Halet Group Research

Signaling in mouse oocytes 

Our research is focused on the study of signaling cascades that contribute to oocyte meiosis, fertilization and preimplantation development to blastocyst. In particular, we have a strong interest in the role of Rho GTPases and actin dynamics during oocyte meiotic divisions.

Actin dynamics play a key role during oocyte maturation (progression from prophase I to metaphase II) and the accomplishment of asymmetric meiotic divisions (emission of the polar bodies). We have shown that Cdc42 GTPase is activated in a polarized fashion in the cortical area overlying the meiotic spindle. Cdc42 recruits its effector N-WASP at the oocyte cortex, thus promoting the formation of the actin cap that characterizes polarized oocytes. Cdc42 inhibition results in a loss of the actin cap, associated with defects in polar body emission. The polarized activation of Cdc42 is induced by maternal chromosomes, in a Ran GTPase-dependent manner. We are now studying the mechanism by which Ran promotes polarized Cdc42 activation. We are also investigating whether Cdc42 regulates other effectors at the oocyte cortex.

Cdc42 recruits N-WASP in the polarized cortex (Dehapiot et al, 2013)

We also study the oocyte polarization process, which translates into the cortical accumulation of signaling proteins (Cdc42, Myosin-II…) and so-called polarity proteins (Par3, Par6) in a restricted cortical area overlying the meiotic spindle. This polarization relies on the activation of Ran GTPase in the vicinity of maternal chromosomes, but the mechanism by which activated Ran promotes cortical remodeling remains elusive. We have shown that Ran promotes not only the polarized activation of Cdc42 (Cdc42-GTP), but also the polarized dephosphorylation of proteins of the Ezrin/Radixin/Moesin family. We are now investigating the signaling intermediates acting downstream of Ran to polarize the oocyte cortex.

The implantation project 

Embryo implantation is a dynamic process visible during a limited period of time called implantation window, relying both the quality of the endometrium and of the embryo. In the clinic, in vitro fertilization techniques (IVF) allow to evaluate, on morphological and growth criteria, the quality of the preimplantation embryo, but do not allow to determine the endometrial receptivity, as measuring the thickness of the endometrium by ultrasound is still insufficient to predict the chances of embryo implantation. In this context, the pregnancy rate is currently 25 to 30% following embryo transfer.

Previous research on the genetic causes of congenital utero-vaginal aplasia syndrome (Mayer-Rokitansky-Kuster-Hauser syndrome), lead us to identify a new molecule expressed almost exclusively in the uterus and called ITIH-5 (Inter-α-Trypsin Inhibitor 5). It is a secreted protein and a component of the extracellular matrix, like other members of its family (ITIH-1 to -4). 

In a former study in mice (where the coding parts of the gene show 81% homology with human, and 88% at the protein level), we identified two isoforms of this protein, embryonic and adult. We have shown that during embryogenesis the expression of ITIH-5 increases strongly and specifically in the female genital tract, from the onset of differentiation up to the end of its development. In the adult uterus, its expression is found at low levels in the absence of gestation, and whatever the stage of the estrous cycle. On the other hand, it is greatly enhanced during gestation with a first peak of expression corresponding to the implantation window, and a second peak during the mass growth of the uterus.

A first maturation phase of the project, supported by a Region Bretagne-FEDER grant, allowed to provide the first proof of concept in humans, as well as the development of immunological tools (monoclonal antibodies) dedicated to the identification of the adult ITIH-5 isoform and to the development of a specific ELISA assay. This phase was also part of an extensive patent filing in Europe and the USA (No. WO2013135836).

The “Implantation” project as a whole, focuses on the uterine expression of the ITIH-5 molecule and includes three overlapping aspects. The first one, involving research and development, is focused on the elaboration and validation of a prognostic and diagnostic kit for implantation failure. It takes place in the framework of a strong CNRS-CHU-CRB partnership already in place and opens the possibility of industrial transfer. The second, which received the support of the French Biomedicine Agency, is a preclinical retrospective study aiming at correlating the levels of ITIH-5 and other relevant and validated markers, for a successful implantation. It aims at providing further and definitive proof of ITIH-5 role in the development and quality of the uterine matrix. It also represents the starting point of basic research on the regulation and mechanism of action of ITIH-5, in straight relation to implantation, which is at term the third aspect of the project. This study, in the mouse, will primarily aim at analyzing the process of interaction between the embryo and the uterine matrix, for which only the embryonic side has been described so far. We will first focus on the still controversial mechanisms of adhesion of the embryo to the endometrium and next on those of trophoblastic invasion.




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