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 (HCO₃⁻) 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. HCO₃⁻ induces V-ATPase accumulation in apical microvilli in clear cells via HCO₃⁻-sensitive adenylyl cyclase–dependent cAMP production. HCO₃⁻ 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.

The rate of human fertility has been declining throughout the world during the past 3 decades (Skakkebaek et al, 2006). Consequently, infertility is now a major health problem that affects at least one-sixth of couples worldwide. Although a large percentage of these couples are afflicted by male reproduction dysfunction, the etiology of the fertility problem remains unexplained for most of these men. Male infertility is often caused by sperm that have low motility and interact poorly with the oocyte (Elzanaty et al, 2002; Aitken, 2006). Spermatozoa acquire these crucial functions in the epididymis, where they undergo their final maturation and are stored (Yeung et al, 1993; Hinton et al, 1996; Jones and Murdoch; 1996, Orgebin-Crist, 2003; Robaire et al, 2006; Cornwall, 2009). The epididymis is formed by a highly convoluted tubule that is segmented into morphologically and functionally distinct regions: the initial segment, caput, corpus, and cauda. Each of these regions is further divided into intraregional segments, and 10 and 19 segments were identified in the mouse and rat epididymis, respectively (Johnston et al, 2007). Four cell types are present in the epithelium lining the epididymal lumen: narrow, clear, principal, and basal cells. Although principal and basal cells are present in all epididymal regions, narrow cells are located exclusively in the initial segment, and clear cells are present in the caput, corpus, and cauda epididymidis (Figure 1). Each cell type contributes to the establishment and regulation of a unique luminal environment for the concentration, maturation, storage, and viability of spermatozoa (Wong et al, 2002; Robaire et al, 2006; Da Silva et al, 2007; Cornwall, 2009; Shum et al, 2009). This review will focus on how these cells work in a concerted manner, together with spermatozoa, to acidify the luminal fluid.

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.

Luminal Acidification in the Epididymis

The establishment of a low bicarbonate (HCO₃⁻) concentration and low pH (Levine and Marsh, 1971; Levine and Kelly, 1978) is essential for the maintenance of sperm quiescence during their maturation and storage in the epididymis (Acott and Carr, 1984; Carr et al, 1985; Pastor-Soler et al, 2005). By preventing the activation of sperm-specific Ca²⁺ and K⁺ channels involved in capacitation, acidic pH helps to keep sperm in a dormant state (Kirichok et al, 2006; Navarro et al, 2007, 2008). High HCO₃⁻ levels in seminal vesicle fluids and in the female reproductive tract elevate cAMP in sperm via activation of the HCO₃⁻-sensitive adenylyl cyclase (sAC; also known as SACY; Vishwakarma, 1962; Hamner and Williams, 1964; Okamura et al, 1985; Chen et al, 2000; Sinclair et al, 2000, Hess et al, 2005; Salicioni et al, 2007; Chan et al, 2009; Visconti, 2009). Bicarbonate entry into sperm cells is mediated by the cystic fibrosis conductance transmembrane regulator, (CFTR) (Xu et al, 2007), and an Na⁺/HCO₃⁻ cotrans-porter (Demarco et al, 2003). Protein kinase A (PKA)-dependent phosphorylation of serine and threonine residues of several proteins is one of the key events leading to sperm capacitation (Visconti et al, 1999; Demarco et al, 2003; Hernandez-Gonzalez et al, 2006; Salicioni et al, 2007). For example, inhibition of epithelial Na⁺ channel after phosphorylation was shown to induce the hyperpolarization that accompanies capacitation (Hernandez-Gonzalez et al, 2006). Thus, pH and HCO₃⁻ are key regulators of sperm function, and a defect in acid/base transport in the epididymis, leading to elevation of luminal pH or HCO₃⁻ concentration, might have profound consequences on male fertility by allowing sperm to be prematurely activated.

The importance of luminal acidification was shown in c-ros knockout (KO) male mice, which are infertile and have an abnormally elevated epididymal luminal pH (Yeung et al, 2004). In addition, male mice that are deficient in the transcription factor Foxi1 (Foxi1 KO), which is specifically expressed in clear cells and controls expression of proteins involved in acid secretion, also have a higher epididymal luminal pH compared with wild type and are infertile because of the inability of their spermatozoa to move up the female reproductive tract and fertilize an egg (Blomqvist et al, 2006; Vidarsson et al, 2009). In addition, environmental pollutants that inhibit luminal acidification, such as tobacco smoke and heavy metals (Caflisch and DuBose, 1991; Herak-Kramberger et al, 2000), induce a reduction in male fertility (Parizek and Zahor, 1956; Tas et al, 1996).

Luminal acidification is achieved by epithelial cells that have specific roles depending on their location along the epididymis. In the initial segments, principal cells reabsorb HCO₃⁻ (Levine and Marsh, 1971; Caflisch, 1992; Pastor-Soler et al, 2005; Shum et al, 2009). In the rest of the epididymis and proximal vas deferens, clear cells are involved in net proton secretion via the proton-pumping ATPase (V-ATPase), which is highly expressed in their apical plasma membrane (Brown et al, 1992; Breton et al, 1996; Herak-Kramberger et al, 2000; Pastor-Soler et al, 2003, 2005, 2008; Beaulieu et al, 2005; Da Silva et al, 2007; Shum et al, 2008). Clear cells are significantly more numerous in the cauda epididymidis compared with the caput region (Figure 2), indicating that they have a more predominant acidifying role in the distal region, where spermatozoa are stored. The V-ATPase is composed of several subunits, and a particular set of subunit isoforms, including subunits B1, a4, A, and E2, are expressed exclusively or are highly enriched in clear cells (Figure 3; Miller et al, 2005; Pietrement et al, 2006). In addition to mice and rats, clear cells of the human epididymis also express the V-ATPase (Herak-Kramberger et al, 2001; Da Silva et al, 2007), indicating that the acidifying role of these cells occurs across species. The a4 promoter is under the control of the Foxi1 transcription factor, and several V-ATPase subunits are absent in clear cells from Foxi1 KO mice (Blomqvist et al, 2006; Vidarsson et al, 2009), further illustrating the important role of the V-ATPase in male fertility.

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.

Regulation of Proton Secretion via Recycling in Clear Cells

Clear cells have a very high level of endocytosis, and they were proposed to play a role in the removal of factors from the lumen (Moore and Bedford, 1979; Hermo et al, 1988, 1991). We developed a surgical procedure that allows the microperfusion of the lumen of the cauda epididymidis in vivo (Figure 4A). By perfusing the lumen with fluid phase markers, we showed that under resting control conditions, V-ATPase colocalizes with endosomes (Figure 4B; Breton et al, 2000a; Pastor-Soler et al, 2003; Beaulieu et al, 2005), indicating that V-ATPase recycling significantly contributes to the high endocytic activity of clear cells.

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.

Proton secretion in clear cells is increased following the redistribution of V-ATPase from subapical vesicles to the apical membrane (Pastor-Soler et al, 2005; Da Silva et al, 2007; Shum et al, 2009), a process that is accompanied by the formation and extension of apical V-ATPase–rich microvilli (Breton et al, 1996, 1998, 2000a; Brown et al, 1997; Herak-Kramberger et al, 2000; Beaulieu et al, 2005; Shum et al, 2008). The microtubule-disrupting agent, colchicine, induces a complete redistribution of the V-ATPase from the apical pole to numerous vesicles scattered throughout the cell, and cleavage of the v-SNARE protein cellubrevin decreases V-ATPase–dependent proton secretion (Breton et al, 2000a). Microtubules play an important role in exocytic events, and therefore these results indicated that exocytosis controls the number of V-ATPase molecules that are inserted into the apical membrane. V-ATPase recycling also depends on dynamic remodeling of the actin cytoskeleton (Beaulieu et al, 2005). Several V-ATPase subunits (B1, B2, and C) interact directly or indirectly with the actin cytoskeleton (Breton et al, 2000b; Holliday et al, 2000; Vitavska et al, 2003; Chen et al, 2004), and we showed that the actin-severing and actin-capping protein, gelsolin, is a key regulator of V-ATPase recycling (Beaulieu et al, 2005).

V-ATPase Recycling Is Regulated by the cAMP/PKA and Phospholipase C Signaling Pathways

We showed that luminal perfusion with the cAMP permeant analogue, cpt-cAMP, in a bicarbonate-containing solution induces V-ATPase apical membrane accumulation in clear cells (Figure 4C; Pastor-Soler et al, 2003), a process that is dependent on the activity of PKA but is independent of the exchange protein activated by cAMP (Epac) (Pastor-Soler et al, 2008). We later showed that the cAMP/PKA pathway also regulates V-ATPase recycling in renal intercalated cells, which are analogous to clear cells (Paunescu et al, 2010). A large-scale phosphoproteomics analysis showed that some V-ATPase subunits (a2, B2, G3, H, and A) are phosphorylated in mouse kidney (Paunescu et al, 2010). In addition, phosphorylation of the V-ATPase C and A subunits by PKA was demonstrated in the blowfly salivary gland (Voss et al, 2007) and HEK-293T cells (Hallows et al, 2009; Alzamora et al, 2010), respectively. In addition, we have previously shown that V-ATPase accumulation to the apical membrane requires intracellular calcium and is dependent on phospholipase C activity (Beaulieu et al, 2005).

Intercellular Communication Networks in the Epididymis

As mentioned above, principal, clear, and basal cells, together with spermatozoa, work in a concerted manner to establish the appropriate luminal environment for sperm maturation and storage. We will describe here the intercellular communication networks that exist between these cell types to regulate V-ATPase–dependent proton secretion in clear cells.

Principal Cell-Clear Cell Cross Talk

Basolateral stimulation of the epididymal epithelium triggers bicarbonate secretion by principal cells (Leung et al, 1992; Wong, 1998; Sedlacek et al, 2001; Carlin et al, 2003; Hagedorn et al, 2007; Pierucci-Alves and Schultz, 2008; Pierucci-Alves et al, 2010). This leads to a subsequent rise in luminal bicarbonate concentration, which was proposed to prime spermatozoa before ejaculation (Carlin et al, 2003; Pierucci-Alves et al, 2010). However, a sustained elevation in luminal bicarbonate concentration might be harmful to spermatozoa because it would maintain them in a preactivated state during storage. We postulated that clear cells might contribute to the reestablishment of an acidic resting luminal pH following principal cell activation. Indeed, we showed that luminal perfusion with a bicarbonate-containing solution induced the apical accumulation of the V-ATPase in clear cells compared with a phosphate-based solution (Pastor-Soler et al, 2003). We demonstrated that activation of sAC, which is enriched in clear cells, is essential for the bicarbonate stimulation of clear cells. We proposed that, following bicarbonate secretion by principal cells, clear cells respond to luminal bicarbonate by increasing their rate of V-ATPase–dependent proton secretion. This would ensure that the luminal environment is kept at the acidic pH that is required for keeping sperm in a dormant state during their storage in the cauda.

Clear cells are also “activated” at the luminal alkaline pH of 7.8, compared with the physiologic pH of 6.6 (Pastor-Soler et al, 2003). The alkaline pH–induced apical accumulation of V-ATPase is abolished in the presence of the calcium chelator, BAPTA-AM. However, the link between alkaline pH stimuli and intracellular calcium has remained unknown. Interestingly, ATP stimulation via purinergic receptors can be modulated by extracellular pH. In search of a potential link between the effects of luminal alkaline pH and the role of calcium in V-ATPase recycling, we characterized the response of clear cells to purinergic activation (Belleannee et al, 2010). Activation of P2 receptors by ATP induces an increase in intracellular calcium, either from the release from intracellular stores (mediated by P2Y receptors), or from an influx of calcium via the P2X receptors, which act as calcium channels. Purinergic receptors are expressed in primary cultures of rat epididymal cells and epithelial cells of mouse epididymis (Wong, 1988; Shariatmadari et al, 2003). In fact, the epididymis was the first intact tissue in which the role of ATP in the modulation of its function was studied, and Wong (1988) showed that P2 receptor stimulation increases chloride and water secretion. In addition, we showed that ATP induced the V-ATPase apical accumulation in rat epididymal clear cells, an effect that was abolished by BAPTA-AM, confirming the participation of P2 receptors in this response. Messenger RNA transcripts specific for several purinergic receptors, including ATP P2 receptors and adenosine P1 receptors, were detected in epididymal epithelial cells isolated by laser cut microdissection (Belleannee et al, 2010). Among these receptors, the P2X4 receptor is potentiated at alkaline pH (Zsembery et al, 2003). Future studies will be required to determine whether P2X4 is the receptor involved in the modulation of clear cells following ATP secretion by activated principal cells, and whether the response is amplified by a bicarbonate-induced pH increase. Adenosine, the hydrolysis product of ATP, also triggered the apical accumulation of the V-ATPase. ATP is rapidly degraded via the activity of ectonucleotidases located in the epididymal luminal fluid and epithelial cell apical membranes. Activation of P1 receptors by adenosine results in either a decrease (A1 and A3 receptors) or increase (A2B receptor) in intracellular cAMP. We showed that the PKA inhibitor mPKI abolished the response elicited by adenosine, indicating the participation of the P1 A2B receptor in the activation of clear cells.

The mechanisms of ATP release from cells remain for the most part unknown. ATP can be released via exocytosis of ATP-containing vesicles, or ATP efflux can be mediated via nonselective channels, such as CLC-3 and CLC-5 (Schwiebert and Zsembery, 2003; Praetorius and Leipziger, 2009; Ransford et al, 2009). In the epididymis, spermatozoa together with epithelial cells are potential sources of luminal ATP (Wong, 1988, 1990). Epididymal principal cells express CLC-3 in their apical membrane, and CLC-5 colocalizes with the V-ATPase in clear cells (Isnard-Bagnis et al, 2003). We recently showed that the immortalized epididymal cell lines originating from the proximal (PC1) and distal (DC2) caput (kindly provided by Marie-Claire Orgebin-Crist and colleagues) secrete significant amounts of ATP (Ruan et al, unpublished data). We are currently investigating the potential role of principal cells in the release of ATP into the epididymal lumen in situ.

Previous studies have described the role of the CFTR either as a direct mediator of ATP release or as a regulator of ATP efflux mechanisms (Schwiebert et al, 1995; Jiang et al, 1998; Cantiello, 2001; Schwiebert and Zsembery, 2003; Reigada and Mitchell, 2005; Fitz, 2007). CFTR is expressed in several tissues involved in active secretion, including the upper airway tract, pancreas, and epididymis. In the epididymis, it is located in the apical membrane of epididymal principal cells (Ruz et al, 2004; Pietrement et al, 2008; Shum et al, 2009). Male infertility has been linked to several mutations of the CFTR gene (van der Ven et al, 1996; Wong, 1998; Hernandez-Gonzalez et al, 2007; Xu et al, 2007). We recently found that known activators of CFTR (eg, forskolin) stimulate ATP release from DC2 cells, and that the forskolin activation was abolished in the presence of CFTR inhibitors (Ruan et al, unpublished data), indicating the participation of CFTR in ATP secretion in the epididymis. CFTR was also shown to mediate bicarbonate secretion in primary cultures of epididymal principal cells (Leung and Wong, 2000). Thus, CFTR might be an important mediator of the regulation of clear cells following activation of principal cells.

In summary, current results indicate the presence of cross talk between principal cells (via bicarbonate and ATP secretion) and clear cells for the regulation of V-ATPase–dependent proton secretion. We propose that during sexual arousal, basolateral activation of principal cells triggers CFTR-mediated ATP and bicarbonate secretion into the lumen. This leads to the subsequent increase in proton secretion by clear cells, which would allow luminal pH to be restored to its resting acidic value. This model predicts that clear cells can respond to variations in the luminal environment in a hormone-independent manner. The following section will describe how luminal hormones can also regulate clear cells following activation of basal cells.

Basal Cell-Clear Cell Cross Talk

We showed that in vivo luminal perfusion of the cauda epididymis with angiotensin II (ANGII) triggers V-ATPase apical accumulation and activates V-ATPase–dependent proton secretion in clear cells (Shum et al, 2008). The epididymal lumen contains all components of the renin-angiotensin system, including ANGI and ANGII (Wong and Uchendu, 1990; Krege et al, 1995; Esther et al, 1996; Zhao et al, 1996; Hagaman et al, 1998; Ramaraj et al, 1998; Speth et al, 1999; Leung and Sernia, 2003; Saez et al, 2004). ANGII is produced from ANGI by the angiotensin I–converting enzyme (ACE), which exists in two forms, testicular (tACE, also known as germinal ACE) and somatic (Langford et al, 1993; Sibony et al, 1994; Corvol et al, 1995). Absence of tACE in mice results in male infertility due to a defect in sperm function, but not production, indicating dysfunction of the posttesticular tract (Krege et al, 1995; Esther et al, 1996; Hagaman et al, 1998). Testicular ACE is bound to immature sperm and is released into the luminal fluid as sperm move along the epididymal tubule (Gatti et al, 1999; Metayer et al, 2002; Thimon et al, 2005). The enzymatic activity of newly released tACE is intact, and tACE was proposed to regulate the epithelium. This would indicate the presence of cross talk between maturing spermatozoa and epithelial cells lining the epididymal lumen. The activation of clear cells that we showed after luminal ANGII exposure (Shum et al, 2008) is compatible with this model. Luminal ANGII stimulates clear cells by interacting with the ANGII type II receptor (AGTR2) followed by activation of the nitric oxide (NO)-cGMP pathway. Surprisingly, AGTR2 was absent from clear cells, as demonstrated by immunofluorescence and by reverse transcriptase–polymerase chain reaction analysis of clear cells isolated by fluorescence-activated cell sorting from B1-transgenic mice, which express green fluorescent protein in these cells. Instead, AGTR2 was detected exclusively in basal cells, which were found to extend narrow body projections between adjacent epithelial cells toward the lumen (Figure 5A). Interestingly, basal cells reach the lumen at the tripartite junctions between epithelial cells (Figure 5B through B″), where they have the ability to form a new tight junction between themselves and adjacent cells (Figure 6A). Basal cells can send their projections between 2 principal cells (Figure 6A) or between a principal and a clear cell (Figure 6B). In addition, we found that basal cells from the trachea and coagulating gland also have the property to reach the lumen, and future studies will be required to characterize this new function in these organs. In the epididymis, the “lumen-reaching” property of basal cells now places them in a new central position within the epithelium. We proposed the presence of cross talk mechanisms between basal cells and clear cells (Figure 7). Based on this model, basal cells sense luminal ANGII via the AGTR2 receptor, which triggers the production of NO. NO then diffuses out of basal cells to reach clear cells, where it activates soluble guanylate cyclase, which is enriched in these cells (Shum et al, 2008). Subsequent elevation of cGMP triggers the apical accumulation of V-ATPase in well-developed microvilli. Interestingly, ANGII production depends on the presence of tACE, which originates from spermatozoa. Thus, luminal hormone sensing by basal cells would provide an indirect mechanism by which clear cells are modulated by luminal spermatozoa. Although our model predicts that basal cells can regulate the activity of clear cells via sampling of luminal factors, stimulation of basal cells by basolateral lysylbradykinin has also been shown to increase anion secretion in principal cells (Leung et al, 2004; Cheung et al, 2005). Thus, a complex intercellular network between basal cells and both clear cells and principal cells modulates apical and basolateral regulation of the epididymal epithelium.

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.

Conclusion

Several cases of male infertility are still labeled “idiopathic.” Some of these cases are related to dysfunctional spermatozoa, which are otherwise produced in sufficient numbers, eliminating the testis as a likely candidate. Thus, there is a clear need for novel diagnostic and therapeutic interventions for the cure of male infertility related to posttesticular malfunction. On the other hand, the epididymis is thought to be the ideal posttesticular contraceptive target for agents that would not affect normal hormone production and spermatogenesis per se. Up to 50% of pregnancies in the world are still unintended, and studying posttesticular factors regulating male fertility might also reveal potential targets for the development of new methods for male contraception.

Although significant progress has been made toward the characterization of the role of the different cell types that line the epididymal lumen in the establishment of the optimal environment for sperm maturation and storage, several of the transepithelial transport mechanisms involved and their regulation still remain unknown. Novel approaches to study the integrated functions of the epididymis are now required to bring our knowledge to a new level. These include microsurgical procedures combined with functional and intravital imaging techniques that will allow the characterization of the dynamic interplay between different cell types, including spermatozoa and epididymal epithelial cells, while they reside in their native environment. Monitoring and decoding intercellular communication networks that are established in the epididymis will help define the physiologic mechanisms that control the luminal environment in which sperm acquire their fertilizing power.