Reproductive biology and Embryology Brno, March 2018 • Early embryo cleavages • Implantation • Somatic nuclear transfer– cloning • Gastrulation • Extraembryonal structures • Fetal membranes Lecture 4 Fertilization Zygote formation and the first cleavages Gap junctionsZygote pronuclei fused (Syngamy) DNA synthesis Sperm • DNA • Cenriol • Oocyte-activating factor Ooocyte • DNA • Mitochondria • Cytoplasm ~6-8 hrs ~12-14 hrs ~3-4 days~8-12 hrs O v i d u c t U t e r u s Blastocyst formation Compacted morula Hatched blastocyst Hatching blastocyst Expanded blastocyst 220-280 mm Trophoblast Blastocoel Embryoblast Day 5 – 6 (60 to 8O cells) Early embryogenesis of human embryo Dr. Zuzana Holubcová REPROFIT, Brno PB PN 20 44 G1 S G2/M G1 S G2/M Translation of maternal mRNA Translation of zygotic mRNA Zygotic transcription Repression of transcription Significance of „enhancers“ Activation of embryonal genome A potency of oocyte cytoplasm It is not a single discrete event (first signs occur in zygote, in man it reaches its maximum in 4- to 8-cell embryo) Transcrips that replace degraded maternal mRNAs Novel transcripts that underlie new pattern of gene expression Two types of transcripts It is „responsible“ for establishment of totipotency of blastomeres It represents phenomenon known as genome REPROGRAMMING & Activation of embryonal genome Effectivity of cloning is very low (1-3%) Reprogramming is slow and most likely incomplete (as the result, gene expression is often abnormal) Effectivity of reprogamming depends on many factors (type of somatic cells, position in cell cycle phase, …) Nuclear transfer (cloning) - principle Egg Somatic cell Reprogramming „Normal“ development Activation Blastocyst implantation 0 5 14 28 Menstrual phase Proliferative phase (estrogens) Secretory phase (progesteron) Ovulation followed by Fertlizaion Functional zone of endometrium compact + spongious + basal Stroma Syncytiotrophoblast Embryoblast Epithelia Trophoblast Molecular players Selectin on trophoblast + glycoproteins on epithelia Integrins + Laminin, Fibronectin IGF1, IGF2, VEGF Metaloproteinases + their tissue inhibitors Progesteron hCH Prostaglandins http://myselfishgenes.blogspot.hu/2013/05/what-happens-to-my-embryos-if-they-do.html Blastocyst implantation Blastocyst impantation – place of implantation Normal (posterior / anterior wall of uterus) Abnormal (0,25 až 1%of all implantations) Early development – The second week (1) Completion of implantation + Further embryo development • Furtner invasion into endometrium • Damage to capilaries and glands • Engulfment of apoptosing cells (Fas/Fas ligand) Trophoblast Cytotrophoblast • Mitoticaly dividing cell (single layer) • Supply for syncytiotrophoblast Syncytiotrophoblast • Fused cells (nondividing, multinucleated) • Produces hCG (stimulates CL)Trophoblastic lacuna Syncytiotrophoblast Uterine gland Endometrial capilary Cytotrophoblast Amniotic cavity Hypoblast Blastocyst cavity Embryonal pole Abembryonal pole Status at day 8 Early development – The second week (2) Vessel number/density increases Fibroblasts differeniate into glycogen- and lipid-containing cells Decidual reaction of endometrium Status at day 10 Status at day 12 Syncytiotrophoblast Cytotrophoblast Epithelization of the implantation site 2-3 mm Sponge-like appearence Arterial branch Venous branch Lacuna Uterine vessels erode Fused lacunae create a network + Uteroplacental circulation Human Embryonic Stem (hES) Cells (Thompson et al, 1998) Early embryo at blastocyst stage Isolated embryoblast (ICM - Inner Cell Mass) Isolated embryoblast after placing to in vitro conditions (+ feeder cells + FGF2) Propagation in culture by enzymatic disaggregation (repeated passaging) Derivation of postmeiotic germ cells from hESC Prof. Harry Moore, University of Sheffield, 2009 B) C-KIT C) I-97 antigen D) Cells with condensed chromatin and signs of flagellum Structures that are highly reminiscent to oocyte-granulosa complexes (zona pellucida is not developed) Gastrulation – establishment of three germ layers Actual size Beginning of the 2. week Epiblast Hypoblast Syncytiotrofoblast Cytotrofoblast = embryonic disk Day 8 to 9 Hypoblast Epiblast Cytotrophoblast Primary yolk sac Mesoderm = ectoderm = endoderm Amnionic cavity Gastrulation – establishment of three germ layers Embronic disk – first at day 6 to 7 Caudal Prechorodal ploate Primitive streak Primitive node Cloacal membrane Notochord Rostral Caudal Rostral Dorsal view Transversal section Mesodem specification (expanded view) Primitive streak (groove) Ectodemal epithelial cells Mesodermal cells Endoderm = epithelo-mesenchymal transition Gastrulation – establishment of three germ layers Day 9 – primary yolk sac Gastrulation – establishment of three germ layers Day 12 - 13 Cytotrophoblast Secondary youlk sac Extraembryonic coelom = chorionic cavity Yolk cyst Mesoderm Endoderm Ectoderm Future embryonal stalk Extraembryonal splanchnopleure (Heuser´s membrane) Extraembryonal somatopleure Amnionic cavity Gastrulation – establishment of three germ layers Extraembryonal structures – yolk sac 1 End of the 2. week Wall of yolk sac = endoderm + mesoderm Heuser´s membrane Extraembryonal structures – yolk sac 2 approx 6. week approx 8. week Functions of yolk sac: • does not contain yolk (oligolecithal egg) • 3. week – hematopoesis (since 6. week in liver) • 3.- 4. week– PGC • 4. week – incorporation into primitive gut • since 6. week – loss of link to gut – obliteration • abnormal persistence - Meckel diverticleEmbryonal stalk Embryonal stalk Embryonal stalk Yolk sac Yolk sac Yolk sac Amnionic cavity Amnionic cavity Amnionic cavity Ductus omphaloentericus Umbilical cord Beginning of the 3. week – diverticle of caudal wall of yolk sac Functions: • In humans rudimentar structure • vessels of alantois – umbilical vein and arteries • 2. month – extraembryonal part degenerates • 3. month – intraembryonal part – urachus (link to urinary bladder) Extraembryonal coelom Embryonal stalk with alantois Extraembryonal structures – amnion (internal fetal membrane) Amnion thin, transparent = flat simple ectodermal epithelium + extraembryonal mesoderm (somatopleure) 4,5 weeks 3,5 weeks 2 months Amnion Amnion Amnion Amnionic cavity Amnionic cavity (amnionic fluid) Amnionic fluid: • 99% water; 800 až 1000 ml in the last month of pregnancy • source = diffusion from endometrium through amniochorion • source = transfer through placenta • source = transfer through skin of fetus • source j = excretion by fetal kidneys (since 11. week) • very fast turnover – passage via fetus (digestive + respiratory tracts) • function – free movement without adhesion (space for symmetrical growth) • function – barrier (temperature, mechanical damage, infection) • function – chemical homeostasis Extraembryonal structures – amnion (internal fetal membrane) Extraembryonální struktury – chorion (zevní plodový obal) Syncytiotrophoblast invades into surrounding stroma Implantation Extraembryonal structures – chorion (external fetal membrane) Extraembryonal structures – chorion – chorionic villi Day 11 až 13 Day 16 End of the 3. week primary villus secondary villus tertiary villus Blood vesselsMesodermal center Amnionic cavity Amnionic cavity Chorion Chorion + Amnion Extraembryonal structures – chorion – expansion of amnion Extraembryonal structures – growth of amnion and chorion 4 week embryo 8 week embryo Growth of amnionic and chorionic cavities Chorionic cavity Amnionic cavity Extraembryonal structures – chorion – frondosum x laeve Chorion frondosum Chorion laeve Ch. frondosum – vilous Ch. laeve - smooth Extraembryonal structures – chorion – decidua D. basalis D. capsularis D. parietalis Chorion laeve Chorion frondosum Decidua basalis – between embryo and myometrium Decidua capsularis – between embryo and uterine cavity (becomes thinner) Decidua cparietalis – opposite wall of uterus Decidua – according to its locality • basalis • capsularis • parietalis Extraembryonal structures – chorion – decidua Chorionic villi - finger like projection of embryonic tissue that come in contact with bleeding endometrium Decidual cells – fibroblast of endometrium (large, cuboidal, very active proteosynthesis) Placenta – thick disk made by decidua and chorionic villi (formed at the start of month 4) Extraembryonal structures – chorion – placenta Bazální ploténka Placenta • pars fetalis – chorion plate, chorion villi (anchored, free = terminal) • pars materna – decidua basalis • intervilous spaces – develop from lacunes discoid 15 – 20 cm 400 – 600 g Chorion plateFree villi Ancored villi Intervillous space Discoidalis + Hemochorialis Basal plate Placenta – blood circulation Umbilical arteries – deoxygenated blood Umbilical vein – oxygenated blood Placenta – terminal villi Terminal villi – human – end of pregnancy Placenta - fetomaternal barrier 1 Fetal capilaries Intervilous spaces (mother´s blood) Trophoblast Mesenchyme Barrier = 5 elements • Endothelium of fetal capilaries • Basal membrane of endothelium • Mesenchyme of villi (extraembr. mesoderm) • Basal membrane of trophoblast cells • Cells of cyto- a syncytio-trophoblast since month 5 cytotrophoblast looses its continuity Terminal villi – human – end of pregnancy Barrier = 5 elements • Endothelium of fetal capilaries • Basal membrane of endothelium • Mesenchyme of villi (extraembr. mesoderm) • Basal membrane of trophoblast cells • Cells of cyto- a syncytio-trophoblast since month 5 cytotrophoblast looses its continuity Placenta - fetomaternal barrier 2 Placenta - cotyledons Fetal surface Maternal surface Placenta – umbilical cord 1 Amnionic ectoderm • diameter 1,5 to 2 cm • length 50 to 60 cm • 1x vein + 2x artery (spiral organization) • Wharton´s jelly – loose connective tissue 4,5 weeks 3,5 weeks 2 months Amnion Amnion Amnion Amniová dutina Amnionic cavity (amnionic fluid)  1-Connecting stalk: – Allantois – Umbilical vessels (two arteries & one vein), they all embedded in – Wharton’s jelly (extra embryonic mesoderm)  2-Yolk stalk (Vitello-intestinal duct):  (Ductus omphaloentericus) – A narrow, elongated duct which connects gut to yolk sac – It contains Vitelline Vessels – (Later on , it is obliterated and the vitelline vessels disappear). Placenta – umbilical cord 2 Attachment of umbilical cord to placenta Insertio centralis (norm) Insertio marginalis Insertio veluminosa (to chorion laeve) • Short umb. cord < 40 cm • Long umb. cord > 60 cm • Absence of one artery – fetal hypotrophy True knot Fetal strangulation Umbilical prolapsus False knot True knot Umbilical cord - anomalies Placenta – anomalies 1 Attachment of placenta (related to myometrium) • Placenta accreta attached to myometrium • Placenta increta grown into myometrium • Placenta percreta grown through myometrium Normal Location of placenta in uterus (1 to 5 according to frequency) Lateral wall Uterine fundus Anterior / posterior wall Placenta – anomalies 2 Shape and formation of placenta Placenta fenestrata Placenta tripartita Placenta triplex Placenta duplex Placenta membranacea Placenta succenturiata Normal placenta Placenta – multiparous pregnancy 1 2 oocytes + 2 sperms 2x amnion + 2x chorion + 2x placenta TWINS Dizygotic Monozygotic Placenta – multiparous pregnancy 2 1 oocyte + 1 sperm 2x amnion + 2x chorion + 2x placenta Embryo splits at 2-cell stage (as dizygotic twins) TWINS Dizygotic Monozygotic Trophoblast common to both embryos Most frequent twins – 65 % 1 oocyte + 1 sperm 2x amnion + 1x chorion + 1x placenta (monochorial, diamniotic) Placenta – multiparous pregnancy 3 TWINS Dizygotic Monozygotic Embryo splits at blastocyst stage Placenta – multiparous pregnancy 4 Trophoblast and amnion are common to both embryos 1 oocyte + 1 sperm 1x amnion + 1x chorion + 1x placenta (monochorial, monamniotic) TWINS Dizygotic Monozygotic Embryo splits at bilaminr stage Thank you for your attention ! 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