Page 1Page 2Page 3Page 4Page 5Page 6Page 7Page 8Page 9Page 10Page 11Page 12Page 13Page 14Page 15Page 161Biology 4361Developmental BiologyFertilizationOctober 19, 2006Fertilization - fusion of sperm and egg - accomplishes: A) sex (combining genes derived from two genomes) B) reproduction - transmits genes from parents to offspring - initiates reactions in the egg cytoplasm that allows development to proceed Generally, four major events occur:1) Contact and recognition between sperm and egg.- also ensures that egg and sperm are of the same species2) Regulation of sperm entry into the egg- only one sperm can fertilize- usually, only one sperm is allowed to enter the egg; others are excluded3) Fusion of the genetic material of the sperm and egg.4) Activation of egg metabolism to start development.Structure of the GametesSperm - rather obscure; took a bad rap for a long time, e.g. - von Leeuwenhoek (co-discoverer of sperm) - considered sperm to be parasites in thesemen- this “parasite” idea was popular through many generations of scientists- note that sperm were discovered in 1676, but their role in fertilization was not fullyelucidated until 1876 (200 years! a long time to wait!; Oscar Hertwig, Herman Fol(independently) demonstrated sperm entry into the egg and union of cells’ nuclei) Sperm structure: - head contains:- streamlined haploid nucleus- DNA in pseudo-crystalline form with protamines- transcriptionally inactive- acrosome (acrosomal vesicle)- Golgi-derivative- contains enzymes that digest proteins and complex sugars; used to lyse outercoverings of the egg- modified secretory vesicle- many species contain region of globular actin proteins between sperm nucleus andacrosomal vesicle2- g-actin used to extend acrosomal process (filamentous actin) during early stagesof fertilization- recognition between sperm and egg (i.e. species specificity) molecules located inon the acrosomal process - Flagellum (tail) contains:- axoneme- microtubules (tubulin) emanate form the basal centriole- 9 + 2 construction- 2 central microtubules- 9 doublet microtubules- one mt of doublet is complete (13 protofilaments)- second mt of doublet is incomplete; C-shaped (11protofilaments)- NOTE - 9 +2 arrangement with dynein arms conserved inaxonemes throughout the eukaryotic kingdoms- dynein attached to microtubules- motor protein- ATPase- dynein activity causes outer mt to slide past inner, causingflagellar bending- midpiece- mitochondria- mammals - several in a spiral around axoneme- sea urchins - one large mitochondrion in a ring around axonemeEgg (ovum) - all material necessary for the beginning of growth and development must be stored in the egg - eggs actively accumulate material as they develop - NOTE - volume of eggs v. sperm; sea urchin egg: 2 x 10 mm (200 picoliters); > 200X-4 3volume of the sea urchin sperm- proteins- yolk (made in other organs; mostly liver)- ribosomes and tRNA- burst of protein synthesis after fertilization- mRNA- encodes proteins for use in the early stages of development- messages remain repressed until after fertilization- morphogenic factors- direct the differentiation of cells into certain types- transcription factors, paracrine factors- localized regionally; segregated into different cells during cleavage- protective chemicals3- DNA repair- UV filters- antibodies- alkaloids (defensive) - in many (most) species, the final stages of egg meiosis take place while the sperm’s nuclearmaterial (male pronucleus) is traveling toward what will become the female pronucleusRecognition of egg and spermThe interaction of egg and sperm generally proceeds according to five basic steps1. The chemoattraction of the sperm to the egg by soluble molecules secreted by the egg.2. The exocytosis of the acrosomal vesicle to release its enzymes.3. The binding of the sperm to the extracellular envelope (vitelline layer or zona pellucida)of the egg.4. The passage of the sperm through the extracellular envelope.5. Fusion of egg and sperm cell membranes.NOTE - sometimes steps 2 and 3 are reversed (e.g. mammalian fertilization); sperm bindsto extracellular matrix of the egg before releasing acrosomal contends - after these 5 steps, sperm and egg pronuclei meet and development is initiatedExternal Fertilization in Sea UrchinsFertilization challenges:- how to bring two very small cell together in a large place- how to ensure that only sperm and eggs of the same species joinSperm attraction: Action at a distance Species-specific sperm attraction documented in numerous species, including cnidarians, molluscs, echinoderms, urochordates chemotaxis - sperm are attracted towards eggs of their species- sperm follow chemoattractant gradient- Orthopyxis caliculata (cnidarian) regulates taxis (movement) and timing of sperm- prior to 2 meiotic division = no chemotaxisnd- after 2 meiotic division = chemotaxisnd chemotaxis mechanisms - molecular agents can be different even in closely related species (need to be!)- sea urchins - motility acquired shortly after exposure to seawater2- in testes, pH is kept low (pH ~7.2) by high CO content- in seawater, pH elevated to ~7.6- results in activation of dynein ATPase (flagellar motor protein)4- chemotaxis: guided by egg-derived peptides, e.g. resact- Resact - 14aa peptide isolated from egg jelly of Arbacia punctulata- sperm in sea water swim in circles- add resact - sperm congregate- resact specific for A. punctulata; will not activate sperm from Strongylocentrotus purpuratus- S. purpuratus chemotactic peptide - speract- A. punctulata sperm have resact receptors in membrane- resact binding activates guanylyl cyclase activity in cytoplasmic side of receptor- cGMP activates a calcium channel- Ca influx provides directional cue2+NOTE - a single resact molecule can provide directional information for sperm;swim up a concentration gradient until they reach the egg- resact also acts as a sperm-activating peptide (resact = respiration activating)- increased mitochondrial respiration and sperm motility- increase in cGMP and Ca activates mitochondrial ATP generating apparatus2+and dynein ATPase that stimulates flagellar movement Acrosome reaction- in marine invertebrates AR has two components:1. Fusion of the acrosomal vesicle with the sperm cell membrane (exocytosisresults in the release of acrosomal contents2. Extension of the acrosomal process- sea urchin AR initiated by contact of sperm with egg jelly- specific