Browsing by Author "Rodrigues, Bernardo Coutinho"
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- Aquaporin-9 as a molecular partner of CFTR in the Blood-Testis BarrierPublication . Rodrigues, Bernardo Coutinho; Oliveira, Carlos Pedro Fontes de; Silva, Branca Maria Cardoso Monteiro da; Alves, Marco Aurélio Gouveia; Calamita, GiuseppeThe lack of knowledge regarding the etiology of male infertility boosted several investigations focused on male reproductive structures, particularly on male gonads - the testes. These paired organs are pivotal not only for the correct maintenance of steroidogenesis, but also to allow the occurrence of spermatogenesis, the process through which immature germ cells grow and differentiate into spermatozoa. Within testes, Sertoli cells perform an essential role in the control of spermatogenesis by: (1) developing a structural and nutritional support; (2) establishing a protective barrier to germ cells, the blood-testis barrier; and (3) controlling the balance of the seminiferous tubular fluid composition. To achieve this balanced environment, which is critical to the maintenance of membrane potential, osmotic balance and to allow the fluid movement, Sertoli cells are supported by several membrane proteins, including Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Aquaporin-9. CFTR is a glycoprotein responsible for transepithelial salt and water transport, regulating Cl-/HCO3- exchange. Malfunctions in this protein have been associated with severe clinical outcomes, such as Congenital Bilateral Absence of Vas Deferens in male reproductive tract. In addition to its role, CFTR often interacts with several other plasma-membrane proteins, including Aquaporin-9. Aquaporin-9 is an Aquaglyceroporin permeable to water and to a variety of other solutes, including lactate, glycerol, urea, adenine and uracil. Several studies identified the close interaction between CFTR and Aquaporin-9 in the male reproductive tract, including in rat Sertoli cells. This specific interaction was further proved to be pivotal in water permeability and ionic balance, without which male infertility may be a reality. With that said, the full enlightenment of the molecular mechanisms behind these channels function in the human reproductive system is crucial. Herein, we evaluated the physiological significance of Aquaporin-9 and CFTR mRNA and protein expression in human Sertoli cells primary cultures. We also investigated the repercussions of inhibiting each one of the proteins with specific inhibitors for Aquaporin-9 (Phloretin) and CFTR (CFTR(inh)-172) in human Sertoli cells viability and function. Our results showed, for the first time, that Aquaporin-9 is highly expressed in human Sertoli cells. We were also able to demonstrate that Phloretin, at a concentration of 0.05 mM, significantly reduces GATA4 and SOX9 mRNA expression which, in accordance with a reduction on glutamine consumption, suggest that this inhibitor triggers an undifferentiation process of human Sertoli cells, highlighting the vital role of Aquaporin-9 in human male reproductive tract. Further investigation unveiled that CFTR is present in human Sertoli cells and that it may exist in a different isoform from the ones previously described. Protein inhibition by CFTR(inh)-172 (3 µM) also decreased significantly GATA4 and SOX9, similarly to what happened with the Phloretin-treated cells, which uncovers the possibility of a crucial interaction between Aquaporin-9 and CFTR, as it has been described in rat Sertoli cells. We can conclude that this innovative investigation allowed the discovery of pivotal information regarding Aquaporin-9 and CFTR implications in human Sertoli cells. A lot of questions were raised with this study and the respective answers should be unveiled with future investigation.