HGNC Approved Gene Symbol: CCIN
Cytogenetic location: 9p13.3 Genomic coordinates (GRCh38) : 9:36,169,388-36,171,334 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 9p13.3 | Spermatogenic failure 91 | 620838 | Autosomal recessive | 3 |
In mammalian sperm, the dense cytoplasmic webs surrounding the nuclei contain a complex structure called the perinuclear theca. Longo et al. (1987) determined that 2 kinds of basic proteins are the major constituents of the thecal structure: calicin, a 60-kD protein localized almost exclusively to the calyx, and a group of multiple-band polypeptides (MBPs or cylicins; see 300768) that are found both in the calyx and the subacrosomal layer.
Von Bulow et al. (1995) purified calicin from bull sperm and determined a partial protein sequence. By PCR analysis of testis cDNA with oligonucleotide primers based on the calicin protein sequence, they isolated bovine and human cDNAs encoding calicin. The predicted bovine protein contains 588 amino acids and has a calculated pI of 8.1. The partial human cDNA encodes a protein that starts at a position corresponding to the fourth amino acid of bovine calicin. Overall, the 2 calicins are 91% identical. There are 3 consecutive repeats in the middle of the calicin protein. Sequence comparisons revealed that calicin shares homology with the Drosophila kelch protein, which is associated with the actin-rich intercellular bridges (ring canals) in the egg chamber that connect the oocyte and the nurse cells. Von Bulow et al. (1995) noted that calicin is missing or is arranged in a dramatically different pattern in the heads of malformed human spermatozoa such as teratozoospermias with 'round-headed' sperm or with other 'postacrosomal sheath defects.'
By immunoblot analysis, Lecuyer et al. (2000) showed that calicin was present in protein extracts from boar and human sperm heads. Immunoblot analysis revealed that calicin was present in human spermatogenic cells. Immunofluorescence analysis showed a calyx structure in both boar and human sperm cells. In boar and human testis, calicin was present with actin (see 102560) in the acrosomal region of round spermatids and localized mainly in the postacrosomal region of late spermatids and spermatozoa. HPLC gel filtration analysis of purified boar calicin demonstrated that calicin formed homomultimers in vitro.
Using immunofluorescence assays for calicin on control human spermatozoa, Fan et al. (2022) observed localization to the calyx structure surrounding the posterior portion of the nucleus. In mice, qPCR revealed high expression of Ccin in adult testes. However, the authors noted that expression was not detected in testes at 7, 14, or 21 days postpartum (dpp), a time when germ cells are undergoing meiosis, but became apparent in testes at 28 dpp, coinciding with the process of spermiogenesis in male gonads. The authors concluded that Ccin is preferentially expressed and functions during spermatogenesis in mice.
Gross (2014) mapped the CCIN gene to chromosome 9p13.3 based on an alignment of the CCIN sequence (GenBank AF333334) with the genomic sequence (GRCh37).
Using purified calicin from boar sperm heads, Lecuyer et al. (2000) showed that calicin bound to F-actin with high affinity and with a stoichiometry of approximately 1:12.
By exome sequencing in a cohort of 15 infertile men with globozoospermia or acrosomal hypoplasia (SPGF91; 620838) who were negative for mutation in the known globozoospermia-associated genes SPATA16 (609856) and DPY19L2 (613893), Oud et al. (2020) identified a Lebanese man (GL-12) with globozoospermia who was homozygous for a missense mutation in the CCIN gene (G285S; 603960.0001). The proband was reported to have 3 infertile brothers, but segregation analysis was not performed.
In 2 Chinese brothers (family F1) with infertility and abnormal sperm head morphology, Fan et al. (2022) identified homozygosity for a missense mutation in the CCIN gene (H42L; 603960.0002) that segregated with disease and was not found in public variant databases. Sequencing the coding regions of CCIN in 126 infertile Chinese men with teratozoospermia identified another man (family F2) who was compound heterozygous for a missense mutation (R432W; 603960.0003) and a nonsense mutation (C447X; 603960.0004). Immunofluorescence assays in the probands of both families revealed near-total absence of calicin protein in patient sperm, indicating that all 3 variants represent null mutations.
By whole-exome sequencing in a 30-year-old Han Chinese man with infertility due to asthenoteratozoospermia, He et al. (2023) identified homozygosity for a missense mutation in the CCIN gene (L77P; 603960.0005). Sanger sequencing confirmed the mutation and its presence in heterozygosity in the proband's first-cousin parents; the variant was not found in public variant databases. Functional analysis revealed near-total absence of the mutant protein in patient sperm, with abnormal localization in the sperm head.
Fan et al. (2022) generated knock-in mice with mutations in the Ccin gene corresponding to those detected in infertile men (603960.0002-603960.0004). Both female and male Ccin H42L homozygotes and Ccin R432W/C447X compound heterozygotes developed normally, indicating that Ccin is dispensable for embryonic and postnatal development. Male mutants were infertile, whereas mutant female mice showed no defects in fertility. Immunofluorescence assays, Western blot, and mass spectrometry demonstrated near-total absence of Ccin in mouse spermatozoa. Ectopic expression in HEK293T cells revealed that the mutant proteins were much less soluble than wildtype, suggesting that the mutations impair the stability of the calicin protein. Histologic examination of mutant testes showed normal spermatogenesis with epididymides filled with spermatozoa. However, mutant sperm heads appeared abnormally shaped, and the head defects were confirmed by transmission and scanning electron microscopy, which revealed an increase in length and width, apical defects of the acrosome, and nuclear lacunae devoid of chromatin. In addition, the flagella showed mitochondrial sheath misassembly and tail coiling around the nucleus, and both homozygous and compound heterozygous mutant sperm exhibited a substantial decrease in progressive motility. Ultrastructural quantification of sperm head malformation during development showed that mutant spermatids failed to form an elongated nucleus in the acrosomal phase, and the acrosomal opening was irregular in shape rather than oval. The microtubule bundles of the manchette skirt were unevenly distributed and showed a 'raggedy' appearance, and thus the normal shrinking of the perinuclear ring resulted in nuclear distortion. In vitro fertilization assays showed that mutant spermatozoa failed to adhere to the zona pellucida, and no 2-cell embryos were observed. Intracytoplasmic sperm injection (ICSI) enabled mutant sperm to successfully fertilize the oocytes, and the resultant zygotes developed into 2-cell embryos and then blastulas in vitro; transplantation into pseudopregnant mice resulted in implantation and development into pups with normal fertility.
In a Lebanese man (GL-12) with infertility due to globozoospermia (SPGF91; 620838), Oud et al. (2020) identified homozygosity for a c.853G-A transition (c.853G-A, NM_005893.2) in the CCIN gene, resulting in a gly285-to-ser (G285S) substitution at a conserved residue within a Kelch domain. The proband, who was born of consanguineous parents, was reported to have 3 infertile brothers, but segregation analysis was not performed. The variant was present in the non-Finnish European population of the gnomAD database at a minor allele frequency of 0.0001.
In 2 Chinese brothers (family F1) with infertility due to sperm head defects (SPGF91; 620838), Fan et al. (2022) identified homozygosity for a c.125A-T transversion in the CCIN gene, resulting in a his42-to-leu (H42L) substitution within the highly conserved BTB domain. Their consanguineous parents were heterozygous for the mutation, which was not found the 1000 Genomes Project, ExAC, or gnomAD databases.
In a 30-year-old Chinese man (family F2) with infertility due to sperm head defects (SPGF91; 620838), Fan et al. (2022) identified compound heterozygosity for mutations in the CCIN gene: a c.1294C-T transition, resulting in an arg432-to-trp (R432W) substitution within an evolutionarily conserved Kelch motif, and a c.1341C-A transversion, resulting in a cys447-to-ter (C447X; 603960.0004) substitution. The proband's unaffected parents were each heterozygous for 1 of the mutations. The C447X mutation was not found in public variant databases, whereas the R432W substitution was present at low minor allele frequency in the ExAC and gnomAD databases.
For discussion of the c.1341C-A transversion in the CCIN gene, resulting in a cys447-to-ter (C447X) substitution, that was found in compound heterozygous state in a 30-year-old Chinese man (family F2) with infertility due to sperm head defects (SPGF91; 620838) by Fan et al. (2022), see 603960.0003.
In a 30-year-old Han Chinese man with infertility due to asthenoteratozoospermia (SPGF91; 620838), He et al. (2023) identified homozygosity for a c.230T-C transition in the CCIN gene, resulting in a leu77-to-pro (L77P) substitution. Sanger sequencing confirmed the mutation and its presence in heterozygosity in the proband's first-cousin parents; the variant was not found in the 1000 Genomes Project or gnomAD databases. Immunostaining showed near-total absence of the mutant protein in patient sperm, with abnormal clustering in the sperm head. Transfected HEK293T cells showed significantly reduced expression of the L77P mutant compared to wildtype CCIN.
Fan, Y., Huang, C., Chen, J., Chen, Y., Wang, Y., Yan, Z., Yu, W., Wu, H., Yang, Y., Nie, L., Huang, S., Wang, F., Wang, H., Hua, Y., Lyu, Q., Kuang, Y., Lei, M. Mutations in CCIN cause teratozoospermia and male infertility. Sci. Bull. (Beijing) 67: 2112-2123, 2022. [PubMed: 36546111] [Full Text: https://doi.org/10.1016/j.scib.2022.09.026]
Gross, M. B. Personal Communication. Baltimore, Md. 3/31/2014.
He, J., Liu, Q., Wang, W., Su, L., Meng, L., Tan, C., Zhang, H., Zhang, Q., Lu, G., Du, J., Lin, G., Tu, C., Tan, Y. Q. Novel homozygous variant of CCIN causes male infertility owing to the abnormal sperm head with a nuclear subsidence phenotype. Clin. Genet. 103: 495-497, 2023. [PubMed: 36527329] [Full Text: https://doi.org/10.1111/cge.14281]
Lecuyer, C., Dacheux, J.-L., Hermand, E., Mazeman, E., Rousseaux, J., Rousseaux-Prevost, R. Actin-binding properties and colocalization with actin during spermiogenesis of mammalian sperm calicin. Biol. Reprod. 63: 1801-1810, 2000. [PubMed: 11090452] [Full Text: https://doi.org/10.1095/biolreprod63.6.1801]
Longo, F. J., Krohne, G., Franke, W. W. Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: a novel class of cytoskeletal elements. J. Cell Biol. 105: 1105-1120, 1987. [PubMed: 3308904] [Full Text: https://doi.org/10.1083/jcb.105.3.1105]
Oud, M. S., Okutman, O., Hendricks, L. A. J., de Vries, P. F., Houston, B. J., Vissers, L. E. L. M., O'Bryan, M. K., Ramos, L., Chemes, H. E., Viville, S., Veltman, J. A. Exome sequencing reveals novel causes as well as new candidate genes for human globozoospermia. Hum. Reprod. 35: 240-252, 2020. [PubMed: 31985809] [Full Text: https://doi.org/10.1093/humrep/dez246]
von Bulow, M., Heid, H., Hess, H., Franke, W. W. Molecular nature of calicin, a major basic protein of the mammalian sperm head cytoskeleton. Exp. Cell Res. 219: 407-413, 1995. [PubMed: 7641791] [Full Text: https://doi.org/10.1006/excr.1995.1246]