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First published online 16 February 2005
doi: 10.1242/dev.01658

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Ovol1 regulates meiotic pachytene progression during spermatogenesis by repressing Id2 expression

¹ Department of Biological Chemistry, University of California, Irvine, CA 92697, USA
² Developmental Biology Center, University of California, Irvine, CA 92697, USA
³ Department of Biomedical Science, Cornell University, Ithaca, NY 14852, USA

View larger version (82K): [in a new window]   Fig. 1. Ovol1 RNA expression in adult and prepubertal testis. (A-F) In situ hybridization of testis from 8-week-old (A-D) or 16-day-old (E,F) mice using a Ovol1 cRNA probe (A,C,E), or counterstained with an -Ldhc4 antibody (B,D,F). The following stages were identified according to cell sizes, positions within the tubule and relative intensities of -Ldhc4 staining (Hintz and Goldberg, 1977): p, pachytene spermatocytes; r, round spermatids; e, elongate spermatids. No hybridization signal was observed using a sense probe (not shown). (G) Northern blot analysis detecting multiple Ovol1 transcripts in testis during prepubertal development. Numbers at the top of each lane indicate the postnatal ages at which testis samples were taken. The same blots were then stripped and hybridized with a Gapdh probe. Scale bar: 25 µm in A-D; 30 µm in E-F.

View larger version (136K): [in a new window]   Fig. 2. Proliferation, morphology and apoptosis in juvenile Ovol1-/- testis. (A,B) BrdU labeling of testis from P14 wild-type (A) and Ovol1-/- (B) mice. Arrows indicate seminiferous tubules that contain multiple rows of BrdU-positive germ cells. Arrowheads indicate the labeled Leydig cells. (C-J) PAS staining of testis from control (wild-type or Ovol1+/-) (C,E,G) and Ovol1-/- (D,F,H-J) mice at P14, P16 and P21. E/MP, early/mid-pachytene spermatocytes; DP, diplotene spermatocytes. (I) High-magnification image of the boxed area in H. Black arrowheads in F,I,J indicate apoptotic cells intermingled with late-pachytene spermatocytes or more advanced germ cells. (K,L) TUNEL assays on wild-type (K) and mutant (L) testis from 21-day-old mice. Scale bar: 80 µm in A,B; 35 µm in C-H,J; 9 µm in I; 100 µm in K,L.

View larger version (100K): [in a new window]   Fig. 3. Accumulation of Gcna1+ germ cells and reduction of Ldhc4+/histone H1t+ germ cells in testis of juvenile Ovol1-/- mice. (A-D) Immunohistochemical detection of the Gcna1 protein in testis from Ovol1+/+ (A,C) and Ovol1-/- (B,D) mice at P14 and P16 using an -Gcna1 antibody. (E-L) Immunofluorescence staining of testis from Ovol1+/+ (E,G,I,K) and Ovol1-/- (F,H,J,L) mice at P14 and P16 using an -Ldhc4 antibody (E-H) and an -histone H1t antibody (I,J). (K,L) Merged images between G and I, and H and J, respectively. Scale bar: 55 µm in A-F; 60 µm in G-L.

View larger version (89K): [in a new window]   Fig. 4. Expression and localization of cyclin B1 protein in wild-type (A,C,D,F,G,I) and Ovol1-/- (B,E,H) testis. Shown are results of immunostaining experiments using -cyclin B1 (A-C) and -Ldhc4 (D-F) antibodies on P16 (A,B,D,E,G,H) and P21 (C,F,I) testis. (G-I) Merged images. Asterisk in G-I indicates mitotic spermatogonia at the tubule periphery that stained positive for cyclin B1. Arrows and arrowheads indicate cytoplasmic and nuclear cyclin B1 in primary spermatocytes, respectively. Scale bar: 25 µm.

View larger version (85K): [in a new window]   Fig. 5. Spermatocyte spread preparations from Ovol1-/- males showing normal cytological progression through prophase I (A) and normal accumulation of MLH1 and MLH3 at pachynema of prophase I (B). In the top panels (A), the accumulation of SCP3 (in green) along chromosomes begins in leptonema and continues through zygonema, at which time SCP1 (in red) begins to accumulate and results in homologous chromosome synapsis (double staining resulted in `yellow' chromosomes). By pachynema, all autosomes are fully synapsed, and the XY bivalent is synapsed only at the pseudoautosomal region. At diplonema, the central element of the synaptonemal complex breaks down and the SCP3-associated homologs move apart, but remain attached at sites of chiasmata (these sites are still associated with SCP1 at this time). In the bottom panels (B), the progression of recombination is associated with the accumulation of MLH1 (green in the last panel) and MLH3 (red) on the SCP3-positive (fainter green) chromosome cores in a manner identical to that in the wild-type control littermates (not shown). Colocalization of MLH1 and MLH3 is evident (as indicated by bright yellow spots along the chromosomes). In all panels, the telocentric centromere is indicated by CREST immunostaining (blue).

View larger version (81K): [in a new window]   Fig. 6. Altered gene expression in Ovol1-/- testis. (A) Reduced expression of known pachytene differentiation markers in P16 Ovol1-/- testis. (B) Northern blot analysis showing apparently normal levels of cyclin B1 and Cdc2 transcripts in P16 Ovol1-/- testis. (C,D) Novel pachytene or testis markers were downregulated in P16 Ovol1-/- testis (C) and showed temporal expression patterns similar to that of Ovol1 during wild-type prepubertal testis development (D). RNA samples were pooled from three to five mice of the same genotype and used to make multiple blots. All blots were stripped and reused. Only one representative hybridization experiment with a Gapdh probe was shown as a loading control. (E-H) In situ hybridization of P16 wild-type (E,F) and Ovol1-/- (G,H) testis using a Ovol2 cRNA probe (E,G) or counterstained with an -Ldhc4 antibody (F,H). Ovol2 transcripts are present in Ldhc4-postive cells. No signal was detected when a sense probe was used (not shown). Scale bar: 30 µm.

View larger version (43K): [in a new window]   Fig. 7. Id2 transcription is upregulated in Ovol1-deficient germ cells and is repressed by Ovol1 protein in vitro. (A) Increased levels of Id2 mRNA in P16 Ovol1-/- testis. (B) In situ hybridization of P16 wild-type (A',C') and Ovol1-/- (B',D') testis using an Id2 cRNA probe. (C' and D') High magnification images of individual tubules showing the strongest Id2 expression. Arrowheads and arrows indicate the Id2-expressing primary spermatocytes and the non-expressing spermatogonia, respectively. (C) Temporal expression of Id2 during normal prepubertal testis development. (D) Repression of Id2 promoter (Id2-P)-luciferase reporter expression by Ovol1. (E) Activation of Id2-P-luciferase expression by VP16-Ovol1. The triangles indicate increasing concentrations of expression vectors. The VP16 alone control is at a concentration corresponding to the highest one used for VP16-Ovol1. (F) Repression or activation is partially dependent on the CCGTTA sequence in Id2 promoter. Each bar represents the average of triplicate samples in a single experiment, and results are representative of several independent experiments. Luciferase activities are normalized for transfection efficiency by using a ß-actin promoter driving lacZ as an internal control.