The solute carrier 26 (SLC26) family, which encodes 11 anion transporters, is a relatively young gene family which is characterised based on rare human diseases (Mount and Romero, 2004; Kere, 2006; Dorwart et al., 2008; Ohana et al., 2009). SLC26 family members transport a variety of monovalent and divalent anions, including sulfate (SO42−), chloride (Cl−), iodide (I−), bicarbonate (HCO3−), hydroxy ion (OH−) and oxalate (Ox2−), with different affinities (Mount and Romero, 2004; Dorwart et al., 2008; Ohana et al., 2009). Of the 11 SLC26 family members, SLC26A3/Slc26a3 and SLC26A6/Slc26a6 are electrogenic obligatory Cl−/HCO3− exchangers expressed in the luminal membrane of many epithelia and play a central role in Cl− absorption and HCO3− secretion (Mount and Romero, 2004; Ohana et al., 2009). These Cl−/HCO3− exchangers have different stoichiometries, with 2Cl−/HCO3− and Cl−/2HCO3− for Slc26a3 and Slc26a6, respectively (Shcheynikov et al., 2006a, 2006b, 2008). Recent work from our laboratory and others has revealed the involvement of these exchangers in reproductive processes, particularly, uterine HCO3− secretion and sperm activation.
It has been known for more than sixty years that the luminal fluid of the female reproductive tract has a HCO3− content two to four times higher than that of the plasma (Vishwakarma, 1962; Murdoch and White, 1968; Maas et al., 1977). It is also known that HCO3− is essential to a number of reproductive events occurring in the female reproductive tract (Chan et al., 2006, 2009, 2012; Liu et al., 2012), including sperm motility (Mann and Lutwak-Mann, 1982; Tajima et al., 1987; Jones and Murdoch, 1996; Abaigar et al., 1999; Holt and Harrison, 2002; Wennemuth et al., 2003; Wennemuth, 2004; Mannowetz et al., 2011), capacitation (Boatman and Robbins, 1991; Shi and Roldan, 1995; Zhou et al., 2005), a sperm activation process by which sperm acquire their ability to fertilise the egg, and early embryo development (Chen et al., 2010; Lu et al., 2012). The questions as to how HCO3− is secreted into the lumen of the female reproductive tract and how HCO3− is transported into sperm and embryo have not been fully addressed. The emergence of the SLC26 transporter family, and particularly, the work on Slc26a3 and Slc26a6 anion exchangers in rodent models has shed light on our understanding of the molecular mechanisms underlying uterine HCO3− secretion and sperm capacitation. This review will re-examine the results from early studies working on anion secretion by the endometrium, finding clues for the involvement of Cl−/HCO3− exchangers in electrogenic anion transport, and summarise the related results obtained from the authors' laboratory and reported in the literature on Slc26 members, particularly, Slc26a3 and Slc26a6, in uterine epithelial cells and sperm. We will also provide an analysis on the working of the two exchangers with a cAMP-activated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR), in uterine epithelial cells and sperm, indicating possible role of these two ion exchangers in reproduction and their implications for infertility.
Uterine bicarbonate secretion and fertilising capacity of sperm
The understanding of cellular mechanisms of electrogenic anion transport, such as Cl− and HCO3− secretion, across uterine epithelium was greatly advanced by the use of reconstituted endometrial epithelia from of a number of species, including the mouse, pig and human, in conjunction with the short-circuit current (Isc) technique in Using chamber experiments (Matthews et al., 1993; Vetter and O'Grady, 1996; Chan et al., 1997a, 1997b, 1997c; Fong et al., 1998). In these cultured endometrial epithelia, it was reported that the Isc, which represents active and electrogenic ion transport across the epithelium, could be stimulated by a number of neuronal and hormonal agonists, such as adrenaline, prostaglandins and prolactin, and that the agonist-induced current could be reduced by removal of Cl− or HCO3− from the bathing solutions, indicating active anion secretion upon stimulation. Most of the studies reporting electrogenic anion transport across endometrial epithelia attributed it to epithelial anion channels, such as CFTR, mutations of which are known to cause cystic fibrosis with infertility as one of the hallmark defects apart from lung disease (Quinton, 1999; Chan, 2007; Chan et al., 2009; Chen et al., 2012a), and Ca2+-activated Cl− channels. However, re-examination of the results from these early studies also reveal possible involvement of other transport mechanisms in electrogenic ion transport across uterine epithelium. In cultured mouse endometrial epithelia, for example, over 60% of the Isc induced by adrenaline (Chan et al., 1997c) or PGE2 (Fong and Chan, 1998) could be abolished by the removal of extracellular Cl−; while removal of extracellular HCO3− could also produced 60% reduction in the PGE2-induced Isc (Fong and Chan, 1998), suggesting that there must be some portion of the Isc measured depends on both Cl− and HCO3−. In a more recent study on a porcine endometrial epithelial culture, up to 86% and 92% of the prolactin-induced Isc could be abolished by removal of either Cl− or HCO3−, respectively (Deachapunya et al., 2008), indicating the majority of the prolactin-induced Isc depended on both Cl− and HCO3−. The best explanation for these observations would be the involvement of Cl−/HCO3− exchanger(s). However, the traditional knowledge of the known anion exchangers being electro-neutral then precluded the consideration of their possible involvement in the electrogenic anion transport process.
A clear demonstration of the involvement of a Cl−/HCO3− exchanger in HCO3− secretion by endometrial epithelium was performed on cultured mouse endometrial epithelial cells using intracellular pH measurement (Wang et al., 2003). A pH sensitive dye, BCECF-AM, was used to measure pH recovery after cellular alkalisation, which reflects HCO3− extrusion. However, when extracellular Cl− was removed, the rate of pH recovery was greatly attenuated, suggesting the involvement of a Cl−/HCO3− exchanger in the HCO3− extrusion process. However, the exact molecular identity of the exchanger was unclear. Interestingly, in the absence of extracellular Cl− when the anion exchanger is not in operation, challenging the cells with adenylyl cyclase activator, forskolin, could still increase the rate of pH recovery, which could be reversed by a CFTR inhibitor. These results suggest that uterine HCO3− secretion may be mediated by the anion exchanger and CFTR channel.
We recently examined the expression of Slc26a6 in murine endometrial epithelial cells. Interestingly, the expression of Slc26a6 in mouse uterus exhibits a cyclic pattern, similar to that of CFTR, with a maximal level detected at oestrus by Western blot (He et al., 2010). The cyclic expression pattern of Slc26a6 and CFTR was also seen in rats with minimal expression levels of both mRNA and protein at dioestrous (Gholami et al., 2012). The involvement of oestrogen (He et al., 2010) and progesterone (Gholami et al., 2012) in regulating the expression of these HCO3− transporters was also demonstrated, indicating hormonal regulation of uterine HCO3− secretion during oestrous cycle. Indeed, luminal surface pH measurement using the pH-sensitive fluorescent dye HAF revealed a significantly higher luminal pH, as reflected by a higher fluorescent ratio, in oestrous uteri, compared to that of dioestrous uteri, indicating greater HCO3− secretory activity at oestrus. This is consistent with the finding obtained by the lead nitrate precipitation method showing a two- to fourfold higher bicarbonate content in oestrous uteri compared that of dioestrous uteri (Mannowetz et al., 2011). Interestingly, the luminal surface pH in oestrous uteri has a greater sensitivity to inhibitors of either the anion exchanger or CFTR compared to that of dioestrous uteri, consistent with the maximal expression levels of Slc26a6 and CFTR at oestrus and their involvement in uterine HCO3− secretion. More importantly, sperm recovered after incubation in oestrous uteri had elevated beat frequencies compared to that recovered from dioestrous uteri (Mannowetz et al., 2011), suggesting that uterine HCO3− content may have a physiological consequence on sperm motility. The importance of uterine HCO3− on the fertilising capacity of sperm was also noted using a sperm-endometrial epithelial cell co-culture system, where knockdown of CFTR resulted in reduced number of capacitated sperm and IVF rate (Wang et al., 2003). This is consistent with a critical role of CFTR in uterine HCO3− secretion that is essential to sperm activation. This also provides a new explanation to the cause of reduced fertility seen in CF women (Wang et al., 2003). While a direct role of CFTR in transporting HCO3− has been found by the pH recovery after cellular alkalisation when the anion exchanger is inactivated by removal of extracellular Cl− (Wang et al., 2003), it is not clear whether CFTR also plays an indirect role in working with Slc26a6 by providing a recycling pathway for the Cl− exit required for the operation of the anion exchanger or by regulating the activation of Slc26a3 and Slc26a6 through protein–protein interaction, as seen in pancreatic duct cells (Ko et al., 2002). Of note, Slc26a6 knockout mice are fertile (Peter Arronson, personal communication). This may suggest that the defect in the Slc26a6-mediated uterine HCO3− secretion may be either compensated by CFTR or by up-regulation of Slc26a3, as seen in the pancreatic duct of Slc26a6-null mice (Ishiguro et al., 2007; Song et al., 2012). Future work on endometrial epithelial cells from Slc26a6-null mice may provide the answer.
Bicarbonate entry and sperm capacitation
It is well established that HCO3− is essential to sperm functions by activating a soluble form of adenylyl cyclase (sAC) in sperm (Okamura et al., 1985, 1986; Chen et al., 2000) and subsequently cAMP-dependent events, including protein tyrosine phosphorylation that leads to sperm motility, sperm capacitation and acrosome reaction (Austin, 1951; Lefièvre et al., 2002; Fraser, 2010). However, the cellular pathway(s) by which HCO3− enters into sperm remain incompletely understood. We tested the possible involvement of CFTR by examining its expression in mouse and human sperm and found its localisation to the equatorial segment of sperm head in both species (Xu et al., 2007). CFTR inhibitor or antibody significantly reduce sperm capacitation as well as related HCO3−-dependent events, including membrane hyperpolarization, intracellular pH increase, cAMP production, indicating the involvement of CFTR in mediating the HCO3− entry necessary for sperm capacitation. This notion was supported by the results obtained from sperm collected from heterozygous CF mutant mice showing reduced HCO3−-induced cAMP production and fertility rate in vitro and in vivo (Xu et al., 2007), indicating an dispensable role of CFTR in determining the fertilising capacity of sperm. However, it was unclear whether CFTR directly conducts HCO3− or works with an anion exchanger for uptake of HCO3− into sperm. We later tested the possible involvement of an anion exchanger in guinea pig sperm (Chen et al., 2009). Depletion of Cl−, even in the presence of HCO3−, abolished sperm capacitation and vice versa, indicating the involvement of both anions, and thus an anion exchanger, in the process. Capacitation-associated events, such as increase in intracellular pH, cAMP production and protein tyrosine phosphorylation were also dependent on extracellular Cl−. Interestingly, the capacitation-induced hyperactivated sperm motility was inhibited by inhibitors of CFTR and anion exchangers, with higher sensitivity towards niflumate, which has a more potent effect on Slc26a3 compared to DIDS, which is a more effective inhibitor of Slc26a6. An antibody against Slc26a3 also had an inhibitory effect in a concentration-dependent manner, indicating the involvement of Slc26a3, in addition to CFTR, in mediating the HCO3− entry required for sperm capacitation. The importance of both CFTR and Slc26a3 in the process of sperm capacitation was confirmed in mouse sperm (Chávez et al., 2012). However, the localisation of CFTR and the anion exchangers were associated with the mid-piece of sperm tail, rather than sperm head reported by Xu et al. (2007), and further investigation is required to resolve the discrepancy. It might be plausible that these proteins exhibit dynamic localisation pattern, as for the P2X receptor (Banks et al., 2010).
The involvement of both Slc26a3 and CFTR in sperm capacitation suggests that defects in these transporters leads to male infertility, which is consistent with the infertility of male CF patients (Xu et al., 2007; Chen et al., 2012a) and reduced male fertility in patients with congenital chloride diarrhoea caused by mutations of SLC26A3 (Hihnala et al., 2006; Höglund et al., 2006). Of note, we have recently examined the expression of SLC26A3 in human sperm, confirming its involvement in sperm function in humans. Although Slc26a3 deficient mice are available, detailed analysis of sperm function from these mice is lacking.
Slc26 anion exchangers are involved in uterine bicarbonate secretion and sperm capacitation, and suggesting that these anion exchangers working closely with CFTR (Figure 1). However, what we have learned so far on Slc26 anion exchangers in the reproductive system is only the beginning and many questions remain, the answers to which will undoubtedly shed new light on our understanding of the molecular mechanisms underlying many HCO3−-dependent cellular responses in the reproductive system. For example, are both SLC26A3/Slc26a3 and SLC26A6/Slc26a6 involved in similar functional processes in uterine epithelial cells and sperm? Although both anion exchangers have been found in sperm, it is unclear whether both are involved in transporting HCO3− into sperm for capacitation. We recently detected both SLC26A3 and SLC26A6 in human endometrial epithelial cells, suggesting their involvement in uterine HCO3− secretion and/or Cl− reabsorption. However, the working relationship between the two remains to be elucidated. Are Slc26 members other than Slc26a3 and Slc26a6 also expressed in uterine epithelial cells and sperm? If yes, what role do they play? In fact, Slc26a4 (also known as pendrin), mutations of which are known to affect thyroid function and hearing, as seen in Pendred syndrome (Everett et al., 1997, 1999), is expressed in human endometrial epithelial cells, whose expression and localisation change progressively with the menstrual cycle (Suzuki et al., 2002). Since there is no evidence of endometrial abnormalities in patients with Pendred syndrome, it is unclear whether it plays a significant role in endometrial function or there are compensatory mechanisms for its function in the uterus. Another Slc26 member, Slc26a8 (also known as Tat1), exclusively expressed in adult human testis and which reportedly transports SO42−, Cl−, Ox2− (Toure et al., 2001; Lohi et al., 2002), has also been found in human and mouse sperm (Rode et al., 2012). In that study, more interestingly, Slc26a8 was found to physically interact with CFTR and affect intracellular cAMP production upon its inactivation, which is consistent with the sperm defects, non-motile and impaired capacitation, observed in Slc26a8 (Tat1)-null sperm (Touré et al., 2007). In general, Slc26a8 (Tat1) may form a complex with CFTR in regulation of Cl− and HCO3− fluxes during sperm capacitation, but the exact details remain elusive. Are Slc26 anion exchangers involved in other HCO3−-dependent reproductive events? Apart from uterine HCO3− secretion and sperm capacitation, preimplantation embryo development is also dependent on HCO3− and our recent work shows the involvement of CFTR in linking extracellular HCO3− to the activation of mir-125b required for embryo development (Lu et al., 2012). However, it is unclear whether CFTR works alone or together with another anion exchanger for uptaking HCO3− into the embryo. Interestingly, no study has been reported on the expression of Slc26 anion exchangers in preimplantation embryos reproductive processes. Future study using Slc26a3 or Slc26a6 knockout mice to examine whether there is a defect in HCO3−-dependent mir-125b activation may help us understand whether they are also involved in the process of embryo development in addition to CFTR. If the answer is yes, how does CFTR work with these anion exchangers? Does it work to provide a recycling pathway for Cl− required for the operation of the anion exchanger(s)? Or does CFTR regulate these anion exchangers' activity through protein–protein interaction? Are Slc26 members involved in the signalling network of CFTR that regulates various reproductive events (Chen et al., 2012b; Lu et al., 2012)? The answers to these questions may not only advance our understanding of how HCO3− is transported in and out of the cells in the reproductive system but also provide new insight into the molecular signalling mechanisms governing HCO3− dependent reproductive events, and thus, possible causes of infertility.
Figure 1. Working model of SLC26A3/6 in uterine epithelium and sperm. Bicarbonate (HCO3−) is secreted by endometrial epithelial cells into the lumen and taken up by sperm through SLC26A3/6, with CFTR providing the recycling pathway for Cl−. The question mark indicates that the function of the transporter has not been demonstrated. CFTR, cystic fibrosis transmembrane conductance regulator; NBC, Na+–HCO3− cotransporter; sAC, soluble adenylyl cyclase.
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