Maternal Caffeine Intake Disrupts Eggshell Integrity and Retards Larval Development by Reducing Yolk Production in a Caenorhabditis elegans Model

Abstract

During pregnancy, most women are exposed to caffeine, which is a widely consumed psychoactive substance. However, the consequences of maternal caffeine intake on the child remain largely unknown. Here, we investigated the intergenerational effects of maternal caffeine intake on offspring in a Caenorhabditis elegans model. We treated a young mother (P0) with 10 mM of caffeine equivalent to 2-5 cans of commercial energy drinks and examined its reproduction and growth rate from P0 to F2 generation. The fertility decreased and embryonic lethality increased by defective oocytes and eggshell integrity in caffeine-ingested mothers, and F1 larval development severely retarded. These results were due to decreased production of vitellogenin protein (yolk) in caffeine-ingested mothers. Furthermore, effects of RNA interference of vitellogenin (vit) genes, vit-1 to vit-6, in P0 mothers can mimic those by caffeine-ingested mothers. In addition, RNA interference (RNAi) depletion of unc-62 (human Meis homeobox), a transcriptional activator for vit genes, also showed similar effects induced by caffeine intake. Taken together, maternal caffeine intake reduced yolk production mediated by the UNC-62 transcription factor, thereby disrupting oocyte and eggshell integrity and retarding larval development. Our study suggests the clinical significance of caffeine intake for prospective mothers.

Keywords: 1,3,7-trimethylxanthine; Caenorhabditis elegans; UNC-62; caffeine; eggshell integrity; intergenerational effect; maternal effect; reproduction; vitellogenin; yolk protein.

Figure 1

Caenorhabditis elegans P0 mothers were fed single compound caffeine (1,3,7-trimethylxanthine).

Figure 2

Caffeine intake caused a reduction in fertility of P0 mothers and an increase in F1 embryonic lethality in Caenorhabditis elegans. (A) Total number of progenies by caffeine-ingested mothers (5, 10, and 30 mM) compared to mothers with a caffeine-free diet (0 mM). * p < 0.05. (B) The percentage of embryonic lethality among the total number of progenies produced by caffeine-ingested P0 mothers that were fed 0, 5, 10, and 30 mM of caffeine at the L4 stage for 24 h. * p < 0.05. (C) A scheme of assays for intergenerational effects of caffeine intake by P0 mothers. The P0 mothers were fed 10 mM of caffeine, and reproduction and growth were measured from P0 to F2 generation for analysis of intergenerational effects of caffeine intake by P0 mothers. (D) The total number of progenies produced by caffeine-ingested P0 mothers and their offspring (black bars) compared to caffeine-free diet P0 mothers and their offspring (white bars). * p < 0.05. (E) The percentage of embryonic lethality among the total number of progenies produced by caffeine-ingested P0 mothers and their offspring (black bars), and caffeine-free diet P0 mothers and their offspring (white bars). Error bars represent SD. * p < 0.05.

Figure 3

Caffeine intake by P0 mothers affected development in the subsequent generation of Caenorhabditis elegans. (A,B) Synchronized L4-stage animals (n = 30) of C. elegans wild-type were fed either 0 or 10 mM of caffeine for 24 h, and embryos were transferred to respective OP50-seeded nematode growth medium plates and further cultured at 20 °C. The developmental stage of each individual in the F1 and F2 generations was determined based on its size and stage-specific morphological characteristics (see Section 2 for details) during development by either caffeine-ingested (10 mM) or caffeine-free diet P0 mother (0 mM).

Figure 4

Maternal caffeine intake reduces vitellogenin (yolk proteins) in Caenorhabditis elegans. (A) VIT-6::GFP transgenic animals synchronized at the L4-stage were exposed to caffeine for 24 h at 20 °C. VIT-6::GFP expresses in the intestine. The reduced level of VIT-6::GFP in the intestine was observed in caffeine-ingested mothers. (B) Western blot analysis of VIT-6::GFP protein levels in caffeine-free diet mother (0 mM) and caffeine-ingested mother (10 mM). Respective GFP band intensities were normalized against those of α-tubulin in the same lane. Then the normalized GFP band intensity was converted to a relative value compared to the normalized GFP band intensity of 0 mM, as shown in the right graph with mean ± SD values. These GFP band intensity values were obtained from three independent Western blot analyses. Statistical significance was calculated using Student’s t-test. * p < 0.05. (C,D) VIT-2::GFP transgenic animals synchronized at the L4-stage were exposed to caffeine for 24 h at 20 °C. The caffeine-ingested mother showed a reduced level of VIT-2::GFP intensity in oocytes and embryos, as shown in the right graph with mean ± SD values. (E) Synchronized L4-stage animals (n = 30) of wild-type were treated with RNA interference (RNAi) of vit-3 gene for 24 h and recovered to OP50-seeded nematode growth medium (NGM) plates for 24 h. The embryos produced by P0 mothers were transferred to respective NGM plates and further cultured at 20 °C. The developmental stage of each individual in the progenies was determined based on its size and stage-specific morphological characteristics (see Section 2 for details) during development by either the vit-3 RNAi-treated P0 mother (10 mM) or non-treated (mock) P0 mother (0 mM). (F) VIT-6::GFP transgenic animals synchronized at the L4-stage were exposed to caffeine for 24 h at 20 °C. Then their F1 generation grew under the caffeine-free diet (0 mM) condition, and VIT-6::GFP transgenic F1 animals were observed at the adult stage. Statistical significance was calculated using Student’s t-test. n.s., p > 0.05.

Figure 5

Maternal caffeine intake disrupted eggshell integrity in Caenorhabditis elegans. (A) Oocytes at the –1 position in a gonad arm of wild-type adult hermaphrodites grown without or with 10 mM caffeine. DNA was stained with TO-PRO-3 and then six bivalents were examined. The percent of intact six bivalents is shown in a bar graph on the right. (B–D) DIC images (B) and lipophilic dye FM4-64 staining images (C,D) of embryos from the dissected caffeine-free diet (0 mM) and caffeine-ingested mothers (10 mM). The caffeine-free diet (0 mM) mother produced intact and ovoid embryos and only polar bodies (white arrowheads) were stained, but the caffeine-ingested mother produced ruptured embryos (C) and their eggshells were permeable to FM4-64, and the cell membranes of the embryos were stained (D). Statistical significance was calculated using Student’s t-test. * p < 0.05.

Figure 6

Vitellogenin is required for eggshell and oocyte integrity in Caenorhabditis elegans. (A) Eggshell permeability was examined by lipophilic dye FM4-64 staining in the embryos produced by mock RNAi- or vit-1 to vit-6 RNAi-treated mothers. Statistical significance was calculated using Student’s t-test. * p < 0.05 against mock RNAi-treated animals. (B) Eggshell permeability was examined by FM4-64 staining in the embryos produced by vit-1(ok2616), vit-2(ok3211), and vit-5(ok3239) mutants. Statistical significance was calculated using Student’s t-test. * p < 0.05 against N2 control. (C) Oocyte permeability examined with lipophilic dye FM4-64 staining in the caffeine-ingested mother (10 mM) and caffeine-free diet mother (0 mM). Gonads were extruded by dissecting adult animals in 150 mM of KCl. Statistical significance was calculated using Student’s t-test. * p < 0.05. (D) Oocyte permeability was examined by FM4-64 staining after treatment with RNAi of the vitellogenin genes from vit-1 to vit-6. Statistical significance was calculated using Student’s t-test. * p < 0.05 against mock RNAi-treated animals.

Figure 7

Caffeine intake reduces unc-62 expression and the reduced level of unc-62 exhibits defects in embryo and oocyte integrity in Caenorhabditis elegans. (A) UNC-62 binding sites in the genomic structures of vitellogenin genes. The thin black lines indicate the promoter and introns of each of six vitellogenin loci, and the thick black lines indicate exons of six vitellogenin genes. The blue lines indicate UNC-62 binding sites, and the red bars indicate VPE-1 (ATTGACA) vitellogenin regulatory motif previously described [25,27]. (B) Fold induction of mRNA level of unc-62 in caffeine-ingested mothers (10 mM) than caffeine-free diet mothers (0 mM). The mRNA level of unc-62 was determined by three independent qRT-PCR using the mRNA level of act-1 in each sample as an internal control for normalization. T-bars represent SD. Approximately 150 adult animal individuals were used to prepare total RNA for respective conditions. Statistical significance was calculated using Student’s t-test. * p < 0.05. (C) VIT-2::GFP transgenic animals synchronized at the L4-stage were treated with unc-62 RNAi. The unc-62 RNAi-treated mothers showed the reduced level of VIT-2::GFP intensity both in oocytes and embryos, as shown in the right graph with mean ± SD values. (D) Eggshell permeability examined by lipophilic dye FM4-64 staining of the embryos produced by unc-62 RNAi-treated mothers. Statistical significance was calculated using Student’s t-test. * p < 0.05 against mock RNAi-treated animals. (E) Oocyte permeability examined by lipophilic dye FM4-64 staining in the dissected gonad from the unc-62 RNAi-treated mothers. Statistical significance was calculated using Student’s t-test. * p < 0.05 against mock RNAi-treated mothers.

Figure 8

Model of the intergenerational effects of maternal caffeine intake in Caenorhabditis elegans. Caffeine intake decreased the production of yolk proteins by reducing unc-62 expression. The decreased levels of yolk proteins disrupted oocyte and eggshell integrity and induced embryonic lethality and growth retardation of the next generation.