Interaction of structure-specific and promiscuous G-protein-coupled receptors mediates small-molecule signaling in Caenorhabditis elegans

A chemically diverse family of small-molecule signals, the ascarosides, control developmental diapause (dauer), olfactory learning, and social behaviors of the nematode model organism, Caenorhabditis elegans. The ascarosides act upstream of conserved signaling pathways, including the insulin, TGF-β, serotonin, and guanylyl cyclase pathways; however, the sensory processes underlying ascaroside function are poorly understood. Because ascarosides often are multifunctional and show strongly synergistic effects, characterization of their receptors will be essential for understanding ascaroside biology and may provide insight into molecular mechanisms that produce synergistic outcomes in small-molecule sensing. Based on DAF-8 immunoprecipitation, we here identify two G-protein-coupled receptors, DAF-37 and DAF-38, which cooperatively mediate ascaroside perception. daf-37 mutants are defective in all responses to ascr#2, one of the most potent dauer-inducing ascarosides, although this mutant responds normally to other ascarosides. In contrast, daf-38 mutants are partially defective in responses to several different ascarosides. Through cell-specific overexpression, we show that DAF-37 regulates dauer when expressed in ASI neurons and adult behavior when expressed in ASK neurons. Using a photoaffinity-labeled ascr#2 probe and amplified luminescence assays (AlphaScreen), we demonstrate that ascr#2 binds to DAF-37. Photobleaching fluorescent energy transfer assays revealed that DAF-37 and DAF-38 form heterodimers, and we show that heterodimerization strongly increases cAMP inhibition in response to ascr#2. These results suggest that that the ascarosides' intricate signaling properties result in part from the interaction of highly structure-specific G-protein-coupled receptors such as DAF-37 with more promiscuous G-protein-coupled receptors such as DAF-38.     

Contrasting responses within a single neuron class enable sex-specific attraction in Caenorhabditis elegans

Animals find mates and food, and avoid predators, by navigating to regions within a favorable range of available sensory cues. How are these ranges set and recognized? Here we show that male Caenorhabditis elegans exhibit strong concentration preferences for sex-specific small molecule cues secreted by hermaphrodites, and that these preferences emerge from the collective dynamics of a single male-specific class of neurons, the cephalic sensory neurons (CEMs). Within a single worm, CEM responses are dissimilar, not determined by anatomical classification and can be excitatory or inhibitory. Response kinetics vary by concentration, suggesting a mechanism for establishing preferences. CEM responses are enhanced in the absence of synaptic transmission, and worms with only one intact CEM show nonpreferential attraction to all concentrations of ascaroside for which CEM is the primary sensor, suggesting that synaptic modulation of CEM responses is necessary for establishing preferences. A heterogeneous concentration-dependent sensory representation thus appears to allow a single neural class to set behavioral preferences and recognize ranges of sensory cues.The chemical senses of taste and smell are an important source of sensory input for organisms from worms to humans, and elements of the olfactory system are evolutionarily conserved across metazoa (1, 2). The neural mechanisms of olfactory processing are a subject of active research (3), and much is known about the encoding of odor identity and concentration (4–6). However, the issue of ranges of favorable odor concentrations has been less studied. A reasonable general hypothesis is that physical sensory limitations set perceptual boundaries, limiting the range of an animal to respond favorably. However, there are instances where differences in odor concentrations can have different meanings: For example, both male and female rodents produce the same pheromone at different concentrations (7), and so males need to be able to distinguish between low and high concentrations. Similarly, a very high concentration might signal an adverse environment with overcrowding, in which case the animal is better off looking elsewhere. In such cases, the concentration preferences of the animals are tuned to some optimal value that has a higher probability of a successful outcome. Here, we show that Caenorhabditis elegans exhibits a striking tuning of pheromone concentration preferences, and that this concentration tuning is actively built and maintained by a single class of male-specific neurons, the cephalic sensory neurons (CEMs).The nervous system of C. elegans is famously compact, with 302 hermaphrodite neurons grouped into 118 classes based on morphology and connectivity (8), and 385 male neurons (9–11). Some classes of neurons are sex-specific (Fig. 1A). Members of a class are typically distinguished from each other by their relative anatomical position, such as left/right and dorsal/ventral. Although initially it was thought that members of a class were functionally similar, several studies have revealed asymmetry in the responses of members of a class, in particular the sensory neurons (12, 13).Open in a separate windowFig. 1.Male-specific CEM neurons respond to multiple sex-specific ascarosides. (A) Sex-specific neurons in hermaphrodites (Top) and male C. elegans (Bottom). (B) Two ascarosides that are produced by the hermaphrodite, ascr#8 and ascr#3. (C) Behavioral assay used to determine attractiveness of ascarosides. (D and E) Behavioral tuning curve showing the mean behavioral dwell time at different concentrations of ascr#8 and ascr#3, respectively. Asterisks indicate responses significantly greater than controls with buffer, P < 0.0001, Student’s t test. Concentrations without asterisks were not significantly different from controls. Green arrowheads indicate concentrations of ascaroside used for electrophysiological analyses. (F) Ablation of male-specific CEM neurons abolishes attraction to ascr#8 (Left) and reduces attraction to ascr#3 (Right). Data for ascr#3 has been reported by us (23). (G) CEM neurons labeled with green fluorescent protein (GFP) in adult male worm expressing pkd-2::gfp. (H) CEM neurons show responses to ascr#8 and ascr#3 but not to water. Black, example traces.The four male-specific CEM neurons are considered members of a single class based on substantial evidence: their fourfold symmetric location of cell bodies (14), the morphology of their processes (15), the morphology of their nuclei (16) and their cilia (17), and their gene expression (15, 18, 19). Presumptive CEMs die in the hermaphrodite (20) and are under coordinated genetic control, although the ventral CEMs are less sensitive to sex-specific apoptosis (16).Chemical analyses of hermaphrodite secretions by mass spectroscopy and 2D NMR spectroscopy have discovered a novel family of small molecules called ascarosides (21–23), which serve diverse biological functions (24). Certain ascarosides secreted by hermaphrodites are attractive exclusively to males, which exhibit strong concentration preferences (23). We mapped the behavioral concentration tuning curve and ablated individual neurons to identify the mediators of this response. We next performed electrophysiological, calcium imaging, and genetic analyses to uncover the sensory coding strategy that allows C. elegans to develop and maintain its concentration preferences. We find that C. elegans employs a novel mechanism of heterogeneous responses combined with concentration-dependent kinetics within a primary sensory neuron class to build a concentration tuning curve and likely uses synaptic modulation to do so.     

dissatisfaction, a gene involved in sex-specific behavior and neural development of Drosophila melanogaster

Few mutations link well defined behaviors with individual neurons and the activity of specific genes. In Drosophila, recent evidence indicates the presence of a doublesex-independent pathway controlling sexual behavior and neuronal differentiation. We have identified a gene, dissatisfaction (dsf), that affects sex-specific courtship behaviors and neural differentiation in both sexes without an associated general behavioral debilitation. Male and female mutant animals exhibit abnormalities in courtship behaviors, suggesting a requirement for dsf in the brain. Virgin dsf females resist males during courtship and copulation and fail to lay mature eggs. dsf males actively court and attempt copulation with both mature males and females but are slow to copulate because of maladroit abdominal curling. Structural abnormalities in specific neurons indicate a role for dsf in the differentiation of sex-specific abdominal neurons. The egg-laying defect in females correlates with the absence of motor neuronal innervation on uterine muscles, and the reduced abdominal curling in males correlates with alteration in motor neuronal innervation of male ventral abdominal muscles. Epistasis experiments show that dsf acts in a tra-dependent and dsx-independent manner, placing dsf in the dsx-independent portion of the sex determination cascade.     

Biosynthesis of the Caenorhabditis elegans dauer pheromone

To sense its population density and to trigger entry into the stress-resistant dauer larval stage, Caenorhabditis elegans uses the dauer pheromone, which consists of ascaroside derivatives with short, fatty acid-like side chains. Although the dauer pheromone has been studied for 25 years, its biosynthesis is completely uncharacterized. The daf-22 mutant is the only known mutant defective in dauer pheromone production. Here, we show that daf-22 encodes a homolog of human sterol carrier protein SCPx, which catalyzes the final step in peroxisomal fatty acid β-oxidation. We also show that dhs-28, which encodes a homolog of the human d-bifunctional protein that acts just upstream of SCPx, is also required for pheromone production. Long-term daf-22 and dhs-28 cultures develop dauer-inducing activity by accumulating less active, long-chain fatty acid ascaroside derivatives. Thus, daf-22 and dhs-28 are required for the biosynthesis of the short-chain fatty acid-derived side chains of the dauer pheromone and link dauer pheromone production to metabolic state.     

The quantitative genetics of sex differences in parenting

Sex differences in parenting are common in species where both males and females provide care. Although there is a considerable body of game and optimality theory for why the sexes should differ in parental care, genetics can also play a role, and no study has examined how genetic influences might influence differences in parenting. We investigated the extent that genetic variation influenced differences in parenting, whether the evolution of differences could be constrained by shared genetic influences, and how sex-specific patterns of genetic variation underlying parental care might dictate which behaviors are free to evolve in the burying beetle Nicrophorus vespilloides. Females provided more direct care than males but did not differ in levels of indirect care or the number of offspring they were willing to rear. We found low to moderate levels of heritability and evolvability for all 3 parenting traits in both sexes. Intralocus sexual conflict was indicated by moderately strong intersex genetic correlations, but these were not so strong as to represent an absolute constraint to the evolution of sexual dimorphism in care behavior. Instead, the pattern of genetic correlations between parental behaviors showed sex-specific tradeoffs. Thus, differences in the genetic correlations between parental traits within a sex create sex-specific lines of least evolutionary resistance, which in turn produce the specific patterns of sex differences in parental care. Our results therefore suggest a mechanism for the evolution of behavioral specialization during biparental care if uniparental and biparental care behaviors share the same genetic influences.     

The species, sex, and stage specificity of a Caenorhabditis sex pheromone

Four species in the ELEGANS group of subgenus the Caenorhabditis are distinguished by two very different mating systems: androdioecy in C. elegans and Caenorhabditis briggsae with males and self-fertilizing hermaphrodites and dioecy in Caenorhabditis remanei and Caenorhabditis sp. strain CB5161 with males and females. Using chemotaxis assays, we demonstrate that females secrete a potent sex pheromone that attracts males from a distance, whereas hermaphrodites do not. The female sex pheromone is not species-specific, with males of all four species attracted to both the C. remanei and Caenorhabditis sp. female sex pheromones. The pheromone is, however, sex-specific, with only females secreting the pheromone and attracting only males. Furthermore, the sex pheromone is stage-specific, with female secretion and male detection of the pheromone beginning near adulthood. Females lose their attractiveness immediately after mating but regain it several hours after mating ceases. Finally, the female somatic gonad is required for sex-pheromone production, and the male-specific cephalic neurons (CEM) are required for male response.     

Acyl-CoA oxidase complexes control the chemical message produced by Caenorhabditis elegans

Caenorhabditis elegans uses ascaroside pheromones to induce development of the stress-resistant dauer larval stage and to coordinate various behaviors. Peroxisomal β-oxidation cycles are required for the biosynthesis of the fatty acid-derived side chains of the ascarosides. Here we show that three acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, form different protein homo- and heterodimers with distinct substrate preferences. Mutations in the acyl-CoA oxidase genes acox-1, -2, and -3 led to specific defects in ascaroside production. When the acyl-CoA oxidases were expressed alone or in pairs and purified, the resulting acyl-CoA oxidase homo- and heterodimers displayed different side-chain length preferences in an in vitro activity assay. Specifically, an ACOX-1 homodimer controls the production of ascarosides with side chains with nine or fewer carbons, an ACOX-1/ACOX-3 heterodimer controls the production of those with side chains with seven or fewer carbons, and an ACOX-2 homodimer controls the production of those with ω-side chains with less than five carbons. Our results support a biosynthetic model in which β-oxidation enzymes act directly on the CoA-thioesters of ascaroside biosynthetic precursors. Furthermore, we identify environmental conditions, including high temperature and low food availability, that induce the expression of acox-2 and/or acox-3 and lead to corresponding changes in ascaroside production. Thus, our work uncovers an important mechanism by which C. elegans increases the production of the most potent dauer pheromones, those with the shortest side chains, under specific environmental conditions.The nematode Caenorhabditis elegans secretes the ascarosides, structurally diverse derivatives of the 3,6-dideoxy-l-sugar ascarylose, as chemical signals to control its development and behavior. Under favorable growth conditions, C. elegans progresses from the egg through four larval stages (L1–L4) to the reproductive adult. At high nematode population densities, however, specific ascarosides, which are together known as the dauer pheromone, trigger entry into a specialized L3 larval stage called the dauer (Fig. 1A) (1–5). Dauers have a thickened cuticle, do not feed, derive energy from fat stores, and up-regulate stress-resistance pathways (6). Certain ascarosides also influence behaviors, including male attraction to hermaphrodites, hermaphrodite attraction to males, avoidance, and aggregation (7–12). C. elegans senses the ascarosides using several classes of G protein-coupled receptors (GPCRs), which are expressed in specific chemosensory neurons (13, 14). The dauer pheromone ascarosides induce dauer formation by downregulating the insulin/insulin-like growth factor 1 (IGF-1) and TGF-β pathways, which control not only dauer development but also metabolism and lifespan in C. elegans (15).Open in a separate windowFig. 1.Ascaroside pheromones and β-oxidation enzymes implicated in their biosynthesis. (A) Dauer-inducing ascaroside pheromones. (B) Modular structure of the ascarosides. The ascarosides can be named according to the number of carbons in their side chains using the following rubric: head group-asc-(ω)(Δ)C#-terminus group. Head groups can be attached via the 4′-hydroxyl of ascarylose (e.g., indole-3-carbonyl group) or the 2′-hydroxyl of ascarylose (e.g., glucosyl group). Modifications to the terminus of the side chain include para-aminobenzoic acid and methylketone. R = H for the ω-ascarosides and R = CH₃ for the (ω-1)-ascarosides. (C) The β-oxidation enzymes implicated in ascaroside biosynthesis. The red dot tracks the position of the β-carbon during the β-oxidation cycle.All ascarosides have a simple, modular structure with a fatty acid-derived side chain (Fig. 1B). The fatty acid side chain is attached to the ascarylose sugar at either its penultimate (ω-1) or terminal (ω) carbon, and is sometimes unsaturated (Δ) at the α-β position. The ascarosides can be named according to the number of carbons in their side chains using the following rubric: head group-asc-(ω)(Δ)C#-terminus group (16). Head groups attached to the ascarylose sugar include the indole-3-carbonyl (IC) and glucosyl (Glu) modifications, and terminus groups on the fatty acid terminus include the methylketone (MK) and para-aminobenzoic acid (PABA) modifications (Fig. 1B). The dauer pheromone consists of at least five ascarosides, asc-C6-MK (C6; ascr#2), asc-ΔC9 (C9; ascr#3), asc-ωC3 (C3; ascr#5), IC-asc-C5 (C5; icas#9), and asc-ΔC7-PABA (ascr#8) (Fig. 1A) (2–5). Of the dauer pheromone ascarosides, only asc-ωC3 has a side chain that is attached to the ascarylose sugar at its ω-carbon [as opposed to its (ω-1)-carbon]. Intriguingly, this ascaroside works synergistically with the others to induce dauer formation (3), and targets a unique class of GPCRs (13, 14).Biosynthesis of the side chains of the ascarosides requires peroxisomal β-oxidation cycles that shorten the side chains by two carbons per cycle (11, 17–19). Four peroxisomal enzymes, an acyl-CoA oxidase (ACOX-1), enoyl-CoA hydratase (MAOC-1), (3R)-hydroxyacyl-CoA dehydrogenase (DHS-28), and 3-ketoacyl-CoA thiolase (DAF-22), have been implicated in the β-oxidation cycles for ascaroside biosynthesis (Fig. 1C) (11, 17–19). These enzymes are homologous to mammalian peroxisomal enzymes that process very long chain fatty acids, branched-chain fatty acids, and bile acid intermediates (20). Worms with mutations in maoc-1, dhs-28, or daf-22 do not produce any of the short-chain, dauer-inducing ascarosides, but instead accumulate ascarosides with long-chain side chains, stalled at different steps in the β-oxidation process (11, 17). This phenotype suggests the following three models for the role of β-oxidation in ascaroside biosynthesis: (i) β-Oxidation shortens long-chain ω/(ω-1)-ascarosides to short-chain ω/(ω-1)-ascarosides; (ii) β-oxidation shortens long-chain ω/(ω-1)-hydroxylated fatty acids to short-chain ω/(ω-1)-hydroxylated fatty acids, which are then attached to the ascarylose sugar; or (iii) β-oxidation shortens long-chain fatty acids to short-chain fatty acids, which are then ω/(ω-1)-hydroxylated and attached to the ascarylose sugar (Fig. S1). Worms with mutations in acox-1 have a less severe phenotype; they produce very little asc-ωC3, asc-C5, asc-ΔC7, and asc-ΔC9 and, instead, accumulate asc-ωC5 and asc-C9, implicating ACOX-1 in the β-oxidation of both ω- and (ω-1)-ascarosides (11). This result suggests that fatty acid side-chain hydroxylation precedes β-oxidation during the biosynthetic process and that the third model is not correct.Ascaroside production has been shown to be influenced by a variety of factors, including developmental stage (21), temperature (3, 19), and sex (8), although interpretation of these results is often complicated by the fact that they were obtained by changing multiple variables at the same time (21) or by using long-term, mixed-larval stage cultures (3, 19). Starvation has been suggested to increase the ratio of asc-ΔC9 to asc-ωC3 in long-term, mixed-stage cultures (11). On the other hand, starved L1 larvae, in comparison with those that are fed, have been shown to secrete a higher ratio of ascarosides with shorter, 5-carbon side chains to those with longer, 9-carbon side chains (12).Here we show that specific acyl-CoA oxidases enable C. elegans to modulate the composition of the ascaroside pheromones that it produces under different environmental conditions. Specifically, an ACOX-1 homodimer acts on the CoA-thioester of an ascaroside with a 9-carbon (ω-1)-side chain, an ACOX-1/ACOX-3 heterodimer acts on one with a 7-carbon (ω-1)-side chain, and an ACOX-2 homodimer acts on one with a 5-carbon ω-side chain. Using quantitative (q)RT-PCR, we show that acox-2 and acox-3, which act as gatekeepers for the production of shorter-chain ascarosides, are regulated by changes in temperature and food availability, leading to corresponding changes in ascaroside production. Studies of the ascaroside biosynthetic pathway and how it is modulated by different factors to control pheromone production will shed light on chemical communication in the C. elegans model system, as well as in other free-living and parasitic nematode species, which also use ascarosides as pheromones (22, 23).     

Male fitness increases when females are eliminated from gene pool: Implications for the Y chromosome

Because the two sexes share a common gene pool while performing many different biological functions, mutations benefiting one sex may not accumulate due to counter selection in the other sex. In these experiments 99% of a haploid genome of Drosophila melanogaster was constrained to segregate like a male-limited Y chromosome for 41 generations, thereby eliminating potential counter selection in females. The synthetic Y chromosomes rapidly accumulated genetic variation that increased male fitness and decreased female fitness. The survival and fertility of females declined when they were mated to males expressing the synthetic Y chromosomes. These results suggests that opposing selection between the sexes may substantially interfere with sex-specific adaptation. They also demonstrate how intersexual evolutionary conflict can lead to perpetual degeneration of the Y via genetic hitchhiking of deleterious mutations.     

Enhanced angiotensin II type 2 receptor mechanisms mediate decreases in arterial pressure attributable to chronic low-dose angiotensin II in female rats

The renin-angiotensin system is a far more complex enzymatic cascade than realized previously. Mounting evidence suggests sex-specific differences in the regulation of the renin-angiotensin system and arterial pressure. We examined the hemodynamic responses, angiotensin II receptor subtypes, and angiotensin-converting enzyme 2 gene expression levels after graded doses of angiotensin II in males and females. Mean arterial pressure was measured via telemetry in male and female rats in response to a 2-week infusion of vehicle, low-dose (50 ng/kg per minute SC) or high-dose (400 ng/kg per minute SC) angiotensin II. The effect of concurrent infusion of the angiotensin II type 2 receptor (AT(2)R) blocker (PD123319) was also examined. The arterial pressure response to high-dose angiotensin II was attenuated in females compared with males (24+/-8 mm Hg versus 42+/-5 mm Hg; P for the interaction between sex and treatment <0.002). Remarkably, low-dose angiotensin II decreased arterial pressure (11+/-4 mm Hg; P for the interaction between sex and treatment <0.02) at a dose that did not have an effect in males. This decrease in arterial pressure in females was abolished by AT(2)R blockade. Renal AT(2)R, angiotensin-converting enzyme 2, and left ventricular AT(2)R mRNA gene expressions were markedly greater in females than in males with a renal angiotensin II type 1a receptor:AT(2)R ratio of approximately 1 in females. Angiotensin II infusion did not affect renal AT(2)R mRNA expression but resulted in significantly less left ventricular mRNA expression. Renal angiotensin-converting enzyme 2 mRNA expression levels were greater in females than in males treated with high-dose angiotensin II (approximately 2.5 fold; P for the interaction between sex and treatment <0.05). In females, enhancement of the vasodilatory arm of the renin-angiotensin system, in particular, AT(2)R and angiotensin-converting enzyme 2 mRNA expression, may contribute to the sex-specific differences in response to renin-angiotensin system activation.     

Environmental influence on primary sex ratio in a dioecious plant

The proximity of mates can influence mating opportunities and the quantity and quality of offspring, especially in dioecious plant species. Progeny sex ratios modulated by environmental conditions is one of the most radical ways in which offspring quality may be influenced, yet it has rarely been reported in plants. A mechanism proposed to influence progeny sex ratios in dioecious plants involves competition between female- and male-determining microgametophytes (certation) as a result of variation in pollination intensity. However, the role of selective fertilization in dioecious plants is controversial and has not been demonstrated under field conditions. Here we investigate whether natural variation in the spatial arrangement of females and males influences pollination intensity and progeny sex ratios in the wind-pollinated herb Rumex nivalis. Based on previous experimental manipulation of pollination intensity in this species, we predicted that maternal parents in close proximity to males would produce more strongly female-biased progeny sex ratios. We tested this prediction in six alpine populations in Switzerland by measuring the distance between focal females and neighboring males and assessing pollen loads and seed sex ratios of maternal parents. In four of the six populations, females positioned in close proximity to males captured more pollen and exhibited more female-biased sex ratios. Our results demonstrate that demographic aspects of the maternal mating environment can influence progeny sex ratios. The most probable explanation for biased primary sex ratios in Rumex is selective fertilization resulting from pollen tube competition.     

Low short-term and long-term cardiovascular and all-cause mortality in absence of coronary artery calcium: A 22-year follow-up observational study from large cohort

ObjectivesWe sought to evaluate the gender-specific predictive value of coronary artery calcium (CAC) score on all-cause mortality and cardiovascular disease (CVD) mortality in individuals with and without diabetes mellitus (DM).BackgroundCAC score is a robust predictor of CVD and all-cause mortality during long-term follow-up in large cohorts in adults with DM. However, less is known about its sex-specific impact on all-cause mortality in DM.MethodsWe evaluated 25,563 asymptomatic participants with no known history of coronary artery disease (CAD) who underwent clinically indicated CAC. 1999 (7.8%) individuals had diabetes. CAC was characterized as an Agatston score of 0, 1–99, 100–300, and ?300. We evaluated the association between CAC and all-cause mortality and CVD mortality.ResultsOverall, 1345 individuals died (5.3%) from all causes during a mean follow-up of 14.7 ± 3.8 years. CAC score was 0 in 57.5% females and 34.4% of males without DM, while 36.6% females and 20.3% males with DM had CAC-0. The frequency of CAC ? 300 was 18% and 36% in females and males with DM, respectively. CAC score of zero was associated with low all-cause mortality event rate in females and males with diabetes (1.7 and 2.5 events per 1000 person-years, respectively). Cardiovascular mortality per 1000 person years was ?1 in females and males with CAC score of 0 irrespective of their diabetes. Adjusted multivariable analysis, compared to CAC-0, HR for all-cause mortality associated with CAC 1–99, 100–299 and ?300 were 1.74(95% CI 0.65, 4.63, P-0.20), 5.54(95% CI 2.16, 14.22, P ? 0.001) and 5.75(95% CI 2.30, 14.37, P ? 0.001) in females with DM respectively; in males with DM HR associated with CAC 1–99, 100–299 and ?300 were 1.87(95% CI 0.95, 3.66, P-0.06), 2.15(95% CI 1.05, 4.38, P-0.035) and 2.60(95% CI 1.34, 5.0, P-0.004), respectively.ConclusionPresence of subclinical atherosclerosis varies among individuals with DM. The absence of CAC was associated with very low cardiovascular as well as all-cause mortality events in all subgroups during long term follow-up.     

Identifying sexual differentiation genes that affect Drosophila life span

Background

Sexual differentiation often has significant effects on life span and aging phenotypes. For example, males and females of several species have different life spans, and genetic and environmental manipulations that affect life span often have different magnitude of effect in males versus females. Moreover, the presence of a differentiated germ-line has been shown to affect life span in several species, including Drosophila and C. elegans.

Methods

Experiments were conducted to determine how alterations in sexual differentiation gene activity might affect the life span of Drosophila melanogaster. Drosophila females heterozygous for the tudor[1] mutation produce normal offspring, while their homozygous sisters produce offspring that lack a germ line. To identify additional sexual differentiation genes that might affect life span, the conditional transgenic system Geneswitch was employed, whereby feeding adult flies or developing larvae the drug RU486 causes the over-expression of selected UAS-transgenes.

Results

In this study germ-line ablation caused by the maternal tudor[1] mutation was examined in a long-lived genetic background, and was found to increase life span in males but not in females, consistent with previous reports. Fitting the data to a Gompertz-Makeham model indicated that the maternal tudor[1] mutation increases the life span of male progeny by decreasing age-independent mortality. The Geneswitch system was used to screen through several UAS-type and EP-type P element mutations in genes that regulate sexual differentiation, to determine if additional sex-specific effects on life span would be obtained. Conditional over-expression of transformer female isoform (traF) during development produced male adults with inhibited sexual differentiation, however this caused no significant change in life span. Over-expression of doublesex female isoform (dsxF) during development was lethal to males, and produced a limited number of female escapers, whereas over-expression of dsxF specifically in adults greatly reduced both male and female life span. Similarly, over-expression of fruitless male isoform A (fru-MA) during development was lethal to both males and females, whereas over-expression of fru-MA in adults greatly reduced both male and female life span.

Conclusion

Manipulation of sexual differentiation gene expression specifically in the adult, after morphological sexual differentiation is complete, was still able to affect life span. In addition, by manipulating gene expression during development, it was possible to significantly alter morphological sexual differentiation without a significant effect on adult life span. The data demonstrate that manipulation of sexual differentiation pathway genes either during development or in adults can affect adult life span.     

Androgens during development in a bird species with extremely sexually dimorphic growth, the brown songlark, Cinclorhamphus cruralis

In birds, early exposure to androgens has been shown to influence offspring growth and begging behaviour, and has been proposed as a mechanism for the development of sexual size dimorphism (SSD). Sex specific effects during development can occur due to sex-specific allocation of maternal androgens, sensitivity to, or synthesis of, androgens. In addition, maternal hormones have been suggested as a mechanism to skew brood sex ratio. This study uses one of the world’s most extreme SSD species, the brown songlark Cinclorhamphus cruralis, to investigate (1) sex-specific differences of androgens in yolk and chick plasma and (2) the relationship between androgens and sex ratio bias. The study reveals no indication of sex-specific maternal allocation, but a modest sex effect during the later stages of incubation when the embryo starts to produce its own androgens. Moreover, there was a strong seasonal sex ratio bias: female-biased early and male-biased later in the season, but yolk testosterone (T) did not show a seasonal trend. Taken together these results suggest that if androgens, from any source, have a significant role in development of SSD in this species it is most likely via sex-specific sensitivity or synthesis rather than differential maternal transfer to the egg.     

Müllerian inhibiting substance contributes to sex-linked biases in the brain and behavior

Many behavioral traits and most brain disorders are common to males and females but are more evident in one sex than the other. The control of these subtle sex-linked biases is largely unstudied and has been presumed to mirror that of the highly dimorphic reproductive nuclei. Sexual dimorphism in the reproductive tract is a product of Müllerian inhibiting substance (MIS), as well as the sex steroids. Males with a genetic deficiency in MIS signaling are sexually males, leading to the presumption that MIS is not a neural regulator. We challenge this presumption by reporting that most immature neurons in mice express the MIS-specific receptor (MISRII) and that male Mis^−/− and Misrii^−/− mice exhibit subtle feminization of their spinal motor neurons and of their exploratory behavior. Consequently, MIS may be a broad regulator of the subtle sex-linked biases in the nervous system.     

Effects of sex and COMT genotype on environmentally modulated cognitive control in mice

Cognitive functioning differs between males and females, likely in part related to genetic dimorphisms. An example of a common genetic variation reported to have sexually dimorphic effects on cognition and temperament in humans is the Val/Met polymorphism in catechol-O-methyltransferase (COMT). We tested male and female wild-type mice (^+/+) and their COMT knockout littermates (^+/− and ^−/−) in the five-choice serial reaction time task (5CSRTT) to investigate the effects of sex, COMT genotype, and their interactions with environmental manipulations of cognitive functions such as attention, impulsivity, compulsivity, motivation, and rule-reversal learning. No sex- or COMT-dependent differences were present in the basic acquisition of the five-choice serial reaction time task. In contrast, specific environmental manipulations revealed a variety of sex- and COMT-dependent effects. Following an experimental change to trigger impulsive responding, the sexes showed similar increases in impulsiveness, but males eventually habituated whereas females did not. Moreover, COMT knockout mice were more impulsive compared with wild-type littermates. Manipulations involving mild stress adversely affected cognitive performance in males, and particularly COMT knockout males, but not in females. In contrast, following amphetamine treatment, subtle sex by genotype and sex by treatment interactions emerged primarily limited to compulsive behavior. After repeated testing, female mice showed improved performance, working harder and eventually outperforming males. Finally, removing the food-restriction condition enhanced sex and COMT differences, revealing that overall, females outperform males and COMT knockout males outperform their wild-type littermates. These findings illuminate complex sex- and COMT-related effects and their interactions with environmental factors to influence specific executive cognitive domains.     

Evolution of spatial cognition: sex-specific patterns of spatial behavior predict hippocampal size.

In a study of two congeneric rodent species, sex differences in hippocampal size were predicted by sex-specific patterns of spatial cognition. Hippocampal size is known to correlate positively with maze performance in laboratory mouse strains and with selective pressure for spatial memory among passerine bird species. In polygamous vole species (Rodentia: Microtus), males range more widely than females in the field and perform better on laboratory measures of spatial ability; both of these differences are absent in monogamous vole species. Ten females and males were taken from natural populations of two vole species, the polygamous meadow vole, M. pennsylvanicus, and the monogamous pine vole, M. pinetorum. Only in the polygamous species do males have larger hippocampi relative to the entire brain than do females. Two-way analysis of variance shows that the ratio of hippocampal volume to brain volume is differently related to sex in these two species. To our knowledge, no previous studies of hippocampal size have linked both evolutionary and psychometric data to hippocampal dimensions. Our controlled comparison suggests that evolution can produce adaptive sex differences in behavior and its neural substrate.     

Association between polymorphisms of APE1 and OGG1 and risk of colorectal cancer in Taiwan

AIM: To evaluate the effects of OGG1(Ser326Cys, 11657A/G, and Arg154His) and APE1(Asp148Glu, and T-656G) polymorphisms on colorectal cancer(CRC) risk.METHODS: We enrolled 727 cases newly diagnosed with colorectal adenocarcinoma and 736 age- and sex-matched healthy controls from a medical center in Taiwan. Genomic DNA isolated from the buffy coat was used for genotyping through polymerase chain reaction. Unconditional logistic regressions were used for calculating ORs and 95%CIs to determine the association between the genetic polymorphisms and CRC risk. Haplotype frequencies were estimated using PHASE software. Moreover, stratification analyses onthe basis of sex, age at diagnosis, and tumor subsite and stage were performed.RESULTS: The CRC risk was higher in patients with the OGG1 326Ser/Cys + Cys/Cys genotype(OR = 1.38, 95%CI: 1.03-1.85, P = 0.030), particularly high in patients with stage Ⅲ + Ⅳ cancer(OR = 1.48, 95%CI: 1.03-2.13) compared with patients with the Ser/Ser genotype. In addition, OGG1 11657 G allele carriers had a 41% reduced CRC risk among stage 0-Ⅱ patients(OR = 0.59, 95%CI: 0.35-0.98). The CRC risk was significantly higher among females with the APE1 Glu allele(OR = 1.41, 95%CI: 1.02-1.96). The APE1 148Glu/-656 G haplotype was also associated with a significant CRC risk in females(OR = 1.36, 95%CI: 1.03-1.78).CONCLUSION: OGG1 and APE1 polymorphisms are associated with stage- and sex-specific risk of CRC in the Taiwanese population.     

Reproductive endocrinology of wild, long-lived raptors

The last decades have witnessed a surge of studies analyzing the role of sex hormones on the behavior and ecology of wild bird populations, allowing a more integrated view of the evolution of avian physiology and life histories. Despite a marked progress, field studies show a considerable bias towards research on specific phylogenetic groups, neglecting a significant fraction of the class Aves. Here we analysed changes in the circulating levels of sex steroids in relation to reproductive behaviour in wild black kites (Milvus migrans), a long-lived and socially monogamous Accipitridae raptor. Males and females displayed a single seasonal peak of circulating testosterone (males) and estradiol (females) during pre-laying and laying. Absolute male testosterone levels were low even at the seasonal maximum and remained below detection limits in females. The latter results supports the idea that avian species establishing long-term pair bonds require lower amounts of circulating androgens for reproduction. Circulating progesterone showed a single seasonal peak in females and males, but their timing (during Incubation and Post-brooding respectively) did not overlap. The fact that females black kites perform the majority of incubation and males provide the majority of care to fledglings suggests that progesterone is involved in the expression of parental behaviors.     

Attraction of phlebotomine sand flies to baited and non-baited horizontal surfaces

Female phlebotomine sand flies (Diptera: Psychodidae) transmit leishmaniasis as they engorge on vertebrate blood required for egg production. Phlebotomus (Phlebotomus) papatasi (Scopoli, 1786), the vector of Leishmania major (Yakimoff & Schokhor, 1914), the causative agent of cutaneous leishmaniasis (CL) were not attracted to large horizontal sticky traps (LHSTs) unless these were baited with CO₂ derived from dry ice or from fermenting sugar/yeast mixture (SYM). Attraction of P. papatasi males by CO₂ may indicate their tendency to mate on or near the blood-host. Male P. (Larroussius) orientalis (Parrot, 1936), the vector of visceral leishmaniasis (VL) in Ethiopia, were collected on LHSTs in large numbers. Although the number of females remained low, augmentation with SYM, increased the number of females by 800% while the number of males increased by only about 40%. Apparently, male P. orientalis utilize the horizontal surfaces for forming mating swarms. P. (Paraphlebotomus) sergenti (Parrot, 1917), is the vector of CL caused by Leishmania tropica. Although approximately twice as many P. sergenti males were caught on LHSTs as females, it appears that LHSTs were attractive to both sexes. Use of SYM baits is potentially useful for monitoring phlebotomine sand flies in places where dry ice is unobtainable or prohibitively expensive. LHSTs can provide an inexpensive alternative to CDC traps for monitoring some species of sand flies. Unfortunately, the numbers of female sand flies, crucial for estimating transmission of Leishmania, is usually low on LHSTs.