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. Author manuscript; available in PMC: 2008 Apr 15.
Published in final edited form as: Exp Cell Res. 2007 Feb 3;313(7):1373–1392. doi: 10.1016/j.yexcr.2007.01.017

Fig. 3. Changes in CAR expression in rat testes during testicular maturation.

Fig. 3

Fig. 3

Fig. 3

A: Immunofluorescent staining of CAR (H-300) was performed on frozen sections of rat testes of different ages: 15-day (a-d), 25-day (e-h), and 60-Day (i-l) v.s. adult rat kidney (body weight, 300 gm) (m-p). A mouse antibodyagainst ZO-1 conjugated with FITC (green fluorescence) was used to localize the blood-testis barrier. Sections were then incubated with a donkey anti-rabbit Cy3 conjugated antibody to visualize CAR (red fluorescence). (m-p): Cross-section of frozen rat kidney was included to demonstrate the specificity of CAR staining. Signals of CAR staining co-localized with ZO-1 to tight junctions in the collecting tubules of rat kidney. At 15 day, when blood-testis- barrier was absent and only spermatogonia were found in the seminiferous tubules, CAR staining was detected at the Sertoli-Sertoli and Sertoli-germ cell interface, surrounding cell nuclei. At day 25, CAR staining was observed to be co-localizing with ZO-1 to the newly formed blood-testis barrier, along with staining surrounding the acrosome region of late round spermatids. At day 60, when the rat testis matured with complete cycles of spermatogenesis, CAR staining were found at germ cells of all stages, yet the signals were most intense in early stage VIII tubules, where elongated spermatids anchor their heads to Sertoli cells in the epithelium. Signals were also detected at the blood-testis barrier, but not of the same intensity compared to those at the apical ectoplasmic specialization. Scale bar = 25 μm in (a), which also applies to (b-d) & (m-p). Scale bar = 50 μm in (e), which also applies to (f-l). B: Immunohistochemical localization of CAR in the seminiferous epithelium was performed on frozen sections of testes from rats of different ages: 15- (a-c), 20- (d-f), 25-day-old rats (g-i) a,d,g are negative controls using normal rabbit IgG for each sample group. Scale bar = 50 μm in (b), which also applies to (e,h) and upper-right corner of (a,d,g). Scale bar = 25 μm in (a), which also applies to (c,d,f,g,i). C: Immunoblot of CAR using testis lysates from rats of different ages. Brain lysate of 20-day-old rats served as a positive control. Bar graph summarizes results of three sets of immunoblots using separate batches of testis lysate preparation. The level of CAR in testes from 90-day-old rats was arbitrarily set at 1.