Germline expression of Imp-L2 in Drosophila females enhances reproductive activity and longevity

Abstract

The Imaginal morphogenesis protein-Late 2 (Imp-L2) in Drosophila is recognized as a functional homolog of the insulin-like growth factor (IGF) binding protein family. In this study, we report that Imp-L2 expression in germline cells during oogenesis simultaneously enhances both fecundity and lifespan in female Drosophila. Loss of Imp-L2, either through knockout or germline-specific knockdown, resulted in decreased reproductive activity, as evidenced by reduced ovary size and fecundity, along with a higher proportion of infertile flies. Conversely, overexpression of Imp-L2 specifically in germline cells enhanced reproductive activity. Imp-L2 appears to regulate germline stem cell proliferation and differentiation independently of IGF signaling. Interestingly, germline-specific knockdown of Imp-L2 shortened the lifespan of female flies, whereas its overexpression extended it. Thus, Imp-L2 expression in the germline promotes both reproductive activity and longevity, presenting an exception to the typical trade-off between reproduction and lifespan.

Keywords: Drosophila; Imp-L2; Insulin-like growth factor binding protein; lifespan; reproductive aging.

Figure 1.

Modulation of Imp-L2 expression affects ovary size and fecundity of female flies. (A) Female germline-specific knockdown of Imp-L2 using otu-Gal4 decreased ovary size. One-way ANOVA (F[2, 117] = 15.46; P < 0.0001), tukey’s multiple comparisons test; ***P < 0.001. (B) Imp-L2-deficient mutant flies exhibited a significant decrease in ovary size. One-way ANOVA (F[4, 81] = 20.43; P < 0.0001), tukey’s multiple comparisons test; *P < 0.05; **P < 0.01. (C) Germline-specific overexpression of Imp-L2 did not significantly affect ovary size. One-way ANOVA (F[2, 70] = 0.1241; P = 0.8835), tukey’s multiple comparisons test. (D) Knockdown of Imp-L2 decreased fecundity (Left) and increased infertility rates (Right). (E) Imp-L2-deficient mutant flies showed reduced fecundity (Left) and increased infertility rates (Right), similar to Imp-L2 knockdown flies. (F) Germline-specific overexpression of Imp-L2 increased fecundity (Left) and reduced infertility rates (Right). n.s.: not statistically significant. Error bars indicate S.E.M. Magnification: 25x.

Figure 2.

Modulation of Imp-L2 expression affects the number of germline stem cells (GSCs) and subsequent oogenesis. (A, B) Both female germline-specific Imp-L2 knockdown (A) and Imp-L2-deficient (B) flies showed a reduction in the average number of GSCs per germarium, along with an increased proportion of germaria containing fewer GSCs. (C) Germline-specific overexpression of Imp-L2 increased the average number of GSCs per germarium and the proportion of germaria containing a greater number of GSCs. (D) Germline-specific knockdown of Imp-L2 decreased Bag of marbles (Bam) expression in cystoblasts (CBs). (E) Imp-L2-deficient flies exhibited diminished Bam expression in CBs. (F) Germline-specific overexpression of Imp-L2 increased Bam expression in CBs. Hts (a marker for GSCs) and Bam staining are shown in green, while DAPI staining (nuclei) is shown in blue. Arrows in panels (A – C) indicate GSCs. Scale bar: 5 μm. Magnification: 400x.

Figure 3.

Modulation of Imp-L2 expression affects egg ovulation. (A, B) Both Imp-L2-deficient (A) and germline-specific Imp-L2 knockdown (B) flies exhibited a reduced frequency of ovulation. Ovulation was assessed based on the presence of eggs retained within the lateral oviduct (lOd), with the proportion of flies ovulating from one or both oviducts being determined. (C) Germline-specific overexpression of Imp-L2 showed a slight increasing trend in ovulation frequency, though this was not statistically significant. *p < 0.05; ***p < 0.001 compared to control values (t-test). n.s.: not statistically significant. Error bars indicate S.E.M.

Figure 4.

Modulation of Imp-L2 expression affects the lifespan of female flies. (A, B) Germline-specific overexpression of Imp-L2 significantly extended the lifespan of female flies (green) compared to control flies (blue) (A), whereas no effect was observed in males (B). One-way ANOVA (F[2, 6] = 27.31; P = 0.001), tukey’s multiple comparisons test; **P < 0.001 (A). One-way ANOVA (F[2, 6] = 1.194; P = 0.3659), tukey’s multiple comparisons test (B). (C, D) Germline-specific knockdown of Imp-L2 reduced the lifespan of female flies (green) compared to controls (blue) (C), with no effect observed in males (D). *p < 0.05 compared to control values (t-test). Note that flies in panels (A) and (B), but not in (C) and (D), carried one copy of the yw gene, which appears to negatively affect lifespan. A total of n = 450 flies were analyzed across all groups. Survival rates were assessed in groups of 150 individuals, and the data represent the results from three independent replicates. n.s.: not statistically significant. Error bars indicate S.E.M.