Norene A. Buehner

Seminal fluid protein allocation and male reproductive success.

Postcopulatory sexual selection can select for sperm allocation strategies in males [1, 2], but males should also strategically allocate nonsperm components of the ejaculate [3, 4], such as seminal fluid proteins (Sfps). Sfps can influence the extent of postcopulatory sexual selection [5-7], but little is known of the causes or consequences of quantitative variation in Sfp production and transfer. Using Drosophila melanogaster, we demonstrate that Sfps are strategically allocated to females in response to the potential level of sperm competition. We also show that males who can produce and transfer larger quantities of specific Sfps have a significant competitive advantage. When males were exposed to a competitor male, matings were longer and more of two key Sfps, sex peptide [8] and ovulin [9], were transferred, indicating strategic allocation of Sfps. Males selected for large accessory glands (a major site of Sfp synthesis) produced and transferred significantly more sex peptide, but not more ovulin. Males with large accessory glands also had significantly increased competitive reproductive success. Our results show that quantitative variation in specific Sfps is likely to play an important role in postcopulatory sexual selection and that investment in Sfp production is essential for male fitness in a competitive environment.

A plant signal peptide–hepatitis B surface antigen fusion protein with enhanced stability and immunogenicity expressed in plant cells

The use of transgenic plants to express orally immunogenic protein antigens is an emerging strategy for vaccine biomanufacturing and delivery. This concept has particular suitability for developing countries. One factor that has limited the development of this technology is the relatively modest levels of accumulation of some antigenic proteins in plant tissues. We used fusion protein design to improve expression of the hepatitis B surface antigen (HBsAg) by attempting to mimic the process of HBsAg targeting to the endoplasmic reticulum of human liver cells during hepatitis B virus infection. We created a gene encoding a recombinant HBsAg modified to contain a plant signal peptide fused to its amino terminus. The signal peptide from soybean vegetative storage protein vspA (VSPαS) directed endoplasmic reticulum targeting of HBsAg in plant cells, but was not cleaved and resulted in enhanced VSPαS-HBsAg fusion accumulation. This product was more stable and presented the protective “a” antigenic determinant to significantly higher levels than unmodified native HBsAg expressed in plant cells. It also showed a greater extent of intermolecular disulfide bond formation and formation of virus-like particles. Moreover, VSPαS-HBsAg stimulated higher levels of serum IgG than native HBsAg when injected into mice. We conclude that HBsAg tolerates a polypeptide fusion at the amino terminus and that VSPαS-HBsAg is an improved antigen for plant-based expression of a subunit vaccine for hepatitis B virus.

Calcium waves occur as Drosophila oocytes activate

Significance This paper reports the first visualization of calcium dynamics in Drosophila eggs in vivo and in vitro, demonstrating that a calcium wave is a conserved feature of egg activation (the process by which a mature egg becomes able to initiate embryo development). In vertebrates and echinoderms, the fertilizing sperm triggers egg activation by inducing calcium release from the egg’s internal stores, causing wave(s) of increased calcium to sweep across the egg. However, insect eggs activate without fertilization. We show that a wave of increased calcium occurs during activation of Drosophila eggs. The wave is induced during ovulation by influx of calcium into the egg through mechanosensitive ion channels. Release of calcium from intracellular stores is required for wave propagation. 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.

Modulation of MAPK activities during egg activation in Drosophila.

The mitogen-activated protein kinases (MAPKs) play essential roles during oocyte maturation and egg activation and are also active in somatic cell cycle regulation in many animals. In clams, starfish, ascidians, mice, and frogs, the species-specific timing of MAPK activity during oocyte maturation and egg activation correlates with the different meiotic arrest points of these various organisms. Furthermore, MAPKs have been shown to regulate the meiotic cell cycle in marine invertebrates and vertebrates. The initial trigger for egg activation in insects is different from that of marine invertebrates and vertebrates, and it was not previously known whether changes in MAPK activity accompany egg activation in insects. To examine the regulation of MAPKs during Drosophila egg activation and early embryogenesis, we quantified the levels of phosphorylated (active) forms of ERK, p38 kinase, and JNK by western blotting with antibodies specific to the phospho- forms of these kinases. Levels of phospho-ERK, phospho-p38 kinase, and phospho-JNK are high in Drosophila oocytes. Upon egg activation, levels of all these phospho- (active) forms of MAPKs decrease. Fertilization is not required for this decrease, consistent with the independence of egg activation from fertilization in Drosophila. The decrease in levels of phospho-MAPK occurs normally in embryos laid by sterile females mutant in the egg activation genes cortex, sarah, and prage. We present a model in which the decrease in MAPK activity is an intermediate step in the pathway leading from the calcium signal that initiates egg activation to the downstream events of activation.

Immortal coils: conserved dimerization motifs of the Drosophila ovulation prohormone ovulin.

Dimerization is an important feature of the function of some proteins, including prohormones. For proteins whose amino acid sequences evolve rapidly, it is unclear how such structural characteristics are retained biochemically. Here we address this question by focusing on ovulin, a prohormone that induces ovulation in Drosophila melanogaster females after mating. Ovulin is known to dimerize, and is one of the most rapidly evolving proteins encoded by the Drosophila genome. We show that residues within a previously hypothesized conserved dimerization domain (a coiled-coil) and a newly identified conserved dimerization domain (YxxxY) within ovulin are necessary for the formation of ovulin dimers. Moreover, dimerization is conserved in ovulin proteins from non-melanogaster species of Drosophila despite up to 80% sequence divergence. We show that heterospecific ovulin dimers can be formed in interspecies hybrid animals and in two-hybrid assays between ovulin proteins that are 15% diverged, indicating conservation of tertiary structure amidst a background of rapid sequence evolution. Our results suggest that because ovulins self-interaction requires only small conserved domains, the rest of the molecule can be relatively tolerant to mutations. Consistent with this view, in comparisons of 8510 proteins across 6 species of Drosophila we find that rates of amino acid divergence are higher for proteins with coiled-coil protein-interaction domains than for non-coiled-coil proteins.

Functional male accessory glands and fertility in Drosophila require novel ecdysone receptor

In many insects, the accessory gland, a secretory tissue of the male reproductive system, is essential for male fertility. Male accessory gland is the major source of proteinaceous secretions, collectively called as seminal proteins (or accessory gland proteins), which upon transfer, manipulate the physiology and behavior of mated females. Insect hormones such as ecdysteroids and juvenoids play a key role in accessory gland development and protein synthesis but little is known about underlying molecular players and their mechanism of action. Therefore, in the present study, we examined the roles of hormone-dependent transcription factors (Nuclear Receptors), in accessory gland development, function and male fertility of a genetically tractable insect model, Drosophila melanogaster. First, we carried out an RNAi screen involving 19 hormone receptors, individually and specifically, in a male reproductive tissue (accessory gland) for their requirement in Drosophila male fertility. Subsequently, by using independent RNAi/ dominant negative forms, we show that Ecdysone Receptor (EcR) is essential for male fertility due to its requirement in the normal development of accessory glands in Drosophila: EcR depleted glands fail to make seminal proteins and have dying cells. Further, our data point to a novel ecdysone receptor that does not include Ultraspiracle but is probably comprised of EcR isoforms in Drosophila male accessory glands. Our data suggest that this novel ecdysone receptor might act downstream of homeodomain transcription factor paired (prd) in the male accessory gland. Overall, the study suggests novel ecdysone receptor as an important player in the hormonal regulation of seminal protein production and insect male fertility.

Long-term interaction between Drosophila sperm and sex peptide is mediated by other seminal proteins that bind only transiently to sperm

Seminal fluid proteins elicit several post-mating physiological changes in mated Drosophila melanogaster females. Some of these changes persist for over a week after mating because the seminal protein that causes these changes, the Sex Peptide (SP), binds to sperm that are stored in the female reproductive tract. SPs sperm binding is mediated by a network of at least eight seminal proteins. We show here that some of these network proteins (CG1656, CG1652, CG9997 and Antares) bind to sperm within 2 h of mating, like SP. However, while SP remains bound to sperm at 4 days post-mating, none of the other network proteins are detectable at this time. We also observed that the same network proteins are detectable at 2 h post-mating in seminal receptacle tissue from which sperm have been removed, but are no longer detectable there by 4 days post-mating, suggesting short-term retention of these proteins in this female sperm storage organ. Our results suggest that these network proteins act transiently to facilitate the conditions for SPs binding to sperm, perhaps by modifying SP or the sperm surface, but are not part of a long-acting complex that stably attaches SP to sperm.

Correction: Functional male accessory glands and fertility in Drosophila require novel ecdysone receptor

[This corrects the article DOI: 10.1371/journal.pgen.1006788.].