Project description:Aims of study: (1) To identify systemic differences in osteoarthritic (OA) bone that contribute to OA pathogenesis. (2) Identify novel osteoporotic (OP) bone-related disease genes. Study involved comparison of trabecular bone extracted from the intertrochanteric (IT) region of the proximal femur (PF) from OA, OP and normal/control (CTL) individuals. Bone was obtained from OA and OP individuals at surgery for total hip replacement and from CTL individuals at autopsy. Keywords: Bone tissue comparison, diseased versus non-diseased, factorial design, linear modelling Four sets of sample comparisons (39 comparisons in total) were made in this study. These comprised 10 OA-CTL female, 10 OA-CTL male, 10 OA-OP female and 9 OP-CTL female comparisons. A Compugen Human 19K-oligo library spotted onto glass slides by the Adelaide Microarray facility (AMF) was used in this study. The slides were interrogated by competitive hybridisation of Cy3 and Cy5 labelled pairs of OA-CTL, OA-OP or OP-CTL amplified RNA samples. Sample pairs were age-matched as closely as possible. A biological dye-swap strategy was employed. After hybridisation and washing of the slides they were scanned using a GenePix 4000B Scanner driven by GenePix Pro 4.0. All analyses were performed using the statistical programming and graphics environment R. The “SPOT” software package was used to identify spots by adaptive segmentation method and subtract backgrounds utilising morphological opening approach. Data analysis was performed in R using Bioconductor. Loess print tip method was used to correct for dye-bias and intensity within each group of adjacent spots printed by one pin. Linear modelling was performed with the Limma package of Bioconductor. Linear models were used to incorporate all available data into a single analysis. This allowed the use of both direct and indirect comparisons in calculation of expression ratios as well as improved accuracy in estimation of variance for each gene. Factorial design utilised the following factors: medical condition (OA or OP or CTL); sex (male or female) for OA patients only.

Project description:Mammalian spermatozoa acquire their fertilizing ability during epididymal transit. Gene expression patterns along the epididymis are established by specific transcription factor networks that coordinate region-specific functions. The epididymis is usually divided into 3 segments: caput, corpus, and cauda. The human epididymis anatomy does not allow clear distinction between these three segments. To determine to which extent gene expression is segmented along the human epididymis, transcriptome profiling was performed on 8 distinct epididymal regions from 3 donors. Microarray analysis was performed on a Gene Chip Human Clariom S (Affymetrix®) array representing 337 100 transcriptional variants encoded by 20 800 genes. Proximal segments 1 to 3 were distinguishable from the distal epididymal segments (4 to 8) as shown by unsupervised Principal Component Analysis. Transcripts from each segment with differentially expressed genes (DEGs) > 2-fold change and FDR < 0.05 were clustered in relation to their intensity profiles. While no DEGs were detected between segments 1–3 corresponding to the efferent ducts, 1140 DEGs were detected between efferent ducts (1–3) and the epididymis (4–8), 400 between caput (4–6) vs. corpus/cauda (7–8) and none between corpus (7) and cauda (8). Gene Ontology annotation revealed that up-regulated DEGs in the efferent ducts (1–3) were predominantly related to cilium assembly/movement and cell differentiation. The biological process terms fertilization, defense and immune responses were associated with caput epididymis (4–6) while spermatogenesis and protein binding were found all along the epididymis (4–8). In conclusion, the proximal human epididymis is exclusively occupied by efferent ducts with a distinct DEG profile compared with the downstream epididymal segments. Moreover, gene expression profiling revealed two regions in the human epididymis; the caput and the distal corpus/cauda region. Taken together, analysis of the human epididymal transcriptome reveals a limited DEG profile.

Project description:The role of estrogen and testosterone in the regulation of gene expression in the proximal reproductive tract is not completely understood. To address this question, mice were treated with testosterone or estradiol and RNA from the efferent ducts and caput epididymis was processed and hybridized to Affymetrix MOE 430 2.0 microarrays. Analysis of array output identified probe sets in each tissue with altered levels in hormone treated versus control animals. Hormone treatment efficacy was confirmed by determination of serum hormone levels pre- and post-treatment and observed changes in transcript levels of previously reported hormone-responsive genes. Tissue-specific hormone sensitivity was observed with 2867 and 3197 probe sets changing significantly in the efferent ducts after estrogen and testosterone treatment, respectively. In the caput epididymis, 117 and 268 probe sets changed after estrogen and testosterone treatment, respectively, demonstrating a greater response to hormone in the efferent ducts than the caput epididymis. Transcripts sharing similar profiles in the intact and hormone-treated animals compared with castrated controls were also identified. Ontological analysis of probe sets revealed a significant number of hormone-regulated transcripts encode proteins associated with lipid metabolism, transcription and steroid metabolism in both tissues. Real-time RT-PCR was employed to confirm array data and investigate other potential hormone-responsive regulators of proximal reproductive tract function. The results of this work reveal previously unknown responses to estrogen in the caput epididymis and to testosterone in the efferent ducts as well as tissue specific hormone sensitivity in the proximal reproductive tract. Adult animals were castrated or sham-castrated, allowed to recover for 14 days, and then treated with 0.015 mg estradiol (castrated), 0.015 mg testosterone propionate (castrated), or vehicle (castrated and sham-castrated as biological controls) in duplicate. Efferent duct and caput epididymis was collected from each sample and analyzed. Duplicates are included in the provided data and numbered 1 or 2 for each treatment regimen.

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Microarray on proximal epididymis from Ctl and cKO mouse

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