Neprilysins: an evolutionarily conserved family of metalloproteases that play important roles in reproduction in Drosophila

Abstract

Members of the M13 class of metalloproteases have been implicated in diseases and in reproductive fitness. Nevertheless, their physiological role remains poorly understood. To obtain a tractable model with which to analyze this protein family's function, we characterized the gene family in Drosophila melanogaster and focused on reproductive phenotypes. The D. melanogaster genome contains 24 M13 class protease homologs, some of which are orthologs of human proteases, including neprilysin. Many are expressed in the reproductive tracts of either sex. Using RNAi we individually targeted the five Nep genes most closely related to vertebrate neprilysin, Nep1-5, to investigate their roles in reproduction. A reduction in Nep1, Nep2, or Nep4 expression in females reduced egg laying. Nep1 and Nep2 are required in the CNS and the spermathecae for wild-type fecundity. Females that are null for Nep2 also show defects as hosts of sperm competition as well as an increased rate of depletion for stored sperm. Furthermore, eggs laid by Nep2 mutant females are fertilized normally, but arrest early in embryonic development. In the male, only Nep1 was required to induce normal patterns of female egg laying. Reduction in the expression of Nep2-5 in the male did not cause any dramatic effects on reproductive fitness, which suggests that these genes are either nonessential for male fertility or perform redundant functions. Our results suggest that, consistent with the functions of neprilysins in mammals, these proteins are also required for reproduction in Drosophila, opening up this model system for further functional analysis of this protein class and their substrates.

Keywords: Drosophila; neprilysins; neuropeptides; proteolysis; reproduction.

Figure 1

Phylogeny and expression of Drosophila neprilysins. A phylogenetic tree of the 24 known D. melanogaster M13 class proteases based on protein sequence similarity. The proteins fall into three distinct clades. Mapping the reproductive tract expression of each gene onto the tree reveals broad expression in the female RT (shaded and solid) and enrichment of male expression (open) in the clade that contains Nep1–5. The same clade also demonstrates enrichment for brain (red) and abdominal-thoracic ganglion expression (hatched).

Figure 2

Conserved binding motifs in Drosophila, human, and locust neprilysins. (A) Schematic representation of neprilysin. Solid, cytoplasmic domain; light shading, transmembrane domain; dark shading, extracellular domain. NAYY/F, important for substrate binding; HExxH, zinc-binding domain; ExxxD, zinc-binding domain; CxxW: sequence critical for protein folding and maturation of the enzyme. (B) Alignment of NAY/FY, HExxH, ExxxD, and CxxW sequences of D. melanogaster Nep1-5, L. migratoria ECE, and H. sapiens ECE1-2.

Figure 3

Embryonic expression pattern of neprilysin genes. (A and B) Embryonic stage 12 Nep4 expression in muscle founder cells (arrows in A) and in pericardial cells (pc). (C) Embryonic stage 13 Nep3 expression in the ventral nerve cord (vnc). (D) Embryonic stage 14 Nep4 expression in brain (br) and ventral nerve cord (vnc). (E and F) Embryonic stage 17 Nep1 expression in peripheral nervous system (arrows), antenno-maxillary complex (circled), and cells in the pharynx (open arrowheads) and midgut (solid arrowheads). (G–I) Embryonic stage 17 Nep2 expression in dorsal trunk (dt), dorsal branches (db), foregut (fg), and hindgut (hg). (J) Embryonic stage 17 Nep3 expression in brain (br) and ventral nerve cord (vnc). (K) Embryonic stage 17 Nep4 expression in brain (br), ventral nerve cord (vnc), and tracheal dorsal trunk (dt). (L) Embryonic stage 17 Nep5 expression in antenno-maxillary complex (circled) and in the pharynx (open arrowheads).

Figure 4

Larval expression pattern of neprilysin genes. (A–D) Expression of Nep1–4 in larval CNS. (A) Nep1 in mushroom bodies (mb), pars intercerebralis (pi), and ventral ganglia (vg). (B) Nep2 in few cells in the brain (br) and in the ventral ganglia (vg). (C) Nep3 in central brain (cbr) and thoracic ventral ganglia (tvg). (D) Nep4 in glia of the larval CNS. (E–H) Expression of Nep1–3 in the larval gastrointestinal system. (E) Nep1 in cells in the midgut. (F–G) Nep2 in the proventriculus (pv) and in the stellate cells of the Malpighian tubules (arrowheads). (H) Nep3 in scattered cells in the larval midgut (arrowheads). (I–L) Expression of Nep1 and Nep2 in larval imaginal discs. (I) Nep1 in leg disc. (J) Nep1 in dorsal and ventral wing pouch (dwp–vwp) and ventral pleura (vp) of the wing disc. (K) Nep2 in the eye disc anterior (arrowhead) to the morphogenetic furrow (mf) and in the second antennal segment (arrow). (L) Nep2 in the leg disc femur (fe) and tarsus (ta).

Figure 5

Adult expression pattern of neprilysin genes. (A–C) Expression of Nep1–2 in the adult CNS. (A) Nep1 in the adult brain mushroom bodies (mb) and pars intercerebralis (pi). (B) Nep2 in cells in the pars intercerebralis (pi), central brain (cb), and optic lobes (ol). (C) Nep2 in the third thoracic (t3) and abdominal (abd) neuromere. (D) Nep1 in adult midgut cells. (E) Nep2 in adult stellate cells of the Malpighian tubules (arrowhead). (F) Nep2 in border cells (bc) and posterior polar cells (ppc) of a stage 10 ovarian follicle. (G–M) Expression of neprilysin genes in the male reproductive tract. (G–H) Nep1 in the testicular tube (te) and the seminal vesicles (sv). (I) Nep 2 in the part of the testis (te) close to the seminal vesicle (sv). (J–L) Nep4 in the somatic cyst cells (arrowheads) and in other cells (arrows) in the part of the testes close to the seminal vesicle (sv). (M) Nep5 in the seminal vesicle.

Figure 6

Egg laying in mates of Nep RNAi males. (A) The mean number of eggs laid per female mated to either control males (gray line) or RNAi/null males (black line) over a 10-day period. Only mates of Nep1 RNAi males laid fewer eggs than mates of control males (Nep1: rmANOVA P = 0.0041, control N = 16, Nep1 RNAi N = 17). Mates of Nep2–5 RNAi laid comparable numbers of eggs as control mated females (Nep2—rmANOVA P = 0.095, control N = 11, Nep2 RNAi N = 14; Nep3—rmANOVA P = 0.7556, control N = 17, Nep3 RNAi N = 21; Nep4—rmANOVA P = 0.6972, control N = 16, Nep4 RNAi N = 16; Nep5—rmANOVA P = 0.8986, control N = 22, Nep5 RNAi N = 25; Nep2 null—rmANOVA P = 0.3448, control N = 18, Nep2 null N = 21). (B) The mean hatchability (#progeny/#eggs) per female for mates of control or RNAi/null males for the egg-laying assays in A. None of the Neps had a significant effect on hatching rate (Nep2—rmANOVA P = 0.4326 ; Nep3—rmANOVA P = 0.1494; Nep5—rmANOVA P = 0.1909; Nep2 null—rmANOVA P = 0.3673). (C) The mean observed (black line) and expected (dashed line) progeny ((# eggs) × (expected survival rate) × (average hatchability of eggs laid by control females)) for mates of Nep1 and Nep4 males. No difference in observed vs. expected progeny was seen for mates of either Nep1 or Nep4 RNAi males (Nep1, rmANOVA P = 0.3714; Nep4, rmANOVA P = 0.2874).

Figure 7

Egg laying in Nep RNAi females. (A) The mean number of eggs laid by control (gray line) or RNAi/null females (black line) mated to WT males over a 10-day period. Nep1, Nep2, and Nep4 RNAi and Nep2 null females lay fewer eggs than controls (Nep1—rmANOVA P = 0.0015, control N = 20, Nep1 RNAi N = 18; Nep2—rmANOVA P = <0.0001*, control N = 12, Nep2 RNAi N = 12; Nep4: rmANOVA P = 0.0207*, control N = 16, Nep4 RNAi N = 16; Nep2 null—rmANOVA P = <0.0001*, control N = 18, Nep2 null N = 15). (B) The mean observed (black line) and expected (dashed line) progeny ((# eggs) × (expected survival rate) × (average hatchability of eggs laid by control females)) for Nep1 and Nep4 females. No difference in overall observed vs. expected progeny was seen for either RNAi female (Nep1, rmANOVA P = 0.2853; Nep4, rmANOVA P = 0.4325). Nep1 RNAi females showed a trend for reduced observed progeny on day 1 (P = 0.0686) and a significant reduction on day 2 (P = 0.0253) after mating. (C) The mean hatchability (#progeny/#eggs) per female for Nep2. Both Nep2 RNAi and the Nep2 null females show drastically reduced hatchability (Nep2, rmANOVA P = <0.0001*; Nep2 null, rmANOVA P = <0.0001*) suggesting that Nep2 plays an essential role in this process.

Figure 8

Eggs laid by Nep2 null females arrest during early embryogenesis. (A) Eggs laid by Nep2 null females are fertilized at the same rate as eggs laid by control females (WRST P = 0.1593, control N = 37, Nep2 null N= 39) based on sperm tail staining. (B) DAPI staining of 1.5- to 3.5-hr-old eggs laid by Nep2⁺ control or Nep2 null females were sorted into two categories: developing or nondeveloping. All nondeveloping embryos contained a polar body rosette (C), whereas developing embryos were all at stage 4+ (D) of development consistent with the time point chosen. Eggs laid by Nep2 null females are significantly more likely to fall into the nondeveloping category than eggs laid by control females (WRST P < 0.0001*, control N= 43, Nep2 null N = 48). Since the fertilization rate between Nep2 null and control females is not different this result suggests that Nep2 may be critical for early embryogenesis.

Figure 9

(A) Counts of sperm stored in both sets of sperm storage organs (Total), the seminal receptacle (SR), and the paired spermatheca (SP), of Nep2 null (solid) vs. control females (shaded) at 2 hr and 4 days after the start of mating (ASM). Overall Nep2 null females store more sperm at 2 hr ASM (ANOVA, F= 4.8029, P = 0.0398, control N = 13, Nep2 null N = 10) and fewer sperm at 4 days ASM (ANOVA, F = 6.0175 P = 0.0215*, control N = 13, Nep2 null N = 14) than control females. Within the SR, Nep2 null females store the same number of sperm at 2 hr ASM (ANOVA, F = 0.71 P = 0.4061, control N = 17, Nep2 null N = 15) and marginally fewer sperm at 4 days ASM (ANOVA, F = 3.920 P = 0.0580, control N = 14, Nep2 null N = 15) than controls. Within the SP, Nep2 null females store the same number of sperm at both 2 hr ASM (ANOVA, F = 3.1304. P = 0.0901, control N = 13, Nep2 null N = 12) and 4 days ASM (ANOVA, F = 0.5584 P = 0.4614, control N = 14, Nep2 null N = 15). (B) For sperm competition assays Nep2 null or control females were first mated to a Canton-S male and then allowed to mate a second time with a bw^D male. The proportion of female progeny sired by the first male (Canton-S) referred to as P1 (# progeny from first male/total progeny) was significantly reduced in Nep2 null females compared to control females (WRST P = <0.0001*, control N = 76, Nep2 null N = 72). This difference is most apparent in the first 4 days ASM (WRST P = <0.0001*) compared to days 5–8 (WRST P = 0.1886)