Quantitative Proteomics Identification of Seminal Fluid Proteins in Male Drosophila melanogaster

Abstract

Seminal fluid contains some of the fastest evolving proteins currently known. These seminal fluid proteins (Sfps) play crucial roles in reproduction, such as supporting sperm function, and particularly in insects, modifying female physiology and behavior. Identification of Sfps in small animals is challenging, and often relies on samples taken from the female reproductive tract after mating. A key pitfall of this method is that it might miss Sfps that are of low abundance because of dilution in the female-derived sample or rapid processing in females. Here we present a new and complementary method, which provides added sensitivity to Sfp identification. We applied label-free quantitative proteomics to Drosophila melanogaster, male reproductive tissue - where Sfps are unprocessed, and highly abundant - and quantified Sfps before and immediately after mating, to infer those transferred during copulation. We also analyzed female reproductive tracts immediately before and after copulation to confirm the presence and abundance of known and candidate Sfps, where possible. Results were cross-referenced with transcriptomic and sequence databases to improve confidence in Sfp detection. Our data were consistent with 125 previously reported Sfps. We found nine high-confidence novel candidate Sfps, which were both depleted in mated versus, unmated males and identified within the reproductive tract of mated but not virgin females. We also identified 42 more candidates that are likely Sfps based on their abundance, known expression and predicted characteristics, and revealed that four proteins previously identified as Sfps are at best minor contributors to the ejaculate. The estimated copy numbers for our candidate Sfps were lower than for previously identified Sfps, supporting the idea that our technique provides a deeper analysis of the Sfp proteome than previous studies. Our results demonstrate a novel, high-sensitivity approach to the analysis of seminal fluid proteomes, whose application will further our understanding of reproductive biology.

BMP-signalling inhibition inDrosophilasecondary cells remodels the seminal proteome, and self and rival ejaculate functions

“…To this end, we performed label-free quantitative proteomics on the accessory glands of Dad and control males dissected either before or immediately after mating. This pre- and post-mating approach has previously been shown to provide a deep analysis of the seminal proteome, sensitive to low abundance proteins, while exposing patterns of differential SFP production, depletion, and transfer (19, 51). We detected 1194 proteins on the basis of at least 2 unique peptides (as in 19, 59), of which 88 are SFPs known to be transferred to females (see Materials and Methods ).…”

“…We ran this experiment five times in order to produce five independent biological replicates. Our quantitative proteomics analysis was conducted in accordance with the gel-aided sample preparation (GASP) protocol outlined in detail elsewhere (19, 63). Details of this method, the LC-MS/MS platform, and the data processing and normalization are given in SI Materials and Methods .…”

“…Our assessment of whether a protein was a SFP was based on a reference list provided by Mariana Wolfner (Cornell University, NY) and Geoff Findlay (College of the Holy Cross, MA) and updated to include the high confidence SFPs from Sepil et al (19). We also included Intrepid (intr), despite it not having been conclusively shown to be transferred to females, as we find it at significantly lower abundance in mated glands and because it is known to function in the sex peptide network (16).…”

“…1A). The majority of the ∼200 SFPs known to be transferred to females are produced and stored in the accessory glands (19). Each of the two lobes of the glands is composed of two distinct cell-types (20).…”

DrosophilaSex Peptide Controls the Assembly of Lipid Microcarriers in Seminal Fluid

“…This glandular epithelial tube surrounds a large lumen. The AG secretome and its functions have been extensively characterised and multiple bioactive Accessory gland proteins (Acps) identified (7, 8). Several of these induce behavioural and physiological changes in mated females.…”