LINC in Sperm Head Formation Figure 2. Sun3 is a testis specific protein showing a postmeiotic expression profile. (A) Total RNA was isolated from different tissues and used for RT-PCR. cDNA was amplified using Sun3-specific primers and a GAPDH-PCR served as control for RNA fidelity. (B) Samples from the same tissues were homogenized in SDS sample buffer and separated by SDS-PAGE (56105 cells; 30 mg protein/lane). Sun3 protein was detected with an affinity-purified anti-Sun3 antiserum (Arrow). (C) Total RNA was isolated from testicular cells from pubertal mice of increasing ages (8-25 days p.p.) and used for RT-PCR. cDNA was amplified using primers for Sun3, Cage1 (postmeiotic expression) and Sycp3 (meiotic expression). (D) To analyze stage-specific expression of protein Sun3 testes from pubertal mice were homogenized in SDS sample buffer and separated by SDS-PAGE (46105 cells/lane). Sun3 protein was detected with affinity-purified anti-Sun3 antiserum. For comparison pAbs against Lamin B3 and Cage1 were used. (E) Localization of Sun3 within seminiferous tubules was analyzed by indirect immunofluorescence microscopy. Testis paraffin sections of adult mice were stained using affinity-purified anti-Sun3 antiserum (green) and mAb 13d4 against LAP2 (red). DNA was labeled with 33258-Hoechst (blue). Sun3 was only detectable in spermatids (Sp) but completely absent from somatic cells (S), spermatogonia (Sg) and spermatocytes (Sc). Scale bar: 15 mm. doi:10.1371/journal.pone.0012072.g002 elongated stages. Instead, Sun3 covers only lateral regions excluding the implantation fossa during entire sperm head elongation (Figure 3, Figure S2). Absence of Sun3 at the very posterior pole could be confirmed by double-label immunofluorescences with antibodies to a- or b-tubulin. Both, a- and b-tubulin are the major structural components of the spermatid-specific manchette, a bundle of cytoplasmic microtubules which covers the posterior part of the nucleus throughout spermiogenesis [52] (see below). As shown in Figure 3G,H, Sun3 exclusively localizes to regions where microtubule bundles contact the NE. This fact is quite remarkable since it suggests a coherency between manchette formation and posterior localization of Sun3 and, moreover, provides a first hint for a putative involvement of Sun3 in nucleo-cytoskeletal linkage. Following up this issue, we next asked whether Sun3 is part of a, thence, spermiogenesis-specific LINC complex. To this end, we PLoS ONE | www.plosone.org 4 August 2010 | Volume 5 | Issue 8 | e12072