Male zebrafish, Brachydanio rerio, have paired testes and no additional reproduction glands. Incubation experiments with 3H-labeled steroid precursors showed the capacity of testes to synthesize seven steroid glucuronides. Enzyme histochemical studies demonstrated interstitial (Leydig) cells as steroid and steroid glucuronide producing sites. Male holding water, testis homogenates, and testis fractions containing steroid glucuronides were able to induce ovulation in female zebrafish. Deglucuronidation of these fractions led to a loss of ovulation inducing potency, indicating steroid glucuronides as ovulation inducers. The chemical substances are perceived by the recipients by means of olfaction, as anosmic females do not have a n ovulatory response after administration of male holding water. In African catfish, Clarias gariepinus, the male reproductive organ system consists of two paired structures, the testis and the seminal vesicle. Histochemical enzyme investigations pointed to interstitial cells as sites of steroid and steroid glucuronide production both in testis and seminal vesicle. Glucuronidation of steroids may also take place in the epithelium lining and seminal vesicle tubules. Biochemical studies showed the seminal vesicle as the main source of steroid glucuronides. Eight conjugated steroids were identified. Electrophysiological studies demonstrated that these compounds, especially 5P-pregnane-3a,l7au-dio1-20-oneglucuronide are olfactory stimulants in female conspecifics. Males with enlarged seminal vesicles, caused by compensatory growth after castration, were more attractive to ovulated female catfish in a two-choice test, using a U-shaped tank. Males lacking seminal vesicles were less attractive. The steroid glucuronide fraction of seminal vesicle fluid appeared to be responsible for the attraction effect. A synthetic mixture of steroid glucuronides resulted in a dose dependent attraction effect. The data are indicative of a pheromonal function for steroid glucuronides that are produced by the reproductive organ system of male zebrafish and African catfish.
