296 M H ABEL and others . Effects of FSH on testicular transcript levels exception of Igfbp3 which has been shown to be of Sertoli cell origin (Smith et al. 1990). In order to identify more of the differentially expressed transcripts on the arrays, which may be of a Sertoli cell origin, up- and down- regulated transcripts were compared with those ident- iﬁed as being of likely Sertoli cell origin by Chalmel et al. (2007) using a cell isolation, GeneChip and clustering approach (Supplementary Table 2A and B). A degree of caution is required as some known Sertoli cell transcripts (e.g. Rhox5) are missing from the list generated by Chalmel et al. (2007) probably due to Chip sensitivity or the subsequent ﬁltering process. Nevertheless, of the transcripts up-regulated at 12 h, 44% matched to the data from Chalmel et al. (2007). Interestingly, this number declined to 35% at 24 h and 27% at 72 h after the start of FSH treatment (Supplementary Table 2A). The number of down-regulated transcripts that matched the Sertoli cell list was 27, 21 and 28% at 12, 24 and 72 h respectively (Supplementary Table 2B). Real-time PCR Leydig cell genes To conﬁrm results from the array studies, real-time PCR was used to measure the effect of FSH treatment on testicular expression of selected transcripts which are known to be expressed exclusively in the Leydig cells (O’Shaughnessy et al. 2002). Eight mRNA species were tested which had shown an increase in transcript levels on the arrays after FSH (Star, Cyp17a1, Hsd17b3, Akr1b7, Lhr, Cyp11a1, Insl3 and Ren1) (Fig. 2A). Results from the real-time PCR studies conﬁrmed that seven of these transcripts are regulated by FSH in the hpg testis, although no change in Lhr was seen. Two other Leydig cell mRNA species (Hsd3b6 and Sult1e1) that had not shown any response to FSH on the arrays were also tested by real-time PCR (Fig. 2A). Levels of Hsd3b6 did not show a response to FSH but there was a signiﬁcant, if variable, increase in Sult1e1 after 72 h. Androgen-dependent genes The array studies showed clearly that a number of androgen-dependent Sertoli cell transcripts were altered after FSH treatment. Real-time PCR was used to conﬁrm changes in selected transcripts (Rhox5, Tsx, Drd4, Spinlw1 (Eppin) and Igfbp3) shown previously to be androgen-regulated (Lindsey & Wilkinson 1996, Denolet et al. 2006, O’Shaughnessy et al. 2007; Fig. 2B). In agreement with results from the array studies four transcripts (Rhox5, Tsx, Drd4 and Spinlw1) showed increased expression 24–72 h after FSH treatment while one transcript (Igfbp3) showed a signiﬁcant decrease in expression (Fig. 2B). Journal of Molecular Endocrinology (2009) 42, 291–303 Sertoli cell genes In addition to androgen-dependent Sertoli cell genes described above, 16 other Sertoli cell transcripts were measured by real-time PCR following FSH treatment of adult hpg mice (Fig. 3). Of these transcripts, seven had shown signiﬁcantly increased expression on the arrays (Tesc, Lgals1, Aqp5, Dhh, Pappa, Wnt4 and Shbg), six had not shown any signiﬁcant change (Trf, Wt1, Amh, Spata2, Tjp1 and Gdnf), two had shown a signiﬁcant decrease (Fshr and Rgs11) after FSH treatment and one transcript (Defb19) was not on the array. Results from real-time studies conﬁrmed increased transcript levels for six out of the seven mRNA species identiﬁed on the array (the exception was Shbg) and for the one transcript (Defb19) not on the array (Fig. 3A). Both transcripts decreased on the arrays after FSH treatment also showed a signiﬁcant decrease by real-time PCR (Fig. 3A). Interestingly, however, the real-time PCR data showed there was a signiﬁcant increase in levels of three out of the six transcripts that were not signiﬁcantly changed on the arrays (Trf, Wt1 and Amh; Fig. 3A). Two of these transcripts (Trf and Amh) had shown a greater than twofold increase on the arrays but had not reached signiﬁcance. Differences between results from real-time PCR and arrays may be a matter of sensitivity and variability of the two techniques or may be due to the choice of primers, away from the 30 region targeted by these arrays. The inhibin subunits are expressed in a number of cell types in the testis (Barakat et al. 2008) although it might be expected that initial responsiveness to FSH would be predominantly localised in the Sertoli cells. On the arrays, both Inha and Inhbb were signiﬁcantly increased by FSH (Table 1 and Supplementary Table 2) while there was no effect on Inhba. Results from the real- time PCR studies reﬂected the same pattern of results (Fig. 3B). Germ cell genes The number of known germ cell transcripts on the array affected by FSH, within the time-span of the study, was not high. This was, therefore, investigated further using real-time PCR. Expression of nine germ cell transcripts known to be expressed predominantly in spermatogonia (Stra8, Pou5f1, Dkkl1 and Spo11), spermatocytes (Mybl1, Zfp541) or spermatids (1700021K02Rik, Hdc and Tp1) was measured following FSH treatment (Fig. 4). Expression levels of Stra8, Spo11, Mybl1 and Tp1 were unaffected by treatment but there was a transient increase in Pou5f1 at 12 h while Dkkl1 and Zfp541 were signiﬁcantly increased at 72 h. Expression of Hdc and 1700021K02Rik (shown pre- viously to be increased on the array) was increased at all times after treatment. www.endocrinology-journals.org