| Male mice deficient in ERalpha (ERalphaKO mice) are infertile and sperm recovered from the cauda epididymis exhibit reduced motility and fail to fertilize eggs in vitro. These effects on sperm appear to result from defective epididymal function and not a direct effect on spermatogenesis, as ERalphaKO germ cells transplanted into wildtype testes yield normal offspring. It has been previously shown that the absence of a functional ERalpha leads to defects in efferent duct fluid reabsorption, which we hypothesized would have consequences on the downstream luminal milieu. Analysis of the epididymal fluid revealed that the ERalphaKO maintains a higher luminal pH throughout the epididymis, confirming an inability of the epididymis to properly acidify the luminal contents. Similarly, the osmolality of the ERalphaKO epididymal fluid is reduced relative to wildtype, confirming inappropriate water absorption. Subsequent studies showed that these abnormalities were not the result of global defects in epididymal function, since protein secretion by the ERalphaKO epididymis appeared normal as judged by SDS-PAGE and immunoblotting of secreted proteins. To gain insight into the basis of the aberrant fluid homeostasis in the ERalphaKO epididymis, the expression of several enzymes and transporters known to be involved in acid/base regulation were analyzed. The levels of NHE3, as well as carbonic anhydrase XIV and NBC1 were all reduced in the initial segment of the ERalphaKO epididymis, while other components appeared unaffected, including other ion transporters and V-ATPase. To determine the effect of the altered epididymal milieu on ERalphaKO sperm, the internal sperm pH was assayed and shown to be markedly elevated in ERalphaKO sperm relative to control. Since pH and bicarbonate ions are critical regulators of sperm cAMP levels and motility, we attempted to bypass the abnormal luminal and intracellular environment by supplementing sperm with exogenous cAMP. This treatment rescued all defective motility parameters, as assayed by CASA, demonstrating that motility defects are not intrinsic to the sperm, but rather, result from the abnormal epididymal milieu. Furthermore, ERalphaKO sperm recovered from the epididymis were found to have abnormal axoneme coiling and increased incidence of spontaneous acrosome reactions, both of which are consistent with defective regulation of the epididymal fluid. This was confirmed by incubating ERalphaKO sperm in a more wildtype-like osmotic environment, which significantly rescued the abnormal flagellar coiling. This work demonstrates that ERalpha activity in the efferent ducts and epididymis is essential for maintaining the luminal environment necessary for proper sperm maturation and function. |
