Bloom's syndrome protein, BLM, colocalizes with replication protein A in meiotic prophase nuclei of mammalian spermatocytes

Abstract

Bloom's syndrome (BS) is a rare autosomal recessive disorder of humans characterized by severe pre- and postnatal growth deficiency, immunodeficiency, genomic instability, and a predisposition to a wide variety of neoplasms. The genomic instability is evidenced in BS somatic cells as a high incidence of gaps and breaks, chromatid exchanges, chromosome rearrangements, and locus-specific mutations. BS arises from a mutation in BLM, a gene encoding a protein with homology to the RecQ helicase family. Men with BS are sterile; women have reduced fertility and a shortened reproductive span. The current immunocytological study on mouse spermatocytes shows that the BLM protein is first evident as discrete foci along the synaptonemal complexes (SCs) of homologously synapsed autosomal bivalents in late zygonema of meiotic prophase. BLM foci progressively dissociate from the synapsed autosomal axes during early pachynema and are no longer seen in mid-pachynema. BLM colocalizes with the single-stranded DNA binding replication protein A, which has been shown to be involved in meiotic synapsis. However, there is a temporal delay in the appearance of BLM protein along the SCs relative to replication protein A, suggesting that BLM is required for a late step in processing of a subset of genomic DNA involved in establishment of interhomologue interactions in early meiotic prophase. In late pachynema and into diplonema, BLM is more dispersed in the nucleoplasm, especially over the chromatin most intimately associated with the SCs, suggesting a possible involvement of BLM in resolution of interlocks in preparation for homologous chromosome disjunction during anaphase I.

Figure 1

Comparison of the distribution of BLM (Left, red) and RPA (Right, green) at different times during meiotic prophase in mouse spermatocytes. The axial elements are stained with SCP3 (white). (A and B) Zygonema. Only some synapsed SCs in zygonema have BLM foci (A), whereas all synapsed SCs have RPA foci (B). (C and D) Early pachynema. SCs have multiple BLM foci (C) and RPA foci (D) along their length. (E and F) Mid-pachynema. Most BLM foci have disappeared from the SCs, and a more diffuse chromatin reaction is beginning to increase throughout the nucleoplasm. The build-up is most evident over the sex body (intense red area at 11 o’clock) and around the SCs (E). At a similar stage, the majority of RPA foci have disappeared (F).

Figure 2

Colocalization of RPA (green) and BLM (red) on prophase spermatocytes. Colocalization produces a yellow signal. (A–C) Mid-zygotene nucleus with RPA (A), BLM (B), and RPA/BLM (yellow). Note that most of the foci in C are green, indicating that BLM is not yet present. (D–F) Early pachytene nucleus with RPA (D), BLM (E), and RPA/BLM (F). Note that most foci are yellow, indicating near perfect colocalization at this stage.

Figure 3

Diagram showing the change in RPA and BLM localization on a bivalent as it progresses from early to late zygonema and into early pachynema.