Transcriptional regulation of genes involved in oogenesis in european sea bass (dicentrarchus labrax)

Resumen

Ovarian maturation in European sea bass, as in other teleosts, is a highly synchronized event. Photoperiod and temperature variations are integrated by the brain that will communicate with the gonads through the gonadotropin hormones. By binding to their receptors in the gonads, the Fsh and Lh will activate different intracellular pathways and modify the transcription of several genes, leading to a change in steroidogenesis and finally to the maturation of the ovarian follicles. The initial aim of this doctoral thesis was to study the transcriptional regulation of some crucial genes involved in this process: the gonadotropin receptors (fshr and lhr), two steroidogenic enzymes (cyp19a1a and star) and a crucial ovarian transcription factor (foxl2). For this purpose, one of the main objectives was to establish a stable line of ovarian follicular cells. Although this purpose was not reached, the methodology of cell isolation was substantially improved in terms of quantity of cells obtained and proliferative activity. This technique allows the obtainment of numerous follicular cells that maintain follicle-specific gene expression and steroidogenic capacities, which can be available all year long and can be sent to any laboratory. The conservation of numerous cells in liquid nitrogen provides homogeneous material to perform a given study, strongly reducing the heterogeneity generally observed within these systems. A stimulation protocol was established to avoid interferences of medium changes in cell dynamics and responsiveness. Gene overexpression was possible in freshly extracted cells, but this technique was not successful in frozen-thawed follicular cells, and an effective transfection system still has to be developed for them. These cells were not responsive to gonadotropin stimulation, in terms of estradiol production and enhancement of cyp19a1a and star, as they probably require interactions with the oocyte for many of their derived actions. Finally, during this doctoral thesis, these cells were used to study in vitro regulation of gene transcription by exogenous factors. The cAMP/PKA pathway is one of the main intracellular pathways controlled by the activation of G-protein coupled receptors such as Fshr or Lhr. In the sea bass FTF cell system, the 8Br-cAMP, a cAMP analog compound, was able to modify the transcription of numerous genes involved in follicular physiology. Various kinds of responses were observed following exposure to 8Br-cAMP. A primary increase of cyp19a1a transcription could be observed after 2 h, followed by a secondary surge after 6 to 24 h of exposure, which could be mediated by the corresponding up regulation of Foxl2 and Foxl3 transcription factors. cyp19a1a up-regulation leads to an increase in estradiol levels 6 h after stimulation. These estradiol levels were sufficient to significantly modify lhr transcription at 12 h, which is also clearly upregulated by direct addition of exogenous estradiol. The transcription of fshr and star were quicky and strongly up-regulated by exposure to the 8Br-cAMP. These observations suggest an involvement of the cAMP/PKA pathway in the direct and indirect transcriptional control of several genes involved in follicular physiology. The promoter regions of lhr, star and foxl2 were cloned to study their transcription regulation together with those of cyp19a1a, fshb and fshr, which were isolated in previous studies. FSHR/Fshr transcriptional regulation is partially elucidated in mammals but very few information is available in teleosts. The involvement of the human USF transcription factors in the activation of the sea bass fshr promoter was shown by studying their effect on promoter-driven luciferase activity. Next, three different sea bass genes coding for Usf proteins and named sbsusf1, sbsusf2a and sbsusf2b were isolated from sea bass ovarian follicular cells and for the first time in a teleost species. The phylogenetic position of usf2a and usf2b points to their possible origin during the teleost-specific whole genome duplication. Despite their ubiquitous expression, they are involved in the regulation of fshr, a tissue specific gene. Indeed, sbsUsf2a and sbsUsf2b are able to enhance the luciferase transcription driven by fshr 5’-flanking region, and the consequent luciferase activity. This effect was mediated by the binding of both proteins to an E-box (CACGTG) located in the 5’ UTR of the fshr gene. These data suggest that at least two Usf proteins are fully functional in the sea bass ovarian follicular layer that could play a crucial role in its reproductive success as they directly regulate fshr expression. Foxl2 transcription factor, described as critical for ovarian development and function in mammals, was also investigated during this work. foxl2 and foxl3, two ancient paralogs which emerged before the teleost whole genome duplication, were isolated in sea bass. Gonadal expression patterns point to a strong sexual dimorphism, and the mRNA levels of foxl3 in testis vary significantly during the reproductive cycle. The results obtained in this work highlight the conserved role, across vertebrates, of Foxl2 in ovarian development and physiology, but also in reproduction at the pituitary level, through its interactions with Nr5a family members to control cyp19a1a and fshb transcription. Overexpression of exogenous foxl2 in follicular cells showed its involvement in the transcription, at least indirectly, of fshr, lhr, star and cyp17a1. Both Fsh and Lh enhanced foxl2 expression in a primary culture of a post-vitellogenic ovaries while foxl3 was up-regulated only after short exposure to Lh. Unexpectedly, foxl2 transcription was not enhanced by estradiol stimulation in vitro. A putative binding site and role for Foxl3 is reported also for the first time in a vertebrate species. Similar to Foxl2, Foxl3 might be involved in ovarian development and interact in an analogue fashion with Nr5a family members through the same DNA binding sites, as the one located in the cyp19a1a promoter region. However, the different regulation of their expression, suggests that the functions of Foxl2 and Foxl3 might be determined by a different transcriptional regulation and interaction with other transcription factors. In summary, this doctoral thesis includes the improvement and characterization of an in vitro system to study European sea bass ovarian follicular physiology. It also contains the description of putative roles for Foxl, Nr5a and Usf family members in the regulation of some genes involved in ovarian maturation in sea bass. Foxl and Nr5a families could interact in vivo to regulate cyp19a1a transcription. Both Foxl2 and Folx3 can potentially regulate the transcription of similar targets but their different expression pattern might indicate different functions. Foxl2 is probably involved with Nr5a family in fshb expression control. The fshr transcription might be regulated in vivo by Usf family members, in the same way as it is in vitro. These results give new insight in the complex regulation processes involved in the synchronization of the ovarian maturation.