Abstract Carbonic anhydrases (CAs) are a group of zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate (CO2 + H2O ⇔ HCO3- + H+). They are present in almost all organs and are implicated in various biological functions, the most important of which is participation in the regulation of ion, water, and acid-base balance. Recently, some members of the CA gene family have been suggested to promote cell proliferation and to act as trophic growth factors. The present study was undertaken to examine the distribution of CA isoenzymes in the reproductive system, to attain a more detailed view on their linkage to the reproductive processes and to neonatal development. The expression of membrane-bound CA IX and CA XII was studied in the female and male reproductive tracts by immunohistochemistry and western blotting. CA XII was found to be expressed in the basolateral plasma membrane of luminal and glandular epithelia in human uterus. In human efferent ducts, it was located in the basolateral plasma membrane of luminal epithelium, where it coexpressed with Aquaporin-1. In epididymal duct, CA XII was only expressed in occasional epithelial cells. These cells coexpressed CA II, suggesting that they represent apical mitochondria-rich cells (AMRC). CA IX was also expressed in the basolateral plasma membrane of luminal epithelium in human efferent ducts, but its expression was not uniform among the tubules. These findings suggest that basolateral plasma membrane-associated CA IX and CA XII contribute, along with CA II and CA IV, to the regulation of acid-base balance and water transport in the reproductive tract. Western blotting of rat Leydig tumor cells and testis for CA II revealed an unidentified 66-kDa polypeptide band. The polypeptide was successfully purified from several rat tissues using CA inhibitor affinity chromatography. The amino acid sequence of the polypeptide showed it to be identical to NonO/p54nrb, a non-POU domain-containing octamer-binding protein previously implicated in transcriptional regulation. The recombinant NonO/p54nrb was shown to display CA activity, and the antibody to it predominantly immunostained the nuclei in lymphocytes, where CA activity was also detected histochemically. Accordingly, the nuclear Leydig cell CA immunoreactivity represents NonO/p54nrb. It is classified as a novel, nonclassical CA, and it may participate in pH-related events in the nucleus. Human and rat milk was found to contain CA VI by immunohistochemistry and western blotting. The enzyme purified from human milk by CA inhibitor affinity chromatography was confirmed by PNGase F digestion and amino acid sequence as CA VI. The CA VI concentrations in human colostral milk were approximately eight times higher than those in mature milk (34.7 mg/l vs. 4.5 mg/l). Secretion of CA VI into milk is suggested by its localization in the alveolar epithelium of the rat mammary gland. The structural and functional stability of CA VI in an acidic milieu, its suggested growth-supporting function in taste bud stem cells, and its high concentration in colostrum suggest that it is an essential factor for the growth and development of the newborn alimentary canal.

Orientadores: Antonio Marcos Orsi, Sergio Luis Felisbino / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T10:45:56Z (GMT). No. of bitstreams: 1 Domeniconi_RaquelFantin_D.pdf: 10868266 bytes, checksum: 8bdfe8a912508f6afd1b6131f9f8934e (MD5) Previous issue date: 2007 / Resumo: Estudos recentes têm identificado família de proteínas denominadas aquaporinas (AQP), relacionadas à alta permeabilidade de água em várias membranas biológicas. As AQP1, AQP2, AQP7, AQP8 e AQP9 são as principais AQPs identificadas no sistema genital masculino, sendo a sua localização espécie-específica e região-específica. Em vista da importância do fluido luminal na via espermática para a integridade morfofuncional dos espermatozóides, bem como dos componentes que os constituem, tais como a água e proteínas, é importante estudar a distribuição das AQPs ao longo da via espermática. Assim, este trabalho teve como objetivos principais estudar no cão as AQP1, AQP2, AQP7, AQP8 e AQP9, visando identificá-las e localizá-las, através de imuno-histoquímica e ¿Western blotting¿ na via espermática. No cão, a AQP1 foi notada na rede testicular, ductos eferentes e em vasos, sugerindo sua importância na rápida absorção de fluido testicular. Pela primeira vez a AQP2 foi detectada na rede testicular, ductos eferentes e epidídimo, e a AQP7 no epitélio epididimário e ducto deferente em mamíferos. Porém, o papel funcional dessas AQPs no sistema genital masculino do cão permanece desconhecido. A AQP8 não foi detectada ao longo dos ductos extratesticulares do cão. A AQP9 foi abundantemente expressada ao longo da via espermática do cão, que representa um importante caminho apical para o fluxo transmembrana de água e solutos. Portanto, os resultados confirmam o padrão de expressão espécie-específica e região-específica das AQPs, sugerindo variações de atividades de absorção de fluidos e solutos ao longo da via espermática. O conhecimento destas variações torna-se relevante para estudos clínicos de infertilidade, bem como para tecnologias de reprodução assistida / Abstract: Recent studies have identified proteins called aquaporins (AQP) related to the fast water permeability in some biological membranes. AQPs are small, intrinsic membrane proteins that are present in many cell types involved in fluid transport. AQP1, AQP2, AQP7, AQP8 and AQP9 had been the main AQPs identified in the male reproductive tract, being their localization species-specific and region-specific. In view of the importance of the luminal fluid to sperm maturation and integrity of the spermatozoa, it is important to study the distribution of the AQPs throughout the spermatic way. Thus, the aim of this study was to examine the expression of AQP1, AQP2, AQP7, AQP8 e AQP9 in epithelial cells in the adult dog efferent ducts, epididymis and vas deferens, using immunohistochemistry and estern blotting methods to characterize the aquaporins in male reproductive tract. In dog, AQP1 was noted in rete testis, efferent ducts and in vessels in intertubular space, suggesting that AQP1 is important for rapid absorption of testicular fluid. For the first time the AQP2 was detected in rete testis, efferent ducts and epididymis and the AQP7 was expressed in the epithelium epididymidis and in vas deferens in mammals. But its functional role in the male dog reproductive tract, remain unknown. No specific staining for AQP8 was detected in epithelial cells of excurrent ducts in dog testis. AQP9 was abundantly expressed in dog male reproductive tract, in which it is an important apical pathway for transmembrane flow of water and neutral solutes. Thus the results confirm that the AQPs are species-specific and region-specific, suggesting activity variations related with the fluid and solute absorption throughout male excurrent ducts. Investigations of AQP biology could be relevant to clinical studies of the male reproductive tract, as well as to technologies for assisted procreation / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural