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First published online July 10, 2003
doi: 10.1242/10.1242/jcs.00650

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Developmental expression of BMP4/ALK3/SMAD5 signaling pathway in the mouse testis: a potential role of BMP4 in spermatogonia differentiation

Dipartimento di Sanita' Pubblica e Biologia Cellulare, Sezione di Anatomia, Universita' di Roma Tor Vergata, Rome, Italy

View larger version (26K): [in a new window]   Fig. 1. Expression pattern of BMP4, Alk3 and BMPIIR in the testicular cell types. (A) Northern blot analyses of BMP4 expression in Sertoli cells obtained at different ages of postnatal development (upper panel), compared to ethidium bromide staining of the same gel (bottom panel). (B) Northern blot analyses of BMP4 (upper panel) and Alk3 (middle panel) expression in different cell types from testis at different ages of development (sptogonia=spermatogonia at 4 and 7 dpn; sptocytes=spermatocytes at 60 dpn; sptids=spermatids at 60 dpn), compared to actin expression (lower panel). (C) Northern blot analyses of BMPIIR expression (upper panel) within the different cell types of the testis, compared to actin expression (lower panel).

View larger version (76K): [in a new window]   Fig. 2. Alk3 is specifically expressed in PGCs and postnatal spermatogonia. Testes from 4 and 7 dpn mice were analyzed for Alk3 expression by immunofluorescence. (A) Representative images of Toluidine Blue stained frozen sections from 4 and 7 dpn testes (arrowheads point to spermatogonia within the tubules; 400x magnification). (B) Alk3 immunostainings in testes of the same ages as in (A) (arrowheads point to spermatogonia within the tubules; 400x magnification). (C) Alk3 immunostainings in isolated spermatogonia at 7 dpn (1000x magnification). Two classes of Alk3-positive cells are shown: strongly labeled cells with decondensed chromatin (arrowheads) and weakly labeled cells with more condensed chromatin. (D) Western blot analysis (20 µg of proteins) shows that 7 dpn spermatogonia, but not spermatocytes, express Alk3 protein. (E) Immunofluorescence analysis for Alk3 in frozen sections of genital ridges from a 10.5 dpc embryo: left panel shows alkaline phosphatase (APase) histochemistry to identify PGCs (200x magnification; the inset shows the area where the immunostainings have been performed); right panel shows Alk3 immunostaining only in PGCs (arrowheads) (400x magnification). Kit immunostainings in (B) and (E) were included to identify spermatogonia and PGCs, respectively (400x magnification).

View larger version (15K): [in a new window]   Fig. 3. BMP4 induces [3H]thymidine incorporation in postnatal spermatogonia. Proliferative response to BMP4 and Kitl in 4 and 7 dpn isolated spermatogonia. Mean of the fold increase of [3H]thymidine incorporating cells for at least four independent experiments. Bars represent the standard deviation. Asterisks indicate the presence of statistical significance (P<0.001).

View larger version (87K): [in a new window]   Fig. 4. Smads expression in postnatal testis and in PGCs. (A) Frozen sections from testes at 7 dpn were analyzed by immunofluorescence for the expression of Smad4 and Smad8, which shows ubiquitous immunostaining. (B) Frozen sections from testes at 4 and 7 dpn mice, (C) from 12.5 dpc fetal testis (200x magnification) and from 10.5 dpc genital ridges were analyzed for Smad5 expression, which shows a nuclear immunostaining pattern only in the germ cell compartment (arrowheads point to germ cells, within the seminiferous tubules or within the genital ridge; 400x magnifications).

View larger version (38K): [in a new window]   Fig. 5. Nuclear translocation of Smad4 upon BMP4 stimulation in postnatal spermatogonia. Immunofluorescence analysis for Smad4 in control and BMP4-treated spermatogonia isolated at 4 dpn with (A) Smad4, (B) Smad5, and (C) Smad8 (400x magnifications). The merged images show the nuclear localization of Smad4 and Smad5, but not of Smad8. (D) Western blot analysis for Smad4, Smad5 and Smad8 in nuclear and cytoplasmic extracts from control and BMP4-treated 7 dpn spermatogonia. Twenty µg of proteins were loaded in each lane.

View larger version (20K): [in a new window]   Fig. 6. BMP4 stimulation induces Smad4/Smad5/CBP association and DNA-binding activity in spermatogonia. (A) Eighty µg of total cell protein extracts from control or BMP4-treated spermatogonia were immunoprecipitated for Smad4 (lower panel) and immunoblotted for Smad5 and CBP (upper and middle panel, respectively). (B) DNA-binding activity in nuclear extracts from control and BMP4-treated spermatogonia probed with a 3xGCCG-[-32P]ATP double-stranded oligonucleotide, in the presence of 100x molar excess of competitors (wild-type or mutated 3xGCCG double-stranded oligonucleotides) or in the presence of an anti-Smad4 antibody. (C) DNA-binding activity in nuclear extracts from control and BMP4-treated spermatogonia probed with a 3xGCCG-[-32P]ATP double-stranded oligonucleotide, in the presence or absence of anti-Smad5 and anti-Smad8 antibodies.

View larger version (28K): [in a new window]   Fig. 7. BMP4 treatment induces Kitl sensitivity and Kit expression in Kit-negative and Kit-positive spermatogonia. (A) Mean of the fold increase of [3H]thymidine incorporating cells from three independent experiments. Asterisks indicate the presence of statistical significance (P<0.001). (B) Kit immunoprecipitation and in vitro autophosphorylation from 4 dpn spermatogonia total extracts, treated or untreated with BMP4 for 24 hours. (C) Northern blot analysis for c-kit expression in 7 dpn spermatogonia RNAs, treated or untreated with BMP4 for 24 hours.