immunofluorescence microscopy reveal a coherency between the manchette and the posterior Sun3/Nesprin1 polarization. Inter- estingly, Russell and collegues [34] previously reported that rod- like elements of constant length were linking the innermost microtubules of the manchette to the outer surface of the NE and, even more, to the inner leaflet of the NE across the PNS. Furthermore, they and also others [38,52] share the opinion that these links between the manchette and the NE provide a structural basis for the transmission of forces to elongate nuclei. Sun3/ Nesprin1 LINC complexes could finely reflect these NE- manchette-connecting filaments. Moreover, Nesprin1 has poten- tial binding ability to KIF3B, a subunit of kinesin II [57], and the dynein/dynactin complex [58]. Therefore its interaction with manchette microtubules via kinesin II and/or dynein/dynactin would be conceivable (Figure 7). However, Kierszenbaum and Tres [35] demonstrated that F-actin, to which Nesprin1 is rather related, is also present in the manchette. Therefore, actin could be the primary cytoskeletal element to which Sun3/Nesprin1 LINC complexes are connected as well. The Sun1g/Nesprin3 LINC complex at the anterior sperm head pole Though Sun1/Nesprin3 initially polarize to the posterior pole, a distinct proportion is also found at the anterior pole in early round spermatids. With progressing differentiation anterior localization becomes more and more prominent while the posterior proportion disappears. Most striking, at the anterior pole Sun1/Nesprin3 does not localize to the NE but appears outside the nucleus likely being part of the acrosomal membrane system. This behavior, however, is quite remarkable since Sun1 as an INM protein has never been observed before to localize purposefully outside the NE. What might be the reason and the functional significance of this exceptional Sun1 localization? As shown in our study, the appearance of the non-nuclear LINC complex closely correlates with the expression of a new, spermiogenesis-specific Sun1 isoform, Sun1g, which lacks exons seven to ten. Loss of these exons, however, implies that Sun1g does not contain hydrophobic sequences H1 and H2 in part. Recently, Liu and colleges [12] reported that these hydrophobic sequences are required for INM targeting and nuclear retention. Since the NE is profoundly restructured during spermiogenesis including redistribution and disappearance of certain components (see above) it is plausible that the long Sun1 variant is initially recruited to the posterior pole but successively disappears in the course of differentiation. Sun1g however, due to absence of N-terminal sequences required for efficient INM retention [12] could fail to associate with nuclear interaction partners and, as a consequence, relocates to the non- nuclear acrosomal membrane system. Accordingly, by direct interaction of their respective SUN or KASH domains (this study and [5]), Sun1g has the competence to recruit Nesprin3 to form a functional, however non-nuclear LINC complex at the anterior pole of the sperm head. In somatic cells Nesprin3 apparently links the nucleus to the intermediate filament system via its interaction with plectin [18,21], a cytoskeletal cross-inker protein which is suggested to influence cytoskeletal organization and mechanical strengthening of cells [59–61]. Plectin for its part is further able to interact with actin [62] which in spermatids is enriched within the acroplaxome and at specialized membranous junctions of spermatids and Sertoli cells at the anterior sperm surface [35,63,64]. Notably, both anterior occurring structures appear, like the posterior manchette to be involved in shaping of the sperm head. Interestingly, plectin was also reported to be concentrated at those junctions between Sertoli cells and elongating spermatids potentially linking to LINC in Sperm Head Formation adjacent actin filaments [65,66]. For that reason, we propose a model in which Sun1g/Nesprin3 LINC complexes anchor via plectin to these actin containing structures (Figure 7). This linkage in turn would provide a structural basis for the location of the Sun1g/Nesprin3 LINC complex to the anterior pole and, in addition, via closely connected NE-acrosomal-plasma membrane systems to the outer surface of the acrosome (Figure 7). In this way spermatid nuclei could become tethered to anterior cytoskeletal structures by the spermiogenesis-specific LINC complex presented here. Taken together we conclude that mammalian sperm head formation involves two novel opposed polarizing LINC complexes which connect differentiating nuclei to surrounding sperm-specific cytoskeletal elements. Thus, our findings provide a first hint how spermatid nuclei could be anchored to cytoskeletal structures at opposite sides of the sperm head to enable its gradual shaping and elongation. Materials and Methods Ethics Statement All animal care and experimental protocols were conducted in accordance with the guidelines provided by the German Animal Welfare Act (German Ministry of Agriculture, Health and Economic Cooperation). Animal housing and breeding was approved by the regulatory agency of the city of W u¨rzburg (Reference ABD/OA/Tr; according to 111/1 No. 1 of the German Animal Welfare Act). Personnel from our laboratory carried out all aspects of the mouse work under strict guidelines to insure careful, consistent and ethical handling of mice. Animals and tissue preparation Tissues used in this study were obtained from C57BL/6 and CD1 mice. Somatic tissues as well as testis tissue samples from adult and pubertal mice (8–25 days p.p.) were processed for either RNA isolation or protein analysis as previously described [48]. Mature spermatozoa were obtained from epididymes of adult mice and sperm heads were fractioned as described in Longo et al. [67]. cDNA cloning and generation of plasmid constructs The putative human SUN3 protein sequence (GenBank accession number AAH26189) served as query to search for murine homologous sequences in the NCBI database by the TBLASTN algorithm. This search identified one cDNA clone of 1180 bp (GenBank accession number AK132922) containing the complete coding region of murine Sun3. For cloning mouse Sun3 full-length cDNA, total RNA was isolated from a testicular suspension of an adult mouse using TriFASTTM (Peqlab Biotechnology) according to manufacturer’s protocol. Reverse transcription was performed on 1 mg of total RNA with oligo(dT) primer and M-MLV reverse transcriptase (Promega). Based on the database sequence a 5’ oligonucleotide, corresponding to a sequence upstream of the start codon (Sun3-5’; primers and PCR-conditions are denoted in Table S1) and a 3’ oligonucleotide, complementary to the 3’ end of the coding region (Sun3-3’), were selected for PCR amplification. The obtained Sun3 cDNA was cloned into pCRH2.1-TOPOH vector (Invitrogen) and sequenced according to standard protocols. cDNA encoding full-length murine Sun1 (GenBank accession number BC047928) was obtained from RZPD (Deutsches Ressourcenzentrum fu¨ r Genom- forschung GmbH). For antibody production, expression vectors coding for Sun3 and Sun1 antigens fused to C-terminal His–tags were generated. A Sun3 fragment coding for amino acids 31–148 was amplified by PCR from full-length Sun3 (Sun3AB-5’Nde; PLoS ONE | www.plosone.org 10 August 2010 | Volume 5 | Issue 8 | e12072