BEFORE I WAS AN EMBRYO,I WAS A PRE-EMBRYO: OR WAS I?

Issues surrounding human embryos are poignant and profound. Should research be conducted on them? Should they be discarded? Should they be donated to infertile couples? The Warnock Report was a landmark in providing guidelines limiting experimentation on human embryos to the first 14 days after fertilization, at which time implantation of the embryo is complete and the primitive streak has appeared.² However, these embryological features were not considered sufficiently distinctive to bestow upon this 14-day period a separate classification. This situation changed when, in 1986, Anne McLaren suggested the use of the terms ‘pre-embryo’ or ‘conceptus' to designate “the entire product of the fertilized egg up to the end of the implantation stage” and the term ’embryo‘ for “that small part of the pre-embryo or conceptus, first distinguishable at the primitive streak stage, that later develops into the foetus.”³ In this paper we look critically at the term ‘pre-embryo’, and we shall present the case for an alternative set of terms, namely, embryo-placenta and embryo-fetus. We consider this latter to be biologically-based terminology, that does not have any connotation of restricted moral value as the term pre-embryo does for some.     

The Beginning of Personhood: A Thomistic Biological Analysis

‘When did I, a human person, begin to exist?’ In developing an answer to this question, I utilize a Thomistic framework, which holds that the human person is a composite of a biological organism and an intellective soul. Eric Olson and Norman Ford both argue that the beginning of an individual human biological organism occurs at the moment when implantation of the zygote in the uterus occurs and the ‘primitive streak’ begins to form. Prior to this point, there does not exist an individual human organism, but a cluster of biological cells which has the potential to split and develop as one or more separate human organisms (identical twinning). Ensoulment (the instantiation of a human intellective soul in biological matter) does not occur until the point of implantation. This conception of the beginning of human personhood has moral implications concerning the status of pre‐implantation biological cell clusters. A new understanding of the beginning of human personhood entails a new understanding of the morality of certain medical procedures which have a direct affect on these cell clusters which contain human DNA. Such procedures discussed in this article are embryonic stem cell research, in vitro fertilization, procured abortion, and the use of abortifacient contraceptives.     

Rapid mitochondrial DNA segregation in primate preimplantation embryos precedes somatic and germline bottleneck

The timing and mechanisms of mitochondrial DNA (mtDNA) segregation and transmission in mammals are poorly understood. Genetic bottleneck in female germ cells has been proposed as the main phenomenon responsible for rapid intergenerational segregation of heteroplasmic mtDNA. We demonstrate here that mtDNA segregation occurs during primate preimplantation embryogenesis resulting in partitioning of mtDNA variants between daughter blastomeres. A substantial shift toward homoplasmy occurred in fetuses and embryonic stem cells (ESCs) derived from these heteroplasmic embryos. We also observed a wide range of heteroplasmic mtDNA variants distributed in individual oocytes recovered from these fetuses. Thus, we present here evidence for a previously unknown mtDNA segregation and bottleneck during preimplantation embryo development, suggesting that return to the homoplasmic condition can occur during development of an individual organism from the zygote to birth, without a passage through the germline.     

Spatial separation of parental genomes in preimplantation mouse embryos

We have used two different experimental approaches to demonstrate topological separation of parental genomes in preimplantation mouse embryos: mouse eggs fertilized with 5-bromodeoxyuridine (BrdU)-labeled sperm followed by detection of BrdU in early diploid embryos, and differential heterochromatin staining in mouse interspecific hybrid embryos. Separation of chromatin according to parental origin was preserved up to the four-cell embryo stage and then gradually disappeared. In F1 hybrid animals, genome separation was also observed in a proportion of somatic cells. Separate nuclear compartments during preimplantation development, when extreme chromatin remodelling occurs, and possibly in some differentiated cell types, may be associated with epigenetic reprogramming.     

PTEN抑制胚胎原肠胚形成期EMT的过程

PTEN(Phosphatase and tensin homolog)是一种重要的抑癌基因,具有非常广泛的生物学活性,例如在细胞的生长发育、迁移、凋亡和信号传导等均发挥重要作用。PTEN基因表达始于在胚胎早期的上胚层,而后主要出现在神经外胚层和胚胎中胚层结构,表明PTEN可能参与胚胎早期发育过程的细胞迁移、增殖和分化。文章主要应用在体改变早期胚胎PTEN的表达水平来观察其对上胚层至中胚层细胞转换—EMT(Epithe-lial-mesenchymal transition)的作用。首先,原位杂交结果提示,内源性PTEN表达在原条以及之后的中胚层细胞结构如体节等。在体PTEN转染实验,体外培养至HH3期的鸡胚胎,转染Wt PTEN-GFP或移植Wt PTEN-GFP原条组织至未转染的同时期的宿主胚胎相同部位后,观察到PTEN转染细胞大都由上胚层迁移至原条并滞留于原条,不再参与中胚层细胞形成。移植实验也得到相似结果,发现在Wt PTEN-GFP阳性原条组织移植后很少细胞迁移出原条。另外在原肠胚期PTEN siRNA降调胚胎一侧PTEN基因后,降调侧中胚层细胞数明显少于正常侧。上述研究结果均提示PTEN基因在胚胎原肠胚期三胚层形成过程中可能具有抑制EMT的作用。     

Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo

Gastrulation in higher vertebrate species classically commences with the generation of mesoderm cells in the primitive streak by epithelio-mesenchymal transformation of epiblast cells. However, the primitive streak also marks, with its longitudinal orientation in the posterior part of the conceptus, the anterior-posterior (or head-tail) axis of the embryo. Results obtained in chick and mouse suggest that signals secreted by the hypoblast (or visceral endoderm), the extraembryonic tissue covering the epiblast ventrally, antagonise the mesoderm induction cascade in the anterior part of the epiblast and thereby restrict streak development to the posterior pole (and possibly initiate head development anteriorly). In this paper we took advantage of the disc-shape morphology of the rabbit gastrula for defining the expression compartments of the signalling molecules Cerberus and Dickkopf at pre-gastrulation and early gastrulation stages in a mammal other than the mouse. The two molecules are expressed in novel expression compartments in a complementary fashion both in the hypoblast and in the emerging primitive streak. In loss-of-function experiments, carried out in a New-type culturing system, hypoblast was removed prior to culture at defined stages before and at the beginning of gastrulation. The epiblast shows a stage-dependent and topographically restricted susceptibility to express Brachyury, a T-box gene pivotal for mesoderm formation, and to transform into (histologically proven) mesoderm. These results confirm for the mammalian embryo that the anterior-posterior axis of the conceptus is formed first as a molecular prepattern in the hypoblast and then irrevocably fixed, under the control of signals secreted from the hypoblast, by epithelio-mesenchymal transformation (primitive streak formation) in the epiblast.Edited by D. Tautz     

Prenatal fate of parthenogenetic cells in mouse aggregation chimaeras

Parthenogenetically activated BCF1 and fertilized BALB/c embryos were aggregated to form chimaeras. The fate of the parthenogenetic component was followed in the conceptus during the second half of gestation. The results indicate an early strong selection against parthenogenetic cells in the extra-embryonal part, which is presumably complete by term, and a weaker selective process in the embryo. During early development, parthenogenetic cells have nearly normal developmental potency in the embryo, which allows their balanced contribution in the chimaeras on day 12. Later, this contribution declines significantly resulting in an unbalanced relation to the advantage of the fertilized counterpart. From the results, we suggest that gametic imprinting may play a role not only in the key steps of preimplantation and early postimplantation development, but later in cell and tissue differentiation.     

Evolution of germ cell development in tetrapods: comparison of urodeles and amniotes

SUMMARY. The embryonic development of germ cells in tetrapods is described, focusing on groups with the inductive mode of germ cell specification. In mammals PGCs are induced early in the gastrulation process, they are internalized with future extraembryonic mesoderm in the early posterior primitive streak, and specified soon thereafter. Strong evidence indicates that a similar process occurs in turtles and some other reptiles. In amniotes, the PGCs appear well before formation of the gonad in the posterior trunk, resulting in a period in which they are located outside the embryo before their migration to the gonad. In contrast, in urodeles the PGCs appear relatively late, and throughout development maintain a position close to precursors of the somatic cells of the gonad so that migration is not required. In lampreys early development of germ cells is strikingly similar to that in urodeles, suggesting this is the primitive process. As amniotes evolved large yolky eggs and better access to nutrition, development of the posterior half of the trunk became more dependent on cell proliferation; this was followed or accompanied by a shift of early germ cell development to the equivalent of the early primitive streak. A similar process may have occurred as some basal vertebrates developed large yolky eggs.     

Chromosome studies in human in vitro fertilization

Summary The chromosome constitution of 22 human preimplantation embryos from donor oocytes fertilized in vitro by donor sperm was studied to assess the contribution of lethal chromosome anomalies to the high failure rate of implantation of in vitro fertilized embryos after embryo transfer in infertile women. Evidence was found of nondisjunction, resulting in trisomy, monosomy, and nullosomy; structural abnormalities; haploidy; and triploidy. Despite the lethality of their chromosome complements, these embryos could not be distinguished morphologically from those with normal chromosomes.     

Post-hatching development of the porcine and bovine embryo--defining criteria for expected development in vivo and in vitro

Particular attention has been paid to the pre-hatching period of embryonic development although blastocyst development is a poor indicator of embryo viability. Post-hatching embryonic development in vitro would allow for establishment of more accurate tools for evaluating developmental potential without the need for transfer to recipient animals. Such a system would require (1) definition of milestones of expected post-hatching embryonic development in vivo; and (2) development of adequate culture systems. We propose a stereomicroscopical staging system for post-hatching embryos defining the following stages: (1) Expanded hatched blastocyst stage where the embryo presents an inner cell mass (ICM) covered by trophoblast. (2) Pre-streak stage 1 where the embryonic disc is formed. (3) Pre-streak stage 2 where a crescent-shaped thickening of the caudal portion of the embryonic disk appears. (4) Primitive streak stage where the primitive streak has developed as an axis of cell ingression of cells for meso- and endoderm formation. (5) Neural groove stage where the neural groove is developing from the rostral pole of the embryo along with a proportional shortening of the primitive streak; and (6) Somite stage(s) where paraxial mesoderm gradually condensates to form somites. Post-hatching development of bovine embryos in vitro is compromised and although hatching occurs and elongation can be physically provoked by culture in agarose tunnels, the embryonic disk characterizing the pre-streak stage 1 is never established. Thus, particular focus should be placed on establishing culture conditions that support at least some of the above-mentioned critical phases of development that in vivo occur within the initial two (pig) to three (cattle) weeks.     

Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development

Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.     

Amino acid transport regulation and early embryo development

Amino acids are essential components of media utilized to culture fertilized human eggs to the blastocyst stage in vitro. Use of such media has led to a significant increase in the proportion of embryos that implant upon transfer to the uterus and to a decrease in the number that need to be transferred to achieve pregnancy. Little is known about the mechanisms by which amino acids foster development of healthy human blastocysts. Indications are, however, that many of these mechanisms are the same in human and mouse embryos. Both essential and nonessential amino acid transport benefit preimplantation mouse embryo development, albeit at different stages. Nonessential amino acid transport improves development primarily during cleavage, whereas essential amino acid transport supports development of more viable embryos, especially subsequent to the eight-cell stage. This review discusses likely mechanisms for these beneficial effects.     

The embryo pancreas is a source of increased yolk amylase in the fertilized eggs of domestic fowls.

The amylases were studied in the yolk of fertilized eggs and in the pancreases of the embryos of domestic fowls. The amylase activity in the yolk increased markedly from 13 days of incubation until hatching, but the activity decreased when the embryos were taken out of the eggs. The isoamylases in the yolk and in the pancreas of the embryo were identical electrophoretically. The amylase occurs mainly in the pancreas of the embryo. We think that the increase in amylase activity in the yolk of fertilized eggs during incubation depends upon the accumulation of pancreatic amylase synthesized by the developing embryo in the egg.     

Adaptive responses by mouse early embryos to maternal diet protect fetal growth but predispose to adult onset disease

Poor maternal nutrition during pregnancy can alter postnatal phenotype and increase susceptibility to adult cardiovascular and metabolic diseases. However, underlying mechanisms are largely unknown. Here, we show that maternal low protein diet (LPD), fed exclusively during mouse preimplantation development, leads to offspring with increased weight from birth, sustained hypertension, and abnormal anxiety-related behavior, especially in females. These adverse outcomes were interrelated with increased perinatal weight being predictive of later adult overweight and hypertension. Embryo transfer experiments revealed that the increase in perinatal weight was induced within blastocysts responding to preimplantation LPD, independent of subsequent maternal environment during later pregnancy. We further identified the embryo-derived visceral yolk sac endoderm (VYSE) as one mediator of this response. VYSE contributes to fetal growth through endocytosis of maternal proteins, mainly via the multiligand megalin (LRP2) receptor and supply of liberated amino acids. Thus, LPD maintained throughout gestation stimulated VYSE nutrient transport capacity and megalin expression in late pregnancy, with enhanced megalin expression evident even when LPD was limited to the preimplantation period. Our results demonstrate that in a nutrient-restricted environment, the preimplantation embryo activates physiological mechanisms of developmental plasticity to stablize conceptus growth and enhance postnatal fitness. However, activation of such responses may also lead to adult excess growth and cardiovascular and behavioral diseases.     

The hamster as a model for embryo implantation: insights into a multifaceted process

Defects in preimplantation embryonic development, uterine receptivity, and implantation are the leading cause of infertility, pregnancy problems and birth defects. Significant progress has been made in our basic understanding of these processes using the mouse model, where implantation is ovarian estrogen-dependent in the presence of progesterone. However, an animal model where implantation is progesterone-dependent must also be studied to gain a full understanding of the embryo and uterine events that are required for implantation. In this regard, the hamster is a useful model and this review summarizes the information currently available regarding mechanisms involved in synchronous preimplantation embryo and uterine development for implantation in this species.     

Limited left-right cell migration across the midline of the gastrulating avian embryo

During avian development the earliest phase in which the avian embryo expresses axial features of a left-right axis is at the primitive streak stage. Until the stage of definitive primitive streak (streak 4 H&H), the axis seems to possess morphological bilateral symmetry. Morphological asymmetry begins only during the next few hours of incubation, with development of overt morphological and molecular asymmetry within Hensen's node (stage 5 H&H). In this report, we present an experimental study aimed at following the pattern of cell movements during primitive streak formation and gastrulation of specific left-right regions from earlier stages of the avian embryo. To determine the origin of cells contributing to each side of the primitive streak, we applied the dye Lysinated-Rodamine-Dextran (LRD) to one half, either left or right, of the pre-streak blastoderm (stages X–XIII, EG&K). We tried to estimate the relative cell contribution to primitive streak formation, and to the three germ layers evolving during gastrulation in the context of the left-right axis. Moreover, we asked whether the midline serves as a border, that is, as a physiological barrier preventing cell passing during gastrulation. Our results demonstrate that on each side of the axis, either the right or the left, most of the cells originate from the same half of a pre-streak blastoderm, populate the same half of the PS and contribute to tissues largely confined to that particular side. However, along the primitive streak, a few cells were detected on the opposite side of the midline. Moreover, variation in the number of cells crossing the midline at specific regions along the primitive streak was found. Most crossing cells were located near the mid rostrocaudal extent of the primitive streak, from 25–85% of its length. At the posterior end of the primitive streak, fewer crossing cells were detected. At the anterior region of the PS, that is, within Hensen's node, cells do not cross the midline. These results suggest that differences occur in the process of ingression along the rostrocaudal extent of the PS. Dev. Genet. 23:175–184, 1998. © 1998 Wiley-Liss, Inc.     

Polarity of the mouse embryo is anticipated before implantation

In most species, the polarity of an embryo underlies the future body plan and is determined from that of the zygote. However, mammals are thought to be an exception to this; in the mouse, polarity is generally thought to develop significantly later, only after implantation. It has not been possible, however, to relate the polarity of the preimplantation mouse embryo to that of the later conceptus due to the lack of markers that endure long enough to follow lineages through implantation. To test whether early developmental events could provide cues that predict the axes of the postimplantation embryo, we have used the strategy of injecting mRNA encoding an enduring marker to trace the progeny of inner cell mass cells into the postimplantation visceral endoderm. This tissue, although it has an extraembryonic fate, plays a role in axis determination in adjacent embryonic tissue. We found that visceral endoderm cells that originated near the polar body (a marker of the blastocyst axis of symmetry) generally became distal as the egg cylinder formed, while those that originated opposite the polar body tended to become proximal. It follows that, in normal development, bilateral symmetry of the mouse blastocyst anticipates the polarity of the later conceptus. Moreover, our results show that transformation of the blastocyst axis of symmetry into the axes of the postimplantation conceptus involves asymmetric visceral endoderm cell movement. Therefore, even if the definitive axes of the mouse embryo become irreversibly established only after implantation, this polarity can be traced back to events before implantation.