Developmental Bio

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Optic Vesicle experiment: Eye comes from optic vesicle and lens placode...lens placode is a raised part of the ectoderm that sinks inside to form the lens of the eye. Optic vesicle forms a cup off the side of the brain, forms retina of the eye. These two things form with just precision that it was thought the optic vesicle must send signal to lens placode to form in the exact right spot. So Hans Spemann removed one optic vesicle from frog tadpoles brain and left other as control. He used a glass needle to cut ectodermal layer over the optic vesicle and folded it back with a hair loop. The optic vesicle was cut and removed. No lens formed on that side.
Embryology: -descriptive study of the series of events that occur when various types of embryos form..more "what" questions than "how" questions
-classical methods such as dissection, light and electroc microscopy
-dev biologists have relied heavily on work of embryologists
developmental biology: -study of how organisms form, including postembyonic processes
-more newer methods such as molecular genetics techniques, mutagenesis, biochem, laser ablation
-reductionist
epigenesis: the generation of structures from preexisting ones
aristotle
preformation: -up to 17th century
-ovist: little person inside egg
-spermist: little person inside sperm
-develop only by increasing size
-evidence against this when wolff showed that blood vessels, gut and kidneys in chicken embryo looked diff than adult chicken (change in size AND features)
ontogeny: embryonic development
phylogeny: evolutionary development of organisms in time or the set of ancestory to descendent relationsihps of all things that ever lived
gill arches: -pharyngeal puches
-mammalian embryos have these but they develop into middle ear,tonsils, thymus and thyroid...fish develop into gill slits
Ontogeny recapitulate phylogeny?: No, humans do not go through "fish" or "tadpole" stages. Embyros may have certain structures in common btu mutations allowed them to develop into different things. This relationship is not simple, especially because early embryos can look quiet different between species
-Kaltohoff, natures seems to prefer the "modification of what is has over the construction of novelties"
basic but important events for embryo formation: -humans are expection
-egg is not a homogenous bag of macromolecules
-certain molecules, such as rna and proteins called cytoplasmic determinants, are distrbuted asymmetrically so that after cleavage different cells contain different determinates which lead to different genes being expressed in the nuclei, these cells then interact to form organs and tissues
fertilization: union of egg and sperm to give rise to a 2N zygote
cleavage: -series of divisions that gives rise to smaller cells called blastomeres
-by the end of cleavate,a hollow ball of blastomeres surring a fluid filled cavity (blastocoel) is formed and is called a blastula
gastrulation: -stage during which cells (either as indiv or sheets) begin to move with respect to one another
-embryo called gastrula at this point
-end result is formation of three germ layers
ectoderm: outermost germ layer, will give rise to epidermis and nervous system
endoderm: inner most germ layerk, will give rise to the inner lining of the digestive tract and its appendages (live, pancreas)
mesoderm: middle germ layer, will give rise to bone, muscle, heart, blood and blood vessels, kidneys and reproductive organs
organogenesis: further cellular movements and interactions between layers lead to formation of rudimentary organs, basic body plan is established by the end of this phase
-neurulation
-migration of ectodermal cells gives rise to a raised neural plate
-margins of this plate fuse to form the neural tube (eventually form brain and spinal cord)
-archenteron (rudimentary gut) formed by endodermis
-mesoderm gives rise to regions that will become the backbone, dorsal musculature, kidneys, heart, etc.
neurulation: part of organogenesis, formation of the rudimentary nervous system
histogenesis: tissues begin to mature as the cells therein continue to differentiate and specialize
controlled interference: -changing one parameter (genetic, cellular, environmental) at a time and looking at the developmental outcome
-early days, these were crude but elegant and informative experiments
-ablation, ligation,isolation, removal, transplantation
ablation: -stabbing a cell with a hot needle or can use a laser
-ablation of one cell in 2-cell stage of frog development results in a half embryo on one side and ball of undifferentiated cells on the other
-useful for cell autonomy: what can one cell do in absence of its neighbors
ligation: -uses hair loop to completely separate two blastomeres from one another
-Hertwig discovered that separating amphibian embryos at the 2-cell stage leads to 2 viable embryos only if the ligation allows each half to get a portion of the gray crescent
-in terms of genetic material in each blastomere, it's identical in both
-in terms of cytoplasmic inheritance,it's important for normal dev., you can't just divide it any way you want
isolation: -portion of the embryo is excised and placed into tissue culture
-helps decide whether the isolate (cell or organ)is committed
committed: when a cell's fate becomes restricted, even if it does not appear phenotypically diff from neighbors
specification: -first stage of commitment
-cell or tissue is specified if when its isolated and cultured in a neutral environ (lacking inducing signals) it carries out its original fate
-reversible
determination: -second stage of commitment
-when fate is fixed
-when differentiates according to original fate even when placed in a diff part of embryo
-irreversible
what does it tell you if a cell or tissue cannot carry out its fate in isolation?: lacking growth signals, lacking proper info to continue growth
removal: -how will the remainder of a embryo develop without a particular part
-this tells you how that removed piece affects the rest of the embryo
coculture experiments: tells whether there are diffusible signals in the dev process
ectopic or heterotpic transplantation: embryonic part removed from the donor and implanted into a diff place in a comparable recipient
-ex.Spemann and Mangold's "organizer" experiments
heterochronic transplantation: -there is an age diff between the host and recipient
-addresses the issue of competence-question of exactly when a cell or tissue is capable of responding to an inductive signal
-competent when capable of responding to such a signal
-may occur for only a brief window of time
-molecular level, are proper receptors displayed? when disappear do they endocytose back in?
heterospecific transplantation: between species; this helps decide whether signaling is the same between species, what factors may be different, different pathways or same pathways; kind of like plug and play to see what happens
null mutations: complete loss of gene or function; knock out proteins ability to function or absense of functional gene product
loss-of-function mutation: reduction in function; still function bu at lower rate
gain-of-function allele: gene becomes active at a time when, or in ap lace where, it is normally silent; allele loses a regulatory region that normally binds to a repressor, and therefore, it is expressed at a time or place when it would normally not be expressed
-example: if the attennapedia gene, which in wildtype Drosophila embryos, is normally expressed in regions that are destined to give rise to legs, is mutated in such a way that it is expressed in the head, then the attennae are converted into legs
maternal effect genes: synthesized and deposited in the egg during oogenesis; guides development until the zygotic genes can take over; mutations in certain genes of the mother can have profound effects on development; offspring of bicoid-mutants lack a head and thorax and show partial duplication of the abdomen at the anterior end-2-butt embryos
epistasis: -when one gene masks the expression of another
-double mutant analysis can help determine the order of function of gene products in a given pathway
-single mutant strains are cross and give rise to double mutants
-asses the phenotype of the resulting offspring
-example: recessive gene apterous in Drosopholis produces wingless homozygotes. If these are crossed with an other mutants with abnormal wings (curled wings) the action of the apterous masks the curled wings.
-apterous is said to be epistatic to curled wing
Spemann and Mangold's "organizer" experiment: -a piece of tissue from teh dorsal lip of the blastopore of a newt gastrula is grafted to the opposite side of a grastrula from anotehr, pigmented, newt species.
-the grafted tissue induces a new body axis containing neural tube and somites.
-the unpigmented graft tissue forms a notochord at its new site but the neural tube and the other structures of the new site have been induced from teh pigmented host tissue
cell fate mapping: performed by adding lipophilic dyes (bind to plasma membranes) or microinjection of high molecular weight dyes (these all are restricted to the labeled cells and their progeny)
-mutation of DNA can also be used if it results in a color change
-nematode C.elegans has been entirely mapped
-mammals cant be so easily mapped because c.elegans are entirely invariable (mosaic development when cells are determined) and mammals not so precise (regulative development when cells arent determined)
gray crescent: -found in some frogs and amphibians
-product of the fact that fertilization causes rearrangement of the cytoplasm
-the grey crescent forms opposite of the site of fertilization, because after fertilization occurs, the cortical cytoplasm (pigmented) rotates 30 degrees relative to the internal cytoplasm
-this rotation exposes an area of more diffuse pigment beneath the cortical cytoplasm
-does mark future site of blastopore and therefore dorsal structures
-"inducers" of the gray crescent have been considered one of the holy grails of devel biology
-beta-catenin (a protein that can act both as transcription factor and to anchor actin to plasma membranes)is thought to be involved but it is unclear the exact role it is playing
-gastrulation will begin here
-pigmented region on the dorsal (back side) of the embryo
-both ligated cells must get a portion of this in order to form 2 viable embryos
gametogenesis: occurs in gonads and results in haploid gametes; occurs in a manner that is related to the distinct functions of the two types of gametes
-primordial germ cells to dnot form in gonads but must migrate to the developing gonads during embryogenesis
sperm highly specialized-features related to its mission?: -cocked and loaded weapon
-mobile-adapted to deliver males chroms to egg
-"stripped down cells" with strong flagellum, no ER, Golgi, ribosomes, etc.
-many mitos to power motility
-head and tail covered by single plasma membrane
-head contains condensed haploid nucleus and an acrosomal vescile so that will exocytose hydrolytic enzymes to penetrate egg coat, help sperm bind to egg
-sperm tail-anterior part (midpiece)full of mitos to generate ATP, flagellum with a central axoneme
egg before fert.: -largest cell made my a given species
-initial purpose is to sit and wait to be fertilized
-specializations related to fert. include elaborate protective envelopes
once fert. occurs the eggs contains a variety of resources to...: -nourish the new developing org (glycoproteins, carbs and lipids)
-initiate cleavage (tubulin, actin, histones)
-provide spatial info(tubulin, actin and mRNAs)
-seems to be the case (but not in animals) that the egg is literally pumped full of these molecules during meitoic arrest
the germ line concept: -special lineage of cells (the germ line) gives rise to gametes
-germline includes the zygote and those blastomeres that give rise to both gametes and somatic cells
-further along in germ line exist primordial germ cells (give rise to exclusively gametes)
-in most animals (except mammals)the determination of PGC's is brought about by cytoplasmic localization of specific determinants; some prevent mitosis or repress transcription of certain genes during migration; in other species, cell-cell interactions play more of a roll in fate specification
-conflicts with Lamarckism because acquired trains are not heritable-only germ line cells, not somatic cells, pass on genetic info
-a mutation is not necessarily always heritable change in DNA (can have a mutation in a cell that will never divide again)
-germ cells are associated with somatic cells in the formation of gonads-somatic portions of gonads are derived from the mesoderm
-the PGC must migrate to the developing gonad
PGC'S migrate in humans how? what molecules?: migrate around the hindgut and up the mesentery of the hindgut from an appendage of the embryonic gut known as the yolk sac
-focal adhesions-integrins
-integrins connect to laminins
oogonia and spermatogonia: PGCs first divide mitotically in the gonads to produce diploid oogonia and spermatogonia
primary oocytes and primary spermatocytes: the oogonia and spermatogonia undergo DNA replication...now ready to undergo meiosis
secondary oocytes and secondary spermatocytes: after meiosis I
spermatids and eggs: after completion of meiosis
spermiogenesis: -part of spermatogenesis
-maturation of spermatids into sperm
-formation of the acrosome from the golgi
-a flagellum grows (constructed from the MTs of the centrioles)
-mito. aggregrate around the base of the flagellum, forming a sheath
-the chroms. condense
-the spermatozoa become individual cells and are released into the tubule lumen
ovulation: at some point DURING meiosis the egg is released from the ovary....metaphase II in humans and amphibians
-"egg" is used loosely after this point since it's not really an egg til after fert...so its still secondary oocyte
in human females meiosis begins before birth: -3-8 mos gestation in
-meiosis not complete til after fert. so some eggs can be in arrest in meiosis I for 50 years
in human males, spermatocytes start entering meiosis at puberty: and continue until death
spermatogenesis: -occurs in seminiferous tubules of the testes
-developing sperm are connected by cytoplasmic bridges because products of the X chrom need to be shared
-sertoli cells span the seminiferous tubule wall and effectively compartmentalize (bc connected by tight junctions) the tubule into 2 compartments forming a blood-testes barrier (outer contains spermatogonia and inner contains spermatocytes and spermatids underoing spermiogensis)
-this means that the interior compartment milieu is controlled by the sertolli cells themselves, which secrete signals and nutrients
-before puberty the sertoli cells inhibit proliferation of spermatogonia and prevent their entry into meiosis
-after pub. they stimulate celldivision and provide nutrients
-during fetal dev., they secrete anti-Mullarian duct hormone, which prevents the formation of female repro. parts
oogenesis: meiotic divisons produce one large gamete and 3 polar bodies that mark the site of the animal pole
during meiotic arrest, eggs become endowed with numerous abundant RNAs, how?: can make its own and get it from other cells
-the nucleus becomes very enlarged andis called the germinal vesicle
-in many species, the chroms whcih are actively being transcribed, assume a bushy conformation consisting of many loops, eggs also devleop numerous nucleoli so that they can produce sufficient rRNAs
-in insects and inverts, the egg is pumped full of RNAs by nurse cells; these cells are essentially sister cells and stay connected to the oocyte by cytoplasmic bridges in a nurse cell-oocyte complex
-nurse cells are helper cells that are polyploid so they can synthesize large amounts of RNA
-during early and mid-oogenesis RNAs are transported into the oocyte by microtubules and through the aforementioned cytoplasmic bridges
-during mid-late oogenesis, nurse cells contract and pump cytoplasm into the oocyte; then a MT dependent mixing of the cytoplasm occurs; this will result in homogeneity of some cytoplasmic components but certain mRNAs anchored to the cytoskeleton will be asymmetrically distributed (example: bicoid mRNA from nurse cells remains anchored at anterior part of egg and is nec.for head dev)
drosophilia ovary: -each contain 16 ovarioles
-most developed egg chamber near base of the ovariole
-anterior pole of oocyte is that facing the germarium and nurse cells
-follicle cells surround the nurse cell-oocyte complex and mediate the uptake of yolk proteins and they also synthesize eggshell proteins
what is a yolk?: a mixture of proteins, lipids and glycogen used to nourish the developing embryo; minimal in mammals and most abundant in birds, reptiles and sharks
how does yolk accumulate?: -accumulate during meiotic arrest
-precursors of the yolk proteins (or vitellins) are called vitellogenins-made in the liver in vertebrates or in the fat body + follicle cells in insects
-released into the blood (verts) or hemalymph (inverts)
-taken up by the oocyte by receptor mediated endocytosis and modified into vitellins
-mircovilli (very elaborate extensions of plasma membranes) and/or follicle cell-created passage ways (temp. form channels between themselves) allow upteake
-yolk synth. is hormonally controlled
-once inside the egg, the vitellogenins are modified into their less soluble vitellin forms and then sequestered into crystalline-like arrays in membrane-bound yolk bodies
oocyte maturation: -adjustment of membrane permeability and receptivity, chromatin condensation, etc
-in most species, germinal vescile breakdown is an early event, prior to fert.
-oocyte proceeds through meiosis until it reaches metaphase II,when it is arrested again (caused by cytostatic factor
(mosplus other unidentified factors))
-arrest broken by fert.
germinal vesicle breakdown in oocytes: -prior to fert.
-hormonally controlled (stimulated by progesterone)
-progesterone binds to surface receptor, leads to decrease in cAMP levels
-leads to translation of certain maternal mRNAs including ones that encode the mos protein (a kinase)
-leads to phosphorylation of histones (leading to condensation) as well as nuclear lamins...leading to germinal vesicle breakdown
when oocyte arrest is broken by fert.: -leads to rapid increase in intracellular Ca2+
-leading to destruction of MPF and c-mos (perhaps via Ca2+ dependent kinases)
-egg is thus released from the second arrest and is "free" to begin cleavage
if you want to see if cAMP levels are imp. in oocyte maturation...: microinject cAMP and see if the cascade of events follows...it shouldnt.
protective envelopes: -vitelline envelope: layer of glycoprotein called the vitelline envelope in most animals (or zona pellucida in mammals); protective and contains sperm receptors
-additional egg coats (if present) provide additional protection and can provide nutrition for hatchlings
-hard shell of bird eggs, deposited after fert, is mad eout of calcium carbonate but contains collagen-filled pores-prevents dessication while allowing resp
-insect eggs have chorions; hard if laid in open air and can contain special chimney-like structures call aeropyles, for aeration, as well as an opening to allow fertilization (micropyle)
test idea that low levels of cAMP leads to mos production: 1. inject cAMP to see if prevents cascade
2. inject phosphodiesterase which inhibits cAMP to see if it prevents the cascade
3. inject phosphodiesterase in absence of progesterone to see if prevents cascade
test idea that breaking of arrest involves increase in calcium: 1.photolabile caged antibody against IP3 receptors; normally IP3 binds to receprot to release more calcium but antibody would blcok binding so calcium not released
2.caged Ca2+ injection causes release of meiotic arrest
3. caged IP3 causes release of meiotic arrest
5 basic steps of fertilization: 1. sperm approach
2. penetration of egg envelopes
3. plasma membrane contact and fusion
4. egg activation
5.fusion of genetic material
capacitation: -process by which sperm become able to penetrate the zona pellucida/vitelline envelope
-in mammals, occurs in the female genital tracts
-involves an increase in metabolic activity leading to more rapid flagellar movements
-plasma membrane undergoes changes in composition (lower choleserol levels) to facilitate fusion
-possible cytoskeletal changes and unmasking of egg binding sites as well
-overall, poorly understood, but thought that removal of cholesterol from the membrane increases it's permeability to Ca2+ and bicarbonate ions, leading to increased cAMP production by adenylyl cyclase and increased tyrosine phosphorylation of sperm proteins
what about IVF and capacitation?: capacitate sperm in presence of things like glucose and albumin; mimic conditions of female gen. tract well enough to capacitate sperm
chemoattraction: -attraction of sperm to egg via chemical signals
-chemotaxis:oriented movement in resonseto an external signal, occurs in the uterus and/or oviduct (also in aquatic or marine environs)
-may be released by the eggs themselves or by the egg-containing structure
some chemoattratants have been purified: 1. resact-from the jelly layer of the eggs of species of sea urchin-is a 14 amino acid peptide bound by receptors on the sperm plasma membrane. causes sperm to swim faster and from areas of lower concentration to areas of higher concent.; works by increasing sperm respoiration rate in a guanylyl cyclase dependant manner, so the secondary messenger, cyclic GMP is involved
what experiment first implicated receptors in the first place?: preincubated with resac; put it in presence of egg; no longer has ability to chemotax
chemotaxis in mammals: evidence in humans, sperm respond to a substance released in the follicular fluid during ovulation,chemical nature not yet known
-only a fraction of sperm are receptive to it maybe because selects those at peak of ability to fertilize
-they are looking for an orthogue of Xenopus protein known as (no joke) alluring that may be involved
fert. in detail in sea urchins: -sperm approaches by chemotaxis (to resact)
-reaches the egg jelly and must penetrate it
-acrosome reaction
acrosome reaction in fert of sea urchins: -one component of the jelly coast binds to a receptor on the sperm plasma membrane
-leads to a transient rise in Ca2+ in the sperm
-leads to the acrosomal reaction, which is carried out by the acrosome
-first step is exocytosis,the outer acrosomal membrane and the overlying portion of the plasma membrane vesiculate thereby releasing the acrosomal contents
-enzymes lyse a hole in the egg jelly
-acrosomal process (long extension) forms at the tip of the sperm via polymerization of the G-actin in the subacrosomal space-allows the sperm to reach the egg's vitelline envelope
-adherence in sea urchin requires the acrosomal protein bindin-is species specific,will only stick to egg of own species
-immunostaining indicated that bindin appears on the surface of sperm that had undergone the acrosomal reaction
-Glabe and Lennarz put bindin aggregates into a plastic well containing eggs, shook for 2-5 mins and foudn that each bindin bound to an agglutinated only eggs from its own species
-adherence of the sperm and egg membranes leads to egg activation
-it also leads to formation of fertilization cone (eggs form this structure which engulfs the sperm and formaton is dependent upon actin polymerization)
the acrosome of the sperm: -consists of an outer and inner acrosomal membrane which surround the acrosomal contents
-acrosomal contents include hydrolytic enzymes and the protein bindin
-subacrosomal space occurs below the inner acrosomal membrane and contains globular actin-capable of rapid self-assembly into F-actin
fert in detail in mammals: 1.first obstacle encounterd by sperm is the layer of granulose=follicle cells surrounding the oocyte (these cells, along with the egg, are released from the ovary at ovulation)
-sperm has an enzyme known as PH20 which can degrade hyaluronic acid, the principle ECM protein between granulosa cells
-the sperm also makes burrowing movements to reach the zona pellucida
-acrosomal reaction occurs, in most mammals, when the ZP is reached
-the ZP glycoprotein called ZP3 is thought to bind a receptor on the sperm head in a species specific manner and this leads to ...
-fusion and vesiculation of the outer acrosomal membrane with the plasma membrane=the acrosomal reaction
-bc the vesiculation only occurs part of the way down, a collar of membrane is left around the sperm head; the collar is known as the equatorial segment; also note that the anterior sperm membrane now consists of only the inner acrosomal membrane; the region of the sperm plasma membrane below the equatorial segement is called the postequitorial region
-mammalian sperm DO NOT form an acrosomal process (dont have massive polymerization of actin)
-sperm forms a hole in the ZP (due to the release of lytic enzymes) and wriggles its way between the ZP and egg PM (the space in between is called the perivitellin space)
-the region of the sperm PM that binds the egg PM is either the equatorial or postequatorial region of the head; even though these regions look structurally similar to how they looked before the acrosomal reaction, they ahve apparently been somehow modified during the acrosomal reaction such they become competent to bind (sperm that have not undergone the acrosomal reaction cannot bind)
-subsequent membrane fusion between sperm and egg was thought to involve the protein fertilin, which has metalloproteinase activity
-infact it was shown that fertilin binds to integrins on the surface of the egg; the problem is that integrin mutant eggs can be fertilized; the current thinking ont his mess is that only initial binding involves fertilin (prob without integrins); GPI linked proteins may be involved with membrane fusion but this is still unclear
-sperm nucleus enters the egg
-
follicle cells vs. oocytes producing ZP3: 1. immunocytochemistry
-antibody against ZP3
-antibody plus colloidal gold
2.insitu hybridization
-antisense probes (labeled RNA) to see if oocyte makes ZP3 of if transported there (saw labeling in oocytes)
3. in vivo labeling
-cultures oocytes separately from follicle cells
-fed them to 3H-fucose (incorprated into ZP3 bc its glycoprotein) and into 35S-Met
-homoegenize each one, run SDS-PAGE
-in oocyte they saw band of correct size
egg activation accomplishes two major goals: 1. block to polyspermy
2. release from the second meiotic arrest
fast block to polyspermy: -occurs in inverts and amphs and is temporary
-established as soon as first sperm has fused with the egg and is due to an alteration in the membrane potential of the egg
-as is the case for most animal cells, the egg membrane is electrically polarized and in sea urchin eggs the resting potential is approx -75mv; this is due to the relative concents of sodium and potassium in the cytoplasm and outside of the cell (sea water)
-as soon as first sperm has fused with the egg, plasma membrane channels open so that small amount of sodium enter the egg, altering the membrane potiential to 20mv for about 1 min before returning to the resting potential; in the case of frog eggs, the change in potential comes about due to efflux of Cl- ions. Membranes fusion cannot occur at such high positive membrane potentials
-this positive so called "fert potential" is necessary for the first sperm to fully enter into the egg
slow block of polyspermy: -follwing the return of its membrane to resting potential, the egg would be at risk of fusing with another sperm hence the need for additional block
-in inverts, this block is physical in nature as one of the consequences is lifting away of vitelline env and any sperm attached to it away from egg surface
-release of proteases cleave connections between VE and plasma membrane
-cortical granules also release GAGs,this results in hyaline layer between VE and plasma membrane
-peroxidases harden the VE by interconnection proteing therein; this is now called fert. evnelope
-sperm receptors modified so no other sperm can attach,a nd attached sperm fall off
-cortical granule exocytosis takes place in mammalian eggs in order to block polyspermy (called zona reaction)
-enzymes released during exocytosis remove carb groups from ZP3 so that the sperm can no longer bind tot he ZP and the acrosome reaction is not stimulated; in addition proteins in the ZP are cross linked such that it becomes impermabnle to sperm
what actually triggers the slow reaction and the resumption of the cell cycle?: -one thing is clear and constant between species,even plants, a wave of elevated calcium crosses the egg originating from the point at which the sperm and egg fused (in mammals the calcium levels continue to oscillate for hours)
-coritcal granule cytosis is caused by an increase in cytosolic calicum ion concentration-can articially induce this increase in using a calcium ionophore (poke holes in ER, form channels and calicum comes ou) that cuases releases from intracellular stores; also, in many species, one can microinject IP3 and exocytosis will occur (so calcium release is via the IP3 pathway in some spcies)
-some species, not mammals, the intracellular pH of the egg changes as well; DAG activates PKC(protein kinase C) which phosphorylates a Na+/H+ antiporter; this pumps sodium inot the cell and expels hydrogen leading to an incrase in cytosolic ph from 6.8 to 7.2; this ph change leads to increased protein and lipid synthesis
-3 models of what initiates these signaling events
the receptor model for initiation of signaling events in slow reaction: activation is initiated at the cell surface (by binding of sperm to a surface receptor, perhaps a G-protein-associated receptor, a receptor tyrosine kinase and or integrins?) and prior to fusion of egg and sperm
-really not a lot of evidence
-heterotrimeric G-protein involvement in acivation in some species, but G-protein function does not appear to be necessary for egg activation in mammalian systems
-also, purified putative surface ligands from sperm cannot bring about the whole, complete activation pathway
the fusion model for initiation of signaling events in slow reaction: the fusion of gametes is what leads to egg activation, perhaps via release of small tyrosine kinase of perhaps phopholipase C from the cytosol of the sperm
-evidence: calcium oscillations can be induced in several species by the mircoinjection of soluble sperm proteins
The nitric oxide model: -a stanford team showed that in sea urchin, nitric oxide released by the sperm induces calicum release as well as nitric oxide synthase activity in the egg which leads to even more calcium release
-the inital NO release from teh sperm appears to arise when NOS is activated in the sperm follwing contanct with the vitelline envelope
-press releases are still touting the finding as a possibly solution to human male infertility bc many infertile males have defective NOS enzymes in their sperm
-but other fert researchers remain skeptical-were unable to detect changes in NO levels during mammalian fert.,yet NOS inhibition cause reduced in vitro fert rate
-not clear that direct contact would be necessary to get thigns started in systems that use NO-would the closest spermatozoan win?
egg's release from Meiotic block: calcium release leads to activation of Calmodulin-dependent kinase II which leads to degradation of the cyclin portion of MPF as well as to the degradationof mos, then meiosis can be completed
formation of diploid nucleus in sea urchins: occurs by syngamy-that is, pronuclear fusion (faciliated by motor proteins on microtubules)
-mitosis begins, first cleavage
formation of diploid nucleus in mammals: membranes of pronuclei never fuse,but rather, they break down just before the first mitosis and the diploid nucleus is not formed until after the first mitosis
-each pronucleus migrates toward each other, replicated DNA asit travels; when meet the nuclear envs break down but instead of forming a common zygote nucleus, the chromatin condenses into chroms that orient themselves on a common mitotic spindle
-a true diploid nucleus in mammals is first seen not in the zygote but at the 2-cell stage
-mitosis begins,first cleavage
cleavage accomplishes...: -generation of a large number of cells
-generation of many copies of the genome/many nuclei
-segregation of cytoplasmic components into diff blastomeres
-increasing of the nucleocytoplasmic ration(nuclear volume/cytoplasmic volume increases over that which existed in the egg)-imp for the turnover of RNA and proteins which have short life spans,the smaller this ratio the harder to transcribe enough RNA to replace protein losses from a large cytoplasm
-second and third are imp so that cells can and will start to express diff genes
cleavage accomplishes..: -generation of large number of cells
-generation of many copies of genome; imp so that cells can and will begin to express diff genes
-segregation of the cytoplasmic components into diff blastomeres; imp so can express diff genes
-increasing of the nucleocytoplasmic ration (nuclear volume/cytoplasmic volume icrease over that which existed in teh egg)-imp for turnover of RNA and proteins, which habve short life spans; the smaller the ratio the more difficult it becomes to transcribe enough RNA to replace the protein losses froma large cytoplasm
Cleavage divisions are distinct from later cell division in two respects...: -blastomeres do not grow between cleavages
-divisoins occur at a rapid pace, although the pace is slowerin mammals because cell cycle takes longer and because they don't all divide at same time
Patternsof yolk distribution: isolethical-small amount of evenly distributed yolk; sea urchin, humans and other mammals

mesolethical-moderate amount of yolk, mostly in the vegetal hemisphere; amphibians

telolethical- large amounts filling the entire egg except for a small area near the vegetal pole; fish, reps and birds

centrolethical-yolk concentrated in the central cytoplasm
major classes of cleavage: holoblastic-egg is completely cleaved;in isolethical and mesolethical

meroblastic-egg is incompletely cleaved
Radial holoblastic cleavage in sea urchins: radial-blastomeres are arranged with radial symmetry around the a-v axis

-first 2 cleavages are meridonial (cleavage furrows run through the a and v poles)followed by an equitorial cleavage (the 3rd cleavage)

-fourth cleavage is in the animal hemi, occurs with a slightly oblique orientation so that two tiers of blastomeres (mesomeres) are slightly offset such that the spinldes of the v blastomeres are tilted toward the v pole
-the blastomeres of the v pole divide unequally to give four very small cells, micromeres, at the very bottom of the v pole and four very large, macromeres.

-fifth cleavage gives rise to 8 blastomeres in each of two layers, an1 and an2 in the animal hemi because of meridonial cleavage
-meridonial cleavege in the v hemi also leads to 8 macromeres
-finally the micromeres divide unequally to give rise to four larage and four small micromeres.

-sixth cleavage, macromeres, mesomeres and large micromeres also divide equatorially
-result is top tier (an1-derived), a second tier (an-2 derived), 2 layersof macromeres (veg 1 and veg 2) and a set of large and small mircormeres
-thats the blastula with 60 cells and a blasocoel.

-still surrounded by and adhere to the hyaline layer and are also adhered to one another on their apical surfaces
holoblastic radial cleavage in amphs: look on packet!
what roles do the desmosomes and tight junctions play in the blastula?: desmosomes-keep it together

-tight junctions-keep stuff out
gap junctions in blastula: all adjacent cells in blastula iterconnected by gap junctions to allow ions to flow, etc

-good for communication
holoblastic spiral: occurs on snails and worms with isolethical eggs

look in packet
RDA: radial, deuterostone, anus=blastopore
SPM: spiral, protostone, mouth=blastopore

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Deck Info

Number of cards 101