but was completely absent form the posterior pole (Figure 4A d-f, Figure S3). These findings clearly evidence that Sun1 in fact is expressed in all spermatogenic stages. In spermatids, Sun1 initially displays a dual localization; however, in the course of differentiation it gradually accumulates at the anterior pole. Sperm head formation involves expression of Sun1g, a novel spermiogenesis-specific Sun1 isoform The anterior polarization and rather unexpected non-nuclear localization lead us to investigated Sun1 expression with more detail. Recently, it was shown that the N-terminal, nucleoplasmic domain of Sun1 is critical for NE retention [12,22]. Therefore we speculated that non-nuclear Sun1 localization in spermatids could be due to expression of a distinct Sun1 splice variant that lacks sequences required for NE retention. To clarify this, we performed RT-PCR analysis to amplify N-terminal regions of all Sun1 isoforms described so far [12] (for primer details see Table S1). We used total RNA from different tissues in order to monitor the general expression profile of Sun1 isoforms that vary within their nucleoplasmic domain. We were able to identify three to seven distinguishable Sun1 messages depending on the tissue tested (Figure 6A). Sequencing of all these isoforms revealed that the LINC in Sperm Head Formation different Sun1 transcripts lacked specifically one or more exons in the part coding for the nucleoplasmic domain (Figure 6D). Remarkably, in our approach we could identify a novel so far undescribed transcript lacking exons seven to ten, that we called Sun1g. Expression of this isoform appears to be restricted to the testis since it could not be amplified in any other tissue (Figure 6A). To verify testis-specificity of Sun1g, we used an additional primer set specific for the Sun1g isoform. Thereby, we could detect only one single message in the testis tissue sample confirming that expression of Sun1g is indeed restricted to the testis. To examine the expression profile of Sun1g during mammalian spermatogenesis, we next performed RT-PCR on total RNA of pubertal testes [46,47]. Using the same primers as above, Sun1g was only present in postmeiotic stages of spermatogenesis (Figure 6B). In testes that only contain spermatogonia and spermatocytes; i.e. 8- and 12-day-old animals, Sun1g was not detectable at all. However, at days 15 and 18 p.p., when first spermatids appear within seminiferous tubules, a faint Sun1g specific signal could be amplified. With accumulation of round and elongated spermatids (days 21–25 p.p.) the Sun1g signal became significantly stronger. Using Sycp1 and Cage1 as meiotic or postmeiotic marker, respectively, we could find that expression of Sun1g differs from meiotic Sycp1, but resembles that of Figure 6. Novel Sun1 isoform Sun1g is testis-specific and exclusively expressed in postmeiotic stages of sperm development. (A) Presence of N-terminal Sun1 variants in different mouse tissues was analyzed by RT-PCR. To amplify predicted N-terminal Sun1 isoforms primers annealing in exon 6 and 11 were used. To verify testis-specificity and expression profile of Sun1g a primer pair that specifically amplifies Sun1g was applied. GAPDH served as control for RNA fidelity. (B) Appearance of Sun1g during mouse sperm differentiation was determined by RT-PCR. Total RNA from pubertal mice of increasing ages (8-25 days p.p.) served as template. cDNA was amplified using the same primers as in (A). Cage1 (postmeiotic expression), Sycp1 (meiotic expression) and GAPDH (RNA fidelity) were used for control. (C) To analyze the expression of protein Sun1g, suspensions from fractioned sperm heads (16106 cells) and whole testicular suspensions (56105 cells) were homogenized in SDS sample buffer and separated by SDS-PAGE. Affinity-purified anti-Sun1 antiserum was used for detection of respective Sun1 isoforms. (D) Schematic representation of the Sun1 isoforms yet identified in different mouse tissues. Epitope used for antibody production is indicated. doi:10.1371/journal.pone.0012072.g006 PLoS ONE | www.plosone.org 7 August 2010 | Volume 5 | Issue 8 | e12072