No-RT controls for each sample were screened to check for the presence of residual genomic DNA. The primers and probes used for real-time SYBR PCR are shown in Supplementary Table 1, see supplementary data in the online version of the Journal of Molecular Endo- crinology at http://jme.endocrinology-journals.org/ content/vol42/issue4/. Different animals were used to provide RNA for real-time PCR and microarray studies. Hormone assay In a separate study adult hpg mice were treated with rhFSH as above and intratesticular levels of testosterone measured by RIA following ethanol extraction as previously described (O’Shaughnessy & Shefﬁeld 1990). The limit of detection of the assay was 25 fmol/testis. Statistical analysis With the exception of the array studies described above, the effects of FSH treatment were analysed initially by single-factor ANOVA followed by post hoc analysis using Fisher’s test. Results Testicular weight and histology after rhFSH treatment There was a signiﬁcant increase in testis weight within 12 h of the start of FSH treatment and weight continued to increase up to 24 h (Fig. 1A). This weight increase was accompanied by an apparent increase in tubular diameter with clear formation of a tubular lumen (Fig. 1B). On the semi-thin light micrographs, there was also an apparent increase in vacuolation of the Sertoli cell cytoplasm by 24 h which became more marked by 72 h (Fig. 1B). This was conﬁrmed on electron micrographs with several small vacuoles apparent within the cytoplasm at 24 h and larger vacuoles present at 72 h (Fig. 1C). There was no clear advancement of spermatogenesis within the timescale of the experiment. Hormone levels Intratesticular testosterone levels were undetectable in control hpg mice (!25 fmol/testis (!12 fmol/mg tissue), nZ8) and increased to low but consistently detectable levels 24 h after the start of treatment with FSH (65.0G12.4 fmol/testis (19.1G3.6 fmol/mg), nZ4) and remained detectable up to 72 h (76.2G 45.0 fmol/testis (19.5G11.5 fmol/mg), nZ4). www.endocrinology-journals.org Effects of FSH on testicular transcript levels . M H ABEL and others 293 Figure 1 Effect of rhFSH on testis weight and morphology in hpg mice. A) Testis weights of control adult hpg mice and mice treated every 12 h with FSH (n for each group is shown in the histogram). B) Semi-thin sections of testes from control adult hpg mice and mice treated with FSH for 12, 24 and 72 h. Note the appearance of vacuoles within the cytoplasm of the Sertoli cell at 24 and 72 h post-treatment. C) Electron micrographs at 24 and 72 h, arrows indicate the progression from multiple small vacuoles to fewer large vacuoles. Microarray data Analysis of the array data showed that there were 182, 164 and 203 transcripts signiﬁcantly (O2-fold) increased in the hpg testis 12, 24 and 72 h after the start of FSH treatment and 162, 411 and 215 signiﬁcantly decreased at the same times. Transcripts with the highest fold changes in expression at each time during treatment are listed in Table 1 and the complete list of signiﬁcantly altered transcripts (O2-fold) is shown in Supplementary Table 2, see supplementary data in the online version of the Journal of Molecular Endocrinology at http://jme.endocrinology-journals. org/content/vol42/issue4/. At 12 h after the start of FSH treatment, there was a clear increase in the levels of a number of transcripts known to be expressed in the Sertoli cells (e.g. Fabp5, Lgals1, Tesc and Scara5; Journal of Molecular Endocrinology (2009) 42, 291–303