J Mol Neurosci DOI 10.1007/s12031-014-0304-9

Gene Expression Profiling in the Hippocampus of Orchidectomized Rats Telma Quintela & Helena Marcelino & Isabel Gonçalves & Filipa M. Patriarca & Cecília R. A. Santos

Received: 13 March 2014 / Accepted: 7 April 2014 # Springer Science+Business Media New York 2014

Abstract Evidence from the literature suggests that testosterone (T) plays an important role in the neural structure, physiology, and function of the hippocampus (HP). However, many of the genes involved and underlying mechanisms remain to be elucidated. To shed light on this issue, we explored the transcriptome of HP in orchidectomized (OOX) rats to identify T-dependent gene expression in rat HP. RNA from OOX and sham HP animals were processed and measured by the Applied Biosystems microarray platform. The results showed a total of 271 genes differentially expressed between OOX vs. sham animals. Overall, T depletion resulted in the upregulation of 98 genes, including genes associated with neurogenesis and behavior. Of particular interest was the downregulation of 173 genes, with known functions, including signal transduction or neurological system processes. Our data shows that T depletion results in significantly altered hippocampal gene expression profiles and constitutes a starting tool to elucidating the molecular mechanisms involved in the action of androgens in the physiology of the HP. Keywords Hippocampus . Testosterone depletionm Microarrays

Introduction Testosterone (T) exerts neurotrophic and neuroprotective effects playing a major role in the regular functioning of the Electronic supplementary material The online version of this article (doi:10.1007/s12031-014-0304-9) contains supplementary material, which is available to authorized users. T. Quintela (*) : H. Marcelino : I. Gonçalves : F. M. Patriarca : C. R. A. Santos CICS-UBI—Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, Covilhã 6200-506, Portugal e-mail: [email protected]

central nervous system (Janowsky 2006). T levels in men begin to decline with advancing age at a steady rate (Vermeulen 1991). This age-related T decline is associated with loss of balance, lethargy, depression, irritability, and impairments in certain aspects of cognitive performance and synaptic plasticity. Although many alterations in brain pathways resulting from T deficiency have been reported, the full range of consequences of the gradual loss of T remains incompletely defined. Within the brain, T is thought to target several regions, a concept consistent with the widespread distribution of androgen receptors (Simerly et al. 1990): choroid plexus, hypothalamus, amygdala, prefrontal cortex, and hippocampus (HP) (Sar et al. 1990; Beyenburg et al. 2000). Indeed, AR expression in HP, measured by androgen receptors messenger RNA (mRNA) concentration, is of the same order of magnitude as in the prostate (Beyenburg et al. 2000). Studies using cell or animal models show that T modifies the neural structure, physiology, and function of the HP (Janowsky 2006), demonstrating powerful neuroprotective and homeostatic effects. For instance, androgen deprivation dramatically reduces the number of spine synapses in the HP in rodents that is restored with either T or its metabolite dihydrotestosterone supplementation (Leranth et al. 2003). A significant positive effect of T on hippocampal volume has been reported in meadow voles (Galea and McEwen 1999). Depletion of T via gonadectomy in male rats resulted in the accumulation of beta amyloid, a risk factor for Alzheimer’s disease, that was reversed by dihydrotestosterone supplementation (Ramsden et al. 2003). Moreover, beta amyloid neurotoxicity was significantly reduced by T in cultured hippocampal neurons (Pike 2001). Although androgens have been frequently associated with several mechanisms altered by the gradual decrease in the availability of T in the hippocampal tissue, it is worth to identify other mechanisms and genes responsive to androgens. Thus, the present study was conducted to identify, by gene

J Mol Neurosci

microarray technology, relevant genes that could be involved in the action of androgens in rat HP. Here, we report a comparison of hippocampal gene expression between controls and orchidectomized (OOX) male rats, revealing a number of novel gene expression changes and thereby increasing our understanding of the biological processes that are regulated by androgens in this tissue.

Materials and Methods Ethics Statement Animals were handled in compliance with the NIH guidelines and the European Union rules for the care and handling of laboratory animals (Directive 2010/63/EU). Animal experiments were also carried out according to the Portuguese law for animal welfare, and the protocol was approved by the Committee on the Ethics of Animal Experiments of the Health Science Research Centre of the University of Beira Interior (DGV/2011). Moreover, all efforts were made to minimize animal suffering. Animals and Tissue Collection Animals were handled in compliance with the NIH guidelines and the European Union rules for the care and handling of laboratory animals (Directive 2010/63/EU). Wistar rats were housed under a 12-h light and 12-h dark cycle, with food and water ad libitum during the course of the experiment. Male rats (2 months±2 weeks) were OOX, and 2 weeks after surgery, castrated animals were euthanized under anesthesia (mixture of ketamine and medetomidine). Sham controls involved the exposure of the testicle without isolation. The brain was removed, and the HP was collected by dissection of each hemisphere. The samples were then immediately frozen with liquid nitrogen and stored at −80 °C until RNA isolation. Measurement of Hormone Levels A blood sample was collected from the right atrium of the heart immediately after the anesthesia and just before decapitation. T levels in plasma were measured by radioimmunoassays following the methodology described in the study of Scott and Canario (1992). Intra-assay and inter-assay precisions (coefficient of variation) were 7.5 and 12.4 % for T. The limit of detection of assays was 200 pg ml−1 for T. Microarray Experimental Design Rat HP were homogenized and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following

the manufacturer’s instructions. RNA was quantified using a NanoDrop spectrophotometer (NanoDrop Technologies), and RNA integrity was assessed by Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Equal amounts of RNA extract (200 ng) from the HP of sham or OOX animals, in a total of three biological replicates, were amplified and Cy3-labeled using the Low Input Quick Amp Labeling kit (Agilent Technologies). Hybridizations were carried out on an Agilent-based microarray platform using custom-designed whole genome Rat GE 4x4K v3 microarrays. Scanned images on an Agilent microarray scanner were analyzed by Feature extraction software (Agilent Technologies) using GE1_105_Dec08 protocol. The signal intensity was normalized by centering the median of the signal distribution using BRB-ArrayTools v3.8.1 (http://linus.nci.nih.gov/BRB-ArrayTools.html). All further processing was carried out according to Quintela et al. (2013). Microarray Data Analysis Differentially expressed genes were identified by pairwise comparison using a Student’s t test, with a p value cutoff of 0.05. Only genes with a fold change above 1.5 were considered differentially expressed for further analyses. In order to identify Gene Ontology (GO) terms with a significant number of differentially expressed genes, we used the database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 (http://david.abcc.ncifcrf.gov/) (da Huang et al. 2009). Significantly enriched Kyoto Encyclopedia of Genes and Genome (KEGG) pathways and GO terms for biological processes (BP), molecular functions (MF), and cellular components (CC) were identified using a p value cutoff of 0.05. Redundant terms and categories comprising less than five genes were omitted. Real-Time RT-PCR Validation Microarray verification was performed by real-time RT-PCR analysis of selected genes using SYBR Green assay and the iCycler iQ™ system (Bio-Rad). Complementary DNA was synthesized using 1 μg of total RNA in a reverse transcription reaction. The PCR conditions were denaturation at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s and 1 min of annealing and extension at 56 °C. To normalize the complementary DNA content of the samples, we used the comparative threshold cycle method, which consists of the normalization of the number of target gene copies versus the endogenous reference gene cyclophilin A, as described in the study of Quintela et al. (2013). Primers for target genes were designed using Primer-Blast-NCBI-NIH and are listed in Table 1. In addition, the resulting PCR products were run on a

J Mol Neurosci Table 1 Validation of microarray data by real-time quantitative RT-PCR Gene name

Gene ID symbol

Microarray data RT-PCR validation Primer forward and reverse fold change fold change

Product size (bp)

Apelin

Apln

NM_031612.3

4.32

1.15

101

Fibroblast growth Fgf9 factor 9 Melanoregulin Mreg

NM_012952.1

2.93

1.2

NM_001192002 .1 0.24

0.62

Presenilin 1

NM_019163.3

0.87

Psen1

0.76

1.5 % agarose gel and were sequenced to verify the sequence specificity.

Results Characterization of the Hormonal Status of the Animals The effectiveness of gonadectomy was confirmed by the measurement of T serum levels in sham (2.625±0.445) and in OOX animals (0.238±0.0534). A Mann-Whitney U test indicated an overall steep effect on T depletion (p

Gene expression profiling in the hippocampus of orchidectomized rats.

Evidence from the literature suggests that testosterone (T) plays an important role in the neural structure, physiology, and function of the hippocamp...
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