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doi: 10.1242/10.1242/jcs.00163

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Essential role of citron kinase in cytokinesis of spermatogenic precursors

Ferdinando Di Cunto^1,*,, Sara Imarisio^1,*, Paola Camera¹, Carla Boitani², Fiorella Altruda¹ and Lorenzo Silengo¹

¹ Department of Genetics, Biology and Biochemistry, Via Santena 5 bis, Torino, Italy
² Department of Histology and Medical Embryology, University of Rome "La Sapienza", Rome, Italy

View larger version (73K): [in a new window]   Fig. 1. Expression of CIT-K during testicular development. (A) Protein extracts from mouse wild-type tissues were probed by western blotting using anti-CIT-K (upper panel) or anti GAPDH (lower panel) antibodies. The samples were cerebellum at P4 (1), testis at P4 (2) and P40 (3), ovary at P4 (4). (B-C) Sections from mouse wild-type testes at P4 (B) and P40 (C) were analyzed by immunohistochemistry with affinity-purified anti-Citron antibodies. Bars, 50 µm.

View larger version (105K): [in a new window]   Fig. 2. Specific testicular impairment in CIT-K-/- mice. (A) Frontal view of P14 CIT-K+/+ and -/- testes. (B-E) Histological sections of P14 testes (B,C) and ovaries (D,E) from P14 CIT-K+/+ (B,D) and -/- (C,E) mice. Staining hematoxylin and eosin, bar, 50 µm.

View larger version (95K): [in a new window]   Fig. 3. Analysis of the spermatogenic block in CIT-K-/- mice. (A-B) Expression of the indicated spermatogenic markers was analyzed by RT-PCR under semi-quantitative conditions. The assay was performed on total RNA extracted from CIT-K knockout (-) and littermate control (+) mice at P8 (A) and P14 (B). (C,D) Primordial germ cells were identified by alkaline phosphatase staining of comparable longitudinal sections of E12.5 CIT-K+/+ (C) and -/- (D) embryonic testes. (E,F) Gonocytes and undifferentiated spermatogonia were identified on sections of P4 CIT-K+/+ (E) and -/- (F) testes by anti-cyclinD1 immunostaining. (G) The total number of gonocytes and undifferentiated spermatogonia was determined on hematoxylin- and eosin-stained histological sections of testes from CIT-K+/+ (open bars) and -/- (closed bars) mice at the indicated times. Values are expressed as a percentage of the control. Error bars are standard deviation, P<0.01.

View larger version (132K): [in a new window]   Fig. 4. Apoptotic loss of spermatogenic precursors in CIT-K-/- mice. E14.5 embryos from CIT-K heterozygous intercrossing were labeled for 1 hour in utero with BrdU and then processed for immunohistochemistry. (A,B) Apoptotic gonocytes were identified by immunohistochemistry for activated Caspase-3, as intratubular-positive cells (red arrowheads). The assay was performed on comparable sections from CIT-K+/+ (A) and -/- (B) testes. Interstitial cells displayed a background cytoplasmic positivity in both control and knockout samples. (C,D) The tubular proliferative index was determined on sections adjacent to those used for the apoptosis assay, labeled by anti-BrdU immunostaining. (E) Graphic representation of the apoptotic (Cas-3) and proliferative (BrdU) indexes at E14.5 in testes of CIT-K+/+ (open bars) and -/- (closed bars). Values are expressed as the relative increase compared with the control. Error bars showstandard deviation, P<0.01.

View larger version (140K): [in a new window]   Fig. 5. Defective cytokinesis in CIT-K-/- undifferentiated male germ cells. (A,B) Semi-thin sections of P4 CIT-K+/+ (A) and -/- (B) testes were analyzed by optical microscopy. Red arrowheads point to type A spermatogonia displaying more than one nucleus. (C) The percentage of undifferentiated germ cells displaying a binucleate/multinucleate morphology was determined on semi-thin sections by optical microscopy. Error bars show standard deviation, P<0.005. (D) Electron microscopy picture of a P4 CIT-K-/- type A spermatogonium, displaying three nuclei surrounded by the same cytoplasm. (E) Anti-cyclinD1 immunohistochemistry performed on P4 CIT-K-/- testis, showing a multinucleated spermatogonium expressing high levels of this protein. (F) High-power field of anti-activated Caspase-3 immunostaining, performed on P4 CIT-K-/- testis. Red arrowhead indicates an early apoptotic, binucleate cell. Bars, 20 µm.