Daniel S. Johnston

The Mouse Epididymal Transcriptome: Transcriptional Profiling of Segmental Gene Expression in the Epididymis

Abstract Maturation of spermatozoa, including the acquisition of motility and the ability to undergo capacitation, occurs during transit through the dynamic environment of the epididymis. The microenvironments created along the length of the epididymal tubule are essential to the molecular modifications of spermatozoa that result in fertile gametes. The secretory and resorptive processes of the epithelial cells that line this tubule generate these microenvironments. In the current study, 10 morphologically distinct segments of the mouse epididymis were identified by microdissection. We hypothesized that the changing environments of the epididymal lumen are established by differential gene expression among these segments. RNA isolated from each of the 10 segments was analyzed by microarray analysis. More than 17 000 genes are expressed in the mouse epididymis, compared with about 12 000 genes identified from whole epididymal samples. Screening a panel of normal mouse tissues identified both epididymal-selective and epididymal-specific transcripts. In addition, this study identified 2168 genes that are up-regulated or down-regulated by greater than 4-fold between at least two different segments. The expression patterns of these genes identify distinct patterns of segmental regulation. Using principal component analysis, we determined that the 10 segments form 6 different transcriptional units. These analyses elucidate the changes in gene expression along the length of the epididymis for 17 000 expressed transcripts and provide a powerful resource for the research community in future studies of the biological factors that mediate epididymal sperm maturation.

The Rat Epididymal Transcriptome: Comparison of Segmental Gene Expression in the Rat and Mouse Epididymides

Abstract Regional differences along the epididymis are essential for the establishment of the luminal environment required for sperm maturation. In the current study, 19 morphologically distinct segments of the rat epididymis were identified by microdissection. Total RNA was isolated from each segment and subjected to microarray analysis. Segmental analysis of epididymal gene expression identified more than 16 000 expressed qualifiers, whereas profiling of RNA from whole rat epididymis identified approximately 12 000 expressed qualifiers. Screening a panel of normal rat tissues identified both epididymal-selective and epididymal-specific transcripts. In addition, more than 3500 qualifiers were shown to be present and differentially upregulated or downregulated by more than fourfold between any two segments. The present study complements our previous segment-dependent analysis of gene expression in the mouse epididymis and allows for comparative analyses between datasets. A total of 492 genes was shown to be present on both the MOE430 (mouse) and RAE230_2 (rat) microarrays, expressed in the epididymis of both species, and differentially expressed by more than fourfold in between segments in each species. Moreover, in-depth quantitative RT-PCR analysis of 36 members of the beta defensin gene family showed highly conserved patterns of expression along the lengths of the mouse and rat epididymides. These analyses elucidate global gene expression patterns along the length of the rat epididymis and provide a novel evaluation of conserved and nonconserved gene expression patterns in the epididymides of the two species. Furthermore, these data provide a powerful resource for the research community for future studies of biological factors that mediate sperm maturation and storage.

Analysis of the Human Sperm Proteome

Abstract: As part of our effort to identify putative protein targets for the development of male contraceptives, we performed an in‐depth proteomic analysis of human sperm by liquid chromatography and tandem mass spectrometry. Motile sperm were collected from a single fertile individual and fractionated into detergent‐soluble and detergent‐insoluble fractions. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis separation of these fractions, followed by manual cutting of the gel, yielded 35 gel sections for each fraction to include proteins across the full range of electrophoretic mobility. Proteomic analysis of these gel sections identified more than 1,760 proteins with high confidence, with 1,350 proteins identified in the soluble fraction, 719 identified in the insoluble fraction, and 309 identified in both fractions. This characterization of the human sperm proteome provides a high‐resolution, physiologically relevant index of the proteins that comprise human sperm.

Identification of Rat Cysteine-Rich Secretory Protein 4 (Crisp4) as the Ortholog to Human CRISP1 and Mouse Crisp4

Abstract Cysteine-rich secretory proteins (CRISPs) are present in a diverse population of organisms and are defined by 16 conserved cysteine residues spanning a plant pathogenesis related-1 and a C-terminal cysteine-rich domain. To date, the diversification of mammalian CRISPs is evidenced by the existence of two, three, and four paralogous genes in the rat, human, and mouse, respectively. The current study identifies a third rat Crisp paralog we term Crisp4. The gene for Crisp4 is on rat chromosome 9 within 1 Mb of both the Crisp1 and Crisp2 genes. The full-length transcript for this gene was cloned from rat epididymal RNA and encodes a protein that shares 69% and 91% similarity with human CRISP1 and mouse CRISP4, respectively. Expression of rat Crisp4 is most abundant in the epididymis, with the highest levels of transcription observed in the caput and corpus epididymis. In contrast, rat CRISP4 protein is most abundant in the corpus and cauda regions of the epididymis. Rat CRISP4 protein is also present in caudal sperm extracts, appearing as a detergent-soluble form at the predicted MWR (26 kDa). Our data identify rat Crisp4 as the true ortholog to human CRISP1 and mouse Crisp4, and demonstrate its interaction with spermatozoa in the epididymis.

Differential Gene Expression among the Proximal Segments of the Rat Epididymis Is Lost after Efferent Duct Ligation

Abstract The epididymis has traditionally been divided into the caput, corpus, and cauda regions, which are further organized into intraregional segments. In the rat and mouse, these segments have high degrees of transcriptional differentiation, and what has traditionally been called the initial segment of the rat epididymis actually consists of three transcriptionally different intraregional segments. These segments are regulated by endocrine, lumicrine, and paracrine factors, whose relative importance remains a topic of investigation. In the present study, 15-day unilateral efferent duct ligation (EDL) was used to deprive ipsilateral rat epididymides of lumicrine regulation. Segments 1–4 of EDL epididymides and contralateral, sham-operated tissues were collected individually. Microarray analysis of gene expression was used to determine the effect of lumicrine factor deprivation on the transcriptome-wide gene expression of each segment studied. More than 11 000 genes were detected as being expressed in each of the four segments examined. More than 2000 genes responded significantly to EDL in segment 1, although this number of genes declined in each succeeding segment. Segments 1 and 2 of control tissues were the most different transcriptionally and the most affected by EDL. In the absence of lumicrine factors, the four segments regressed to a transcriptionally undifferentiated state, which was consistent with the less-differentiated histology seen after EDL. Interestingly, for an individual gene, lumicrine factor deprivation could stimulate expression in some segments and suppress expression in other segments. These results reveal a higher complexity to the regulation of rat epididymal segments than heretofore appreciated.

Stem Leydig Cell Differentiation: Gene Expression During Development of the Adult Rat Population of Leydig Cells

ABSTRACT Leydig cells are the testosterone-producing cells in the adult male. Adult Leydig cells (ALCs) develop from stem Leydig cells (SLCs) through at least two intermediate cells, progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs). Microarray gene expression was used to identify the transcriptional changes that occur with the differentiation of SLCs to PLCs and, thus, with the entry of SLCs into the Leydig cell lineage; to comprehensively examine differentiation through the development of ALCs; and to relate the pattern of gene expression in SLCs to that in a well-established stem cell, bone marrow stem cells (BSCs). We show that the pattern of gene expression by SLCs was more similar to the expression by BSCs, an established stem cell outside the male reproductive tract, than to any of the cells in the Leydig cell developmental lineage. These results indicated that the SLCs have many of the molecular characteristics of other stem cells. Pathway analysis indicated that development of Leydig cells from SLCs to PLCs was associated with decreased expression of genes related to adhesion and increased expression of genes related to steroidogenesis. Gene expression changes between PLCs and ILCs and between ILCs and ALCs were relatively minimal, suggesting that these cells are highly similar. In contrast, gene expression changes between SLCs and ALCs were quite distinct.

Approaches and limitations of phosphatidylinositol-3-kinase pathway activation status as a predictive biomarker in the clinical development of targeted therapy

The central role played by the class IA phosphatidylinositol-3-kinase (PI3K) signaling node in human cancer is highlighted in the multiple mechanisms by which these signals become dysregulated. Many studies suggest that constitutive PI3K activation in human cancer contributes to drug resistance, including targeted agents and standard cytotoxic therapy. The combination of activation mechanisms and the multiple downstream cascades that emanate from the PI3K node contributes to the difficulty in measuring PI3K activation as a biomarker. Although many agents suppress the pathway in models, the challenge remains to translate this biology into a patient selection strategy (i.e., identify patients with “PI3K activated” tumors) and subsequently link this biomarker definition to drug responses in patients. The various genetic and epigenetic lesions resulting in pathway activation necessitate combined approaches using genetic, genomic, and protein biomarkers to accurately characterize “PI3K activated” tumors. Such a combined approach to pathway status can be assessed using a statistical stratification of patients in a randomized trial into “pathway on” and “pathway off” subsets to compare the treatment effect in each arm. Instead of considering individual biomarkers for their predictive ability, this strategy proposes the use of a collection of biomarkers to identify a specific “pathway on” patient population predicted to have clinical benefit from a pathway inhibitor. Here, we review the current understanding of the mechanisms of PI3K activation in breast cancer and discuss a pathway-based approach using PI3K as a predictive biomarker in clinical development, which is currently in use in a global phase 3 setting.

Gene expression during development of fetal and adult Leydig cells.

Abstract: In rats and mice, Leydig cells are formed as two morphologically and functionally different generations. The first generation develops in utero, from undifferentiated stem Leydig cells (SLCs) that differentiate into fetal Leydig cells (FLCs). After birth, SLCs that may differ from the fetal SLCs undergo lineage‐specific commitment and give rise to adult Leydig cells (ALCs). The intermediates of ALCs first become apparent by day 11 postpartum. These first‐appearing intermediates, progenitor Leydig cells (PLCs), are spindle shaped and identifiable as steroidogenic because they express luteinizing hormone receptor (LHR) and 3β‐hydroxysteroid dehydrogenase (3βHSD). The next step in the transition of PLCs to ALCs is the appearance of the immature Leydig cells (ILCs), most commonly seen in the testis during days 28 to 56 postpartum. ILCs have a more abundant smooth endoplasm reticulum (SER), the network of membranes providing a scaffold for steroidogenic enzyme localization, compared to PLCs, but are considered immature because they secrete higher levels of 5α‐reduced androgen than testosterone. ILCs undergo a final division before ALC steroidogenic function matures by postnatal day 56. ALCs mark the point of maximum differentiation, and at this stage, the Leydig cell secretes testosterone at the highest rate. In this review, trends of gene expression during development of the two Leydig‐cell generations, and recent information from gene profiling by microarray, are evaluated. The expression profiles are distinct, indicating that FLCs and ALCs may originate from separate pools of stem cells.

Identification of testis-specific male contraceptive targets: Insights from transcriptional profiling of the cycle of the rat seminiferous epithelium and purified testicular cells

Abstract: In an effort to identify novel targets for the development of nonhormonal male contraceptives, genome‐wide transcriptional profiling of the rat testis was performed. Specifically, enzymatically purified spermatogonia plus early spermatocyctes, pachytene spermatocytes, round spermatids, and Sertoli cells was analyzed along with microdissected rat seminiferous tubules at stages I, II–III, IV–V, VI, VIIa,b, VIIc,d, VIII, IX– XI, XII, XIII–XIV of the cycle of the seminiferous epithelium using RAE 230_2.0 microarrays. The combined analysis of these studies identified 16,971 expressed probe sets on the array. How these expression data, combined with additional bioinformatic data analysis and quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR) analysis, led to the identification of 58 genes that have 1000‐fold higher expression transcriptionally in the testis when compared to over 20 other nonreproductive tissues is described. The products of these genes may play important roles in testicular and/or sperm function, and further investigation on their utility as nonhormonal contraceptive targets is warranted. Moreover, these microarray data have been used to expedite the identification of a mutation in RIKEN cDNA 2410004F06 gene as likely being responsible for spermatogenic failure in a line of infertile mice generated by N‐ethyl‐N‐nitrosourea (ENU) mutagenesis. The microarray data and the qRT‐PCR data described are available in the Mammalian Reproductive Genetics database (http://mrg.genetics.washington.edu/).

Gene Expression Profiling and its Practice in Drug Development

The availability of sequenced genomes of human and many experimental animals necessitated the development of new technologies and powerful computational tools that are capable of exploiting these genomic data and ask intriguing questions about complex nature of biological processes. This gave impetus for developing whole genome approaches that can produce functional information of genes in the form of expression profiles and unscramble the relationships between variation in gene expression and the resulting physiological outcome. These profiles represent genetic fingerprints or catalogue of genes that characterize the cell or tissue being studied and provide a basis from which to begin an investigation of the underlying biology. Among the most powerful and versatile tools are high-density DNA microarrays to analyze the expression patterns of large numbers of genes across different tissues or within the same tissue under a variety of experimental conditions or even between species. The wide spread use of microarray technologies is generating large sets of data that is stimulating the development of better analytical tools so that functions can be predicted for novel genes. In this review, the authors discuss how these profiles are being used at various stages of the drug discovery process and help in the identification of new drug targets, predict the function of novel genes, and understand individual variability in response to drugs.