1. FERTILIZATION

2. Fertilization What are the factors that affect the sperm’s potential for successfully fertilizing the egg? The process by which the sperm initiates and participates, with the egg, in the development of the embryo 1. Capacitation - occurs in the female’s vagina. Vaginal secretions cause a molecular change in the sperm plasmalemma (removal of decapacitating factor - semen proteins, results in increased membrane fluidity,). Takes 4-5 hr in humans, 1 hr in mice, 6 hr in rabbits. http://www.erin.utoronto.ca/~w3bio380/l ecture/Lect07/L7.htm

3. Increased rate of metabolism Flagellum beats more rapidly; Result: Sperm are more motile Changes in sperm plasmalemma proteins allow sperm-egg binding and occurrence of the acrosome reaction Pro-Acrosin (inactive) is converted to acrosin (active) Sperm become capable of chemotaxis http://www.erin.utoronto.ca/~w3bio380/l ecture/Lect07/L7.htm Galactosyltransferase is a protein in the sperm plasmalemma that MAY act as the receptor that binds to the zona pellucida and initiates the acrosome reaction. Effects of Capacitation on Sperm

4. Factors that affect the sperm’s potential for successfully fertilizing the egg? (cont.) 3. Egg secretions - importance varies among species - fertilizins - F.R. Lille (1919) activating factors, agglutination, chemoattraction 2. Dilution Marine invertebrates - Free spawning of sperm into surrounding ocean water. Mammals - Dilution: 40 - 400 million spermatozoa in vagina, only a few hundred to a thousand reach the upper oviduct. http://www.erin.utoronto.ca/~w3bio3 80/lecture/Lect07/L7.htm

5. http://www.talbotcentral.ucr.edu/mammalianfert.htm http://arbl.cvmbs.colostate.edu/hbooks/pathphys/reprod/fert/gxport.html Factors that affect the sperm’s potential for successfully fertilizing the egg? (cont.) 4. Structures surrounding the egg - barriers Follicle cells Zona pellucida (vitelline membrane in non- mammals) Oolemma (plasmalemma of egg)

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7. Acrosome Reaction

8. How does the sperm get through the barriers surrounding the egg? 1.Used to be thought that the acrosome enzymes did it all. 2.More recent data a.Sperm arrives at zona pellucida with the acrosome still intact. So, how does it get through the follicle cells that surround the egg? b.Has to break down the intercellular cement that holds the follicle cells together - hyaluronic acid c.Lin and co-investigators (1994) demonstrated that a protein found in the sperm plasmalemma (PH-20) had hyaluronidase activity. http://arbl.cvmbs.colostate.edu/hbooks/pathphys/reprod/fert/gxport.html

9. Getting through the zona pellucida What happens when the sperm gets to the zona pellucida? 1.Attachment - loose association 2.Binding - strong attachment 3.Acrosome reaction - release of enzymes 4.Penetration of the zona pellucida by the sperm http://www.talbotcentral.ucr.edu/mammalianfert.htm

10. Wassermann and co-workers (1980, 1985, 1987, 1988) Found that zona pellucida is composed of 3 glycoproteins ZP1, ZP2, ZP3 Repeating subunits of ZP2 and ZP3 form filaments that are bound together by ZP1 How is this accomplished?

11. 1.Wassermann et al. found that when the sperm binds to ZP3 it causes a change in Ca +2 and Na + flux across the sperm plasmalemma that results in the acrosome reaction. (ZP1 and ZP2 will not cause this to happen). 2.The sperm actually binds to an o-linked oligosaccharide that is part of ZP3 - (a carbohydrate component of the glycoprotein) 3.The identity of the receptor in the sperm plasmalemma that binds to this oligosaccharide is, as yet, uncertain. It has been suggested that a part of PH-20 is the receptor; however, more recent work indicates this may not be the case.

12. When the acrosome reaction occurs, a number of proteolytic enzymes are exposed or released. One or more of these enzymes is responsible for digesting the hole through the zona pellucida through which the sperm enters the perivitelline space.

13. 1.The male nucleus enters the egg cytoplasm and becomes the male pronucleus. 2.As a result of the sperm fusing with the egg plasmalemma, the oocyte nucleus, which is at metaphase of the second meiotic division, completes that division giving rise to another polar body. 3.Following the second meiotic division, what is now the nucleus of the ovum becomes the female pronucleus. 4.The haploid male and female pronuclei move toward one and other, meet, and fuse to form the diploid nucleus of the zygote. Following fusion of the sperm with the egg, http://www.talbotcentral.ucr.edu/mammalianfert.htm Male and female pronuclei in a fertilized hampster egg Cleavage http://arbl.cvmbs.colostate.edu/hbooks/pathphys/reprod/fert/cleavage.html 5. The zygote will now proceed to undergo cleavage. http://www.molbiolcell.org/cgi/content/full/9/7/1609/F6 In mammals:

14. Blocks to polyspermy 1.Dilution 2.Fertilizin like secretions cause agglutination of sperm in some invertebrate species (Does not occur in mammals) 3.“Hardening” of vitelline membrane or zona pellucida that results from cortical granule rupture - - slow block to polyspermy, takes 1 - 5 min http://www.molbiolcell.org/cgi/content/full/9/7/1609

15. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=dbio.figgrp.1397

16. 4.Depolarization of the oolemma - membrane potential changes from negative to positive, sperm cannot fuse with positively charged oolemma - fast block to polyspermy, takes 1 - 2 seconds. Caused by influx of sodium ions. Blocks to polyspermy Sea urchinsFrogs Before sperm fusion -70 mv -28 mv After sperm fusion +10 mv + 8 mv Depolarization lasts longer than 5 min, but eventually the potential difference across the oolemma returns to its original value. The change in membrane potential happens in many species, but does not occur in mammals

17. Blocks to polyspermy 5. Hydrogen peroxide release by the eggs of some invertebrate species (e.g. sea urchins) - inactivates sperm - Does not occur in mammalian eggs. 6. Unknown mechanisms that cause degeneration of extra sperm nuclei after they have entered the egg cytoplasm. Seen in salamanders. As a result, even though more than one sperm fuses with the egg, only one male pronucleus survives to fuse with the female pronucleus. - Does not occur in mammalian eggs. Thus, only 1) dilution of the sperm and 2) hardening of the zona pellucida due to the contents of the cortical granules act to prevent polyspermy in most mammals, including humans.

18. Egg Activation A series of morphological, physiological and molecular changes that occur in the egg in response to fusion of the sperm with the egg. 1.Frees egg from constraints that kept it from developing prior to fertilization. 2.Prepares the egg for development. Why do I say “frees the egg” ? Why not “frees the egg and sperm”? 1.Egg activation does not require the sperm nucleus 2.In some species, just pricking the egg with a needle, shocking it, or simply changing the pH of the solution it’s in will cause activation. 3.In some species eggs that are activated this way will start to develop. Thus, we have a mechanism that could allow for selection of parthenogenesis. What does egg activation accomplish?

19. 1.Release of Ca++ (calcium) stored in the egg endoplasmic reticulum - appears to be the critical step in the process. 2.Cortical reaction - rupture of cortical granules that occurs concurrently with the Ca++ release. Contents of granules are released into perivitelline space and cause “hardening” of the vitelline membrane or zona pellucida. Causes vitelline/fertilization membrane to rise away from surface of egg in some species. The seven events that characterize egg activation following fusion of the sperm with the egg: http://www.molbiolcell.org/cgi/content/full/9/7/1609

20. “ The fertilization transient can be seen at the very start of the animation. Later there is a rise in calcium starting at the point of sperm entry which correlates with the start of the movement of the female pronucleus to the center of the egg and then a further general rise in calcium which correlates with the fusion of the two pronuclei. ” This movie shows A. calcium transients that occur firstly at fertilization B. then as the female pronucleus starts to move toward the male pronucleus C. and finally general rise at the fusion of the pronuclei The data shown is the ratio of a calcium sensitive dye at the time of the event with the background calcium dye signal. http://petrus.ncl.ac.uk/urchins/fertilizationb.html Sea Urchin

21. The seven events that characterize egg activation following fusion of the sperm with the egg: 3. In many species, an influx of Na + (sodium) into the egg cytoplasm that causes a change in membrane potential - fast block to polyspermy. 4. In many species a reorganization of the egg cytoplasm. 5. In most cases, completion of meiosis by the egg. 6. An efflux of H + (hydrogen) ions causing an increase in cytoplasmic pH - this activates previously inhibited synthetic pathways. 7. Increase in metabolism - zygote gears up for development. Events that occur soon after egg activation: a.DNA replication as male and female pronuclei approach each other b.Male and female pronuclei merge c.Preparation for first cleavage