Dietary restriction enhances germline stem cell maintenance

Summary

Dietary restriction (DR) increases lifespan in species ranging from yeast to primates, maintaining tissues in a youthful state and delaying reproductive senescence. However, little is known about the mechanisms by which this occurs. Here we demonstrate that, concurrent with extending lifespan, DR attenuates the age-related decline in male germline stem cell (GSC) number in Drosophila. These data support a model whereby DR enhances maintenance of GSCs to extend the reproductive period of animals subjected to adverse nutritional conditions. This represents the first example of DR maintaining an adult stem cell pool and suggests a potential mechanism by which DR might delay aging in the tissues of higher organisms.

Dietary restriction (DR), reducing food intake without malnutrition, increases longevity and maintains animals in a youthful state in species ranging from yeast to primates (reviewed in Mair & Dillin 2008). This conserved phenomenon is thought to be an adaptation to survive famine, redirecting limited resources from reproduction towards somatic maintenance until plentiful times return (Shanley & Kirkwood 2000). In mammals, although DR decreases fecundity, previously restricted animals that are re-fed maintain the ability to reproduce at advanced ages when ad libitum fed controls have become sterile (Holehan & Merry 1985). Similarly, male Drosophila become less fertile with age (Economos et al. 1979), yet those fed a DR diet early in life father more progeny in sperm competition assays compared to chronically fully-fed (FF) controls (Fricke et al. 2008). Therefore in flies, as it does in mammals, DR extends reproductive capacity. However, little is known about the mechanism by which this occurs.

The dramatic reduction in spermatogenesis seen in aging male fruit flies is attributable to a decrease in both number and proliferation of germline stem cells (GSCs) (Boyle et al. 2007). Female GSC maintenance and proliferation are sensitive to nutritional status (Drummond-Barbosa & Spradling 2001; LaFever & Drummond-Barbosa 2005; Hsu & Drummond-Barbosa 2009) and in male flies, protein starvation leads to progressive loss of GSCs and early germ cells, which are replaced upon re-feeding (McLeod et al., unpublished). Similarly, C. elegans enter an adult reproductive diapause upon starvation, during which much of the germline degenerates. However, a small population of GSCs is maintained. When feeding resumes, GSCs re-populate the germline, and the worms regain fertility (Angelo & Van Gilst 2009). Therefore, we wanted to determine whether the ability of DR to extend lifespan and delay the aging of reproductive tissues was accompanied by increased maintenance of GSCs.

In Drosophila, male GSCs are located at the tip of the testis in contact with the hub, a cluster of somatic cells that send self-renewal signals to the adjacent GSCs to specify their maintenance (Figure 1A). GSCs in this system can be readily quantified, allowing us to analyse the effect of DR both on the lifespan of male flies and on the stem cells within the testis. As described previously (Partridge et al. 2005), DR extended the median and maximal lifespan of male flies (Figure 1B, Figure S1). Interestingly, males on DR had significantly more GSCs per testis than controls at each time point analysed subsequent to the mortality curves diverging, indicating that indeed DR enhances maintenance of GSCs (Figure 1B-D; Figure S1). Furthermore, at every sampling interval, a greater proportion of the DR cohort had testes containing Fasciclin III positive hub cells than controls (Table S1), suggesting that DR also delays age-related changes to the stem cell niche (Boyle et al. 2007).

Figure 1. DR extends lifespan in Drosophila males and increases average number of germline stem cells.

(A) Schematic of the apical tip of the Drosophila testis (B) Lifespan of wild-type Drosophila males on fully fed (FF) or DR feeding paradigms. DR significantly extends lifespan of male flies (P<0.0001, Log Rank Test). Median Lifespan; Control = 57 days, DR = 66 days, 16% extension. Average number of GSCs per testis is shown (for time points indicated by dashed vertical lines) with standard error and number of testes analysed in parentheses. Significant increases in average number of GSCs per testis were observed in DR males compared to controls (Students T-test, asterisk p<0.05, double asterisk p<0.01). (C, D) Immunofluorescent images of testes from FF males (C) or males on DR feeding paradigm (D) stained with antibodies against Vasa (germ cells; green) and FasIII (hub; red). GSCs were scored as Vasa⁺ cells in contact with the hub (dots). Scale bars, 20 μm.

The effects of DR on GSCs could result from either an acute increase of GSC number at all time points or by attenuating age-related GSC loss. To distinguish between these possibilities we measured the average number of GSCs in fully fed (FF) and DR flies early in life, before their survival rates had diverged. At days 10 and 20 after the instigation of a DR regime, there was no significant difference between GSC number in males on either food regime (Table S2), with significantly more GSCs at day 30 in testes from the DR cohort, as seen previously (Figure 1 B). These data are consistent with the hypothesis that DR enhances stem cell maintenance and attenuates age-related depletion of GSCs (Figure 2).

Figure 2. DR delays the rate of GSC loss with age.

Combined average GSC number per testes in fully-fed (FF) and dietary restricted (DR) animals with age in three experiments. Linear regression for FF (blue, R² = 0.95) and DR (red, R² = 0.96) data points. DR significantly reduces the rate of GSC loss with age, analysis of covariance (ANCOVA) P=0.037, F=5.39.

This is the first demonstration that DR concurrently extends lifespan and maintenance of adult stem cells. Although currently it is unknown if sustained maintenance of GSCs in Drosophila directly translates into enhanced male reproductive fitness later in life, these results provide a potential mechanism by which DR could extend reproductive capability when previously restricted animals are re-fed. Many adult tissues in higher organisms are maintained and repaired throughout life by a pool of adult stem cells; therefore, it will be intriguing to determine whether DR attenuates aging of mammalian tissues by enhancing maintenance of adult stem cell pools.