Uteri Of Foster Mothers Research Articles

Long-term liquid nitrogen vapor storage of mouse embryos cryopreserved using vitrification or slow cooling

To store embryos in LN(2) vapor, which eliminates direct contact with LN(2), is presented as an alternative method to protect embryos cryopreserved by vitrification or slow cooling from LN(2)-borne pathogens. Basic animal research. University-affiliated hospital. Two-cell mouse embryos collected from superovulated ICR mice. Embryos cryopreserved by vitrification or slow cooling were stored in LN(2) or LN(2) vapor and then thawed after 1 week, 1 month, or 6 months. Thawed embryos cultured to the blastocyst stage in vitro were evaluated or transferred into the uterus of foster mothers. Survival, apoptosis, and in vitro and in vivo development of freeze-thawed mouse embryos as a function of cryopreservation and cryostorage methods were determined. After short- and long-term storage of vitrified and slow-cooled embryos, embryonic survival and development rate were the same for embryos stored in LN(2) vapor and in direct contact with LN(2). Cell numbers and apoptosis frequency were similarly indistinguishable between the two groups after storage of embryos for 6 months, and there were no differences in delivery rate or litter size after ET. The present study shows that embryos stored in LN(2) vapor retain full developmental potential. This storage system thus represents a useful alternative for safe and effective long-term storage of embryos that avoids direct contact with LN(2).

Pax6 Regulation in Retinal Cells by CCCTC Binding Factor

A previous study demonstrated that CTCF (CCCTC binding factor) regulates homeobox Pax6 gene expression in early embryonic stages and plays a dominant role in eye development. The purpose of the present study was to explore further the mechanism of CTCF controlling Pax6 gene expression in human retinoblastoma (Rb) cells and in the development of chicken and mouse retinas. Northern and Western analyses were used to detect expressions of CTCF and Pax6 in Rb cells. Pax6 transcription reporter and deletion mutants were used to study the regulatory interaction between CTCF and Pax6 in Rb cells and in the retina of chicken embryos. CTCF transgenic chicken embryos and mice were established by lipofection and microinjection of linearized cytomegalovirus (CMV)-CTCF construct into fertilized eggs and mouse oocytes, respectively. Injected oocytes were implanted in the uterus of foster mothers through microinjection into the ovarian duct. The expression of CTCF and Pax6 was determined in embryo sections by immunochemistry. Stimulation of Rb cells with 10% FBS resulted in an increase in CTCF expression and a decrease in Pax6 expression. To study the regulatory mechanism, the Pax6 reporter and its deletion mutant activities were determined in transfected Rb cells and chicken embryonic retinas, revealing that CTCF interacts with the Pax6 gene in Rb cells through transcription control in the 5'-flanking region upstream from the Pax6 P0 promoter. Overexpression of CTCF in Rb cells suppressed Pax6 reporter activity and downregulated endogenous Pax6 expression. In contrast, downregulation of CTCF expression by knockdown of CTCF mRNA using specific small interfering (si)RNA markedly enhanced Pax6 expression in Rb cells. Further study in CTCF transgenic mouse embryos verified that overexpression of CTCF suppressed Pax6 gene expression in the retina. CTCF plays an important role in regulating Pax6 expression in Rb cells and in the developmental retina, and the regulation of Pax6 gene expression by CTCF in the retina is through transcriptional regulation.

Whole embryo culture and the study of postimplantation mammalian development.

In the past decade, striking advances have been made in the field of gene introducing/disrupting technology including generation of transgenic and knockout mice, which have enabled us to elucidate roles of specific genes in development. In this technology, embryos introduced with exogenous genes or chimeric embryos aggregated/injected with embryonic stem (ES) cells carrying targeted genes are allowed to develop in the uterus of foster mothers. The uterus, however, is like a black box for researchers investigating postimplantation development of mammalian embryos. Embryo culture is one of the powerful techniques that can open this black box. In this review, we focus on the applicable aspects of the whole embryo culture in the study of mammalian development and discuss the future possibilities of this technique.

Cryopreservation of Rat Blastocysts: A Comparative Study of Different Cryoprotectants and Freezing/Thawing Methods

Cryopreservation has proved to be a highly successful method for long-term storage of viable embryos. The objective of this study on rat blastocysts was to define conditions for their cryopreservation. Three cryoprotectants, dimethyl sulfoxide, glycerol, and plopanediol/sucrose, were compared in two cooling programs (to -30 or -80°C) and two thawing protocols. The cooling was followed by immersion in liquid nitrogen. Programmed thawing was at the rate of 8°C per minute; fast thawing consisted of direct exposure of the frozen embryos to the ambient laboratory temperature. The survival after the freeze/thaw was assessed from the post-thaw embryo morphology and ability to develop into apparently normal offspring in uteri of foster mothers (embryonic survival). The best method for preservation of rat blastocysts proved to be programmed cooling to - 80°C followed by fast thawing with glycerol as cryoprotectant (embryonic survival of 28.1%). In all the experimental groups, the proportion of embryos with good to excellent preservation of morphology was high. With dimethyl sulfoxide, after programmed cooling to -80deg;C, embryonic survival was 9.9%'(programmed thawing) and 17.5% (fast thawing). No embryos survived after programmed cooling to -30°C. However, when the cryoprotectant was propanediol/sucrose, no difference was observed between programmed cooling to - 80°C with either method of thawing and programmed cooling to - 30°C and fast thawing (12.3, 6.2, and 8.0%, respectively).

The effect of experimental infection of mouse preimplantation embryos with paramyxovirus Sendai.

Zona-intact and zona-free mouse embryos at the morula stage were exposed to Sendai virus in vitro, and then transferred to the uteri of recipient foster mothers. Both embryos developed into expanded blastocyst stage after 48 hr of culture. Zona-intact embryos were resistant to infection and subsequent transfer resulted in the development of fetuses, indicating that the zona pellucida plays a role of a barrier to virus infection. On the other hand, zona-free embryos were susceptible to infection and only one fetus out of 64 transfers developed to term. Implantation sites were scarcely observed in the uteri of the foster mothers that received zona-free embryos, suggesting that most of the embryos did not develop after embryo transfer. Sendai virus was shed in the culture fluid of the zona-free embryos indicating viral replication in the embryonic cells. By immunofluorescence assay, viral antigens were detected in the embryos, tissues of the fetus and implantation site derived from the zona-free embryos. These findings indicate that replication of Sendai virus in the embryonic cells interfere with early embryonic development and fetal growth of the embryo.

Genetically identical parthenogenetic mouse embryos produced by inhibition of the first meiotic cleavage with cytochalasin D

The microfilament inhibitor cytochalasin D inhibits extrusion of the first polar body when present during the first meiotic division of mouse oocytes; however, it does not interfere with anaphase movement of chromosomes, and thus induces the formation of tetraploid oocytes. After the separation of chromosomes in anaphase, two spindles start to assemble. However, they merge rapidly and a single meiotic spindle forms. During the transition between metaphase I and metaphase II, in the presence of cytochalasin D, a drop in histone kinase activity takes place demonstrating a transitional decrease in the activity of the maturation promoting factor. These oocytes can be activated parthenogenetically a few hours after washing out the inhibitor. After completion of the second meiotic division and extrusion of a polar body, they contain a diploid number of chromosomes. They are genetically identical to each other and to their mother. Such eggs develop to the blastocyst stage and can implant in the uteri of foster mothers. Most of these fetuses die before the 9th day of gestation, as do diploid control fetuses treated with cytochalasin D during the second meiotic division. The heterozygous state of the experimental embryos obtained after activation of eggs recovered from heterozygous females and treated with cytochalasin D during the first meiotic division was confirmed using a glucose-phosphate isomerase assay. This technique allows the production of genetic clones of parthenogenetic embryos by simple means.

Embryonic development of fertilized rat oocytes induced to mature by an analogue of gonadotrophin-releasing hormone

A gonadotrophin-releasing hormone analogue (GnRHa) was administered to hypophysectomized immature rats. Postovulatory mature oocytes obtained under these conditions were exposed in vitro to a sperm suspension for fertilization. Developmental ability of the fertilized ova was studied by transfer of the 2-cell stage embryos to oviducts of foster mothers. The potential of oocytes, undergoing maturation in response to GnRHa, to develop into 2-cell embryos was similar to that of oocytes stimulated by hCG (76.4% and 83.1% respectively). The 2-cell stage embryos obtained from such oocytes were equally able to implant in the uteri of foster mothers (25.7% and 21.2% respectively) and subsequently develop into live embryos (15.3% and 15.2%, respectively, at Day 20 of pregnancy).

Active ras and myc oncogenes can be compatible, but Sv40 large T antigen is specifically suppressed with normal differentiation of mouse embryonic stem cells

The pathobiological effects of oncogenes on normal differentiation of mouse embryonic stem cells from 4-day embryos were examined by introducing active ras, myc, and SV40 large T genes, all driven by mouse metallothionein I enhancer and promoter. Stem cell clones R5, M3, and T2 for ras, myc, and SV40 T genes, respectively, were particularly chosen for analyses because of their higher levels of transgene expression and their diploid chromosomal constitutions. These stem cells were then introduced into host 4-day embryos and the embryos were allowed to develop in the uterus of foster mothers. The stem cells colonized the tissues as extensively as the parent cells and gave rise to adult chimera with no apparent loss or abnormality of the embryos. The active ras and myc oncogenes introduced were expressed not only in the stem cells, but also in the developing embryos and in a variety of tissues of adult chimeras. However, although T antigen was originally expressed in the stem cells, it was not expressed in either developing embryos or tissues of adult chimeras. Induced by retinoic acid treatment in vitro or by subcutaneous grafting, this suppression of T-gene expression was also confirmed in differentiated progeny cells from several stem cell clones expressing T antigen. Permanent lines of fibroblast-like cells could be established at higher frequency from primary cultures of tissues of chimera, subcutaneous differentiated cells, and in vitro differentiated cells derived from T2 cells, and all these clones reexpressed T antigen. The results suggest that active myc and ras genes can be compatible with normal differentiation of the stem cells, but the expression of T antigen is specifically suppressed with recognition of its coding domain.

Role of the mitochondrial genome during early development in mice. Effects of ethidium bromide and chloramphenicol.

The role of the mitochondrial genome in early development and differentiation was studied in mouse embryos cultured in vitro from the two to four cell stage to the blastocyst (about 100 cells). During this period the mitochondria undergo morphological differentiation: progressive enlargement followed by an increase in matrix density, in number of cristae, and in number of mitochondrial ribosomes. Mitochondrial ribosomal and transfer RNA synthesis occurs from the 8 to 16 cell stage on and contributes to the establishment of a mitochondrial protein-synthesizing system. Inhibition of mitochondrial RNA- and protein-synthesis by 0.1 microg/ml of ethidium bromide or 31.2 microg/ml of chloramphenicol permits essentially normal embryo development and cellular differentiation. Mitochondrial morphogenesis is also nearly normal except for the appearance of dilated and vesicular cristae in blastocyst mitochondria. Such blastocysts are capable of normal postimplantation development when transplanted into the uteri of foster mothers. Higher concentrations of these inhibitors have general toxic effects and arrest embryo development. It is concluded that mitochondrial differentiation in the early mouse embryo occurs through the progressive transformation of the preexisting mitochondria and is largely controlled by the nucleocytoplasmic system. Mitochondrial protein synthesis is required for the normal structural organization of the cristae in blastocyst mitochondria. Embryo development and cellular differentiation up to the blastocyst stage are not dependent on mitochondrial genetic activity.