were investigated immunohistochemically using a specific antibody against CaM and an indirect immunoenzyme method. Immunoreactive staining was first observed in the marginal layer of the cranial neural tube after 9.5 days of gestation; thereafter, the amount of stained structures increased rapidly. Particularly intense staining was observed in the long neuronal processes extending from or into the brain and spinal cord primordia. Intense immunostaining was also observed in the optic nerve layer of early retinae from 12.5 days of gestation. The appearance of CaM immunoreactivity is thus an early event during neuronal differentiation, apparently concomitant with the initiation of axon extension and the appearance of neurofilament proteins.     

The sex-chromosome constitution and early postimplantation development of diandric triploid mouse embryos

Diandric triploid mouse embryos were produced by standard micromanipulatory techniques, using eggs isolated from female mice with a normal chromosome constitution that had been mated to homozygous Rb(1.3)1Bnr males (which carry a large metacentric "marker" chromosome, viz., a Robertsonian translocation involving chromosomes 1 and 3). The tripronucleate embryos were transferred to the oviducts of pseudopregnant mice, which were subsequently autopsied at about midday on the 10th day of gestation. Although a relatively small number of the isolated conceptuses consisted of morphologically abnormal egg-cylinder-like structures or empty gestational sacs, most were at clearly distinguishable embryonic stages, from the primitive streak stage to embryos with about 20 pairs of somites present. These embryos all appeared to be morphologically normal but were substantially smaller than normal (diploid) fertilized embryos analyzed at similar stages of development. A total of 63 diandric triploid conceptuses were recovered and analyzed cytogenetically. They were G-banded to determine their sex-chromosome constitution and confirm their diandric triploid status. No obvious difference was observed in the developmental potential of the 58,XXX class of diandric triploids, compared to that of the 58,XXY class. The ratio of 58,XXX to 58,XXY embryos was close to the expected ratio of 1:2, assuming that unfertilized eggs have an equal chance of becoming fertilized by an X- or a Y-bearing spermatozoon and that the additional (i.e., "donor") male pronucleus also has an equal chance of having either an X or a Y sex chromosome present. However, the development of the 58,XYY class appeared to be restricted, even at the stage of gestation analyzed, in that no embryos with this genetic constitution were observed that had progressed beyond the early somite stage. The present findings are discussed in relation to the cytogenetic findings in human triploid conceptuses, the majority of which are spontaneously aborted during the first half of pregnancy. In man, the 69,XYY class (equivalent to the 58,XYY class in our study) is only rarely encountered, and it has been assumed that these triploid embryos are probably lost at a very early stage of gestation.     

Involvement of insulin in early development of mouse one-cell stage embryos

Recent studies have suggested that growth factors and hormones play important roles in cell prolif-eration and differentiation during early embryonic development. In the present study, we examined the expression and localization of insulin in the mouse oocytes and one-cell stage embryos by quantitative ELISA, RT-PCR, Western blot and immunofluorescence. In the mouse oocytes and one-cell stage em-bryos, expression of insulin was uniformly distributed in the cytoplasm. We also examined the expres-sion, activity and localization of mTOR (mammalian target of rapamycin) and p70S6K. The expression of mTOR and p70S6K was not significantly different at the cell cycle of mouse one-cell stage embryos. mTOR and S6K were distributed evenly in the cytoplasm at G1, G2 and M phase phase, but at S phase, the distribution of mTOR and S6K was around the pronucleus. At different phases, the activity of mTOR fluctuated. We also used the PI3K specific inhibitor-Wortmannin to investigate the cleavage rate of eggs. The result showed that the rate obviously decreased. When the mTOR specific inhibitor Rapa-mycin was used, the first mitotic division of the mouse one-cell stage embryo was delayed. These re-sults suggested that insulin was expressed both in mouse oocytes and one-cell stage embryos, and may play functional roles in regulation of mouse early embryogenesis by activating the signal pathway of PI3K/PKB/mTOR/S6K.     

自分泌生长因子对早期胚胎体外发育的影响（简报）

Two-cell-stage embryos were flushed from the oviducts on Day 2. Zygotes were collected from oviducts on Day 1 (Fertilization In Situ, ISF) or derived from fertilization in vitro (IVF). 2-cell embryos had a high rate of blastocyst development to each embryo concentration from 1 embryo/microliter to 1 embryo/1000 microliters. The zygotes produced by either ISF or IVF were adversely affected by reducing the embryo concentration over this range (P < 0.001), with approximately 82.5% of ISF zygotes developing to blastocysts at highest concentration but only 22.3% at the lowest. For IVF zygotes the corresponding results were 46.3% and 5.2%. The number of cells in each blastocyst from 2-cell embryos was significantly higher than that from ISF and IVF group. The media supplementing Platelet-activating factor (PAF) caused a significant increase in the rate of blastocyst development of IVF zygotes at embryo concentration of 1 embryo/10 microliters (10 ng/ml) and 1 embryo/100 microliters (100 ng/ml). Insulin-like growth factor (IGF) (10 ng/ml) also stimulated development of IVF zygotes when they were cultured at the concentration of 1 embryo/10 microliters. Epidermal growth factor (EGF) was no effect over range of 1-1000 ng/ml to embryo development. The results show that factors necessary for normal embryo development are diluted to suboptimal levels during culture at low embryo concentration. The PAF, IGF-I partially compensate the effects of low embryo concentration during culture and play important roles as autocrine embryotrophic factors.     

Stimulatory effect of epidermal growth factor on the development of mouse early embryos in vitro.

Effects of epidermal growth factor (EGF) on the development of mouse 2-cell embryos cultured in vitro were investigated. The addition of EGF at a concentration of 0.5 ng/ml enhanced the development of 2-cell embryos during 24 h of incubation. As expected, EGF stimulated the synthesis of DNA in the 2-cell embryos about 4-fold over the control. The growth-promoting effect of EGF seemed to be specific in that other growth factors, such as transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), nerve growth factor (NGF) and fibroblast growth factor (FGF) had no effect on the embryonal development. The addition of EGF also increased the rate of RNA synthesis in a dose-related manner between 0.1 and 50 ng/ml. However, protein synthesis was unaffected by EGF. These results raise the possibility that EGF may participate in the process of early embryogenesis in vivo.     

The development of XO gynogenetic mouse embryos

Diploid gynogenetic embryos, which have two sets of maternal and no paternal chromosomes, die at or soon after implantation. Since normal female embryos preferentially inactivate the paternally derived X chromosome in certain extraembryonic membranes, the inviability of diploid gynogenetic embryos might be due to difficulties in achieving an equivalent inactivation of one of their two maternally derived X chromosomes. In order to investigate this possibility, we constructed XO gynogenetic embryos by nuclear transplantation at the 1-cell stage. These XO gynogenones showed the same mortality around the time of implantation as did their XX gynogenetic counterparts. This shows that the lack of a paternally derived autosome set is sufficient to cause gynogenetic inviability at this stage. Autosomal imprinting and its possible relation to X-chromosome imprinting is discussed.     

Glucose transporter gene expression in early mouse embryos.

The glucose transporter (GLUT) isoforms responsible for glucose uptake in early mouse embryos have been identified. GLUT 1, the isoform present in nearly every tissue examined including adult brain and erythrocytes, is expressed throughout preimplantation development. GLUT 2, which is normally present in adult liver, kidney, intestine and pancreatic beta cells is expressed from the 8-cell stage onward. GLUT 4, an insulin-recruitable isoform, which is expressed in adult fat and muscle, is not expressed at any stage of preimplantation development or in early postimplantation stage embryos. Genetic mapping studies of glucose transporters in the mouse show that Glut-1 is located on chromosome 4, Glut-2 on chromosome 3, Glut-3 on chromosome 6, and Glut-4 on chromosome 11.     

Effects of tunicamycin upon glycoprotein synthesis and development of early mouse embryos

Tunicamycin, an antimetabolite which inhibits the N-glycosylation of proteins, does not block the initial cleavages of mouse embryos, even at relatively high concentrations. However, it can interfere with compaction and blastocyst formation. Although tunicamycin treatment from the two-cell or eight-cell stage can cause developmental arrest prior to hatching from the zona pellucida, much higher (sublethal) concentrations of the antimetabolite added at the morula or blastocyst stage do not specifically affect hatching of blastocysts, their attachment to the substratum, or outgrowth of trophoblast cells. The consequence of continuous exposure of embryos to moderate amounts (0.05 to 0.1 μg/ml) of tunicamycin through peri-implantation stages is death of trophoblast cells with little effect upon the cells of the inner cell mass (ICM). The latter give rise to apparently normal early endoderm cells in the presence of the antimetabolite. The incorporation of leucine, mannose, and fucose into acid-insoluble material by ICM cells is only minimally inhibited by tunicamycin. On the other hand, the antimetabolite causes a severe inhibition of incorporation of not only mannose, but also leucine, into acid-insoluble material in trophoblast cells. Thus, trophoblast cells resemble transformed cells by their extreme sensitivity to tunicamycin.     

Differential hippo signaling in early mouse embryos

Download : Download video (15MB)Hiroshi Sasaki discusses his group's recent findings on the role of differential Hippo signaling in regulating cell fate during trophectoderm formation.     

Effect of maternal parity on aneuploidy in early mouse embryos.

An attempt to evaluate the incidence of chromosomal aneuploidy in mouse blastocysts recovered from females of various ages and parity levels revealed an insignificant regression coefficient for aneuploidy on age of the female and its square, and an insignificant correlation coefficient for aneuploidy with the number of previous offspring born to the dam. However, significant regression coefficients were obtained for aneuploidy on parity of the dam and its square. These results indicate that not only does aneuploidy increase with parity level, but the rate of increase accelerates as parity level increases. Possible explanations for the increase in aneuploidy and its detrimental effect on reproductive efficiency were discussed.