Kansas University Medical Center. Kansas City, USA

Transcription

1 Follicle l Regulation Of Oocyte Maturation David F. Albertini, Ph.D. Kansas University Medical Center Kansas City, USA

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3 Acknowledgements Albertini Lab Susan Barrett Patrícia Rodrigues Lynda McGinnis Karla Hutt Stephanie Fiedler Darlene Limback Josh Ecklund Gloria Lee KUMC University of South Florida David Keefe and Lin Liu Center for Reproductive Sciences Bill Kinsey, Paul Terranova, and Brian Petrov Kumar and Christenson Labs University of Lisbon Portugal Carlos C. Plancha Alexandra Sanfins COLLABORATORS Technobios/ os/ Bologna Italy Gio Coticchio Lucia DeSantis Sandra Cecconi NIH HD, ESHE Fund, Hall Family Foundation Fundação Ciência e Tecnologia Lisbon, Portugal Woscester Polytechnic Institute Eric Overström Elena Ibañez

4 OOGENESIS months HOURS FOLLICULOGENESIS EMBRYOGENESIS DAYS Temporal Coordination Optimizes Egg Quality From 2 cell/2 gonadotropin theory to 3cell/3 factors Paradigm shift: from endocrine to oocyte maturation (fewer follicles for egg retrieval, decrease multiples)

5 OUTLINE The importance of oocyte maturation to egg and embryo quality The acquisition and maintenance of meiotic competence during early folliculogenesis The acquisition and maintenance of developmental competence Gonadotropin and Growth Factor Actions during Cytoplasmic Maturation: Why In Vitro Matured Oocytes are developmentally deficient.

6 From simplicity to complexity The Biological Questions: 1. How is meiotic resumption and completion synchronized with ovulation? Hunter shows offset compromises egg quality (1970s) Release ease from inhibition/ Induction/ or both.factorology.mis, o ogy FFFs, FF-MAS 2. Are the rules governing nuclear and cytoplasmic maturation equivalent or distinct? 3.Is the trigger simplistic or complex? 4.Is the oocyte preprogrammed ed to support post LH dependent cytoplasmic c maturation? Along come the knock out mice that establish that these events are coupled and require both oocyte and granulosa cell specific genes

7 The Problems Humans exhibit 30-40% rates of embryonic or early fetal mortality(noyes y et al 1952, AJOG; Edwards et al 1982 Fert Ster) Human preimplantation embryos account for more than half of this loss and exhibit high rates of chromosome imbalance( Munne et al 2003 Reprod Biomed Online) Most IVF cycles cles fail after ET BUT before implantation (Schieve, e CDC) In Vitro procedures(ivm,ivf,icsi,ec) have been linked to deficiencies in embryo quality evidenced by decreased cell numbers,cell allocation and ES cell derivation(tieleus et al 2006), and altered gene expression and translation Rinaudo and Schultz, 2004; Corcoran et al 2006). Suggests that maintaining follicular regulation of oocyte in situ improves pregnancy outcome

8 From Matzuk

9 Genetic Determinants in Oogenesis Preantral Fig α Cxn 37 ZP2 & 3 GDF9 FSH Ovulatory Cmos Cdc25 formin2 BMP-15 PTX-3 HAS-2 PR COX-2 TCL1 BUB3 Cyclin A2 Dnmt 1 Mater NPM-2 Tubg1 Embryonic Preovulatory FSHR Cyclin D2 ArKO IRS-2 LH Oocyte Granulosa

10 Oocyte Maturation Nuclear and Cytoplasmic Maturation GV M-1 M-2 Oocyte Growth Developmental Competence

11 Communication Breakdown oogenesis folliculogenesis Control FSH-β GDF-9 Cx-37 O=F O=F O>F O<F

12 The Oocyte As Dominatrix Dictates rate of follicle development Eppig et al (2002) Proc. Nat. Acad. Sci. 99:2890 Secretes dominant paracrine effectors (GDF9, BMP-15) Expresses circadian clock Dong components et al (1996) Nature 383:531 Galloway et al (2000) Nature Genetics 25:279 Juengel et al (2002) Biol Reprod. 67:1777 Klementiev and Tischkau (2002) Biol. Reprod. 66:101

13 Stages of IVM:mouse oocytes T0 GV T4h Prometaphase 1 T7h Metaphase 1 T8h anaphase 1 T12h telophase 1 T14h metaphase 2

14 Oocyte Maturation is more than chromosomes

15 Oocyte Quality Requires In Vivo Hormone Stimulation Both cytoplasmic and nuclear maturation are deficient after In Vitro Maturation (IVM) Cytoplasmic defects affect the timing of the first embryonic cell cycle causing delays in the 1 to 2 cell transition Nuclear defects are manifest in failed congression on the meiotic spindle and displacement of the spindle from the oocyte cortex What causes loss PhosphoHis3 spindle of oocyte quality? displaced Spindle MTs PB

16 Ovulation Induction Optimizes Oocyte Quality Allows propagation of physiological signals to induce and sustain oocyte maturation Maintains the microenvironment within the cumulus complex LH "EGFs" Cumulus Expansion Oocyte Maturation Prevent Luteinization Metabolic Loading Sets Egg Clock MURAL CUMULUS OUTCOME

17 Significant differences shown across weeks of stimulation within each oocyte category; Mann-Whitney, p < 0.05 from Combelles and Albertini, BOR

18 Kinetic Analysis of In Vitro Maturation & Incidence of Spontaneous Activation

19 Human Oocytes Are in G-2 Phase of Cell Cycle ph3 - MPM-2 + Interphase Microtubules

20 Repeated stimulation had no effect on embryo quality but oocyte was progressively diminished e.g. ATP content. Thus even compromised mouse oocytes are able to recover when replaced into an in vivo environment.

21 Methods to Establish Kinetics of Embryo Cleavage IVMb IVM+ IVO Embryos fixed and stained for Rhodamine-phalloidin DAPI Scored for cell number and stage % ICM

22 Follicle Development Primordial follicle formation Follicle recruitment Antral cavity Growth and and growth formation maturation of Graafian follicle (After Picton et al. 98) Acquiring Competencies for: MPF activation Genomic imprinting i i Ca 2+ oscillations Histone transfer activity Oocyte Development Mature GV Oocyte (meiotically competent)

23 Contingencies to address FSH E2 Acquires Meiotic Competence Imprinted Time in PA follicle Dmnt1 0 GDF9 Cyclins,cdc25 Activates IF. A.. Primordial Follicle l And all these things happen on time and in the right place Acquires Devtl competence LH Connexin 37 TUBg1 Resumes meiosis cmos Completes meiosis THEN a good oocyte may emerge

24 Conclusions The axis from within the gonad involves interactions between germ line and soma Ovulation induction preserves microenvironment for meiotic resumtion and cumulus activation But endocrine profiling does not guarantee quality eggs as folliculogenesis is accelerated at the expense of oogenesis Negative Regulation In Follicle is key Prevent oocyte from resuming Cell cycle Prevent follicle from Prevent follicle from Differentiating (steroidogensis)too soon!

25 Oocyte Contact Limits Granulosa Cell Proliferative Capacity GDF-9 FSH GDF-9 FSH Status Proliferation OFF ON OFF Differentiation OFF OFF ON

26 Consequences of LH Surge on COC Reception of EGF-ligands from mural compartment Restricted access of LH to the COC due to mucification and junctional uncoupling Metabolic up-regulation for ATP and GSH production and delivery to the oocyte Asymmetric contacts with corona that drive completion of Asymmetric contacts with corona that drive completion of nuclear and cytoplasmic maturation