Figures and data
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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.
(C) Fertility analysis of Cylicin-deficient mice visualized by mean litter size and pregnancy rate (%) in comparison to wild type 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.
(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. Scale bar: 5 µm.
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Loss of Cylicins impairs spermiogenesis
(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) PAS staining of testicular sections from WT, Cylc1−/y, Cylc2+/-, Cylc2−/-, Cylc1 −/y Cylc2+/- and Cylc1−/y Cylc2−/- mice. Scale bar: 100 µm.
(D) 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.
(E) 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.
(F) Motility of the epididymal sperm of WT, Cylc1−/y, Cylc2+/-, Cylc2−/-, Cylc1 −/y Cylc2+/- and Cylc1−/y Cylc2−/- males activated in TYH medium.
(G) 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.
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Sperm morphology is severely altered in Cylicin deficient mice
(A) TEM micrographs of WT 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. Scale bar: 1 µm.
(B) Immunofluorescence staining of epididymal sperm acrosomes with PNA lectin (green) and tails with MITOred (red). Nuclei were counterstained with DAPI. Scale bar: 5 µm.
(C) 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.
(D) 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. Scale bar: 5 µm.
(E) 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.
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 upper panel. Middle panel shows cap phases on round spermatids (stage V-VIII). In the bottom panel acrosomal phase is shown (stage IX-XI. Nuclei were counterstained with DAPI. Scale bar: 10 µm.
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Cylc2 deficiency causes delay in manchette removal
(A) Immunofluorescence staining of Tubulin manchette structure in the squash testis samples of WT, Cylc1−/y, Cylc2+/-, Cylc2−/-, Cylc1 −/y Cylc2+/- and Cylc1−/y Cylc2−/- mice. Spermatids in different stages of spermiogenesis were shown, for step-to-step comparison Scale bar: 5 µm.
(B) Co-staining of the manchette with HOOK1 and acrosome with PNA-lectin is shown in round, elongating and elongated spermatids of WT (upper panel) and Cylc2−/- mice (lower panel). Schematic representation shows acrosomal structure (green) and manchette filaments (red).
(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 Scale bar: 1 µm.
(D) TEM micrographs of degrading damaged spermatids in testicular sections of Cylc2−/- mice. Scale bar: 5 µm.
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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.
(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 who carry cytoplasmatic residues without nuclei. Heads were counterstained with DAPI.
(D) Immunofluorescence staining of the CCIN in the human sperm cells. While in healthy sperm CCIN locates in the funnel shaped calyx, sperm from M2270 show irregular expression of CCIN and lack of calyx.
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Semen analysis of the patient M2770 carrying variants in the CYLC1 and CYLC2 genes
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Protospacer sequences
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PCR primer sequences
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qRT primer sequences
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