A public resource of single cell transcriptomes and multiscale networks from persons with and without Alzheimer's disease.

The emergence of technologies that can support high-throughput profiling of single cell transcriptomes offers to revolutionize the study of brain tissue from persons with and without Alzheimer's disease (AD). Integration of these data with additional complementary multiomics data such as genetics, proteomics and clinical data provides powerful opportunities to link observed cell subpopulations and molecular network features within a broader disease-relevant context. We report here single nucleus RNA sequencing (snRNA-seq) profiles generated from superior frontal gyrus cortical tissue samples from 101 exceptionally well characterized, aged subjects from the Banner Brain and Body Donation Program in combination with whole genome sequences. We report findings that link common AD risk variants with CR1 expression in oligodendrocytes as well as alterations in peripheral hematological lab parameters, with these observations replicated in an independent, prospective cohort study of ageing and dementia. We also observed an AD-associated CD83(+) microglial subtype with unique molecular networks that encompass many known regulators of AD-relevant microglial biology, and which are associated with immunoglobulin IgG4 production in the transverse colon. These findings illustrate the power of multi-tissue molecular profiling to contextualize snRNA-seq brain transcriptomics and reveal novel disease biology. The transcriptomic, genetic, phenotypic, and network data resources described within this study are available for access and utilization by the scientific community.

Viral anti-inflammatory serpin reduces immuno-coagulopathic pathology in SARS-CoV-2 mouse models of infection.

SARS-CoV-2 acute respiratory distress syndrome (ARDS) induces uncontrolled lung inflammation and coagulopathy with high mortality. Anti-viral drugs and monoclonal antibodies reduce early COVID-19 severity, but treatments for late-stage immuno-thrombotic syndromes and long COVID are limited. Serine protease inhibitors (SERPINS) regulate activated proteases. The myxoma virus-derived Serp-1 protein is a secreted immunomodulatory serpin that targets activated thrombotic, thrombolytic, and complement proteases as a self-defense strategy to combat clearance. Serp-1 is effective in multiple animal models of inflammatory lung disease and vasculitis. Here, we describe systemic treatment with purified PEGylated Serp-1 as a therapy for immuno-coagulopathic complications during ARDS. Treatment with PEGSerp-1 in two mouse-adapted SARS-CoV-2 models in C57Bl/6 and BALB/c mice reduced lung and heart inflammation, with improved outcomes. PEGSerp-1 significantly reduced M1 macrophages in the lung and heart by modifying urokinase-type plasminogen activator receptor (uPAR), thrombotic proteases, and complement membrane attack complex (MAC). Sequential changes in gene expression for uPAR and serpins (complement and plasminogen inhibitors) were observed. PEGSerp-1 is a highly effective immune-modulator with therapeutic potential for severe viral ARDS, immuno-coagulopathic responses, and Long COVID.

Nuclear Export Inhibitor Selinexor Enhances Oncolytic Myxoma Virus Therapy against Cancer.

Oncolytic viruses exploited for cancer therapy have been developed to selectively infect, replicate, and kill cancer cells to inhibit tumor growth. However, in some cancer cells, oncolytic viruses are often limited in completing their full replication cycle, forming progeny virions, and/or spreading in the tumor bed because of the heterogeneous cell types within the tumor bed. Here, we report that the nuclear export pathway regulates oncolytic myxoma virus (MYXV) infection and cytoplasmic viral replication in a subclass of human cancer cell types where viral replication is restricted. Inhibition of the XPO-1 (exportin 1) nuclear export pathway with nuclear export inhibitors can overcome this restriction by trapping restriction factors in the nucleus and allow significantly enhanced viral replication and killing of cancer cells. Furthermore, knockdown of XPO-1 significantly enhanced MYXV replication in restrictive human cancer cells and reduced the formation of antiviral granules associated with RNA helicase DHX9. Both in vitro and in vivo, we demonstrated that the approved XPO1 inhibitor drug selinexor enhances the replication of MYXV and kills diverse human cancer cells. In a xenograft tumor model in NSG mice, combination therapy with selinexor plus MYXV significantly reduced the tumor burden and enhanced the survival of animals. In addition, we performed global-scale proteomic analysis of nuclear and cytosolic proteins in human cancer cells to identify the host and viral proteins that were upregulated or downregulated by different treatments. These results indicate, for the first time, that selinexor in combination with oncolytic MYXV can be used as a potential new therapy. Significance: We demonstrated that a combination of nuclear export inhibitor selinexor and oncolytic MYXV significantly enhanced viral replication, reduced cancer cell proliferation, reduced tumor burden, and enhanced the overall survival of animals. Thus, selinexor and oncolytic MYXV can be used as potential new anticancer therapy.

Humanization and expression of IgG and IgM antibodies in plants as potential diagnostic reagents for Valley Fever.

Monoclonal antibodies (mAbs) are important proteins used in many life science applications, from diagnostics to therapeutics. High demand for mAbs for different applications urges the development of rapid and reliable recombinant production platforms. Plants provide a quick and inexpensive system for producing recombinant mAbs. Moreover, when paired with an established platform for mAb discovery, plants can easily be tailored to produce mAbs of different isotypes against the same target. Here, we demonstrate that a hybridoma-generated mouse mAb against chitinase 1 (CTS1), an antigen from Coccidioides spp., can be biologically engineered for use with serologic diagnostic test kits for coccidioidomycosis (Valley Fever) using plant expression. The original mouse IgG was modified and recombinantly produced in glycoengineered Nicotiana benthamiana plants via transient expression as IgG and IgM isotypes with human kappa, gamma, and mu constant regions. The two mAb isotypes produced in plants were shown to maintain target antigen recognition to CTS1 using similar reagents as the Food and Drug Administration (FDA)-approved Valley Fever diagnostic kits. As none of the currently approved kits provide antibody dilution controls, humanization of antibodies that bind to CTS1, a major component of the diagnostic antigen preparation, may provide a solution to the lack of consistently reactive antibody controls for Valley Fever diagnosis. Furthermore, our work provides a foundation for reproducible and consistent production of recombinant mAbs engineered to have a specific isotype for use in diagnostic assays.

Functional Diversity and Evolution of the Drosophila Sperm Proteome.

Spermatozoa are central to fertilization and the evolutionary fitness of sexually reproducing organisms. As such, a deeper understanding of sperm proteomes (and associated reproductive tissues) has proven critical to the advancement of the fields of sexual selection and reproductive biology. Due to their extraordinary complexity, proteome depth-of-coverage is dependent on advancements in technology and related bioinformatics, both of which have made significant advancements in the decade since the last Drosophila sperm proteome was published. Here, we provide an updated version of the Drosophila melanogaster sperm proteome (DmSP3) using improved separation and detection methods and an updated genome annotation. Combined with previous versions of the sperm proteome, the DmSP3 contains a total of 3176 proteins, and we provide the first label-free quantitation of the sperm proteome for 2125 proteins. The top 20 most abundant proteins included the structural elements α- and ß-tubulins and sperm leucyl-aminopeptidases. Both gene content and protein abundance were significantly reduced on the X chromosome, consistent with prior genomic studies of X chromosome evolution. We identified 9 of the 16 Y-linked proteins, including known testis-specific male fertility factors. We also identified almost one-half of known Drosophila ribosomal proteins in the DmSP3. The role of this subset of ribosomal proteins in sperm is unknown. Surprisingly, our expanded sperm proteome also identified 122 seminal fluid proteins (Sfps), proteins originally identified in the accessory glands. We show that a significant fraction of 'sperm-associated Sfps' are recalcitrant to concentrated salt and detergent treatments, suggesting this subclass of Sfps are expressed in testes and may have additional functions in sperm, per se. Overall, our results add to a growing landscape of both sperm and seminal fluid protein biology and in particular provides quantitative evidence at the protein level for prior findings supporting the meiotic sex-chromosome inactivation model for male-specific gene and X chromosome evolution.

Comparative Proteomics of Coxiella like Endosymbionts (CLEs) in the Symbiotic Organs of Rhipicephalus sanguineus Ticks.

Maternally transmitted obligatory endosymbionts are found in the female gonads as well as in somatic tissue and are expected to provide missing metabolite to their hosts. These deficiencies are presumably complemented through specific symbiotic microorganisms such as Coxiella-like endosymbionts (CLEs) of Rhipicephalus ticks. CLEs are localized in specialized host tissue cells within the Malpighian tubules (Mt) and the ovaries (Ov) from which they are maternally transmitted to developing oocytes. These two organs differ in function and cell types, but the role of CLEs in these tissues is unknown. To probe possible functions of CLEs, comparative proteomics was performed between Mt and Ov of R. sanguineus ticks. Altogether, a total of 580 and 614 CLE proteins were identified in Mt and Ov, respectively. Of these, 276 CLE proteins were more abundant in Mt, of which 12 were significantly differentially abundant. In Ov, 290 CLE proteins were more abundant, of which 16 were significantly differentially abundant. Gene Ontology analysis revealed that most of the proteins enriched in Mt are related to cellular metabolic functions and stress responses, whereas in Ov, the majority were related to cell proliferation suggesting CLEs function differentially and interdependently with host requirements specific to each organ. The results suggest Mt CLEs provide essential nutrients to its host and Ov CLEs promote proliferation and vertical transmission to tick progeny. IMPORTANCE Here we compare the Coxiella-like endosymbionts (CLEs) proteomes from Malpighian tubule (Mt) and the ovaries (Ov) of the brown dog tick Rhipicephalus sanguineus. Our results support the hypothesis that CLEs function interdependently with host requirements in each of the organs. The different functional specificity of CLE in the same host suggest that metabolic capabilities evolved according to the constrains imposed by the specific organ function and requirements. Our findings provide specific CLE protein targets that can be useful for future studies of CLE biology with a focus on tick population control.

A cross-species approach for the identification of Drosophila male sterility genes.

Male reproduction encompasses many essential cellular processes and interactions. As a focal point for these events, sperm offer opportunities for advancing our understanding of sexual reproduction at multiple levels during development. Using male sterility genes identified in human, mouse, and fruit fly databases as a starting point, 103 Drosophila melanogaster genes were screened for their association with male sterility by tissue-specific RNAi knockdown and CRISPR/Cas9-mediated mutagenesis. This list included 56 genes associated with male infertility in the human databases, but not found in the Drosophila database, resulting in the discovery of 63 new genes associated with male fertility in Drosophila. The phenotypes identified were categorized into six distinct classes affecting sperm development. Interestingly, the second largest class (Class VI) caused sterility despite apparently normal testis and sperm morphology suggesting that these proteins may have functions in the mature sperm following spermatogenesis. We focused on one such gene, Rack 1, and found that it plays an important role in two developmental periods, in early germline cells or germline stem cells and in spermatogenic cells or sperm. Taken together, many genes are yet to be identified and their role in male reproduction, especially after ejaculation, remains to be elucidated in Drosophila, where a wealth of data from human and other model organisms would be useful.

The Drosophila Neprilysin 4 gene is essential for sperm function following sperm transfer to females.

Sperm are modified substantially in passing through both the male and the female reproductive tracts, only thereafter becoming functionally competent to fertilize eggs. Drosophila sperm become motile in the seminal vesicle; after ejaculation, they interact with seminal fluid proteins and undergo biochemical changes on their surface while they are stored in the female sperm storage organs. However, the molecular mechanisms underlying these maturation processes remain largely unknown. Here, we focused on Drosophila Neprilysin genes, which are the fly orthologs of the mouse Membrane metallo-endopeptidase-like 1 (Mmel1) gene. While Mmel1 knockout male mice have reduced fertility without abnormality in either testis morphology or sperm motility, there are inconsistent results regarding the association of any Neprilysin gene with male fertility in Drosophila. We examined the association of the Nep1-5 genes with male fertility by RNAi and found that Nep4 gene function is specifically required in germline cells. To investigate this in more detail, we induced mutations in the Nep4 gene by the CRISPR/Cas9 system and isolated two mutants, both of which were viable and female fertile, but male sterile. The mutant males had normal-looking testes and sperm; during copulation, sperm were transferred to females and stored in the seminal receptacle and paired spermathecae. However, following sperm transfer and storage, three defects were observed for Nep4 mutant sperm. First, sperm were quickly discarded by the females; second, the proportion of eggs fertilized was significantly lower for mutant sperm than for control sperm; and third, most eggs laid did not initiate development after sperm entry. Taking these observations together, we conclude that the Nep4 gene is essential for sperm function following sperm transfer to females.

Seminal fluid protein divergence among populations exhibiting postmating prezygotic reproductive isolation.

Despite holding a central role in fertilization, reproductive traits often show elevated rates of evolution and diversification. The rapid evolution of seminal fluid proteins (Sfps) within populations is predicted to cause mis-signalling between the male ejaculate and the female during and after mating resulting in postmating prezygotic (PMPZ) isolation between populations. Crosses between Drosophila montana populations show PMPZ isolation in the form of reduced fertilization success in both noncompetitive and competitive contexts. Here we test whether male ejaculate proteins produced in the accessory glands or ejaculatory bulb differ between populations using liquid chromatography tandem mass spectrometry. We find more than 150 differentially abundant proteins between populations that may contribute to PMPZ isolation, including a number of proteases, peptidases and several orthologues of Drosophila melanogaster Sfps known to mediate fertilization success. Males from the population that elicit the stronger PMPZ isolation after mating with foreign females typically produced greater quantities of Sfps. The accessory glands and ejaculatory bulb show enrichment for different gene ontology (GO) terms and the ejaculatory bulb contributes more differentially abundant proteins. Proteins with a predicted secretory signal evolve faster than nonsecretory proteins. Finally, we take advantage of quantitative proteomics data for three Drosophila species to determine shared and unique GO enrichments of Sfps between taxa and which potentially mediate PMPZ isolation. Our study provides the first high-throughput quantitative proteomic evidence showing divergence of reproductive proteins between populations that exhibit PMPZ isolation.

Evolutionary Proteomics Reveals Distinct Patterns of Complexity and Divergence between Lepidopteran Sperm Morphs.

Spermatozoa are one of the most strikingly diverse animal cell types. One poorly understood example of this diversity is sperm heteromorphism, where males produce multiple distinct morphs of sperm in a single ejaculate. Typically, only one morph is capable of fertilization and the function of the nonfertilizing morph, called parasperm, remains to be elucidated. Sperm heteromorphism has multiple independent origins, including Lepidoptera (moths and butterflies), where males produce a fertilizing eupyrene sperm and an apyrene parasperm, which lacks a nucleus and nuclear DNA. Here we report a comparative proteomic analysis of eupyrene and apyrene sperm between two distantly related lepidopteran species, the monarch butterfly (Danaus plexippus) and Carolina sphinx moth (Manduca sexta). In both species, we identified ∼700 sperm proteins, with half present in both morphs and the majority of the remainder observed only in eupyrene sperm. Apyrene sperm thus have a distinctly less complex proteome. Gene ontology (GO) analysis revealed proteins shared between morphs tend to be associated with canonical sperm cell structures (e.g., flagellum) and metabolism (e.g., ATP production). GO terms for morph-specific proteins broadly reflect known structural differences, but also suggest a role for apyrene sperm in modulating female neurobiology. Comparative analysis indicates that proteins shared between morphs are most conserved between species as components of sperm, whereas morph-specific proteins turn over more quickly, especially in apyrene sperm. The rapid divergence of apyrene sperm content is consistent with a relaxation of selective constraints associated with fertilization and karyogamy. On the other hand, parasperm generally exhibit greater evolutionary lability, and our observations may therefore reflect adaptive responses to shifting regimes of sexual selection.

The Old and the New: Discovery Proteomics Identifies Putative Novel Seminal Fluid Proteins in Drosophila.

Seminal fluid proteins (SFPs), the nonsperm component of male ejaculates produced by male accessory glands, are viewed as central mediators of reproductive fitness. SFPs effect both male and female post-mating functions and show molecular signatures of rapid adaptive evolution. Although Drosophila melanogaster, is the dominant insect model for understanding SFP evolution, understanding of SFP evolutionary causes and consequences require additional comparative analyses of close and distantly related taxa. Although SFP identification was historically challenging, advances in label-free quantitative proteomics expands the scope of studying other systems to further advance the field. Focused studies of SFPs has so far overlooked the proteomes of male reproductive glands and their inherent complex protein networks for which there is little information on the overall signals of molecular evolution. Here we applied label-free quantitative proteomics to identify the accessory gland proteome and secretome in Drosophila pseudoobscura,, a close relative of D. melanogaster,, and use the dataset to identify both known and putative novel SFPs. Using this approach, we identified 163 putative SFPs, 32% of which overlapped with previously identified D. melanogaster, SFPs and show that SFPs with known extracellular annotation evolve more rapidly than other proteins produced by or contained within the accessory gland. Our results will further the understanding of the evolution of SFPs and the underlying male accessory gland proteins that mediate reproductive fitness of the sexes.

Identification and characterization of the zebra finch (Taeniopygia guttata) sperm proteome.

Spermatozoa exhibit remarkable variability in size, shape, and performance. Our understanding of the molecular basis of this variation, however, is limited, especially in avian taxa. The zebra finch (Taeniopygia guttata) is a model organism in the study of avian sperm biology and sperm competition. Using LC-MS based proteomics, we identify and describe 494 proteins of the zebra finch sperm proteome (ZfSP). Gene ontology and associated bioinformatics analyses revealed a rich repertoire of proteins essential to sperm structure and function, including proteins linked to metabolism and energetics, as well as tubulin binding and microtubule related functions. The ZfSP also contained a number of immunity and defense proteins and proteins linked to sperm motility and sperm-egg interactions. Additionally, while most proteins in the ZfSP appear to be evolutionarily constrained, a small subset of proteins are evolving rapidly. Finally, in a comparison with the sperm proteome of the domestic chicken, we found an enrichment of proteins linked to catalytic activity and cytoskeleton related processes. As the first described passerine sperm proteome, and one of only two characterized avian sperm proteomes, the ZfSP provides a significant step towards a platform for studies of the molecular basis of sperm function and evolution in birds. SIGNIFICANCE: Using highly purified spermatozoa and LC-MS proteomics, we characterise the sperm proteome of the Zebra finch; the main model species for the avian order Passeriformes, the largest and most diverse of the avian clades. As the first described passerine sperm proteome, and one of only two reported avian sperm proteomes, these results will facilitate studies of sperm biology and mechanisms of fertilisation in passerines, as well as comparative studies of sperm evolution and reproduction across birds and other vertebrates.

Contrasting patterns of evolutionary constraint and novelty revealed by comparative sperm proteomic analysis in Lepidoptera.

BACKGROUND: Rapid evolution is a hallmark of reproductive genetic systems and arises through the combined processes of sequence divergence, gene gain and loss, and changes in gene and protein expression. While studies aiming to disentangle the molecular ramifications of these processes are progressing, we still know little about the genetic basis of evolutionary transitions in reproductive systems. Here we conduct the first comparative analysis of sperm proteomes in Lepidoptera, a group that exhibits dichotomous spermatogenesis, in which males produce a functional fertilization-competent sperm (eupyrene) and an incompetent sperm morph lacking nuclear DNA (apyrene). Through the integrated application of evolutionary proteomics and genomics, we characterize the genomic patterns potentially associated with the origination and evolution of this unique spermatogenic process and assess the importance of genetic novelty in Lepidopteran sperm biology. RESULTS: Comparison of the newly characterized Monarch butterfly (Danaus plexippus) sperm proteome to those of the Carolina sphinx moth (Manduca sexta) and the fruit fly (Drosophila melanogaster) demonstrated conservation at the level of protein abundance and post-translational modification within Lepidoptera. In contrast, comparative genomic analyses across insects reveals significant divergence at two levels that differentiate the genetic architecture of sperm in Lepidoptera from other insects. First, a significant reduction in orthology among Monarch sperm genes relative to the remainder of the genome in non-Lepidopteran insect species was observed. Second, a substantial number of sperm proteins were found to be specific to Lepidoptera, in that they lack detectable homology to the genomes of more distantly related insects. Lastly, the functional importance of Lepidoptera specific sperm proteins is broadly supported by their increased abundance relative to proteins conserved across insects. CONCLUSIONS: Our results identify a burst of genetic novelty amongst sperm proteins that may be associated with the origin of heteromorphic spermatogenesis in ancestral Lepidoptera and/or the subsequent evolution of this system. This pattern of genomic diversification is distinct from the remainder of the genome and thus suggests that this transition has had a marked impact on lepidopteran genome evolution. The identification of abundant sperm proteins unique to Lepidoptera, including proteins distinct between specific lineages, will accelerate future functional studies aiming to understand the developmental origin of dichotomous spermatogenesis and the functional diversification of the fertilization incompetent apyrene sperm morph.

Comparative Sperm Proteomics in Mouse Species with Divergent Mating Systems.

Sexual selection is the pervasive force underlying the dramatic divergence of sperm form and function. Although it has been demonstrated that testis gene expression evolves rapidly, exploration of the proteomic basis of sperm diversity is in its infancy. We have employed a whole-cell proteomics approach to characterize sperm divergence among closely related Mus species that experience different sperm competition regimes and exhibit pronounced variation in sperm energetics, motility and fertilization capacity. Interspecific comparisons revealed significant abundance differences amongst proteins involved in fertilization capacity, including those that govern sperm-zona pellucida interactions, axoneme components and metabolic proteins. Ancestral reconstruction of relative testis size suggests that the reduction of zona pellucida binding proteins and heavy-chain dyneins was associated with a relaxation in sperm competition in the M. musculus lineage. Additionally, the decreased reliance on ATP derived from glycolysis in high sperm competition species was reflected in abundance decreases in glycolytic proteins of the principle piece in M. spretus and M. spicilegus. Comparison of protein abundance and stage-specific testis expression revealed a significant correlation during spermatid development when dynamic morphological changes occur. Proteins underlying sperm diversification were also more likely to be subject to translational repression, suggesting that sperm composition is influenced by the evolution of translation control mechanisms. The identification of functionally coherent classes of proteins relating to sperm competition highlights the utility of evolutionary proteomic analyses and reveals that both intensified and relaxed sperm competition can have a pronounced impact on the molecular composition of the male gamete.

Sperm Proteome Maturation in the Mouse Epididymis.

In mammals, transit through the epididymis, which involves the acquisition, loss and modification of proteins, is required to confer motility and fertilization competency to sperm. The overall dynamics of maturation is poorly understood, and a systems level understanding of the complex maturation process will provide valuable new information about changes occurring during epididymal transport. We report the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. This study identified 765 proteins that are present in sperm obtained from all three segments. We identified 1766 proteins that are potentially added (732) or removed (1034) from sperm during epididymal transit. Phenotypic analyses of the caput, corpus and cauda sperm proteomes identified 60 proteins that have known sperm phenotypes when mutated, or absent from sperm. Our analysis indicates that as much as one-third of proteins with known sperm phenotypes are added to sperm during epididymal transit. GO analyses revealed that cauda sperm are enriched for specific functions including sperm-egg recognition and motility, consistent with the observation that sperm acquire motility and fertilization competency during transit through the epididymis. In addition, GO analyses revealed that the immunity protein profile of sperm changes during sperm maturation. Finally, we identified components of the 26S proteasome, the immunoproteasome, and a proteasome activator in mature sperm.

Panning for sperm gold: Isolation and purification of apyrene and eupyrene sperm from lepidopterans.

We describe a simple and straightforward procedure for the purification and separation of apyrene and eupyrene forms of lepidopteran sperm. The procedure is generally applicable to both butterfly and moth species with results varying according to the relative amounts of sperm produced and size of sperm storage organs. The technique relies upon inherent differences between eupyene sperm bundles and free apyrene sperm morphology. These differences allow for separation of the sperm morphs by repeated "panning" of sperm bundles into the center of a plastic dish. The purified eupyrene sperm bundles can then be removed and apyrene sperm collected from the supernatant by centrifugation. Efficacy of the purification process was confirmed by light microscopy and gel electrophoresis of the resulting fractions. Both one- and two-dimensional gel electrophoresis identified significant protein differences between the fractions further suggesting that the panning procedure effectively separated eurpyrene from apyrene sperm. The panning procedure should provide a convenient and accessible technique for further studies of sperm biology in lepidopterans.

The Rhesus macaque (Macaca mulatta) sperm proteome.

Mass spectrometry based proteomics has facilitated sperm composition studies in several mammalian species but no studies have been undertaken in non-human primate species. Here we report the analysis of the 1247 proteins that comprise the Rhesus macaque (Macaca mulatta) sperm proteome (termed the MacSP). Comparative analysis with previously characterized mouse and human sperm proteomes reveals substantial levels of orthology (47% and 40% respectively) and widespread overlap of functional categories based on Gene Ontology analyses. Approximately 10% of macaque sperm genes (113/1247) are significantly under-expressed in the testis as compared with other tissues, which may reflect proteins specifically acquired during epididymal maturation. Phylogenetic and genomic analyses of three MacSP ADAMs (A-Disintegrin and Metalloprotease proteins), ADAM18-, 20- and 21-like, provides empirical support for sperm genes functioning in non-human primate taxa which have been subsequently lost in the lineages leading to humans. The MacSP contains proteasome proteins of the 20S core subunit, the 19S proteasome activator complex and an alternate proteasome activator PA200, raising the possibility that proteasome activity is present in mature sperm. Robust empirical characterization of the Rhesus sperm proteome should greatly expand the possibility for targeted molecular studies of spermatogenesis and fertilization in a commonly used model species for human infertility.

Segmental dataset and whole body expression data do not support the hypothesis that non-random movement is an intrinsic property of Drosophila retrogenes.

BACKGROUND: Several studies in Drosophila have shown excessive movement of retrogenes from the X chromosome to autosomes, and that these genes are frequently expressed in the testis. This phenomenon has led to several hypotheses invoking natural selection as the process driving male-biased genes to the autosomes. Metta and Schlötterer (BMC Evol Biol 2010, 10:114) analyzed a set of retrogenes where the parental gene has been subsequently lost. They assumed that this class of retrogenes replaced the ancestral functions of the parental gene, and reported that these retrogenes, although mostly originating from movement out of the X chromosome, showed female-biased or unbiased expression. These observations led the authors to suggest that selective forces (such as meiotic sex chromosome inactivation and sexual antagonism) were not responsible for the observed pattern of retrogene movement out of the X chromosome. RESULTS: We reanalyzed the dataset published by Metta and Schlötterer and found several issues that led us to a different conclusion. In particular, Metta and Schlötterer used a dataset combined with expression data in which significant sex-biased expression is not detectable. First, the authors used a segmental dataset where the genes selected for analysis were less testis-biased in expression than those that were excluded from the study. Second, sex-biased expression was defined by comparing male and female whole-body data and not the expression of these genes in gonadal tissues. This approach significantly reduces the probability of detecting sex-biased expressed genes, which explains why the vast majority of the genes analyzed (parental and retrogenes) were equally expressed in both males and females. Third, the female-biased expression observed by Metta and Schlötterer is mostly found for parental genes located on the X chromosome, which is known to be enriched with genes with female-biased expression. Fourth, using additional gonad expression data, we found that autosomal genes analyzed by Metta and Schlötterer are less up regulated in ovaries and have higher chance to be expressed in meiotic cells of spermatogenesis when compared to X-linked genes. CONCLUSIONS: The criteria used to select retrogenes and the sex-biased expression data based on whole adult flies generated a segmental dataset of female-biased and unbiased expressed genes that was unable to detect the higher propensity of autosomal retrogenes to be expressed in males. Thus, there is no support for the authors' view that the movement of new retrogenes, which originated from X-linked parental genes, was not driven by selection. Therefore, selection-based genetic models remain the most parsimonious explanations for the observed chromosomal distribution of retrogenes.

Proteomic discovery of diverse immunity molecules in mammalian spermatozoa.

Ongoing proteomic analyses are providing a wealth of new data on the composition of the sperm proteome across a range of mammals and other taxa. Although molecular evolution and functional genomic analyses of the proteome have only begun recently, we now broadly understand the molecular composition of sperm. Systems level analysis has revealed a variety of molecular insights into sperm evolution and function, including a remarkable diversity of immunity-related proteins within the proteome. Using existing mammalian sperm proteomes as a starting point, we provide an overview of this important class of sperm proteins and what is known about their function in sperm maturation, sperm quality, sperm competition, and fertilization. The recent observation that many sperm immunity proteins are rapidly evolving, presumably under the influence of positive selection, suggests that they may be responding not only to selection associated with host immunity defense but also with pleiotropic functions in sperm. In addition to the documented role of sperm in the mediation of female immune response, we propose that the fundamental mechanisms involved in cell-cell recognition and binding in both immune processes and fertilization may underlie the multi-functionality of proteins in immunity and reproductive systems.