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Recently, we cloned and sequenced the cDNA of allurin, a sperm chemoattractant isolated from the jelly of Xenopus laevis eggs [Proc. Natl. Acad. Sci. U.S.A. 78 (2001) 11205]. In this report, we demonstrate that allurin mRNA is expressed almost exclusively in the oviduct and that its expression is increased 2.5-fold by human chorionic gonadotropin over a 12-h period. Both dot blots and immunocytochemistry show that allurin is secreted from the upper two thirds of the oviduct that includes the pars recta and the proximal pars convoluta. Allurin appears to be deposited on the ciliated surfaces of luminal epithelial cells that come in direct contact with eggs as they move through the oviduct. Immune staining also demonstrates the presence of allurin in the serosal capsule of the oviduct. In contrast, allurin is not found within the tubular jelly-secreting glands or ducts that constitute a major portion of the oviduct wall. Therefore, we hypothesize that allurin is synthesized by nonciliated secretory cells in the luminal epithelium of the oviduct, is displayed on the ciliary layer and then mechanically mixed with jelly, and applied to eggs as they progress down the oviduct. This hypothesis is consistent with the fact that eggs progressing down the oviduct initially show evidence of allurin being incorporated into the J1 layer. Subsequently, allurin within J1 diffuses outward to J3 and eggs stored in the uterus now demonstrate a J3 localization of this chemoattractant.

Previously we reported the identification of the first vertebrate sperm chemoattractant, allurin, in the frog Xenopus laevis (Xl) and demonstrated that it was a member of the CRISP family of proteins. Here we report identification, purification, and characterization of Xenopus tropicalis (Xt) allurin, a homologous protein in X. tropicalis. “Egg water” as well as purified allurin from both species exhibit efficient cross-species sperm chemoattractant activity. Western blots show that Xt egg water contains a single anti-allurin cross-reactive protein whose molecular weight (20,497 Da by MALDI MS) agrees well with the molecular weight of the hypothetical gene product for a newly recognized “Crisp A” gene in the X. tropicalis genome. A recombinant form of the protein, expressed in 3T3 cells, exhibits chemoattraction for both Xt and Xl sperm and cross reacts with anti-allurin antibodies. Examination of Crisp protein expression in the Xt oviduct using RT-PCR showed that of five documented Xt Crisp genes (Crisps 2, 3, LD1, LD2 and A) only Crisp A was expressed. In contrast, Crisp 2, Crisp 3, Crisp LD1, and Crisp LD2, but not Crisp A, were all found to be expressed in the Xt testes while subsets of Crisp proteins where expressed in the Xt ovary. These data suggest that Crisp proteins in amphibians may play multiple roles in sperm production, maturation and guidance just as they are thought to in mammals indicating that Crisp protein involvement in reproduction may not be limited to mammals.

Allurin, a 21kDa protein isolated from egg jelly of the frog Xenopus laevis, has previously been demonstrated to attract frog sperm in two-chamber and microscopic assays. cDNA cloning and sequencing has shown that allurin is a truncated member of the Cysteine-Rich Secretory Protein (CRISP) family, whose members include mammalian sperm-binding proteins that have been postulated to play roles in spermatogenesis, sperm capacitation and sperm–egg binding in mammals. Here, we show that allurin is a chemoattractant for mouse sperm, as determined by a 2.5-fold stimulation of sperm passage across a porous membrane and by analysis of sperm trajectories within an allurin gradient as observed by time-lapse microscopy. Chemotaxis was accompanied by an overall change in trajectory from circular to linear thereby increasing sperm movement along the gradient axis. Allurin did not increase sperm velocity although it did produce a modest increase in flagellar beat frequency. Oregon Green 488-conjugated allurin was observed to bind to the sub-equatorial region of the mouse sperm head and to the midpiece of the flagellum. These findings demonstrate that sperm have retained the ability to bind and respond to truncated Crisp proteins over 300million years of vertebrate evolution.