Concerted evolution in an egg receptor for a rapidly evolving abalone sperm protein

Gamete interactions during fertilization exhibit species specificity. In abalone, the sperm protein lysin species-specifically creates a hole in the egg envelope. Lysin evolves rapidly by positive Darwinian selection. Evolution of the egg receptor for lysin provides the selective pressure for lysin's divergence. The egg receptor for lysin is a tandemly repeated sequence that evolves by concerted evolution. Concerted evolution in the egg receptor could explain the rapid, adaptive evolution in sperm lysin and may provide an underlying molecular mechanism that gives rise to species-specific fertilization.     

The molecular evolution of sperm bindin in six species of sea urchins (Echinoida: Strongylocentrotidae)

The acrosomal protein bindin attaches sperm to eggs during sea urchin fertilization. Complementary to ongoing functional biochemical studies, I take a comparative approach to explore the molecular evolution of bindin in a group of closely related free-spawning echinoid species. Two alleles of the mature bindin gene were sequenced for each of six species in the sea urchin family Strongylocentrotidae. The nucleotide sequences diverged by at least 1% per Myr at both silent and replacement sites. Two short sections flanking the conserved block show an excess of nonsynonymous substitutions. Each is homologous to a region that had been identified as a target of selection in other sea urchin comparisons. A large proportion of the bindin-coding sequence consists of a highly variable repeat region. Bindin sequences, even including the large intron, could not resolve the branching order among five of the species.     

Identification of a new sea urchin vitelline envelope sperm binding glycoprotein

The sea urchin egg vitelline envelope (VE) is composed of eight major glycopolypeptides that are heavily mannosylated and contain fucose and N-acetylglucosamine moieties based on lectin staining. In the present study, the macromolecular composition of the VE and the potential role of a purified VE glycoprotein in initial gamete binding was investigated. The VE components were solubilized from the surface of intact, dejellied eggs with dithiothreitol in divalent cation-free seawater, and analyzed using native, reduced electrophoresis and immunoblotting. Three major VE glycoproteins, VE-A, VE-B and VE-C, and one minor component, VE-D, were identified with antisera against whole VE preparations and against glutaraldehyde-fixed, unfertilized eggs. The electrophoretically purified glycoproteins resolved into a common subunit doublet and one unique subunit each of decreasing size on blots of sodium dodecylsulfate polyacrylamide gels. Lectin affinity chromatography was used for analysis and purification of reduced VE components; a glycoprotein eluted from Con A columns with methyl-mannoside comigrated with VE-B when analyzed by immunoblotting. Whole VE preparations and VE-B obtained from Con A columns were found to inhibit fertilization when preincubated with sperm, thus directly establishing a role for VE-B in gamete binding.     

PDLLA microspheres containing steroids: spray-drying, o/w and w/o/w emulsifications as preparation methods

Sea urchins of the genus Arbacia (order Stirodonta) have discontinuous allopatric distributions ranging over thousands of kilometers. Mitochondrial DNA (mtDNA) sequences were used to reconstruct phylogenetic relationships of four Arbacia species and their geographic populations. There is little evidence of genetic structuring of populations within species, except in two cases at range extremes. The mtDNA sequence differentiation between species suggests that divergence occurred about 4-9 MYA. Gene sequences encoding the sperm protein bindin and its intron were obtained and compared with the mtDNA phylogeny. Sea urchins among the well-studied echinoid order Camarodonta, with degrees of mtDNA divergence similar to those of Arbacia species, are known to have remarkable variation in bindin. However, in Arbacia, little variation in deduced amino acid sequences of bindin was found, indicating that purifying selection acts on the protein. In contrast, bindin intron sequences showed much differentiation, including numerous insertion/deletions. Fertilization experiments performed between a divergent pair of Arbacia species from the Atlantic and Pacific Oceans revealed no evidence of blocks to gamete recognition. In Arbacia, fertilization specificities may have evolved relatively slowly as a result of extensive gene flow within species, greater functional constraint on the bindin polypeptide, or reduced selective pressure for species recognition in singly occurring species.     

Evolution of gamete recognition proteins

REVIEW Although fertilization has been studied for more than a century, the cell surface proteins mediating the process are only now becoming known. Gamete interaction in animals appears to be molecularly complex. Although it is difficult to generalize at present, diversity of structure may be a recurring theme in the evolution of fertilization proteins. Examples of rapid evolution of fertilization proteins by positive selection are known, and concerted evolution can influence the differentiation of gamete recognition proteins between closely related species.     

Participation of sperm proteasome in fertilization of the phlebobranch ascidian Ciona intestinalis

Sperm proteasomes are thought to be involved in sperm binding to and in sperm penetration through the vitelline coat of the eggs of the stolidobranch ascidian Halocynthia roretzi. However, it is not known whether they are involved in the fertilization of eggs of other ascidians. Therefore, we investigated whether sperm proteasomes are also involved in the fertilization of the eggs of the primitive phlebobranch ascidian Ciona intestinalis. Fertilization of the eggs of C. intestinalis was potently inhibited by the proteasome inhibitors MG115 and MG132 but not by the cysteine protease inhibitor E-64-d. On the other hand, neither fertilization of the vitelline coat-free eggs nor sperm binding to the vitelline coat was inhibited by the two proteasome inhibitors at a concentration sufficient to inhibit fertilization of intact eggs. These results indicate that the proteasome plays an essential role in sperm penetration through the vitelline coat rather than in sperm binding to the coat or in sperm-egg membrane fusion. The proteasome activity, which was detected in the sperm extract using Suc-Leu-Leu-Val-Tyr-MCA as a substrate, was strongly inhibited by both MG115 and MG132, and was weakly inhibited by chymostatin, whereas neither leupeptin nor E-64-d inhibited the activity. The molecular mass of the enzyme was estimated to be 600-kDa by Superose 12 gel filtration, and the activity in sperm extract was immunoprecipitated with an anti-proteasome antibody. These results indicate that the proteasome present in sperm of C. intestinalis is involved in fertilization, especially in the process of sperm penetration through the vitelline coat, probably functioning as a lysin.     

Positive Darwinian selection after gene duplication in primate ribonuclease genes

Evolutionary mechanisms of origins of new gene function have been a subject of long-standing debate. Here we report a convincing case in which positive Darwinian selection operated at the molecular level during the evolution of novel function by gene duplication. The genes for eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) in primates belong to the ribonuclease gene family, and the ECP gene, whose product has an anti-pathogen function not displayed by EDN, was generated by duplication of the EDN gene about 31 million years ago. Using inferred nucleotide sequences of ancestral organisms, we showed that the rate of nonsynonymous nucleotide substitution was significantly higher than that of synonymous substitution for the ECP gene. This strongly suggests that positive Darwinian selection operated in the early stage of evolution of the ECP gene. It was also found that the number of arginine residues increased substantially in a short period of evolutionary time after gene duplication, and these amino acid changes probably produced the novel anti-pathogen function of ECP.     

AM67, a secretory component of the guinea pig sperm acrosomal matrix, is related to mouse sperm protein sp56 and the complement component 4-binding proteins

The guinea pig sperm acrosomal matrix is the dense core of the acrosome and is likely to be important in acrosome biogenesis and fertilization. Isolated acrosomal matrices are composed of a limited number of major bands when analyzed by SDS-polyacrylamide gel electrophoresis, among which is a Mr 67,000 protein that we have termed AM67. Indirect immunofluorescence demonstrated that AM67 is localized to the apical segment of the cauda epididymal sperm acrosome. Immunoelectron microscopy further refined the localization of AM67 to the M1 (dorsal bulge) domain within the acrosome. Using a polymerase chain reaction product based upon tryptic peptide sequences from AM67, a lambdagt11 guinea pig testis cDNA library was screened to yield two cDNA clones that encode the AM67 peptides. Northern analysis revealed that AM67 is transcribed as a 1. 9-kilobase testis-specific mRNA. The complete AM67 sequence encodes a prepropolypeptide of 533 amino acids with a calculated Mr of 59, 768. Following cleavage of a probable signal sequence, the polypeptide was predicted to have a Mr of 56,851 and seven consensus sites for asparagine-linked glycosylation. The deduced amino acid sequence of AM67 is most similar to those of the mouse sperm protein sp56 and the alpha-subunits of complement component 4-binding proteins from various mammalian species. Although mouse sp56 has been reported to be a cell-surface receptor for the murine zona pellucida glycoprotein ZP3, standard immunoelectron microscopy using the anti-sp56 monoclonal antibody 7C5 detected sp56 within the mouse sperm acrosome, but failed to detect sp56 on the surface of acrosome-intact mouse sperm. Furthermore, acrosomal labeling was detected in mouse sperm prepared for immunofluorescence using paraformaldehyde fixation, but was not observed with live unfixed sperm. Thus, the finding that sp56 is present within the acrosome provides further support that sp56 and AM67 are orthologues and suggests that sp56 may function in acrosomal matrix-zona pellucida interactions during and immediately following the acrosome reaction in the mouse.     

Isolation, physicochemical properties, and the macromolecular composition of the vitelline and fertilization envelopes from Xenopus laevis eggs

As a step toward defining in molecular terms the sperm-triggered block to polyspermy reaction established by the egg at fertilization, vitelline (VE) and fertilization (FE) envelopes were isolated from eggs of the Sounth African clawed toad Xenopus laevis and some of their physicochemical properties determined. Envelopes were isolated after lysis of the fertilized or unfertilized eggs by sieving techniques; isolated envelopes retained their in situ morphology as determined by electron microscopy. The isolated envelopes had different solubility properties and, in general, VE was more readily dissolved by aqueous solvents than FE, although both could be completely dissolved by detergents or chaotropic agents. Changes in envelope solubility correlated with the progression of the cortical reaction implicating a role for cortical granule material in modifying the solubility properties of the envelope. The VE and FE were composed of protein and carbohydrate with no lipid components detected. As determined by immunodiffusion experiments, the FE contained the same antigens as the VE plus components derived from the cortical granules and the innermost jelly layer, J. The macromolecular composition of the envelopes was determined by sodium dodecyl sulfate gel electrophoresis. The VE contained at least 11 glycoproteins with molecular weights ranging from 125 000 to less than 16 000 with two components (40 000 and 33 000) accounting for almost two-thirds of the total stainable material. The FE contained ten glycoproteins that had the same molecular weights as those in the VE. One glycoprotein component underwent a reduction in molecular weight from 77 000 to 67 500 when the VE was converted to the FE. This molecular weight change was interpreted as the probable result of limited proteolysis. In addition, the FE gel electrophoresis patterns contained macromolecular components derived from the cortical granules and jelly layer, J, consistent with the immunodiffusion experiments. These components were absent when the FE was prepared in the absence of Ca2+, suggesting a role for Ca2+ in binding the VE, cortical granules, and J components together. We concluded that the conversion of the glycoproteinaceous VE to FE at fertilization is caused by interaction of the VE with components from the cortical granules and jelly layer J. These interactions are of both a chemical and physical nature.     

Production of reactive oxygen species by and hydrogen peroxide scavenging activity of spermatozoa in an IVF program

PURPOSE: Reactive oxygen species (ROS) including H2O2 produced by spermatozoa have been suggested, on one hand, to be associated with idiopathic male infertility and, on the other hand, to stimulate certain sperm function leading to fertilization. The influence of ROS on fertilization was investigated in 75 IVF patients by correlating fertilization rates with the production of ROS and the H2O2-scavenging activity of swim-up spermatozoa prepared in parallel with the IVF samples. RESULTS: Low rates of ROS production by the swim-up sperm was detected by the luminol-dependent chemiluminescence assay. They were not correlated with fertilization rates. The hydrogen peroxide scavenging capacity of these spermatozoa, measured as the removal of exogenous H2O2 assayed spectrophotometrically, decreased stepwise in groups of patients achieving higher fertilization rates, suggesting a positive effect of this ROS on fertilization. An alternative explanation of this correlation is plausible in view of the association of both high scavenging activities and poor fertilization rates with poor sperm morphology. CONCLUSIONS: ROS produced by spermatozoa selected by swim-up plays no negative, if not a positive, role in fertilization.     

Tumour size measured by transrectal ultrasonography in the staging of prostate cancer before radical prostatectomy

Fluoroquinolone-resistant mutants of Mycoplasma hominis were selected in vitro from the PG21 susceptible reference strain either by multistep selection on increasing concentrations of various fluoroquinolones or by one-step selection on agar medium with ofloxacin. The quinolone resistance-determining regions (QRDR) of the structural genes encoding the A and b subunits of DNA gyrase were amplified by PCR, and the nucleotide sequences of eight multistep-selected resistant strains were compared to those of susceptible strain PG21. Four high-level resistant mutants that were selected on norfloxacin or ofloxacin contained a C-to-T transition in the gyrA QRDR, leading to substitution of Ser-83 by Leu in the GyrA protein. Analysis of the sequence of the gyrB QRDR of the eight multistep-selected mutants did not reveal any difference compared to that of the gyrB QRDR of the reference strain M. hominis PG21. Similar analyses of eight one-step-selected mutants did not reveal any base change in the gyrA and gyrB QRDRs. These results suggest that in M. hominis, like in other bacterial species, a gyrA mutation at Ser-83 is associated with fluoroquinolone resistance.     

Modified sperm stress test: a simple assay that predicts sperm-related abnormal in-vitro fertilization

Loss of sperm motility is associated with the process of sperm senescence and occurs at different rates within a given normal or abnormal sperm population. Reactive oxygen species attack cell membrane phospholipids, generating fatty acid peroxides and other degradation products, that also have deleterious effects on sperm motility and fertilizing ability. The objective of this investigation was to study a modification of the original sperm stress test (MOST), changing the culture medium to one offering transitional metals and shortening the total test time, to ascertain whether it can predict fertilization under these laboratory conditions. A total of 41 semen samples was obtained from patients undergoing in-vitro fertilization (IVF) at our institution. Semen samples were grouped into those producing total fertilization rates (FR) within normal limits (>50%) and those showing low total FR (<50%). The normal FR group had a significantly greater MOST mean value than the low FR group (0.71 versus 0.44). Furthermore, there was a statistically significant correlation between the MOST score and ungrouped fertilization rates (r = 0.53, P = 0.0004). Diagnostic statistics for MOST ratio values predicting <50% FR showed an optimal threshold of 0.39. Collectively, sensitivity, specificity, positive predictive value and negative predictive value have their largest values at this threshold. Taking into account the above mentioned threshold figures, there is a significant association between MOST and FR categories (P = 0.0009). In conclusion, MOST is a simple assay that has significant predictive value for sperm related IVF abnormalities.     

Fractal landscapes and molecular evolution: modeling the myosin heavy chain gene family

Mapping nucleotide sequences onto a "DNA walk" produces a novel representation of DNA that can then be studied quantitatively using techniques derived from fractal landscape analysis. We used this method to analyze 11 complete genomic and cDNA myosin heavy chain (MHC) sequences belonging to 8 different species. Our analysis suggests an increase in fractal complexity for MHC genes with evolution with vertebrate > invertebrate > yeast. The increase in complexity is measured by the presence of long-range power-law correlations, which are quantified by the scaling exponent alpha. We develop a simple iterative model, based on known properties of polymeric sequences, that generates long-range nucleotide correlations from an initially noncorrelated coding region. This new model-as well as the DNA walk analysis-both support the intron-late theory of gene evolution.     

Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection

Disease resistance genes in plants are often found in complex multigene families. The largest known cluster of disease resistance specificities in lettuce contains the RGC2 family of genes. We compared the sequences of nine full-length genomic copies of RGC2 representing the diversity in the cluster to determine the structure of genes within this family and to examine the evolution of its members. The transcribed regions range from at least 7.0 to 13.1 kb, and the cDNAs contain deduced open reading frames of approximately 5. 5 kb. The predicted RGC2 proteins contain a nucleotide binding site and irregular leucine-rich repeats (LRRs) that are characteristic of resistance genes cloned from other species. Unique features of the RGC2 gene products include a bipartite LRR region with >40 repeats. At least eight members of this family are transcribed. The level of sequence diversity between family members varied in different regions of the gene. The ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitutions was lowest in the region encoding the nucleotide binding site, which is the presumed effector domain of the protein. The LRR-encoding region showed an alternating pattern of conservation and hypervariability. This alternating pattern of variation was also found in all comparisons within families of resistance genes cloned from other species. The Ka /Ks ratios indicate that diversifying selection has resulted in increased variation at these codons. The patterns of variation support the predicted structure of LRR regions with solvent-exposed hypervariable residues that are potentially involved in binding pathogen-derived ligands.     

Expression of glucose transporters in rat brain following transient focal ischemic injury

To better understand genetic diversity of mammalian reoviruses, we studied sequence variability in the S3 gene segment of 17 field-isolate reovirus strains and prototype strains of the three reovirus serotypes. Strains studied were isolated over a 37-year period from different mammalian hosts and geographic locations. A high degree of variability was observed in the nucleotide sequences of the S3 gene, whereas the deduced amino acid sequences of the S3 gene product, sigma NS, were highly conserved. When variability among the S3 nucleotide sequences was analyzed using pairwise comparisons, we found that 5' and 3' noncoding regions were significantly more conserved than the remainder of the gene. This high degree of sequence conservation was also observed within the first 15 nucleotides of the 5' coding region. Phylogenetic analyses showed that multiple alleles of the S3 gene cocirculate and that genetic diversity in the S3 gene does not correlate with host species, geographic locale, or date of isolation. Phylogenetic trees constructed from variation in the S3 sequences are distinct from those previously generated from sequences that encode attachment protein sigma 1, core protein sigma 2, and outer capsid protein sigma 3, which supports the hypothesis that reovirus gene segments reassort in nature. These findings suggest that reovirus gene segments are well-adapted to mammalian hosts and that reovirus evolution has reached an equilibrium.     

Psychological and Physiological Adaptations to Sperm Competition in Humans.

Postcopulatory competition between males, in the form of sperm competition, is a widespread phenomenon in many animal species. The extent to which sperm competition has been an important selective pressure during human evolution remains controversial, however. The authors review critically the evidence that human males and females have psychological, behavioral, and physiological adaptations that evolved in response to selection pressures associated with sperm competition. The authors consider, using evidence from contemporary societies, whether sperm competition is likely to have been a significant adaptive problem for ancestral humans and examine the evidence suggesting that human males have physiological and psychological mechanisms that allow for "prudent" sperm allocation in response to variations in the risk of sperm competition. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The comparative amino acid sequences, substrate specificities and gene or cDNA nucleotide sequences of some prokaryote and eukaryote amidinotransferases: implications for evolution

The amino acid sequences of the amidinotransferases and the nucleotide sequences of their genes or cDNA from four Streptomyces species (seven genes) and from the kidneys of rat, pig, human and human pancreas were compared. The overall amino acid and nucleotide sequences of the prokaryotes and eukaryotes were very similar and further, three regions were identified that were highly identical. Evidence is presented that there is virtually zero chance that the overall and high identity regions of the amino acid sequence similarities and the overall nucleotide sequence similarities between Streptomyces and mammals represent random match. Both rat and lamprey amidinotransferases were able to use inosamine phosphate, the amidine group acceptor of Streptomyces. We have concluded that the structure and function of the amidinotransferases and their genes has been highly conserved through evolution from prokaryotes to eukaryotes. The evolution has occurred with: (1) a high degree of retention of nucleotide and amino acid sequences; (2) a high degree of retention of the primitive Streptomyces guanine + cytosine (G + C) third codon position composition in certain high identity regions of the eukaryote cDNA; (3) a decrease in the specificities for the amidine group acceptors; and (4) most of the mutations silent in the regions suggested to code for active sites in the enzymes.     

Stimulation of oxidant generation by human sperm suspensions using phorbol esters and formyl peptides: relationships with motility and fertilization in vitro

OBJECTIVES: To investigate the influence of reactive oxygen species generated by human spermatozoa and contaminating leukocytes on sperm movement and fertilization in vitro. DESIGN: A chemiluminescence technique, using luminol and peroxidase, was used to monitor the generation of reactive oxygen species by human sperm suspensions and the results were correlated with sperm movement and the fertilization of human ova in vitro. SETTING: Diagnostic Andrology Laboratory and IVF Clinic. PATIENTS: Infertile couples undergoing IVF therapy. RESULTS: An N-formyl-methionyl-leucyl-phenylalanine (FMLP) provocation test was used to demonstrate that the presence of leukocytes in 28.5% of the sperm preparations was associated with elevated levels of spontaneous reactive oxygen species production, impaired movement, and a reduced capacity for fertilization in vitro. In the absence of leukocytes, exposure to phorbol ester stimulated a burst of reactive oxygen species generation by human spermatozoa, the magnitude of which was correlated highly with a loss of sperm motility but not with fertilization rates observed in the concurrent IVF cycle. CONCLUSION: Leukocyte contamination of human sperm preparations can be detected readily by FMLP-induced, luminol-dependent chemiluminescence and the results have an important bearing on the fertilizing capacity of the spermatozoa in vitro.