Differential expression of Rfx1-4 during mouse spermatogenesis

The regulatory factor X (RFX) family of transcription factors has been recently implicated in gene regulation during spermatogenesis. However, the relative expression of individual members during this developmental process is not completely characterized, particularly in the case of Rfx4, which has multiple transcript variants in the testis. We used reverse transcriptase-dependent real-time PCR, 5'-RACE cloning, and Western blotting to compare transcripts and protein levels for this family in cell populations from the three major phases of spermatogenesis (mitotic, meiotic, and haploid). Transcripts for Rfx1-4 were present at trace to low levels in spermatogonia prepared from 8-day-old mice. Transcripts for both Rfx2 and Rfx4 were elevated in mid-late pachytene spermatocytes; however, the dominant Rfx4 transcript present begins at a downstream exon and lacks the DNA binding domain. Transcripts for all four genes were elevated in early haploid cells (round spermatids). In these cells Rfx4 transcripts originate primarily from a newly described promoter with intron 1 but are expected to be translationally compromised due to a poorly situated start codon. Western blotting confirmed that RFX2 is greatly elevated beginning in meiosis and also confirmed that full-length RFX4 protein is not prevalent in mouse testis at any stage. These results imply that RFX2 is the most likely X box binding factor to influence novel gene expression during meiosis, that RFX1-3 may all play roles in haploid cells but that RFX4 is much less prevalent than implied by its high transcript levels.