An acid pH is established in the lumen of the excurrent duct of the male reproductive tract, which is important for sperm maturation as well as for maintaining sperm in a quiescent state during their passage through, and storage in, the epididymis/vas deferens. One major component of the complex series of events that trigger sperm motility during ejaculation is neutralization by the alkaline prostatic fluid, followed by an increase in intracellular pH. The ultimate aim of this research is to understand how defective acidification may impair reproductive function in pathologic states, and how intervention to manipulate acidification may eventually be used to control male fertility. A novel physiological approach using the non-invasive self-referencing proton-selective electrode, in collaboration with Peter Smith, had allowed us to identify the major proton secretory mechanism in the initial part of the vas deferens. We have shown that a bafilomycin-sensitive, proton-pumping H+-ATPase (proton pump - PP) is concentrated in the apical membrane of a subpopulation of epithelial cells (PP-rich cells) and is a major contributer to luminal acidification (1, 2, 3).
Our specific aims are 1) to define and characterize the complete pathways of proton secretion by the PP-rich cells; 2) to elucidate the mechanisms of vesicle recycling underlying the regulation of proton secretion; and 3) to examine the effect of some environmental pollutants on proton secretion. 1) Proton secretion pathways: PP-rich cells are enriched in the cytoplasmic carbonic anhydrase type II (CAII) indicating that bicarbonate transport is involved in proton secretion. We have shown, using the proton-selective electrode, that the CAII inhibitor, acetazolamide, markedly inhibits proton secretion by the vas deferens. Subsequent addition of bafilomycin after acetazolamide produced no further inhibitory effect on luminal acidification. The bicarbonate transport inhibitor, SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid), also inhibits proton secretion to the same extent as bafilomycin. These results suggest that H+ secretion by the PP-rich cells depends on the reabsorption of bicarbonate. Two bicarbonate transporters have been described in various organs including the kidney, the Na-HCO3 cotransporter and the Cl-HCO3 exchanger. We will identify which of these transporters is involved in the vas deferens by examining the effect of SITS in the absence of Na (replaced by N-methyl-D-glucamine) or Cl (replaced by cyclamate). 2) Proton pump recycling: We have shown that endocytic vesicles (labelled with the fluid phase marker, FITC-dextran) contain H+ATPase and are probably involved in the regulation of H+ secretion by the epididymis/vas deferens. In addition, the vesicle-associated protein, cellubrevin, is highly expressed in the PP-rich cells providing further evidence of a regulated endo/exocytotic apparatus in these cells. We have observed a significant inhibition of the proton flux measured using the proton-selective probe after cleavage of cellubrevin by tetanus toxin. This result suggests that H+ATPase activity is modulated, at least partly, via exocytosis of PP-containing vesicles followed by insertion of PP into the apical membrane. 3) Effect of heavy metals on proton secretion: Exposure to lead, cadmium. manganese and mercury has been shown to reduce male reproductive capacity in a variety of species including human. We are examining whether this reduction of male fertility can be attributed to a defect of the acidification capacity of the epididymis and vas deferens. Using the proton-selective probe, we found a marked inhibition of proton secretion in vas deferens accutely exposed in vitro to a heavy metal, cadmium (500mM). This inhibition is of the same amplitude as that observed with either acetazolamide, SITS or bafilomycin, and the latter had no additional effect indicating inhibition of the proton pump by cadmium. Breton, S, Smith, P. J. S., Lui, B. and Brown, D. 1996. Acidification of the male reproductive tract by a proton-pumping ATPase. Nature Medicine 2: 470-472. Breton, S, Smith, P. J. S. and Brown, D. 1997. Presence de cellules acidificantes dans l'epididyme et le canal deferent: implication de la pompe a protons, H+ATPase. Medecine Sciences. (Revue internationale de biologie et de medicine.) 13: 57-61. Brown, D., Smith, P. J. S. and Breton, S. 1997. Role of V-ATPase rich cells in acidification of the male reproductive tract. J. exp. Biol. 200 (2): 257-262. Patent pending: Brown, D., Breton, S., and Smith, P. J. S. 1996. Method of reversibly controlling male fertility. U. S. Patent Application No. 08/826,019.
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