292 M H ABEL and others . Effects of FSH on testicular transcript levels used arrays to examine the short-term effects (up to 24 h) of a single injection of FSH on testicular gene expression in vivo (Sadate-Ngatchou et al. 2004). From these studies, we can now identify a number of transcripts acutely regulated by FSH but we continue to lack a clear understanding of how FSH acts to regulate testicular development and function over the longer term. To address this issue, we have carried out a comprehensive review of the effects of more prolonged FSH treatment (multiple injections up to 72 h) on transcript levels in the testis of the hpg mouse. Materials and methods Animals and treatments hpg mice from the original colony ﬁrst identiﬁed at the MRC Laboratories, Harwell, Oxford (Cattanach et al. 1977) were bred at Oxford. The hpg mutation was identiﬁed by PCR analysis of tail DNA as previously reported (Lang 1995). All procedures were carried out in accordance with the UK Animals (Scientiﬁc Procedures) Act 1986 and with the approval of a local ethical review committee. Male hpg mice, 10 weeks of age and in group sizes of 3–4, were injected subcutaneously with 8 IU recombi- . nant human FSH (rhFSH) (Serono Ltd) in 0 2 ml PBS (PBS, pH 7.4, Sigma Aldrich) at the start of the experiment and every 12 h thereafter for 12, 24 or 72 h. This dose of recombinant hormone had previously been shown to induce a signiﬁcant increase in testis weight in hpg mice when given for 1 week (Abel and Charlton unpublished). Mice were killed 1 h after the last injection, testes removed, snap frozen in liquid nitrogen and stored at K70 8C. Testicular histology Three hpg mice treated as above were killed at each time point. The testes were weighed and one testis from each animal was ﬁxed in 1% glutaraldehyde, 4% paraformaldehyde, in phosphate buffer, 0 . 1 M, pH 7.2 for 24 h at 4 8C, and embedded in araldite. Semi-thin, 1 mm sections were cut and stained with toluidine blue. DNA microarray Three or four animals from FSH-treated or control hpg groups were killed at each time point and the RNA from testes of individual animals extracted on RNeasy columns (Qiagen). RNA was quantiﬁed using a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) and RNA quality was checked using the Agilent bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Samples of total RNA (8 mg) from individual animals Journal of Molecular Endocrinology (2009) 42, 291–303 were reverse transcribed and then in vitro transcribed and hybridised to mouse MOE430A arrays (Affymetrix, Santa Clara, CA, USA) (nZ3 or 4 for each group) according to the GeneChip expression technical manual (Affymetrix) as previously reported (Baban & Davies 2008). All the experiments were designed and information compiled in compliance with MIAME guide lines. Gene transcript levels were determined from data image ﬁles using algorithms in Gene Chip Operating Software (GCOS1.2, Affymetrix). The array data were generated in two batches. In the ﬁrst experiment control, 12 and 72 h FSH groups were extracted and hybridised to the arrays and in a subsequent experiment control and 24 h FSH groups were processed in the same way. Each treatment group was analysed against its own control. Differentially expressed genes were identiﬁed using the Welch t-test, variance not assumed equal, P!0.05. Analysis of canonical pathways was carried out using Ingenuity Pathways Analysis (www.ingenuity.com). The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession GSE8924. Real-time PCR Total RNA was extracted from individual testes of control or FSH-treated hpg mice using Trizol (Life technologies) and residual genomic DNA was removed by DNAse treatment (DNA-free, Ambion Inc., Austin, TX, USA, supplied by AMS Biotechnology, Abingdon, UK). RNA (1 mg) was reverse transcribed using random hexamers (Ambion) and Moloney murine leukaemia virus reverse transcriptase (Life Technologies) as previously described (Hirst et al. 2004). Quantitative real-time PCR was used to conﬁrm changes in selected mRNA transcripts identiﬁed from the microarray analysis or to examine other transcripts of potential interest. The real-time PCR used either the Taqman (Inha, Inhba, Inhbb and Hdc) or the SYBR green (all other transcripts) method in a 96-well plate format. For Taqman, Universal Taqman master mix, and optimised primer and probe sets were purchased from Applied Biosystems (Warrington, UK) and used accord- ing to the manufacturer’s recommendations in a 25 ml volume. For SYBR green, each reaction contained 5 ml 2!SYBR mastermix (Stratagene, Amsterdam, Nether- lands), primer (100 nM) and template in a total volume of 10 ml. The thermal proﬁle used for ampliﬁcation was 95 8C for 8 min followed by 40 cycles of 95 8C for 25 s, 63 8C for 25 s and 72 8C for 30 s. At the end of the ampliﬁcation phase a melting curve analysis was carried out on the products formed. All primers were designed by Primer Express 2.0 (Applied Biosystems) using para- meters previously described (Czechowski et al. 2004). www.endocrinology-journals.org