Abstract
BackgroundMammalian spermatogenesis involves formation of haploid cells from the male germline and then a complex morphological transformation to generate motile sperm. Focusing on meiotic prophase, some tissue-specific transcription factors are known (A-MYB) or suspected (RFX2) to play important roles in modulating gene expression in pachytene spermatocytes. The current work was initiated to identify both downstream and upstream regulatory connections for Rfx2.ResultsSearches of pachytene up-regulated genes identified high affinity RFX binding sites (X boxes) in promoter regions of several new genes: Adam5, Pdcl2, and Spag6. We confirmed a strong promoter-region X-box for Alf, a germ cell-specific variant of general transcription factor TFIIA. Using Alf as an example of a target gene, we showed that its promoter is stimulated by RFX2 in transfected cells and used ChIP analysis to show that the promoter is occupied by RFX2 in vivo. Turning to upstream regulation of the Rfx2 promoter, we identified a cluster of three binding sites (MBS) for the MYB family of transcription factors. Because testis is one of the few sites of A-myb expression, and because spermatogenesis arrests in pachytene in A-myb knockout mice, the MBS cluster implicates Rfx2 as an A-myb target. Electrophoretic gel-shift, ChIP, and co-transfection assays all support a role for these MYB sites in Rfx2 expression. Further, Rfx2 expression was virtually eliminated in A-myb knockout testes. Immunohistology on testis sections showed that A-MYB expression is up-regulated only after pachytene spermatocytes have clearly moved away from the tubule wall, which correlates with onset of RFX2 expression, whereas B-MYB expression, by contrast, is prevalent only in earlier spermatocytes and spermatogonia.ConclusionWith an expanding list of likely target genes, RFX2 is potentially an important transcriptional regulator in pachytene spermatocytes. Rfx2 itself is a good candidate to be regulated by A-MYB, which is essential for meiotic progression. If Alf is a genuine RFX2 target, then A-myb, Rfx2, and Alf may form part of a transcriptional network that is vital for completion of meiosis and preparation for post-meiotic differentiation.
Highlights
Mammalian spermatogenesis involves formation of haploid cells from the male germline and a complex morphological transformation to generate motile sperm
We identified high quality X box motif matches in promoter regions for phosducin-like 2 (Pdcl2), sperm antigen 6 (Spag6) and a disintegrin and metalloproteinase 5 (Adam5) (Fig. 1)
In the first part of this work we identified three new potential Regulatory factor X 2 (RFX2) target genes that are sharply up-regulated during pachytene, a point in meiosis at which the fundamental events of chromosome pairing and the initiation of recombination events have already occurred, and early expression of genes that will eventually generate the distinctive features of a sperm has begun
Summary
Mammalian spermatogenesis involves formation of haploid cells from the male germline and a complex morphological transformation to generate motile sperm. Spermatogenesis encompasses the complex differentiation that converts cells of the male germline into haploid, motile spermatozoa. Germ cells become spermatocytes as they enter meiosis, which will generate haploid progeny. The renewal of the germinal stem cells, the proliferation of differentiating spermatogonia, and the commitment to enter meiosis are controlled by extracellular signaling molecules provided by somatic cells of the testis [see [1,2,3] for recent reviews]. While initiation and completion of meiosis in the testis normally depend on external cues and perhaps the physiological environment of the tubule, much of male meiosis is undoubtedly controlled by an intrinsic genetic program
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