Spermatozoa acquire their motility and fertilization capacity as they traverse the epididymis. The lumen of the epididymis is acidic and has a low bicarbonate concentration: both factors are important for keeping sperm quiescent during their maturation and storage. In the previous funding periods, we showed that luminal acidification is achieved by clear cells, via the proton pumping V-ATPase, and that it can be regulated via crosstalk with surrounding epithelial cells. We now plan to dissect the intercellular communication networks between principal cells and clear cells using a systems biology approach that links molecular events in signaling pathways to cellular events in the intact epididymis in vivo. We propose that stimulation of principal cells by basolateral paracrine factors during sexual arousal leads to subsequent activation of clear cells via ATP and bicarbonate that are secreted into the epididymal lumen. This would allow luminal pH to be restored to its resting acidic value. Alternatively, some of these factors might directly stimulate clear cells to secrete protons.
In Aim 1 A, multiphoton intravital microscopy (IVM), functional ion flux assays of proton secretion and intracellular calcium measurements will be used to examine clear cell activation following stimulation of principal cells by basolateral hormones and agonists. These effects will be compared with those elicited by luminal ATP, adenosine and bicarbonate.
Aim 1 B will identify and characterize the apical purinergic receptors (e.g. A2B, P2X4), and bicarbonate transporters (e.g. NBC3) involved in the response to luminal mediators (ATP, adenosine and bicarbonate), using LC-MS/MS, RT-PCR, in situ hybridization, western blotting and immunofluorescence. Their role in clear cell activation will be analyzed, as described in Aim 1A.
Aim 2 will monitor luminal ATP and bicarbonate secretion by principal cells by IVM following basolateral stimulation, using a novel luciferase assay and BCECF-dextran, respectively. The role of critical apical transporters and regulators involved in ATP (e.g. ClC-3, CFTR) and bicarbonate (e.g. SLC26A6) secretion will be examined. The role of CFTR, which plays a critical role in male fertility, will be further dissected in immortalized epididymal cell lines, and the consequences of cystic fibrosis-associated CFTR mutations will be determined.
Aim 3 will explore the direct activation of clear cells by hormones and agonists, without principal cell involvement. Cell morphology, V-ATPase apical recruitment, V-ATPase-dependent proton secretion and intracellular calcium will be examined in EGFP-expressing clear cells isolated by FACS from our unique transgenic mice. These studies will provide new insights into the mechanisms by which epithelial cells work in a concerted manner to establish and maintain an acidic luminal environment that is permissive for sperm maturation and storage in a quiescent state. In summary, we propose to use a multidisciplinary approach to address a problem that is central to our understanding of post-testicular regulation of male fertility. Data generated here will provide novel insights into the male reproductive tract in particular, as well as the physiology of acid/base transporting epithelia in general.

Public Health Relevance

The epididymis, which connects the testis to the vas deferens, is the site where freshly made spermatozoa acquire their fertilizing capacity, and therefore, plays a crucial role in male fertility. Our laboratory studies how cells that line the epididymal tube work together to establish an optimal environment for sperm maturation and viability. These results will provide new frameworks for the evaluation and treatment of male infertility.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD040793-11
Application #
8446938
Study Section
Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
Program Officer
Moss, Stuart B
Project Start
2005-08-03
Project End
2016-02-29
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
11
Fiscal Year
2013
Total Cost
$379,995
Indirect Cost
$161,154
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
P?unescu, Teodor G; Shum, Winnie W C; Huynh, Chuong et al. (2014) High-resolution helium ion microscopy of epididymal epithelial cells and their interaction with spermatozoa. Mol Hum Reprod 20:929-37
Ruan, Ye Chun; Wang, Yan; Da Silva, Nicolas et al. (2014) CFTR interacts with ZO-1 to regulate tight junction assembly and epithelial differentiation through the ZONAB pathway. J Cell Sci 127:4396-408
Shum, Winnie W C; Hill, Eric; Brown, Dennis et al. (2013) Plasticity of basal cells during postnatal development in the rat epididymis. Reproduction 146:455-69
Breton, Sylvie; Brown, Dennis (2013) Regulation of luminal acidification by the V-ATPase. Physiology (Bethesda) 28:318-29
Vedovelli, Luca; Rothermel, John T; Finberg, Karin E et al. (2013) Altered V-ATPase expression in renal intercalated cells isolated from B1 subunit-deficient mice by fluorescence-activated cell sorting. Am J Physiol Renal Physiol 304:F522-32
Roy, Jeremy W; Hill, Eric; Ruan, Ye Chun et al. (2013) Circulating aldosterone induces the apical accumulation of the proton pumping V-ATPase and increases proton secretion in clear cells in the caput epididymis. Am J Physiol Cell Physiol 305:C436-46
Wagner, Carsten A; Mohebbi, Nilufar; Uhlig, Ulrike et al. (2011) Angiotensin II stimulates H?-ATPase activity in intercalated cells from isolated mouse connecting tubules and cortical collecting ducts. Cell Physiol Biochem 28:513-20
Shum, Winnie Waichi; Da Silva, Nicolas; Belleannee, Clemence et al. (2011) Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells. Am J Physiol Cell Physiol 301:C31-43
Shum, Winnie W C; Ruan, Ye Chun; Da Silva, Nicolas et al. (2011) Establishment of cell-cell cross talk in the epididymis: control of luminal acidification. J Androl 32:576-86
Paunescu, Teodor G; Ljubojevic, Marija; Russo, Leileata M et al. (2010) cAMP stimulates apical V-ATPase accumulation, microvillar elongation, and proton extrusion in kidney collecting duct A-intercalated cells. Am J Physiol Renal Physiol 298:F643-54

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