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Gametogenesis Lecture 5 Gametogenesis Gametogenesis is the development of gametes – sperm, egg, pollen, ova, spores etc. Germ cells can derive from diverse tissues depending upon organism: Pole Plasm Plants: any stem cell wi...

Gametogenesis Lecture 5 Gametogenesis Gametogenesis is the development of gametes – sperm, egg, pollen, ova, spores etc. Germ cells can derive from diverse tissues depending upon organism: Pole Plasm Plants: any stem cell will do specialized cytoplasmic region zygote - or eggof the Insects, fish, nematodes, frogs: containsmmminants aa - specialized cytoplasm is localized to one pole of the egg, or the freshly fertilized embryo – called pole plasm During early embryonic cell division, only some cells inherit that Pole Cells cytoplasm – pole cells - begin - cells that form germ polar the adult egg at the cells ends of Pole cells are the precursors to primordial germs cells & GIVE RISE TO GAMETES Insects (Drosophila melanogaster – fruit fly) Why Drosophila? functionality conserved very Cheap to feed, store, rear within genes network to are breeds quickly and in great quantities provide Function nuclei divide internally Short reproductive cycle (can breed traits quickly) Few chromosomes, well documented, mapped Sequenced Can knock out, mutate, knock in genes incomplete partioning Many strains, probes, and reagents available to study Pole plasm segregates early Tale of Karl Illmansee (from teratocarcinoma, to fly, to mouse clones by nuclear transfer). http://www.zoology.ubc.ca/ Nematodesfruit only one to cell receives granules cell become like fys germ curoplasmdifwhereyou on a 3 p granules are only included in Pell ↳ become confined to Germ line is lineage the P-cell - lineage restricted We will come back Mosaic Development to this when we talk ↳ highly regulative about embryonic develoament potentials p. 400, Fig 9.3  : During gastulation Mouse to cells end move of posterior above the embryo primitive steah PGCs first detectable & inproximate PGCs to the Start migrate to gonads Cells move from the tube gut into the genital ridge via dorsal p. 402 fig. 9.5 mesentery Primordial Germ Cells (PGCs) migrate from outside the developing body (in the posterior primitive streak - discussed later during lectures on gastrulation), into the PGCs p. 403 fig 9.6 developing body when the gut begins to roll up to form a tube. in populate the genital ridge migrate through GI tract eventually become enclosed in developing gonadal tissue (testes or through body ovary) cality rest on two ridges overstrap Lord  dep on organisms ↳ develop at diff times Spermatogenesis cells enter germ testes where embryonic will become they Sperm PGC differentiates into spermatogonia (puberty) unipotent Spermatogonia serve as stem cells for Spermatogenesis ↳ divide mitotically Golgi orient to tip of head and coalesce into acrosome Form top again - on forming a of population cells of Stem nucleus (spermatogonia) Globular actin between nucleus and Golgi globular and activ Mitochondria and centrosome with tubulin segregate 2NP diploid meiosis haploid to towards tail Stem cells G Flagella grows specialized form of Cilliq Spermatogonia d Cytoplasm sloughed off & Spermatocyte ↓ Spermatics ↓ Mature Sperm Hogarth and Griswold J Clin Invest. 2010;120(4):956-962 p. 406 Fig 9.9 Sperm become motile in epididymis  Acrosome : atanterioendofthespensed to digest the protective coats around the a Simplified Structure of Spermatozoa Sperm moves by single - Flagellum powered by Mitochondria L actin hat the sitting Over head of nucleus haploid chromosome Set 40 80 - (Axoneme) p. 413 Fig 9.14 Meiosis produces four symmetrically divided haploid cells Acrosome forms from Golgi, is membrane-bound, encapsulates some of nucleus Mitochondria move down to axoneme – 40-80 depending upon species  outside of sperm receptors Sperm structure adhere to help egg 9+2 arrangement of microtubules forms Flagella power movement of flagella Globular actin between nucleus and acrosome Head Axoneme Tail End Piece Adapted from: https://en.wikipedia.org/wiki/Acrosome#/media/File:Simplified_spermatozoon_diagram.svg Globular actin concentrates between membranes of acrosome and nucleus External sperm head and interior membrane of acrosome hold receptors to stick to cumulous cells and/or oocyte At fertilization, all internal components transferred to oocyte Mitochondria degenerate – all mitochondria in zygote thought to derive from oocyte (mother) Mitochondria mutations from Father Symmetric cell division yield 4 haploid spermatozoa  Flagella and Cilia ↓Faganas I https://en.wikipedia.org/wiki/Flagellum#/media/File:Eukaryotic_flagellum.svg AMD Dynein - men Mutations locking action https://upload.wikimedia.org/wikipedia/commons/thumb/d/de/Chlamydomonas_TEM_17.jpg/ 1280px-Chlamydomonas_TEM_17.jpg lung infections receptiveenq can't occur 3 Flagella don't swim- accumulation neighboring in mis - immotile - infertile lungs Dynein Cytoskeleton motor protein ATP-dependent Mutation can cause immotility of flagella – also of cilia Immotile sperm = sterility meeting of sperm and Immotile cilia is egg absent Absence of bronchial tractoring of mucous – predisposition to lung infection Left-right asymmetry issues (laterality defects – situs inversus) Sperm Maturation Sperm must then mature via passage through epididymis – makes motile Maturation: is hormone mediated Sperm endocytose epididymosomes (epigenetic information in form of miRNA at least) diff miRNAs metabolic programming deposited in the Sperm Mammalian sperm require further (drinking smoring) affect way , processing in female reproductive tract sperm are armed Capacitation destabilizes acrosome - now consequences ready to fertilize on contact -allowsto penetrate for offsprin gas of egg Hyperactivation induced by near-oocyte A. Head of epididymus, B. Body of epididymus, 1 environment: ZP3 (zona pellucida ligand, progesterone (cumulus cells). Sperm flagella C. Tail of epididymis, and amplitude and frequency increases, sperm D. Vas deferens may be chemotactic at this point of required for penetration pellucida zoa https://en.wikipedia.org/wiki/Epididymis#/media/File:Epididymis-KDS.jpg  After cells enter the germ , where they embryonic ovary will form oocytes they divide mitotically a few times and Oogenesis (Egg) thenenter theprophasa serision No more cell multiplication Further in In mice ; development occurs the adult female don't start eggs highly variable ovulating have sex until they Progression of oocyte through oogenesis varies from one species to the next Some arrest at oogonial stage, others at primary oocyte stage, others at secondary etc. g Oocytes shed at species-specific times ie; rabbits ovulate only after stimulus of coitus, human on a monthly cycle I oave giving rise to legg  cells Polar Bodies : small formed meiosis by during of development an Examples of Patterns of Oogenesis the oocyte an into egg. highly assymetic Egg growing in size - RNA - miRNA life proteinse support - until hatch yolk - or placental support complete cell division Arrest : p. 405 Box 9A, Fig 1  Oogenesis Common Oogenesis steps Oogonia increases in size as it grows and as maternal cells deposit goods Mitochondria amplify to the 1000s - - fertilized it ready When is RNAs (ribosomal, mRNA, miRNA) require genetic transc trans behaviour days before takes messages polypep , - stage embryo deposit early on wall Proteins including those necessary to support first few cell divisions – histones, enzymes, telomerases, yolk granules Full of Golgi forms thousands of vesicles that disperse to periphery (cortex) of cell – cortical granules viscous Fluid and proteins Cortical granules contain hyaline, glycosaminoglycans, proteoglycans, enzymes to cleave receptors, cross- attract water link vitelline coat Globular actin accumulates in the cortex just under the plasma memberane and around cortical granules Oocyte starts to deposit external protective materials: Sheath of protein called vitelline coat and also zona pellucida (glycoprotein) tough after fertilization ↳ zone around Jelly, albumen, shell depending upon animal Off peripher ↳ protection egg clear ↳ Area of interdigitation (villi) with associated nursing cells in mammals all membrane Undergoes highly asymmetric cell divisions – 1 becomes 2 (one big cell, one polar body), becomes 4 (I big cell, 2-3 polar bodies) Meiosis reduces chromosomal complement to haploid in the remaining large oocyte cell. Cortical Granules Nilsson, B et al.(1981). Upsala journal of medical sciences 86: 225-232 > - around peripheyis Ovarian Context Thecal cells Cumulus cells Oocyte 3o or Graafian Follicle Mehlmann (2005). Reproduction. Vol 130 (6): 791–799 https://doi.org/10.1530/rep.1.00793 Mammalian Zona Pellucida Cumulus Cells Zona Pellucida Perivitelline Space Oocyte Kere et al. (2020) Animals 10(4), 664  Spermatogenesis Oogenesis PUBERTY BIRTH Spermatogonium an 2n Oogonium ↓ MITOSIS X2 ↓ X2 Primary Spermatocyte &↓ an ↓ PrimarySee pubert STAGE Of MELOSIS Secondary Spermatocyte n M * Secondary Oocyte No dy N OFERTILAO STAGE 11 Spermatics Egg Cell (orum) MELOSIS N nn N motile ↓ ↓ d ↓ X8 sperm Spermatozoa 92 & X2 of meiosis Stage I and 11 divides cytoplasm unequally to form I and smaller egg bodies polar Syngamy: The Start of You ↓ Lecture 6 fusing of gametes  Less in sperm masturbation Fertilization – It’s Just Sex???! than when having sex commutingspose Getting to fertilization is complex and involves: ↳ sperm count very high Environment of female genitalia environment of If having Cycle-dependent pH women internal reproductive affair tract change ↳ keep sperm in Cycle- and tract area-dependent Y body shed usual mucopolysaccharides (cervical mucous) capaciated partners sperm Women Select Mucopolysaccharides form fern-like branching huperactivation to < sperm inseminate with prime) & structures – packing and density alters Orientation changes viscosity and degree of barrier tail helpsspegive directionali w G-mucous – Sticky, dense block to bacteria 6mucous o and sperm preventinsemination wis L-mucous L-mucous – stretchy but opaque mucous – spem Loser Frapped traps ineffective sperm hyperactive sperm not mucous S-mucous – stretchy and clear – oriented S and loosely packed – lend directionality to - swin sperm flagellar action Cortes (2014). Revista de Medicina Veterinaria 28(28):103-108 Fertilization – It’s Just Sex (Cont’d) Cervical activity (orgasm – 1 minute before insemination ideal) Pole-ax theory (Desmond Morris “The Naked Ape”) when vibrating during orgasm Sperm “up-suck” theory Hormonal priming theory: in pre-industrial times – sex-induce hormonal priming of reproductive cycles in subsistence societies women may norbe noti Ciliary action in uterus, fallopian tubes where are eggs Storage in ampullae? (Bovines accept sperm and save them in a sort of suspended animation – shed slowly over a week or so) femalephysiologybuilttoselectsperm from viablea set Fertilization For Sperm important factors include: > go - right direction in Time and vaginal/uterine environment for sperm length of reproductive time in tract elongated - Window Sperm count and quality Maturation, Capacitation, Hyperactivation Length of time in reproductive tract before exposure to oocyte Ciliary tractoring up fallopian tubes sweep sperm up to where it needs to go Pub-Worthy Reproductive Stories Cambridge/Oxford (Oxbridge for short) Study Large number of commuting couples – one lives in the university town, the other work in/stays in London for few day or work week, or commutes daily Committed couples asked to keep private intimate diaries Collect samples after sex – tested sperm count Men Sperm count varies proportional to prospect of conceiving, especially if cost-free Ejaculate from masturbation had low sperm count Ejaculate during protected sex had low sperm count Perform foreplay – higher sperm count Unprotected sex had higher sperm count Illicit unprotected sex had high sperm count Pub-Worthy Reproductive Stories(Cont’d) Women Post-sex specimen sperm count high if from regular partner Post-sex specimen sperm count low if from illicit lover Remember ampulla storage? Or perhaps has to do with reproductive tract mucous, cilia, dynamics/condition/orgasm? Selection of gametes from most attractive mate? 1. Both genders are regulating sperm count 2. In males, this depends upon how much is committed to ejaculate 3. In females, sperm retention is regulated - this is called sperm selection or sperm gating 4. Physiology and “head space” more important than commonly acknowledged  Steps to Syngamy Syngamy – the moment that the sperm meets and fuses with the oocyte Sperm nears the cumulus cells surrounding egg Sperm undergo hyperactivation Undergoes hypercapacitation – directed and vigorous flagellar activity swimming Enzymes on sperm head (hyaluronidase) help sperm to penetrate between cumulus cells Sperm hits Zona Pellucida – contact with these glycoproteins stimulates acrosome reaction receptorsonhead of Sperm help to undergo Acrosome reaction T 6 acrosome acrosomal membrane fuses with sperm plasma membrane Fuses with acrosomal contents are released into immediate environment external membrane Acrosomal enzymes digest a tunnel through the zona pellucida of sperm Receptors inside the acrosome are now exposed and can bind to oocyte membrane Actin polymerization under the acrosome begins to polymerize to form a filament Filament extends to oocyte surface – inner acrosomal receptors can contact oocyte  After sperm have been deposited in the Female reproductive tract they undergo - , hypercapacitation which facilitates fertilization. first In mammalian the layer is sticky layer of hylauronic acid and - eggs , a embedded somatic follide cells Surround the when released These , egg. cells are called Cumulus cells form a 'cloud' around it , of the head helps it to - Hyaluronidase surface activity on the sperm penetrate this cumulas layer. - Sperm next encounters the Zona pellucida , a layer of fibrous glycoproteins barrier to Secreted by the oocyte. Acts as a physical , but sperm are helped it the penetrate by acrosomal reaction of enzymes contained in Acrosomal reaction is the release the acrosome ,a Golgi - derived reside located in the head Sperm. The break down the Oligosaccharide chains on the acrosome side - enzymes zona hole in that the pellucida glycoproteins making , the zona pellucida a helps to the membrane sperm approach egg plasma Sea Urchin Acrosome Reaction outward grows remodel to makes contact with make contact membrane surface Sinauer 2000 Receptor stimulus causes cross-membrane influx of Na+ Acrosomal membrane fuses with plasma membrane to release acrosomal contents Actin remodels to form filamentous process Receptors on inner face of what was acrosomal membrane bind egg The picture can't be displaye displayed. Specificity of Bindin in Urchin d. no interaction aggregation no aggregation R bindin sticks to eggs Sea Urchin Sperm Penetration Human Fertilization Spermatozoa attach side-on Actin plays less prominent role Still have acrosomal reaction https://biotexcom.com/wp-content/uploads/2017/03/32-1024x730.jpg Syngamy Induces Cortical Reaction cross-link - sperm can't get through protect egg - from infection actin > filaments  Fertilization and activation are associated egg - with release of free Cad+ from within the a of Ca2+ across it egg - Wave Cast release triggered by sperm entry - Initiation of Cortical Reaction Fertilizinga TRIGGER - WAVE Sperm Lionel Jaffe’s Work p. 417 Fig 9.18 1. Na+ influx at syngamy initiates release of free Ca2+ in cortex 2. Ca2+ wave spreads across cortex of oocyte (zygote now) 3. Ca2+ initiates remodeling of actin in cortex 4. Wave of actin remodeling moves cortical granules to the surface 5. Cortical granules fuse with plasma membrane 6. Cortical granule contents released into perivitelline space Cortical Reaction (Continued) CROSS-LINK p. 416 Fig 9.17 Cortical Granules Release: Water swelling agents (glycosaminoglycans (GAGs), hyaluronate, proteoglycans) Enzymes Cleave sperm receptors to prevent additional sperm binding Cross link external vitelline coat - becomes impermeable Perivitelline Space Swells Lifts vitelline coat Increases perivitelline thickness Cortical Actin Remodels Villi grow to lift vitelline coat (tent poles)  Egg is surrounded vitelline - which lies by a membrane outside the , ↳ to plasma membrane corresponds in mammalian zona pellucida oocytes Membrane-bound cortical just beneath granules lie the egg plasma - membrane At Fertilization the cortical fuse with the membrane granules plasma - , , and some of the contents are extruded by exocytosis. Cortical form Fertilization granule material and vitelline membrane tough - a membrane which then lifts off the surface further , egg and prevents sperm entry. Other to hyaline layer granules give Cortical rise which surrounds the - a , egg under the Fertilization membrane. Syngamy Completes Totality of sperm absorbed Oocyte/embryo rendered resistant to further fertilization Maternal and paternal pronuclei begin to migrate to fuse How Is Polyspermy Prevented? The problem: there are millions of sperm. How is it that only one enters the egg??? Statistical analysis indicated that cortical reaction (5-10 minutes duration) too slow to prevent multiple sperm from entering Is there another mechanism also at work? How is polyspermy prevented??? How Is Polyspermy Prevented? The problem: there are millions of sperm. How is it that only one enters the egg??? Statistical analysis indicated that cortical reaction (5-10 minutes duration) too slow to prevent multiple sperm from entering Is there another mechanism also at work? How is polyspermy prevented???  Block to Polyspermy block to Rapid polyspermy of in sea urchins is triggered Within seconds by - a transient depolarization the egg plasma membrane that occurs on Fusion sperm-egg The electrical from membrane potential across the plasma goes - membrane 70mV to 20mV within few seconds of + a sperm entry - Membrane returns to normal potential level - If depolarization is prevented polyspermy - occurs , As the plasma membrane repolarizes impenetrable called - an membrane , the Fertilization membrane is formed around the egg. Slow of polyspermy ; triggered by block to - a wave calcium release in due to leads to the cortical the egg sperm entry and granules their contents to releasing by the outside membrane plasma exocytosis Fast Block to Polyspermy Laurinda Jaffe (daughter of Lionel Jaffe) Sea Urchin Fertilization Used electrophysiology to test membrane potential You can measure current or voltage change relative to an external electrode You can insert a second electrode and pump/measure current to maintain a stead state You can artificially clamp a membrane at specific potential difference (voltage)  blook activation at Fertilization Try to egg of sea by clamping urchin membrane at voltage the a very positive potential - cab I egg can't enter egg Fast Block to Polyspermy Laurinda Jaffe depolarizes Membrane potential alters immediately at fertilization Voltage clamping can either prevent or facilitate fertilization, depending on where voltage is clamped High voltage prevents, low voltage facilitates polyspermy↳ Cart can't enter the egg  Cortical the Nicotine allows reaction to occur Nicotine inhibits fast block Laurinda Jaffe – took work of the Hertwigs (1887) that nicotine induced polyspermy Found that nicotine inhibits amplitude of electrical block – polyspermy HAPPENS ensues. Normal Nicotine & Fertilization membrane formed at normal line but was lower ↳ First clearage - polyspermy ↳ Second Step identified fertilization amplitudeisreducedNicotines impairmentf of the fertilization pot amplitaa Generalized Response: frogs, echinoderms, marine worms, mammals depolarization occurs +15 mV -70 mV few seconds 30 minutes time Final Proof Test Voltage clamp high: Fertilization occurs = depolarization: No fertilization Fast Block to Polyspermy commences +15 mV -70 mV Release Voltage clamp: Fertilization possible time Nicotine inhibits fast block Artificial Depression of potential difference (Sodium Iodide) permits visualization of multiple inseminations (polyspermy) fertilizations -70 mV Other mechanisms to prevent polyspermy Micropyle – Fish Insects D-G are goldfish, H is loach, I is Zebrafish Yanagimachi ety al (2017). Chemical and Physical Guidance of Fish Spermatozoa into the Egg through the Micropyle1. Biology of Reproduction. 96. 10.1093/biolre/iox015. Polyspermy as the norm: urodeles, birds, reptiles Molecular Reproduction and Development, Volume: 87, Issue: 3, Pages: 358-369, First published: 16 July 2019, DOI: (10.1002/mrd.23235) Basal Body from sperm and egg each nucleate microtubule growth Mitotic spindles grow Maternal and paternal chromosomes align, diploidy is restored Peripheral or late spindles and associated chromosomes bulldozed out of the game and degenerate Cleavage (not what you think!) Lecture 8 ↳ first step be followed into smaller Cells (blastomeres) can & division of fertilized egg of cells the blastula by formation of hollow sphere  Cleavage: Cell Division without Growth of mass embryo does not increase 24GOTE ↓ BLASTOMERES Cleavage divisions are the first mitotic divisions of the embryo. They divide up the single celled zygote into increasingly more and increasingly smaller cells (blastomeres). - Cleavage stage ends at the blastula stage - Cleavage: Altered Mitosis – no G1 G2 kinase phosphorylates SINACTIVE After Nigg et al, 1995 Cdk1-cyclinB (MPF – maturation promotion factor - Masui) Kinase promotes mitosis Cdk1-cyclinB normally active at M-phase, cyclin degraded after M-phase and resynthesized (new transcription) before next M- phase. Gap phase = low cyclin levels Cleavage embryo is filled with Cdk1-cyclinB. After mitosis, only cyclinB that was associated with nucleus is destroyed. Levels decline almost unnoticeably. No gap phase because Cdk1- - cyclinB is always present and active -  Cleavage: Characteristics ↑ Cleavage divisions After Nigg et al, 1995 very rapid compared to other cell divisions lack defined G1 and G2 phases due to constitutive Cdk1-cyclinB activity no cell growth – cells get smaller each division cells are transcriptionally silent – Zygotic genome inactive still – embryo relies on oocyte stores Cell divisions are often synchronous pattern of cleavage divisions is stereotypical for an organism, and affected by yolk thickness/distribution Cleavage: Characteristics Different organisms exploit different environments at different stages of life. Cleavage can be described using a few different parameters: Amount and distribution of yolk Whether or not early cleavage is complete Whether or not cleavage is symmetrical (ie; daughter cells are identical) How planes of cell division contribute to distribution of blastomeres HUMANS Mesolecithal - Cleavage divisions in frogs Egg yolk is concentrated in lower half of egg Complete divisions = holoblastic > - complete divisions First 2 divisions \vertical, 3rd is horizontal and closer to top subsequently top cells divide faster (less yolk) After first two cell divisions (4 cell stage) cleavage is asymmetric  Meroblastic cleavage divisions in fish, birds Yolk is very thick and cell divisions cannot pass through Only upper region of egg is free of yolk first 5 cell divisions are vertical and incomplete - Meroblastic ↳ partial cleavage furrows stop when they reach yolk cleavage not blastomeres are still not separated from yolk completevision 6th division is horizontal and separates yolk from upper blastomeres  Telolecithal - Cephalopod Meroblastic Cleavage Galis et al. (2002). Divergence and convergence in early embryonic stages of metazoans. Contributions to Zoology. 71. 101-113. 10.1163/18759866-0710103008. connected partially are still - blastomeres of bilateral meroblastic cleavage on top a undergo - , large yolk Cleavage divisions in mammals Holoblastic cleavage Egg has little yolk that is evenly distributed (isolecithal) Cleavage divisions go completely through the egg/blastomeres each blastomere is of equal size different than other animals in that cleavage divisions are slower and zygotic transcription starts at minimal level early in embryo 24-48 hours Cleavage divisions, Planes, and Rotation Holoblastic cleavage 2nd cleavage is at right angles to 1st – rotational cleavage Rotational or Meridional  Pattern of cleavage division is determined by orientation of mitotic spindle mitotic will spindle - segregate the duplicated chromosomes into two identical sets of mis that composed - come out of two centrosomes M positioned two opposite or - of - poles spindle Spindle Mis grow from centrosome and w the meets overlap from the spindle mis opp. pole midzone cells spindle - = divide astral mis anchors each - Centrosome to cell cortex Cleavage divisions in C. elegans Holoblastic and asymmetric IE; only P cell and its progeny inherit P granules Fates of cells are very lineage restricted Ablate a cell, future vulva, or specific neuron not present. Centrosomes, Spindles, cleavage orientation 90% 3 same plane as unequal division first cell anterior AB larger division cell smaller Pi cell posterior Plane of cell division depends on orientation of mitotic spindles This depends on centrosome positioning Centrosome positioning depends on proteins at cell cortex that interact with centrosomes (usually via astral microtubules) “Meroblastic” Fruitfly Cleavage 1. Initially, DNA replication and nuclear division without cell cleavage 2. Nuclei migrate to periphery 3. Pole plasm-containing cells collect posteriorly (presumptive germ cells) 4. Cells partially partition but remain open at bottom (syncytial) 5. Finally, around division 13, cellular partition completes  Cleavage divisions Cleavage divisions divide up the zygote into multiple cells that will each eventually have different fates in the embryo Are cell fates established during the cleavage divisions? in one model, cell fate determinants are present in the zygote and are asymmetrically distributed at each cell division each successive cleavage division further restricts the fate of a cell Answer: Depends on organism Some are highly mosaic with regard to distribution of cytoplasmic components (determinative) Some are very flexible (regulative or indeterminate)