The Caenorhabditis elegans Female-Like State: Decoupling the Transcriptomic Effects of Aging and Sperm Status

Abstract

Understanding genome and gene function in a whole organism requires us to fully comprehend the life cycle and the physiology of the organism in question. Caenorhabditis elegans XX animals are hermaphrodites that exhaust their sperm after 3 d of egg-laying. Even though C. elegans can live for many days after cessation of egg-laying, the molecular physiology of this state has not been as intensely studied as other parts of the life cycle, despite documented changes in behavior and metabolism. To study the effects of sperm depletion and aging of C. elegans during the first 6 d of adulthood, we measured the transcriptomes of first-day adult hermaphrodites and sixth-day sperm-depleted adults, and, at the same time points, mutant fog-2(lf) worms that have a feminized germline phenotype. We found that we could separate the effects of biological aging from sperm depletion. For a large subset of genes, young adult fog-2(lf) animals had the same gene expression changes as sperm-depleted sixth-day wild-type hermaphrodites, and these genes did not change expression when fog-2(lf) females reached the sixth day of adulthood. Taken together, this indicates that changing sperm status causes a change in the internal state of the worm, which we call the female-like state. Our data provide a high-quality picture of the changes that happen in global gene expression throughout the period of early aging in the worm.

Keywords: RNA-seq; aging; epistasis; genetic interactions; germline sex determination; life cycle.

Figure 1

Experimental design to identify genes associated with sperm loss and aging. Studying the wild-type worm alone would measure time- and sperm-related changes at the same time, without allowing us to separate these changes. Studying the wild-type worm and a fog-2(lf) mutant would enable us to measure sperm-related changes but not time-related changes. By mixing both designs, we can measure and separate both modules.

Figure 2

(A) Differentially expressed isoforms in the aging category. We identified a common aging expression signature between N2 and fog-2(lf) animals, consisting of 6193 differentially expressed isoforms totaling 5592 genes. The volcano plot is randomly down-sampled 30% for ease of viewing. Each point represents an individual isoform. ${\beta }_{\text{Aging}}$ is the regression coefficient. Larger magnitudes of β indicate a larger log-fold change. The y-axis shows the negative logarithm of the q-values for each point. Green points are differentially expressed isoforms; orange points are differentially expressed isoforms of predicted transcription factor genes (Reece-Hoyes et al. 2005). An interactive version of this graph can be found on our website. (B) Enriched tissues in aging-associated genes. Tissue enrichment analysis (Angeles-Albores et al. 2016) showed that genes associated with muscle tissues and the nervous system are enriched in aging-related genes. Only statistically significantly enriched tissues are shown. Enrichment fold change is defined as $\text{Observed}/\text{Expected}.$ hmc, head mesodermal cell.

Figure 3

Explanation of linear regressions with and without interactions. (A) A linear regression with two variables, age and genotype. The expression level of a hypothetical gene increases by the same amount as worms age, regardless of genotype. However, fog-2(lf) has higher expression of this gene than the wild type at all stages (blue arrow). (B) A linear regression with two variables and an interaction term. In this example, the expression level of this hypothetical gene is different between wild-type worms and fog-2(lf) (blue arrow). Although the expression level of this gene increases with age, the slope is different between wild type and fog-2(lf). The difference in the slope can be accounted for through an interaction coefficient (red arrow).

Figure 4

fog-2(lf) partially phenocopies early aging in C. elegans. The β on each axis is the regression coefficient from the GLM, and can be loosely interpreted as an estimator of the log-fold change. Loss of fog-2 is associated with a transcriptomic phenotype involving 1881 genes. 1040/1881 of these genes are also altered in wild-type worms as they progress from young adulthood to old adulthood, and 905 change in the same direction. However, progression from young to old adulthood in a fog-2(lf) background results in no change in the expression level of these genes. (A) We identified genes that change similarly during feminization and aging. The correlation between feminization and aging is almost 1:1. (B) Epistasis plot of aging vs. feminization. Epistasis plots indicate whether two genes (or perturbations) act on the same pathway. When two effects act on the same pathway, this is reflected by a slope of –0.5. The measured slope was –0.51 ± 0.01.

Figure 5

Phenotype and GO enrichment of genes involved in the female-like state. (A) Phenotype enrichment analysis. (B) Gene ontology enrichment analysis. Most of the terms enriched in PEA reflect the abundance of ribosomal subunits present in this gene set.

Figure 6

(A) A substrate-dependent model showing how fog-2 promotes sperm generation, whereas aging promotes sperm depletion, leading to entry into the female-like state. Such a model can explain why fog-2 and aging appear epistatic to each other. (B) The complete C. elegans life cycle. Recognized stages of C. elegans are marked by black arrows. States are marked by red arrows to emphasize that at the end of a state, the worm returns to the developmental time point it was at before entering the state. The L2d state is an exception. It is the only stage that does not return to the same developmental time point; rather, the L2d state is a permissive state that allows entry into either dauer or the L3 stage. We have presented evidence of a female-like state in C. elegans. At this point, it is unclear whether the difference between hermaphrodites and females is reversible by males. Therefore, it remains unclear whether it is a stage or a true state.