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      Aquaporins Are Differentially Regulated in Canine Cryptorchid Efferent Ductules and Epididymis

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          Abstract

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          The distribution and expression of aquaporins (AQPs) in the testes and spermatozoa of several animal species play important roles in spermatogenesis and spermatozoon transit in this region. The aim of this study was to evaluate AQP7, AQP8, and AQP9 localization and expression in the efferent ductules and epididymal regions (the caput, corpus, and cauda) of normal and cryptorchid dogs. The results from immunohistochemistry, Western blotting, and real-time reverse transcription polymerase chain reaction (RT-PCR) show regional tissue distributions, particularly at the level of the epithelium of efferent ductules and both the regions caput and cauda of the canine cryptorchid epididymis. These findings support the hypothesis that these channel proteins respond differently to multiple stimuli that cause cryptorchidism (hormones, heat, osmolarity, etc.) and participate in the mechanisms of cell “resilience” or apoptosis taking place in the epididymis.

          Abstract

          The efferent ductules and the epididymis are parts of the male reproductive system where spermatozoa mature. Specialized epithelial cells in these ducts contribute to the transport of fluids produced by spermatozoa’s metabolic activity. Aquaporins (AQPs) have been demonstrated to be expressed in the spermatozoan membrane and testis epithelial cells, where they contribute to regulating spermatozoan volume and transit through environments of differing osmolality. Due to the lack of detailed literature regarding AQP expression in the canine male genital tract, the aim of this study was to investigate both the distribution and expression of AQP7, AQP8, and AQP9 in the efferent ductules and epididymal regions (caput, corpus, and cauda) from normal and cryptorchid dogs by using immunohistochemistry, Western blotting, and real-time reverse transcription polymerase chain reaction (RT-PCR). Our results show different patterns for the distribution and expression of the examined AQPs, with particular evidence of their upregulation in the caput and downregulation in the cauda region of the canine cryptorchid epididymis. These findings are associated with a modulation of Hsp70 and caspase-3 expression, suggesting the participation of AQPs in the luminal microenvironment modifications that are peculiar characteristics of this pathophysiological condition.

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          Structure and function of aquaporin water channels

          The aquaporins (AQPs) are a family of small membrane-spanning proteins (monomer size ∼30 kDa) that are expressed at plasma membranes in many cells types involved in fluid transport. This review is focused on the molecular structure and function of mammalian aquaporins. Basic features of aquaporin structure have been defined using mutagenesis, epitope tagging, and spectroscopic and freeze-fracture electron microscopy methods. Aquaporins appear to assemble in membranes as homotetramers in which each monomer, consisting of six membrane-spanning α-helical domains with cytoplasmically oriented amino and carboxy termini, contains a distinct water pore. Medium-resolution structural analysis by electron cryocrystallography indicated that the six tilted helical segments form a barrel surrounding a central pore-like region that contains additional protein density. Several of the mammalian aquaporins (e.g., AQP1, AQP2, AQP4, and AQP5) appear to be highly selective for the passage of water, whereas others (recently termed aquaglyceroporins) also transport glycerol (e.g., AQP3 and AQP8) and even larger solutes (AQP9). Evidence for possible movement of ions and carbon dioxide through the aquaporins is reviewed here, as well as evidence for direct regulation of aquaporin function by posttranslational modification such as phosphorylation. Important unresolved issues include definition of the molecular pathway through which water and solutes move, the nature of monomer-monomer interactions, and the physiological significance of aquaporin-mediated solute movement. Recent results from knockout mice implicating multiple physiological roles of aquaporins suggest that the aquaporins may be suitable targets for drug discovery by structure-based and/or high-throughput screening strategies.
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            Regulation of epithelial function, differentiation, and remodeling in the epididymis

            The epididymis is a single convoluted tubule lined by a pseudostratified epithelium. Specialized epididymal epithelial cells, the so-called principal, basal, narrow, and clear cells, establish a unique luminal environment for the maturation and storage of spermatozoa. The epididymis is functionally and structurally divided into several segments and sub-segments that create regionally distinct luminal environments. This organ is immature at birth, and epithelial cells acquire their fully differentiated phenotype during an extended postnatal period, but the factors involved in this complex process remain incompletely characterized. In the adult epididymis, the establishment of an acidic luminal pH and low bicarbonate concentration in the epididymis contributes to preventing premature activation of spermatozoa during their maturation and storage. Clear cells are proton-secreting cells throughout the epididymis, but principal cells have distinct acid/base transport properties, depending on their localization within the epididymis. Basal cells are located in all epididymal segments, but they have a distinct morphology depending on the segment and species examined. How this structural plasticity of basal cells is regulated is discussed here. Also, the role of luminal factors and androgens in the regulation of epithelial cells is reviewed in relation to their respective localization in the proximal versus distal regions of the epididymis. Finally, we describe a novel role for CFTR in tubulogenesis and epithelial cell differentiation.
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              Establishment of cell-cell cross talk in the epididymis: control of luminal acidification.

              Male infertility is often caused by sperm that have low motility and interact poorly with the oocyte. Spermatozoa acquire these crucial functions in the epididymis. A low luminal bicarbonate (HCO(3)(-)) concentration and low pH keep sperm quiescent during their maturation and storage in this organ. This review describes how epididymal epithelial cells work in a concerted manner, together with spermatozoa, to establish and maintain this acidic luminal environment. Clear cells express the proton-pumping ATPase (V-ATPase) in their apical membrane and actively secrete protons. HCO(3)(-) induces V-ATPase accumulation in apical microvilli in clear cells via HCO(3)(-)-sensitive adenylyl cyclase-dependent cAMP production. HCO(3)(-) is secreted from principal cells following basolateral stimulation, to transiently "prime" spermatozoa before ejaculation. Luminal ATP and adenosine also induce V-ATPase apical accumulation in clear cells via activation of P2 and P1 receptors, respectively. ATP is released into the lumen from sperm and principal cells and is then metabolized into adenosine by local nucleotidases. In addition, the V-ATPase is regulated by luminal angiotensin II via activation of basal cells, which can extend narrow body projections that cross the tight junction barrier. Basal cells then secrete nitric oxide, which diffuses out to stimulate proton secretion in clear cells via activation of the cGMP pathway. Thus, an elaborate communication network is present between principal cells and clear cells, and between basal cells and clear cells, to control luminal acidification. Monitoring and decoding these "intercellular conversations" will help define pathophysiologic mechanisms underlying male infertility.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                Animals (Basel)
                Animals (Basel)
                animals
                Animals : an Open Access Journal from MDPI
                MDPI
                2076-2615
                25 May 2021
                June 2021
                : 11
                : 6
                : 1539
                Affiliations
                [1 ]Department of Veterinary Medicine and Animal Productions, University of Napoli Federico II, 80100 Naples, Italy; caterina.squillacioti@ 123456unina.it (C.S.); nicola.mirabella@ 123456unina.it (N.M.); giuseppe.germano@ 123456unina.it (G.G.)
                [2 ]Department of Advanced Biomedical Sciences, University of Napoli Federico II, 80100 Naples, Italy; alessandra.pelagalli@ 123456unina.it
                [3 ]Institute of Biostructures and Bioimages, National Research Council, 80145 Naples, Italy
                Author notes
                [†]

                These authors contributed equally to this work as first authors.

                Author information
                https://orcid.org/0000-0003-0482-7681
                Article
                animals-11-01539
                10.3390/ani11061539
                8227126
                34070358
                7a61a42f-2317-4806-bd06-d57907a4b6ec
                © 2021 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/).

                History
                : 14 April 2021
                : 24 May 2021
                Categories
                Article

                aquaporins,epididymis,dog,cryptorchidism,homeostasis,male reproduction

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