One-cell Mouse Embryos Research Articles

Expression of a mouse metallothionein- Escherichia coli β-galactosidase fusion gene (MT-βgal) in early mouse embryos

We have microinjected DNA containing the inducible mouse metallothionein-I (MT-I) promoter, coupled to the structural gene for Escherichia coli β-galactosidase ( lacZ), into the pronuclei of one-cell mouse embryos. A qualitative histochemical assay, with 5-bromo-4-chloro-3-indolylβ- d-galactopyranoside (X-Gal) as a substrate, was used to detect expression of lacZ at several preimplantation stages. We observed staining indicative of exogenous β-galactosidase activity in 5–17% of DNA-injected embryos assayed at preimplantation stages after 16–24 h treatment with ZnSO 4. Thus, lacZ can be used as an indicator gene for promoter function during early mouse embryogenesis, and the incorporation of the MT-I promoter into fusion genes can be a useful means of controlling the expression of exogenous genes in preimplantation mouse embryos.

Dynamics of chromatin changes in live one-cell mouse embryos: A continuous follow-up by fluorescence microscopy

Conditions of minimal dye concentration and minimal irradiation which allow the continuous observation of pronuclei in live unicellular mouse eggs by fluorescence microscopy have been found with the use of Hoechst 33342 as fluorophore and a camera of high sensitivity coupled with an image processing system allowing true integration of weak fluorescent signals and further treatment and analysis. Under these conditions the developmental potential of the embryos is not affected. Using such an approach, which avoids eventual artifacts due to fixation procedure, we describe the changes in the nuclear organization and chromatin structure, from formation of pronuclei to mitosis, with particular attention to the chromatin associated with nucleoli and the timing process of chromatin condensation.

Quality control in the in vitro fertilization laboratory: comparison of bioassay systems for water quality.

Three mouse embryo bioassays [mouse one-cell and two-cell embryos and in vitro fertilization (IVF)] were tested for their ability to discriminate between three sources of water for medium preparation: tap water, high-performance liquid chromatography (HPLC)-grade water, and Milli-Q purified water. No differences could be detected using these assays. The lack of sensitivity of the mouse bioassays could not be attributed to the protein source or medium type. The hamster sperm motility assay (HSMA) permitted quantitative discrimination between water sources (Milli-Q greater than HPLC greater than tap). Media prepared for use in human IVF using water that exceeded minimal HSMA quality standards resulted in pregnancy rates that were greater than those attained with a lot of HPLC water that did not meet these standards. The HSMA can serve as a basis for a quality-control program in the human IVF laboratory.

Cold culture of different stage mouse embryos in bicarbonated and bicarbonate-free media

Mouse embryos of different stages of development were cultured to expanded blastocysts following storage (1 to 8 d) at 4°C in the presence or absence of HCO 3 −. The effect of oxygen tension on the cold storage of one- and two-cell mouse embryos at 4°C was evaluated by 37°C culture and transfer to pseudopregnant recipients. Survival at 4°C of early, one- to four-cell mouse embryos was improved with HCO 3 − in the medium. The presence of HCO 3 − was not of benefit for morulae or blastocyst survival following cold storage. Reducing the oxygen atmosphere from 20 to 5% O 2 improved survival of one-cell mouse embryos stored at 4°C. The survival of two- and four-cell embryos, morulae and blastocysts at 4°C was similar in 90% N 2, 5% CO 2 and 5% CO 2 in air, but it was significantly poorer in air alone. The collapse of morulae and blastocysts during cold storage up to 5 d was reduced with HCO 3 − in the storage medium. Blastocysts stored for 6 d at 4°C failed to survive following immediate transfer to pseudopregnant recipients. Blastocyst survival was improved compared to controls (direct transfer of unstored blastocysts to recipients) when cultured for 36 h at 37°C following 6 d of cold storage. This result suggests that cold-stored mouse blastocysts may require a metabolic period of readjustment to survive following transfer to synchronized recipients.

The temporal effects of changes in in vitro fertilization culture media on the one-cell mouse embryo system.

The one-cell mouse embryo system has previously been shown to be more sensitive than the two-cell system to mild changes in in vitro fertilization (IVF) culture media. To determine whether this greater sensitivity is related to the developmental stage or to the length of exposure, one-cell embryos were collected and cultured in control media (Ham's F-10, 282 mOsm/liter), in media of altered osmolality (260, 300, and 316 mOsm/liter), or in media containing Cidex diluted 1:100,000. The one-cell embryos were exposed to control or altered media in four patterns: control group--control medium for 96 hr; Group A--altered medium for the first 24 hr followed by control medium for 72 hr; Group B--control medium for the first 24 hr followed by altered medium for 72 hr; and Group C--control medium for the first 24 hr, altered medium for the next 24 hr, and control medium again for 48 hr. The percentage of embryos developing to blastocysts in Group A (exposed to adverse conditions only for the first 24 hr of culture) was significantly lower than in the control group under all conditions studied. In contrast, the percentage of blastocysts developing in Group B was significantly lower than in the control group only in medium of 315 mOsm/liter and was not different from that in controls under the other conditions studied. There was no difference between Group C and the control group. We conclude that the higher sensitivity of the one-cell system is an inherent property of the one-cell stage, as exposure of the embryo during this critical first 24-hr period proved to have the most profound consequences.

The One-cell Mouse Embryo: Cell Cycle-dependent Radiosensitivity and Development of Chromosomal Anomalies in Postradiation Cell Cycles

One-cell mouse embryos were irradiated with X-rays at different cell cycle stages. Examination of structural chromosomal anomalies and of micronucleus formation in postradiation mitoses and interphases demonstrated cell cycle-dependent radiosensitivities in the order: late G2 phase greater than G1 phase greater than S phase greater than early G2 phase greater than stage of decondensing nuclei. Comparison of the quality and quantity of chromosomal aberrations from the first to the third mitosis led to the conclusion that new chromosomal anomalies were formed in the course of postirradiation cell cycles. This hypothesis was supported by an increasing number of micronuclei from 24 to 48 h post-conception. In addition to structural chromosomal aberrations, radiation-induced chromosome loss was observed with a frequency that was obviously independent of the exposed cell cycle phase. Loss of acentric chromosome fragments and of single chromosomes contributed to the micronucleus formation.

Analysis of structural and numerical chromosomal anomalies at the first, second, and third mitosis after irradiation of one-cell mouse embryos with X-rays or neutrons.

One-cell mouse embryos were irradiated with X-rays or neutrons. Analysis of the first, second, and third postradiation mitoses revealed that the yields of structural aberrations increased linearly after exposure to both radiation qualities. For X-rays the aberration frequency decreased from the first to the third mitosis, whereas after neutrons it decreased from the first to the second mitosis but then increased in the third mitosis. RBEs of 4.7, 4.8, and 7.4 were calculated for the corresponding mitoses. It was clearly demonstrated that new aberrations were produced after the first postradiation mitosis and expressed during the second and third mitosis. Chromosome loss also increased with increasing radiation dose at the second mitosis. An RBE of 2 was calculated for this effect. Comparing the presented data with previous investigations on embryonic and fetal death after prenatal irradiation, it was concluded that the high radiosensitivity of the one-cell embryo is due to the induction of structural as well as of numerical chromosome aberrations.

Lower sodium lactate in Whitten's medium improves in vitro developmental capacity of one-cell mouse embryos

A modification of Whitten's medium, involving a reduced content of Na-lactate syrup (0.2 ml/100 ml; 11.65 mM) and osmolarity (251 mOsm), was compared with normal Whitten's medium (0.37 ml/100 ml; 21.6 mM) for ability to support mouse embryonic development in vitro from one-cell to the blastocyst stage. In a pilot study utilizing 10 ICR donor female mice, in vitro developmental capacity (IVDC; percentage of fertilized one-cell embryos developing to blastocysts in vitro per female donor) was significantly enhanced by the modified medium (68.0 versus 24.0%; P<0.001). In the main study, utilizing 134 ICR and 17 ICR x C57BL/6J F 1 donor females, the modified medium supported increased IVDC for both ICR (67.9 versus 51.1%; P<0.001) and F 1 females (98.5 versus 89.4%; P<0.05). A large degree of among donor-female variation in IVDC was observed for both media in the ICR stock (SD = 30.0). The beneficial role of the reduction of Na-lactate in Whitten's medium may be related to an improved provision of energy requirements for first cleavage and/or a more suitable osmolarity for development.

Differential effects of activators of cAMP-dependent protein kinase and protein kinase C on cleavage of one-cell mouse embryos and protein synthesis and phosphorylation in one- and two-cell embryos

Membrane-permeable cAMP analogs or elevation of intracellular cAMP by cyclic nucleotide phosphodiesterase (PDE) inhibitors activates cAMP-dependent protein kinase. Biologically active phorbol esters or diacylglycerol activate the calcium-, phospholipid-dependent protein kinase, protein kinase C (PK-C). We report that membrane-permeable cAMP analogs, PDE inhibitors, biologically active phorbol esters, or a synthetic diacylglycerol inhibited cleavage of 1-cell mouse embryos to the 2-cell stage. The cAMP analogs and PDE inhibitors were effective only when added prior to S of the first cell cycle, whereas PK-C activators inhibited cleavage when added up until late G2 M . The PDE inhibitor Ro 20 1724/1 inhibited both DNA and protein synthesis in 1-cell embryos, whereas the phorbol ester, 12- O-tetradecanoylphorbol-13 acetate, or α-amanitin did not. In addition, 1-cell embryos prevented from cleaving by PDE inhibitors did not show specific changes in the pattern of protein phosphorylation associated with the 2-cell embryo, whereas such changes occurred in 1-cell embryos inhibited from cleaving with PK-C activators. Transcription in the 2-cell embryo results in the synthesis of a specific set of proteins, which is inhibited by α-amanitin. Although treatment of 1-cell embryos with aphidicolin or PK-C activators during G1 did not inhibit the synthesis of these proteins, treatment with cAMP analogs or PDE inhibitors during G1 inhibited the appearance of these proteins. These results are discussed in terms of how the synthesis of transcription-dependent proteins in the 2-cell embryo may be regulated by protein phosphorylation.

Production of transgenic mice following deoxyribonucleic acid microinjection and embryo freezing

Experiments with mouse embryos were designed to assess the feasibility of freezing embryos after DNA microinjection. One-cell pronuclear stage mouse embryos were microinjected with cloned deoxyribonucleic acid (DNA) and cultured in vitro to the late eight-cell stage. Microinjected and matched control embryos were frozen and stored in liquid nitrogen. Following thawing, embryos were cultured for 8 h and transferred to recipient females. In a separate set of experiments, embryos were transferred to recipients immediately following DNA microinjection. Control (uninjected) embryos developed to the late eight-cell stage significantly better than surviving microinjected embryos. Of the embryos thawed, 76% of the microinjected and 60% of the control embryos survived to be transferred to recipients. Progeny were obtained with similar survival rates from both groups following embryo transfer with transgenic mice identified among the progeny from microinjected embryos. Mouse embryos can be microinjected with DNA, cultured in vitro, frozen, thawed, transferred to recipients and transgenic progeny can be obtained.

The effect of inhibiting DNA replication in the one-cell mouse embryo.

Inhibition of DNA replication in the 1-cell mouse embryo causes arrest in interphase. In such arrested cells the pattern of protein synthesis progresses as normal through the first cell cycle but does not show the characteristic changes that are associated with mitosis. Synthesis does occur of the 68/70 kd heat shock-like proteins, on the earliest detectable embryonic transcripts synthesised shortly after first cleavage in control embryos. However, the quantitative change in the pattern of protein synthesis, indicative of the major activation of the embryonic genome that normally occurs during G2 of the second cell cycle, is prevented.

An improved culture medium for one-cell mouse embryos: [formula omitted] and [formula omitted]. [formula omitted]. Lab of Radiobiol. and Environ. Hlth. and Dept. of Ob/Gyn., Univ. of Calif., San Francisco, CA., 94143, U.S.A.

An improved culture medium for one-cell mouse embryos: [formula omitted] and [formula omitted]. [formula omitted]. Lab of Radiobiol. and Environ. Hlth. and Dept. of Ob/Gyn., Univ. of Calif., San Francisco, CA., 94143, U.S.A.

Inability of mouse blastomere nuclei transferred to enucleated zygotes to support development in vitro.

More than 90 percent of enucleated one-cell mouse embryos receiving pronuclei from other one-cell embryos successfully develop to the blastocyst stage in vitro. In this investigation, nuclei from successive preimplantation cleavage stages were introduced into enucleated one-cell embryos and the embryos were tested for development in vitro. Although two-cell nuclei supported development to the morula or blastocyst stage, four-cell, eight-cell, and inner cell mass cell nuclei did not. The inability of cell nuclei from these stages to support development reflects rapid loss of totipotency of the transferred nucleus and is not the result of simultaneous transfer of membrane or cytoplasm.

Spontaneous heteroploidy in one-cell mouse embryos.

First cleavage divisions were analyzed, after chromosome banding, in 321 zygotes recovered from superovulated, outbred ICR mice 33-35 h after injection of human chorionic gonadotropin. With 72.1% of all zygotes at metaphase and late prophase analyzed, there was 87% diploidy, 3.6% trisomy, 2.5% monosomy, 0.4% structural rearrangement, and 0.93% triploidy. Aneuploid zygotes in which there were 37 or 38 chromosomes accounted for 6.5% of the population with both haploid complements analyzable. In 45 diploid zygotes in which only one complement was analyzable, there was 93.4% haploidy, 2.2% disomy, and 4.4% hypoploidy. The chromosome most often involved in aneuploidy was the 19. The parental origin of the chromosome anomalies was determined on the basis of differential condensation of the chromosomes. The source of trisomy was shown to be the male in three and the female in five of the eight cases determined. This indicates a paternal contribution to trisomy and a female nondisjunction frequency of 1.5% (five of 321). For monosomy where the origin was identified in five of the cases, the male complement was implicated in three zygotes. The single break was seen in a sperm-derived chromosome. Two of the three triploids were dispermic and one was digynic. The outbred Swiss mouse might be a useful model for studying factors responsible for the induction of cytogenetic anomalies in early development of mammals.

RNA synthesis and cytoplasmic polyadenylation in the one-cell mouse embryo.

The time of onset of transcriptional activity in the early mammalian embryo is unclear. RNA synthesis has been shown to occur at the two-cell stage in the mouse1–4 and at the two- to four-cell stage in the rabbit5, but the period immediately following fertilization has proved largely inaccessible to biochemical characterization of transcription because of the low permeability of early embryos to exogenous precursors such as 3H-uridine1–7. This difficulty, and the failure to detect RNA polymerase activity in the pronuclei of one-cell mouse embryos8, suggested that the embryonic genome was transcriptionally inactive until some time after the first cleavage. To investigate this issue further, we have now incubated one-cell pronuclear embryos with 3H-adenosine, which is taken up about 1,000 times faster than uridine3. Three labelled RNA species could be identified. The major product is large, heterodisperse, poly(A)− RNA. High-molecular-weight poly(A)+ RNA is also heavily labelled but this is mostly due to cytoplasmic polyadenylation of previously non-polyadenylated, stored RNA. A significant amount of label is incorporated into the -CCA termini of transfer RNA but some new synthesis of tRNA also takes place. No label was found in ribosomal RNA at the one-cell stage but synthesis of mature rRNA species was evident in the two-cell embryo.

The transient appearance of specific proteins in one-cell mouse embryos

Using the technique of two-dimensional gel electrophoresis, a detailed study of the protein synthetic patterns of [ 35S]methionine-labeled preimplantation mouse embryos was undertaken. Autoradiographs of gels from the unfertilized egg to the 8- to 16-cell stage were examined. Of special interest was the finding that, during the first day after fertilization, the pattern of newly synthesized protein changed dramatically, with some proteins appearing and disappearing within a matter of a few hours. The significance of these proteins is discussed, particularly with regard to their possible use as a tool for studying the role of maternal mRNA in early development.

Program of early development in the mammal: Changes in patterns and absolute rates of tubulin and total protein synthesis during oogenesis and early embryogenesis in the mouse

The absolute rates of total protein synthesis and tubulin synthesis during oogenesis and early embryogenesis in the mouse have been determined by measuring specific activities of the endogenous methionine pool and rates of incorporation of [ 35S]methionine into total protein and tubulin. The absolute rate of protein synthesis decreases from 43 to 33 pg/hr/oocyte during meiotic maturation, while the size of the endogenous methionine pool remains essentially unchanged at 65 fmole/oocyte ( R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978, Proc. Nat. Acad. Sci. USA, 75, 4160 ). The one-cell mouse embryo synthesizes protein at a rate of 45 pg/hr/embryo, so that fertilization is accompanied by about a 40% increase in the absolute rate of total protein synthesis. The eight-cell compacted embryo synthesizes protein at the rate of 51 pg/hr/embryo. The size of the endogenous methionine pool increases dramatically during early embryogenesis, from 74 fmole in the unfertilized ovum to 137 and 222 fmole in the one-cell embryo and eight-cell compacted embryo, respectively. Tubulin is one of the major proteins synthesized by the mouse oocyte and embryo since the absolute rate of tubulin synthesis is, on the average, 1.3% that of total protein synthesis. The absolute rate of tubulin synthesis decreases from 0.61 to 0.36 pg/hr/oocyte during meiotic maturation and then increases to 0.60 pg/hr/embryo in the one-cell embryo and to 0.66 pg/hr/embryo in the eight-cell compacted embryo. During meiotic maturation and early embryogenesis the direction and magnitude of changes in the rate of tubulin synthesis closely parallel those of total protein synthesis. Although equimolar amounts of tubulin subunits are present in microtubules, the ratio of the absolute rate of synthesis of the β subunit to that of the α subunit is about 2.0 throughout meiotic maturation and early embryogenesis. High-resolution two-dimensional gel electrophoretic analysis of [ 35S]methionine-labeled proteins reveals that many of the newly synthesized proteins that first appear during meiotic maturation of the oocyte continue to be synthesized in the one-cell embryo. Nearly all of the proteins synthesized in the one-cell embryo are also synthesized in the unfertilized ovum, although some changes in the pattern of protein synthesis are associated with fertilization. Therefore, the developmental program for early embryogenesis in the mouse appears to be activated during meiotic maturation of the oocyte. These results are compared with those obtained using oocytes and embryos from nonmammalian animal species.

Uridine and guanosine incorporation by the mouse one-cell embryo

ABSTRACT The activity of the embryonic genome prior to the first cleavage has been assessed by studying the uptake of [3H]uridine, its phosphorylation and incorporation into RNA by mouse one-cell embryos. One-cell embryos incorporated [3H]uridine linearly into cold trichloracetic acid (TCA) insoluble material at a low level 1-9 h post fertilization. The incorporation of [3H]guanosine was also low but followed a biphasic curve which had a steeper slope at 1-3 h than during the period 4-9 h post fertilization. Unfertilized mouse ova incorporated very little [3H]uridine or [3H]guanosine into TCA insoluble material, and much of this was RNase insensitive. Dimethyl sulfoxide (DMSO) enhanced the uptake of [3H]thymidine and its incorporation into pronuclear DNA by one-cell embryos, but had no effect on the incorporation of [3H]uridine by them, or of [3H]uridine and [3H]guanosine by unfertilized ova. The uptake and incorporation of [3H]guanosine by one-cell embryos were enhanced by DSMO, but only during the period 1-3 h post fertilization. Sugar derivatives of UDP, and UMP, UDP, UTP, CMP, CDP and CTP have been identified in the soluble fraction obtained from mouse one-cell embryos incubated with [3H]uridine 1-3 h post fertilization. Very little of the [3H]uridine taken up by the embryos is present as [3H]UTP, or [3H]CTP; most is found as [3H]UMP or [3H]UDP or as the sugar derivatives. Alkaline or ribonuclease (A, T1 and T2)hydrolysis of the 3H-labeled ethanol insoluble material precipitated from the lysate of one-cell embryos incubated with [3H]uridine 1-3 h post fertilization liberated radioactive cytidine and uridine-3’-phosphates. This demon strates that [3H]uridine is incorporated into an internal position in RNA and suggests that RNA synthesis does occur in the one-cell embryo 1-3 h post fertilization. Since pronuclei of one-cell embryos incubated with [3H]uridine were not labeled it appears, however, that the RNA synthesized at the one-cell stage is not a product of the embryonic genome.

Expression of lactate dehydrogenase isozyme 5 (LDH-5) in cultured mouse blastocysts in the absence of implantation and outgrowth.

Extensive extraction studies with Triton X-100 revealed only LDH-1 (B4) but no trace of LDH-5 (A4) in one-cell and two-cell mouse and rat embryos. The LDH isozyme pattern of preimplantation mouse embryos changes from the maternally inherited B subunit isozyme (LDH-1) to a pattern dominated by LDH-5 when mouse blastocysts are cultured under conditions that prevent hatching but allow trophoblast giant cell transformation. During differentiation of mouse blastocysts in vitro, implantation is therefore not essential for the appearance of the A subunit form of LDH (LDH-5) coded for by the embryonic genome. Mechanisms controlling the expression of LDH-5 in mouse blastocysts during in vivo development are discussed.

The beneficial effect of EDTA on development of mouse one-cell embryos in chemically defined medium

An improved methology for culturing noninbred (ICR) mouse one-cell embryos is described. The successful development of one-cell embryos into blastocysts in chemically defined (Whitten's) medium was significantly enhanced by the presence of EDTA. More than 70% of ICR one-cell embryos developed into blastocysts in Whitten's medium in the presence of 10.8 μ M EDTA, while, without EDTA, only 15–30% of embryos reached blastocyst stage. A concentration of 10.8 μ M EDTA also promoted the development of 65–90% of inbred C57BL 6 one-cell embryos in Whitten's medium. This beneficial role of EDTA is probably related to the chelation of some metal ion(s) other than Ca 2+ or Mg 2+.