Architecture of the nuclear periphery of rat pachytene spermatocytes: distribution of nuclear envelope proteins in relation to synaptonemal complex attachment sites

Abstract

The nucleus of spermatocytes provides during the first meiotic prophase an interesting model for investigating relationships of the nuclear envelope (NE) with components of the nuclear interior. During the pachytene stage, meiotic chromosomes are synapsed via synaptonemal complexes (SCs) and attached through both ends to the nuclear periphery. This association is dynamic because chromosomes move during the process of synapsis and desynapsis that takes place during meiotic prophase. The NE of spermatocytes possesses some peculiarities (e.g., lower stability than in somatic cells, expression of short meiosis-specific lamin isoforms called C2 and B3) that could be critically involved in this process. For better understanding of the association of chromosomes with the nuclear periphery, in the present study we have investigated the distribution of NE proteins in relation to SC attachment sites. A major outcome was the finding that lamin C2 is distributed in the form of discontinuous domains at the NE of spermatocytes and that SC attachment sites are embedded in these domains. Lamin C2 appears to form part of larger structures as suggested by cell fractionation experiments. According to these results, we propose that the C2-containing domains represent local reinforcements of the NE that are involved in the proper attachment of SCs.

Figure 1

Immunolocalization of pore complex protein p62 (A), lamin B1 (B), LAPs2 (C) as well as lamin C2 (D). Labeled rat pachytene spermatocytes were investigated by confocal laser scanning microscopy. Bar, 10 μm.

Figure 2

Double-label immunolocalization with antibodies against pore complex protein p62 and SC-protein SCP3 (A–A′), lamin B1 and SCP3 (B–B′), LAPs2 and SCP3 (C–C′), lamin C2 and SCP3 (D–D′), and C2 and LAPs2 (E–E′). Labeled rat pachytene spermatocytes were investigated by confocal laser scanning microscopy. Overlays are shown in A"–E". Some of the attachment sites of the SCs are denoted by arrows. Bar, 10 μm.

Figure 3

Double-label immunolocalization with antibodies against lamin C2 (A–C) and protein SCP3 (A′–C′). The labeled rat pachytene spermatocyte was investigated by confocal laser scanning microscopy. Three consecutive optical sections are shown (A–A′, B–B′, C–C′). Overlays are presented in A"–C". A‴–C‴, Schematic representation of the arcs displayed by SCs #1 (blue) and #2 (red). The arrowheads in C′–C" point at an XY body. Bar, 10 μm.

Figure 4

Electron microscopical immunolocalization of lamins B1 (A, B) and C2 (C–F) in pachytene spermatocytes of the rat using peroxidase-conjugated secondary antibodies. The patchy pattern obtained with C2 antibodies is denoted by brackets (C–F). Frontal (B, D) and lateral (F) views of SC attachment sites at the NE are shown. XY, XY body; Cy, cytoplasm. XY body axial elements (including an insertion at the NE) are denoted by arrows (E). Bars, 1 μm.

Figure 5

Two-dimensional PAGE of whole RV-SMC cells (A; 5 × 10⁶ cells) and pachytene spermatocytes of the rat (B; 5 × 10⁶ cells). After transfer to nitrocellulose, the proteins were incubated with mAb 13d4 against the LAPs2 α, β, and γ.

Figure 6

Two-dimensional PAGE of whole RV-SMC cells (A; 5 × 10⁶ cells) and pachytene spermatocytes of the rat (B, C; 5 × 10⁶ cell/gel). After transfer to nitrocellulose, the proteins were incubated with mAb PKB8 against lamins A, B1, and C (A and B) or mAb R27 against lamins A, C, and C2 (C).

Figure 7

Immunoblotting of NE proteins of RV-SMC cells (A–C; 2.5 × 10⁴ cell equivalents/lane) and pachytene spermatocytes of the rat (A′–C′; 2.5 × 10⁴ cell equivalents/lane) that were fractionated with buffers containing 1% Triton X-100 and 250 mM or 2 M salt. mAbs 13d4 (A–A′; against LAPs2 α, β, and γ), PKB8 (B–B′; against lamins A, B1, and C) as well as R27 (C–C′; against lamins A, C, and C2) were used to detect the respective antigens. S, Supernatant fraction; P, pellet fraction.