We wished to establish whether any of our candidate testosterone-responsive genes showed a dose-dependent response to testosterone stimulation. We, therefore, filtered the list of second-phase genes to select for probes showing a significant change in expression across the dose-response sample series time course (ANOVA P < 0.05 after FDR correction) and where the direction of change was consistent with the existing data. Table 2 indicates the number of genes in each category that passed this final filter. Most of the genes in the middle and late categories did not show any significant dose sensitivity.
Early Androgen-Responsive Genes in Spermatogenesis Regulation
This is almost certainly because most of these genes only change in expression after 48 h of testosterone stimulation, and the dose-response sample series was taken at 24 h after the treatment. However, all of the remaining six genes in the early down-regulated category passed this filter. These genes—Defb45, Drd4, Lmcd1, Pla1a, Rhox5, and Spinlw1—were activated in the normal testis relative to LuRKO from Day 8 onward, they responded to androgen treatment within 12 h, and they were sensitive to low dosages of androgen. They are thus key candidates for a regulatory role in the androgen-dependent progression of spermatogenesis. It is interesting to note that these changes occurred well before the spermatogenic block became evident. This suggests that these genes have an upstream role in preparing the somatic cell compartment to support the subsequent germ cell development.
Known Leydig Cell Genes: Developmental Profile and Testosterone Sensitivity
Finally, we examined the expression of the known Leydig cell genes identified by O’Shaughnessy et al.. Table 3 shows the log2 ratio expression data in LuRKO mice relative to control testes across the time course and the log2 expression ratio for the sample from the maximally treated animals (2.5 mg of testosterone for 48 h) relative to the untreated control. Of the genes they identified, the Lhcgr data is not shown here because the expression is negligible at all ages in LuRKO. Estrogen sulfotransferase (Sult1e1) was not present in our data set. Multiple measurements for the same gene indicated the presence of multiple probes on the array. In the case of Hsd17b3, probes directed at different exons showed differing expression patterns, potentially indicating differential regulation splice variants for this transcript.
Of these known Leydig cell genes, Cyp11a1, Cyp17a1, and Hsd3b1 were down-regulated at Day 3, indicating LH dependence in fetal Leydig cells. Insl3, Star, and Srd5a1 also showed moderate down-regulation at early ages, although they were not significant. The above six genes were all significantly down-regulated at later ages, indicating LH dependence in adult-type Leydig cells. Ephx1 appeared to be LH dependent in adults but not in fetal Leydig cells, while Vcam1 was specific to adult cells and was LH dependent. Akr1c14 was down-regulated at Day 3 but not at later ages.
