Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human

Schneider S, Kovacevic A, Mayer M, Dicke AK, Arévalo L, Koser SA, Hansen JN, Young S, Brenker C, Kliesch S, Wachten D, Kirfel G, Strünker T, Tüttelmann F, Schorle H, 28.11.2023

Abstract

Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

Simon Schneider Andjela Kovacevic Michelle Mayer Ann-Kristin Dicke Lena Arévalo Sophie A Koser Jan N Hansen Samuel Young Christoph Brenker Sabine Kliesch Dagmar Wachten Gregor Kirfel Timo Strünker Frank Tüttelmann Hubert Schorle (2023) Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human eLife 12:RP86100

Publication: https://doi.org/10.7554/eLife.86100.3

Disclaimer

The publication Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human by Schneider S, Kovacevic A, Mayer M, Dicke AK, Arévalo L, Koser SA, Hansen JN, Young S, Brenker C, Kliesch S, Wachten D, Kirfel G, Strünker T, Tüttelmann F, Schorle H is published under an open access license: https://creativecommons.org/licenses/by-nc-nd/4.0/. Permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

Curation by the MFGA team
Relevant data sets presented in the publication have been identified. If possible, annotations (title, general information, conditions, processed tissue types and processed cell types) have been added based on information from the publication. Data tables and images that provide a good overview on the publication's findings on the data set have been extracted from the publication and/or supplement. If not stated otherwise, images are depicted with title and description exactly as in the publication. Tables have been adjusted to the MFGA table format. Conducted adjustments are explained in the detailed view of the tables. However, titles and descriptions have been adopted from the publication.

Data set 1: Cylicins are indispensable for male fertility in mice

Genome: Genotyping

Species

Species
Mouse

Tissue Types

BRENDA tissue ontology	Maturity	Description	Species	Replicates
BTO_0001363: testis	sexually mature males at the age of 8–15 weeks	A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity.	Mouse

Cell Types

Cell ontology	Maturity	Description	Species	Replicates	Cells per replicate
CL_0000018: spermatid		A male germ cell that develops from the haploid secondary spermatocytes. Without further division, spermatids undergo structural changes and give rise to spermatozoa.	Mouse

Images

Figure 1: 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 (WT) 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 quantitative reverse transcription-polymerase chain reaction (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 three 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.

Source: Simon Schneider Andjela Kovacevic Michelle Mayer Ann-Kristin Dicke Lena Arévalo Sophie A Koser Jan N Hansen Samuel Young Christoph Brenker Sabine Kliesch Dagmar Wachten Gregor Kirfel Timo Strünker Frank Tüttelmann Hubert Schorle (2023) Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human eLife 12:RP86100

Licensed under: https://creativecommons.org/licenses/by-nc-nd/4.0/

Data set 2: Sperm morphology is severely altered in Cylicin-deficient mice

Proteome: Immunofluorescence

Species

Species
Mouse

Tissue Types

BRENDA tissue ontology	Maturity	Description	Species	Replicates
BTO_0001230: semen	sexually mature males at the age of 8–15 weeks	A viscid whitish fluid of the male reproductive tract consisting of spermatozoa suspended in secretions of accessory glands.	Mouse

Cell Types

Cell ontology	Maturity	Description	Species	Replicates	Cells per replicate
CL_0000019: sperm		A mature male germ cell that develops from a spermatid.	Mouse

Images

Figure 2: Sperm morphology is severely altered in Cylicin-deficient mice.

(A) Testis weight (mg) and sperm count (×107) of wildtype (WT), Cylc1-/y, Cylc2+/-, Cylc2-/-, Cylc1-/y Cylc2+/-, and Cylc1-/y Cylc2-/- males. Mean values ± SD are shown; black 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 (×107) of WT, Cylc1-/y, Cylc2+/-, Cylc2-/-, Cylc1-/y Cylc2+/-, and Cylc1-/y Cylc2-/- males. Mean values ± SD are shown; black dots represent data points for individual males. (D) Viability of the epididymal sperm stained with Eosin-Nigrosin. Percentage of Eosin negative (viable) and Eosin positive (inviable) sperm is shown. Data represented as mean ± SD. Staining was performed on three 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 peanut agglutinin (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 three 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 perinuclear theca (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 three 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.

Licensed under: https://creativecommons.org/licenses/by-nc-nd/4.0/

Data set 3: Cylicins are required for normal sperm morphology in human

Exome: Whole Exome Sequencing

Species

Species
Human

Tissue Types

BRENDA tissue ontology	Maturity	Description	Species	Replicates
BTO_0000553: peripheral blood	adult	Blood circulating throughout the body.	Human	2066

Cell Types

Cell ontology	Maturity	Description	Species	Replicates	Cells per replicate
CL_0000542: lymphocyte	adult	A lymphocyte is a leukocyte commonly found in the blood and lymph that has the characteristics of a large nucleus, a neutral staining cytoplasm, and prominent heterochromatin.	Human	2066

Data set 4: Cylicins are required for normal sperm morphology in human Part 2

Proteome: Immunofluorescence

Species

Species
Human

Tissue Types

BRENDA tissue ontology	Maturity	Description	Species	Replicates
BTO_0001230: semen		A viscid whitish fluid of the male reproductive tract consisting of spermatozoa suspended in secretions of accessory glands.	Human	2

Cell Types

Cell ontology	Maturity	Description	Species	Replicates	Cells per replicate
CL_0000019: sperm		A mature male germ cell that develops from a spermatid.	Mouse	2

Images

Figure 3: 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.

Licensed under: https://creativecommons.org/licenses/by-nc-nd/4.0/

Data set 5: Cylc2-/- sperm cells have altered flagellar beat

Other: Transmission electron microscopy

Species

Species
Mouse

Tissue Types

BRENDA tissue ontology	Maturity	Description	Species	Replicates
BTO_0001230: semen		A viscid whitish fluid of the male reproductive tract consisting of spermatozoa suspended in secretions of accessory glands.	Mouse

Cell Types

Cell ontology	Maturity	Description	Species	Replicates	Cells per replicate
CL_0000019: sperm		A mature male germ cell that develops from a spermatid.	Human

Images

Figure 4: Cylc2-/- sperm cells have altered flagellar beat.

(A) Transmission electron microscopy (TEM) micrographs of wildtype (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.

Licensed under: https://creativecommons.org/licenses/by-nc-nd/4.0/