Two cleavage products of the Drosophila accessory gland protein ovulin can independently induce ovulation

Abstract

Proteins and peptides in Drosophila melanogaster seminal fluid induce mated females to increase their rates of egg deposition. One seminal-fluid protein, ovulin (Acp26Aa), stimulates an early step in the egg-laying process, the release of oocytes by the ovary. Ovulin, upon transfer to females, is cleaved sequentially within the mated female's reproductive tract. Here, we show that systemic ectopic expression of ovulin is sufficient to stimulate ovulation in unmated females. By using this assay to assess the functionality of ovulin's cleavage products, we find that two of the four cleavage products of ovulin can stimulate ovulation independently. Thus, ovulin's cleavage in mated females is not destructive and instead may liberate additional functional products with potential to modulate ovulation independently.mating ͉ processing ͉ prohormone ͉ seminal proteins E ven after mating, a male can continue to affect the reproductive success of his mate. One way this effect is accomplished results from the male's donation of seminal proteins. For example, in Drosophila melanogaster, Ϸ83 accessory gland proteins (Acps) are produced in the male's accessory glands (refs. 1 and 2; reviewed in ref.3). In the mated female, Acps increase egg production, ovulation, and egg deposition; decrease receptivity to remating; promote the storage of sperm; and decrease the lifespan of the mated female (reviewed in refs. 3-8). Individual Acps have been shown to cause specific changes in mated females: Acp26Aa (ovulin) and Acp70A (sex peptide) affect the egg-laying process (9-15), Acp70A affects receptivity (12-15), Acp36DE is needed for sperm storage (16,17), and Acp62F has been suggested to contribute to the decrease in the mated female's life span (18).Here, we focus on ovulin, an Acp that stimulates ovulation. Egg-laying in Drosophila is a multistep process (ref. 10; reviewed in ref. 19). First, eggs produced in the ovaries must pass checkpoints during oogenesis (refs. 20-22; reviewed in ref. 23). Mature oocytes are then released from ovaries (ovulation), a process that also triggers oocyte activation (24). Oocytes move through the oviducts to the uterus, where they can be fertilized by sperm released from storage, and, finally, they are released from the uterus and deposited onto the substratum (Fig. 1A). Mates of males who do not provide ovulin show decreased ovulation and egg deposition (9-11). Within the mated female, some ovulin from the male enters the circulatory system (hemolymph) and some localizes to the base of the ovary (10, 25). Thus, ovulin could conceivably induce ovulation in the female by direct interaction with neuromuscular targets along the upper region (lateral oviducts) of the genital tract or indirectly by affecting the activity of the neuroendocrine system (26).The sequence of ovulin suggests that it is a prohormone. The primary translation product is 264 aa long, including a predicted signal sequence of 18 aa that is expected to be cleaved to allow secretion of ovulin (27). Ovulin contains amino acid sequence...

Evidence for structural constraint on ovulin, a rapidly evolving Drosophila melanogaster seminal protein

“…12) were subcloned by using the Gateway system (Invitrogen, Carlsbad, CA) into Gateway-compatible yeast two-hybrid vectors (original vectors from Clontech, modified by K. Ravi Ram, A. Garfinkel, and M.F.W., unpublished data). All possible pairwise interactions were then tested by using histidine and adenine prototrophy, and X-Gal staining, as markers.…”

“…[6][7][8][9][10][11]. After transfer to the female, ovulin is sequentially cleaved into four smaller peptides (CP1, CP2, CP3C, and CP3N) (12,13), and increases ovulation in females during the first 24 h after mating (14)(15)(16). Full-length ovulin, as well as its two C-terminal cleavage products, are each individually capable of inducing ovulation in ectopic expression assays (12).…”

“…After transfer to the female, ovulin is sequentially cleaved into four smaller peptides (CP1, CP2, CP3C, and CP3N) (12,13), and increases ovulation in females during the first 24 h after mating (14)(15)(16). Full-length ovulin, as well as its two C-terminal cleavage products, are each individually capable of inducing ovulation in ectopic expression assays (12). Ovulin may therefore act as a prohormone, in that its cleavage may release active products; however, that uncleaved ovulin is also active contrasts with other prohormones (17,18).…”

“…To fully understand ovulin's molecular evolution and function as a hormone (or prohormone), structure/function relationships within the ovulin polypeptide need to be clarified. To date, cleavage products of ovulin sufficient to induce ovulation have been identified (12). Further work will be required to determine the functional roles of rapidly evolving regions and amino acid residues, as well as those domains and residues that are more highly conserved.…”

Predicted seminal astacin-like protease is required for processing of reproductive proteins in Drosophila melanogaster

“…Ovulin (Acp26Aa) increases the mated female's rate of ovulation (20). Ovulin is cleaved into several peptides (21)(22)(23), two of which can independently induce ovulation (24). Acp36DE, a large glycoprotein whose cleaved forms are seen in the mated female, is essential for sperm storage by mated females (25).…”