Fig. 4.

JWH133 promotes differentiation of male germ cells from 7 days post partum (dpp) mice. (A) Histogram representing the percentage of nuclei with meiotic SCP3 staining in control testicular germ cells from 7 dpp or in cells stimulated with JWH133, AM630, or pretreated with AM630 and then treated with JWH133, after 24 h of culture. Bars represent s.d.. (B) Representative immunofluorescence images showing SCP3 (green) organization on nuclear spreads at the stages of early leptotene, leptotene, zygotene and zygo-pachytene of meiotic prophase, observed in 24 h cultured spermatogonia. (C) Percentage of leptotene, zygotene and zygo-pachytene nuclei in control cultures of germ cells from 7 dpp or in cells treated for 24 h with JWH133. Bars represent s.d. (D) qRT-PCR were performed as described in Methods in JWH133-treated premeiotic germ cells (JWH133) and untreated cells (Ctr). The expression levels in Ctr, i.e., the reference condition (*), were considered as 1 for all of the targets. The means of threshold cycles for the reference condition were 25.76, 23.21, 26.25, and 29.79 for c-Kit (first bar from the left), Stra-8 (second bar from the left) DMC-1 (third bar from the left), Lhx8 (fourth bar from the left), and SPO11 (last bar from the left), respectively. Standard deviations were calculated by the Gene expression module of iQ5 real-time PCR. The differences (except for Spo11 target) were significant (P < 0.05) as evaluated according to Pfaffl et al. (40). A typical experiment (R.I.N. > 8.5, see Methods) is depicted.