Recent evidence suggested the involvement of calcium-binding protein regucalcin (RGN) in testicular apoptosis. Herein, we investigated the role of RGN controlling apoptotic pathways in the testis by using a transgenic rat model overexpressing RGN (Tg-RGN). Seminiferous tubules (SeT) from Tg-RGN and their wild-type (Wt) counterparts were cultured ex vivo in presence or absence of apoptosis inducers thapsigargin (Thap, 10(-7) and 10(-6) m) and actinomycin D (Act D, 0.5 and 1 μg/mL). Expression levels of key regulators of apoptosis in SeT of Tg-RGN and Wt animals were determined by quantitative real-time PCR and Western blot analysis. Measurement of caspase-3 enzymatic activity was included as an end point of apoptosis. Tg-RGN SeT treated with 10(-6) m of Thap or 1 μg/mL of Act D showed a diminished enzymatic activity and gene transcription of caspase-3, along with increased mRNA and protein expression of antiapoptotic Bcl-2. Bcl-2/Bax (antiapoptotic/proapoptotic) protein ratio was also enhanced in these SeT. Although caspase-9 mRNA was increased in the SeT of Tg-RGN treated with Thap, no differences were observed at protein level, and no differences were also found on protein levels of apoptosis-inducing factor. mRNA expression of proapoptotic p53 and p21 was strongly decreased in Tg-RGN SeT treated with Thap (10(-6) m) or Act D (1 μg/mL). These findings demonstrated that RGN suppresses Thap- and Act D-induced apoptosis in SeT by modulating the expression and activity of key apoptotic and antiapoptotic factors. Moreover, results indicate that RGN overexpression protects germ cell from apoptosis induced by noxious stimuli, which could be a relevant mechanism for fertility preservation in situations of oncological treatments.
Keywords: actinomycin D; apoptosis; calcium; germ cells; rat; regucalcin; seminiferous tubules; spermatogenesis; testis; thapsigargin.
© 2014 American Society of Andrology and European Academy of Andrology.