Fertilization & First Week of Development PDF

FERTILIZATION & FIRST WEEK OF DEVELOPMENT Gizem SÖYLER, PhD Near East University Faculty of Medicine Department of Histology & Embryology Ovulation Ovulation is the hormone-driven process in which a mature oocyte is released from the ovary, typically occurring around the midpoint of a 28-day...

FERTILIZATION & FIRST WEEK OF DEVELOPMENT Gizem SÖYLER, PhD Near East University Faculty of Medicine Department of Histology & Embryology Ovulation Ovulation is the hormone-driven process in which a mature oocyte is released from the ovary, typically occurring around the midpoint of a 28-day menstrual cycle. Triggered by a surge in luteinizing hormone (LH) stimulated by rising estrogen levels, ovulation involves the final maturation and release of the oocyte from the dominant follicle. Before ovulation, the oocyte completes its first meiotic division, forming a secondary oocyte and a polar body. The oocyte then begins the second meiotic division, which is arrested at metaphase and will only be completed if fertilization occurs. The stigma, an ischemic area on the follicle, ruptures, releasing the oocyte along with follicular fluid into the peritoneal cavity. Oocyte Transport The first step in egg transport is capture of the ovulated egg by the uterine tube. By ovulation, the fimbriae of the uterine tube move closer to the ovary and captures the ovulated egg complex. If the fimbriated end of the tube has been removed, the ovulated egg would have to travel free in the pelvic cavity for a considerable distance before entering the ostium of the uterine tube on the other side (Ectopic Pregnancy).  When inside the uterine tube, the egg is transported toward the uterus, mainly as the result of contractions of the smooth musculature of the tubal wall. Although the cilia lining the tubal mucosa may also play a role in egg transport. Tubal transport of the egg usually takes 3 to 4 days, whether or not fertilization occurs. By roughly 80 hours after ovulation, the ovulated egg or embryo has passed from the uterine tube into the uterus. If fertilization has not occurred, the egg degenerates and is phagocytized. Sperm Transport Sperm transport occurs in both the male reproductive tract and the female reproductive tract. After spermiogenesis in the seminiferous tubules, the spermatozoa are morphologically mature but are nonmotile and incapable of fertilizing an egg. Spermatozoa are passively transported via testicular fluid from the seminiferous tubules to the caput (head) of the epididymis through the rete testis and the efferent ductules. Spermatozoa spend about 12 days in the highly convoluted duct of the epididymis during which time they undergo biochemical maturation. By the time the spermatozoa have reached the cauda (tail) of the epididymis, they are capable of fertilizing an egg.  On ejaculation, the spermatozoa rapidly pass through the ductus deferens and become mixed with fluid secretions from the seminal vesicles and prostate gland. Prostatic fluid is rich in citric acid, acid phosphatase, zinc, and magnesium ions, whereas fluid of the seminal vesicle is rich in fructose (the principal energy source of spermatozoa) and prostaglandins. Despite the numerous spermatozoa (>100 million) normally present in an ejaculate, a number as small as 25 million spermatozoa per ejaculate may be compatible with fertility.  In the female reproductive tract, sperm transport begins in the upper vagina and ends in the ampulla of the uterine tube, where the spermatozoa make contact with the ovulated egg. During copulation, the seminal fluid is normally deposited in the upper vagina, where its composition and buffering capacity immediately protect the spermatozoa from the acid fluid found in the upper vaginal area.  There are two main modes of sperm transport through the cervix. One is a phase of initial rapid transport, by which some spermatozoa can reach the uterine tubes within 5 to 20 minutes of ejaculation. Such rapid transport relies more on muscular movements of the female reproductive tract than on the motility of the spermatozoa themselves. The second, slow phase of sperm transport involves the swimming of spermatozoa through the cervical mucus (traveling at a rate of 2 to 3 mm/hour), their storage in cervical crypts, and their final passage through the cervical canal as much as 2 to 4 days later. At this point, the spermatozoa enter one of the uterine tubes. According to some more recent estimates, only several hundred spermatozoa enter the uterine tubes, and most enter the tube containing the ovulated egg. Capacitation Once inside the uterine tube, the spermatozoa collect in the isthmus and bind to the epithelium for about 24 hours. During this time, they are influenced by secretions of the tube to undergo the capacitation reaction. One phase of capacitation is removal of cholesterol from the surface of the sperm. Cholesterol is a component of semen and acts to inhibit premature capacitation. The next phase of capacitation consists of removal of many of the glycoproteins that were deposited on the surface of the spermatozoa during their tenure in the epididymis. Capacitation is required for spermatozoa to be able to fertilize an egg (specifically, to undergo the acrosome reaction.  After the capacitation reaction, the spermatozoa undergo a period of hyperactivity and detach from the tubal epithelium. Hyperactivation helps the spermatozoa to break free of the bonds that held them to the tubal epithelium. It also assists the sperm in penetrating isthmic mucus, as well as the corona radiata and the zona pellucida, which surround the ovum. Fertilization of the egg normally occurs in the ampullary portion (upper third) of the uterine tube. Fertilization Fertilization is a series of processes rather than a single event. The phases of fertilization include the following: Phase 1, penetration of the corona radiata Phase 2, penetration of the zona pellucida Phase 3, fusion of the oocyte and sperm cell membranes  When sperm reach the egg in the ampulla of the uterine tube, they encounter the corona radiata and remnants of the cumulus oophorus. Fertilization begins when spermatozoa penetrate the corona radiata, the outer layer surrounding the egg. The process concludes with the mixing of maternal and paternal chromosomes inside the egg. Hyaluronidase, an enzyme from the sperm head, play a significant role in penetrating the corona radiata, though the active movement of sperm is also crucial. The zona pellucida, consists principally of four glycoproteins—ZP1 to ZP4.  After they have penetrated the corona radiata, spermatozoa bind tightly to the zona pellucida by means of the plasma membrane of the sperm head. Spermatozoa bind specifically to the ZP3 molecule. Molecules on the surface of the sperm head are specific binding sites for the ZP3 sperm receptors on the zona pellucida. Acrosome Reaction When mammalian sperm bind to the zona pellucida, they undergo the acrosomal reaction. This reaction involves the fusion of the outer acrosomal membrane with the sperm's plasma membrane, resulting in the release of enzymes stored in the acrosome. A significant influx of calcium (Ca++) into the sperm head initiates this process. The fusion of membranes leads to the shedding of vesicles, freeing the enzymes (acrosin & serine proteinases) that help the sperm penetrate the zona pellucida. Membrane Fusion Once the sperm penetrates the zona and enters the perivitelline space, it can make direct contact with the egg's plasma membrane. After entering the perivitelline space, the sperm binds to and then fuses with the egg's plasma membrane in two distinct steps. The binding occurs when the equatorial region of the sperm head contacts the microvilli on the egg’s surface. The acrosomal reaction is essential for this fusion; without it, the sperm cannot fuse with the egg. Zona Reaction Following sperm entry into the egg, waves of Ca++ spread through the egg's cytoplasm, triggering important events. The initial waves help complete the egg's second meiotic division, while later waves activate maternal RNAs and affect the cortical granules. These granules then fuse with the egg's plasma membrane, releasing hydrolytic enzymes and polysaccharides into the perivitelline space. These secretory products diffuse into the zona pellucida and hydrolyze sperm receptor molecules (like ZP3), preventing other sperm from binding and penetrating the zona. This process, known as the zona reaction, along with alterations in sperm receptor molecules on the egg's plasma membrane, helps ensure that no additional sperm can fertilize the egg, contributing to the slow block to polyspermy. Pronucleus Formation After the sperm enters the egg, Ca++ released within the egg stimulates an increase in respiration, metabolism, and intracellular pH. As the sperm head penetrates the egg, the permeability of its nuclear membrane increases, allowing egg cytoplasmic factors to influence the sperm's nuclear contents. The tightly packed chromatin in the sperm, held together by disulfide (—SS—) cross-links in protamine molecules, begins to loosen. These disulfide bonds are reduced to sulfhydryl (—SH) groups by reduced glutathione in the egg's cytoplasm, causing the protamines to detach from the sperm's DNA. Consequently, the chromatin spreads out within the sperm nucleus, forming the male pronucleus, which then moves closer to the egg's nuclear material in preparation for fertilization. Zygote Formation After a short period during which the male chromosomes are naked, histones begin to associate with the chromosomes. Simultaneously, the egg completes the second meiotic division, releasing a second polar body. Approximately 6 to 8 hours after sperm entry, pronuclei form and persist for about 10 to 12 hours. During this time, the maternal and paternal chromosomes organize around a mitotic spindle, which is derived from the sperm's centrosome, in preparation for the first mitotic division. This marks the completion of fertilization, and the fertilized egg is now referred to as a zygote.  Cleavage After fertilization, the zygote undergoes a significant metabolic shift and begins the process of cleavage. During this period, the embryo, still enclosed in the zona pellucida, is transported through the uterine tube to the uterus. The zygote starts dividing mitotically, increasing the number of cells known as blastomeres, which become progressively smaller with each division. Morula Formation Initially, the blastomeres form a loosely arranged cluster, but after the third cleavage, they compact tightly together, forming a ball of cells held by tight junctions. This compaction marks a critical stage in early embryonic development. This process, compaction, segregates inner cells, which communicate extensively by gap junctions, from outer cells. Approximately 3 days after fertilization, cells of the compacted embryo divide again to form a 16-cell morula (mulberry). Inner cells of the morula constitute the inner cell mass, and surrounding cells compose the outer cell mass. Inner cell mass gives rise to tissues of the embryo proper, and the outer cell mass forms the trophoblast, which later contributes to the placenta.  Blastocyst Formation As the morula enters the uterine cavity, fluid penetrates through the zona pellucida into the spaces within the inner cell mass. These spaces gradually merge to form a single cavity known as the blastocele, marking the embryo's development into a blastocyst. The cells of the inner cell mass, now called the embryoblast, cluster at one pole, while the outer cell mass, or trophoblast, forms the epithelial wall of the blastocyst. Embryo Hatching After floating in the uterine secretions for about 2 days, the zona pellucida surrounding the blastocyst begins to degenerate and eventually disappears. This shedding of the zona pellucida, which has been observed in vitro, allows the blastocyst to rapidly increase in size. During this time, while still in the uterus, the embryo derives nourishment from the secretions of the uterine glands.  The disappearance of the zona pellucida allows the blastocyst to begin implantation. Around the sixth day, trophoblastic cells at the embryoblast pole start penetrating the uterine mucosa. Initial attachment of the blastocyst to the uterus is mediated by interactions between L-selectin on trophoblast cells and its carbohydrate receptors on the uterine epithelium. Following the initial capture, integrins on the trophoblast interact with extracellular matrix molecules like laminin and fibronectin to promote attachment and migration, facilitating implantation. By the end of the first week, the blastocyst is implanted in the uterine mucosa, having progressed through the morula and blastocyst stages. EPIBLAST, HYPOBLAST, AND AXIS FORMATION During the early blastocyst stage, under the influence of fibroblast growth factors (FGFs), cells in the embryoblast differentiate into two distinct layers: the epiblast and the hypoblast. Initially, these cells are scattered within the embryoblast, but as implantation approaches, they organize into specific layers—epiblast cells dorsally and hypoblast cells ventrally, adjacent to the blastocele. This organization establishes the embryo's dorsal-ventral polarity.  Some hypoblast cells differentiate further to form the anterior visceral endoderm (AVE), which migrates to the future cranial end of the embryo. AVE cells, being part of the endoderm, secrete nodal antagonists such as cerberus and lefty1. These inhibitors act on nearby epiblast cells to specify the cranial end of the embryo. In the absence of these inhibitors, nodal signaling establishes the primitive streak at the caudal end. Thus, the cranial-caudal axis of the embryo is established near the time of implantation, around days 5.5 to 6.