Biology 4361 Developmental Biology. Fertilization. June 24, PDF Free Download

Biology 4361 Developmental Biology Fertilization June 24, 2009

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: 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. Lennart Nilsson

Fertilization Overview 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 Mammalian fertilization NOTE Tremendous variation among species - models: sea urchin, mouse, chick

Sperm Formation

Sperm Axoneme

The Egg All materials necessary to begin development are stored in the egg. 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 - some localized regionally Morphogenic factors - initiate differentiation - e.g. transcription factors, paracrine factors bicoid mrna nucleus nanos mrna Protective chemicals - UV filters - DNA repair enzymes - antibodies - alkaloids (and other protective molecules)

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 Sea Urchin Volume: 2 x 10-4 mm 3 (>200 X sperm volume) (200 picoliters) egg jelly - glycoprotein meshwork - attract or activate sperm vitelline envelope - extracellular (inverts) - fibrous mat - sperm-egg recognition - contains glycoproteins egg cell membrane - binds sperm - fuses with sperm cell membrane

Egg Membrane Structure egg jelly actin microvilli filamentous (f-actin) cortex globular (g-actin) Cortical granules: Golgi-derived - proteolytic enzymes - mucopolysaccharides - hyaline protein - adhesive glycoproteins - cortex layer

Interactions Between Egg and Sperm 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 - 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 Pronuclear fusion: sperm and egg nuclei (pronuclei) meet, fuse; development initiated

Sea Urchin Fertilization Challenges for sea urchins (and others): 1) Bring two very small cells together in a very large space. 2) Ensure that only sperm and eggs of the same species join.

Sperm Chemoattraction Chemoattraction: eggs produce chemical attractant for sperm, e.g. Arbacia punctulata eggs produce resact A. 0 sec B. 20 sec Resact - 14 aa peptide - source egg jelly - species-specific - A.p. sperm - membrane resact receptors - binding: guanylyl cyclase - cgmp activates Ca 2+ channel - Ca 2+ i provides directional cues resact C. 40 sec D. 90 sec

Sea Urchin Acrosome Reaction Egg jelly stimulates the sperm acrosome reaction Acrosome reaction: fusion of acrosome and cell membranes - releases acrosome contents Ionic changes stimulate actin polymerization; forms acrosomal process Acrosome contains enzymes that digest jelly layer 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 - + /H + exchanger - pumps + 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 egg 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 Why? 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 Tim Watkins

Seconds Fast Block to Polyspermy Cell membranes provide a selective ionic barrier: - seawater: high +, low K + (relatively) - cytoplasm: low +, high K + (relatively) This ionic imbalance is maintained by membrane pumps, exchangers Ionic imbalance creates electrical potential across the membrane; ~ -70 mv = -70 mv (inside) K K K K K K K resting membrane potential plasma membrane

Seconds Fast Block to Polyspermy Cell membranes provide a selective ionic barrier: - seawater: high +, low K + (relatively) - cytoplasm: low +, high K + (relatively) This ionic imbalance is maintained by membrane pumps, exchangers Ionic imbalance creates electrical potential across the membrane; ~ -70 mv Sperm binding (or fusion) causes + influx 1-3 sec after sperm binding, membrane potential shifts to ~+20 mv Depolarization K K K K K K K - sperm cannot bind to eggs with positive membrane potential

Seconds Fast Block to Polyspermy Cell membranes provide a selective ionic barrier: - seawater: high +, low K + (relatively) - cytoplasm: low +, high K + (relatively) This ionic imbalance is maintained by membrane pumps, exchangers Ionic imbalance creates electrical potential across the membrane; ~ -70 mv Sperm binding (or fusion) causes + influx 1-3 sec after sperm binding, membrane potential shifts to ~+20 mv K K K K K K K Depolarization transient; membrane re-polarizes - sperm cannot bind to eggs with positive membrane potential

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 egg s cell membrane. CG contents released into the space between the cell membrane and vitelline envelope (perivitelline space)

Slow Block to Polyspermy Cortical Granule 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 Elevation of vitelline envelope Cortical granule fusion; release of CG contents

Cortical Granule Exocytosis 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 - at fertilization, egg cytoplasmic [Ca 2+ ] 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 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

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 )

Egg Activation Early Responses

Late Responses

Egg Activation 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 - results in sperm centriole between the sperm and egg pronuclei - sperm centriole acts as a microtubule organizing center; forms 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 Many similarities with sea urchin; 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

Mammalian Egg Cumulus ovarian follicular cells Inner-most layer corona radiata

Gamete Translocation 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 Fertilization takes place at the ampulla of the fallopian tube - sperm are transported by the female reproductive tract via uterine muscle contractions Sperm motility is not sufficient to move sperm to ampulla - sperm transport slows at ampulla (timed-release mechanism?) - sperm motility important within the oviduct

Gamete Translocation - hyperactivated motility in the vicinity of the oocyte or cumulus - directional cues from temperature gradients (thermotaxis)

Sperm Capacitation (Mammals) Freshly ejaculated mammalian sperm cannot fertilize the egg - fresh sperm 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 (protein) 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?

Sperm Capacitation (Mammals) 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

Acrosome Reaction - Mouse, cont. Sperm galactosyltransferase crosslinks ZP3 N-acetylglucosamine - crosslinking activates specific G-proteins in sperm membrane - initiates a cascade that opens membrane Ca 2+ channels - results in Ca 2+ -mediated exocytosis of the acrosomal vesicle

Mammalian Gamete Fusion Mammalian sperm enter egg tangentially - contact on the side of the sperm - membrane fusion at the junction of the inner acrosomal and cell membrane = equatorial region - egg cortical actin polymerizes in the region of sperm binding - extends microvilli to sperm Equatorial region Mitochondria 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

Sperm Contribution Sperm contributes 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 are brought into the egg Also, micrornas imported; may down-regulate receptors involved in early cell division

Egg Activation Pathway Early responses Late responses

Biology 4361 Developmental Biology. Fertilization. June 24, 2009