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The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster.

ABSTRACT: Seminal proteins from the Drosophila male accessory gland induce post-mating responses (PMR) in females. The PMR comprise behavioral and physiological changes that include increased egg laying, decreased receptivity to courting males, and changes in the storage and use of sperm. Many of these changes are induced by a "sex peptide" (SP) and are maintained by SP's binding to, and slow release from, sperm. The accessory gland contains two secretory cell types with distinct morphological and developmental characteristics. Products of these "main" and "secondary" cells work interdependently to induce and maintain the PMR. To identify individual genes needed for the morphology and function of secondary cells, we studied iab-6(cocu) males, whose secondary cells have abnormal morphology and fail to provide products to maintain the PMR. By RNA-seq, we identified 77 genes that are downregulated by a factor of >5× in iab-6(cocu) males. By functional assays and microscopy, we tested 20 candidate genes and found that at least 9 are required for normal storage and release of SP in mated females. Knockdown of each of these 9 genes consequently leads to a reduction in egg laying and an increase in receptivity over time, confirming a role for the secondary cells in maintaining the long-term PMR. Interestingly, only 1 of the 9 genes, CG3349, encodes a previously reported seminal fluid protein (Sfp), suggesting that secondary cells may perform essential functions beyond the production and modification of known Sfps. At least 3 of the 9 genes also regulate the size and/or abundance of secondary cell vacuoles, suggesting that the vacuoles' contents may be important for the machinery used to maintain the PMR.

SUBMITTER: Sitnik JL

PROVIDER: S-EPMC4788108 | biostudies-other | 2016 Mar

REPOSITORIES: biostudies-other

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The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster.

Sitnik Jessica L JL Gligorov Dragan D Maeda Robert K RK Karch François F Wolfner Mariana F MF

Genetics 20160108 3

Seminal proteins from the Drosophila male accessory gland induce post-mating responses (PMR) in females. The PMR comprise behavioral and physiological changes that include increased egg laying, decreased receptivity to courting males, and changes in the storage and use of sperm. Many of these changes are induced by a "sex peptide" (SP) and are maintained by SP's binding to, and slow release from, sperm. The accessory gland contains two secretory cell types with distinct morphological and develop ...[more]

PMID: 26746709

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Project description:In Drosophila, the accessory gland proteins (Acps) secreted from the male accessory glands (MAGs) and transferred along with sperm into the female reproductive tract have been implicated in triggering postmating behavioral changes, including refractoriness to subsequent mating and propensity to egg laying. Recently, Acps have been found also in Anopheles, suggesting similar functions. Understanding the mechanisms underlying transcriptional regulation of Acps and their functional role in modulating Anopheles postmating behavior may lead to the identification of novel vector control strategies to reduce mosquito populations. We identified heat-shock factor (HSF) binding sites within the Acp promoters of male Anopheles gambiae and discovered three distinct Hsf isoforms; one being significantly up-regulated in the MAGs after mating. Through genome-wide transcription analysis of Hsf-silenced males, we observed significant down-regulation in 50% of the Acp genes if compared to control males treated with a construct directed against an unrelated bacterial sequence. Treated males retained normal life span and reproductive behavior compared to control males. However, mated wild-type females showed a ?46% reduction of egg deposition rate and a ?23% reduction of hatching rate (?58% combined reduction of progeny). Our results highlight an unsuspected role of HSF in regulating Acp transcription in A. gambiae and provide evidence that Acp down-regulation in males leads a significant reduction of progeny, thus opening new avenues toward the development of novel vector control strategies. Total RNA from 4-d-old dsLacZ-treated controls and HSF-silenced males was extracted using TRIzol reagent (Life Technologies), following protocols set according to manufacturer’s instructions. RNA preparation, labelling, hybridization and data analysis were performed by the Oxford Gene Technology (OGT) company. In particular, RNA was further cleaned up using the RNeasy Mini Kit (Qiagen) followed by ethanol precipitation. Sample passing the purity metrics (260/280 and 260/230 ratio) were considered for labeling and hybridization procedures. Labeling was done with cyanine 3 (dsHSF1 and dsHSF123) and cyanine 5 (control dsLacZ). Samples and controls were hybridized in triplicates to the Agilent Mosquito Gene expression arrays (4x44,000, Agilent Technologies, Santa Clara, CA, USA). Data analysis was performed by OGT according to a standardized procedure, as implemented by the software applications Feature Extraction (version 9.5.3.1.), and Genespring GX (version 11.0). Expression data was first normalised using linear and loess normalization in Feature Extraction, then it was imported into Genespring where it was converted into normalised log ratios. The distribution of the data between and within the treatments was checked via boxplots and hierarchical clustering to confirm consistency. Intensity measures from spots flagged as being of poor quality were excluded. Then, the control probes were removed for the statistical analysis: t-tests were carried out for each condition with null hypothesis of log ratio being equal to zero. This was done on the normalised log ratios from the 3 technical replicates. Probes meeting a cut-off of p<0.05 with Benjamini-Hochberg multiple testing correction and with a fold change cutoff greater than 2 were considered statistically and biologically relevant and were used to generate lists of differentially expressed targets.

Project description:In insects, products of the male reproductive tract are essential for initiating and maintaining the female post-mating response (PMR). The PMR includes changes in egg laying, receptivity to courting males, and sperm storage. In Drosophila, previous studies have determined that the main cells of the male accessory gland produce some of the products required for these processes. However, nothing was known about the contribution of the gland's other secretory cell type, the secondary cells. In the course of investigating the late functions of the homeotic gene, Abdominal-B (Abd-B), we discovered that Abd-B is specifically expressed in the secondary cells of the Drosophila male accessory gland. Using an Abd-B BAC reporter coupled with a collection of genetic deletions, we discovered an enhancer from the iab-6 regulatory domain that is responsible for Abd-B expression in these cells and that apparently works independently from the segmentally regulated chromatin domains of the bithorax complex. Removal of this enhancer results in visible morphological defects in the secondary cells. We determined that mates of iab-6 mutant males show defects in long-term egg laying and suppression of receptivity, and that products of the secondary cells are influential during sperm competition. Many of these phenotypes seem to be caused by a defect in the storage and gradual release of sex peptide in female mates of iab-6 mutant males. We also found that Abd-B expression in the secondary cells contributes to glycosylation of at least three accessory gland proteins: ovulin (Acp26Aa), CG1656, and CG1652. Our results demonstrate that long-term post-mating changes observed in mated females are not solely induced by main cell secretions, as previously believed, but that secondary cells also play an important role in male fertility by extending the female PMR. Overall, these discoveries provide new insights into how these two cell types cooperate to produce and maintain a robust female PMR.

Dataset Information

The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster.

Publications

The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster.

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