Winnie W. C. Shum

Transepithelial Projections from Basal Cells Are Luminal Sensors in Pseudostratified Epithelia

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.

Regulation of luminal acidification in the male reproductive tract via cell–cell crosstalk

SUMMARY In the epididymis, spermatozoa acquire their ability to become motile and to fertilize an egg. A luminal acidic pH and a low bicarbonate concentration help keep spermatozoa in a quiescent state during their maturation and storage in this organ. Net proton secretion is crucial to maintain the acidity of the luminal fluid in the epididymis. A sub-population of epithelial cells, the clear cells, express high levels of the proton-pumping V-ATPase in their apical membrane and are important contributors to luminal acidification. This review describes selected aspects of V-ATPase regulation in clear cells. The assembly of a particular set of V-ATPase subunit isoforms governs the targeting of the pump to the apical plasma membrane. Regulation of V-ATPase-dependent proton secretion occurs via recycling mechanisms. The bicarbonate-activated adenylyl cyclase is involved in the non-hormonal regulation of V-ATPase recycling, following activation of bicarbonate secretion by principal cells. The V-ATPase is also regulated in a paracrine manner by luminal angiotensin II by activation of the angiotensin II type 2 receptor (AGTR2), which is located in basal cells. Basal cells have the remarkable property of extending long and slender cytoplasmic projections that cross the tight junction barrier to monitor the luminal environment. Clear cells are activated by a nitric oxide signal that originates from basal cells. Thus, a complex interplay between the different cell types present in the epithelium leads to activation of the luminal acidifying capacity of the epididymis, a process that is crucial for sperm maturation and storage.

Cell-cell interaction underlies formation of fluid in the male reproductive tract of the rat.

The epithelia lining the epididymides of many species consists of several cell types. We have provided evidence that the basal cells are essential to the integrated functions of the epithelium. Basal cells, but not principal cells, and other cells in the epididymis express TRPC3 and COX-1. We have isolated basal cells from intact rat epididymis using antibody-coated Dynabeads and subjected them to whole-cell patch-clamp measurement of nonselective cation channel activity, a feature of TRPC3 protein, and Fluo-3 fluorescence measurement of intracellular Ca2+ concentration. The results show that a nonselective cation current blockable by La3+ (0.1 mM), Gd3+ (0.1 mM), or SKF96365 (20 μM) could be activated by lysylbradykinin (200 nM). In cells loaded with Fluo-3, addition of lysylbradykinin (100 nM) caused a sustained increase of intracellular Ca2+. This effect was blocked by Gd3+ (0.1 mM) or SKF96365 (20 μM) and was not observed in Fluo-3–loaded principal cells. Stimulation of basal cell/principal cell cocultures with lysylbradykinin (200 nM) evoked in principal cells a current with CFTR-Cl− channel characteristics. Isolated principal cells in the absence of basal cells did not respond to lysylbradykinin but responded to PGE2 (100 nM) with activation of a CFTR-like current. Basal cells, but not principal cells, released prostaglandin E2 when stimulated with lysylbradykinin (100 nM). The release was blocked by SKF96365 (20 μM) and BAPTA-AM (0.05 or 0.1 mM). Confluent cell monolayers harvested from a mixture of disaggregated principal cells and basal cells responded to lysylbradykinin (100 nM) and PGE2 (500 nM) with an increase in electrogenic anion secretion. The former response was dependent on prostaglandin synthesis as piroxicam blocked the response. However, cell cultures obtained from principal cells alone responded to PGE2 but not to bradykinin. These results support the notion that basal cells regulate principal cells through a Ca2+ and COX signaling pathway.

Epithelial Basal Cells Are Distinct from Dendritic Cells and Macrophages in the Mouse Epididymis

ABSTRACT 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.

ATP secretion in the male reproductive tract: essential role of CFTR

CFTR is expressed in principal cells but not clear cells in mouse cauda epididymis. Inhibition or knockdown of CFTR inhibits ATP release from mouse epididymal principal cells. Inhibition of CFTR reduces ATP release into the lumen of cauda epididymis in mice in vivo. These results show the involvement of CFTR in the regulation of ATP release from epithelial principal cells in the cauda epididymidis. Defective ATP signalling in the epididymis might contribute to dysfunction of the male reproductive tract associated with CFTR mutations.

Angiotensin II and hypertonicity modulate proximal tubular aquaporin 1 expression

Aquaporin 1 (AQP1) is the major water channel in the renal proximal tubule (PT) and thin descending limb of Henle, but its regulation remains elusive. Here, we investigated the effect of ANG II, a key mediator of body water homeostasis, on AQP1 expression in immortalized rat proximal tubule cells (IRPTC) and rat kidney. Real-time PCR on IRPTC exposed to ANG II for 12 h revealed a biphasic effect AQP1 mRNA increased dose dependently in response to 10(-12) to 10(-8) M ANG II but decreased by 50% with 10(-7) M ANG II. The twofold increase of AQP1 mRNA in the presence of 10(-8) M ANG II was abolished by the AT(1) receptor blocker losartan. Hypertonicity due to either NaCl or mannitol also upregulated AQP1 mRNA by three- and twofold, respectively. Immunocytochemistry and Western blotting revealed a two- to threefold increase in AQP1 protein expression in IRPTC exposed concomitantly to ANG II (10(-8)M) and hypertonic medium (either NaCl or mannitol), indicating that these stimuli were not additive. Three-dimensional reconstruction of confocal images suggested that AQP1 expression was increased by ANG II in both the apical and basolateral poles of IRPTC. In vivo studies showed that short-term ANG II infusion had a diuretic effect, while this effect was attenuated after several days of ANG II infusion. After 10 days, we observed a twofold increase in AQP1 expression in the PT and thin descending limb of Henle of ANG II-infused rats that was abolished when rats were treated with the selective AT(1)-receptor antagonist olmesartan. Thus ANG II increases AQP1 expression in vitro and in vivo via direct interaction with the AT(1) receptor, providing an important regulatory mechanism to link PT water reabsorption to body fluid homeostasis via the renin-angiotensin system.

Segmental Expression of the Bradykinin Type 2 Receptor in Rat Efferent Ducts and Epididymis and Its Role in the Regulation of Aquaporin 9

Abstract Water and solute transport in the efferent ducts and epididymis are important for the establishment of the appropriate luminal environment for sperm maturation and storage. Aquaporin 9 (AQP9) is the main water channel in the epididymis, but its regulation is still poorly understood. Components of the kinin-kallikrein system (KKS), leading to the production of bradykinin (BK), are highly expressed in the lumen of the male reproductive tract. We report here that the epididymal luminal fluid contains a significant amount of BK (2 nM). RT-PCR performed on epididymal epithelial cells isolated by laser capture microdissection (LCM) showed abundant BK type 2 receptor (Bdkrb2) mRNA expression but no type 1 receptor (Bdkrb1). Double-immunofluorescence staining for BDKRB2 and the anion exchanger AE2 (a marker of efferent duct ciliated cells) or the V-ATPase E subunit, official symbol ATP6V1E1 (a marker of epididymal clear cells), showed that BDKRB2 is expressed in the apical pole of nonciliated cells (efferent ducts) and principal cells (epididymis). Triple labeling for BDKRB2, AQP9, and ATP6V1E1 showed that BDKRB2 and AQP9 colocalize in the apical stereocilia of principal cells in the cauda epididymidis. While uniform Bdkrb2 mRNA expression was detected in the efferent ducts and along the epididymal tubule, marked variations were detected at the protein level. BDKRB2 was highest in the efferent ducts and cauda epididymidis, intermediate in the distal initial segment, moderate in the corpus, and undetectable in the proximal initial segment and the caput. Functional assays on tubules isolated from the distal initial segments showed that BK significantly increased AQP9-dependent glycerol apical membrane permeability. This effect was inhibited by BAPTA-AM, demonstrating the participation of calcium in this process. This study, therefore, identifies BK as an important regulator of AQP9.

High-resolution helium ion microscopy of epididymal epithelial cells and their interaction with spermatozoa

We examined the rat and mouse epididymis using helium ion microscopy (HIM), a novel imaging technology that uses a scanning beam of He(+) ions to produce nanometer resolution images of uncoated biological samples. Various tissue fixation, sectioning and dehydration methods were evaluated for their ability to preserve tissue architecture. The cauda epididymidis was luminally perfused in vivo to remove most spermatozoa and the apical surface of the epithelial lining was exposed. Fixed epididymis samples were then subjected to critical point drying (CPD) and HIM. Apical stereocilia in principal cells and smaller apical membrane extensions in clear cells were clearly distinguishable in both rat and mouse epididymis using this technology. After perfusion with an activating solution containing CPT-cAMP, a permeant analog of cAMP, clear cells exhibited an increase in the number and size of membrane ruffles or microplicae. In contrast, principal cells did not exhibit detectable structural modifications. High-resolution HIM imaging clearly showed the ultrastructure of residual sperm cells, including the presence of concentric rings on the midpiece, and of cytoplasmic droplets in some spermatozoa. Close epithelium-sperm interactions were also detected. We found a number of sperm cells whose heads were anchored within the epididymal epithelium. In certain cases, the surface of the sperm cytoplasmic droplet was covered with vesicle-like structures whose size is consistent with that of epididymosomes. In conclusion, we describe here the first application of HIM technology to the study of the structure and morphology of the rodent epididymis. HIM technology represents a major imaging breakthrough that can be successfully applied to study the epididymis and spermatozoa, with the goal of advancing our understanding of their structure and function.

CCNYL1, but Not CCNY, Cooperates with CDK16 to Regulate Spermatogenesis in Mouse.

Cyclin Y-like 1 (Ccnyl1) is a newly-identified member of the cyclin family and is highly similar in protein sequences to Cyclin Y (Ccny). However, the function of Ccnyl1 is poorly characterized in any organism. Here we found that Ccnyl1 was most abundantly expressed in the testis of mice and was about seven times higher than the level of Ccny. Male Ccnyl1-/- mice were infertile, whereas both male and female Ccny-/- mice displayed normal fertility. These results suggest that Ccnyl1, but not Ccny, is indispensable for male fertility. Spermatozoa obtained from Ccnyl1-/- mice displayed significantly impaired motility, and represented a thinned annulus region and/or a bent head. We found that the protein, but not the mRNA, level of cyclin-dependent kinase 16 (CDK16) was decreased in the testis of Ccnyl1-/- mice. Further study demonstrated that CCNYL1 interacted with CDK16 and this interaction mutually increased the stability of these two proteins. Moreover, the interaction increased the kinase activity of CDK16. In addition, we observed an alteration of phosphorylation levels of CDK16 in the presence of CCNYL1. We identified the phosphorylation sites of CDK16 by mass spectrometry and revealed that several phosphorylation modifications on the N-terminal region of CDK16 were indispensable for the CCNYL1 binding and the modulation of CDK16 kinase activity. Our results therefore reveal a previously unrecognized role of CCNYL1 in regulating spermatogenesis through the interaction and modulation of CDK16.

Role of Testicular Luminal Factors on Basal Cell Elongation and Proliferation in the Mouse Epididymis

ABSTRACT 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.