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Our laboratory examines physiological and pathophysiological processes involved in the regulation of male fertility.
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Our laboratory examines physiological and pathophysiological processes involved in the regulation of male fertility. In the United States, millions of couples are infertile. A significant number of these couples are affected by male fertility defects that are still unexplained. A major cause of male infertility is the production of sperm that have reduced function, including low motility and poor interaction with oocytes. Spermatozoa acquire their ability to become motile and to fertilize an egg in the lumen of the tubules that form the epididymis, an organ that is located downstream of the testis. Thus, the study of epididymal function might provide clues to understand better the cause of some unexplained cases of male infertility.
One critical feature of the epididymal luminal fluid is that it is maintained acidic (9). Acidic pH and low bicarbonate concentration are involved in maintaining sperm in an immotile, dormant state during their storage period in the epididymis. Thus, a defect in the acidification capacity of the epididymis might have important consequences for male reproductive physiology. A second major role of the epididymis is related to its concentrating capacity (2). Significant fluid reabsorption occurs in this organ, which results in a marked increase in spermatozoa concentration.
Our research is divided into four major themes: a) luminal acidification and its regulation; b) modeling of basal cell function in pseudostratified epithelia; c) fluid and solute transport; and d) interaction between the various cell types that are present in the epithelium, and that work in a concerted manner to establish and maintain the appropriate environment for sperm maturation, storage and concentration in the epididymis. These four NIH-funded studies focus on the intact organ. By using a multidisciplinary apporach including high-resolution laser scanning confocal microscopy and intravital multiphoton microscopy, 3D reconstructions of single cells, and luminal perfusion of the epididymis in vivo, we are studying the function of epithelial cells while they reside in their native environment.
1) Luminal acidification in the epididymis We have identified a novel pathway for the sensing and regulation of extracellular pH via a physiological link between the bicarbonate activated soluble adenylyl cyclase (sAC) and the vacuolar H+ATPase (V-ATPase). This discovery was made using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage. Proton secretion in a sub-population of epididymal epithelial cells, the clear cells, is regulated via recycling of V-ATPase (1, 9, 11, 12), a process that is modulated by luminal pH and bicarbonate, via changes in sAC activity (7). We have identified PKA as a down-stream effector of sAC-dependent elevation of cAMP (8). As sAC is expressed in other acid/base transporting epithelia, including the kidney, this cAMP-dependent signal transduction pathway may represent a widespread mechanism that allows cells to sense and modulate extracellular pH. These results represent a “paradigm shift” as they provide a potential route by which cAMP can be elevated within the cell without the participation of neurotransmitters or hormones. We have, therefore, uncovered a sensitive feedback mechanism by which epithelial cells in the epididymis can respond to variations in the acid/base status of their luminal environment. We are also examining the potential regulation of V-ATPase via assembly-disassembly of the various subunits that form the holoenzyme.
2) Three-dimensional modeling of basal cell function in pseudostratified epithelia Many organs in the body, including those of the reproductive tract and the lungs, are comprised of a system of tubules lined by epithelial cells. The prevailing view is that so-called “basal cells” in these epithelia are never in contact with the fluid or air-filled cavity (known as the lumen), but we showed recently that these cells in fact extend long, slender projections that scan the lumen and modulate organ function by communicating their findings to adjacent cells (13). We propose to create new model systems in which the three-dimensional relationship and functions of different epithelial cell types (identified in live animals by the presence of different colored fluorescent markers) can be monitored in real time as the basal cells detect and respond to various drugs, hormones, chemicals and pathogens that appear in the cavity of the organ; the data we generate will promote new diagnostic and therapeutic strategies for diseases including male infertility, chronic obstructive airway disease and cystic fibrosis.
3) Water and solute transport in the epididymis The composition of the luminal environment of the epididymis and vas deferens is tightly regulated. Major fluid reabsorption occurs in the epididymis and leads to a significant increase in sperm concentration. Therefore, the process of water movement in the excurrent duct is a crucial step for the establishment of male fertility. However, very little is known about the mechanisms responsible for water permeability in the epididymis. We and others have identified AQP9 as a major apical aquaporin in the epididymis (4,6). AQP9 is an aquaglyceroporin that can transport neutral solutes in addition to water. This study is aimed at characterizing the function and regulation of this protein in the epididymis. We are also searching for novel aquaporins in the basolateral membrane of epididymal epithelial cells. In the distal epididymis, water transport is linked to CFTR-dependent chloride secretion and is an important step that helps control the final fluidity of the luminal content. One of the major causes of male infertility in the human population is cystic fibrosis (CF). Although the epididymis is one of the most affected organs in CF, very little is known about the mechanisms that lead to its malfunction. We showed that AQP9 and CFTR co-localize in the apical membrane of principal cells of the epididymis and that they are part of the same co-immunoprecipitated complex (10). We also showed that CFTR is a key regulator of AQP9-dependent permeability. Obstructive pathologies followed by atrophy of the male reproductive tract occur in men with cystic fibrosis. We are currently examining the possibility that impairment of water transport following disruption of a functional complex between AQP9 and CFTR might contribute to the pathogenesis of male infertility in cystic fibrosis.
4) Genomic and proteomic profiling in specific cell types, and cell-cell cross-talk in the epididymis The male reproductive tract is composed of highly heterogeneous tissues that consist of many morphologically and functionally distinct cell types, which play a vital role in establishing the optimal environment for the maturation and storage of spermatozoa. Transepithelial transport in the epididymis involves a concerted interaction between the different cell types that constitute the epithelium (clear, principal and basal cells). We are currently dissecting the factors involved in the establishment and maintenance of the mature phenotypes of these cells, as well as factors that coordinate the ion transport activities of these differentiated cells. This information is critical to our understanding of the control of male reproductive physiology.
We are using novel molecular biology tools (including laser cut microdissection, in vitro mRNA amplification, quantitative PCR) and model animals (including transgenic B1-EGFP mice) to bring together an integrated approach that can be applied to male reproductive physiology (3-5). These tools and models are being used in our laboratory to characterize the patterns of gene expression in clear cells and principal cells of the epididymis, during postnatal and pre-pubertal development, under normal and pathophysiological conditions. In particular, we are examining how the cellular elements for luminal acidification are established by cell-specific differentiation, and how transepithelial acid/base transport is subsequently modulated via cell signaling processes in the final differentiated epithelium. The ultimate goal of this research is to determine how abnormal acidification may impair male fertility, and how interventions to affect acidification may eventually be used to control male fertility.
Sylvie Breton, PhDBreton.Sylvie@mgh.harvard.edu
Clémence Belleannée, PhDBelleannee.Clemence@mgh.harvard.edu
Nicolas Da Silva, PhD (Instructor in Medicine)Dasilva.Nicolas@mgh.harvard.edu
Eric Hill, BSHill.Eric@mgh.harvard.edu
Marija Ljubojevic, PhDLjubojevic.Marija@mgh.harvard.edu
Yechun Ruan, PhDyruan1@partners.org
Winnie Shum, PhD (Instructor in Medicine)Shum.Winnie@mgh.harvard.edu
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