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Stem cell factor activates telomerase in mouse mitotic spermatogonia and in primordial germ cells

¹ Dipartimento di Sanita' Pubblica e Biologia Cellulare, Sezione di Anatomia, Rome, Italy
² Istituto Dermopatico dell'Immacolata (IDI, IRCCS), Rome, Italy
³ Dipartimento di Neuroscienze, Universita' di Roma Tor Vergata, Rome, Italy

View larger version (86K): [in a new window]   Fig. 1. Telomerase activity is induced by Kitl in mitotic spermatogonia. (A) Spermatogonial cell extracts were obtained from cells that were freshly collected (T0) or after 24 hour in culture without (control; 24 hr) or with the addition of Kitl. Enzyme activity was assessed by the TRAP assay in the presence (+) or absence (-) of RNase A. (B) Spermatogonial cell extracts were cultured for 24 hours (24h) in the absence of Kitl and then for further 24 hours in culture without (48h- Kitl) or with the addition of Kitl (48h+ Kitl). Enzyme activity was assessed by the TRAP assay in the presence (+) or absence (-) of RNase A.

View larger version (26K): [in a new window]   Fig. 2. TR and TERT expression are induced by Kitl treatment. RT-PCR from spermatogonia RNA after 24 hours of culture without (ctrl) or with the addition of Kitl (Kitl) using mTR- (A) and mTERT- (B) specific primers. Hprt amplification has been used to verify RNA integrity. (C) Kit amplification was performed to show that equal amounts of Kit-expressing cells were analyzed.

View larger version (76K): [in a new window]   Fig. 3. In situ hybridization of hTR in mouse spermatogonia. A 35S-riboprobe obtained from the hTR template was hybridized to spermatogonia that were either freshly withdrawn (A,E) or withdrawn after 24 hours in culture without (B,F) or with (C,G) Kitl. A sense probe (D,H) was used in T0 cultures to verify the specificity of hybridization. (A-D, represent brightfield images; E,F, represent darkfield.)

View larger version (47K): [in a new window]   Fig. 4. The P13K inhibitor LY294002 abolishes the Kitl-induced increase of telomerase activity. Spermatogonial cell extracts were obtained from cells after 24 hours in culture in the presence or absence of 10 µM LY294002 with or without the addition of Kitl. Enzyme activity was assessed by the TRAP assay.

View larger version (47K): [in a new window]   Fig. 5. Telomerase activity in fetal germ cell is induced by Kitl. Telomerase activity was assessed from cell extracts that were equivalent to 200 viable cells in 12.5 dpc male germ cells. These extracts were freshly withdrawn (lane 1), or withdrawn after 24 hours in culture in the presence (lane 2) or absence of Kitl (lane 3) or from 14.5 and 15.5 dpc male germ cells (lanes 5 and 6, respectively). Cell extracts corresponding to 200 spermatogonia were analysed for comparison (lane 4).

View larger version (62K): [in a new window]   Fig. 6. Telomerase activity in growing oocytes is not influenced by Kitl. Telomerase activity was assessed from oocyte extracts at T0 or after 24 hours of culture in the absence or presence of Kitl. Cell extracts were obtained from 15 growing oocytes. Enzyme activity was assessed by the TRAP assay in the presence (+) or absence (-) of RNase A.