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• W3012002136 abstract "Sperm head shaping is a key event in spermiogenesis and is tightly controlled via the acrosome–manchette network. Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of Sad1 and UNC84 domain–containing (SUN) and Klarsicht/ANC-1/Syne-1 homology (KASH) domain proteins and form conserved nuclear envelope bridges implicated in transducing mechanical forces from the manchette to sculpt sperm nuclei into a hook-like shape. However, the role of LINC complexes in sperm head shaping is still poorly understood. Here we assessed the role of SUN3, a testis-specific LINC component harboring a conserved SUN domain, in spermiogenesis. We show that CRISPR/Cas9-generated Sun3 knockout male mice are infertile, displaying drastically reduced sperm counts and a globozoospermia-like phenotype, including a missing, mislocalized, or fragmented acrosome, as well as multiple defects in sperm flagella. Further examination revealed that the sperm head abnormalities are apparent at step 9 and that the sperm nuclei fail to elongate because of the absence of manchette microtubules and perinuclear rings. These observations indicate that Sun3 deletion likely impairs the ability of the LINC complex to transduce the cytoskeletal force to the nuclear envelope, required for sperm head elongation. We also found that SUN3 interacts with SUN4 in mouse testes and that the level of SUN4 proteins is drastically reduced in Sun3-null mice. Altogether, our results indicate that SUN3 is essential for sperm head shaping and male fertility, providing molecular clues regarding the underlying pathology of the globozoospermia-like phenotype. Sperm head shaping is a key event in spermiogenesis and is tightly controlled via the acrosome–manchette network. Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of Sad1 and UNC84 domain–containing (SUN) and Klarsicht/ANC-1/Syne-1 homology (KASH) domain proteins and form conserved nuclear envelope bridges implicated in transducing mechanical forces from the manchette to sculpt sperm nuclei into a hook-like shape. However, the role of LINC complexes in sperm head shaping is still poorly understood. Here we assessed the role of SUN3, a testis-specific LINC component harboring a conserved SUN domain, in spermiogenesis. We show that CRISPR/Cas9-generated Sun3 knockout male mice are infertile, displaying drastically reduced sperm counts and a globozoospermia-like phenotype, including a missing, mislocalized, or fragmented acrosome, as well as multiple defects in sperm flagella. Further examination revealed that the sperm head abnormalities are apparent at step 9 and that the sperm nuclei fail to elongate because of the absence of manchette microtubules and perinuclear rings. These observations indicate that Sun3 deletion likely impairs the ability of the LINC complex to transduce the cytoskeletal force to the nuclear envelope, required for sperm head elongation. We also found that SUN3 interacts with SUN4 in mouse testes and that the level of SUN4 proteins is drastically reduced in Sun3-null mice. Altogether, our results indicate that SUN3 is essential for sperm head shaping and male fertility, providing molecular clues regarding the underlying pathology of the globozoospermia-like phenotype. Spermiogenesis involves drastic morphological changes of spermatids, including acrosome formation, elongation and condensation of the nucleus, and disposal of residual cytoplasm, to differentiate into mature spermatozoa consisting of a head and a tail (1Jan S.Z. Hamer G. Repping S. de Rooij D.G. van Pelt A.M. Vormer T. Molecular control of rodent spermatogenesis.Biochim. Biophys. Acta. 2012; 1822 (22366765): 1838-185010.1016/j.bbadis.2012.02.008Crossref PubMed Scopus (122) Google Scholar, 2Lehti M.S. Sironen A. Formation and function of the manchette and flagellum during spermatogenesis.Reproduction. 2016; 151 (26792866): R43-R5410.1530/REP-15-0310Crossref PubMed Scopus (96) Google Scholar). During this complex process, a transient cytoskeletal structure called the manchette appears concurrently with spermatid nucleus elongation. The manchette, which is first evident in step 8 spermatids in mice (3O'Donnell L. O'Bryan M.K. Microtubules and spermatogenesis.Semin. Cell Dev. Biol. 2014; 30 (24440897): 45-5410.1016/j.semcdb.2014.01.003Crossref PubMed Scopus (120) Google Scholar), is composed of parallel arrays of microtubules aligned with the long axis of the nucleus and a belt-like perinuclear ring made of actins where the microtubules are anchored, forming a sleeve-like structure that encircles the spermatid nucleus (4Kierszenbaum A.L. Spermatid manchette: plugging proteins to zero into the sperm tail.Mol. Reprod. Dev. 2001; 59 (11468770): 347-34910.1002/mrd.1040Crossref PubMed Scopus (62) Google Scholar, 5Courtens J.L. Loir M. The spermatid manchette of mammals: formation and relations with the nuclear envelope and the chromatin.Reprod. Nutr. Dev. 1981; 21 (7349538): 467-47710.1051/rnd:19810312Crossref PubMed Scopus (15) Google Scholar, 6Kierszenbaum A.L. Tres L.L. The acrosome-acroplaxome-manchette complex and the shaping of the spermatid head.Arch. Histol. Cytol. 2004; 67 (15700535): 271-28410.1679/aohc.67.271Crossref PubMed Scopus (213) Google Scholar). It is believed that manchette microtubules emanate from perinuclear rings, as these microtubules appear from the post-acrosomal region in mouse and bovine spermatids (7Moreno R.D. Schatten G. Microtubule configurations and post-translational α-tubulin modifications during mammalian spermatogenesis.Cell Motil. Cytoskeleton. 2000; 46 (10962478): 235-24610.1002/1097-0169(200008)46:4%3C235::AID-CM1%3E3.0.CO;2-GCrossref PubMed Scopus (39) Google Scholar). As spermatids differentiate, the microtubular structure and the perinuclear ring move caudally to the posterior pole of spermatid nucleus, sculpting the nucleus into a hook-like morphology. The important role of the manchette in sperm head shaping has been demonstrated in several studies in which disruption of manchette-related proteins resulted in various forms of sperm head abnormalities and eventually impaired male fertility. For example, loss of functional HOOK1 causes ectopic positioning of the manchette microtubular structure within spermatids, resulting in abnormal sperm head morphologies, such as club-shaped and crescent forms (8Mendoza-Lujambio I. Burfeind P. Dixkens C. Meinhardt A. Hoyer-Fender S. Engel W. Neesen J. The Hook1 gene is non-functional in the abnormal spermatozoon head shape (azh) mutant mouse.Hum. Mol. Genet. 2002; 11 (12075009): 1647-165810.1093/hmg/11.14.1647Crossref PubMed Scopus (114) Google Scholar). Similarly, disruption of RIM-BP3, a HOOK1-interacting protein located in the manchette, also causes ectopic positioning of manchette and sperm head abnormalities similar to those of Hook1 mutant mice (9Zhou J. Du Y.-R. Qin W.-H. Hu Y.-G. Huang Y.-N. Bao L. Han D. Mansouri A. Xu G.-L. RIM-BP3 is a manchette-associated protein essential for spermiogenesis.Development. 2009; 136 (19091768): 373-38210.1242/dev.030858Crossref PubMed Scopus (50) Google Scholar). The microtubule plus-end-tracking protein CLIP-170 localizes prominently to the manchette rings during manchette formation, and male mice with knockout of Clip-170 are subfertile because of defective sperm head shaping and abnormal elongation of the manchette tubules (2Lehti M.S. Sironen A. Formation and function of the manchette and flagellum during spermatogenesis.Reproduction. 2016; 151 (26792866): R43-R5410.1530/REP-15-0310Crossref PubMed Scopus (96) Google Scholar, 10Akhmanova A. Mausset-Bonnefont A.L. van Cappellen W. Keijzer N. Hoogenraad C.C. Stepanova T. Drabek K. van der Wees J. Mommaas M. Onderwater J. van der Meulen H. Tanenbaum M.E. Medema R.H. Hoogerbrugge J. Vreeburg J. et al.The microtubule plus-end-tracking protein CLIP-170 associates with the spermatid manchette and is essential for spermatogenesis.Gene Dev. 2005; 19 (16230537): 2501-251510.1101/gad.344505Crossref PubMed Scopus (87) Google Scholar). Despite the essential role of the manchette in spermiogenesis, how the microtubule manchette is assembled and regulated remains poorly understood. Sad1/UNC84 (SUN) 6The abbreviations used are: SUNSad1 and UNC84KASHKlarsicht/ANC-1/Syne-1 homologyLINClinker of nucleoskeleton and cytoskeletonPNApeanut agglutininPASperiodic acid-Schiff. homology proteins are a family of nuclear membrane proteins that share a conserved C terminus, the SUN domain. SUN proteins and Klarsicht/ANC-1/Syne-1 homology (KASH) proteins form a linker of nucleoskeleton and cytoskeleton (LINC) complex that functions like a bridge across the inner and outer nuclear membranes to physically connect the nucleus to the cytoskeleton (11Tzur Y.B. Wilson K.L. Gruenbaum Y. SUN-domain proteins: “velcro” that links the nucleoskeleton to the cytoskeleton.Nat. Rev. Mol. Cell Biol. 2006; 7 (16926857): 782-78810.1038/nrm2003Crossref PubMed Scopus (191) Google Scholar). This complex is responsible for various important cellular functions, such as mechanotransduction, cellular signaling, nuclear anchorage, and positioning. So far, five SUN proteins have been described in mammals and have been demonstrated or suggested to play roles in germ cell development (12Hodzic D.M. Yeater D.B. Bengtsson L. Otto H. Stahl P.D. Sun2 is a novel mammalian inner nuclear membrane protein.J. Biol. Chem. 2004; 279 (15082709): 25805-2581210.1074/jbc.M313157200Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 13Padmakumar V.C. Libotte T. Lu W. Zaim H. Abraham S. Noegel A.A. Gotzmann J. Foisner R. Karakesisoglou I. The inner nuclear membrane protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope.J. Cell Sci. 2005; 118 (16079285): 3419-343010.1242/jcs.02471Crossref PubMed Scopus (315) Google Scholar, 14Göb E. Schmitt J. Benavente R. Alsheimer M. Mammalian sperm head formation involves different polarization of two novel LINC complexes.PLoS ONE. 2010; 5 (20711465): e1207210.1371/journal.pone.0012072Crossref PubMed Scopus (104) Google Scholar, 15Calvi A. Wong A.S. Wright G. Wong E.S. Loo T.H. Stewart C.L. Burke B. SUN4 is essential for nuclear remodeling during mammalian spermiogenesis.Dev. Biol. 2015; 407 (26417726): 321-33010.1016/j.ydbio.2015.09.010Crossref PubMed Scopus (41) Google Scholar, 16Pasch E. Link J. Beck C. Scheuerle S. Alsheimer M. The LINC complex component Sun4 plays a crucial role in sperm head formation and fertility.Biol. Open. 2015; 4 (26621829): 1792-180210.1242/bio.015768Crossref PubMed Scopus (43) Google Scholar, 17Yassine S. Escoffier J. Abi Nahed R. Pierre V. Karaouzene T. Ray P.F. Arnoult C. Dynamics of Sun5 localization during spermatogenesis in wild type and Dpy19l2 knock-out mice indicates that Sun5 is not involved in acrosome attachment to the nuclear envelope.PLoS ONE. 2015; 10 (25775128): e011869810.1371/journal.pone.0118698Crossref PubMed Scopus (28) Google Scholar). SUN1 and SUN2 are ubiquitously expressed in various tissues (12Hodzic D.M. Yeater D.B. Bengtsson L. Otto H. Stahl P.D. Sun2 is a novel mammalian inner nuclear membrane protein.J. Biol. Chem. 2004; 279 (15082709): 25805-2581210.1074/jbc.M313157200Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 13Padmakumar V.C. Libotte T. Lu W. Zaim H. Abraham S. Noegel A.A. Gotzmann J. Foisner R. Karakesisoglou I. The inner nuclear membrane protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope.J. Cell Sci. 2005; 118 (16079285): 3419-343010.1242/jcs.02471Crossref PubMed Scopus (315) Google Scholar) and mediate meiotic telomere attachment to the nuclear envelope (18Morimoto A. Shibuya H. Zhu X. Kim J. Ishiguro K. Han M. Watanabe Y. A conserved KASH domain protein associates with telomeres, SUN1, and dynactin during mammalian meiosis.J. Cell Biol. 2012; 198 (22826121): 165-17210.1083/jcb.201204085Crossref PubMed Scopus (146) Google Scholar, 19Ding X. Xu R. Yu J. Xu T. Zhuang Y. Han M. SUN1 is required for telomere attachment to nuclear envelope and gametogenesis in mice.Dev. Cell. 2007; 12 (17543860): 863-87210.1016/j.devcel.2007.03.018Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 20Penkner A.M. Fridkin A. Gloggnitzer J. Baudrimont A. Machacek T. Woglar A. Csaszar E. Pasierbek P. Ammerer G. Gruenbaum Y. Jantsch V. Meiotic Chromosome homology search involves modifications of the nuclear envelope protein Matefin/SUN-1.Cell. 2009; 139 (19913286): 920-93310.1016/j.cell.2009.10.045Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 21Lei K. Zhang X. Ding X. Guo X. Chen M. Zhu B. Xu T. Zhuang Y. Xu R. Han M. SUN1 and SUN2 play critical but partially redundant roles in anchoring nuclei in skeletal muscle cells in mice.Proc. Natl. Acad. Sci. U.S.A. 2009; 106 (19509342): 10207-1021210.1073/pnas.0812037106Crossref PubMed Scopus (170) Google Scholar, 22Link J. Leubner M. Schmitt J. Göb E. Benavente R. Jeang K.-T. Xu R. Alsheimer M. Analysis of meiosis in SUN1 deficient mice reveals a distinct role of SUN2 in mammalian meiotic LINC complex formation and function.PLoS Genet. 2014; 10 (24586178): e100409910.1371/journal.pgen.1004099Crossref PubMed Scopus (42) Google Scholar). SUN3, SUN4, and SUN5 are specifically expressed in the testes (14Göb E. Schmitt J. Benavente R. Alsheimer M. Mammalian sperm head formation involves different polarization of two novel LINC complexes.PLoS ONE. 2010; 5 (20711465): e1207210.1371/journal.pone.0012072Crossref PubMed Scopus (104) Google Scholar, 15Calvi A. Wong A.S. Wright G. Wong E.S. Loo T.H. Stewart C.L. Burke B. SUN4 is essential for nuclear remodeling during mammalian spermiogenesis.Dev. Biol. 2015; 407 (26417726): 321-33010.1016/j.ydbio.2015.09.010Crossref PubMed Scopus (41) Google Scholar, 16Pasch E. Link J. Beck C. Scheuerle S. Alsheimer M. The LINC complex component Sun4 plays a crucial role in sperm head formation and fertility.Biol. Open. 2015; 4 (26621829): 1792-180210.1242/bio.015768Crossref PubMed Scopus (43) Google Scholar, 17Yassine S. Escoffier J. Abi Nahed R. Pierre V. Karaouzene T. Ray P.F. Arnoult C. Dynamics of Sun5 localization during spermatogenesis in wild type and Dpy19l2 knock-out mice indicates that Sun5 is not involved in acrosome attachment to the nuclear envelope.PLoS ONE. 2015; 10 (25775128): e011869810.1371/journal.pone.0118698Crossref PubMed Scopus (28) Google Scholar). SUN5 localization is restricted to the head–tail junctions of sperm and is essential for anchoring the sperm head to the tail (23Shang Y. Zhu F. Wang L. Ouyang Y.C. Dong M.Z. Liu C. Zhao H. Cui X. Ma D. Zhang Z. Yang X. Guo Y. Liu F. Yuan L. Gao F. et al.Essential role for SUN5 in anchoring sperm head to the tail.Elife. 2017; 6 (28945193): e2819910.7554/eLife.28199Crossref PubMed Scopus (52) Google Scholar). SUN4 localizes to the spermatid nuclear envelope in close association with manchette microtubules (15Calvi A. Wong A.S. Wright G. Wong E.S. Loo T.H. Stewart C.L. Burke B. SUN4 is essential for nuclear remodeling during mammalian spermiogenesis.Dev. Biol. 2015; 407 (26417726): 321-33010.1016/j.ydbio.2015.09.010Crossref PubMed Scopus (41) Google Scholar, 16Pasch E. Link J. Beck C. Scheuerle S. Alsheimer M. The LINC complex component Sun4 plays a crucial role in sperm head formation and fertility.Biol. Open. 2015; 4 (26621829): 1792-180210.1242/bio.015768Crossref PubMed Scopus (43) Google Scholar). Knockout of Sun4 in mice disrupts the lateral interactions of the manchette to the nucleus, and the nucleus thus fails to elongate, eventually resulting in a globozoospermia-like phenotype and male infertility (15Calvi A. Wong A.S. Wright G. Wong E.S. Loo T.H. Stewart C.L. Burke B. SUN4 is essential for nuclear remodeling during mammalian spermiogenesis.Dev. Biol. 2015; 407 (26417726): 321-33010.1016/j.ydbio.2015.09.010Crossref PubMed Scopus (41) Google Scholar, 16Pasch E. Link J. Beck C. Scheuerle S. Alsheimer M. The LINC complex component Sun4 plays a crucial role in sperm head formation and fertility.Biol. Open. 2015; 4 (26621829): 1792-180210.1242/bio.015768Crossref PubMed Scopus (43) Google Scholar). SUN3 protein expression begins at postnatal day 15, and, similar to SUN4, its localization is closely associated with the manchette in developing spermatids (14Göb E. Schmitt J. Benavente R. Alsheimer M. Mammalian sperm head formation involves different polarization of two novel LINC complexes.PLoS ONE. 2010; 5 (20711465): e1207210.1371/journal.pone.0012072Crossref PubMed Scopus (104) Google Scholar). However, because of the lack of animal models, whether Sun3 indeed plays a role in spermiogenesis is not clear. Sad1 and UNC84 Klarsicht/ANC-1/Syne-1 homology linker of nucleoskeleton and cytoskeleton peanut agglutinin periodic acid-Schiff. To investigate the physiological functions of Sun3 during mammalian spermatogenesis, we generated a Sun3 knockout mouse model using CRISPR/Cas9 genome editing technology. We found that Sun3−/− male mice are infertile, displaying abnormal sperm head morphology and irregular acrosome localization, likely resulting from disruption of manchette assembly. These findings demonstrate that Sun3 is essential for sperm head shaping during spermiogenesis. Consistent with previous findings (14Göb E. Schmitt J. Benavente R. Alsheimer M. Mammalian sperm head formation involves different polarization of two novel LINC complexes.PLoS ONE. 2010; 5 (20711465): e1207210.1371/journal.pone.0012072Crossref PubMed Scopus (104) Google Scholar, 16Pasch E. Link J. Beck C. Scheuerle S. Alsheimer M. The LINC complex component Sun4 plays a crucial role in sperm head formation and fertility.Biol. Open. 2015; 4 (26621829): 1792-180210.1242/bio.015768Crossref PubMed Scopus (43) Google Scholar), SUN3 was expressed testis-specifically (Fig. S1A) and was detected in spermatids, localizing to the nuclear pole distal to the acrosome and overlapping with α-tubulin and SUN4 (Fig. S1B). To investigate the biological function of SUN3, we generated Sun3-null mice using CRISPR/Cas9 genome editing technology (Fig. 1A). Sanger sequencing of genomic DNA from the mutant mice revealed that a thymidine was inserted between nucleotide position 184 and 185 (c.184_185insT) in exon 4 of Sun3, predicted to result in premature translation termination (p.Pro62Leufs*2) (Fig. 1B). Western blotting analyses further confirmed that full-length SUN3 proteins were absent in the testes of Sun3−/− mice (Fig. 1C and Fig. S2). Mice lacking Sun3 appeared normal, displaying no obvious abnormalities in development and behavior. Given the testis-restricted expression pattern of SUN3, we studied fertility in Sun3−/− male mice. Mating attempts of Sun3−/− males with WT females did not produce any offspring, indicating that Sun3−/− males are infertile. Sun3−/− females showed no overt abnormalities in fertility. Sun3−/− mice had smaller testes (Fig. 2, A and B) and sharply declined epididymal sperm numbers (0.17 ± 0.03 million/ml) compared with WT mice (12.80 ± 0.36 million/ml) (Fig. 2C). To further characterize the spermatogenic defects in Sun3−/− mice, H&E staining of testis and epididymis sections was performed. All types of spermatogenic cells were present in an orderly way in Sun3+/+ seminiferous tubules and mature spermatozoa, and a canonical hook-shaped head could be seen in the lumen of tubules (Fig. 2D, a and b). However, in Sun3−/− mice, all elongating and elongated spermatids as well as spermatozoa were observed to have a noncanonical round head (Fig. 2D, c and d). Consistent with the result of sperm counting per epididymis, the numbers of these noncanonical spermatids and spermatozoa were apparently lower in seminiferous tubules and in the cauda epididymides of Sun3−/− mice compared with those in Sun3+/+ mice (Fig. 2D, e and f). Furthermore, a TUNEL assay in combination with a germ cell–specific marker (MVH) was performed, and the results indicated apoptosis in cells with a small amorphous nuclear shape, which corresponds to the noncanonical spermatids in Sun3−/− mice (Fig. 2E). The frequency of TUNEL+ tubules (11.99% ± 0.75% versus 32.00% ± 3.17%) and the number of TUNEL+ cells per TUNEL+ tubule (1.70% ± 0.15% versus 2.52% ± 0.15%) in Sun3−/− mice were significantly increased compared with Sun3+/+ mice (Fig. 2F), suggesting that the spermatids of Sun3−/− mice underwent apoptosis. These results indicate that Sun3 is essential for spermatogenesis and particularly for the development of spermatids. We further analyzed the sperm morphology in the epididymis and found that, in contrast to the typical hook-shaped appearance of sperm heads in Sun3+/+ mice, all sperm heads in Sun3−/− mice were amorphous with a smaller and more rounded shape (Fig. 3, A and B). Additionally, ∼89.67% ± 2.02% of spermatozoa from Sun3−/− mice also displayed various midpiece defects, such as irregular caliber, bending, coiling, and/or cracking (Fig. 3, A and C, and Fig. S3). Immunofluorescence staining of peanut agglutinin (PNA), a marker of acrosomes, was performed on sperm smears and seminiferous tubules. In Sun3+/+ mice, acrosomes with a typical crescent shape were found on top of the nucleus in the anterior dorsal part of the sperm head. However, acrosomes of spermatozoa from Sun3−/− mice were missing, mislocalized, or fragmented (Fig. 3D). Because failure of sperm head elongation usually leads to a rounded head appearance, to understand the specific step at which head abnormalities occur in Sun3−/− mice, we compared spermatids of Sun3+/+ and Sun3−/− mice at different steps of spermiogenesis. PAS staining revealed that the morphology of spermatids until steps 7 and 8 were comparable in Sun3+/+ and Sun3−/− mice, indicating that the development of round spermatids was normal in Sun3−/− mice (Fig. 4). At step 9, the nuclei of spermatids became flattened and started to elongate along with condensation of chromatin in Sun3+/+ mice, whereas in Sun3−/− mice, spermatids still had a round nucleus although the chromatin had been condensed. At step 10, spermatid nuclei in Sun3+/+ mice became thinner and more elongated; however, the nuclei of spermatids were small and deformed and remained round in Sun3−/− mice (Fig. 4). We also noted that the number of spermatids was markedly reduced from step 10. Altogether, these findings demonstrate that abnormal morphology of Sun3−/− spermatids occurs when spermatids start to elongate. Because the microtubule manchette is essential for elongation of spermatids, and SUN3 localization is closely associated with the manchette (4Kierszenbaum A.L. Spermatid manchette: plugging proteins to zero into the sperm tail.Mol. Reprod. Dev. 2001; 59 (11468770): 347-34910.1002/mrd.1040Crossref PubMed Scopus (62) Google Scholar, 14Göb E. Schmitt J. Benavente R. Alsheimer M. Mammalian sperm head formation involves different polarization of two novel LINC complexes.PLoS ONE. 2010; 5 (20711465): e1207210.1371/journal.pone.0012072Crossref PubMed Scopus (104) Google Scholar), we next wanted to find out whether the structure of the manchette was disrupted after Sun3 deletion. Coimmunofluorescence staining of testis sections with antibodies against α-tubulin, a marker of manchette microtubules, and PNA was conducted. In Sun3+/+ mice, manchette microtubules were found to be tightly attached to the nuclear periphery at the caudal region opposite the acrosome of spermatids; however, these structures were not detected in Sun3−/− mice despite some weak and diffuse labeling that was ectopically positioned (Fig. 5A). To confirm loss of manchette microtubules in Sun3 knockout spermatids, immunofluorescence staining on testis cell smears was performed. In Sun3+/+ mice, spermatids from step 1 to step 16 could be distinguished based on the morphology of the acrosome, and the acrosome covering the anterior side of the nucleus as well as the microtubules of the manchette tightly surrounding the caudal region could be seen from step 7/8 to step 14/15 in Sun3+/+ spermatids. In Sun3−/− mice, we did not detect any morphological abnormalities in spermatids until step 7/8, and acrosomes covering the anterior side of the nucleus appeared to be normal in round spermatids. However, the typical microtubule arrays of the manchette were not observed in all spermatids from Sun3−/− mice. Aberrantly polymerized microtubule bundles that were dissociated from the nucleus were observed in about 5% of spermatids (Fig. 5B). Moreover, the perinuclear ring, a belt-like structure surrounding the nucleus where manchette microtubules insert, was also absent in spermatids from Sun3−/− mice (Fig. S4). Transmission EM (TEM) further revealed the presence of manchette microtubule bundles that were closely associated with the nuclear envelope in elongating spermatids from Sun3+/+ testes; however, these recognizable structures were not seen in elongating spermatids from Sun3−/− testes, although some disorganized microtubule bundles that had lost their interaction with the nucleus were observed (Fig. 5C). Thus, we conclude that SUN3 is not only indispensable for manchette formation but also likely required for the organization of manchette during sperm head shaping in mice. Deletion of SUN4, another SUN protein that associates with the manchette, also leads to defects in acrosome and manchette formation in mice (15Calvi A. Wong A.S. Wright G. Wong E.S. Loo T.H. Stewart C.L. Burke B. SUN4 is essential for nuclear remodeling during mammalian spermiogenesis.Dev. Biol. 2015; 407 (26417726): 321-33010.1016/j.ydbio.2015.09.010Crossref PubMed Scopus (41) Google Scholar), similar to the findings in Sun3-null mice. To explore whether SUN3 interacts with SUN4 in testes, we performed immunoprecipitation using an anti-SUN3 antibody with testis lysates from Sun3+/+ mice. Western blotting detected SUN3 and SUN4 in lysates immunoprecipitated by the anti-SUN3 antibody but not in IgG-immunoprecipitated lysates (Fig. 6, A and B). Immunofluorescence staining in Sun3+/+ and Sun3−/− testicular sections revealed clear signals of SUN4 proteins surrounding the caudal region of the nucleus opposite the acrosome, where the manchette locates in step 7/8 spermatids, elongating spermatids, and elongated spermatids in Sun3+/+ mice, whereas only unspecific diffuse signals were observed in the cytoplasm of spermatids from Sun3−/− mice (Fig. 6C). Western blotting of testicular lysates revealed that the level of SUN4 proteins was drastically reduced in adult Sun3−/− mice compared with Sun3+/+ mice (Fig. 6D). These findings demonstrated that SUN3 and SUN4 interact and that SUN3 is required to maintain the level of SUN4 proteins in vivo. Sperm head shaping is a key event during spermiogenesis, and misshaping of sperm heads often leads to male infertility (24Yan W. Male infertility caused by spermiogenic defects: lessons from gene knockouts.Mol. Cell Endocrinol. 2009; 306 (19481682): 24-3210.1016/j.mce.2009.03.003Crossref PubMed Scopus (133) Google Scholar). In this study, we generated mice lacking Sun3 through CRISPR/Cas9 technology and investigated the function of SUN3 during spermiogenesis. We found that loss of SUN3 leads to a drastic reduction in sperm numbers, a globozoospermia-like phenotype accompanied by multiple sperm tail defects resulting from failure of manchette formation during sperm head shaping, and, ultimately, male infertility. Additionally, we also demonstrated that SUN3 interacts with SUN4 in vivo and is required to maintain the level of SUN4 proteins in testes. We report, for the first time, that Sun3 is indispensable for sperm head shaping and particularly required for manchette formation. A typical manchette is characterized by highly organized microtubule bundles attached to the perinuclear ring, forming a sleeve-like structure surrounding the posterior part of the spermatid nucleus (5Courtens J.L. Loir M. The spermatid manchette of mammals: formation and relations with the nuclear envelope and the chromatin.Reprod. Nutr. Dev. 1981; 21 (7349538): 467-47710.1051/rnd:19810312Crossref PubMed Scopus (15) Google Scholar, 25Kierszenbaum A.L. Rivkin E. Tres L.L. Cytoskeletal track selection during cargo transport in spermatids is relevant to male fertility.Spermatogenesis. 2011; 1 (22319670): 221-23010.4161/spmg.1.3.18018Crossref PubMed Google Scholar). This transient structure appears when spermatids are going to elongate and disappears when the sperm heads are properly shaped. Several genes have been reported to be implicated in manchette function in mice, including Hook1, Katnb1, Lrguk1, Meig1, Pacrg, and Spef2 (8Mendoza-Lujambio I. Burfeind P. Dixkens C. Meinhardt A. Hoyer-Fender S. Engel W. Neesen J. The Hook1 gene is non-functional in the abnormal spermatozoon head shape (azh) mutant mouse.Hum. Mol. 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