Zonula occludens-1 (ZO-1) is involved in morula to blastocyst transformation in the mouse

It is unknown whether or not tight junction formation plays any role in morula to blastocyst transformation that is associated with development of polarized trophoblast cells and fluid accumulation. Tight junctions are a hallmark of polarized epithelial cells and zonula occludens-1 (ZO-1) is a known key regulator of tight junction formation. Here we show that ZO-1 protein is first expressed during compaction of 8-cell embryos. This stage-specific appearance of ZO-1 suggests its participation in morula to blastocyst transition. Consistent with this idea, we demonstrate that ZO-1 siRNA delivery inside the blastomeres of zona-weakened embryos using electroporation not only knocks down ZO-1 gene and protein expressions, but also inhibits morula to blastocyst transformation in a concentration-dependent manner. In addition, ZO-1 inactivation reduced the expression of Cdx2 and Oct-4, but not ZO-2 and F-actin. These results provide the first evidence that ZO-1 is involved in blastocyst formation from the morula by regulating accumulation of fluid and differentiation of nonpolar blastomeres to polar trophoblast cells.     

Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 μg/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.     

Na+, K+, ATPase activity in the human and bovine preimplantation embryo

Blastocyst formation is associated with a marked increase in ATP production, much of which is thought to be associated with the active transport of ions across the trophectoderm mediated by the sodium pump (Na+, K+, ATPase) resulting in the vectorial transport of water into the blastocoel. In this study, the biochemical activity of the sodium pump was measured directly in single human and bovine embryo extracts by monitoring the conversion of ATP to ADP in the presence and absence of ouabain. ATP and ADP were assayed by HPLC. In both species, there was a transient, significant increase in sodium pump activity while the blastocyst was actively expanding. The oxygen consumption of single human blastocysts was measured in order to estimate the proportion of total ATP used by the Na+, K+, ATPase. The results suggest that approximately 60 and 36% of the ATP produced is used by the sodium pump during blastocoel expansion in the human and bovine blastocyst, respectively.     

Spatial arrangement of individual 4-cell stage blastomeres and the order in which they are generated correlate with blastocyst pattern in the mouse embryo

In the unperturbed development of the mouse embryo one of the 2-cell blastomeres tends to contribute its progeny predominantly to the embryonic and the other to the abembryonic part of the blastocyst. However, a significant minority of embryos (20-30%) do not show this correlation. In this study, we have used non-invasive lineage tracing to determine whether development of blastocyst pattern shows any correlation with the orientation and order of the second cleavage divisions that result in specific positioning of blastomeres at the 4-cell stage. Although the orientation and order of the second cleavages are not predetermined, in the great majority (80%) of embryos the spatial arrangement of 4-cell blastomeres is consistent with one of the second cleavages occurring meridionally and the other equatorially or obliquely with respect to the polar body. In such cleaving embryos, one of the 2-cell stage blastomeres tends to contribute to embryonic while the other contributes predominantly to abembryonic part of the blastocyst. Thus, in these embryos the outcome of the first cleavage tends to correlate with the orientation of the blastocyst embryonic-abembryonic axis. However, the order of blastomere divisions predicts a specific polarity for this axis only when the earlier 2-cell blastomere to divide does so meridionally. In contrast to the above two groups, in those embryos in which both second cleavage divisions occur in a similar orientation, either meridionally or equatorially, we do not observe any tendency for the 2-cell blastomeres to contribute to specific blastocyst parts. We find that all these groups of embryos develop to term with similar success, with the exception of those in which both second cleavage divisions occur equatorially whose development can be compromised. We conclude that the orientations and order of the second cleavages are not predetermined; they correlate with the development of blastocyst patterning; and that the majority, but not all, of these cleavage patterns allow equally successful development.     

Effect of Gossypol on Bovine Blastocyst Attachment and Trophoblastic outgrowth in Vitro

The presont study was carried out to investigate the effect of different amounts of gossypol on bovineblastocyst attachment and trophoblastic outgrowth in vitro. Bovine oocyte were collected from theovaries of slaughtered cows and were matured and fertilized in vitro. Cleaved oocyte were culturedin CRlaa + BOEC and TC-199 + 10% FCS combined in an 1:1 ratio. After 8 days of co-culture,the hatched blastocysts were randomly allotted to different treatment groups. All were cultured ona fetal fibroblast monolaycr (prepared from bovine fetuses) in TC-199 culture medium supplementedwith 10% fetal calf serum (TCFCS). But the groups differed from one another in the dose ofgossypol given: 0.01 μg, 0.1 μg, 1 μg, 10 μs/ml, and no gossypol as control. All cultures wereperformed in 24-well culture plates at 39℃ with 5% CO_2 in air. The results indicate that the ratesof attached and outgrowing blastocysts in the medium containing 1 μg/ml gossypol were significantlylowcr than the control group (p<0.01) and outgrowth were inhibited by gossypol in a dose-dependent manner.     

从早期胚胎多能干细胞生成的嵌合鼠

本文利用囊胚注射法将小鼠胚胎多能干细胞－ＣＣＥ细胞注射到发育３天半的昆明和Ｃ３７ＢＬ／６Ｊ小鼠受体囊胚腔内,经假孕鼠借腹怀胎,获３只ＣＣＥ细胞毛色嵌合鼠。实验共注射胚胎６５４个,经培养其恢复成活率７３．８％,胚胎移植后,假母受孕率及产仔率分别为３２．９％和５３％。在所获３只嵌合鼠中,２只为ＣＣＥ－昆明毛色嵌合鼠,１只为ＣＣＥ－Ｃ３７ＢＬ／６Ｊ毛色嵌合鼠,这是国内首次利用胚胎多能干细胞获得嵌合鼠。为     

Mucin-like glycoproteins in the equine embryonic capsule

The equine embryonic capsule replaces the zona pellucida and envelops the conceptus during the second and third weeks of pregnancy. Although this capsule was described more than 100 years ago, its molecular structure has not been characterized. Here we present evidence that the glycoprotein(s) of the equine capsule resembles those of the mucin glycoprotein family. The resistance of the capsule to chemical and enzymatic solubilization was confirmed, and, as in mucins, protein constituted only 35–40% of its total dry mass. Determination of the sugar composition of the capsule using colorimetric assays and high-performance anion-exchange chromatography also showed it to have mucin-like characteristics. Gal, GalNAc, sulfated sugars, and sialic acid make up a high proportion of the capsular carbohydrate, while GlcNAc, Glc, and Man are minor components. These findings were verified using lectin histochemical staining of frozen sections of conceptuses. The results of amino acid analysis were also consistent with the proposal that the capsular glycoproteins belong to the mucin family. Removal of the covalently bound carbohydrate by b?-elimination under reducing conditions demonstrated that the capsule is O-glycosylated mainly on threonine residues. Affinity chromatography on jacalin-agarose confirmed that, like mucins, the capsular glycoproteins are heavily O-glycosylated. SDS-PAGE analysis revealed a prominent 21-kDa band, specific to the capsule, in preparations solubilized by trypsin but not by other proteases. Characterization of its constituent glycoprotein(s) should be helpful in elucidating the role of the capsule (and analogous blastocyst coverings in other species) during early pregnancy. © 1993 Wiley-Liss, Inc.     

Early embryo development in the Siberian hamster (Phodopus sungorus)

Development of preimplantation embryos of the Siberian hamster (Phodopus sungorus) in vivo and in vitro was examined. The timing of early development in vivo was found to be slower than that reported for the golden hamster. Progression through the cleavage stages, cavitation, and hatching from the zona pellucida occurred later, with blastocyst formation beginning on the afternoon of day 4 and uterine attachment occurring early on day 5. In vitro, morulae, and early blastocysts collected on day 4 and cultured in serum-containing medium formed expanded blastocysts and some began to hatch from the zona pellucida. With extended culture, blastocysts attached and formed trophoblast outgrowths. Outgrowth was characterized by an initial migration of small cells from the blastocyst, followed by formation of a sheet of trophoblast giant cells. Differences in the morphology of outgrowth between the hamster and mouse suggest that further comparative studies with the Siberian hamster may be useful.