Loss of Cylc1 or Cylc2 results in impaired male fertility.

(A) Schematic representation of the Cylc1 and Cylc2 gene structure and targeting strategy for CRISPR/Cas9-mediated generation of Cylc1- and Cylc2-deficient alleles. Targeting sites of guide RNAs are depicted by red arrows. Genotyping primer binding sites are depicted by black arrows.

(B) Representative genotyping PCR of Cylc1- and Cylc2-deficient mice. N=3

(C) Fertility analysis of Cylicin-deficient mice visualized by mean litter size and pregnancy rate (%) in comparison to wildtype matings. Black dots represent mean values obtained for each male included in fertility testing. Columns represent mean values +/− standard deviation (SD). Total number of offspring per total number of pregnancies as well as total number of pregnancies per total number of plugs are depicted above each bar.

(D) Expression of Cylc1 and Cylc2 in testicular tissue of WT, Cylc1−/y, Cylc2+/−, Cylc2−/−, Cylc1−/y Cylc2+/− and Cylc1−/y Cylc2−/− mice analyzed by qRT PCR. Biological replicate of 3 was used.

(E) Immunofluorescent staining of testicular tissue and cauda epididymal sperm from WT, Cylc1−/y, Cylc2+/−, Cylc2−/−, Cylc1−/y Cylc2+/− and Cylc1−/y Cylc2−/− males against CYLC1 and CYLC2. Cell nuclei were counterstained with DAPI. Staining was performed on 3 animals from each genotype. Scale bar: 5 µm.

(F) Schematic illustration of CYLC localization during spermiogenesis. CYLC localization (green) is shown for round and elongating spermatids as well as mature sperm.

(G) Representative immunoblot against CYLC1 and CYLC2 on cytoskeletal protein fractions from WT, Cylc1−/y, Cylc2+/− and Cylc2−/− testes. α-Tubulin was used as load control.

Sperm morphology is severely altered in Cylicin-deficient mice

(A) Testis weight [mg] and sperm count (x107) of WT, Cylc1−/y, Cylc2+/−, Cylc2−/−, Cylc1−/y Cylc2+/− and Cylc1−/y Cylc2−/− males. Mean values +/− SD are shown; block dots represent data points for individual males.

(B) Comparable photographs of the testes of WT, Cylc1−/y, Cylc2+/−, Cylc2−/−, Cylc1−/y Cylc2+/− and Cylc1−/y Cylc2−/− mice.

(C) Epididymal sperm count (x107) of WT, Cylc1−/y, Cylc2+/−, Cylc2−/−, Cylc1−/y Cylc2+/− and Cylc1−/y Cylc2−/− males. Mean values +/− SD are shown; block dots represent data points for individual males.

(D) Viability of the epididymal sperm stained with Eosin Nigrosine. Percentage of Eosin negative (viable) and Eosin positive (inviable) sperm is shown. Data represented as mean +/− SD. Staining was performed on 3 animals from each genotype.

(E) Bright field microscopy pictures of epididymal sperm from WT, Cylc1−/y, Cylc2+/− and Cylc2−/− mice. Scale bar: 10 μm.

(F) Immunofluorescence staining of epididymal sperm acrosomes with PNA lectin (green) and tails with MITOred (red). Nuclei were counterstained with DAPI. Scale bar: 5 µm.

(G) Quantification of abnormal sperm of WT, Cylc1−/y, Cylc2+/−, Cylc2−/−, Cylc1−/y Cylc2+/− and Cylc1−/y Cylc2−/− mice is shown. Acrosome aberrations and tail coiling were counted separately. Staining was performed on 3 animals from each genotype.

(H) Nuclear morphology analysis of WT, Cylc1−/y, Cylc2+/−, Cylc2−/−, Cylc1−/y Cylc2+/− and Cylc1−/y Cylc2−/− sperm. Number of cells analyzed for each genotype is shown.

(I) Representative pictures of immunofluorescent staining against PT proteins CCIN (upper panel) and CAPZa3 (lower panel) in WT, Cylc1−/y, Cylc2+/−, Cylc2−/−, Cylc1−/y Cylc2+/− and Cylc1−/y Cylc2−/− sperm. Nuclei were counterstained with DAPI. Staining was performed on 3 animals from each genotype. Scale bar: 5 µm. (J-K) Quantification of sperm with abnormal calyx integrity in WT, Cylc1−/y, Cylc2+/−, Cylc2−/−, Cylc1−/y Cylc2+/− and Cylc1−/y Cylc2−/− mice based on CCIN and CapZA staining patterns.

Cylc2−/− sperm cells have altered flagellar beat

(A) TEM micrographs of WT, Cylc1−/y and Cylc2−/− epididymal sperm. Acrosome appears detached from the nucleus in Cylc2−/− sperm (green arrowheads), while the calyx is missing entirely (red arrowheads). The head-tail connecting piece shifted from the basal plate is shown by white arrowheads causing the looping of the flagellum and formation of a cytoplasmatic sac. Cylc1−/y sperm appears comparable to WT. Scale bar: 1 µm.

(B) Motility of the epididymal sperm of WT, Cylc1−/y, Cylc2+/−, Cylc2−/−, Cylc1−/y Cylc2+/− and Cylc1−/y Cylc2−/− males activated in TYH medium.

(C) Full and half-beat cycle plots of the flagellar beat are shown for WT and Cylc2−/− spermatozoa. Half beat cycle shows the stiffness of the midpiece (upper arrow) and high oscillations (lower arrow) in Cylc2−/− sperm in one direction of the beat.

Cylicins are required for acrosome attachment to the nuclear envelope

(A) PNA-FITC lectin immunofluorescence staining of the acrosome in testicular tissue of WT, Cylc1−/y, Cylc2+/−, Cylc2−/−, Cylc1−/y Cylc2+/− and Cylc1−/y Cylc2−/− mice (green). Golgi phase of acrosome biogenesis at round spermatid stage (I-IV) is visible in the left panel. Middle panel shows cap phases on round spermatids (stage V-VIII). In the right panel acrosomal phase is shown (stage IX-XI). Nuclei were counterstained with DAPI. Staining was performed on 3 animals from each genotype. Scale bar: 10 µm. Insets show representative single spermatids at higher magnification (scale bar: 2 µm).

(B) PAS staining of testicular sections from WT, Cylc1−/y, Cylc2+/−, Cylc2−/−, Cylc1−/y Cylc2+/− and Cylc1−/y Cylc2−/− mice. Representative spermatids at different steps of spermiogenesis are shown. Scale bar: 3 µm.

(C) TEM micrographs of testicular tissues of WT and Cylc2−/− mice. Single spermatids from step 6 to step 16 are shown. nu: nucleus; av: acrosomal vesicle; pr: perinuclear ring; m: manchette microtubules; cy: cytoplasm; green arrowheads: developing acrosome; red arrowheads: manchette; white arrowhead: cytoplasm; yellow arrowhead: remaining microtubules in mature sperm. Scale bar: 1 µm.

Cylc2 deficiency causes delay in manchette removal

(A) Immunofluorescence staining of α-Tubulin to visualize manchette structure in squash testis samples of WT, Cylc1−/y, Cylc2+/−, Cylc2−/−, Cylc1−/y Cylc2+/− and Cylc1−/y Cylc2−/− mice. Spermatids in different steps of spermiogenesis were shown, for step-to-step comparison. Scale bar: 5 µm.

(B) Quantification of manchette length in WT, Cylc1−/y, Cylc2+/−, Cylc2−/−, Cylc1−/y Cylc2+/− and Cylc1−/y Cylc2−/− α-Tubulin stained spermatids at step 10-13.

(C) Co-staining of the manchette with HOOK1 (red) and acrosome with PNA-lectin (green) is shown in round, elongating and elongated spermatids of WT (upper panel) and Cylc2−/− mice (lower panel). Scale bar: 3 µm. Schematic representation shows acrosomal structure (green) and manchette filaments (red).

Species phylogeny with branch length representing number of nucleotide substitutions per codon with schematic representation of (A) CYLC1 and (B) CYLC2 amino acid alignment used in the PAML CodeML analysis. Alignments were stripped of columns with gaps in more than 80% of species. Evolutionary rate (ω) obtained by CodeML models M0 is shown for the whole alignment. The graph on top shows the evolutionary rate (ω) per codon sites across the whole tree (CodeML model M2a). Significantly positively selected sites are marked by asterisks. Sites with a probability of higher than 0.95 to belonging to the conserved or positively selected site class are marked in green and red respectively below the graph.

Cylicins are required for human male fertility

(A) Pedigree of patient M2270. His father (M2270_F) is carrier of the heterozygous CYLC2 variant c.551G>A and his mother (M2270_M) carries the X-linked CYLC1 variant c.1720G>C in a heterozygous state. Asterisks (*) indicate the location of the variants in CYLC1 and CYLC2 within the electropherograms.

(B) Immunofluorescence staining of CYLC1 in spermatozoa from healthy donor and patient M2270. In donor’s sperm cells CYLC1 localizes in the calyx, while patient’s sperm cells are completely missing the signal. Scale bar: 5 µm.

(C) Bright field images of the spermatozoa from healthy donor and M2270 show visible head and tail anomalies, coiling of the flagellum as well as headless spermatozoa, which carry cytoplasmatic residues without nuclei (white arrowhead). Heads were counterstained with DAPI. Scale bar: 5 µm.

(D-E) Quantification of flagellum integrity (D) and headless sperm (E) in the semen of patient M2270 and a healthy donor.

(F-G) Immunofluorescence staining of CCIN (F) and PLCz (G) in sperm cells of patient M2270 and a healthy donor. Nuclei were counterstained with DAPI. Scale bar: 3 µm.

Semen analysis of the patient M2770 carrying variants in the CYLC1 and CYLC2 genes

Protospacer sequences

PCR primer sequences

qRT primer sequences

Antibodies