Showing papers by "Mariana F. Wolfner published in 2015"

Sexual Conflict and Seminal Fluid Proteins: A Dynamic Landscape of Sexual Interactions

Abstract: Both sexes benefit from successful reproduction, but the different reproductive strategies adopted by males and females may result in differential costs and benefits. This can result in sexual conflict before, during, and after mating. Conflict in the more familiar form of competition can also occur between females and between males, with the latter situation including interejaculate competition. Of the many “weapons” in these conflicts and competitions, this article focuses on the seminal fluid proteins (SFPs) that are made by males and transferred to females during mating. These proteins represent a crucial interface of functional activity between male and female. Transfer of SFPs can affect physiology and, in some animals, the behavior and life span of mated females (reviewed in Chapman 2001; Gillott 2003; Poiani 2006; Avila et al. 2011; Rodriguez-Martinez et al. 2011). Because SFPs have important effects on the most intimate of interactions between the sexes, they are prime candidates to become subject to sexually antagonistic selection (Arnqvist and Rowe 2005). With increasing knowledge of the functions of SFPs, their roles in inter- and intrasexual conflict and their evolutionary responses to conflict are becoming ever more apparent. Here, we explore the roles, evolution, and significance of these male-derived players in sexual conflict. We refer the reader to previous reviews for much of the detailed functional information on SFPs (e.g., Chapman 2001; Gillott 2003; Kubli 2003; Arnqvist and Rowe 2005; Poiani 2006; Sirot et al. 2009; Avila et al. 2011; Rodriguez-Martinez et al. 2011) and focus here instead on selected examples, drawn largely from the study of insects.

Integrated 3D view of postmating responses by the Drosophila melanogaster female reproductive tract, obtained by micro-computed tomography scanning

Abstract: Physiological changes in females during and after mating are triggered by seminal fluid components in conjunction with female-derived molecules. In insects, these changes include increased egg production, storage of sperm, and changes in muscle contraction within the reproductive tract (RT). Such postmating changes have been studied in dissected RT tissues, but understanding their coordination in vivo requires a holistic view of the tissues and their interrelationships. Here, we used high-resolution, multiscale micro-computed tomography (CT) scans to visualize and measure postmating changes in situ in the Drosophila female RT before, during, and after mating. These studies reveal previously unidentified dynamic changes in the conformation of the female RT that occur after mating. Our results also reveal how the reproductive organs temporally shift in concert within the confines of the abdomen. For example, we observed chiral loops in the uterus and in the upper common oviduct that relax and constrict throughout sperm storage and egg movement. We found that specific seminal fluid proteins or female secretions mediate some of the postmating changes in morphology. The morphological movements, in turn, can cause further changes due to the connections among organs. In addition, we observed apparent copulatory damage to the female intima, suggesting a mechanism for entry of seminal proteins, or other exogenous components, into the female’s circulatory system. The 3D reconstructions provided by high-resolution micro-CT scans reveal how male and female molecules and anatomy interface to carry out and coordinate mating-dependent changes in the female’s reproductive physiology.

Calcium waves occur as Drosophila oocytes activate

Abstract: Egg activation is the process by which a mature oocyte becomes capable of supporting embryo development. In vertebrates and echinoderms, activation is induced by fertilization. Molecules introduced into the egg by the sperm trigger progressive release of intracellular calcium stores in the oocyte. Calcium wave(s) spread through the oocyte and induce completion of meiosis, new macromolecular synthesis, and modification of the vitelline envelope to prevent polyspermy. However, arthropod eggs activate without fertilization: in the insects examined, eggs activate as they move through the female’s reproductive tract. Here, we show that a calcium wave is, nevertheless, characteristic of egg activation in Drosophila. This calcium rise requires influx of calcium from the external environment and is induced as the egg is ovulated. Pressure on the oocyte (or swelling by the oocyte) can induce a calcium rise through the action of mechanosensitive ion channels. Visualization of calcium fluxes in activating eggs in oviducts shows a wave of increased calcium initiating at one or both oocyte poles and spreading across the oocyte. In vitro, waves also spread inward from oocyte pole(s). Wave propagation requires the IP3 system. Thus, although a fertilizing sperm is not necessary for egg activation in Drosophila, the characteristic of increased cytosolic calcium levels spreading through the egg is conserved. Because many downstream signaling effectors are conserved in Drosophila, this system offers the unique perspective of egg activation events due solely to maternal components.

On a matter of seminal importance.

Abstract: Egg and sperm have, understandably, been the "stars" of mammalian fertilization biology, particularly because artificial reproductive technologies allow for fertilization to occur outside of the female reproductive tract without other apparent contributions from either sex. Yet, recent research, including an exciting new paper, reveals unexpected and important contributions of seminal plasma to fertility. For example, seminal plasma proteins play critical roles in modulating female reproductive physiology, and a new study in mice demonstrates that effects of some of these proteins on the female can even affect the health of her progeny. Furthermore, although several actions of seminal plasma have been conserved across taxa, male accessory glands and their products are diverse - even among mammals. Taken together, these studies suggest that the actions of seminal plasma components are important to understand, and also to consider in future development of assisted reproductive technologies (ART) for humans, farm species and endangered species of mammals.

Heritable variation in courtship patterns in Drosophila melanogaster.

Abstract: Little is known about the genetic basis of naturally occurring variation for sexually selected behavioral traits. Drosophila melanogaster, with its rich repertoire of courtship behavior and genomic and genetic resources, is an excellent model organism for addressing this question. We assayed a genetically diverse panel of lines with full genome sequences, the Drosophila Genetic Reference Panel, to assess the heritability of variation in courtship behavior and mating progression. We subsequently used these data to quantify natural variation in transition probabilities between courtship behaviors. We found heritable variation along the expected trajectory for courtship behaviors, including the tendency to initiate courtship and rate of progression through courtship, suggesting a genetic basis to male modulation of courtship behavior based on feedback from unrelated, outbred, and genetically identical females. We assessed the genetic basis of variation of the transition with the greatest heritability—from copulation to no engagement with the female—and identified variants in Serrate and Furin 1 as well as many other polymorphisms on the chromosome 3R associated with this transition. Our findings suggest that courtship is a highly dynamic behavior with both social and genetic inputs, and that males may play an important role in courtship initiation and duration.

Retention of Ejaculate by Drosophila melanogaster Females Requires the Male-Derived Mating Plug Protein PEBme

Abstract: Within the mated reproductive tracts of females of many taxa, seminal fluid proteins (SFPs) coagulate into a structure known as the mating plug (MP). MPs have diverse roles, including preventing female remating, altering female receptivity postmating, and being necessary for mated females to successfully store sperm. The Drosophila melanogaster MP, which is maintained in the mated female for several hours postmating, is comprised of a posterior MP (PMP) that forms quickly after mating begins and an anterior MP (AMP) that forms later. The PMP is composed of seminal proteins from the ejaculatory bulb (EB) of the male reproductive tract. To examine the role of the PMP protein PEBme in D. melanogaster reproduction, we identified an EB GAL4 driver and used it to target PEBme for RNA interference (RNAi) knockdown. PEBme knockdown in males compromised PMP coagulation in their mates and resulted in a significant reduction in female fertility, adversely affecting postmating uterine conformation, sperm storage, mating refractoriness, egg laying, and progeny generation. These defects resulted from the inability of females to retain the ejaculate in their reproductive tracts after mating. The uncoagulated MP impaired uncoupling by the knockdown male, and when he ultimately uncoupled, the ejaculate was often pulled out of the female. Thus, PEBme and MP coagulation are required for optimal fertility in D. melanogaster. Given the importance of the PMP for fertility, we identified additional MP proteins by mass spectrometry and found fertility functions for two of them. Our results highlight the importance of the MP and the proteins that comprise it in reproduction and suggest that in Drosophila the PMP is required to retain the ejaculate within the female reproductive tract, ensuring the storage of sperm by mated females.

Induction of excessive endoplasmic reticulum stress in the Drosophila male accessory gland results in infertility.

Abstract: Endoplasmic reticulum (ER) stress occurs when misfolded proteins accumulate in the lumen of the ER. A cell responds to ER stress with the unfolded protein response (UPR), a complex program of transcriptional and translational changes aimed at clearing misfolded proteins. Secretory tissues and cells are particularly well adapted to respond to ER stress because their function requires high protein production and secretory load. The insect male accessory gland (AG) is a secretory tissue involved in male fertility. The AG secretes many seminal fluid proteins (SFPs) essential for male reproduction. Among adult Drosophila tissues, we find that genes upregulated by ER stress are most highly expressed in the AG, suggesting that the AG is already undergoing high levels of ER stress due to its normal secretory functions. We hypothesized that induction of excessive ER stress in the AG above basal levels, would perturb normal function and provide a genetic tool for studying AG and SFP biology. To test this, we genetically induced excessive ER stress in the AG by conditional 1) expression of a misfolded protein or 2) knockdown of the UPR regulatory protein, BiP. Both genetic manipulations induced excessive ER stress in the AG, as indicated by the increase in Xbp1 splicing, a marker of ER stress. Both models resulted in a large decrease in or loss of SFP production and male infertility. Sperm production, motility, and transfer appeared unaffected. The induction of strong ER stress in the insect male AG may provide a simple way for studying or manipulating male fertility, as it eliminates AG function while preserving sperm production.

The genetic architecture of the genome-wide transcriptional response to ER stress in the mouse.

Abstract: Endoplasmic reticulum (ER) stress occurs when misfolded proteins accumulate in the ER. The cellular response to ER stress involves complex transcriptional and translational changes, important to the survival of the cell. ER stress is a primary cause and a modifier of many human diseases. A first step to understanding how the ER stress response impacts human disease is to determine how the transcriptional response to ER stress varies among individuals. The genetic diversity of the eight mouse Collaborative Cross (CC) founder strains allowed us to determine how genetic variation impacts the ER stress transcriptional response. We used tunicamycin, a drug commonly used to induce ER stress, to elicit an ER stress response in mouse embryonic fibroblasts (MEFs) derived from the CC founder strains and measured their transcriptional responses. We identified hundreds of genes that differed in response to ER stress across these genetically diverse strains. Strikingly, inflammatory response genes differed most between strains; major canonical ER stress response genes showed relatively invariant responses across strains. To uncover the genetic architecture underlying these strain differences in ER stress response, we measured the transcriptional response to ER stress in MEFs derived from a subset of F1 crosses between the CC founder strains. We found a unique layer of regulatory variation that is only detectable under ER stress conditions. Over 80% of the regulatory variation under ER stress derives from cis-regulatory differences. This is the first study to characterize the genetic variation in ER stress transcriptional response in the laboratory mouse. Our findings indicate that the ER stress transcriptional response is highly variable among strains and arises from genetic variation in individual downstream response genes, rather than major signaling transcription factors. These results have important implications for understanding how genetic variation impacts the ER stress response, an important component of many human diseases.

Don't pull the plug! the Drosophila mating plug preserves fertility

Abstract: Mating plugs are hardened structures--typically a coagulation of seminal fluid components--that are transferred to, or formed within, the female reproductive tract of numerous animal species (both mammals and insects). Analysis of the role(s) of the mating plug in reproduction has been conducted in a wide array of diverse species. These structures have been proposed to have a multitude of functions, which include altering female re-mating rate, acting as a barrier to re-mating and being required for sperm storage or sperm movement to occur in mated females. A recent analysis of the Drosophila melanogaster mating plug has shown that proper formation of the structure is required for optimal fertility in flies: the Drosophila mating plug is required to retain the ejaculate within the female reproductive tract once mating has terminated. Here, we discuss the possible implications of the Drosophila mating plug in the fertility of this species in light of these new results.

Who’s Zooming Who? Seminal Fluids and Cryptic Female Choice in Diptera

Abstract: Dipteran females have many opportunities to influence the reproductive success of their mates. After each mating, females may influence their mates’ post-copulatory reproductive success by choosing whether and where to store sperm, whether and when to remate and lay eggs, and how much to invest in eggs fertilized by different males. Female neural, endocrine, and muscular mechanisms are necessary for these processes to occur. We review physiological experiments that have borne this out. Further evidence from many Diptera shows that seminal fluid proteins (Sfps) also influence female post-copulatory processes including ones that affect sperm use. The most comprehensive evidence comes from Drosophila melanogaster , whose seminal proteome is well characterized. In this species, studies of sequence variation, including in natural populations, and of gene-specific knockdown in the laboratory, have identified male and female genes whose actions influence and/or correlate with post-copulatory processes in the female. Furthermore, co-evolution between Sfps and female reproductive proteins suggests their involvement in common functional pathways. We review the evidence for the interaction of Sfp-mediated effects and cryptic female choice (CFC), with a focus on D. melanogaster and evidence from other Diptera as available. Finally, we conclude by assessing what is known and as yet unknown about the interface between CFC and Sfps and by suggesting avenues for further research in this fascinating area.

Corrigendum. Heritable Variation in Courtship Patterns in Drosophila melanogaster.

Abstract: Little is known about the genetic basis of naturally occurring variation for sexually selected behavioral traits. Drosophila melanogaster, with its rich repertoire of courtship behavior and genomic and genetic resources, is an excellent model organism for addressing this question. We assayed a genetically diverse panel of lines with full genome sequences, the Drosophila Genetic Reference Panel, to assess the heritability of variation in courtship behavior and mating progression. We subsequently used these data to quantify natural variation in transition probabilities between courtship behaviors. We found heritable variation along the expected trajectory for courtship behaviors, including the tendency to initiate courtship and rate of progression through courtship, suggesting a genetic basis to male modulation of courtship behavior based on feedback from unrelated, outbred, and genetically identical females. We assessed the genetic basis of variation of the transition with the greatest heritability—from copulation to no engagement with the female—and identified variants in Serrate and Furin 1 as well as many other polymorphisms on the chromosome 3R associated with this transition. Our findings suggest that courtship is a highly dynamic behavior with both social and genetic inputs, and that males may play an important role in courtship initiation and duration.