Fertilization and Cleavage Zona pellucida, compaction, blastomers, infertility Fertilization and Cleavage Dr. Nandor Nagy Semmelweis University

Process of fertilization -maturation of sperms: capacitation -The chemoattraction of sperm to the egg -attachment to and penetration of the zona pellucida The exocytosis of acrosomal vesicle to release enzymes The binding of the sperm to the extracellular envelope The passage of the sperm through the envelope The membrane fusion between sperm and egg -the process end with the intermingling of maternal and paternal chromosomes

Spermatozoa Undergo a Terminal Step of Functional Maturation Called Capacitation devbio8e-fig-07-02-3.jpg -freshly ejaculated sperm are unable to fertilize oocytes

Increased membran fluidity and Increased motility capacitated sperm is metabolically more active and beats its flagellum rapidly Thermotaxis: capacitated sperm can sense thermal gradient between isthmus of oviduct and ampulla (about 2oC). devbio8e-fig-07-02-4.jpg Acrosome (Proacrosin-GFP) Nucleus/mitochondria/mictotubule Increased membran fluidity and Increased motility albumin and lipid transfer protein (LTP-1; both protein present in female genital organ)  decrease the level of cholesterol more fluid spermatozoa cell membrane

Chemotaxis: secreted molecules from cumulus cells and oocyte. obstacles… First obstacle: -granulosa cells embedded in a loose extracellular matrix (hyaluronic acid) Before the encounter

second obstacle: Zona pellucida (13 µm)

Events Leading to the Fusion of Egg and Sperm Plasma Membranes Recognition of egg and sperm: 1. Chemoattraction of the sperm to the egg by soluble molecules 2. The binding of the sperm to the extracellular envelop of the egg 3. The exocytosis of the acrosomal vesicle to release its enzyme 4. The passage of the sperm through this envelop 5. Fusion of egg and sperm cell membrane Figures\Chapter07\DevBio7e07081.jpg Events Leading to the Fusion of Egg and Sperm Plasma Membranes

zona pellicida 3 protein of zona pellucida : ZP1 (200 kDa), ZP2 (120 kDa), ZP3 (83 kDa). When a spermatozoon reaches the tough zona pellucida surrounding the oocyte, it binds in a species- (that is, human-) specific interaction with a glycoprotein sperm receptor molecule in the zona (ZP3, one of three glycoproteins composing the zona pellucida).

A zona pellucida fehérjéi: ZP1 (200 kDa) ZP2 (120 kDa) ZP3 (83 kDa, ebből csak 44 kDa a polipeptidváz, a többi a piros „dobverőként” illusztrált oligoszaccharid oldallácok révén) galactosyltransferase-I -binds to ZP-3 –activate G protein—open Ca channel—acrsomal rxn- Release proteases that lyse the ZP-make a hole in egg Binding to ZP3 is mediated by a sperm surface protein called SED1

The acrosome reaction -The exocytosis of the acrosomal vesicle to release its enzyme -Enzymes are released from the acrosome [esterases, collagenase, acrosin and neuraminidase] -Enzymes cause lysis of the zona pellucida

As a result of this binding, the acrosome is induced to release degradative enzymes that allow the sperm to penetrate the zona pellucida.

The cell membrane of the two cells fuse. Molecules required for this event: -CD9 -Fertilin -tetraspanin -Izumo

Robert Edwards

ART – assisted reproductive technology ZIFT - Zygote Intra-Fallopian Transfer But: laparascopy IUI - Intra-Uterine Insemination IVF – In Vitro Fertilization Nobel prize 2010 Edwards Ovarian hyperstimulation ICSI - Intra-Cytoplasmatic Sperm Injection GIFT – Gamete Intra-Fallopian Transfer But: laparascopy

Number of months attempting to get pregnant Infertility Statistics on conception in normal fertile couples: within a month: 20–25%. within a year: 90% Definition of infertility: the inability of a couple to conceive after a year of regular intercourse without contraception. Number of months attempting to get pregnant Figure from www.ivf.indiana.com website Data from HFEA (human fertilization embryology authority) 2

Binding and fusion of sperm cell and egg -in normal monospermy: only one sperm enters a haploid sperm nucleus + a haploid egg nuleus combine: diploid nucleus of the fertilized egg (zygote) http://www.advancedfertility.com/triploid.htm Restoration of the chromosome number Polyspermy leads to disastrous consequences in most organism -two sperm entry results in triploid nucleus -each sperm’centriole divides to form the two poles of mitotic apparatus -triploid chromosomes -such cells either die or develop abnormally (triploid embryos nearly always abort) -amorphous, nodular embryos 3 pronuclei are seen in the center of the cell Each pronucleus contains 23 chromosomes This embryo has 69 chromosomes instead of the normal 46 Zona pellucida (shell) is visible as a halo around periphery Sperm are visible at 1 and 8 o'clock - outside of the zona

polyspermy block Membrane fusion immediately causes two events to occur: formation of a calcium wave that radiates over the surface of the egg from the point of sperm contact; and release of the contents of thousands of small cortical granules, located just beneath the oocyte cell membrane, into the perivitelline space between the oocyte and the zona pellucida. These two events alter the sperm receptor molecules, causing the zona to become impenetrable by additional spermatozoa. – - These changes prevent polyspermy or the fertilization of the oocyte by more than one spermatozoon.

polyspermy block: step 1 Wave of Ca2+ release across a sea urchin egg during fertilization induces cortical granule reaction polyspermy block: step 1 devbio8e-fig-07-20-0.jpg

Cortical granule exocytosis polyspermy block: step 2 devbio8e-fig-07-19-3.jpg

Morula Early blastomeres lack well developed intercellular junctions. Cells are in contact through microvilli and other cellular projections Morula microvilli

Compaction E-cadherin                                                                                                                                                                          E-cadherin E-cadherin=uvomorulun: 54,000 D cell adhesion molecule

Compacting Human Morula Biology of Reproduction Nikas et al. 1996, Biol. Reprod. 55:32-37

compaction Before and after

Blastocyst cavity forms within the morula (cavitation) - Sodium/potassium ATPase pumps sodium interior of the morula and water follows through osmosis

Early Embryonic Development Location Day Development Ampullary- 0-2 One cell Isthmic Junction All cell divisions are mitotic Smaller cell size with each cleavage division Ampullary- Isthmic Junction 1-3 Two cell Isthmus 2-3 Four cell Isthmus 3-5 Eight cell Uterus 4-5 Sixteen cell Uterus 5-8 Morula

Day Development Compact Morula form tight cell contacts so that Blastocyst formation is possible 6-7 Tight Morula 7-8 Early Blastocyst Fills with fluid in center - Cells have established polarity 7-9 Blastocyst Blastocoel Inner Cell Mass - Forms Embryo Trophoblast - Forms Placenta