Stephen A. Krawetz

Global functional profiling of gene expression.

The typical result of a microarray experiment is a list of tens or hundreds of genes found to be differentially regulated in the condition under study. Independent of the methods used to select these genes, the common task faced by any researcher is to translate these lists of genes into a better understanding of the biological phenomena involved. Currently, this is done through a tedious combination of searches through the literature and a number of public databases. We developed Onto-Express (OE) as a novel tool able to automatically translate such lists of differentially regulated genes into functional profiles characterizing the impact of the condition studied. OE constructs functional profiles (using Gene Ontology terms) for the following categories: biochemical function, biological process, cellular role, cellular component, molecular function, and chromosome location. Statistical significance values are calculated for each category. We demonstrate the validity and the utility of this comprehensive global analysis of gene function by analyzing two breast cancer datasets from two separate laboratories. OE was able to identify correctly all biological processes postulated by the original authors, as well as discover novel relevant mechanisms.

Spermatozoal RNA profiles of normal fertile men

BACKGROUND Findings from several studies support the conclusion that spermatozoa contain a complex repertoire of mRNAs. Even though these mRNAs are thought to provide an insight into past events of spermatogenesis, their complexity and function have yet to be established. Our aim was to determine whether we could use spermatozoal mRNAs to generate a genetic fingerprint of normal fertile men. METHODS We used a suite of microarrays containing 27016 unique expressed sequence tags (ESTs) to investigate cDNAs from a pool of 19 testes, cDNAs from a pool of nine individual ejaculate spermatozoal mRNAs, and cDNAs constructed from spermatozoal mRNAs from a single ejaculate. We also used ontological data mining to determine the function of the genes identified in each EST profile. FINDINGS The cDNAs from the testes, pooled ejaculate, and single ejaculate hybridised to 7157, 3281, and 2780 ESTs, respectively. The testicular population contained all of the ESTs identified by the cDNAs from the pooled and individual ejaculate. The pooled ejaculate population contained all but four ESTs identified from the individual ejaculate. A subset of the spermatozoal mRNAs was associated with embryo development. INTERPRETATION The microarray data from testes and spermatozoa (pooled and individual) were concordant, supporting the view that a spermatozoal mRNA fingerprint can be obtained from normal fertile men. Thus, profiling can be used to monitor past events-ie, gene expression of spermatogenesis. Moreover, the data suggest that, in addition to delivering the paternal genome, spermatozoa provide the zygote with a unique suite of paternal mRNAs. Ejaculate spermatozoa can now be used as a non-invasive proxy for investigations of testis-specific infertility.

Reproductive biology: Delivering spermatozoan RNA to the oocyte

Even though the genetic fingerprint of human sperm has been defined, its role in orchestrating fertilization and the development of the early embryo remains vague. Here we show that human male gametes pass over more to the oocyte than just the haploid male genome — paternal messenger RNAs are also delivered to the egg at fertilization. If these transcripts, previously thought to be left-overs from spermatogenesis, are important in early development, our findings may have implications for the success of somatic-cell nuclear transfer in cloning technology and the identification of components leading to unexplained male-factor infertility.

Bioinformatics Methods and Protocols

Part 1. Sequence Analysis Packages GCG: The Wisconsin Package of Sequence Analysis Programs David D. Womble Web-Based Interfaces for the GCG Sequence Analysis Programs David D. Womble Omiga: A PC-Based Sequence Analysis Tool Jeffrey A. Kramer MacVector: Integrated Sequence Analysis for the Macintosh Promila A. Rastogi DNASTARs Lasergene Sequence Analysis Software Timothy G. Burland PepTool(TM) and GeneTool(TM): Platform-Independent Tools for Biological Sequence Analysis David S. Wishart, Paul Stothard, and Gary H. Van Domselaar The Staden Package, 1998 Rodger Staden, Kathryn F. Beal, and James K. Bonfield Building a Multiuser Sequence Analysis Facility Using Freeware Brian Fristensky Part 2. Molecular Biology Software Free Software in Molecular Biology for Macintosh and MS Windows Computers Appendix: Software Listings Don Gilbert Flexible Sequence Similarity Searching with the FASTA3 Program Package William R. Pearson The Use of CLUSTAL W and CLUSTAL X for Multiple Sequence Alignment Ashok Aiyar Phylogenetic Analysis Using PHYLIP Jacques D. Retief Annotating Sequence Data Using Genotator Nomi L. Harris Low Cost Gel Analysis Jeffry A. Reidler Part 3. Web-Based Resources Computer Resources for the Clinical and Molecular Geneticist Yuval Yaron and Avi Orr-Urtreger The NCBI: Publicly Available Tools and Resources on the Web Jack P. Jenuth Resources at EBI Patricia Rodriguez-Tome Computer-Assisted Analysis of Transcription Control Regions: MatInspector and Other Programs Thomas Werner Computational Approaches for Gene Identification Gautam B. Singh Primer3 on the WWW for General Users and for Biologist Programmers Steve Rozen andHelen Skaletsky Using the WWW to Supply the Molecular Biology Lab MaryAnn Labant and Roger Anderson Part 4. Computers and Molecular Biology: Issues and Constraints Network Computing: Restructuring How Scientists Use Computers and What We Get Out of Them Brian Fristensky Computing with DNA Lila Kari and Laura F. Landweber Detecting Biological Patterns: The Integration of Databases, Models, and Algorithms Gautam B. Singh Part 5. Teaching Bioinformatics and Keeping Up-to-Date with the Literature Design and Implementation of an Introductory Course for Computer Applications in Molecular Biology and Genetics Stephen A. Krawetz The Virtual Library I: Searching MEDLINE Keir Reavie The Virtual Library II: Science Citation Index and Current Awareness Services Keir Reavie The Virtual Library III: Electronic Journals, Grants, and Funding Information Keir Reavie

Onto-Tools, the toolkit of the modern biologist: Onto-Express, Onto-Compare, Onto-Design and Onto-Translate

Onto-Tools is a set of four seamlessly integrated databases: Onto-Express, Onto-Compare, Onto-Design and Onto-Translate. Onto-Express is able to automatically translate lists of genes found to be differentially regulated in a given condition into functional profiles characterizing the impact of the condition studied upon various biological processes and pathways. OE constructs functional profiles (using Gene Ontology terms) for the following categories: biochemical function, biological process, cellular role, cellular component, molecular function and chromosome location. Statistical significance values are calculated for each category. Once the initial exploratory analysis identified a number of relevant biological processes, specific mechanisms of interactions can be hypothesized for the conditions studied. Currently, many commercial arrays are available for the investigation of specific mechanisms. Each such array is characterized by a biological bias determined by the extent to which the genes present on the array represent specific pathways. Onto-Compare is a tool that allows efficient comparisons of any sets of commercial or custom arrays. Using Onto-Compare, a researcher can determine quickly which array, or set of arrays, covers best the hypotheses studied. In many situations, no commercial arrays are available for specific biological mechanisms. Onto-Design is a tool that allows the user to select genes that represent given functional categories. Onto-Translate allows the user to translate easily lists of accession numbers, UniGene clusters and Affymetrix probes into one another. All tools above are seamlessly integrated. The Onto-Tools are available online at http://vortex.cs.wayne.edu/Projects.html.

Short communicationGlobal functional profiling of gene expression

The typical result of a microarray experiment is a list of tens or hundreds of genes found to be differentially regulated in the condition under study. Independent of the methods used to select these genes, the common task faced by any researcher is to translate these lists of genes into a better understanding of the biological phenomena involved. Currently, this is done through a tedious combination of searches through the literature and a number of public databases. We developed Onto-Express (OE) as a novel tool able to automatically translate such lists of differentially regulated genes into functional profiles characterizing the impact of the condition studied. OE constructs functional profiles (using Gene Ontology terms) for the following categories: biochemical function, biological process, cellular role, cellular component, molecular function, and chromosome location. Statistical significance values are calculated for each category. We demonstrate the validity and the utility of this comprehensive global analysis of gene function by analyzing two breast cancer datasets from two separate laboratories. OE was able to identify correctly all biological processes postulated by the original authors, as well as discover novel relevant mechanisms.

Paternal contribution: new insights and future challenges.

It has been widely held that all that fathers essentially contribute to the next generation is half their genome. However, recent progress towards understanding biological processes such as sperm maturation and fertilization now indicates that the paternal contribution has been underestimated. To tackle some of the misconceptions surrounding the paternal contribution, the factors that are actually delivered by the sperm at fertilization and their potential developmental functions will be discussed using data from humans and animal models. Although still in their infancy, the practical applications of using sperm RNAs have already emerged in reproductive medicine as markers that are indicative of successful vasectomy. They are also beginning to appear in the forensic sciences and, within the next decade, might appear in the environmental sciences.

Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences.

During the haploid phase of mammalian spermatogenesis, nucleosomal chromatin is ultimately repackaged by small, highly basic protamines to generate an extremely compact, toroidal chromatin architecture that is critical to normal spermatozoal function. In common with several species, however, the human spermatozoon retains a small proportion of its chromatin packaged in nucleosomes. As nucleosomal chromatin in spermatozoa is structurally more open than protamine-packaged chromatin, we considered it likely to be more accessible to exogenously applied endonucleases. Accordingly, we have used this premise to identify a population of endonuclease-sensitive DNA sequences in human and murine spermatozoa. Our results show unequivocally that, in contrast to the endonuclease-resistant sperm chromatin packaged by protamines, regions of increased endonuclease sensitivity are closely associated with gene regulatory regions, including many promoter sequences and sequences recognized by CCCTC-binding factor (CTCF). Similar differential packaging of promoters is observed in the spermatozoal chromatin of both mouse and man. These observations imply the existence of epigenetic marks that distinguish gene regulatory regions in male germ cells and prevent their repackaging by protamines during spermiogenesis. The ontology of genes under the control of endonuclease-sensitive regulatory regions implies a role for this phenomenon in subsequent embryonic development.

A survey of small RNAs in human sperm

BACKGROUND There has been substantial interest in assessing whether RNAs (mRNAs and sncRNAs, i.e. small non-coding) delivered from mammalian spermatozoa play a functional role in early embryo development. While the cadre of spermatozoal mRNAs has been characterized, comparatively little is known about the distribution or function of the estimated 24,000 sncRNAs within each normal human spermatozoon. METHODS RNAs of <200 bases in length were isolated from the ejaculates from three donors of proved fertility. RNAs of 18-30 nucleotides in length were then used to construct small RNA Digital Gene Expression libraries for Next Generation Sequencing. Known sncRNAs that uniquely mapped to a single location in the human genome were identified. RESULTS Bioinformatic analysis revealed the presence of multiple classes of small RNAs in human spermatozoa. The primary classes resolved included microRNA (miRNAs) (≈ 7%), Piwi-interacting piRNAs (≈ 17%), repeat-associated small RNAs (≈ 65%). A minor subset of short RNAs within the transcription start site/promoter fraction (≈ 11%) frames the histone promoter-associated regions enriched in genes of early embryonic development. These have been termed quiescent RNAs. CONCLUSIONS A complex population of male derived sncRNAs that are available for delivery upon fertilization was revealed. Sperm miRNA-targeted enrichment in the human oocyte is consistent with their role as modifiers of early post-fertilization. The relative abundance of piRNAs and repeat-associated RNAs suggests that they may assume a role in confrontation and consolidation. This may ensure the compatibility of the genomes at fertilization.

Letrozole versus Clomiphene for Infertility in the Polycystic Ovary Syndrome

BACKGROUND Clomiphene is the current first-line infertility treatment in women with the polycystic ovary syndrome, but aromatase inhibitors, including letrozole, might result in better pregnancy outcomes. METHODS In this double-blind, multicenter trial, we randomly assigned 750 women, in a 1:1 ratio, to receive letrozole or clomiphene for up to five treatment cycles, with visits to determine ovulation and pregnancy, followed by tracking of pregnancies. The polycystic ovary syndrome was defined according to modified Rotterdam criteria (anovulation with either hyperandrogenism or polycystic ovaries). Participants were 18 to 40 years of age, had at least one patent fallopian tube and a normal uterine cavity, and had a male partner with a sperm concentration of at least 14 million per milliliter; the women and their partners agreed to have regular intercourse with the intent of conception during the study. The primary outcome was live birth during the treatment period. RESULTS Women who received letrozole had more cumulative live births than those who received clomiphene (103 of 374 [27.5%] vs. 72 of 376 [19.1%], P=0.007; rate ratio for live birth, 1.44; 95% confidence interval, 1.10 to 1.87) without significant differences in overall congenital anomalies, though there were four major congenital anomalies in the letrozole group versus one in the clomiphene group (P=0.65). The cumulative ovulation rate was higher with letrozole than with clomiphene (834 of 1352 treatment cycles [61.7%] vs. 688 of 1425 treatment cycles [48.3%], P<0.001). There were no significant between-group differences in pregnancy loss (49 of 154 pregnancies in the letrozole group [31.8%] and 30 of 103 pregnancies in the clomiphene group [29.1%]) or twin pregnancy (3.4% and 7.4%, respectively). Clomiphene was associated with a higher incidence of hot flushes, and letrozole was associated with higher incidences of fatigue and dizziness. Rates of other adverse events were similar in the two treatment groups. CONCLUSIONS As compared with clomiphene, letrozole was associated with higher live-birth and ovulation rates among infertile women with the polycystic ovary syndrome. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT00719186.).