Reproductive biology and Embryology Brno, April 2016 Gametes • Meiosis • Structure and development • Differences between oogenesis and spermatogenesis • Regulation of gametogenesis • Ovarian and menstrual cycles • Ovulation • Transport of gametes, sperm capacitation, acrosome reaction Fertilization and Early Embryogenesis • Cortical reaction • Cleavage, morula, blastocyst • Activation of embryonal genome • Embryonic stem cells, nuclear transfer (cloning) Zygote Multicellular embryo Dynamic multicellular organism CELLDIFFERENTIATION and MORPHOGENESIS STABLE GENOME Genomic equivalence (= equal amount of DNA and the same nucletide sequence in all cells of a organism – cloning) VARIABLE TRANSCRiPTOME Transcription Regulators X Embryology: what does it cover? Embryonal x Fetal Development All the organs are established Embryo Fetus Week 8Fertilization Week 39-40 Parturition The primitive heart starts beating at 4 weeks. 7 weeks Early embryo before implantation Any use of understanding principles of reproduction and embryonal/fetal development? • Infertility treatments • Contraception • Avoidance of developmental abnormalities Genetic basis of gamete development Examination of genetic status (amniotic fluid) Understanding the effects of teratogenic compounds Intrauterine examination - sonography Intrauterine surgeries Others to come Instruments Cell culture Molecular biology and genetics Reproduction • allows for continuity of a given species via propagation of its individuals • key element in reproduction is the transfer of DNA duplicate from parents onto progeny Indviduals of different sex produce different gametes Key element in sexual reproduction Sexual reproduction mediated by gametes may seem to be too complicated and much less effective than asexual but serves very significant adaptation role. This adaptation role realizes via unique genetic processes, which take place during development of gametes – eggs and sperm. Although development of eggs and sperm differ in many morphogenetic details, key genetic processes taking place in both types of gamates are principally the same. Genetic processes that are crucial for gametogenesis take place during meiotic cell division - MEIOSIS These genetic processes include: • Reduction of the number of chromosomes • Independent segregation chromosomes • „Crossing over“ Reduction of the number of chromosomes Why? Somatic cell Somatic cell 2n 2n Progeny 4n Gametes have to contain haploid number of chromosomes (n) in order to prevent multiplication of chromosomes in progeny above a diploid number (2n) In principle, the number of chromosomes could be reduced in one step by just separating homologous chromosomes without preceding replication of DNA (DNA synthesis) 2n 1n 1n Meiosis – two divisions instead of one DNA replication establishment of bivalents „crossing over“ 1. meiotic division Independent segregation of chromosomes 2. meiotic division Separation of chromatids 2n 2C 2n 4C 1n 2C 1n 2C 1n 1C 1n 1C Fully functional germ cells • Independent segregation of chromosomes • „Crossing over“ • Fertilization are sources of genetical diversity, that underlies adaptation of living organisms. Sperm Eggs Significance for embryogenesis (reproduction) Morphological and physiological properties Development and underlying regulatory mechanisms Genetic function D I F F E R E N T S A M E X • numbers of ocytes (follicles) in ovary is given at the time of birth and do not increase (in woman ~500 000) • only small number of oocytes develop into fertilizable eggs (in woman ~400) • at the time of menopause, ovary contains only small number of remaining oocytes (in woman ~100-1000) • after reaching puberty, sperm cells are produced in testes continuosly until high age (two testes of man produce about 1000 spermatozoa every second) Primordial germ cells – PGC • stem cells, which are common to both sperm cells and oocytes • originate in yolk sac (extraembryonally) • divide mitotically while migrating into gonad anlagen (genital ridges) (due to signals from surrounding environment – laminin, kit-ligand, TGF-beta1, …) • in man PGCs are sexually indifferent until the 6th week of embryonic development + DEVELOPMENTAL PROCESSES Ooocyte ~0,12 mm One of the biggest and most „precious“ (by both number and significance) cells. Paradoxical cell Highly specialized cell. The only cell in the female body that can undergo meiosis and fertilization, and thus give rise to a new individual. „Totipotent“ cell It can generate all the cellular diversity that si typical for multicellular organism. & Even the era of cloning did not replace the functions of egg ! Key periods of oocyte development G2/M block mitotic division growth (~year) meiotic maturation (hours) PGC & oogonia MI MII Resumption of meiosis Block of meiosis in diplotene of the 1. meiotic division Fertilizable egg, capable of supporting early embryonal development Where and how the oocyte development is achieved ? (1) DiploteneoftheMeiosisI Inside the ovary Preantral follicle (early stage) Recruited follicle Antral follicle (medium size) Preovulatory fol. (large) Primordial follicle30 mm 20 mm Selection of the Dominant follicle (the one most sensitive to FSH) ~14 days Growth FSH independent Massive hormone production Androstendione Theca folliculi interna Estradiole Granulosa cell Takes place in ovary (along with the growth of follicle) It is fully dependent on the contact of oocyte with granulosa cells of the follicle (mediated for example by the gap junction protein connexin-37) Signal that initiates growth is not known (it is not FSH - hypophysectomy does not prevent growth) Communication between oocyte and granulosa cells is bidirectional & & & kit- ligand GDF-9 Where and how the oocyte development is achieved ? (2) Ooocyte growth 100x increase in volume – accumulation of organelles a molecules providing egg with the ability to support early embryogenesis until reaching autonomy (about 105 mitochondria accumulated in oocyte supports embryogenesis until blastocyst stage) Slow process (several months in woman) Intensive transcription – accumulation of mRNA in dormant state (regulated by polyadenylation and ?) oocyte growth transcription Fully grown oocyte – ~2,5 ng total mRNA Intensive translation – many proteins (very limited knowledge) Example: ZP1, ZP2, ZP3 – proteins of zona pellucida Fully grown oocyte – ~120 ng total protein Transkriptome and proteome – underlie unique properties of oocyte Where and how the oocyte development is achieved ? (3) Ooocyte growth Reactivation of X chromosome • somatic cells – one X chromosome is inactivated by hypermethylation of cytosine residues in molecule of DNA • growing oocyte – both X chromosomes are active (crucial for oocyte development – karyotype 45, X0 results in an abnormal development of ovaries) Genomic imprinting • epigenetic modification of autosomal chromosomes that leads to monoallelic expression of genes – due to activity of enzyme DNA methyltransferase • PGCs are globally demethylated • imprinting is newly established during oocyte growth (about 70-80 genes) & Abnormalities in imprinting may result in spontanneus abortions in assisted reproduction !!! (in vitro manipulation with gametes and embryos may produce abnormalities in imprinting) Epigenetic changes occuring during oocyte growth Where and how the oocyte development is achieved ? (4) MPFactivity fully grown oocyte (G2/MI) oocyte at MI oocyte in interphase egg at MII zygoteSperm penetration GVBD LH 25 hours Transcription inhibited Translation activatedX drop of cAMP Where and how the oocyte development is achieved ? (5) The last hours before ovulation – meiotic maturation The final look into the ovary Where and how the oocyte development is achieved ? (6) Hormonal regulation Ovarial cycle Hypothalamus Pituitary gland Gonadotropin-releasing Hormone (pulses) Follicullar growth Meiotic maturation + Ovulation Corpus luteum Gonadotropins FSH LH Menstrual ph. Proliferative phase Secretory (lutal) phase Ischemic Menstrual ph. 0 5 14 28 527 Estrogen Progesteron + Estrogen Sperm cell development - Spermatogenesis (1) Minimal ejaculate (WHO) • Volume - 1.5 ml • Sperm number - 15.1 millions/ml • Motility - 40% Sperms on the oocyte ~4 mm ~60 mm ~0.5 mm Sperm cell development (2) Before puberty Slowly mitotically dividing spermatogonia in genital ridges After puberty ~0.25 mm ~0.5 km Lumen Spermatocytogenesis (mitotic) Meiotic phase Spermiogenesis Sperm cell development (3) A0 Spermatogonia – Stem cells A1 Spermatogonia A2 Spermatogonia A3 Spermatogonia B Spermatogonia • Mitotic divisions • Connected to basal membrane • 2N, 4C Primary Spermatocytes Secondary Spermatocytes - 1N, 2C 1. Meiotic division 2. Meiotic division Spermatides – 1N, 1C Spermiogenesis • No division • Differentiation Sperm cell development (4) - Spermiogenesis Histones to Protamines Genome inactivation Loss of cytoplasm Sperm production • 1 million sperms every hour • Spermatogenesis takes ~70 days • Transport through epididimis ~8-17 days • Cyclic character (Cycle of the seminiferous epithelium – 16 days – the same developmental stage at the same place) Sperm cell development (5) - Regulation Sertoli cells •Support , protect, and nourish •Phagocyte •Blood-testis barrier (zon. occlud.) •Produce anti-mullerian hormone •Produce fructose •Produce inhibin (inh. FSH prod.) Leydig cells •In interstitium •10 % of testis •Produce testosteron Fertilization (1) = the process that culminates in the union of one sperm nucleus with the egg nucleus within the activated egg cytoplasm Where do the gametes meet ? Ovary Uterus Oviduct Ampula Fertilization (2) Oocyte makes itself ready for being penetrated LH surge Graafian follicle 35-40 hrs Preovulatory stage Ovulation Fertilization Fimbriae Infundibulum Oocyte is fertilizable for only 12 to 16 hours Fertilization (3) Travel of sperm to the site of fertilization Testis Seminal gland (vesicles) Ductus deferrens Epididymis Bulbourethral gland Prostate Urin. bladder Urethra EJACULATE (2-6 ml) Sperms 200 – 600 million Products of accessory sex glands • Cholesterol (in prostasomes) • Prostaglandin • Fructose • Vesiculase (coagulates semen) Fertilization (4) Travel of sperm to the site of fertilization Vagina 200-600 millions of sperms Oviduct 100-1000 sperms Cervix Uterus 2 – 7 hours • Cleans sperm • Cervical mucus stabilizes sperms • Initiates capacitation • Acid environment • Sperms move actively • Removal of glycoproteins from the head • Change to composition of cell membrane • Increase of motility Fertilization (5) Entry of sperm into the oocyte Acrosome reaction Initiated by the contact with ZP proteins Release of acrosin, neuraminidase, esterases Zona pellucida Perivitelline space Entry into ooplasm Fusion with oolemma Release of hyaluronidase Penetration of ZP Release of lysosomal enzymes from cortical granules Modification of ZP Block of polyspermy Completion of meiosis II Fertilization (6) 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) 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 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) Thank you for your attention ! Questions and comments at: ahampl@med.muni.cz