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First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01196

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Novel expression of resistin in rat testis: functional role and regulation by nutritional status and hormonal factors

¹ Department of Physiology, University of Santiago de Compostela, c/San Francisco s/n, 15705 Santiago de Compostela, Spain
² Department of Medicine, University of Santiago de Compostela, c/San Francisco s/n, 15705 Santiago de Compostela, Spain
³ Department of Cell Biology, Physiology and Immunology, University of Córdoba, Avda. Menendez Pidal s/n, 14004 Córdoba, Spain
⁴ Department of Physiology and Department of Pediatrics, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland

View larger version (32K): [in a new window]   Fig. 1. (A,B) Analysis of resistin gene expression in random testis specimens, as evaluated by RT-PCR and Southern blot (A), and optimization of RT-PCR conditions for semi-quantitative evaluation of relative expression levels of resistin mRNA in testis samples (B). For comparative purposes, expression of resistin gene in white adipose tissue (WAT) is presented. (C) The developmental profile of expression of the resistin gene in rat testis throughout postnatal maturation. A representative semi-quantitative RT-PCR assay of expression levels of resistin mRNA in testicular samples from 5-, 15-, 30-, 60- and 90-day-old rats. Parallel amplification of L19 ribosomal protein mRNA served as internal control. Semi-quantitative values are the mean±s.e.m. of at least four independent determinatio ns. a,b and c denote groups that are statistically different (P<0.01; ANOVA followed by Tukey's test).

View larger version (39K): [in a new window]   Fig. 2. Real-time RT-PCR analysis of testicular resistin mRNA levels throughout post-natal development and in aging. Total RNA samples were obtained from 5-day, 15-day, 30-day, 60-day, 90-day, and 17-month-old rats. Quantitative data in terms of resistin expression levels were normalized to those of the internal control RP-L19. Values are the mean±s.e.m. of at least four determinations. a,b and c denote groups that are statistically different (P<0.01; ANOVA followed by Tukey's test).

View larger version (76K): [in a new window]   Fig. 3. Immunolocalization of resistin protein in adult rat testis sections. (A) A representative reaction after immunolabeling with the specific anti-resistin primary antibody. At higher magnification (B), specific resistin immunoreactivity was clearly evident on interstitial Leydig cells and, at a lower intensity, in Sertoli cells within the seminiferous tubules. In addition, in some tissue sections, resistin peptide was also detected in macrophages present in the testicular interstitium. Scale bars: (A) 50 µm; (B) 20 µm.

View larger version (51K): [in a new window]   Fig. 4. Effects of hypophysectomy (HPX) and gonadotropin replacement on testicular expression of resistin. (Left panels) A representative RT-PCR assay of resistin mRNA levels in testes from long-term (4-week) HPX rats, with or without replacement of hCG (10 IU/rat/24 hours for 7 days) or FSH (7.5 IU/rat/24 hours for 7 days). Semi-quantitative values are the mean±s.e.m. of at least four independent determ inations. a,b and c denote groups that are statistically different (P<0.01; ANOVA followed by Tukey's test). (Right panels) Representative photomicrographs of resistin immunoreactivity in testis tissue from HPX rats, with or without replacement of hCG. (A) A representative control section. (B,C) Long-term HPX resulted in clear-cut suppression of resistin immunoreactivity (B), whereas hCG replacement partially restored this signal (C). Scale bar: 20 µm.

View larger version (32K): [in a new window]   Fig. 5. Acute regulation of testicular resistin mRNA expression by gonadotropins. (A) A representative RT-PCR assay of the testicular expression levels of resistin mRNA at different time-points (2-24 h) after exposure to 25 IU hCG in vivo. (B) A representative RT-PCR assay is shown of testicular resistin mRNA levels at different times (2-24 hours) after exposure to 12.5 IU recombinant FSH in vivo. Semi-quantitative values are the mean±s.e.m. of at least four independent determinations. a,b and c denote groups that ar e statistically different (P<0.01; ANOVA followed by Tukey's test).

View larger version (38K): [in a new window]   Fig. 6. Expression profile of resistin gene in seminiferous tubules segments at different stages of the seminiferous epithelial cycle, in basal conditions and after stimulation with FSH. A representative RT-PCR assay of the expression levels of resistin mRNA in tubule preparations at stages II-VI, VII-VIII, IX-XII, and XIII-I of the cycle, in three experimental situations: immediately after isolation (C-0), after 24-hour culture in the presence of medium alone (C-24), or after 24-hour culture in the presence of FSH (10 ng/ml) (FSH-24). Semi-quantitative values are the mean±s.e.m. of at least four independent determinations. a,b and c denote groups that are statistically different (P<0.01; ANOVA followed by Tukey's test).

View larger version (35K): [in a new window]   Fig. 7. Regulation of resistin mRNA expression in rat testis by the selective ligand of PPAR, rosiglitazone. (A) A representative RT-PCR assay of the testicular expression levels of resistin mRNA after a 1-week exposure to rosiglitazone (TZD; 5 mg/kg/day), in vivo. (B) A representative RT-PCR assay of testicular expression levels of resistin mRNA after in vitro exposure to 10–10-10–4 M rosiglitazone (TZD). Semi-quantitative values are the mean±s.e.m. of at least four independent determinations. In A, *P<0.05 versus the vehicle-treated (VEH) group; in B, a,b and c denote groups that are statistically different (P<0.01; ANOVA followed by Tukey's test).

View larger version (41K): [in a new window]   Fig. 8. Regulation of resistin mRNA expression in rat testis by the nutritional status and the adipocyte-derived hormone, leptin. (A) A representative RT-PCR assay is presented of the testicular expression levels of resistin mRNA in male rats fed ad libitum and after short-term (48 hours) fasting. Animals were infused centrally with recombinant human leptin (Lep; 15 µg/day) or vehicle (C), for 7 days. (B) Quantitative values of resistin mRNA levels in the experimental groups, determined by real-time RT-PCR, are presented as the mean±s.e.m. of at least four independent determinations. a,b and c denote groups that are statistically different (P<0.01; ANOVA followed by Tukey's test).

View larger version (17K): [in a new window]   Fig. 9. Regulation of in vitro testicular testosterone secretion by resistin. Testicular slices were incubated with increasing doses of resistin-(23-42) (10–10-10–6 M) in the absence (–hCG; basal conditions) or the presence (+hCG; stimulated conditions) of 10 IU/ml hCG. The concentration of testosterone in the media was monitored after 90 and 180 minutes incubation; only data from the 90-minute incubation are presented. Values are normalized per gram of incubated tissue. Data are expressed as mean±s.e.m. (n=10-12 samples/group). **P<0.01 vs. corresponding controls (ANOVA followed by Tukey's test).

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