Post-mating change in excretion by mated Drosophila melanogaster females is a long-term response that depends on sex peptide and sperm

Drosophila seminal fluid proteins elicit physiological and behavioral changes in the female after mating. For example, the seminal protein sex peptide (SP) causes females to lay more eggs, reduce receptivity to re-mating, consume more food and produce more concentrated excreta upon mating. It has been reported that SP indirectly increases food consumption as a result of its stimulation of egg production, but its role in producing more concentrated excreta in the mated female was reported to be independent of egg production. Additionally, it has been shown that SP’s effect on food consumption persists for several days after mating, while it is unknown whether this is true for its effect on excretion.     

Octopamine receptor OAMB is required for ovulation in Drosophila melanogaster

Octopamine is a major monoamine in invertebrates and affects many physiological processes ranging from energy metabolism to complex behaviors. Octopamine binds to receptors located on various cell types and activates distinct signal transduction pathways to produce these diverse effects. We previously identified one of the Drosophila octopamine receptors named OAMB that produces increases in cAMP and intracellular Ca2+ upon ligand binding. It is expressed at high levels in the brain. To explore OAMB's physiological roles, we generated deletions in the OAMB locus. The resultant oamb mutants were viable without gross anatomical defects. The oamb females displayed normal courtship and copulation; however, they were impaired in ovulation with many mature eggs retained in their ovaries. RT-PCR, in situ hybridization, and expression of a reporter gene revealed that OAMB was also expressed in the thoracicoabdominal ganglion, the female reproductive system, and mature eggs in the ovary. Moreover, analysis of various alleles pinpointed the requirement for OAMB in the body, but not in the brain, for female fecundity. The novel expression pattern of OAMB and its genetic resource described in this study will help advance our understanding on how the neuromodulatory or endocrine system controls reproductive physiology and behavior.     

Multiple morphs of sperm were required for the evolution of the Sex Ratio trait in Drosophila

In Sex Ratio males of D. subobscura Y sperm degenerate. Nevertheless, Sex Ratio males can produce as many offspring as non-Sex Ratio males. In order to find an explanation for the high number of offspring of Sex Ratio males we analysed spermatogenesis and sperm storage in females. Because D. subobscura has two morphs of sperm, short and long sperm, we studied the relationship of sperm heteromorphism to the Sex Ratio trait in this species. Spermatids of both lengths developed simultaneously in the testes. The first mature sperm produced by a young male were nearly all short sperm. This result indicates that short cysts need less time to mature than long cysts. Thus in a given time more short cysts than long cysts can mature. In the testes of Sex Ratio males more short cysts and fewer long cysts were formed than in the tests of non-Sex Ratio males. Thus in a unit time Sex Ratio males can produce as many or more sperm than non-Sex Ratio males. Females mated to Sex Ratio males had more sperm in their storage organs than those which were mated to non-Sex Ratio males. From the transferred sperm females stored selectively more long than short sperm after mating to a Sex Ratio male than after mating to a non-Sex Ratio male. The effect of the high amount of sperm in Sex Ratio males is twofold: the loss of Y sperm is compensated, and probably in nature a second copulation is prevented by the complete filling of the female storage organs. From these results we conclude that the development and persistence of the Sex Ratio trait in natural populations depends on the presence of sperm heteromorphism.     

Sperm of the wasted mutant are wasted when females utilize the stored sperm in Drosophila melanogaster

Females of many animal species store sperm after copulation for use in fertilization, but the mechanisms controlling sperm storage and utilization are largely unknown. Here we describe a novel male sterile mutation of Drosophila melanogaster, wasted (wst), which shows defects in various processes of sperm utilization. The sperm of wst mutant males are stored like those of wild-type males in the female sperm storage organs, the spermathecae and seminal receptacles, after copulation and are released at each ovulation. However, an average of thirteen times more wst sperm than wild type sperm are released at each ovulation, resulting in rapid loss of sperm stored in seminal receptacles within a few days after copulation. wst sperm can enter eggs efficiently at 5 hr after copulation, but the efficiency of sperm entry decreases significantly by 24 hr after copulation, suggesting that wst sperm lose their ability to enter eggs during storage. Furthermore, wst sperm fail to undergo nuclear decondensation, which prevents the process of fertilization even when sperm enter eggs. Our results indicate that the wst gene is essential for independent processes in the utilization of stored sperm; namely, regulation of sperm release from female storage organs, maintenance of sperm efficiency for entry into eggs, and formation of the male pronucleus in the egg at fertilization.     

The putative signal peptide of glucagon-like peptide-1 receptor is not required for receptor synthesis but promotes receptor expression

GLP-1R (glucagon-like peptide-1 receptor) mediates the ‘incretin effect’ and many other anti-diabetic actions of its cognate ligand, GLP-1 (glucagon-like peptide-1). It belongs to the class B family of GPCRs (G protein-coupled receptors) and possesses an N-terminal putative SP (signal peptide). It has been reported that this sequence is required for the synthesis of GLP-1R and is cleaved after receptor synthesis. In the present study, we conducted an in-depth exploration towards the role of the putative SP in GLP-1R synthesis. A mutant GLP-1R without this sequence was expressed in HEK293 cells (human embryonic kidney 293 cells) and displayed normal functionality with respect to ligand binding and activation of adenylate cyclase. Thus the putative SP does not seem to be required for receptor synthesis. Immunoblotting analysis shows that the amount of GLP-1R synthesized in HEK293 cells is low when the putative SP is absent. This indicates that the role of the sequence is to promote the expression of GLP-1R. Furthermore, epitopes tagged at the N-terminal of GLP-1R are detectable by immunofluorescence and immunoblotting in our experiments. In conclusion, the present study points to different roles of SP in GLP-1R expression which broadens our understanding of the functionality of this putative SP of GLP-1R and possibly other Class B GPCRs.     

A functioning ovary is not required for sex peptide to reduce receptivity to mating in D. melanogaster

In many species of invertebrates that mate multiply, mating induces a temporary reduction in sexual receptivity and an increase in the rate of egg laying. These processes often appear to be co-ordinately regulated, and triggered by the passage of seminal fluid components. However, little is known about the mechanisms of the links between these processes. In Drosophila melanogaster females, post-mating sexual receptivity is decreased and egg laying increased by the actions of the male ejaculate-derived sex peptide (SP). Effects of SP on egg laying and receptivity have not been observed separately, which has led to the suggestion that the reduction in receptivity is at least partially dependent on the status of egg development or egg laying, with the presence of an egg in the uterus being a strong predictor of receptivity state. Here, we examine the response to SP of females in which egg development is arrested at an early, pre-vitellogenic stage. We find that females in which egg development is arrested mate normally and that normal receptivity responses to SP are independent of early egg arrest. Among fertile control females that laid eggs, a significant effect of SP on receptivity was also observed, independent of whether an egg was present in the uterus. The results show that the effects of SP on receptivity are not dependent upon a fully functional ovary, and hence that egg development or laying is not causal in the SP receptivity response.     

Characterization of the short neuropeptide F receptor from Drosophila melanogaster

A seven transmembrane G-protein coupled receptor has been cloned from Drosophila melanogaster. This receptor shows structural similarities to vertebrate Neuropeptide Y(2) receptors and is activated by endogenous Drosophila peptides, recently designated as short neuropeptide Fs (sNPFs). sNPFs have so far been found in neuroendocrine tissues of four other insect species and of the horseshoe crab. In locusts, they accelerate ovarian maturation, and in mosquitoes, they inhibit host-seeking behavior. Expression analysis by RT-PCR shows that the sNPF receptor (Drm-sNPF-R) is present in several tissues (brain, gut, Malpighian tubules and fat body) from Drosophila larvae as well as in ovaries of adult females. All 4 Drosophila sNPFs clearly elicited a calcium response in receptor expressing mammalian Chinese hamster ovary cells. The response is dose-dependent and appeared to be very specific. The short NPF receptor was not activated by any of the other tested arthropod peptides, not even by FMRFamide-related peptides (also ending in RFamide), indicating that the Arg residue at position 4 from the amidated C-terminus appears to be crucial for the response elicited by the sNPFs.     

Post-thaw boar sperm motility is affected by prolonged storage of sperm in liquid nitrogen. A retrospective study

Owing to the quick genetic turnover of the pig industry, most AI-boar sires live 2-3 yr, a period during which for 1-2 yr their semen is extended and used in liquid form for AI. Despite showing low cryosurvival, affecting fertility after AI, boar semen is frozen for easiness of transport overseas and reposition of valuable genetics. For the latter, semen is stored in liquid nitrogen (LN₂, cryostorage) for many years, a controversial practice. Here we studied how length of cryostorage could affect sperm quality. Straws (0.5 mL) frozen using the same cryopreservation protocol at one specific location from AI- sires of proven fertility were stored in LN₂ for up to 8 yr. Post-thaw sperm quality was evaluated after 2, 4 or 8 yr of cryostorage, always compared to early thawing (15 d after freezing). Sperm motility and kinematics were evaluated post-thaw using CASA and sperm viability was cytometrically evaluated using specific fluorophores. Sperm viability was not affected by length of cryostorage, but total and progressive sperm motility were lower (p < 0.01) in sperm samples cryostored for 4 or 8 yr compared to those thawed 15 d after freezing. Cryostorage time affected sperm kinetics, but with greater intensity in the samples cryostored for 4 yr (p < 0.001) than in those for 2 yr (p < 0.01). The fact that the major phenotypic characteristic of boar spermatozoa, motility, is constrained by time of cryostorage should be considered when building cryobanks of pig semen. Attention should be placed on the finding that >2 yr of cryostorage time can be particularly detrimental for the post-thaw motility of some sires, which might require increasing sperm numbers for AI.     

Suppression of cold ischemic injury in stored kidneys by the antimicrobial peptide bactenecin

BACKGROUND: Cold ischemic injury plays an important role in short- and long-term function of kidneys after transplant. Antimicrobial peptides have not previously been studied for their impact on cold ischemia in transplanted kidneys. METHODS: Bactenecin (L- and D-forms) was added to University of Wisconsin (UW) preservation solution for 3-day cold storage of dog kidneys. Effects on membrane permeability were studied in synthetic liposomes and in kidney cortex tissue slices. The role of bactenecin as a tissue mitogen and direct cytoskeletal stabilizer were studied with cultured cells and in vitro. RESULTS: Bactenecin (both L- and D- forms) resulted in significant decreases in postoperative serum creatinine and time required for return of creatinine to the normal range showing the effect was independent of chirality. Bactenecin permeabilized synthetic liposomes and altered kidney cortex tissue slice membrane permeability characteristics, irrespective of chirality. Neither did bactenecin act as a mitogen for either primary renal tubule or Madin-Darby canine kidney (MDCK) cells stored in UW solution, nor did it appear to directly affect cytoskeletal dynamics. CONCLUSIONS: These results show that the antimicrobial peptide bactenecin can improve the quality of static cold storage of kidneys. The mechanism of its action is independent of receptor binding and does not appear to involve either an effect on the cytoskeleton or via activity as a mitogen. Current evidence best supports the hypothesis that bactenecin protects against cold ischemic injury by a controlled permeabilization of the membranes of the kidney during cold storage.     

Oxidized low-density lipoprotein-induced foam cell formation is mediated by formyl peptide receptor 2

The increased level of LDL and its modification into oxLDL has been regarded as an important risk factor for the development of cardiovascular diseases such as atherosclerosis. Although some scavenger receptors including CD36 and RAGE have been considered as target receptors for oxLDL, involvement of other receptors should be investigated for oxLDL-induced pathological responses. In this study, we found that oxLDL-induced foam cell formation was inhibited by formyl peptide receptor 2 (FPR2) antagonist WRW⁴. oxLDL also stimulated calcium signaling and chemotactic migration in FPR2-expressing RBL-2H3 cells but not in vector-expressing RBL-2H3 cells. Moreover, oxLDL stimulated TNF-α production, which was also almost completely inhibited by FPR2 antagonist. Our findings therefore suggest that oxLDL stimulates macrophages, resulting in chemotactic migration, TNF-α production, and foam cell formation via FPR2 signaling, and thus likely contributes to atherogenesis.     

Orphan nuclear receptor betaFTZ-F1 is required for muscle-driven morphogenetic events at the prepupal-pupal transition in Drosophila melanogaster

In Drosophila melanogaster, fluctuations in 20-hydroxyecdysone (ecdysone) titer coordinate gene expression, cell death, and morphogenesis during metamorphosis. Our previous studies have supported the hypothesis that betaFTZ-F1 (an orphan nuclear receptor) provides specific genes with the competence to be induced by ecdysone at the appropriate time, thus directing key developmental events at the prepupal-pupal transition. We are examining the role of betaFTZ-F1 in morphogenesis. We have made a detailed study of morphogenetic events during metamorphosis in control and betaFTZ-F1 mutant animals. We show that leg development in betaFTZ-F1 mutants proceeds normally until the prepupal-pupal transition, when final leg elongation is delayed by several hours and significantly reduced in the mutants. We also show that betaFTZ-F1 mutants fail to fully extend their wings and to shorten their bodies at the prepupal-pupal transition. We find that betaFTZ-F1 mutants are unable to properly perform the muscle contractions that drive these processes. Several defects can be rescued by subjecting the mutants to a drop in pressure during the normal time of the prepupal-pupal transition. Our findings indicate that betaFTZ-F1 directs the muscle contraction events that drive the major morphogenetic processes during the prepupal-pupal transition in Drosophila.     

血管钠肽对低氧大鼠心脏钠尿肽C受体表达的影响

目的 :探讨低氧对大鼠心脏钠尿肽C受体 (NPR C)表达的调节作用 ,以及血管钠肽 (VNP)对这一过程的影响。方法 :将大鼠随机分为 3组 :对照组、低氧组 (3～ 2 8d)和VNP(2 5～ 75 μg/kgbw) 低氧组 ,采用放射免疫的方法测定大鼠血浆心房钠尿肽 (ANP)的浓度 ,并采用定量PCR的方法分析NPR C的mRNA水平。结果 :低氧 2 8d大鼠血浆ANP浓度显著高于正常大鼠 (P <0 .0 5 ) ,而且每天注射 75 μg/kgbw的VNP使ANP浓度进一步升高 (P <0 .0 1)。低氧 3d对大鼠心脏NPR C的mRNA的量没有显著影响 ;低氧 7d使大鼠心脏NPR C的mRNA的拷贝数显著升高 (P <0 .0 5 ) ;低氧 14d、2 8d使大鼠心脏NPR C的mRNA的拷贝数进一步升高 (P <0 .0 1)。每日注射 2 5μg/kgbw的VNP对低氧诱导的大鼠心脏NPR C表达没有显著影响 ;5 0 μg/kgbw的VNP显著降低低氧大鼠心脏NPR C的表达 (P <0 .0 5 ) ;75 μg/kgbw的VNP进一步降低低氧大鼠心脏NPR C的表达 (P <0 .0 1)。 结论 :VNP可以升高低氧大鼠的血浆ANP水平 ;低氧可以使大鼠心脏NPR C表达增加 ,而且具有时间依赖性 ,而VNP对这一过程有抑制作用 ,并且呈剂量依赖性     

Rab11 is required for tubulogenesis of Malpighian tubules in Drosophila melanogaster

Intracellular vesicular trafficking is one of the important tools in maintaining polarity, adhesion, and shape of epithelial cells. Rab11, a subfamily of the Ypt/Rab gene family of ubiquitously expressed GTPases and a molecular marker of recycling endosomes, transports different components of plasma membrane. Here, we report that Rab11 affects tubulogenesis of Malpighian tubules (MTs). MTs are simple polarized epithelial tubular structures, considered as functional analogue of human kidney. Rab11 has pleiotropic effects on MTs development as down‐regulation of Rab11 in principal cells (PCs) of MTs from embryonic stages of development results in reduced endoreplication, clustering of cells, disorganized cytoskeleton, and disruption of polarity leading to shortening of MTs in third instar larvae. Rab11 is also required for proper localization of different transporters in PCs, essential for physiological activity of MTs. Collectively, our data suggest that Rab11 plays a key role in the process of tubulogenesis of MTs in Drosophila.     

Coupling of growth to nutritional status: The role of novel periphery-to-brain signaling by the CCHa2 peptide in Drosophila melanogaster

ABSTRACT

The coupling of growth to nutritional status is an important adaptive response of living organisms to their environment. For this ability, animals have evolved various strategies, including endocrine systems that respond to changing nutritional conditions. In animals, nutritional information is mostly perceived by peripheral organs, such as the digestive tract and adipose tissues, and is subsequently transmitted to other peripheral organs or the brain, which integrates the incoming signals and orchestrates physiological and behavioral responses. In Drosophila melanogaster, adipose tissue, known as the fat body, functions as an endocrine organ that communicates with the brain. This fat body-brain axis coordinates growth with nutritional status by regulating the secretion of Drosophila insulin-like peptides (Dilps) from the brain. However, the molecular nature of the fat body-brain axis remains to be elucidated. We recently demonstrated that a small peptide, CCHamide-2 (CCHa2), expressed in the fat body and gut, directly stimulates its receptor (CCHa2-R) in the brain, leading to Dilp production. Notably, the expression of CCHa2 is sensitive to the presence of nutrients, particularly sugars. Our results, together with the results of previous studies, show that signaling between peripheral organs and the brain is a conserved strategy that couples nutritional availability to organismal physiology.     

Regulation of Commissureless by the ubiquitin ligase DNedd4 is required for neuromuscular synaptogenesis in Drosophila melanogaster

Muscle synaptogenesis in Drosophila melanogaster requires endocytosis of Commissureless (Comm), a binding partner for the ubiquitin ligase dNedd4. We investigated whether dNedd4 and ubiquitination mediate this process. Here we show that Comm is expressed in intracellular vesicles in the muscle, whereas Comm bearing mutations in the two PY motifs (L/PPXY) responsible for dNedd4 binding [Comm(2PY-->AY)], or bearing Lys-->Arg mutations in all Lys residues that serve as ubiquitin acceptor sites [Comm(10K-->R)], localize to the muscle surface, suggesting they cannot endocytose. Accordingly, aberrant muscle innervation is observed in the Comm(2PY-->AY) and Comm(10K-->R) mutants expressed early in muscle development. Similar muscle surface accumulation of Comm and innervation defects are observed when dNedd4 is knocked down by double-stranded RNA interference in the muscle, in dNedd4 heterozygote larvae, or in muscles overexpressing catalytically inactive dNedd4. Expression of the Comm mutants fused to a single ubiquitin that cannot be polyubiquitinated and mimics monoubiquitination [Comm(2PY-->AY)-monoUb or Comm(10K-->R)-monoUb] prevents the defects in both Comm endocytosis and synaptogenesis, suggesting that monoubiquitination is sufficient for Comm endocytosis in muscles. Expression of the Comm mutants later in muscle development, after synaptic innervation, has no effect. These results demonstrate that dNedd4 and ubiquitination are required for Commissureless endocytosis and proper neuromuscular synaptogenesis.