Sexual pheromones in lipids and other fractions from urine of the male mole rats, Spalax ehrenbergi

Spalax ehrenbergi mole rats are blind, solitary, territorial, aggressive, subterranean rodents with a yearly breeding season that peaks in December and January. We confirm here an earlier report that estrous females are attracted to substances present in the urine of homospecific as compared to heterospecific adult males. We have also found that nonestrous female mole rats show avoidance behavior to the same homospecific urine. Our objective was to ascertain the nature of the pheromone(s) and gain insight as to its possible role in reproductive isolation and speciation. An active principle, detected in either two- or three-choice behavior tests, was found to be extractable from urine by methylene chloride (CH₂Cl₂) and mainly found in the neutral lipid fraction. Total lipids were chromatographed by thin layer chromatography on silica gel G60 plates. Most of the activity was found in a zone bounded by R_fs 0.2 and 0.7. Cholesterol, other sterols, and ethyl esters of fatty acids chromatographed in this zone as determined by standards and staining. Ethyl esters of fatty acids were also detected in this fraction by GC/MS analysis. Although a large amount of activity was found in lipids, it only accounted for about 1% of that found in urine. Some activity may have been destroyed or lost during the extraction procedure and some may remain in a lipid insoluble form. Preliminary tests of lipid extracts of various portions of the male urogenital tract revealed pheromonal activity present, particularly in tissues associated with testes, epididymis, prostate, and bladder.