Resumen de Molecular physiology of a teleost oocyte aquaporin: evolution, regulation and role during oocyte hydration / Fisiología molecular de una acuaporina ovocitaria de teleósteos: Evolución, regulación y papel durante la hidratación del oocito
Cinta Zapater Cardona
In marine teleosts that spawn pelagic eggs (pelagophils), the process of oocyte hydration that occurs during meiosis resumption is a key physiological process for the survival of the eggs in the ocean. Previous studies have discovered the role of a teleost-specific aquaporin water channel (Aqp1b) during fish oocyte hydration, but direct experimental evidence for the function of Aqp1b in oocytes is still lacking. In addition, the molecular regulation of the Aqp1b-mediated mechanism remains poorly understood. In this context, the main objectives of the present thesis were to investigate the evolutionary origin of aqp1ab in teleosts, to provide functional evidence of the role of Aqp1b during oocyte hydration, and to begin to dissect the molecular mechanisms involved in the transcriptional regulation of aqp1b in the oocyte of marine teleosts. By integrating the molecular phylogeny with synteny and structural analyses we show that the teleost aqp1aa and -1ab paralogs (previously annotated as aqp1a and -1b, respectively) arose by tandem duplication, and that the Aqp1ab C-terminus is the most rapidly evolving subdomain within the vertebrate aquaporin superfamily. The functional role of Aqp1ab was investigated in Atlantic halibut (Hippoglossus hippoglossus), a marine acanthomorph teleost that spawns one of the largest pelagic eggs known. Using immunological inhibition of Aqp1ab in halibut oocytes and artificial expression of the halibut paralog Aqp1aa, we demonstrate that Aqp1ab is required for full hydration of oocytes undergoing meiotic maturation. To investigate the aqp1ab transcriptional regulation in oocytes, we isolated the 5’-flanking region of the gilthead seabream (Sparus aurata) aqp1ab gene which contains regulatory cis-elements for the nuclear progestin receptor (Pgr) and SOX transcription factors. The Pgr, as well as sox3 and -8b transcripts, are co-expressed in seabream oo-gonia, whereas in primary growth oocytes, when aqp1ab mRNA and protein are synthesized, the Pgr is translocated into the nucleus. In contrast, sox9b is highly expressed in more advanced oocytes showing the depletion of aqp1ab transcripts. In the seabream, four different pgr transcript variants are expressed in primary growth ovaries which are generated by alternative pre-mRNA splicing. Seabream wild-type Pgr shows the highest transactivation response to progestins such as 17?,20?-dihydroxy-4-pregnen-3-one (17,20?-P) and 17?,20?,21-trihydroxy-4-pregnen-3-one (17,20?,21-P), whereas two of the N-terminally truncated Pgr isoforms regulate novel nuclear and cytosolic mechanisms of dominant-negative repression of Pgr-mediated transcription. Transactivation assays on the aqp1ab promoter demonstrated that aqp1ab transcription is dependent on wild-type Pgr, with Sox3 and -8b acting synergistically, while Sox9b acts as a repressor. Incubation of primary ovarian explants in vitro with 17,20?-P, followed by chromatin immunoprecipitation, confirmed that 17,20?-P-activated Pgr enhanced aqp1ab promoter activation. The production of 17,20?-P in seabream primary growth ovaries in vivo was consistent with the expression of P450c17-II (Cyp17a2) and 20?-hydroxysteroid dehydrogenase (Cbr1), enzymes needed for progestins synthesis, in granulosa cells associated with primary growth oocytes, and with a high concentration of 17,20?-P. Incubation of primary ovarian explants with recombinant piscine follicle-stimulating hormone (rFsh) in vitro stimulated 17,20?-P synthesis, which was reduced in the presence of Cbr1 inhibitors. The rFsh-mediated production of 17,20?-P correlated with the up-regulation of cyp17a2 and cbr1 transcription, as well as of wild-type pgr mRNA and protein levels. Altogether, these data suggest that aqp1ab transcription in seabream primary growth oocytes is under Fsh regulation through the synthesis of progestins. The results of this thesis show that the Aqp1ab mediated mechanism for oocyte hy-dration is likely conserved in marine teleosts. In addition, the tight transcriptional reg-ulation of Aqp1ab during oogenesis highlights the essential physiological role of this water channel and opens new research avenues for understanding the molecular basis of egg formation in marine fish.
