directly involved in dynamic processes concerning anchorage and migration of nuclei (reviewed in [3]) but also positioning and movement of nuclear structures (i.e. meiotic telomeres) [23–24]. Mutations in either SUN or KASH proteins that result in defective LINC complex assembly lead to severe failures in nuclear migration, anchorage and organization [24–29]. Moreover, LINC complexes have been proposed to play a role in nuclear deformation and shaping [30–32]. Nuclear restructuring is very pronounced during spermiogen- esis, a highly complex process which ensures the differentiation from haploid male germ cells into mature, fertilization competent spermatozoa. A most prominent feature in this process is the shaping of the sperm nucleus from spherical to elongated [33]. Nuclear remodeling during sperm head formation requires an elaborate cooperation of different cellular mechanisms that involve assembly of sperm-specific cytoskeletal structures, nuclear move- ment and chromatin compaction [34–38]. Thereby the initially round cell nuclei reshape to elongate. Nuclear elongation, however, is well-directed and leads to striking polarization concerning nuclear and cellular shape [39,40]. Noteworthy, failures in sperm head shaping and formation in effect is a major cause of male infertility [41]. Recent studies indicate that elongation of the sperm nucleus involves an extensive modulation of the NE. This, in particular, concerns its general composition as well as the behavior of the single components. A central characteristic during sperm head elongation is the remarkable redistribution and polarization of a variety of NE components (e.g. lamins B1 and B3, Lap2 and lamin B receptor) [42–44]. At present, the functional significance of this redistribution is unclear. However, it was speculated that NE remodeling contributes to sperm-specific nuclear shaping and chromatin reorganization [43–45]. In a variety of cell types SUN and KASH proteins function as nucleo-cytoplasmic linkers that directly contribute to nuclear positioning and shaping. Therefore, LINC complexes are good candidates for playing a key role in nuclear deformation and elongation that is overt during mammalian sperm differentia- tion. In the present study, we investigated the expression of LINC complex components in the male postmeiotic germ line. We describe Sun3 as a novel spermiogenesis-specific member of the SUN protein family. In addition, we could find Sun1 showing an exceptional behavior in postmeiotic cells. We further were able to identify a novel testis-specific, non-nuclear Sun1 isoform which is exclusively expressed in spermatids and mature spermatozoa. Remarkably, analyzing the subcellular localization of SUN proteins and nesprins in differentiating spermatid nuclei we detected two specifically polarizing novel LINC complexes consisting of differentially interacting SUN and KASH pairs. Thus, we propose a model for a well-directed sperm head formation that involves the presence of two spermiogenesis-specific LINC complexes at opposite poles of the sperm head. Results Murine Sunc1 codes for Sun3, a short testis-specific member of the SUN domain protein family Previous studies described a short human SUN domain protein, SUN3, which was suggested to be expressed mainly in the testis [4,5,12]. However, up to the present the actual expression pattern as well as its subcellular distribution within the cells naturally expressing SUN3 is not known. To close this gap, we investigated SUN3 expression and localization in detail. LINC in Sperm Head Formation To identify the murine homolog of human SUN3, we per- formed a TBLASTN search using human SUN3 peptide sequence (gb|AAH26189) as probe. This led to the identification of a cDNA derived from adult testis (gb|AK132922) encoded by the murine Sunc1 gene (Sad1 and UNC84 domain containing 1; Gene ID: 194974). To isolate full-length murine Sun3 cDNA, we used an oligonu- cleotide corresponding to the 5’-untranslated region adjacent to the putative start codon and a 3’-oligonucleotide complementary to the end of the coding region. Using total testis RNA as template for RT-PCR, we were able to amplify a 985 bp cDNA with an open reading frame of 963 bp. The corresponding protein consists of 320 amino acid residues (Figure 1A) with a calculated molecular weight of approx. 36,800 and a pI of about 7.67. On the amino acid level murine Sun3 shows 71% identity (78% similarity) to the previously described putative human SUN3 [4]. PSORTII and SMART prediction of protein structural motifs revealed that Sun3 protein comprises distinct N- and C- terminal regions (Figure 1B). A short N- and a long C-terminal domain are separated by one predicted TM region thus characterizing Sun3 as a type II transmembrane protein, similar to other SUN proteins [12]. The presumptive lumenal C-terminus contains one putative coiled-coil region and a distal SUN domain representing the region of homology with the mammalian paralogues Sun1 and Sun2 (Figure 1B). With the exception of the SUN domain, Sun3 does not share any homology with other mammalian proteins. Thus, Sun3 is a typical member of the SUN protein family consistent with the basic protein conformation representative for SUN domain proteins. According to database entries the Sunc1 gene appears mainly expressed in the testis as indicated by high representation as testis ESTs. To confirm the presumptive expression of Sun3 we first performed sensitive RT-PCR analysis on a broad range of different mouse tissues. Using sequence specific primers we could substantiate presence of Sun3 mRNA in the testis (Figure 2A). In contrast, we could not amplify Sun3 in any other somatic tissue or in the ovarian tissue sample. To verify tissue-specificity of Sun3 protein we performed western-blot analysis using an affinity- purified Sun3-antibody raised against a Sun3-specific peptide (Figure 1). The antibody recognized only one single protein band with the expected molecular mass of 37 kDa in the testis (Figure 2B). A corresponding signal, however, was clearly absent in all other tissues tested. Sun3 expression correlates with sperm head formation We next performed a detailed analysis of Sun3 expression during spermatogenesis. Therefore, we used whole RNA fractions of 8- to 25- day-old mice that cover the first wave of spermatogenesis and consequently allow for exact timing of Sun3 appearance [42,46,47]. In 8- and 12-day old animals when testes only contain spermatogonia and spermatocytes, Sun3 message was not detectable (Figure 2C). However, with the appearance of first postmeiotic stages at days 15 and 18 postpartum (p.p.) the Sun3 message could be detected as a faint signal. As spermatogenesis progresses round and elongated spermatids begin to accumulate (i.e. days 21 and 25 p.p.) and, concomitantly, the Sun3 signal became stronger. For control, we performed RT-PCR analysis using primers for spermiogenesis- specific protein Cage1 (a component of the acrosome [48]) and meiotic protein Sycp3 (a structural component of the synaptone- mal complex [49]). As expected for an acrosomal protein, Cage1 message first appeared in testes at day 15 p.p. with an increasing expression level thereafter which resembles the expression pattern of Sun3. By contrast, the Sycp3 message was detectable as early as PLoS ONE | www.plosone.org 2 August 2010 | Volume 5 | Issue 8 | e12072