Gametogenesis and Fertilisation PDF

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Gametogenesis and FertilisationTypical Sequence of Gametogenesis Germ cells develop outside of the gonads and have to migrate in  Male germ cells develop into spermatogonia  Female germ cells develop into oogoniaPrimordial Germ Cells Migrate from their site of origin into the gonad during early ...

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Gametogenesis and Fertilisation

Typical Sequence of Gametogenesis   

Germ cells develop outside of the gonads and have to migrate in Male germ cells develop into spermatogonia Female germ cells develop into oogonia

Primordial Germ Cells      

Migrate from their site of origin into the gonad during early development Originate in the extra-embryonic tissue Migrate into the embryo via the gut and into the genital ridge PGCs multiply by mitosis as they migrate The genital ridge gives rise to the somatic cells of the gonad (testis or ovary) The PGCs divide by meiosis to produce the gametes

Meiosis   

Reduces the chromosome number by half Introduces genetic variability Has the ability to pause at any stage

Spermatogenesis    

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PGC’s multiply by mitosis during migration to generate spermatogonia that rest in the G1 phase After birth – diploid spermatogonia multiply by mitosis to generate spermatocytes Spermatocytes enter meiosis Incomplete cytokinesis yields syncytia – haploid spermatids connected by cytoplasmic bridges - Allows synchronisation of maturation and sharing of gene products Spermatids differentiate into spermatozoa - 4 gametes (n) from each primary spermatocyte

Sperm Development       

Golgi apparatus develops into acrosomal cap Flagellum develops Cytoplasm extruded Mitochondria coalesce near base of the flagellum Arginine rich protamines replace histones Nucleus condenses Cytoplasmic bridges collapse

Sperm Head 

The acrosome contains lytic enzymes for: - Protein digestion e.g. acrosin - Carbohydrate digestion - Lipid digestion

Sperm Tail   

Axonme contains 2 central single microtubules 9 doublet circles surrounding the central one Dynein is attached to the microtubules – enables the sperm to swim

Dynein  

Uses ATP hydrolysis to slide the microtubules past one another Allows the movement of the sperms tail

Oogenesis     

Meiosis occurs after differentiation Embryo: PGCs multiply by mitosis during migration to generate oogonia that continue to divide by mitosis Diploid oogonia enter meiosis and arrest in prophase of meiosis I as primary oocytes Upon ovulation, meiosis I is completed; secondary oocytes arrest in metaphase II Meiosis II is completed after fertilisation - 1 ovum/egg (n) and 2 polar bodies from each primary oocyte

Eggs        

Specialised to generate a new individual Contain nutrient reserves and an elaborate coat Nutritive yolk proteins Protein synthesis machinery (ribosomes and tRNA’s) MRNA’s encoding proteins needed for early development Morphogenetic factors to direct early development Protective chemicals e.g. UV filters, enzymes for DNA repair Extracellular glycoprotein coat

Secondary Oocyte 

Surrounded by the zona pellucida

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Traslucent layer of 3 glycoproteins - ZP1 - ZP2 - ZP3

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ZP3 is the sperm receptor; its O-linked polysaccharide determines species specificity ZP3 ‘knockout’ mice produce oocytes lacking the zona pellucida, and are therefore infertile Human ZP3 gene ‘knock-in’ rescues zona formation and fertility, but human sperm is not able to bind this ZP3 because it does not recognise the murine polysaccharide

ZP3

Cortical Granules  

Derived from the Golgi apparatus Contain proteases and glycosidases

Penetration of the Cumulus Cell Layer  

The oocyte is surrounded by cumulus cells from the follicle, in a matrix of hyaluronic acid Hyaluronidase activity on the sperm head enables it to penetrate this layer

Sperm galactosyltransferase (GalT) recognizes Nacetylglucosamine residues on ZP3    

GalT-ZP3 crosslinking causes GalT proteins to cluster, triggering G protein activation The change in membrane potential opens voltage-gated calcium channels, increasing intracellular Ca2+ Calcium-mediated exocytosis of the acrosomal vesicle is initiated: the acrosomal reaction Acrosomal enzymes, including b-Nacetylglucosaminidase (digests oligosaccharide side chains) and acrosin (serine protease), lyse the zona pellucida

Second Recognition Event  

Sperm Izumo binds oocyte Juno, recruiting oocyte CD9, causing the plasma membranes to fuse, and the sperm enters the oocyte The oocyte (egg) responds in three ways: - Exocytosis of cortical granules - Completion of the second meiotic division, producing the definitive oocyte and second polar body - Metabolic activation

Sperm Entry 

Sperm entry triggers release of calcium from the ER within the egg

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A wave of Ca2+ release crosses the egg at 5-10 µm s-1, followed by Ca2+ oscillations The sharp increase in free Ca2+ is essential for egg activation and for the initiation of development

Calcium release triggers the cortical reaction    

Actin polymerises into microfilaments, which transport cortical granules to the plasma membrane Cortical granule contents released by exocytosis Enzymes partially digest ZP2 and remove carbohydrate from ZP3 The ZP hardens, and further sperm cannot bind, blocking polyspermy

The increase in free Ca2+ initiates development   

Ca2+ activates a kinase that leads to proteolysis of cyclin The metaphase II arrested oocyte nucleus completes meiosis A centrosome forms around the sperm centriole, which becomes the MT organising centre for the sperm aster

Sperm and egg pronuclei approach each other and prepare for the first mitotic division of the zygote   

Both pronuclei undergo DNA replication as they migrate along microtubules towards one another The pronuclear envelopes break down, the centrosome replicates and organises a mitotic spindle The chromosomes align on a common metaphase plate

The Sperm Provides  

A haploid genome A centriole

The Egg Provides   

A haploid genome Mitochondria and other organelles MRNAs and proteins needed for early development

The Main Results of Fertilisation Are 

Restoration of the diploid number of chromosomes

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Sex determination of new individual (XX or XY) Initiation of cleavage

Development Involves Cell Division 

Cleavage division can set up asymmetries by segregating determinants

Development Involves the Emergence of Pattern    

The process by which special and temporal pattern of cellular activities is organised within an embryo so that a well ordered structure develops An early step is allocation to different germ layers Cells must ‘know’ where they are and differentiate accordingly: regional specification Positional information may be in the form of inherited cytoplasmic determinants, or short or long-range signals

Development Involves Morphogenesis      

Creation of structure or form Differential proliferation Cell movement Cell fusion Cell death Gastrulation moves the germ layer relative to one another

Development Involves Growth  

Generally there is little growth in early development, while the basic body plan is being established Differential growth rates can result in a change in body proportions

Totipotent Zygote 

Generates all the cell types of the body and extra-embryonic tissues

Development involves progressive cell commitment    

Early embryonic cells are pluripotent Their potential gradually becomes restricted Differentiation is the process of cells becoming structurally and functionally specialised Reflecting activation and maintenance of a particular pattern of gene expression

Cleavage produces a cluster of blastomeres

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Divisions occur in the absence of growth Spherical blastomeres form a loose clump

Morulla     

Forms 3 days after fertilisation Undergoes compaction E-cadherin becomes restricted to regions of intercellular contact Increased cell-cell adhesion maximises contact between blastomeres Forming a compact ball of cells held together by tight junctions

Subsequent tangential cleavages    

Produce one polarised and one non-polarised daughter cell The outer cells have distinct apical and basal surfaces The non-polarised cells form the inner cell mass The inner cells communicate extensively through gap junctions

Fates of Cells  

The inner and outer cells have different fates The segregation of trophectoderm and inner cell mass (ICM) lineages is the first differentiation event in mammalian embryonic development

Blastocyst     

A fluid-filled cavity develops, and the embryo is now called a blastocyst The inner cell mass will give rise to the embryo proper (and some extra-embryonic structures) The zona pellucida prevents implantation in the oviduct Flattened epithelial cells of the trophectoderm will form extra-embryonic tissues Fluid-filled blastocyst cavity (or blastocoel)

Active transport of sodium ions leads to fluid accumulation in the blastocoel    

Tight junctions between outer cells act as a permeability barrier Na+ is actively transported into the blastocoel As the ion concentration in the blastocoel increases, water flows in by osmosis The resulting hydrostatic pressure inflates the blastocoel

Enzymes digest through the zona pellucida and the embryo hatches   

Zona pellucida Trophoblast (derived from trophectoderm) Hypoblast (part of inner cell mass)

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Blastocyst cavity Epiblast (part of inner cell mass)

Implantation   

Requires interaction between trophoblast cells and uterine cells Trophoblast cells express integrin proteins These interact with extracellular matrix proteins expressed by the epithelial cells of the uterine mucosa: - Integrin/laminin interactions promote attachment - Integrin/fibronectin interactions promote migration - The inner cell mass is now called the embryoblast

The embryoblast forms a flat disc of two layers, epiblast and hypoblast 

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Epiblast - Columnar cells adjacent to newly formed amniotic cavity - Will form the embryo proper Hypoblast - Small cuboidal cells adjacent to the blastocyst cavity - Will form extra-embryonic structures that will connect to the mother’s circulation

The primitive streak forms after two weeks   

The primitive streak forms on the surface of the epiblast in the region that will become the posterior of the embryo This is the first sign of the anteroposterior axis During gastrulation, epiblast cells migrate towards the primitive streak and invaginate (move inwards), displacing the hypoblast

Gastrulation      

Epiblast cells migrate through the primitive streak to create the three germ layers The first cells to invaginate form endoderm The next cells to invaginate form mesoderm The remaining epiblast cells form ectoderm The germ layers are morphologically distinct and have different fates After gastrulation, the flat embryo rapidly folds into a three-dimensional body

Neurulation   

The ectoderm folds along its central axis to form the neural tube The neural folds elevate and fuse Neural crest cells form near the site of fusion and migrate away

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The epidermis fuses above the neural tube

Paraxial Mesoderm  

Segments into somites Somites generate trunk and limb muscles, dermis and vertebrae

Endoderm  

Is internalised and gives rise to the epithelial lining of the gastrointestinal tract Endoderm also provides the stomach, liver and pancreas, and the epithelial lining of the respiratory tract

Organogenesis   

During the embryonic period (weeks 3-8) the germ layers give rise to tissues and organ systems Stem cell populations are establishing each of the organ primordia, and are sensitive to genetic and environmental influences This is the period when most structural birth defects are induced

Stages     

Fertilisation Cleavage to form the blastula Gastrulation to reorganise the structure of the embryo and generate the germ layers Neurulation Organogenesis...