Lecture 9 - Gametogenesis PDF

Lecture 9 – gametogenesis Gametogenesis - Development of haploid sex cell – mature occyte and sperm cell in a diploid organism by meiosis o Oogenesis o Spermatogenesis Origin of gametes - At about 4 weeks of pregnancy - Sex cell precursors – PGC (primord...

Lecture 9 – gametogenesis Gametogenesis - Development of haploid sex cell – mature occyte and sperm cell in a diploid organism by meiosis o Oogenesis o Spermatogenesis Origin of gametes - At about 4 weeks of pregnancy - Sex cell precursors – PGC (primordial germ cells) rise in the yolk sac - Alkaline phosphatase – marker of PGC - 4. – 6. Week of pregnancy PGC migrate to genital ridge – bisexual gonads Origin of gametes - Early migration of PCGs is dependent on the expression of interferon-induced transmembrane proteins 1 and 3 (IFITM1 and IFITM3) in the surrounding mesoderm Formation of spermatogonia and oogonia 45 X and Y chromosome genes involved in sex determination and differentiation The Male Determining Pathway: SRY - SRY – sex determining region of the Y chromosome - Main role of SRY = upregulating the expression of SOX9 during a very narrow critical time window - In humans SRY is expressed in both Sertoli cells and germ cells at fetal and adult stages The Male determining pathway: SOX9 – A target of SRY - SOX 9, an autosomal member of the HMG-box protein superfamily - master regulator of Sertoli cell differentiation - SOX9 is expressed at low levels in the bipotential gonads of both sexes under SF1 regulation, but persists only in testicular Sertoli cells after SRY expression has peaked Epigenetic regulation of gametogenesis General considerations - Some oocyte and sperm genes are imprinted differently - Because germ cell genes but no somatic cell genes, are passed on the next generation: o Imprinted marks present in the zygote must be erased o Reset at some point during germ cell development ▪ gametes of developing embryo need to be imprinted with proper marks according to the sex of the developing embryo o in the somatic cells of the developing embryo, these same imprinted marks must be retained Epigenetic changes in PGC - Widespread chromatin modifications: o PGCs undergo genome-wide demethylation – reaching a “ground state” in terms of epigenetic marks o Second wave of DNA demethylation occurs and erases the methylation marks of imprinted genes in the PGC genomes, when they reach the gonads o Remethylation of germ cell genome occurs later during fetal life 46 Epigenetic changes in migrating PGCs of the developing embryo of mice - During the time of migration, the epigenetic marks of the PGCs begin to change X chromosome re-activation - In female PGC (2n) - 2 X chromosomes active (inactivated x is reactivated) - Reduction of Xist RNA levels Epigenetic control during gametogenesis – in meiosis (see graphic in lecture) - Male and female germ cells share certain epigenetic marks, some of which may be very important for gametogenesis, e.g.: o Enzymes, important for synapsis: ▪ Histone methyltransferase, PRDM9 transfers a methyl group to H3K4, an epigenetic mark generally thought to open up chromatin for transcription ▪ Euchromatic histone-lysine N-methyltransferase 2 protein (EHLM/Ga9), which is important for H3K9me or H3K9me2 repressive marks o Histone acetylation, is also important for proper chromosome segregation within developing gametes Timing of gametogenesis - males – starts at puberty and continues throughout adult life - females – starts during embryonic life and ends for each particular oocyte after fertilization 47 Oogenesis - By mitosis and meiosis - Modified meiosis: o 2 arrest (stops): ▪ In meiosis I (prophase I, diplotene) ▪ In meiosis II (metaphase II) o Meiosis II can be finished only after fertilization Periods of oogenesis - Proliferation period - Growth period - Maturation period Prenatal period (before the birth) Proliferation period - approx. 3rd month of embryonic development - From PGC → oogonia (2n) - Primary oogonia → secondary oogonia (approx. 7 million) Growth period - Approx. 4. – 6. Month of embryonic development - Secondary oogonia → primary oocytes - By the 5th month approx. 7 million (degeneration begins) 48 Maturation period - Approx. 7. – 8. month of embryonic development – with meiosis I (initiation of meiosis in the fetal ovary is heralded by the increase in retinoic acid levels) o Leptotene, zygotene, pachytene and diplotene stops - Primary oocytes in embryonic ovaries are surrounded by follicular cells and form an embryonic follicle (see graphic) Postnatal period (after birth) – at the time of birth - Primary oocytes (700 000) remain in diplotene till the puberty - The rest of primary oocytes degenerate Postnatal period – at the puberty - 400 000 primary oocytes - Embryonic (primordial) follicles → primary follicles - Maturation of primary oocytes Primary follicle - Oocyte I + follicular cells - Develop from embryonic follicles - Structure: - Oocyte I: Plastic membrane, nucleus, cytoplasm - Zona pellucida - One layer of follicular cells 49 At the puberty - Every month 5-15 oocytes I start maturation - Only one mature - Primary follicles → secondary (developing) follicles - Why only few primordial follicles commence folliculogenesis each month? o Not clear o One possibility – some follicles become progressively more sensitive to the stimulating effects of FSH as they advance in development or o selection process is regulated by a complex system of feedback between the pituitary and ovarian hormones and growth factors Secondary (developing follicle) - Develops from the primary follicle as follicular cells become large - Structure: o Oocyte I o Zona pellucida o Several layers of follicular cells o Anthrum enlarges → Fluid-filled vesicles develop among granulosa cells and theca becomes apparent Mature follicle (Graafian follicle) - Develops from the secondary follicle - Structure: o Oocyte I → is located in cumulus mass o Zona pellucida o Differentiation of follicular cells: ▪ Corona radiata ▪ Cumulus oophorus - Anthrum is enlarged more Maturation period - Only one oocyte I finishes meiosis I and develops in o One oocyte II (n), that continues with meiosis II and stops in metaphase II and o One first polar body (n) - Oocyte II from ovaries enters the fallopian tube 50 Cytokinesis - By the help of actin – myosis contractile ring - Cytokinesis is unequal, but complete: o One large cell – oocyte II (n) o One small cell – polar body (n) Regulation of meiosis in oogenesis - Unique features that are responsible for metaphase II arrest - Meiosis of oocytes is controlled my MPF (maturation promoting factor) – complex of Cdc2 and cyclin B) - MPF then induces chromosome condensation, nuclear envelope breakdown, and formation of the spindle - Activation of the anaphase-promoting complex B and decrease in MPF levels Regulation of meiosis in oogenesis cont. - Mos protein kinase maintains metaphase II arrest by inhibiting anaphase-promoting complex - action of Mos is mediated by MEK, ERK and Rsk protein kinases After fertilization - Oocyte II continues meiosis II - One large cell – mature oocyte II (egg) and small cell – polar body (n) In absence of fertilization - Oocyte II stays in meiosis II metaphase II - Mature follicle shrinks, corpus luteus is formed – secreting hormones, promotes development of the next oocyte - Approx. 450 – 500 mature oocytes II develop during one female’s life 51 The structure of mature oocyte – during ovulation - Oocyte is released from the follicle with some surrounding granulosa cells of the cumulus mass = corona radiata Follicular cells - Granulosa cells: o Produce sex hormones – estrogen and progesterone (depending on the phase of the menstrual cycle) o React to different hormones (e.g., LH and FSH) o Produce different growth factors - Theca cells: o Internal – Theca interna – produce androgens and progesterone o External – Theca externa – connective tissue, important in the ovulation 52 Abnormal female gametes Summary of oogenesis - Result of oogenesis is one mature oocyte II (n) and polar bodies, which are not used in fertilization - main function of polar bodies = reduce chromosome number and “donate” cytoplasm to oocyte II - Oogenesis occurs from 3rd month of embryonic development till the menopause Spermatogenesis - Starts at puberty and continues through lifetime - One cycle of spermatogenesis lasts 64 – 72 days - Occurs in testes (seminiferous tubules) - Process of sperm production Prenatal period (before the birth) - Males are born with a finite number of spermatogonia Why don’t spermatogonia enter meiosis? - mesonephros from the indifferent gonad, the lung and adrenal gland, synthesize retinoic acid, that acts as meiosis inducer - Mouse Sertoli cells express two factors that prevent meiosis onset: FGF9 and CYP26B1, an enzyme that catabolizes retinoic acid - NANOS2 I = another meiosis-preventing protein 53  - In human fetal testis, CYP26B1 does not seem to be expressed and the mechanism underlying the inhibition of germ cell entry into meiosis needs to be elucidated Postnatal period (after the birth) - At the time of birth sex cells are located in sex chords Sertoli cells - Tall columnar epithelial cells: o large nucleus o Abundant in chromatin o Well-developed Golgi complex o Many mitochondria (Mt) o Extended smooth ER Sertoli cells (functions) - Surrounded developing sperm cells – tight junctions; hemato-testicular barrier → spermatogenesis is occurring between them - Secrete proteins and fluids - Trophic function - Serete growth and other factors, needed for development of sex cells - Synchronize the events of spermatogenesis, associated with sperm production Serminiferous tubules - Direction: from side to centre - Sertoli cells are associated with them Spermatogenesis – periods - Proliferation - Growth - Maturation - Spermiogenesis 54 Spermatogenesis - Spermatogenesis occurs between sertoli cells (two compartments: basal, lumenal) → - spermatogonia (2n) undergo mitosis (divide indefinitely after puberty) - Secondary spermatogonia grow and develop in primary spermatocytes (2n) - With meiosis 1 they produce secondary spermatocytes (n) → they undergo meiosis II to become spermatids (n) - Spermatids (n) undergo maturation and development stage to become spermatozoa (n) (male gametes) Proliferation period - Spermatogonia Mitosis (9-10 times) - Begins in embryonic life and continues through life time - During embryonic life Sertoli cells produce inhibitor of meiosis – PG D2 - PGC → primary (A) spermatogonia (2n) - Primary spermatogonia → secondary (B) spermatogonia (2n) Growth period - Begins in puberty - Secondary spermatogonia grow → primary spermatocytes (2n) - Growth period is weak comparing to oogenesis Maturation period - Two meiosis - Lasts for 22 – 24 days - Cytoplasmic bridges – spermatogonial syncytium: o Synchronize spermatogenesis in one seminiferous tubule o E.g. AKAP82 – protein, used for tail formation 55 Role of syncytium - Akap82 protein – important in tail formation - Akap82 gene is located on X chromosome Cytokinesis - By help of actin – myosine contractile ring (cleavage furrow) - Cytokinesis is equal, but incomplete: o Two cells of identical size o Male’s sex cells precursors remain joined by cytoplasmic bridges, which resolve only in the end of spermatogenesis Spermiogenesis Spermiogenesis – steps - Formation of an acrosome - Nuclear morphogenesis - Formation of tail structures - Rearrangement of organelles - Sheding most of the cytoplasm Spermiogenesis: Formation of an acrosome - Formed from golgi complex - Specialised lysosome, contains digestive enzymes (hydrolases), e.g. hyaluronidase, acrosin, neuraminidase - Enzymes allow sperm to penetrate oocyte Nuclear morphogenesis - Changes in shape (from round to oval) - Becomes more dense - Chromatin condensation - Sperm specific histones are produced 56 Rearrangement of organelles – Formation of tail structures - Elongation of microtubules - Distal centriole elongates Rearrangement of organelles - Mitochondria fuse and form spiral around the tail Sheding most of the cytoplasm Basic structure of human sperm cell - Sperm (Spermatozoa) functions: o To deliver its set of genes to the egg o To activate the egg - Basic knowledge: sperm: haploid (n) → 22 autosomes, 1 sex chromosome; acrosome contains enzymes necessary for fertilization; mitochondria → produce ATP needed for moving; flagellum moves in a whip like motion to propel the sperm forward Abnormal male gametes 57 Abnormal sperm and sperm production syndromes - Azoospermia: no sperm in semen (ejaculate) - Oligozoospermia: low sperm count (< 20 million spermatozoa per ml of ejaculate) - Sertoli cell only syndrome: in seminiferous tubules only Sertoli cells - Kartegener syndrome: hereditary condition affecting the motility of spermatozoa flagella Spermatogenesis and oogenesis in comparison 58 Molecular aspects of fertilization 59 Acrosomal reaction - Acrosome undergoes exocytosis – sperm releases digestive enzymes into the zona pellucida Sperm-Egg recognition & binding - sperm migrates through the coat of the follicle cells (corona radiata) and binds to a receptor molecule in the zona pellucida of the egg (secondary oocyte) Sperm-Egg recognition & binding Receptors of Zona pellucida - ZP3 mediates sperm-specific egg binding - ZP2 mediates subsequent sperm binding - ZP1 important for structural integrity of zona pellucida Acrosomal reaction: Reception of sperm and egg membrane receptors - With the help of acrosomal reaction the sperm reaches the egg, and a membrane protein (ADAM) of the sperm binds to a receptor (ZP2) on the egg membrane 60 Contact and fusion of sperm and egg membranes - plasma membranes fuse making it possible for sperm cell to enter the egg Contact and fusion of sperm and egg membranes – cont. - A key signal from the binding – an increase in the level of Ca2+ in the egg cytoplasm o Signals the completion of meiosis in the oocyte, is triggered by Ca2+ - dependent on activation of the anaphase-promoting complex o Ca2+ induced exocytosis of secretory vesicles Cortical reaction - Enzymes released from corticular granules harden Zona pellucida - Block the polyspermy (allow monospermy) Summary: Fertilization 61 After sperm cell enters the egg… - Basal body of the sperm’s flagellum divides and forms the centrioles of the zygote - Male and female pronuclei form synkaryon From fertilization till cleavage Cleavage - Series of mitotic divisions and cytokinesis that transform the zygote Significance of fertilization - Changes in the egg’s cytoplasm are induced - The diploid chromosome number is re-established - Mixing of paternal and maternal chromosomes take place 62