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. 2011 May 9;6(5):e19120. doi: 10.1371/journal.pone.0019120

Figure 1. Defective spermatogenesis and altered expression of CREB in CF mice testis.

Figure 1

(A) Significantly reduced testis size and weight seen in heterozygous S489X mice compared with wild-type mice (n = 3; *:p<0.05). (B) Reduced daily sperm production (DSP) values retrieved from heterozygous S489X testes (n = 4; *:p<0.05). (C) Reduced sperm numbers recovered from the epididymis of heterozygous S489X mice (+/+ n = 6, +/− n = 4; *:p<0.05). (D) H&E staining of the cross sections of wild−type, heterozygous and homozygous S489X CF mice testes. Seminiferous tubules remain intact in +/− and −/− CF mice, but looser tubule contacts with larger interstitial space are observed in −/− testes (right) when compared with wild type (left). Leydig cells and peritubular myloid cells are detached from most tubules in −/− testes. Bar  = 20 µm (upper panel) or 50 µm (lower panel). (E) Statistic shows slight decrease in tubular diameter is observed in −/− testes. (F) Realtime PCR of protamine-2 in S489X CF mice testes. Protamine-2 mRNA level is significantly lowered in heterozygous and homozygous S489X CF mice compared to their wildtype littermates. (G) Immunofluorescent staining of Protamine-2 in +/+, +/− and −/− S489X CF mice testes (stage VII–VIII). In +/+ testes, Protamine-2 positive signals are located in elongated spermatids (ES) with strong immunoreactivity (red arrow). In +/− testes, Protamine-2 is found in ES with moderate immunoreactivity (red arrow). In −/− testes, there is weak intensity in the pachytene spermatocytes (P) (white arrow), ES (red arrow) and round spermatids (RS) (yellow arrow). Bar  = 50 µm. (H) Immunofluorescent staining of CREM in S489X CF mice testes (stage VIII–I). In wild-type testes, CREM positive signals are localized in RS (white arrow), with strong immunoreactivity. In +/− and −/−testis, CREM showed weak immunoreactivity. Bar  = 50 µm. (I) Immunofluorescent staining of CREB in S489X CF mice testes shows stronger immunoactivity in +/+ and +/− mice Sertoli cell compared to that of −/−. Bar  = 50 µm. (J) Western blot analysis of CREM expression in S489X CF testes. CREM expression is decreased in +/− and −/− testes compared to wild-type (n = 3; *:p<0.05). (K) Western blot analysis of CREB expression in S489X CF testes. CREB expression is decreased in −/− testes compared to +/− and wild-type (+/+ n = 2, +/− and −/− n = 3; **:p<0.01). Statistic data are shown as mean±SEM.