Relative contribution of clear cells and principal cells to luminal pH in the mouse epididymis†

Following testicular spermatogenesis, mammalian sperm continue to mature in a long epithelial tube known as the epididymis, which plays key roles in remodeling sperm protein, lipid, and RNA composition. To understand the roles for the epididymis in reproductive biology, we generated a single-cell atlas of the murine epididymis and vas deferens. We recovered key epithelial cell types including principal cells, clear cells, and basal cells, along with associated support cells that include fibroblasts, smooth muscle, macrophages and other immune cells. Moreover, our data illuminate extensive regional specialization of principal cell populations across the length of the epididymis. In addition to region-specific specialization of principal cells, we find evidence for functionally specialized subpopulations of stromal cells, and, most notably, two distinct populations of clear cells. Our dataset extends on existing knowledge of epididymal biology, and provides a wealth of information on potential regulatory and signaling factors that bear future investigation.

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An atlas of cell types in the mammalian epididymis and vas deferens

10.1101/2020.01.24.918979 ◽

2020 ◽

Cited By ~ 2

Author(s):

Vera D. Rinaldi ◽

Elisa Donnard ◽

Kyle J. Gellatly ◽

Morten Rasmussen ◽

Alper Kucukural ◽

...

Keyword(s):

Vas Deferens ◽

Immune Cell ◽

Cell Types ◽

Receptor Expression ◽

Basal Cells ◽

Regional Specialization ◽

Clear Cells ◽

Principal Cells ◽

Mammalian Sperm ◽

Genomic Clusters

ABSTRACTFollowing spermatogenesis in the testis, mammalian sperm continue to mature over the course of approximately 10 days as they transit a long epithelial tube known as the epididymis. The epididymis is comprised of multiple segments/compartments that, in addition to concentrating sperm and preventing their premature activation, play key roles in remodeling the protein, lipid, and RNA composition of maturing sperm. In order to understand the complex roles for the epididymis in reproductive biology, we generated a single cell atlas of gene expression from the murine epididymis and vas deferens. We recovered all the key cell types of the epididymal epithelium, including principal cells, clear cells, and basal cells, along with associated support cells that include fibroblasts, smooth muscle, macrophages and other immune cells. Moreover, our data illuminate extensive regional specialization of principal cell populations across the length of the epididymis, with a substantial fraction of segment-specific genes localized in genomic clusters of functionally-related genes. In addition to the extensive region-specific specialization of principal cells, we find evidence for functionally-specialized subpopulations of stromal cells, and, most notably, two distinct populations of clear cells. Analysis of ligand/receptor expression reveals a network of potential cellular signaling connections, with several predicted interactions between cell types that may play roles in immune cell recruitment and other aspects of epididymal function. Our dataset extends on existing knowledge of epididymal biology, and provides a wealth of information on potential regulatory and signaling factors that bear future investigation.

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Effect of castration and testosterone replacement on high glucose 6-phosphatase activity in principal cells of the mouse epididymis

The Anatomical Record ◽

10.1002/ar.1092070207 ◽

1983 ◽

Vol 207 (2) ◽

pp. 289-295 ◽

Cited By ~ 8

Author(s):

Kazuo Kanai ◽

Shinsuke Kanamura ◽

Jun Watanabe ◽

Mari Asada-Kubota ◽

Motoko Yoshikawa

Keyword(s):

Phosphatase Activity ◽

High Glucose ◽

Testosterone Replacement ◽

Principal Cells ◽

Mouse Epididymis

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Detection of ClC-3 and ClC-5 in epididymal epithelium: immunofluorescence and RT-PCR after LCM

AJP Cell Physiology ◽

10.1152/ajpcell.00374.2001 ◽

2003 ◽

Vol 284 (1) ◽

pp. C220-C232 ◽

Cited By ~ 25

Author(s):

Corinne Isnard-Bagnis ◽

Nicolas Da Silva ◽

Valérie Beaulieu ◽

Alan S. L. Yu ◽

Dennis Brown ◽

...

Keyword(s):

Vas Deferens ◽

Chloride Transport ◽

The Body ◽

Transepithelial Transport ◽

Rt Pcr ◽

Clear Cells ◽

Principal Cells ◽

Endosome Trafficking ◽

Cauda Epididymidis ◽

Laser Capture

Epithelial cells of the epididymis and vas deferens establish an optimum luminal environment in which spermatozoa mature and are stored. This is achieved by active transepithelial transport of various ions including Cl−and H+. We investigated the localization of three closely related members of the ClC family, ClC-3, ClC-4, and ClC-5, in the epididymis and vas deferens. RT-PCR using mRNA isolated by laser capture microdissection (LCM)-detected ClC-3 and ClC-5 transcripts but did not detect any ClC-4-specific transcript. Western blot and immunofluorescence analysis demonstrated that ClC-3 and ClC-5 proteins are present in all regions of the epididymis and in the vas deferens. ClC-5 is expressed exclusively in H+-ATPase-rich cells (narrow and clear cells). Confocal microscopy showed that ClC-5 partially colocalizes with the H+-ATPase in the subapical pole of clear cells. ClC-3 is strongly expressed in the apical membrane of principal cells of the caput epididymidis and the vas deferens and is less abundant in principal cells of the body and cauda epididymidis. These findings are consistent with a potential role for ClC-3 in transepithelial chloride transport by principal cells and for ClC-5 in the acidification of H+-ATPase-containing vesicles in narrow and clear cells. ClC-5 might facilitate endosome trafficking in the epididymis, as has been proposed in the kidney.

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A mathematical model of the rat kidney IV: whole-kidney response to hyperkalemia

AJP Renal Physiology ◽

10.1152/ajprenal.00413.2021 ◽

2022 ◽

Author(s):

Alan Mark Weinstein

Keyword(s):

Rat Kidney ◽

Macula Densa ◽

Tubuloglomerular Feedback ◽

Na Transport ◽

Ample Evidence ◽

Principal Cells ◽

Relative Contribution ◽

K Secretion ◽

Luminal Flow ◽

The Impact

The renal response to acute hyperkalemia is mediated by increased K secretion within connecting tubule (CNT), flux that is modulated by tubular effects (e.g. aldosterone) in conjunction with increased luminal flow. There is ample evidence that peritubular K blunts Na reabsorption in proximal tubule, thick ascending Henle limb, and distal convoluted tubule (DCT). While any such reduction may augment CNT delivery, the relative contribution of each is uncertain. The kidney model of this lab was recently advanced with representation of cortical labyrinth and medullary ray. Model tubules capture the impact of hyperkalemia to blunt Na reabsorption within each upstream segment. However, this forces the question of the extent to which increased Na delivery is transmitted past macula densa and its tubuloglomerular feedback (TGF) signal. Beyond increasing macula densa Na delivery, peritubular K is predicted to raise cytosolic Cl and depolarize macula densa cells, which may also activate TGF. Thus, although upstream reduction in Na transport may be larger, it appears that the DCT effect is critical to increasing CNT delivery. Beyond the flow effect, hyperkalemia reduces ammoniagenesis and reduced ammoniagenesis enhances K excretion. What this model provides is a possible mechanism. When cortical NH4 is taken up via peritubular Na,K(NH4)-ATPase, it acidifies principal cells. Consequently, reduced ammoniagenesis increases principal cell pH, thereby increasing conductance of both ENaC and ROMK, enhancing K excretion. In this model, aldosterone's effect on principal cells, diminished DCT Na reabsorption, and reduced ammoniagenesis, all provide relatively equal and additive contributions to renal K excretion.

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Expression of the rhesus glycoproteins, ammonia transporter family members, RHCG and RHBG in male reproductive organs

Reproduction ◽

10.1530/rep-13-0154 ◽

2013 ◽

Vol 146 (3) ◽

pp. 283-296 ◽

Cited By ~ 11

Author(s):

Hyun-Wook Lee ◽

Jill W Verlander ◽

Mary E Handlogten ◽

Ki-Hwan Han ◽

Paul S Cooke ◽

...

Keyword(s):

Male Fertility ◽

Reproductive Tract ◽

Immunoblot Analysis ◽

Reproductive Organs ◽

Normal Male ◽

Male Reproductive Tract ◽

Clear Cells ◽

Principal Cells ◽

Cellular Expression ◽

Transporter Family

The rhesus glycoproteins, Rh B glycoprotein (RHBG) and Rh C glycoprotein (RHCG), are recently identified ammonia transporters. Rhcg expression is necessary for normal male fertility, but its specific cellular expression is unknown, and Rhbg has not been reported to be expressed in the male reproductive tract. This study sought to determine the specific cellular expression of Rhcg, to determine whether Rhbg is expressed in the male reproductive tract, and, if so, to determine which cells express Rhbg using real-time RT-PCR, immunoblot analysis, and immunohistochemistry. Both Rhbg and Rhcg were expressed throughout the male reproductive tract. In the testis, high levels of Rhbg were expressed in Leydig cells, and Rhcg was expressed in spermatids during the later stages of their maturation (steps 13–16) in stages I–VIII of the seminiferous epithelium cycle. In the epididymis, basolateral Rhbg was present in narrow cells in the initial segment, in principal cells in the upper corpus, and in clear cells throughout the epididymis. Apical Rhcg immunolabel was present in principal cells in the caput and upper corpus epididymidis and in clear cells in the middle and lower corpus and cauda epididymidis. In the vas deferens, apical Rhcg immunolabel and basolateral Rhbg immunolabel were present in some principal cells and colocalized with H+-ATPase immunolabel. We conclude that both Rhbg and Rhcg are highly expressed in specific cells in the male reproductive tract where they can contribute to multiple components of male fertility.

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Relocalization of the V-ATPase B2 subunit to the apical membrane of epididymal clear cells of mice deficient in the B1 subunit

AJP Cell Physiology ◽

10.1152/ajpcell.00596.2006 ◽

2007 ◽

Vol 293 (1) ◽

pp. C199-C210 ◽

Cited By ~ 35

Author(s):

Nicolas Da Silva ◽

Winnie W. C. Shum ◽

Jaafar El-Annan ◽

Teodor G. Păunescu ◽

Mary McKee ◽

...

Keyword(s):

Apical Membrane ◽

Wild Type Mouse ◽

Immunogold Electron Microscopy ◽

Western Blots ◽

Pixel Intensity ◽

Clear Cells ◽

Intracellular Compartments ◽

Luminal Ph ◽

Cauda Epididymidis ◽

Renal Tubular

An acidic luminal pH in the epididymis contributes to maintaining sperm quiescent during their maturation and storage. The vacuolar H+ATPase (V-ATPase), located in narrow and clear cells, is a major contributor to luminal acidification. Mutations in one of the V-ATPase subunits, ATP6v1B1 (B1), cause distal renal tubular acidosis in humans but surprisingly, B1−/− mice do not develop metabolic acidosis and are fertile. While B1 is located in the apical membrane of narrow and clear cells, the B2 subunit localizes to subapical vesicles in wild-type mouse, rat and human epididymis. However, a marked increase (84%) in the mean pixel intensity of B2 staining was observed in the apical pole of clear cells by conventional immunofluorescence, and relocalization into their apical membrane was detected by confocal microscopy in B1−/− mice compared with B1+/+. Immunogold electron microscopy showed abundant B2 in the apical microvilli of clear cells in B1−/− mice. B2 mRNA expression, determined by real time RT-PCR using laser-microdissected epithelial cells, was identical in both groups. Semiquantitative Western blots from whole epididymis and cauda epididymidis showed no variation of B2 expression. Finally, the luminal pH of the cauda epididymidis was the same in B1−/− mice as in B1+/+ (pH 6.7). These data indicate that whereas overall expression of B2 is not affected in B1−/− mice, significant redistribution of B2-containing complexes occurs from intracellular compartments into the apical membrane of clear cells in B1−/− mice. This relocation compensates for the absence of functional B1 and maintains the luminal pH in an acidic range that is compatible with fertility.

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