Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Postmating transcriptional changes in reproductive tracts of con- and heterospecifically-mated Drosophila mojavensis females

ABSTRACT: In internally fertilizing organisms, mating involves a series of highly coordinated molecular interactions between the sexes that occur within the female reproductive tract. In species with promiscuous mating systems, traits involved in postcopulatory interactions are expected to evolve rapidly, potentially leading to postmating-prezygotic (PMPZ) reproductive isolation between diverging populations. Here, we use a novel study design to investigate the postmating transcriptional response of Drosophila mojavensis female reproductive tracts following copulation with either conspecific or heterospecific (D. arizonae) males at three time points postmating. Relatively few genes (15 total) were transcriptionally regulated in the female lower reproductive tract in response to conspecific mating. Heterospecifically-mated females exhibited significant perturbations in the expression of the majority of these genes, and also downregulated transcription of a number of others, including several involved in mitochondrial function. These striking regulatory differences indicate failed postcopulatory molecular interactions between the sexes consistent with the strong PMPZ isolation observed for this cross. We also report, for the first time, the transfer of male mRNA transcripts to females during copulation. These included transcripts from male accessory-gland proteins (ACPs), a finding with potentially broad implications for understanding postcopulatory molecular interactions between the sexes. Dataset from Postmating transcriptional changes in reproductive tracts of con- and heterospecifically-mated Drosophila mojavensis females Bono, JM, Matzkin,LM, Kelleher, ES, and Markow, MA, Proceedings of the National Academy of Sciences, USA. The treatments were con- and heterospecific mated D. mojavensis females. Three different post-copulation times points were assayed, 15 minutes, 2 hours and 6 hours. For each time point there were two independent replicates. Additionally, two replicates of a virgin D. mojavensis female control were assayed. A total of 14 arrays. All RNA extractions were from the lower reproductive tract of females.

ORGANISM(S): Drosophila mojavensis

SUBMITTER: Luciano Matzkin

PROVIDER: E-GEOD-27454 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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.

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Postmating transcriptional changes in reproductive tracts of con- and heterospecifically-mated Drosophila mojavensis females

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