Cleavage of the Drosophila seminal protein Acp36DE in mated females enhances its sperm storage activity

Abstract

Sperm storage in the mated female reproductive tract (RT) is required for optimal fertility in numerous species with internal fertilization. In Drosophila melanogaster, sperm storage is dependent on female receipt of seminal fluid proteins (SFPs) during mating. The seminal fluid protein Acp36DE is necessary for the accumulation of sperm into storage. In the female RT, Acp36DE localizes to the anterior mating plug and also to a site in the common oviduct, potentially "corralling" sperm near the entry sites into the storage organs. Genetic studies showed that Acp36DE is also required for a series of conformational changes of the uterus that begin at the onset of mating and are hypothesized to move sperm towards the entry sites of the sperm storage organs. After Acp36DE is transferred to the female RT, the protein is cleaved by the astacin-metalloprotease Semp1. However, the effect of this cleavage on Acp36DE's function in sperm accumulation into storage is unknown. We used mass spectrometry to identify the single cleavage site in Acp36DE. We then mutated this site and tested the effects on sperm storage. Mutations of Acp36DE's cleavage site that slowed or prevented cleavage of the protein slowed the accumulation of sperm into storage, although they did not affect uterine conformational changes in mated females. Moreover, the N-terminal cleavage product of Acp36DE was sufficient to mediate sperm accumulation in storage, and it did so faster than versions of Acp36DE that could not be cleaved or were only cleaved slowly. These results suggest that cleavage of Acp36E may increase the number of bioactive molecules within the female RT, a mechanism similar to that hypothesized for Semp1's other substrate, the seminal fluid protein ovulin.

Keywords: Acp36DE; Drosophila; Proteolytic cleavage; Sperm storage.

Figure 1

Extracted ion chromatogram (XIC) of peptides from (A) Acp36DE’s first cleavage product (CP1; 68kDa) and (B) Acp36DE’s second cleavage product (CP2; 50kDa) in the range of residues 476–547. The specific targeted peptides are shown. The identified targeted peptide is marked with (**) when not the base peak. The red arrows in (B) identify peaks of 3 forms of the predicted peptides that constitute the N-terminus of CP2.

Figure 2. Mutation of residue 517 of Acp36DE eliminates cleavage of the protein in the female RT, and mutation of residue 516 slows cleavage

Western blot of RTs from females mated to males expressing the indicated version of Acp36E at 35min and 1hr ASM. Arrows indicate full-length Acp36DE (122kDa) and the first and second cleavage products (CP1 and CP2; 68kDa and 50kDa, respectively). Cleavage of Acp36DE^FL (lanes 5,9) appears to occur at a slightly reduced efficiency than cleavage of endogenous Acp36DE transferred by Canton S (CS; lanes 1, 10)) at both time points. The Acp36DE^S516A (lanes 3,7) construct is cleaved at a slower rate than Acp36DE^FL whereas cleavage of Acp36DE^D17A (lanes 4, 8) is essentially undetectable. TM3 males (lane 2) are null for Acp36DE. For females mated to transgenic males, protein from 5 RTs is loaded in each lane, for females mated to CS males, protein from 1 RT is loaded into each lane. The increased amount of tissue needed to detect transgenic Acp36DE led to increased background in those lanes.

Figure 3. Mutation of Acp36DE’s cleavage site results in less sperm in the SR at 35min relative to the number stored in the presence of full-length Acp36DE or its N-terminal-cleavage product

Females that received Acp36DE^FL, Acp36DE^CP1 and Acp36DE^S516A each stored significantly more sperm than females mated to Acp36DE null males at 35min ASM: Acp36DE^FL (N_FL = 14, N_Cont = 17, p = 0.0002), Acp36DE^CP1 (N_CP1 = 20, N_Cont = 17, p < 0.0001), Acp36DE^S516A (N_S516A = 18, N_Cont = 17, p = 0.0133), Acp36DE^D517A (N_D517A =12, N_Cont = 17, p = 0.138). Females that received Acp36DE^CP1 stored significantly more sperm than females that received Acp36DE^S516A, the slow-cleaving mutant (N_CP1 = 20, N_S516A = 18, p = 0.0366). * p < 0.05, ** p < 0.001

Figure 4. Receipt of each version of Acp36DE is sufficient to mediate uterine conformation

Receipt of Acp36DE^CP1 (N_CP1 = 20, N_FL = 20, p = 0.51), Acp36DE^S516A (N_S516A = 23, N_FL = 20, p = 0.27) and Acp36DE^D517A (N_D517A = 20, N_FL = 20, p = 0.99) did not significantly differ from Acp36DE^FL in their ability mediate progression of the uterine conformational changes. All four transgenic constructs are significantly different from females who did not receive Acp36DE at mating; Acp36DE^CP1: (N_CP1 = 20, N_Cont = 19, p < 0.0001); Acp36DE^FL: (N_FL = 20, N_Cont = 19, p = 0.0002); Acp36DE^D517A: (N_D517A = 20, N_Cont = 19, p < 0.0003); (N_S516A = 23, N_Cont = 19, p = 0.0001); Acp36DE^D516A: (N_D517A = 20, N_Cont = 19, p < 0.0003). Uterine stage classification is described in Adams and Wolfner (2007). In the box plots, the middle horizontal line represents the median, the lower and upper margins of the box represent the 25th and 75th quartiles, and the whiskers extend to the minimum and maximum of the data (excluding outliers, shown as points (•) outside the whiskers).