Preimplantation development

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CHAPTER 8 Preimplantation development

Understanding the spatial and temporal developmental processes that take place within an embryo as it develops from a single cell into a recognizable human is the challenge of embryology. The control of these processes resides within the genome: fundamental questions remain concerning the genes and interactions involved in development.

STAGING OF EMBRYOS

For the purposes of embryological study, prenatal life is divided into an embryonic period and a fetal period. The embryonic period covers the first 8 weeks of development (weeks following ovulation and fertilization resulting in pregnancy). The ages of early human embryos have previously been estimated by comparing their development with that of monkey embryos of known postovulatory ages. Because embryos develop at different rates and attain different final weights and sizes, a classification of human embryos into 23 stages occurring during the first 8 weeks after ovulation was developed most successfully by Streeter (1942), and the task was continued by O’Rahilly & Müller (1987). An embryo was initially staged by comparing its development with that of other embryos. On the basis of correlating particular maternal menstrual histories and the known developmental ages of monkey embryos, growth tables were constructed so that the size of an embryo (specifically, the greatest length) could be used to predict its presumed age in postovulatory days (synonymous to postfertilizational days). O’Rahilly & Müller (2000) emphasize that the stages are based on external and internal morphological criteria and are not founded on length or age. Ultrasonic examination of embryos in vivo has necessitated the revision of some of the ages related to stages, and embryos of stages 6–16 are now thought to be up to 3 to 5 days older than the previously used embryological estimates (O’Rahilly & Müller 1999). Within this staging system, embryonic life commences with fertilization at stage 1; stage 2 encompasses embryos from two cells, through compaction and early segregation, to the appearance of the blastocele. The developmental processes occurring during the first 10 stages of embryonic life are shown in Fig. 8.1.

Much of our knowledge of the early developmental processes is derived from experimental studies on amniote embryos, particularly the chick, mouse and rat. Figure 8.2 shows the comparative timescales of development of these species and human development up to stage 12. The size and age, in postovulatory days, of human development from stage 10 to stage 23 is given in Fig. 8.3.

Information on developmental age after stage 23 (8 weeks postovulation) is shown in Fig. 14.3, where the developmental staging used throughout this text is juxtaposed with the obstetric estimation of gestation that is used clinically. A critique of staging terminology and the hazards of the concurrent use of gestational age and embryonic age is given in Chapter 14; sizes and ages of fetuses towards the end of gestation are illustrated in Fig. 14.7.

FERTILIZATION

The central feature of reproduction is the fusion of the two gamete pronuclei at fertilization. In humans the male gametes are spermatozoa, which are produced from puberty onwards. Female gametes are released as secondary oocytes in the second meiotic metaphase, usually singly, in a cyclical fashion. The signal for the completion of the second meiotic division is fertilization, which stimulates the cell division cycle to resume, completing meiosis and extruding the second polar body (the second set of redundant meiotic chromosomes).

Fertilization normally occurs in the ampullary region of the uterine tube, probably within 24 hours of ovulation. Very few spermatozoa reach the ampulla to achieve fertilization. They must undergo capacitation, a process which is still incompletely understood, and which may involve modifications of membrane sterols or surface proteins. They traverse the cumulus oophorus and corona radiata, then bind to specific glycoprotein receptors on the zona pellucida, ZP3 and ZP2. Interaction of ZP3 with the sperm head induces the acrosome reaction, in which fusion of membranes on the sperm head releases enzymes, such as acrosin, which help to digest the zona around the sperm head, allowing the sperm to reach the perivitelline space. In the perivitelline space, the spermatozoon fuses with the oocyte microvilli, possibly via two disintegrin peptides in the sperm head and integrin in the oolemma (Fig. 8.4 and Fig. 8.5A).

Fusion of the sperm with the oolemma causes a weak membrane depolarization and leads to a calcium wave, which is triggered by the sperm at the site of fusion and crosses the egg within 5–20 seconds. The calcium wave amplifies the local signal at the site of sperm–oocyte interaction and distributes it throughout the oocyte cytoplasm. The increase in calcium concentration is the signal that causes the oocyte to resume cell division, initiating the completion of meiosis II and setting off the developmental programme that leads to embryogenesis. The pulses of intracellular calcium that occur every few minutes for the first few hours of development also trigger the fusion of cortical granules with the oolemma. The cortical secretory granules release an enzyme that hydrolyses the ZP3 receptor on the zona pellucida and so prevents other sperm from binding and undergoing the acrosome reaction, thus establishing the block to polyspermy. The same cortical granule secretion may also modify the vitelline layer and oolemma, making them less susceptible to sperm–oocyte fusion and providing a further level of polyspermy block.

The sperm head undergoes its protamine → histone transition as the second polar body is extruded. The two pronuclei grow, move together and condense in preparation for syngamy and cleavage after 24 hours (Fig. 8.5B). Nucleolar rRNA, and perhaps some mRNA, is synthesized in pronuclei. A succeeding series of cleavage divisions produces eight even-sized blastomeres at 2.5 days, when embryonic mRNA is transcribed.

Several examples of cells which contain male and female pronuclei, termed ootids, have been described. Pronuclear fusion as such does not occur: the two pronuclear envelopes disappear and the two chromosome groups move together to assume positions on the first cleavage spindle. No true zygote containing a membrane-bound nucleus is formed.

The presence of the pronuclei from both parental origins is crucial for spatial organization and the controlled growth of cells, tissues and organs. In the mouse, embryos in which the paternal pronucleus has been removed and replaced with a second maternal pronucleus develop to a relatively advanced state (25 somites), but with limited development of the trophoblast and extraembryonic tissues. In contrast, embryos in which the maternal pronucleus has been replaced by a second paternal pronucleus develop very poorly, forming embryos of only six to eight somites, but with extensive trophoblast. Thus it seems that the maternal genome is relatively more important for the development of the embryo, whereas the paternal genome is essential for the development of the extraembryonic tissues that would lead to placental formation.

This functional inequivalence of homologous parental chromosomes is called parental imprinting. The process causes the expression of particular genes to be dependent on their parental origin: some genes are expressed only from the maternally inherited chromosome and others from the paternally inherited chromosome. The genes involved are called imprinted genes. The requirement for both parental genomes is limited to a subset of the chromosomes. Uniparental disomy can arise through meiotic and mitotic non-disjunction events, and results in individuals who are completely disomic or who exhibit mosaicism of disomic and non-disomic cells. If imprinted genes reside on the affected chromosomes, then the uniparental disomic cells will either express a double dose of the gene or have both copies repressed. For example, the gene encoding the embryonal mitogen insulin-like growth factor II is expressed from the paternally inherited chromosome, and repressed when maternally inherited.

In vitro fertilization

Fertilization of human gametes in vitro (IVF) is a successful way of overcoming most forms of infertility. Controlled stimulation of the ovaries (e.g. pituitary downregulation using gonadotrophin-releasing hormone superactive analogues, followed by stimulation with purified follicle stimulating hormone or urinary menopausal gonadotrophins) enables many preovulatory oocytes (often 10 or more) to be recruited and matured, and then aspirated either by laparoscopy or transvaginally using ultrasound guidance, 34–38 hours after injection of human chorionic gonadotrophin (which is given to mimic the luteinizing hormone surge). These oocytes are then incubated overnight with motile spermatozoa in a specially formulated culture medium, to achieve successful fertilization in vitro. In cases of severe male-factor infertility, in which there are insufficient normal spermatozoa to achieve fertilization in vitro, individual spermatozoa can be directly injected into the oocyte in a process known as intracytoplasmic injection of sperm, which is as successful as routine in vitro fertilization. In cases in which there are no spermatozoa in the ejaculate, suitable material can sometimes be directly aspirated from the epididymis or surgically retrieved from the testes, and the extracted sperm are then used for intracytoplasmic injection of sperm. It is also now possible, in some cases, to test embryos for the presence of a particular genetic or chromosomal abnormality in a process known as preimplantation genetic diagnosis. A sample (biopsy) is removed from either the oocyte polar body, the embryo itself (a blastomere) or the blastocyst (small piece of trophectoderm), and subjected to a specific genetic test. Unaffected embryos can then be identified for transfer to the patient. Embryos that are surplus to immediate therapeutic requirements can also be cryopreserved in liquid nitrogen for later use. Propanediol or dimethylsulphoxide is used as a cryoprotectant for early embryos, and glycerol is used for blastocysts. Conception rates per cycle using ovarian stimulation, in vitro fertilization and successive transfers of fresh and cryopreserved embryos, far outstrip those obtained during non-assisted conception.

PREIMPLANTATION DEVELOPMENT

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