Methionine Supplementation Alleviates the Germ Cell Apoptosis Increased by Maternal Caffeine Intake in a C. elegans Model

Caffeine (1,3,7-trimethylxanthine) is a widely consumed bioactive substance worldwide. Our recent study showed that a reduction in both reproduction and yolk protein production (vitellogenesis) caused by caffeine intake were improved by vitamin B12 supplementation, which is an essential co-factor in methionine metabolism. In the current study, we investigated the role of methionine in the reproduction of caffeine-ingested animals (CIAs). We assessed the effect of methionine metabolism on CIAs and found that caffeine intake decreased both methionine levels and essential enzymes related to the methionine cycle. Furthermore, we found that the caffeine-induced impairment of methionine metabolism decreased vitellogenesis and increased germ cell apoptosis in an LIN-35/RB-dependent manner. Interestingly, the increased germ cell apoptosis was restored to normal levels by methionine supplementation in CIAs. These results indicate that methionine supplementation plays a beneficial role in germ cell health and offspring development by regulating vitellogenesis.


Introduction
Caffeine is the most popularly consumed psychoactive stimulant in a variety of foods such as tea, coffee, soda, and some drugs [1].Its neurological effects are well known from in vivo studies of caffeine consumption.It acts as an antagonist of A1 and A2 adenosine receptors; affects the central nervous system; increases adrenaline, dopamine, and noradrenaline; alters fat and carbohydrate metabolism by mobilizing blood circulation and intracellular calcium stores; and stimulates lipolysis in humans [2][3][4].In addition, 400 mg of caffeine a day appears to be generally safe, enhances physical performance, and confers health benefits, with improvement of chronic liver disease, Type 2 diabetes, Parkinson's disease, and Alzheimer's disease [5][6][7].However, there are growing concerns about caffeine consumption in pregnant women, owing to the increased risk of pregnancyrelated adverse effects, including low birth weight and pregnancy loss [8,9].However, studies on the effects of maternal caffeine intake are limited.
Methionine is necessary for cell growth and differentiation as an essential amino acid and functions as an initiator of protein synthesis across species [10].It also plays major roles in metabolism, including the methionine cycle, transsulfuration pathway, and salvage cycle, which are well conserved from Escherichia coli to humans [10].The first step in methionine metabolism is the methionine cycle, which is regulated by S-adenosylmethionine synthetase (sams-1), which leads to the biosynthesis of S-adenosylmethionine (SAM), a major methyl donor, from methionine (Figure 1A).The Met/SAM cycle is involved in the one-carbon metabolism and produces many important building blocks for the maintenance of the body [11].Methionine deficiency induces renal apoptosis and cell cycle arrest [12].It has the one-carbon metabolism and produces many important building blocks for the maintenance of the body [11].Methionine deficiency induces renal apoptosis and cell cycle arrest [12].It has also been reported that methionine supplementation improves reproduction, egg quality, antioxidant status, and the immune response [13].Therefore, the balance of methionine composition in the body is important for maintaining healthy reproduction.
C. elegans has been used as the most powerful model organism to research the relationships between diet, metabolism, and reproduction [14,15].Adult hermaphrodite gonads consist of two gonad arms, which are U-shaped and connected to the uterus.Approximately half of the germ cells die to support the development of oocytes, the so-called physiological germ cell apoptosis, during oogenesis at the adult stage [16,17].Germ cell apoptosis is induced by the core apoptotic machinery, including anti-apoptotic protein CED-9/BCL-2 [16], caspase activator CED-4/APAF-1 [18], and the caspase CED-3/Caspase [18] (Figure 1B).The germ cell apoptosis physiologically induced by LIN-35/RB (tumor suppressor retinoblastoma) occurs in response to starvation stress, the RAS/MAPK signaling pathway, or E2F components [16,17,19].Meanwhile, the induction of an increase in apoptosis in the germline via DNA damage is mediated by CEP-1, which is thought to be mammalian p53 [20] (Figure 1B).Germ cell apoptosis is important for maintaining germ cell health, oocyte quality, and offspring viability [21,22].Thus, the question of how germ cell apoptosis is regulated by metabolic variation, specifically maternal diet, to maintain germ cell quality and offspring development was investigated in this study, using a caffeine-ingested C. elegans model.The examined enzymes of the methionine cycle (A), core apoptotic genes (CED-9, CED-4, and CED-3), and its related genes (B) are indicated in blue.The scheme is modified from [17].

Germ Cell Apoptosis Assay
Germ cell apoptosis was measured either using the reporter CED-1::GFP for engulfed apoptotic nuclei or by counting germ cell corpses, as previously described [30,31].We used two methods to measure germ cell death for following reasons: (1) measuring the number of CED-1-positive germ cells using the CED-1::GFP transgenic animals to observe the engulfing cell during apoptosis and (2) measuring the number of cell corpses in ced-1 mutant background, an engulfment defective mutant to check whether caffeine intake affects apoptosis through a defect in the engulfment pathway.The synchronized L4-stage animals were exposed to 10 mM of caffeine for 24 h at 20 • C and the adult hermaphrodites were prepared for live imaging with 10 mM levamisole in M9 buffer.The animals were mounted on a 4% agarose pad with a cover slip and images were captured using a fluorescence microscope (Zeiss Axioscope, Oberkochen, Germany).One gonad arm was measured per animal.Fifteen to twenty animals were examined in each condition.

RNA Interference (RNAi) Assay
The soaking RNAi method was performed as described previously [32].The dsRNAs of cep-1, mpk-1, lin-35, dpl-1, sams-1, metr-1, mtrr-1, and vit-2 genes were synthesized in vitro using the respective cDNA template.The synchronized L4-stage animals were soaked in dsRNA solution for 24 h at 20 • C, transferred onto either caffeine-containing or caffeine-free NGM agar plates, and incubated for 24 h at 20 • C. Table 2 shows the primers used for the RNAi assay.

Western Blot Analysis
Approximately 100 adult animals for each condition, with three independent sets of experiments, were prepared and a total of 300 adult animals were used for whole-worm protein extraction for Western blotting.The ECL Western blotting detection kit (Amersham, Pittsburgh, PA, USA) was used for visualization and the LAS-300 (Fuji Film, Tokyo, Japan) was used for quantification.Table 3 shows the antibodies used in this study.Live animals in each condition were observed and imaged as previously described [24].In brief, GFP-tagged fluorescent proteins were visualized in living worms by mounting them on 2% agarose pads with M9 buffer with 10 mM levamisole.Live fluorescent imaging was performed using a Zeiss microscope at 20× magnification.ImageJ software (Version 1.54c) was used for the quantification of fluorescence intensity.

Observation of Developmental Stages of Animals
To observe the growth rate of the offspring, the percentage of larval development was determined as the percentage of larvae of the total number of hatched embryos that reached each developmental stage, as previously described [33].We observed the characteristics of each developmental stage, as shown in Table 4.

Statistical Analysis
GraphPad Prism 10.2.1 software was applied for statistical analyses and data graphs.The scatter dot plots with bars displays the standard deviation from the mean value.n values for each experiment are denoted in the corresponding figure legends.p-values were calculated using either Student's t-test (between the two groups) or a one-way ANOVA test (between more than two groups) for the statistical evaluation of the data.We considered p values lower than 0.05 to be statistically significant.

Maternal Caffeine Intake Causes Alterations in Methionine Metabolism in C. elegans
We previously showed that the decrease in reproduction and vitellogenesis caused by caffeine intake was improved by vitamin B12 supplementation, which acts as a co-factor in the Met/SAM cycle in C. elegans [34].Considering the important role of methionine in cellular metabolism, we investigated its role in reproduction in caffeine-ingested animals (CIAs).We performed GC-TOF-MS chromatography to analyze the changes in methionine levels due to caffeine intake.The level of methionine in CIAs was significantly decreased by approximately 2-fold (0.408) compared to that in the caffeine (−) group (Figure 2A).This result indicates that caffeine intake causes a reduction in methionine levels in C. elegans.Given the decrease in methionine level due to caffeine intake, we investigated whether the methionine metabolic genes are affected by caffeine intake through measuring the mRNA level of sams-1 (S-adenosyl methionine synthetase), metr-1 (5-methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase)), mtrr-1 (methyltransferase reductase (methionine synthase reductase)), mel-32 (serine hydroxymethyltransferase), ahcy-1 (S-adenosylhomocysteine hydrolase), and mthf-1 (methylenetetrahydrofolate reductase) genes in CIA using qRT-PCR (Figures 1A and 2B).We found that the mRNA levels of sams-1, metr-1, and mtrr-1 genes significantly decreased in CIAs, while the mRNA levels of mel-32, ahcy-1, and mthf-1 genes were not significantly altered (Figure 1B).These results suggest that maternal caffeine intake causes a reduction in methionine levels by lowering the mRNA levels of mtrr-1, metr-1, and sams-1 methionine cycle genes.ure 2A).This result indicates that caffeine intake causes a reduction in methionine l in C. elegans.Given the decrease in methionine level due to caffeine intake, we investi whether the methionine metabolic genes are affected by caffeine intake through me ing the mRNA level of sams-1 (S-adenosyl methionine synthetase), metr-1 (5-methyl hydrofolate-homocysteine methyltransferase (methionine synthase)), mtrr-1 (me transferase reductase (methionine synthase reductase)), mel-32 (serine hydroxyme transferase), ahcy-1 (S-adenosylhomocysteine hydrolase), and mthf-1 (methylenetet drofolate reductase) genes in CIA using qRT-PCR (Figures 1A and 2B).We found th mRNA levels of sams-1, metr-1, and mtrr-1 genes significantly decreased in CIAs, the mRNA levels of mel-32, ahcy-1, and mthf-1 genes were not significantly altered (F 1B).These results suggest that maternal caffeine intake causes a reduction in methio levels by lowering the mRNA levels of mtrr-1, metr-1, and sams-1 methionine cycle g

Caffeine-Induced Impaired Methionine Cycle Increases Germ Cell Apoptosis in an LIN-35/RB-Dependent Manner
Based on the decreased amount of methionine caused by caffeine intake, we investigated whether caffeine intake affects germ cell apoptosis or proliferation due to the caffeine-induced impairment of the methionine cycle.We observed the level of engulfment using the transgenic animals carrying the transgene bcIs39 [Plim-7::ced-1::gfp], which expresses GFP-tagged CED-1 in gonadal sheath cells [31].The level of engulfment was higher in CIAs than in the caffeine-free diet group and a noticeable increase was observed after 24 h of caffeine treatment (Figure 3A).We also scored the germ cell corpses in CIAs and confirmed that CIA gonads contained more germ cell corpses than the caffeine-free diet group at 24 h post-caffeine treatment (Figure 3B), indicating that caffeine intake increased the level of germ cell apoptosis, possibly through an altered methionine cycle.Next, we examined the effects of caffeine intake on germ cell proliferation.CIAs were immunostained with anti-PH3 (late G2 and m-phase markers) to examine mitotic germ cells.The number of PH3-positive germ cells in CIAs was not significantly altered compared with that in the control group (Figure 3C).These findings suggest that caffeine intake mainly affects germ cell apoptosis but not cell proliferation.To confirm that the increased level of germ cell apoptosis caused by caffeine intake was due to activation of the core apoptotic machinery, we performed ced-4 and ced-3 RNAi depletion.Caffeine treatment did not increase the number of apoptotic germ cells in the depletion of both ced-4 and ced-3 (Figure 3D).Western blot analysis was performed using anti-CED-4 and anti-CED-9 antibodies.CIAs contained higher levels of CED-4 and lower levels of CED-9 than the control group (Figure 3E).These results demonstrate that caffeine intake significantly increases germ cell apoptosis by inactivating CED-9, thereby activating CED-4, a core apoptotic machinery protein.
Next, we examined the pathways involved in caffeine-induced germ cell apoptosis.As shown in Figure 1B, the core apoptotic machinery is regulated by several pathways, including the activity of cep-1 in response to DNA damage, mpk-1 in response to physiological cytoplasmic stress, lin-35 in response to physiological condition/starvation, and dpl-1, which regulates CED-4.Each pathway was examined using the RNAi depletion of each gene in CIAs.We found that germ cell apoptosis increased with the depletion of either cep-1 or mpk-1 RNAi (Figure 3F).In contrast, the RNAi depletion of lin-35 and dpl-1 did not result in a significant increase in germ cell apoptosis after caffeine intake (Figure 3F).These results suggest that lin-35 and dpl-1, but not cep-1 and mpk-1, are required to increase the germ cell apoptosis induced by caffeine intake.We further confirmed that the lin-35 mRNA level, but not dpl-1, was increased by caffeine intake (Figure 3G,H).Our results indicate that caffeine intake increases germ cell apoptosis in an LIN-35/RB-dependent manner.

Methionine Metabolism Is Involved in Physiological Germ Cell Apoptosis and Vitellogenesis
Next, we confirmed whether the activity of genes essential for the methionine cycle affects germ cell apoptosis by RNAi depletion of sams-1, metr-1, and mtrr-1, which showed reduced mRNA transcript levels after caffeine intake (Figure 2B).We found that the RNAi depletion of sams-1, metr-1, or mtrr-1 increased germ cell apoptosis (Figure 4A), suggesting that methionine metabolism is involved in physiological germ cell apoptosis.Next, we investigated whether vitellogenesis is affected by alterations in methionine metabolism in CIAs.We measured the level of vitellogenesis in transgenic animals carrying an integrated VIT-2::GFP transgene after the simultaneous depletion of sams-1, metr-1, or mtrr-1 to suppress methionine metabolism.As reflected in the germ cell apoptosis phenotype due to impaired methionine metabolism, we observed that vitellogenesis was also significantly reduced by the RNAi depletion of sams-1, metr-1, and mtrr-1 compared to the control group (Figure 4B).Furthermore, when CED-1::GFP transgenic animals with vit-2 RNAi depletion were examined, the number of apoptotic germ cells significantly increased.However, the number of apoptotic germ cells in CIAs with vit-2 RNAi depletion was comparable to that in the control group in CIAs (Figure 4C).These results suggest that vitellogenesis also affects physiological germ cell apoptosis and that caffeine intake and vitellogenesis share the same pathway to increase germ cell apoptosis.In a previous study, caffeine intake reduces vitellogenesis [24].We found that caffeine intake increases lin-35 activity and the same pathway as vitellogenesis to induce germ cell apoptosis.To examine the mutual effects of vitellogenesis and lin-35 activity on germ cell apoptosis induced by caffeine intake, we observed vitellogenesis expression with lin-35 RNAi in CIAs.We found that the RNAi depletion of lin-35 and dpl-1 did not affect vitellogenesis in CIAs (Figure 4D).We also examined the lin-35 activity in a vit-2 (ok3211) mutant background and found that this did not significantly alter the lin-35 mRNA transcript level in the vit-2 mutant compared to that in wild-type animals (Figure 4E).To elucidate the genetic interaction between lin-35 and vit-2 in the germ cell apoptosis pathway induced by caffeine intake, we performed double RNAi of lin-35 and vit-2 in CIAs and the control groups.Germ cell apoptosis induced by vit-2 RNAi was abolished by lin-35 RNAi depletion, suggesting that vit-2 functions upstream of lin-35 to negatively regulate the activity of lin-35 (Figure 4F).vit-2 and lin-35 double RNAi showed a phenotype similar to that of lin-35 single RNAi under both caffeine-free and caffeine-intake conditions (Figure 4F).Taken together, the decreased level of vitellogenesis in CIAs activates lin-35 and, thus, increases germ cell apoptosis in CIAs (see Discussion).

Methionine Supplementation Alleviates the Effects of Caffeine Intake
We found that the reduction in both reproduction and vitellogenesis by caffeine intake was improved by vitamin B12 supplementation, which is an important co-factor in methionine metabolism in a previous study.Considering these previous findings, we tested whether methionine supplementation plays a beneficial role in caffeine-induced germ cell apoptosis.We first tested whether methionine supplementation recovers the level of germ cell apoptosis in CIAs and found that the elevated level of germ cell apoptosis induced by caffeine intake was significantly reduced in CIAs with methionine supplementation (Figure 5A, Supplementary Materials Figure S3).We also confirmed that the increased level of lin-35 mRNA transcript caused by caffeine intake was significantly decreased by methionine supplementation (Figure 5B), indicating that methionine supplementation plays a beneficial role in decreasing the level of germ cell apoptosis induced by caffeine intake.Maternal caffeine intake has been shown to retard offspring development by reducing vitellogenesis in C. elegans [24].Therefore, we tested whether methionine supplementation could contribute to offspring growth in CIAs.We evaluated the subsequent F1 generation in CIAs with methionine supplementation.Interestingly, caffeine-fed mothers displayed a significant improvement in offspring developmental growth rate (approximately 59.5% adults with caffeine and 92.0%adults with caffeine + methionine supplementation at 72 h of growth).These results suggest that methionine supplementation has a beneficial effect on both caffeine-fed mothers and their subsequent generation.

Methionine Supplementation Alleviates the Effects of Caffeine Intake
We found that the reduction in both reproduction and vitellogenesis by caffeine intake was improved by vitamin B12 supplementation, which is an important co-factor in methionine metabolism in a previous study.Considering these previous findings, we tested whether methionine supplementation plays a beneficial role in caffeine-induced germ cell apoptosis.We first tested whether methionine supplementation recovers the level of germ cell apoptosis in CIAs and found that the elevated level of germ cell apoptosis induced by caffeine intake was significantly reduced in CIAs with methionine supplementation (Figure 5A, Supplementary Materials Figure S3).We also confirmed that the increased level of lin-35 mRNA transcript caused by caffeine intake was significantly decreased by methionine supplementation (Figure 5B), indicating that methionine supplementation plays a beneficial role in decreasing the level of germ cell apoptosis induced by caffeine intake.Maternal caffeine intake has been shown to retard offspring development by reducing vitellogenesis in C. elegans [24].Therefore, we tested whether methionine supplementation could contribute to offspring growth in CIAs.We evaluated the subsequent F1 generation in CIAs with methionine supplementation.Interestingly, caffeine-fed mothers displayed a significant improvement in offspring developmental growth rate (approximately 59.5% adults with caffeine and 92.0%adults with caffeine + methionine supplementation at 72 h of growth).These results suggest that methionine supplementation has a beneficial effect on both caffeine-fed mothers and their subsequent generation.

Discussion
The previous study showed that maternal caffeine intake negatively affected offspring development in a C. elegans model [24].In this study, we investigated the physiological alterations affecting germ cell health and offspring development following maternal caffeine intake.We found that maternal caffeine intake decreased methionine levels, which seemed to be due to the reduced expression of sams-1, metr-1, and mtrr-1, which are essential for the methionine cycle.The decreased amount of methionine resulted in a reduction in vitellogenesis, thus increasing the level of apoptosis in the germline and decreasing developmental growth in the offspring (Figure 6).Surprisingly, when methionine was supplemented in CIAs, the negative effects of maternal caffeine intake were alleviated.
Germ cell apoptosis in C. elegans occurs during oogenesis at the adult stage to maintain oocyte quality so that the subsequent offspring development is normally proceeded [21,22].A proper supply of yolk protein is essential for oocyte quality [24,26].We previously found a reduction in vitellogenesis in CIAs [24].Vitellogenesis in C. elegans at the adult stage is regulated by methionine supplementation [35].Considering these findings, we propose that the reduction in vitellogenesis in CIAs is due to alterations in the methionine cycle.We found that caffeine intake significantly reduced methionine levels and vitellogenesis and, thus, increased germ cell apoptosis.Recently, it has been reported that maternal caffeine intake causes mitochondrial stress in the germ line of C. elegans [34], but the mechanism by which caffeine intake induced mitochondrial stress in the germline remains unclear.Interestingly, some studies have demonstrated an interrelationship between methionine metabolism and mitochondrial function with direct relevance [36][37][38].Furthermore, the function of mitochondria is essential for maintaining oocyte quality [39][40][41].Based on the previous findings and this study, it raises the possibility that maternal caffeine intake targets mitochondrial function by altering methionine metabolism, which induces an increased level of germ cell apoptosis during oogenesis.Hence, it is necessary to investigate the genetic interactions between mitochondrial stress response and methionine cycle genes during the germ cell apoptosis induced by maternal caffeine intake in future studies.
which seemed to be due to the reduced expression of sams-1, metr-1, and mtrr-1, which are essential for the methionine cycle.The decreased amount of methionine resulted in a reduction in vitellogenesis, thus increasing the level of apoptosis in the germline and decreasing developmental growth in the offspring (Figure 6).Surprisingly, when methionine was supplemented in CIAs, the negative effects of maternal caffeine intake were alleviated.The tumor suppressor retinoblastoma (RB) is a transcriptional regulator essential for normal cell cycle progression and apoptosis [42].The RB is located in the outer mitochondrial membrane and promotes apoptosis [43][44][45].It was reported that lin-35, a C. elegans homolog of Rb, is increased under starvation conditions to suppress the expression of ced-9, thereby holding open CED-4; then, it interacts with CED-3 to induce germ cell apoptosis [46].Starvation conditions also promote dpl-1, which increases CED-4 accumulation [46].In this study, we showed that maternal caffeine intake increased germ cell apoptosis in an LIN-35/RB-dependent manner.Although maternal caffeine intake appears to partially require DPL-1 activity, germ cell apoptosis induced by caffeine intake appears to be distinct from increased germ cell apoptosis induced by starvation.There is a distinct difference between starvation and caffeine-induced germ cell apoptosis, in that starvation caused an increase in the expression of both lin-35 and dpl-1, whereas caffeine intake exclusively increased the lin-35 mRNA level (Figure 3H).This suggests that the increased germ cell apoptosis resulting from caffeine intake is regulated somewhat differently from the starvation-induced LIN-35 pathway.There are intriguing questions thar remain to be answered, as follows: how does maternal caffeine intake induce LIN-35 activity, how do alterations in the methionine cycle and vitellogenesis regulate LIN-35 activity, and, in particular, the possibility that C. elegans LIN-35 functions in mitochondria to promote apoptosis in the germ line?Maternal nutritional status is considered a critical factor in the interaction with major metabolic pathways, shaping both health and individual phenotypes during fetal development [47][48][49].Methionine is an essential methyl donor for DNA methylation, which affects gene expression by epigenetic alterations and is linked to the offspring transcriptome, which is mediated by changes in DNA methylation [50].Therefore, maternal methionine metabolism is important for embryonic development, growth, and offspring health.We found that impaired methionine metabolism caused by maternal caffeine intake reduced vitellogenesis.Interestingly, the decrease in vitellogenesis due to caffeine intake caused an upregulation in germ cell apoptosis; however, unlike the increased lin-35 due to caffeine intake in the wild-type background, there was no change in lin-35 mRNA level in the vit-2 mutant (Figure 4E).This may be due to redundancy, as the vit-2 single mutation is not sufficient to increase the mRNA level of lin-35, and in C. elegans there are six orthologous genes from vit-1 to vit-6 for vitellogenesis [26].Considering the beneficial effect of methionine on egg and milk production reported in previous studies [51,52], it is thought to play a specific role in nutrient metabolism and reproduction.When methionine was supplemented in CIAs, both reduced vitellogenesis and the retarded developmental growth of offspring caused by caffeine intake were improved, consistent with its beneficial role in reproduction.
C. elegans is popular as an excellent animal model to investigate chemical toxicology.We showed that maternal caffeine intake led to the alterations in methionine metabolism and germ cell apoptosis in C. elegans.However, there are limitations in directly applying it to human physiology.Moreover, the delivery methods for chemicals in C. elegans are indirect and can vary [53][54][55][56] and the cuticle of C. elegans also hinders the uptake of chemicals.Therefore, the concentration of caffeine treatment in this study using C. elegans may be at a low level.
Collectively, these results indicate that methionine alleviated the harmful effects of maternal caffeine consumption.Our study suggests new insights into the negative effects of maternal metabolic alterations on reproductive and offspring health.

Conclusions
This study showed that maternal caffeine intake decreased both methionine levels and expression levels of genes for essential enzymes modulating methionine cycle, such as sams-1, metr-1, mtrr-1.We also found that the caffeine-induced impairment of methionine metabolism decreased vitellogenesis and increased germ cell apoptosis in an LIN-35/RBdependent manner.Methionine supplementation in caffeine-ingested animals alleviated caffeine-induced negative effects on reproduction.Our findings suggest that methionine supplementation has a beneficial effect on germ cell health and offspring development through the regulation of vitellogenesis.

Nutrients 2024 , 17 Figure 3 .
Figure 3. Caffeine intake increased germ cell apoptosis in an LIN-35/RB-dependent manner.(A) The number of apoptotic germ cells in MD701 transgenic animals treated with or without caffeine at 20 °C for 24 h, 48 h, and 72 h after the L4 stage.n > 30 in each condition.**, p < 0.005.*, p < 0.01 (oneway analysis of variance (ANOVA) with Tukey's post hoc test).(B) The number of germ cell corpses in ced-1(e1735) mutants treated with or without caffeine at 20 °C for 24 h after the L4 stage.n > 30 in each condition.***, p < 0.001 (Student's t-test).(C) The number of PH3-positive germ cells in wildtype animals treated with or without caffeine at 20 °C for 24 h after the L4 stage.n > 30 in each condition.n.s., not significant (Student's t-test).(D) The number of apoptotic germ cells by RNAi depletion of mock, ced-4, and ced-3 in MD701 transgenic animals treated with or without caffeine at 20 °C for 24 h after the L4 stage.n > 30 in each condition.**, p < 0.005.n.s., not significant (ANOVA with Tukey's post hoc test).(E) Western blot analysis of CED-4 and CED-9 in wild-type animals treated with or without caffeine at 20 °C for 24 h after the L4 stage.n > 300 in each condition.***, p < 0.001.*, p < 0.01 (Student's t-test).(F) The number of apoptotic germ cells by RNAi depletion of mock, cep-1, mpk-1, lin-35, and dpl-1 in MD701 transgenic animals treated with or without caffeine at 20 °C for 24 h after the L4 stage.n > 30 in each condition.**, p < 0.005.n.s., not significant (ANOVA with Tukey's post hoc test).(G,H) Fold induction in mRNA of lin-35 (G) and dpl-1 (H) in CIAs.p values were calculated using Student's t-test.*, p < 0.01.n.s., not significant.Error bars represent SD.

Figure 3 .
Figure 3. Caffeine intake increased germ cell apoptosis in an LIN-35/RB-dependent manner.(A) The number of apoptotic germ cells in MD701 transgenic animals treated with or without caffeine at 20 • C for 24 h, 48 h, and 72 h after the L4 stage.n > 30 in each condition.**, p < 0.005.*, p < 0.01 (one-way analysis of variance (ANOVA) with Tukey's post hoc test).(B) The number of germ cell corpses in ced-1(e1735) mutants treated with or without caffeine at 20 • C for 24 h after the L4 stage.n > 30 in each condition.***, p < 0.001 (Student's t-test).(C) The number of PH3-positive germ cells in wild-type animals treated with or without caffeine at 20 • C for 24 h after the L4 stage.n > 30 in each condition.n.s., not significant (Student's t-test).(D) The number of apoptotic germ cells by RNAi depletion of mock, ced-4, and ced-3 in MD701 transgenic animals treated with or without caffeine at 20 • C for 24 h after the L4 stage.n > 30 in each condition.**, p < 0.005.n.s., not significant (ANOVA with Tukey's post hoc test).(E) Western blot analysis of CED-4 and CED-9 in wild-type animals treated with or without caffeine at 20 • C for 24 h after the L4 stage.n > 300 in each condition.***, p < 0.001.*, p < 0.01 (Student's t-test).(F) The number of apoptotic germ cells by RNAi depletion of mock, cep-1, mpk-1, lin-35, and dpl-1 in MD701 transgenic animals treated with or without caffeine at 20 • C for 24 h after the L4 stage.n > 30 in each condition.**, p < 0.005.n.s., not significant (ANOVA with Tukey's post hoc test).(G,H) Fold induction in mRNA of lin-35 (G) and dpl-1 (H) in CIAs.p values were calculated using Student's t-test.*, p < 0.01.n.s., not significant.Error bars represent SD.

Figure 4 .
Figure 4. Methionine metabolism is involved in physiological germ cell apoptosis and vitellogenesis.(A) The number of apoptotic germ cells by RNAi depletion of mock, sams-1, metr-1, and mtrr-1 in MD701 transgenic animals.n > 30 in each condition.p values were calculated using one-way analysis of variance (ANOVA) with Tukey's post hoc test.***, p < 0.001.Error bars represent SD. (B) VIT-2::GFP expression in DH1033 transgenic animals treated with mock, sams-1, metr-1, and mtrr-1 RNAi.n > 30 in each condition.Relative intensity was examined using ImageJ analysis.p values were calculated using ANOVA with Tukey's post hoc test.***, p < 0.001.(C) The number of apoptotic germ cells by RNAi depletion of vit-2 in CED-1::GFP transgenic animals with caffeine treatment.n > 30 in each condition.p values were calculated using ANOVA with Tukey's post hoc test.***, p < 0.001.n.s., not significant.Error bars represent SD. (D) VIT-2::GFP expression in DH1033 transgenic animals by RNAi depletion of lin-35 and dpl-1 with caffeine treatment.n > 30 in each condition.p values were calculated using ANOVA with Tukey's post hoc test.n.s., not significant.Error bars represent SD. (E) Fold induction in mRNA of lin-35 in N2 and vit-2(ok3211) mutants with caffeine treatment.n > 300 in each condition.p values were calculated using ANOVA with Tukey's post hoc test.*, p < 0.01.n.s., not significant.Error bars represent SD. (F) The number of apoptotic germ cells by RNAi depletion of lin-35 and vit-2 in MD701 transgenic animals with caffeine treatment.n > 30 in each condition.***, p < 0.001.n.s., not significant (ANOVA with Tukey's post hoc test).Error bars represent SD.

Figure 4 .
Figure 4. Methionine metabolism is involved in physiological germ cell apoptosis and vitellogenesis.(A) The number of apoptotic germ cells by RNAi depletion of mock, sams-1, metr-1, and mtrr-1 in MD701 transgenic animals.n > 30 in each condition.p values were calculated using one-way analysis of variance (ANOVA) with Tukey's post hoc test.***, p < 0.001.Error bars represent SD. (B) VIT-2::GFP expression in DH1033 transgenic animals treated with mock, sams-1, metr-1, and mtrr-1 RNAi.n > 30 in each condition.Relative intensity was examined using ImageJ analysis.p values were calculated using ANOVA with Tukey's post hoc test.***, p < 0.001.(C) The number of apoptotic germ cells by RNAi depletion of vit-2 in CED-1::GFP transgenic animals with caffeine treatment.n > 30 in each condition.p values were calculated using ANOVA with Tukey's post hoc test.***, p < 0.001.n.s., not significant.Error bars represent SD. (D) VIT-2::GFP expression in DH1033 transgenic animals by RNAi depletion of lin-35 and dpl-1 with caffeine treatment.n > 30 in each condition.p values were calculated using ANOVA with Tukey's post hoc test.n.s., not significant.Error bars represent SD. (E) Fold induction in mRNA of lin-35 in N2 and vit-2(ok3211) mutants with caffeine treatment.n > 300 in each condition.p values were calculated using ANOVA with Tukey's post hoc test.*, p < 0.01.n.s., not significant.Error bars represent SD. (F) The number of apoptotic germ cells by RNAi depletion of lin-35 and vit-2 in MD701 transgenic animals with caffeine treatment.n > 30 in each condition.***, p < 0.001.n.s., not significant (ANOVA with Tukey's post hoc test).Error bars represent SD.

Figure 5 .
Figure 5. Methionine supplementation alleviates the effects of maternal caffeine intake.(A) The number of apoptotic germ cells in MD701 transgenic animals treated with caffeine and methionine.n > 30 in each condition.p values were calculated using one-way analysis of variance (ANOVA) with Tukey's post hoc test.***, p < 0.001.Error bars represent SD. (B) Fold induction in mRNA of lin-35 in N2 with caffeine and methionine treatment.n > 300 in each condition.p values were calculated

Figure 5 .
Figure 5. Methionine supplementation alleviates the effects of maternal caffeine intake.(A) The number of apoptotic germ cells in MD701 transgenic animals treated with caffeine and methionine.n > 30 in each condition.p values were calculated using one-way analysis of variance (ANOVA) with Tukey's post hoc test.***, p < 0.001.Error bars represent SD. (B) Fold induction in mRNA of lin-35 in N2 with caffeine and methionine treatment.n > 300 in each condition.p values were calculated using ANOVA with Tukey's post hoc test.***, p < 0.001.Error bars represent SD. (C) Synchronized wild-type L4-stage mothers (P0) were treated with caffeine and methionine for 24 h.The embryos (F1) produced by each group of mothers were cultured at 20 • C and their developmental stages were measured until they reached adult stage.Each developmental stage in the F1 generation was determined by its size-and stage-specific morphological characteristics during development in caffeine-ingested P0 mother supplemented with methionine.n > 40 in each condition.

Figure 6 .
Figure 6.Working model.Maternal caffeine intake impairs the methionine cycle by reducing the level of methionine.This alteration decreases vitellogenesis and induces physiological germ cell apoptosis in an LIN-35/RB-dependent manner.When methionine is supplemented in CIAs, the level of vitellogenin and lin-35-depedent germ cell apoptosis recovers to normal levels.Additionally, delayed offspring growth due to maternal caffeine intake is also improved by methionine supplementation.

Figure 6 .
Figure 6.Working model.Maternal caffeine intake impairs the methionine cycle by reducing the level of methionine.This alteration decreases vitellogenesis and induces physiological germ cell apoptosis in an LIN-35/RB-dependent manner.When methionine is supplemented in CIAs, the level of vitellogenin and lin-35-depedent germ cell apoptosis recovers to normal levels.Additionally, delayed offspring growth due to maternal caffeine intake is also improved by methionine supplementation.

:
Experimental schemes for caffeine treatment and methionine supplementation in C. elegans; Figure S2: Survival assay of C. elegans treated with caffeine; and Figure S3: Vitellogenin (VIT-2::GFP) expression by caffeine treatment.Author Contributions: Conceptualization, H.M. and Y.-H.S.; methodology, H.M., J.K., M.L. and S.K.; software, H.M. and J.K.; validation, H.M., J.K., M.L. and S.K.; formal analysis, H.M. and J.K.; investigation, J.K. and S.K.; resources, Y.-H.S.; data curation, H.M. and J.K.; writing-original draft preparation, H.M., J.K. and Y.-H.S.; writing-review and editing, H.M. and Y.-H.S.; supervision, Y.-H.S.; project administration, Y.-H.S.; funding acquisition, Y.-H.S. and H.M. All authors have read and agreed to the published version of the manuscript.Funding: This study was supported by the National Research Foundation of Korea (NRF), funded by the Korean Ministry of Science and ICT (NRF-2021R1A2C1011658 to Y.-H.S.), and by the Basic Science Research Program through the NRF funded by the Ministry of Education (RS-2023-00246465 to H.M.).Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).

Table 1 .
The primers used for qRT-PCR and the GenBank database accession number for C. elegans mRNA.

Table 2 .
The primers used for the RNAi assay.

Table 3 .
The antibodies used for this study.

Table 4 .
Characteristics of each developmental stage in C. elegans.