Oxygen transport to embryos in microdrop cultures

The standard method for culturing small preimplantation mammalian embryos is a system in which they are placed into microdrops of culture medium under oil. Thus the source of oxygen for the embryos lies beyond two liquid phases--medium and oil. If transport of oxygen is not sufficiently rapid to replace that consumed by the embryos, the medium could become depleted of oxygen. Over small distances, the dominant means by which oxygen is transported through liquid is by diffusion. Our calculations show that diffusion alone is sufficient to supply oxygen to mouse embryos; there is virtually no perturbation of the oxygen concentration when there are up to 10 embryos in the drop, and 50 embryos produce a drop in oxygen tension that is not large enough to have a deleterious effect. Furthermore, diffusion is probably not the dominant mechanism by which oxygen is transported to the embryos; on the scale of these microdrops, convection is faster and would serve to mix the drop so that anoxic regions cannot develop. Therefore, we conclude that even a relatively large number of embryos in a culture drop do not significantly deplete oxygen.     

Morphological and physiological effects of beta-hydroxybutyrate on rat embryos grown in vitro at different stages

Diabetic women are more likely to give birth to infants with congenital malformations than are nondiabetic women. Rodent embryos have been used as a model for the study of abnormal fetal development associated with maternal diabetes, and some of the metabolic factors which are altered in diabetes, such as raised glucose and ketones, have been shown to cause abnormal development of rodent embryos in vitro. The present work explores further the teratogenicity of beta-hydroxybutyrate to rat embryos. To determine the sensitivities of rat embryos at different stages of their development, rat embryos at 9.5 days of gestation have been cultured in vitro for 24 or 48 h, with or without 4 x 10(-2) M beta-hydroxybutyrate for all or part of the culture period. The embryos have been examined by scanning electron microscopy, and a detailed morphometric analysis of one tissue, the neuroepithelium, has been undertaken. The results confirm that beta-hydroxybutyrate causes abnormal development of rat embryos. The results of experiments in which embryos were exposed to beta-hydroxybutyrate for only part of a 48 h culture show that embryos exposed to beta-hydroxybutyrate for a complete 48 h culture are more severely affected than embryos exposed to beta-hydroxybutyrate for only part of the culture and that embryos are more vulnerable to beta-hydroxybutyrate during the first half of a 48 h culture (equivalent to 9.5 to 10.5 days of gestation) than during the second half of a 48 h culture (10.5 to 11.5 days of gestation). The results of experiments in which embryos were cultured with beta-hydroxybutyrate from 9.5 days of gestation for 24 h (equivalent to 9.5 to 10.5 days of gestation) showed that some effects of beta-hydroxybutyrate are already apparent after 24 hours in culture. Many of the abnormalities produced by beta-hydroxybutyrate can be classified as embryonic retardations rather than malformations--that is, embryos show features characteristic of normal, but younger, embryos. Embryos exposed to beta-hydroxybutyrate for the complete 48 h culture period consume less glucose and produce less lactate than control embryos on a per embryo basis, but not on a per microgram protein basis, suggesting that the reduced metabolism is an effect of beta-hydroxybutyrate-induced developmental delay rather than a cause of it.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sensitivity of fragmented and stratified sea urchin embryos to cytotoxic neuropharmacological preparations

The red half embryos and related quarter embryos (yolk and pigment) of Arbacia lixula, obtained by means of centrifugation of the eggs in sucrose gradient, retain the normal level of sensitivity and supersensitivity to cytotoxic neuropharmaca, antagonists of biogenic monoamines. The white half embryos and clear quarter embryos practically lack supersensitivity whereas the granular quarter embryos restore it to the initial level. The non-pigmented blastomers of stratified embryos are characterized by somewhat weakened supersensitivity. A suggestion is put forward that the supersensitive embryos of A. lixula possess a sensibilizing factor which couples the supersensitivity receptors with the processes of cell division and moves together with the yolk granules upon centrifugation. This factor is not observed in the Strongylocentrotus granularis embryos lacking evident supersensitivity.     

Metabolism and cell allocation during parthenogenetic preimplantation mouse development

Diploid parthenogenetic postimplantation mouse embryos, containing two maternal genomes, are characterized by poor development of extraembryonic membranes derived from the trophectoderm and primitive endoderm of the blastocyst. This is thought to be caused by a deficiency of expression of paternally derived imprinted genes. Here we have compared the inner cell mass, from which the primitive endoderm and fetal lineages are derived, and the trophectoderm, which forms a major component of the placenta, in parthenogenetic and fertilized preimplantation embryos. We have also studied the metabolism from the 1-cell to the blastocyst stage. Cell numbers were reduced in the ICM and TE of parthenogenetic blastocysts compared to fertilized blastocysts. This was thought to be due to the increased levels of cell death observed in these lineages. Pyruvate and glucose uptake by parthenogenetic embryos was similar to that by fertilized embryos throughout preimplantation development. However, at the expanded blastocyst stage glucose uptake by parthenogenetic embryos was significantly higher than by fertilized embryos. The implications of the actions of imprinted genes and of X-inactivation is discussed. © 1996 Wiley-Liss, Inc.     

Eugenic Selection Benefits Embryos

The primary question to be addressed here is whether pre‐implantation genetic diagnosis (PGD), used for both negative and positive trait selection, benefits potential supernumerary embryos. The phrase ‘potential supernumerary embryos’ is used to indicate that PGD is typically performed on a set of embryos, only some of which will be implanted. Prior to any testing, each embryo in the set is potentially supernumerary in the sense that it may not be selected for implantation. Those embryos that are not selected, and hence destroyed or frozen, are ‘actually supernumerary’. The argument to be advanced is hypothetical: If embryos may be said to benefit or be harmed by our actions, then PGD used to select for an embryo or embryos with the highest expected Wellbeing benefits potential supernumerary embryos. The argument shows that the ‘non‐identity’ problem is not sufficient to show that eugenic selection does not benefit supernumerary embryos.     

N^7＋重离子注入和贯穿后小麦种胚的期外DNA合成效应

用＾３ＨＴｄＲ掺入法研究了经Ｎ＾７＋重离子注入贯穿处理的８２５７９小麦和８８１２小麦种胚的ＤＮＡ合成动态。结果发现,未经Ｎ＾７＋重离子任何处理的两个小麦品种的对照种胚,在萌发早期（２０ｈ内）仅存在一个ＤＮＡ合成峰（于萌发的第１４ｈ）,而经过Ｎ＾７＋重离子注入和贯穿处理的小麦种胚则存在两个ＤＮＡ合成峰（分别于萌发的８－１０ｈ和１４－１６ｈ）,该种子经ＤＮＡ修复合成的抑制剂咖啡因处理后,第一个ＤＮＡ合     

Osmotic stress and abscisic acid induce expression of the wheat Em genes

The early-methionine-labelled (Em) polypeptide is the single most abundant cytosolic protein of dry wheat embryos. It is encoded by messenger RNA which accumulates during the later (maturation) stages of embryogenesis. The accumulation of Em mRNA can be induced in isolated developing embryos, in culture, by the application of the plant growth regulator, abscisic acid, which prevents precocious germination. Precocious germination is also inhibited by the culture of embryos under conditions of osmotic stress when accumulation of Em mRNA is induced. This induction occurs in the absence of any significant increase in the endogenous levels of embryonic abscisic acid although there is a requirement for the continued presence of the growth regulator. Additionally, expression of Em genes can be repeated during early germination, if imbibing embryos are subjected to osmotic stress. Induction of Em-gene expression by osmotic stress is consistent with the proposed role of the Em polypeptide in mediating the remarkable tolerance of cereal embryos to the programmed desiccation undergone during their maturation.     

小鼠四倍体早期胚胎基因组甲基化模式

利用小鼠抗5-甲基胞嘧啶(5MeC)单克隆抗体检测了体外培养小鼠四倍体早期胚胎的基因组甲基化模式。结果表明: 利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程, 在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样, 呈现高度甲基化状态; 在细胞核开始融合的时候, 甲基化水平急速下降, 在细胞核完全融合的时候甲基化水平达到最低点; 随着胚胎继续分裂, 胚胎甲基化水平逐渐增加, 在桑葚胚期甲基化水平最高; 但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别, 这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。因此, 小鼠体外培养四倍体胚胎的甲基化模式是不正常的, 这可能是四倍体小鼠难以发育到妊娠足月的原因之一。这是对小鼠四倍体早期胚胎基因组甲基化模式的首次报道。     

Evolution of predetermined germ cells in vertebrate embryos: implications for macroevolution

The germ line is established in animal embryos with the formation of primordial germ cells (PGCs), which give rise to gametes. Therefore, the need to form PGCs can act as a developmental constraint by inhibiting the evolution of embryonic patterning mechanisms that compromise their development. Conversely, events that stabilize the PGCs may liberate these constraints. Two modes of germ cell determination exist in animal embryos: (a) either PGCs are predetermined by the inheritance of germ cell determinants (germ plasm) or (b) PGCs are formed by inducing signals secreted by embryonic tissues (i.e., regulative determination). Surprisingly, among the major extant amphibian lineages, one mechanism is found in urodeles and the other in anurans. In anuran amphibians PGCs are predetermined by germ plasm; in urodele amphibians PGCs are formed by inducing signals. To determine which mechanism is ancestral to the tetrapod lineage and to understand the pattern of inheritance in higher vertebrates, we used a phylogenetic approach to analyze basic morphological processes in both groups and correlated these with mechanisms of germ cell determination. Our results indicate that regulative germ cell determination is a property of embryos retaining ancestral embryological processes, whereas predetermined germ cells are found in embryos with derived morphological traits. These correlations suggest that regulative germ cell formation is an important developmental constraint in vertebrate embryos, acting before the highly conserved pharyngula stage. Moreover, our analysis suggests that germ plasm has evolved independently in several lineages of vertebrate embryos.     

Ontogeny of low molecular weight stress protein p26 during early development of the brine shrimp, Artemia franciscana

Embryogenesis in the brine shrimp, Artemia sp., occurs by one of two pathways: (i) the direct, uninterrupted development of nauplius larvae within the female or (ii) the production of embryos that arrest development at the gastrula stage and enter diapause. Diapause embryos are released from females into the aqueous environment where they remain in diapause until activated by appropriate environmental cues and resume development. These encysted embryos possess at least one low molecular weight stress protein, which we refer to as p26 and which has been implicated previously in the stress response of activated embryos. We investigated the appearance of p26 in developing diapause embryos in utero and looked for its presence in embryos developing directly into nauplii. We found p26 to be specific to diapause-destined embryos; it was not detected in direct-developing embryos. We conclude that p26 is not required for the basic developmental program that produces the nauplius. In diapause-destined embryos, p26 was first detectable after 3 days of development, at which time the embryos were late gastrulae. This protein continues to increase in amount until the encysted embryos are released, approximately 5 days after fertilization. At the time of release almost all p26 is located in the low speed supernatant fraction, but as released embryos continue diapause, p26 transfers to the pelleted nuclear fraction in increasing amounts. Our working hypothesis views p26 as a molecular chaperone preventing protein denaturation and aggregation under conditions associated with metabolic arrest and other stressful states, which these encysted embryos encounter.     

寒武纪宽川铺生物群中球状胚胎化石表面刺状结构及其意义*

以宽川铺生物群中获取的磷酸盐化立体保存的胚胎化石为研究材料,对胚胎化石表面超微结构和内部解剖结构进行观察并对比分析。结果显示,大部分胚胎化石表面被一单层光滑卵膜结构所包裹,卵膜之下胚胎形态多样;刺状结构位于卵膜之下,为原生质胚胎分泌而成的保护层,出现在不同大小和不同类型的胚胎中。结果表明:1)单一依靠胚胎大小或者表面结构不能作为胚胎的分类依据;2)与现生海葵等动物胚胎表面微绒毛相似,Olivooides表面刺状结构可能具有保护胚胎和生态扩散的功能;3)带刺Olivooides很可能发育成为动物演化早期最简单的带刺幼虫形式,是动物间接发育的证据。     

Response to heat shock of different sea urchin species

It is demonstrated that sea urchin embryos of the species Sphaerechinus granularis are able to respond to heat shock by producing heat shock proteins at the same stage as embryos of Paracentrotus lividus, i.e. after hatching. Arbacia lixula embryos are able to synthesize heat shock proteins already at the stage of 64-128 blastomeres. Embryonic survival is observed if the embryos are heated at the stages at which they can synthesize the heat shock proteins. The inhibition of the bulk protein synthesis after heating at 31 degrees C is never less than 50%.     

Acquisition of a defended resource: a benefit of group foraging for the neotropical wrasse,Thalassoma lucasanum

Synopsis The neotropical Pacific wrasse, Thalassoma lucasanum, forms groups of 30 to 300 individuals that overwhelm the defense of embryos by the Pacific sergeant major, Abudefduf troschelii, and thereby gain access to a food resource from which solitary individuals are effectively excluded by paternal defense. The duration of feeding by the wrasse groups and the fraction of embryos eaten in a nest are positively correlated with group size. The benefit of group foraging is probably derived from a reduction in the frequency with which group members experience attack by defending sergeant majors, as reflected in the positive correlation of group size with feeding duration. Large foraging groups only form when sergeant major embryos are present in nests, indicating that this is a primary benefit of participation in these groups for this wrasse. In contrast, in the Caribbean large foraging groups of the congeneric bluehead wrasse, T. bifasciatum, fail to form despite the presence of defended embryos of the Caribbean sergeant major, A. saxatilis. This might be due to the relatively low population densities of the Caribbean wrasse in comparison to those of the Pacific wrasse or to the relatively low densities of Caribbean sergeant major nests containing embryos at any time.     

A novel cytochrome P450, zebrafish Cyp26D1, is involved in metabolism of all-trans retinoic acid

Retinoid signaling is essential for development of vertebrate embryos, and its action is mainly through retinoic acid (RA) binding to its RA receptors and retinoid-X receptors, while the critical concentration and localization of RA in embryos are determined by the presence and activity of retinal dehydrogenases (for RA synthesis) and cytochrome P450 RAs (Cyp26s) (for degradation of RA). Previously, we identified a novel cyp26 gene (cyp26d1) in zebrafish that is expressed in hindbrain during early development. Using reverse-phase HPLC analyses, we show here that zebrafish Cyp26D1 expressed in 293T cells could metabolize all-trans RA, 9-cis RA, and 13-cis RA, but could not metabolize retinol or retinal. The metabolites of all-trans RA produced by Cyp26D1 were the same as that produced by Cyp26A1, which are mainly 4-hydroxy-all-trans-RA and 4-oxo-all-trans-RA. Performing mRNA microinjection into zebrafish embryos, we demonstrated that overexpression of Cyp26D1 in embryos not only caused the distance between rhombomere 5 and the first somite of the injected embryos to be shorter than control embryos but also resulted in left-right asymmetry of somitogenesis in the injected embryos. These alterations were similar to those caused by the overexpression of cyp26a1 in zebrafish embryos and to that which resulted from treating embryos with 1 microm 4-diethylamino-benzaldehyde (retinal dehydrogenase inhibitor), implying that cyp26d1 can antagonize RA activity in vivo. Together, our in vitro and in vivo results provided direct evidence that zebrafish Cyp26D1 is involved in RA metabolism.