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. 2010 Jul 20;30(18):4391–4403. doi: 10.1128/MCB.00002-10

FIG. 2.

FIG. 2.

In wild-type mouse spermatocytes Spo11 transcripts peak past the stage in prophase in which meiotic DSBs are introduced. (A) Total Spo11 transcripts quantified in juvenile mice peak in late prophase I. Total Spo11 transcripts were quantified in testes of juvenile mice at 1 to 18 dpp. Total RNA was reverse transcribed and subjected to quantitative PCR (qPCR) using a TaqMan assay targeting the exon 11-12 boundary. Data were normalized to the 18-dpp sample. The scheme below the x axis indicates the predominant cell type corresponding to each age in wild-type juvenile mice, according to reference 15. Most prophase I mutants are arrested at stage IV (corresponding to early pachytene spermatocytes); therefore, their cell type composition would be comparable to that of a 13-dpp wild-type pup, whereas mutants arrested in metaphase I contain spermatocytes in all stages of prophase I. MR, meiotic replication; L, leptonema; Z, zygonema; EP, early pachynema; LP, late pachynema; D, diplonema. (B) Mutants arrested in prophase I display a 10-fold reduction in total Spo11 transcripts. Total Spo11 transcripts were quantified by reverse transcription-qPCR (RT-qPCR) in testes of several mutants arrested in prophase I. The ΔSpo11 strain was used as a negative control. Transcript levels for each homozygous mutant were normalized to those of its wild-type (wt) littermate. (C) Mutants arrested in metaphase I do not display the drastic reduction in total Spo11 transcript levels observed for prophase I mutants. Total Spo11 transcript levels in the testes of Mlh1−/− mutant mice were compared to those of a wild-type littermate. (D) The low levels of total Spo11 transcripts detected in mutants arrested in prophase I are not a reflection of lower overall transcription levels due to the arrest. Cot-1 DNA consists of repetitive elements ubiquitously represented throughout the genome that can be used to detect nascent RNA transcripts in the nucleus by RNA FISH. The intensity of the nuclear Cot-1 signal correlates to the rate of transcription. Combined Cot-1 RNA FISH and SCP3 immunostaining were performed on wild-type and mutant spermatocyte spreads to assess overall transcription levels in leptotene and more advanced spermatocytes. Spermatocytes were classified as early (a, c, e, and g) or pachytene-like (b, d, f, and h) according to the SCP3 staining pattern.