Quantitative Proteomics Identification of Seminal Fluid Proteins in Male Drosophila melanogaster - PubMed (original) (raw)
Quantitative Proteomics Identification of Seminal Fluid Proteins in Male Drosophila melanogaster
Irem Sepil et al. Mol Cell Proteomics. 2019.
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.
Keywords: Animal models*; Drosophila melanogaster*; Label-free quantification; Protein Identification*; Tandem Mass Spectrometry.
© 2019 Sepil et al.
Figures
Fig. 1.
Experimental design. Males are expected to lose seminal fluid proteins (Sfps) from the accessory glands (AGs) and ejaculatory duct (ED) at copulation as they are transferred to females. By analyzing protein abundance in the AGs and ED immediately after copulation versus, in unmated males we can infer Sfps that are likely transferred. Sfps should be significantly more abundant in unmated males than in mated males.
Fig. 2.
Volcano plot displaying all proteins detected in (A,) Male Data Set 1 and (B,) Male Data Set 2 that were identified by at least two unique peptides. The log2 fold change [log2 (unmated) - log2 (newly mated)] is shown on the x, axis and significance is displayed on the y, axis as the negative logarithm (log10 scale) of the fdr corrected p, value. Known Sfps are colored in blue. The candidate Sfps identified in this study are displayed as triangles and colored in red. The high-confidence candidate Sfps are named. The rest of the proteins are colored black. The significance cutoff (p, < 0.05) is highlighted with a dashed line.
Fig. 3.
Venn diagram displaying the protein overlap between Male Data Set 1 and Male Data Set 2 (A,) for known Sfps significantly higher (p, ≤ 0.035) in unmated samples and (B,) for the rest of the proteins significantly higher (p, ≤ 0.049) in unmated samples.
Fig. 4.
Boxplot of the relative abundance of known Sfps and candidate Sfps found in (A,) Male Data Set 1 and (B,) Male Data Set 2. Protein abundances were averaged across all the samples in the experiment and were sorted by decreasing order. Known Sfps are colored in blue and candidate Sfps are colored in red.
Fig. 5.
Volcano plot displaying all proteins detected in the Female Data Set that were identified by at least two unique peptides. The log2 fold change [log2 (newly mated) - log2 (virgin)] is shown on the x, axis and significance is displayed on the y, axis as the negative logarithm (log10 scale) of the fdr corrected p, value. Known Sfps are colored in blue; sperm proteins are colored in orange and high-confidence candidate Sfps are named, displayed as triangles and colored in red. The significance cutoff (p, < 0.05) is highlighted with a dashed line.
Fig. 6.
Boxplot of the relative abundance of functionally important Sfps and the rest of the known Sfps found in (A,) Male Data Set 1 and (B,) Male Data Set 2. Protein abundances were averaged across all the samples in the experiment and were sorted by decreasing order. Functionally important Sfps are colored in yellow and the rest of the known Sfps are colored in blue.
Fig. 7.
Boxplot of the abundances of putative ejaculatory duct specific proteins in accessory gland only samples (AG), ejaculatory duct only samples (DU) and samples containing both the ejaculatory duct and accessory gland (BO). The 11 previouly known Sfps were significantly more abundant (p, ≤ 0.05) both in DU and in BO compared with AG.
References
- Poiani A. (2006) Complexity of seminal fluid: A review. Behav. Ecol. Sociobiol. 60, 289–310
- Hopkins B. R., Sepil I., and Wigby S. (2017) Seminal fluid. Curr. Biol. 27, R404–R405 - PubMed
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