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Equipes de recherche - Biologie du Développement de Villefranche sur mer
Unité Mixte de Recherche CNRS 7009

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Ovogenèse et maturation méiotique

Oogenesis and oocyte maturation

 

Clytia is well suited for studies of oogenesis and oocyte maturation. Under laboratory conditions each medusa produces eggs daily, spawning being precisely controlled by the light-dark cycle such that unfertilised eggs can be reliably collected 2h after the beginning of a light period, following at least 2h darkness (see Amiel & Houliston, 2009)

Remarkably, Clytia gonads isolated from the adult by simple dissection and cultured in filtered seawater undergo successive cycles of oocyte growth and ovulation for several days, responding normally to the light cue that induces spawning and maturation. These processes, are illustrated in these  time-lapse movies of oocyte growth in an isolated Clytia female gonad (18h period) and of the same gonad under going meiotic maturation  (2 h period).

 

 

 

 

 

 

 

Isolated female gonad,

with rhodamine dextran injected into

a growing oocyte (top left) and into the

gastroendodermal cavity

 

 

Oogenesis


The Clytia female gonad consists of a collection of germline precursors, meiotic cells and vitellogenic oocyte stages, sandwiched between a layer of columnar endodermal cells, and a thin overlying ectoderm layer. Early differentiating stages are positioned close to the radial canals, and vitellogenic stages more distally.
Cohorts of small Stage I oocytes embark on their final growth phase each day following spawning. Vitellogenesis (yolk accumulation) is completed after approx.13-18 hours.

 

 

 

 

 

 In Clytia, the animal-vegeal polarity of the unfertilised egg  directs development of the future oral-aboral axis of the larva, as a consequence of RNA localisation for the maternal determiants CheFz1, CheFz3 and CheWnt3 (Momose and Houliston, 2007, Momose et al 2008).  
Animal-vegetal polarity first starts to develop during the latter stages of vitellogenesis, when the large nucleus (or GV for Germinal Vesicle) loses its central position and becomes positioned progressively closer to the future animal pole (Amiel and Houliston, 2009).
CheFz1, CheFz3 and CheWnt3 mRNAs  become localised to different sites along the AV axis via mechanistically and temporally disting localisation pathways during vitellogeneis and oocyte maturation.

 

 

 

 

 

 Figure adapted from Amiel and Houliston, 2009

 

Oocyte maturation and spawning


As in other animals, the meiotic division cycle in hydrozoans is arrested in prophase of first meiosis during oocyte growth. Meiosis resumes at the time of spawning, as part of the maturation process by which oocytes acquire the ability to be fertilised. After completion of meiosis and emission of two polar bodies, the cell cycle arrests again in G1 until fertilisation  At the end of the maturation period oocytes are released through rupturing of the overlying epithelium. Maturation and spawning are generally triggered in relation to the day–night cycle, either by a light cue after a dark period or by darkness after light (Honegger et al., 1980). The light/dark stimulus causes the tissues of the gonad to release a diffusible factor, probably a peptide, which acts rapidly on the oocyte (Ikegami et al., 1978). The immediate intracellular consequence is a rapid rise in cAMP concentrations (Takeda et al., 2006). Elevated cAMP in turn leads to germinal vesicle breakdown (GVDB), due to activation of the universal M-phase kinase Cdk1-CyclinB (MPF).
In Clytia, spawning occurs about 120 minutes after the light signal, with GVBD, typically occurring after 15-20 minutes. The first polar body forms after about 50-60 minutes and the second after 80-90 minutes. Maturation of isolated oocyes can be conveniently be triggered experimentally by treatment with the cell-permeable cAMP analogue Br-cAMP (Freeman and Ridgway, 1988; Amiel and Houliston, 2009;

 

 


The kinase Mos in Clytia oocyte maturation (Amiel, Leclère et al, 2009 ).
We  found that as in vertebrate and starfish oocytes, Mos synthesis during oocye maturation triggers activation of the MAP kinase cascade and  mediates the cytostatic arrect of the unferilised egg.
Curiously, two distinct Mos genes were found in our EST collection. Both these Mos kinases had cytotatic activity when tested in Xenopus or Clytia embryos, and their expression was detected exclusively in germ cells.

We tested Clytia Mos function by injecting isolated oocytes with antisense morpholino oligonucleotides prior to Br-cAMP treatment. The oocytes spontaneous activation indicating that Mos plays an evolutionarily conserved role in occyte cytostatic arrest. A second striking phenotype  was suppression of polar body formation, reflecting the failure of the first meiotic spindle to position correctly at the oocyte cortex and the second spindle to adopt a correct bipolar morphology. We propose that spindle positioning at the cortex as is also an ancestral and conserved role for the Mos/MAP kinase cascade. These functions in Clytia is mostly accounted for by one of the two genes, CheMos1. The CheMos2 gene may rather have adopted during evolution an earlier role in oogenesis.

Mohamed Khamla - 13/03/13