Cleavage and Implantation

The mammalian embryo undergoes a series of mitotic cell divisions to form 2-cell, 4-cell, 8-cell, 16-cell, morula and blastocyst stages called cleavage. The zygote is termed an embryo when it starts mitotic division.

23.2.1 Cleavage

The first rapid series of mitotic cell division of the zygote is called cleavage (Table 23.7). The first cleavage results in two-cell embryo, and the daughter cells are called blastomeres. The cells become progressively smaller throughout the cleavage with no net increase in the size up to successive three divisions (up to the eight-cell stage). It resulted in the decreased weight of the embryo from the single-cell zygote and is called a negative growth. The

Table 23.7 Stage of early embryonic development before implantation (period considered after fertilisation)

Source: Lopes and Mummery (2014), Soom et al.

Fig. 23.4 Fertilisation to implantation. The fertilisation occurs at the ampullary-isthmus junction. The zygote undergoes a series of mitotic cell divisions (cleavage) to form 2-cell, 4-cell, 8-cell, 16-cell, morula and blastocyst. Morula proceeds towards the uterus and gradually

transforms into blastocyst after developing a cavity called blastocoele. The blastocyst then attaches to the endometrial surface through implantation

proportion of decrease in the cellular mass is nearly 20% and 40% in cows and sheep, respectively. The cell’s nuclei size increases to maintain the appropriate amount of nucleic acid. The first stage of cell division is occurred longitudinally, followed by the second longitudinal cell division at 90° to the plane of the first. The third division occurs in the perpen­dicular plane of the first two divisions. The rates of cleavage vary among species. It takes about 144 h (6 days), 96 h (4 days) and 120-140 h (5 days) in cows, sheep and goats, respectively, to reach the morula stage. The cleavage process continues until a solid mass of cells called morula is formed.

23.2.1.1 Morula

The 16-cell stage of an embryo is called morula (singular morulae), which develops after four rounds of cleavage divisions. The weight of the embryo starts increasing from the morula stage compared to the one-cell stage. Initially, the cells of the morula are totipotent. It becomes pluripotent and gives rise to two layers of cells. The outer layer is called the trophoblast. The inner cell mass remains in a cluster. The cells of the morulae undergo compaction due to cell-to-cell adhesion, and the shape of the cells turns spherical to polyg­onal. This stage is called a tight morula, and the size is reduced compared to the early morula stage. The cells of tight morula could be as many as 32.

Most of the embryos are generally evaluated during this stage. The evaluation criteria include the number and shape of the cells, the degree of compaction, appearance of excluded blastomeres, character of peri-vitelline space and regions of degeneration. Embryos are graded based on these criteria as excellent, good, fair, poor, degenerative and unfertilised (details in Chap. 24). Excellent and good quality embryos are generally used for embryo transfer technology (ETT). The embryo with an appropriate category may also preserve, but their sustainability is poor.

23.2.1.2 Blastocyst Formation or Blastulation

The tight morula gradually acquires fluids to form a cavity (blastocoele or exocalome) in between an outer layer of the blastocyst (trophoblast or trophectoderm) and inner cell mass (the embryoblast) at one pole. The inner cell layer of the trophectoderm is known as cytotrophoblast, and the outer one is called syncytiotrophoblast. The structure thus formed is called the blastocyst. The time of blastulation varies between species. It takes 6 days in pigs and horses, 7 days in sheep and 8 days in cattle after fertilisation. The trophoblast becomes flat and makes the epithelial wall of the blastocyst. The internal secretion of fluid forms the blas- tocoele by the blastomeres. This process is called cavitation.

The blastocoele pumps the fluid between the cells, and the blastocyst is turned into an expanded blastocyst. The blasto­cyst is still under the covering of zona pellucida. The sperm receptors at the zona pellucida are lost after fertilisation, and the gelatinous capsule protects the embryo from the invasion of pathogens and the maternal immune system.

23.2.2 Maternal Recognition of Pregnancy

Maternal recognition of pregnancy (MRP) is the biological process by which conceptus (elongated blastocyst) signals its presence to the mother and prevents the luteolytic mechanism from sustaining the life span of the corpus luteum. The ultimate goal of maternal recognition of pregnancy is to ensure the continuous release of progesterone to maintain the pregnancy. The major signalling agents that cause the MRP are generally species specific (Table 23.8).

23.2.2.1 Interferon Tau (IFN_h) and Inhibition of Luteolytic Mechanism

Trophoblast-derived interferon Tau (IFNT) acts as an anti- luteolytic factor in domestic ruminants. IFN^t is a Type IIFN family member that acts through Type I IFN receptors (IFNAR) in the LE, GE and stroma. Upon binding with its receptor, IFN^t inhibits the transcription of the ESR1 gene

Table 23.8 Pregnancy recognition signals in mammals

Table 23.9 IFN-stimulated genes and their role in implantation

through a signalling pathway via IFN regulatory factor (IRF) 2.

23.2.2.2 Estradiol and MRP in Pigs

The pregnancy recognition signal in pigs is the oestrogen secreted by the conceptuses. The release of oestrogen by the conceptus is biphasic. The oestrogen appears first at 11-12 of pregnancy, followed by a sustained release at days 15-30 for conceptus attachment and placental development. The anti-luteolytic action of oestrogen in pigs can be explained by the endocrine/exocrine hypothesis. According to this model, the oestrogen secreted from the conceptus directed endometrial-derived PGF2α away from uterine vas­culature (endocrine) and sequestrated into the uterine lumen (exocrine). In the uterine lumen, PGF2α inactivates into its inactive 13,14-dihydro-15-keto prostaglandin F2α metabo­lite. In addition to the anti-luteolytic mechanism, oestrogen also helps in the migration and spacing of blastocysts. To establish a pregnancy, the presence of at least two conceptuses in each uterine horn is mandatory initially to establish a pregnancy. Oestrogen also increases fibroblast growth factor 7 (FGF-7) expression in the endometrium for proliferation and differentiation of trophectoderm.

23.2.2.3 Chorionic Gonadotropin (CG) and MRP in Horses and Primates

CG is the key maternal recognition signal in horses and primates. It is a glycoprotein hormone secreted from the trophoblast. CG acts like an LH agonist and performs anti- luteolytic actions together with induction of steroidogenesis in CL. The anti-luteolytic actions of CG include (1) maintaining stable luteal blood flow, (2) increases in the Bcl2/Bax ratio to prevent apoptosis and (3) preventing tissue remodelling by modulating matrix metalloproteinase- 2 (MMP-2) functions and recruitment of macrophages.

23.2.2.4 Pregnancy Recognition in Rodents

Semicircadian prolactin surges due to cervical stimulation are required for pregnancy in rodents. These surges are responsi­ble for converting the corpus luteum (CL) of the cycle into the corpus luteum of pregnancy (CLP).

23.2.2.5 Pregnancy Recognition in Dogs and Cats In dogs and cats, MRP is not essentially required to establish pregnancy as the life span of CL usually is about 60 days, irrespective of whether conception occurred or not.

23.2.3 Implantation

Implantation is a biological process of attachment of the embryo to the endometrial surface followed by the invasion of the epithelium to form the placenta (Fig. 23.4, Tables 23.7 and 23.10). Both the embryo and the uterus undergo a series of physiological process that ultimately favours implantation. Implantation requires crosstalk between a receptive uterus and the conceptus for a limited period called the “window of implantation”. The time for initiation of the implantation process is species-specific. In sow, the process starts after 2 days of fertilisation; in sheep, 2.5-3 days, cattle, 3-4 days; and in horses, 5.5-6 days. In humans, it starts the 8-10 days after ovulation and continues till the second week. Endome­trial differentiation is an essential prerequisite for the initia­tion of the implantation process. Oestrogen induces endometrial differentiation. Progesterone also acts over the oestrogen primed endometrium to reinforce further differen­tiation to make a suitable environment for embryo implanta­tion. The process of implantation can be divided into six distinct phases: (1) shedding of the zona pellucida or zona hatching, (2) blastocyst elongation orientation and spacing, (3) intrauterine migration and spacing, (4) apposition, (5) adhesion and (6) endometrial invasion.

23.2.3.1 Zona Hatching

The release of the blastocyst from the zona pellucida is called zona hatching, and the expanded blastocyst is transformed into a hatched blastocyst. Zona pellucida prevents premature

Table 23.10 Period of implantation and gestation period of some mammals

Compiled from various sources ^a Days count after standing oestrus ^b Days after mating

implantation of the embryo. Usually, the zona pellucida collapses by some enzymatic reaction. It usually occurs 9-11 days post ovulation in the cow. The blastocyst generally comes out from its embryonic pole, the side opposite the inner cell mass (ICM). The zona hatching occurs by two forces. The mechanical pressure exerted by the growing blastocyst and the enzymatic lysis of the zona pellucida. Depending on the species, several proteases are involved in the zona lysis, such as serine proteases, cysteine proteases and metalloproteinases. Cathepsins are a cysteine protease actively engaged in zona hatching. Ovastacin also helps in the zona hatching process by removing cortical granules. The zona hatching is regulated by hormones, growth factors, cytokines and transcription factors. The predominant growth factors involved in zona hatching are heparin-binding epider­mal growth factor (HB-EGF), transforming growth factor­beta (TGF-β) and leukaemia inhibitory factor (LIF). The cyclooxygenase-2 (COX-2) inhibitors, prostaglandins (PGs, E2 or I2), plasmin and trypsin play an essential role in the hatching process. Calcium is also required for zona hatching as some of the mechanisms of zona hatching are calcium dependent.

23.2.3.2 Blastocyst Elongation and Orientation The shedding of zona pellucida is followed by the rapid growth of the blastocyst. The hatched blastocyst gradually becomes elongated and moves from the oviducts to the uter­ine horns for implantation. In cow, the spherical blastocyst (3 cm) is transformed into a filamentous thread-like structure (25 cm) called conceptus from day 13 to day 25 post fertilisation. This elongation occurs through continual hyper­plasia of the trophectoderm and entire endoderm. The blasto­cyst does not elongate in the horse but somewhat increases in diameter by 2-3 mm/day to become a spherical baseball-like form. At this stage, the trophoblast secretes a hormone called pregnancy serum protein B (PSPB or PAG). It influences the corpus luteum survivability and helps to secret progesterone for embryo development and maintenance of gestation. The level of PSPB is gradually increased during the gestation period and reaches a maximum during the day of parturition.

The conceptus develops a specific orientation concerning the uterus. In most domestic species, the early conceptus is arranged so that the yolk sac is found on the endometrial side of the uterine lumen and the embryonic disc lies on the anti- mesenteric side.

23.2.3.3 Intrauterine Migration and Spacing

In polytocous species, intrauterine migration and equidistant spacing are essentially required for embryo survival. The embryos of polytocous species (rabbit, pig, rat and mouse) enter the uterine horn at late morula and early blastocyst. They orient themselves at the longitudinal axis so that the inner cell mass (ICM) is situated at the mesometrial side of the uterus. In the ruminant embryo, the migration is limited, and the embryo rarely passes through the body of the uterus into the contralateral horn. Sheep embryo tends to migrate when multiple ovulations occur in the same ovary. There is no correlation between the side of ovulation and the side of

Table 23.11 Stages of trans-uterine migration and associated mechanisms

embryo attachment in the horse. Intrauterine migration and spacing occur in three stages (Table 23.11).

23.2.3.4 Apposition

The trophectoderm adheres with the adhesive receptors of the endometrial luminal epithelium. The presence of an anti­adhesive substance such as mucin 1 (MUC1) prevents the blastocyst adhesion. The progesterone facilitates the declin­ing of MUC1 from the endometrial luminal epithelium, and the adhesive receptors such as integrins are exposed to tro­phoblast for initial apposition. The endometrial glandular epithelium secretes histotroph under the influence of proges­terone that nourishes the developing blastocysts.

23.2.3.5 Adhesion

The adhesion between blastocyst trophectoderm and endo­metrial luminal epithelial is achieved through the interaction between cell adhesion molecules, such as glycosylated cell adhesion molecule (GLYCAM1) 1, galectin 15 (LGALS15) and secreted phosphoprotein 1 (SPP1 or osteopontin) with their receptors (integrins and glycoconjugates). The cell adhesion molecules are expressed on the trophoblast apical surface and interact with their receptors at the luminal epithelium.

23.2.3.6 Endometrial Invasion

In this phase, the giant binucleate cells (BNC) of the tropho­blast fuse with the LE to form multinucleated syncytial plaques. The BNC develops from the mononuclear trophectoderm cells through mitotic polyploidy (nuclear divisions without cytokinesis). The giant BNC migrate to the trophoblast and fuse with the individual luminal epithelium to form trinucleated foetomaternal hybrid cells. The remaining luminal epithelium, unable to form hybrid cells, undergoes apoptosis. The migration and fusion of BNC are continued till the syncytial plaques are limited in size to 20-25 nuclei. Then no further nuclear divisions occur, and syncytial plaques and linked with tight junctions to form caruncular syncytia. The caruncular syncytia expand from cotyledons. The giant BNC serve two essential functions: (1) formation of foetomaternal syncytial plaques that give rise to cotyledon of placentome and (2) synthesis of chorionic somatomammotropin hormone 1 (placental lactogen), pregnancy-associated glycoproteins (PAGs) and progester­one that facilitates the growth of endometrial glands and differentiation of endometrium. In ruminants, the endoge­nous retroviruses (ERV) are involved in the fusion of BNC and LE.

In pigs, the trophoblast and uterine epithelium undergo loose apposition immediately after the blastocyst elongation. There is interdigitation between the microvilli of two epithe­lial surfaces (trophectoderm and LE), and later the tropho­blastic surface becomes modified to form an absorptive surface called areolae. The nutrient uptake by the developing conceptus is facilitated through these areolae. In this species, the attachment begins on day 13 and is completed around days 18-24.

In ruminants, an initial transitory attachment occurs between trophoblast and LE. Trophoblast develops finger­like villi that penetrate the lumen of uterine glands and act as a temporary anchor. The centres of caruncles become depressed and cytoplasmic protrusions are developed from the trophoblast epithelium. The permeability of the caruncular capillaries increases on day 15. Between days 16-19, the effective attachment occurs through the interpen­etration of uterine microvilli and cytoplasmic projections of trophectoderm (Fig. 23.4).

In the mare, the attachment occurs between the surface epithelium of the embryonic vesicle and uterine lining through interdigitations.

23.2.3.7 Types of Implantation

Implantation can classify as invasive or non-invasive based on the degree of invasion or penetration. In primates and rodents, the blastocyst penetrates the uterine mucosa, followed by the phagocytosis of uterine LE. The blastocyst migrates the uterine stroma. In this type of implantation, the endometrial stromal cells and endothelial cells of the blood vessels undergo decidualisation in the presence of leukocytes under the influence of progesterone to form a special kind of tissue called decidua. It suppresses the mother’s immune response to prevent the immune rejection of foetuses. The decidua also secretes various growth factors, cytokines, insulin-like growth factor binding protein 1 (IGFBP1), pro­lactin and different extracellular matrix proteins like fibro­nectin and laminin, favouring the invasion process. The decidua becomes the part of placenta with the advancement of pregnancy.

In contrast, the implantation in domestic ruminants, carnivores, pigs and horses is non-invasive. The conceptus remains within the uterine lumen and is embedded in the uterine wall.

Based on blastocyst orientation, implantation can be of three types centric, eccentric and interstitial. In the centric type of implantation, the embryo(s) remains at the centre of the uterus and its size increases before implantation. It is non- invasive implantation seen in all domestic ruminants, carnivores, pigs and horses. In an eccentric pattern, the small-sized blastocyst(s) invades one side (generally the reverse side of the mesometrium) of the uterus. This implan­tation pattern is generally invasive and occurs in some rodents like rats and mice. In guinea pigs, humans and other primates, the small-sized blastocyst(s) is entered deep into the endometrial epithelium and attached to the subepithelial connective tissue of the endometrium. It is called interstitial or nidation or nest making pattern of implantation. It is also of invasive type.

In humans, a cellular structure called pinopode or uterodome is thought to be involved in uterine receptivity. Pinopode is a large cellular protrusion on the uterine epithe­lial surface under the influence of hormones. Other than humans, pinopodes are seen in mice and rats. Pinopods act as a clinical marker of endometrial receptivity in such spe­cies. Pinopods are involved in regulating uterine luminal contents and regulation of implantation associated proteins.

23.2.4 Gastrulation

Gastrulation is the formation of three germ layers, viz. ecto­derm, endoderm and mesoderm. In blastocyst, the embryonic cells differentiate into outer trophoblast and inner cell mass (embryoblast) (Fig. 23.5). Dramatic changes occur at the embryoblast, giving rise to epiblast (outer layer) and hypo­blast (inner layer). The hypoblast is small and cuboidal in shape. It provides the nutrients to all the embryo cells by forming a yolk sac until the development of the placenta is functional. In the next stage, the inner cell mass cells are organised into a sheet of columnar epithelium called an embryonic disc. The trophoblast and embryonic disc are collectively called ectoderm, the first primary germ layer. Delamination of the cells from the inner surface leads to forming a second layer. The third and final germ layer forms between the first and second layers as a wave of cellular emigration from the embryonic disc called meso­derm. The parts of primary germ layers beyond the embry­onic disc are extraembryonic parts that give rise to the extraembryonic membrane. Blastulation ends with the forma­tion of three primary germ layers.

23.2.5 Organogenesis

A thick structure develops along the midline of the embry­onic disk, called a primitive streak. The primitive streak determines the major axis, and accordingly, the embryo’s left, right, cranial and caudal regions are designated. Thus, three body axes are developed in the embryo anterior- posterior (head-tail), left-right (lateral-medial) and dorsal- ventral (back-belly). A node (primitive node) is extended from cranial to caudal end through its midline, called a primitive groove. The outer layer cells start to fold up towards the inside and gradually separate along with the primitive streak. This process is called invagination. The invagination causes the replacement of the outer cells with new cells and forms definitive endoderm. Similarly, the internalised cells form definitive ectoderm, and the cells that remain between definitive ectoderm and endoderm are called definitive mesoderm. In cattle, it occurs within the third week after fertilisation.

Three different germ layers have the potency to give rise to all tissues and organs of the organism (Table 23.12). The heart is the first organ that develops from the endoderm. The process by which the germ layers of the embryo into tissues and organs is called organogenesis. The embryo is termed as foetus when organogenesis is initiated and continued. In cattle, it occurs around the end of the fourth week.

Fig. 23.5 Gastrulation. The blastocyst consists of outer trophoblast and inner cell mass (ICM). At the time of implantation, the trophoblast differentiates into two distinct layers. The outer Syncytiotrophoblast assists in the implantation by releasing hydrolytic enzymes and hCG. The inner cytotrophoblast surrounds the somatic mesoderm. The inner cell mass gives rise to hypoblast and epiblast. Hypoblast is differentiated from extraembryonic mesoderm, which gives rise to amnion, allantois, chorion, and visceral yolk sac. The epiblast forms three primary germ layers: ectoderm, mesoderm, and endoderm

Table 23.12 Organs developed from germ layers

Germ

The formation of an extraembryonic membrane starts with separating the mesoderm into an inner and outer layer by a narrow cavity. The outer mesoderm is called somatic meso­derm, and the inner layer is called splanchnic mesoderm. The intervening cleft between the outer and inner mesoderm is called embryonic coelom. The splanchnic mesoderm is associated with viscera formation, and the somatic mesoderm develops connective tissue of the body wall. The somatic mesoderm fuses with the underlying ectoderm to form somatopleure, and the splanchnic mesoderm fuses with the underlining ectoderm to form splanchnopleure. The formation of amnion, chorion and yolk sac develops due to the folding of somotopluerae and splanchnicplurae. Amnion and chorion are developed from somatopleure, whereas allantois and yolk sac are derived from splanchnopleure. The amnion and chorion are fused to form an amniochorion.

Further invaginations of splanchnicplureae subdivide the blastocoel into primitive gut and yolk sac. The yolk sac comprises extraembryonic endoderm on its inner surface and splanchnic mesoderm on its outer surface. The splanchnic mesoderm is the site of primitive haematopoiesis in the developing embryo. In domestic animals, the yolk sac is small and absorptive function during early pregnancy in mares and carnivores.

The allantois develops from the ventral part of the hindgut. The internal layer of the allantois forms from the endoderm, and the external layer originates from the splanchnic meso­derm. Allantois serves as temporary storage of urine in developing foetuses. The fusion of the outer layer of allantois with overlaying chorion leads to the formation of allantochorion. The formation of blood vessels at the allantochorion acts as a transient organ for gas exchange.

The internal epithelium cell lining of the chorion develops from vascularised mesenchyme tissue, which covers the exocelema, amnion, allantois and yolk sac. The amnion is made up of squamous epithelium cells and makes a membra­nous layer containing mesenchyme connective tissue. It guards the foetus against mechanical pressure along with the nutrient exchange process.

The allantois comprises a squamous epithelial cell layer with a basement membrane and extraembryonic mesenchyme developed from the embryonic intestine as an extraembry- onic urinary bladder. The yolk sac is involved in the exchange process at the early embryonic stage; later, it merges with chorion when the placenta develops in most mammals. It is rudimentary in pigs, guinea pigs and humans.

23.3