BIOS 30342 Developmental Biology Exam 1 Study Notes 2024 PDF

BIOS 30342 DEVELOPMENTAL BIOLOGY EXAM 1 STUDY NOTES 2024 UNIT 1 CONTENT INDEX OVERVIEW INTRODUCTION TO DEVELOPMENTAL BIOLOGY Making the zygote à embryo à birth of organism à adult sexual maturity Cell diversification, o...

BIOS 30342 DEVELOPMENTAL BIOLOGY EXAM 1 STUDY NOTES 2024 UNIT 1 CONTENT INDEX OVERVIEW INTRODUCTION TO DEVELOPMENTAL BIOLOGY Making the zygote à embryo à birth of organism à adult sexual maturity Cell diversification, organization, behavior, recipe, flavoring the recipe = evolution Reasons to study development o fascinating scientific crossroads o medical applications Historical perspectives Use of model organisms to study development Overview of the animal life cycle LECTURE 1 MECHANISMS OF DEVELOPMENTAL PATTERNING Pattern formation Genetic control of development à genomic equivalence & differential gene expression Development is progressive o use of fate maps to track destiny o stages of commitment à specification à determination à differentiation Modes of development o mosaic = determinants o regulative = interactions Communication during development o cell-cell signaling o induction = inducer interacts with responder that is competent to respond LECTURES 2,3 MAJOR APPROACHES TO STUDYING DEVELOPMENT Rules of evidence o correlative o functional = loss and gain Staging series Correlative evidence o tracking cell fate = stains and genetic labels o assessing gene expression = RNA, protein & transgenic reporters Experimental embryology à physical manipulations to test loss and gain of function Genetic manipulations o forward genetics à screens for dominant, recessive, and maternal effect genes o reverse genetics à gene targeting and knockdown strategies LECTURES 4,5 GAMETOGENESIS & FERTILIZATION Primordial germ cells Gametogenesis strategies o spermatogenesis o oogenesis Fertilization & zygote activation in the sea urchin o acrosomal reaction o fast and slow block to polyspermy o jumpstart of metabolism Fertilization & zygote activation in mammals o involves similar acrosomal reaction, blocks to polyspermy & metabolism start o sperm capacitation INTRODUCTION TO DEVELOPMENTAL BIOLOGY WHAT IS DEVELOMENTAL BIOLOGY? Making the zygote à embryo à birth of organism à adult sexual maturity o gametes are produced and are brought together to make the ZYGOTE = fertilized egg o 1-cell ZYGOTE à becomes a complex EMBRYO with dozens to hundreds of cells fashioned into tissues, organs and organ systems o development does not end with BIRTH/HATCHING event à continues through SEXUAL MATURITY when gametes can be produced to create a 1-cell ZYGOTE Questions developmental biologists seek to answer: o How do cells arising from 1 cell become different from each other?...........CELL DIVERSIFICATION o How are cells assembled into complex structures like a limb?................... ORGANIZATION o What activities drive individual cells to make highly organized patterns?... BEHAVIOR o How are the instructions embedded within the zygote?.............................. RECIPE o How have changes in developmental programs evolved animal form?...... FLAVORING THE RECIPE Development is a fascinating scientific crossroads = intersect of life and physical sciences ! Medical applications of developmental biology o understand congenital birth defects (aka congenital anomaly or malformation) = alterations to normal events which arise from genetic and/or environmental effects (e.g. infection, maternal health) o provides insights to fundamental mechanisms of genetics and physiology o origin of juvenile & adult conditions = developmental gene defects à initiate acquired, chronic diseases o therapy = regenerative medicine & stem cell technologies to treat diverse medical conditions HISTORICAL PERSPECTIVES Humans have been fascinated with the steps of animal and plant development for thousands of years, first known record dating back to Aristotle in 350 BC in his book detailing variations in how animals are born: oviparity = from external eggs; viviparity = live birth (placental); ovoviviparity = hatched from internal eggs Conceptual debate about development before the molecular age: o EPIGENESIS = concept of generating each animal from scratch versus o PREFORMATION = concept that all animals were already generated and were within an original egg or sperm = envisioned as the homunculus, or little person fashioned inside = Russian nesting doll analogy Critical advances in thought from 1800’s onward: o cell theory = notion that cells are the building block of the body o discovery of germ layers = separate embryonic zones of ectoderm, mesoderm, endoderm o nature of inheritance = nuclear = DNA = passage of hereditary material through gametes o genetic basis of development = precise DNA sequences direct cell identity & behavior USE OF MODEL ORGANISMS TO STUDY DEVELOPMENT Invertebrate and vertebrate species provide clues about the basis of human development PHYLOTYPIC STAGE = period of time when vertebrate embryos share a high resemblance o basis is the shared genetic program of development o common features: § distinct embryonic axis = discernible head-tail character § neural tube = prefigures the central nervous system & runs length of embryo axis § notochord = feature of chordate species § somites = transitory blocks of mesoderm that prefigure musculoskeletal system OVERVIEW OF THE ANIMAL LIFE CYCLE GAMETOGENESIS o produce gametes by meiosis = reductive division of inherited material = chromosomes of DNA o gametes are highly specialized = produced by differentiation § their unique features enable proper gamete joining & species recognition § features also support normal zygote development FERTILIZATION o steps involved with getting the GAMETES fuse à ZYGOTE ! CLEAVAGE o early rounds of zygote division o rapidly partition the ZYGOTE à BLASTULA = ball of cells o turn 1 cell in several hundred that are used to start making layers of the body o creation of a BLASTOCOEL à hollow cavity filled with watery fluid used during gastrulation o REGIONAL SPECIFICATION à spatial organization will be performed to create body plan GASTRULATION o reorganize BLASTULA à GASTRULA § move cells around to actually construct the body plan and then progressively refine it § body plan = emergence of anterior-posterior and dorsal-ventral axes, e.g. head-trunk-tail o partitioning of fates into 4 distinct lineages: § 3 GERM LAYERS = ECTODERM, MESODERM, ENDOGERM § reproductive GERM CELL lineage = PRIMORDIAL GERM CELLS CELL LINEAGE DEFINITIONS § ECTODERM = outer layer Þ Examples: make skin, nervous system, neural crest (contributes to all 3 germ layers) Þ Note: the nervous system & neural crest become internalized (we will study this in unit 2) § MESODERM = middle layer Þ Examples: make muscle, bone, blood, kidney, heart, limbs § ENDODERM = innermost layer Þ Examples: make primitive gut tube, gut accessory organs, derivatives like the lungs § GERM CELL LINEAGE = segregated to a special location (typically posterior) until gonads form Þ can make either gamete depending on gonad destination (see Lecture 4 notes, this unit) ORGANOGENESIS o body plan of layers à now make subsets of cells into organs! “germ layersà organs” via MORPHOGENESIS and GROWTH = involving cell proliferation and regulated cell death/pruning Examples over developmental time from early to later stages: (we will see these in units 2-4 !) § NEURULATION = make central nervous system and disperse neural crest throughout the body to make peripheral nervous system and contributions to all the germ layers § establish LEFT-RIGHT axis § CARDIOVASCULAR = make the heart, blood, blood vessels to oxygenate growing fetus § emergence of UROGENITAL, GASTROINTESTINAL, & RESPIRATORY systems HATCHING à BIRTH EVENT o emergence from embryonic membrane(s), egg, or uterus PROGRESSION TO SEXUAL MATURITY o may involve METAMORPHOSIS o emergence of secondary sex characteristics and the sequence of complex hormonal changes will initiate gametogenesis and release of gametes during mating ritual or coitus LECTURE 1. MECHANISMS OF DEVELOPMENTAL PATTERNING PATTERN FORMATION PATTERNING = mechanisms that create the embryonic form o involves a stepwise series of choices that direct cells from the ZYGOTE TOTIPOTENT STATE to the final destination = DIFFERENTIATED STATE = possess unique structure & physiological function Examples § establishing the body plan à defining the axes (anterior-posterior, dorsal-ventral, left-right) § allocation of cells to the germ layers and germ cell lineage o differentiation is a gradual process that transpires typically over many cell divisions FUNDAMENTAL CONCEPTS o STEM CELL = precursor = unspecialized = immature = traits of the zygote for example versus o DIFFERENTIATED CELL = mature = “terminally differentiated” = choice to become is finalized § exhibit unique traits = size, shape, color, motile, stationary, mesenchymal, epithelial, resting membrane potential, organelles, molecular components like RNA and protein § animals are comprised of several hundred (e.g. range of 100-250, or more) § differentiated state is stable § terminal = end of development path = last stop = only options left are to function or die o POTENCY = all the fates possible for a cell § development is a process of potency restriction = limiting what a cell can become § totipotent à pluripotent à multipotent à sometimes as specific as unipotent GENETIC CONTROL OF DEVELOPMENT GENOMIC EQUIVALENCE = most cells contain the same (complete) set of genetic materials DIFFERENTIAL GENE EXPRESSION = different cells express different sets of genes o HOUSEKEEPING = for fundamental cell processes = e.g. proteins that make nuclear envelope o SIGNATURE = enable cell to perform discrete, unique set of physiological functions o cells share expression of housekeeping genes and are different due to signature gene expression o gene expression is regulated at 5 major levels: § transcription, RNA processing, RNA transport, translation, protein modification § transcriptional regulation is extremely important o regulatory or control regions dictate gene expression and are bound by transcription factor proteins § promoters = near gene coding region § enhancers = distant from gene coding region DEVELOPMENT IS PROGRESSIVE FATE MAPS = track destiny of cells over time Examples o make a map of the blastula à what do each of the blastomere cells become? o make a map of the gastrula à what do each of the germ layers become? STAGES OF COMMITMENT o SPECIFICATION = are cells capable of becoming their fate in a neutral environment? § Example: test by culturing cells in saline/simple culture medium o DETERMINATION = are cells capable of becoming their fate in a non-neutral environment? § Example: test by culturing cells in another region of the embryo = expose to other proteins o DIFFERENTIATION = executing identity = exhibit final differential gene expression profile o POTENCY IS TYPICALLY GREATER THAN FATE = cells can do more than what they are normally triggered to do during embryogenesis § along the continuum of commitment, cells are groomed to adopt a unique differentiated identify § but cell types across the germ layers share many characteristics, and thus cells can be made into many related cell types depending on the context they find themselves in! MODES OF DEVELOPMENT o MOSAIC = cells operate independently to self-differentiate = direct themselves without inputs from other cells or the environment § molecular determinants = e.g. certain mRNAs, proteins à specify or determine cell identity o REGULATIVE = cross-talk between cells and/or their environment direct differentiation § interactions dictate changes in gene expression & cellular activities based on cell signaling o most embryos display both modes during different times of development COMMUNICATION DURING DEVELOPMENT o CELL-CELL SIGNALING § diffusible signal docks on receiving cell § cells make direct contact with one another to relay messages o INDUCTIVE INTERACTIONS = cell signaling events that direct commitment and differentiation § INDUCER = source of directions § RESPONDER = receiver of directions § COMPETENCE = ability to respond Examples Þ depend on the expression of a membrane receptor Þ depend on the expression of a particular transcription factor LECTURES 2 & 3. MAJOR APPROACHES TO STUDYING DEVELOPMENT RULES OF EVIDENCE CORRELATIVE = implicates the involvement of a factor in a process Example: gene transcript expression correlates with formation of a particular organ FUNCTIONAL o LOSS OF FUNCTION = When a factor is removed, what happens? Is the factor NECESSARY for a process? Example: interfere with normal expression of a gene à prevent creation of a normal gene product o GAIN OF FUNCTION = When a factor is added, what happens? Is the factor SUFFICIENT for a process? Example: addition of transcripts to a group of cells à addition of protein where it would not normally be STAGING SERIES DOCUMENTATION OF TIME & SEQUENCE OF DEVELOPMENTAL EVENTS FOR A GIVEN SPECIES o each model organism has an atlas based on different time intervals, e.g. hours, days o the atlas could be based on physical features of the embryo, e.g. number of somites at a given time, time organs appear, or pigmentation pattern COLLECTION OF CORRELATIVE EVICENCE TRACKING CELL FATE = mark the origin of a cell and its destiny at later time points o NATURAL FATE MAPPING = pigment(s) in an embryo o VITAL DYES = non-toxic chemical transferred to the embryo Examples: § Nile blue sulfate, India ink, neutral red, Bismark brown, Janus green § diI or diO = modern fluorescent molecules that associates with cell membrane o AUTORADIOGRAPHY = introduce radioactive substance to cells o GENETIC FATE MAPPING = cells with genetic differences that can be distinguished with antibodies, genotyping (PCR, sequencing) or morphological characteristics Example: § CHIMERAS = exchange cells from related species, such as quail and chick  ASSESSING GENE EXPRESSION o mRNA TRANSCRIPTS Examples § RT-PCR = reverse transcriptase PCR = assess presence of an mRNA transcript & quantify it § WISH = whole mount in situ hybridization = visualize mRNA transcripts “in place” = spatial 3D o PROTEINS Example § immunohistochemistry = antibody staining = see location of proteins in spatial 3D o TRANSGENIC REPORTERS Example § use a tissue specific promoter to drive expression of a visual marker or stainable gene product § need to have isolated DNA sequence for a control region that drives expression in particular cell group at particular developmental times § use control region to drive expression of a reporter gene whose product is easy to stain or see in real time, like green fluorescent protein EXPERIMENTAL EMBRYOLOGY PHYSICAL MANIPULATIONS TO TEST LOSS AND GAIN OF FUNCTION o SURGICAL PROCEDURES TO TRANSPLANT, ABLATE OR PUT BARRIERS BETWEEN TISSUES Examples § transplant of dorsal blastopore lip at gastrula stage in amphibian embryo à twinned axis § Gurdon’s in vitro experiments with animal caps and vegetal caps à to assess features of mesoderm induction like the proximity of the vegetal signal, whether it can cross a piece of filter paper, and the effect of different amounts of vegetal cells on a given amount of animal cap § Gurdon’s discovery of the “COMMUNITY EFFECT” = a critical mass of vegetal cells were necessary to induce mesoderm formation à indicates interactions amongst a group of cells § role of the apical ectodermal ridge (AER) in vertebrate limb development = removal of the thin layer of ectoderm leads to stunted limb outgrowth from the trunk à indicates signal(s) from the ectoderm are necessary for full appendage growth § modern microinjection methods à example of how researchers manually deliver molecules to the 1 cell stage embryo to interrogate gene function à may deliver copies of mRNA, protein, DNA or agents to manipulate gene expression like blocking mRNA expression or protein activity GENETIC MANIPULATIONS observations of “SPONTANEOUS” aka random/naturally occurring genetic mutants à lead to systematic efforts to create & study mutations that alter developmental events genetic manipulation = manipulate model organism at the level of inherited DNA Examples § introduce mutations randomly by altering nucleotides in the chromosomes § introduce a DNA sequence (construct) engineered in the lab that can change gene expression (e.g. encode a truncated protein due to introduction of a premature stop codon) o each model organism has different amenabilities to genetic manipulations o desirable traits would include: § breed rapidly = short generation time § large brood size = number of offspring § able to maintain large colony in lab environment = such as size of animal § knowledge of genome FORWARD GENETICS o basic strategy: generate animal colony harboring HERITABLE mutations à search for animals with phenotype of interest à identify the responsible genetic defect = lesion o MUTAGEN = agent used to change chromosome Examples § chemical = alkylating agents = introduce single base (point) mutations in chromosomes § electromagnetic radiation = x-ray, gamma ray = introduces large anomalies in chromosomes (deletions, translocations, inversions) § DNA insertion = retroviruses, transposable elements  o SCREEN FOR DOMINANT GENES à mutate F0 adult and breed to wild-type partner à each F1 individual carries 1 possible heritable mutation (based on the amount of mutagen they were exposed to) à examine F1 offspring to screen for mutations that alter a developmental process à dominant alleles will display a phenotype with 1 copy of the allele § rapid screen because only breeding 1 generation § dominant mutations à typically strong phenotypes à will embryo survive to pass on allele? o SCREEN FOR RECESSIVE GENES à mutate F0 adult and breed to wild-type partner à each F1 individual carries 1 possible heritable mutation (based on the amount of mutagen they were exposed to) à breed the F1 to a wild-type partner à raise the F2 offspring in order to create a family with multiple carriers à intercross the F2 adults and look at their F3 offspring to screen for mutations that alter a developmental process à if you mate two F2 carriers: offspring are 25% wild-type; 50% heterozygote; and 25% homozygous with a phenotype due to inheritance of 2 copies of the recessive allele § homozygous recessive state may be embryonic lethal = many genes are necessary for multiple developmental events = are PLEIOTROPIC = multiple functions § homozygous recessive state may not show a phenotype because of REDUNDANCY = genes and pathways that have overlapping, redundant functions § homozygous recessive state may not show a phenotype because of MATERNAL EFFECT = the F2 mom is a heterozygous carrier of the recessive allele Examples § F3 screen for body plan alterations in the fruit fly, Drosophila melanogaster = invertebrate = insect revealed genes needed to make a proper pattern of body segments § F3 screens for developmental alterations in the zebrafish, Danio rerio = vertebrate = teleost fish revealed genes needed for gastrulation, body plan, organogenesis o SCREEN FOR MATERNAL EFFECT GENES à maternal production of oocytes involves addition of many materials to the differentiating gamete à to identify these genetically encoded agents, need to look at offspring of a mother who harbors two mutant alleles à in a recessive screen strategy (above) the F3 homozygous mutant offspring are raised à F3 adults are mated and the offspring examined. § If the F3 homozygous female harbors a maternal effect gene and is mated to a wild-type male, all the embryos will show a phenotype due to the lack of maternal contribution to the oocyte. § Researchers also examine the combined result of homozygous defects in the zygotic AND maternal genomes by mating the F3 homozygous female to an F3 heterozygous male. In this cross, half of the embryos are heterozygous for the recessive allele and have no maternal contribution; other half are homozygous for the recessive allele and have no maternal contribution. REVERSE GENETICS o GENE TARGETING = modify the DNA = creation of designer or specific mutations § KNOCK OUT = remove code and obliterate gene function § KNOCK IN = switch sequence to introduce alternative gene code o performed by Þ HOMOLOGOUS RECOMBINATION = exchange or swap a region of DNA to modify chromosomes in embryonic stem (ES) cells which are obtained from the inner cell mass of the mouse blastocyst = ES cells are pluripotent and retain the ability to make all cell types of the embryo Þ introduce the modified ES cells into a wild-type blastocyst à CHIMERA will possess a mixture of wild-type and modified cells and thus a mixed genotype Þ if the modified cells contribute to the germ lineage of the CHIMERA, then offspring of the chimera will be heterozygous for the modified, mutant gene and can be bred to obtain homozygous mutants Þ when homozygous state is embryonic lethal, researchers turn to making CONDITIONAL or tissue specific knockouts in order to disrupt gene in a specific location at a specific time Þ we will revisit conditional alleles later in the course § gene editing = use of CRISPR/Cas9 system to target a specific sequence *details not discussed o GENE KNOCKDOWN = use of a transient method = various approaches available in different models Example § morpholino in zebrafish = *details not discussed LECTURES 4 & 5. GAMETOGENESIS & FERTILIZATION PRIMORDIAL GERM CELLS (PGCs) = GERM STEM CELLS BIOPOTENTIAL PRECURSORS that are set aside early in development o if come to reside in testis à produce sperm o if come to reside in ovary à produce eggs o exhibit self-renewal and divide by MITOSIS o when they differentiate à offspring switch to MEIOSIS = REDUCTIVE DIVISION = halve PLOIDY number PGCs undergo migration o in mammals, reside in the posterior of the early embryo (epiblast) o migrate into the ENDODERM = region of the early gastrointestinal tract precursors o travel to reach the GENITAL RIDGE = bilateral gonad rudiment o expanding tissue mass located medial to the primitive kidney structures o genitourinary systems form in close proximity and interchange or repurpose some parts over time postulated cascade of gonad formation involves transcription factors followed by hormones o hormones will dictate genesis of the gonad & accessory plumbing o XX genotype à express estrogen § promotes Müllerian duct survival à become oviduct § primitive renal tube, Wolffian duct, will degenerate § drives formation of the uterus, cervix, vagina from surrounding mesoderm o XY genotype à 2 hormones § AMH = anti-Müllerian hormone à induces degeneration of the Müllerian duct § testosterone à blocks Wolffian duct degeneration à become the epididymis & vas deferens § also directs scrotum and penis formation GAMETOGENESIS STRATEGIES (focus on mammalian gonads & structures) prepare the nuclear material and make particular cytoplasmic components and organelles tenets of successful fertilization = ensure fidelity = PREVENT MISTAKES o proper ploidy number = chromosomes copied & segregated during meiosis § pair duplicated homologous chromosomes in first division, when recombination is possible § separating sister chromatids in second division o identify correct partner gamete = compatible = usually the same species SPERMATOGENESIS = differentiate to make small shuttle that carries the DNA o within testis, sperm housed in SEMINIFEROUS TUBULES = specialized tunnels o supported by SERTOLI CELLS = resemble a jigsaw puzzle piece in shape § wraps PGCs and their offspring and makes protective blood-testes barrier § phagocytoses the RESIDUAL BODY = cytoplasm jettisoned from the mature sperm cell o steps in sperm production: PGC = spermatogonia that divide by MITOSIS à when they initiate MEIOSIS the offspring are termed primary spermatocytes (4n) that divide to make à secondary spermatocytes (2n) that divide to make à spermatid (1n) à finish differentiation into the sperm § divisions of the spermatogonia and spermatocytes connect cells = CYTOPLASMIC BRIDGES § PGCs at basement membrane (basal lamina) of the seminiferous tubule and offspring are distributed in layers approaching the lumen § sperm production is regulated through influence of hormones o features of the mature sperm = organized into 3 regions, the head-midpiece-tail § CONDENSED NUCLEUS mostly packed with PROTAMINES à some packing with histones and those regions are more accessible to cell machinery § ACROSOMAL VESICLE = Golgi derivative = contains digestive enzymes to make path to egg § centriole which organizes the FLAGELLUM = tail § MITOCHONDRIA in midpiece provide ATP for dynein ATPase motor protein in the flagellum  OOGENESIS = differentiate to make a cell capable of supporting zygotic life & embryo form o eggs are HUGE cells § house all the materials necessary for beginning development and early growth § animals that develop ex utero will contain more materials than those that develop in utero § receive help in differentiation from many other cells, such as local neighbors and distant organs like the liver o features of the mature egg § NUCLEUS Þ nucleus is not condensed = may be at any stage of MEIOSIS at the time of fertilization Þ will be in NUCLEAR ARREST = suspended animation at time of fertilization Þ species vary with regard to whether PGCs = OOGONIA divide mitotically throughout life Þ in humans, millions of oogonia are made and degenerate during gestation, then continue to decline in number as we age Þ meiosis will conserve cytoplasm = put nuclear material into small POLAR BODIES § CYTOPLASM Þ generate a massive, complex cytoplasm with materials that support Þ cell metabolism (e.g. mitochondria, yolk proteins) Þ DNA production (e.g. DNA polymerases and nucleotides) Þ gene expression (RNA polymerases, ribosomes, transfer RNAs, amino acids) Þ some have innate defense mechanisms Þ chemicals that block ultraviolet light or distasteful (disincentivize predation) § MITOCHONDRIA = truckloads !!! most abundant of any animal cell known to date § METABOLIC ARREST = activities like protein synthesis are dormant once the gamete reaches the stage when it would interact with the sperm = again, this stage varies across animal species; in mammals it is metaphase II of meiosis II FERTILIZATION & ZYGOTE ACTIVATION IN THE SEA URCHIN premier model for fertilization studies o large eggs that can be seen with a light microscope (compound) o easily accessible gametes that are released into the water o large numbers of gametes that have been used to purify proteins and determine identity of factors that are necessary & sufficient for fertilization EGG structure = jelly coat, vitelline envelope, cell membrane, cortical granules, cytoplasm and nucleus o JELLY COAT contains = CHEMOTAXIC PEPTIDES = example of resact, a small peptide that attracts sperm and physically binds a membrane protein on sperm that triggers opening of Ca++ channels to induce faster swimming = CHEMOKINESIS o vitelline envelope & cell membrane contain species-specific proteins used for molecular recognition o CORTICAL GRANULES = prevent polyspermy and change chemical composition of membranes at time of fertilization FERTILIZATION occurs through the ACROSOMAL REACTION o sperm contacts jelly and interacts with complex sugars and proteins in this coat o species match triggers Ca++ influx on sperm à dumps the acrosomal vesicle contents à digest jelly o sperm extends the ACROSOMAL PROCESS by polymerizing actin to reach out and touch the egg cell membrane o specific proteins on sperm cell membrane interact with receptors on the vitelline envelope and egg cell membrane à competent sites for interaction are not ubiquitous on the egg o sperm will enter the cell with help from the egg: egg makes fertilization cone and grabs sperm with microvilli PREVENTION OF POLYSPERMY o FAST BLOCK § sperm binding to egg cell membrane triggers rapid opening of Na+ gated channels § surge of Na+ influx raises resting membrane potential of egg cell from -70 mV to +20 mV § positive resting membrane potential deters sperm binding !  o SLOW BLOCK § sperm binding to egg cell membrane ALSO triggers internal signal transduction events § tyrosine kinase is activated and activates phospholipase C = a membrane associated enzyme that splits PIP2 into 2 messengers: IP3 and DAG § IP3 travels to the endoplasmic reticulum and opens Ca++ ligand gated channels § Ca++ surge in cytoplasm triggers fusion of CORTICAL GRANULES with egg cell membrane § release of proteases that cut connections between egg cell membrane and vitelline envelope § vitelline membrane lifts off the egg cell and space filled with water à at this time, the vitelline envelope is now named the FERTILIZATION ENVELOPE § fertilization envelope puts critical barrier around egg and is chemically changed (e.g. peroxidases released by cortical granules) and egg is chemically changed as well…compatibility with sperm is eliminated EGG ACTIVATION à JUMPSTART METABOLISM o Ca++ surge in cytoplasm from the slow block has a second agent, DAG, which targets activation of: Na+/H+ exchange pump on the egg cell membrane § activation of the pump removes H+ ions, thus raising pH to alkaline § Ca++ and pH change alter many molecules à triggers onset of DNA replication, protein synthesis § proteins are made from maternally deposited transcripts § all initial proteins come from the maternal genome ! § activation of zygotic gene transcription occurs at a later timepoint (varies in species) o the pronuclei will be brought together using microtubule tracts § assisted by sperm: contributes its CENTRIOLE to form the first mitotic spindle in the zygote FERTILIZATION & ZYGOTE ACTIVATION IN MAMMALS EGG maturation release = OVULATION o triggered by ovarian cycle which operates due to a hormonal cascade o hypothalamus produces gonadotropin releasing hormone, which stimulates the anterior pituitary to release follicle stimulating hormone (FSH) and luteinizing hormone (LH) o FSH and LH stimulate the maturation of the primary oocyte within its follicle o LH surge triggers actual ovulation EGG structure = cumulus made of cells from follicle that protects the egg à zona pellucida = complex layer of glycoproteins à cell membrane à cortical granules à cytoplasm and nucleus FERTILIZATION o sperm burrows through cumulus through swimming action o sperm interacts with specific components of the zona pellucida for species match o upon matching, the acrosomal reaction is triggered to digest path to egg cell membrane o side of the sperm head makes actual contact with the egg cell membrane o entire sperm brought inside with microvilli helping à most mitochondria degraded o as in the sea urchin, a phospholipase C is at work: however, a soluble form enters the egg from the sperm cell and splits egg PIP2 into 2 messengers: IP3 and DAG o IP3 travels to the endoplasmic reticulum and opens Ca++ ligand gated channels o Ca++ surge in cytoplasm triggers fusion of CORTICAL GRANULES o Ca++ surge & raise in pH cause similar activation of egg metabolism as in sea urchin, initiating cell cycle, DNA replication and protein synthesis SPERM CAPACITATION newly ejaculated sperm are not competent to fertilize an egg o cells in the oviduct prompt sperm maturation = CAPACITATION o sperm interacting with the oviduct are exposed to albumin and bicarbonate ions o bicarbonate ions hyperactivate the flagellum à increased swimming by entry of Ca++ ions attraction to egg o THERMOTAXIS = heat gradient in female reproductive tract gives ‘direction’ to the swimming sperm! o CHEMOTAXIS = progesterone is secreted by the cumulus cells around the egg