When C. elegans hermaphrodites are deprived of food during the mid-L4 larval stage and throughout adulthood, they enter an alternative stage termed “adult reproductive diapause (ARD)” in which they halt reproduction and extend their lifespan. During ARD, germ cell proliferation stops; oogenesis is slowed; and the gonad shrinks progressively, which has been described as the “oogenic germline starvation response”. Upon refeeding, the shrunken gonad is regenerated, and animals recover fertility and live out their remaining lifespan. Little is known about the effects of ARD on oocyte quality after ARD. Thus, the aim of this study was to determine how oocyte quality is affected after ARD by measuring brood size and embryonic lethality as a reflection of defective oocyte production. We found that ARD affects reproductive capacity. The oogenic germline starvation response protects oogenic germ cells by slowing oogenesis to prevent prolonged arrest in diakinesis. In contrast to a previous report, we found that germ cell apoptosis is not the cause of gonad shrinkage; instead, we propose that ovulation contributes to gonad shrinkage during the oogenic germline starvation response. We show that germ cell apoptosis increases and continues during ARD via lin-35/Rb and an unknown mechanism. Although apoptosis contributes to maintain germ cell quality during ARD, we demonstrated that apoptosis is not essential to preserve animal fertility. Finally, we show that IIS signaling inactivation partially participates in the oogenic germline starvation response.
Keywords: Research Article; Biology and life sciences; Cell biology; Cellular types; Animal cells; Germ cells; Anatomy; Reproductive system; Genital anatomy; Gonads; Medicine and health sciences; Cell processes; Cell death; Apoptosis; Ova; Oocytes; Developmental biology; Embryology; Embryos; Sperm; Nutrition; Malnutrition; Starvation; Endocrinology; Endocrine physiology; Menstrual cycle; Ovulation; Physiology; Reproductive physiology
To ensure species continuity, animals have developed mechanisms for protecting germ cells during stressful conditions. The C. elegans hermaphrodite germline serves as an excellent model for studying cell biology. In C. elegans hermaphrodites, 2 identical U-shaped gonad arms contain germ cells (Fig 1A). Under control conditions, L4 hermaphrodites (Fig 1C and 1E) produce approximately 40 germ cells that give rise 160 spermatids per gonad arm, which are stored within each spermatheca. Thereafter, during the adult stage, the remaining germ cells either differentiate into oocytes or are eliminated by physiological germline apoptosis [[
The short, highly stereotyped reproductive cycle of Caenorhabditis elegans can be altered when animals are deprived of food and enter into reversible states of growth arrest or diapause, depending on the stage in which they are deprived of food [[
When mid-L4 larvae or adult hermaphrodites face starvation conditions, they enter into adult reproductive diapause (ARD), characterized by delayed reproduction and an extended lifespan [[
Remarkably, gonad shrinking and delayed reproduction are reversible [[
ARD extends not only the total lifespan but also the reproductive period of animals [[
In this report, we use brood size and embryonic lethality to reflect defective oocyte production to investigate the effects of ARD on oocyte quality. We found that ARD affects gametes' reproductive capacity and prevents oogenic germ cells from undergoing prolonged arrest in diakinesis. During ARD, germ cell apoptosis is very active; however, in contrast to a previous report, we found that it is not important for gonad shrinking. We observed that increased germ cell apoptosis during ARD depends partially on lin-35/Rb and an unknown mechanism. We propose that ovulation causes gonad shrinking by exhausting gonad contents when a few oocytes are produced. Finally, DAF-2 inactivation causes gonad shrinking in the presence of food suggesting that it may partially participate in this pathway. Here, we describe the effects of ARD on fertility and the regulation of germ cell apoptosis under starvation conditions.
C. elegans strains were maintained as described previously [[
Animals were mounted with 10 μl of 0.01% tetramisole in M9 on 2% agarose pads and observed using a Nikon Eclipse E600 microscope equipped with an AxioCam MRc camera (Zeiss). Images were obtained using Axio Vision software (Zeiss) and processed with ImageJ software.
We performed the starvation protocol described by Seidel and Kimble (2011) [[
Cell corpses were counted using the MD701 bcIs39 [Plim-7: :ced-1: :gfp;lin-15(+)] transgenic strain. After the starvation protocol, animals were placed in seeded plates as the control condition or in unseeded plates as the starvation condition. Animals were transferred daily until they ceased laying eggs and were then picked, anesthetized, mounted and visualized under an epifluorescence microscope.
We used DAPI staining to quantify the germ cells in each gonad arm as described by Silva-García and Navarro (2013) with some modifications [[
We determined the brood size and embryonic lethality, resulting from self-fertilization and mating as described by Bukhari et al., 2012 [[
We determined the temporal progression of germ cell corpse clearance using a method described by Kim et al., 2013 [[
We followed the methodology described by Huang et al., 2012 [[
The t-test or Mann-Whitney U test was used for comparisons with controls in fertility assays. For multiple comparisons in apoptosis, fertility assays and germ cell corpse clearance assays, the data were analyzed using one way ANOVA and Dunn's test for multiple comparisons.
We wanted to study how fertility and germ cell quality are affected after exposing animals to ARD. Although it has been reported that wild-type animals survive up to 30 days in ARD, their self-fertility is severely impaired after 15 days of starvation [[
Table 1: Fertility assays in diverse genetic backgrounds used in this study.
Genotype Brood Size Dead Embryos N WT 231.8 ± 2.3 2.1 ± 0.1 144 Recovered WT 67.0 ± 2.9 *** 3.8 ± 0.3 *** 69 Control WT 424.9 ± 8.3 2.6 ± 0.3 22 x WT males Recovered WT 344.8 ± 26.3 n.s 2.8 ± 0.6 n.s. 17 x WT males Control fog-2(q71) 324.5 ± 6.5 2.4 ± 0.3 46 x WT males Recovered fog-2(q71) 183.2 ± 5.6 * 4.0 ± 0.3 * 42 x WT males Prolonged diakinesis fog-2(q71) 55.8 ± 2.3 * 4.8 ± 0.2 * 51 x WT males Control fog-1(q253) 302.3 ± 7.0 2.5 ± 0.1 31 x WT males Recovered fog-1(q253) 177.1 ± 3.3 * 4.4 ± 0.2 * 35 x WT males Prolonged diakinesis fog-1(q253) 65.0 ± 1.6 * 5.6 ± 0.1 * 39 x WT males Control ced-3(n717) 141.0 ± 2.6 8.1 ± 0.7 43 Recovered ced-3(n717) 53.1 ± 1.8 * 7.3 ± 0.5 * 41 Control ced-3(n1286) 143.8 ± 3.2 7.5 ± 0.4 88 Recovered ced-3(n1286) 52.0 ± 2.3 * 12.3 ± 0.8 * 58 Control ced-3(n717) 315.6 ± 8.6 19.8 ± 3.0 11 x WT males Recovered ced-3(n717) 248.9 ± 12.3 ** 19.3 ± 1.9 n.s. 20 x WT males
- 4 Hermaphrodites with the different genetic backgrounds were individually selected at the mid-L4 stage and transferred to new plates every 24 h until they ceased laying embryos. Recovered wild-type hermaphrodites were selected at the mid-L4 stage, starved for 5 days and transferred to new plates every 24 h until they ceased laying embryos. t-test (control vs. recovered). Control fog-1(q253) and fog-2(q71) animals were individually selected in the mid-L4 stage, mated with well-fed males and transferred to fresh plates daily until they ceased laying embryos. Recovered virgin fog-1(q253) and fog-2(q71) animals were selected in the mid-L4 stage and starved for 5 days, then individually refed for 1 day, mated with well-fed males and transferred to fresh plates daily until they ceased laying embryos. Virgin fog-1(q253) and fog-2(q71) animals were selected in the mid-L4 stage and placed on food for 6 days, then mated with well-fed males and transferred to fresh plates daily until they ceased laying embryos. Plates were scored for dead embryos and total progeny. Embryos that did not hatch within 24 h after being laid were scored as dead. Dunn's test (wild-type values as control). n.s. non significant.
- 2 * P ≤ 0.05
- 3 ** P ≤ 0.01
1 *** P≤ 0.001
When we analyzed the progeny produced by the control and recovered animals by day, we observed that recovered animals produced fewer offspring during the first two days after refeeding, after which progeny production peaked at the third day and ceased at the fifth day (Fig 2B). It is likely that the delay in offspring production in recovered animals was due to the gonad regeneration process, which usually takes two days.
One explanation for the low fertility after ARD is that because the starvation experiments started at the mid-L4 larval stage (approx. 4 h after L4 molting), when spermatogenesis has not yet been completed, insufficient sperm production could occur. To discard this possibility, we quantified sperm production in starved animals once they were close to completing the L4 larval stage (approx. 8 h after L4 molting) by DAPI staining. Control wild-type animals produced an average of 105.11± 1.1 sperm (N = 36, Fig 2C) and starved wild-type animals produced a similar number of 101.89 ± 2.5 (N = 37). After 5 days of L4 larval molting, the control wild-type animals did not have any more sperm in their spermatheca, while 5-day-starved wild-type animals still harbored an average of 102.5 ± 0.68 sperm (N = 28, Fig 2C). We conclude that even during prolonged starvation sufficient sperm are produced, therefore this is not a factor that explains low progeny numbers after ARD.
We also quantified the embryonic lethality of self-fertilizing recovered hermaphrodites after ARD and found that recovered animals exhibited significantly higher embryonic lethality than control animals (3.8 ± 0.3 dead embryos/worm in recovered animals vs. 2.1 ± 0.1 dead embryos/worm in control animals, i.e., 1.8-fold; Table 1, Fig 2D).
To counteract the effect of ARD on sperm, we quantified the brood size and embryonic lethality of wild-type hermaphrodites that were exposed to ARD and later crossed with well-fed wild-type males. Under control conditions, mid-L4 wild-type hermaphrodites were crossed with 4 wild-type males overnight and then transferred daily to new Petri dishes until they ceased laying embryos. For ARD, mid-L4 wild-type animals were deprived of bacteria for 5 days, then transferred to plates with food and immediately crossed with 4 well-fed wild-type males. We observed that the fertility of recovered wild-type animals crossed with well-fed males improved considerably (81% of that in control wild-type animals) even though their brood size never reached that of the control (Fig 2E). Additionally, we did not observe significant differences in embryonic lethality between these two groups of animals (2.6 ± 0.3 dead embryos in control mated animals vs. 2.8 ± 0.6 dead embryos in recovered mated animals; Table 1, Fig 2F). Our results suggest that ARD affects fertility and embryonic survival due to defects in oogenic germ cells; however, we were not able to rule out the possibility that sperm quality is impaired under starvation conditions.
To test the effect of ARD exclusively on oogenic germ cells, we used fog-1(q253) and fog-2(q71) mutant animals, which have feminized germlines. Female fog-2(q71) animals are unable to produce sperm and only reproduce when they are crossed with males [[
To confirm our findings we compared the brood size and embryonic lethality of control and recovered fog-1(q253) mutant animals. To do so, a group of synchronized hermaphrodite fog-1(q253) animals were grown from L1-mid-L4 at 25°C and then individually transferred to a plate with bacteria and 4 wild-type males overnight as a control. Another group of fog-1(q253) animals was transferred to plates without bacteria and incubated for 5 days at 25°C. On the fifth day, the animals were recovered for 24 h in a plate with bacteria and then individually transferred to plates with bacteria and crossed with 4 wild-type males overnight. Recovered fog-1(q253) mutant animals showed significantly fewer progeny than control animals (Fig 3A and Table 1). Additionally, recovered fog-1(q253) mutant animals showed higher embryonic lethality (1.7-fold; 2.5 ± 0.1 dead embryos/worm in fog-1 control animals vs. 4.4 ± 0.2 dead embryos/worm in fog-1 recovered animals; Table 1, Fig 3B). Our results demonstrate that ARD affects oogenic germ cells to an extent that could interfere with embryo survival long after starvation has been ended. Since we observed that the effect of ARD was stronger in feminized mutants, we conclude that some genetic backgrounds could be more sensitive than others.
Well-fed virgin fog-2 mutant animals' do not produce sperm; instead, all of their germ cells develop as oocytes, and the most proximal remain arrested in diakinesis within the gonad [[
To continue testing whether the oogenic germline starvation response during ARD exerts a protective effect on germ cells, we compared the quality of germ cells when they were exposed to prolonged arrest in meiosis under well-fed conditions vs. ARD. We crossed well-fed 6-day-old virgin fog-2 and fog-1 mutant animals with well-fed wild-type males and determined their brood size and embryonic lethality, which were compared to those of fog-2(q71) and fog-1(q253) mutant animals crossed under control and recovered conditions. We found that well-fed 6-day-old fog-2 and fog-1 mutant animals produced smaller broods (by 17% and 22%, respectively) than the controls and even smaller broods (by 30.5% and 36.7%, respectively) than animals recovered after 5 days of ARD (Fig 3A, Table 1).
Additionally, well-fed 6-day-old fog-2 and fog-1 mutant animals exhibited higher embryonic lethality than control (by 2- and 2.2-fold, respectively) and recovered animals (1.2- and 1.3-fold, respectively) (Fig 3B and Table 1). We observed that dead embryos were present mainly during the first day in well-fed 6-day-old fog-2 and fog-1 mutant animals (Fig 3C). Similarly, in fog-2 and fog-1 mutant recovered animals, the main peak of dead embryos was observed on the first day after the cross (Fig 3C). Apparently, the dead embryos were produced from the oocytes that were arrested in diakinesis for the longest period during ARD. We observed that embryonic lethality in fog-2(q71) and fog-1(q253) mutant animals was mainly caused by embryos that resulted from the fertilization of the oocytes that were already present or produced during ARD and presumably were arrested for a long period of time. We conclude that because the oogenic germline starvation response during ARD slows oocyte production, oogenic germ cells are prevented from undergoing diakinesis arrest, which preserves oocyte quality. We suggest that this could explain why feminized germline mutant animals are more sensitive to ARD than wild-type animals.
During the course of these experiments, we observed that the gonads of fog-2 and fog-1 animals did not shrink during ARD (Fig 4D and 4F). Our results, as well as those of other similar approaches reported previously [[
Table 2: Germ cell quantification per gonad arm in different genetic backgrounds under control and starvation conditions.
5 Days post mid-L4 mid-L4 N Control N Starvation N WT 141.9 ± 1.8 49 468.2 ± 2.0 38 ** 36.0 ± 0.5 54 ** 20°C fog-2(q71) 143.1 ± 2.0 42 315.9 ± 2.6 47 ** 141.0 ± 1.5 48 ** WT 144.7 ± 2.2 30 386.9 ± 1.6 20 ** 35.5 ± 0.8 24 ** 15°C fog-1(q253) 143.5 ± 1.8 30 386.2 ± 1.6 30 ** 36.0 ± 0.6 25 ** WT 135.3 ± 1.5 20 388.5 ± 1.8 25 ** 34.2 ± 0.7 25 ** 25°C fog-1(q253) 133.5 ± 0.8 30 338.5 ± 1.8 30 ** 141.8 ± 1.6 25 n.s.
5 The gonads of animals under control conditions or starvation were dissected, stained and scored for the number of germ cells using epifluorescence microscopy. Control animals remained on NGM plates seeded with OP50. For starvation conditions, animals were grown on food from L1 to the mid-L4 larval stage and then starved for 5 days. Animals were subsequently picked and dissected. The dissected gonads were stained with DAPI and the number of germ cells per gonad arm was scored under fluorescence microscopy.
Caspase CED-3 is required for germ cell apoptosis under control and starvation conditions [[
To verify that the gonads of ced-3 mutant animals shrink similarly to those of wild-type animals, we quantified the number of germ cells per gonad arm by DAPI staining from the mid-L4 stage over the next 5 days under control and starvation conditions in animals with the wild-type and ced-3(
Table 3: Germ cells per gonad arm under control, starvation and recovered conditions.
WT Days post mid-L4 Control N Starvation N Recovering N 0 150.4 ± 2.5 36 150.4 ± 2.5 36 339.2 ± 3.5 25 1 346.1 ± 2.0 35 95.1 ± 1.0 36 2 431.3 ± 1.9 35 74.5 ± 0.9 36 3 475.6 ± 2.7 25 45.6 ± 0.7 35 4 485.3 ± 1.4 30 35.0 ± 0.6 36 5 463.7 ± 4.7 24 35.5 ± 0.9 35 ced-3(n717) Days post mid-L4 Control N Starvation N Recovering N 0 145.5 ± 4.6 35 145.5 ± 4.6 35 342.9 ± 2.8 30 1 340.9 ± 1.9 36 93.7 ± 1.9 35 2 427.8 ± 1.7 36 78.9 ± 1.2 35 3 475.8 ± 2.7 24 46.1 ± 0.8 35 4 484.6 ± 2.4 25 38.6 ± 0.8 36 5 454.7 ± 3.7 25 38.1 ± 0.8 35 ced-3(n1286) Days post mid-L4 Control N Starvation N Recovering N 0 153.7 ± 2.1 36 153.7 ± 2.1 36 342.6 ± 2.7 30 1 351.8 ± 1.8 25 96.8 ± 1.3 35 2 427.1 ± 3.3 26 78.8 ± 0.9 36 3 471.2 ± 3.9 25 46.1 ± 0.6 35 4 479.7 ± 2.2 25 37.7 ± 1.0 36 5 459.2 ± 2.9 25 36.7 ± 0.8 35
6 The gonads of animals under control conditions, starvation and recovery were dissected, stained and scored for the number of germ cells using epifluorescence microscopy. Control animals were placed in NGM plates seeded with OP50 and transferred to fresh plates daily until they ceased laying eggs. For starvation conditions, animals were placed in NGM plates unseeded and transferred to fresh plates daily, similar to the control animals. Animals were picked and dissected. The dissected gonads were stained with DAPI, and the number of germ cells per gonad arm was scored under fluorescence microscopy. For recovery, animals that had spent 5 days in ARD were transferred to plates seeded with OP50 for 3 days. Animals were then picked and dissected.
Angelo and van Gilst (2009) also reported that ced-3(n1286) caspase mutant animals were unable to produce progeny after 15 days of prolonged starvation and suggested that apoptosis was essential to maintain fertility after fasting [[
We quantified the progeny produced by well-fed and recovered ced-3 animals (alleles n717 and n1286) and compared the number to that in wild-type animals after spending 5 days in ARD. Andux and Ellis (2008) previously reported that ced-3 mutant animals (with the n718, n2439, n2921 alleles) produce fewer progeny and exhibit higher embryonic lethality than wild-type animals in control conditions [[
As reported by Andux and Ellis (2008), the embryonic lethality of ced-3 mutant animals under normal conditions was higher than that of the wild-type (3.9-fold higher than the wild-type for the ced-3(
To counteract the effect of ARD on sperm, we quantified the brood size and embryonic lethality of ced-3(
We investigated germ cell apoptosis dynamics during ARD. To quantify apoptosis, we used a germ cell apoptosis reporter P
Table 4: Germ cell apoptosis dynamics under control and starvation conditions.
ced-1: :gfp Days post mid-L4 Control N Starvation N 0 0.0 ± 0.00 141 0.0 ± 0.01 n.s. 140 1 5.4 ± 0.21 139 8.8 ± 0.20 ** 140 2 11.4 ± 0.26 129 6.5 ± 0.15 ** 141 3 13.6 ± 0.34 111 6.3 ± 0.24 ** 129 4 16.3 ± 0.55 103 5.6 ± 0.18 ** 123 5 13.7 ± 0.40 111 5.2 ± 0.16 ** 143 6 11.6 ± 0.26 121 4.5 ± 0.10 ** 135 7 8.5 ± 0.22 110 4.2 ± 0.11 ** 129 8 8.2 ± 0.25 119 4.1 ± 0.10 ** 144 9 7.9 ± 0.30 76 3.9 ± 0.11 ** 124 10 8.0 ± 0.21 72 3.6 ± 0.08 ** 105
- 8 The number of germ cell corpses was scored using ced-1: :gfp animals under epifluorescence microscopy. Control mid-L4 hermaphrodites were placed in NGM plates seeded with OP50 and transferred to fresh plates daily until they ceased laying eggs; the animals were picked daily for up to 10 days and mounted with 20 μl of 0.01% tetramisole in M9 on 2% agarose pads. For starvation conditions, mid-L4 animals were placed in unseeded NGM plates and transferred to fresh plates daily similar to the control animals; the animals were picked daily for up to 10 days and mounted with 20 μl of 0.01% tetramisole in M9 on 2% agarose pads. n.s. non significant
- 9 * P ≤ 0.05
- 7 ** P ≤ 0.01,.
It is remarkable that germ cell apoptosis continues and is very active during prolonged starvation particularly because germ cell proliferation stops after 30 minutes of fasting [[
Table 5: The number of germ cells per gonad arm under control and starvation conditions.
ced-1: :gfp Days post mid-L4 Control N Starvation N 0 172.2 ± 4.57 45 172.2 ± 4.57 45 1 359.6 ± 21.88 15 138.4 ± 4.72 42 2 406.1 ± 21.23 15 89.4 ± 3.04 30 3 487.3 ± 20.82 17 56.0 ± 1.83 31 4 506.8 ± 10.94 15 48.4 ± 3.02 27 5 505.1 ± 19.53 18 43.6 ± 1.37 35 6 521.3 ± 14.82 18 37.2 ± 1.18 30 7 509.6 ± 11.76 16 37.8 ± 0.75 24 8 545.9 ± 13.44 17 37.8 ± 1.04 38 9 548.6 ± 8.19 17 38.4 ± 1.19 34 10 574.7 ± 6.56 15 38.4 ± 1.19 38
10 The gonads of animals under control conditions and starvation were dissected, stained and scored for the number of germ cells using epifluorescence microscopy. Control animals were placed in NGM plates seeded with OP50 and transferred to fresh plates daily until they ceased laying eggs. For starvation conditions, animals were placed in unseeded NGM plates and transferred to fresh plates daily, similar to the control animals. Animals were picked and dissected. The dissected gonads were stained with DAPI and the number of germ cells per gonad arm was scored under fluorescence microscopy.
We quantified the percentage of germ cells eliminated by apoptosis (Table 4) and compared it to the number of germ cells present in the gonad (Table 5) under control conditions and during starvation. We found that under control conditions, approximately 1.91% of the total number of germ cells appeared as germ cell corpses at each time point. However, during starvation the percentage of germ cell corpses was considerably higher at each point compared to the control conditions (average of 9.27% of the total number of germ cells) (Fig 7C). Our data show that germ cell apoptosis increases up to 4.8-fold during the oogenic germline starvation response and continues under these conditions.
To test whether germ cell corpse clearance is affected during starvation, we quantified germ cell corpse engulfment in animals exposed to 1–5 days of starvation by time-lapse microscopy (see methods). We found that the timing of germ cell corpse clearance was not extended during the first 5 days of prolonged starvation compared to the control (approx. 55 minutes (prolonged starvation), similar to the control time of 58 minutes) (Fig 7D, Table 6). Our results demonstrate that the process of germ cell corpse engulfment remains active during prolonged starvation.
Table 6: Germ cell corpse clearance under control and starvation conditions.
WT Days post mid-L4 Control N Starvation N 0 No corpses detected 25 No corpses detected 25 1 60.7 ± 1.51 9 54.6 ± 1.47 * 9 2 57.4 ± 1.89 9 58.7 ± 1.74 n.s. 9 3 57.3 ± 2.63 9 52.9 ± 1.61 * 9 4 59.4 ± 1.74 8 55.6 ± 2.15 n.s. 8 5 59.9 ± 1.97 8 52.0 ± 2.50 * 8
- 12 The timing of germ cell corpse clearance under control and starvation conditions was scored using a long-term immobilization technique and Nomarski microscopy at 60x magnification. Control mid-L4 hermaphrodites were placed in NGM plates seeded with OP50, picked daily for up to 5 days and immobilized with 0.5 μl of a polystyrene bead suspension on 10% agarose pads to observe an individual germ cell corpse and determine its clearance time in minutes. For starvation conditions, mid-L4 hermaphrodites were placed in unseeded NGM plates, picked daily for up to 5 days and then immobilized with 0.5 μl of a polystyrene bead suspension on 10% agarose pads to observe an individual germ cell corpse and determine its clearance time in minutes. The Holm-Sidak test for comparisons within groups was used. n.s. = non significant
- 11 * P < 0.05.
Germ cell apoptosis can be triggered by DNA damage or meiosis defects via CEP-1, the C. elegans p53 homologue [[
In our previous work, we found that lin-35/Rb is essential for inducing germ cell apoptosis during starvation [[
In an attempt to reveal the molecular mechanisms that govern the oogenic germline starvation response, we subjected mid-L4 hermaphrodites of different genetic backgrounds to starvation for 5 days, followed by refeeding for 3 days, to test the capacity of their gonad to shrink and regenerate. We chose the gene candidates based on their known roles in the stress response or in germ cell proliferation. We tested the C. elegans retinoblastoma ortholog lin-35/Rb because it is essential for inducing apoptosis in short-term starvation [[
Table 7: The oogenic germline starvation response in different genetic backgrounds.
Genotype Relevant function for this study References Human ortholog Starvation response Re-feeding WT Control [7, 8] - Shrunken gonad Completely recovered ced-3(n1286) The only effector caspase [7, 20] Isoform 1 Caspase-2 Shrunken gonad Completely recovered Not required Not required ced-3(n717) The only effector caspase [7, 20] Isoform 1 Caspase-2 Not required Not required ced-3(ok2734) The only effector caspase Isoform 1 Caspase-2 Not required Not required lin-35(n745) Causes starvation-induced germline apoptosis [26] Rb Not required Not required daf-16(mgDf50) Regulates dauer diapause [28] FoxO Not required Not required skn-1(zu135) Dietary restriction and other stresses response [30] Nrf Not required Not required pha-4(zu225) Dietary restriction and other stresses response [30] FoxA Not required Not required alg-1(gk214) miRNA pathway and proliferation [13] Argonaut Not required Not required rsks-1(ok1255) Increases resistance to starvation [31] Putative ribosomal protein S6K Not required Not required ife-1(bn127) Regulates germline progenitor number [32] eIF4e Not required Not required gla-3(op212) Increased germline apoptosis [33] - Not required Not required cep-1(gk138);ced-1: :gfp Regulates DNA-damage-induced germline apoptosis [34] p53 transcription factor Not required Not required
13 Hermaphrodites from the different backgrounds were selected at the mid L4, starved for 5 days and observed daily under Nomarski microscopy to compare their gonad size to that under the oogenic germline starvation response using WT hermaphrodites as a control. At day 5, the hermaphrodites were refed for 3 days and observed daily under Nomarski microscopy to determine their gonad recovery, using WT hermaphrodites as a control.
The insulin/insulin receptor signaling (IIS) pathway has been established as a link between nutrient availability and homeostasis in species ranging from worms to humans. This pathway is maintained through the activity of insulin-like molecules that bind to the insulin receptor. In C. elegans, more than 40 peptides related to insulin bind to the insulin receptor/DAF-2 to negatively regulate the DAF-16/FoxO transcription factor. Thus, mutations in the insulin receptor/DAF-2 promote the transcription of target genes through the activity of DAF-16/FoxO to cope with nutrient scarcity and other types of stress [[
To test the role of daf-2 in the oogenic germline starvation response, we used the temperature-sensitive mutant daf-2(e1370) [[
Unexpectedly, we observed that the well-fed control mid-L4 daf-2 mutant animals grown at the restrictive temperature for 5 days exhibited shrunken gonads in the presence of food (Fig 9F) when their gonads were compared to those of the wild-type animals (Fig 9C). We decided to quantify the germ cell number per gonad arm at the mid-L4 stage and 5 days after IIS pathway inactivation in the presence of food using daf-2(e1370) mutant animals and the wild-type as a control. We found that in wild-type animals, the germ cell number increased from 140.6 ± 1.2 at mid-L4 to 380.8 ± 3.4 5 days post-mid-L4. However, daf-2(e1370) mutant animals did not show the same increase; instead, the number of germ cell nuclei per gonad arm was reduced from 137.6 ± 1.0 at mid-L4 to 77.2 ± 1.5 5 days post-mid-L4 (Fig 9I and Table 8).
Table 8: Quantification of germ cell number per gonad arm in different genetic backgrounds on food.
5 Days post mid-L4 mid-L4 N 15°C N 25°C N WT 140.6 ± 1.2 37 339.0 ± 2.0 35 380.8 ± 3.4 20 daf-2(e1370) 137.6 ± 1.0 42 337.0 ± 1.4 44 77.2 ± 1.5 41 daf-16(m26);daf-2(e1370) 136.4 ± 0.7 43 342.3 ± 1.7 43 382.2 ± 2.5 22
14 The gonads of animals were dissected, stained and scored for the number of germ cells using epifluorescence microscopy. Animals were grown in NGM plates seeded with OP50 from hatching to mid-L4 at 15°C and then incubated on food at 15°C or 25°C for 5 days. Animals were picked and dissected. The dissected gonads were stained with DAPI and the number of germ cells per gonad arm was scored under fluorescence microscopy.
The transcription factor daf-16/FoxO is activated when the daf-2 signal is absent [[
To test whether IIS pathway inactivation could mimic entry to ARD even in the presence of food, we determined the ovulation rate in 1-day-old wild-type and daf-2(e1370) and daf-16(m26);daf-2(e1370) mutant animals grown from L1 to mid-L4 at 15°C that were late upshifted to 25°C in the presence of food. It has been reported that ARD causes a delay in the ovulation rate [[
Another feature of the oogenic germline starvation response is that the ovulation rate is limited by the rate of oocyte growth [[
Angelo and van Gilst (2009) reported a novel type of adult reproductive diapause for the first time while studying reproduction when nutrients are limited. They showed that crowded populations of mid-L4 larval stage animals subjected to starvation delay their reproductive cycle and are able to live for up to 30 days without food. Furthermore, when conditions are restored, these animals resume their reproductive cycle and live out a normal lifespan. During ARD, the gonad experiences reversible germ cell loss and size reduction; when animals are returned to food, the gonad regenerates in terms of both germ cell number and size [[
Later, Seidel and Kimble (2011) studied ARD in depth. They showed that germline shrinkage occurs in all oogenic germlines, from the mid-L4 to adult stages, and practically all shrunken gonads regenerate upon refeeding if bagging is prevented. Seidel and Kimble (2011) showed that embryo production continues during prolonged starvation, and it is possible to distinguish one or two viable-looking embryos within the uterus, which are a product of recent fertilizations, indicating that oocyte production and fertilization are active but considerably delayed in starved animals; however, embryo viability is severely impaired during starvation. They also showed that population density is not a prerequisite for inducing or maintaining the starvation response in starved animals and suggested that this phenomenon is not a form of diapause [[
Despite not supporting the existence of the ARD proposed by Angelo and van Gilst (2009), Seidel and Kimble (2011) coined the term "oogenic germline starvation response" referring to the germline plasticity controlled by nutritional cues [[
Our results support the following conclusions: Long-term exposure to fasting compromises the animals' reproductive capacity, and the oogenic germline starvation response prevents oogenic germ cells from undergoing prolonged arrest in diakinesis by slowing oocyte production. In contrast to Angelo and van Gilst (2009), we demonstrated that apoptosis is not the cause of gonad shrinkage during ARD and that it is not essential for fertility recovery after ARD. Instead, we propose that once germ cell proliferation stops, ovulation contributes to gonad shrinking during the oogenic germline starvation response. Germ cell apoptosis is increased during ARD and continues during prolonged starvation. Germ cell apoptosis during ARD is not induced by DNA damage or chromosome segregation errors during meiosis but is partially regulated by lin-35/Rb. We also found that during ARD an unknown mechanism triggers germ cell apoptosis. Finally, we suggest that inactivation of the IIS signaling pathway triggers gonad shrinking in the presence of food that might resemble the oogenic germline response.
In the present work, we showed that fertility is affected after ARD (Figs 2, 3 and 6). Although recovered animals exhibit restoration of fertility upon refeeding, none of the genetic backgrounds tested (N2, fog-1, fog-2 and ced-3) produced broods that were as large as those of the controls by self-fertilization or mating. Angelo and van Gilst (2009) proposed that the fecundity of self-fertilizing animals depended on the survival of functional sperm during starvation [[
Several authors have used embryonic lethality to reflect oocyte quality [[
In the C. elegans germline, the presence of food and sperm promotes meiotic progression and oogenesis. Sperm depletion causes prolonged diakinesis arrest in the proximal gonad due to oocyte stacking, and starvation halts meiotic progression in the pachytene region [[
In this study, we demonstrated that ced-3 mutant animals respond to starvation by shrinking their gonads during ARD and partially recover their fertility upon refeeding (Figs 5 and 6). In contrast to Angelo and van Gilst (2009), who reported that apoptosis-deficient ced-3(n1286) animals subjected to 15 days of starvation did not show loss of germ cell nuclei or gonad shrinkage, and did not produce progeny after ARD [[
As previously reported [[
Our work shows that apoptosis is increased during prolonged starvation and continues to be high during ARD. Germ cell apoptosis is required during oogenesis for properly allocating resources within the gonad and ensuring oocyte quality [[
Furthermore, germ cell apoptosis can be triggered when animals face different types of stress [[
Why is germ cell apoptosis elevated during ARD? Our results support the notion proposed by Andux and Ellis (2008) that germ cell death serves as a mechanism to redistribute resources in the gonad [[
We sought to investigate the molecular mechanism that governs the oogenic germline starvation response during ARD. We tested some genes involved in the regulation of dauer diapause, L1 arrest and other genes implicated in germ cell apoptosis and proliferation and demonstrated that only IIS in the mid-L4 larval stage caused a reduction in gonad size even in the presence of food (Table 8 and Fig 9). We propose that IIS pathway inactivation might trigger a response that mimics the entry into ARD in well-fed conditions, since animals show a reduced gonad size (Fig 9). Similarly, daf-2 animals slow ovulation when they reduced their gonad size suggesting that daf-2 might participate in ARD signaling; however, the shrunken gonads of daf-2 animals do not form one single oocyte at a time as that of the ARD gonads, and daf-2 is not essential for the gonad shrinking during ARD. Although our results provide some evidence of the pathway that governs the oogenic germline starvation response, further information is still needed for a better understanding of the precise molecular mechanism that controls reproduction under starvation conditions.
DIAGRAM: Fig 1: Comparison between well-fed and starved adult hermaphrodite gonad arms in Caenorhabditis elegans . Schematic representation of well-fed (A) and starved (B) adult hermaphrodites. (C, E) Nomarski image of mid-L4 gonad arms. (D) Nomarski image of a hermaphrodite that was well-fed for 3 days. (F) Nomarski image of a hermaphrodite that was starved for 3 days from mid-L4. In all images one gonad arm is outlined in white; the distal gonad is marked with an asterisk (*); and the arrow points to the proximal gonad. Scale bar = 20 μm.
DIAGRAM: Fig 2: Animals subjected to ARD do not recover their fertility due to defects in germ cells. (A) The graph represents the brood size produced by self-fertilizing control (black) and recovered (red) wild-type animals. Mid-L4 hermaphrodites were allowed to self-fertilize (black) or were starved for 5 days and then refed (red). The data represent the mean brood size (±SEM) per animal. Statistical significance was determined by the Student's t-test (P ≤ 0.001). (B) Quantification of the progeny produced on each day by self-fertilizing control (black line) and recovered (red line) wild-type animals. Time-course data are displayed as the mean (±SEM) number of progeny per time point. (C) Quantification of sperm produced by wild-type animals under control conditions (black line) or subjected to ARD (red line) at several time points: the late-L4 larval stage and 1, 3 and 5 days after the mid-L4 larval stage. The data are displayed as the mean (±SEM) number of sperm per time point. (D) The number of dead embryos within the progeny of self-fertilizing wild-type animals under control (black) and recovered conditions (red) was calculated. Data represent the mean number of dead embryos (±SEM) per animal. Statistical significance was determined by the Mann-Whitney rank sum test (P ≤ 0.001). (E) The graph represents the brood size produced by mating control (black) and recovered (red) wild-type animals. Well-fed mid-L4 hermaphrodites were individually mated to 4 well-fed wild-type males overnight and were then transferred individually to fresh plates until they ceased laying eggs (black). For the recovered animals, mid-L4 hermaphrodites were starved for 5 days, then recovered on food and mated to 4 well-fed 1-day-old wild-type males overnight, then transferred individually to fresh plates until they ceased laying eggs (red). The data represent the mean brood size (±SEM) per animal. Statistical significance was determined by the Mann-Whitney rank sum test (P ≤ 0.024). (F) The number of dead embryos within the progeny produced by mating wild-type animals under control (black) and recovered conditions (red) to well-fed wild-type males was calculated. Data represent the mean number of dead embryos (±SEM) per animal. Statistical significance was determined by the Mann-Whitney rank sum test, and the difference was not significant (n.s.).
DIAGRAM: Fig 3: Germlines from feminized mutant backgrounds are more sensitive to ARD that those of wild-type animals. (A) The graph represents the brood size produced by mating virgin fog-2(q71) and fog-1(q253) mutant animals to well-fed wild-type males. Mid-L4 virgin animals from different genetic backgrounds were mated to 4 well-fed wild-type males overnight, then transferred individually to fresh plates until they ceased laying eggs (black). Mid-L4 virgin mutant animals were starved for 5 days, recovered on food for 24 h and then mated with 4 well-fed 1-day-old wild-type males overnight, after which they were transferred individually to fresh plates until they ceased laying eggs (red). Well-fed 6-day-old virgin mutant animals, which exhibited stacked oocytes arrested in prolonged diakinesis within the gonad, were mated with 4 well-fed 1-day-old wild-type males overnight and then transferred individually to fresh plates until they ceased laying eggs (green). The data represent the mean brood size (±SEM) per animal. Statistical significance was determined by one-way ANOVA on ranks, followed by Dunn's test (P<0.05). (B) The graph shows the number of dead embryos within the progeny produced by mating with well-fed wild-type males under control conditions (black), recovered conditions (red) and prolonged arrest in diakinesis (green). The data represent the mean number of dead embryos (±SEM) per animal. Statistical significance was determined by one-way ANOVA on ranks, followed by Dunn's test (P<0.05). (C) Quantification of dead embryos within the progeny produced each day by mating with wild-type males under control (black line) and recovered (red line) conditions and prolonged arrest in diakinesis (green line). Time course data are displayed as the mean (±SEM) number of dead embryos per time point. (D) Number of germ cells per gonad arm in wild-type and fog-2 (q71) and fog-1(q253) mutant animals. The graph represents the average number of germ cells scored using DAPI staining in dissected gonads during the mid-L4 stage (0) and 5 days after the mid-L4 stage under starvation conditions.
DIAGRAM: Fig 4: The gonads of feminized germline mutant animals do not shrink during ARD. Nomarski gonad images of the indicated genetic backgrounds and conditions. (A) Representative Nomarski image of a well-fed 5-day-old wild-type hermaphrodite. (B) Nomarski image of a wild-type hermaphrodite that had spent 5 days in ARD. (C) A well-fed 5-day-old virgin fog-2(q71) mutant animal. (D) A virgin fog-2(q71) animal that had been starved for 5 days. (E) A well-fed 5-day-old virgin fog-1(q253) mutant animal. (F) A virgin fog-1(q253) animal that had been starved for 5 days. In all images one gonad arm and the oocytes within it are outlined in white; the distal gonad is marked with an asterisk (*); the arrow points to the proximal gonad. Scale bar = 20 μm.
DIAGRAM: Fig 5: Apoptosis is not required to reduce gonad size during the oogenic germline starvation response. Nomarski images of gonads of mid-L4 wild-type animal and animals with the three different alleles of ced-3 (A, D, G and J). Nomarski images of gonads of wild-type and ced-3 mutant animals starved for 5 days (B, E, H and K). Nomarski images of gonads of wild-type and ced-3 animals that spent 5 days under starvation and were recovered on food for 3 days (C, F, I and L). In all images one gonad arm is outlined in white; the distal gonad is marked with an asterisk (*); the arrow points to the proximal gonad. Scale bar = 20 μm. (M) Germ cell nuclei per gonad arm scored in wild-type animals and two ced-3 defective mutants by DAPI staining. The graph shows the average number of germ cell nuclei per gonad arm in control conditions (solid lines) and during starvation (dotted lines) ±SEM at each point.
DIAGRAM: Fig 6: Apoptosis is as important to preserve oocyte quality after ARD as in control conditions but it is not essential for recovering fertility after ARD. (A) The graph represents the brood size produced by control (black) and recovered (red) wild-type and ced-3 mutant animals. Mid-L4 hermaphrodites of the different genetic backgrounds were allowed to self-fertilize (black) or were starved for 5 days and then refed (red). The data represent the mean brood size (±SEM) per animal. Statistical significance was determined by the t-test (WT control vs. recovered) or by ANOVA on ranks and Dunn's test for multiple comparisons. (B) The number of dead embryos within the progeny of self-fertilizing wild-type and ced-3 mutant animals under control (black) and recovered conditions (red) was calculated. The data represent the mean number of dead embryos (±SEM) per animal. Statistical significance was determined by the Mann-Whitney rank sum test (P ≤ 0.001) (WT control vs. recovered) or by ANOVA on ranks and Dunn's test for multiple comparisons. (C) The graph represents the brood size produced by mating control (black) and recovered (red) animals of the different genetic backgrounds. Well-fed mid-L4 hermaphrodites were individually mated with 4 well-fed wild-type males overnight, then transferred individually to fresh plates until they ceased laying eggs (black). For the recovered animals, mid-L4 hermaphrodites were starved for 5 days, then recovered on food and immediately mated with 4 well-fed 1-day-old wild-type males overnight and transferred individually to fresh plates until they ceased laying eggs (red). The data represent the mean brood size (±SEM) per animal. Statistical significance was determined by the Mann-Whitney rank sum test (P ≤ 0.024) in wild-type populations and by the Student's t-test (P ≤ 0.001) in ced-3(
DIAGRAM: Fig 7: Germ cell apoptosis is increased and continues during ARD. (A) The ced-1: :gfp transgene was used to visualize the number of germ cell corpses per gonad arm (see ) for 10 days in control (black line) and starvation conditions (red line). The average number of germ cell corpses per gonad arm is shown with SEM. (B) DAPI staining was used to quantify the number of germ cells per gonad arm (see ) for 10 days in control (black line) and starvation (red line) conditions. The average number of germ cells per gonad arm is shown with SEM. (C) The percentage of germ cell corpses among the total germ cells per gonad arm in fed animals (black line) and during starvation (red line) was calculated. (D) The time of germ cell corpse clearance was recorded every day after 1–5 days post-mid-L4 (see ) in control (black bars) and starvation (red bars) conditions.
DIAGRAM: Fig 8: Germ cell apoptosis is p53 independent and partially regulated by lin-35/Rb during ARD. (A-B) cep-1(gk38);ced-1: :gfp, lin-35(
DIAGRAM: Fig 9: Long-term IIS pathway inactivation mimics the entry to ARD in the presence of food. Mid-L4 wild-type, daf-2(e1370) and daf-16(m26); daf-2(e1370) larvae were fed until mid-L4 at 15°C (A, D and G, respectively). Wild-type and daf-2(e1370) mid-L4 larvae starved for 5 days at 25°C (B, E). Nomarski images of well-fed wild-type, daf-2(e1370) and daf-16(m26); daf-2(e1370) animals grown until mid-L4 at 15°C, then upshifted to 25°C for 5 days (C, F and H). In all images one gonad arm is outlined in white; the distal gonad is marked with an asterisk (*); and the arrow points to the proximal gonad. Scale bar = 20 μm. (I) Quantification of the number of germ cell nuclei per gonad arm during prolonged IIS pathway inactivation. Animals were grown from L1 to the mid-L4 stage at the permissive temperature and then upshifted to the restrictive temperature for 5 days. Dissected gonads were stained using DAPI in each condition. The graph shows the average number of germ cell nuclei per gonad arm (±SEM). (J) Ovulation rate determination during IIS pathway inactivation. Animals were grown from L1 to the mid-L4 stage at the permissive temperature and then either upshifted to the restrictive temperature or maintained in the permissive temperature for 1 day. Animals were individually picked and scored for the number of embryos in utero, then placed in plates for 4 h and finally scored for the number of embryos in utero. The ovulation rate was calculated as follows: (final number of embryos—initial number) / (2 * 4 h). The graph shows the average number of ovulations per gonad arm per hour (±SEM).
We thank the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440) for kindly providing the strains used in this work. We thank WormBase release WS224 for gathering information and making it available. We also thank Laura Silvia Salinas for technical assistance, members of the Navarro Lab and Dr. Jesús Chimal from the Instituto de Investigaciones Biomédicas-UNAM and Dr. Wilhem Hansberg and Dr. Félix Recillas from the Instituto de Fisiología Celular-UNAM for helpful discussion and comments on this project. Emilio Carranza-García received a CONACyT doctoral fellowship (361899) and data in this work are part of his doctoral dissertation in the Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México.
By E. Carranza-García, Writing – review & editing and R. E. Navarro, Writing – review & editing