Animal Fertilization Lectures 10 & 11 PDF

Be familiar with the processes of spermatogenesis and oogenesis. Know what the beginning components are as compared to the endpoints. N. Hartsoeker - - Scientist who created an early version of the microscope in 1965 who aimed to understand the mechanisms behind the early stages of development. Exam...

Be familiar with the processes of spermatogenesis and oogenesis. Know what the beginning components are as compared to the endpoints. N. Hartsoeker - - Scientist who created an early version of the microscope in 1965 who aimed to understand the mechanisms behind the early stages of development. Examined sperm under a microscope. Developed the first spermists' theory, the pre-formist theory: inside each sperm was a Homunculus, a microscopic-sized human, fully-developed organism, just very small Model systems we use to examine animal development - Medaka egg Sea urchin Frog Chick Floyd Frog Life Cycle - Begins with egg and sperm; forms zygote through fertilization - Cells divide through a process called cleavage - Eventually, the divided cells form a ball of relatively-similar cell called Blastula - Blastula devil's three distinct cell layers through Gastrulation (the ball of cells is called Gastrula) - The cells begin to form organs, differentiate, and become specialized through Organogenesis - In Histogenesis, the organs reach a state of maturity where they can start to function - Hatching is when the organism is released from the protective layer that has been housing it until now - Following hatching, the frog enters Postembryonic development which can include larval development, substantial changes to morphology (metamorphosis), and other changes. Here, the tadpoles will develop traits reminiscent of an adult frog such as losing its tail, growing limbs, and developing gonads (produce eggs or sperm) - Reaches adulthood once it can produce its own gametes (sperm or egg) Spermatogenesis - the making of sperm cells from stem cells - occurs from puberty to the end of the male's life - Starts with diploid (2n) stem cell Stem cell divides through mitosis to make a lot of cells called Spermatogonia (2n) that are the precursor to sperm Spermatogonia become Primary Spermatocytes (2n) and can undergo meiosis In Meiosis I, chromosome pairs are separated and are now the haploid Secondary Spermatocyte or 2C [2 chromosomes (since the chromatids are still together)] (1n) In Meiosis II, the chromatids are separated becoming Spermatid or 1C (1n) The final stage of sperm production takes these haploid cells and has them undergo Spermatogenesis which causes physiological changes such as elongating and giving it a tail creating Spermatozoa (sperm). Spermiogenesis - Differentiation and transformation of spermatids to form spermatozoa Spermatogenesis vs Spermiogenesis Spermatogenesis is the process for stem cells becoming sperm Spermiogenesis is the final step of Spermatogenesis 2C = 2 chromatids attached 1C = 1 chromatid Oogenesis - the making of egg cells from stem cells - similar process to spermatogenesis, except the initial mitotic divisions of stem cells (the first step) all happen before the female is born. Therefore, all the cells that a female has to make eggs have all already been made by the time she is born In Utero (before birth) part: Starts with diploid (2n) stem cell Stem cell divides through mitosis to make a lot of cells called Oogonia (2n) that are the precursor to eggs At the start of puberty (all Oogonia (2n) that the female will have already been produced before she was born) [RECHECK] Cells grow, RNA and yolk accumulation in Primary oocyte (2n) Undergo Meiosis 1 to become Secondary oocyte or 2C (1n). One of the cells is much larger than the other [intentionally done to give that cell a better chance of surviving] Undergoes Meiosis II to make one big egg (1C) and three polar bodies polar bodies - each of the small cells that bud off from an oocyte at the two meiotic divisions and do not develop into ova. Know the major steps involved in fertilization, the union of sperm and egg to produce a zygote. Be prepared to discuss the details of the acrosome reaction and the process of sperm adhesion and fusion with the egg. What is the role of G actin in these processes? When does fertilization occur? - occurs at different points in egg development depending on the species - fertilization can occur at any stage of meiosis depending on the species - Ex. while in the primary oocyte [before meiosis], first metaphase, second metaphase (e.g. humans), or after meiosis has competed (e.g. sea urchins) germinal vesicle - nucleus of the primary oocyte - contains the DNA first metaphase - aka metaphase I in meiosis I second metaphase - aka metaphase II in meiosis II - where fertilization happens in humans, fish, amphibians, and most mammals When does fertilization happen in sea urchins? - once meiosis is completed Response of sperm to fertilization - acrosomal reaction Responses of egg to fertilization 1) Slow polyspermy block - Sea Urchin egg - Madako egg 2) Fast polyspermy block - Sea Urchin egg - Madako egg Diagram of seas urchin sperm Plasma membrane - first barrier of protection Mitochondria - energy generated for power and speed of sperm; "motor" Tail - controls movement Nucleus - "driver" of sperm; takes up most of the cell space (since the whole purpose of the sperm is to carry DNA) Acrosomal vesicle - head of sperm; first part of sperm to interact with egg G-actin acrosomal vesicle - vesicle at the head of sperm that helps penetrate the egg - contains enzymes that are important for fertilization - globular actin (g-actin) is stored underneath the acrosomal membrane G-actin - aka globular actin - The individual actin molecules which have polarity which gives overall polarity to the actin filament (F-actin) - monomers of F-actin - oriented in a way that the F-actin has a distinct plus end and a minus end - Stored underneath the acrosomal vesicle in its unpolymerized stage (has not formed F-actin yet) F-actin - A fibrous protein made of a long chain of G actin molecules twisted into a helix - has distinct positive and negative ends What happens when the sperm makes initial contact with the egg? - When the sperm interacts with the egg, Ca(2+) [Calcium] is released inside the sperm cell which causes the plasma membrane to be pulled back from the sperm head which releases the content of the acrosomal vesicle onto the outer layer of the egg [process called acrosome reaction] - The Ca(2+) that was released also activates the polymerization of G-actin into F-actin, resulting in the formation of actin microfilaments. The F-actin takes the shape of a long stalk. The purpose of this is to change the morphology of the sperm so that it pushes out the head of the sperm, causing an increase in the surface area of the sperm. This leads to the greater presence of the surface protein Bindin which are important for the further interactions between the sperm and the egg. acrosome reaction - When the contents of the acrosome are released and proteases digest a route for the sperm. Bindin - receptor proteins on the sperm head pushed out by F-actin the reacts with the vitelline envelope sea urchin egg outer layer structure Plasma Membrane Vitelline envelope - protein layer directly on top of the plasma membrane that acts as a barrier to sperm Jelly coat - flexible, fluid layer made of glycoproteins; the initial barrier to sperm Vitelline envelope - protein layer on egg directly on top of the plasma membrane that acts as a barrier to sperm - contain receptors for Bindin (surface protein on sperm) Sea Urchin Fertilization Step 1: Chemotaxis Chemotaxis: the movement of sperm is guided towards the egg by the release of substances by the egg into the environment Sperm interacts with jelly coat, which triggers the acrosome reaction Step 2: Acrosome Reaction The contents of the acrosomal vesicle are released and start to degrade the jelly coat Calcium is released inside the sperm which causes the formation of the long, stalk-like F-actin made up of G-actin The contents of the acrosomal vesicle continue to degrade the jelly coat around the sperm head until the head reaches the Vitelline envelope Step 3: Sperm-egg Adhesion The binding proteins on the Vitelline envelope of the egg interact with Bindin on the sperm head resulting in a stable connection between the sperm and the egg Step 4: Plasma membrane Contact The acrosomal content pushes further and reaches the plasma membrane Step 5: Gamete Fusion The plasma membranes of the sperm and egg have come together and fused into one, continuous membrane - becomes one cell The DNA inside the sperm can now be pushed into the cell SEM of the fusion of the acrosomal membrane with the egg plasma membrane Chemotaxis - Cell movement that occurs in response to chemical stimulus - In sperm, it is the the movement of sperm guided towards the egg by the release of substances by the egg into the environment vitelline envelope - protective layer over the plasma membrane of egg cells - becomes part of the fertilization membrane sperm-egg adhesion - The binding proteins on the Vitelline envelope of the egg interact with Bindin on the sperm head resulting in a stable connection between the sperm and the egg Gamete fusion - gametic exchange between individuals in which haploid gametes fuse to produce a diploid offspring Be able to explain the contributions of Ca++ and of cortical granules to the process of fertilization. What are the responses of the egg to the sperm? - In all species that have been studied, there is a rapid increase in cytosolic Ca2+ following the fusion of the sperm membrane with the egg's membrane. - - - If cytosolic Ca2+ is increased by other means, in the absence of sperm, many of the events of normal fertilization are initiated, and under certain circumstances, normal development of an embryo can occur [increase of Ca+2 is a trigger for development, but it will eventually cease since you need the DNA from the sperm to continue this process] Evidence for calcium levels after fertilization comes from the paper "a free calcium wave traverses the activating egg of the medaka [Japanese rice fish]" - Geroge T. Reynold from PURDUE measured calcium levels shortly after fertilization occurs Reynold found that a wave of Ca2+ sweeps across the fertilized egg of fish, frogs, and sea urchins. A rise in Ca 2+ occurs in all eggs at fertilization. dye showed calcium increase gets bigger and bigger and eventually dies down in waves Medaka egg fertilization - Japanese rice fish Common in fertilization research because of their availability/noticeable responses/similarity to humans In Reynold's experiment, eggs were injected with a light-emitting calcium indicator egg is mostly yolk (nutrients for zygote); very little cytoplasm 1.2 mm in diameter Chorion: protective outer layer over the plasma membrane Micropyle: opening in chorion specifically for sperm to enter; ensures only one sperm fertilizes the egg Reynold found that a wave of Ca2+ sweeps across the fertilized egg of fish, frogs, and sea urchins. A rise in Ca 2+ occurs in all eggs at fertilization. polyspermy - fertilization by more than one sperm - BAD Know the role that Ernest Everett played in improving our understanding of fertilization. Ernest Everett - Studied sea urchin fertilization at the Marine Biological Lab in Woods Hole, MA - described the event that took place when a sea urchin egg is exposed to sperm - looked at a microscope and drew what he saw Ernest Everett's pictures - In 1, the egg is surrounded by sperm - You can see in 2 that most of the sperm have disappeared and there is only the one predominant sperm; there is a raised line in close association to where the sperm is [fertilization cone, leads to the adhesion and fusion of plasma membrane] - In 5 and 6, you begin to see a rather large space forming where the sperm made contact that continues to get wider and wider till in 9 it's made its way all the way around the egg [turned out to be fertilization membrane - Later on, as microscope techniques improved we began to more clearly view what was happening cortical vesicles (CV) - aka cortical granule - Several large vesicles directly underneath the plasma membrane of the egg in sea urchin. - When the sperm's plasma membrane fuses with the egg's plasma membrane, the cortical vesicle fuses with the egg's plasma membrane releasing its content (exocytosis) into the perivitelline space [between the plasma membrane and the vitelline envelope]. The content released from these vesicles are responsible for the growing spaces that were seen in Everett's pictures, called fertilization membrane, which prevents additional sperm from getting up (preventing polyspermy) - called slow block to polyspermy During fertilization, the plasma membrane of the egg fuses with....the plasma membrane of the sperm + cortical granules Why does the fertilization membrane grow once the cortical granules dump their content into the region? - When the contents of the cortical granules are dumped into the perivitelline space, the content of solute is very high, which means the water potential is negative and so water will flow into the fertilization membrane creating a higher hydrostatic pressure. This is was generates the force to push the membrane further from the egg Perivitelline space - space in the egg between the plasma membrane and the vitelline envelope - size increases following the exocytosis of cortical vesicles fertilization membrane - refers to vitelline envelope + perivitelline space after it has been pushed out by the exocytosis of cortical vesicles following the fusion of the egg and sperm - barrier that prevents a second sperm from entering the egg - When the contents of the cortical granules are dumped into the perivitelline space, the content of solute is very high, which means the water potential is negative and so water will flow into the fertilization membrane creating a higher hydrostatic pressure. This is was generates the force to push the membrane further from the egg - aka slow block to polyspermy - after sperm fuses with the egg, its centrioles and pronucleus (contains its DNA) are released into the egg Understand the mechanisms involved in the Blocks to polyspermy. Be familiar with the difference between the slow and the fast blocks. slow block to polyspermy - The formation of the fertilization envelope and other changes in an egg's surface that prevent fusion of the egg with more than one sperm. The slow block begins about 1 minute after fertilization. - 30 years later, Lord Rothschild measured the time to block polyspermy. Lord Rothschild - Measured the time needed for the block of polyspermy. Found that there was two blocks: one that occurred in a couple seconds [fast block], and one that took up to a minute [slow block]. Rindy Jaffe Experiment - purdue student that unraveled fast block a Purdue undergraduate in biology and then got her PhD under Susumu Hagiwara at UCLA. This paper was part of her PhD thesis. Experiment - In the paper, they measured the membrane potential (charge of cell; calculated using Nernst equation) of the egg and charges that occurred after contact with sperm using two electrodes, one that was embedded into the egg and one that was in the bath [the difference between the two electrodes were used to calculate membrane potential] - Before the membrane was added the membrane potential was -70 mV [resting potential] - After the sperm was added, the potential remained at -70 mV - 10 seconds after the sperm was added, the membrane potential quickly jumped to +10 mV (depolarization [negative number to pos/less neg number) - After around a minute, the fertilization membrane occurred [slow block], and the membrane potential slowly dipped back to resting potential - Upon encountering the sperm, the membrane potential of the sea urchin egg depolarizes from -70 mV to +10 mV Further experiments showed that: - If the membrane potential is artificially depolarized by passing current through a microelectrode, sperm cannot fuse with the egg’s membrane. - If Na+ is removed from the sea water, the depolarization does not occur and the eggs become polyspermic - These experiments support the idea that sperm fusion to the egg leads the activation of sodium channels in the egg. When Na+ enter the egg, depolarization occurs fast block to polyspermy - The depolarization of the egg plasma membrane upon fertilization, (-70 to +10) designed to prevent the entry of more than one sperm into the egg. - require activation of sodium channels (Na+ ions enter the cell) - happens with seconds of sperm contact Post fertilization events 1) Pronuclear fusion 2) Centrioles from sperm divide and move to opposite sides of the newly formed nucleus 3) Centrioles pull the chromosomes apart promoting the first mitotic division pronuclear fusion - The merging of the sperm pronuclear and egg pronuclear in a fertilized egg to fuse and produce a single zygotic genome - forms one nucleus [in pic, egg pronucleus is the larger indentation; sperm pronucleus is dark splotch] - before they come together, the centrioles from the sperm divide and form spindles from - now diploid - can take up to 800 seconds - Now it can start undergoing mitosis Animal Pole - The hemisphere of the egg where the sperm entered - Eventually it is where the least yolk is concentrated; opposite of the vegetal pole. Vegetal Pole - Opposite of the animal pole; the portion of the egg where most yolk is concentrated Elevating the fertilization envelope is the slow block to polyspermy, while the changes in the plasma membrane potential is the fast block to polyspermy