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First published online 27 July 2004
doi: 10.1242/jcs.01283

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Ryanodine receptors are expressed and functionally active in mouse spermatogenic cells and their inhibition interferes with spermatogonial differentiation

¹ Department of Histology and Medical Embryology and Centro di Eccellenza Biologia e Medicina Molecolare, University of Rome "La Sapienza", Via A. Scarpa 14, 00161 Roma, Italy
² Department of Neuroscience, Section of Molecular Medicine, University of Siena, Via A. Moro, 53100 Siena, Italy

View larger version (52K): [in a new window]   Fig. 1. Analysis of RyR1 expression by immunoblot and RT-PCR. (A) RyR1 detection in microsomal proteins prepared from adult mouse skeletal muscle (SM) (10 µg), and mouse testis (100 µg) at different ages of postnatal development between 10 and 60 days. (B) Immunoblot of microsomal proteins prepared from adult mouse skeletal muscle (SM), 60-day-old testis (T60), 10-day-old testis (T10), mixed populations of germ cells (gc), round spermatids (spt), pachytene spermatocytes (spc), highly purified spermatogonia (spg), epididymal spermatozoa (spz), Sertoli cells (SC). 10 µg of proteins were loaded for SM, whereas 100 µg for all other samples. (C) RTPCR analysis of RyR1 expression in adult skeletal muscle (SM), 60-day-old testis (T60), 7-day-old testis (T7), mixed germ cells (gc), highly purified spermatogonia (spg) and Sertoli cells (SC). Using isoform-specific primers, a single 435 bp product was identified. Lane marked `-' is' the negative control, where cDNA has been omitted in the PCR reaction.

View larger version (25K): [in a new window]   Fig. 2. RyR2 expression by western blotting and RT-PCR in the developing testis and isolated germ cells. (A) Immunoblot of microsomal proteins prepared from adult heart (10 µg), and total testis (100 µg) at different ages of postnatal development between 10 and 60 days. (B) RT-PCR analysis of RyR2. Using isoform-specific primers, a single 635 bp product was identified in 7-day-old testis (T7) and in an enriched (approximately 60%) population of spermatogonia (spg*), but not in highly purified spermatogonia (spg) and in Sertoli cells (SC). Lane marked `-' is the negative control.

View larger version (20K): [in a new window]   Fig. 3. Analysis of RyR3 expression by western blotting and RTPCR. (A) Immunoblot of 100 µg microsomal proteins extracted from diaphragm (D), 60-day-old testis (T60), mixed germ cells (gc) probed with antibody against RyR3. -RyR3 recognized a single specific band in diaphragm and a lower molecular weight band in testis and germ cells, which was proven to be nonspecific. (B) RyR3 detection by RT-PCR using isoform-specific primers that gives a single 505 bp product. Lane marked `-' is the negative control.

View larger version (20K): [in a new window]   Fig. 4. Caffeine-induced Ca2+ release in spermatogonia, spermatocytes and spermatids. Effects of 10 mM caffeine on [Ca2+]i release in Fura-2-loaded spermatogonia (A,D), pachytene spermatocytes (B,E), round spermatids (C,F). A single cell was equilibrated in KHH, then caffeine was added as pointed by the arrow. Panels (A,B,C) represent response to caffeine following cell preincubation in plain MEM. Panels (D,E,F) represent caffeine-induced Ca2+ release following cell preincubation in MEM additioned with 10 mM CaCl2. The signal was calibrated with 5 µM ionomycin. [Ca2+] values are representative of six different experiments for each cell type.

View larger version (10K): [in a new window]   Fig. 5. Dose response curve of germ cells to caffeine under conditions of increased extracellular calcium. Stimulation with caffeine (1-10 mM) induced a concentration dependent increase in Ca2+ release. Values are expressed in terms of fluorescence ratio at the two excitation wavelengths and are the mean ± s.d. of three independent experiments.

View larger version (15K): [in a new window]   Fig. 6. Treatment with 300 µM ryanodine abolishes caffeine induced Ca2+ release in germ cells. Cells were preloaded with 10 mM CaCl2, washed and either incubated (A) with or (B) without 300 µM ryanodine for 1 hour with before measuring caffeine-dependent Ca2+-release. Ionomycin (IMC) was added to elicit further release of intracellular Ca2+. The traces are typical of three independent measurements.

View larger version (40K): [in a new window]   Fig. 7. Effects of blocking RyR channels on spermatogonia proliferation and differentiation. Testicular fragments were cultured for 3 days with 100 µM or 300 µM ryanodine (Ry) or 20 ng/ml FSH (Ry) alone, and combinations of 20 ng/ml FSH with 100 µM Ry, 300 µM ryanodine or 300 µM 8-Br-cADPR, and eventually labelled with BrdU for 5 hours. (A) Percentages of tubules with BrdU-labelled spermatogonia; (B) percentages of tubules containing meiotic cells. Cell counting was performed as described in Materials and Methods. Values are the mean ± s.e.m. of three independent experiments. FSH+Ry 300 µM versus FSH: P<0.001; FSH+300 µM 8-Br-cADPR versus FSH: P<0.001.

View larger version (25K): [in a new window]   Fig. 8. Spermatogonial proliferation and differentiation are independently affected by RyR inhibition. Testicular fragments were cultured for 24, 48 and 72 hours in the presence of 20 ng/ml FSH, with or without 300 µM ryanodine. (A) Percentages of tubules displaying BrdU-labelled spermatogonia. (B) Percentages of tubules displaying meiotic cells. Labelling and counting were performed as described in Materials and Methods. Each value represents the mean ± s.e.m. of at least 200 tubules for each experimental condition, obtained in two independent experiments. (A) FSH+Ry versus FSH at 72 hours: P<0.001; (B) FSH+Ry versus FSH at 48 and 72 hours: P<0.001.