SUN5 interacts with nuclear membrane LaminB1 and cytoskeletal GTPase Septin12 mediating the sperm head-and-tail junction.

Acephalic spermatozoa syndrome (ASS) is a severe teratospermia with decaudated, decapitated, and malformed sperm, resulting in male infertility. Nuclear envelope protein SUN5 localizes to the junction between the sperm head and tail. Mutations in the SUN5 gene have been identified most frequently (33-47%) in ASS cases, and its molecular mechanism of action is yet to be explored. In the present study, we generated Sun5 knockout mice, which presented the phenotype of ASS. Nuclear membrane protein LaminB1 and cytoskeletal GTPases Septin12 and Septin2 were identified as potential partners for interacting with SUN5 by immunoprecipitation-mass spectrometry in mouse testis. Further studies demonstrated that SUN5 connected the nucleus by interacting with LaminB1 and connected the proximal centriole by interacting with Septin12. The binding between SUN5 and Septin12 promoted their aggregation together in the sperm neck. The disruption of the LaminB1/SUN5/Septin12 complex by Sun5 deficiency caused separation of the Septin12-proximal centriole from the nucleus, leading to the breakage of the head-to-tail junction. Collectively, these data provide new insights into the pathogenesis of ASS caused by SUN5 deficiency.

SUN5, a testis-specific nuclear membrane protein, participates in recruitment and export of nuclear mRNA in spermatogenesis.

SUN5, a testis-specific gene, is associated with acephalic spermatozoa syndrome (ASS). Here, we demonstrate that Sun5 is involved in mRNA export. In Sun5-knockout mice ( Sun5 -/-), poly(A) + RNA accumulates in the nuclei of germ cells, leading to reduced sperm counts, decreased sperm motility and disrupted sperm head-to-tail junctions. Additionally, in the GC-2 germ cell line with RNA interference of Sun5, heterogeneous nuclear ribonucleoproteins (hnRNPs) and poly (A) + RNA (mainly mRNA) are retained in the nucleus. Further mechanistic studies reveal that Sun5 interacts with Nxf1 (nuclear RNA export factor 1) and nucleoporin 93 (Nup93). Interference with Nup93 inhibits mRNA export. Treatment with leptomycin B to block the CRM1 pathway indicates that Sun5 regulates mRNA export through an Nxf1-dependent pathway. In Sun5 -/- mice, the binding of Nxf1 and Nup93 decreases due to loss of Sun5 function, and the process of submitting Nxf1-binding mRNPs to Nup93 is inhibited, resulting in abnormal spermatogenesis. Together, these data may elucidate a novel pathway for mRNA export in male germ cells.

Genetic basis of acephalic spermatozoa syndrome, and intracytoplasmic sperm injection outcomes in infertile men: a systematic scoping review.

PURPOSE: Acephalic spermatozoa syndrome (ASS) is known as a severe type of teratozoospermia, defined as semen composed of mostly headless spermatozoa that affect male fertility. In this regard, this systematic review aimed to discuss gene variants associated with acephalic spermatozoa phenotype as well as the clinical outcomes of intracytoplasmic sperm injection (ICSI) treatment for the acephalic spermatozoa-associated male infertility. METHODS: A systematic search was performed on PubMed, Embase, Scopus, and Ovid databases until May 17, 2020. This systematic scoping review was reported in terms of the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) statement. RESULTS: Twenty articles were included in this systematic review. Whole-exome and Sanger sequencing have helped in the identification of variants in SUN5, PMFBP1, BRDT, TSGA10, DNAH6, HOOK1, and CEP112 genes as possible causes of this phenotype in humans. The results of the ICSI are conflicting due to both positive and negative reports of ICSI outcomes. CONCLUSION: ASS has a genetic origin, and several genetic alterations related to the pathogenesis of this anomaly have been recently identified. Notably, only SUN5 and PMFBP1 mutations are well-known to be implicated in ASS. Accordingly, more functional studies are needed to confirm the pathogenicity of other variants. ICSI could provide a promising treatment for acephalic spermatozoa-associated male infertility. Besides the importance of sperm head-tail junction integrity, some other factors, whether within the sperm cell or female factors, may be involved in the ICSI outcome.

Identification and Characterization of the Most Common Genetic Variant Responsible for Acephalic Spermatozoa Syndrome in Men Originating from North Africa.

Acephalic spermatozoa syndrome (ASS) is a rare but extremely severe type of teratozoospermia, defined by the presence of a majority of headless flagella and a minority of tail-less sperm heads in the ejaculate. Like the other severe monomorphic teratozoospermias, ASS has a strong genetic basis and is most often caused by bi-allelic variants in SUN5 (Sad1 and UNC84 domain-containing 5). Using whole exome sequencing (WES), we investigated a cohort of nine infertile subjects displaying ASS. These subjects were recruited in three centers located in France and Tunisia, but all originated from North Africa. Sperm from subjects carrying candidate genetic variants were subjected to immunofluorescence analysis and transmission electron microscopy. Moreover, fluorescent in situ hybridization (FISH) was performed on sperm nuclei to assess their chromosomal content. Variant filtering permitted us to identify the same SUN5 homozygous frameshift variant (c.211+1_211+2dup) in 7/9 individuals (78%). SUN5 encodes a protein localized on the posterior part of the nuclear envelope that is necessary for the attachment of the tail to the sperm head. Immunofluorescence assays performed on sperm cells from three mutated subjects revealed a total absence of SUN5, thus demonstrating the deleterious impact of the identified variant on protein expression. Transmission electron microscopy showed a conserved flagellar structure and a slightly decondensed chromatin. FISH did not highlight a higher rate of chromosome aneuploidy in spermatozoa from SUN5 patients compared to controls, indicating that intra-cytoplasmic sperm injection (ICSI) can be proposed for patients carrying the c.211+1_211+2dup variant. These results suggest that the identified SUN5 variant is the main cause of ASS in the North African population. Consequently, a simple and inexpensive genotyping of the 211+1_211+2dup variant could be beneficial for affected men of North African origin before resorting to more exhaustive genetic analyses.

[Genetic analysis of three cases of acephalic spermatozoa syndrome caused by SUN5 mutation and the outcome of assisted reproductive technology].

To explore the genetic causes of 3 male infertility patients with acephalospermia and the outcome of assisted reproductive technology. Clinical diagnosis, sperm morphology examination, sperm transmission electron microscopy examination were performed on 3 patients, and the whole exome sequencing technology was used for screening, Sanger sequencing verification, mutation pathogenicity analysis, and protein sequence homology comparison. Assisted reproductive technology was implemented to assist pregnancy treatment. The 3 patients were all sporadic infertile men, aged 25, 42 and 26 years, and there was no obvious abnormality in the general physical examination. Male external genitalia developed normally, bilateral testicles were normal in volume, and bilateral epididymis and spermatic vein were palpated without nodules, cysts, and tenderness. Repeated semen analysis showed that a large number of immature sperm could be seen, and they had the ability to move. The SUN5 gene of the 3 male infertile patients was a case of homozygous missense mutation c.7C>T (p.Arg3Trp), a case of compound heterozygous missense mutation c.1067G>A (p.Arg356His) and nonsense mutation c.216G>A (p.Trp72*) and a case of homozygous missense mutation c.1043A>T (p.Asn348Ile), of which c.7C>T (p.Arg3Trp) and c.1067G>A (p.Arg356His) were new variants that had not been reported. SIFT, Mutation Taster and PolyPhen-2 software function prediction results were all harmful, the nonsense mutation c.216G>A (p.Trp72*) led to the premature termination of peptide chain synthesis which might have a greater impact on protein function. The homology regions in the protein sequence homology alignment were all highly conserved.The 3 male patients and their spouses obtained 4 biological offspring through intracytoplasmic sperm injection, all of which were boys, and one of them was a twin.Three male infertile patients might be caused by SUN5 gene mutations. Such patients could obtain their biological offspring through assisted reproductive technology. It was still necessary to pay attention to the genetic risk of ASS, it was recommended that both men and women conduct genetic counseling and screening at the same time. In clinical diagnosis, whole exome sequencing technology could be used to perform auxiliary examinations to determine the treatment plan and assisted reproductive methods as soon as possible to reduce the burden on the family and society. The newly discovered mutation sites of SUN5 gene provided clues and directions for elucidating the pathogenic mechanism, and at the same time expanded the pathogenic mutation spectrum of ASS.

Mechanistic insights into acephalic spermatozoa syndrome-associated mutations in the human SUN5 gene.

Acephalic spermatozoa syndrome has been reported for many decades; it is characterized by very few intact spermatozoa and tailless sperm heads in the semen and causes severe male infertility. The only gene in which mutations have been found to be associated with this syndrome encodes Sad1 and UNC84 domain-containing 5 (SUN5), a testis-specific nuclear envelope protein. The functional role of SUN5 has been well-studied in mouse models, but the molecular basis for the pathogenic effects of mutations in the human SUN5 gene remains elusive. Here, we report a new SUN5 mutation (c.475C→T; p.Arg159*), and explore the pathogenic effects of all known SUN5 mutations on acephalic spermatozoa syndrome. Using an artificial splicing system, we found that the intronic mutation affects the splicing of SUN5 mRNA, yielding a premature stop codon that results in a truncated SUN5 protein. We also found that SUN5 interacts with the coupling apparatus protein DnaJ heat shock protein family (Hsp40) member B13 (DNAJB13) during spermatogenesis, and the substitutions in the SUN5 SUN domain impair its interaction with DNAJB13. Furthermore, we observed that many SUN5 mutations affect the secondary structure of the protein and influence its folding and cellular localization. In summary, our findings indicate an interaction of SUN5 with DNAJB13 during spermatogenesis, provide mechanistic insights into the functional role of this interaction in sperm head-tail integration, and elucidate the molecular etiology of acephalic spermatozoa syndrome-associated SUN5 mutations.

[Advances in the molecular genetic studies of acephalic spermatozoa syndrome].

Acephalic spermatozoa syndrome (ASS) is characterized by a predominance of headless spermatozoa with abnormal head-tail junction in the ejaculate, which causes severe male infertility. The pathogenic mechanism of ASS remained unclarified for a long time until recent identification of the four ASS-associated genes SUN5, PMFBP1, TSGA10, and BRDT and their mutations due to the development of high-throughput sequencing technology. This review summarizes the advances in the genetic studies of ASS, focusing on its pathogenic molecular mechanisms, which provide an important basis for the molecular diagnosis of the disease as well as for assisted reproductive technology.

Molecular Cloning of Porcine SUN5 Gene and Association between a SNP with Litter Size Trait.

SUN domain-containing protein 5 (SUN5) is an important reproduction related gene. In this study, we cloned the full-length coding sequence of porcine SUN5 gene through RT-PCR. Sequence analysis of this gene revealed that the pig SUN5 gene encodes a protein of 383 amino acids that has high homology with the SUN5 protein of eight species: wild Bactrian camel (95%), alpaca (95%), Yangtze River dolphin (94%), sperm whale (94%), sheep (93%), black flying fox (93%), goat (92%), and horse (91%). This gene is structured into 13 exons and 12 introns as revealed by computer-assisted analysis. The prediction of transmembrane helices showed that pig SUN5 protein might be a transmembrane protein. PCR-Taq I-RFLP was established to detect the GU475008:c.138 G>A substitution of porcine SUN5 gene coding sequence and eight pig breeds displayed obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n = 200) and Landrace (n = 200) pig populations, and the results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White and Landrace sows (P < 0.05). Therefore, the SUN5 gene could be a useful candidate gene for increasing the litter size in pigs.

Nuclear Membrane Protein SUN5 Is Highly Expressed and Promotes Proliferation and Migration in Colorectal Cancer by Regulating the ERK Pathway.

SUN5 was first identified as a nuclear envelope protein involved in spermatocyte division. We found that SUN5 was highly expressed in some cancers, but its function and mechanism in cancer development remain unclear. In the present study, we demonstrated that SUN5 was highly expressed in colorectal cancer (CRC) tissues and cells, as indicated by bioinformatics analysis, and SUN5 promoted cell proliferation and migration in vitro. Moreover, the overexpression of SUN5 upregulated phosphorylated ERK1/2 (pERK1/2), whereas the knockdown of SUN5 yielded the opposite results. PD0325901 decreased the level of pERK1/2 to inhibit cell proliferation and migration, which was partially reversed by SUN5 overexpression, indicating that drug resistance existed in patients with high SUN5 expression. The xenograft transplantation experiment showed that SUN5 accelerated tumor formation in vivo. Furthermore, we found that SUN5 regulated the ERK pathway via Nesprin2 mediation and promoted the nuclear translocation of pERK1/2 by interacting with Nup93. Thus, these findings indicated that highly expressed SUN5 promoted CRC proliferation and migration by regulating the ERK pathway, which may contribute to the clinical diagnosis and new treatment strategies for CRC.

Novel Mutation and Deletion in SUN5 Cause Male Infertility with Acephalic Spermatozoa Syndrome.

Acephalic spermatozoa syndrome (ASS) is a severe form of teratozoospermia, previous studies have shown that SUN5 mutations are the major cause of acephalic spermatozoa syndrome. This study is to identify the pathogenic mutations in SUN5 leading to ASS. PCR and Sanger sequence were performed to define the breakpoints and mutations in SUN5. Whole genome sequencing (WGS) was performed to detect heterozygous deletion. Western blotting and immunofluorescence analysis detected the expression level and localization of SUN5. Furthermore, the pathogenicity of the mutant SUN5 was predicted in silico and was verified by the experiments in vitro. We identified one novel homozygous missense mutation (c.775G>A; p.G259S) and one compound heterozygous including one reported missense mutation (c.1043A>T; p.N348I) and a large deletion that contains partial EFCAB8 ( NM_001143967 .1) and BPIFB2 ( NM_025227 ) and complete SUN5 ( NM_080675 ), and one recurrent homozygous splice-site mutation (c.340G>A; p.G114R) in SUN5 in three patients with ASS. Our results showed that SUN5 could not be detected in the patients' spermatozoa and the exogenous expression level of the mutant protein was decreased in transfected HEK-293T cells. This study expands the mutational spectrum of SUN5. We recommended a clinical diagnostic strategy for SUN5 genomic deletion to screen heterozygous deletions and indicated that the diagnostic value of screening for SUN5 mutations and deletions in infertile men with ASS.

SUN5 Interacting With Nesprin3 Plays an Essential Role in Sperm Head-to-Tail Linkage: Research on Sun5 Gene Knockout Mice.

Acephalic spermatozoa syndrome is a rare genetic and reproductive disease. Recent studies have shown that approximately 33-47% of patients with acephalic spermatozoa syndrome have SUN5 mutations, but the molecular mechanism underlying this phenomenon has not been elucidated. In this study, we generated Sun5 knockout mice and found that the head-to-tail linkage was broken in Sun5-/- mice, which was similar to human acephalic spermatozoa syndrome. Furthermore, ultrastructural imaging revealed that the head-tail coupling apparatus (HTCA) and the centrosome were distant from the nucleus at steps 9-10 during spermatid elongation. With the manchette disappearing at steps 13-14, the head and the tail segregated. To explore the molecular mechanism underlying this process, bioinformatic analysis was performed and showed that Sun5 may interact with Nesprin3. Further coimmunoprecipitation (Co-IP) and immunofluorescence assays confirmed that Sun5 and Nesprin3 were indeed bona fide interaction partners that formed the linker of the nucleoskeleton and cytoskeleton (LINC) complex participating in the connection of the head and tail of spermatozoa. Nesprin3 was located posterior and anterior to the nucleus during spermiogenesis in wild-type mice, whereas it lost its localization at the implantation fossa of the posterior region in Sun5-/- mice. Without correct localization of Nesprin3 at the nuclear membrane, the centrosome, which is the originator of the flagellum, was distant from the nucleus, which led to the separation of the head and tail. In addition, isobaric tag for relative and absolute quantitation results showed that 47 proteins were upregulated, and 56 proteins were downregulated, in the testis in Sun5-/- mice, and the downregulation of spermatogenesis-related proteins (Odf1 and Odf2) may also contribute to the damage to the spermatozoa head-to-tail linkage. Our findings suggested that Sun5 is essential for the localization of Nesprin3 at the posterior nuclear membrane, which plays an essential role in the sperm head-tail connection.

Genetic contribution of SUN5 mutations to acephalic spermatozoa in Fujian China.

Acephalic spermatozoa is an extremely rare disease associated with primary infertility. A recent study showed that genetic alterations in the SUN5 gene lead to this disease, and SUN5 mutations could explain the disease in about half of the patients. Therefore, in the present study, to re-visit the genetic contribution of SUN5 mutations to acephalic spermatozoa, we recruited 15 unrelated affected individuals and screened the SUN5 gene for mutations by whole-exome sequencing (WES) and Sanger sequencing. Five of the 15 (33.33%) subjects were found to carry the same homozygous mutation in the SUN5 gene c.381delA (p.V128Sfs*7). Neither homozygous nor compound heterozygous mutations in SUN5 were found in the other 10 patients. The c.381delA mutation resulted in the truncation of the SUN5 protein and decreased the expression and altered the distribution of the outer dense fiber 1 (ODF1) protein. Thus, in our study SUN5 mutations accounted for only one-third of the patients in our cohort, which is lower than the percentage reported previously. Thus, our study suggests that the contribution of SUN5 mutations to acephalic spermatozoa might not be as high as described previously. These results will help in the genetic counseling of patients with acephalic spermatozoa.

Essential role for SUN5 in anchoring sperm head to the tail.

SUN (Sad1 and UNC84 domain containing)-domain proteins are reported to reside on the nuclear membrane playing distinct roles in nuclear dynamics. SUN5 is a new member of the SUN family, with little knowledge regarding its function. Here, we generated Sun5-/- mice and found that male mice were infertile. Most Sun5-null spermatozoa displayed a globozoospermia-like phenotype but they were actually acephalic spermatozoa. Additional studies revealed that SUN5 was located in the neck of the spermatozoa, anchoring sperm head to the tail, and without functional SUN5 the sperm head to tail coupling apparatus was detached from nucleus during spermatid elongation. Finally, we found that healthy heterozygous offspring could be obtained via intracytoplasmic injection of Sun5-mutated sperm heads for both male mice and patients. Our studies reveal the essential role of SUN5 in anchoring sperm head to the tail and provide a promising way to treat this kind of acephalic spermatozoa-associated male infertility.