Reproduksjonsbiologi Arne Sunde IC 2002

Transkript

1 Reproduksjonsbiologi

2 Hormoner og Reproduksjon Sperm Spermatogenesis Oogensis Meiosis Fertilisation The first week

3 The Testis Tunica albuginea Seminiferous tubuli Epidiymis Vas Deference

4 The Testis Seminiferous tubuli Spermatozoa Germinal epithelium

5 The Testis The different compartments: Seminiferous tubuli Interstitial cells The blood-testis barrier

6 The Testis - The compartments

7 The Testis The compartments Tubuli: Sertoli cells Spermatogenesis Estrogens InhibinA Interstitial cells Blood, lymph Leydig cells Testosterone

8 Testis- Production of hormones LDL-Cholesterol LH FSH Estradiol OH O Testosterone Leydig - cell Sertoli - cell

9 Hormoner og reproduksjon De viktigste aktørene Luteniserende hormon Follikkel stimulerende hormon humant Chorion Gonadotropin - LH - FSH - hcg Glykoproteiner bestående av to kjeder (α β) α - kjedene er like β - kjedene er ulike Sterkt glykosylert (40%) av vekt

10 Hormoner og reproduksjon De viktigste aktørene LH hcg FSH α-kjede 92 aa 92 aa 92 aa β-kjede 115 aa 131 aa 118 aa β - kjeden til LH og hcg er lik fram til aminosyre 115. Forskjell i glykosylering mellom LH og hcg

11 Testis- Production of hormones Regulation trough Negative Feedback Brain TRH Hypothalamus Hypofyse GnRH LH Cholesterol Testosteron FSH Østradiol Inhibin Leydig celle - Peritubular-celle - Sertoli-celle - Sædceller

12 Hormoner og reproduksjon Gonadotropin frigjøringshormon GnRH Dekapeptid pyroglu GLU-HIS-TRP-SER-TYR-GLY-LEU-ARG-PRO-GLT-NH 2

13 Spermcellens historie Primordiale kjønnceller migrerer fra plommesekken til gonaden 3-5 fosteruke. Primordale kjønncellerligger i dvale i primitive sædkanaler omgitt av umodene Sertoliceller ligger fram til pubertet. Sædkanlene differensierer og spermatogenesen starter under puberteten. Spermatogenesen fortsetter i prinsipper til mannen dør. (litt strikkmotorpreget)

14 The sperm cell Structure and basic components Break-in tools Target identification systems Amplitude modulators Genetic packet Motor Whip

15 The spermatozoa - The tail Amplitude modulators Dense fibres Mitochondria in a spiral Microtubules + axonem Whip Middle piece Principal piece Terminal piece)

16 The spermatozoa - The head Break in tools Target identification system Genetics packet Acrosome Equatorial ring Receptors Highly condensed DNA Packed with protamines Instead of histones

17 Meiose Dannelse av haploide (n) kjønnsceller

18 Dipoid celle med 4 kromosompar 1 p 1 m 2 p 2 m 3 p 3 m 4 p 4 m

19 1 1 Dipoid celle med 4 kromosompar 2 4 = 16 mulige kombinasjoner 1 1 p p p p p p p p p 2 p 2 p 2 p 2 m 2 m 2 m 2 m 3 p 4 p 3 m 4 p 3 p 4 m 3 m 4 m 3 p 4 p 3 m 4 p 3 m 4 m 3 p 4 m 1 m 1 m 1 m 1 m 1 m 1 m 1 m 1 m 2 p 2 p 2 p 2 p 2 m 2 m 2 m 2 m 3 p 3 m 3 p 3 m 3 p 3 m 3 m 3 p 4 p 4 p 4 m 4 m 4 p 4 p 4 m 4 m

20 Diploid celle med 23 kromosompar 2 23 = kombinasjoner bare basert på tilfeldig fordeling av kromosomer Rekombinering gir en mulighet for ~ ulike kjønnceller

21 Sperm: Epididymis -Vas deference Temperature control o CNOT 37 o C Epididymis Spermatozoa training camp Progressive motility Zona Pellucida receptors Storage (Cauda Epididymis) Mobilised by rhythmic contractions of Vas deference

22 Spermtransport

23 The first 30 hours- Signalling across membranes Membrane receptors

24 Sperm transport trough the cervix Sperm transport from the cervix Liquefaction Coagulation after ejaculation Liquefy after approx 30 min (proteases from the prostate) Propulsion trough the cervix Progressive linear movement important May remain in the cervix for days The spermatozoa swim along the longitudinal microstructure of the mucus During the preovulatory period the uterus contains fluid facilitating sperm movements In many species, a sperm depot is formed at the isthmus (uterus/fallopian tube) and may stay there for days.

25 Sperm capacitation Capacitation As spermatozoa ascends the female genital tract they are said to undergo a physiological transformation resulting in the attainment of a state of capacitation Ca 2+ increase, camp increase Sperm plasma membrane increase fluidity (cholesterol loss) In the capacitated state, the sperm cell can undergo the acrosome reaction in response to appropriate stimulus. De-capacitating factors The seminal plasma contains factors that prevent capacitation Mask receptors, Cholesterol source Calmodulin like proteins (Ca 2+ binder)

26 Oocytten Metafase-II oocytt Perivitelline rom Pol-legeme Polbody Zona pellucida Oocyttmembran

27 Oogenesis Nuclear maturation Completion of meiosis Cytoplasmic maturation Acquire the functional capacity of an mature oocyte

28 The history of the oocyte Primordial germ cells migrate form the Yolk sac to the primitive gonad in foetal week 3-5. Differentiates to oogonia and thereafter to primary oocytes Starts first meiotic division and is surrounded by a layer of follicular cells (7. Month)

29 Number of oocytes in the ovary

30 Preantral follicles Crosstalk between oocyte and surrounding follicular cells Default pathway (FSH): Mural granulosa cells

31 Oocyte Cumulus cell crosstalk Follicle Formation Proliferation Differentiation Steroidogenesis Cumulus Expansion Ovulation

32 Oocyte Cumulus cell crosstalk Growth Meiotic arrest Transcription Maturation

33 Growth of the oocytes 20 µm ~30 follicles/day start development 60 days 120 µm

34 Growth vs competence 20 µm Ability to undergo Resumption of meiosis 120 µm

35 Oocyte maturation Nuclear maturation Achievement of meiotic competence GVBD, resumption of meiosis Progression to MII Easy to detect by morphological markers Germinal Vesicle Metaphase I stage Metaphase II stage

36 The ovary

37 Produksjon av steroider i ovariet i follikelfasen 2 - celle konseptet LDL-Cholesterol LH FSH Østradiol O O Androstendion Theca - celle Granulosa - celle

38 Hormoner og reproduksjon Cholesterol Hypothalamus Hypofyse LH FSH GnRH Activin Inhibin Androstendion Østradiol Progesteron Theca - celle Granulosaceller Oocytter

39 Hormoner og reproduksjon Produksjon av steroider i ovariet i lutealfasen LDL-Cholesterol LH (hcg) Progesteron Theca lutein - celle Granulosa lutein - celle

40 Oocytten: den omnipotente Egget: Nøkkelen til livet DNA-fra mor (kan byttes ut) Metabolsk niste Mitochondrier (kan byttes ut) Umodent (GV) egg m-rna (informasjonsbærere) Transkripsjonsfaktorer (regulatorer)

41 Meiotic arrest Meiosis arrested at prophase I (chiasmata formed) The arrest actively maintained by corona cells who pump in and maintain a high camp level in the oocyte

42 Meiotic arrest Meiosis arrested at prophase I (chiasmata formed) The arrest actively maintained by corona cells who pump maintain a high camp level in the oocyte Connexin43 expression in corona cells downregulated by LH

43 Nuclear maturation GV-stage foetal life to 1 day prior to ovulation Metaphase I Chromosomes aligned on a metaphase plate Still 4 sets of chromosomes Metaphase II Chromosomes aligned on a (MII) metaphase plate Half the chromosomes expelled in the polar body Ready for fertilization

44 Extracellular matrix Interaction with the cumulus mass Extracellular matrix Mainly hyaluronic acid In animals, only hypercativated, acrosome intact spermatozoa can penetrate the cumulus mass. The sperm have receptors for hyaluronic acid which might modulate sperm movements

45 Binding to the Zona pellucida The Zona pellucida comprises three major glycoprotein species, ZP1 and ZP2 forms an open porous matrix formed by interconnecting filaments (ZP2 filaments held together by ZP1) ZP3 plays a role in sperm recognition The sperm receptor ZP3 -/- female mice infertile

46 The acrosome reaction As a consequence of the spermatozoa to the ZP3 receptor, the cell undergo a secretory event known as the acrosome reaction. focal fusion of the plasma membrane and the acrosomal membrane releases of the most soluble components of the acrosome vesicle vigorous movements of the sperm drives the sperm trough the zona pellucida into the perivitelline space

47 The acrosome reaction

48 Sperm-oocyte fusion During the course of the acrosome reaction, a discrete band of plasma membrane around the equatorial segment of the sperm head suddenly acquires the capacity to recognise and fuse with the vitelline membrane of the oocyte Prior to the acrosome reaction, the spermatozoa has no capacity to interact with the oocyte

49 Sperm-oocyte fusion Several molecules implicated in the fusion process that are similar to viral fusion proteins

50 The first 30 hours- Ca-oscillations Sperm entry induces oscillations in the intracellular free Ca 2+ level The model:

51 The first 30 hours- Oocyte activation Cortical granule release Appears to be induced by the calcium transients Prevents polyspermy by inducing changes in the zona pellucida The defence against polyspermy: The slow zona block The fast vitelline block

52 Pronuclear formation The sperm aster

53 Sperm aster (centrosome) formation Unfertilised oocyte 3-6,5 h post insemination sperm astral microtubules assemble around the base of the sperm head

54 Sperm aster (centrosome) formation Duplication of the centrosome (15 h post insemination) Sperm aster enlarge and seek out the female pronuclei Prometaphase Bipolar array of microtubules marks the first mitotic spindle Sperm axoneme still visible

55 The sperm The sperm provides 3 essential components The paternal chromosomes The centrosome The oocyte activating factor(s) inducing Ca 2+ oscillations

56 Sperm nucleus decondensation The sperm chromatin is packed using protamines The sperm chromatin must be reorganised Protamines must be replaced with histones

57 The first 30 hours- Oocyte activation Following sperm-oocyte fusion, the oocyte becomes activated and initiates the cascade of events that culminate in the initiation of embryonic differentiation The release of cortical granules The resumption of meiosis The formation of male and female pronuclei

58 Germinal vesicle Cellekjerne med 4n DNA Fra fosterliv Follikkelfase Germinal vesicle breakdown Kjernemembranen forsvinner Meiosen starter opp igjen Metafase-I oocytt Metafase-II-oocytt LH Ved eggløsning

59 Metafase-II oocytt Befruktning Siste meiotiske deling Dannelse av pronuklei 6-8 timer Fusjon av pronuklei Mitose timer timer

60 Maternelle vekstfaktorer, kofaktorer m-rna etc (oocytt-niste) Totipotente celler Ekspresjon av fetale genom

61 Human embryo development Expression of embryonal genes Day 2 Day 3 Day 4 Day 5 Day 5/6

62 Feil under fertilisering 3PN zygoter Feil kromosom segregering Kan utvikle seg til termin Enkelte zygoter kan reorganisere til normal 2n genotyp eller til 2n/3n mosaikk Bare kvinnenes arvemateriale Placenta dannes ikke og utviklingen stopper ved implantasjonen Bare mannens Arvemateriale Selve embryoet dannes ikke kun anlegg til placenta Blæremola

63 Hva skjer? What can I do? Who am I? We won! We are friends Anybody here

64 Det er fysiologisk med store genetiske avvik tidlig i embryoutviklingen hos mennesker Frequency of gametes/zygotes/embryos with wrong number of chromosomes 30% in all cells 80% one or more cells 40% 20% in all cells 100% one or more cells 30% oocytes 10% spermatozoa

65 The old view Gametogenesis A process designed to produce the best sperm and oocytes 200 million sperm cells a day 1/ chance of winning the lottery on a lucky day oocytes a day oocytes lost each month One dominant follicle ovulates the winner

66 The old view the winner is

67 The old view Fertilization and early embryo cleavage A biological selection procedure Only the best embryos will develop and implant

68 The old view Gametogenesis, gamete transport, fertilization and early embryo development Designed to weed out the bad ones

69 Gametogenesis Are genetically abnormal gametes deselected during gametogenesis? Yes to a certain extent Balanced/unbalanced translocations Klinefelter - sperm. No selection of oocytes during the final maturation Follicular dominance not correlated to genetic health of the oocytes

70 Gamete transport Genetically abnormal gametes deselected? Sperm Prior to ejaculation unknown After ejaculation the female genital tract Strong selection of correct phenotype Weak selection of normal haploid sperm Oocytes - unknown

71 Fertilization Are genetically abnormal gametes deselected during fertilization? Seemingly only on phenotype

72 Conventional IVF vs ICSI ICSI bypass a lot of biological checkpoints Seemingly without creating problems

73 ICSI vs IVF Major differences between IVF- ICSI The oocyte is a robust little machine Can use different biological programs to start the embryo development

74 Hints from the past Average fecundity in humans 19-20%. Young fertile couples trying to conceive: In 60% of the cycles hcg could be detected in serum in the luteal phase hcg secreting embryos In 60% of these cases, only transient synthesis of hcg Embryos dying Large embryo wastage at the time around implantation

75 The CEMAS II Study København, Gøteborg Embryo culture until day 3 Blastomere separation FISH analysis 127 embryos 474 blastomerer FISH analysis of X, Y 13, 16, 18, 21, 22

76 The CEMAS II Study- Results Number of with all blastomeres normal Normal embryos (%) Abnormal embryos (%) Inconclusive embryos (%) 47 (37 %) 67 (53 %) 13 (10 %)

77 The first cleavages: A dangerous game No checkpoint control during the first mitosis Number of abnormal cells increase during the first cleavages

78 Aneuploidy rate 30% 10-15% Fertilization Zygote 4 cells, 2 days 35% 50% 8-cells, 3 days >50% 0,6 % 10-15% Blastocyst 5/6 days 100%? 30-50%

79 Aneuploidy rate What happens to all the aneuploid cells? Dependent on the cell type? Germ line stem cell markers as early as 8-cell stage Dies out? What if it was an important stem cell? Other stem cells can be recruited? Rerouted to less important functions? Placenta, membranes.. Persists.mosaicism

80 Phenotype vs genotype What if some of the phenotypic variation is secondary to early embryo aneuploidy Chromosomes involved Frequency i.e. proportion of cells affected Cell lineages affected Mosaicism

81 Placing the quilt Humans have a low fecundity (20%) Humans have low genetic quality of their gametes Early human embryo development imperfect Who shall we blame for this?

82 I m dreaming of a guy walking on his two feet and with brains A. afarensis

83

84 The big brain Because we needed it? Or because it was attractive?

85 Larger brain Narrow pelvis Premature birth Child care Needs a father How to keep him Bond him with sex A lot of sex A few pregnancies Bad sperm, bad eggs Quality control downstream

86 My hypothesis Humans are not designed to be efficient breeders on the contrary Low genetic quality of gametes and embryos Synchronisation of menses Bonding was more important to establish sufficient large cooperative groups Sex and small talk

87 Implications for ART Weak correlation between genotype and phenotype in human gametes. Biological quality control at/after implantation Human gametes and embryos can tolerate a lot of abuse without this leading to malformations in the offspring

88 Embryokloning - embryosplitting Embryo splitting Skjer naturlig (eneggede tvillinger/trillinger) Kan også utnyttes bevisst Naturens egen metode

89 Eneggede flerlinger Separate morkaker og fosterhule (diamnionale/dichorionale - di/di ) Forskjellig morkake, samme fosterhule (mo/di) Deler fosterhule og morkake mo/mo Deler organer (siamesiske tvillinger)

90 Flerlinger

91 Inneholder totipotente celler Befruktning Pronuklei stadiet 2-celler, et døgn 8-celler, 3 dager 4 celler, 2 dager Trofoektoderm Indre cellemasse Morula, 4 dager Blastocyst, 5/6 dager Inneholder multipotente celler

92 Generering av Embryonale stamceller (ES-celler) Trofoektoderm Indre cellemasse Isolert indre cellemasse Dyrkning og stadige nye omsettinger av cellekulturen i løper av 3-4 uker Fibroblaster som feeder celler Kultur av Embryonale stamceller (ES-celler)

93 Kloning somatisk cellekjerne overføring Cellekultur av somatiske celler Isolert cellekjerne overføres til egg uten eget DNA Donor egg DNA Egg uten eget arvemateriale Fusjonering av cellekjerne og egg 8-celles pre-embryo Embryonale stamceller Indre cellemasse Blastocyst