Dataset Information

Elucidation of the protein composition of mouse seminal vesicle fluid

ABSTRACT: The seminal vesicles are male accessory sex glands that contribute the bulk of the seminal plasma in which mammalian spermatozoa are bathed during ejaculation. In addition to helping convey sperm through the ejaculatory duct, seminal vesicle secretions support sperm survival after ejaculation and influence the female reproductive tract to promote receptivity to pregnancy. Despite its biological importance, there is limited understanding of seminal vesicle fluid (SVF) composition with only 75 proteins having been identified in publicly available proteomic analyses. To address this limitation, here we report new preparative methodology involving sequential solubilization of mouse SVF in guanidine hydrochloride, prior to acetone precipitation and analysis by label-free quantitative mass spectrometry. This strategy identified 126 proteins, including 83 previously undetected in SVF. Members of the seminal vesicle secretory protein family were the most abundant, accounting for 79% of all peptide spectrum matches. Functional analysis of SVF proteins identified inflammation and formation of the vaginal plug as the two most prominent biological processes. Notable additional processes included modulation of sperm function and regulation of the female reproductive tract immune environment. Together, these findings provide a robust methodological framework for future SVF studies and identify novel proteins with the potential to influence both male and female reproductive health.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Seminal Vesicle Fluid

SUBMITTER: David Skerrett-Byrne

LAB HEAD: David Skerrett-Byrne

PROVIDER: PXD028564 | Pride | 2022-01-20

REPOSITORIES: Pride

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Elucidation of the protein composition of mouse seminal vesicle fluid.

Smyth Shannon P SP Nixon Brett B Anderson Amanda L AL Murray Heather C HC Martin Jacinta H JH MacDougall Lily A LA Robertson Sarah A SA Skerrett-Byrne David A DA Schjenken John E JE

Proteomics 20220119 9

The seminal vesicles are male accessory sex glands that contribute the major portion of the seminal plasma in which mammalian spermatozoa are bathed during ejaculation. In addition to conveying sperm through the ejaculatory duct, seminal vesicle secretions support sperm survival after ejaculation, and influence the female reproductive tract to promote receptivity to pregnancy. Analysis of seminal vesicle fluid (SVF) composition by proteomics has proven challenging, due to its highly biased prote ...[more]

PMID: 35014747

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Project description:Male-derived accessory gland proteins (Acps) that are transferred to females during mating have profound effects on female reproductive physiology including increased ovulation, mating inhibition, and effects on sperm utilization and storage. The extreme rates of evolution seen in Acps may be driven by sperm competition and sexual conflict, processes which may ultimately drive complex interactions between female- and male-derived molecules and sperm. However, little is known of how gene expression in female reproductive tissues changes in response to the presence of male molecules and sperm. To characterize this response, we conducted parallel genomic and proteomic analyses of gene expression in the reproductive tract of 3-day-old unmated and mated female Drosophila melanogaster. Using DNA microarrays, we identified 539 transcripts that are differentially expressed in unmated vs. mated females and revealed a striking peak in differential expression at 6 hrs postmating and a marked shift from primarily down-regulated to primarily up-regulated transcripts within 3 hrs after mating. Combining two-dimensional gel electrophoresis and liquid chromatography mass spectrometry analyses, we identified 84 differentially expressed proteins at 3 hrs postmating, including proteins which appeared to undergo post-translational modification. Together, our observations define transcriptional and translational response to mating within the female reproductive tract and suggest a bimodal model of postmating gene expression initially correlated with mating and the final stages of female reproductive tract maturation and later with the declining presence of male reproductive molecules and with sperm maintenance and utilization. Experiment Overall Design: Three-day-old mated and unmated females were dissected to remove the lower reproductive tract (upper uterus, sperm-storage organs, and accessory glands). Mated females were dissected either immediately following mating (0 hr) or at 3, 6, or 24 hrs following the termination of mating. Tracts of 12-40 females of like category were pooled and total RNA extracted via a TRIzol-based protocol. Processing and labeling of transcript was performed by the Molecular Biology Core Facility at the Medical College of Georgia. Arrays from mated females at the different timepoints were compared to unmated females.

Project description: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 behaviour. 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 due to dilution in the female-derived sample or rapid processing in females. Here we present a new and complimentary 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 analysed 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 was consistent with 124 previously reported Sfps. We found 8 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 31 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.

			Action	DRS
	DSB_SVF_Proteome.mzML	Mzml
	DSB_SVF_Proteome.mzid.gz	Mzid
	DSB_SVF_R1.raw	Raw
	DSB_SVF_R2.raw	Raw
	DSB_SVF_R3.raw	Raw