lecture 8 Jan29 pdf - BIOL 303 Developmental Biology...

Info icon This preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: BIOL 303 - Developmental Biology Beginnings of Development: Fer3liza3on 1.  Gamete structure & recogni2on 2.  External fer2liza2on -­‐ Sea urchin 3.  Internal fer2liza2on -­‐ Mammals © Tamara Western 2013! Beginnings of Development: Fertilization Need sperm and egg! Steps of fertilization 1) Sperm needs to find the egg 2) Sperm needs to bind the egg (species specificity) 3) Sperm needs to get into the egg (only 1 sperm! Prevention of polyspermy) 4) Egg activation: egg metabolism has to be activated 5) Male and female pronuclei have to fuse 6) First zygotic division Accomplishes 2 things: 1)  Mix of two haploid genomes 2)  Egg ac2va2on Fertilization - sperm Bull sperm Mouse sperm Sperm needs to: 1) Provide DNA à male pronucleus 2) Move à MT based propulsion system called flagellum 3) Enter the egg à acrosome Acrosomal enzyme labelled with GFP DNA mitochondria MTs Egg needs to: Fertilization - egg 1)  Provide DNA à female pronucleus (either already haploid or arrested in different stages of meiosis – species dependent) 2 ) control sperm entry -­‐ sperm receptor -­‐ only one sperm à prevent polyspermy 3) Provide supply for early embryonic development -­‐ Nutri2ve proteins: supply of energy and amino acids (Yolk) -­‐ Ribosomes + tRNA for protein synthesis -­‐ mRNAs (no embryonic TS in early stages) -­‐ Morphogene2c factors ac2ng as cell fate determinants -­‐ Protec2ve chemicals (UV protec2on, an2bodies, “bad taste” chemicals) Sea urchin egg at fer2liza2on Fertilization - egg Anatomy of sea urchin egg External fer2liza2on Extracellular matrix: -­‐  Egg jelly (glycoprotein meshwork), a]racts and/or ac2vates sperm -­‐  Vitelline envelope (made of glycoproteins), involved in species specific sperm binding Cell membrane: -­‐  fuses with sperm cell membrane, -­‐  Controls ion flows during fer2liza2on Cortex: -­‐  “s2ff” cytoplasm underneath the cell membrane -­‐  Contains lots of ac2n (microvilli, cell division) -­‐  Contains cor2cal granules (comp: acrosome), helps prevent polyspermy Sea urchin egg at fer2liza2on Fertilization - egg Anatomy of a mouse egg -­‐ Internal fer2liza2on Extracellular matrix: -­‐  Cumulus: ovarian follicular cells nurturing the egg un2l ovula2on (=release from the ovary) -­‐  Zona pellucida: (made of glycoproteins), involved in species specific sperm binding Cell membrane: -­‐  fuses with sperm cell membrane, -­‐  Controls ion flows during fer2liza2on 1) Sperm needs to find egg Sea urchin – sperm attraction External fer2liza2on: sea urchin In the ocean: how does sperm find egg?? 1 sec 20 sec 40 sec 90 sec 1) Sperm needs to find egg Resact binds Receptor Guanylyl cyclase (RGC) à produc2on of cGMP à Ca+ influx Signal transduc2on induced by Resact in sperm Highly species specific! 1 molecule of Resact is enough! Both cGMP and Ca+ ac2vate mitochondrial respira2on à ATP produc2on à feeds dynein motor 2) Sperm needs to bind egg + 3) sperm needs to enter egg Sperm – Egg interac2ons that lead to successful fer2liza2on 1) Species specific! 2) Acrosome reac2on (species specific!) -­‐  Exocytosis to release diges2ve enzymes -­‐  Presenta2on of the acrosomal process 4) An acrosomal protein called “bindin” mediates binding of the sperm to the vitelline membrane of the egg (species specific!) 2) Sperm needs to bind egg + 3) sperm needs to enter egg Sea urchin – acrosome reaction Acrosome reac2on Species specific recogni2on event Fusion of acrosome membrane with sperm membrane (=exocytosis) à Release of diges2ve enzymes Globular ac2n starts to polymerize à “Acrosomal process” Leads to de-­‐masking and presenta2on of bindin Sea urchin – sperm binding How was Bindin iden2fied? 2) Sperm needs to bind egg + 3) sperm needs to enter egg 2) Sperm needs to bind egg + 3) sperm needs to enter egg Sea urchin – sperm binding Where is Bindin located? 2) Sperm needs to bind egg + 3) sperm needs to enter egg Sea urchin – sperm binding What egg protein does Bindin bind to? Sea urchin egg saturated with sperm Should be EBR1! Sea urchin – sperm entry Acrosomal process (Bindin binds EBR1) 3) sperm needs to enter egg Fer2liza2on cone (via ac2n polymeriza2on) Helps engulf the sperm Bindin has a second role: helps egg-­‐sperm membrane fusion 3) Preven2on of polyspermy Sea urchin – prevention of polyspermy Eggs needs to prevent polyspermy Dispermic sea urchin egg Dispermic human egg See also: http://www.nature.com/news/2007/070326/full/news070326-1.html Sea urchin – prevention of polyspermy Fast Block to polyspermy -­‐  -­‐  -­‐  -­‐  Within 1-­‐3 seconds Change in membrane poten2al Transient (about 1 min) In sea urchins, marine worms, frogs (external fer2liza2on) Slow Block to polyspermy -­‐  Within 30 seconds -­‐  Physical block through changes of the vitelline membrane (hardens, becomes the “fer2liza2on envelope”) -­‐  Permanent -­‐  Most animal, including mammals (external and internal fer2liza2on) 3) Preven2on of polyspermy Sea urchin – prevention of polyspermy 3) Preven2on of polyspermy Fast Block Res2ng membrane poten2al of the egg: About -­‐70mV (inside is neg) Sperm entry Influx of Na+ 490mM Na+ in the medium 120mM Na+ in the medium membrane poten2al of the egg: About +20mV (inside is pos) No more sperm binding Sea water: 600mM Na+ 3) Preven2on of polyspermy Sea urchin – prevention of polyspermy Fast Block to polyspermy -­‐  -­‐  -­‐  -­‐  Within 1-­‐3 seconds Change in membrane poten2al Transient (about 1 min) In sea urchins, marine worms, frogs (external fer2liza2on) ✓ Slow Block to polyspermy -­‐  Within 30 seconds -­‐  Physical block through changes of the vitelline membrane (hardens, becomes the “fer2liza2on envelope”) -­‐  Permanent -­‐  Most animal, including mammals (external and internal fer2liza2on) 3) Preven2on of polyspermy Sea urchin – prevention of polyspermy Slow Block Cor2cal reac2on: forma2on of a fer2liza2on envelope 10 sec 25 sec forma2on of the fer2liza2on envelope starts at the point of sperm entry 35 sec fer2liza2on envelope is almost complete fer2liza2on envelope is complete Mediated by the “cor2cal granule reac2on” Sea urchin – prevention of polyspermy 3) Preven2on of polyspermy Slow Block Cor2cal granules release: -­‐ Cor2cal granule serine protease Digests connec2on between vitelline membrane and cell membrane; clips off sperm receptors+sperm -­‐ Transgluaminases and ovoperoxidase harden the vitelline envelope à fer2liza2on envelope -­‐ Glycoaminoglycans Glycoproteins that are very hydrophilic à absorb water and so expand the space between the fer2liza2on membrane and the cell membrane -­‐ Hyaline layer à Provides support during cleavage 3) Preven2on of polyspermy 4.1 Sea Urchin Fer2liza2on 3) Preven2on of polyspermy Sea urchin – prevention of polyspermy Slow Block – Ca+ waves 3) Preven2on of polyspermy 4.2 Calcium Wave in Sea Urchin Fer2liza2on 3) Preven2on of polyspermy 4) Ac2va2on of egg metabolism Release of internal Ca+ How is that mediated? Diacylglycerol (DAG) Phospha2dylinositol 4,5-­‐bisphophate (PIP2) Inositol 1,4,5-­‐trisphophate (IP3) Key point: PLC mediated cleavage of PIP2 IP3 Ca+ release from ER DAG Increase of pH Summery Fer2liza2on in sea urchins Sperm chemotaxis Egg Acrosome reac2on sperm entry Egg response to sperm entry: Na+ influx PIP2 PLC IP3 + DAG FAST BLOCK Release of Ca+ from the ER Cor2cal granule reac2on SLOW BLOCK Increase of pH Ac3va3on of egg metabolism -­‐  Start zygo2c mitosis -­‐  Protein synthesis -­‐  Cytoplasmic movement of morphogene2c material Internal fer2liza2on in mammals Very hard to study: -­‐ Happens in the oviduct -­‐ Only 200 out of 280 x 106 sperm make it to the vicinity of the egg -­‐ Sperm is heterogeneous (spermatozoa of different stages of matura2on) -­‐ Probably more than 1 way to induce the acrosomal reac2on Both gametes have to meet in the oviduct – transloca2on: egg: -­‐ fimbriae of the oviduct transport cumulus-­‐oocyte complex to the ampulla Sperm -­‐  Flagellar mo2lity does NOT get the sperm into the oviduct (becomes hyperac2ve only once in vicinity of the egg) -­‐  Uterine contrac2ons are the means of transport -­‐  Thermotaxis and chemotaxis (progesterone) as direc2onal clues -­‐  Some uterine regions slow sperm downà gradual release increases fer2liza2on rate -­‐  A sperm’s journey trough the female reproduc2ve tract is required for CAPACITATION Mammals – sperm capacitation Capacita2on = physiological changes mediated by the oviduct that make sperm competent to fer2lize an egg (not the fastest makes the race!!) Oviduct binds and capacitated sperm over ~ 6 days (human), releases “packets” of capacitated sperm à prolongs “window of opportunity” Capacita2on involves: 1) Changes in sperm membrane composi2on 2) De-­‐masking of receptors 3) Protein ac2va2on via phosphoryla2on 4) Changes in membrane poten2al Capacitated sperm become hyperac2ve Mammals – acrosome reaction Old model: Acrosomal reac2on happens as sperm binds to the Zona Pellucida protein ZP3 Recent data: Successful sperm has undergone the acrosomal reac2on BEFORE reaching the Zona Pellucida (in the Cumulus) What do sperm bind to once at the ZP? Mammals – sperm binding Q: where does human sperm bind to? Mammals – prevention of polyspermy Fast block: -­‐ none Slow block: -­‐ cor2cal granule reac2on mediates (among other reac2ons) the cleavage of ZP2 -­‐ cleavage is mediated by cor2cal granule protease ovastacin Q: Is the preven2on of polyspermy dependent of ZP2 cleavage? Conclusion: yes! ZP2 cleavage is a key event in the preven2on of polyspermy ...
View Full Document

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern