Inhibitory roles of the mammalian GnIH ortholog RFRP3 in testicular activities in adult mice
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Figure 1
Light microscopic histological analysis of seminiferous tubules (STs) showing cellular alterations in different spermatogenic stages in control and treated mice. (A and B, stage VII) Control STs showed spermatogonia, preleptotene spermatocytes, pachytene spermatocytes round spermatids, and elongated spermatids. (C, D and E, stages VII and VIII) Low dose (0.02 μg RFRP3) showed loosening of germinal epithelium in ST. (F, G and H, stage VIII) Moderate dose (0.2 μg RFRP3) showed giant cell and degenerating germ cell. (I, J and K, stages VI and VIII) High dose (2.0 μg RFRP3) showed chromatin condensation in round spermatids and exfoliation of elongated spermatids in lumen. All the figures are shown in 40× and 100× magnification. Pl, preleptotene; P, pachytene; S, spermatogonia; RS, round spermatids; ES, elongated spermatids; DGC, degenerated germ cell; GC, giant cell; Ch CRS, chromatin condensation of round spermatids; Ex ES, exfoliation of elongated spermatids. Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-14-0333.
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Figure 2
(A) Changes in the serum testosterone level in RFRP3-treated mice showed a significant decrease in the circulating testosterone level with different doses (low dose=0.02 μg; b, P<0.05: moderate dose=0.2 μg; c, P<0.05, and high dose=2.0 μg/day; d, P<0.05) of RFRP3 when compared with the control (a, P<0.05). Changes in testicular 3β-hydroxysteroid dehydrogenase (3β-HSD) enzyme activity in treated mice showed a significant decrease in 3β-HSD enzyme activity with different doses (low dose=0.02 μg; b, P<0.05: moderate dose=0.2 μg; c, P<0.05, and high dose=2.0 μg/day; d, P<0.05) of RFRP3 when compared with the control (a, P<0.05). Values are expressed as means±s.e.m. Different letters indicate a significant difference at P<0.05. (B) The densitometric analysis of the immunoblot of testicular STAR protein in mice treated with different doses of RFRP3 (low dose=0.02 μg; b, P<0.05: moderate dose=0.2 μg, c, P<0.05, and high dose=2.0 μg/day, d, P<0.05) showed a significant decrease in the expression of testicular STAR protein when compared with the control (a, P<0.05) and (C). P450scc proteins in mice treated with different doses of RFRP3 (moderate dose=0.2 μg; b, P<0.05 and high dose=2.0 μg/day; d, P<0.05) showed significant decreases in the expressions of P450scc proteins when compared with the control (a, P<0.05) and low dose of RFRP3 (0.02 μg; a, P<0.05). Values are expressed as means±s.e.m. Different letters indicate a significant difference at P<0.05.
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Figure 3
(A) The densitometric analysis of the immunoblot of testicular LHR protein in mice treated with different doses of RFRP3 (low dose=0.02 μg; b, P<0.05: moderate dose=0.2 μg; c, P<0.05, and high dose=2.0 μg/day; c, P<0.05) showed a significant decrease in LHR when compared with the control (a, P<0.05). (B) The immunoblot of testicular GNRHR protein in mice treated with different doses of RFRP3 (low dose=0.02 μg, moderate dose=0.2 μg, and high dose=2.0 μg/day) showed a significant decrease in the expression of GNRHR protein in low, moderate (b, P<0.05), and high doses of RFRP3 (c, P<0.05) when compared with the control (a, P<0.05).
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Figure 4
Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the testis with different doses of RFRP3 (low dose=0.02 μg, moderate dose=0.2 μg, and high dose=2.0 μg/day). (A and B) In controls, PCNA-positive cells were strongly detected in spermatogonia and early-stage spermatocyte (mainly in the spermatogonia B (B Sg), preleptotene (pl Sc)/leptotene (L), and pachytene (P Sc) stage) of the spermatogenic cycle in mice. Mice treated with different doses, i.e. low dose (C and D), moderate dose (E and F), and high dose (G and H), of RFRP3 showed low PCNA-positive staining in germinal cell when compared with the control. Negative control for PCNA protein was shown (I). Arrowhead indicates spermatogonia and early-stage spermatocytes. All the figures are shown in 40× and 100× magnification. Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-14-0333.
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Figure 5
The densitometric analysis of the immunoblot of BCL2 protein in mice treated with different doses of RFRP3 (low dose=0.02 μg; b, P<0.05: moderate dose=0.2 μg; c, P<0.05, and high dose=2.0 μg/day; c, P<0.05) showed a significant decrease in the expression of BCL2 when compared with the control (a, P<0.05) (A). The immunoblot of PCNA protein in mice treated with different doses of RFRP3 (low dose=0.02 μg; b, P<0.05: moderate dose=0.2 μg; c, P<0.05, and high dose=2.0 μg/day; d, P<0.05) showed a significant decrease in the expression of PCNA when compared with the control (a, P<0.05) (B). The immunoblot of caspase 3 protein in mice treated with different doses of RFRP3 (low dose=0.02 μg; b, P<0.05: moderate dose=0.2 μg; c, P<0.05, and high dose=2.0 μg/day; d, P<0.05) showed a significant increase in the expression of Caspase 3 when compared with the control (a, P<0.05) (C). The immunoblot of PARP protein in mice treated with different doses of RFRP3 (low dose=0.02 μg; b, P<0.05: moderate dose=0.2 μg; c, P<0.05, and high dose=2.0 μg/day; d, P<0.05) when compared with the control (a, P<0.05) in mice (D). Values are expressed as means±s.e.m. Different letters indicate a significant difference at P<0.05.
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Figure 6
Changes in testosterone level in media with different doses of RFRP-3 (low=1 ng/ml and high dose=10 ng/ml) with or without LH (low dose=10 ng/ml and high dose=100 ng/ml) showed a significant decrease in the testosterone level in both dose of RFRP-3 (b and c, P<0.05) vs control (a, P<0.05) and significantly increased in the testosterone level in both doses of LH (d and e, P <0.05) vs control (a, P<0.05), whereas, high dose of LH together with high dose of RFRP-3 showed a remarkable significant (f, P<0.05) increase in testosterone level vs control (a, P<0.05) (A). Immunoblot analysis of an in vitro treatment of RFRP-3 (low dose=1 ng/ml and high dose=10 ng/ml) with or without LH (low dose = 10 ng/ml and high dose = 100 ng/ml) showed a significantly decrease in the expression of StAR protein in both doses of RFRP-3 (b, P<0.001) vs control (a, P<0.001) and both doses of LH alone showed a significant increased the expression StAR protein (c and d, P<0.001) vs control (a, P<0.001), whereas, high dose of RFRP-3 with high dose of LH showed a significant (e, P<0.001) increase the expression of StAR protein vs control (a, P<0.001) (B). Immunoblot analysis of an in vitro treatment of RFRP-3 (low dose=1 ng/ml and high dose =10 ng/ml) with or without LH (low dose=10 ng/ml and high dose=100 ng/ml) showed a significant decrease in the expression of LH-R protein with both doses of RFRP-3 (b and c, P<0.05) vs control (a, P<0.05) and both the doses of LH alone showed a significant increased the expression LH-R protein (d and e, P<0.05) vs control (a, P<0.05), whereas, high dose of RFRP-3 with high dose of LH showed a significant (e, P<0.05) increase the expression of LH-R protein vs control (a, P<0.05) (C). Values are the means±SEM. Different letters indicate a significant difference at P<0.05.
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Figure 7
Western blot/slot blot analysis of in vitro treatment of RFRP3 (low dose=1 ng/ml and high dose=10 ng/ml) with or without LH (low dose=10 ng/ml and high dose=100 ng/ml) showed significant decreases in the expressions of GNRHR (A) and GNRH (B) proteins at both doses of RFRP3 (b, P<0.05) when compared with the control (a, P<0.05), both doses of LH alone showed a significant decrease in the expression of GNRHR protein (c and d, P<0.05) when compared with the control (a, P<0.05), whereas a high dose of RFRP3 with a high dose of LH showed significant (e, P<0.05) increases in the expressions of GNRHR and GNRH proteins when compared with the control (a, P<0.05). Values are expressed as means±s.e.m. Different letters indicate a significant difference at P<0.05.
- © 2014 Society for Endocrinology
