Blastulation

About the formation of blastula and peri-implantation events. Also discusses Carneige staging for embryos.

1. Blastulation Dr. Komal Parmar

2. Fertilization Fertilization normally occurs in the ampullary region of the uterine tube , probably within 24 hours of ovulation . The two pronuclei grow, move together and condense in preparation for syngamy and cleavage in about 24 hours after that. Division of the zygote into blastomeres begins approximately 30 hours after fertilization. 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 .

3. Clevage Cleavage consists of repeated mitotic divisions of the zygote , resulting in a rapid increase in the number of cells ( blastomeres ). These embryonic cells become smaller with each successive cleavage division. Cleavage occurs as the zygote passes along the uterine tube toward the uterus. During cleavage , the zygote is within the zona pellucida

4. Division of the zygote into blastomeres begins approximately 30 hours after fertilization. Subsequent cleavage divisions follow one another, forming progressively smaller blastomeres . The cell cycle is quite long, the first two cell cycles being around 24 hours each, thereafter reducing to 12–18 hours.

5. Cell division is asynchronous Daughter cells may retain a cytoplasmic link through much of the immediately subsequent cell cycle via a midbody , as a result of the delayed completion of cytokinesis. No centrioles are present until 16 to 32 cells are seen , but amorphous pericentriolar material is present and serves to organize the mitotic spindles. All cleavage divisions after fertilization are dependent upon continuing protein synthesis which up to the 8 cell stage is derived from the available maternal mRNA and thereafter from the embryonic mRNA. Hence the initial cell divisions are independent of the mRNA synthesis. Early cleavage, up to the formation of eight cells, requires pyruvate or lactate as metabolic substrates, but thereafter more glucose is metabolized and may be required . Up to the formation of eight cells, cells are essentially spherical, touch each other loosely , and have no specialized intercellular junctions or significant extracellular matrix

6. After the nine-cell stage, the blastomeres change their shape and tightly align themselves against each other to form a compact ball of cells: Compaction . Cells flatten on each other to maximize intercellular contact, initiate the formation of gap and focal tight junctions. As a result of the process of compaction, the embryo forms a primitive protoepithelial cyst , which consists of eight polarized cells, in which the apices face outward and basolateral surfaces face internally . C ell surface and the calcium dependent cell–cell adhesion glycoprotein, E-cadherin (also called LCAM or uvomorulin ).

7. Morula T wo-cell populations are formed in the 16-cell embryo that differ in phenotype (polar, apolar ) and position (superficial , deep ). The outer polar cells contribute largely to the trophectoderm , whereas the inner apolar cells contribute almost exclusively to the inner cell mass in most embryos. T he generation of cell diversity, to either trophectoderm or inner cell mass, occurs at the time of 4 th /5 th day in the 16-cell morula During the next cell division (16 to 32 cells), a proportion of polar cells again divide differentiatively as in the previous cycle, each yielding one polar and one apolar progeny.

8. After division to the 32 cells, the outer polar cells complete their differentiation into a functional epithelium, display structurally complete zonular tight junctions and begin to form desmosomes . The nascent trophectoderm engages in vectorial fluid transport from uterine cavity to the inside of embryo to generate a cavity that expands in size during the 32- to 64-cell cycles and converts the ball of cells, the morula , to a sphere , the blastocyst . Once the blastocyst forms, the diversification of the trophectoderm and inner cell mass lineages is complete, and trophectoderm differentiative divisions no longer occur

9. Blastocyst The blastocyst ‘hatches’ from its zona pellucida at 6–7 days, possibly assisted by an enzyme similar to trypsin. Early pregnancy factor , an immunosuppressant protein , is secreted by the trophoblastic cells and appears in the maternal serum within 24 to 48 hours after fertilization. EPF forms the basis of a pregnancy test during the first 10 days of development . The outer cells of the blastocyst, the trophoblast or trophectoderm , are flattened polyhedral cells, which possess ultrastructural features typical of a transporting epithelium . The trophoblast covering the inner cell mass is the polar trophoblast and that surrounding the blastocyst cavity is the mural trophoblast .

10. Implantation occurs during a period of 7–12 days postovulation . Even at this early stage, cells of the inner cell mass are already arranged into an upper layer (i.e. closest to the polar trophoblast ), the epiblast , which will give rise to the embryonic cells, and a lower layer, the hypoblast, which has an extraembryonic fate. D orsoventral axis of the developing embryo and a bilaminar arrangement of the inner cell mass are both established at or before implantation .

11. Attachment to the uterine wall Implantation includes the following stages: Dissolution of the zona pellucida ; orientation and adhesion of the blastocyst onto the endometrium ; trophoblastic penetration into the endometrium; migration of the blastocyst into the endometrium; spread and proliferation of the trophoblast , which envelops and specifically disrupts and invades the maternal tissues . The site of implantation is normally in the endometrium of the posterior wall of the uterus, nearer to the fundus than to the cervix and may be in the median plane or to one or other side.

12. Formation Of Extraembryonic Tissues Epiblast and amniotic cavity closest to the implanting face of the trophoblast , A basal lamina surrounds a spherical cluster of epiblast cells, and isolates them from all other cells . E piblast cells adjacent to the hypoblast become taller and more columnar than those adjacent to the trophoblast , and this causes the epiblast sphere to become flattened and the centre of the sphere to be shifted towards the polar trophoblast . Amniotic fluid accumulates and the amniotic cavity is formed.

13. Hypoblast and yolk sac Hypoblast is the term used to delineate the lower layer of cells of the early bilaminar disc. During early implantation, the hypoblast extends beyond the edges of the epiblast and can now be subdivided into those cells in contact with the epiblast basal lamina, the visceral hypoblast, and those cells in contact with the mural trophoblast , the parietal hypoblast . T he parietal hypoblast cells are proliferating and spreading along the mural trophoblast . The cavity that the layer initially surrounds is termed the primary yolk sac, or alternatively the primary umbilical vesicle

14. At the same time, a space appears between the parietal hypoblast ( Heuser’s membrane) and the mural trophoblast that limits the circumference of the hypoblastic cavity. The resultant smaller cavity inside lined by hypoblast is termed the secondary yolk sac . The visceral hypoblast cells are believed to be important in many aspects of the early specification of cell lines. The cells induce the formation of the primitive streak, thus establishing the first axis of the embryonic disc. They are also believed to be necessary for successful induction of the head region and for the successful specification of the primordial germ cells.

15. After the formation of the secondary yolk sac, a diverticulum of the visceral hypoblast, the allantois, forms towards one end of the embryonic region and extends into the local extraembryonic mesoblast . It passes from the roof of the secondary yolk sac to the same plane as the amnion .

16. Extraembryonic mesoblast E xtraembryonic tissues encompass all tissues that do not contribute directly to the future body of the definitive embryo. Extraembryonic mesoblast : It will come to cover the amnion, secondary yolk sac and the internal wall of the mural trophoblast , and will form the connecting stalk of the embryo with its contained allanto -enteric diverticulum. The first mesoblastic extraembryonic layer gives rise to the layer known as extraembryonic mesoblast , arranged as a mesothelium with underlying extraembryonic mesenchymal cells.

17. Later extraembryonic mesoblast populations mushroom beneath the cytotrophoblastic cells at the embryonic pole, forming the cores of the developing villus stems, and villi and the angioblastic cells that will give rise to the capillaries within them and the earliest blood cells . .

18. Initially, the extraembryonic mesoblast connects the amnion to the chorion over a wide area. Continued development and expansion of the extraembryonic coelom means that this attachment becomes increasingly circumvented to a connecting stalk, which is a permanent connection between the future caudal end of the embryonic disc and the chorion . The connecting stalk forms a pathway along which vascular anastomoses around the allantois establish communication with those of the chorion

19. Clinical Correlates

20. MOSAICISM If nondisjunction (failure of a chromosome pair to separate) occurs during an early cleavage division of a zygote, an embryo with two or more cell lines with differentchromosome complements is produced . Example: Down’s Syndrome

21. PREIMPLANTATION GENETIC DIAGNOSIS C an be carried out 3 to 5 days after IVF of the oocyte. One or two cells ( blastomeres ) are removed from the embryo known to be at risk for a single gene defect or chromosomal anomaly.

22. ABNORMAL EMBRYOS AND SPONTANEOUS ABORTIONS Many zygotes, morulae , and blastocysts abort spontaneously. Early implantation of the blastocyst is a critical period of development that may fail to occur owing to inadequate production of progesterone and estrogen by the corpus luteum . Early spontaneous abortions occur for a variety of reasons , one being the presence of chromosomal abnormalities. More than half of all known spontaneous abortions occur because of these abnormalities.

23. Ectopic implantation The conceptus may be arrested at any point during its migration through the uterine tube and implant in its wall. Previous pelvic inflammation damages the tubal epithelium and may predispose to such delay in tubal transport. The presence of an intrauterine contraceptive device or the use of progesterone-based oral contraceptives may also predispose to ectopic pregnancy, probably because of alteration in the normal tubal transport mechanisms.

24. Abnormal Implantation within Uterus Implantation near the internal os results in the condition of placenta praevia , with its attendant risk of severe antepartum haemorrhage .

25. Twinning: Identical Twins

26. Staging and Study of Embryo

27. Embryology The study of spatial and temporal developmental processes that take place within an embryo as it develops from a single cell into a recognizable organism is known as embryology. Much of our knowledge of the early developmental processes is derived from experimental studies on amniote embryos, particularly the chick, mouse and rat . 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.

28. Embryonic Staging Fetal Staging The embryonic period has been defined by Streeter as 8 weeks postfertilization , or 56 days. This timescale is divided into 23 Carnegie stages, a term introduced by O’Rahilly & Müller (1987) to replace developmental ‘horizons ’. The designation of stage is based on external and internal morphological criteria and not on length or age. Staging of fetal development and growth is based on an estimate of the duration of a pregnancy . Whereas development of a human from fertilization to full term averages 266 days, or 9.5 lunar months (28 day units), the start of pregnancy is traditionally determined clinically by counting days from the last menstrual period; estimated in this manner , pregnancy averages 280 days, or 10 lunar months (40 weeks ). In obstetric practice the duration of the period of gestation is regarded as nine calendar months, which is approximately 270 days. The period of pregnancy is divided into thirds, termed trimesters. The first and second trimesters each cover a period of 12 weeks, and the third trimester covers the period from 24 weeks to delivery.