VAP1, with cystatin C motif, an oocyte protein encoded by a novel ovarian-specific gene during oogenesis in the common brushtail possum (Trichosurus vulpecula)

In the brushtail possum oocyte, vesicles accumulate in a polarized fashion at the vegetal pole and cytoplasm rich in mitochondria and containing the germinal vesicle comprise the animal pole. During cleavage to early blastocyst stages, animal pole cytoplasm locates to the cells of the embryonic hemisphere (pluriblast) and vegetal pole vesicular cytoplasm to cells of the abembryonic hemisphere (trophoblast). Previously identified 16 amino acid residues, associated with the vesicle-rich cytoplasm were used for molecular cloning and characterization of a vesicle associated protein, VAP1. The degenerate primer was used in a 3'RACE for vap1 gene cloning. The cDNA encoding VAP1 was 516 bp in length with no significant homologies and coded for 172 amino acid residues for the mature protein. The N-terminal domain of VAP1 showed a structural homology to the cysteine protease inhibitor, Cystatin. Gene expression studies during oogenesis revealed that vap1 had an ovary-specific, possibly oocyte-specific expression, which occurs during follicle formation and growth and in adult ovaries. Recombinant VAP1 fusion protein generated polyclonal antibodies in the mouse and in the brushtail possum.     

Cloning and characterization of a new gene from the PAT protein family,in a marsupial,the stripe‐faced dunnart (Sminthopsis macroura)

Recent studies of PAT proteins in Drosophila and Xenopus have revealed significant roles for this family of proteins in the polarized transport of lipid droplets and maternal determinants during early embryogenesis. In mammals, PAT proteins are known to function mainly in lipid metabolism, yet research has yet to establish a role for PAT proteins in mammalian embryogenesis. Oocytes and early cleavage stages in Sminthopsis macroura show obvious polarized cytoplasmic distribution of organelles, somewhat similar to Drosophila and Xenopus, suggesting that a PAT protein may also be involved in S. macroura embryonic development. In the present study, we identified a new marsupial gene for PAT family proteins, DPAT, from S. macroura. Expression analyses by RT‐PCR and whole mount fluorescent in situ hybridization revealed that DPAT expression was specific to oocytes and cleavage stage conceptuses. Analysis of the localization of lipid droplets during S. macroura early embryonic development found a polarized distribution of lipid droplets at the two‐ and four‐cell stage, and an asymmetric enrichment in blastomeres on one side of conceptuses from two‐ to eight‐cell stage. Lipid droplets largely segregate to pluriblast cells at the 16‐cell stage, suggesting a role in pluriblast lineage allocation. Mol. Reprod. Dev. 77: 373–383, 2010. © 2010 Wiley‐Liss, Inc.     

凹耳臭蛙胚胎发育及其分类学意义(英文)

通过人工受精的方法获得的凹耳臭蛙(Odorrana tormota)的早期胚胎及胚后幼体的发育过程,根据胚胎发育过程中的形态及生理特征变化规律进行分期。把凹耳臭蛙的发育过程分成两个阶段:1)早期胚胎发育阶段,即从蛙卵受精到鳃盖完成期,在18～23℃水温下,凹耳臭蛙早期胚胎发育阶段历时324h;2)蝌蚪发育阶段,即从鳃盖完成期结束到尾部被完全吸收,本阶段在20～24℃水温条件下历时1207h。凹耳臭蛙蝌蚪未发现腹吸盘特征,从形态特征上支持了分子系统分类学将之从湍蛙属划出的观点。实验中发现,多数胚胎在8细胞期为纬裂,16细胞期为经裂,同时有小部分胚胎(1.5%)在8细胞期为经裂,16细胞期为纬裂。该文进一步讨论了影响卵裂率、孵化率、发育速度,以及生态适应的因素。     

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.     

Inner cell allocation in the mouse morula: the role of oriented division during fourth cleavage

Two populations of blastomeres become positionally distinct during fourth cleavage in the mouse embryo; the inner cells become enclosed within the embryo and the outer cells form the enclosing layer. The segregation of these two cell populations is important for later development, because it represents the initial step in the divergence of placental and fetal lineages. The mechanism by which the inner cells become allocated has been thought to involve the oriented division of polarized 8-cell blastomeres, but this has never been examined in the intact embryo. By using the technique of time-lapse cinemicrography, we have been able for the first time to directly examine the division planes of 8-cell blastomeres during fourth cleavage, and find that there are three, rather than two, major division plane orientations; anticlinal (perpendicular to the outer surface of the blastomere), periclinal (parallel to the outer surface of the blastomere), and oblique (at an angle between the other two). The observed frequencies of each type of division plane orientation provide evidence that the inner cells of the morula must derive from oriented division of 8-cell blastomeres, in accordance with the polarization hypothesis. Analysis of fourth cleavage division plane orientation with respect to either lineage or division order reveals that it is not associated with lineage from either the 2- or the 4-cell stage, but has a slight statistical association with fourth cleavage division order. The lack of association between division plane orientation and lineage supports the prediction that packing patterns and intercellular interactions within the 8-cell embryo during compaction play a role in determining fourth cleavage division plane orientation and thus, the positional fate of the daughter 16-cell blastomeres.     

Blastomeres arising from the first cleavage division have distinguishable fates in normal mouse development

Two independent studies have recently suggested similar models in which the embryonic and abembryonic parts of the mouse blastocyst become separated already by the first cleavage division. However, no lineage tracing studies carried out so far on early embryos provide the support for such a hypothesis. Thus, to re-examine the fate of blastomeres of the two-cell mouse embryo, we have undertaken lineage tracing studies using a non-perturbing method. We show that two-cell stage blastomeres have a strong tendency to develop into cells that comprise either the embryonic or the abembryonic parts of the blastocyst. Moreover, the two-cell stage blastomere that is first to divide will preferentially contribute its progeny to the embryonic part. Nevertheless, we find that the blastocyst embryonic-abembryonic axis is not perfectly orthogonal to the first cleavage plane, but often shows some angular displacement from it. Consequently, there is a boundary zone adjacent to the interior margin of the blastocoel that is populated by cells derived from both earlier and later dividing blastomeres. The majority of cells that inhabit this boundary region are, however, derived from the later dividing two-cell stage blastomere that contributes predominantly to the abembryonic part of the blastocyst. Thus, at the two-cell stage it is already possible to predict which cell will contribute a greater proportion of its progeny to the abembryonic part of the blastocyst (region including the blastocyst cavity) and which to the embryonic part (region containing the inner cell mass) that will give rise to the embryo proper.     

Localized axis determinant in the early cleavage embryo of the goldfish, Carassius auratus

 The teleost dorsoventral axis cannot be distinguished morphologically before gastrulation. In order to examine whether the yolk cell affects axis determination, we bisect early cleavage embryos of the goldfish, Carassius auratus. When the vegetal yolk hemisphere is removed by bisection along the equatorial plane at the 2-cell stage, the embryos develop abnormally and exhibit a symmetrical morphology. No dorsal structures, such as notochord, somites and neural tube, differentiate and no embryonic shield is formed during gastrulation. In addition, no goosecoid mRNA is expressed before gastrulation. The frequency of abnormality decreases as the age at which the vegetal yolk hemisphere is removed increases. Most embryos removed at the 32-cell stage develop normally. Their morphological phenotype is similar to that of a Xenopus ventralized embryo generated by ultraviolet irradiation on the vegetal hemisphere soon after fertilization. We also observed that, when the embryos were bisected along the first cleavage plane at the 2-cell stage, the proportion of pairs of embryos of which one embryo developed normally was 44.8%. These results indicate that the vegetal yolk hemisphere of the early cleavage embryo of the goldfish contains axis determination factor(s), which are necessary for generation of dorsal structures. Furthermore, it is suggested that these determinant(s) are distributed asymmetrically within the vegetal yolk hemisphere. Received: 25 May 1996 / Accepted: 19 September 1996     

Origin of the inner cell mass in mouse embryos: cell lineage analysis by microinjection

The mouse inner cell mass is established by cells that are allocated to internal positions after the 8-cell stage. We analyzed the timing of this allocation by microinjecting two cell lineage markers, horseradish peroxidase and rhodamine-conjugated dextran, into mouse blastomeres at the 8- to 32-cell stage. Prospective analysis was performed by coinjection of peroxidase and dextran, followed by 12-22 hr of culture and staining for peroxidase activity; retrospective analysis was performed by injection of peroxidase alone and localization of sister cells without further culture. Both approaches indicated that cells are allocated to internal positions during the fourth and fifth cleavage divisions, but not the sixth cleavage division, of the mouse embryo. Thus, outer cells can have inner descendants until the late morula/early blastocyst (32-cell) stage, but cells remaining outside after the fifth cleavage division are restricted to a trophectoderm fate. This information about cell lineage indicates that the previously observed totipotency of the cleaving mammalian embryo's cells is a regulative attribute that is used in normal development.     

Allocation of cells in mouse blastocyst is not determined by the order of cleavage of the first two blastomeres

The second cleavage of the mouse embryo is asynchronous. Some recent investigators have proposed that the sequence of division of blastomeres in two-cell embryos may predict the ultimate location of the descendants of these blastomeres within the blastocyst. To verify this model, we tracked the cells derived from two-cell stage blastomeres using tetramethylrhodamine-conjugated dextran as a lineage tracer. In the first variant of the experiment, we labeled one of two blastomeres in two-cell embryos and subsequently recorded which blastomere cleaved first. In the second variant of the experiment, fluorescent dextran was injected at the three-cell stage into the blastomere that had not yet cleaved. Subsequently, the fate of the progeny of labeled and unlabeled blastomeres was followed up to the blastocyst stage. Our results suggest that allocation of cells into the embryonic and abembryonic parts of the blastocyst is not determined by the order of cleavage of the first two blastomeres.     

Placentation in the Mexican lizard Sceloporus mucronatus (Squamata: Phrynosomatidae)

We used light microscopy to study placental structure of the lizard Sceloporus mucronatus throughout 6 months of embryonic development. Three stages of placental development could be assigned to embryos based on the arrangement of the extraembryonic membranes. A highly vascular choriovitelline placenta was present in the embryonic hemisphere and a nonvascular bilaminar omphalopleure covered most of the abembryonic hemisphere of the egg during embryonic Stages 10-28. A chorioallantoic placenta replaced the choriovitelline placenta by embryonic Stage 29 and an omphaloplacenta covered the abembryonic hemisphere at this stage. The combination of these two placental types occurred in Stage 29-36 embryos. The final stage of placentation, embryonic Stages 37-40, was characterized by an omphalallantoic placenta in the abembryonic hemisphere and a chorioallantoic placenta in the embryonic hemisphere of the egg. The choriovitelline and chorioallantoic placentae are well vascularized, with closely apposed maternal and embryonic blood vessels. These structures are the most likely sites of respiratory exchange. In contrast, the omphaloplacenta and omphalallantoic placentae contain cuboidal or columnar epithelia and these structures may function in histotrophic exchange. Placentation of S. mucronatus is similar to that of predominantly lecithotrophic species in other squamate lineages suggesting that the evolution of this placental morphology is a response to similar factors and is independent of phylogeny.     

Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos

Inner cell mass (ICM) and trophectoderm cell lineages in preimplantation mouse embryos were studied by means of iontophoretic injection of horseradish peroxidase (HRP) as a marker. HRP was injected into single blastomeres at the 2- and 8-cell stages and into single outer blastomeres at the 16-cell and late morula (about 22- to 32-cell) stages. After injection, embryos were either examined immediately for localization of HRP (controls) or they were allowed to develop until the blastocyst stage (1 to 3.5 days of culture) and examined for the distribution of labeled cells. In control embryos, HRP was confined to one or two outer blastomeres. In embryos allowed to develop into blastocysts, HRP-labeled progeny were distributed into patches of cells, showing that there is limited intermingling of cells during preimplantation development. A substantial fraction of injected blastomeres contributed descendants to both ICM and trophectoderm (95, 58, 44, and 35% for injected 2-cell, 8-cell, 16-cell, and late morula stages, respectively). Although more than half of the outer cells injected at 16-cell and late morula stages contributed descendants only to trophectoderm (53 and 63%, respectively), some outer cells contributed also to the ICM lineage even at the late morula stage. Although the mechanism for allocation of outer cells to the inner cell lineage is unknown, our observation of adjacent labeled mural trophectoderm and presumptive endoderm cells implicated polarized cell division. This observation also suggests that mural trophectoderm and presumptive endoderm are derived from common immediate progenitors. These cells appear to separate into inner and outer layers during the fifth cleavage division. Our results demonstrate the usefulness of HRP as a cell lineage marker in mouse embryos and show that the allocation of cells to ICM or trophectoderm begins after the 2-cell stage and continues into late cleavage.     

Cleavage and gastrulation of the euphausiacean<Emphasis Type="Italic"> Meganyctiphanes norvegica</Emphasis> (Crustacea,Malacostraca)

According to the Articulata hypothesis the cleavage of arthropods must be derived from spiral cleavage. However, arthropods show a great variety of cleavage modes with a widespread occurrence of superficial cleavage. In the Malacostraca, holoblastic cleavage occurs in some taxa such as Amphipoda, Euphausiacea and Dendrobranchiata. In particular, the cleavage of euphausiaceans has been proposed to be a modified spiral cleavage. The cell lineage of early stages up to blastoderm formation of the euphausiacean Meganyctiphanes norvegica is reconstructed using recent methods of fluorescent staining. Only the oblique angle of the mitotic spindles during the transition from the 2- to the 4-cell stage resembles the spiral cleavage mode. At the 8-cell stage, four cells each form a pattern of two interlocking bands which is preserved until the 122-cell stage. One blastomere is delayed in division and shows an oblique division from the fourth cleavage on. It is the precursor cell of two enlarged and cleavage-arrested cells at the 32-cell stage. At the 62-cell stage, these two cells are surrounded by eight cells following a specific cell division pattern during the subsequent division cycles. The cleavage pattern of M. norvegica occurs in two mirror images. A comparative approach reveals distinct similarities between the early cleavage patterns of Euphausiacea and Dendrobranchiata which are suggested to be homologous. Furthermore, the relationships to non-malacostracan cleavage patterns are discussed. It is shown that the early cleavage pattern of M. norvegica does not offer an example of a spiral cleavage within arthropods.     

Trophoblast and hypoblast in the monotreme, marsupial and eutherian mammal: evolution and origins

The pregastrula stage mammalian conceptus consists of both embryonic and non-embryonic components. The latter forms the bulk of the tissues, provides nutrition for the developing embryo and also contributes developmental signals that influence events within the embryo itself. Understanding the origins and relationships between the embryonic and extraembryonic cell lineages is thus central to understanding development in mammals. Despite the apparent gross differences in early developmental strategy and form, the conceptuses of eutherian, marsupial and monotreme mammals show some remarkable similarities in the lineage allocation to trophoblast and hypoblast and in the emergent properties of the two cell types. We suggest that the gross differences can be explained by two relatively small evolutionary timing changes affecting cell adhesion patterns and the polarisation of developmentally significant information. These changes result in the conversion of a unilaminar blastocyst to a morula form composed of blastomeres with increased regulatory capacity.     

On the classification of the cleavage patterns in amphibian embryos

This paper presents a brief survey and preliminary classification of embryonic cleavage patterns in the class Amphibia. We use published data on 41 anuran and 22 urodele species concerning the character of the third cleavage furrow (latitudinal or longitudinal) and the stage of transition from synchronous to asynchronous blastomere divisions in the animal hemisphere (4–8-celled stage, 8–16-celled stage or later). Based on this, four patterns of amphibian embryonic cleavage are recognized, and an attempt to elucidate the evolutionary relationships among these patterns is undertaken. The so-called “standard” cleavage pattern (the extensive series of synchronous blastomere divisions including latitudinal furrows of the third cleavage) with the typical model species Ambystoma mexicanum and Xenopus laevis seems to be derived and probably originated independently in the orders Anura and Caudata. The ancestral amphibian cleavage pattern seems to be represented by species with longitudinal furrows of the third cleavage and the loss of synchrony as early as the 8-celled stage (such as in primitive urodele species from the family Cryptobranchidae).     

Early patterning of the mouse embryo--contributions of sperm and egg

The first cleavage of the fertilised mouse egg divides the zygote into two cells that have a tendency to follow distinguishable fates. One divides first and contributes its progeny predominantly to the embryonic part of the blastocyst, while the other, later dividing cell, contributes mainly to the abembryonic part. We have previously observed that both the plane of this first cleavage and the subsequent order of blastomere division tend to correlate with the position of the fertilisation cone that forms after sperm entry. But does sperm entry contribute to assigning the distinguishable fates to the first two blastomeres or is their fate an intrinsic property of the egg itself? To answer this question we examined the distribution of the progeny of early blastomeres in embryos never penetrated by sperm - parthenogenetic embryos. In contrast to fertilised eggs, we found there is no tendency for the first two parthenogenetic blastomeres to follow different fates. This outcome is independent of whether parthenogenetic eggs are haploid or diploid. Also unlike fertilised eggs, the first 2-cell blastomere to divide in parthenogenetic embryo does not necessarily contribute more cells to the blastocyst. However, even when descendants of the first dividing blastomere do predominate, they show no strong predisposition to occupy the embryonic part. Thus blastomere fate does not appear to be decided by differential cell division alone. Finally, when the cortical cytoplasm at the site of sperm entry is removed, the first cleavage plane no longer tends to divide the embryo into embryonic and abembryonic parts. Together these results indicate that in normal development fertilisation contributes to setting up embryonic patterning, alongside the role of the egg.     

Contributions to the study of oviparity–viviparity transition: Placentary structures of Liolaemus elongatus (Squamata: Liolaemidae)

Liolaemus elongatus (Liolaemidae) is a viviparous, mainly lecithotrophic species with placental structures specialized for uptake of oxygen and inorganic nutrient transport. An allantoplacenta and an omphaloplacenta are present during early embryonic stages (25–28) and there is a moderate distension of the uterine wall and major glandular activity in the uterine mucosa and submucosa compared with nonpregnant females. The uterine epithelium increases in height, first as a growth in the height of some dispersed cells localized in all regions of the placenta, and later as groups of cells localized in the periembryonic and central‐abembryonic regions. At embryonic stage 39, the allantoplacenta reaches its maximum extension around the yolk sac. Omphaloplacenta is restricted to the abembryonic zone, the yolk cleft limiting the newly formed isolated vitelline mass. At more advanced embryonic stages (39–42), the blood supply to the allantoplacenta's periembryonic zone increases, matching the profuse allantoic vascularization. At embryonic stage 42, a secondary cleft opens in the main vitelline mass, above the first yolk cleft, and allantoic blood vessels enter into this secondary cleft. This secondary cleft subdivides the vitelline mass into a large embryonic region connected to a much smaller abembryonic region. In L. elongatus most nutritional resources seem to be provided by the yolk that remains attached to the newborn for 2 or 3 days as an external supply. The embryo's wet weight doubles the weight of the decrease observed in vitelline mass. But the dry wet diminishes, evidencing the importance of the exchange of water and inorganic nutrients. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.