James E. Shima

The Murine Testicular Transcriptome: Characterizing Gene Expression in the Testis During the Progression of Spermatogenesis

Abstract One of the most promising applications of microarrays is the study of changes in gene expression associated with the growth and development of mammalian tissues. The testis provides an excellent model to determine the ability of microarrays to effectively characterize the changes in gene expression as an organ develops from birth to adulthood. To this end, a developmental testis gene expression time course profiling the expression patterns of ∼36 000 transcripts on the Affymetrix MGU74v2 GeneChip platform at 11 distinct time points was created to gain a greater understanding of the molecular changes necessary for and elicited by the development of the testis. Additionally, gene expression profiles of isolated testicular cell types were created that can aid in the further characterization of the specific functional actions of each cell type in the testis. Statistical analysis of the data revealed 11 252 transcripts (9846 unique) expressed differentially in a significant manner. Subsequent cluster analysis produced five distinct expressional patterns within the time course. These patterns of expression are present at distinct chronological periods during testis development and often share similarities with cell-specific expression profiles. Analysis of cell-specific expression patterns produced unique and characteristic groups of transcripts that provide greater insight into the activities, biological and chronological, of testicular cell types during the progression of spermatogenesis. Further analysis of this time course can provide a distinct and more definitive view into the genes implicated, known and unknown, in the maturation, maintenance, and function of the testis and the integrated process of spermatogenesis.

Postmeiotic Sex Chromatin in the Male Germline of Mice

In mammals, the X and Y chromosomes are subject to meiotic sex chromosome inactivation (MSCI) during prophase I in the male germline, but their status thereafter is currently unclear. An abundance of X-linked spermatogenesis genes has spawned the view that the X must be active . On the other hand, the idea that the imprinted paternal X of the early embryo may be preinactivated by MSCI suggests that silencing may persist longer . To clarify this issue, we establish a comprehensive X-expression profile during mouse spermatogenesis. Here, we discover that the X and Y occupy a novel compartment in the postmeiotic spermatid and adopt a non-Rabl configuration. We demonstrate that this postmeiotic sex chromatin (PMSC) persists throughout spermiogenesis into mature sperm and exhibits epigenetic similarity to the XY body. In the spermatid, 87% of X-linked genes remain suppressed postmeiotically, while autosomes are largely active. We conclude that chromosome-wide X silencing continues from meiosis to the end of spermiogenesis, and we discuss implications for proposed mechanisms of imprinted X-inactivation.

Profiling Gene Expression During the Differentiation and Development of the Murine Embryonic Gonad

Abstract The application of microarray technology to the study of mammalian organogenesis can provide greater insights into the steps necessary to elicit a functionally competent tissue. To this end, a temporal profile of gene expression was generated with the purpose of identifying changes in gene expression occurring within the developing male and female embryonic gonad. Gonad tissue was collected from mouse embryos at 11.5, 12.5, 14.5, 16.5, and 18.5 days postcoitum (dpc) and relative steady-state levels of mRNA were determined using the Affymetrix MGU74v2 microarray platform. Statistical analysis produced 3693 transcripts exhibiting differential expression during male and/or female gonad development. At 11.5 dpc, the gonad is morphologically indifferent, but at 12.5 dpc, transitions to a male or female phenotype are discernible by the appearance of testicular cords. A number of genes are expressed during this period and many share similar expression profiles in both sexes. As expected, the expression of two well-known sex determination genes, specifically Sry and Sox9, is unique to the testis. Beyond 12.5 dpc, differential gene expression becomes increasingly evident as the male and female tissue morphologically and physiologically diverges. This is evident by two unique waves of transcriptional activity occurring after 14.5 dpc in the male and female. With this study, a large number of transcripts comprising the murine transcriptome can be examined throughout male and female embryonic gonad development and allow for a more complete description of gonad differentiation and development.

Androgen-Regulated Transcripts in the Neonatal Mouse Testis as Determined Through Microarray Analysis

Abstract Androgens are required for normal spermatogenesis in mammalian testes. These hormones directly regulate testicular somatic cells that, in turn, support germ cell differentiation. However, the identity of genes under androgen regulation in the testis are not well known. In the present study, neonatal male mice (8 days postpartum) treated by testosterone propionate (TP) were used to study androgen action in the testis as evidenced by alterations in gene expression. Mice were treated with 0.5 mg of TP or dihydrotestosterone (DHT) or vehicle (oil), and testes were harvested 4, 8, and 16 h after treatment. Global gene expression was monitored by microarray analysis. Real-time reverse transcription-polymerase chain reaction was performed to confirm the microarray results. The methodology was verified by confirming the presence of previously characterized TP-regulated genes, including Pem in Sertoli cells and Cyp17a1 in Leydig cells. No significant differences in gene expression were found between TP- and DHT-treated samples. Microarray analysis identified 141, 119, and 109 up-regulated genes at 4, 8 and 16 h after TP treatment, respectively, and 83, 99, and 111 down-regulated genes at the same corresponding time points. The androgen regulation of the selected gene was verified further using testes from flutamide-treated adult mice and isolated Sertoli cells in culture. The data generated in the present study may serve as a foundation for hypothesis-driven research and provide insights regarding gene networks and pathways under androgen control in the testis.

Non-Muscle Cofilin Is a Component of Tubulobulbar Complexes in the Testis

Abstract Tubulobulbar complexes are finger-like structures that form at the interface between maturing spermatids and Sertoli cells prior to sperm release and at the interface between two Sertoli cells near the base of the seminiferous epithelium. They originate in areas previously occupied by actin filament-associated intercellular adhesion plaques known as ectoplasmic specializations. Actin filaments also are associated with tubulobulbar complexes where they appear to form a network, rather than the tightly packed bundles found in ectoplasmic specializations. Cofilin, a calcium-independent actin-depolymerizing protein, previously has been identified in the testis, but has not been localized to specific structures in the seminiferous epithelium. To determine if cofilin is found in Sertoli cells and is concentrated at actin-rich structures, we reacted fixed frozen sections of rat testis, fixed fragmented tissue, and blots of seminiferous epithelium with pan-specific and non-muscle cofilin antibodies. In addition, GeneChip microarrays (Affymetrix, Santa Clara, CA) were utilized to determine the abundance of mRNA for all cofilin isoforms in Sertoli cells. Using the monoclonal pan-specific cofilin antibody, we found specific labeling exclusively at tubulobulbar complexes and not at ectoplasmic specializations. On one-dimensional (1D) Western blots this antibody reacted monospecifically with one band, and on 2D blots reacted with two dots, which we interpret as phosphorylated and nonphosphorylated forms of a single cofilin isotype. Messenger RNA for non-muscle cofilin in Sertoli cells is about 8.5-fold higher than for muscle-type cofilin. To confirm that the non-muscle isoform of cofilin is present at tubulobulbar complexes, we used antibodies specific to non-muscle cofilin for immunofluorescent localization. As with the pan-specific antibody, we found that the non-muscle cofilin antibody exclusively labeled tubulobulbar complexes. Results presented here indicate that non-muscle cofilin is concentrated at tubulobulbar complexes. Our results also indicate that cofilin is not concentrated at ectoplasmic specializations.

Human KIT ligand promoter is positively regulated by HMGA1 in breast and ovarian cancer cells.

KIT ligand (KL) and its receptor, c-kit, are coexpressed in many types of cancer cells and have been implicated in tumor growth and angiogenesis. While Sertoli cell-specific regulation of the KL promoter has been well characterized, regulation in cancer cells remains to be elucidated. We recently reported microarray results demonstrating that increased high-mobility group (HMG) A1a protein expression correlates with increased KL transcription in MCF-7 human breast cancer cells. Sequence analysis indicates a potential for multiple HMGA1 binding sites within the human KL promoter. In order to better define the underlying molecular mechanisms that HMGA1 uses to facilitate malignant transformation of cancer cells, we have used a variety of methods to determine whether HMGA1a directly regulates the human KL promoter in breast and ovarian cancer cells. Our results indicate that: (i) KL promoter activity is significantly higher in MCF-7 cells overexpressing HMGA1a; (ii) HMGA1a protein binds to AT-rich regions of the KL promoter DNA both in vitro and in vivo; (iii) mutation of the AT-rich regions inhibits HMGA1a binding in vitro; and (iv) HMGA1a-specific inhibition significantly decreases transcription of KL in OCC1 human ovarian cancer cells. In addition, MCF-7 cells with transgenic HMGA1 overexpression stained positive for the KL protein by immunocytochemistry and immunohistochemistry, and were growth-inhibited by KL neutralization. The cumulative evidence indicates that HMGA1 positively regulates the human KL promoter in breast and ovarian cancer cells and implicates serum KL as a diagnostic marker for HMGA1-positive carcinomas.

Erratum: The murine testicular transcriptome: Characterizing gene expression in the testis during the progression of spermatogenesis (Biology of Reproduction (2004) 71 (319-330))

Inhibin alpha GDP dissociation inhibitor 1 Insulin-like growth factor 1 Jun proto-oncogene related gene dl Kit ligand X55957 U07950 X04480 J04509 M57647 A A A A B [13] [14] [15] [16] [17] Bcl2-associated X Mitochondrial insertional mutation Desert hedgehog Solute carrier family 12, member 2 Claudin 11 L22472 AI183160 AI666359 U13174 U19582 B B B B C [18] [19] [20] [21] [22] Kit receptor Ataxia telangiectasia mutated homolog Dax1 Double-sex and mab-3 related transcription factor 1 Y00864 U43678 U41568 AL133300 C C C C [23] [24] [25] [26] Cyclin A1 Heat shock protein 70-2 TATA box binding protein-like 1 Calmegin X84311 L27086 AB017697 U08373 D D D D [27] [28] [29] [30] A disintegrin and metalloprotease domain 2 MutS homolog 4 Transition protein 1 Transition protein 2 Protamine 1 U16242 AV277719 X12521 M60254 X07625 D D E E E [31] [32] [33] [34] [35] Seven in absentia 1a Hormone-sensitive lipase Phosphoserine/threonine/tyrosine interaction protein Testicular haploid expressed gene AA492653 U69543 U34973 AJ011834 E E E E [36] [37] [38] [39]