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Macrophages (MΦ) and dendritic cells (DCs) are heterogeneous families of functionally and developmentally related immune cells that play crucial roles in tissue homeostasis and the regulation of immune responses. During the past 5 years, immunologists have generated a considerable amount of data that challenge dogmas about the ontogeny and functions of these highly versatile cells. The male excurrent duct system plays a critical role in the establishment of fertility by allowing sperm maturation, transport and storage. In addition, it is challenged by pathogens and must establish a protective and tolerogenic environment for a continuous flow of autoantigenic spermatozoa. The post-testicular environment and, in particular, the epididymis contain an intricate network of DCs and MΦ; however, the immunophysiology of this intriguing and highly specialized mucosal system is poorly understood. This review summarizes the current trends in mouse MΦ and DC biology and speculates about their roles in the steady-state epididymis. Unraveling immune cell functions in the male reproductive tract is an essential prerequisite for the design of innovative strategies aimed at controlling male fertility and treating infertility.

The onslaught of foreign antigens carried by spermatozoa into the epididymis, an organ that has not demonstrated immune privilege, a decade or more after the establishment of central immune tolerance presents a unique biological challenge. Historically, the physical confinement of spermatozoa to the epididymal tubule enforced by a tightly interwoven wall of epithelial cells was considered sufficient enough to prevent cross talk between gametes and the immune system and, ultimately, autoimmune destruction. The discovery of an intricate arrangement of mononuclear phagocytes (MPs) comprising dendritic cells and macrophages in the murine epididymis suggests that we may have underestimated the existence of a sophisticated mucosal immune system in the posttesticular environment. This review consolidates our current knowledge of the physiology of MPs in the steady state epididymis and speculates on possible interactions between auto-antigenic spermatozoa, pathogens and the immune system by drawing on what is known about the immune system in the intestinal mucosa. Ultimately, further investigation will provide valuable information regarding the origins of pathologies arising as a result of autoimmune or inflammatory responses in the epididymis, including epididymitis and infertility.

A subset of basal cells (BCs) in the initial segment (IS) of the mouse epididymis has a slender body projection between adjacent epithelial cells. We show here that these projections occasionally cross the apical tight junctions and are in contact with the luminal environment. Luminal testicular factors are critical for the establishment of the IS epithelium, and we investigated their role in the regulation of this luminal sensing property. Efferent duct ligation (EDL) was performed to block luminal flow from the testis without affecting blood flow. Cytokeratin 5 (KRT5) labeling showed a time-dependent reduction of the percentage of BCs with intercellular projections from 1 to 5 days after EDL, compared to controls. Double labeling for caspase-3 and KRT5 showed that a subset of BCs undergoes apoptosis 1 day after EDL. Ki67/KRT5 double labeling showed a low rate of BC proliferation under basal conditions. However, EDL induced a marked increase in the proliferation rate of a subset of BCs 2 days after EDL. A 2-wk treatment with the androgen receptor antagonist flutamide did not affect the number of BCs with intercellular projections, but reduced BC proliferation. Flutamide treatment also reduced the increase in BC proliferation induced 2 days after EDL. We conclude that, in the adult mouse IS, 1) luminal testicular factors play an important role in the ability of BCs to extend their body projection towards the lumen, and are essential for the survival of a subset of BCs; 2) androgens play an important role in the proliferation of some of the BCs that survive the initial insult induced by EDL; and 3) the formation and elongation of BC intercellular projections do not depend on androgens., (© 2015 by the Society for the Study of Reproduction, Inc.)

The epithelium that lines the epididymal duct establishes the optimal milieu in which spermatozoa mature, acquire motility, and are stored. This finely tuned environment also protects antigenic sperm against pathogens and autoimmunity, which are potential causes of transient or permanent infertility. The epididymal epithelium is pseudostratified and contains basal cells (BCs) that are located beneath other epithelial cells. Previous studies showed that in the mouse epididymis, BCs possess macrophage-like characteristics. However, we previously identified a dense population of cells belonging to the mononuclear phagocyte (MP) system (comprised of macrophages and dendritic cells) in the basal compartment of the mouse epididymis and showed that a subset of MPs express the macrophage marker F4/80. In the present study, we evaluate the distribution of BCs and MPs in the epididymis of transgenic CD11c-EYFP mice, in which EYFP is expressed exclusively in MPs, using antibodies against the BC marker keratin 5 (KRT5) and the macrophage marker F4/80. Immunofluorescence labeling for laminin, a basement membrane marker, showed that BCs and most MPs are located in the basal region of the epithelium. Confocal microscopy showed that in the initial segment, both BCs and MPs project intraepithelial extensions and establish a very intricate network. Flow cytometry experiments demonstrated that epididymal MPs and BCs are phenotypically distinct. BCs do not express F4/80, and MPs do not express KRT5. Therefore, despite their proximity and some morphological similarities with peritubular macrophages and dendritic cells, BCs do not belong to the MP system.

Extracellular ATP is essential for the function of the epididymis and spermatozoa, but ATP release in the epididymis remains uncharacterized. We investigated here whether epithelial cells release ATP into the lumen of the epididymis, and we examined the role of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) and HCO(3)(-) conducting ion channel known to be associated with male fertility, in this process. Immunofluorescence labelling of mouse cauda epididymidis showed expression of CFTR in principal cells but not in other epithelial cells. CFTR mRNA was not detectable in clear cells isolated by fluorescence-activated cell sorting (FACS) from B1-EGFP mice, which express enhanced green fluorescent protein (EGFP) exclusively in these cells in the epididymis. ATP release was detected from the mouse epididymal principal cell line (DC2) and increased by adrenaline and forskolin. Inhibition of CFTR with CFTR(inh172) and transfection with CFTR-specific siRNAs in DC2 cells reduced basal and forskolin-activated ATP release. CFTR-dependent ATP release was also observed in primary cultures of mouse epididymal epithelial cells. In addition, steady-state ATP release was detected in vivo in mice, by measuring ATP concentration in a solution perfused through the lumen of the cauda epididymidis tubule and collected by cannulation of the vas deferens. Luminal CFTR(inh172) reduced the ATP concentration detected in the perfusate. This study shows that CFTR is involved in the regulation of ATP release from principal cells in the cauda epididymidis. Given that mutations in CFTR are a leading cause of male infertility, we propose that defective ATP signalling in the epididymis might contribute to dysfunction of the male reproductive tract associated with these mutations.

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.

Luminal acidification in the epididymis is critical for sperm maturation and storage. Clear cells express the vacuolar H(+)-ATPase (V-ATPase) in their apical membrane and are major contributors to proton secretion. We showed that this process is regulated via recycling of V-ATPase-containing vesicles. We now report that RhoA and its effector ROCKII are enriched in rat epididymal clear cells. In addition, cortical F-actin was detected beneath the apical membrane and along the lateral membrane of "resting" clear cells using a pan-actin antibody or phalloidin-TRITC. In vivo luminal perfusion of the cauda epididymal tubule with the ROCK inhibitors Y27632 (10-30 μM) and HA1077 (30 μM) or with the cell-permeable Rho inhibitor Clostridium botulinum C3 transferase (3.75 μg/ml) induced the apical membrane accumulation of V-ATPase and extension of V-ATPase-labeled microvilli in clear cells. However, these newly formed microvilli were devoid of ROCKII. In addition, Y27632 (30 μM) or HA1077 (30 μM) decreased the ratio of F-actin to G-actin detected by Western blot analysis in epididymal epithelial cells, and Y27632 also decreased the ratio of F-actin to G-actin in clear cells isolated by fluorescence activated cell sorting from B1-enhanced green fluorescence protein (EGFP) transgenic mice. These results provide evidence that depolymerization of the cortical actin cytoskeleton via inhibition of RhoA or its effector ROCKII favors the recruitment of V-ATPase from the cytosolic compartment into the apical membrane in clear cells. In addition, our data suggest that the RhoA-ROCKII pathway is not locally involved in the elongation of apical microvilli. We propose that inhibition of RhoA-ROCKII might be part of the intracellular signaling cascade that is triggered upon agonist-induced apical membrane V-ATPase accumulation.

One of the most intriguing aspects of male reproductive physiology is the ability to generate spermatogenic cells - which are 'foreign' to the host - without triggering immune activation. After leaving the testis, spermatozoa enter the epididymis where they mature and are stored. In this study, we report a previously unrecognized dense network of dendritic cells (DCs) located at the base of the epididymal epithelium. This network was detected in transgenic mice expressing CD11c-EYFP and CX3CR1-GFP reporters. Epididymal DCs (eDCs) establish intimate interactions with the epithelium and project long dendrites between epithelial cells toward the lumen. We show that isolated eDCs express numerous leukocyte markers described previously in other organs that are in contact with the external environment, and present and cross-present ovalbumin to T cells in vitro. eDCs are, therefore, strategically positioned to regulate the complex interplay between immune tolerance and activation, a balance that is fundamental to male fertility.

Proton-transporting cells are located in several tissues where they acidify the extracellular environment. These cells express the vacuolar H(+)-ATPase (V-ATPase) B1 subunit (ATP6V1B1) in their plasma membrane. We provide here a comprehensive catalog of the proteins that are expressed in these cells, after their isolation by enzymatic digestion and fluorescence-activated cell sorting (FACS) from transgenic B1-enhanced green fluorescent protein (EGFP) mice. In these mice, type A and B intercalated cells and connecting segment cells of the kidney, and narrow and clear cells of the epididymis, which all express ATP6V1B1, also express EGFP, while all other cell types are negative. The proteome of renal and epididymal EGFP-positive (EGFP(+)) cells was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and compared with their respective EGFP-negative (EGFP(-)) cell populations. A total of 2,297 and 1,564 proteins were detected in EGFP(+) cells from the kidney and epididymis, respectively. Out of these proteins, 202 and 178 were enriched by a factor greater than 1.5 in EGFP(+) cells compared with EGFP(-) cells, in the kidney and epididymis respectively, and included subunits of the V-ATPase (B1, a4, and A). In addition, several proteins involved in intracellular trafficking, signaling, and cytoskeletal dynamics were identified. A novel common protein that was enriched in renal and epididymal EGFP(+) cells is the progesterone receptor, which might be a potential candidate for the regulation of V-ATPase-dependent proton transport. These proteomic databases provide a framework for comprehensive future analysis of the common and distinct functions of V-ATPase-B1-expressing cells in the kidney and epididymis.

Extracellular purinergic agonists regulate a broad range of physiological functions via P1 and P2 receptors. Using the epididymis as a model system in which luminal acidification is essential for sperm maturation and storage, we show here that extracellular ATP and its hydrolysis product adenosine trigger the apical accumulation of vacuolar H(+)-ATPase (V-ATPase) in acidifying clear cells. We demonstrate that the epididymis can hydrolyze luminal ATP into other purinergic agonists such as ADP via the activity of nucleotidases located in the epididymal fluid and in the apical membrane of epithelial cells. Alkaline phosphatase activity and abundant ecto-5'-nucleotidase protein were detected in the apical pole of principal cells. In addition, we show that nine nucleotidase genes (Nt5e, Alpl, Alpp, Enpp1, 2, and 3, and Entpd 2, 4, and 5), seven ATP P2 receptor genes (P2X1, P2X2, P2X3, P2X4, P2X6, P2Y2, P2Y5), and three adenosine P1 receptor genes (A1, A2B, and A3) are expressed in epithelial cells isolated by laser cut microdissection (LCM). The calcium chelator BAPTA-AM abolished the apical V-ATPase accumulation induced by ATP, supporting the contribution of P2X or P2Y in this response. The PKA inhibitor myristoylated protein kinase inhibitor (mPKI) inhibited adenosine-dependent V-ATPase apical accumulation, indicating the participation of the P1 A2B receptor. Altogether, these results suggest that the activation of P1 and P2 purinergic receptors by ATP and adenosine might play a significant role in luminal acidification in the epididymis, a process that is crucial for the establishment of male fertility.

An acidic luminal pH in the epididymis contributes to maintaining sperm quiescent during their maturation and storage. The vacuolar H(+)ATPase (V-ATPase), located in narrow and clear cells, is a major contributor to luminal acidification. Mutations in one of the V-ATPase subunits, ATP6v1B1 (B1), cause distal renal tubular acidosis in humans but surprisingly, B1(-/-) mice do not develop metabolic acidosis and are fertile. While B1 is located in the apical membrane of narrow and clear cells, the B2 subunit localizes to subapical vesicles in wild-type mouse, rat and human epididymis. However, a marked increase (84%) in the mean pixel intensity of B2 staining was observed in the apical pole of clear cells by conventional immunofluorescence, and relocalization into their apical membrane was detected by confocal microscopy in B1(-/-) mice compared with B1(+/+). Immunogold electron microscopy showed abundant B2 in the apical microvilli of clear cells in B1(-/-) mice. B2 mRNA expression, determined by real time RT-PCR using laser-microdissected epithelial cells, was identical in both groups. Semiquantitative Western blots from whole epididymis and cauda epididymidis showed no variation of B2 expression. Finally, the luminal pH of the cauda epididymidis was the same in B1(-/-) mice as in B1(+/+) (pH 6.7). These data indicate that whereas overall expression of B2 is not affected in B1(-/-) mice, significant redistribution of B2-containing complexes occurs from intracellular compartments into the apical membrane of clear cells in B1(-/-) mice. This relocation compensates for the absence of functional B1 and maintains the luminal pH in an acidic range that is compatible with fertility.

Luminal acidification in the epididymis is an important process for the regulation of male fertility. Low pH and low bicarbonate concentration are among key factors that keep spermatozoa in a dormant state while they mature and are stored in this organ. Although significant bicarbonate reabsorption is achieved by principal cells in the proximal regions of the epididymis, clear and narrow cells are specialized for net proton secretion. Clear cells express very high levels of the vacuolar proton pumping ATPase (V-ATPase) in their apical membrane and are responsible for the bulk of proton secretion. In the present paper, selected aspects of V-ATPase regulation in clear cells are described and potential pathologies associated with mutations of some of the V-ATPase subunits are discussed.

The male reproductive tract and accessory glands comprise a complex but interrelated system of tissues that are composed of many distinct cell types, all of which contribute to the ability of spermatozoa to carry out their ultimate function of fertilizing an oocyte. Spermatozoa undergo their final steps of maturation as they pass through the male excurrent duct, which includes efferent ducts, the epididymis and the vas deferens. The composition of the luminal environment in these organs is tightly regulated. Major fluid reabsorption occurs in efferent ducts and in the epididymis, and leads to a significant increase in sperm concentration. In the distal epididymis and vas deferens, fluid secretion controls the final fluidity of the luminal content. Therefore, the process of water movement in the excurrent duct is a crucial step for the establishment of male fertility. Aquaporins contribute to transepithelial water transport in many tissues, including the kidney, the brain, the eye and the respiratory tract. The present article reviews our current knowledge regarding the distribution and function of aquaporins in the male excurrent duct.

The mammalian aquaporins (AQPs) are a family of 13 transmembrane channel proteins that are involved in the transport of water in numerous organs. In the male excurrent duct, the movement of fluid and solutes across the epithelium is essential for establishing the proper luminal environment in which sperm mature and are stored. AQP9 is abundantly expressed in the efferent ducts, the epididymis, and the vas deferens, where it could represent an important apical pathway for transmembrane water and solute movement. However, other organs in which water transport is critical, including the kidney, the lung, or the eye, express several different AQPs with a cell-specific pattern. To undertake a systematic analysis of the expression of known AQPs in the postnatal and adult rat epididymis, we examined the expression of their respective mRNAs in epithelial cells isolated by laser capture microdissection (LCM), and we determined their corresponding protein expression pattern by immunofluorescence and Western blotting. Our data show that, whereas AQP9 is the main AQP of the epididymis, the mRNA specific for Aqp2, 5, 7, and 11 are also expressed in epididymal epithelial cells. AQP5 protein colocalizes with AQP9 in the apical membrane of a subpopulation of principal cells in the corpus and cauda regions. Aqp2 mRNA was detected in epithelial cells after the second postnatal week and the amount significantly increased up to adulthood. However, AQP2 protein was detected only in the distal cauda of young rats (between the second and fourth postnatal week). No AQP2 protein was detected in the adult epididymis, indicating that posttranscriptional mechanisms are involved in the regulation of AQP2 expression. In addition, epididymal epithelial cells express significant amounts of the mRNAs coding for AQP7 and 11. No mRNA or protein for AQPs 0, 4, 6, and 8 were detectable in epithelial cells, and Aqp1 was detected in whole epididymal samples, but not in epithelial cells. Thanks to the recent development of microdissection technologies, our observations suggest that epididymal epithelial cells express several members of the AQP family with a region-specific pattern. AQPs may be involved not only in the transepithelial transport of water in the epididymis but also in the postnatal development of this organ, as suggested by the differential expression of AQP2.

B1 and B2 are two highly homologous isoforms of the vacuolar H(+)-ATPase (V-ATPase) 56-kDa B subunit. We investigated whether the B2 subunit is expressed alongside B1 in proton-secreting cells of the rodent kidney collecting duct (intercalated cells, IC) and epididymis (clear cells) by using antibodies against distinct COOH-terminal peptides from the two B isoforms. B2 was detected not only in the kidney proximal tubule, thick ascending limb, distal convoluted tubule, and connecting segment but also in A- and B-type IC of collecting ducts (CD) in both rat and mouse. B2 had a predominant cytoplasmic localization in most IC but was clearly located in a tighter apical band together with the V-ATPase 31-kDa E subunit in some A-IC, especially in the medulla. Apical membrane staining was confirmed by immunogold electron microscopy. B2 was very weakly expressed on the basolateral membranes of B-IC in control kidney CD, but some connecting segment B-IC had more distinct basolateral staining. In response to chronic carbonic anhydrase inhibition by acetazolamide, many A-IC showed a strong apical membrane localization of B2, where it colocalized with E and B1. In rat and mouse epididymis, B2 isoform expression was detected in clear cells, where it was concentrated in subapical vesicles. Unlike B1, B2 did not colocalize with the E subunit in the apical microvilli. These findings indicate that in addition to its role in the acidification of intracellular organelles, the B2 isoform could also contribute to transepithelial proton secretion and the maintenance of acid-base homeostasis.

Epithelial cells of the epididymis and vas deferens establish an optimum luminal environment in which spermatozoa mature and are stored. This is achieved by active transepithelial transport of various ions including Cl(-) and H(+). We investigated the localization of three closely related members of the ClC family, ClC-3, ClC-4, and ClC-5, in the epididymis and vas deferens. RT-PCR using mRNA isolated by laser capture microdissection (LCM)-detected ClC-3 and ClC-5 transcripts but did not detect any ClC-4-specific transcript. Western blot and immunofluorescence analysis demonstrated that ClC-3 and ClC-5 proteins are present in all regions of the epididymis and in the vas deferens. ClC-5 is expressed exclusively in H(+)-ATPase-rich cells (narrow and clear cells). Confocal microscopy showed that ClC-5 partially colocalizes with the H(+)-ATPase in the subapical pole of clear cells. ClC-3 is strongly expressed in the apical membrane of principal cells of the caput epididymidis and the vas deferens and is less abundant in principal cells of the body and cauda epididymidis. These findings are consistent with a potential role for ClC-3 in transepithelial chloride transport by principal cells and for ClC-5 in the acidification of H(+)-ATPase-containing vesicles in narrow and clear cells. ClC-5 might facilitate endosome trafficking in the epididymis, as has been proposed in the kidney.

Summary: Basal cells are by definition located on the basolateral side of several epithelia, and they have never been observed reaching the lumen. Using high-resolution 3D confocal imaging, we report that basal cells extend long and slender cytoplasmic projections that not only reach toward the lumen but can cross the tight junction barrier in some epithelia of the male reproductive and respiratory tracts. In this way, the basal cell plasma membrane is exposed to the luminal environment. In the epididymis, in which luminal acidification is crucial for sperm maturation and storage, these projections contain the angiotensin II type 2 receptor (AGTR2). Activation of AGTR2 by luminal angiotensin II, increases proton secretion by adjacent clear cells, which are devoid of AGTR2. We propose a paradigm in which basal cells scan and sense the luminal environment of pseudostratified epithelia and modulate epithelial function by a mechanism involving crosstalk with other epithelial cells. [Copyright &y& Elsevier]

Water and solute transport across the plasma membrane of cells is a crucial biological function that is mediated mainly by aquaporins and aquaglyceroporins. The regulation of these membrane proteins is stifi incompletely understood. Using the male reproductive tract as a model system in which water and glycerol transport are critical for the establishment of fertility, we now report a novel pathway for the regulation of aquaporin 9 (AQP9) permeability. AQP9 is the major aquaglyceroporin of the epididymis, liver, and peripheral leukocytes, and its COOH-terminal portion contains a putative PDZ binding motif (SVIM). Here we show that NHERF1, cystic fibrosis transmembrane conductance regulator (CFTR), and AQP9 co-localize in the apical membrane of principal cells of the epididymis and the vas deferens, and that both NHERF1 and CFTR co-immunoprecipitate with AQP9. Overlay assays revealed that AQP9 binds to both the PDZ1 and PDZ2 domains of NHERF1, with an apparently higher affinity for PDZ1 versus PDZ2. Pull-down assays showed that the AQP9 COOH-terminal SVIM motif is essential for interaction with NHERF1. Functional assays on isolated tubules perfused in vitro showed a high permeabffity of the apical membrane to glycerol, which is inhibited by the AQP9 inhibitor, phioretin, and is markedly activated by cAMP. The CFTR inhibitors DPC, G1yH-101 and CFTR1nh-172 all significantly reduced the cAMP-activated glycerol-induced cell sweffing. We propose that CFTR is an important regulator of AQP9 and that the interaction between AQP9, NHERF1, and CFTR may facilitate the activation of AQP9 by cAMP. [ABSTRACT FROM AUTHOR]

B1 and B2 are two highly homologous isoforms of the vacuolar H+-ATPase (V-ATPase) 56-kDa B subunit. We investigated whether the B2 subunit is expressed alongside B1 in proton-secreting cells of the rodent kidney collecting duct (intercalated cells, IC) and epididymis (clear cells) by using antibodies against distinct COOH-terminal peptides from the two B isoforms. B2 was detected not only in the kidney proximal tubule, thick ascending limb, distal convoluted tubule, and connecting segment but also in A- and B-type IC of collecting ducts (CD) in both rat and mouse. B2 had a predominant cytoplasmic localization in most IC but was clearly located in a tighter apical band together with the V-ATPase 31-kDa E subunit in some A-IC, especially in the medulla. Apical membrane staining was confirmed by immunogold electron microscopy. B2 was very weakly expressed on the basolateral membranes of B-IC in control kidney CD, but some connecting segment B-IC had more distinct basolateral staining. In response to chronic carbonic anhydrase inhibition by acetazolamide, many A-IC showed a strong apical membrane localization of B2, where it colocalized with E and BI. In rat and mouse epididymis, B2 isoform expression was detected in clear cells, where it was concentrated in subapical vesicles. Unlike B1, B2 did not colocalize with the E subunit in the apical microvilli. These findings indicate that in addition to its role in the acidification of intracellular organelles, the B2 isoform could also contribute to transepithelial proton secretion and the maintenance of acid-base homeostasis. [ABSTRACT FROM AUTHOR]

Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H[sup +]ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate, sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH. [ABSTRACT FROM AUTHOR]