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 (603960), a 60-kD protein localized almost exclusively to the calyx, and a group of multiple-band polypeptides (MBPs, or cylicins) that are found both in the calyx and the subacrosomal layer.

By screening a human testis cDNA expression library with antibodies against calyx proteins, Hess et al. (1993) cloned partial cDNAs encoding the MBP cylicin-1, named for the Greek word for 'cup' or 'beaker.' The deduced protein contains numerous lysine dipeptides followed by a third variable amino acid (KKX), which in most cases is aspartic acid; these KKX tripeptides occur throughout the molecule, with the exception of the C-terminal tail, which contains proline-rich segments. The central portion of the protein is arranged as a series of repeating units that are predicted to form individual short alpha helices, which are interrupted by short linker segments. Antibodies against human cylicin-1 reacted with 2 polypeptides of 60 kD and 80 kD by immunoblot analysis and stained specifically the calyx of human and bovine spermatozoa by immunofluorescence microscopy. Northern blot analysis of bovine tissues detected a cylicin-1 transcript only in testis.

Schneider et al. (2023) used polyclonal antibodies to visualize the localization of cylicins during spermiogenesis in mice. Immunofluorescence staining showed the presence of both Cylc1 and Cylc2 (604035) from the round spermatid stage onward, first as a cap-like structure lining the developing acrosome in the subacrosomal region, then moving toward the caudal part of the cell as the spermatids elongated. As localization intensified in the postacrosomal calyx region, staining in the subacrosome faded.

Using a rabbit anti-CYLC1 antibody, Jin et al. (2024) observed initial expression of cylicin-1 at the subacrosomal layer in round and elongating mouse spermatids, with later translocation to the postacrosomal region in sperm.

The CYLC1 sequence reported by Hess et al. (1993), GenBank Z22780, maps to Xq21.1 (Amberger, 2009).

Using coimmunoprecipitation assays in HEK293T cells, Jin et al. (2024) demonstrated that CYLC1 interacts with itself as well as several other proteins of the perinuclear theca, including ACTRT1 (300487), ACTRT2 (608535), ACTL7A (604303), CAPZA3 (608722), and CCIN (603960). The ACTL7A-CYLC1 interaction was confirmed by coimmunoprecipitation assays in mouse testis extracts, and microscale thermophoresis revealed a dose-dependent binding affinity between CYLC1 and ACTL7A. In addition, the endogenous interaction between ACTRT1 and ACTL7A was partially disrupted by loss of CYLC1, indicating an assistant role for CYLC1 on the ACTRT1-ACTL7A connection. CYLC1 was also shown to connect with the nuclear envelope (NE) protein FAM209 and inner acrosomal membrane (IAM) protein SPACA1 (612739), with a dose-dependent binding affinity between SPACA1 and CYLC1.

Schneider et al. (2023) reported a 40-year-old German man with infertility due to morphologic abnormalities of the sperm (SPGFX8; 301119) who was hemizygous for a missense mutation in exon 4 of the CYLC1 gene (c.1720G-C; E574Q) and heterozygous for a missense mutation in exon 5 of the CYLC2 gene (c.551G-A; G184D). Both variants were present at low minor allele frequency in the gnomAD database, and both were classified as variants of unknown significance (VUS) by ACMG criteria. The proband's father, who reported difficulties with fertility, was also heterozygous for the G184D variant in CYLC2. No other potentially pathogenic variants in genes associated with sperm morphologic defects were identified by analysis of the proband's exome data.

By whole-exome sequencing in more than 500 Chinese men with infertility due to sperm head deformities, Jin et al. (2024) identified 18 men who were hemizygous for a missense mutation in the CYLC1 gene (K459N; 300768.0001) that was present in gnomAD at a minor allele frequency of 0.001386. The authors suggested that variation in CYLC1 contributes to male infertility, possibly in combination with variation in other male infertility-associated genes.

Using CRISPR/Cas9 gene editing, Schneider et al. (2023) generated mice deficient in Cylc1 and/or Cylc2. Cylc1 -/y male hemizygotes showed significantly reduced pregnancy rates and litter sizes. Cylc2 -/- males were infertile, whereas Cylc2 +/- males showed no significant differences in fertility parameters compared to wildtype mice. Intercrossing mouse lines generated Cylc1 -/y;Cylc2 +/- and Cylc1 -/y;Cylc2 -/- mice; males with either genotype were infertile. A strong decline in sperm counts was observed in all cylicin-deficient males, with the greatest reduction (to only 15% of wildtype) in Cylc1 -/y;Cylc2 -/- mice. Sperm morphology was severely altered in cylicin-deficient mice, with coiling of sperm tails, kinked sperm heads, and acrosomal malformations. Mislocalization of other calyx-specific proteins, such as Ccin (603960) and Capza3 (608722), was also observed. Transmission electron microscopy (TEM) confirmed the structural defects observed by light microscopy, showing tail coiling, dislocation of the head-tail connecting piece from the basal plate, excess cytoplasm, absent posterior portion of the perinuclear theca calyx, and loosening of the periacrosomal region. In addition, motility of Cylc2 -/- sperm was markedly reduced (7% motile sperm), and that of Cylc1 -/7;Cylc2 -/- sperm was even lower (2%); the little motility observed was not progressive, but circular. Analysis of spermiogenesis in cylicin-deficient males showed that Cylc1- and Cylc2-null mice had gaps in the forming acrosome, as well as an irregular shape of the cap. In Cylc1 -/y;Cylc2 +/- and Cylc1 -/y;Cylc2 -/- mice, most round spermatids were deformed or displayed irregularly localized caps, and Cylc2 -/- and Cylc1 -/y;Cylc2 -/- mice showed detachment of the acrosome from the NE. Immunofluorescence staining to analyze formation and development of the manchette revealed abnormal elongation and disassembly in cylicin-deficient spermatids, associated with loosening of the acrosome.

Using CRISPR/Cas9 technology, Jin et al. (2024) generated Cylc1 knockout (KO) male mice. The KO mice exhibited severe subfertility, with significantly reduced pregnancy rates and litter sizes. Papanicolaou staining of KO sperm showed abnormal head morphologies, with varying degrees of a 'fan-shaped' head. Scanning electron microscopy confirmed the fan-shaped sperm heads. Examination of the acrosome structure using peanut agglutinin (PNA)-FITC staining revealed varying degrees of acrosome detachment from the NE in KO spermatids. TEM confirmed the acrosome detachment in KO sperm, and showed an enlarged space between the IAM and the NE in elongating spermatids within the testis. In addition, there was perturbation of the acrosome reaction (AR) in KO sperm, as shown by significantly reduced rates of calcium ionophore-induced AR in KO sperm and lower percentages of 2-cell embryos using KO sperm for in vitro fertilization compared to wildtype sperm. The authors generated Cylc1-mutant mice carrying a mutation equivalent to the human K459N variant (300768.0001), and observed that the mutant males were subfertile. Similar to the Cylc1-KO mice, the mutants produced sperm with abnormal head morphologies, exhibiting varying degrees of fan-shaped heads. Mutant spermatids showed varying degrees of acrosome detachment, confirmed by TEM, which also showed an enlarged space between the IAM and the NE. Filamentous (F)-actin bundles of mutant spermatids showed uneven, thin, or ectopic distribution. Interactions between Cylc1 and its partners were altered by the mutation, including attenuation of the endogenous interaction between Cylc1 and Actl7a and between Cylc1 and the IAM protein Spaca1 in mutant testis lysates. In addition, the endogenous interaction between Actl7a and Spaca1 was affected by mutation in Cylc1.