Females translate male mRNA transferred during mating

Abstract

Although RNA is found in the seminal fluid of diverse organisms, it is unknown whether this RNA is functional within females. Here, we develop an experimental proteomic method called VESPA (Variant Enabled SILAC Proteomic Analysis) to test the hypothesis that Drosophila male seminal fluid RNA is translated by females. We find strong evidence for 67 male-derived, female-translated proteins (mdFTPs) in female lower reproductive tracts at six hours postmating, many with predicted functions relevant to reproduction. Gene knockout experiments indicate that genes coding for mdFTPs play diverse roles in postmating interactions, with effects on fertilization efficiency, and the formation and persistence of the insemination reaction mass, a trait hypothesized to be involved in sexual conflict. These findings advance our understanding of reproduction by revealing a novel mechanism of postmating molecular interactions between the sexes that strengthens and extends male influences on reproductive outcomes in previously unrecognized ways. Given the diverse species known to carry RNA in seminal fluid, this discovery has broad significance for understanding molecular mechanisms of cooperation and conflict during reproduction.

Keywords: Drosophila; Fertility; Proteomics; Reproductive incompatibility; Seminal fluid composition.

Figure 1.. Overview of VESPA.

VESPA differentiates proteins made by males and females by isotopic labeling, and the source of RNA transcripts by fixed nucleotide differences between species. Peptides that match the sequence of the male but carry the heavy label of the female are diagnostic for mdFTPs.

Figure 2.. mdFTPs are supported by multiple lines of evidence.

(A) All mdFTPs were identified by a minimum of two heavy peptides, with at least one being diagnostic. mdFTPs inside broken lines were identified in multiple replicates or by multiple diagnostic peptides. The most strongly supported mdFTPs include those identified by multiple diagnostic peptides, in two or more replicates, and/or by both MaxQuant and MSFragger. (B) Mean RNA transfer index (RTI) per gene was higher for high support mdFTPs (yellow in panel ‘A’, n=67) and low support mdFTPs (n=99) compared to all other genes with RNA-seq data (n=9727). (GLMM: gene category χ²=812.8, P= 2.2e⁻¹⁶). Post hoc tests were performed using Tukey’s method (****P≤ 0.0001). Error bars represent standard error of the mean. (C) Venn diagram showing overlap of mdFTPs with D. arizonae SFPs. Low overlap suggests mdFTPs may perform different functions from other proteins in the ejaculate.

Figure 3.. mdFTPs have functional significance to reproduction.

(A) Enrichment of protein domains and GO terms for biological process and molecular function link mdFTPs to processes important for reproduction. This includes enrichment of CAP domains, which are linked to reproduction in diverse organisms, and two domains (serpin family and terpenoid cyclases/protein prenyltransferase alpha-alpha toroid) that have predicted involvement in coagulation. GO terms associated with energy production, oxidative stress response, and immunity were also enriched. (B) List of mdFTPs with protein domains associated with coagulation. Additional domains not necessarily linked to coagulation are also noted. High support mdFTPs refer to the 67 in yellow from figure 1A, while low support mdFTPs represent the remaining 99.

Figure 4.. Gene KO experiments demonstrate mdFTPs have functional effects on diverse reproductive processes.

(A) Drosophila arizonae females mated to D. arizonae males with a ARI26694 KO mutation had a smaller reaction mass initially, as measured by the average perimeter. However, it degraded more slowly compared to the reaction mass in females mated to WT males (Two-way ANOVA: genotype x time interaction, F= 16.5, P= 0.0001). Post hoc comparisons were performed using Tukey’s method (**P≤ 0.01). The dashed line indicates the mean perimeter of unmated female lower reproductive tract. Error bars represent 95% confidence intervals of estimated marginal means. KO-0 hr: n=26; WT-0 hr: n=35; KO-6 hr: n=15. (b) D. arizonae females mated to D. arizonae males with a ARI11629 KO mutation laid more unfertilized eggs on days one and three postmating compared to females mated to WT males (GLM: genotype x day interaction, χ²= 17.2, P= 0.0002). Post hoc comparisons were performed using Tukey’s method (***P> 0.001). Error bars represent 95% confidence intervals of estimated marginal means. WT-D1: n=119; KO-D1: n=44; WT-D3: n=104; KO-D3 n=360; WT-D5 n=131; KO-D5 n=90.