Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Mating induced transcriptional changes in the female lower reproductive tract (LRT) of Anopheles gambiae mosquitoes at three time points (3, 12 and 24 hours) post mating.

ABSTRACT: To gain better insight into the molecular link between the post-mating transcriptional and morphological changes and the behavioral and physiological shift induced by copulation in An. gambiae females, we performed an in-depth transcriptional analysis of the female lower reproductive tract (LRT), comprising the atrium and the spermatheca, to identify the molecular pathways specifically regulated in these female reproductive tissues. We analyzed three time points post mating (3, 12 and 24 hours post mating) in order to capture transcriptional changes occurring within a broad time window after copulation. For each time point, tissues were collected from mated and age-matched virgin females and the transcriptional profile compared across 4 biological replicates using Agilent one-color microarrays.

ORGANISM(S): Anopheles gambiae

SUBMITTER: Sara Mitchell

PROVIDER: E-MTAB-3022 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description: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.

Project description:In Drosophila melanogaster, mating radically transforms female physiology and behavior. Post-mating responses include an increase in the oviposition rate, a reduction in female receptivity, and an activation of the immune system . The fitness consequences of mating are similarly dramatic – females must mate once in order to produce fertile eggs, but additional matings have a clear negative effect. Previously, microarrays have been used to examine gene expression of females differing in their reproductive status with the aim of identifying genes influenced by mating. However, since only virgin and single mated females were compared, transcriptional changes associated with reproduction (under natural selection) and the effects of male-induced harm (under sexually antagonistic selection) cannot be disentangled. We partitioned these fundamentally different effects by instead examining the expression profiles of virgin, single mated and double mated females. We found substantial effects relating to reproduction and further effects that are only attributable to a second mating. Immune response genes dominate this male-induced harm effect indicating that the cost of mating may be due partly to this system's activation. We propose that both sexually antagonistic and natural selection have been important in the evolution of the innate immunity genes, thereby contributing to the sexual dimorphismand rapid evolution at these loci. Keywords: Female response to mating Female flies were flash frozen in liquid nitrogen either as virgins or 6 hours after mating and stored at -80°C until RNA extraction was performed (not more than 2 days). 8 whole flies – randomly selected within each treatment – were pooled for each extraction. Total RNA was extracted using Trizol (Invitrogen) and purified with an RNeasy Mini Kit (Qiagen). RNA quantity and quality was checked with an Agilent Bioanalyzer. According to the manufacturer's instructions, samples were prepared and hybridized to Affymetrix GeneChip Drosophila Genome 2.0 (Affymetrix, Santa Clara, CA, USA) by the Uppsala Array Platform (Uppsala, Sweden). Each experimental treatment consisted of 4 independent RNA extractions and hybridizations, giving a total of 12 arrays.

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.

Dataset Information

Mating induced transcriptional changes in the female lower reproductive tract (LRT) of Anopheles gambiae mosquitoes at three time points (3, 12 and 24 hours) post mating.

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