RESUMO
Sex peptide (SP), a seminal fluid protein of Drosophila melanogaster males, has been described as driving a virgin-to-mated switch in females, through eliciting an array of responses including increased egg laying, activity, and food intake and a decreased remating rate. While it is known that SP achieves this, at least in part, by altering neuronal signaling in females, the genetic architecture and temporal dynamics of the female's response to SP remain elusive. We used a high-resolution time series RNA-sequencing dataset of female heads at 10 time points within the first 24 h after mating to learn about the genetic architecture, at the gene and exon levels, of the female's response to SP. We find that SP is not essential to trigger early aspects of a virgin-to-mated transcriptional switch, which includes changes in a metabolic gene regulatory network. However, SP is needed to maintain and diversify metabolic changes and to trigger changes in a neuronal gene regulatory network. We further find that SP alters rhythmic gene expression in females and suggests that SP's disruption of the female's circadian rhythm might be key to its widespread effects.
Assuntos
Relógios Circadianos , Proteínas de Drosophila , Animais , Masculino , Feminino , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Espermatozoides/metabolismo , Relógios Circadianos/genética , Fatores de Tempo , Peptídeos/metabolismo , Perfilação da Expressão Gênica , Comportamento Sexual Animal/fisiologiaRESUMO
SignificanceIn species with internal fertilization, sperm spend an important part of their lives within the female. To examine the life history of the sperm during this time, we used semiquantitative proteomics and sex-specific isotopic labeling in fruit flies to determine the extent of molecular continuity between male and female reproductive tracts and provide a global catalog of sperm-associated proteins. Multiple seminal fluid proteins and female proteins associate with sperm immediately after mating. Few seminal fluid proteins remain after long-term sperm storage, whereas female-derived proteins constitute one-fifth of the postmating sperm proteome by then. Our data reveal a molecular "hand-off" from males to females, which we postulate to be an important component of sperm-female interactions.
Assuntos
Drosophila/fisiologia , Genitália , Espermatozoides/metabolismo , Animais , Drosophila/crescimento & desenvolvimento , Feminino , Estágios do Ciclo de Vida , Masculino , Proteoma , Proteômica , Reprodução , Proteínas de Plasma Seminal/metabolismo , Comportamento Sexual AnimalRESUMO
Mosaic animals have provided the platform for many fundamental discoveries in developmental biology, cell biology, and other fields. Techniques to produce mosaic animals by mitotic recombination have been extensively developed in Drosophila melanogaster but are less common for other laboratory organisms. Here, we report mosaic analysis by gRNA-induced crossing-over (MAGIC), a new technique for generating mosaic animals based on DNA double-strand breaks produced by CRISPR/Cas9. MAGIC efficiently produces mosaic clones in both somatic tissues and the germline of Drosophila. Further, by developing a MAGIC toolkit for 1 chromosome arm, we demonstrate the method's application in characterizing gene function in neural development and in generating fluorescently marked clones in wild-derived Drosophila strains. Eliminating the need to introduce recombinase-recognition sites into the genome, this simple and versatile system simplifies mosaic analysis in Drosophila and can in principle be applied in any organism that is compatible with CRISPR/Cas9.
Assuntos
Sistemas CRISPR-Cas/genética , Troca Genética/genética , Mosaicismo/embriologia , RNA Guia de Cinetoplastídeos/fisiologia , Animais , Animais Geneticamente Modificados , Clonagem Molecular/métodos , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero , Feminino , Edição de Genes/métodos , Marcação de Genes/métodos , Vetores Genéticos , Genoma de Inseto , Masculino , FenótipoRESUMO
Drosophila melanogaster females experience a large shift in energy homeostasis after mating to compensate for nutrient investment in egg production. To cope with this change in metabolism, mated females undergo widespread physiological and behavioral changes, including increased food intake and altered digestive processes. The mechanisms by which the female digestive system responds to mating remain poorly characterized. Here, we demonstrate that the seminal fluid protein Sex Peptide (SP) is a key modulator of female post-mating midgut growth and gene expression. SP is both necessary and sufficient to trigger post-mating midgut growth in females under normal nutrient conditions, and likely acting via its receptor, Sex Peptide Receptor (SPR). Moreover, SP is responsible for almost the totality of midgut transcriptomic changes following mating, including up-regulation of protein and lipid metabolism genes and down-regulation of carbohydrate metabolism genes. These changes in metabolism may help supply the female with the nutrients required to sustain egg production. Thus, we report a role for SP in altering female physiology to enhance reproductive output: Namely, SP triggers the switch from virgin to mated midgut state.
Assuntos
Sistema Digestório/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fertilidade/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptores de Peptídeos/metabolismo , Reprodução/fisiologia , Transcriptoma/genética , Animais , Copulação , Sistema Digestório/anatomia & histologia , Sistema Digestório/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Fertilidade/genética , Ontologia Genética , Intestinos/anatomia & histologia , Intestinos/crescimento & desenvolvimento , Intestinos/fisiologia , Masculino , Receptores de Peptídeos/genética , Reprodução/genética , Sêmen/metabolismo , Comportamento Sexual Animal/fisiologia , Transcriptoma/fisiologiaRESUMO
CRISPR/Cas9 has emerged as a powerful technology for tissue-specific mutagenesis. However, tissue-specific CRISPR/Cas9 tools currently available in Drosophila remain deficient in three significant ways. First, many existing gRNAs are inefficient, such that further improvements of gRNA expression constructs are needed for more efficient and predictable mutagenesis in both somatic and germline tissues. Second, it has been difficult to label mutant cells in target tissues with current methods. Lastly, application of tissue-specific mutagenesis at present often relies on Gal4-driven Cas9, which hampers the flexibility and effectiveness of the system. Here, we tackle these deficiencies by building upon our previous CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) tools. First, we significantly improved gRNA efficiency in somatic tissues by optimizing multiplexed gRNA design. Similarly, we also designed efficient dual-gRNA vectors for the germline. Second, we developed methods to positively and negatively label mutant cells in tissue-specific mutagenesis by incorporating co-CRISPR reporters into gRNA expression vectors. Lastly, we generated genetic reagents for convenient conversion of existing Gal4 drivers into tissue-specific Cas9 lines based on homology-assisted CRISPR knock-in. In this way, we expand the choices of Cas9 for CRISPR-TRiM analysis to broader tissues and developmental stages. Overall, our upgraded CRISPR/Cas9 tools make tissue-specific mutagenesis more versatile, reliable, and effective in Drosophila These improvements may be also applied to other model systems.
Assuntos
Sistemas CRISPR-Cas , Drosophila/genética , Técnicas Genéticas , Mutagênese , Animais , Feminino , Masculino , RNA Guia de CinetoplastídeosRESUMO
Even in well-characterized genomes, many transcripts are considered noncoding RNAs (ncRNAs) simply due to the absence of large open reading frames (ORFs). However, it is now becoming clear that many small ORFs (smORFs) produce peptides with important biological functions. In the process of characterizing the ribosome-bound transcriptome of an important cell type of the seminal fluid-producing accessory gland of Drosophila melanogaster, we detected an RNA, previously thought to be noncoding, called male-specific abdominal (msa). Notably, msa is nested in the HOX gene cluster of the Bithorax complex and is known to contain a micro-RNA within one of its introns. We find that this RNA encodes a "micropeptide" (9 or 20 amino acids, MSAmiP) that is expressed exclusively in the secondary cells of the male accessory gland, where it seems to accumulate in nuclei. Importantly, loss of function of this micropeptide causes defects in sperm competition. In addition to bringing insights into the biology of a rare cell type, this work underlines the importance of small peptides, a class of molecules that is now emerging as important actors in complex biological processes.
Assuntos
Infertilidade Masculina/genética , Mutação com Perda de Função , Espermatozoides/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Masculino , Peptídeos/genética , Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: The female reproductive tract is exposed directly to the male's ejaculate, making it a hotspot for mating-induced responses. In Drosophila melanogaster, changes in the reproductive tract are essential to optimize fertility. Many changes occur within minutes after mating, but such early timepoints are absent from published RNA-seq studies. We measured transcript abundances using RNA-seq and microRNA-seq of reproductive tracts of unmated and mated females collected at 10-15 min post-mating. We further investigated whether early transcriptome changes in the female reproductive tract are influenced by inhibiting BMPs in secondary cells, a condition that depletes exosomes from the male's ejaculate. RESULTS: We identified 327 differentially expressed genes. These were mostly upregulated post-mating and have roles in tissue morphogenesis, wound healing, and metabolism. Differentially abundant microRNAs were mostly downregulated post-mating. We identified 130 predicted targets of these microRNAs among the differentially expressed genes. We saw no detectable effect of BMP inhibition in secondary cells on transcript levels in the female reproductive tract. CONCLUSIONS: Our results indicate that mating induces early changes in the female reproductive tract primarily through upregulation of target genes, rather than repression. The upregulation of certain target genes might be mediated by the mating-induced downregulation of microRNAs. Male-derived exosomes and other BMP-dependent products were not uniquely essential for this process. Differentially expressed genes and microRNAs provide candidates that can be further examined for their participation in the earliest alterations of the reproductive tract microenvironment.
Assuntos
Proteínas de Drosophila , MicroRNAs , Animais , Feminino , Masculino , Drosophila melanogaster/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Reprodução/genética , Fertilidade/fisiologia , Genitália , Comportamento Sexual Animal , Proteínas de Drosophila/genéticaRESUMO
Declining ejaculate performance with male age is taxonomically widespread and has broad fitness consequences. Ejaculate success requires fully functional germline (sperm) and soma (seminal fluid) components. However, some aging theories predict that resources should be preferentially diverted to the germline at the expense of the soma, suggesting differential impacts of aging on sperm and seminal fluid and trade-offs between them or, more broadly, between reproduction and lifespan. While harmful effects of male age on sperm are well known, we do not know how much seminal fluid deteriorates in comparison. Moreover, given the predicted trade-offs, it remains unclear whether systemic lifespan-extending interventions could ameliorate the declining performance of the ejaculate as a whole. Here, we address these problems using Drosophila melanogaster. We demonstrate that seminal fluid deterioration contributes to male reproductive decline via mating-dependent mechanisms that include posttranslational modifications to seminal proteins and altered seminal proteome composition and transfer. Additionally, we find that sperm production declines chronologically with age, invariant to mating activity such that older multiply mated males become infertile principally via reduced sperm transfer and viability. Our data, therefore, support the idea that both germline and soma components of the ejaculate contribute to male reproductive aging but reveal a mismatch in their aging patterns. Our data do not generally support the idea that the germline is prioritized over soma, at least, within the ejaculate. Moreover, we find that lifespan-extending systemic down-regulation of insulin signaling results in improved late-life ejaculate performance, indicating simultaneous amelioration of both somatic and reproductive aging.
Assuntos
Envelhecimento , Drosophila melanogaster , Proteínas de Plasma Seminal , Espermatozoides , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Feminino , Fertilidade/genética , Fertilidade/fisiologia , Infertilidade Masculina/genética , Infertilidade Masculina/fisiopatologia , Masculino , Proteoma/análise , Proteoma/genética , Proteoma/fisiologia , Proteínas de Plasma Seminal/análise , Proteínas de Plasma Seminal/fisiologia , Comportamento Sexual Animal/fisiologia , Espermatozoides/química , Espermatozoides/fisiologiaRESUMO
Neuronal networks are the standard heuristic model today for describing brain activity associated with animal behavior. Recent studies have revealed an extensive role for a completely distinct layer of networked activities in the brain-the gene regulatory network (GRN)-that orchestrates expression levels of hundreds to thousands of genes in a behavior-related manner. We examine emerging insights into the relationships between these two types of networks and discuss their interplay in spatial as well as temporal dimensions, across multiple scales of organization. We discuss properties expected of behavior-related GRNs by drawing inspiration from the rich literature on GRNs related to animal development, comparing and contrasting these two broad classes of GRNs as they relate to their respective phenotypic manifestations. Developmental GRNs also represent a third layer of network biology, playing out over a third timescale, which is believed to play a crucial mediatory role between neuronal networks and behavioral GRNs. We end with a special emphasis on social behavior, discuss whether unique GRN organization and cis-regulatory architecture underlies this special class of behavior, and review literature that suggests an affirmative answer.
Assuntos
Comportamento , Encéfalo/fisiologia , Redes Reguladoras de Genes , Animais , Encéfalo/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , HumanosRESUMO
BACKGROUND: Male-derived seminal fluid proteins (SFPs) that enter female fruitflies during mating induce a myriad of physiological and behavioral changes, optimizing fertility of the mating pair. Some post-mating changes in female Drosophila melanogaster persist for ~10-14 days. Their long-term persistence is because the seminal protein that induces these particular changes, the Sex Peptide (SP), is retained long term in females by binding to sperm, with gradual release of its active domain from sperm. Several other "long-term response SFPs" (LTR-SFPs) "prime" the binding of SP to sperm. Whether female factors play a role in this process is unknown, though it is important to study both sexes for a comprehensive physiological understanding of SFP/sperm interactions and for consideration in models of sexual conflict. RESULTS: We report here that sperm in male ejaculates bind SP more weakly than sperm that have entered females. Moreover, we show that the amount of SP, and other SFPs, bound to sperm increases with time and transit of individual seminal proteins within the female reproductive tract (FRT). Thus, female contributions are needed for maximal and appropriate binding of SP, and other SFPs, to sperm. Towards understanding the source of female molecular contributions, we ablated spermathecal secretory cells (SSCs) and/or parovaria (female accessory glands), which contribute secretory proteins to the FRT. We found no dramatic change in the initial levels of SP bound to sperm stored in mated females with ablated or defective SSCs and/or parovaria, indicating that female molecules that facilitate the binding of SP to sperm are not uniquely derived from SSCs and parovaria. However, we observed higher levels of SP (and sperm) retention long term in females whose SSCs and parovaria had been ablated, indicating secretions from these female tissues are necessary for the gradual release of Sex Peptide's active region from stored sperm. CONCLUSION: This study reveals that the SP-sperm binding pathway is not entirely male-derived and that female contributions are needed to regulate the levels of SP associated with sperm stored in their storage sites.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Masculino , Feminino , Drosophila melanogaster/fisiologia , Proteínas de Drosophila/metabolismo , Sêmen/metabolismo , Espermatozoides/fisiologia , Comportamento Sexual Animal/fisiologia , Peptídeos/metabolismoRESUMO
In many animal species, females undergo physiological and behavioral changes after mating. Some of these changes are driven by male-derived seminal fluid proteins and are critical for fertilization success. Unfortunately, our understanding of the molecular interplay between female and male reproductive proteins remains inadequate. Here, we analyze the postmating response in a Drosophila species that has evolved strong gametic incompatibility with its sister species; Drosophila novamexicana females produce only â¼1% fertilized eggs in crosses with Drosophila americana males, compared to â¼98% produced in within-species crosses. This incompatibility is likely caused by mismatched male and female reproductive molecules. In this study, we use short-read RNA sequencing to examine the evolutionary dynamics of female reproductive genes and the postmating transcriptome response in crosses within and between species. First, we found that most female reproductive tract genes are slow-evolving compared to the genome average. Second, postmating responses in con- and heterospecific matings are largely congruent, but heterospecific matings induce expression of additional stress-response genes. Some of those are immunity genes that are activated by the Imd pathway. We also identify several genes in the JAK/STAT signaling pathway that are induced in heterospecific, but not conspecific mating. While this immune response was most pronounced in the female reproductive tract, we also detect it in the female head and ovaries. These results show that the female's postmating transcriptome-level response is determined in part by the genotype of the male, and that divergence in male reproductive genes and/or traits can have immunogenic effects on females.
Assuntos
Drosophila/genética , Evolução Molecular , Isolamento Reprodutivo , Animais , Copulação , Drosophila/metabolismo , Feminino , Genitália Feminina/metabolismo , Masculino , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteínas de Plasma Seminal , Transcrição Gênica , TranscriptomaRESUMO
Seminal fluid proteins (Sfps) have striking effects on the behaviour and physiology of females in many insects. Some Drosophila melanogaster Sfps are not highly or exclusively expressed in the accessory glands, but derive from, or are additionally expressed in other male reproductive tissues. The full suite of Sfps includes transferred proteins from all male reproductive tissues, regardless of expression level or presence of a signal peptide.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Secreções Corporais/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Feminino , Masculino , Proteínas de Plasma Seminal/metabolismoRESUMO
Egg activation is the process in which mature oocytes are released from developmental arrest and gain competency for embryonic development. In Drosophila and other arthropods, eggs are activated by mechanical pressure in the female reproductive tract, whereas in most other species, eggs are activated by fertilization. Despite the difference in the trigger, Drosophila shares many conserved features with higher vertebrates in egg activation, including a rise of intracellular calcium in response to the trigger. In Drosophila, this calcium rise is initiated by entry of extracellular calcium due to opening of mechanosensitive ion channels and initiates a wave that passes across the egg prior to initiation of downstream activation events. Here, we combined inhibitor tests, germ-line-specific RNAi knockdown, and germ-line-specific CRISPR/Cas9 knockout to identify the Transient Receptor Potential (TRP) channel subfamily M (Trpm) as a critical channel that mediates the calcium influx and initiates the calcium wave during Drosophila egg activation. We observed a reduction in the proportion of eggs that hatched from trpm germ-line knockout mutant females, although eggs were able to complete some egg activation events including cell cycle resumption. Since a mouse ortholog of Trpm was recently reported also to be involved in calcium influx during egg activation and in further embryonic development, our results suggest that calcium uptake from the environment via TRPM channels is a deeply conserved aspect of egg activation.
Assuntos
Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Desenvolvimento Embrionário , Fertilização/fisiologia , Oócitos/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Sistemas CRISPR-Cas , Sinalização do Cálcio/fisiologia , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Feminino , Masculino , Oócitos/citologia , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genéticaRESUMO
BACKGROUND: Mating induces behavioral and physiological changes in the arbovirus vector Aedes aegypti, including stimulation of egg development and oviposition, increased survival, and reluctance to re-mate with subsequent males. Transferred seminal fluid proteins and peptides derived from the male accessory glands induce these changes, though the mechanism by which they do this is not known. RESULTS: To determine transcriptome changes induced by seminal proteins, we injected extract from male accessory glands and seminal vesicles (MAG extract) into females and examined female lower reproductive tract (LRT) transcriptomes 24 h later, relative to non-injected controls. MAG extract induced 87 transcript-level changes, 31 of which were also seen in a previous study of the LRT 24 h after a natural mating, including 15 genes with transcript-level changes similarly observed in the spermathecae of mated females. The differentially-regulated genes are involved in diverse molecular processes, including immunity, proteolysis, neuronal function, transcription control, or contain predicted small-molecule binding and transport domains. CONCLUSIONS: Our results reveal that seminal fluid proteins, specifically, can induce gene expression responses after mating and identify gene targets to further investigate for roles in post-mating responses and potential use in vector control.
Assuntos
Aedes , Aedes/genética , Animais , Feminino , Masculino , Mosquitos Vetores/genética , Reprodução/genética , Comportamento Sexual Animal , TranscriptomaRESUMO
The biogenic monoamine octopamine (OA) is a crucial regulator of invertebrate physiology and behavior. Since its discovery in the 1950s in octopus salivary glands, OA has been implicated in many biological processes among diverse invertebrate lineages. It can act as a neurotransmitter, neuromodulator and neurohormone in a variety of biological contexts, and can mediate processes including feeding, sleep, locomotion, flight, learning, memory, and aggression. Here, we focus on the roles of OA in female reproduction in insects. OA is produced in the octopaminergic neurons that innervate the female reproductive tract (RT). It exerts its effects by binding to receptors throughout the RT to generate tissue- and region-specific outcomes. OA signaling regulates oogenesis, ovulation, sperm storage, and reproductive behaviors in response to the female's internal state and external conditions. Mating profoundly changes a female's physiology and behavior. The female's OA signaling system interacts with, and is modified by, male molecules transferred during mating to elicit a subset of the post-mating changes. Since the role of OA in female reproduction is best characterized in the fruit fly Drosophila melanogaster, we focus our discussion on this species but include discussion of OA in other insect species whenever relevant. We conclude by proposing areas for future research to further the understanding of OA's involvement in female reproduction in insects.
Assuntos
Insetos/fisiologia , Octopamina/fisiologia , Comportamento Sexual Animal/fisiologia , Animais , Feminino , Reprodução/fisiologiaRESUMO
In almost all animals studied to date, the crucial process of egg activation, by which an arrested mature oocyte transitions into an actively developing embryo, initiates with an increase in Ca2+ in the oocyte's cytoplasm. This Ca2+ rise sets off a series of downstream events, including the completion of meiosis and the dynamic remodeling of the oocyte transcriptome and proteome, which prepares the oocyte for embryogenesis. Calcineurin is a highly conserved phosphatase that is activated by Ca2+ upon egg activation and that is required for the resumption of meiosis in Xenopus,, ascidians, and Drosophila. The molecular mechanisms by which calcineurin transduces the calcium signal to regulate meiosis and other downstream events are still unclear. In this study, we investigate the regulatory role of calcineurin during egg activation in Drosophila melanogaster,. Using mass spectrometry, we quantify the phosphoproteomic and proteomic changes that occur during egg activation, and we examine how these events are affected when calcineurin function is perturbed in female germ cells. Our results show that calcineurin regulates hundreds of phosphosites and also influences the abundance of numerous proteins during egg activation. We find calcineurin-dependent changes in cell cycle regulators including Fizzy (Fzy), Greatwall (Gwl) and Endosulfine (Endos); in protein translation modulators including PNG, NAT, eIF4G, and eIF4B; and in important components of signaling pathways including GSK3ß and Akt1. Our results help elucidate the events that occur during the transition from oocyte to embryo.
Assuntos
Calcineurina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Óvulo/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Feminino , Células Germinativas/metabolismo , Masculino , Oócitos/metabolismo , Fosfopeptídeos/metabolismo , Fosforilação , Biossíntese de Proteínas , Proteômica , Regulação para CimaRESUMO
The yellow fever mosquito, Aedes aegypti,, transmits several viruses causative of serious diseases, including dengue, Zika, and chikungunya. Some proposed efforts to control this vector involve manipulating reproduction to suppress wild populations or to replace them with disease-resistant mosquitoes. The design of such strategies requires an intimate knowledge of reproductive processes, yet our basic understanding of reproductive genetics in this vector remains largely incomplete. To accelerate future investigations, we have comprehensively catalogued sperm and seminal fluid proteins (SFPs) transferred to females in the ejaculate using tandem mass spectrometry. By excluding female-derived proteins using an isotopic labeling approach, we identified 870 sperm proteins and 280 SFPs. Functional composition analysis revealed parallels with known aspects of sperm biology and SFP function in other insects. To corroborate our proteome characterization, we also generated transcriptomes for testes and the male accessory glands-the primary contributors to Ae. aegypti, sperm and seminal fluid, respectively. Differential gene expression of accessory glands from virgin and mated males suggests that transcripts encoding proteins involved in protein translation are upregulated post-mating. Several SFP transcripts were also modulated after mating, but >90% remained unchanged. Finally, a significant enrichment of SFPs was observed on chromosome 1, which harbors the male sex determining locus in this species. Our study provides a comprehensive proteomic and transcriptomic characterization of ejaculate production and composition and thus provides a foundation for future investigations of Ae. aegypti, reproductive biology, from functional analysis of individual proteins to broader examination of reproductive processes.
Assuntos
Aedes/genética , Aedes/metabolismo , Proteínas de Insetos/metabolismo , Sêmen/metabolismo , Proteínas de Plasma Seminal/metabolismo , Espermatozoides/metabolismo , Estruturas Animais/metabolismo , Animais , Cromossomos/genética , Ejaculação , Ontologia Genética , Masculino , Proteoma , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Plasma Seminal/genética , Comportamento Sexual Animal , Transcriptoma/genéticaRESUMO
Although thousands of long non-coding RNAs (lncRNA) have been identified in the genomes of higher eukaryotes, the precise function of most of them is still unclear. Here, we show that a >65 kb, male-specific, lncRNA, called male-specific abdominal (msa) is required for the development of the secondary cells of the Drosophila male accessory gland (AG). msa is transcribed from within the Drosophila bithorax complex and shares much of its sequence with another lncRNA, the iab-8 lncRNA, which is involved in the development of the central nervous system (CNS). Both lncRNAs perform much of their functions via a shared miRNA embedded within their sequences. Loss of msa, or of the miRNA it contains, causes defects in secondary cell morphology and reduces male fertility. Although both lncRNAs express the same miRNA, the phenotype in the secondary cells and the CNS seem to reflect misregulation of different targets in the two tissues.
Assuntos
Drosophila/fisiologia , Organogênese/genética , RNA Longo não Codificante/fisiologia , Animais , Animais Geneticamente Modificados , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Fertilidade/genética , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação , Oviposição/fisiologia , Fenótipo , Comportamento Sexual Animal/fisiologiaRESUMO
The transition from a developmentally arrested mature oocyte to a developing embryo requires a series of highly conserved events, collectively known as egg activation. All of these events are preceded by a ubiquitous rise of intracellular calcium, which results from influx of external calcium and/or calcium release from internal storage. In Drosophila, this calcium rise initiates from the pole(s) of the oocyte by influx of external calcium in response to mechanical triggers. It is thought to trigger calcium responsive kinases and/or phosphatases, which in turn alter the oocyte phospho-proteome to initiate downstream events. Recent studies revealed that external calcium enters the activating Drosophila oocyte through Trpm channels, a feature conserved in mouse. The local entry of calcium raises the question of whether Trpm channels are found locally at the poles of the oocyte or are localized around the oocyte periphery, but activated only at the poles. Here, we show that Trpm is distributed all around the oocyte. This requires that it thus be specially regulated at the poles to allow calcium wave initiation. We show that neither egg shape nor local pressure is sufficient to explain this local activation of Trpm channels.
Assuntos
Sinalização do Cálcio/fisiologia , Fertilização/fisiologia , Oócitos/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Drosophila melanogaster , Feminino , Mecanotransdução Celular/fisiologia , Oogênese/fisiologiaRESUMO
Egg activation is the essential process in which mature oocytes gain the competency to proceed into embryonic development. Many events of egg activation are conserved, including an initial rise of intracellular calcium. In some species, such as echinoderms and mammals, changes in the actin cytoskeleton occur around the time of fertilization and egg activation. However, the interplay between calcium and actin during egg activation remains unclear. Here, we use imaging, genetics, pharmacological treatment, and physical manipulation to elucidate the relationship between calcium and actin in living Drosophila eggs. We show that, before egg activation, actin is smoothly distributed between ridges in the cortex of the dehydrated mature oocytes. At the onset of egg activation, we observe actin spreading out as the egg swells though the intake of fluid. We show that a relaxed actin cytoskeleton is required for the intracellular rise of calcium to initiate and propagate. Once the swelling is complete and the calcium wave is traversing the egg, it leads to a reorganization of actin in a wavelike manner. After the calcium wave, the actin cytoskeleton has an even distribution of foci at the cortex. Together, our data show that calcium resets the actin cytoskeleton at egg activation, a model that we propose to be likely conserved in other species.