A role of trophoblastic cells in regulation of mouse blastocyst survival in vitro after microinjection and osmotic stress

We have evaluated the morphology of the mouse preimplantation embryos at developmental stages from morula to late blastocyst after two different impacts: microinjection of modified Witten’s medium and osmotic stress in physiological osmolarity (310 mOsM), in 5% glucose (560 mOsM) at high concentration of NaCl (614 mOsM). Results of our research showed that these stresses caused similar changes in embryo morphology: volume was reduced followed by its recovery in culture medium (osmolality was less than a physiological value, 260 mOsM). The ability of embryos to recover the volume and morphology up to the initial level depends on a stage of embryo development and consequently competence of TB cells. In this study it was revealed that a key role in regulation of volume homeostasis after microinjection and after short-time (30–60 min) osmotic stress belongs to TB cells. Both physical effects induce the further embryo development in vitro up to the formation of primary colonies of embryonic and trophoblastic cells. These data could be used to develop the morphological criteria for a prediction of blastocyst-stage embryonic implantation potential.     

Developmentally regulated cell cycle dependence of swelling-activated anion channel activity in the mouse embryo

Anion channels activated by increased cell volume are a nearly ubiquitous mechanism of cell volume regulation, including in early preimplantation mouse embryos. Here, we show that the swelling-activated anion current (I(Cl,swell)) in early mouse embryos is cell-cycle dependent, and also that this dependence is developmentally regulated. I(Cl,swell) is present both in first meiotic prophase (germinal vesicle stage) mouse oocytes and in unfertilized mature oocytes in second meiotic metaphase, and it persists after fertilization though the 1-cell and 2-cell stages. I(Cl,swell) was found to remain unchanged during metaphase at the end of the 1-cell stage. However, I(Cl,swell) decreased during prophase and became nearly undetectable upon entry into metaphase at the end of the 2-cell stage. Entry into prophase/metaphase was required for the decrease in I(Cl,swell) at the end of the 2-cell stage, since it persisted indefinitely in 2-cell embryos arrested in late G(2). There is considerable evidence that the channel underlying I(Cl,swell) is not only permeable to inorganic anions, but to organic osmolytes as well. We found a similar pattern of cell cycle and developmental dependence in the 1-cell and 2-cell stages for the swelling-induced increase in permeability to the organic osmolyte glycine. Thus, entry into metaphase deactivates I(Cl,swell) in embryos, but only after developmental progression through the 2-cell stage.     

Beta-alanine but not taurine can function as an organic osmolyte in preimplantation mouse embryos cultured from fertilized eggs

Early preimplantation mouse embryos are susceptible to the detrimental effects of increased osmolarity and, paradoxically, their in vitro development is significantly compromised by osmolarities near that of oviductal fluid. In vitro development can be restored, however, by several compounds that are accumulated by 1-cell embryos to act as organic osmolytes, providing intracellular osmotic support and cell volume regulation. Taurine, a substrate of the beta-amino acid transporter that functions as an organic osmolyte transporter in other cells, had been proposed to function as an organic osmolyte in mouse embryos. Here, however, we found that taurine is neither able to provide protection for in vitro embryo development against increased osmolarity nor is it accumulated to higher intracellular levels as osmolarity is increased, indicating that it cannot function as an organic osmolyte in early preimplantation embryos. In contrast, beta-alanine, the other major substrate of the beta-amino acid transporter, both protects against increased osmolarity and is accumulated to somewhat higher levels as osmolarity is increased, indicating that it is able to function as an organic osmolyte in embryos. However, we also found that beta-alanine is displaced from embryos by glycine-the most effective organic osmolyte in embryos previously identified-and beta-alanine does not increase protection above that afforded by glycine at concentrations near those in vivo. Thus, the beta-amino acid transporter is likely present in early preimplantation embryos to supply beta-amino acids such as taurine for purposes other than to serve as organic osmolytes.     

The organic osmolytes betaine and proline are transported by a shared system in early preimplantation mouse embryos

Betaine and proline protect preimplantation mouse embryos against increased osmolarity and decreased cell volume, implying that they may function as organic osmolytes. However, the transport system(s) that mediates their accumulation in fertilized eggs and early embryos was unknown, and previously identified mammalian organic osmolyte transporters could not account for their transport. Here, we report that there is a single saturable transport component shared by betaine and proline in 1-cell mouse embryos. A series of inhibitors had nearly identical effects on both betaine and proline transport by this system. In addition, K(i) values for reciprocal inhibition of betaine and proline transport were approximately 100-300 microM, similar to K(m) values ( approximately 200-300 microM) for their transport, and both had similar maximal transport rates (V(max)). The K(i) values for inhibition of betaine and proline transport by dimethylglycine were similar ( approximately 2 mM), further supporting transport of both substrates by a single transport system. Finally, betaine and proline transport each required Na(+)- and Cl(-). These data were consistent with a single, Na(+)- and Cl(-)-requiring, betaine/proline transport system in 1-cell mouse embryos. While betaine was only transported by a single saturable system, we found an additional, less conspicuous proline transport route that was betaine-insensitive, Na(+)-sensitive, and inhibited by alanine, leucine, cysteine, and methionine. Furthermore, we showed that betaine, like proline, is present in the mouse oviduct and thus could serve as a physiological substrate. Finally, accumulation of both betaine and proline increased with increasing osmolarity, consistent with a possible role as organic osmolytes in early embryos.     

"生存还是毁灭",发育还是阻滞--小鼠早胚体外发育2-细胞阻滞产生机制初析

在体外培养条件下,小鼠受精卵往往经一次分裂后就停滞在２－细胞,不能完成到达囊胚的后续发育过程,称作２－细胞阻滞。氧自由基伤害、培养液成分不平衡等外界因素都能引起阻滞。小鼠的２－细胞阻滞现象受细胞质内母型物质制约,具有品系依赖性。在阻滞品系小鼠胚胎的细胞质内可能缺乏某些重要的蛋白因子,在无外源信号的培养体系内不能继续分裂。发生阻滞的胚胎细胞内ＭＰＦ前体物虽然储备充足,但因无法去磷酸化激活而最终导致发     

Differentiation of 2-cell and 8-cell mouse embryos arrested by cytoskeletal inhibitors

Mouse embryos at the 2-cell stage were cultured in the presence of cytochalasin B (CB), cytochalasin D (CD), colchicine (COL) or colcemid (COM) for up to 72 h. Cleavage was arrested in the 2-cell and 8-cell embryos cultured in CB or CD but the blastomeres continued to differentiate, since chromosome replication occurred in the blastomeres at approximately the same time as control embryos underwent cleavage; an increase in the incorporation of [³H]uridine into RNA was also detected. Furthermore, the cleavage-arrested embryos acquired the necessary information to undergo morphogenesis; these embryos when explanted to fresh medium after 48 h culture in CB or CD underwent compaction within 15–60 min and started to cavitate to produce trophoblastic vesicles within 5–6 h at the same time as when the control embryos were undergoing compaction and beginning to form blastocoelic cavities. In contrast, the embryos arrested in the presence of COM or COL showed none of these differentiative, biochemical or morphogenetic changes. Hence, differentiation of blastomeres and morphogenesis is apparently coupled with nuclear divisions and the information does not reside within the blastomeres at the 2-cell or 8-cell stage. The trophoblastic vesicles produced after cleavage arrest subsequently gave rise to only trophoblast giant cells and no embryonic derivatives were detected.     

Betaine is a highly effective organic osmolyte but does not appear to be transported by established organic osmolyte transporters in mouse embryos

Betaine protects early preimplantation mouse embryos against increased osmolarity in vitro, functioning as an organic osmolyte. Betaine is effective at very low external concentrations, with half-maximal protection of 1-cell embryo development to blastocysts at approximately 50 microM, making it one of the best osmoprotectants for mouse preimplantation embryos. We performed studies designed to determine whether known high-affinity organic osmolyte transporters could account for the ability of betaine to act as an organic osmolyte in preimplantation embryos. We found no evidence in 1-cell embryos of transport by a betaine/GABA transporter (BGT1), the osmoregulated betaine transporter found in a number of cell types, as betaine and GABA did not inhibit each other's transport. Instead, all saturable GABA transport in embryos was apparently via the beta-amino acid transporter. We also found that the glycine transporter, GLY, which mediates osmoprotective transport of glycine in early preimplantation embryos, does not appear to transport betaine. Finally, increased osmolarity did not induce any detectable System A amino acid transporter activity, which is osmotically-inducible in other cells and can transport betaine. There does appear, however, to be a saturable betaine transporter in 1-cell mouse embryos, as considerable 14C-betaine transport was measured which was substantially inhibited by excess unlabeled betaine. Our data imply that betaine functions as an organic osmolyte in embryos due to its saturable transport via a mechanism distinct from known osmolyte transporters. We propose that an unidentified high-affinity betaine transporter may be expressed in early embryos and mediate transport of betaine as an organic osmolyte.     

Cleavage pattern and survivin expression in porcine embryos by somatic cell nuclear transfer

Mammalian embryos produced in vitro show a high rate of early developmental failure. Numerous somatic cell nuclear transfer (SCNT) embryos undergo arrest and show abnormal gene expression in the early developmental stages. The purpose of this study was to analyze porcine SCNT embryo development and investigate the cause of porcine SCNT embryo arrest. The temporal cleavage pattern of porcine SCNT embryos was analyzed first, and the blastocyst origin at early developmental stage was identified. To investigate markers of arrest in the cleavage patterns of preimplantation SCNT embryos, the expression of survivin—the smallest member of the inhibitor of apoptosis (IAP) gene family, which suppresses apoptosis and regulates cell division—was compared between embryos showing normal cleavage and arrested embryos.A total of 511 SCNT embryos were used for cleavage pattern analysis. Twenty-four hours post activation (hpa), embryos were classified into five groups based on the cleavage stage as follows; 1-cell, 2-cell, 4-cell, 8-cell and fragmentation (frag). In addition, 48 hpa embryos were more strictly classified into 15 groups based on the cleavage stage of 24 hpa; 1-1 cell (24 hpa-48 hpa), 1-2 cell, 1-4 cell, 1-8 cell, 1 cell-frag, 2-2 cell, 2-4 cell, 2-8 cell, 2 cell-frag, 4-4 cell, 4-8 cell, 4 cell-frag, 8-8 cell, 8 cell-frag, and frag-frag. These groups were cultured until 7 d post activation, and were evaluated for blastocyst formation. At 24 hpa, the proportion of 2-cell stage was significantly higher (44.5%) than those in the other cleavage stages (1-cell: 13.4%; 4-cell: 17.9%; 8-cell: 10.3%; and frag: 13.9%). At 48 hpa, the proportion of embryos in the 2-4 cell stage was significantly higher (32.4%) than those in the other cleavage stages (2-8 cell: 8.2%; 4-8 cell: 12.1%; and frag-frag: 13.9%). Some embryos arrested at 48 hpa (1-1 cell: 5.8%; 2-2 cell: 2.8%; 4-4 cell: 3.8%; 8-8 cell: 6.5%; and total arrested embryos: 18.9%). Blastocyst formation rates were higher in 2-4 cell cleavage group (20.2%) than in other groups. SCNT embryos in 2-4 cell stage showed stable developmental competence. In addition, we investigated survivin expression in porcine SCNT embryos during the early developmental stages. The levels of survivin mRNA in 2-cell, 4-cell stage SCNT embryos were significantly higher than those of arrested embryos. Survivin protein expression showed a similar pattern to that of survivin mRNA. Normally cleaving embryos showed higher survivin protein expression levels than arrested embryos. These observations suggested that 2-4 cell cleaving embryos at 48 hpa have high developmental competence, and that embryonic arrest, which may be influenced by survivin expression in porcine SCNT embryos.     

Phosphate induced developmental arrest of hamster two-cell embryos is associated with disrupted ionic homeostasis.

Culture of hamster embryos with 0.35 mM inorganic phosphate results in developmental arrest at the 2-cell stage. These arrested 2-cell embryos were found to have significantly elevated levels of both intracellular pH and intracellular free calcium. Culture of 2-cell embryos with both glucose and phosphate did not further alter intracellular ionic homeostasis. Developmental arrest of 2-cell embryos was dependent on the concentration of phosphate used. Culture with 1.25 microM phosphate did not alter development, while concentrations of 2.5 microM and 5.0 microM resulted in a percentage of embryos arresting development at the 2-cell stage. Analysis of intracellular levels of pH and calcium after culture with different phosphate concentrations revealed a significant negative correlation between intracellular calcium levels and development beyond the 2-cell stage. There was no correlation between the increase in intracellular pH and embryo development in the presence of phosphate. The increase in intracellular calcium levels after culture with phosphate appears to be derived from intracellular pools, as preventing the influx of extracellular calcium did not alter development beyond the 2-cell stage. Therefore, it is apparent that a disruption in ionic homeostasis is associated with developmental arrest of hamster embryos cultured with phosphate.     

Platelet derived growth factor (PDGF) stimulates development of bovine embryos during the fourth cell cycle.

In vitro produced, 2-cell bovine embryos were cultured in serum-free medium supplemented with various combinations of growth factors to test the hypothesis that these polypeptide factors are able to signal preimplantation development. The developmental arrest that occurs during the 8-cell stage with typical culture methods might be relieved by a growth factor-dependent mechanism that would stimulate expression of the embryonic genome, thereby mimicking events that occur in vivo in the oviduct during the fourth cell cycle (8- to 16-cell stage). Subsequently, other growth factors might promote compaction and blastulation, processes which normally occur in the uterus. The effects of growth factors on early embryos were evaluated using phase contrast microscopy to monitor progression to the 8-cell stage, completion and duration of the fourth cell cycle, and blastocyst formation. Platelet derived growth factor (PDGF) promoted development beyond the 16-cell stage in 39.1% of the 2-cell embryos examined in all experiments. The duration of the fourth cell cycle among these embryos was approximately 26 hours. During development after the 16-cell stage, PDGF reduced the proportion of embryos bastulating from 12.7% to 5.8%; in contrast, transforming growth factor alpha (TGF alpha), acting during the same developmental time period, increased the proportion of embryos blastulating from 8.6% to 40.6%. These results, using serum-free medium, indicated that PDGF signalled completion of the fourth cell cycle. TGF alpha, and perhaps basic fibroblast growth factor (bFGF), promoted blastulation of 16-cell embryos during subsequent culture.     

Gene expression and in vitro development of inter-species nuclear transfer embryos

This study examined the chromatin morphology, in vitro development, and expression of selected genes in cloned embryos produced by transfer of mouse embryonic fibroblasts (MEF) into the bovine ooplasm. After 6 hr of activation, inter-species nuclear transfer (NT) embryos (MEF-NT) had one (70%) or two pronuclei (20%), respectively. After 72 hr of culture in vitro, 62.6% of the MEF-NTs were arrested at the 8-cell stage, 31.2% reached the 2- to 4-cell stage, and only 6.2% had more than eight blastomeres, but none of these developed to the blastocyst stage. Whereas, 20% of NT embryos derived from bovine embryonic fibroblast fused with bovine ooplasm (BEF-NT) reached the blastocyst stage. Donor MEF nuclei expressing an Enhanced Green Fluorescent Protein (EGFP) transgene resulted in 1- to 8-cell stage MEF-NT that expressed EGFP. The expression of selected genes was examined in 8-cell MEF-NTs, 8-cell mouse embryos, enucleated bovine oocytes, and MEFs using RT-PCR. The mRNA for heat shock protein 70.1 (Hsp 70.1) gene was detected in MEF-NTs and MEF, but not in mouse embryos. The hydroxy-phosphoribosyl transferase (HPRT) mRNA was found in normal mouse embryos and MEF but not in MEF-NTs. Expression of Oct-4 and embryonic alkaline phospatase (eAP) genes was only detected in normal mouse embryos and not in the inter-species NT embryos. Abnormal gene expression profiles were associated with an arrest in the development at the 8-cell stage, but MEF-NT embryos appeared to have progressed through gross chromatin remodeling, typical of intra-species NT embryos. Therefore, molecular reprogramming rather than chromatin remodeling may be a better indicator of nuclear reprogramming in inter-species NT embryos.     

多能性转录因子OCT4和SOX2在体外发育的小鼠2-细胞胚胎的表达与定位

为探讨多能性转录因子OCT4和SOX2在昆明小鼠(Mus musculus)2-细胞胚胎发育过程中与2-细胞胚胎阻滞发生的相关性,本研究应用实时荧光定量PCR技术检测了小鼠卵母细胞及在M16培养液中培养的不同发育阶段体外受精胚Oct4和Sox2基因的表达,并利用实时荧光定量PCR和免疫荧光技术比较了2-细胞胚、2-细胞阻滞胚和4-细胞胚的OCT4和SOX2的表达与定位。采用ANOVA对实验所得的数据进行分析,P0.05被认为是具有显著性差异。研究结果显示,2-细胞胚只有24.8%发育成4-细胞胚,75.2%的2-细胞胚发生了阻滞。Sox2和Oct4的m RNA在MⅡ期卵母细胞、原核胚、2-细胞胚、4-细胞胚、桑椹胚和囊胚中都有表达。Oct4 m RNA的表达水平在4-细胞胚显著高于2-细胞胚和2-细胞阻滞胚(P0.05),Sox2 m RNA的表达水平在2-细胞胚显著高于2-细胞阻滞胚和4-细胞胚(P0.05),而后两者之间没有差异(P0.05)。OCT4蛋白在2-细胞胚和4-细胞胚中与核共定位,但在2-细胞阻滞胚中弥散存在于胞质中。SOX2蛋白在以上3类胚胎中始终定位于细胞核。上述结果提示,转录因子OCT4和SOX2的表达和定位与小鼠2-细胞胚胎发育阻滞相关,母源性SOX2表达的维持对胚胎合子基因组激活(ZGA)的发生具有重要作用,母源性OCT4的异常定位可能影响了合子基因组激活相关基因的激活,而合子中Oct4的表达影响合子基因组激活后胚胎的发育。     

The regulation of porin expression in Escherichia coli: effect of turgor stress

The OmpF and OmpC porins are major outer membrane proteins of Escherichia coli. Their expression is affected by medium osmolarity such that OmpF is normally produced at low osmolarity and OmpC at high osmolarity. Potassium ion accumulation is a major means by which cells maintain their internal osmolarity in high osmolarity medium in the absence of organic osmolytes such as glycine-betaine. Starvation for potassium causes cells to become turgor stressed. The effect of turgor stress and potassium ion concentration on OmpF and OmpC expression was examined. It was found that ompF gene expression was switched off by turgor stress but there was no concomitant increase in OmpC. Instead, ompC expression responded to the accumulation of potassium ions by the cell in high osmolarity medium.     

Characterization of embryos derived from calf oocytes: kinetics of cleavage, cell allocation to inner cell mass, and trophectoderm and lipid metabolism

Embryos derived from calf oocytes were compared with adult cow oocyte-derived embryos (1) by studying the kinetics of embryo development using time-lapse cinematography (2) by evaluating the ratio between inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts (3) by measuring the triglyceride content of the blastocysts. The rate of calf oocyte-derived embryos reaching the blastocyst stage was reduced (26 vs. 46% for adult derived embryos). Calf oocyte-derived embryos preferably arrested their development before the 9-cell stage. Those that developed into blastocysts had cleaved earlier to reach the 2-cell or 3-cell stages than embryos that arrested before the 9-cell stage. The 9-cell stage tended to appear later in calf oocyte-derived embryo that reached the blastocyst stage than in adult-derived embryos. This difference became significant at the morula stage. Accordingly, the fourth cell cycle duration was longer for calf oocyte-derived embryos. Day 8 blastocysts from both sources had similar total cell numbers (calf: 89 +/- 20; cow: 100 +/- 30) and cell distribution between TE and ICM. The triglyceride content of day 7 blastocysts was similar for both sources (64 +/- 15 vs. 65 +/- 6 ng/embryo, respectively). In conclusion, calf oocyte-derived embryos are characterized by a higher rate of developmental arrest before the 9-cell stage and by a longer lag phase preceding the major onset of embryonic genome expression. These changes might be related to insufficient "capacitation" of the calf oocyte during follicular growth. Despite these differences, modifications in the quality of the resulting blastocysts were not detected.     

Nuclear structure in early mouse embryos: A comparative ultrastructural and immunocytochemical study with special emphasis on the "2-cell block in vitro"

The embryos from many outbred and inbred strains of mice are arrested at the late 2-cell stage when cultured in vitro in simple culture media. This phenomenon is referred to as the "2-cell block in vitro". The ultrastructural morphology of the nuclei of the blocked embryos is not yet well described. In the present paper we documented the results of a comparative study on the nuclei of mouse embryos, both normally developing and arrested at the 2-cell stage. The blocked embryos maintain the morphological integrity of their nuclei. Main nuclear domains (nucleolus precursor bodies, interchromatin granule clusters, perichromatin granules, and perichromatin fibrils), typical for the control embryos, are observed in the blocked ones. A number and morphological characteristics of these nuclear substructures are not changed significantly in the blocked embryos. At the same time, RNA polymerase II and pre-mRNA splicing factors are redistributed in the nucleus of the blocked embryos. Although something goes to show that nuclear organization of the blocked embryos differ from that of the control, we could not reveal in the blocked embryos distinct signs of degeneration which might characterize aged or dying cells. Our data confirm a peculiar functional state of the 2-cell blocked embryos.     

Oviduct Epithelial Cell Co-culture of early Porcine Embryos

One- to 16-cell porcine embryos were cultured in either Whittens medium supplemented with bovine serum albumin and fetal calf serum (WM) or in the same medium with porcine oviduct epithelial cell co-culture (WM-Poec). All stages of embryos cultured in WM-POEC had higher cell counts after 144–168 h of development than did embryos in WM. There was however, no significant difference in blastocyst formation rate of embryos cultured in WM-POEC over those cultured in WM. A high proportion of the embryos entering culture at the 1-2-cell were able to pass the 4-cell block stage in both WM and WM-POEC, 81% and 77%, respectively. In both media, most of the 1-2-cell embryos arrested their development at the compacted morula stage and failed to blastulate while embryos initiating culture at the 4-and 8-16-cell embryos formed blastocysts in culture at a rate of 80–90%.     

Survival of Salmonella paratyphi B and Pseudomonas aeruginosa in seawater after incubation or washing in the presence of osmolytes]

The authors have compared the survival in seawater of Salmonella paratyphi B and Pseudomonas aeruginosa cells grown at low or high osmolarity, in the presence of organic osmolytes: glycine betaine, choline, proline, and glutamate. The four substrates enhanced the survival potential of S. paratyphi B while only glycine betaine protected P. aeruginosa. In addition only S. paratyphi B cells were more resistant after a preliminary growth at high osmolarity. Both bacteria were sensitive to osmotic down-shock, sensitization of S. paratyphi B being inversely proportional (p greater than or equal to 0.01) to the osmolarity of the medium used to wash cells. The transit in wastewater, at low osmolarity, can therefore modify the behavior of these pathogens in the marine environment.     

Metabolic and epigenetic dysfunctions underlie the arrest of in vitro fertilized human embryos in a senescent-like state

Around 60% of in vitro fertilized (IVF) human embryos irreversibly arrest before compaction between the 3- to 8-cell stage, posing a significant clinical problem. The mechanisms behind this arrest are unclear. Here, we show that the arrested embryos enter a senescent-like state, marked by cell cycle arrest, the down-regulation of ribosomes and histones and down-regulation of MYC and p53 activity. The arrested embryos can be divided into 3 types. Type I embryos fail to complete the maternal-zygotic transition, and Type II/III embryos have low levels of glycolysis and either high (Type II) or low (Type III) levels of oxidative phosphorylation. Treatment with the SIRT agonist resveratrol or nicotinamide riboside (NR) can partially rescue the arrested phenotype, which is accompanied by changes in metabolic activity. Overall, our data suggests metabolic and epigenetic dysfunctions underlie the arrest of human embryos.

Human embryos develop poorly in vitro, and as much as 60% of embryos will irreversibly arrest. This study shows that the arrested embryos enter a senescent-like state, and reveals that several mechanisms related to zygotic genome activation problems and erroneous metabolism can at least partly explain some of the reasons behind this arrest.