Age-related diseases, such as cardiovascular disease, cancer, and neurodegenerative syndromes can display sex-specific differences. Since mitochondrial dysfunction is central to most age-related diseases, we examined the mitochondrial unfolded protein response (UPR ^mt ), a conserved, broad-range transcriptional response that, among other functions, aids in the refolding of mitochondrial matrix proteins (Kim, Ramalho, and Haynes 2024). In C. elegans , UPR ^mt activation is largely localized to the intestine, the major metabolic tissue in the nematode. In addition to digestion, the intestine also carries out many functions similar to the liver, such as detoxification, immunity, and fat metabolism (Dimov and Maduro 2019). We and others recently discovered that the activation of the intestinal UPR ^mt depends on the function of another tissue: the germline (Foulger et al. 2025; Charmpilas et al. 2024; Shen et al. 2024; Zhou et al. 2024) ^. Specifically, we found that adult nematodes with actively proliferating germlines, such as hermaphrodites or mated females, can activate a robust UPR ^mt in the intestines when challenged with either a high dose of the metal manganese (Mn) or RNAi of the OSCP/ atp-3 subunit of complex V (Foulger et al. 2025). Conversely, adult nematodes with mutations that lead to a lack germline stem cells or lack of sperm, such glp-1 or fem-1 mutants, cannot activate the intestinal UPR ^mt . Additionally, we found that loss of the transcription factor FOXO/ daf-16 fully restored the UPR ^mt in fem-1 mutants (Foulger et al. 2025). Here, we probe whether vitellogenesis, which occurs in the intestines and is dependent on FOXO/ daf-16 , impacts the UPR ^mt .

Vitellogenins are precursor yolk proteins that provide nutrients to developing embryos. In C. elegans , the highly conserved genes vit-1 through vit-6 are responsible for encoding these proteins (Perez and Lehner 2019). In hermaphrodites, they are synthesized in the intestines and transported into the pseudocoelom and to maturing oocytes (Perez and Lehner 2019). Young adult fem-1 mutants display elevated yolk content compared to wild-type hermaphrodites at Day 1 of adulthood (DePina et al. 2011), presumably due to a lack of vitellogenin transport out of the intestines to maturing oocytes. We used RNAi to knockdown vit-1 , vit-3 , vit-5 , or all three genes simultaneously ( vit-1 ,3,5 ) and monitored intestinal activation of the UPR ^mt in young adult nematodes. We used the UPR ^mt reporter in which the GFP expression is driven by the promoter of the mitochondrial chaperone mtHsp70/ hsp-6 (Yoneda et al. 2004). We additionally used RNAi of OSCP/ atp-3 to induce the UPR ^mt in young adult nematodes as previously described (Angeli et al. 2021). We found that in hermaphroditic nematodes, knockdown of vit-1 trended toward elevating the intestinal UPR ^mt (p=0.0504) and knockdown of vit-3 , vit-5 , or vit-1 ,3,5 all significantly elevated the UPR ^mt ( Figure 1A- 1D ). In contrast, knockdown of vit-1 , vit-3 , or vit-5 did not significantly alter the UPR ^mt of fem-1 mutants ( Figure 1A- 1D ). The knockdown of vit-1 ,3,5 did significantly enhance the UPR ^mt of the fem-1 mutants, but not to wild-types levels ( Figure 1D ). One caveat of this study is that we were not able to procure RNAi clones for vit-2 , vit-4 , or vit-6 . However, since vit-2 is over 80% identical to vit-1 and vit-3 / vit-4 are duplicated genes that are 99% identical (Perez and Lehner 2019), we would expect to observe more impact from individual RNAi knockdown on females if these vitellogenins played an outsized role on the UPR ^mt . The vit-6 gene is the most divergent vitellogenin, so it remains possible that it plays an untested role in the UPR ^mt . Overall, while it does appear that vitellogenins can mildly suppress the adult UPR ^mt , especially in hermaphroditic nematodes, we conclude that additional germline-to-intestinal signals are responsible for the potent suppression of the UPR ^mt in female nematodes.

Strains. Bristol N2 (wild type) nematodes were obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota) and cultured using standard conditions (Sulston J 1988). The following strains were used: GL347 ( SJ4100 p hsp-6 ::GFP backcrossed 6× to N2 ) and GL364 ( fem-1 ( hc17 ); p hsp-6 ::GFP).

Nematode and bacterial culture conditions. Nematodes were maintained on nematode growth medium (NGM) plates. NGM plates were seeded with Escherichia coli OP50 obtained from CGC that was grown in LB at 37°C for 18 hours shaking at 225 rpm. Seeded plates were dried for 48 hours at room temperature before use. For RNAi experiments, HT115 (DE3) bacteria obtained from the Horizon Biosciences RNAi library were used. All RNAi clones were verified via sequencing. RNAi plates were prepared by cooling NGM to 55°C and supplementing with a final concentration of 50μg/ml carbenicillin and 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). RNAi bacteria were inoculated with one colony of RNAi bacteria into LB with 50μg/ml carbenicillin and were grown shaking overnight for 18 hours at 37° at 225 rpm. RNAi cultures were seeded on RNAi plates and allowed to grow for 48 hours at room temperature. Plates were stored at 4 °C for no longer than 2 weeks.

Microscopy. Nematodes were anesthetized with 2 - 5mM levamisole and mounted on 2% agarose pads on glass slides. Fluorescence micrographs of GFP were taken using a Zeiss Axioscope 5 fluorescent compound microscope equipped with Zen microscopy imaging program. GFP expression was enhanced using the brightness/contrast tool in Adobe Photoshop. The same parameters were used for all images. GFP intensity of nematodes was quantified using ImageJ 1.54G. The “integrated density” of GFP expression and length of nematodes was measured using ImageJ tools. Integrated density value was normalized by number of nematodes and average length of nematodes. The final value is in arbitrary units.

Statistics. Significance between control and experimental groups was determined by using a two-tailed Student's t -test. Asterisks denote corresponding statistical significance: * p <  0.05; ** p  < 0.01; *** p  < 0.001. Error bars were generated using the standard error of the mean (SEM), typically from three or more pooled biological replicates.