Sex-peptide is the molecular basis of the sperm effect in Drosophila melanogaster

Mating elicits two major changes in the reproductive behavior of many insect females. The egg-laying rate increases and the readiness to accept males (receptivity) is reduced. These postmating responses last approximately 1 week in Drosophila melanogaster. Males that do not transfer sperm but transfer seminal fluid during mating induce a short-term response of 1 day. The long-term response of 1 week requires the presence of sperm (sperm effect). Hence, sperm is essential for the long-term persistence of the postmating responses. Three seminal fluid peptides elicit postmating responses: ovulin, sex-peptide (SP), and DUP99B. Using the technique of targeted mutagenesis by homologous recombination, we have produced males with mutant SP genes. Here, we report that males lacking functional SP elicit only a weak short-term response. However, these males do transfer sperm. Thus, (i) SP is the major agent eliciting the short-term and the long-term postmating responses and (ii) sperm is merely the carrier for SP. The second conclusion is supported by the finding that SP binds to sperm. The 36-aa-encoding SP gene is the first small Drosophila gene knocked out with the method of homologous recombination.     

Nucleotide diversity and linkage disequilibrium in loblolly pine

Outbreeding species with large, stable population sizes, such as widely distributed conifers, are expected to harbor relatively more DNA sequence polymorphism. Under the neutral theory of molecular evolution, the expected heterozygosity is a function of the product 4N(e)mu, where N(e) is the effective population size and mu is the per-generation mutation rate, and the genomic scale of linkage disequilibrium is determined by 4N(e)r, where r is the per-generation recombination rate between adjacent sites. These parameters were estimated in the long-lived, outcrossing gymnosperm loblolly pine (Pinus taeda L.) from a survey of single nucleotide polymorphisms across approximately 18 kb of DNA distributed among 19 loci from a common set of 32 haploid genomes. Estimates of 4N(e)mu at silent and nonsynonymous sites were 0.00658 and 0.00108, respectively, and both were statistically heterogeneous among loci. By Tajima's D statistic, the site frequency spectrum of no locus was observed to deviate from that predicted by neutral theory. Substantial recombination in the history of the sampled alleles was observed and linkage disequilibrium declined within several kilobases. The composite likelihood estimate of 4N(e)r based on all two-site sample configurations equaled 0.00175. When geological dating, an assumed generation time (25 years), and an estimated divergence from Pinus pinaster Ait. are used, the effective population size of loblolly pine should be 5.6 x 10(5). The emerging narrow range of estimated silent site heterozygosities (relative to the vast range of population sizes) for humans, Drosophila, maize, and pine parallels the paradox described earlier for allozyme polymorphism and challenges simple equilibrium models of molecular evolution.     

Evidence for a causal relationship between the structure, size, and load of calcium pyrophosphate dihydrate crystals, and attacks of pseudogout.

OBJECTIVE--To investigate any relationship between the nature, size, and numbers of synovial fluid (SF) calcium pyrophosphate dihydrate (CPPD) crystals, and attacks of pseudogout. METHODS--Knee SF was aspirated from nine selected patients, first during an attack of pseudogout (acute sample) and again later when the attack had subsided (interval sample). CPPD crystals were extracted, weighed, examined by high resolution transmission electron microscopy (HRTEM), and characterised by size and crystal habit (monoclinic or triclinic). Structural analysis was carried out by x ray powder diffraction (XRD) and the proportions of monoclinic to triclinic CPPD were estimated from densitometric measurements of selected key reflections. RESULTS--The mean crystal size, by HRTEM, indicated that the crystals in the acute sample were larger than those in the interval sample. The ratio of monoclinic to triclinic CPPD, whether estimated from their morphological appearance by HRTEM, or from XRD, was greater in the acute than in the interval sample in all nine patients. The total amount of extracted mineral varied, but in every patient the concentration of CPPD per ml of fluid, and the total mineral per joint, were greater in the acute sample than in the interval sample. CONCLUSION--In this highly selected group of patients, the large numbers of CPPD crystals associated with attacks of pseudogout included a greater proportion of monoclinic crystals, and larger crystals, than those present when inflammation had subsided. A special, phlogistic population of crystals may exist, originating in different joint tissues, or cleared in a different manner, than the more common populations of smaller crystals with a greater proportion of triclinic CPPD, seen in chronic disease.     

Direct electrophoretic detection of the allelic state of single DNA molecules in human sperm by using the polymerase chain reaction.

We have developed a procedure that allows the detection of polymerase chain reaction (PCR) products derived from a single target DNA molecule in a human sperm without using radioactive probes. With this method, three genetic loci present in a single sperm can be amplified simultaneously. The amplification procedure is specific as well as efficient and permits detection of the PCR product by ethidium bromide staining after polyacrylamide gel electrophoresis. When allele-specific PCR primers that differ in length are used, the size of the PCR products of different alleles also vary in length, allowing the allelic state at each locus to be determined electrophoretically. Studies on individual sperm by using this procedure should facilitate the measurement of genetic recombination in humans over small physical distances. The ability to directly analyze the allelic state of PCR products from one cell rapidly and simply will also be useful for the prenatal diagnosis of genetic disease, especially in the analysis of single blastomeres taken from in vitro fertilized eggs prior to implantation.     

Resolving variation in the reproductive tradeoff between sperm size and number

Spermatozoa are amongst the most variable cells, and three factors are thought to account for this variation in design: fertilization mode, phylogeny, and postcopulatory sexual selection. In addition, it has long been assumed that a tradeoff exists between sperm size and number, and although postcopulatory sexual selection affects both traits, empirical evidence for a tradeoff has so far been elusive. Our recent theoretical model predicts that the nature of a direct tradeoff between sperm size and number varies with sperm competition mechanism and sperm competition risk. We test these predictions using a comparative approach in two very different taxa with different sperm competition mechanisms: passerine birds (mechanism: simple raffle) and Drosophila fruit flies (sperm displacement). We show that in both groups, males increase their total ejaculate investment with increasing sperm competition risk, but whereas passerine birds allocate disproportionately to sperm number, drosophilids allocate disproportionately to sperm size. This striking difference between the two groups can be at least partly explained by sperm competition mechanisms depending on sperm size relative to the size of the female reproductive tract: in large animals (passerines), sperm numbers are advantageous in sperm competition owing to dilution inside the female tract, whereas in small animals (drosophilids), large sperm are advantageous for physical competition (sperm displacement). Our study provides two important results. First, we provide convincing evidence for the existence of a sperm size-number tradeoff. Second, we show that by considering both sperm competition mechanism and dilution, can we account for variation in sperm size between different taxa.     

Gamete plasticity in a broadcast spawning marine invertebrate

Sperm competition has classically been thought to maintain anisogamy (large eggs and smaller sperm) because males are thought to maximize their chance of winning fertilizations by trading sperm size for number. More recently it has been recognized that sperm quality (e.g., size, velocity) can also influence sperm competition, although studies have yielded conflicting results. Because sex evolved in the sea, debate has continued over the role of sperm competition and sperm environment in determining both sperm and egg size in externally fertilizing broadcast spawners. Remarkably, however, there have been no direct tests of whether broadcast spawners change the traits of their gametes depending on the likelihood of sperm competition. We manipulated the density (and thus, sperm environment) of a broadcast spawning ascidian (Styela plicata) in the field and then determined whether the phenotype of eggs and sperm changed. We found that sperm from adults kept at high density were larger and more motile than sperm from low-density adults. In vitro fertilizations revealed that sperm from high-density adults also lived longer and induced less polyspermy. Adult density also affected egg traits: eggs from high-density adults were smaller targets for sperm overall but produced larger ovicells than eggs from low-density adults. This suggests that broadcast spawning mothers balance (potentially conflicting) pre- and postzygotic selection pressures on egg size. Overall, our results suggest that sperm competition does not represent a strong force maintaining anisogamy in broadcast spawners. Instead, sperm limitation seems to select for large eggs and smaller, more numerous sperm.     

Enhanced meiotic recombination on the smallest chromosome of Saccharomyces cerevisiae

Chromosome I is the smallest chromosome in Saccharomyces cerevisiae and contains a DNA molecule that is only 250 kilobases (kb). Approximately 75% of this DNA molecule has been cloned. A restriction map for the entire DNA molecule from chromosome I was determined and most of its genetically mapped genes were located on this physical map. Based on the average rate of recombination (centimorgans/kb) found for other S. cerevisiae chromosomes, the outermost markers on the genetic map of chromosome I were expected to be close to the ends of the DNA molecule. While the rightmost genetic marker was 3 kb from the end, the leftmost marker, CDC24, was located near the middle of the left arm, suggesting that the genetic map would be much longer. To extend the genetic map, a copy of the S. cerevisiae URA3 gene was integrated in the outermost cloned region located 32 kb centromere distal to CDC24, and the genetic map distance between these two genes was determined. The new marker substantially increased the genetic map length of chromosome I. In addition, we determined the relationship between physical and genetic map distance along most of the length of the chromosome. Consistent with the longer genetic map, the average rate of recombination between markers on chromosome I was greater than 50% higher than the average found on other yeast chromosomes. Owing to its small size, it had been estimated that approximately 5% of the chromosome I homologues failed to undergo meiotic recombination. New measurements of the zero-crossover class indicated that the enhanced rate of recombination ensures at least one genetic exchange between virtually every pair of chromosome I homologues.     

Large-male advantages associated with costs of sperm production in Drosophila hydei, a species with giant sperm.

Males of the fruit fly Drosophila hydei were found to produce 23.47 +/- 0.46-mm-long spermatozoa, the longest ever described. No relationship was found between male body size and sperm length. We predicted that if these giant gametes are costly for males to produce, then correlations should exist between male body size, rates of sperm production, and fitness attributes associated with the production of sperm. Smaller males were found to make a greater relative investment in testicular tissue growth, even though they have shorter and thinner testes. Smaller males were also found to (i) be maturing fewer sperm bundles within the testes at any point in time than larger males, (ii) require a longer period of time post-eclosion to become reproductively mature, (iii) mate with fewer females, (iv) transfer fewer sperm per copulation, and (v) produce fewer progeny. The significance of these findings for body size-related fitness and the question of sperm size evolution are discussed.     

Phenotypic variations among paternal centrosomes expressed within the zygote as disparate microtubule lengths and sperm aster organization: correlations between centrosome activity and developmental success.

This study describes a paternal effect on sperm aster size and microtubule organization during bovine fertilization. Immunocytochemistry using tubulin antibodies quantitated with confocal microscopy was used to measure the diameter of the sperm aster and assign a score (0-3) based on the degree of radial organization (0, least organized; 3, most organized). Three bulls (A-C) were chosen based on varying fertility (A, lowest fertility; C, highest fertility) as assessed by nonreturn to estrus after artificial insemination and in vitro embryonic development to the blastocyst stage. The results indicate a statistically significant bull-dependent difference in diameter of the sperm aster and in the organization of the sperm astral microtubules. Insemination from bull A resulted in an average sperm aster diameter of 101.4 microm (76.3% of oocyte diameter). This significantly differs (P < or = 0.0001) from the average sperm aster diameters produced after inseminations from bull B (78.2 microm; 60.8%) or bull C (77.9 microm; 57.8%), which themselves displayed no significant differences. The degree of radial organization of the sperm aster was also bull-dependent. Sperm asters organized by bull A-derived sperm had an average quality score of 1.8, which was higher than that of bull B (1.4; P < or = 0.0005) or bull C (1.2; P < or = 0.0001). Results with bulls B and C were also significantly different (P < or = 0.025). These results indicate that the paternally derived portion of the centrosome varies among males and that this variation affects male fertility, the outcome of early development, and, therefore, reproductive success.     

Two-year comparison of testicular responses to pulsatile gonadotropin-releasing hormone and exogenous gonadotropins from the inception of therapy in men with isolated hypogonadotropic hypogonadism

Men with the complete form of isolated hypogonadotropic hypogonadism (initial mean testes volume less than 4 mL) require 2 or more yr of exogenous gonadotropin therapy combining hCG and human menopausal gonadotropin (hMG) to achieve maximal, but subnormal, testis size and sperm output. To test whether pulsatile GnRH therapy, which more closely mimics normal hormonal stimulation, would accelerate or further augment testicular growth, hasten the onset of sperm production, and/or increase sperm output more than occurs during conventional exogenous gonadotropin therapy, we administered either hCG/hMG or GnRH from the inception of therapy to 2 comparable groups of men with complete IHH (initial testicular volume, less than 4 mL) and compared their testicular responses during the first 2 hr of therapy. Five men were treated with pulsatile GnRH in doses of 143-714 ng/kg every 2 h, sc, while 11 other men received hCG (2000 IU) and hMG (75 IU FSH and 75 IU LH) im 3 times/week. In the GnRH-treated men, the mean plasma total and free testosterone levels during therapy rose to within the normal range, but were significantly lower (P less than 0.01 and P less than 0.02, respectively) than those in the hCG/hMG-treated men. The mean plasma estradiol concentrations during therapy were within the high normal range and were similar in the two groups. The mean plasma FSH levels achieved in the GnRH-treated men were significantly (P less than 0.01) and 1.3- to 3.2-fold higher than those in the hCG/hMG-treated men. The mean testicular size achieved in the GnRH-treated men was not significantly different from that in the hCG/hMG-treated men (P = 0.08); the mean testicular volumes after 2 yr were 4.8- and 4.3-fold the pretreatment values in the GnRH and hCG/hMG groups, respectively. After 12 months of therapy, sperm production had occurred in one man in the GnRH group and in no subject in the hCG/hMG group. After 24 months, two men in the GnRH group and eight men in the hCG/hMG group produced sperm. Thus, 40% of the GnRH-treated men and 80% of the hCG/hMG-treated men (P = NS) produced sperm after 2 yr of therapy. The sperm concentrations in all men were below 5 million/mL and were comparable in the two groups (P = NS). These results suggest that pulsatile sc GnRH therapy for the first 2 yr does not accelerate or enhance testicular growth, hasten the onset of sperm production, or increase sperm output significantly compared to hCG/hMG.     

Estimating Design Effect and Calculating Sample Size for Respondent-Driven Sampling Studies of Injection Drug Users in the United States

Respondent-driven sampling (RDS) has become increasingly popular for sampling hidden populations, including injecting drug users (IDU). However, RDS data are unique and require specialized analysis techniques, many of which remain underdeveloped. RDS sample size estimation requires knowing design effect (DE), which can only be calculated post hoc. Few studies have analyzed RDS DE using real world empirical data. We analyze estimated DE from 43 samples of IDU collected using a standardized protocol. We find the previous recommendation that sample size be at least doubled, consistent with DE = 2, underestimates true DE and recommend researchers use DE = 4 as an alternate estimate when calculating sample size. A formula for calculating sample size for RDS studies among IDU is presented. Researchers faced with limited resources may wish to accept slightly higher standard errors to keep sample size requirements low. Our results highlight dangers of ignoring sampling design in analysis.     

Sperm quantity and size variation in un-irradiated and irradiated males of the malaria mosquito Anopheles arabiensis Patton

Anopheles mosquitoes are important candidates for genetic control strategies. However, little is known about sperm quality and quantity as determinants of male reproductive success. In this study, sperm quantity and length variation were assessed in testes of un-irradiated and irradiated Anopheles arabiensis.Male reproductive organs were dissected for sperm and an estimate of the total number of spermatozoa was made. Sperm lengths were measured using imaging software. The effects of irradiation were evaluated for males exposed in the pupal or adult stage to a full (120 Gy) or partially sterilising dose (70 Gy). Sperm length variation in the laboratory strain was compared to the distribution observed in wild males. We also determined the size distribution of sperm lengths in spermathecae of inseminated females compared to those observed in male testes.Sperm quantity increased with age, and 12-day-old males had significantly more sperm in their testes (8214 ± 467) than males aged 3 days (5022 ± 375). Mosquitoes irradiated in the pupal stage had significantly fewer sperm (2982 ± 125) than un-irradiated males (4950 ± 848) although for adult stage irradiation similar amounts of sperm were observed compared to un-irradiated males. Sperm length variation was detected with sperm lengths ranging between <50 and 500 μm. There were no differences in sperm length distribution compared to wild males. Sperm length distributions were similar to those reported for the closely related sibling species An. gambiae s.s. There was no major effect of irradiation on the distribution of sperm lengths in the testes, with the exception that pupal irradiation resulted in a significant increase in sperm numbers in the category of 100-200 μm. Sperm length distributions in spermathecae were different to those measured directly from sperm in the testes and harboured less cells of the smaller (<100-200 μm), and more cells of the larger category (300-400 μm).The finding that testes of pupal irradiated males produce fewer and smaller sperm in comparison to un-irradiated and adult irradiated testes are discussed in the context of genetic control strategies, in particular the Sterile Insect Technique (SIT).     

Variation in recombination rate across the X chromosome of Anopheles gambiae

The M and S molecular forms of Anopheles gambiae are considered to be incipient species, despite residual gene exchange. Of the three small genome regions that are strongly differentiated between the molecular forms ("speciation islands"), two are located near centromeres, on the left arm of chromosome 2 and the X chromosome. To test the prediction of reduced recombination in these islands, we estimated recombination rates between microsatellite loci on the X chromosome using two M-form strains. Across most of the chromosome, recombination occurred at approximately 1 centimorgan per megabase (cM Mb(-1)), a value closely matching the genome-wide average estimated for A. gambiae and for other eukaryotes. Recombination was much higher at the telomeric end, > 7 cM Mb(-1). In the speciation island at the centromeric end, recombination was sharply reduced to approximately 0.2 cM Mb(-1), consistent with a role for reduced recombination in maintaining differentiation between nascent species despite gene flow.     

The optimal measure of allelic association

Allelic association between pairs of loci is derived in terms of the association probability rho as a function of recombination theta, effective population size N, linear systematic pressure v, and time t, predicting both rho(rt), the decrease of association from founders and rho(ct), the increase by genetic drift, with rho(t) = rho(rt) + rho(ct). These results conform to the Malecot equation, with time replaced by distance on the genetic map, or on the physical map if recombination in the region is uniform. Earlier evidence suggested that rho is less sensitive to variations in marker allele frequencies than alternative metrics for which there is no probability theory. This robustness is confirmed for six alternatives in eight samples. In none of these 48 tests was the residual variance as small as for rho. Overall, efficiency was less than 80% for all alternatives, and less than 30% for two of them. Efficiency of alternatives did not increase when information was estimated simultaneously. The swept radius within which substantial values of rho are conserved lies between 385 and 893 kb, but deviation of parameters between measures is enormously significant. The large effort now being devoted to allelic association has little value unless the rho metric with the strongest theoretical basis and least sensitivity to marker allele frequencies is used for mapping of marker association and localization of disease loci.     

Estimate of effective recombination rate and average selection coefficient for HIV in chronic infection

HIV adaptation to a host in chronic infection is simulated by means of a Monte-Carlo algorithm that includes the evolutionary factors of mutation, positive selection with varying strength among sites, random genetic drift, linkage, and recombination. By comparing two sensitive measures of linkage disequilibrium (LD) and the number of diverse sites measured in simulation to patient data from one-time samples of pol gene obtained by single-genome sequencing from representative untreated patients, we estimate the effective recombination rate and the average selection coefficient to be on the order of 1% per genome per generation (10(-5) per base per generation) and 0.5%, respectively. The adaptation rate is twofold higher and fourfold lower than predicted in the absence of recombination and in the limit of very frequent recombination, respectively. The level of LD and the number of diverse sites observed in data also range between the values predicted in simulation for these two limiting cases. These results demonstrate the critical importance of finite population size, linkage, and recombination in HIV evolution.     

Evolutionary trade-off between weapons and testes

It has long been recognized that male mating competition is responsible for the evolution of weaponry for mate acquisition. However, when females mate with more than one male, competition between males can continue after mating in the form of sperm competition. Theory predicts that males should increase their investment in sperm production as sperm competition is increased, but it assumes that males face a trade-off between sperm production and other life-history traits such as mate acquisition. Here, we use a genus of horned beetle, Onthophagus, to examine the trade-off between investment in testes required for fertilizations and investment in weapons used to obtain matings. In a within-species study, we prevented males from developing horns and found that these males grew larger and invested relatively more in testes growth than did males allowed to grow horns. Among species, there was no general relationship between the relative sizes of horns and testes. However, the allometric slope of horn size on body size was negatively associated with the allometric slope of testes size on body size. We suggest that this reflects meaningful evolutionary changes in the developmental mechanisms regulating trait growth, specifically in the degree of nutrition-dependent phenotypic plasticity versus canalization of traits. Finally, we show how this resource allocation trade-off has influenced the evolutionary diversification of weapons, revealing a rich interplay between developmental trade-offs and both pre- and postmating mechanisms of sexual competition.     

Recombination within and between the human insulin and beta-globin gene loci.

We detected a large number of polymorphic insulin restriction fragments in black Americans. These different size fragments were probably generated by unequal recombination on both sides of the human insulin gene. Population genetic analysis indicates that recombination occurred 33 times more frequently than expected to generate this large number of polymorphic fragments. Specific properties of the unique repeated 14- to 16-base-pair sequences 5' to the insulin gene suggest that this sequence would promote increased unequal recombination. Additional pedigree analysis showed that the recombination rate between the structural insulin and beta-globin gene loci was 14% with strong evidence for linkage. Since both insulin and beta-globin have been mapped to the short arm of human chromosome 11, this study establishes that the genetic map distance between these genes is 14.2 centimorgans.     

Gene conversions and unequal crossovers between CYP21 (steroid 21-hydroxylase gene) and CYP21P involve different mechanisms.

Most cases of congenital adrenal hyperplasia, the inherited inability to synthesize cortisol, are caused by mutations in the steroid 21-hydroxylase gene (CYP21). Steroid 21-hydroxylase deficiency is unusual among genetic diseases in that approximately 95% of the mutant alleles have apparently been generated by recombination between a normally active gene (CYP21) and a linked pseudogene (CYP21P). Approximately 20% of mutant alleles carry DNA deletions of 30 kb that have presumably been generated by unequal meiotic crossing-over, whereas 75% carry one or more mutations in CYP21 that are normally found in the CYP21P pseudogene. These latter mutations are termed "gene conversions," although the mechanism by which they are generated is not well understood. To assess the frequency at which these different recombination events occur, we have used PCR to detect de novo deletions and gene conversions in matched sperm and peripheral blood leukocyte DNA samples from normal individuals. Deletions with breakpoints in a 100-bp region in intron 2 and exon 3 were detected in sperm DNA samples with frequencies of approximately 1 in 10(5)-10(6) genomes but were never detected in the matching leukocyte DNA. Gene conversions in the same region occur in approximately 1 in 10(3)-10(5) genomes in both sperm and leukocyte DNA. These data suggest that whereas deletions occur exclusively in meiosis, gene conversions occur during both meiosis and mitosis, or perhaps only during mitosis. Thus, gene conversions must occur by a mechanism distinct from unequal crossing-over.     

轮状病毒VP4蛋白与肠产毒性大肠杆菌热稳定肠毒素蛋白的融合表达

利用DNA重组技术将轮状病毒（Rotavirus RV)VP4蛋白主要抗原编码区基因与产肠毒素大肠杆菌(Enteotoxigenic Escherichia coli ETEC)热稳定肠毒素（Heat-stable enteotoxin Escherichia coli ST)基因融合，插入原核表达质粒pET-28a( )中，经PCR、酶切、测序分析表明已将vp4-st片段克隆入PET－28a( ),而且具有正确的读码框和方向性，正确构建了VP4－ST的融合表达载体pET28-VP4-ST。表达质粒转化受体菌BL21（DE3）plysS，取经IPTG诱导后表达的目的蛋白进行SDS－PAGE、凝胶薄层扫描分析。结果表明：表达的目的蛋白以包涵体的形式存在，大小约40．2kDa，表达的蛋白量占菌体总蛋白的13．048％。     

A population genetics model with recombination hotspots that are heterogeneous across the population

Both sperm typing and linkage disequilibrium patterns from large population genetic data sets have demonstrated that recombination hotspots are responsible for much of the recombination activity in the human genome. Sperm typing has also revealed that some hotspots are heterogeneous in the population; and linkage disequilibrium patterns from the chimpanzee have implied that hotspots change at least on the separation time between these species. We propose a population genetics model, inspired by the double-strand break model, which features recombination hotspots that are heterogeneous across the population and whose population frequency changes with time. We have derived a diffusion approximation and written a coalescent simulation program. This model has implications for the "hotspot paradox."