Biology 4361 Developmental Biology Fertilization October 18, 2007
Fertilization Fertilization accomplishes two things: Sex (combining genes from two genomes) Reproduction (initiates reactions in the egg cytoplasm that allow development to proceed) Major Events: Lennart Nilsson 1. Contact and recognition between sperm and eggs. must be species specific 2. Regulation of sperm entry into egg. 3. Fusion of genetic material of sperm and egg. 4. Activation of egg metabolism to start development.
Fertilization Overview Many variations among species models: sea urchin, mouse Sperm formation and structure Egg structure and function Interactions between sperm and eggs Chemoattraction Acrosome reaction Binding and fusion Prevention of polyspermy Egg activation Pronuclear fusion
Sperm Formation
Sperm Axoneme
The Egg All material necessary to begin development and growth is stored in the egg. Eggs actively accumulate material as they develop: Proteins yolk (made in other organs (liver, fat bodies), transported to egg Ribosomes and trna burst of protein synthesis after fertilization mrna encode proteins for use in early development Morphogenic factors direct differentiation of cells into certain types transcription factors, paracrine factors some localized regionally; segregated into different cells during cleavage Protective chemicals UV filters DNA repair enzymes bicoid antibodies alkaloids (and other protective molecules) nucleus nanos
Egg Maturation at Sperm Entry Most eggs are not fully mature at the time of fertilization; sperm entry activates metabolism and relieves meiotic arrest
Egg Maturation at Sperm Entry Most eggs are not fully mature at the time of fertilization; sperm entry activates metabolism and relieves meiotic arrest
Egg Structure e.g. Sea Urchin Volume: 2 x 10 4 mm 3 (200 picoliters) >200 X sperm volume egg jelly glycoprotein meshwork attract or activate sperm extracellular envelope inverts vitelline envelope fibrous mat sperm egg recognition contains glycoproteins (vertebrates zona pellucida) cell membrane functions: fusion with sperm cell membrane regulates ion flow
Mammalian Egg cumulus ovarian follicular cells inner most layer corona radiata
Egg Membrane Structure egg jelly actin microvilli filamentous (f actin) cortex globular (g actin) cortex Cortical granules: Golgi derived; contain: proteolytic enzymes hyaline protein mucopolysaccharides adhesive glycoproteins
Interactions Between Egg and Sperm General steps: 1. Chemoattraction of sperm to egg soluble molecules released by egg 2. Exocytosis of the acrosome stimulated by binding of egg molecules 3. Binding of sperm to the extracellular envelope usually a multi step process involves binding molecules and receptors located on each gamete 4. Passage of sperm through the extracellular envelope 5. Fusion of the egg and sperm cell membranes sperm and egg pronuclei meet, fuse; development initiated
Sea Urchin Fertilization Challenges for sea urchins (and others): how to bring two very small cells together in a very large space how to ensure that only sperm and eggs of the same species join
Sperm Chemoattraction Chemoattraction: eggs produce chemical attractant for sperm e.g. urchin Arbacia punctulata eggs produce resact A. 0 sec B. 20 sec Resact 14 aa peptide source egg jelly species specific sperm have membrane resact receptors C. 40 sec D. 90 sec binding: guanylyl cyclase cgmp activates Ca 2+ channel Ca 2+ i provides directional cues
Sperm Egg Interaction Sea Urchin Egg jelly stimulates the sperm acrosome reaction Acrosome reaction: fusion of acrosome and cell membranes releases acrosome contents Ionic changes stimulate actin polymerization acrosomal process Acrosome contains enzymes that digest jelly layer The exposed sperm membrane contains proteins that bind to egg receptors Sperm acrosomal process membrane fuses with egg membrane
Acrosome Reaction Sea Urchin AR stimulated by contact with egg jelly species specific stimulatory molecules in S. purpuratus fucose sulfate fucose sulfate binding to sperm receptor activates: Ca 2+ transport channel allows Ca 2+ into sperm head Na + /H + exchanger pumps Na + in/h + out phospholipase produces inositol trisphosphate (IP 3 ) elevated Ca 2+ and basic cytoplasm triggers fusion of acrosomal and cell membranes proteolytic enzymes digest a path through jelly coat to surface
Acrosome Reaction Sea Urchin Ca 2+ influx stimulates g actin polymerization to f actin acrosomal process adheres to vitelline envelope via bindin protein Bindin species specific binding to egg receptor on vitelline envelope Bindin Actin microfilaments
Vitelline Membrane Bindin Receptors Note regular sperm distribution suggests regular bindin receptor distribution species specificity
Fusion of Sperm and Egg Membranes acrosome reaction acrosomal process adheres to egg membrane microvilli membranes fuse (fusogenic protein?) causes egg actin polymerization fertilization cone formed actin from both gametes form connections sperm nucleus and tail pass through cytoplasmic bridge
Prevention of Polyspermy More than one sperm entering an egg results in polyploidy; usually eventual death Fast block to polyspermy electrical sea urchins, frogs, not in most mammals (why not??) Slow block to polyspermy chemical, physical most species, including mammals
Fast Block to Polyspermy Cell membranes provide a selective ionic barrier: cytoplasm/extracellular seawater: high Na +, low K + (relatively) cytoplasm: low Na +, high K + (relatively) This ionic imbalance is maintained by membrane pumps, exchangers Ionic imbalance creates electrical potential across the membrane;~ 70 mv 1 3 sec after sperm binding, membrane potential shifts to ~+20 mv Depolarization sperm cannot bind to eggs with positive membrane potential NOTE transient
Slow Block to Polyspermy Cortical granule reaction chemical and mechanical block active ~ 1 min after sperm egg fusion R. Bowen Cortical granules just beneath plasma membrane ~ 15,000 granules/sea urchin egg ~ 1 μm diameter Sperm entry initiates fusion of cortical granule membrane with cell membrane (like Acrosome Reaction) CG contents released into the space between the cell membrane and vitelline envelope (perivitelline space)
Slow Block to Polyspermy CG contents: 1. serine protease dissolves protein connections between envelope and membrane clips off bindin receptors & connected sperm 2. mucopolysaccharides sticky compounds; produce osmotic pressure water rushes in, vitelline envelope raises (fertilization envelope) 3. peroxidases oxidizes and crosslinks tyrosines hardens fertilization envelope 4. hyaline (protein) forms a coating around the egg: hyaline layer
Cortical Granule Exocytosis 1 Elevation of vitelline envelope Cortical granule fusion; release of CG contents
Cortical Granule Exocytosis 2 Hyaline layer
Fertilization Envelope Sea urchins Time after sperm addition: 10 sec 25 sec 35 sec
Ca 2+ Role in Cortical Granule Reaction Cortical granule reaction mechanism similar to acrosome reaction mechanism at fertilization, egg cytoplasmic Ca 2+ concentration rises high Ca 2+ causes cortical granule membranes to fuse with cell membrane internal Ca 2+ released as a self propagating wave Ca 2+ causes advancing cortical granule exocytosis, fertilization envelope, etc. 1 2 t=0 3 4 t=30 sec
Activation of Egg Metabolism Fertilization results in: 1. merging of two haploid nuclei 2. initiating the processes that start development these events happen in the cytoplasm occur without nuclear involvement Sperm fusion activates egg metabolism stimulates a preprogrammed set of metabolic events into action early responses occur within seconds of cortical reaction late responses start within minutes after fertilization
Egg Activation Early Responses
Egg Activation Late Responses
Early Responses Ca 2+ released from internal store at fertilization increases concentration from 0.1 1.0 μm Ca 2+ activates metabolic reactions; e.g. NAD + kinase burst of O 2 reduction (to H 2 O 2 )
Late Responses
Events After Membrane Fusion In sea urchins, fertilization occurs after 2 nd meiotic division therefore, a haploid female pronucleus is already present at fertilization After cell membrane fusion, sperm nucleus and centriole separate from mitochondria and flagellum sperm flagellum and mitochondria disintegrate sperm nuclear envelope vesiculates sperm DNA decondenses transcription and replication can start The sperm pronucleus rotates 180 so that sperm centriole is between the sperm and egg pronuclei sperm centriole acts as a microtubule organizing center forms an aster Aster microtubules extend throughout the egg; contact female pronucleus Pronuclei migrate towards one another Pronuclear fusion forms a diploid zygotic nucleus
Pronuclear Fusion
Mammalian Fertilization Sea urchin v. mammalian fertilization: many similarities, some differences: internal fertilization heterogeneity of sperm population translocation of gametes transport of both gametes to the oviduct sperm motility sperm capacitation chemotaxis, thermotaxis, hyperactivation of motility recognition at the zona pellucida (vitelline envelope in urchin eggs) gamete adhesion sperm egg binding acrosome reaction prevention of polyspermy fusion of genetic material
Sperm Translocation and Capacitation The ovulated egg (surrounded by cumulus cells) is picked up by the oviduct fimbriae ciliary beating and muscle contractions move oocyte cumulus complex into oviduct Sperm are deposited at the cervix; fertilize the egg at the ampulla of the fallopian tube motility not sufficient to move sperm to the ampulla sperm are transported by the female reproductive tract uterine muscle contractions move sperm to the oviduct sperm transport slows at ampulla (sperm time release?) sperm motility important within the oviduct hyperactivated motility in the vicinity of the oocyte or cumulus directional cues from temperature gradients (thermotaxis)
Mammalian Sperm Capacitation Freshly ejaculated mammalian sperm cannot fertilize the egg non capacitated sperm are held up in the cumulus matrix Capacitation a series of physiological maturation events that take place in the vaginal tract, uterus, and oviduct conditions for capacitation vary among species can be accomplished in vitro for many species using: oviduct fluid culture medium albumin Capacitation involves changes in: membrane lipid carbohydrates, proteins, membrane potential (becomes more negative), protein phosphorylation, internal ph, and enzyme activation Capacitation is transient; sperm become uncapacitated after a period WHY? Timing: nearly all human pregnancies result from sexual intercourse during a 6 day period ending on the day of ovulation. fertilizing sperm may take a long as 6 days to reach the ampulla
Hyperactivation, Thermotaxis, Chemotaxis Hyperactivation Motility patterns change in the oviduct in some species hyperactivated motility higher velocity, greater force suited for viscous oviduct fluid Thermotaxis Sperm may be able to sense a thermal gradient ampulla of oviduct is 2 C warmer than isthmus only capacitated sperm can respond thermotactically Chemotaxis Oocytes and cumulus cells may secrete chemotactic agents follicular fluid shows some chemotactic ability only fertilizable follicles had chemotactic activity only capacitated sperm respond
Recognition at the Zona Pellucida Mammalian Zona Pellucida analogous to vitelline envelope sperm binding relatively species specific e.g. mouse zona composed of 3 glycoproteins: ZP1, ZP2, ZP3 (and some internal accessory proteins) ZP matrix is synthesized by oocyte Sequential interactions between sperm proteins and zona components 1. Weak binding between sperm and peripheral egg protein 2. Stronger binding between zona and sperm SED1 protein 3. Sperm protein binds strongly to ZP3 ZP3 stimulates acrosome reaction
Acrosome Reaction Mouse Sperm Acrosome reaction induced when ZP3 crosslinks sperm membrane receptors. [sperm that undergo AR before reaching the zona unable to penetrate] sperm galactosyltransferase binds to ZP3 N acetylglucosamine crosslinking activates specific G proteins in sperm membrane this initiates a cascade that opens membrane Ca 2+ channels resulting in Ca 2+ mediated exocytosis of the acrosomal vesicle
Mammalian Gamete Fusion Mammalian sperm enter egg tangentially contact takes place on the side of the sperm membrane fusion at the junction of the inner acrosomal and cell membrane = equatorial region Equatorial region Mitochondria egg cortical actin polymerizes in the region of sperm binding extends microvilli to sperm cortical granules Cortical granules release enzymes that modify ZP so that it can no longer bind sperm N acetylglucosiminidase cleaves part of ZP3 carbohydrate chain ZP2 is also clipped; loses ability to bind sperm
Mammalian Pronuclear Fusion Essentially the same as sea urchin. mammalian pronuclear migration takes far longer (12 h v. ~ 1 h) glutathione from egg cytoplasm reduces disulfide bonds in sperm protamines (protamines replace histones in the sperm nucleus) allows uncoiling of sperm chromatin replication and transcription allowed protamine S S protamine GSH GS protamine SH + HS protamine Mammalian oocyte nucleus is arrested in metaphase of 2 nd meiotic division when sperm enters Sperm entry initiates Ca 2+ oscillations in the oocyte Ca 2+ i stimulates the cell cycle (i.e. cell division pathways) e.g. Ca 2+ inactivates MAP kinase (MEK) allows DNA synthesis
Pronuclear Fusion, cont. In mammals DNA synthesis occurs separately in male and female pronuclei male and female pronuclear chromatin condenses into chromosomes that orient themselves on a common mitotic spindle a true diploid nucleus in mammals is not seen in the zygote, but at the two cell stage (NOTE sea urchins produce a common zygote nucleus) Sperm contribution to zygote: nucleus, centriole, mitochondria, cytoplasm (minor) however, mitochondria and mitochondrial DNA are degraded therefore, all embryonic mitochondria are derived from the mother basis for mtdna tracing of geneology/phylogenetics several sperm proteins and mrnas for transcription and paracrine factors brought into the egg also, micrornas imported; may down regulate receptors involved in early cell division
Egg Activation Pathway Early responses Late responses