蛋白激酶Cα在小鼠孤雌及四倍体胚胎早期发育过程中的分布和作用

为了观察蛋白激酶Cα(protein kinase Cα,PKCα在昆明白小鼠受精卵、孤雌激活和四倍体胚胎早期发育阶段的亚细胞定位和致密化进程中的表达变化,本实验利用免疫荧光化学染色与激光共聚焦显微镜观察相结合的方法,对受精卵、孤雌激活和四倍体胚胎早期发育阶段PKCα的表达进行了定位观察,并利用Western blot对三组胚胎致密化进程中PKCα的表达进行定量分析.结果显示,PKCα在上述三组胚胎发育的2-细胞期至囊胚期均有表达,虽然不同胚胎PKCα的分布在同一发育阶段存在差异,却表现出在各胚胎期主要分布于卵裂球核染色质内,以及在胚胎致密化开始,PKCα在卵裂球连接处发生重新分布的共同特点.此外,三组胚胎PKCα在致密化进程中的表达呈升高趋势,即致密化后的表达高于敛密化前.结果表明,PKCct对胚胎致密化的调节具有重要作用,其在8-细胞/4-细胞期的重新分布是胚胎进入桑椹胚期的必然事件,是胚胎致密化的前提,同时伴随蛋白表达增多.此外,PKCα在囊胚期发生了植入前的第二次重新分布.PKCα在三组胚胎各发育阶段表达情况各不相同,它对小鼠胚胎发育的影响体现在整个早期发育阶段.PKCα在小鼠受精卵早期发育阶段的两次重新分布可能与在致密化开始时启动的细胞黏附事件存在某种必然联系.     

Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation

Apicolateral tight junctions (TJs) between epithelial cells are multiprotein complexes regulating membrane polarity and paracellular transport and also contribute to signalling pathways affecting cell proliferation and gene expression. ZO-2 and other ZO family members form a sub-membranous scaffold for binding TJ constituents. We investigated ZO-2 contribution to TJ biogenesis and function during trophectoderm epithelium differentiation in mouse preimplantation embryos. Our data indicate that ZO-2 is expressed from maternal and embryonic genomes with maternal ZO-2 protein associated with nuclei in zygotes and particularly early cleavage stages. Embryonic ZO-2 assembled at outer blastomere apicolateral junctional sites from the late 16-cell stage. Junctional ZO-2 first co-localised with E-cadherin in a transient complex comprising adherens junction and TJ constituents before segregating to TJs after their separation from the blastocyst stage (32-cell onwards). ZO-2 siRNA microinjection into zygotes or 2-cell embryos resulted in specific knockdown of ZO-2 mRNA and protein within blastocysts. Embryos lacking ZO-2 protein at trophectoderm TJs exhibited delayed blastocoel cavity formation but underwent normal cell proliferation and outgrowth morphogenesis. Quantitative analysis of trophectoderm TJs in ZO-2-deficient embryos revealed increased assembly of ZO-1 but not occludin, indicating ZO protein redundancy as a compensatory mechanism contributing to the mild phenotype observed. In contrast, ZO-1 knockdown, or combined ZO-1 and ZO-2 knockdown, generated a more severe inhibition of blastocoel formation indicating distinct roles for ZO proteins in blastocyst morphogenesis.     

Compaction in preimplantation mouse embryos is regulated by a cytoplasmic regulatory factor that alters between 1- and 2-cell stages in a concentration-dependent manner.

Present studies were performed to investigate what factors affect the morphogenesis of preimplantation mouse embryos, and to find the action mechanism of that factor by using cytoplasm removal and its reconstitution from a different developmental stage embryo. Half (HP group) or one-third of cytoplasm (TP group) was removed from 1-cell mouse embryos by micromanipulation, and their morphogenesis and genome expression were compared with sham-operated embryos (SP group). The compaction and blastocoel formation of embryos in both the HP and TP groups were accelerated in time and cell stage when compared with those of the SP group. However, the total activity and time of RNA synthesis, and gene expression of ZO-1alpha+ isoform were not different. To change the cytoplasm composition without altering the nucleus/cytoplasmic ratio, half a 1-cell embryo with both pronuclei was reconstituted with the half enucleated cytoplasm of 1-cell embryo (P + P group), 2-cell (P + 2 group) or 4-cell (P + 4 group) by electrofusion. Embryonic compaction, timing of RNA synthesis, and stage-specific gene expression of the ZO-1alpha(+) isoform in the P + 2 and P + 4 groups were accelerated in time and cell stage than that in the P + P group, but not different between the P + 2 and P + 4 groups. In addition, a blastomere of 2-cell embryo was reconstituted with the enucleated cytoplasm of 1-cell embryo (2 + P group) or 2-cell (2 + 2 group) in equal volume by electrofusion. Also, the karyoplast of 2-cell was fused with the enucleated 1-cell embryo (2 + PP group). Embryonic development, total activity of RNA synthesis, and gene expression of the ZO-1alpha(+) isoform of embryos in the 2 + P and 2 + PP groups were delayed when compared with those of the 2 + 2 group. Also, the phenomena of compaction and blastocoel formation were delayed in the development time and cell stage. From these results, the nucleus/cytoplasm ratio was found to have no direct effect on the regulation of embryonic morphogenesis, although it accelerated compaction and blastocoel formation. However, cytoplasmic factors that altered between 1- and 2-cell stages regulate embryonic morphogenesis, especially compaction, of preimplantation mouse embryos in concentration-dependent manner.     

Cell specific loss of polarity-inducing ability by later stage mouse preimplantation embryos

The individual blastomeres of the preimplantation mouse embryo become polarized during the 8-cell stage. Microvilli become restricted to the free surface of the embryo and this region of the membrane shows increased labeling with FITC-Con A and trinitrobenzenesulfonate (TNBS). Previous studies have shown that this polarity develops in response to asymmetric cell-cell contact with stage specific induction competent blastomeres. In the present study, the ability of later stage embryos to induce 8-cell polarization has been investigated. Newly-formed, nonpolar 8-cell stage blastomeres (1/8 cells) were isolated, then aggregated with morulae, inner cell clusters (from morulae), blastocysts, or inner cell masses (ICM) and cultured for 8 hr. Aggregates were then assayed for polarity. The results show a hierarchy of inducing ability, with the ICM and IC cluster possessing greater activity than the morula and polar trophectoderm of the early blastocyst, while the mural trophectoderm shows very little inducing activity. Furthermore, the inducing ability of the polar trophectoderm decreases with complete expansion and hatching of the blastocyst. These results indicate that the ability to induce 8-cell blastomere polarization is retained by the embryo beyond the 8-cell stage and that this ability is lost with further differentiation.     

Embryonic cytoplasmic extracts rescue murine androgenones to the blastocyst stage.

Androgenones (paternally derived genome) show a significant inability to form a blastocoele cavity. Eighty percent of these embryos die or arrest at earlier stages. Factor(s) from both normal and parthenogenetic late preimplantation embryos injected into each blastomere of androgenetic 4-cell stage can rescue more than twice as many to the blastocyst stage (47.2% versus 19.2% for non-injected androgenones). This factor(s) becomes available beginning at the 4-cell stage and is titratable. Injected total cytoplasmic mRNA will also cause a rescue response. Isolating this specific factor message(s) will permit the eventual cloning of possibly the earliest parentally imprinted gene(s) expressed during development.     

Characterization of intercellular junctions in the preimplantation mouse embryo by freeze-fracture and thin-section electron microscopy

Intercellular junction formation in preimplantation mouse embryos was investigated with thin-section and freeze-fracture electron microscopy. At the four-cell stage, regions of close membrane apposition with focal points of membrane contact and occasional underlying cytoplasmic densities were observed between blastomeres of thin-sectioned embryos. Corresponding intramembrane specializations were not, however, observed in freeze-fractured embryos. At the 8- to 16-cell stage, small gap and macula occludens junctions and complexes of these junctions were observed at all levels between blastomeres of freeze-fractured embryos. As development progressed from the early to mid 8- to 16-cell stage, the size of the occludens/gap junction complexes increased, forming fascia occludens/gap junction complexes. At the morula stage, gap junctions and occludens/gap junction complexes were observed on both presumptive trophoblast and inner cell-mass cells. Zonula occludens junctions were first observed at the morula stage on presumptive trophoblast cells of freeze-fractured embryos. The number of embryos possessing zonula occludens junctions increased at the mid compared to the early morula stage. At the blastocyst stage, junctional complexes consisting of zonula occludens, macula adherens, and gap junctions were observed between trophoblast cells of freeze-fractured and thin-sectioned embryos. Isolated gap and occludens junctions, adherens junctions, and occludens/gap junction complexes were observed on trophoblast and inner cell-mass cells.     

The presence of gap junctions during early Patella embryogenesis: An electron microscopical study

The presence of gap junctions has been examined up to the sixth cleavage in the early Patella embryo. Gap junctions are located all over the blastomere borders. In 2-, 4-, and 8-cell embryos they were also observed at peripheral contacts. The frequency and size of the gap junctions increase at the 32-cell stage. The structure of gap junctions is similar in all stages investigated with hexagonally arranged equal-sized particles (11 nm) having a constant center-to-center spacing (13.0 nm). At the 32-cell stage formation plaques were observed, indicating an increase of gap junctions.     

Chronological appearance of spontaneous and induced apoptosis during preimplantation development of rabbit and mouse embryos

This study was undertaken to obtain specific information on the characteristics of spontaneous and induced apoptosis during preimplantation development of rabbit in vivo and in vitro developed embryos and mouse in vitro embryos. After reaching appropriate developmental stages, embryos were transferred into culture media with or without apoptotic inductor (actinomycin D 500 ng/mL) and cultured for 10 h. The identification of apoptotic cells was based on morphological assessment of nuclei and on detection of specific DNA degradation, phosphatidylserine redistribution and active caspase-3 under fluorescence microscope.Our experiments proved that apoptosis is a frequent physiological event occurring during normal preimplantation development. A high number of untreated rabbit and mouse blastocysts contained at least one apoptotic cell. Rabbit embryos showed a lower incidence of spontaneous apoptosis. Treated blastocysts of both species responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and significant increase in the incidence of apoptotic cell death. The occurrence of spontaneous apoptosis during earlier preimplantation development was sporadic and its presence was observed only at stages following embryonic genome activation (at 4-cell stage and later in mouse, at 16-cell and morula stage in rabbit). The susceptibility of embryos at early stages to the apoptotic inductor was much lower. The presence of actinomycin D did not increase the incidence of apoptotic embryos or apoptotic cells. Nevertheless, it slowed down embryo growth and triggered earlier appearance of some apoptotic features (at the 6-cell stage in rabbit). The results show that the occurrence of both spontaneous and induced apoptosis in preimplantation embryos is stage- and species-specific.     

Dynamics of zonula occludens-2 expression during preimplantation embryonic development in the hamster

The objective was to study the expression of zonula occludens-2, a tight junction protein, during preimplantation hamster embryonic development, to predict its possible localization, source, and roles in trophectoderm differentiation and blastocyst formation in this species. Comparison of zonula occludens-2 expression pattern between the hamster and mouse preimplantation embryos from the zygote up to the blastocyst stage was also an objective of this study. Zonula occludens-2 localization was noted in nuclei of blastomeres in all stages of hamster and mouse embryonic development. Compared to mice, where zonula occludens-2 was first localized in the interblastomere membrane at the morula stage, hamster embryos had membranous zonula occludens-2 localization from the 2-cell stage onwards. Based on combined results of immunolocalization study in parthenogenic embryos and ovarian and epididymal sections, and quantitative PCR done in oocytes and all developmental stages of preimplantation embryos, perhaps there was a carry-over of zonula occludens-2 proteins or mRNA from the dam to the embryo. Based on these findings, we inferred that maternally derived zonula occludens-2 was involved in nuclear functions, as well as differentiation of blastomeres and blastocoel formation during preimplantation embryonic development in the hamster.     

Chronological appearance of spontaneous and induced apoptosis during preimplantation development of rabbit and mouse embryos

This study was undertaken to obtain specific information on the characteristics of spontaneous and induced apoptosis during preimplantation development of rabbit in vivo and in vitro developed embryos and mouse in vitro embryos. After reaching appropriate developmental stages, embryos were transferred into culture media with or without apoptotic inductor (actinomycin D 500 ng/mL) and cultured for 10 h. The identification of apoptotic cells was based on morphological assessment of nuclei and on detection of specific DNA degradation, phosphatidylserine redistribution and active caspase-3 under fluorescence microscope. Our experiments proved that apoptosis is a frequent physiological event occurring during normal preimplantation development. A high number of untreated rabbit and mouse blastocysts contained at least one apoptotic cell. Rabbit embryos showed a lower incidence of spontaneous apoptosis. Treated blastocysts of both species responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and significant increase in the incidence of apoptotic cell death. The occurrence of spontaneous apoptosis during earlier preimplantation development was sporadic and its presence was observed only at stages following embryonic genome activation (at 4-cell stage and later in mouse, at 16-cell and morula stage in rabbit). The susceptibility of embryos at early stages to the apoptotic inductor was much lower. The presence of actinomycin D did not increase the incidence of apoptotic embryos or apoptotic cells. Nevertheless, it slowed down embryo growth and triggered earlier appearance of some apoptotic features (at the 6-cell stage in rabbit). The results show that the occurrence of both spontaneous and induced apoptosis in preimplantation embryos is stage- and species-specific.     

U2 small nuclear RNA localization and expression during bovine preimplantation development.

This study describes the localization of the U2 small nuclear RNA (snRNA) and the major U snRNA group ribonucleoproteins (snRNPs) during bovine preimplantation development. In vitro maturation, fertilization, and oviductal epithelial cell coculture methods were employed to produce several developmental series totalling over 2,000 preimplantation-stage bovine oocytes and embryos. These oocytes and preimplantation embryos were processed for in situ hybridization, immunofluorescence and Northern blotting methods. The U2 snRNA and the major U group snRNPS were localized initially over the germinal vesicle (GV) of preovulatory oocytes but following GV breakdown were released throughout the ooplasm. They subsequently reassociated with both pronuclei during fertilization. From the two-cell to the blastocyst stages, the U2 snRNA and U snRNPs were localized to the interphase nucleus of each blastomere. The levels of U2 snRNA throughout bovine preimplantation development were determined by probing a Northern blot containing total RNA isolated from the following preimplantation bovine embryo stages: one to two cell, eight to 16 cell, early morula (greater than 32 cell), and late morula/early blastocysts. The levels of U2 snRNA remained constant between the one-cell and eight- to 16-cell bovine embryo stages but increased 4.4-fold between the eight- to 16-cell stage and the late morula/early blastocyst stages. The results suggest that a maternal pool of snRNAs is maintained in mammalian preimplantation embryos regardless of the duration of maternal control of development.     

Synthesis and phosphorylation of uvomorulin during mouse early development.

The cell adhesion molecule, uvomorulin, is synthesised in both the 135 x 10(3) M(r) precursor and 120 x 10(3) M(r) mature forms on maternal mRNA templates in unfertilized and newly fertilized mouse oocytes. Synthesis on maternal message ceases during the 2-cell stage to resume later on mRNA encoded presumptively by the embryonic genome. Uvomorulin is detectable by immunoblotting at all stages upto the blastocyst stage, but shows variations in its total amount and processing with embryonic stage. Whilst only trace levels of phosphorylated uvomorulin are detectable in early and late 4-cell embryos, uvomorulin in 8-cell embryos is phosphorylated.