Meiotic cohesin REC8 marks the axial elements of rat synaptonemal complexes before cohesins SMC1beta and SMC3

Abstract

In meiotic prophase, the sister chromatids of each chromosome develop a common axial element (AE) that is integrated into the synaptonemal complex (SC). We analyzed the incorporation of sister chromatid cohesion proteins (cohesins) and other AE components into AEs. Meiotic cohesin REC8 appeared shortly before premeiotic S phase in the nucleus and formed AE-like structures (REC8-AEs) from premeiotic S phase on. Subsequently, meiotic cohesin SMC1beta, cohesin SMC3, and AE proteins SCP2 and SCP3 formed dots along REC8-AEs, which extended and fused until they lined REC8-AEs along their length. In metaphase I, SMC1beta, SMC3, SCP2, and SCP3 disappeared from the chromosome arms and accumulated around the centromeres, where they stayed until anaphase II. In striking contrast, REC8 persisted along the chromosome arms until anaphase I and near the centromeres until anaphase II. We propose that REC8 provides a basis for AE formation and that the first steps in AE assembly do not require SMC1beta, SMC3, SCP2, and SCP3. Furthermore, SMC1beta, SMC3, SCP2, and SCP3 cannot provide arm cohesion during metaphase I. We propose that REC8 then provides cohesion. RAD51 and/or DMC1 coimmunoprecipitates with REC8, suggesting that REC8 may also provide a basis for assembly of recombination complexes.

Figure 1.

The 14 successive cell associations (numbered I–XIV) in testicular tubules of the rat. Each column represents one cell association, with the cell types found in that association. A, In, and B, A-type, intermediate, and B-type spermatogonia; PL, L, Z, P, and Di, preleptotene, leptotene, zygotene, pachytene and diplotene spermatocytes; MDs, meiotic divisions; 1–19, successive differentiation stages of spermatids; 19*, mature spermatozoa. The top of the figure shows the life span of each cell association in Wistar rats (Hilscher and Hilscher, 1969). The horizontal lines indicate when various proteins appear and when they disappear. For RAD50 and MRE11, the continuous line indicates when these proteins are abundant throughout the nuclei, whereas the broken line shows in which stages of meiosis these proteins are only abundant in sex vesicles (XY bivalents) (Eijpe et al., 2000b). REC8 is present until anaphase II and reappears in spermatids.

Figure 2.

Western blot analyses using anti-REC8 antibodies. (A) Peptides of mouse REC8 that were used for immunization. (B) Binding of MαSMC3 and affinity-purified antibodies from RαN, RαC, ChαN, or RαSMC3 (see Materials and methods) to immunoblot strips carrying proteins from purified SCs (s), spermatocyte nuclei (c), or liver nuclei (l). (C) Dephosphorylation of REC8; isolated SCs were treated with AP (right panel), AP and a specific inhibitor of this enzyme (middle panel), or AP buffer only (left panel) and analyzed by Western blot. Strips of the resulting blot were stained with Ponceau S (a) and probed with the affinity-purified anti-REC8 antibodies RαN (b), RαC (c), or ChαN (d). (D) Abundance of various proteins in premeiotic S phase/preleptotene (p) or midprophase (= pachytene and diplotene) (m), analyzed by Western blot, using antibodies recognizing SCP2 (serum 493), RαSMC3, SMC1β (Mab 76), REC8 (RαN), RAD50, or SCP3 (serum A1). MW, molecular weight markers.

Figure 3.

Appearance of cohesins in premeiotic S-phase cells. Immunofluorescence double labeling of frozen sections of rat testis with anti-BrdU and an antibody against a cohesin. The Roman numerals refer to the cell associations present in the tubules (Fig. 1). The three panels in each row represent the same section. We have indicated in the upper right corners what the different colors represent. SMC3 was detected by RαSMC3. g(a), spermatogonium type A; lp, late pachytene; pl, preleptotene; pl(S), preleptotene (premeiotic S phase); t, spermatid; sz, spermatozoa. Bars: (A) 50 μm; (D, G, and J) 25 μm.

Figure 4.

Appearance of AE components and RAD51 in premeiotic S-phase cells. Immunofluorescence double and triple labeling of frozen sections of rat testis. The Roman numerals refer to the cell associations (Fig. 1). The three panels in each row represent the same section. We have indicated in the upper right corners what the different colors represent. In D–F, we detected REC8 using RαC, which recognizes the COOH terminus of REC8 (REC8 C); in panels A–D, we used RαN. di, diplotene; g(a), spermatogonium type A; lp, late pachytene; pl, preleptotene; pl(S), preleptotene (premeiotic S phase); t, spermatid; z, zygotene. Bars: (A and J) 50 μm; (D, G, and M) 25 μm.

Figure 5.

REC8 in early spermatocytes. Spread early rat spermatocytes were labeled with anti-REC8 (RαN) in combination with (A–D) anti-SCP3 (II52F10), (E–H) anti-SMC3 (MαSMC3), (I–L) anti-BrdU, and (M–P) monoclonal anti-RAD51/DMC1. The cells in I–L originate from a BrdU-treated rat (see Materials and methods). In B, J, and K, some of the first short stretches of REC8-AEs have been enlarged. They appear already during premeiotic S phase (J and K). Arrows in D point at sites where SCP3 has a broader distribution along AEs than REC8. Arrow in L points at BrdU incorporation along an AE in early zygotene. Bars, 10 μm.

Figure 6.

REC8 in spread spermatocytes in pachtytene to metaphase I. We detected REC8 using affinity-purified antibodies from RαN, except in panel A, where we used affinity-purified antibodies from ChαN. (A and B) Pachytene nuclei (XY, XY bivalent). (C–L) Diplotene SCs. (F–L) Diplotene SCs with bridges between desynapsed AEs (the bridges contain SCP3, but not REC8). (K and L) Cdk2 marks the position of these bridges on the AEs. (M and N) Diakinesis; the boxed bivalent is enlarged in O–Q to show that SCP3 and REC8 do not colocalize precisely anymore. (R and S) Metaphase I bivalents labeled with RαSMC3 and anti-SCP3 (II52F10). (X and Y) Metaphase I cell; the boxed bivalent is shown in detail in Z–AB to show that SCP3 is absent from the chromosome arms. (AC–AE) Bivalent from another metaphase I cell to show absence of SCP3 and SMC1β, but not REC8, from the chromosome arms. Bars: (A–C, M, and X) 10 μm; (all other panels) 2 μm.

Figure 7.

REC8 in spread metaphase I/anaphase I to anaphase II spermatocytes. For all panels, we used affinity-purified rabbit anti-REC8 antibodies (RαN). (A and B) Cell at the metaphase I to anaphase I transition; the homologues of at least one bivalent (boxed) have separated; this bivalent is shown in detail in C–E. (F–H) Detail of the centromeric region of a chromosome from another anaphase I cell (for an interpretation, see Fig. 9). (I and J) Anaphase II cell. (K–M) Centromeric region of one of the chromosomes in a metaphase II cell (for an interpretation, see Fig. 9). Bars: (A and I) 10 μm; (all other panels) 2 μm.

Figure 8.

Coimmunoprecipitation of proteins with REC8. (A) Coimmunoprecipitation of RAD50 and RAD51/DMC1 with REC8. REC8 was precipitated from spermatocyte extract by affinity-purified RαN antibodies, and the immunoprecipitate, in parallel with various controls, was analyzed by immunoblotting. Strips of the blots were probed with blocking buffer (−; negative control) and affinity-purified antibodies recognizing REC8 (RαN; positive control for the immunoprecipitation), RAD50, or RAD51/DMC1 (serum 2308). The strips in each subpanel carry (a) control immunoprecipitate (obtained without anti-REC8 [RαN] antibodies), (b) the supernatant of the control immunoprecipitate, (c) the REC8 immunoprecipitate, (d) supernatant of the REC8 immunoprecipitate, (e) purified SCs, or (f) spermatocyte lysate. (B) Coimmunoprecipitation of REC8 with RAD51/DMC1. We precipitated RAD51/DMC1 from spermatocyte extract using affinity-purified antibodies from serum 2308 and analyzed the immunoprecipitate on Western blots, using affinity-purified anti-REC8 antibodies (RαN). For comparison, we precipitated in parallel REC8 from the same spermatocyte extract using RαN. In each subpanel, the strips carry (a) the unbound fraction of the spermatocyte lysate, (b) the last wash of the immunoprecipitate, and (c) the RAD51/DMC1 or REC8 immunoprecipitate. Strip d carries synaptonemal complex proteins, analyzed in parallel with the immunoprecipitates, and strip e carries proteins of the spermatocyte extract that was used for immunoprecipitation. (C) No detectable coimmunoprecipitation of SCP2 and SCP3 with REC8. The strips in each subpanel carry (a) control immunoprecipitate (obtained without RαN or RαC), (b) the supernatant of the control immunoprecipitate, (c) immunoprecipitate obtained with RαN, (d) supernatant of the RαN immunoprecipitate, (e) immunoprecipitate obtained with RαC, (f) supernatant of the RαC immunoprecipitate, (g) proteins of purified SCs, and (h) (left subpanel only) spermatocyte lysate. The position of various proteins on the blots is indicated to the left of the strips. SCP3 dm indicates the position of SCP3 dimers on the strips (Lammers et al., 1994). The fuzzy bands indicated by Ab result from reaction of the goat anti–rabbit IgG (conjugated to AP) with monomers or dimers of the heavy chain of the rabbit anti-REC8 antibodies that were used for immunoprecipitation. MW, molecular weight markers.

Figure 9.

Interpretative drawing showing the localization of REC8, SMC1β, SMC3, SCP2, and SCP3 along AEs and chromosomes throughout meiosis. Diffusely distributed protein has not been indicated.