Reference : PhD Norbert Ghyselinck
Differentiation of male germ cells, or spermatogenesis, is a highly compartmentalized and strictly coordinated process with the aim of ensuring a constant production of spermatozoa, necessary for sustainability of the species. It requires complex, sequential interactions between stem germ cells (SGC) capable of self-renewal and somatic supporting cells, the Sertoli cell (SC), during whcih SGC are amplified by mitosis and directed towards meiosis. These interactions are set up at the time of the puberty under the control of vitamin A. This vitamin circulates in the blood in the form of a complex with a transporter protein whose receptor, STRA6, is present in CS. Vitamin A is transformed in CS into its active metabolite, retinoic acid (RA) by retinaldehyde dehydrogenases (RALDH). RA acts, similarly to steroid hormones, through binding to nuclear receptors. Our previous work indicates that RA controls the balance between SGC self-renewal and differentiation, as well as the transition between mitosis and meiosis, from the first wave of spermatogenesis (or pubertal spermatogenesis) onwards.
The thesis work will at first focus on determining the chronology of appearance of RA target cells during pubertal spermatogenesis and to characterize their progeny. This will be done using a transgenic mouse line allowing to label RA-responsive cells, and then to track their fate. In parallel, the candidate will identify genes controlled by RA, in vivo, in SGC and SC during a period of time comprised between the onset of spermatogenesis and the appearance of the 1st meiotic cells, 4 days later. To satisfy this objective, the candidate will proceed to comparative studies of transcriptomes through DNA microarrays using mouse testes containing synchronized germ cell populations. If needed, SGC populations will be purified by FACS.
The analysis will be carried on:
(1) mutant mice carrying null mutations of RALDH in SGC and/or in SC; and
(2) mutant mice carrying null mutations of STRA6 in SC. All these mutants are developed in the host laboratory and are available. Secondly, the physiological relevance of genes highlighted by the transcriptome analysis will be tested by in situ hybridization, immunohistochemistry and functional genomic approaches (gain and/or loss-of-function by testicular infection of SGC or CS using recombinant viral vectors).
The experimental model used by the host team and the related thesis project will allow unravelling molecular mechanisms which underlie:
(1) the initiation of spermatogenesis, which represents a privileged target of endocrine disruptors responsible for the reduction of male fertility in industrialized countries; and
(2) the capacity of RA to promote SGC differentiation. This stem cell population is of therapeutic interest (for gene therapy) and is at the origin of numerous cancers.
EXPERTISES WHICH WILL BE ACQUIRED AND METHODS USED DURING THE TRAINING : Techniques in molecular and cellular biology, and analysis of gene expression (organ culture, cell culture and transfection, shRNA, purification of cells by FACS, histology, immunohistochemistry, in situ hybridization, DNA, RNA and protein extraction, quantitative real-time PCR, immunoprecipitation of chromatin, recombinant lentiviral and retroviral vectors).
Application Deadline : Nov. 1, 2018