A new meiosis-specific cohesin complex implicated in the cohesin code for homologous pairing

Abstract

We identify a new mammalian cohesin subunit, RAD21-like protein (RAD21L), with sequence similarity to RAD21 and REC8. RAD21L localizes along axial elements in early meiotic prophase, in a manner that is spatiotemporally different to either REC8 or RAD21. Remarkably, RAD21L and REC8 have symmetrical, mutually exclusive localization on the not-yet-synapsed homologues, implying that the cohesin patterning could provide a code for homologue recognition. RAD21 transiently localizes to axial elements after the dissociation of RAD21L and REC8 in late pachytene, a period of recombination repair. Further, we show that the removal of cohesins and synaptonemal complex during late meiotic prophase is promoted by Polo-like kinase 1, which is similar to the mitotic prophase pathway.

Figure 1

Identification of a new cohesin subunit RAD21L. (A) Amino-acid sequence alignment of mouse RAD21L, RAD21 and REC8. The RAD21L sequence has been submitted to the DDBJ, EMBL and GenBank databases under accession number AB602048. (B) Schematic showing the amino-acid similarity and identity of RAD21L compared with REC8 and RAD21. (C) Tissue specificity of mRNA expression for the indicated genes was analysed by RT–PCR. (D) Immunoprecipitates from spermatocyte extracts obtained using RAD21L, REC8 and RAD21 antibodies or control immunoglobulin G (IgG) were analysed by western blotting using antibodies against the indicated proteins. Input: 1 and 0.1% of whole soluble extract. E, embryonic day; IgG, immunoglobulin G; P, postnatal; RT–PCR, reverse transcription PCR; RT(–), control PCR; W, week.

Figure 2

RAD21L cohesin localizes along axial elements in both spermatocytes and oocytes during meiosis. (A) Chromosome spreads of spermatocytes were stained with RAD21L and SCP3 antibodies. Arrow: RAD21L aggregates. Arrowhead: XY body. NIH3T3 mitotic cells were stained with RAD21L and CENP-C antibodies. (B) Chromosome spreads of fetal oocytes were stained as shown in (A). Oocytes at 2 h (Prometaphase I) and 6 h (Metaphase I) after GVBD were stained with RAD21L and CENP-C antibodies as indicated. Scale bars, 5 μm. CENP-C, centromere protein; GVBD, germinal vesicle breakdown.

Figure 3

Comparative localization of RAD21L and REC8 cohesins during meiosis. (A) Chromosome spreads of spermatocytes were stained with RAD21L and REC8 antibodies. Arrow: RAD21L aggregates. Arrowhead: XY body. (B,C) Immunostaining of chromosome spreads of (B) pachytene spermatocytes and (C) pachytene fetal oocytes. Magnified images are shown on the right. Line scans of green/red signals along paired chromosomes (dashed arrow) were performed and the plots are shown (bottom right). (D) Immunostaining of chromosome spreads of spermatocytes at zygotene/pachytene transition with RAD21L and SCP1, a component of central element of synaptonemal complex, antibodies. Magnified images of the two not-yet-synapsed homologous chromosomes are shown on the right. (E, F) Immunostaining of chromosome spreads of (E) spermatocytes and (F) fetal oocytes at zygotene/pachytene transition with RAD21L and REC8 antibodies. Magnified images of the two not-yet-synapsed homologous chromosomes are shown on the right. Scale bars, 5 μm.

Figure 4

RAD21 cohesin transiently localizes on axial element during late meiotic prophase. (A) Chromosome spreads of spermatocytes were stained with RAD21L and RAD21 antibodies. Metaphase I spermatocytes (far right) were stained with CENP-C and RAD21 antibodies. Arrow: RAD21L aggregates. Arrowhead: XY body. (B) Late pachytene spermatocytes were stained with RAD21L and RAD21 antibodies or (C) REC8 and RAD21 antibodies. Magnified images are shown on the right. (D) Chromosome spreads of diakinesis/metaphase I cells with or without GW843682X (GW) were stained with SCP3 and CENP-U pT78 antibodies. The staining patterns were classified into two categories and quantified (n=32, 39 for cells with and without GW, respectively). (E) Chromosome spreads of diakinesis/metaphase I cells with or without GW were stained with SCP3 and REC8 or RAD21L antibodies. (F) Schematic illustration of the localization and dissociation of RAD21L, REC8 and RAD21 cohesins along the axial element during meiotic prophase. Scale bars, 5 μm. PLK1, Polo-like kinase 1.