Targeted gene deletion and phenotypic analysis of the Drosophila melanogaster seminal fluid protease inhibitor Acp62F

Abstract

Internally fertilizing organisms transfer a complex assortment of seminal fluid proteins, a substantial fraction of which are proteolysis regulators. In mammals, some seminal protease inhibitors have been implicated in male infertility and these same molecular classes of protease inhibitors are also found in Drosophila seminal fluid. Here, we tested the reproductive functions of the Drosophila melanogaster seminal fluid protease inhibitor Acp62F by generating a precise deletion of the Acp62F gene. We did not detect a nonredundant function for Acp62F in modulating the egg laying, fertility, remating frequency, or life span of mated females. However, loss of Acp62F did alter a male's defensive sperm competitive ability, consistent with the localization of Acp62F to sperm storage organs. In addition, the processing of at least one seminal protein, the ovulation hormone ovulin, is slower in the absence of Acp62F.

Figure 1.—

(A) Targeting an Acp62F null allele to the Acp62F locus. FLP and I-SceI induction mediates excision of the donor construct and introduces a double-strand break. Homologous recombination within the 3′ flanking region results in a tandem duplication of endogenous Acp62F adjacent to the targeted null Acp62F allele. The targeting strategy presented here is based upon the gene-targeting technique of Rong and Golic (2000). (B) Reduction of Acp62F tandem duplicates to a single copy. (Left) Recombination events between the 5′ flanking regions of the duplicates leaves only the Acp62F null allele. (Right) Recombination between the 3′ flanking regions leaves only the wild-type Acp62F. The two reduction events can be differentiated with primers Acp62Fscreen-1 and Acp62Fscreen-2 (arrows in 5′ and 3′ flanking regions and supplemental Table 1). The reduction strategy presented here is based upon the previous technique of Xie and Golic (2004). (C) Crossing scheme used to generate a deletion of the Acp62F coding region. Targeted donor constructs that map the w^hs marker to the third chromosome were used for targeting events. Progeny of males carrying the donor construct P[Acp62F^del] crossed to females carrying hsp70-FLP and hsp70-SceI transgenes were heat-shocked to excise the donor construct. Stably integrated targeting events at the Acp62F locus were identified by the w^hs marker segregating with the third chromosome and nonmosaics when crossed to eyeless-FLP. Females carrying the duplicated Acp62F alleles were crossed to hsp70-Cre transgenic males to induce Cre-mediated double-strand-break recombinase and thus reduction events. Lines that lost the w^hs marker were candidate Acp62F null alleles.

Figure 1.—

(A) Targeting an Acp62F null allele to the Acp62F locus. FLP and I-SceI induction mediates excision of the donor construct and introduces a double-strand break. Homologous recombination within the 3′ flanking region results in a tandem duplication of endogenous Acp62F adjacent to the targeted null Acp62F allele. The targeting strategy presented here is based upon the gene-targeting technique of Rong and Golic (2000). (B) Reduction of Acp62F tandem duplicates to a single copy. (Left) Recombination events between the 5′ flanking regions of the duplicates leaves only the Acp62F null allele. (Right) Recombination between the 3′ flanking regions leaves only the wild-type Acp62F. The two reduction events can be differentiated with primers Acp62Fscreen-1 and Acp62Fscreen-2 (arrows in 5′ and 3′ flanking regions and supplemental Table 1). The reduction strategy presented here is based upon the previous technique of Xie and Golic (2004). (C) Crossing scheme used to generate a deletion of the Acp62F coding region. Targeted donor constructs that map the w^hs marker to the third chromosome were used for targeting events. Progeny of males carrying the donor construct P[Acp62F^del] crossed to females carrying hsp70-FLP and hsp70-SceI transgenes were heat-shocked to excise the donor construct. Stably integrated targeting events at the Acp62F locus were identified by the w^hs marker segregating with the third chromosome and nonmosaics when crossed to eyeless-FLP. Females carrying the duplicated Acp62F alleles were crossed to hsp70-Cre transgenic males to induce Cre-mediated double-strand-break recombinase and thus reduction events. Lines that lost the w^hs marker were candidate Acp62F null alleles.

Figure 2.—

Western blot analyses of Acp62F^1a–d null lines of whole male flies, dissected male accessory glands, and mated female flies. The solid arrow at the right marks Acp62F, which runs at ∼14 kDa (Lung and Wolfner 1999). Acp62F protein is produced in Acp62F^1a–d/TM3, Sb males and is not produced in Acp62F^1a–d/Acp62F^1a–d males (lanes 4–11). Acp62F is transferred in Canton-S females mated to Acp62F^1a/TM3, Sb males (lane 12), but not in Canton-S females mated to Acp62F^1a/Acp62F^1a males.

Figure 3.—

Expression, transfer, and cleavage of ovulin in null Acp62F males. Lane 1, male accessory gland. Lane 2, Canton-S males. Lane 3, virgin Canton-S females. Lane 4, Acp62F^1a/Acp62F^1a males. Lane 6, Canton-S females mated to Acp62F^1a/Acp62F^1a males, 10 min after mating, receive normal levels of ovulin. Ovulin full-length and processed products can be detected in mates of Acp62F^1a/TM3, Sb and Acp62F^1a/Acp62F^1a males. Ovulin processing appears to be differentially regulated in mates of Acp62F^1a/TM3, Sb (lane 7) and Acp62F^1a/Acp62F^1a males (lane 6).

Figure 4.—

Effect of Acp62F on female life span. Survival of females continuously mated to null (Acp62F^1bB and Acp62F^1c) or control (Acp62F^Tandem and Acp62F^1cCtrl) males against time (days).