Embryology: Early Development, Week 1 PDF

Kubalak – 2022-2024 Embryology: Week 1 Embryology: Early Development, Week 1 Steven W. Kubalak, PhD Department of Regenerative Medicine and Cell Biology Basic Science Building (BSB), Room 615C Email: [email protected] Office Phone: 2-0624 OUTLINE I. II. III. IV. V. Fertilization and Formation...

Kubalak – 2022-2024 Embryology: Week 1 Embryology: Early Development, Week 1 Steven W. Kubalak, PhD Department of Regenerative Medicine and Cell Biology Basic Science Building (BSB), Room 615C Email: [email protected] Office Phone: 2-0624 OUTLINE I. II. III. IV. V. Fertilization and Formation of the Zygote a. Capacitation b. Acrosome reaction c. Zona reaction Cleavage a. Compaction b. Blastocyst c. Twinning Implantation a. Cytotrophoblast b. Syncytiotrophoblast Clinical Implications Embryo manipulation OBJECTIVES 1. Describe the process of fertilization including the key features of the acrosomal reaction and the zona reaction during fertilization. 2. Define which cells represent the inner cell mass and its immediate derivatives. 3. Define which cells represent outer cell mass and its immediate derivatives. [Image: Cochard LR. Netter’s Atlas of Human Embryology. 2012. Updated edition] 4. Discuss what trophoblast cells differentiate into and the importance of these newly differentiated cells including what they produce if a blastocyst successfully implants. 5. Identify at what stage and at what time implantation occurs. 6. Identify the normal and abnormal sites of implantation and indicate which are most common. 7. Describe the basic difference between a transgenic embryo and a gene-targeted embryo. Know how these embryos are created and how they are used to understand the function of a gene. READING REFERENCES Text: Langman’s Medical Embryology, 14th ed. by Sadler © 2018 Ch. 3 pp 34-49 Ch. 4 pp 50-58 Note: To reinforce specific concepts, there are also a few select images taken from Histology and Cell Biology by Kierszenbaum. The histology reading material is optional. The key concepts from each of these specific slides will be presented in lecture and is contained in this printed syllabus. References to this text are given on each slide where they appear. 1 Kubalak – 2022-2024 Embryology: Week 1 INTRODUCTION The next three lectures present the very early development of the embryo—the first four weeks of development after fertilization (development of the egg and sperm will be discussed later in the course in the Reproductive Block). Each week of development will be covered separately showing the major changes that take place in both embryonic and extraembryonic tissues. The clinical significance of the developmental processes will also be presented for each week, where relevant. FERTILIZATION Fertilization normally takes place in the ampulla of uterine tube. Location Implantation at the end of the first week normally takes place in the posterior wall of the uterus. Figure 3.12 Embryonic development begins at fertilization. This figure (Figure 3.12) is shows the first 5-6 days (week 1) of development after fertilization and the normal location of the conceptus in the female reproductive tract during this time frame. Fertilization normally takes place in the ampulla of the uterine tube and marks day “0”. Notice the conceptus continues to develop while traveling through the uterine (Fallopian) tubes. The embryo (normally) implants in the posterior wall of the uterus at the end of the first week. Fundamental to understanding fertility issues is a foundational knowledge of mechanistic details regarding fertilization. For fertilization to occur normally, a specific series of events must take place. These events include capacitation and the acrosome which Events(3) reaction, are in reference to the sperm, and the zona reaction, which occurs in the next sequence of events is shown Figure 3.5 in Figure 3.5 and detailed on the next page.) When sperm are deposited in the vagina, they are not capable of fertilizing the egg. 2 Events important for fertilization of the egg (defined on the next page): • Capacitation • Acrosome reaction • Zona reaction Kubalak – 2022-2024 Embryology: Week 1 The outer cell membrane of the sperm needs to be “conditioned” by removal of a glycoprotein layer: this process is called capacitation. The environment of the uterus accomplishes this. One important result of capacitation is an increase in the motility of the sperm, which the sperm uses to its advantage by facilitating travel through both the uterus and uterine tubes. Upon arrival at the egg, the sperm must work its way through the corona radiata cells that are surrounding the egg and then penetrate both the zona pellucida and perivitelline space. These latter two layers are regions of extracellular matrix that are superficial to the egg cell membrane. The sperm plasma membrane displays hyaluronidase activity, which facilitates the breakdown of the extracellular matrix around the corona radiata cells. This allows the sperm to penetrate between the cells in the corona radiata cell layer (note: some texts say sperm pass freely through the corona radiata cells). To accomplish passage through the zona pellucida, the sperm undergoes what is called the acrosome reaction. The acrosome reaction takes place in the head of sperm where the acrosome is located (see below two figures from the histology text, Histology and Cell Biology, by Kierszenbaum, © 2008 by Mosby Pub). The acrosome contains the enzymes hyaluronidase and acrosin, among several other enzymes. Hyaluronidase and acrosin are needed to penetrate the corona radiate and zona pellucida, respectively. Once the sperm reach the zona pellucida, ZP3 receptors on the plasma membrane of the sperm recognize and bind to ZP3 in the zona pellucida. This interaction stimulates a massive influx of Ca2+, which then initiates the 3 EVENTS ARE LISTED IN ORDER OF OCCURANCE: Capacitation: • Removal of a glycoprotein layer from the cell membrane of the sperm Hyaluronidase release: • released by sperm allows for the penetration of sperm through the corona radiata cells Sperm binding: • sperm plasma membrane ZP3 receptors bind to ZP3 in zona pellucida • Initiates the acrosome reaction Acrosome reaction: • Triggered by ZP3 binding • Release of enzymes, particularly acrosin, that allows for the sperm to penetrate the zona pellucida Kubalak – 2022-2024 Embryology: Week 1 acrosome reaction. The acrosome reaction results from the fusion of the sperm plasma membrane with the outer acrosomal membrane, which allows for the release of the contents of the acrosome. Within the released enzymes is acrosin, which then breaks down the zona pellucida and allows the sperm to penetrate the zona layer and reach the egg cell membrane. Subsequently, fertillin / on the sperm plasma membrane interact with egg integrins and CD9 on the egg plasma membrane. This interaction results in the fusion of the two cell’s membranes. Importantly therefore, mutations in any of these proteins would prevent fertilization because they would interfere with the sperm-egg interaction. The zona reaction occurs immediately following fusion of the sperm and oocyte plasma membranes. This reaction is a Ca 2+-dependent exocytosis of cortical granules into the perivitelline space. The cortical granules contain enzymes that breakdown ZP2 and ZP3 in the zona pellucida and thereby prevent other sperm from fertilizing the same oocyte (polyspermy). 4 Zona Reaction: • Binding of sperm fertilin / with egg integrins and CD9 on the egg plasma membrane with oocyte plasma membrane triggers the zona reaction • Prevents subsequent sperm from fertilizing the same oocyte Kubalak – 2022-2024 Embryology: Week 1 The following summarizes these important steps: Capacitation – is a process that prepares the acrosome to release enzymes that breakdown zona. Cholesterol, the glycoprotein coat, and seminal plasma proteins are removed Important steps(3) from the plasma membrane. Acrosome reaction - after contact with the zona, perforations in acrosomal wall result in the release of enzymes to allow penetration of the zona pellucida and perivitelline space. Zona reaction - a change in the properties of the zona pellucida that make it impermeable to other sperms. Occurs via the release of cortical granules from the oocyte. Fertilization by more than one sperm is called polyspermy. Penetrating sperm triggers the second meiotic division Once the paternal DNA enters the cell, 2N is restored and the conceptus is called a zygote. Figure 3.6 Once there is contact between the sperm and egg plasma membranes, a receptor-mediated interaction causes the two membranes to fuse allowing the sperm cellular contents (predominantly DNA) to enter the cell. The penetrating sperm triggers the second meiotic division, which occurs in both the oocyte and the first polar body. (Figure 3.6) Note the use of the term zygote once the DNA complement has been restored to 2N. 5 Kubalak – 2022-2024 Embryology: Week 1 CLEAVAGE . Figures 3.7AB and 3.10A The zygote undergoes successive cell divisions (cleavages) (Figure 3.7, 3.10A and 3.8). Importantly, there is initially an increase in the number of cells without an overall increase in size – this is called compaction. This results in the cells becoming tightly associated with each other. At this stage the mass of cells is referred to as a morula. Compaction: • Increase in cell number (proliferation) but, decrease in cell size. Blastocyst: • Inner and out cell masses with a cavity called the blastocoele. Inner cell mass: • Embryoblast • Stem cells Outer cell mass: • Trophoblast Figure 3.8 Around the time of the 56-cell stage, a cavity forms in the morula called a blastocoele (also called a blastocyst cavity) and the mass of cells is referred to as a blastocyst. The blastocyst now has an outer cell mass and inner cell mass, each with distinct cell fates. Figure 3.10BC About this time (end of week 1), the blastocyst reaches the uterine cavity where the zygote “hatches” from degenerating zona pellucida and is now ready for implantation in the posterior wall of the uterus. What are alternative names for the inner and outer cell masses? TWINNING An important clinical correlate to normal cleavage and formation of the blastocyst is the prospect of twinning. Twinning can occur with no consequences to the developing embryos and occurs at an incidence of 32.6 per 1,000 births (2008 statistics). However, this is not always the case. Shown on the next page are various modes of dizygotic (two eggs each becoming fertilized by separate sperm, Figure 8.18) and monozygotic (one original egg, Figure 8.19) twinning. Splitting of the inner cell mass results in monozygotic twinning. If the inner cell mass does not completely separate, conjoined twins may result with any of a variety of configurations. 6 Dizygotic twinning • Separate eggs ovulate • Fertilization by separate sperm Monozygotic twinning • Single egg fertilized • Splitting after fertilization Kubalak – 2022-2024 Embryology: Week 1 Figures 8.18 Figure 8.19 Twin frequencies(4) Approximately 90% of twins are dizygotic, which are shown in Figure 8.18 above. Dizygotic twins result in two separate fetuses, each with their own amnion and chorion (Panel A above); however, occasionally they can share a chorion (Panel B above) if they implant very close together. Monozygotic twins occur at a rate of 3 to 4 per 1,000 birth. They result from the splitting of the zygote after fertilization. Example are shown in Figure 8.19. Triplets occur at a rate of approximately 1 per 7,600 pregnancies where the inner cell mass splits into three separate groups of cells. Conjoined twins occur when the inner cell mass does not completely separate and some part of each fetus develops in contact with the other. Sharing of tissue can be simple or very complex. Figure 3.14 from Carlson 5e, 2014 7 Twinning frequency rates • Overall: 32 per 1,000 birth • Dizygotic: ~30 per 1,000 • Monozygotic: ~ 3-4 per 1,000 • Triplets: 1 per 7,600 Kubalak – 2022-2024 Embryology: Week 1 IMPLANTATION Embryo implants at embryonic pole. Figure 3.10 Trophoblast gives rise to: • cytotrophoblast • syncytiotrophoblast Syncytiotrophoblast invades endometrium and underlying stroma. Hypoblast forms on the surface of inner cell mass Figure 4.1 The blastocyst attaches to the endometrial epithelium at the embryonic pole of the zygote (inner cell mass side of the blastocyst) (Figure 3.10). This is the beginning of implantation. Trophoblast cells (outer cell mass) then proliferate rapidly and give rise to two new cell layers, the cytotrophoblast and syncytiotrophoblast (Figure 4.1). Once the syncytiotrophoblast forms, cells of the trophoblast are now referred to as cytotrophoblast cells. The syncytiotrophoblast penetrates the endometrial epithelium and starts to invade the stroma. A new layer of cells called the hypoblast forms on the surface of the inner cell mass. Conceptus differentiates into: • Embryonic tissues (from inner cell mass) • Extra-embryonic tissues (from trophoblast) Conceptus differentiates(2) Figure 4.3 Implantation is almost complete at 9-10 days (Figure 4.3). The closing plug (the portion of the conceptus visible in the wall of the uterus) remains but proliferating epithelial cells soon covers the area. When this occurs, implantation is complete. 8 Kubalak – 2022-2024 Embryology: Week 1 Figure 4.4 Syncytiotrophoblast continue to form from cytotrophoblast cells and invade the endometrium. Cytotrophoblast gives rise to primary chorionic villi during the end of the second week into the third week of development PCV -sig(2) Figure 4.6 Some cytotrophoblast cells begin to organize in discrete pyramid-shaped structures called primary chorionic villi and mark the beginning the placenta and fetal-maternal circulation (see next page). Other new cell types also begin to emerge and will be discussed in detail in the next lecture. 9 Kubalak – 2022-2024 Embryology: Week 1 Anatomical Summary: This image (Figure 1.1) taken from Netter’s Atlas of Human Embryology provides a excellent summary of many of these early stages of development. Fig 10 Kubalak – 2022-2024 Embryology: Week 1 ECTOPIC PREGNANCIES Anything that delays transport of zygote through uterine tube could result in an ectopic pregnancy. Figure 4.8 An important clinical correlate to implantation is the possibility that implantation takes place in an abnormal location. If this occurs, it is referred to as an ectopic pregnancy (Figure 4.8). As alluded to above, implantation normally takes place in the posterior wall of the uterus. However, ectopic pregnancies can occur and are in general rare, occurring at a frequency of 0.25 – 1.0%. These occur at various locations but the most common (9597%) site is within the uterine tube (i.e. a tubal pregnancy). Ectopic pregnancies can be due to various factors including a delay or prevention of transport of the cleaving zygote as a result of mucosal adhesions or blockages caused by infections. Most ectopic pregnancies (9597%) occur in the ampulla and adjoining uterine tube. Ectopic causes(2) Figure 4.10 (4) INHIBITION OF IMPLANTATION Large doses of progestin compounds often referred to as “morning-after pills” (levonorgestrel: Trade names: Plan B One Step® or Next Choice®, Mirena®) can prevent pregnancies in four ways. (1) Inhibit implantation by disrupting the balance between estrogen and progesterone and the uterus is “not ready” so, implantation does not occur, (2) Delay ovulation, (3) Inhibit ovulation, (4) Prevent fertilization by altering transport of sperm/ova. 11 Large doses of progestin can inhibit implantation in multiple ways: • Disrupting balance between estrogen and progesterone so uterus is not receptive to conceptus • Delay ovulation • Inhibit ovulation • Prevent fertilization Kubalak – 2022-2024 Embryology: Week 1 EMBRYO MANIPULATION Panel A: Transgenic mice are created by injecting DNA into pronucleus of fertilized egg. Endogenous genes are theoretically not altered. Panel B: Gene-targeted mice are created by transferring genes into DNA of stem cells and injecting cells into blastocyst. Transferred DNA takes the place of, or disrupts the endogenous gene. Figure 3.12 The ability to alter the fate of the developing embryo has the potential for widespread application. This has been the subject of intense research using mouse models for several years. In fact, much of what has been learned about the developmental properties of the early embryo has come about as a result of being able to manipulate the genome. Various techniques have been developed that study the effects of either adding or subtracting specific genes within the genome. Mice have become an extremely valuable model for these experiments and many of the papers you will read will involve “knocking out” genes in mice. As a result of generating either transgenic or gene-targeted mice a vast amount of information has been acquired over the past several years as to the function of specific genes. Transgenic mice are created by injecting the pronucleus with a DNA fragment that then becomes randomly incorporated into the cell’s genome. The key here is that the DNA not only gets integrated into the genome but it is randomly integrated. Panel A in this figure (Figure 3.12) is an example of this, which shows the generation of a transgenic mouse by pronuclear 12 Kubalak – 2022-2024 Embryology: Week 1 injection of DNA (the gene) into a fertilized egg. Gene-targeted mice are created by constructing a DNA fragment in such a way that the gene is not only incorporated into the genome but also replaces part of the endogenous gene that “matches” specific regions of the DNA fragment. The DNA fragment is incubated with a large number of embryonic stem cells and a process called transfection allows the incorporation of the DNA into the cell/genome. In a small fraction of the cells, the DNA will swap itself with the matching DNA sequence (i.e. the matching gene): this is called homologous recombination. In this way, gene “knockouts” can be created if the engineered DNA fragment carries a mutation. Panel B in this figure (Figure 3.12) schematically shows this process. In both cases, mice are being generated that contain genetically-engineered DNA fragments in their genomes. Transgenic mice have the DNA fragment randomly integrated in the genome, which allows for a functional understanding of the inserted gene after it is expressed. The DNA fragment has two main features, the promoter region and the gene sequence that codes for the protein of interest. The point of generating this kind of mouse is to study the function of the protein encoded by the DNA fragment. The location (in the body) where the gene is expressed is determined by the promoter on the DNA fragment - these promoters are called "tissue-specific". Therefore, one can use a promoter for a different, unrelated gene in order to have the protein of interest expressed in a specific location in the body. These kinds of mice are not considered knockouts because theoretically no genes are disrupted (including the endogenous gene that codes for the same protein as the DNA fragment). Example: Let's say you want to investigate the over-expression of a specific Ca2+ channel in the myocytes of the heart. One way to do that might be to make a transgenic mouse carrying a DNA fragment that has a cardiomyocyte-specific promoter (e.g. cardiac myosin light chain-2) and the gene sequence for the desired Ca2+ channel. In this mouse, the Ca2+ channel would be expressed wherever cardiac myosin light chain-2 is normally expressed. Gene-targeted mice have the DNA fragments specifically incorporated in the genome where their matching endogenous genes are located. Thus, the DNA fragments swap out the normal gene in the genome (or part of the normal gene). These DNA fragments only carry sequences for the protein of interest (i.e. there are no promoter sequences). The initial mouse is considered chimeric because not all the cells contain the mutated gene. After a round of breeding with normal mice (non-mutated) you can isolate mice that have both alleles of the gene mutated. So, for example if the DNA fragment encodes a mutation that renders the resulting protein nonfunctional and both alleles carry the new mutated gene, the result is a knockout animal. This is because the mutated fragment swaps itself into the normal gene's location and there is no longer a normal gene present in the genome. This allows for the study of the mouse in the complete absence of that particular protein. Example: In this case let's say you want to study the effects of removal of a specific Ca2+ channel. That is, you want to knockout the Ca2+ channel to study the effects of not having the channel present. The DNA fragment is engineered such that it swaps itself into the exact location in the genome that has the normal Ca2+ channel gene (thus, removing it). The initial mice are chimeric but, after a couple generations of mice you can obtain a knockout and you are now able to study the effects of a lack of that particular Ca2+ channel. 13 Importantly: The above-described experimental approaches to studying the roles for various proteins is quickly be replaced by CRISPR-Cas9 technology. This relatively new, but extremely exciting and promising technology is allowing researchers and clinical scientists to manipulate the genome in a variety of ways. This technology will be presented in the Reproductive Block of the curriculum.