Early Development I: Cleavage Lecture PDF

 Early Development I: Cleavage Lecture Outline  Four Major Developmental Events  The Timing of Early Development  Cleavage: Functions and Events  Mitotic vs Normal Cell Cycle  The Purposes of Cleavage  The Stages of Early Cleavage  Cleavage, Compaction, Blastocyst F...

 Early Development I: Cleavage Lecture Outline  Four Major Developmental Events  The Timing of Early Development  Cleavage: Functions and Events  Mitotic vs Normal Cell Cycle  The Purposes of Cleavage  The Stages of Early Cleavage  Cleavage, Compaction, Blastocyst Formation and Hatching  Mammalian Compaction  Formation of the Blastocyst  Hatching of the Blastocyst Four Major Developmental Events There are 4 major developmental processes that occur in human embryogenesis. Cell Division (Cleavage) - Converts 1 cell to many; the egg is one cell, the embryo is multicellular; the conversion from one cell into many involves an initial cleavage (embryonic mitoses) phase followed by regular mitotic cell divisions. Cell Differentiation - formation of different, specialized cell types; the egg is one cell type, the embryo contains hundreds of cell types; understanding how cells specialize is a fundamental problem of developmental biology Morphogenetic Events - literally the "generation of shape", morphogenesis results in the embryonic organization, the pattern & polarity to cells, organs & tissues; the egg is round while the embryo has a specific organization of multiple layers of different cells; the terms morphogenesis and differentiation are not synonymous although many researchers today make this mistake in terminology; cell differentiation is just one component of morphogenesis  Growth - Increases size of organism; the embryo increases dramatically in size from the next to invisible egg to the full grown fetus. Of course there are other component events that also occur such as apoptosis (controlled cell death) which underlies many morphogenetic events in the formation of tissues and organs.  The Timing of Early Development Here we will focus on the events that occur during the first 7 days after fertilization. This will take us to the hatched blastocyst which will have arrived at the uterus which will be ready to accept it for implantation. Cleavage: Functions & Events  Initial divisions of zygote to form the multicellular embryo  During cleavage the cells are called "Blastomeres"  They are special mitotic divisions  Since they are mitotic divisions, they maintain the 2N complement  They are rapid cell divisions with no intervening growth (G1 & G2) phases  Cells become smaller with time  Holoblastic Cleavage: cells are completely separate Mitotic vs. Normal Cell Cycle Cleavage is characterized by a shortened cell cycle During cleavage G1 and G2 stages are by-passed so cells simply progress from S (DNA synthesis) to M (mitosis) without the intervening growth phases As a result cleavage cells continue to decrease in size until they approximate the size of somatic cells The Purposes of Cleavage  Converts unicellular zygote to multicellular embryo  Produces many cells that can interact and be moved around  Maintains diploid complement of cells--all are genetically identical  Human cleavage is not synchronous; all of the cells do not cleave at precisely the same time, as a result embryos with odd numbers of cells can be seen at various times  Slow cleavage; takes approximately 12-24h between each cell division  No growth occurs during early cleavage, so the total embryo will remain ~100 microns (0.1mm) in diameter The Stages of Early Cleavage Cleavage begins about 24h after the egg has been fertilized once the pronuclei have fused. 1st Cleavage Resulting embryo is 2 cells (i.e., 2 blastomeres or 2 cleavage cells) DNA has been synthesized: Embryo has double the DNA content of zygote Embryo has made membrane to surround both cells Growth has not occured so the 2 blastomeres together are approximately the same size as the original zygote 2nd Cleavage Embryo is 4 cells DNA has been synthesized: Embryo now has 4x DNA content Embryo has made membrane to surround all four cells Growth has not occurred so the 4 blastomeres together are about the same size as the original zygote 3rd Cleavage Embryo is 8 cells More DNA has been synthesized: Embryo now has 8x DNA content Embryo has made membrane to surround all 8 cells DNA duplication and membrane synthesis of will continue as cells continue to divide Growth has not occurred so the 8 blastomeres together are approximately the same size as the zygote Embryo will now undergo compaction just prior to next division leading to 16 cell stage Cleavage, Compaction, Blastocyst Formation and Hatching The following diagram takes the human embryo from the zygote surrounded by the zona pellucida to the hatched blastocyst stage where it is ready for implantation in the uterus. As cleavage continues (compaction is not shown), fluid appears in between the cells after the morula stage resulting in the early blastocyst. This fluid accumulates in an increasingly larger cavity called the blastocoel as differences in the cell populations become evident. The blastocyst hatches from the zona pellucida preparing it for implantation.  At the 8 cell stage the human embryo undergoes a process called compaction. It results from cells adhering together more tightly The embryo becomes more compact, but the cells still remain separate from each other The cell adhesion protein, E-cadherin appears at the time of compaction and causes the cells to adhere together more tightly than previously. Scanning Electron Microscope image Phase Contrast Microscope image Uncompacted Embryo Compacted Embryo The following diagram shows the process of compaction. Here's how cadherin interacts homophilically (same molecules bind together) to cause cell adhesion:  Cadherin & Compaction As demonstrated by the following experimental results, the appearance of cadherin has been detected at the surfaces of cells of compacted embryos using immunofluorescence microscopy with cells stained with anti-cadherin antibodies (green staining). If the cells are treated with anti-cadherin prior to compaction, they do not undergo compaction. This occurs because the anti- cadherin binds to any cadherin molecules that appear preventing them from being detected or from binding to other cadherin molecules. This work reveals the importance of cadherin in the process.  Formation of the Blastocyst  Fluid begins to appear between blastomeres  Process is called "Cavitation"  Begins ~4 Days after fertilization  Produces "Blastocoele": the fluid-filled cavity of blastocyst  Hatching of the Blastocyst Hatching occurs just prior to implantation Embryo breaks through ZP due to proteases secreted by blastocyst Inability to hatch is a reason for infertility; could be due to altered zona pellucida or to the absence of essential protease for zona digestion Often assisted hatching is done in vitro during ART procedures EMBRYO IMPLANTATION, PROCESS & THEIR STAGES What is Implantation? Implantation, also known as nidation, is a crucial stage in the development of mammals where the blastocyst, a hollow ball of cells, attaches and invades the wall of the female’s uterus. This process allows the embryo to establish a connection with the mother’s body and receive the necessary oxygen and nutrients for further growth and development. Successful implantation is a significant milestone, as it marks the beginning of pregnancy. In women, the presence of increased levels of human chorionic gonadotropin (hCG) in a pregnancy test is an indication of an implanted embryo. This hormone is produced by the developing placenta and serves as a marker for pregnancy. Conditions for implantation The proper uterine environment needs to be created, where the endometrium and embryo can interact. Thus, implantation is also not 100% safe in assisted reproduction cycles, even though fertilization has taken place in the laboratory and good-quality embryos are transferred. Factors related to the embryo For an embryo to be able to join the endometrium, it must be in the blastocyst stage. At this point in its development, it has about 200-400 cells and is made up of two well-differentiated parts: Internal cell mass cells that will give rise to the embryo itself. Trophoectoderm are the outermost cells that will form the placenta and other embryonic attachments. Additionally, prior to implantation, the blastocyst must also have detached from its zona pellucida, the outer layer that surrounds it, and have reached its maximum degree of expansion: the hatched blastocyst Development and implantation of embryo in the uterus Factors related to the endometrium The endometrium is the innermost layer of the uterus, which is renewed in each menstrual cycle in order to accommodate the embryo during pregnancy. For this reason, if implantation does not take place, the endometrium is shed and eliminated each month in the form of menstruation. Throughout the menstrual cycle, the endometrium thickens little by little and undergoes changes thanks to the action of the female sex hormones: estrogens and progesterone. For embryo implantation to occur, the endometrium must be receptive. This is achieved when its endometrial thickness is between 7-10 mm and its appearance is trilaminar. The site of implantation refers to the specific location within the uterus where the blastocyst becomes attached and implanted. While the fundus of the uterus is the most common site for implantation, the blastocyst can potentially implant in other areas of the uterus as well. The site of implantation is influenced by various factors, and it plays a critical role in the establishment and development of a healthy pregnancy.  Endometrial growth and characteristics for implantation The main qualities of the receptive endometrium are: Trilaminar aspect in the ultrasound we can see three parallel lines. Thickness should be between 6 and 10 mm approximately. The passage from the nonreceptive to the receptive uterus occurs only under the hormonal influence. For this reason, it is essential that the woman to whom the embryo or embryos are to be transferred receives estrogen and progesterone supplements to allow the endometrium to move from its nonreceptive state to its deciduous or receptive state When does implantation occur? As we have already said, the nesting of the embryo will only take place when the endometrium is receptive. This time of the menstrual cycle is known as the implantation window and has an approximate duration of 4 days. In most women, the implantation window runs from day 19 to day 21 of the menstrual cycle. At this time, if there has been fertilization, the blastocyst will be about 6 or 7 days old and ready to implant. However, there are women with the What is the timing of the implementation window? implantation window displaced, which can lead to implantation failure and sterility. In short, implantation occurs at a specific time in the menstrual cycle, when the endometrium changes from a nonreceptive to a receptive state under the hormonal influence and there is a synchrony between embryo and endometrium. Phases of implantation Once the dialogue between the embryo and the maternal endometrium has been established, the embryo implantation or nesting, which usually takes place in the middle third of the posterior face of the uterus  1.Migration and Hatching: After reaching the uterus, the blastocyst undergoes migration within the uterine cavity. It moves and floats freely before hatching from the protective zona pellucida, a glycoprotein shell that surrounded the early embryo during its development in the fallopian tube. 2.Pre-Contact: During this stage, the blastocyst approaches the receptive endometrium, which is the lining of the uterus that has reached its optimal state for implantation. The blastocyst starts to make contact with the endometrial surface but does not yet attach. 3.Attachment: In this stage, the blastocyst firmly attaches to the endometrial lining. Specialized cells called trophoblasts, derived from the outer layer of the blastocyst, interact with the endometrium. These trophoblasts initiate the formation of the placenta, which will provide oxygen and nutrients to the developing embryo. 4.Adhesion: Once attached, the blastocyst further adheres to the endometrium, establishing a stronger connection. This adhesion is mediated by specific molecules and cell-to-cell interactions between the trophoblasts and the endometrial cells. 5.Invasion: The final stage of implantation involves the invasion of the trophoblasts into the endometrial tissue. This invasion allows the blastocyst to establish a close relationship with the maternal blood vessels and facilitate the exchange of nutrients and waste products.. It’s important to note that these stages occur within a limited timeframe known as the window of implantation. The uterus is only receptive to implantation during this specific period, which is influenced by hormonal changes and the cyclic nature of the endometrial lining 1. Detachment of the zona pellucida The first step for the embryo to implant is to leave its shell: the zona pellucida. It's what's known as hatching. It consists of the breakage of the zona pellucida and the exit of the embryo, both from the ICM and from the trophoectoderm. 2. Pre-contact and apposition Approximately between day 5 and 6 of embryonic development, the fertilized egg is positioned in the endometrial tissue and remains immobile in the acquired position. It only directs the embryonic pole (where the ICM is) towards the epithelium of the endometrium. In this phase, the so-called pinopods, projections of the endometrial cells that help the blastocyst at the junction with the endometrial epithelium, are fundamental. 3. Adhesion This is the moment when the trophoectoderm cells strongly bind to endometrial cells through adhesion molecules such as integrins, L-selectins, proteoglycans, fibronectins, etc. 4. Invasion This usually occurs from day 8-9 of embryonic development. Little by little, the cells of the trophoectoderm proliferate towards the endometrium and thus manage to displace and replace the endometrial cells. This eventually leads to complete invasion of the endometrial stroma by the trophoblast, which becomes totally embedded in the endometrium. Eclosion of embryo and precontact Embryo adhesion Embryo invasion Apposition of embryo Process of Implantation of the Blastocyst in the Uterus The process of implantation of the blastocyst in the uterus involves several stages that are essential for successful pregnancy. Let’s explore these stages based on the provided information: 1. Formation of the Morula: After fertilization occurs, the zygote undergoes rapid cell division , forming a solid ball of cells called the morula. This transformation takes place within the fallopian tube 2. Transport to the Uterus: The morula takes approximately 3 to 5 days to travel through the fallopian tube and reach the uterine cavity. This transport is facilitated by a combination of fluid currents created by epithelial secretions and the beating motion of cilia lining the fallopian tube, which propel the morula toward the uterus. Weak contractions of the fallopian tube may also aid in this process. 3. Transformation into a Blastocyst: Within the uterus, the morula further develops into a blastocyst, which consists of approximately 100 cells. The blastocyst is a fluid-filled structure with an outer layer of cells called the trophoblast and an inner cell mass. 4. Implantation: The blastocyst spends 3 to 6 days freely floating in the uterine cavity before it becomes implanted in the uterine endometrium. During this time, the blastocyst relies on the nutritive secretions of the uterine endometrium, often referred to as “uterine milk.” 5. Trophoblast Action: Implantation occurs due to the activity of the trophoblast cells on the surface of the blastocyst. These trophoblast cells secrete proteolytic enzymes that digest and liquefy the adjacent cells of the uterine endometrium. This process allows the blastocyst to penetrate and anchor itself to the endometrium. Some of the fluid and nutrients released during this action are actively transported by the trophoblast cells into the blastocyst, providing additional nourishment for its growth. 6. Proliferation and Placenta Formation: Once implantation takes place, the trophoblast cells and adjacent cells from both the blastocyst and the uterine endometrium rapidly proliferate. This proliferation leads to the formation of the placenta and various membranes of pregnancy, which play crucial roles in supporting fetal development and maintaining pregnancy. Receptivity of Uterus The receptivity of the uterus plays a crucial role in the process of implantation. It involves various changes in the endometrial cells and tissues that prepare the uterus to receive and support the conceptus. Here are the key aspects of uterine receptivity based on the provided information: 1. Plasma membrane Transformation: Receptivity involves changes in the endometrial cells known as plasma membrane transformation. These changes include the formation of pinopodes, which are mushroom-like protrusions from the apical cell membrane of uterine epithelial cells. Pinopodes are ultrastructural markers of receptivity and are fully formed during the window of implantation, typically between days 19 and 21 of gestational age. 2. Decidualization: Decidualization refers to the process of the endometrium becoming prepared for implantation. It includes an increase in endometrial thickness, vascularization, and growth of the glands. The endometrium also produces decidual cells, which form a new layer called the decidua. The decidua plays a crucial role in supporting and nourishing the developing embryo. 3. Window of Implantation: The window of implantation is a limited timeframe during which the endometrium is optimally receptive for the attachment of the blastocyst. In humans, this window occurs between days 20 and 24 of the secretory phase of the menstrual cycle, when luteinizing hormone levels are at their peak. The window of implantation lasts only 24 to 36 hours, emphasizing the importance of timing for successful implantation 4. Receptor-Ligand Interactions: During the window of implantation, there is significant communication between the blastocyst and the endometrium. Receptor-ligand interactions, specifically involving integrin- matrix and proteoglycan receptors, play a role in this communication. Proteoglycan receptors are found on the surface of the decidua, while the blastocyst’s trophoblast cells have corresponding proteoglycans. These interactions contribute to the adhesion and invasion of the blastocyst into the endometrium. 5. Pinopodes: Pinopodes, which are formed during the window of implantation, aid in the receptivity of the uterus. They enhance the absorption of uterine fluid, reducing the volume of the uterus and bringing the blastocyst closer to the endometrium. Pinopodes also facilitate direct contact and adherence between the blastocyst and the uterine epithelial cells, promoting implantation. 6. Decidualization and Embryonic Influence: Decidualization not only prepares the endometrium for implantation but also responds to signals from the developing embryo. Factors released by the blastocyst trigger the final formation of decidual cells and further changes in the endometrium. This interaction highlights the co-dependence between the viability of the embryo and the receptivity of the uterus. Symptoms of implantation The implantation of the fertilized egg does not always give rise to specific symptoms by which we can confirm that the embryos have implanted in the uterus. However, there are women who experience certain symptoms or signs on the days of nesting that may make us suspect that implantation has occurred. Some of the most common are: Implantation bleeding (light, light-colored, low-intensity bleeding) Abdominal pain Breast swelling Increased urge to urinate Gastrointestinal disorders (diarrhea or constipation) Nausea and/or vomiting Cramps Abhorrence of certain odors and/or foods Main symptoms of embryo implantation  Placenta Development The embryo enters the uterus 4 days after fertilization and transitions from a morula to a blastocyst.  It then undergoes hatching where the outer zona pellucida is removed allowing it to attach to the endometrium.  It undergoes invasive interstitial implantation, where the cells of the blastocyst invade into the uterine lining.  This invasion, and the merging of the syncitiotrophoblasts and endometrial cells allows the placenta to form. The placenta is a discoid organ which is composed of two plates: i. Chorionic plate : faces the foetus and has the umbilical cord attached ii. Basal plate : opposed to the decidua basalis and maternal blood enters through it The placenta is formed in a number of steps, which are key to its role in achieving efficient nutrient exchange 1. Trophoblast differentiation: The trophoblast (outer layer of blastocyst) differentiates into the: a) Syncitiotrophoblast (STB) : outer multinucleated layer b) Cytotrophoblast (CTB): inner deeper layer The Syncitiotrophoblast is non-proliferative and is generated by continual fusion of the Cytotrophoblast cells 2. Erosion of decidua: The Syncitiotrophoblast erodes into the decidua (endometrium) – This breaks the endometrial glands and superficial capillaries – Spaces appear within the STB and coalesce to form lacunae – Glandular secretions and maternal blood fill the lacunae. 3. Early villus formation: Cytotrophoblast cells and extra-embryonic mesoderm from the embryo penetrate into the trabeculae of the Syncitiotrophoblast between the lacunae – This forms the earliest placental villi – A vascular network develops in the mesoderm – This connects back to the foetus via the connecting stalk 4. Formation of villous tree: Side branches extend from the early villi into the lacunae – They gradually branch to become more complex – Repeated branching forms the placental villous tree – The lacunae are then referred to as the intervillous space 5. Regression of villi: In early pregnancy, placental villi form over the entire chorionic sac – Later however, the villi regress over the superficial pole to leave the discoid placenta. (i.e., they only remain in one pole, forming the placenta) – The remainder of the sac forms the placental membranes (these rupture to a provide a route of exit at birth) 6. Remodelling of circulation: The general anatomical organization of the placenta is achieved by 3-4 weeks – However, it is not fully functional until maternal circulation has been remodelled which occurs by 10-12 weeks. – CTB cells, known as extravillous trophoblasts (EVTs), invade the maternal spiral arteries which supply the endometrium – The EVTs replace the endothelium of the arteries, resulting in vessel dilation and loss of vasoreactivity – This results in blood flow to the intervillous spaces that is low pressure and low velocity – Failure to convert the spiral arteries is associated with complications like growth restriction and pre-eclampsia 7. Placental maturation: By the 4th month, the placenta has two components: – The decidua basalis (the maternal portion) – The chorion frondosum (the foetal portion) During the 4th and 5th months: – The decidua form decidual septa – These divide the placenta into compartments called cotyledons – Cotyledons receive their blood supply through 80-100 spiral arteries 8. Full-term placenta: By the end of pregnancy, the placenta has a thickness of about 3cm, diameter 15-25cm and weighs 500-600g. – The foetal side is covered by chorionic plate – The maternal side is covered by a thin layer of decidua basalis. Umbilical cord The umbilical cord is formed from the connecting stalk, which connects the blastocyst to the placenta. – It is made of 3 blood vessels that carry foetal blood: i) 2 umbilical arteries: – These are branches from the internal iliac arteries – Carry deoxygenated blood from the fetus to the placenta ii) 1 umbilical vein: – This joins the inferior vena cava via the ductus venosus – Carries oxygenated blood from the placenta to the fetus Amniotic fluid This describes the protective fluid within the amniotic sac that cushions the fetus and serves as a transport medium for nutrients and metabolites. – It is formed by maternal plasma diffusing through placenta and foetal urine – It is around 850–1500 mL by the end of pregnancy – The amniotic fluid is completely exchanged every 3 hours – It is drained by the fetus (swallows fluid) + reabsorbed by umbilical cord into maternal circulation – It contains proteins, glucose, urea, hair, dead skin, sebum and foetal urine