LINC in Sperm Head Formation Figure 1. Sun3 is a novel member of the mammalian SUN protein family. (A) Schematic illustration of SUN domain proteins yet identified in mice. Relative positions of zinc finger (ZnF), hydrophobic regions (H1-H3), transmembrane domains (TM), coiled-coils (CC) and SUN domains are indicated. (B) Amino acid sequence of murine Sun3. Predicted structural motifs (TM, CC and SUN) are highlighted. The peptide sequence used for immunization is underlined. doi:10.1371/journal.pone.0012072.g001 day 12 p.p., when leptotene/zygotene spermatocytes are the most abundant cells within seminiferous tubules. To confirm our results, we investigated the expression profile of Sun3 during spermatogenesis at the protein level as well. For this purpose, we prepared whole testicular suspensions from testes of pubertal mice at corresponding time points. Immunoblotting revealed that Sun3 was first detectable at day 25 p.p. when spermatids are most frequent within seminiferous tubules (Figure 2D). For control, we again used Sycp3 and Cage1 as marker for either meiotic or postmeiotic expression, respectively [50]. Summarizing, our observations clearly evidence that testis- specific Sun3 is expressed exclusively in postmeiotic stages of male germ cell development. Sun3 defines a novel LINC complex that colocalizes with the manchette To study the distribution within the cells naturally expressing Sun3 (i.e. spermatids) we performed immunofluorescence micro- scopy on testis paraffin sections. In agreement with the previous results, somatic cells of the testis as well as spermatogonia and spermatocytes were negative for Sun3 (Figure 2E). In contrast, we could detect a strong Sun3-labeling at the NE of round and elongated spermatids (Figure 2E, Figure S1). Notably, Sun3 was not homogenously distributed within the nuclear periphery; it rather showed a highly polarized cap-like distribution. In double-label immunofluorescence experiments we compared the distribution of Sun3 with that of Lap2, another group of integral INM proteins [51] that gradually localize to the posterior pole of spermatid nuclei [43]. Both, Sun3 and Lap2 antibodies label the posterior pole of spermatid nuclei (Figure 3A,B). Similarly and consistent with previous findings, also lamin B3, a spermiogenesis-specific isoform of the Lmnb2 gene [42], can be detected like Sun3 solely at the posterior pole (Figure 3C,D). However, Sun3 and the acrosome-specific protein Cage1 [48] localized to opposite poles of spermatid nuclei (Figure 3E,F). Closer inspection of the spatial distribution of Sun3 and certain NE proteins revealed that the localization of Sun3 is unique and differs from that of the other proteins. Although Sun3 localizes like Lap2 and lamin B3 to the posterior pole, their local distribution within the NE shows remarkable differences; this implies that different NE proteins occupy distinguishable territories within the spermatid NE. At beginning of spermiogenesis lamin B3 and Lap2, both are distributed throughout the entire spermatid NE, but are locally enriched at the posterior half (Figure 3A,C). Sun3, by contrast, shows as early as cells are haploid complete posterior polarization, a distribution which is retained during complete spermiogenesis (Figure 2E, Figure 3). As sperm head formation progresses, lamin B3 and Lap2 gradually redistribute to con- gregate in a small region at the very posterior end of the nucleus which corresponds to the implantation fossa (i.e. the site where the flagellum anchors to the nucleus; Figure 3B,D). Although Sun3 colocalizes with lamin B3 and Lap2 in lateral regions of round spermatids, Sun3, however, remarkably appears completely excluded from the very posterior end of nuclei of round and PLoS ONE | www.plosone.org 3 August 2010 | Volume 5 | Issue 8 | e12072