Project description:The vertebrate nuclear hormone receptor steroidogenic factor 1 (SF1; NR5A1) controls reproductive development and regulates the transcription of steroid-modifying cytochrome P450 genes. We find that the SF1-related Drosophila nuclear hormone receptor HR39 is also essential for sexual development. In Hr39 mutant females, the sperm-storing spermathecae and glandular parovaria are absent or defective, causing sterility. Our results indicate that spermathecae and parovaria secrete reproductive tract proteins required for sperm maturation and function, like the mammalian epididymis and female reproductive tract. Hr39 controls the expression of specific cytochrome P450 genes and is required in females both to activate spermathecal secretion and repress male-specific courtship genes such as takeout. Thus, a pathway that, in vertebrates, controls sex-specific steroid hormone production, also mediates reproductive functions in an invertebrate. Our findings suggest that Drosophila can be used to model more aspects of mammalian reproductive biology than previously believed. Experiment Overall Design: Wild type and Hr39(04443) Spermathecae, Wild type and Hr39(04443) Reproductive Tract
Project description:The vertebrate nuclear hormone receptor steroidogenic factor 1 (SF1; NR5A1) controls reproductive development and regulates the transcription of steroid-modifying cytochrome P450 genes. We find that the SF1-related Drosophila nuclear hormone receptor HR39 is also essential for sexual development. In Hr39 mutant females, the sperm-storing spermathecae and glandular parovaria are absent or defective, causing sterility. Our results indicate that spermathecae and parovaria secrete reproductive tract proteins required for sperm maturation and function, like the mammalian epididymis and female reproductive tract. Hr39 controls the expression of specific cytochrome P450 genes and is required in females both to activate spermathecal secretion and repress male-specific courtship genes such as takeout. Thus, a pathway that, in vertebrates, controls sex-specific steroid hormone production, also mediates reproductive functions in an invertebrate. Our findings suggest that Drosophila can be used to model more aspects of mammalian reproductive biology than previously believed. Keywords: mutant/wild-type comparison and tissue comparison
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:Fertility depends on the progression of complex and coordinated postmating processes within the extracellular luminal environment of the female reproductive tract (FRT). To achieve a more comprehensive level of knowledge regarding female-derived proteins available to interact with the ejaculate, we utilized semiquantitative mass spectrometry-based proteomics to study the composition of the FRT tissue and, separately, the luminal fluid, before and after mating in Drosophila melanogaster. Our approach leveraged whole-fly isotopic labelling to delineate between female proteins and those transferred from males in the ejaculate. The dynamic mating-induced proteomic changes in the extracellular FRT luminal fluid further informs our understanding of secretory mechanisms of the FRT and serves as a foundation for establishing the roles of ejaculate-female interactions in fertility.
Project description:Reproductive traits that influence female remating and competitive fertilization rapidly evolve in response to sexual selection and sexual conflict. One such trait, observed across diverse animal taxa, is the formation of a structural plug inside the female reproductive tract, either during or shortly after mating. In Drosophila melanogaster, male seminal fluid forms a mating plug inside the female bursa, which has been demonstrated to influence sperm entry into storage and latency of female remating. Processing of the plug, including its eventual ejection from the female's reproductive tract, influences the competitive fertilization success of her mates and is mediated by female × male genotypic interactions. However, female contributions to plug formation and processing have received limited attention. Using developmental mutants that lack glandular female reproductive tract tissues, we reveal that these glandular tissues are essential for the mating plug to be ejected. We further use proteomics to demonstrate that female glandular proteins, and especially proteolytic enzymes, contribute to mating plug composition and that the absence of glands has a widespread impact of plug formation and composition. Together, these phenotypic and molecular data resolve molecular mechanisms of important postmating, intersexual interactions and cryptic female choice.
Project description:We compared gene expression in oviduct tissues between unmated (control) and mated hen. As spermatozoa are foreign to the female reproductive tract therefore we were interested to look at how spermatozoa survive in the female reproductive tract and keep their fertilization potentiality. To check that we collected tissues from oviduct of both control and mated female chicken and compared if sperm deposition to the oviduct made any gene expression shift related to sperm survival.
Project description:Male Anophele gambiae mosquitoes transfer the steroid hormone 20-hydroxyecdysone (20E) to the female lower reproductive tract during mating. We have shown that this sexually transferred hormone is a key regulator of monandry and oviposition in An. gambiae females. In order to identify possible molecular pathways underlying the effects induced by sexually transferred 20E we investigated the transcriptional response in the atrium and spermatheca at 24 hours after 20E injection in the thorax of virgin females, which delivers to these reproductive tissues 20E levels comparable to those detected after mating. By comparing these results to those of microarrays assessing the post-mating transcriptional response in the female lower reproductive tract (LRT) at 24 hours post copulation, we were able to identify mating responsive genes likely regulated by male transferred 20E. For each experiment atrium and spermatheca were dissected from females 24 hours after 20E or ethanol (10%) injection and transcriptional profiles compared across 4 biological replicates using Agilent two-color microarrays
Project description:We present data using a novel method to simultaneously identify and quantify transferred male seminal proteins and the female reproductive proteome using multiplexed Tandem-Mass-Tag (TMT) isobaric labelling of the lower female reproductive tracts dissected from virgin- or recently mated- females of three species of the virilis group. We identified over 200 putative male ejaculate proteins many of which show differential abundance between species. We also identified over 2000 proteins providing the first description of the Drosophila female reproductive tract proteome outside of the melanogaster group which also shows significant divergence between species. We then assessed the utility of species-specific compared to single species query databases for protein identification and quantification.