Establishment of cell-cell cross talk in the epididymis: control of luminal acidification

Abstract

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.

Figure 1

Schematic view of different epithelial cell types in the epididymis. Several cell types compose the epididymal epithelium. These include narrow, clear, principal, and basal cells. Both narrow and clear cells express the proton-pumping ATPase in their apical membrane. Basal cells have variable morphologic appearances, and they have the property of extending a slender body projection toward the lumen, between adjacent epithelial cells. Although few “apical-reaching” basal cells are present in the initial segment and caput, their numbers increase progressively in the corpus and reach a maximum in the distal corpus and proximal cauda regions. Modified from Shum et al (2009) with permission from the Journal of Experimental Biology.

Figure 2

High number of clear cells in the cauda (B) vs caput (A) epididymidis. Immunofluorescence labeling of rat epididymis sections for the proton-pumping ATPase (V-ATPase; green) and NHERF1, a marker of principal cells (red), showed that V-ATPase–positive clear cells are more numerous in the cauda (B) compared with the caput (A) epididymidis. Nuclei and sperm are stained in blue with 4′,6-diamidino-2-phenylindole. Reproduced from Shum et al (2009) with permission from the Journal of Experimental Biology. Scale bars = 50 μm.

Figure 3

Localization of the B1 and a4 subunits of the proton-pumping ATPase in clear cells. Rat cauda epididymis was stained with antibodies against subunit isoforms B1 (A; green) and a4 (B; red). Panel C shows a merged image from Panels A and B. Both B1 and a4 subunits are located in the apical pole of clear cells. A faint B1 staining is also seen in the cytosol of clear cells. Nuclei and sperm are stained in blue with 4′,6-diamidino-2-phenylindole. Scale bars = 20 μm.

Figure 4

Immunofluorescence labeling of proton-pumping ATPase (V-ATPase) in epididymis after in vivo perfusion. Rat cauda epididymidis was perfused in vivo, and cryostat sections of fixed tissues were stained for the V-ATPase B1 subunit (green). Nuclei were stained in blue with 4′,6-diamidino-2-phenylindole. (A) Luminal sperm are absent from these perfused tubules, and numerous V-ATPase–labeled clear cells are detected. (B) Higher magnification of a clear cell perfused with a control solution adjusted to the “resting” pH of 6.6 and containing the endocytic marker horseradish peroxidase (HRP). Double labeling for the V-ATPase B1 subunit (green) and HRP (red) was performed. The arrows indicate the border between the apical cytoplasm of the cell and microvilli. The V-ATPase is located in subapical vesicles as well as in short microvilli. The yellow staining shows partial colocalization of the V-ATPase in HRP-containing endosomes. (C) After perfusion with an “activating” buffer containing bicarbonate and cpt-cAMP, the V-ATPase is located in longer microvilli (green), and no colocalization with HRP-labeled endosomes is detected (red). Scale bars = 150 μm (A), 5 μm (B, C). Reproduced from Shum et al (2009) with permission from the Journal of Experimental Biology.

Figure 5

Basal cells express angiotensin II type II receptor (AGTR2) and send a slender body projection toward the lumen (Lu). (A) Rat cauda epididymis double labeled for AGTR2 (green) and proton-pumping ATPase (V-ATPase; red). Nuclei and sperm are labeled in blue with 4′,6-diamidino-2-phenylindole. The arrow shows an AGTR2-positive basal cell extending a narrow projection toward the Lu. The arrowhead indicates a clear cell labeled for V-ATPase. (B′, B″, B‴) Three-dimensional reconstruction of an epididymis section showing several basal cells, labeled for claudin-1 (red; arrows), sending their body projections toward the Lu. Double labeling for the tight junction marker, ZO1 (green), showed that these basal cells reach the luminal border of the epithelium at the corner junctions formed by three epithelial cells (arrows). Scale bars = 5 μm (A) and 10 μm (B). Reproduced from Shum et al (2008) with permission from Elsevier.

Figure 6

(A) Basal cells can cross the tight junctions to reach the luminal side of the epithelium. A basal cell, labeled for claudin-1 (red; arrow), forms a tight junction, labeled for ZO1 (green), between adjacent principal cells. This basal cell is in contact with the lumen. The arrowhead indicates a clear cell, labeled for proton-pumping ATPase (V-ATPase; blue). Nuclei are also labeled blue with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar = 5 μm. Reproduced from Shum et al (2008) with permission from Elsevier. (B) A basal cell sending its slender body projection along a clear cell. The cellular body of this basal cell, labeled for keratin 5 (red), is in close contact with a clear cell, labeled for V-ATPase (green). Nuclei and sperm are labeled blue with DAPI. Scale bar = 5 μm.

Figure 7

Schematic representation of cell-cell cross talk in the epididymis. Basolateral activation of principal cells during sexual arousal triggers the secretion of bicarbonate and ATP into the lumen, a process that is mediated by cystic fibrosis transmembrane conductance regulator. Stimulation of bicarbonate-sensitive adenylyl cyclase (sAC) and purinergic receptors in clear cells induces proton-pumping ATPase (V-ATPase) accumulation in apical microvilli, followed by an increase in proton secretion. Basal cells can extend a narrow body projection toward the lumen, and they form a new tight junction with adjacent epithelial cells. Luminal angiotensin II (ANGII) stimulates ANGII type II receptor (AT2) in basal cells and induces the production of nitric oxide (NO), which then diffuses out to produce cGMP in clear cells via activation of guanylate cyclase (sGC). The cGMP triggers the apical accumulation of V-ATPase and increases proton secretion. Simulation of basal cells by basolateral factors, including lysyl bradykinin, was shown to increase bicarbonate and chloride secretion in principal cells via prostaglandin E₂ (PGE₂) stimulation. NBC indicates sodium bicarbonate cotransporter; PKA, protein kinase A.