Sodium Coupled Bicarbonate Influx Regulates Intracellular and Apical pH in Cultured Rat Caput Epididymal Epithelium

Abstract

BackgroundThe epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na+/HCO3 − cotransporter in the pH regulation in rat epididymis.Method/Principal FindingsImmunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit(KH) solution, the intracellular pH (pHi) recovery from NH4Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na+/H+ exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO3 − buffered would cause another pHi recovery. The pHi recovery in HCO3 − buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2, 2-disulfonic acid (DIDS), the inhibitor of HCO3 − transporter or by removal of extracellular Na+. The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH.ConclusionsThe present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.

Epididymal small non‐coding RNA studies: progress over the past decade

“…Given the increasing demands for better resolutions, the progress in the sample preparation for high-throughput studies include the isolation of different epididymal regions (Belleannee et al, 2012;Chu et al, 2015) and the purification of particular cellular components such as epithelial cells (Nixon et al, 2015a), epididymosomes (Hutcheon et al, 2017), spermatozoa (Nixon et al, 2015a(Nixon et al, ,2015bSharma et al, 2016;Chu et al, 2017), and sperm cell parts (Sharma et al, 2018). To remove the spermatozoa and luminal fluid as thoroughly as possible and finally obtain the purified epithelial cells, the epididymal tissue needs to undergo a labor-intensive and timeconsuming process of repeated flushing of the tubule, mincing, vigorous shaking, and digestion, as described in several studies of the epididymal transcriptomes and sncRNA profiles using true epithelial cells (Zuo et al, 2011;Mandon et al, 2015;Nixon et al, 2015a). Then, one can use Percoll gradient centrifugation (Krapf et al, 2012;Nixon et al, 2015b) and/or somatic cell lysis buffer (Peng et al, 2012;Sharma et al, 2016;Chu et al, 2017) to obtain a purified sperm fraction.…”

“…In the studies using small mammals such as mice, due to the limited amount of luminal fluid that can be extracted from each epididymis, the enrichment and purification processes are more susceptible to various contaminations including cell debris and other extracellular vesicles in the blood or lymphatic vessels, and therefore, extra prior steps such as perfusing the vasculature to minimize the blood contaminations are recommended. To remove the spermatozoa and luminal fluid as thoroughly as possible and finally obtain the purified epithelial cells, the epididymal tissue needs to undergo a labor-intensive and timeconsuming process of repeated flushing of the tubule, mincing, vigorous shaking, and digestion, as described in several studies of the epididymal transcriptomes and sncRNA profiles using true epithelial cells (Zuo et al, 2011;Mandon et al, 2015;Nixon et al, 2015a). In addition, the mixed epididymal epithelial cell population can be sorted by flow cytometry to enrich specific cell types, such as basal (Mandon et al, 2015) and clear cells (recent studies from Dr. Sylvie Breton's laboratory), or analyzed by single-cell sequencing techniques, as presented by Dr. Ann Harris's laboratory at the Epididymis 7.…”

Analysis of the small non-protein-coding RNA profile of mouse spermatozoa reveals specific enrichment of piRNAs within mature spermatozoa

“…Following recovery of spermatozoa, epididymal tissue was utilized for epithelial cell isolation using methodology originally described by Zuo et al 65 In brief, the tissue was washed by subjecting it to agitation, prior to being minced with forceps and washed a further three times in sterile PBS. The washed tissue was then digested in 100 mg/mL trypsin (Promega) at 37 C for 30 min with vigorous shaking.…”

The Divergence, Actions, Roles, and Relatives of Sodium-Coupled Bicarbonate Transporters

“…The lumen of the epididymis is a site of Na ϩ reabsorption and H ϩ secretion (572), with low luminal pH being a requirement for storage of viable sperm. NBCe1-B is present in the cells of the epididymis, and cultured epididymal cells exhibit a DIDS-sensitive, Na ϩ -and HCO 3 Ϫ -dependent pH i recovery from an acid load, leading several groups to suggest that NBCe1 might be involved in H ϩ secretion/HCO 3 Ϫ reabsorption by these cells (164,445,729,1119). Mice deficient in the estrogen receptor ESR␣ (or ESR1) are defective in their ability to acidify the epididymal lumen.…”