Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college GE-2T: Developmental Biology/Embryology Gametogenesis: 1. What is spermatogenesis ? Spermatogenesis is the process by which haploid spermatozoa develop from germ cells in the seminiferous tubules of the testis. This process starts with the mitotic division of the stem cells located close to the basement membrane of the tubules. These cells are called spermatogonial stem cells. 2. Describe the process of spermatogenesis. In the process of spermatogenesis, spermatogonial stem cells develop into mature sperms. It occurs in the male gonads testis. Testes are made up of many seminiferous tubules lined by germinal epithelium. Cells of this layer divide to form spermatozoa in the following four steps: (1) Multiplication Phase, (2) Growth Phase, (3) Maturation Phase, and (3) Spermiogenesis. 1) Multiplication Phase: At maturity, the primordial germ cells divide by mitosis to produce a large number of spermatogonia. Type A spermatogonia is the stem cells which divide to form spermatogonia. Type B spermatogonia are the precursors of sperms. Spermatogonia have spherical or oval nuclei, and rest on the basement membrane. 2) Growth Phase: Type B spermatogonium actively grows to a primary spermatocyte. It obtains nourishment from the nursing cells. Cells in prophase of the first meiotic division are primary spermatocytes. They are characterized by highly condensed chromosomes giving the nucleus a coarse chromatin pattern and an intermediate position in the seminiferous epithelium. This is a long stage, so many primary spermatocytes can be seen. 3) Maturation Phase: Each primary spermatocyte undergoes two maturation divisions. The first maturation division is reductional and forms two haploid daughter cells called secondary spermatocytes. Both secondary spermatocytes then undergo second maturation division to form four haploid spermatids. Spermatids are spherical cells with interphase nuclei, positioned high in the epithelium. All of these progeny cells remain attached to each other by cytoplasmic bridges. The bridges remain until sperm are fully differentiated.

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college 4) Spermiogenesis: This is the metamorphosis of spherical spermatids into elongated spermatozoa. The spermatozoa are then known as sperms. No further mitosis or meiosis occurs. During spermiogenesis, the acrosome forms, the flagellar apparatus forms, and most excess cytoplasm (the residual body) is separated and left in the Sertoli cell. Spermatozoa are released into the lumen of the seminiferous tubule. A small amount of excess cytoplasm (the cytoplasmic droplet) is shed later in the epididymis.

Figure: Process of spermatogenesis

3. What is ? Oogenesis is the process of the formation of a mature ovum (female gamete) from the oogonia in females. It takes place in the . Oogenesis is initiated in the embryonic stage. 4. Describe the process of oogenesis. Oogenesis is process by which female gametes ( cells) are produced in the ovaries of the foetus before birth. It begins during fetal development when oogonia are formed from primordial germ cells by mitosis. It occurs in three phases i.e., 1) Multiplication phase, 2) Growth phase and 3) Maturation phase

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college 1) Multiplication phase:  The germinal epithelial cells of divide repeatedly by mitotic division until many diploid oogonia are formed.  The oogonia grow to form primary oocytes. Primary oocytes begin first meiotic division but cell division ceases in Prophase I until on set of puberty.  Primary oocytes surrounded by a layer of follicle cells. 2) Growth phase: In the growth phase, the increase in the size of the primary oocyte is very considerable. It is a slow process and may take a long period. In human female, the primary oocytes are already formed at the time of birth and continue growth only after attaining sexual maturity. It contains a diploid number of chromosomes. 3) Maturation phase:  At puberty some follicles develop each month in response to FSH produced by pituitary gland. Primary oocyte completes first meiotic division to forms two cells of different sizes due to unequal distribution of cytoplasm. The one with less cytoplasm become the first polar body (n) which eventually degenerates. The larger cell becomes secondary oocyte (n).  The follicle cells surrounding the primary follicle develop into the secondary follicle. The follicle layer of the secondary oocyte thickens and folds to form the Graafian follicle. When the Graafian follicle become matures, it will move towards the surface of the ovary wall and rupture to release the secondary oocyte (n).  The secondary oocytes proceeds to meiosis II & stops at prophase II. Meiosis II is completed if cell is fertilized by a sperm (n). The final products of meiosis II are the haploid ootid or ovum and the second polar body.

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college 5. What are the differences between spermatogenesis and oogenesis ? Aspect Spermatogenesis Oogenesis Site of process Entirely in the testes Mostly in the ovaries Cells produced Sperm Ova or egg Size of cells Small Big Cell structure Consist of the head, middle Round pieces and tail Number of Four funcional cells One funcional cells and three gamates non-funcional polar bodies produced. Meiosis Equal division of cells. Unequal division of cytoplasm. Occurs continuously Not continues. Stops at meiosis I. Meiosis II occurs only if sperm penetrates the secondary oocyte. Density of Many mitochondria, less Fewer mitochondria, more organelles cytoplasm. cytoplasm. Parent cells Infinite number of cells can Limited number of cells can become sperm. become ova.

6. What are the similarities of spermatogenesis and oogenesis ? a) Both occur in the reproductive organs. b) Both produces haploid(n) gamates that are involved in fertilization. c) Both involve meiosis. 7. What is Spermiogenesis ? Spermiogenesis is a process of metamorphosis from a round cell with typical organelles to a highly specialized, elongated cell well adapted for traversing the male and female reproductive tracts and achieving fertilization of an egg.

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college

Figure: Transition of spermatid to spermatozoan

8. Describe the ultra structure of sperm. A spermatozoan is a haploid male gamete whose primary function is to fuse with ovum to restore diploid and to transmit paternal characters to the offspring’s. A mammalian sperm is minute, microscopic, flagellated and motile gamete with no nutritive material, protective envelopes and most of cell- organelles like ribosome, endoplasmic reticulum, etc. The whole body of sperm is enveloped by plasma membrane only. It is basically formed of four parts, each performing a specific function: 1. Head: Shape of head varies in different mammals. It is generally oval and flat (in man, bull, rabbit). Basically the head is formed of two parts: Acrosome: It is small cap-like pointed structure present at the tip of nucleus. It is formed from a part of Golgi body of spermatid. During the sperm entry, the acrosome secretes a lytic enzyme, called hyaluronidase, which helps in the penetration of ovum. Nucleus: It is generally long, narrow and pointed but is flat and oval in human sperm. It is formed by condensation of nuclear chromatin of spermatid and loss of RNA, nucleolus and acidic proteins. Chemically, the nucleus is formed of deoxyribonucleoprotein (DNA+basic proteins). It is the carrier of genetic information. Acrosome and anterior half of nucleus are covered by a fibrillar sheath galea.

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college 2. Neck: It is the smallest part of spermatozoan and may be indistinct. It is formed of two centrioles perpendicular to each other and is formed from the centrosome of spermatid. Each centriole is a micro tubular triplet structure having 9+0 arrangement. Proximal centriole lies in a depression in the posterior surface of the nucleus and is perpendicular to main axis of the sperm. Distal centriole is along longitudinal axis of the sperm. Centrioles form spindle for the first cleavage of . Distal centriole acts as basal body and gives rise to axoneme of the sperm-tail. 3. Middle piece: It lies behind the neck and is cylindrical in the human sperm. It is formed of a mitochondrial spiral, nebenkem, around the proximal part of axoneme. The mitochondria are the carriers of the oxidative enzymes and the enzymes which are responsible for oxidative phosphorylation. So the middle piece is the powerhouse of a sperm. Posterior half of nucleus, neck and middle piece of sperm are covered by a sheath, manchette. 4. Tail (flagellum): It is the longest part of sperm. It is slender and tapering part. It is formed of two parts: Central, contractile and micro tubular part called axoneme or axial filament, and outer protoplasmic sheath. Axoneme is formed of 11 proteinous microtubules arranged in 9+2 manner. Sometimes, a ring centriole may be present at the junction of middle piece and flagellum. Tail shows lashing movements which provide forward push to the sperm. Sometimes, the distal part of axoneme is uncovered and is called end piece. Viability: It is the period up to which the sperm is able to fertilize an ovum. Viability of human sperm is about 24 hours.

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college

9. Describe the ultra structure of ovum. Ovum is a maternal haploid gamete and is primarily concerned with receiving the sperm and is determined to develop into a fully developed multicellular organism after fertilization (syngamy) or without fertilization (parthenogenesis). It is generally with reserve food and is genetically programmed. An ovum is generally spherical, non-motile gamete with yolky cytoplasm and enclosed in one or more egg envelopes. Size of ovum varies in different animals and depends upon the amount of . Size of ovum varies from 10 to a few cm. Largest sized egg is of ostrich and is about 170 x 135 mm. Egg size and yolk amount are interdependent. It is about 50 in many polychaete worms, 150 in tunicates but very large sized in birds and reptiles. In mammals, it is generally microlecithal and about 100 .

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college Human ovum is microlecithal with large amount of cytoplasm. Cytoplasm is differentiated into outer, smaller and transparent exoplasm or egg cortex and inner, larger and opaque endoplasm or ooplasm. Egg cortex is with some cytoskeletal structures like microtubules and microfilaments, pigment granules and cortical granules of mucopolysaccharides. Endoplasm is with cell- organelles, informosomes, tRNAs, histones, enzymes etc. Nucleus of ovum is large, bloated with nucleoplasm and is called germinal vesicle. Nucleus is excentric in position so human ovum has a polarity. The side of ovum with nucleus and polar body is called animal pole, while the opposite side is called vegetal pole. Egg envelopes: Human ovum is surrounded by a number of egg envelopes: 1. : It is inner, thin, transparent and is secreted by ovum itself. 2. Zona pellucid: It is middle, thick, transparent and non-cellular. It is secreted partly by follicular cells and partly by the oocyte. 3. Corona radiate: It is outer, thicker coat formed of radially elongated follicular cells. Between the vitelline membrane and zona pellucid there is a narrow perivitelline space.

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college 10. Write down about egg Membranes. Protective formations varying in structure that surround the of most animals. Egg membranes are absent only in the few animals whose eggs are capable of ameboid movement, for example, sponges and certain coelenterates. There are three types of egg membranes. 1) The primary, or vitelline, membrane is produced by the egg itself during the growth period (oocyte). It usually consists of a thin transparent layer; sometimes it attains considerable thickness and is multilayered (Ascaris and certain vertebrates). In most vertebrates the vitelline membrane is striated by numerous radial canals and is hence known as the zona radiata (the zona pellucida in mammals). The canals develop during membrane formation at the points where the villi extend to meet the outgrowths of the surrounding follicle cells. Some biologists believe that the zona radiata is formed by matter secreted not only by the oocyte but also by the follicle cells; for this r eason, they maintain that it is incorrect to regard the membrane as primary. The vitelline membrane is almost always present. Many eggs have an additional, usually secondary, membrane. Some eggs have primary, secondary, and tertiary membranes. 2) The secondary membrane, or chorion, is secreted by the auxiliary cells of the ovary or is formed by the conversion of the auxiliary cells into the material of the membrane. In insects and other arthropods the chorion is permeated by chorionin, a substance similar in composition to keratin; the chorionin imparts toughness to the membrane. 3) The tertiary membranes are secreted by cells of the female’s genital tract when the ovum moves through the . Tertiary membranes include the gelatinous membranes of the eggs of echinoderms, mollusks, fish, and amphibians. They also include the tough protein membranes that are clad in the horny shells of cephalopods and selachians, the fibrous and calcareous shells of reptiles, and the undershell membranes and the calcareous shells of birds. In many invertebrates a tough tertiary shell, called a cocoon, surrounds several eggs. The cocoon contains protein fluid, which serves as a fluid tertiary membrane. If the tough membranes form before the and the spermatozoon unite, special canals known as micropyles form. The micropyles enable the spermatozoon to reach the cytoplasm of the ovum without obstruction.

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college 4) Egg membranes protect eggs from physical injury, penetration by microorganisms and parasites, and desiccation. In many animals the sticky membranes or their appendages also serve to attach the egg to the substrate. Fluid protein membranes are used by developing embryos as nutrient matter. The separation of the membrane from the surface of the egg during fertilizat ion plays an important protective role against penetration by too many sperm atozoa (cortical reaction). 11. Describe the fertilization of Sea-urchin. There are seven events of fertilization in sea-urchin. The events are: 1) Contact and Recognition between Sperm and Egg 2) Acrosomal Reaction and Sperm Penetration 3) Binding of Sperm to the Egg 4) Prevention of Polyspermy 5) Metabolic Activation of the Egg 6) Formation of Pronuclei 7) Migration of Pronuclei and Fusion of the Genetic Material 1) Contact and Recognition between Sperm and Egg: The mode of fertilization in sea urchin being external, the egg and spermatozoa are released into the sea water. The first step is the encounter of spermatozoa and the egg. This encounter is brought about by the swimming movement of the spermatozoa. To ensure the survival of the species, the gametes are produced in large numbers. A single female Arbacia releases about 4 million eggs, while the male releases about 100 billion spermatozoa, during a single spawning. Not- withstanding this, adult sea urchins improve the chances of a sperm meeting an egg by moving into dense aggregates before spawning. However, a successful fertilization depends largely on coordinated timing in the release of gametes and water conditions at that time. Species-specific sperm attractants have been found in sea urchin. Sperms are attracted towards eggs of their own species by chemo-taxis, i.e., by following a gradient of a chemical, secreted by the egg. One such chemotaxin is a 14- amino acid peptide called resect, has been isolated from the egg jelly of a sea urchin Arbacia punctulata. Resact diffuses readily in sea-water. A very low concentration of resact in the sea water can attract sperm of its own species. The sperms of A. punctulata have receptors in their plasma

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college membranes that bind resact. Resact also acts as a sperm-activating peptide that causes drastic and immediate increase in mitochondrial respiration and sperm motility.

2) Acrosomal Reaction and Sperm Penetration: The acrosomal reaction has two parts: a) The fusion of the acrosomal vesicles with the sperm plasma membrane and b) The extension of the acrosomal process. The acrosomal reaction in sea urchin is initiated by contact of the sperm with the egg jelly, that causes exocytosis of the sperm’s acrosomal vesicle and the proteolytic enzyme present in the acrosome gets released. This enzyme digests a path through the jelly coat till it reaches the egg surface. The acrosomal reaction is stimulated by an exchange of extracellular Ca++ with intracellular K+ by sperm plasma membrane resulting in the increase of intracellular pH to more than 7-2. This initiates the localized fusion of the outer acrosomal membrane with the plasma membrane (Fig. 5.19). This releases the soluble enzymes located within the acrosomal vesicle.

The second part of the acrosomal reaction involves the extension of the acrosomal process (Fig. 5.19). This involves the polymerization of the globular actin (g-actin) within the sub acrosomal region, to filament actin (f- actin).

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college The f-actin forms the basis of the acrosomal process, which protrudes from the head of the sperm. The tip of this acrosomal process is covered with a protein called bindin, that helps the sperm to bind to the egg surface. A major species-specific recognition step occurs when the acrosomal process of the sperm, after having penetrated the egg jelly, comes in contact with the egg surface. The bindin that covers the acrosomal process is capable of binding (agglutinizing) to de-jellied eggs of the same species (Fig. 5.20). The vitelline envelope or plasma membrane of the egg has species-specific binding receptors that recognise the bindin of its own species.

After having passed through the egg jelly coat, the spermatozoa encounter the vitelline envelope, which is a tough non-cellular layer present between the jelly coat and the plasma membrane. The vitelline envelope is made up of glycoprotein and polysaccharide molecules. The spermatozoa digest their way through it with the help of acrosomal enzymes with trypsin like activity, commonly called lysin. 3) Binding of Sperm to the Egg:

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college The lysis of the vitelline envelope is followed by the fusion of the sperm plasma membrane with the plasma membrane of the egg. This membrane fusion is a common feature of fertilization in all animals. The binding of sperm with egg membrane appears to cause the formation of several microvilli in the egg plasma membrane. The microvilli seem to engulf the head of the sperm, causing a bulge known as the fertilization cone in the egg (Fig. 5.21). The fertilization cone, like that of the acrosomal process, appears to be extended by the polymerization of actin. The plasma membrane of the sperm is antigenically different from that of the egg. However, it becomes incorporated into the plasma membrane of the egg as a mosaic patch, as the sperm nucleus moves into the interior of the egg. In the sea urchin all region of the egg plasma membrane are capable of fusing with sperm and this fusion is often mediated by specific “fusogenic” proteins. Bindin, however, plays a second role as a fusogenic protein.

4) Prevention of Polyspermy: Normally in sea urchin monospermy results, in which only one sperm enters the egg. The entrance of multiple sperm, i.e., polyspermy, results in the establishment of polyploidy. This leads to disastrous consequences resulting in the early disruption of development and the death of the embryo.

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college Species, therefore, have evolved ways to prevent the union of more than two haploid nuclei and the most common way is to prevent the entry of more than one sperm into the egg. Sea urchin has evolved two blocks to avoid polyspermy. The fast block to polyspermy is accomplished by a temporary electrical change in the egg plasma membrane. This is followed by a slower, more complex but permanent block called the slow block to polyspermy (Fig. 5.22). The fast block is an adaptation for quickly cutting off access to the egg by sperms that are close behind the first one, in penetrating the vitelline mem- brane. It also buys off some time for the egg to set up the permanent block.

a) Fast Block to Polyspermy: The fast block (Fig. 5.22) takes place within 2 to 3 seconds and lasts for about 60 seconds. It is achieved by altering the electric potential of the egg’s plasma membrane, called resting membrane potential. It is generally about 70mV which is usually expressed as -70mV as the inside of the cell is negatively charged with respect to the exterior. The surrounding sea water has high sodium ion (Na+) concentration in comparison to the egg cytoplasm. The reverse is true for potassium ion

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college (K+). Due to small influx of Na+ into the egg, the membrane potential shifts to a positive level (about +20mV), within 1-3 seconds after the binding of the first sperm. This formation of positive resting potential prevents further sperms from fusing to the egg. b) Slow Block to Polyspermy: The fast block to polyspermy initiates the slow block to ensure that multiple sperm do not enter into the egg cytoplasm. The first step in this is the mobilization of Ca++ from the stores present within the egg. Ca++ is first released at the site of sperm entry (Berger, 1992) and a wave of free Ca++ passes through the egg. This wave of released Ca++ initiates the cortical reaction. The sea urchin egg contains about 15,000 cortical granules lying in a layer just beneath the plasma membrane. Each granule have a diameter of about 1 µm. The free calcium ions initiate the cortical granules to move to the inner surface of the plasma membrane and to fuse with it. This ruptures the cortical granules, which then release their contents into the space between the plasma membrane and vitelline envelope, and a regular sequence of event follows (Fig. 5.23). Several proteins are released by the exocytosis of the cortical granules.

The first are proteases, a proteolytic enzyme that breaks the molecular bonds that bind the vitelline envelope to the plasma membrane. They also clip off the bindin receptor and any sperm attached to it. Mucopolysaccharides (a second protein) released by the cortical granules produce an osmotic gradient, that causes water to rush into the space between

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college peri-vitelline envelope and egg membrane. This causes the vitelline envelope to expand and thus form the fertilization envelope. A third protein, peroxidase enzyme, released by the cortical granules, hardens the fertilization envelope. Finally, a fourth protein, hyaline, forms a coating around the egg below the fertilization membrane (Fig. 5.23). This hyaline layer provides support for the blastomeres at the time of cleavage. 5) Metabolic Activation of the Egg: The sea urchins mature egg is a metabolically sluggish cell that is activated by the sperm. This activation is merely a stimulus for undergoing metabolic events that were pre-programmed (at the growth phase of oogenesis). The activation of the egg starts with the two blocks to polyspermy – the fast block initiated by sodium ion influx into the cell and the slow block by the intracellular release of calcium ions. The release of Ca++ appears to be the main stimulus for the activation of egg. Such an increase of Ca++ occurs either through its entry into the egg from out- side, or by its release from the endoplasmic reticulum within the egg. The first mechanism occurs in snails and worms, while in fishes, frogs, sea urchins and mammals, most of the Ca++ probably comes from the endo- plasmic reticulum. Other than the cortical reaction, the events that take place (Fig. 5.22) include: a) A three to five fold increase in oxygen consumption, (probably related to

the formation of H2O2). b) Activation of the enzyme NAD+ kinase which converts NAD+ to NADP+ (Epel et al. 1981). This conversion may facilitate the biosynthesis of new membrane lipids, which is important in the construction of many new cell membranes required during cleavage. c) A second influx of Na+ coupled with an efflux of H+ from the cell, resul- ting in the increase of intracellular pH. This increase of pH leads to an increase in protein synthesis, the activation of transport systems and ultimately the initiation of DNA synthesis, in preparation for the first cleavage division. 6) Formation of Pronuclei: In sea urchin, the nucleus of the sperm enters perpendicular to the egg surface. On fusion of the egg and sperm plasma membrane, the sperm

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college nucleus and centriole separate from the mitochondria and flagellum. Latter the two disintegrate inside the egg cytoplasm. Shortly after the entry of the sperm into the egg cytoplasm (lifting the block to the second meiotic division and releasing the second polar body), the nuclear membrane of the sperm breaks down (Fig. 5.24). The interaction between the nuclear contents of the sperm and the cytoplasm of the egg results in de-condensation of the tightly packed nuclear chromatin. As the chromatin dispersion nears completion, a new nuclear membrane is formed. The nucleus thus becomes vesicular and has an appearance like the interphase nucleus and is called the male pro-nucleus. The nucleus of the egg also undergoes certain changes. After completion of the second meiotic division, the haploid nucleus of the egg forms the female pro-nucleus. 7) Migration of Pronuclei and Fusion of the Genetic Material: When the sperm was entering the egg, the nucleus was in front followed by the centriole. As the sperm moves inward, from the site of the fertilization cone, it soon rotates 180° so that the centriole assumes the leading position and is positioned between the sperm and egg pro-nucleuses. It provides the basis for the formation of the sperm aster that plays a major role in guiding the migrations of the pronuclei. The radiating array of microtubules of the sperm aster pushes against the inner surface of the plasma membrane of the egg and helps to displace the male pro-nucleus towards the centre of the egg. As the rays of the sperm aster reaches the female pro-nucleus, it rapidly moves along the rays toward the male pro-nucleus. Both the pronuclei are then pushed to the center of the egg by the expansion of the sperm aster. In sea urchins, the two pronuclei come into contact with each other, their membranes fuse, resulting in the formation of a single membrane, which encloses both male and female chromosomes. This process is called pronuclear fusion, resulting in the formation of the diploid zygote nucleus. Soon after the pronuclear fusion, the chromosomes replicate their DNA, for the first cleavage division. As the chromosomes prepare for the first cleavage division, the process of fertilization is com- pleted. In case of the mammals as the two pronuclei migrate’ towards each other, it replicates its DNA. The two nuclear envelopes,, on mating, break down and

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college the chromatin condenses into chromosomes that orient themselves on a common mitotic spindle. The nuclear membrane is formed only at the 2-cell stage.

12. Describe the fertilization of mammals. 1) Sperm-egg interactions: a) Fertilization of the primary oocyte occurs in the oviduct. The oocyte is captured by the fimbria (funnel-shaped end) and swept down by the action of cilia in the oviduct lining. Fertility is maintained only for about 24 hours whereas the sperm can live about 2 days in the tract. Sperm must first be capacitated by exposure to the reproductive tract before they can undergo the acrosome reaction. b) Capacitated sperm must move past the follicle cells and then the zona pellucida contact induces the acrosome reaction. The glycoprotein ZP3 and specifically the N-acetylglucosamine sugar on this protein binds to

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college the sperm in a species-specific manner. ZP3 is linked with ZP2 in a fibrous matrix and cross-links are made with ZP1. 2) Fusion and cortical reaction: a) Fusion of the membranes:  In the mouse fertilizin is a protein on the sperm membrane that contains hydrophobic amino acids that may destabilize the egg and sperm membranes so that they can fuse. Fertilizin binds to an integrin of the egg plasma membrane.  The sperm nucleus becomes the pronucleus and the proximal centriole organizes a centrosome in the egg.  A temporary mosaic of sperm and egg plasma membranes exists.  Mitochondria get into the egg cytoplasm but are later lost (by unknown process for unknown reasons). b) Block to polyspermy:  Polyspermy is a disastrous condition whereby more than one sperm enter. The embryo will be polyploid and the extra centriole will set up another set of cleavage planes.  Slow block to polyspermy through the cortical granule reaction. Within one minute of fertilization the cortical granules are exocytosed to release their contents into the space between the plasma membrane and the vitelline membrane. The cortical granule contents include a glycosidase which clips off the carbohydrate of the ZP3 used for sperm binding. This expels other attached sperm.

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college

Figure: fertilization

13. What is Cleavage ? In embryology, cleavage is the division of cells in the early embryo. The process follows fertilization, with the transfer being triggered by the activation of a cyclin-dependent kinase complex. The of many species undergo rapid cell cycles with no significant overall growth, producing a cluster of cells the same size as the original zygote. The different cells derived from cleavage are called blastomeres and form a compact mass called the morula. Cleavage ends with the formation of the blastula. Depending mostly on the amount of yolk in the egg, the cleavage can be holoblastic (total or entire cleavage) or meroblastic (partial cleavage). 14. Explain different types of cleavage planes. An egg can be divided from different planes during cleavage. Depending on the position of the cleavage furrow the planes of cleavage are named. 1) Meridional plane: The plane of cleavage lies on the animal vegetal axis. It bisects both the poles of the egg. Thus the egg is divided into two equal halves.

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college 2) Vertical plane: The cleavage furrows may lie on either side of the meridional plane. The furrows pass from animal to vegetal pole. The cleaved cells may be unequal in size. 3) Equatorial plane: This cleavage plane bisects the egg at right angles to the main axis. It lies on the equatorial plane. It divides the egg into two halves. 4) Latitudinal plane: It is similar to the equatorial plane, but it lies on either side of the equator. It is also called as transverse or horizontal cleavage. 15. Write down the role of yolk in cleavage. Yolk is needed for embryonic development. However the fertilized egg has to undergo all stages of development and result in a suitable ‘young form’ initiating next generation. Somehow with all the influences of yolk the developmental procedures are so adapted and modified that a well formed embryo will result. The initial influence of yolk is felt during the process of cleavage. The amount of the yolk and its distribution affect the process of cleavage. Accordingly several cleavage patterns have been recognized. 1) Total or holoblastic cleavage - In this type the cleavage furrow bisects the entire egg. Such a cleavage may be either equal or unequal. a) Equal holoblastic cleavage - In microlecithal and isolecithal eggs, cleavage leads to the formation of blastomeres of equal size. Eg: Amphioxus and placental mammals. b) Unequal holoblastic cleavage - In mesolecithal and telolocithal eggs, cleavage leads to the formation of blastomeres of unequal size. Among the blastomeres there are many small sized micromeres and a few large sized macromeres. 2) Meroblastic cleavage - In this type the cleavage furrows are restricted to the active cytoplasm found either in the animal pole (macrolecithal egg) or superficially surrounding the egg (centrolecithal egg). Meroblastic cleavage may be of two types. a) Discoidal cleavage - Since the macrolecithal eggs contain plenty of yolk, the cytoplasm is restricted to the narrow region in the animal pole. Hence cleavage furrows can be formed only in 3 the disc-like animal pole region. Such a cleavage is called discoidal meroblastic cleavage. Eg: birds and reptiles.

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college b) Superficial cleavage - In centrolecithal eggs, the cleavage is restricted to the peripheral cytoplasm of the egg. Eg: insects. 16. Explain the cleavage process in mammals. It is not surprising that mammalian cleavage has been the most difficult to study. Mammalian eggs are among the smallest in the animal kingdom, making them hard to manipulate experimentally. The human zygote, for instance, is only 100 μm in diameter—barely visible to the eye and less than one- thousandth the volume of a Xenopus egg. Also, mammalian zygotes are not produced in numbers comparable to sea urchin or frog zygotes, so it is difficult to obtain enough material for biochemical studies. Usually, fewer than ten eggs are ovulated by a female at a given time. As a final hurdle, the development of mammalian embryos is accomplished within another organism, rather than in the external environment. Only recently has it been possible to duplicate some of these internal conditions and observe development in vitro. The unique nature of mammalian cleavage: With all these difficulties, knowledge of mammalian cleavage was worth waiting for, as mammalian cleavage turned out to be strikingly different from most other patterns of embryonic cell division. The mammalian oocyte is released from the ovary and swept by the fimbriae into the oviduct. Fertilization occurs in the ampulla of the oviduct, a region close to the ovary. Meiosis is completed at this time, and first cleavage begins about a day later. Cleavages in mammalian eggs are among the slowest in the animal kingdom—about 12–24 hours apart. Meanwhile, the cilia in the oviduct push the embryo toward the uterus; the first cleavages occur along this journey. In addition to the slowness of cell division, there are several other features of mammalian cleavage that distinguish it from other cleavage types. The second of these differences is the unique orientation of mammalian blastomeres with relation to one another. The first cleavage is a normal meridional division; however, in the second cleavage, one of the two blastomeres divides meridionally and the other divides equatorially. This type of cleavage is called rotational cleavage. The third major difference between mammalian cleavage and that of most other embryos is the marked asynchrony of early cell division. Mammalian blastomeres do not all divide at the same time. Thus, mammalian embryos do not increase exponentially from 2- to 4- to 8-cell stages, but frequently contain

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college odd numbers of cells. Fourth, unlike almost all other animal genomes, the mammalian genome is activated during early cleavage, and produces the proteins necessary for cleavage to occur. In the mouse and goat, the switch from maternal to zygotic control occurs at the 2-cell stage. Most research on mammalian development has focused on the mouse embryo, since mice are relatively easy to breed throughout the year, have large litters, and can be housed easily. Thus, most of the studies discussed here will concern murine (mouse) development. Compaction: The fifth, and perhaps the most crucial, difference between mammalian cleavage and all other types involves the phenomenon of compaction. Mouse blastomeres through the 8-cell stage form a loose arrangement with plenty of space between them. Following the third cleavage, however, the blastomeres undergo a spectacular change in their behavior. They suddenly huddle together, maximizing their contact with one another and forming a compact ball of cells. This tightly packed arrangement is stabilized by tight junctions that form between the outside cells of the ball, sealing off the inside of the sphere. The cells within the sphere form gap junctions, thereby enabling small molecules and ions to pass between them. The cells of the compacted 8-cell embryo divide to produce a 16-cell morula. The morula consists of a small group of internal cells surrounded by a larger group of external cells. Most of the descendants of the external cells become the trophoblast (trophectoderm) cells. This group of cells produces no embryonic structures. Rather, it forms the tissue of the chorion, the embryonic portion of the placenta. The chorion enables the fetus to get oxygen and nourishment from the mother. It also secretes hormones that cause the mother's uterus to retain the fetus, and produces regulators of the immune response so that the mother will not reject the embryo as she would an organ graft. The mouse embryo proper is derived from the descendants of the inner cells of the 16-cell stage, supplemented by cells dividing from the trophoblast during the transition to the 32-cell stage. These cells generate the inner cell mass (ICM), which will give rise to the embryo and its associated yolk sac, allantois, and amnion. By the 64-cell stage, the inner cell mass (approximately 13 cells) and the trophoblast cells have become separate cell layers, neither contributing cells to the other group. Thus, the distinction between trophoblast

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college and inner cell mass blastomeres represents the first differentiation event in mammalian development. This differentiation is required for the early mammalian embryo to adhere to the uterus. The development of the embryo proper can wait until after that attachment occurs. The inner cell mass actively supports the trophoblast, secreting proteins (such as FGF4) that cause the trophoblast cells to divide. Initially, the morula does not have an internal cavity. However, during a process called cavitation, the trophoblast cells secrete fluid into the morula to create a blastocoel. The inner cell mass is positioned on one side of the ring of trophoblast cells. The resulting structure, called the blastocyst, is another hallmark of mammalian cleavage.

Figure: During cleavage, the zygote rapidly divides into multiple cells without increasing in size. (b) The cells rearrange themselves to form a hollow ball with a fluid-filled or yolk-filled cavity called the blastula. The rearrangement of the cells in the mammalian blastula to two layers, the inner cell mass and the trophoblast, results in the formation of the blastocyst.

17. What is Gastrulation ? Gastrulation is the process during embryonic development that changes the embryo from a blastula with a single layer of cells to a gastrula containing multiple layers of cells. Gastrulation typically involves the blastula folding in upon itself or dividing, which creates two layers of cells. Organisms that do not form a third layer are known as diploblastic organisms. These include the

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college jellyfish and related animals. Triploblastic organisms contain a third layer, the mesoderm, which is created from one of the first two layers. Triploblastic organisms account for the majority of higher animals. The layers created by gastrulation become germ layers, or special tissues that give rise to specific parts of the organism. These germ layers always give rise to the same types of tissues. The endoderm will give rise to the gut and associated organs. The ectoderm is the outermost layer, and will create the skin and the nervous system. Between them lies the mesoderm, which will created the connective tissues and musculature in most organisms. 18. Explain the concept of induction. Organs are complex structures composed of numerous types of tissues. In the vertebrate eye, for example, light is transmitted through the transparent corneal tissue and focused by the lens tissue (the diameter of which is controlled by muscle tissue), eventually impinging on the tissue of the neural retina. The precise arrangement of tissues in this organ cannot be disturbed without impairing its function. Such coordination in the construction of organs is accomplished by one group of cells changing the behavior of an adjacent set of cells, thereby causing them to change their shape, mitotic rate, or fate. This kind of interaction at close range between two or more cells or tissues of different history and properties is called proximate interaction, or induction. There are at least two components to every inductive interaction. The first component is the inducer: the tissue that produces a signal (or signals) that changes the cellular behavior of the other tissue. The second component, the tissue being induced, is the responder. Not all tissues can respond to the signal being produced by the inducer. For instance, if the optic vesicle (presumptive retina) of Xenopus laevis is placed in an ectopic location (i.e., in a different place from where it normally forms) underneath the head ectoderm, it will induce that ectoderm to form lens tissue. Only the optic vesicle appears to be able to do this; therefore, it is an inducer. However, if the optic vesicle is placed beneath ectoderm in the flank or abdomen of the same organism, that ectoderm will not be able to respond. Only the head ectoderm is competent to respond to the signals from the optic vesicle by producing a lens.

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college

Figure: Ectodermal competence and the ability to respond to the optic vesicle inducer in Xenopus. (1) The optic vesicle is able to induce lenses in the anterior portion of the ectoderm, but not in the presumptive trunk and abdomen (2). If the optic vesicle is removed (3), the surface ectoderm forms either an abnormal lens or no lens at all. (4) Most other tissues are not able to substitute for the optic vesicle.

Thus, there is no single inducer of the lens. Studies on amphibians suggest that the first inducers may be the pharyngeal endoderm and heart-forming mesoderm that underlie the lens-forming ectoderm during the early- and mid-gastrula stages. The anterior neural plate may produce the next signals, including a signal that promotes the synthesis of Pax6 in the anterior ectoderm. Thus, the optic vesicle appears to be the inducer, but the anterior ectoderm has already been induced by at least two other factors. (The situation is like that of the player who kicks the “winning goal” of a soccer match.) The optic vesicle appears to secrete two induction factors, one of which is BMP4, a protein that induces the transcription of the Sox2 and Sox3 transcription factors (and another, as yet unidentified, signal that induces the appearance of the L-Maf transcription factor. The combination of Pax6, Sox2, Sox3, and L-Maf ensures the production of the lens.

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Figure: Lens induction in embryonic amphibians. (A) The additive effects of inducers, as shown by transplantation and extirpation (removal) experiments on the salamander Tarichosa torosa. The ability to produce lens tissue is first induced by pharyngeal endoderm, then by cardiac mesoderm, and finally by the optic vesicle. The competence of the lens ectoderm to respond to these inducers increases logarithmically from the early gastrula through the tailbud larval stages. (B) Sequence of induction postulated by similar experiments performed on embryos of the frog Xenopus laevis. Unidentified inducers (possibly from the pharyngeal endoderm and heart-forming mesoderm) cause the synthesis of the Otx-2 transcription factor in the head ectoderm during the late gastrula stage. As the neural folds rise, inducers from the anterior neural plate (including the region that will form the retina) induce Pax6 expression in the anterior ectoderm that can form lens tissue. Expression of the Pax6 transcription factor may constitute the competence of the surface ectoderm to respond to the optic vesicle during the late neurula stage. The optic vesicle secretes factors (probably of the BMP family) that induce the synthesis of the Sox transcription factors and initiate observable lens formation.

19. What is competence ? This ability to respond to a specific inductive signal is called competence. Competence is not a passive state, but an actively acquired condition. For example, in the developing chick and mammalian eye, the Pax6 protein appears to be important in making the ectoderm competent to respond to the inductive signal from the optic vesicle. Pax6 expression is seen in the head ectoderm, which can respond to the optic vesicle by forming lenses, and it is not seen in other regions of the surface ectoderm. Moreover, the importance of Pax6 as

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college a competence factor was demonstrated by recombination experiments using embryonic rat eye tissue. The homozygous Pax6-mutant rat has a phenotype similar to the homozygous Pax6-mutant mouse, lacking eyes and nose. It has been shown that part of this phenotype is due to the failure of lens induction. But which is the defective component—the optic vesicle or the surface ectoderm? When head ectoderm from Pax6-mutant rat embryos was combined with a wild-type optic vesicle, no lenses were formed. However, when the head ectoderm from wild-type rat embryos was combined with a Pax6-mutant optic vesicle, lenses formed normally. Therefore, Pax6 is needed for the surface ectoderm to respond to the inductive signal from the optic vesicle. The inducing tissue does not need it. It is not known how Pax6 becomes expressed in the anterior ectoderm of the embryo, although it is thought that its expression is induced by the anterior regions of the neural plate. Competence to respond to the optic vesicle inducer can be conferred on ectodermal tissue by incubating it next to anterior neural plate tissue.

Figure: Induction of optic and nasal structures by Pax6 in the rat embryo. (A, B) Histology of wild-type (A) and homozygous Pax6 mutant (B) embryos at day 12 of gestation shows induction of lenses and retinal development in the wild-type embryo, but neither lens nor retina in the mutant. Similarly, neither the nasal pit nor the medial nasal prominence is induced in the mutant rats. (C) Newborn wild-type rats show prominent nose as well as (closed) eyes. (D) Newborn Pax6 mutant rats show neither eyes nor nose.

20. What is determination in developmental biology? The determination of different cell types (cell fates) involves progressive restrictions in their developmental potentials. When a cell “chooses” a particular fate, it is said to be determined, although it still "looks" just like its

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college undetermined neighbors. Determination implies a stable change - the fate of determined cells does not change. During the development of an embryo, it is not sufficient for all the cell types found in the fully developed individual simply to be created. Each cell type must form in the right place at the right time and in the correct proportion; otherwise, there would be a jumble of randomly assorted cells in no way resembling an organism. The orderly development of an organism depends on a process called cell determination, in which initially identical cells become committed to different pathways of development. A fundamental part of cell determination is the ability of cells to detect different chemicals within different regions of the embryo. The chemical signals detected by one cell may be different from the signals detected by its neighbour cells. The signals that a cell detects activate a set of genes that tell the cell to differentiate in ways appropriate for its position within the embryo. The set of genes activated in one cell differs from the set of genes activated in the cells around it. The process of cell determination requires an elaborate system of cell-to-cell communication in early embryos.

Figure: The ovum contains a small collection of cells in the early stages of human development. As cells divide (A–D), they are separated into different regions of the ovum. Each region of the ovum transmits a unique set of chemical signals to nearby cells. Thus, the signals detected by one cell differ from those detected by its neighbour cells. In this process, known as cell determination, cells are individually programmed to direct them toward development into different cell types.

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college 21. What is differentiation in developmental biology ? Differentiation follows determination, as the cell elaborates a cell-specific developmental program. Differentiation results in the presence of cell types that have clear-cut identities, such as muscle cells, nerve cells, and skin cells. Cellular differentiation is the process in which a cell changes from one cell type to another. Usually, the cell changes to a more specialized type. Differentiation occurs numerous times during the development of a multicellular organism as it changes from a simple zygote to a complex system of tissues and cell types. Differentiation continues in adulthood as adult stem cells divide and create fully differentiated daughter cells during tissue repair and during normal cell turnover. Some differentiation occurs in response to antigen exposure. Differentiation dramatically changes a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes are largely due to highly controlled modifications in gene expression and are the study of epigenetics. Among dividing cells, there are multiple levels of cell potency, the cell's ability to differentiate into other cell types. A greater potency indicates a larger number of cell types that can be derived. A cell that can differentiate into all cell types, including the placental tissue, is known as totipotent. In mammals, only the zygote and subsequent blastomeres are totipotent, A cell that can differentiate into all cell types of the adult organism is known as pluripotent. Embryonic stem cells in animals are the example of pluripotent.

Figure: Stem cell differentiation into various types of tissues

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Compiled and circulated by Dr. Parimal Dua, Assistant Professor, Dept. of Physiology, Narajole Raj college 22. What is organogenesis in human development ? Organogenesis is the process by which three germ layers turn into the internal organs of animals. The outer layer is called ectoderm; the middle layer, the mesoderm; and the inner layer, the endoderm. Organogenesis is the process by which the three germ tissue layers of the embryo, which are the ectoderm, endoderm, and mesoderm, develop into the internal organs of the organism. Organs form from the germ layers through the differentiation: the process by which a less-specialized cell becomes a more- specialized cell type. During organogenesis, the three germ layers of the embryo differentiate and further specialize to form the various organs of the body.  Ectoderm will produce the epidermis (skin) and nervous system of the adult.  Mesoderm will produce many internal organs of the adult such as the muscles, spine and circulatory system.  Endoderm will produce the digestive system and other internal organs of the adult. 23. Describe the development of eye as an example of reciprocal and repeated inductive events. Another feature of induction is the reciprocal nature of many inductive interactions. Once the lens has formed, it can then induce other tissues. One of these responding tissues is the optic vesicle itself. Now the inducer becomes the induced. Under the influence of factors secreted by the lens, the optic vesicle becomes the optic cup, and the wall of the optic cup differentiates into two layers, the pigmented retina and the neural retina. Such interactions are called reciprocal inductions. At the same time, the lens is also inducing the ectoderm above it to become the cornea. Like the lens-forming ectoderm, the cornea-forming ectoderm has achieved a particular competence to respond to inductive signals, in this case the signals from the lens. Under the influence of the lens, the corneal ectodermal cells become columnar and secrete multiple layers of collagen. Mesenchymal cells from the neural crest use this collagen matrix to enter the area and secrete a set of proteins (including the enzyme hyaluronidase) that further differentiate the cornea. A third signal, the hormone thyroxine, dehydrates the tissue and makes it transparent. Thus, there are sequential inductive events, and multiple causes for each induction.

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Figure: Schematic diagram of the induction of the mouse lens. (A) At embryonic day 9, the optic vesicle extends toward the surface ectoderm from the forebrain. The lens placode (the prospective lens) appears as a local thickening of the surface ectoderm near the optic vesicle. (B) By the middle of day 9, the lens placode has enlarged and the optic vesicle has formed an optic cup. (C) By the middle of day 10, the central portion of the lens-forming ectoderm invaginates, while the two layers of the retina become distinguished. (D) By the middle of day 11, the lens vesicle has formed, and by day 13 (E), the lens consists of anterior cuboidal epithelial cells and elongating posterior fiber cells. The cornea develops in front of the lens. (F) Summary of some of the inductive interactions during eye development.

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