Chapter 4 * Figure 4-2 * Modification of a germ cell to form a mammalian sperm. (A) The centriole produces a long flagellum at what will be the posterior end of the sperm, and the golgi apparatus forms the acrosomal vesicle at the future anterior end. The mitochondria collect around the flagellum near the base of the haploid nucleus and become incorporated into the midpiece ("neck") of the sperm. The remaining cytoplasm is jettisoned and the nucleus condenses. The size of the mature sperm has been enlarged relative to the other stages. (B) Mature bull sperm. The DNA is stained blue with DAPI, the mitochondria are stained green, and the tubulin of the flagellum is stained red. (Q Acrosome of mouse sperm, stained green by the fusion protein proacrosin-GFR * Sperm can travel in aqueous environment to reach the egg because of the flagellum * Figure 4-3 * The force for sperm propulsion is provided by dynein, a protein attached to the microtubules (see Figure 4.3B). Dynein is an ATPase, an enzyme that hydrolyzes ATP, converting the released chemical energy into mechanical energy to propel the sperm. * (Bj Interpretive diagram of the axoneme, showing the "9 + 2" arrangement of the microtubules and other flagellar components. The dynein arms contain the ATPases that provide the energy for flagellar movement. (C) Association of tubulin protofilaments into a microtubule double. One portion of the doublet is a fully circular microtubule comprising 13 protofilaments. The second portion of the doublet contains only 11 (occasionally 10) protofilaments. * Figure 4-4, 4-6 * The vitelline envelope contains several different glycoproteins. It is supplemented by extensions of membrane glycoproteins from the cell membrane and by proteinaceous "posts" that adhere the vitelline envelope to the membrane. The vitelline envelope is essential for the species-specific binding of sperm. * Egg jelly- attracts or activates sperm. The egg is specialized for receiving sperm and initiating development. * Sea urchin egg cell surface. (A) Scanning electron micrograph of an egg before fertilization. The cell membrane is exposed where the vitelline envelope has been torn. (B) Transmission electron micrograph of an unfertilized egg, showing microvilli and cell membrane, which are closely covered by the vitelline envelope. A cortical granule lies directly beneath the cell membrane. * Figure 4-7 * Zona pellucida- the extracellular envelope is a separate and thick matrix * Cumulus- a layer of cells that nurture the egg from the time of release from the ovary. * The hamster egg, or ovum, is encased in the zona pellucida
. This in turn, is surrounded by the cells of the cumulus. A polar
 body cell, produced during meiosis, is also visible within the zona pellucida. (B) At lower magnification, a mouse oocyte is shown surrounded by the cumulus. * Figure 4-5 * Stages of egg maturation at the time of sperm entry in different animal species. Note that in most species, sperm entry occurs before the egg nucleus has completed meiosis. The germinal vesicle is the name given to the large diploid nucleus of the primary oocyte. The polar bodies are nonfunctional cells produced by meiosis * Figure 4-8 * (A) Sea urchin fertilization is external. (1)The sperm is chemotactically attracted to and activated by the egg. (2, 3) Contact with the egg jelly triggers the acrosome reaction, allowing the acrosomal process to form and release proteolytic enzymes. (4) The sperm adheres to the vitelline envelope and lyses a hole in it. (5) The sperm adheres to the egg cell membrane and fuses with it. The sperm pro nucleus can now enter the egg cytoplasm. * (B) Mammalian fertilization is internal. (1) The contents of the female reproductive tract capacitate, attract, and activate the sperm. (2) The acrosome-intact sperm binds to the zona pellucida, which is thicker than the vitelline envelope of sea urchins. (3) The acrosome reaction occurs on the zona pellucida. (4) The sperm digests a hole in the zona pellucida. (5) The sperm adheres to the egg, and their cell membranes fuse. * Figure 4-9 * Sperm are attracted toward eggs of their species by chemotaxis— that is, by following a gradient of a chemical secreted by the egg. * Miller found that when sperm were added to oocytes that had not yet completed their second meiotic division, there was no attraction of sperm to eggs. However, after the second meiotic division was finished and the eggs were ready to be fertilized, the sperm migrated toward them. Thus, these oocytes control not only the type of sperm they attract, but also the time at which they attract them. * Resact- sperm migrate to the area of release. It is also species specific. * B-D. Sperm migrate to the center of resact injection * Figure 4-10 * Model for chemotactic peptides in sea urchin sperm. (A) Resact from Arbacia egg jelly binds to its receptor on the sperm. This activates the receptor's guanylyl cyclase (RGC) activity, forming intracellular cGMP in the sperm. The cGMP opens calcium channels in the sperm cell membrane, allowing Ca2+ to enter the sperm. The influx of Ca2* activates sperm motility, and the sperm swims up the resact gradient toward the egg. (B) Ca2* levels in different regions of Strongylocentrotus purpuratus sperm after exposure to 125 nM speract (this species' analog of resact). Red indicates the highest level of Ca2*, blue the lowest. The sperm head reaches its peak Ca2* levels within 1 second * Figure 4-11 * In sea urchins, the acrosome reaction is initiated by the interactions of the sperm cell membrane with a specific complex sugar in the egg jelly. These sulfate-containing polysaccharides bind to specific receptors located on the sperm cell membrane directly above the acrosomal vesicle. The egg jelly factors that initiate the acrosome reaction are often high- ly specific to each species, and egg jelly carbohydrates from one species of sea urchin fail to activate the acrosome reaction even in closely related species * The histograms compare the ability of each polysaccharide to induce the acrosome reaction in the different species of sperm. (B) Chemical structures of the acrosome reaction-inducing sulfated polysaccharides reveal their species-specificity. * Figure 4-12 * Acrosome reaction in sea urchin sperm. The portion of the acrosomal membrane lying directly beneath the sperm cell membrane fuses with the cell membrane to release the contents of the acrosomal vesicle. (D) The actin molecules assemble to produce microfilaments, extending the acrosomal process outward. * Figure 4-13 * Another critical species-specific binding event must occur once the sea urchin sperm has penetrated the jelly and the acrosomal process of the sperm contacts the surface of the egg. The acrosomal protein mediating this recognition in sea urchins is called bindin. Further, its interaction with eggs is often species- specific. Bindin is located specifically on the acrosomal process—exactly where it should be for sperm-egg recognition * Figure 4-14 * Bindin receptors in the egg. Scanning electron micrograph of sea urchin sperm bound to the vitelline envelope of an egg. Although this egg is saturated with sperm, there appears to be room on the surface for more sperm, implying the existence of a limited number of bindin receptors * Figure 4-15 * Scanning electron micrographs of the entry of sperm into sea urchin eggs. (A) Contact or sperm head with egg microvillus through the acrosomal process. (B) Formation of fertilization cone. (C) Internalization of sperm within the egg. * Figure 4-16 * The entrance of multiple sperm—polyspermy—leads to disastrous consequences in most organisms. In the sea urchin, fertilization by two sperm results in a triploid nucleus, in which each chromosome is represented three times rather than twice. Worse, each sperm's centriole divides to form the two poles of a mitotic apparatus; so instead of a bipolar mitotic spindle separating the chromosomes into two cells, the triploid chromosomes may be divided into as many as four cells. Because there is no mechanism to ensure that each of the four cells receives the proper number and type of chromosomes, the chromosomes are apportioned unequally: some cells receive extra copies of certain chromosomes, while other cells lack them * Figure 4-17 * The fast block to polyspermy is achieved by changing the electric potential of the egg cell membrane. * Within 1-3 seconds after the binding of the first sperm, the membrane potential shifts to a positive level, about +20 mV (Longo et al. 1986). This change is caused by a small influx of Na' into the egg. Although sperm can fuse with membranes having a resting potential of -70 mV, they cannot fuse with membranes having a positive resting potential, so no more sperm can fuse to the egg. * Figure 4-18 * This sperm removal is accomplished by the cortical granule reaction, a slower, mechanical block to polyspermy that becomes active about a minute after the first successful sperm-egg fusion * Upon fertilization, the con- centration of free Ca2+ in the egg cytoplasm increases greatly. In this high-calcium environment, the cortical granule membranes fuse with the egg cell membrane, releasing their contents. Once the fusion of the cortical granules begins near the point of sperm entry, a wave of cortical granule exocytosis propagates around the cortex to the opposite side of the egg. * Figure 4-19 * Sperm-Egg fusion * release of IC Ca2+ * Ca2+ causes fusion of cortical granules with plasma membrane. * CG contents released. * Form osmotic gradient. * Convert VE to FE * Raise FE * Harden FE * Figure 4-20, 4-21 * When a sea urchin egg is injected with dye and then fertilized, a striking wave of calcium release propagates across the egg. The release of Ca2* starts at one end of the cell and proceeds actively to the other end, creating a wave of calcium across the egg. * The calcium ions responsible for the cortical granule reaction are stored in the endoplasmic reticulum of the egg. Which are pronounced in the cortex and surrounds the cortical granules. * Figure 4-22 * Probable mechanisms of egg activation. In both cases, a phospholipase C (PLC) is activated and makes IP3 and DAG. (A) Ca-+ release and egg activation by activated PLC from the sperm, or by a substance from the sperm that activates egg PLC. This may be the mechanism in mammals. (B) The bindin receptor (perhaps acting through a G protein) activates an Src kinase, which activates PLC. This is probably the mechanism used by sea urchins. * IP3 is the releaser of Ca2+ ions. * Figure 4-23 * Egg activation * The cortical granules, which fuse with the cell membrane in the presence of high calcium concentrations, respond with a wave of exocytosis that follows the calcium wave. * Figure 4-24 * G protein involvement in Ca2+ entry into sea urchin eggs. * Figure 4-26 * Sperm egg pronuclei migrate toward egg other in the egg * Figure 4-28 * Newly ejaculated mammalian sperm are unable to undergo the acrosome reaction or fertilize an egg until they have resided for some time in the female reproductive tract (Chang 1951; Austin 1952). The set of physiological changes by which sperm become competent to fertilize the egg is called capacitation. * The rise in cAMP activates protein kinase A, causing it to activate the protein tyrosine kinases (while inactivating the protein phosphatases). The kinases phosphorylate proteins that are essential for capacitation. * Figure 4-30 * Interpretive diagram of electron micrographs showing the fusion of the acrosomal and cell membranes in the sperm head. * Figure 4-34 * Pronuclear movements during human fertilization. The microtubules have been stained green, while the DNA is dyed blue. The arrows point to the sperm tail. (A) The mature unfertilized oocyte completes the first meiotic division, budding off a polar body. * B) As the sperm enters the oocyte (left side), microtubules condense around it as the oocyte completes its second meiotic division at the periphery. (C) By 15 hours after fertilization, the two pronuclei have come together, and the centrosome splits to organize a bipolar microtubule array. The sperm tail is still seen (arrow). (D) At prometaphase, chromosomes from the sperm and egg intermix on the metaphase equator and a mitotic spindle initiates the first mitotic division. The sperm tail can still be seen. *