The Seminal fluid proteome of the polyandrous Red junglefowl offers insights into the molecular basis of fertility, reproductive ageing and domestication

Abstract

Seminal fluid proteins (SFPs) are emerging as fundamental contributors to sexual selection given their role in post-mating reproductive events, particularly in polyandrous species where the ejaculates of different males compete for fertilisation. SFP identification however remains taxonomically limited and little is known about avian SFPs, despite extensive work on sexual selection in birds. We characterize the SF proteome of the polyandrous Red junglefowl, Gallus gallus, the wild species that gave rise to the domestic chicken. We identify 1,141 SFPs, including proteins involved in immunity and antimicrobial defences, sperm maturation, and fertilisation, revealing a functionally complex SF proteome. This includes a predominant contribution of blood plasma proteins that is conserved with human SF. By comparing the proteome of young and old males with fast or slow sperm velocity in a balanced design, we identify proteins associated with ageing and sperm velocity, and show that old males that retain high sperm velocity have distinct proteome characteristics. SFP comparisons with domestic chickens revealed both qualitative and quantitative differences likely associated with domestication and artificial selection. Collectively, these results shed light onto the functional complexity of avian SF, and provide a platform for molecular studies of fertility, reproductive ageing, and domestication.

Figure 1. Red junglefowl seminal fluid protein identification and composition.

(A) Histogram displaying the proportion of the seminal fluid proteome identified as a function of the number of males in which proteins were identified. Proteins also identified as components of blood plasma are indicated in red. (B) Histogram comparing the distribution of protein abundance amongst seminal fluid proteins identified (red) and not identified (black) as components of blood plasma. (C) Venn diagram displaying the protein overlap between Red junglefowl SF (red), human SF (black) and human blood plasma (blue). The vast majority of conserved SF proteins are also present in the blood plasma proteome.

Figure 2. Multivariate ordination of seminal fluid protein abundance with respect to male age and sperm velocity.

(A) Principle Component Analysis (PCA1) variables factor map. The direction of the arrows represents the relative loadings of the four categories (Old Fast, Old Slow, Young Fast, Young Slow) on the first and second dimension. All four categories have strong positive loadings on dimension 1; Old Fast has a significant positive loading and Old Slow and Young Fast have significant negative loadings on dimension 2, respectively. (B) Between group PCA (PCA2) biplot. First and second dimension loadings for the four categories of males are displayed, as well as the extent of within category variance (ovals) and loadings of individuals within each category (points at the end of solid lines).

Figure 3. Seminal fluid protein abundance variation associated with sperm velocity in old males.

(A) 3-dimension visualization of Principle Component Analysis (PCA3), using the inclusive protein dataset as variables (Old Fast, blue; Old Slow; black; Young Fast; green; Young Slow, yellow). Dimension 1 captures variance associated with old male sperm velocity, including negative weightings for Old Slow and positive weightings for Old Fast. (B) Histogram displaying the distribution of first dimension weightings for proteins exhibiting significant abundance differences between the Slow and Fast sample of the replicated old male (#308)(abundance increase in fast sample, blue; abundance increase in slow sample, black). (C) Histogram displaying the excess or deficit of blood plasma proteins (red) and exosome markers (black) across first dimension weight ranges.

Figure 4. Comparison of the Red junglefowl and domestic chicken seminal fluid proteomes.

(A) Venn diagram indicating the size and overlap between the seminal fluid proteomes of the Red junglefowl and domestic chicken. (B) Overlap in proteins identified in the domestic chicken in relation to protein abundance in the Red junglefowl proteome. (C) Linear regression identifies a significant correlation in the abundance of proteins identified in both the domestic chicken and Red junglefowl proteomes. Protein abundance is plotted as log transformed APEX values, which represent normalized estimates of protein abundance in each sample. (D) Cumulative protein abundance distribution of the domestic chicken (black) and Red junglefowl (red) proteomes.