300 M H ABEL and others . Effects of FSH on testicular transcript levels study which reported that gonadotrophins reduce transcript levels of Sertoli cell barrier components but that FSH may act at the level of protein organisation to induce barrier functionality (Tarulli et al. 2008). Other transcripts that showed a signiﬁcant decrease in levels after FSH treatment included Fshr and Rgs11. It is well established that FSH will cause down- regulation of its receptor by decreasing transcript levels (O’Shaughnessy 1980, Themmen et al. 1991) and a reduction in Fshr is to be expected. RGS11, in contrast, belongs to the regulator of G protein-signalling family which are GTPase-activating proteins that act to inhibit signal transduction and thus play a role in desensitisa- tion (Chasse & Dohlman 2003). The role of RGSs in normal hormonal signalling is not well established but the declining levels of Rgs11 after FSH treatment may act to enhance signal transduction despite a reduction in receptor levels. In addition to changes in Sertoli cell transcripts induced by FSH, it was clear from the rise in testicular androgen and the array and real-time PCR data that FSH was also acting to induce Leydig cell function. This effect was marked and rapid with a Leydig cell transcript (Ren1) showing the greatest fold change at 12 h (Deschepper et al. 1986). Results from the arrays and real-time PCR show that all components of the androgen biosynthetic pathway were induced at 12 h apart from Hsd3b6. Since Hsd3b1 is already highly expressed in the adult hpg testis (Baker et al. 2003) lack of HSD3B6 is unlikely to affect the steroidogenic potential of the cells. In addition to the steroidogenic enzymes, pathway analysis showed that most com- ponents of the cholesterol biosynthetic pathway were induced 12 h after FSH treatment while Ldlr levels are increased. This shows that FSH is acting to increase the capacity of the Leydig cells to produce and sequester cholesterol and to convert cholesterol to androgen. Interestingly, one of the critical components of the cholesterol biosynthetic pathways (Mvk) also acts to inhibit Lhr translation (Nair & Menon 2004) and this may serve to regulate further Leydig cell sensitivity to LH. The hpg mouse testis is likely to contain both adult and fetal-type Leydig cells (Baker et al. 2003) and Hsd3b6 is a marker of adult Leydig cell differentiation (Baker et al. 1999). This might imply that FSH is acting to induce activity in the fetal Leydig cell population but Sult1e1 is a marker of adult Leydig cells (Song et al. 1997) and is increased 72 h after FSH suggesting that the effects of FSH are probably being mediated through the adult Leydig cells. In the testis, receptors for FSH are only found in the Sertoli cells (Heckert & Griswold 2002) and the effects of FSH must be mediated by a factor or factors released by the Sertoli cells which act on the Leydig cells. In the short-term, the effects of FSH on Leydig cell function in the hpg appear to be more marked than effects of hCG Journal of Molecular Endocrinology (2009) 42, 291–303 (Baker et al. 2003) and the effects are also very rapid since Sadate-Ngatchou et al. (2004) saw a marked increase in Cyp17a1 after only 4 h of FSH treatment. FSH appears, therefore, to be able to induce a powerful and rapid response in Leydig cells presumably through stimulation of release of potent trophic factors by the Sertoli cells. The presence of such factors has been postulated for a number of years since early studies on perfused testes or hypophysectomised animals treated with FSH (Johnson & Ewing 1971, Chen et al. 1976, Vihko et al. 1991). One report has suggested that the active factors are TIMP1 and Procathepsin L (Boujrad et al. 1995) but this has not been conﬁrmed and we saw no evidence of changes in these factors in our study. Following FSH treatment, our array data showed that there was an increase in Igf1 levels and IGF1 has been suggested to play a role in Leydig cell differentiation (Morera et al. 1987). Interestingly, there was a marked decline in Igfbp3 and an increase in Pappa levels after FSH treatment. Increased Pappa would be expected to increase the bioavailability and activity of IGF1 (Conover et al. 2004) although the effect of altered Igfbp3 may be more complex (Modric et al. 2001). The time-course of changes in expression of Igf1 levels does not appear to ﬁt well with a role in the stimulation of Leydig cell function after FSH treatment although it is possible that early changes in Pappa and Igfbp3 may alter early IGF1 bioavailability. Other secreted molecules showing a marked increase in transcript levels after FSH include Wif1, Dkk3 and Dmkn. Both WIF1 and DKK3 act to regulate WNT signalling and the WNT/CTNNB1 pathway is critical for normal Sertoli cell development (Boyer et al. 2008) although its function in the Leydig cell remains uncertain. Following the increase in Leydig cell activity after FSH treatment, there was a signiﬁcant change in the levels of known androgen-dependent Sertoli cell- speciﬁc transcripts. It is possible that changes in these transcripts are due to direct effects of FSH treatment but the known androgen-dependence of the transcripts makes it more likely that changes are related to increased Leydig cell androgen production induced by FSH. The rise in intratesticular androgen after FSH treatment was signiﬁcant but levels remained very low, probably because FSH stimulates synthesis of the components of the steroidogenic pathway without being able to stimulate the pathway itself. The apparent effect of these low levels of androgen on Sertoli cell transcript levels suggests that the Sertoli cells are extremely sensitive to androgen stimulation. Treatment of hpg mice with FSH increased vacuo- lation in the Sertoli cells and induced formation of a lumen within the seminiferous tubules but had little apparent effect on germ cell morphology or pro- gression up to 72 h. Changes in the Sertoli cell and tubule diameter correlate with a marked rise in Aqp5 at www.endocrinology-journals.org