Study of gazomediators role in the meiosis resumption and early development of Xenopus laevis

La famille des gazotransmetteurs, composée du monoxyde d’azote, de l’hydrogène sulfuré et du monoxyde de carbone, est impliquée dans de nombreux processus cellulaires incluant notamment le contrôle du cycle cellulaire. Au cours de ce travail de thèse, nous avons étudié les effets du monoxyde d’azote et de l’hydrogène sulfuré, sur la reprise de méiose, la progression dans le cycle cellulaire et la fécondation des ovocytes de Xénope ainsi que sur le développement précoce des embryons. Le modèle ovocyte de Xénope est reconnu dans le domaine de l’étude du cycle cellulaire. Nous avons pu démontrer que ces deux gazotransmetteurs inhibaient la reprise de méiose des ovocytes de xénope et que la phosphatase Cdc25c serait une cible clé expliquant cet effet perturbateur. De manière spécifique, le monoxyde d’azote influe sur la morphogenèse du fuseau alors que l’hydrogène sulfuré semble avoir un effet inhibiteur de la synthèse protéique. Parallèlement, une étude, menée en collaboration avec l’université de la vie et des sciences de Prague, a démontré de manière surprenante, des effets positifs de l’hydrogène sulfuré sur la reprise de méiose des ovocytes de porc et a tenté de discriminer les acteurs impliqués tels que la voie MAPK. Enfin, l’hydrogène sulfuré induit une baisse du taux de fécondation et semble perturber le développement des embryons de xénope. L’ensemble de ces travaux permettent d’étoffer les connaissances liant les gazotransmetteurs et la reproduction.Gazomediators family is composed by nitric oxyide, hydrogen sulfide and carbon monoxide. This family is involved in many cellular process like cell cycle regulation. During, this thesis we have investigated the effect of nitric oxide and hydrogen sulfide on meiosis resumption, cell cycle progression and fecundation of Xenopus oocytes (a recognized model for cell cycle researchs) and on early development of Xenopus embryos. We have demonstrated that the two gazomediators inhibited the meiosis resumption of Xenopus oocytes. The Cdc25C phosphatase was a key target for this effect. In a specific way, nitric oxide had an effect on spindle morphogenesis while hydrogen sulfide targeted protein synthesis. Surprisingly, a study conducted in collaboration with the University of Prague demonstrated a positive effect of hydrogen sulfide on the meiosis resumption of pig oocytes and discriminated key targets for this effect such as the MAPK pathway. Hydrogen sulfide also induced a decrease of fecundation rate and seemed to disrupt early development of xenopus embryos. This work allows to expand the knowledge linking gazomediators and reproduction

The spatio-temporal dynamics of PKA activity profile during mitosis and its correlation to chromosome segregation

International audienceThe cyclic adenosine monophosphate dependent kinase protein (PKA) controls a variety of cellular processes including cell cycle regulation. Here, we took advantages of genetically encoded FRET-based biosensors, using an AKAR-derived biosensor to characterize PKA activity during mitosis in living HeLa cells using a single-cell approach. We measured PKA activity changes during mitosis. HeLa cells exhibit a substantial increase during mitosis, which ends with telophase. An AKAREV T>A inactive form of the biosensor and H89 inhibitor were used to ascertain for the specificity of the PKA activity measured. On a spatial point of view, high levels of activity near to chromosomal plate during metaphase and anaphase were detected. By using the PKA inhibitor H89, we assessed the role of PKA in the maintenance of a proper division phenotype. While this treatment in our hands did not impaired cell cycle progression in a drastic manner, inhibition of PKA leads to a dramatic increase in chromososme misalignement on the spindle during metaphase that could result in aneuploidies. Our study emphasizes the insights that can be gained with genetically encoded FRET-based biosensors, which enable to overcome the shortcomings of classical methologies and unveil in vivo PKA spatiotemporal profiles in HeLa cells

Dual Effects of Hydrogen Sulfide Donor on Meiosis and Cumulus Expansion of Porcine Cumulus-Oocyte Complexes

<div><p>Hydrogen sulfide (H₂S) has been revealed to be a signal molecule with second messenger action in the somatic cells of many tissues, including the reproductive tract. The aim of this study was to address how exogenous H₂S acts on the meiotic maturation of porcine oocytes, including key maturation factors such as MPF and MAPK, and cumulus expansion intensity of cumulus-oocyte complexes. We observed that the H₂S donor, Na₂S, accelerated oocyte <i>in vitro</i> maturation in a dose-dependent manner, following an increase of MPF activity around germinal vesicle breakdown. Concurrently, the H₂S donor affected cumulus expansion, monitored by hyaluronic acid production. Our results suggest that the H₂S donor influences oocyte maturation and thus also participates in the regulation of cumulus expansion. The exogenous H₂S donor apparently affects key signal pathways of oocyte maturation and cumulus expansion, resulting in faster oocyte maturation with little need of cumulus expansion.</p></div

Effect of Na₂S on HA content in expanded cumulus.

<p>(A) Total and retained HA content in COCs cultivated with 150–900 µM Na₂S for 48 hs, total HA is related to the control group. (B) Total and retained HA content in COCs during <i>in vitro</i> cultivation with 300 µM Na₂S over 12 h time scale, total HA is related to the control group after 48 h cultivation. (C) Total and retained HA content in COCs and OOXs cultivated with or without H₂S donor, total HA is related to the control group of COCs. H₂S: 300 µM Na₂S. ^a,b,cStatistically significant differences among experimental groups in total HA, ^1,2statistically significant differences among experimental groups in retained HA, *statistically significant differences in total HA between control and H₂S groups (P<0.05).</p

Effect of Na₂S on partenogenetic development of porcine oocytes.

<p>Oocytes were matured with or without Na₂S and partenogenetically activated using calcium ionophore. Pronucleus formation after 24 h zygote culture, cleavage rate after 2 days and blastocyst achievement after 7 days presumptive embryos culture were evaluated (%±SE).</p><p>H₂S: 300 µM Na₂S during oocyte maturation.</p><p>*Statistically significant differences between control and H₂S group – in column (P<0.05).</p

Effect of Na₂S on MPF and MAPK activities during oocyte cultivation.

<p>Representative autoradiograms and signal quantifications of phosphorylated histone H1 (A) and MBP (B) reflecting MPF and MAPK activity, respectively. Kinase activity was measured in oocytes cultivated with or without Na₂S over 2 h time scale. The kinase activity was related to oocytes cultivated for 24 hs. C: control; H₂S: 300 µM Na₂S. *Statistically significant differences between control and H₂S groups (P<0.05).</p

Effect of Na₂S on meiotic resumption and transition to meiosis II during oocyte cultivation.

<p>Proportion of GVBD (A) and meiosis I to II transition (B) in oocytes during <i>in vitro</i> cultivation over 2 h time scale. H₂S: 300 µM Na₂S. *Statistically significant differences between control and H₂S groups (P<0.05).</p

Effect of Na₂S on meiosis resumption and transition to meiosis II in DOs.

<p>Proportion of GVBD (A) and meiosis I to II transition (B) during <i>in vitro</i> cultivation after 20 and 30 h <i>in vitro</i> cultivation, respectively. H₂S: 300 µM Na₂S. ^a,b,cStatistically significant differences among experimental groups (P<0.05).</p

Effect of different Na₂S concentrations on meiosis resumption and transition to meiosis II in oocytes.

<p>Proportion of GVBD (A) and meiosis I to II transition (B) during <i>in vitro</i> cultivation after 20 and 30 h <i>in vitro</i> cultivation, respectively. ^a,b,cStatistically significant differences among experimental groups (P<0.05).</p