Fertilization and Cleavage Fertilization, capacitation, cleavage, compaction, blastocyst Fertilization and Cleavage Nóra Fejszák Semmelweis University

Process of fertilization Fertilization is the fusion of gametes to initiate the development of a new individual organism. In humans, the process involves the fusion of an ovum with a sperm. Multi steps, species-specific process Capacitation The chemoattraction of sperm to the egg Attachment to and penetration of the zona pellucida The membrane fusion between sperm and egg The process end with the intermingling of maternal and paternal chromosomes The binding of the sperm to the extracellular envelope The exocytosis of acrosomal vesicle to release enzymes The passage of the sperm through the envelope

Mature human spermatozoon Spermatogenesis (=it is the process in which spermatids are produced from male primordial germ cells by way of mitosis and meiosis) Spermiogenesis (=differentiation from spermatids to mature flagellated sperm) cells) Head -flattened and pointed Nucleus The DNA is tightly compressed Acrosome In front of the compressed haploid nucleus Modified secretory vesicle Contains enzymes that digest prtoteins and complex sugars

Maturation of sperms - capacitation Freshly ejaculated sperm are unable to fertilize oocytes Capacitation: -lasting about few hours in the female genital tract -before capacitation sperm show low activity -capacitated sperm is metabolically more active and beats its flagellum rapidly -molecular changes in sperm head membrane → decrease the level of cholesterol→more fluid spermatozoa cell membrane Acrosome (Proacrosin-GFP) Nucleus/mitochondria/mictotubule

Thermotaxis, chemotaxis - influence the behaviour of spems while they are moving towards the oocyte. Thermotaxis: capacitated sperm can sense thermal gradient between isthmus of oviduct and ampulla (about 2oC). Chemotaxis: secreted molecules from cumulus cells and oocyte.

Before the encounter Second obstacle: Zona pellucida -glycoprotein matrix -produced by growing ovum -species-specific sperm penetration -penetrant block to polyspermy -keep blastomeres together obstacles… First obstacle: -granulosa cells embedded in a loose extracellular matrix hyaluronic acid) -A sperm plasma membrane protein (PH20 ) has an enzymatic activity that digest hyaluronic acid

3 protein of zona pellucida : ZP1 (200 kDa), ZP2 (120 kDa), ZP3 (83 kDa). -it is the specific glycoprotein in the zona pellucida to which sperm binds -initiates the acrosome reaction after sperm have bound to it -interact with galactosyltransferase I. on the sperm head membrane Binding to ZP3 is mediated by a sperm surface protein called SED1

The acrosome reaction 1. The outer acrosomal membrane and the sperm plasma membrane fuse at many points 2. The two membranes break up into small vesicles (remain connected) 3. Massive influx of Ca++ through the plasma membrane of the sperm head 4. Enzymes are released from the acrosome [esterases, acrosin (not diffusible!!), and neuraminidase] 5. 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.

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

Prevention of polyspermy Transiant depolarization (hypothesis) -sodium channels open in the oocyte cytoplasm -resting membrane potential (-70 mV)→ depolarization (+20 mV) -this process hasn’t yet been proved in mammals Cortical reaction -during egg activation the cortical granules undergo exocytosis -upon fertilization, the free Ca++ concentration of the egg increases greatly -in the high-calcium environment, the cortical granule membranes fuse with the egg cell membrane -releasing enzymes which cross-link the zona proteins, thus making the the zona impermeable to sperm -modification of the ZP3 glycoprotein, so that ZP3 no longer binds to sperm nad create a new layer

Binding and fusion of sperm cell and egg I. Oocyte activation -Prior to fertilization the egg is arrested in metaphase of the second meiotic division  Division process is reactivated by the penetration of the sperm (Ca+ influx)  Second meiotic division is completed -  Second polar body is appeared

Binding and fusion of sperm cell and egg II. -in normal monospermy: only one sperm enters a haploid sperm pronucleus + a haploid egg pronuleus 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 http://www.youtube.com/watch?v=7G2rL5Cutd4 http://www.youtube.com/watch?v=BFrVmDgh4v4

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) 2006

Robert G. Edwards

Intracytoplasmic sperm injection (ICSI) -a single sperm is injected directly into an egg. https://www.youtube.com/watch?v=cVmQsv8tVMw

Compaction -Early blastomeres lack well developed intercellular junctions. Cells are in contact through microvilli and other cellular projections - During compaction blastomeres tightly adhere with each other through gap and tight junctions before compaction and after compaction

16-64 cells stage