Reproduction - Biological process by which new individual organisms (or offspring’s) are produced by parents - Fundamental feature of all known life - Each individual organism exists as a result of reproduction Sexual 2 individuals/gametes One from opposite type of sex Increases variability Asexual 1 single parent Reproduction form for single celled organisms (e.g. archaea) Human Life Cycle Diploid Zygote (2n) – Mitosis →Embryo (2n) →multicellular organism (2n) Sex Organs (2n) – Meiosis – Gametes (n) → zygote (2n) Cell Division - All cells (except gametes) are somatic & divide via mitosis - Germ cells – sperm/eggs – undergo meiosis - Homologous chromosomes – chromosomes of same length, centromere & gene location Mitosis Somatic cells Chromosomal # NOT halved Cyclic process 1 division 2 identical diploid cells Meiosis Germ cells Chromosomal # IS halved One way process 2 divisions 4 non-identical haploid cells created Generation of Variation in Meiosis 1. Independent segregation of chromosomes 2. Crossing Over (homologous recombination) Gametogenesis - The process of differentiation of the primordial germ cells (PGCs) into gametes (egg and sperm) - the development of diploid cells (PGCs) into either haploid eggs or sperm - mitosis purpose is for the expansion of the primordial germ cell population - meiosis purpose is for development of diploid germ cells into either haploid eggs or sperms for sexual reproduction Primordial Germ Cells/ Precursor Germ Cells/ Gonocytes - early embryonic cells that acquire the developmental potential to develop into the gametes - still have to reach the gonads - divide repeatedly on their migratory route through the gut to the developing gonads Egg Cell Characteristics - large, non-motile store abundant raw materials and machinery (proteins, RNAs, nucleic acids, ribosomes, mitochondria used to start first cell division cycle) - yolk (energy supply) - egg coat (outer protective coat, also called shell, jelly coat, and zona pellucida in humans) - cortical granules - specialized Golgi structures that protect the egg against polyspermy - totipotent - developmental potential to develop into any cell type of the organism - unique haploid (n) genome Sperm Characteristics small and motile (due to flagella) - few organelles (mitochondria, centrioles) - acrosomal vesicle (specialized golgi structure used by sperm to bind to and penetrate the egg coat) - unique haploid genome Egg Functions - contributes one half of the unique diploid genome of the new organism - provides mechanisms for sperm recognition, binding, fusion and prevention of polyspermy provides the raw materials and machinery to carry out early development without any genomic input from the zygote. Sperm Functions - get to and fuse with egg - contribute one half of their unique diploid genome of the new organism activate the egg Polyspermy – More than one sperm fertilizes the egg Sperm Structure - head region which has a specific organelle called the acrosome (vesicle with enzymes that facilitate egg penetration) - neck region with a lot of mitochondria to provide ATP for tail movement/swimming - long flagella for movement - in humans, the head is 5 μm by 3 μm and the tail is 41 μm long (1 μm =1×10−6 of a meter) Gametogenesis Entails: - Genomic Events - Mitosis - Meiosis - Specialization Events Genomic Event: differential gene expression or cell signaling that happens early on in development that regulates cell determination and migration Example 1: Cell Fate Determination – P Granules in C. elegans - P Granules – Complexes of RNA & proteins – initially distributed throughout the egg then move to posterior end of zygote before first cleavage – later found in adult germ cells in gonads - fate of cells that become germ cells is fixed early in development Development of the Gonads Two structures initially present in undifferentiated gonads – Wolffian Duct & Mullerian Duct - Wolffian -> male internal genitalia (epididymis, vas deferens & seminal vesicles) Mullerian -> female internal genitalia (uterus, oviducts, cervix) Male germs cells commit to spermatogenesis earlier than female germ cells commit to oogenesis Oogenesis - Primary germ cells produce oogonia - Oogonia reproduce (mitosis) to produce primary oocytes - Primary oocytes begin meiosis then arrest at prophase 1 o present in females at birth o reside in a follicle (cavity lined with cells) o at puberty – FSH stimulates follicle to grow & mature o 1 follicle matures/month – oocyte completes meiosis 1 - Cytokinesis of meiosis I is uneven – results in one small cell (1st polar body) & another larger cell (secondary oocyte) - Secondary oocyte progresses to meiosis II but is arrested at metaphase II - Ovulation (breaking open of follicle to release secondary oocyte) occurs - Meiosis II resumes when sperm penetrates secondary oocyte - Cytokinesis of meiosis II is uneven results in one small cell (2nd polar body) & another larger cell (single mature egg (ovum) containing sperm head) Corpus Luteum - Forms from the ruptured follicle - Produces progesterone which thickens uterine lining for pregnancy Polar Body - Small haploid cells formed from uneven division during cytokinesis in oogenesis - Most cytoplasm is in one daughter cell – which becomes egg/ovum - Does not have ability to be fertilized - Usually die (apoptose) Male reproductive anatomy - Testes – descent into scrotum – have temperature 1.5-2.5 below body temperature (required for spermatogenesis) - Seminiferous Tubules – network of tubules in testes - Sertoli cells – form walls of seminiferous tubules – support germ cells - Leydig Cells – adjacent to seminiferous tubules – produce androgens (e.g testosterone) Spermatogenesis - Spermatogonium (stem cell) – 46 single stranded chromosomes - Primary spermatocyte – 46 double stranded chromosomes - Secondary spermatocyte -23 double stranded chromosomes - Spermatids – 23 single stranded chromosomes - Spermatozoa – have distinct heads, midpiece full of mitochondria to move & flagellum Hormonal control of testes Fertilization - Formation of a diploid zygote from ah haploid egg & sperm Key Features o Recognition at a distance o Contact recognition & binding o Egg & sperm fusion o Blocks to Polyspermy o Egg activation Sea Urchin (External Fertilization) Mammals (Internal Fertilization) Zona Pellucida = extracellular matrix of egg Recognition at a distance - Contact recognition & Fusion Binding Resact (soluble glycoprotein derived from jelly layer of the egg) is released into surrounding sea water Sperm recognize & bind to resact & swim in direction of higher concentration Chemotaxis – migration of cells towards soluble concentration gradient of stimulant Capacitation - process by which glycoprotein coat and seminal proteins are removed from the surface of the sperm's acrosome – done by substances secreted by uterus or fallopian tubes o Increases sperm metabolism and motility o Necessary for future sperm and egg binding o Triggered by bicarbonate ions (HCO3–) in the vagina o Requires 5–6 hours in humans Contact recognition: Facilitated by a carbohydrate molecule (fucose sulfate) in egg jelly layer - binds to receptor on sperm plasma membrane Acrosomal Reaction - acrosomal vesicle fuses with plasma membrane – release digestive enzyme – penetrate jelly coat - acrosomal process takes place due to polymerization of actin monomers to form actin filament Microvilli in egg plasma membrane help facilitate fusion - - Bindin – protein molecule on acrosomal membrane that binds to vitelline layer of egg Acrosomal Reaction - - - sperm comes into contact with the oocyte's zona pellucida(zp) - Acrosomal enzymes begin to dissolve zp actin filament comes in contact with zp –> calcium influx – corticle granules from oocyte fuse with outer membrane Zona Pellucida is composed of three proteins, ZP1, ZP2 and ZP3 Sperm plasma membrane receptors bind to ZP3 Blocks to polyspermy Egg activation Fast Block (membrane depolarization) - fusion of egg & sperm triggers depolarization of egg membrane which acts as a fast block to polyspermy - Na+ enters – egg becomes +ve charged – sperm fusion blocked - Depolarization last only 1 min Slow Block (Cortical Reaction) – intracellular calcium release triggers cortical granules to fuse with membrane - Enzymes from granules clip receptors, lifting vitelline layer - Molecules released: o Proteinases and Glycosidases – separate vitelline layer from plasma membrane o Mucopolysaccharides – osmotic gradient o Peroxidases –crosslinks macromolecules of the vitelline membrane o Hyalin protein- modifies the extracellular matrix & coats outer surface of egg Early Events- increase in cell metabolism Late Events – initiation of protein and DNA synthesis in preparation of first cleavage Timing (first cell division) Development 90 mins after fertilization Cortical Reaction - Released contents of cortical granules o remove carbohydrate from ZP3 – cant bind to the sperm plasma membrane partly o cleave ZP2, hardening the zona pellucida. Oocyte undergoes 2nd meiotic division – produces haploid ovum & releases polar body 12-36 hours after fertilization Cleavage - Cell division with no significant growth, producing a cluster of cells that is the same size as the original zygote first stage of early embryonic development after fertilization characterized by a series of mitotic cell divisions Cell divisions during cleavage are rapid: essentially skip G1 and G2 of cell cycle; little to no protein synthesis. Cleavage partitions the embryo into smaller cells = blastomeres Vegetal Pole = region where yolk (nutrients) is concentrated Animal Pole = opposite to vegetal pole Sperm Entry o Sperm enter at animal pole o Critical cue in setting up formation of dorsal & ventricular acis o Triggers rotation of outer cell cortex, exposing grey crescent (exposed non pigmented cytoplasm) - - Grey crescent – has cytoplasmic determinants (in egg) needed for normal development of blastomeres Marks future dorsal side o Sperm entry point establishes location of gastrulation Cleavage Furrow – indentation on surface of developing embryo o First 2 cleavage furrows are parallel to the line connecting the animal and vegetal pole (longitudinal) o Third cleavage (8 celled) is perpendicular to this axis (equatorial) The way in which cleavage occurs can vary & give rise to different body plan polarity o Protostomes (mollusks, arthropods)– blastopore develops into mouth – radial cleavage o Deuterostomes (echinoderms, chordates) – blastopore develops into anus – spiral cleavage Blastula - Formed after 5-7 cleavage divisions hollow ball of cells with a fluid filled cavity (blastocoel) Frog - Blastula has smaller blastomeres at animal side not until blastula has 4,00 cells is there any transcription – all activities controlled by gene products deposited by mother Holoblastic Meroblastic - cleavage furrow passes entirely through the - so much yolk that cleavage furrow does not egg pass through the yolk portion of the embryo - cleavage furrow passes all the way through the - Cell division occurs in a small area embryo - incomplete penetration of cleavage furrow - frogs, mammals, echinoderms - birds, fish, reptiles Gastrulation - cell movement result in a massive reorganization of the embryo from a simple spherical ball of cells, the blastula, into a multi-layered organism - blastocoel cavity allows for exchange of nutrients Ectoderm (outer layer) o Epidermis of skin o Nervous & sensory systems o Pituitary gland, adrenal medulla o Jaws & teeth o Germ cells Mesoderm (middle layer) o Skeletal & muscular system o Circulatory & lymphatic system o Excretory & reproductive system o Dermis of skin o Adrenal cortex Endoderm (Inner layer) o Epithelial lining of digestive tract & associated organs (liver, pancreas) o Epithelial lining of respiratory, excretory & reproductive tracts & ducts o Thymus, thyroid & parathyroid glands Sea Urchin - - - Frog - Chick - Human - - Mesodermal mesenchymal cells migrate from the vegetal pole toward the blastocoel o will eventually secrete calcium carbonate to form the internal skeleton Mesenchymal stem cells are multipotent cells that can differentiate into a variety of cell types, including osteoblasts (bone cells) and chondrocytes (cartilage cells) Vegetal plate folds on itself, forming a cavity Archenteron (future digestive tube) is formed by endodermal cells Some mesenchymal cells extend filopodia o Cellular extensions that facilitate cell attachment & migration o Filopodia contract & extend archenteron Initiated by blastopore formation o Blastopore = create on dorsal side of late blastula o dorsal lip = involuted region of blastopore sheets of cells rollover the dorsal lip & move inward – animal pole cells spread over outer surface of embryo blastopore extends via invagination until it encircles embryo – becomes opening into archenteron embryo consists of upper (epiblast) & lower (hypoblast) layer o all embryo cells come from epiblast o hypoblast cells form part of sac that surrounds yok primitive streak = thickening found at midline due to concentration of migrating cells produces 3 layered embryo with 4 extraembryonic membranes Four extraembryonic membranes: amnion, yolk sac, allantois, chorion o Amnion: membrane that makes the amniotic sac; protection/cushion o Allantois: sac-like structure, involved in nutrition and excretion, webbed with blood vessels, collects liquid waste from the embryo, and exchanges gases used by the embryo o Yolk sac: membrane outside the embryo, connected by a tube (the yolk stalk) through the umbilical opening to the embryo's midgut; helps with circulation Placenta composed of 3 layers – amnion (innermost), allantois (middle), chorion (outermost, in contact with endometrium ) Blastula VS Blastocyst - cleavage has produced 100 cells – embryo = blastula o spherical layer of cells (blastoderm) surrounding a fluid filled yolk filled cavity (blastocoel) - blastocyst implants 7 days after fertilization - in mammals – blastula forms blastocyst o cells in blastula arrange themselves in 2 layers – inner cell mass (develop into embryo) & outer layer (trophoblast), eventually form part of placenta - inner cell mass o epiblast (upper layer of cell) gives rise to 3 germ layers o hypoblast (lower layer) contributes to extra embryonic membranes (such as yolk sac) - Trophoblast forms chorion Extraembryonic membrane between fetus & mother Fetal part of placenta Gives rise to chorionic villi (transfer of nutrients) Gastrulation involves: - Cell motility - Cell shape - Cell adhesion Types of cell movement during gastrulation - Invagination – sheet of cells (epithelial sheet) bends inward - Ingression – individual cells leave epithelial sheet & become freely migrating mesenchyme cells - Involution – epithelial sheet rolls inward to form underlying layer - Epiboly – sheet of cells spread by thinning - Intercalation – rows of cells move between one another, creating array of cells that is longer (in one or more dimensions) but thinner - Convergent extension – rows of cells intercalate but is highly directional Primitive Streak - Formation occurs during beginning of human gastrulation - Analogous to plastopore of xenopus - Distinguishes anterior & posterior (A/P) axis Neurulation - Process of neural tube formation - Steps: o Appearance of neural plate (18 days) – invaginates along central axis to form neural groove with neural folds on each side o Neural folds gradually approach each other in midline & fuse – converting neural groove to neural tube (20 days) - Primary neurulation = neural plates creases inward until edges come in contact & fuse - Secondary neurulation: the tube forms by hollowing out of the interior of a solid precursor - Neural tube differentiates into the spinal cord and brain - Ectoderm thickens to form neural plate due to signals from mesoderm & surrounding cells - Neural plate cells - Change shape and curve the neural plate inward, generating the neural tube. - Neural Crest - cells migrate to other parts of the embryo; become many tissues (peripheral nerves, parts of teeth, skull) - Somites: formed from mesoderm, give rise to vertebrae - Cadherins: transmembrane proteins – play important role in cell adhesion o N-cadherin expressing cells – form primary part of neural tube o o N-cadherin expressing cells – form ectodermal region Developmental info for early embryo - cytoplasmic determinants from mother - cell signaling (induction by nearby vells) - cells make developmental decisions with a temporal & spatial framrework Body Plan - relative position of head/tail, back/front & right/left is determined - In frogs o A/P axis determined during oogenesis o D/V axis determined during fertilization - cortical rotation = plasma membrane rotates towards point of sperm entry enables molecules in vegetal cortex to interact with molecules in animal cortex - In chicks o A/P axis established by gravity as egg moves down oviduct o D/V axis due to differences in pH between 2 sides of blastoderm - In Insects o Pattern formation = development of spatial organization in which tissues & organs of an organism are in right place o Begins in early embryo when body axes are formed o Involves morphogenic gradients Morphogen - Substance governing patter of tissue development & position of various specialized cell types - Spreads from localized source & creates concentration gradient - Gradients generate different cells types in distinct spatial order - Can be product of genes (e.g. Bicoid) or signaling molecules (e.g retinoic acid) o Bicoid Homeobox gene = encodes for transcription factor Maternal effect gene = if mutant in mother, offspring will have mutant phenotype regardless of genotype 2 mutant copies of gene in mum leads to posterior structures at both ends Bicoid is essential for setting up anterior of fly mRNA concentrated in anterior of mature egg – once egg is fertilized, transcribed into protein protein diffuses towards posterior creating gradient binds to enhancers of other genes involved in pattern formation, turning on genes that will direct cells to form appropriate anterior structures. Maternal mRNAs destroyed - embryonic program of gene expression takes over Embryonic genes encode for proteins that serve as transcription factors (engrailed) and signaling molecules (hedgehog, wingless); involved in gene activation or repression. Developmental Genes (Drosophila) - Maternal effect genes – set up A/P & D/V axis - Gap Genes – affect development of contiguous block of segments – aids in development of segmented embryos - e.g., kruppel - Pair-rule genes – control development of adjacent segments (pairs) – e.g. Even skipped - Segment polarity genes - affect individual segments' polarity - Hox genes - affect the identity, characteristics of a particular segment o Expressed along the A/P axis in the embryo in the same order as their gene sare aligned on the chromosome (3’-5’) o Gene duplication and divergence resulted in vertebrates having 4copies of the Hox gene complex o Hox genes specify positional identity, not a specific structure o Hox proteins are transcription factors as they are capable of binding to specific nucleotide sequences on the DNA called enhancers where they either activate or repress genes. o The same Hox protein can act as a repressor at one gene and an activator at another. o The homeodomain is a 60 amino acid long DNA-binding domain (encoded by the homeobox on the DNA). Limb Development – chick limb - Each component of limb develops at specific location due to positional information - Limb Bud = mesodermal tissue covered by ectoderm o 2 main organizing regions AER = Apical Ectodermal Ridge ZPA = zone of polarizing activity - Differential Hox gene expression causes specific cells in the limb bud to react differently to positional cues - Different Hox genes are “turned on” as limb development progresses AER = Apical Ectodermal Ridge - Consist of thickened region of ectoderm at tip of bud - Involved in proximal-distal organization - Cells in AER secrete proteins in the FGF family (fibroblast growth factor) that promote and maintain limb bud outgrowth ZPA = zone of polarizing activity - mesodermal tissue located where the posterior side of the bud is attached to the body - Organizes the A/P axis; indicates “posterior” - Cells nearest to the ZPA give rise to posterior structures - ZPA cells secrete sonic hedgehog (SHH) Sonic Hedgehog (SHH) - Is a signaling molecule and transcription regulator involved in limb development - Specifies digits - shh-/shh (mutation) - yields a loss of digits - Also, if you add cells that express large amounts of Shh, to the anterior region of the limb, result will be “posteriorization” WNT7 - signaling protein found in dorsal limb ectoderm - involved in D/V patterning (also neural tube development, cancer-colon) BMP – Bone Morphogenesis Protein - involved in interdigit programmed cell death (regulates AER- FGFs ) Reorganization of cytoskeleton in neurulation - Microtubules extend and elongate the neural plate cells - Actin filaments at one end of the cellcontract; cells form a curved shape - Changes in cell shape result in a hinge region where the neural tube pinches off Cell Migration - Cell’s “crawl” within an embryo by extending and retracting cellular protrusions - Crawl by disassembly of filaments & rapid diffusion of subunits followed by reassembly of filaments at new site – actin polymerization/depolymerization - Some cells in a developing embryo migrate along specific pathways by matching the orientation of their microfilaments to fibers in the ECM - Cell adhesion molecules such as Integrins - receptor proteins found on the surface of cells o Bind to fibronectin and other ECM glycoproteins o Bind to proteins attached to the microfilaments of the cytoskeleton o Transmit signals between the ECM and cytoskeleton Apoptosis - Programmed cell death - Interdigital tissues die & create space between digits Cell Differentiation - Different cell types result from differential gene expression in cells with same DNA - Differences b/w cells in multicellular organisms come entirely from gene expression, not from cell’s genome - Regulatory mechanisms turn genes on & off - Question arises – are genes irreversibly inactivated during differentiation? Somatic Cell Nuclear Transfer (Reproductive Cloning) - Experiment with frog embryos – transplanted nucleus can often support normal development of egg - Nucleus of unfertilized egg/zygote is replaced with nucleus of differentiated cell (dolly the sheep) o Nuclear transplantation from a differentiated mammary (udder) cell o Used mitosis stimulating inducers Epigenetic Changes - e.g., acetylation of histones or methylation of DNA - must be reversed in nucleus from a donor animal in order for genes to be expressed/repressed appropriately for early stages of development - post translational modifications to histones can impact gene expression by altering chromatin structure – does not alter underlying DNA structure Reasons for low efficiency of cloning - DNA from cells in cloned embryos have more methyl groups – could be cuz differentiated cells have more methylation - Cells from donor nuclei are not completely epigenetically reprogrammed, effect differential gene expression Stem Cells - Relatively unspecialized cell that can reproduce itself indefinitely & differentiate into specialized cells of one or more types Different types of stem cells 1. Totipotent – can give rise to every type of cell in adult body (e.g. zygote is totipotent) 2. Pluripotent – can form many different cell types (e.g. embryonic stem cells – ESC - (taken from inner mass of blastocyst) 3. Multipotent – can differentiate into limited type of cells (e.g. bone marrow cells) Therapeutic Cloning - Uses somatic cell nuclear transfer technique – egg cell (removed nucleus) + adult cell nucleus - Since nucleus is from person who will receive stem cell – it will not be rejected by immune system - Embryonic stems give rise to ectoderm, mesoderm & endoderm Induced Pluripotent Stem Cells (iPSC) - Adult stem cells that have been genetically reprogrammed to an embryonic stem cell like state - Forced to express genes & factors important for maintaining the defining properties of embryonic stem cells - Extra copies of the 4 master regulatory genes (transcription factors = Oct4, Sox2, c-Myc & klf4) were introduced into differentiated cells via a retrovirus Retrovirus o RNA virus replicated in host cell via reverse transcriptase (enzyme) = produce DNA from RNA (e.g. HIV) --- [Coronavirus use RNA dependent RNA polymerase, not reverse transcriptase] o DNA is incorporated into hosts genome by an integrase enzyme o Retroviruses can be packaged for gene delivery o Limitation of using retrovirus Random insertion into genome can cause mutations (and potentially cancer) Lentiviral Vector - Tool used by molecular biologists to deliver genetic material into cells - Delete genes that make virus cause disease - Leave genes that allow virus to package & deliver genetic material you want to get into cell Gene Therapy caveat - Jesse Gelsinger (first person dies of gene therapy trial) – suffered from ornithine transcaramylase deficiency (inability of liver to metabolize ammonia) - Suffered massive immune response – led to organ failure & death CRISPR-Cas 9 - CRISPR = clustered regulatory interspersed short palindromic repeats o Segments of prokaryotic DNA containing short replications of base sequences that are intersperced with sequences derived from viruses- - each repetition is followed by a spacer DNA o Immune defense system found in bacteria & archaea – results in degradation of invading DNA - Cas9 = “CRISPR associated 9” nuclease - CRISPR system can: o Knock out target gene o Knock in – insert donor template into target region o By targeting promotor region, can upregulate or downregulate transcriptional activity - How it works = delivers Cas9 nuclease complex with appropriate guiding RNA’s into cell so DNA can be cut at targeted region The players o Cas9 Nuclease o sgRNA: single guide RNA crRNA = target specific CRISPR RNA binds to targeted DNA tracrRNA (trans-activating crRNA) base pairs with crRNA and enables the Cas9-crRNA complex to locate the targeted DNA - is necessary to activate the enzymatic function of Cas9 o PAM = Protospacer-Adjacent Motif - DNA sequence following the target DNA sequence, necessary for Cas9 to bind and cleave the target DNA;o Repairing double stranded breaks made by Cas9 1) NHEJ = non homologous ends joining o DNA ends are ligated back together, but usually with the introduction of a small insertion or deletion 2) HR = homology directed repair o Donor DNA with homologous sequences at either end can be integrated into the site Development - Cell fate can be manipulated by activating specific endogenous gene expression with CRISPR mediated activator - Modified Cas9 – no longer cuts DNA but still can be guided to & bind with specific sequences – can manipulate endogenous gene expression
