Identification of mutations that delay somatic or reproductive aging of Caenorhabditis elegans

Abstract

Aging is an important feature of animal biology characterized by progressive, degenerative changes in somatic and reproductive tissues. The rate of age-related degeneration is genetically controlled, since genes that influence lifespan have been identified. However, little is known about genes that affect reproductive aging or aging of specific somatic tissues. To identify genes that are important for controlling these degenerative changes, we used chemical mutagenesis to perform forward genetic screens in Caenorhabditis elegans. By conducting a screen focused on somatic aging, we identified mutant hermaphrodites that displayed extended periods of pharyngeal pumping, body movement, or survival. One of these mutations is a novel allele of the age-1 gene. age-1 encodes a phosphatidylinositol-3-kinase (PI3K) that functions in the insulin/insulin-like growth factor-1 (IGF-1) signaling pathway. age-1(am88) creates a missense change in the conserved PIK domain and causes dramatic extensions of the pharyngeal pumping and body movement spans, as well as a twofold extension of the lifespan. By conducting screens focused on reproductive aging in mated hermaphrodites, we identified mutants that displayed increased progeny production late in life. To characterize these mutations, we developed quantitative measurements of age-related morphological changes in the gonad. The am117 mutation delayed age-related declines in progeny production and morphological changes in the gonad. These studies provide new insights into the genetic regulation of age-related degenerative changes in somatic and reproductive tissues.

Figure 1

Design of genetic screens. Wild-type hermaphrodites were mutagenized with EMS, F₁ self-progeny were cultured individually, and the indicated number of F₂ self-progeny was analyzed. F₂ hermaphrodites were self-fertilized (A) or mated to wild-type males on the indicated days (B and C). F₂ animals were analyzed for aging spans (A) or cumulative progeny production from day 10 until cessation (B and C). N indicates the number of F₁ animals analyzed.

Figure 2

The am88 mutation delays multiple age-related degenerative changes. Wild-type (black diamonds) and am88 (open circles) hermaphrodites at the L4 stage were placed on individual Petri dishes and monitored for self-progeny production (A), fast pharyngeal pumping (B), fast body movement (C), any pharyngeal pumping (D), and survival (E). The percentage of animals in the population that displayed the phenotype is graphed vs. age in days (see Table 1 for summary statistics). Total number of animals analyzed was 180 for wild type and 105 for am88. (F) Horizontal bars represent the time in days spent in stages I (gray), II (black), III (white), and IV (stippled) (described in the text). (G) Horizontal bars represent the fraction of the lifespan occupied by each stage, calculated by setting the lifespan of each strain equal to 100%. WT data are from Huang et al. (2004).

Figure 3

The am88 mutation affects a conserved residue in the AGE-1 PI3 kinase. (A) The full-length AGE-1 PI3 kinase is schematized, and conserved domains are shown as stippled boxes and labeled below. The positions of the am88 mutation (E-to-K substitution in the PIK domain) and the hx546 mutation are indicated. (B) The amino acid sequence of the PIK domain of the C. elegans AGE-1 protein is aligned with that of mouse PI3 kinase delta (Chantry et al. 1997; Okkenhaug et al. 2002) and human PI3 kinase delta (Chantry et al. 1997; Vanhaesebroeck et al. 1997). Identical residues are marked by asterisks, similar residues are marked by dots, and gaps are represented by dashes. The am88 change affects the conserved glutamate at residue 725 (shaded gray).

Figure 4

Progeny production of am116 and am117 mutants. (A–C) Average daily progeny production of live hermaphrodites containing the am116 or am117 mutation compared to wild type (WT). Hermaphrodites were self-fertilized (self) or mated for days 1 and 2 to three wild-type males (mated). (D) Percentage of hermaphrodites producing progeny. See Table 4 for values and summary statistics. Number of hermaphrodites at the start of the experiment: WT self = 76, WT mated = 65, am116 self = 25, am116 mated = 19, am117 self = 50, and am117 mated = 24. WT data are from Hughes et al. (2007).

Figure 5

Age-related morphological changes in the hermaphrodite germline. Wild-type hermaphrodites were isolated at the L4 larval stage (day 1), allowed to mature until the indicated day, and visualized by differential interference contrast (DIC) microscopy at ×400 magnification. Images are oriented with ventral below and dorsal above. (A) Day 7, well-organized late-stage oocytes in the proximal gonad (arrow, ventral) and many developing germ cell nuclei in the distal gonad (arrowhead, dorsal). (B) Day 7, late-stage oocytes display “stacking” in the proximal gonad (arrow, ventral), a result of sperm depletion in a self-fertilized hermaphrodite. (C) Day 7, unfertilized eggs in the proximal gonad (arrow, ventral), a result of sperm depletion in a self-fertilized hermaphrodite. (D) Day 8, a narrow distal gonad containing a reduced number of developing germ cells (arrow, dorsal). (E) Day 9, a swollen nucleus in the distal gonad (arrow, dorsal). (F) Day 8, debris in the proximal gonad (arrow, ventral). (G) Day 9, a vacuole-like structure in the proximal gonad (arrow, ventral). (H) Day 8, an opaque mass in the proximal gonad (arrow, ventral).

Figure 6

Quantification of age-related changes in the proximal germline of hermaphrodites. Wild-type (WT) hermaphrodites were self fertilized (self) or mated to males (mated), and am117 mutant hermaphrodites were self-fertilized. Ages of adult hermaphrodites raised at 20° are shown in days: the L4 stage is day 1. (A and B) Average number per gonad arm of large oocytes located between the reflex in the gonad arm and the spermatheca. Compared to WT self, WT mated was significantly different on days 4–7, 9, and 11, and am117 self was significantly different on days 2, 4, 5, and 8–12 (*P ≤ 0.05; N = 10–68, 17–64, and 12–68 gonad arms for WT self, WT mated, and am117 self, respectively). (C and D) Average number per gonad arm of unfertilized and fertilized eggs located between the spermatheca and the vulva. Compared to WT self, WT mated was significantly different on days 4–8, and am117 self was significantly different on days 2, 8, and 10–12 (*P ≤ 0.05; N = 10–64, 24–68, and 12–69 gonad arms for WT self, WT mated, and am117 self, respectively). (E and F) Percentage of gonad arms with one or more masses between the spermatheca and the vulva. Compared to WT self, WT mated was significantly different on day 5, and am117 self was significantly different on days 5, 9, and 10 (*P ≤ 0.05, see Table 5).