1
Q
Fertilization is?
A
Fertilization – fusion of two different gametes (egg + sperm) into a new individual
2
Q
Fertilization accomplishes two things:
A
- sex (combination of genes from two parents)
- reproduction
3
Q
Conception generally consists of four major events:
A
- Contact and recognition between sperm and egg. In most cases, ensures that they are of the same species.
- Prevention of polyspermy.
- Fusion of the genetic material of sperm and egg.
- Activation of egg metabolism to start development.
4
Q
Egg and sperm affects each other how?
A
The egg activates the sperm metabolism that is essential for fertilization
The sperm reciprocates by activating the egg metabolism needed for the onset of development.
5
Q
Sperm anatomy
A
Each sperm cell consists of:
- A haploid nucleus
- A propulsion system, to move the nucleus
- A sac of enzymes, enabling the nucleus to enter the egg
6
Q
Almost all the cell’s cytoplasm is eliminated during sperm maturation, leaving only certain organelles modified for spermatic function:
A
- The sperm’s haploid nucleus becomes very streamlined and the DNA tightly compressed
- Golgi apparatus forms the acrosomal vesicle / acrosome at future anterior end above nucleus, containing enzymes that digest proteins and complex sugars (cut a path through outer coverings of the egg)
- Globular actin proteins in region between nucleus and acrosomal vesicle. Used to extend finger-like acrosomal process during the early stages of fertilization, in many species involved in recognition between sperm and egg
- Centriole produces a long flagellum at posterior end
- Mitochondria collect around the flagellum near the base of the nucleus
7
Q
Sperm head is?
A
acrosome and nucleus
8
Q
Sperm midpiece is?
A
centriole, mitochondria
9
Q
Sperm propulsion varies according to how the species has adapted to environmental conditions. Most species use?
A
whipping of its flagellum
10
Q
Flagellum structure:
A
- Major motor portion is the axoneme, a structure formed by microtubules emanating from the centriole at the base of the sperm nucleus.
- Dynein (ATPase) provides the force for sperm propulsion, attached to the microtubules of axoneme
11
Q
No dynein consequences?
A
No dynein – male sterility, susceptible to bronchial infections etc.
12
Q
In many species, a layer of dense fibres has interposed itself between the mitochondrial sheath and the cell membrane because?
A
Stiffens the tail, the layer decreases towards tail tip
=> prevents sperm head from being whipped around too suddenly
13
Q
Sperm capacitation is?
A
The final stages of sperm maturation.
Sperm can move, but not yet bind to and fertilize an egg.
Do not occur in mammals until the sperm has been inside the female reproductive tract for a certain time period.
14
Q
Oocyte?
A
Developing egg, cannot yet bind sperm or be fertilized
15
Q
The oocyte has what function?
A
Stores all the material necessary for the beginning of growth and development => the developing egg conserves the material it has + actively accumulates more
- The meiotic division that form the oocyte, conserve its cytoplasm rather than giving half of it away
- The oocyte either synthesizes or absorbs proteins such as yolk that act as food reservoirs for the developing embryo
16
Q
Egg cytoplasmic storehouse, contains?
A
- Nutritive proteins. Supply of energy and AA
- Ribosomes and tRNA. Responsible for the burst of protein synthesis soon after fertilization
- Messenger RNAs. Encode proteins for the early stages of development, before fertilized egg can make them itself
- Morphogenetic factors. Often localized in different regions and become segregated into different cells during cleavage
- Protective chemicals. Fx UV filters and DNA repair enzymes, or molecules that potential predators find distasteful
17
Q
Female pronucleus can be:
A
- Already haploid at time of fertilization, fx sea urchins
- Still diploid, fx worms and most mammals, final stages of egg meiosis takes place after the male pronucleus (the sperm’s nuclear material) is already inside the egg
18
Q
Egg membrane + envelope: (ordered from inside to outside)
A
- Cortex
- Cell membrane
- Extracellular matrix
- Egg jelly
- In mammals:
- Cumulus
- Corona radiate
19
Q
Egg membrane + envelope: the cortex
A
Thin layer of gel-like cytoplasm, stiffer than internal cytoplasm, contains high concentrations of globular actin (forms microfilaments necessary for cell division during fertilization), microfilaments also extend egg surface into microvilli (may aid sperm entry)
Contains cortical granules – membrane-bound, Golgi-derived structures containing proteolytic enzymes, mucopolysaccharides, adhesive glycoproteins and hyaline proteins (first two prevent polyspermy, last two surround early embryo, providing support for cleavage stage blastomeres)
20
Q
Egg membrane + envelope: Cell membrane
A
regulates flow of ions during fertilization, capable of fusing with sperm cell membrane
21
Q
Egg membrane + envelope: Extracellular matrix
A
forms a fibrous mat, often involved in sperm-egg recognition.
o Vitelline envelope in invertebrates.
o Zona pellucida: extracellular envelope, separate thick matrix in mammals
22
Q
Egg membrane + envelope: Egg jelly
A
glycoprotein meshwork, numerous functions, most commonly used to either attract or activate sperm
23
Q
Egg membrane + envelope: extra in mammals
A
- Cumulus: layer of cells, made up of the ovarian follicular cells that were nurturing the egg at the time of its release from the ovary.
- Corona radiate: innermost layer of cumulus cells, immediately adjacent to the zona pellucida
24
Q
Interaction between sperm and egg generally proceeds according to five steps:
A
- Chemoattraction of the sperm to the egg by soluble molecules secreted by the egg
- Exocytosis of the sperm acrosomal vesicle and release of its enzymes
- Binding of the sperm to the extracellular matrix (vitelline envelope or zona pellucida) of the egg
- Passage of the sperm through this extracellular matrix
- Fusion of the egg and sperm cell membranes
25
Many marine organisms release their gametes into the environment, facing some problems with solutions:
- Distance => enormous numbers of gametes, species-specific attraction
- “Pollution” from other species => species-specific activation
26
Sperm attraction: action at a distance
Chemotaxis – sperm are attracted toward eggs by following a gradient of an egg secreted chemical, mechanism of which differ among species, and the chemotactic molecules are different even in closely related species
27
Sea urchin sperm activation in water:
Low internal pH (7.1) prevents activation of the dynein ATPase = immotile in gonads
Spawned into seawater => pH elevates (7.6) => activation of ATPase => motility
28
Sperm-activating peptides (SAPs) provides movement direction.
- Diffuses readily from the egg jelly into seawater
- Most are both sperm activating and attracting, though in some species its split in different compounds
- Binding of a single SAP enough to provide direction
- Sperm sense the gradient by curving their tails, interspersing straight swimming with a “turn” to sense the environment
- Sperm have Rs in their membranes that bind these peptides.
29
Binding of SAP by sperm membrane:
Binding of SAP on extracellular side => stimulated flagellar movement
30
The acrosome reaction in most marine invertebrates has two components:
- Fusion of acrosomal vesicle with the sperm cell membrane (exocytosis), caused by contact with egg jelly (sea urchins, highly species specific polysaccharides bind to Rs on sperm membrane)
- Extension of the acrosomal process
31
Acrosome reaction initiated by:
- Polysaccharide on egg binds to R on sperm membrane
- R activates sperm membrane proteins
- Elevated Ca2+ level in a relatively basic cytoplasm triggers fusion of acrosomal membrane with sperm cell membrane
=> release of enzymes
32
Extension of acrosomal process:
- Influx of Ca2+ thought to activate RhoB (GTP binding protein) in acrosomal region and midpiece of sea urchin sperm
- => organize the actin skeleton, thought to be active in polymerizing globular actin molecules into actin filaments
- => extension of acrosomal process
33
Digestion of vitelline envelope proceeds how?
Released enzyme+proteasomes digest a path through the jelly coat to the egg cell surface
=> acrosomal process adheres to the vitelline envelope and tethers sperm to egg
=> proteasome from acrosome coat the acrosomal process, allowing digestion of vitelline envelope at attachment point
=> movement toward egg proceeds
34
Recognition of the egg's extracellular coat
Bindin – acrosomal protein mediating sperm recognition of egg surface.
Rs thought to be aggregated into complexes on vitelline envelope, hundreds of such might be necessary to tether the sperm to the egg.
Species-specific interaction between variations of bindin and their specific Rs.
35
Fusion of the egg and sperm cell membranes, the fertilization cone
Polymerization of actin in the egg, caused by sperm-egg fusion.
Actin from the gametes forms a connection that widens the cytoplasmic bridge between the egg and the sperm, so the sperm nucleus and tail can pass through.
36
Monospermy
normal, one sperm enters the egg and fuses with egg nucleus
37
Centrioles from egg and sperm, double up or?
Centriole provided by sperm divides, centriole provided by egg is degraded
38
Polyspermy
entrance of multiple sperm, leads to disastrous consequences
Fx pga uneven number of chromosomes, multiple centrioles splits cell in too many even cells => some have doubled of certain chromosomes, some lack them entirely, and other problems.
Such cells die or develop abnormally.
39
Two ways out of many to prevent polyspermy in sea urchins:
- An initial, fast reaction, accomplished by an electric change in the egg cell membrane
- Followed by a slower reaction caused by the exocytosis of the cortical granules.
40
Fast block of polyspermy
changes in electric potential of egg cell membrane (through influx of Na+) immediately upon entry of a sperm prevents further sperm from binding.
41
Slow block to polyspermy
Slow block = Cortical granule reaction
Upon sperm entry, cortical granules fuse with the egg cell membrane and release their contents into the space between the cell membrane and the fibrous mat of vitelline envelope proteins.
42
Cortical granule proteins and their function: (5)
- Cortical granule serine protease: cleaves the protein posts that connect the vitelline envelope proteins to the egg cell membrane, also clips off the bindin Rs and any sperm attached to them
- Fertilization envelope: formed of components from the cortical granules binding to the vitelline envelope, starts forming at sperm entry site and continues around the egg (ca 1 min)
- Mucopolysaccharides: elevates the fertilization envelope from the cell membrane, by absorbing water, expanding the space between them.
- Egg specific peroxidase enzymes + transglutaminase: stabilizes the fertilization envelope by crosslinking adjacent proteins allows egg and early embryo to resist the shear forces of the ocean’s intertidal waves
- Hyalin: forms a coating around the egg, the egg then extends elongated microvilli to attach to this layer, providing support for the blastomeres during cleavage.
43
Calcium as the initiator of the cortical granule reaction
Upon fertilization free Ca2+ concentration in egg increases greatly (released from intracellular stores in sea urchins and mammals) cortical granule membranes fuse with egg cell membrane, releasing contents.
Once initiated, the release of calcium is self-propagating.
44
Activation of egg metabolism in sea urchins
Calcium release when sperm enters egg also (besides slow block) critical for activating the egg’s metabolism and initiating development: release inhibitors from maternally stored mRNAs allows translation, release inhibition of nuclear division allows cleavage.
45
IP3: the releaser of Ca2+
IP3= inositol 1,4,5-triphosphate – primary mechanism for releasing calcium from intracellular storage.
46
NAADP = nicotinic acid adenine dinucleotide phosphate: linear dinucleotide derived from NADP, serves as a sperm-borne calcium releaser.
- Frees stored calcium from membrane vesicles during muscle contraction, insulin secretion and neurotransmitter release.
- Upon contact with egg jelly: concentration in sperm increases tenfold, sufficient for stored calcium release
47
The flux of calcium across the egg activates a pre-programmed set of metabolic events.
- ”Early” responses: occur within seconds of cortical granule reaction.
- ”Late” responses: occur several minutes after fertilization begins.
48
"Early" responses
The same release of calcium responsible for the cortical granule reaction is also responsible for the re-entry of the egg into the cell cycle and the reactivation of egg protein synthesis.
49
"Late" responses
resumption of protein and DNA synthesis
50
How come mitochondrial is mainly maternal?
Mitochondria and flagellum disintegrate => few, if any, sperm-derived mitochondria in the organism. => mitochondrial DNA primarily maternal
51
Once inside the egg, the sperm nucleus undergoes a dramatic transformation as it decondenses to form the haploid male pronucleus.
1. Nuclear envelope vesiculates into small packets, exposing the compact sperm chromatin to the egg cytoplasm.
2. Proteins holding the sperm in condensed state (fx sperm-specific histones) are exchanged for proteins derived from the egg cytoplasm. => permitting decondensation
3. Once decondensed, the DNA adheres to the nuclear envelope, where DNA polymerase can initiate replication.
52
Male and female pronucleus meets how?
After sea urchin sperm enters egg cytoplasm, the male pronucleus separates from the tail and rotates 180 degrees so that the sperm centriole is between the sperm pronucleus and the egg pronucleus.
Sperm centriole then acts as a microtubule organizing centre, extending own microtubules + integrating them with eggs to form an aster.
The two pronuclei migrate along these towards each other => fusion, forming the diploid
53
DNA synthesis can begin ?
Either in the pronuclear stage or after the formation of the zygote nucleus, and depends on the level of calcium released earlier in fertilization.
54
Internal fertilization in mammals = difficult to study interactions between mammalian sperm and egg prior to these making contact pga:
- Mammalian fertilization occurs inside the oviducts of the female => hard to mimic these conditions unlike the sea water for sea urchin fertilization.
- The sperm population is probably heterogeneous, containing spermatozoa at different stages of maturation. Since less than 1/10.000 (humans) even get close to egg => difficult to assay molecules that enable the sperm to swim towards the egg and become activated.
- Multiple mechanisms by which mammalian sperm can undergo the acrosome reaction and bind to the zona pellucida.
55
The female reproductive tract is not a passive conduit! But actively regulate the transport and maturity of both gametes.
Both female and male gametes use a combination of small-scale biochemical interactions and large-scale physical propulsion to get to the ampulla.
56
Ampulla is?
Region of the oviduct where fertilization takes place.
57
Translocation of female gamete
Mammalian oocyte just released from ovary is surrounded by a matrix containing cumulus cells. Matrix necessary for ”pick-up” by the fimbriae of the oviduct and entrance to the oviduct.
Once picked up, a combination of ciliary beating and muscle contractions transport the oocyte-cumulus-complex to the appropriate position for its fertilization in the oviduct.
58
Cumulus cells is?
the cells of the ovarian follicle to which the developing oocyte was attached.
59
Sperm translocation from vagina to the oviduct – within 30 minutes (mice, hamsters, guinea pigs, cows, humans) – a time ”too short to have been attained by even the most Olympian sperm relying on their own flagellar power” (Storey 1995).
1. Uterine muscle contractions are critical in getting the sperm into the oviduct.
2. The region of the oviduct before the ampulla may slow down sperm and release them slowly.
3. Sperm (flagellar) motility is important once sperm arrive within the oviduct; sperm become hyperactive in the vicinity of the oocyte.
4. Sperm may receive directional cues from temperature gradients between regions of the oviduct, and from chemical cues derived from the oocyte or cumulus.
5. During this trek (vagina -> ampullary region) the sperm matures so it has the capacity to fertilize the egg when they meet.
60
Sperm incompetent to fertilize before it has resided sometime in the female reproductive tract => Capacitation
set of physiological changes by which mammalian sperm become competent to fertilize the egg.
Acquired as the sperm reach the ampulla, but lost if they stay around too long.
61
”The race isn’t always to the swiftest”
The fertilizing sperm can take as long as 6 days to make the journey to the oviduct.
”Speedy” sperm may not have undergone capacitation = unable to fertilize egg.
62
Capacitation details
1. Sperm cell membrane is altered by removal of cholesterol by female albumin proteins (cholesterol acceptor).
a. This cholesterol efflux is thought to change the location of ”lipid rafts” (isolated regions of cell membrane containing receptor proteins).
b. These lipid rafts now cluster over the anterior sperm head.
c. These lipid domains contain proteins that can bind the zona pellucida and participate in the acrosome reaction.
d. Also allows influx of bicarbonate ions (HCO3-)
2. Particular proteins / carbohydrates are lost, these possibly blocks the recognition sites for the sperm proteins that bind to the zona pellucida.
a. Or this unmasking might be an effect of the cholesterol depletion.
3. Membrane potential become more negative, as K+ leave the sperm
a. Allows opening of calcium channels and entrance of calcium.
b. Ca2+ + HCO3- may be critical in activating cAMP production and facilitating the membrane fusion events of the acrosome reaction
c. Influx of bicarbinate ions and possibly others alkalinizes the sperm, raising its pH (critical in subsequent calcium channel activation).
4. Protein phosphorylation occurs.
a. In particular, two chaperone (heat-shock) proteins migrate to the surface when phosphorylated.
b. One of these, Izumo, is critical in sperm-egg fusion.
5. The outer acrosomal membrane changes and comes into contact with the sperm cell membrane in a way that prepares it for fusion.
63
Capacitation may be a transient event, with a relatively brief window of competence to successfully fertilize the egg. How can this be prolonged?
By binding and capacitating sperm, the oviduct releases ”packets” of capacitated sperm at various intervals --> prolonging the time that successful fertilization can happen.
64
Before entering the ampulla of the oviduct, the uncapacitated sperm bind actively to the membranes of the oviduct cells in the narrow passage (isthmus) preceding it. Temporary binding and broken when capacitated.
Significantly lengthens lifespan and capacitation is slowed down =>
- May function as a block to polyspermy by preventing many sperm from reaching the egg at the same time.
- May maximize the probability that sperm will still be available to meet the egg in the ampulla.
65
In the vicinity of the oocyte: Hyperactivation, thermotaxis and chemotaxis
Hyperactivation - swims faster, enables sperm to digest path through extracellular matrix of cumulus cells
Thermotaxis - thermal gradient between isthmus of oviduct and varmer ampullary region, capacitated sperm can sense tiny temp differences
Chemotaxis - The oocyte and its accompanying cumulus cells secrete molecules that attract capacitated (and only capacitated) sperm toward the egg during the last stages of sperm migration.
66
Zona pellucida (mammals) = vitelline envelope (invertebrates)
Zona pellucida
- Far thicker and denser than vitelline envelope
- Binding of sperm to zona is relatively, but not absolutely, species-specific.
- Made of three major glycoproteins: ZP1, ZP2 and ZP3 (ZP=zona proteins) + accessory proteins that bind to the zona’s integral structure
67
Gamete fusion - sites of binding
In mammals, it is the side of the sperm head that makes contact with the egg (tip in sea urchin).
Sperm do not bind where the polar body has budded off (”bald” spot, without microvilli), microvilli (of polymerized actin) required for binding.
68
Equitorial region is?
junction between inner acrosomal membrane and the sperm cell membrane, where fusion between sperm and egg begins.
69
Mechanism of mammalian gamete fusion still controversial. Thought to involve:
- Integrin-associated CD9 protein on egg: localized to egg microvilli membranes + involved in fusion of myocytes
- Ig-like protein Izumo: found in sperm
Several other candidates => may be several binding systems
Each may be necessary, but insufficient alone
70
Polyspermy blocks
In mammals, no electrical ”fast” block. Possibly pga not needed pga limited number of sperm that reach the ovulated egg.
Slow block – enzymes released by the cortical granules modify the zona pellucida sperm receptor proteins so they can no longer bind sperm.
71
Uncoiling sperm
- The DNA of the sperm pronucleus is bound by protamines, basic proteins that are tightly compacted through disulphide bonds.
- Glutathione (in egg cytoplasm) reduces these disulphide bonds => allowing sperm chromatin to uncoil.
72
Final haploid preparation of egg nucleus
- Mammalian sperm enter the oocyte while its nucleus is ”arrested” in 2. meiotic metaphase
- Calcium oscillations brought about by sperm entry
o inactivate MAP kinase => DNA synthesis allowed(like in sea urchins)
o activate another kinase => eventually results in a haploid female pronucleus
leads to proteolysis of cyclin => continued cell cycle
same for securin, the protein holding the metaphase chromosomes together
73
Meeting of mammalian pronuclei
- DNA synthesis occurs separately in male and female pronuclei
- The centrosome accompanying the male pronucleus produces its asters (largely from proteins stored in the oocyte)
- The microtubules join the two pronuclei and enable them to migrate toward one another
- Upon meeting (15 hrs after fertilization), the two nuclear envelopes break down
- The chromatin then condenses into chromosomes that orient themselves on a common mitotic spindle.
- => in mammals a true diploid nucleus is first seen at the 2-cell stage
74
Sperm mitochondria and centriole
Sperm mitochondria and their DNA are degraded in the egg cytoplasm (both by dilution and actively targeting them for destruction)
Sperm centriole not only survives, in most mammals, but serves as the organizing agent for making the new mitotic spindle.
75
Activation of the mammalian egg
* As in every other animal studied, a transient rise in cytoplasmic calcium is necessary for egg activation in mammals.
* Sperm induces a series of calcium waves that can last for hrs, terminating in egg activation (i.e. resumption of meiosis, cortical granule exocytosis, and release of the inhibition on maternal mRNAs) and the formation of the male and female pronuclei.
* Fertilization triggers intracellular calcium release through the production of IP3 by PLC.
* Unused calcium is pumped back into ER and additional acquired from outside cell => outside recruitment necessary for the egg to complete meiosis.
* Extent (amplitude, duration and number) of calcium oscillations (waves) appears to regulate the timing of mammalian egg activation.
* Calcium influx blocked => no formation of second polar body => two nonviable (triploid) egg pronuclei.
76
Cortical granule exocytosis occurs just before
the resumption of meiosis
77
Intra-cytoplasmic sperm injection (ICSI)
Experimental treatment for curing infertility.
- Sperm are injected directly into oocyte cytoplasm, bypassing any interaction with the egg cell membrane
- fertilization => egg is activated and pronuclei forms
- Unsuccessful sperm – have little or no functional PLCzeta
78
Non-equivalence of mammalian pronuclei - Genomic imprinting
Genomic imprinting can occur in mammals such that the sperm-derived genome and egg-derived genome may be functionally different and play complementary roles during certain stages of development.
Normal development requires both male and female genome.
Genomic imprinting caused by different patterns of cytosine methylation.
79
Imprinted genes
there are approximately 100 mammalian genes expressed only from the sperm-derived or egg-derived chromosomes. A mutation in these genes cannot be compensated for by a wild-type allele on the other parents’ chromosome.
80
Parthogenesis
eggs capable of producing a normal embryo in absence of any spermatic contributions, seen in many invertebrates and some vertebrates ( not mammals).
81
Differentially methylated regions (DMRs)
found near genes that regulate transcription. These methylation patterns are erased during germ cell migration and established during gametogenesis.
- DMRs for maternal-specific methylation (i.e. paternal expression) are usually located at promoters of these genes
- DMRs for paternal-specific methylation (i.e. maternal expression) are usually located in intergenic regions
Developmental Biology
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Chapter 147
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Chapter 235
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Chapter 3 - modified for details44
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Part II12
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Chapter 550
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Chapter 747
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Chapter 481
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Chapter 6 - The genetics of axis specification in drosophila61
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Chapter 9 – Early development in vertebrates (birds and mammals)27
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Chapter 10 - Emergence of the ectoderm68
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Chapter 9b54
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Part 326
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