Aquaporins and male (in)fertility: Expression and role throughout the male reproductive tract

Journal Pre-proof Aquaporins and male (in)fertility: Expression and role throughout the male reproductive tract David F. Carrageta, Raquel L. Bernardino, Graça Soveral, Giuseppe Calamita, Marco G. Alves, Pedro F. Oliveira PII:

S0003-9861(19)30822-7

DOI:

https://doi.org/10.1016/j.abb.2019.108222

Reference:

YABBI 108222

To appear in:

Archives of Biochemistry and Biophysics

Received Date: 19 September 2019 Revised Date:

25 November 2019

Accepted Date: 4 December 2019

Please cite this article as: D.F. Carrageta, R.L. Bernardino, Graç. Soveral, G. Calamita, M.G. Alves, P.F. Oliveira, Aquaporins and male (in)fertility: Expression and role throughout the male reproductive tract, Archives of Biochemistry and Biophysics (2020), doi: https://doi.org/10.1016/j.abb.2019.108222. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

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Title: Aquaporins and male (in)fertility: expression and role throughout the male reproductive tract

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Authors and affiliations: David F. Carrageta1, Raquel L. Bernardino1, Graça Soveral2,3,

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Giuseppe Calamita4, Marco G. Alves1, Pedro F. Oliveira1,5,6
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Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of

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Porto, Porto, Portugal

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Department of Microscopy, Laboratory of Cell Biology, Unit for Multidisciplinary Research in

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de

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Lisboa, Lisbon, Portugal

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Lisboa, Lisbon, Portugal

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Moro”, Bari, Italy

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i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

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Department of Genetics, Faculty of Medicine, University of Porto, Porto, Portugal

Department of Biochemistry and Human Biology, Faculty of Pharmacy, Universidade de

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari “Aldo

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Corresponding author: Pedro F. Oliveira, PhD

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Department of Microscopy, Institute for Biomedical Sciences Abel Salazar, University of Porto.

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Rua de Jorge Viterbo Ferreira n.228, 4050-313 Porto, Portugal.

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Phone: +351 220428000 E-mail:[email protected] Abstract

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Aquaporins (AQPs) are a family of transmembrane channel proteins responsible for the

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transport of water and small uncharged molecules. Thirteen distinct isoforms of AQPs have

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been identified in mammals (AQP0-12). Throughout the male reproductive tract, AQPs greatly

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enhance water transport across all biological barriers, providing a constant and expeditious

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movement of water and playing an active role in the regulation of water and ion homeostasis.

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This regulation of fluids is particularly important in the male reproductive tract, where proper

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fluid composition is directly linked with a healthy and competent spermatozoa production. For

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instance, in the testis, fluid regulation is essential for spermatogenesis and posterior

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spermatozoa transport into the epididymal ducts, while maintaining proper ionic conditions for

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their maturation and storage. Alterations in the expression pattern of AQPs or their dysfunction

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is linked with male subfertility/infertility. Thus, AQPs are important for male reproductive

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health. In this review, we will discuss the most recent data on the expression and function of the

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AQPs isoforms in the human, mouse and rat male reproductive tract. In addition, the regulation

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of AQPs expression and dysfunction linked with male infertility will be discussed.

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Keywords: Aquaporin; Male Reproduction; Spermatozoa; Testis; Water Transport.

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1. Introduction

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Aquaporins (AQPs) are a family of transmembrane channel proteins mainly responsible for the

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transport of water and a series of small uncharged molecules such as glycerol [1], urea [2],

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ammonia [3], hydrogen peroxide [4], some metalloids [5], small metabolites, like lactate and

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certain gases across biological membranes [6]. The discovery of AQPs in 1992, by Peter Agre

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and collaborators, revolutionized the study of cellular water transport. For the first time, a

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protein acting as a selective water pore was identified and characterized, not only in mammals

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but also in plants [7] and bacteria [8]. Meritoriously, Agre was laureated in 2003 with the Nobel 2

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Prize in Chemistry for his detailed study of the structure and function of AQPs [9]. Since then,

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thirteen distinct isoforms have been identified (AQP0-12) in mammals and roughly classified

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into three main groups based on their homology and biophysical properties (Figure 1).

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However, this classification is by now outdated due to the recent discoveries on the real

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transport functions of the AQPs.

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Interestingly, all AQPs are tetramers of four pores, where each monomer establishes an

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independent pore. However, this tetrameric structure also assembles a fifth central pore, which

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is described with hydrophobic nature and whose size and biophysical properties vary among

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groups and even among isoforms of the same group [10]. The first group is constituted by the

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orthodox AQPs. Seven AQPs belong in the orthodox group: AQP0, AQP1, AQP2, AQP4,

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AQP5, AQP6, and AQP8. Orthodox AQPs are described as selective to water molecules,

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although there are few exceptions. For instance, AQP6 was reported to be involved in the

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transport of Cl- under acidic conditions [11] and AQP8 has a role in the transport of ammonia

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and hydrogen peroxide, which justify the designation of ammoniaporin or peroxiporin [12, 13].

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In addition to AQP8, a few other isoforms were also described to facilitate hydrogen peroxide

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permeation, namely AQP3 [14], AQP5 [15], and AQP9 [16], which were recently classified

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also as peroxiporins. The second group concerns to aquaglyceroporins and includes four

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homologues: AQP3, AQP7, AQP9, and AQP10. Aquaglyceroporins have a bigger pore size

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[17] and are permeable not only to water but also to small uncharged solutes, such as glycerol,

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urea, or hydrogen peroxide [16, 18]. The last group is constituted by the so-called unorthodox

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AQPs, AQP11 and AQP12, also known as superaquaporins. Superaquaporins present low

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homology in comparison to orthodox AQPs and aquaglyceroporins [19]. Moreover,

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superaquaporins are often localized in the membrane of intracellular organelles. Thus, it has

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been suggested that the role of AQP11 and AQP12 is mainly linked with intracellular water and

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glycerol transport, regulating organelles volume and homeostasis [20-22]. Further work is

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needed to fully understand the properties and biological relevance of AQP11 and AQP12.

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The transport of water through biological membranes is a vital process in cellular physiology,

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not only for tissue fluid homeostasis but also for intracellular processes. For instance, the

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regulation of water homeostasis is particularly important in the male reproductive tract for a

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healthy and competent spermatozoa production [23, 24]. In the testis, fluid regulation is

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essential for spermatogenesis and posterior transport of spermatozoa into the epididymal ducts,

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while maintaining proper conditions for their maturation. In the seminiferous tubules, fluid

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homeostasis is mainly regulated by Sertoli cells and partly by differentiating germ cells [25].

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Indeed, it is estimated that 70% of cell volume is osmotically eliminated from the cytoplasm of

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spermatids during their differentiation into spermatozoa [26]. Subsequently, the maturation,

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concentration and storage of spermatozoa are associated with the secretion and absorption of

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fluid [27-29]. In this sense, AQPs greatly enhance water transport across all biological barriers,

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including in the male reproductive tract, while providing a constant and expeditious movement

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of water across tight junction barriers and playing an active role in the epithelial regulation of

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water homeostasis [30]. In this review, we will discuss the most recent data concerning the

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expression and function of the different AQPs isoforms in the human and rodent (mouse and

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rat) male reproductive tract. In addition, the regulation of their expression and the association

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with male (in)fertility will be discussed.

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2. Aquaporins expression and functions throughout the male reproductive tract

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AQPs are widely expressed in the human male reproductive tract (Table 1). However, the data

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available concerning some isoforms are not consistent or even scarce. The presence of AQPs

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greatly differs among tissue regions and cell types, evidencing a complex regulatory and

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functional network. Besides, AQPs expression is generally disparate between mammals, with a

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few exceptions. Curiously, rodents (such as mouse and rat) share some similarities with

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humans, which has proven to be helpful as the majority of functional and knockout studies have

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been conducted in these species. Yet, only the first steps have been taken in order to elucidate

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the full extent of AQPs expression and function in human reproductive system. On the

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following topics, the most recent data concerning AQPs expression in the human reproductive

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system will be discussed, highlighting the putative function obtained from studies in humans

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and also rodent animal models.

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2.1. Aquaporin-0 (AQP0)

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Although not yet identified in the testicular environment of humans, the major intrinsic protein

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(MIP) of lens fiber, also known as AQP0, is predicted to be expressed in the human testis [31].

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As an orthodox isoform, AQP0 is reported to mediate mostly water transport, although at a

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lower extent, when compared with other orthodox AQPs [32]. Besides, AQP0 is also reported to

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act as a cell-to-cell adhesion protein in the human eye, more properly in the lens fiber cells. In

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this tissue, AQP0 dysfunction is associated with cataractogenesis, both in humans and in

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knockout mice models [33, 34]. However, the consequences for the male reproductive system

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are unknown as it was not studied in these knockout mice models [34]. Nonetheless, Hermo et

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al. [35] identified AQP0 in rat testis, more specifically in Leydig and Sertoli cells. In this study,

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the authors reported that the expression in Sertoli cells seems to be region-specific and stage-

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dependent, more specifically in a semicircular pattern at stages VI-VIII of the spermatogenic

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cycle. These results suggest a significant role in the transport of water into the lumen of the

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seminiferous tubules during specific stages of spermatogenesis, which are related to the release

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of elongating spermatids. Hence, AQP0 may improve the movement of spermatozoa into the

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epididymis by transporting water into seminiferous tubules. Interestingly, AQP0 is reported to

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be modulated by pH and Ca2+ [36, 37], which are tightly regulated in the testis and suggests that

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AQP0 might be important for proper fluid homeostasis in the seminiferous tubules.

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2.2. Aquaporin-1 (AQP1)

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AQP1 was in fact the first water channel discovered, while also the first described to act as a gas

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channel [38, 39]. AQP1 is widely expressed in humans, where it functions as a major water

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transporter. Besides, AQP1 is reported as crucial for angiogenesis, cell migration and cell

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growth [40]. AQP1 is expressed in the human testis, although restricted to the endothelial cells

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of blood vessels [41]. On the other hand, AQP1 is absent from germ cells and ejaculated

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spermatozoa [42]. Outside the testis, AQP1 was found in noncilitated epithelial cells of human

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efferent ducts and epididymis [43]. Based on its expression, it has been suggested that AQP1

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plays a major role in the re-absorption of water to increase sperm concentration. Almost

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similarly to what was seen in humans, AQP1 is mainly expressed in nonciliated cells and cilia

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of ciliated cells of efferent ducts [44] and nonciliated cells of epididymis in rats [45, 46]. In

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addition, AQP1 was also identified in the epithelial cells of rete testis, vas deferens, prostate,

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and seminal vesicles and in the nonciliated cells of the efferent ducts from mouse [47].

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2.3. Aquaporin-2 (AQP2)

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AQP2 is an arginine vasopressin-regulated AQP that is exclusively permeable to water. AQP2

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is widely expressed in the kidney and it is well known that mutations on the gene encoding

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AQP2 lead to severe forms of nephrogenic diabetes insipidus [48]. Thus, it is suggested that

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AQP2 main function specifies in water reabsorption in the kidney. However, AQP2 might not

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be specific for the kidney as it has been also found to be expressed in the human epididymis and

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seminal vesicles [49].

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In rodents, the expression of AQP2 is rather distinct. In mouse, AQP2 was found in the apical

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and subapical regions of the principal cells of the vas deferens and seminiferous tubules, and

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allegedly on spermatids, although no expression was found in mature sperm [50]. In rats,

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Stevens et al. [51] found that AQP2 is expressed in the ampulla of the vas deferens, namely on

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the apical membrane of principal cells. However, AQP2 is absent from the testes of rats [50].

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This pattern of expression led the authors to suggest that AQP2, similarly to AQP1, may have a

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role in water absorption in order to increase sperm concentration [51]. Besides, Arrighi et al.

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[46] identified AQP2 in the cauda of the epididymis of young rats and its expression appears to

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decrease with the transition into adulthood, which suggests a role in the post-natal development.

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2.4. Aquaporin-3 (AQP3)

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AQP3 is an aquaglyceroporin that is widely expressed in humans. AQP3 is reported to be

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permeable to several molecules besides water, such as glycerol, urea [6], and hydrogen peroxide

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[14]. In the human reproductive system, AQP3 was identified at the basolateral membranes of

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the prostatic tubuloalveolar epithelium, Sertoli cells, pseudostratified ciliated epithelial cells of

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the epididymis, and pseudostratified epithelial cells of seminal vesicles [52]. In human

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spermatozoa, AQP3 is not only present but is also considered essential for its physiology and

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function. AQP3 is found in spermatozoa’s tail membrane and in its absence the male gamete

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exhibits alterations in volume regulation and excessive cell swelling when in the female

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reproductive tract [53, 54]. Therefore, as upon ejaculation spermatozoa experiences an osmotic

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decrease within the transition to the female reproductive tract, AQP3 was found to be crucial for

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spermatozoa osmoregulation. Besides, AQP3 is reported to be responsive to the pH [55], further

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highlighting its function in osmoregulation and fluid dynamics.

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In mouse, AQP3 was also identified in the midpiece of spermatozoa [54]. Moreover, Aqp3

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mRNA was identified in mouse testis [56, 57]. More recently, we also identified AQP3

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expression in the TM4 cells, a cell line derived from a primary culture of mouse Sertoli cells

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[57]. In that study, it was reported that not only AQP3 is expressed in mouse Sertoli cells, but

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also that it is important for the transport of glycerol in these cells. In rats, AQP3 was found to be

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expressed in the prostate [58] and in the epididymis, more specifically in the basal cells,

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suggesting a role in the transport of water and glycerol to the epididymal lumen during sperm

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maturation [35].

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2.5. Aquaporin-4 (AQP4)

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Although present, limited data are available concerning the expression of AQP4 in the human

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male reproductive tract. Data from microarrays identified the expression of AQP4 in human

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seminiferous tubules, seminal vesicles, prostate and epididymis [59]. In rats, AQP4 was

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identified in the prostate [58] and Sertoli cells [60]. Interestingly, our group was able to report

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that AQP4 physically interacts with cystic fibrosis transmembrane conductance regulator

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(CFTR) in Sertoli cells of rats, which suggests that AQP4 may play a role in water and ion

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homeostasis in seminiferous tubule and consequently in the constitution of seminiferous tubular

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fluid ionic content [60].

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2.6. Aquaporin-5 (AQP5)

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In a similar mode to what happens with AQP4, the data available on the presence and function

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of AQP5 in the human male reproductive system are quite scarce. AQP5 is predicted to be

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present in the human testis, more specifically in germ cells and Leydig cells [49]. In rats, AQP5

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is only present in the apical membrane of principal cells in corpus and cauda of epididymis, but

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the relevance for male reproductive potential is not known yet [61].

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2.7. Aquaporin-6 (AQP6)

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Contrariwise to other orthodox AQPs, the function of AQP6 is rather distinct. AQP6 is reported

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to be colocalized with the H+-ATPase in intracellular vesicles in the kidney, but not in the

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plasma membrane, suggesting that AQP6 may act as an intra-vesicle pH regulator [62].

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Interestingly, in this same study Yasui and collaborators reported that AQP6 seems to be

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permeable to water only at acidic conditions or in the presence of HgCl2. Additionally, AQP6 is

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reported to transport urea, glycerol [63], and nitrate [64], which led some authors to classify this

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isoform as an unorthodox AQP. Nonetheless, there is no evidence that AQP6 is expressed in the

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human male reproductive tract. The sole evidence was obtained in rats, with AQP6 mRNA

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transcripts being identified in whole epididymis lysates, while its protein was not detected in the

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epididymal epithelial cells [61].

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2.8. Aquaporin-7 (AQP7)

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AQP7 is an aquaglyceroporin that is able to mediate the transport of water, glycerol, urea [65],

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ammonia, and arsenite [5]. AQP7 is mostly known for its abundant expression in the adipose

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tissue, where it is reported to mediate the efflux of lipolytic glycerol [66, 67]. Herein, abnormal

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regulation of glycerol transport is associated with the development of metabolic disease,

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highlighting that an AQP7 dysfunction may be associated with obesity [68-70].

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In the human reproductive system, Saito et al. [71] identified AQP7 in the testis, more

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specifically in round and elongated spermatids, and in the tail of spermatozoa. Conversely,

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different studies reported different patterns of expression in human spermatozoa. While Moretti

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et al. [72] reported that AQP7 was indeed present in the tail and midpiece, Laforenza et al. [73]

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reported the presence of AQP7 only in the head of human spermatozoa. In fact, there is

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evidence that different patterns of expression or even the absence of AQP7 in human

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spermatozoa are linked with abnormal morphology and decreased motility, and thus infertility,

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which will be further discussed.

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In rats, AQP7 was identified on round and elongated spermatids, spermatozoa, and residual

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bodies [65, 74], and in the epididymis [61, 75]. Due to its pattern of expression, it was

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suggested that AQP7 may play a role in testis development [74] and in the reduction of

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cytoplasm during spermiogenesis [65]. Besides, AQP7 expression in the male reproductive tract

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was also reported to vary with age. Unlike AQP2, AQP7 expression seems to increase with the

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transition to adulthood [74]. In mouse, our group identified the expression of Aqp7 mRNA in

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the testis, although no expression was detected in Sertoli cells [57]. In addition, AQP7 was

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identified in elongated spermatids, testicular and epididymal sperm tail of mouse [27].

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2.9. Aquaporin-8 (AQP8)

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AQP8, a homologue with permeability to ammonia and hydrogen peroxide besides that of

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water, was firstly identified in the intracellular domains of the proximal tubule and the

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collecting ducts cells [76]. Although it is expressed in the plasma membrane, AQP8 is also

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found in the mitochondrial membrane [77, 78], where it facilitates the transport of hydrogen

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peroxide [4, 79]. Besides, it is suggested that AQP8 facilitates the transport of hydrogen

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peroxide and ammonia rather than water in hepatocytes [80, 81].

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In the human male reproductive system, AQP8 is expressed in the cytoplasmic compartment (in

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intracellular organelles or vesicles) of Sertoli cells and of spermatogonia and spermatids [42,

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82]. The presence of this aquaporin in the plasma membrane was observed all germ cells and

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ejaculated spermatozoa, more specifically in the tail and midpiece [42]. Interestingly, in this

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same study the authors reported that the expression of AQP8 was transversal to all donors and it

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was not correlated with motility. As mature spermatozoa exhibits an increased mitochondrial

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activity, and thus high reactive oxygen species and hydrogen peroxide production [83], it was

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postulated that AQP8 is essential to facilitate their efflux and minimize the oxidative stress

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damage.

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Similarly to the human, in rat testes, AQP8 is expressed in primary spermatocytes, elongated

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spermatids, Sertoli cells, and residual bodies [44, 74]. In fact, the expression of AQP8 in the

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plasma membrane of Sertoli cells was found at all stages of the seminiferous epithelium cycle,

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which suggests that AQP8 is relevant for fluid homeostasis in this epithelium [44, 84].

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However, its expression in the epididymis is still inconclusive [44, 76]. Like AQP7, AQP8 has

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been suggested to exert a role in the reduction of cytoplasm during spermiogenesis.

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Additionally, its expression in the testes also seems to vary with age, increasing from 15 to 20

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post-natal days [74, 85]. In mouse, few data are available on AQP8 expression and relevance in

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the male reproductive tract. This AQP was described in the mouse testis, more specifically in

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the residual cytoplasm of elongated spermatids and spermatozoa tail [27]. No functional data

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are available.

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2.10.

Aquaporin-9 (AQP9)

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AQP9 has been the focus of intensive research over the last years. AQP9 is thought to have a

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main role in transporting water, glycerol, and other solutes essential for sperm production and

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maturation [86]. Surprisingly, AQP9 is also permeable to monocarboxylic acids, such as lactic

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acid and acetic acid, probably due to its larger pore size [87]. In the human reproductive system,

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AQP9 was found with low expression in the germinal epithelium, spermatocytes and Sertoli

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cells [42]. As lactate is a vital energetic substrate for germ cells and spermatogenesis [88], the

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presence of AQP9 in Sertoli cells indicates that this channel may help in the transport of lactate

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to the tubular fluid, sharing this role with the high expression of monocarboxylate transporters

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(MCTs) [89]. In addition, AQP9 was identified in the vas deferens, efferent ducts, more

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specifically on the apical membrane of nonciliated cells, and principal cells of the epididymis

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[90]. Based on its pattern of expression in this tissue, AQP9 is suggested to play a role in fluid

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reabsorption and homeostasis. On the other hand, AQP9 is reported to be absent in human

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spermatozoa [42].

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With some similarities with humans, AQP9 is reported to be expressed in Sertoli cells [91],

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Leydig cells, efferent ducts, all regions of epididymis, vas deferens, prostate, and coagulating

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gland of rats [44, 90]. In fact, there is evidence that AQP9 expression is cell-specific in the testis

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and region-specific in the efferent ducts, namely on the microvilli of nonciliated cells, and

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microvilli of the principal cells of epididymis [44]. In mouse, our group recently identified the

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expression of Aqp9 mRNA in both testis and Sertoli cells [57]. Additionally, we showed that

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AQP9 is the major aquaglyceroporin expressed in mouse Sertoli cells, where this isoform is

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essential to regulate the transport of water and glycerol. While widely distributed in the male

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reproductive tract of rodents, its expression in spermatozoa is still a matter of debate. Although

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AQP9 transcripts were found in mouse spermatozoa, immunoblotting did not confirm the result

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[27].

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2.11.

Aquaporin-10 (AQP10)

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AQP10 is an aquaglyceroporin which seems to be specifically expressed in the human

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gastrointestinal tract and adipocytes [92, 93]. AQP10 is mainly responsible for water and

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glycerol transport, being highlighted in pair with AQP7 as a major glycerol transporter of

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adipocytes [94]. However, data concerning AQP10 in male reproductive tissues are still scarce.

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There is no evidence that AQP10 is expressed in the human male reproductive tract. In rats,

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AQP10 is expressed in the microvilli of the nonciliated cells, cilia of the ciliated cells, and

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endothelial cells of vascular channels of efferent ducts and endothelial cells of vascular channels

292

of epididymis [35]. Conversely, AQP10 is a pseudogene in mice and may lead to proteins

293

without functional activity [95].

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2.12.

Aquaporin-11 (AQP11) and Aquaporin-12 (AQP12)

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AQP11 is a relatively recent discovered isoform that has been classified as a superaquaporin.

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AQP11 is described as a water and glycerol channel that is mainly expressed in the vicinity of

299

lipid droplets and in the endoplasmic reticulum membrane [22]. Few studies also addressed

300

AQP11 expression and function in the human male reproductive system. AQP11 mRNA was

301

identified in human testicular tissue [96]. Using immunocytochemistry techniques Laforenza et

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al. [73] identified this channel in granules and vesicles of soma and in the plasma membrane of

303

the tail of ejaculated human spermatozoa. Hence, the subcellular localization of AQP11 remains

304

debatable.

305

In rats, AQP11 was identified in the microvilli of principal cells of epididymis, nonciliated cells

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of efferent ducts [75]. In both mouse and rat testis, AQP11 was identified in elongated

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spermatids, ejaculated spermatozoa tail, and residual bodies inside Sertoli cells [97].

308

Interestingly, Hermo et al. [98] reported that AQP11 expression varies with age, as its

309

expression was observed to decrease in efferent ducts from young to adult rats. However, as

310

knockout Aqp11 animal models are not viable due to the development of polycystic kidney and

311

subsequent premature death due kidney failure [99], few studies addressed AQP11 in the male

312

reproductive tract and new methodologies are needed to further investigate the role of this

313

superaquaporin in the reproductive health. Concerning AQP12, this isoform is reported to be

314

exclusively expressed in acinar cells of the pancreas and, thus, absent from the male

315

reproductive tract [100].

316 317

3. Alteration of aquaporins expression is linked with male infertility

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As aforementioned, AQPs are widely distributed throughout the male reproductive tract and in

319

male gametes. Their main functions comprise absorption/secretion dynamics in order to

320

maintain the homeostasis of the reproductive system to produce healthy and functional

321

spermatozoa. Specific expression patterns in cells, regions, variation with age, and evidence of

322

compensatory mechanisms are indicators of a precise and complex mechanism of regulation and

323

function. Before reaching the caput of the epididymis, 95% of the intraluminal fluid is

324

reabsorbed by the nonciliated cells of the efferent ducts in order to increase sperm concentration

325

[101]. Thus, it is not surprising that AQPs are mainly expressed in the nonciliated epithelium.

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On the other hand, AQPs are less abundant in ciliated cells. The motile cilia present in the male

327

reproductive tract are regarded as essential, as ciliated cells are responsible not only for sperm

328

transport, but also for the maintenance of the suspension of sperm within the intraluminal fluid,

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avoiding sperm aggregation and ensuring proper fluid reabsorption by the nonciliated cells

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[102]. However, to the best of our knowledge, only three homologues were identified in ciliated

331

cells – AQP1 and AQP10 in the efferent ducts of rats, and AQP3 in epididymis of humans. In

332

addition, no studies were performed concerning the role of AQPs on the cilia, which may

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constitute an interesting topic for future research. Moreover, AQPs also play a role in accessory

334

glands, contributing to the composition of seminal fluid [47, 90], and in spermatozoa adaptation

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phenomena after ejaculation, due to the variation of osmotic conditions [53]. Thus, AQPs are of

336

extreme importance for the reproductive health and their dysfunction is related to reproductive

337

disorders.

338

Although data are scarce, there is evidence suggesting that alterations in the expression and

339

function of these transport proteins are associated with subfertility or infertility [103]. For

340

instance, there is some evidence for an association between a deficit in AQP7 expression in

341

spermatozoa and diminished quality and fertilizing ability. In humans, it was found that

342

spermatozoa from infertile individuals’ presents lower amounts or were devoid of AQP7. In

343

addition, spermatozoa lacking AQP7 presented significantly lower motility in comparison with

344

spermatozoa exhibiting positive AQP7 staining [42, 71]. In another study, Moretti et al. [72]

345

also reported that AQP7 expression was diminished in morphologically abnormal spermatozoa.

346

On the other hand, there are studies with a knockout Aqp7 animal model that may jeopardize

347

those findings. Knockout mice for Aqp7 were described as fertile, without differences in testis

348

morphology and with healthy functional spermatozoa [104]. Moreover, other study with

349

knockout mice for Aqp7 reported that an upregulation of Aqp8 expression might have a

350

compensatory effect [27]. Hence, further research is necessary to elucidate the role of AQP7 in

351

the male reproductive health.

352

Like AQP7, alterations in AQP3 and AQP8 expression were associated with reduced

353

reproductive capability. It was found that a deficit of AQP3 led to impaired sperm motility and

354

tail deformations [54]. Concerning AQP8, although few studies addressed its expression and

355

function in spermatozoa, it has been reported that its expression is inversely correlated with the

356

coiling degree of the tail, suggesting that this AQP may play a role in the adaptation to

357

osmolality variations [42]. Additionally, Laforenza et al. [73] proposed that AQP8 malfunction

358

could lead to hydrogen peroxide accumulation in the mitochondria, hence affecting sperm

14

15

359

function. Still, no differences in AQP8 expression were found between fertile and infertile

360

human individuals [42] and it was reported that knockout Aqp8 animal models remain fertile,

361

which suggests a functional compensation [105]. Thus, the importance of AQP8 for the

362

reproductive health requires further investigation.

363

It was reported that Aqp1 knockout mice showed histological alterations in the rete testis due to

364

the obstruction of water reabsorption in tubules lumen [106]. However, these animals are also

365

fertile, as suggested, this can be caused by the existence of compensatory mechanisms like the

366

paracellular movement of water or involvement of other isoforms of aquaporins [47]. Although

367

there are studies with knockout animals for the other AQP isoforms, we found nothing

368

described regarding the fertility of these animals.

369 370

3.1. Hormonal regulation of aquaporins

371

Hormonal alterations are a known factor linked with reduced male reproductive health and those

372

alterations might be associated with altered AQPs expression and function. For instance, there is

373

evidence that AQP1 and AQP9 are regulated by estrogens. In studies with knockout mice for

374

estrogen receptor α (Erα) it was found a reduced expression of AQP1 and AQP9 in the

375

epididymis and efferent ducts, suggesting that estrogens may regulate the fluid absorption in

376

these ducts. These animals also presented a fluid accumulation in efferent ducts with

377

consequently testicular atrophy and infertility [106, 107]. In addition, in a recent study by our

378

group, we found that AQP9 is downregulated by increased estrogen levels in mouse Sertoli cells

379

[57]. Besides, it was also reported that the administration of estrogens to rats reduces the

380

expression of AQP9 in the epididymis, but these effects were reversed with the administration

381

of testosterone [108]. Still in the epididymis, it has been reported that orchiectomized rats were

382

devoid of AQP3 in basal epididymal cells. However, when testosterone was administrated a

383

slightly restoration of AQP3 expression was noticed, suggesting that testosterone can modulate

384

the expression of AQP3 in the epididymis [35]. Curiously, similar effects were also reported for

15

16

385

AQP9 expression [109], suggesting that estrogens would decrease specific isoforms of AQPs

386

expression while testosterone administration would attenuate those effects. Nevertheless, there

387

are contradictory data. Studies showed that while androgens do modulate AQP9 in the initial

388

segment of the epididymis, estrogens did not alter its expression [110], or that estrogens

389

administration increased AQP9 expression in efferent ducts [111]. These inconsistencies in the

390

literature illustrate the complexity surrounding the regulation of AQPs. In sum, while there is

391

evidence to support that AQPs are under hormonal regulation, few data are available and further

392

research on the topic is required.

393 394

3.2. Interactions between AQPs and CFTR

395

CFTR is a transmembrane channel protein responsible for the transport of Cl- and HCO3-,

396

regulating the ionic balance and pH in the tubular fluid [112, 113]. Mutations in CFTR are

397

correlated with cystic fibrosis, a pathology related with infertility scenarios [114]. CFTR is

398

widely expressed and performs an essential role in the male reproductive tract, which requires a

399

tight regulation of water and electrolytes in order to produce healthy spermatozoa [115]. In fact,

400

mutations in CFTR can lead to anatomical abnormalities of the reproductive tract, mainly a

401

congenital bilateral absence of the vas deferens and, consequently, infertility [116]. Although

402

CFTR is not permeable to water, this protein acts as a regulator of other protein channels, such

403

as AQPs. In fact, CFTR acts as a molecular partner of AQPs in epithelial cells, regulating fluid

404

homeodynamics (Figure 2). CFTR’s anion permeability follows a simple ohmic behavior,

405

accordingly to the ionic gradient. As CFTR mediates the transport of Cl- the transport of Na+ is

406

promoted. Ultimately, Na+ is accumulation generates an osmotic driving force for water

407

movement, usually paracellularly and mediated by AQPs [117]. On the other hand, there is

408

evidence suggesting that CFTR may physically modulate AQPs function. For instance, our

409

group described the expression of CFTR, AQP4 and AQP9 in cultured rat primary Sertoli cells

410

[60, 91]. In this study we also investigated the physical interaction between CFTR and AQP4

16

17

411

and AQP9 in Sertoli cells. Taking advantage of co-immunoprecipitation techniques, we

412

observed a molecular interaction between CFTR and both AQP4 and AQP9. Since AQP4 and

413

AQP9 are reported as crucial for water and ion homeostasis in several tissues [27, 42, 108], it is

414

also expectable that both these AQPs regulate water homeostasis in Sertoli cells and

415

consequently inside the seminiferous tubules. Taken together, these results support the

416

hypothesis that CFTR is, at least in Sertoli cells, a regulator of AQPs activity in water

417

homeodynamics. Additionally, there is further evidence that CFTR interacts with AQP9. Both

418

CFTR and AQP9 are co-localized in the luminal membrane of the principal cells in the

419

epididymis of rat and humans [118, 119]. In fact, in vivo and in vitro inhibition of both AQP9

420

and CFTR reduced water permeability [118]. Also in this study, Cheung and collaborators

421

demonstrated a synergic effect between CFTR and AQP9. When expressed together in Xenopus

422

oocytes, the authors reported a significant increase in water permeability which were greater

423

than the sum of the water permeability caused by the expression of either protein alone. Thus, it

424

has been suggested that CFTR may potentiate water permeability of AQP9 in the epididymal

425

epithelium, which is essential for proper sperm maturation and movement. On a side note, it

426

was also described that AQP7 and AQP8 are expressed with in a highly similar pattern with

427

CFTR in the testis, highlighting a possible interaction [103, 120]. In support of this hypothesis,

428

CFTR was co-localized with several other AQPs in different tissues, such as AQP1 and AQP5

429

in the pancreas [121]. Notwithstanding, it is still unclear how CFTR physically regulates AQPs

430

function.

431 432

3.3. Relevance of AQPs in obesity and metabolic diseases

433

Obesity and metabolic syndrome have also been associated with impaired male reproductive

434

function [89, 122, 123]. During obesity scenarios, there is not only an increased aromatization

435

of testosterone to estradiol [124, 125], but also an increased plasmatic concentration of free fatty

436

acids and glycerol (for review [126]). High glycerol concentrations are responsible for the

17

18

437

disruption of the homeostasis of the tubular fluid due to the impaired function of the blood-testis

438

barrier, leading to the apoptosis of germ cells. Thus, while acute exposition to high

439

concentration of glycerol could lead to temporary arrest of spermatogenesis, chronic exposition

440

may result in permanent oligospermia or even azoospermia [127, 128]. Since aquaglyceroporins

441

are responsible for the transport of glycerol, their function or expression might be altered in

442

cases of obesity or metabolic syndrome [129]. In fact, high-fat diet was already associated with

443

increased AQP9 and decreased AQP1 expression in the epididymis [130]. In another study, Pei

444

et al. [58] reported a decreased expression of AQP1, AQP3 and AQP4 in the prostate of diabetic

445

rats. Still, much of the role of AQPs in male reproductive tract in obesity and metabolic

446

syndrome cases is unknown and an interesting matter of investigation.

447 448

3.4. Varicocele and possible role of AQPs

449

It is worth mentioning that AQPs differential expression is also associated with varicocele.

450

When comparing healthy individuals with those who suffer from idiopathic varicocele, it was

451

found that AQP9 expression was reduced or absent in the primary spermatocytes and germ cells

452

[131]. Moreover, AQP1 was identified in Sertoli and germ cells of individuals suffering from

453

varicocele, in opposition to the absense of this AQP in healthy individuals [43].

454 455

4. Conclusion and future perspectives

456

Since the discover of AQPs, the perspective on water transport has been revolutionized. AQPs

457

are now the matter of intense research worldwide, where the elucidation of patterns of

458

expression and function may lead to new therapeutic approaches for several diseases [132].

459

Herein, we highlight that AQPs are widely distributed through the male reproductive tract,

460

where these transmembrane proteins are vital for a healthy spermatogenesis and the

461

maintenance of fluid homeostasis. Expression patterns were found to be specific to cell and

462

tissue regions, or even age of the individual, indicating a precise and complex mechanism of

18

19

463

regulation and function. Indeed, AQPs are of extreme importance for the reproductive health

464

and their malfunction is related to reproductive disorders. Nevertheless, the available data are

465

not always consistent and few are the studies directly addressing the pathogenetic relevance of

466

these channels in the male reproductive tract. In this sense, knockout animal models provide

467

valuable evidence, despite of some differences among species. In sum, AQPs pattern of

468

expression may indeed constitute one important biomarker for male reproductive health. Further

469

studies are essential to clarify the role of AQPs in male fertility and how these channel proteins

470

may be targets for future therapeutic interventions.

471 472

Conflicts of interest

473

None.

474 475

Acknowledgements

476

This work was supported by “Fundação para a Ciência e a Tecnologia” - FCT to David F.

477

Carrageta (SFRH/BD/136779/2018). The work was co-funded by FEDER through the

478

COMPETE/QREN, FSE/POPH to Marco G. Alves (IFCT2015 and PTDC/MEC-

479

AND/28691/2017); Pedro F. Oliveira (IFCT2015 and PTDC/BBB-BQB/1368/2014); UMIB

480

(PEst-OE/SAU/UI0215/2019); co-funded by the EU Framework Programme for Research and

481

Innovation H2020 (POCI/COMPETE2020).

482 483

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874 875

Figure Legend

876

Fig. 1. Aquaporins classification based on their homology and biophysical properties.

877

Currently, thirteen distinct isoforms have been identified in mammals (AQP0-12) and roughly

878

classified into three main groups: orthodox, aquaglyceroporins and superaquaporins. Although

879

high homology exists among homologues of the same group, each homologue may exhibit a

880

different specificity in its permeability to solutes.

881 882

Fig.2. Schematic illustration of the interaction between CFTR and AQPs. CFTR’s mediates the

883

transport of Cl- accordingly to the ionic gradient, which promotes the transport of Na+.

884

Ultimately, the accumulation of Na+ generates an osmotic driving force for water movement,

885

either paracellularly or mediated by AQPs. However, it is also hypothesized that CFTR is able

886

to modulate the AQP-mediated water transport directly through a possible physical interaction

887

between both channels. Abbrevations: AQP – aquaporin; CFTR – cystic fibrosis transmembrane

888

conductance regulator.

35

36

889

Table 1 – Male reproductive tract distribution and suggested functions of all aquaporin isoforms.

Isoform Localization

Putative function in male reproduction

References [31], [35]

AQP0

Human testis* Rat Leydig and Sertoli cells

Homeostasis of the tubular fluid; transport of water into the lumen of seminiferous tubules Absorption of water and regulation of sperm concentration

AQP1

Human testis, efferent ducts and epididymis Rat efferent ducts and epididymis Mouse rete testis, vas deferens, prostate, efferent ducts and seminal vesicles

AQP2

Human epididymis* and seminal vesicles* Rat vas deferens and cauda of epididymis Mouse vas deferens and seminiferous tubules

Absorption of water and regulation of sperm concentration; possible role in post-natal development

AQP3

Human prostate, seminiferous tubules, seminal vesicles*, epididymis*, and spermatozoa Rat epididymis and prostate Mouse Sertoli cells and spermatozoa

Transport of water and glycerol to the epididymal lumen; regulation of spermatozoa volume and osmoadaptation in the uterine cavity

Human seminiferous tubules, seminal vesicles, prostate, and epididymis Rat prostate and Sertoli cells

Water and ionic homeostasis

AQP4

AQP5

Human testis*, germ cells*, and Leydig cells* Rat corpus and cauda of epididymis

Transport of water and small molecules

[41], [43], [45], [46], [47]

[46], [49], [50], [51] [49], [52], [53], [54], [56], [57], [58] [58], [59], [60]

[49], [61]

36

37

AQP6

AQP7

-

No evidence of expression Human testis, round and elongated spermatids, and spermatozoa Rat seminiferous epithelium, round and elongated spermatids, spermatozoa, residual bodies, and epididymis

Transport of water, glycerol and small molecules; reduction of the cytoplasm during spermatogenesis; sperm motility

[61]

[27], [61], [65], [71], [72], [73], [74], [75]

Mouse elongated spermatids and spermatozoa

AQP8

Human germ cells, Sertoli cells, spermatids, and spermatozoa; Rat testis, primary spermatocytes, elongated spermatids, Sertoli cells, and residual bodies

Tubular fluid homeostasis; reduction of cytoplasm during spermiogenesis; release of hydrogen peroxide accumulated in spermatozoa mitochondria

[42], [74], [82], [44], [84], [85]

Mouse spermatozoa Water and glycerol transport; fluid reabsorption; possible role in the transport of lactate to the tubular fluid

AQP9

Human germ cells, spermatocytes, Sertoli cells, vas deferens, efferent ducts, and epididymis Rat Sertoli cells, Leydig cells, efferent ducts, epididymis, vas deferens, and prostate Mouse testis and Sertoli cells

AQP10

Rat efferent ducts and epididymis

Water and glycerol transport Water and glycerol transport

AQP11

Human testis and spermatozoa Rat epididymis, testis, elongated spermatids, and spermatozoa Mouse testis, elongated spermatids and spermatozoa

AQP12

No evidence of expression

[42], [57], [84], [88], [89], [90], [91] [35] [73], [75], [96], [98]

-

[100]

37

38

890

*Predicted based on mRNA microarrays

38

Highlights

•

Aquaporins are widely distributed through the male reproductive tract;

•

Aquaporins greatly enhance water transport across all biological barriers;

•

Regulation of fluids is important for production and transport of spermatozoa;

•

Aquaporins are important for male reproductive health;