Participation of the paternal genome is not required before the eight-cell stage for full-term development of mouse embryos

Differential expression of the paternal and maternal genomes during mouse embryonic development is considered a reason for both genomes being required for development to term. Extending previous studies performed on two-cell embryos, we show here that diploid embryos reconstituted at the four-cell stage from uniparental haploid blastomeres can produce living offspring. This result shows that for normal development to occur, a paternal genome does not need to be associated with a maternal genome within the same nucleus before the eight-cell stage.     

Perturbations in mouse embryo development and viability caused by ammonium are more severe after exposure at the cleavage stages

The presence of ammonium in culture medium has a detrimental effect on embryo physiology and biochemistry; however, the stage at which the embryo is most sensitive to this effect is unknown. The aim of this study was to determine the exact stage at which the embryo is most vulnerable to ammonium by exposing the preimplantation embryo to 300 muM ammonium either at the precompaction stage (between the zygote and two-cell or the two-cell to eight-cell) or at the postcompaction stage (between the eight-cell and blastocyst). This study determined that exposure of embryos to ammonium at the precompaction stage from either the zygote to two-cell stage or from the two-cell to the eight-cell stage did not affect the rate of development to the blastocyst stage; however, the resultant blastocysts had decreased cell numbers and inner cell mass cells. Furthermore, these blastocysts had increased levels of cellular apoptosis and perturbed levels of Slc2a3 expression and glucose uptake. Transfer of these blastocysts revealed that, while implantation was not affected, the number of fetuses was reduced by culture with ammonium at the precompaction stage and fetal development was delayed, as observed by reduced crown-rump length and maturity. In contrast, the later stage embryo was more resistant to the negative effects of ammonium, with only Slc2a3 expression and fetal maturity affected. This raises the possibility that the later stage embryo is more able to protect itself from in vitro-derived stress and that the majority of in vitro-induced damage to mouse embryos is inflicted at the early stages of development.     

Inhibition of endoplasmic reticulum stress improves mouse embryo development

X-box binding protein-1 (XBP-1) is an important regulator of a subset of genes during endoplasmic reticulum (ER) stress. In the current study, we analyzed endogenous XBP-1 expression and localization, with a view to determining the effects of ER stress on the developmental competency of preimplantation embryos in mice. Fluorescence staining revealed that functional XBP-1 is localized on mature oocyte spindles and abundant in the nucleus at the germinal vesicle (GV) stage. However, in preimplantation embryos, XBP-1 was solely detected in the cytoplasm at the one-cell stage. The density of XBP-1 was higher in the nucleus than the cytoplasm at the two-cell, four-cell, eight-cell, morula, and blastocyst stages. Furthermore, RT-PCR analysis confirmed active XBP-1 mRNA splicing at all preimplantation embryo stages, except the one-cell stage. Tunicamycin (TM), an ER stress inducer used as a positive control, promoted an increase in the density of nuclear XBP-1 at the one-cell and two-cell stages. Similarly, culture medium supplemented with 25 mM sorbitol displayed a remarkable increase active XBP-1 expression in the nuclei of 1-cell and 2-cell embryos. Conversely, high concentrations of TM or sorbitol led to reduced nuclear XBP-1 density and significant ER stress-induced apoptosis. Tauroursodeoxycholic acid (TUDCA), a known inhibitor of ER stress, improved the rate of two-cell embryo development to blastocysts by attenuating the expression of active XBP-1 protein in the nucleus at the two-cell stage. Our data collectively suggest that endogenous XBP-1 plays a role in normal preimplantation embryonic development. Moreover, XBP-1 splicing is activated to generate a functional form in mouse preimplantation embryos during culture stress. TUDCA inhibits hyperosmolar-induced ER stress as well as ER stress-induced apoptosis during mouse preimplantation embryo development.     

Paternal cyclophosphamide treatment causes postimplantation loss via inner cell mass-specific cell death.

Treatment of the father with the anticancer alkylating agent cyclophosphamide has negative effects on embryonic development in the rat. Four-week treatment of male rats with a low dose of cyclophosphamide causes a dramatic, dose-dependent increase in postimplantation death of the progeny. Several recent studies have indicated that the paternal genome is required for the development of the extraembryonic tissues. Thus, the purpose of this study was to determine which tissues of the implanting embryo were affected by paternal exposure to cyclophosphamide. Male Sprague-Dawley rats were given cyclophosphamide (6 mg/kg/day) or saline by gavage and bred to untreated female rats after 4 weeks of treatment. Pregnant female rats were killed on day 7 of gestation, and implantation sites were dissected from the uterus, fixed, embedded in Epon for semithin serial sectioning, and stained for subsequent light microscopy. Strikingly, many of the implantation sites of affected embryos sired by treated males displayed an apparently normal trophectoderm enclosing a region of dying cells, containing dark-stained pyknotic nuclei. Very few or no inner cell mass-derived embryonic cells were present in these implantation sites. Therefore, there is a selective death of inner cell mass-derived cells in day 7 implantation sites obtained from the progeny of cyclophosphamide-treated males. The results of this study suggest that treatment of the male with cyclophosphamide can affect paternal genes specifically required for development of the inner cell mass cells of the embryo, without an apparent effect on those genes required for normal trophectoderm.     

Expression of beta-galactosidase in preimplantation ovine and porcine embryos

Knowledge regarding the timing of embryonic expression of the mammalian genome is of relevance for the development of preimplantation diagnostic methods for human genetic diseases. For development of preimplantation diagnosis of lysosomal storage diseases, it will be necessary to know at which embryonic stage the genes for lysosomal enzymes are expressed. In previous studies by other investigators, it has been shown that lysosomal alpha- and beta-galactosidase and beta-glucuronidase in murine embryos increase 50- to 100-fold in activity between the two-cell and late blastocyst stage. We describe here expression of lysosomal beta-galactosidase in preimplantation ovine (two-cell through midblastocyst) and porcine (two-cell through late blastocyst) embryos. Expression of beta-galactosidase in ovine and porcine preimplantation embryos followed a similar rate of increase as that described for murine embryos. Activity of beta-galactosidase increased over 10-fold between the two- to four-cell and midblastocyst stages in ovine embryos, and 300-fold between the two- to four-cell and late blastocyst stages in porcine embryos. Activity expressed on a per cell basis was relatively constant in ovine embryos, as has been described in murine embryos, and increased approximately 5-fold on a per cell basis in porcine embryos. Activity of beta-galactosidase in ovine and porcine embryos initially was greater than 12-fold on a per cell or per embryo basis than in murine embryos evaluated. The knowledge of beta-galactosidase embryonic expression may provide the basis for preimplantation diagnosis of genetic beta-galactosidase deficiency in these species.     

小鼠植入前胚胎GCN5和HDAC1表达模式及体外培养对其表达的影响

为探讨小鼠植入前胚胎组蛋白乙酰化酶GCN5(general control of nucleotide synthesis,GCN5) 和组蛋白去乙酰化酶1(histone deacetyluse1,HDAC1)的表达模式及常规体外培养对它们表达的影响,应用荧光免疫细胞化学技术,检测了体内和体外培养的小鼠2、4、8细胞期卵裂胚胎、桑葚胚和囊胚GCN5和HDAC1的表达。结果显示,GCN5在体内组各细胞期卵裂胚胎和桑葚胚的细胞浆内均呈高表达,细胞核内未见明显表达,而囊胚细胞的细胞浆和细胞核内均无表达:HDAC1在体内组小鼠2细胞期胚胎中以细胞浆内表达为主,在其他各期胚胎均以细胞核内表达为主．囊胚期内细胞团部分细胞的细胞核内未见HDAC1表达。GCN5在体外组小鼠植入前各期胚胎均不表达,而 HDAC1的表达强度明显低于体内组的。提示体外培养抑制小鼠植入前胚胎GCN5和明显降低 HDAC1的表达,影响胚胎基因的正确性表达。     

小鼠植入前胚胎GCN5和HDAC1表达模式及体外培养对其表达的影响

为探讨小鼠植入前胚胎组蛋白乙酰化酶GCN5（general control of nucleotide synthesis,GCN5）和组蛋白去乙酰化酶1（histone deacetylasel,HDAC1）的表达模式及常规体外培养对它们表达的影响,应用荧光免疫细胞化学技术,检测了体内和体外培养的小鼠2、4、8细胞期卵裂胚胎、桑葚胚和囊胚GCN5和HDAC1的表达。结果显示,GCN5在体内组各细胞期卵裂胚胎和桑葚胚的细胞浆内均呈高表达,细胞核内未见明显表达,而囊胚细胞的细胞浆和细胞核内均无表达：HDACl在体内组小鼠2细胞期胚胎中以细胞浆内表达为主,在其他各期胚胎均以细胞核内表达为主。囊胚期内细胞团部分细胞的细胞核内未见HDAC1表达。GCN5在体外组小鼠植入前各期胚胎均不表达。而HDAC1的表达强度明显低于体内组的。提示体外培养抑制小鼠植入前胚胎GCN5和明显降低HDAC1的表达,影响胚胎基因的正确性表达。     

The infection of mouse preimplantation embryos to Sendai virus (parainfluenza I)

To provide information on the susceptibility of mouse embryos to Sendai virus, it was investigated if viral replication occurs in the preimplantation embryo at different stages of development, with or without the zona pellucida (ZP). Mice were induced to superovulate, and embryos were collected on Days 2, 3 and 4 after mating. The ZP was removed by digestion with 0.5% pronase. Embryos were exposed to Sendai virus, washed, and allowed to develop in fresh culture medium. The presence of viral antigen in the embryonic cells was examined by the fluorescent antibody test (FAT). Specific immunofluorescence was demonstrated in the ZP-free morula and ZP-intact blastocyst. However, viral antigen was not detected in the ZP-intact two-cell, four-cell, eight-cell or morula stage embryos. Infected embryos developed normally to expanded blastocysts. These findings show that mouse embryonic cells are permissive hosts to Sendai virus replication and that the ZP played the role of a barrier against the virus.     

Requirement for ERK MAP kinase in mouse preimplantation development

Preimplantation development is a crucial step for successful implantation and pregnancy. Although both compaction and blastocyst formation have been extensively studied, mechanisms regulating the early cell division stages before compaction have remained unclear. Here, we show that extracellular signal regulated kinase (ERK) mitogen-activated protein (MAP) kinase function is required for early embryonic cell division before compaction. Our analysis demonstrates that inhibition of ERK activation in late two-cell-stage embryos leads to a reversible arrest in the G2 phase at the four-cell stage. The G2-arrested four-cell-stage embryos showed weakened cell-cell adhesion as compared with control embryos. Remarkably, microarray analyses showed that most of the programmed changes of upregulated and downregulated gene expression during the four- to eight-cell stages proceeded normally in four-cell-stage-arrested embryos that were subsequently released to resume development; however, the expression profiles of a proportion of genes in these embryos closely paralleled the stages of embryonic rather than normal development. These parallel genes included the genes encoding intercellular adhesion molecules, whose expression appeared to be positively regulated by the ERK pathway. We also show that, whereas ERK inactivation in eight-cell-stage embryos did not lead to cell division arrest, it did cause this arrest when cadherin-mediated cell-cell adhesion was disrupted. These results demonstrate an essential role of ERK function in two-cell to eight-cell-stage embryos, and suggest a loose parallelism between the gene expression programs and the developmental stages before compaction.     

Stage-specific response of preimplantation mouse embryos to W-7, a calmodulin antagonist

Involvement of calmodulin-dependent processes in preimplantation development of mouse embryos was studied with the use of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a specific antagonist of calmodulin. At 25 microM, W-7 interfered with compaction of eight-cell embryos, caused decompaction of compacted eight-cell embryos, inhibited cavitation of late morulae, and caused collapse and degeneration of blastocysts. These effects of W-7 appear to be due to specific inhibition of calmodulin-dependent processes, because W-5, a less active analogue of W-7, was less effective in interfering with development; at 25 microM, W-5 had only a slight effect on compaction and had no effect on blastocyst formation, maintenance of blastocoels, or post-blastocyst development. In addition to the developmental effects just described, W-7 inhibited cell proliferation in four-cell embryos and reduced cell numbers of morulae after treatment at the two- to eight-cell stages. There was a marked increase in embryos' sensitivity to W-7 at the late morula stage, and the sensitivity increased further as embryos developed into blastocysts; the effects of W-7 were largely reversible after treatment at the two-cell through the compacted eight-cell stages, but not after treatment at the late morula or blastocyst stage. At the blastocyst stage, inner cell mass cells appeared to be slightly more resistant to W-7 than trophectoderm cells. This differential sensitivity became more pronounced at the late blastocyst stage: after 3.5-4-h exposure of late blastocysts to 25 microM W-7, all trophectoderm cells degenerated but most of the inner cell masses survived. From these results it appears that calmodulin-dependent processes are involved in development of mouse embryos at all of the preimplantation stages examined.     

Experimental manipulations of compaction and their effects on the phosphorylation of uvomorulin

Compaction of the eight-cell stage mouse embryo is a critical event in the generation of different cell types within the preimplantation embryo. Uvomorulin, a member of the cadherin family of cell adhesion molecules, is important during compaction and its phosphorylation increases early in the eight-cell stage, suggesting that this posttranslational modification may be important for compaction to proceed. We have assessed the importance of the phosphorylation of uvomorulin during compaction by preventing, reversing, or inducing adhesion prematurely. The only condition that affected the overall level of uvomorulin phosphorylation was the prevention of compaction through prolonged exposure of four-cell embryos to low Ca²⁻. This treatment reduced the level of uvomorulin phosphorylation in eight-cell embryos, and perturbed its localization to regions of cell-cell contact. Thus, whilst the phosphorylation of uvomorulin does not appear to regulate directly uvomorulin's adhesive function, it may be associated with the redistribution of uvomorulin during compaction. © 1996 Wiley-Liss, Inc.     

Paternal cyclophosphamide exposure induces the formation of functional micronuclei during the first zygotic division

Paternal exposures to cancer chemotherapeutics or environmental chemicals may have adverse effects on progeny outcome that are manifested in the preimplantation embryo. The objectives of this study were to determine the impact of paternal exposure to cyclophosphamide, an anticancer alkylating agent, on the formation, chromatin origin and function of micronuclei in cleavage stage rat embryos. Male Sprague-Dawley rats were gavaged with saline or cyclophosphamide (6 mg/kg/day) for 4 weeks and mated to naturally cycling females to collect pronuclear zygotes and 2 to 8 cell embryos. Micronuclear chromatin structure was characterized using confocal microscopy to detect immunoreactivities for H3K9me3, a marker for maternal chromatin, and lamin B, a nuclear membrane marker. DNA synthesis was monitored using EdU (5-ethynyl-2'-deoxyuridine) incorporation. Fertilization by cyclophosphamide-exposed spermatozoa led to a dramatic elevation in micronuclei in cleavage stage embryos (control embryos: 1% to 5%; embryos sired by treated males: 70%). The formation of micronuclei occurred during the first zygotic division and was associated with a subsequent developmental delay. The absence of H3K9me3 indicated that these micronuclei were of paternal origin. The micronuclei had incomplete peri-nuclear and peri-nucleolar lamin B1 membrane formation but incorporated EdU into DNA to the same extent as the main nucleus. The formation of micronuclei in response to the presence of a damaged paternal genome may play a role in increasing the rate of embryo loss that is associated with the use of assisted reproductive technologies, parenthood among cancer survivors, and paternal aging.     

In vitro exposure of preimplantation mouse embryos to cocaine and benzoylecgonine inhibits subsequent development.

Cocaine is used by over 20% of women of reproductive age. Although there have been numerous studies focusing on its effects on reproductive processes, none has evaluated its direct effect on preimplantation development. We have investigated the effect of cocaine and its major metabolite, benzoylecgonine, on in vitro preimplantation mouse embryogenesis. One-cell embryos were exposed at the one-, two-, four-, or eight-cell stage for 24 hr to medium containing 0-400 micrograms/ml cocaine or benzoylecgonine and then cultured to the blastocyst stage. Cocaine had its strongest inhibitory effect at the earliest stages of development. At the one- and two-cell stages, there was a significant inhibition of blastocyst formation following exposure to cocaine concentrations of 25-400 micrograms/ml, and at the four-cell stage there was an inhibitory effect at 100 and 400 micrograms/ml cocaine. Benzoylecgonine inhibited the development of embryos to blastocyst only at the one- and two-cell stages, at concentrations of 100-400 micrograms/ml. These findings suggest that cocaine is capable of blocking preimplantation embryogenesis, particularly following exposure at the earliest stages, and that this toxicity may abate as cocaine is biotransformed to benzoylecgonine.     

Relationships between the completion of first cleavage and the chromosomal complement,sex, and developmental rates of bovine embryos generated in vitro

One thousand eighty-four two-cell bovine embryos produced from 1,574 oocytes matured and fertilized in vitro were cultured as groups separated according to the time when they completed their first cleavage (24,30,40,48, or 62 hr postinsemination; hpi). At 5 days after insemination, the proportions of each group that had progressed to the eight-cell stage or beyond were determined and the 350 that had done so were fixed and examined cytogenetically for cell number, chromosomal abnormalities, and sex. Embryos in the “early” cleaving (24 and 30 hpi) and “late” cleaving (40–62 hpi) groups were compared. Early cleaving embryos were more likely to have developed to the eight-cell stage or beyond (52.2% vs. 20%), contained more cells (22 vs. 17), and were more likely to be male (3.6:1 vs. 0.93:1). It is suggested that these phenotypic differences between the sexes begin before the embryonic genome is generally thought to become activated and are due either to differential processing of X- and Y-bearing sperm within the zygote or to very early differential expression of genes derived from X- and Y-bearing sperm. © 1993 Wiley-Liss, Inc.