Development and application of a system for seminolipid metabolism using mouse seminiferous tubules

A convenient tool for studying metabolism of seminolipid in testis was developed by using mouse isolated seminiferous tubules prepared by collagenase treatment. Because more than 99% of [(35)S]sulfate-incorporation was distributed in seminolipid, its metabolism in seminiferous tubules can be analyzed without disturbance of the other sulfolipids in this assay system. Furthermore, the contents of seminolipid and its precursor, galactosylalkylacylglycerol, which were determined by liquid chromatography-electrospray ionization mass spectrometry, did not change within a few hours, indicating that the incorporations of [(35)S]sulfate into seminolipid solely reflects the turnover rate of this sulfolipid. As an initial application of this system, we characterized heat-susceptibility of the seminolipid turnover rate in mouse seminiferous tubules. Severe heating (44 degrees C for 10 min) of the isolated seminiferous tubules suppressed the (35)S-incorporation into seminolipid to 47% of heating at scrotal temperature (32 degrees C for 70 min). In contrast, pretreatment of the testis in vivo under the same condition (44 degrees C for 10 min) did not decrease the seminolipid turnover rate in the isolated seminiferous tubules. In addition, the activity of galactocerebroside sulfotransferase decreased in the temperature-dependent manner in seminiferous tubules as well as crude tubular homogenates, where the activity is significantly more stable in the former than the latter. The newly developed system could provide useful basic data for further analyses of seminolipid metabolism in the testis.