2024.01.11.24301040v1.full-html.html

Disruption in

CYLC1

leads to acrosome

detachment, sperm head deformity, and male

in/subfertility in humans and mice

Hui-Juan Jin,

a,f

Yong Fan,

b,f

Xiao-Yu Yang,

c,f

Yue Dong,

Xiao-Zhen Zhang,

Xin-Yan Geng,

Zheng Yan,

Ling Wu,

Meng Ma,

Bin Li,

Qi-Feng Lyu,

Yun

Pan,

Ming-Xi Liu,

e,*

Yan-Ping Kuang,

b,*

and Su-Ren Chen,

a,*

Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of

Education, Department of Biology, College of Life Sciences, Beijing Normal

University, 100875 Beijing, China;

Department of Assisted Reproduction,

Shanghai Ninth

People’s Hospital, Shanghai Jiao Tong University School of

Medicine, Shanghai 200011, China;

State Key Laboratory of Reproductive

Medicine and Offspring Health, The Center for Clinical Reproductive Medicine,

The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China;

State Key Laboratory of Reproductive Medicine and Offspring Health,

Department of Histology and Embryology, School of Basic Medical Sciences,

Nanjing Medical University, Nanjing 210029, China;

State Key Laboratory of

Reproductive Medicine and Offspring Health, The Affiliated Taizhou People’s

Hospital of Nanjing Medical University, Taizhou School of Clinical Medicine,

Nanjing Medical University, Nanjing 211166, China

These authors contributed equally to this study

*For correspondence:

mingxi.liu@njmu.edu.cn (M-XL)

kuangyanp@126.com (Y-PK)

chensr@bnu.edu.cn (S-RC)

Competing interests:

The authors declare that no competing interests exist.

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NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

Abstract

The perinuclear theca (PT) is a dense cytoplasmic web encapsulating the sperm

nucleus. The physiological roles of PT in sperm biology and the clinical relevance

of variants of PT proteins to male infertility are still largely unknown. We reveal

that cylicin-1, a major constituent of the PT, is vital for male fertility in both mice

and humans. Loss of cylicin-1 in mice leads to a high incidence of malformed

sperm heads with acrosome detachment from the nucleus. Cylicin-1 interacts

with itself, several other PT proteins, the inner acrosomal membrane (IAM)

protein SPACA1, and the nuclear envelope (NE) protein FAM

209 to form an ‘IAM-

cylicins-

NE’ sandwich structure, anchoring the acrosome to the nucleus. WES of

more than 500 Chinese infertile men with sperm head deformities was performed

and a

CYLC1

variant was identified in 19 patients.

Cylc1

-mutant mice carrying

this variant also exhibited sperm acrosome/head deformities and reduced fertility,

indicating that this

CYLC1

variant most likely affects human male reproduction.

Furthermore, the outcomes of assisted reproduction were reported for patients

harbouring the

CYLC1

variant. Our findings demonstrate a critical role of cylicin-

1 in the sperm acrosome-nucleus connection and suggest

CYLC1

variants as

potential risk factors for human male fertility.

Keywords

: Perinuclear theca, CYLC1, Male infertile, Sperm head deformity,

Acrosome detachment

Introduction

In mammalian sperm, the cytoskeletal element encapsulating the nuclei is a

detergent- and high salt-resistant structure called the perinuclear theca (PT) (

Oko

et al., 2009

). Apically, the PT resides between the inner acrosomal membrane

(IAM) and the nuclear envelope (NE) making up the subacrosomal layer (PT-SAL,

also known as the acroplaxome), while caudally, it resides between the cell

membrane and the NE making up the postacrosomal part (PT-PAR, also known

as the calyx). The PT is not composed of traditional cytoskeletal proteins but

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rather of a variety of spermatid-specific cytosolic and nuclear proteins. Longo et

al. determined that two kinds of basic proteins are the major constituents of PT:

calicin and cylicins (

Longo et al., 1987

). After that, more than twenty PT-enriched

proteins, such as fatty acid-binding protein (FABP)9 (

Oko et al., 1994

), actin-

capping protein

(CP) α3/β3 (

Bulow et al., 1997

), transcription factor STAT4

(

Herrada et al., 1997

), actin-related proteins ACTRT1/2 (

Heid et al., 2002

histone H2B variant H2BL1 (

Aul et al., 2002

), sperm-born oocyte activating

factors (PLC

ζ and PAWP) (

Aarabi et al., 2004; Hachem et al., 2017

), and the

glutathione S-transferase GSTO2 (

Hamilton et al., 2017

), have been identified

from mammalian sperm.

The PT is speculated to function as 1) an architectural element involved in the

shape changes of the nucleus during spermiogenesis, 2) a cement linking the

acrosome with the nucleus, and 3) a candidate for sperm-borne oocyte activation.

However, the physiological roles of PT in sperm are largely uncertain due to the

lack of evidence from gene knockout animal models. Our recent studies of

Ccin

and

Actrt1

-knockout mice indicate that PT, at least, physiologically functions to

protect nuclear structure and anchor developing acrosomes to the nucleus

(

Zhang et al., 2022a; Zhang et al., 2022b

). Loss of calicin specifically leads to

the surface subsidence of sperm heads in the nuclear condensation stage

(

Zhang et al., 2022a

). Importantly, we further report that three infertile men with

sperm head deformities carry homozygous pathogenic mutations in

CCIN

, and

the corresponding

Ccin

-mutant mice mimic the phenotype in patients (

Fan et al.,

2022

Actrt1

-KO male mice are severely subfertile as a result of detached

acrosomes and fertilization deficiency (

Zhang et al., 2022b

). ACTRT1 anchors

acrosomes to the nucleus by interacting with some IAM proteins and NE proteins

(

Zhang et al., 2022b

Hess et al. cloned cDNAs encoding cylicin-1, named for the Greek word for

‘cup’ or ‘beaker’ (

Hess et al., 1993

). 74 kDa cylicin-1 contains numerous lysine

dipeptides followed by a third variable amino acid (KKX). The C-terminal tail

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contains proline-rich segments and the central portion of the protein is arranged

as a series of repeating units that are predicted to form short alpha helices.

Northern blotting detects the cylicin-1 transcript only in the testis, and an antibody

against cylicin-1 specifically stains the PT-PAR of human and bovine sperm

(

Hess et al., 1993

Apart from the first expression study of calicin-1 published

30 years ago, the physiological role of cylicin-1 in sperm biology is still unknown.

In this study, we generated a

Cylc1

-KO mouse model and revealed that loss

of cylicin-1 leads to severe male subfertility as a result of sperm head deformities

and acrosome detachment. A

CYLC1

variant was identified from a WES study of

infertile patients with sperm head deformities. The contribution of this variant to

male subfertility with sperm acrosome detachment is further confirmed by animal

evidence from

Cylc1

-mutant mice.

Results

Generation of

Cylc1

-KO mice

Cylicin-1 is a major and specific constituent of the PT of mammalian sperm

(

Longo et al., 1987

). According to the Expression Atlas database, cylicin-1 was

specifically expressed in testis tissues, and restricted to early and late spermatid

subpopulations (

Figure 1A and Figure 1

—figure supplement 1

). Using our

homemade rabbit anti-CYLC1 antibody (

Figure 1

—figure supplement 2

), we

showed that cylicin-1 was initially expressed at the subacrosomal layer (SAL) in

round and elongating spermatids and then translocated to the postacrosomal

region (PAR) in sperm (

Figure 1B

). The dynamic expression pattern of cylicin-1

is quite similar to that of CAPZA3 (

Bulow et al., 1997

), calicin (

Zhang et al.,

2022a

), and ACTRT1 (

Zhang et al., 2022b

), all showing SAL-to-PAR

translocation during spermiogenesis.

To explore the physiological role of cylicin-1, we disrupted its function in mice

by applying CRISPR/Cas9 technology (

Figure 1C

). The

Cylc1

gene has only one

transcript and a 3651 bp region containing exons 3 and 4 was selected as the

knockout region (

Figure 1

—figure supplement 3

). The

Cylc1

gene is located on

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the X-chromosome; accordingly, female

Cylc1

+/-

founder mice were crossed with

WT males to generate

Cylc1

-/Y

(hereafter called

Cylc1

-KO) mice, which were

tested by genotyping PCR of tail genomic DNA (

Figure 1C

). Western blotting

analysis with anti-cylicin-1 antibody confirmed absence of the cylicin-1 in the

testis protein lysates of

Cylc1

-KO mice (

Figure 1C

Severe male subfertility of

Cylc1

-KO mice

Adult

Cylc1

-KO male mice were normal in appearance, healthy and developed

with no identifiable abnormalities. Given that cylicin-1 is a sperm-specific protein,

we studied the fertility of

Cylc1

-KO male mice by mating them with WT females.

Although

Cylc1

-KO male mice were able to mate normally with females, they

suffered from severe male subfertility. Both the pregnancy rates of mating

females and pup

s that were born from

Cylc1

-KO males were significantly

reduced compared with those from WT males

(

Figure 1D

)

. The testis size and

weight were similar between

Cylc1

-KO mice and their littermate WT mice

(

Figure

—figure supplement 4

)

. Histological examination of

Cylc1

-KO testis and cauda

epididymis revealed generally normal spermatogenesis and sperm production

(

Figure 1

—figure supplement 4

)

Both the sperm count and the total motility

were similar between

Cylc1

-KO mice and WT mice (

Figure 1E

We next examined whether

Cylc1

-KO mice suffer from sperm morphological

abnormalities. Sperm were obtained from the cauda epididymis and analysed by

Papanicolaou staining. The sperm of WT mice showed a

typical ‘hook-shaped’

appearance with a streamlined dorsal part (

Figure 1F

), whereas the

Cylc1

-KO

mice produced sperm with abnormal head morphologies, exhibiting varying

degrees of ‘fan-shaped’ heads (

Figure 1F

Mild ‘fan-shaped’ heads showed a

roughly similar morphology to normal sperm heads but with an obvious corner

curve at the dorsal side (

Figure 1F, second row

while severe ‘fan-shaped’

heads exhibited a quite different morphology and looked

like a ‘fan’ when

observed from the head-tail connection (

Figure 1F, third row

). Sperm with mild

and severe

‘fan-shaped’ heads accounted for approximately 60% and 40% of the

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total sperm in

Cylc1

-KO mice, resp

ectively. The phenotype of ‘fan-shaped’ heads

in sperm of

Cylc1

-KO mice was further identified by scanning electron microscopy

(SEM) (

Figure 1G

Acrosome detachment in

spermatids and sperm of

Cylc1

-KO mice

To explore the reason for the sperm head deformities, we examined the acrosome

structure in

Cylc1

-KO mice by peanut agglutinin (PNA)-FITC staining. In

spermatids of WT mice, the developing acrosome was tightly bound to the

nuclear envelope (NE) of the sperm nucleus. S

permatids of

Cylc1

-KO mice, by

contrast, exhibited varying degrees of acrosome detachment from the NE

(

Figure

)

. Statistical analysis indicated that the ratio of acrosome detachment in

spermatids of

Cylc1

-KO mice was significantly higher than that in WT mice

(

Figure 2B

)

. We further examined PT-SAL, an F-actin-containing plate between

the inner acrosomal membrane (IAM) and NE, using an F-actin fluorescence

probe. Compared with WT mice, spermatids of

Cylc1

-KO mice showed

disturbance of the PT-SAL distribution, and sometimes it broke away from the NE

(

Figure 2C

)

. The percentage of spermatids with abnormal PT-SAL structure in

Cylc1

-KO mice was also significantl

y greater than t

hat in WT mice

(

Figure 2D

)

Under a transmission electron microscope (TEM), the phenomenon of acrosome

detachmen

t in

the sperm

Cylc1

-KO mice was clearly observed

(

Figure 2E

)

The number of sperm with detached acrosomes was significantly higher in

Cylc1

KO m

ice than in W

T mice

(

Figure 2F

)

To explore whether acrosome detachment occurs during spermiogenesis,

we performed TEM analysis of elongating spermatids with

in the tes

tis. TEM

analysis clearly revealed an enlarged space area between the IAM and the NE in

spermatids of

Cylc1

-KO mice

(

Figure 2G

)

. The average PT-SAL thickness

(space

between the IAM and the NE) was significantly larger in the spermatids of

Cylc1

-KO mice than those of in WT

mice

(

Figure 2H

)

. Moreover, the manchette

was generally norm

al in the spermatids of

Cylc1

-KO mice, as revealed by tubulin

fluorescence probe staining an

d TEM analysis

(

Figure 2

—figure supplement 1

)

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We next determined whether sperm acrosome detachment in

Cylc1

-KO mice

affects the acrosome reaction (AR) that is required for physiological fertilization.

Intact acrosomes can be lab

elled with PNA-FITC dye, whereas acrosome-

reacted sperm lack

PNA-FITC signals

(

Hao et al., 2014

)

. Ther

e was no difference

in the ratio of spontaneous AR in sperm between WT mice and

Cylc1

-KO mice;

however, the calcium ionophore A23187-induced AR rate in sperm was

significantly lower in

Cylc1

-KO mice than in W

T mice

(

Figure 2I

)

. We next

performed in vitro fertilization (IVF) using sperm

from

Cylc1

-KO mice or WT mice.

The percentage of two-cell embryos using sperm of

Cylc1

-KO mice was

significantly decreased compared with that of WT mice

(

Figure 2J

)

Cylicin-1 interacts with itself and other PT proteins

To explore the molecular mechanisms underlying the

acrosome-nucleus

connection by calicin-1, the interacting partners of calicin-1 were studied using

protein

‒protein interaction technologies. First, w

e found that FLAG-tagged

CYLC1 could be coimmunoprecipitated with Myc-tagged CYLC1 in HEK293T

cells, demonstrating that cylicin-1 interacts with itself

(

Figure 3

—figure

supplement 1

). The interactions between Myc-tagged CYLC1 and several other

FLAG-tagged PT proteins, such as ACTRT1, ACTRT2, ACTL7A, CAPZA3, and

CCIN, were further revealed by co

immunoprecipitation (co-IP)

assays in

HEK293T cells

(

Figure 3A and Figure 3

—figure supplement 1

). The ACTL7A-

CYLC1 interaction was further confirmed by endogenous co-IP assay using

mouse testis extracts (

Figure 3B

). Moreover, microscale thermophoresis (MST)

analysis indicated a binding affinity between GFP-labelled CYLC1 and purified

ACTL7A in a dose-dependent manner (

Figure 3C

). The assembly of these PT

proteins into a large protein complex suggests that they may play a coordinated

role in acrosome attachment. The protein levels of PT proteins, such as ACTRT1,

ACTRT2, ACTL7A, CCIN, CAPZA3, CAPZB, CYLC2, and PLC

ζ, were similar in

the sperm sample between WT mice and

Cylc1

-KO mice, indicating that the

sperm acrosome detachment in

Cylc1

-KO mice may not be due to altered PT

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protein contents

(

Figure 3A and Figure 3

—figure supplement 2

). Previous

studies have indicated that ACTRT1 interacts with ACTL7A to form sperm PT-

specific actin-related proteins (ARPs), and their knockout/mutant mice show

acrosome detachment (

Xin et al., 2020; Fan et al., 2022

). We found that the

endogenous interaction between ACTRT1 and ACTL7A was partially disrupted

by the loss of cylicin-1 (

Figure 3D

), highlighting the assistant role of cylicin-1 on

the ACTRT1-ACTL7A connection.

Cylicin-1 connects with NE protein FAM209 and IAM protein SPACA1

Given that PT-SAL is physically located between the NE and the IAM, cylicin-1

proteins may interact with NE and IAM proteins to anchor the developing

acrosome to the nucleus. We showed that Myc-tagged CYLC1 was

immunoprecipitated with the FLAG-tagged NE protein FAM209 in HEK293T cells

(

Figure 3E

). The interaction between CYLC1 and FAM209 was further confirmed

by endogenous co-IP using mouse testis extracts (

Figure 3F

) and MST analysis

using GFP-FAM209 cell lysates and purified CYLC1 (

Figure 3G

). FAM209 is an

interacting partner of DPY19L2, and loss of the PT-SAL association and abnormal

acrosome development are observed in its knockout mice (

Castaneda et al.,

2021

). We further found that FLAG-tagged CYLC1 was immunoprecipitated with

the Myc-tagged IAM protein SPACA1 in HEK293T cells (

Figure 3H

). The

interaction between CYLC1 and SPACA1 was further confirmed by endogenous

co-IP using mouse testis extracts (

Figure 3I

) and a dose-dependent binding

affinity between GFP-SPACA1 from cell lysates and recombinant CYLC1 by MST

(

Figure 3J

Spaca1

-deficient spermatids exhibit an attenuated association of the

IAM with the NE (

Fujihara et al., 2012

). SPACA1 has also been shown to interact

with ACTL7A to anchor the developing acrosome to the PT-SAL (

Chen et al.,

2021

). Loss of cylicin-1 partially disrupted the IAM and PT-SAL connections

because the interaction between SPACA1 and ACTL7A was weakened in

Cylc1

KO mice, as revealed by an endogenous co-IP assay using mouse testis extracts

(

Figure 3K

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Identification of

CYLC1

variants in infertile patients with sperm head

deformities

To explore the clinical relevance of cylicin-1 deficiency to human male infertility,

we performed WES analysis in a large cohort of more than 500 infertile patients

with sperm head deformities (

Figure 4A

). These patients were recruited from the

Shanghai Ninth

People’s Hospital of Shanghai Jiao Tong University and the First

Affiliated Hospital of Nanjing Medical University. Intron mutations, synonymous

mutations, variants with allele frequency >1% in the Genome Aggregation

Database (gnomAD, total population) (

Gudmundsson et al., 2022

)

, and “benign”

or “likely benign” variants according to the American College of Medical Genetics

and Genomics (ACMG) (

Gonzales et al., 2022

), were excluded. Testis-

specifically or testis-predominantly expressed genes were retained. We identified

some patients carrying homozygous or compound heterozygous mutations in six

known sperm head deformity-associated genes, including

DPY19L2

(

Koscinski

et al., 2021

ZPBP1

(

Yatsenko et al., 2012

PICK1

(

Liu et al., 2010

ACTL7A

(

Xin et al., 2020

ACTL9

(

Dai et al., 2021

), and

CCIN

(

Zhang et al., 2022a

)

(

Figure 4

—source data 1

). Intriguingly, 19 individuals were identified to harbor

a variant of CYLC1 (GenBank: NM_021118.3): c.1377G>T/p.K459N (

Figure 4B

The variant was predicted to be deleterious through utilization of SIFT (

Ng et al.,

2003

), PolyPhen-2 (

Adzhubei et al., 2013

), and CADD (

Rentzsch et al., 2019

)

(

Table 1

). Moreover, the amino acid residue of this variant was conserved

between humans and mice (

Figure 4C

Sperm acrosome detachment in men harbouring the CYLC1 variant

The rates of morphological normality of sperm decreased to zero in 7 of 19

patients harbouring CYLC1 variant, and the remaining affected individuals had a

morphological normality rate of less than 4% (

Barratt et al., 2017

) (

Table 2

). The

morphology of sperm heads and acrosomes in men harboring the CYLC1

variant

was assessed with Papanicolaou staining (

Figure 5A

), immunofluorescence co-

staining (

Figure 5B

), and TEM analysis (

Figure 5C

). In the healthy control, the

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acrosome covered more than one-third of the anterior sperm head; in contrast,

sperm from men harbouring CYLC1 variant displayed deformed acrosomes

(

Figure 5A

). Costaining of PNA-FITC and cylicin-1 indicated that the PT-PAR

distribution of cylicin-1 was disrupted and the acrosomes were deformed in sperm

from patients harbouring CYLC1 variant (

Figure 5B

). TEM analysis clearly

revealed that acrosomes were detached in sperm from patients harbouring

CYLC1 variant (

Figure 5C

Cylc1

-mutant mice exhibit male subfertility with detached acrosomes of

sperm

We next generated

Cylc1

-mutant mice that carried a single amino acid change

equivalent to the variant in human CYLC1 using CRISPR/Cas9 technology

(

Figure 6A and B

Cylc1

-mutant mice grew normally but were male subfertile.

Of the 42 female mice mated with WT males, 35 were pregnant and gave rise to

281 offspring. In contrast, of the 28 female mice mated with

Cylc1

-mutant males,

only 11 were pregnant, giving rise to 49 offspring (

Figure 6C and D

Cylc1

mutant mice and WT mice showed similar testis sizes and weights

(

Figure 6

—

figure supplement 1

Spermatogenesis and sperm production were generally

norm

al, a

s indicated by hematoxylin-eosin staining of the testis and cauda

epididymis from

Cylc1

-mutant

mice and WT mice (

Figure 6

—figure supplement

)

. Further examination showe

d that the sperm count and total motility of

Cylc1

mutant mice were similar to those

indexes in WT mice

(

Figure 6E and F

)

. Similar

Cylc1

-KO mice,

Cylc1

-mutant mice produced sperm with abnormal head

morphologies

, exhibiting varying degrees of ‘fan-shaped’ heads (

Figure 6G

Compared with the closely attached acrosomes in WT mice,

spermatids from

Cylc1

-mutant

mice exhibited varying degrees of acrosome detachment

(

Figure

)

. The F-actin bundles of spermatid heads in WT mice possessed typical

fibrous textures, whereas spermatids of

Cylc1

-mutant

mice showed uneven, thin,

or ectopic F-actin distribution

(

Figure 6H

)

. The phenotype of sperm acrosome

detachment in

Cylc1

-mutant

mice was further observed by TEM technology

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(

Figure 6I

)

. TEM analysis of elongating spermatids wit

hin the tes

tis revealed an

enlarged space area between the IAM and the NE in

Cylc1

-mutant

mice

(

Figure

)

. Hence,

the above animal evidence suggests that this CYLC

1 variant is

generally responsible for the patient phenotypes.

To explore the molecular mechanisms underlying the detached acrosomes in

Cylc1

-mutant mice, we examined the interactions between cylicin-1 variant and

its partners. Although this variant did

not obviously affect the protein expression

of cylicin-1 and its interactors,

the endogenous interaction between cylicin-1 and

ACTL7A was attenuated in

testis lysates of

Cylc1

-mutant

mice

(

Figure 6J

)

. T

protein‒protein interaction of cylicin-1 and IAM protein SPACA1 in testis lysates

was also weakened in

Cylc1

-mutant mice (

Figure 6K

). Moreover, mutation of

cylicin-1 affected the endogenous interaction between ACTL7A and SPACA1

(

Figure 6L

). These biochemistry experiments suggest that this variant is critical

for cylicin-1 to form acrosome attachment protein complexes.

CYLC1-associated male infertility could be rescued by ICSI treatment

We further evaluated whether CYLC1-associated male infertility could be

overcome by assisted reproductive technologies (ART). IVF experiments have

indicated that

the percentage of two-cell embryos using sperm of

Cylc1

-KO mice

was significantly decreased compared with that of WT mice.

We further

conducted intracytoplasmic sperm injection (ICSI) experiments and found that

two-cell embryos and blastocysts could be obtained upon ICSI using sperm from

Cylc1

-KO male mice with a similar efficiency to that from WT male mice (

Table

—source data 1

). Consistent with the observations in mice, ART for patients

carrying CYLC1 variant indicated that ICSI but not IVF could serve as a promising

treatment (

Table 3

). For patients AAW228, AAG547, AAX199, BBB654, BBB841,

AAT812, 3307, and 3256, IVF treatments all failed, whereas ICSI treatment (with

or without assisted oocyte activation (AOA) generated good-quality embryos.

Live birth and pregnancy was finally obtained from individuals AAW228, AAG547,

AAT812, 3307, and 3256. ICSI treatments (with or without AOA) were directly

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performed using sperm of individuals AAX765, BBA344, BBA396, 3086, 3165,

3190, 3203, 3209, and 3010, and good quality embryos were obtained. Finally,

live birth was acquired for individuals AAX765, BBA344, BBA396, 3086, 3165,

3190, 3203, and 3010 after embryo transplantation.

Discussion

In this study, we thoroughly reveal the physiological role of cylicin-1 and explore

its clinical relevance to male infertility. Cylicin-1 is specifically expressed at the

PT-SAL (between the IAM and the NE) in spermatids and is ultimately distributed

to the PT-PAR in sperm.

Cylc1

-KO male mice are severely subfertile with

acrosome detachment and aberrant head morphology. By recruiting a large

cohort (>500 infertile men with sperm head deformities), a

CYLC1

variant was

identified in 19 individuals by WES.

Cylc1

-mutant mice equivalent to the variant

in human

CYLC1

mimicked the phenomena of patients. Our experimental

observations indicate that cylicin-1 mediates the

acrosome-nucleus connection

across species, and importantly,

CYLC1

variants are linked to male in/subfertility.

PT is a cytoskeletal element encapsulating the sperm nucleus. Compared with

other sperm structures, such as acrosome and axoneme-based flagella, our

understanding of the PT is very limited. Although the morphological structures

and protein compositions of PT are well studied, the physiological roles of sperm

PT structure are still largely unknown. Our recent two studies using

Ccin

-KO and

Actrt1

-KO mice have explored two different physiological functions of sperm PT

—

protecting sperm nuclear structure and mediating acrosome-nuclear connection

(

Zhang et al., 2022a; Zhang et al., 2033b

). Loss of calicin specifically causes

‘surface subsidence’ of the sperm nucleus at the nuclear condensation stage,

and sperm from

Ccin

-KO mice exhibit DNA damage and fertilization failure

(

Zhang et al., 2022a

). ACTRT1 forms a large ARP complex with ACTL7A and

ACTL9 (

Zhang et al., 2022b

Actrt1

Actl7a

-, and

Actl9

-deficient mice all exhibit

acrosome detachment and partial/total fertilization failure (

Xin et al., 2020; Dai

et al., 2021; Zhang et al., 2022b

). Studies from

Actrt1

Actl7a

Actl9

-, and

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Cylc1

-deficient mice unequivocally demonstrate the essential role of PT structure

in mediating acrosome-nucleus connections.

Actrt1

-KO mice and

Cylc1

-KO mice

are severely subfertile rather than completely infertile, indicating that acrosome-

nucleus connections may require additive or synergistic effects of several PT

proteins.

In spermatids, the acrosome is tightly bound to the PT-SAL plate, which in

turn is anchored to the NE of the elongating spermatid nucleus (

Zhang et al.,

2022a

). The

‘IAM-SAL-NE’ sandwich structure plays a central role in acrosome

attachment, and perturbations in this structure are conceivably the major causes

of acrosome detachment from the sperm nucleus. The existing evidence is as

follows: (i) no close association of the IAM with the NE is formed in spermatids of

Spaca1

-KO mice (

Fujihara et al., 2012

); (ii) SPACA1 interacts with PT-SAL

proteins (e.g., CCIN, ACTRT1, and ACTL7A) to anchor the acrosome to the PA-

SAL structure (

Xin et al., 2020; Zhang et al., 2022a; Zhang et al., 2022b

); and

(iii) PT-SAL proteins (e.g., CCIN and ACTRT1) could further connect with NE

proteins (e.g., DPY19L2, FAM209, PARP11 and SPATA46) (

Zhang et al., 2022a;

Zhang et al., 2022b

Cylicin-1 interacts with itself and other PT proteins (e.g., CCIN, ACTRT1,

ACTRT2, ACTL7A, CAPZA3, and CAPZB) to form a PT-SAL plate. Cylicin-1

homopolymer further connects the acrosome to the NE, likely by interacting with

IAM proteins (e.g., SPACA1) and NE proteins (e.g., FAM209). Disruption of the

‘IAM-SAL-NE’ sandwich structure is suggested to be the reason for acrosome

detachment in

Cylc1

-deficient spermatids. Deficiency of cylicin-1 also partially

affects the formation or stability of ACTRT1-ACTL7A and ACTL7A-SPACA1.

However, the physiological organization of

‘IAM-SAL-NE’ protein complexes and

how the CYLC1 variant will affect this structure at the molecular or even atomic

level have not been clear until now.

Mutations in genes encoding PT proteins have been shown to be associated

with male infertility with sperm head deformities. Xin et al. identified a

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homozygous missense mutation of

ACTL7A

in two infertile brothers presenting

sperm acrosomal ultrastructural defects and early embryonic arrest (

Xin et al.,

2020

). WES of 21 male individuals with fertilization failure identified homozygous

pathogenic variants in

ACTL9

in three individuals, also showing sperm acrosome

detachment (

Dai et al., 2021

). We report that three infertile men showing

‘surface

subsidence’ of the sperm nucleus carry homozygous pathogenic mutations in

CCIN

, and the corresponding

Ccin

-mutant mice mimic the phenotype in patients

(

Fan et al., 2022

Infertility is a very heterogeneous condition, with genetic causes involved in

approximately half of cases. Taking the percentage of male in/subfertile may

reach more than 20% of the population

into consideration (

Gnoth et al., 2005

the contribution of multiple genetic factors and common variants to male infertility

should not be omitted. It has been suggest that mouse modelling data are critical

to determine the pathogenicity of point mutations (

Singh et al., 2015

). Our mutant

mouse results further proved that the identified CYLC1 variant is harmful for male

fertility. Similar to

Cylc1

-KO mice,

Cylc1

-mutant mice showed severe male

subfertility with sperm acrosome/head deformities. While our study was under

preparation, another laboratory in parallel reported similar findings that

Cylc1

-KO

mice showed sperm acrosome detachment and male subfertility as a reviewed

preprint in eLife (

Schneider et al., 2023

). This report and our current study

unequivocally demonstrate the essential role of cylicin-1 in mediating sperm

acrosome-nucleus connections and mouse male fertility. Intriguingly, Schneider

et al. indicates that deficiency of

Cylc2

(even in heterozygous state) worsened

the fertility of

Cylc1

-KO mice (from severe subfertility to infertility). Extending to

humans, we suggest that CYLC1 variants contribute to male infertility, especially

when combined with certain variants of other male infertility-associated genes.

This notion may improve our multiperspective understanding of male in/subfertility,

which is a very heterogeneous condition.

After analysing the ART outcomes of the patients harbouring CYLC1 variant,

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Materials and Methods

Mice

Animal experiments were approved by the Animal Care and Use Committee of

the College of Life Sciences, Beijing Normal University (CLS-AWEC-B-2023-001).

Wild-type (WT) C57BL/6J mice were obtained from Charles River Laboratory

Animal Technology (Beijing, China). Exons 3~4 were selected as the knockout

region to generate

Cylc1

-KO mice and a G-to-T substitution at nucleotide position

1407 in

Cylc1

was created by CRISPR/Cas9 technology. In brief, single-guide

RNAs

(gRNAs)

and/or

single-stranded oligonucleotides (ssODNs)

were

transcribed in vitro as described previously (

Zhang et al., 2022a

). Cas9 mRNA

was purchased from TriLink BioTechnologies (CA, USA). Mouse zygotes were

coinjected with an RNA mixture of Cas9 mRNA, sgRNAs, and/or ssODNs. The

injected zygotes were transferred into pseudopregnant recipients to obtain the F0

generation. A stable F1 generation was obtained by mating positive F0 generation

mice with C57BL/6JGpt mice. The founder mice were crossed with WT C57BL/6

mice to obtain offspring. Female

Cylc1

+/-

Cylc1

+/mutant

founder mice were

crossed with WT males to generate

Cylc1

-KO or

Cylc1

-mutant mice, which were

tested by genotyping PCR and/or sequencing of tail genomic DNA using the

Mouse Tissue Direct PCR Kit (Tiangen Biotech, Beijing, China). Primers for

genotyping are provided in

Figure 1

—source data 1

Clinical cohorts

The cohort comprised more than 500 infertile patients with sperm head

deformities recruited between 2015 and 2022 from the Reproductive Medicine

Center, Shanghai Ninth People’s Hospital of Shanghai Jiao Tong University

School of Medicine (Shanghai, China) and the Center for Clinical Reproductive

Medicine, The First Affiliated Hospital of Nanjing Medical University (Nanjing,

China). The study regarding the cohorts was approved by the institutional review

boards and signed informed consent was obtained from all subjects participating

in the study (No.20181101).

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WES and bioinformatic analysis

Genomic DNA was extracted from peripheral blood samples of patients using a

QIAamp DNA Blood Mini Kit (QIAGEN, Germany). The exomes of the subject

were captured by Agilent SureSelect Human All Exon V6 Enrichment kits (Agilent,

CA, USA) and then sequenced on a NovaSeq platform (Illumina, CA, USA)

according to the manufacturer’s instructions. All reads were mapped to the

human reference genome (UCSC Genome Browser hg19) using Burrows‒

Wheeler Alignment (

Li et al., 2010

). Single nucleotide variants and indels were

detected by Genome Analysis Toolkit and then annotated by ANNOVAR

(

McKenna et al., 2010; Wang et al., 2010

). Mutations that met the following

criteria were excluded: i) intron mutations and synonymous mutations; ii) variants

with allele frequency >1% in gnomAD (total population) (

Gudmundsson et al.,

2022

)

; iii) “benign” or “likely benign” variants according to the ACMG (

Gonzales

et al., 2022

). Testis-specifically or testis-predominantly expressed genes were

retained. The identified

CYLC1

variant in patients were verified by Sanger

sequencing using the primers listed in

Figure 4

—source data 2

Expression plasmids and transient transfection

Mouse

Cylc1

cDNA was chemically synthesized by GenScript Biotech

Corporation (Suzhou, China) and inserted into FLAG- or Myc-tagged pCMV

vectors (Beyotime, Shanghai, China). Full-length cDNA encoding CCIN, ACTRT1,

ACTL7A, SPACA1, FAM209 and SPATA46 was amplified by PCR and cloned into

FLAG- or Myc-tagged pCMV vectors as described in our previous studies (

Zhang

et al., 2022a; Zhang et al., 2022b

). Primers for CAP

ZA3: 5’-GGTACCATGTCAC

TCAGCGTCTTGAGTAGG-

3’ and 5’-TCTAGATATTATCCAGTTGCACAACACAC

TTC-

3’. HEK293T line was obtained from ATCC (American Type Culture

Collection) and authenticated using STR profiling test by Shanghai Biowing

Applied Biotechnology Co., Ltd. HEK293T cells were cultured at 37°C in a 5%

incubator with Dulbecco’s modified Eagle’s medium (Gibco, NY, USA) with

10% fetal bovine serum (HyClone, UT, USA) and 1% penicillin‒streptomycin

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(Gibco). Transient transfection of HEK293T cells was performed using

Lipofectamine 3000 transfection reagent (Invitrogen, Shanghai, China) following

the manufacturer’s protocol. Cells were then harvested 48 h after transfection.

Generation of anti-CYLC1 antisera

The N-terminal of mouse CYLC1 was cloned into the pET-N-His-C-His vector

(Beyotime, Shanghai, China) and then transfected into the ER2566 E. coli strain

(Weidi Biotechnology, Shanghai, China). CYLC1 protein expression was induced

by 1 mM IPTG (Beyotime) at 24°C for 6 hours. After centrifugation, the bacterial

pellet was resuspended in buffer 1 (50 mM Tris-HCl pH 8.0, 200 mM NaCl), and

the proteins were released by sonication. After centrifugation, anti-His beads

(Beyotime) were added to the supernatant and incubated for 2 hours. After

washing five times with buffer 1, CYLC1-N protein was eluted with 250 mM

imidazole (Beyotime). The protein was concentrated by centrifugation with

ultrafiltration centrifuge tube (Millipore, Shanghai, China).

One hundred

micrograms of CYLC1-N protein was emulsified at a 1:1 ratio (v/v

) with Freund’s

complete adjuvant (Beyotime) and administered subcutaneously into New

Zealand white rabbits (Charles River) at multiple points. For the subsequent three

immunizations, 50 μg CYLC1-N protein was emulsified with incomplete Freund’s

adjuvant (Beyotime) at an interval of 2 weeks. One week after the last

immunization, blood was collected, and the serum was separated.

Mouse fertility testing

To confirm the fertility of male

mice, natural mating tests were conducted. Briefly,

three

Cylc1

-KO (or

Cylc1

-mutant) and three littermate control sexually mature

male mice (8-12 weeks old) were paired with two 8- to 10-week-old C57BL/6J

females (each male was mated with two female mice) for more than 2 months.

The vaginal plugs of the mice were examined every morning. Then, the female

mice with vaginal plugs were separately fed, and the number of pups per litter

was recorded.

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Semen characteristics analysis

Semen samples of human subjects were collected through masturbation after 2

–

7 days of sexual abstinence and analysed in the source laboratories as part of

the routine biological examination according to the 5th World Health Organization

(WHO) guidelines. For mouse models, the backflushing method was used to

retrieve sperm cells from the cauda epididymis (

Baker et al., 2014

). Sperm

counts were determined using a fertility counting chamber (Makler, Haifa, Israel)

under a light microscope, and sperm mobility was assessed via the application of

a computer-assisted sperm analysis (CASA) system. Sperm were collected from

the cauda epididymis and washed three times in PBS buffer. The sperm

suspension was mounted on a glass slide, air-dried, and fixed with 4% PFA for

10 min at room temperature. The slides were stained with Papanicolaou solution

(Solarbio, Beijing, China) and observed using a DM500 optical microscope (Leica,

Germany).

Cell staining

Spermatogenic cells were isolated from adult mouse testes by digestion with 1

mg/ml collagenase IV, 1 mg/ml hyaluronidase, 1 mg/ml trypsin and 0.5 mg/ml

DNase I (Solarbio) for 20 min on a shaker. A cell suspension containing

spermatids was mounted on a glass slide, air-dried, and fixed with 4% PFA for

10 min at room temperature. After permeabilization with 1% Triton X-100 for 30

min, the slides of sperm and spermatids were blocked with 5% goat serum for 45

min. Anti-cylicin-1 antibody was added to the slide and incubated overnight at

4°C. After washing three times with PBS, slides were incubated with Alexa Fluor

484 labelled donkey anti-rabbit IgG for 1 h at room temperature. For the staining

of the acrosome, PT-SAL and manchette, PNA-FITC dye, Actin-Tracker Red-555

dye and Tubulin-Tracker Red dye were used. The nuclei were counterstained

with DAPI dye.

Transmission electron microscope

Precipitation of mouse sperm and testis tissues (

∼

1 mm

) were fixed with 2.5%

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(vol/vol) glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) for 24 h at 4°C.

As a previous protocol (

Zhang et al., 2022a

), the samples were washed four

times in PB and first immersed in 1% (wt/vol) OsO4 and 1.5% (wt/vol) potassium

ferricyanide aqueous solution at 4°C for 2 h. After washing, the samples were

dehydrated through graded alcohol into pure acetone. Samples were infiltrated in

a graded mixture (3:1, 1:1, 1:3) of acetone and SPI-PON812 resin (21 ml

SPOPON812, 13 ml DDSA and 11 ml NMA), and then the pure resin was

changed. The specimens were embedded in pure resin with 1.5% BDMA,

polymerized for 12 h at 45°C and 48 h at 60°C, cut into ultrathin sections (70 nm

thick), and then stained with uranyl acetate and lead citrate for subsequent

observation and photography with a Tecnai G2 Spirit 120 kV (FEI) electron

microscope. All reagents were purchased from Zhongjingkeyi Technology

(Beijing, China).

Scanning electron microscope

Mouse sperm from the cauda epididymis were fixed in 2.5% phosphate-buffered

glutaraldehyde (GA) (Zhongjingkeyi Technology, Beijing, China) at room

temperature for 30 min and then deposited on coverslips. As described in a

previous protocol (

Zhang et al., 2022a

the coverslips were dehydrated via an

ascending gradient of 50%, 70%, 95%, and 100% ethanol and air-dried.

Specimens were then attached to specimen holders and coated with gold

particles using an ion sputter coater before being viewed with a JSM-IT300

scanning electron microscope (JEOL, Tokyo, Japan).

In vitro fertilization

As described in a previous protocol (

Zhang et al., 2022a

), six-week-old C57BL/6J

female mice were superovulated by injecting 5

IU (0.1 ml) of pregnant mare

serum gonadotropin (PMSG), followed by 5 IU (0.1 ml) of human chorionic

gonadotropin (hCG) 48 h later. The

sperm were released from the cauda

epididymis of 10-week-old male mice,

and sperm capacitation was performed for

50 min using TYH solution. Cumulus-oocyte complexes

(COCs) were obtained

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from the ampulla of the uterine tube at 14 h after

hCG injection. The ampulla was

torn with a syringe needle, and the COCs

were gently squeezed onto liquid drops

of HTF medium. COCs were

then incubated with 5-

10 μl sperm suspension

(sperm concentration:

∼

5×10

) in HTF liquid drops at 37°C under 5% CO2. After

6 h, the eggs

were washed several times using HTF medium to remove the

cumulus cells

and then transferred to liquid drops of KSOM medium. Two-cell

embryos were counted at 24 h postfertilization. All lipid drops were covered with

mineral oil and equilibrated overnight at 37°C under 5% CO

. All reagents were

purchased from Aibei Biotechnology (Nanjing, China).

Intracytoplasmic sperm injection

One-month-old C57BL/6J female mice were superovulated by administration of

10 IU PMSG combined with 10 IU hCG (48 hours later). Oocytes were obtained

from the ampulla of the uterine tube at 14 hours after hCG injection. Mouse sperm

heads were separated from sperm tails and injected into mouse oocytes using a

Piezo-driven pipette (PrimeTech, Osaka, Japan). The injected oocytes were

cultured in KSOM medium at 37°C under 5% CO

. All reagents were purchased

from Nanjing Aibei Biotechnology.

Acrosome reaction

Sperm were collected from the cauda epididymis and capacitated for 50 min in

TYH medium at 37°C under 5% CO

. Highly motile sperm were collected from

the upper portion of the medium, and 10 μM calcium ionophore A23187 (Sigma,

Germany) was added to induce the acrosome reaction. After 15 min, sperm were

spotted on a glass microscope slide, dried, and fixed with 4% PFA for 10 min.

Acrosomes were stained with PNA-FITC dye, and nuclei were labelled with DAPI.

Coimmunoprecipitation

As described in a previous protocol (

Zhang et al., 2022a

), 48 hours after

transfection, HEK293T cells were lysed with Pierce™ IP Lysis Buffer (Thermo

Fisher, 87787) with protease inhibitor cocktail for 30 min at 4°C and then

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centrifuged at 12,000 × g for 10 min. Pierce IP Lysis Buffer was composed of 25

mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol. To

prepare input samples, protein lysates were collected and boiled for 5 min in

1.2×SDS loading buffer. The lysates were precleared with 10 μl Pierce™ Protein

A/G-conjugated Agarose (Thermo Fisher) for 1 h at 4°C. Precleared lysates were

incubated overnight with 2 μg anti-Myc antibody or anti-FLAG antibody at 4°C.

The lysates were then incubated with 20 μl Pierce™ Protein A/G-conjugated

Agarose for 2 h at 4°C. The agarose beads were washed four times with Pierce™

IP Lysis Buffer and boiled for 5 min in SDS loading buffer. Input and IP samples

were analysed by western blotting using anti-FLAG or anti-Myc antibodies. For

endogenous co-

IP, adult mouse testis tissues were lysed with Pierce™ IP Lysis

Buffer. Precleared lysates were separated into two groups: one group was treated

with

2 μg anti-ACTL7A antibody, anti-FAM209 antibody, or anti-SPACA1 antibody,

and another group (negative control) was treated with 2 μg rabbit IgG. Other

endogenous co-IP procedures were similar to the co-IP assay in HEK293T cells.

Antibodies used in this study were listed in

Figure 3

—source data 1

Microscale thermophoresis (MST)

The procedure of prokaryotic expression and purification of CYL

C1 is described

in the paragraph ‘generation of anti-CYLC1 antisera’. MST experiments were

conducted on a Monolith NT.115 system (NanoTemper Technologies, Germany).

GFP-labelled

ACTL7A, SPACA1, or FAM209 was transfected into HEK293T cells

and whole proteins were extracted and then added to 16 PCR tubes (10 μl per

tube). The purified ligand CYLC1 was diluted using MST buffer (50 mM Tris-HCl

pH 7.5, 150 mM NaCl, 10 mM MgCl

, 0.05% (v/v) Tween 20) into 16 gradients.

Ten microliter

s of different concentrations of ligand CYLC1 were mixed with 10

μl of

GFP-

ACTL7A,

GFP-

SPAC

A1, and

GFP-

FAM209 cell lysates and reacted in a

dark box at room temperature for 30 min. The samples were added to monolith

capillaries (NanoTemper) and subsequently subjected to MST analysis.

Western blotting

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As described in a previous protocol (

Zhang et al., 2022a

proteins from

HEK293T cells were extracted using RIPA lysis buffer (Applygen, Beijing, China)

containing 1 mM phenylmethylsulfonyl fluoride and protease inhibitors on ice. For

sperm samples, 1% SDS was added to RIPA lysis buffer. The supernatants were

collected following centrifugation at 12,000 × g for 20 min. Proteins were

electrophoresed in 10% SDS‒PAGE gels and transferred to nitrocellulose

membranes (GE Healthcare). The blots were blocked in 5% milk and incubated

with primary antibodies overnight at 4°C, followed by incubation with anti-rabbit

or anti-mouse IgG H&L (HRP) (Abmart) at a 1/10,000 dilution for 1 h. The signals

were evaluated using Super ECL Plus Western Blotting Substrate (Applygen) and

a Tanon5200 Multi chemiluminescence imaging system (Tanon, Shanghai,

China). Antibodies used in this study were listed in

Figure 3

—source data 1

Statistical analysis

The data are presented as the mean±SEM and compared for statistical

significance using GraphPad Prism version 5.01 (GraphPad Software). Unpaired,

two-

tailed Student’s

test was used for the statistical analyses. Statistically

significant differences are represented as *

<0.05, **

<0.01 and ***

<0.001.

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Acknowledgements

We acknowledge Xi-Xia Li and Shuang-Zhong Lv from the Center for Biological

Imaging (CBI), Institute of Biophysics, Chinese Academy of Sciences for their

help in making TEM samples.

Additional information

Funding

Funder

Grant number

Author

National Natural Science

Foundation of China

32370905

Su-Ren Chen

81901531

Yong Fan

81971374

Xiao-Yu Yang

81871163

Qi-Feng Lyu

82171685

Ling Wu

82271732

Yan-Ping Kuang

81971448

Bin Li

National Key Research and

Development Project

2019YFA0802101

Su-Ren Chen

2022YFC2702702

Ming-Xi Liu

Open Fund of Key Laboratory of

Cell Proliferation and Regulation

Biology, Ministry of Education

None

Su-Ren Chen

Fund for Excellent Young

Scholars of Shanghai Ninth

People’s Hospital, Shanghai Jiao

Tong University School of

Medicine

JYYQ004

Yong Fan

Natural Science Foundation of

Jiangsu Province

BK20230004

Ming-Xi Liu

The funders had no role in study design, data collection and interpretation,

or the decision to submit the work for publication.

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Table 1

. 19 individuals carrying a

CYLC1

variant identified from >500

infertile men with sperm head deformities

Variant

cDNA alternation

c.1377G>T

Protein alteration

p.K459N

Patient(s)

AAG547; BBB654; BBB841; AAX199; AAX765; BBA344; BBB396;

AAW228; AAT812; 3086; 3165; 3172; 3190; 3203; 3209; 3307;

3256; 3010; 2431

Allele frequency in human population

gnomAD genome

0.001386

Functional prediction

SIFT

deleterious

PolyPhen-2

damaging

CADD

15.18

Conservation between mouse and human

Yes

NCBI reference sequence number of

CYLC1

is GenBank: NM_021118.3.

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Table 2

Semen characteristics of individuals harbouring the

CYLC1

variant

Individuals

Infertile for

Semen Characteristics

Volume (ml)

Concentration

(10

/ml)

Motility sperm

(%)

Progressive

motility (%)

Morphological

normality (%)

AAG547

5 y

1.8

50.2

49.2%

43.1%

BBB654

3 y

2.7

50.4

54.9%

51%

BBB841

5 y

52.3

81.1%

62.3%

1~2%

AAX199

3 y+

2.7

55.2%

40.8%

AAX765

5 y

3.2

4.4

35.9%

19.2%

BBA344

3 y

41.9%

36.5%

BBB396

6 y

1.6

12.8

30.8%

23.1%

AAW228

5 y

3.9

7.31

71.6%

39.4%

AAT812

2 y

58.7

21.8%

7.6%

3.6%

3086

1 y

0.9

30.8%

23.1%

3165

1 y

1.2

3172

2 y

151.5

14.1%

9.6%

3190

2 y

22.6

29.5%

26.2%

3203

4 y

1.2

24.1

8.9%

6.2%

3209

5 y

6.4

10.6

14.2%

4.1%

3307

6 y

59.9

77.6%

57.1%

3256

1 y

1.4

66.6

67.5%

53%

3010

2 y

3.4

54.4

13%

10.2%

Reference values

>1.5

>15

>40%

>32%

>4%

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Table 3

. Clinical outcomes of ART using sperm from men harbouring the

CYLC1

variant

-, not applicable; N/A, not available; P, pregnant

Individual

IVF/ICSI

Cycles

IVF

ICSI

ICSI+AOA

Transfer

cycles

(embryos)

Live

birth

Fertilization

rate

Good quality

embryo rate

Fertilization

rate

Good quality

embryo rate

Fertilization

rate

Good quality

embryo rate

AAW228

0% (0/10)

30% (3/10)

0% (0/3)

91.3% (21/23) 66.7% (14/21)

2 (4)

AAG547

10.5% (2/19)

50% (1/2)

78.9% (15/19)

46.7 (7/15)

N/A

3 (6)

AAX199

0% (0/2)

83.3% (5/6)

100% (5/5)

100% (4/4)

5 (8)

BBB654

0% (0/14)

88.9% (8/9)

100% (8/8)

N/A

2 (4)

BBB841

0% (0/5)

50% (3/6)

100% (3/3)

75% (3/4)

100% (3/3)

2 (3)

AAT812

35.7% (5/14)

0% (0/5)

80% (8/10)

50% (4/8)

80% (4/5)

75% (3/4)

2 (4)

3307

0% (1/13)

100% (8/8)

37.5% (3/8)

N/A

1 (1)

3256

0% (0/3)

100% (5/5)

60% (3/5)

N/A

1 (1)

3172

33.3% (1/3)

0% (0/1)

N/A

1 (1)

AAX765

N/A

72.7% (8/11)

0% (0/8)

100% (6/6)

50% (3/6)

2 (3)

BBA344

N/A

94.1% (16/17)

37.5% (6/16)

2 (3)

BBA396

N/A

100% (1/1)

75% (3/4)

100% (3/3)

1 (2)

3086

N/A

100% (12/12)

66.7% (8/12)

N/A

2 (2)

3165

N/A

66.7% (4/6)

75% (3/4)

N/A

1 (2)

3190

N/A

93.7% (15/16)

33.3% (5/15)

N/A

1 (1)

3203

N/A

83.3% (5/6)

60% (3/5)

N/A

1 (1)

3209

N/A

71.4% (5/7)

20% (1/5)

N/A

2 (2)

3010

N/A

70% (7/10)

57.1% (4/7)

N/A

1 (2)

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Figure legend

Figure 1.

Severe male subfertility with sperm head deformity in

Cylc1

-KO mice.

(

)

According

the

Expression

Atlas

database

(https://www.ebi.ac.uk/gxa/home), cylicin-1 was specifically expressed in the

testis. Within the testis, cylicin-1 was restricted to the population of early and

late spermatids.

(

) Costaining of cylicin-1 and PNA-FITC (an acrosome dye)

in mouse round and elongating spermatids (left). Scale bars, 2 μm. Costaining

of cylicin-1 and acetylated-tubulin (a flagella marker) in mouse and human

sperm (right). Scale bars, 5 μm. Nuclei were counterstained with DAPI. SAL,

subacrosomal layer; PAR, postacrosomal region.

(

) Schematic illustration of

the targeting strategy used to generate

Cylc1

-KO mice. DNA extracted from

mouse tails was used for PCR genotyping.

Knockout efficiency was determined

by Western blottin

g using testis protein lysates. β-actin served as a loading

control.

(

) Adult

Cylc1

-KO male mice and their littermate WT mice (

=3 each

group) were continuously coupled with WT female mice at a ratio of 1:2 for 2

months. Data are represented as the mean ±

SEM, Student’s

test, ***

<0.001.

(

) Sperm counts were determined using a fertility counting chamber under a

light microscope, and total motility was assessed via computer assisted semen

analysis (CASA). Data are represented as the mean ± SEM (

=6 each group),

Student’s

test, ns, not significant.

(

) Light microscopy analysis of sperm from

WT mice and

Cylc1

-KO mice (

=3 each) with Papanicolaou staining. The

dashed curve indicates the streamline surface at the dorsal side of sperm heads

of WT mice. The dashed broken lines point to the corner curve at the dorsal

side of sperm heads of

Cylc1

-KO mice. Scale bars, 5 µm.

(

)

Scanning electron

microscopy (SEM) images of sperm from WT mice and

Cylc1

-KO

mice. Scale

bars, 1 µm.

Figure 2.

Acrosome detachment in the spermatids and sperm of

Cylc1

-KO

mice. (

)

Visualization of the acrosome (Acr) in spermatids of WT and

Cylc1

KO mice using the fluorescent dye PNA-FITC. The nuclei (Nu) were

counterstained with DAPI dye. Asterisks indicate detached acrosomes. Scale

bars, 2 µm.

(

) Percentage of spermatids with detached Acr in WT and

Cylc1

KO mice.

(

) Subacrosomal layer of perinuclear theca

(PT-SAL) actin bundles

in spermatids of WT and

Cylc1

-KO mice were visible by an F-actin-Tracker Red

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dye. The asterisk indicates the detachment of Apx from Nu. Scale bars, 2 µm.

(

) Percentage of spermatids with abnormal Apx structure in WT and

Cylc1

-KO

mice. In B and D, 100 spermatids in each group (

=3) were counted. Data are

represented as the mean ± SEM, Student’s

test, ***

<0.001.

(

) Transmission

electron microscopy (TEM) analysis showing detachment of Acr from Nu in the

sperm of

Cylc1

-KO mice. The asterisk indicates the enlarged space area

between the Acr and Nu. Scale bars, 200 nm.

(

)

Percentage of sperm with

detached Acr in WT and

Cylc1

-KO mice as revealed by TEM. Twenty-five

sperm in each group (

=3) were counted.

(

) TEM analysis of testis tissues

revealing detachment of the developing Acr from Nu in spermatids of

Cylc1

-KO

mice. The asterisk indicates the enlarged space area between the Acr and Nu.

Scale bars, 200 nm.

(

) Apx thickness in the sperm of WT mice and

Cylc1

-KO

mice. The thickness of the Apx was measured by distance meter software of a

Tecnai G2 Spirit electron microscope in TEM images at a magnification of

30,000×. Five spermatids were measured in each group (

=3). Data in f and h

are represented as the mean ± SEM, Student’s

test, ***

<0.001.

(

)

Spontaneously underwent and A23187-induced acrosome reaction (AR). PNA-

FITC dye was used to label acrosomes, and the sperm nuclei were stained with

DAPI. One hundred sperm in each group (

=3) were counted. Data are

represented as the mean ± SEM, Student’s

test, ns, not significant; ***

<0.001.

(

) An in vitro fertilization assay was performed using sperm from

Cylc1

-KO

mice and WT mice (

=3 for each group). The percentage of two-cell embryos

was calculated at 24 h postfertilization. Data are represented as the mean ±

SEM, Student’s

test, ***

<0.001.

Figure 3.

Roles of cylicin-1 in the formation of NE-SAL-IAM protein complexes.

(

) Myc-tagged CYLC1 was transfected into HEK293T cells with or without

FLAG-tagged ACTL7A. Protein lysates were immunoprecipitated with anti-

FLAG antibody and then detected with anti-

Myc antibody by SDS‒PAGE. (

)

Mouse testis lysates were immunoprecipitated with anti-ACTL7A antibody or

rabbit IgG and then detected with anti-CYLC1 antibody. (

) The binding affinity

between different concentrations of purified CYLC1 and GFP-ACTL7A cell

lysates was measured by microscale thermophoresis (MST). (

) Testis protein

lysates from WT and

Cylc1

-KO mice were immunoprecipitated with anti-

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ACTL7A antibody and then detected with anti-ACTRT1 antibody. (

) Myc-

tagged CYLC1 was immunoprecipitated with FLAG-tagged FAM209 in

HEK293T

cell

extracts.

(

)

Mouse

testis

protein

lysates

were

immunoprecipitated with anti-FAM209 antibody or rabbit IgG and then detected

with anti-CYLC1 antibody. (

) The binding affinity between purified CYLC1 and

GFP-FAM209 cell lysates was revealed by an MST assay. (

) Myc-tagged

SPACA1 was immunoprecipitated with FLAG-tagged CYLC1 in HEK293T cell

extracts. (

) Mouse testis protein lysates were immunoprecipitated with anti-

SPACA1 antibody or rabbit IgG and then detected with anti-CYLC1 antibody.

(

) Binding between GFP-SPACA1 from HEK293T cell lysates and recombinant

CYLC1. (

) Testis protein lysates from WT mice or

Cylc1

-KO mice were

incubated with anti-ACTL7A antibody and then detected with anti-SPACA1

antibody.

Figure 4.

Identification of a

CYLC1

variant in a large cohort of Chinese infertile

men with sperm head deformities. (

)

More than 500 patients with sperm head

deformities were recruited for WES and subsequent bioinformatic analysis. 19

individuals harboured a variant of CYLC1.

(

) Sanger sequencing of the 19

individuals who were affected by the c.1377G>T/p.K459N variant in the

CYLC1

gene. Sanger sequencing of the wild-type (WT) allele in healthy controls is also

shown.

(

) This variant was located at the fourth exon of the

CYLC1

gene.

Conservation analysis was performed by Basic Local Alignment Search Tool

(BLAST). The NCBI protein ID for CYLC1: NP_066941.1 (Human),

XP_001102833.1 (Macaca mulatta), XP_016799824.1 (Pan troglodytes),

NP_776727.1 (Bovin), XP_023489864.1 (Horse), NP_080410.3 (Mouse), and

XP_006229368.1 (Rat). The pathogenicity of variants was analysed by

Polyphen2 and SIFT.

Figure 5.

Sperm morphology and ultrastructure analyses for patients

harbouring the

CYLC1

variant. (

)

Papanicolaou staining of sperm from a fertile

individual and three patients (AAW228, AAX765 and AAG547) harbouring the

CYLC1 variant. Sperm from patients exhibited deformed heads. Scale bars, 10

µm.

(

)

Sperm were costained with anti-cylicin-1 antibody and PNA-FITC (an

acrosome dye). In control sperm, Cylicin-1 was specifically localized to the

postacrosomal region (PAR) and PNA-FITC-labelled acrosomes covering the

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anterior of heads. Both cylicin-1 distribution and acrosome structure were

altered in the sperm of three patients. Scale bars, 2 µm.

(

) TEM analysis of

sperm from a normal control and three patients. Detached acrosomes of sperm

were frequently observed in patient samples. The asterisk indicates the

enlarged space area between the acrosome and the nucleus (Nu). Scale bars,

200 nm.

Figure 6.

Cylc1

-mutant mice show male subfertility with sperm acrosome

detachment. (

)

Mice carrying an SNV (c.1407G>T/p.K469N) in the

Cylc1

gene

equivalent to that in nine patients harboring the CYLC1 variant were generated

using CRISPR/Cas9 technology.

(

) Sanger sequencing of the

Cylc1

gene in

WT mice and

Cylc1

-mutant mice. (

and

) Adult

Cylc1

-mutant male mice and

their littermate controls (

=3 each group) were continuously coupled with WT

female mice at a ratio of 1:2 for 2 months.

(

) Sperm count (10

/ml) of

Cylc1

mutant mice and WT mice.

(

) Total sperm motility (%) of

Cylc1

-mutant mice

and WT mice. In c-

f, data are all represented as the mean ± SEM, Student’s

test, **

<0.01; ns, not significant.

(

) Sperm morphology of

Cylc1

-mutant mice

and WT mice. Scale bars, 5 µm.

(

) Visualization of the acrosome (Acr) in

spermatids using the fluorescent dye PNA-FITC (above). Subacrosomal layer

(SAL) bundles of spermatids were visible by F-actin-Tracker Red staining

(below). The asterisk indicates the enlarged space area between the acrosome

and the nucleus.

Scale bars, 2 µm.

(

) TEM analysis showing detachment of the

acrosome from the sperm nucleus in

Cylc1

-mutant mice (above). TEM analysis

of testis tissues revealing the detachment of the developing acrosome from the

nucleus in spermatids of

Cylc1

-mutant mice (below). The asterisk indicates the

enlarged space area between the acrosome and the nucleus. Scale bars, 200

nm.

(

) Testis protein lysates from WT mice and

Cylc1

-mutant mice were

immunoprecipitated with anti-ACTL7A antibody or rabbit IgG and then detected

with anti-cylicin-1 antibody.

(

) Testis protein lysates were immunoprecipitated

with anti-SPACA1 antibody or rabbit IgG and then detected with anti-cylicin-1

antibody.

(

) Testis protein lysates were immunoprecipitated with anti-ACTL7A

antibody or rabbit IgG and then detected with anti-SPACA1 antibody.

Figure 7.

Schematic diagram showing the physiological role of CYLC1 on

acrosome attachment in humans and mice.

CYLC1

variant is identified in a

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large cohort of >500 Chinese infertile men with sperm head deformities. Both

Cylc1

-KO mice and

Cylc1

-mutant mice exhibited severe male subfertility with

spermatozoa showing detached acrosome and head deformities. Cylicin-1 is

specifically expressed at the acroplaxome (Apx), between nuclear envelope

(NE) and inner acrosomal membrane (IAM), of spermatids. Cylicin-1 interacts

itself and with other PT proteins (including CCIN, ACTRT1, ACTRT2, ACTL7A,

and CAPZA3) in the acroplaxome layer and mediates the acrosome-nucleus

connection through interacting with IAM protein SPACA1 and NE protein

FAM209.

Legends for the supplementary figures:

Figure 1

—figure supplement 1.

Expression pattern of human

CYLC1

mRNA

among different tissues and within the testis.

(

) The tissue data for mRNA

expression

were

obtained

from

the

Expression

Atlas

database

(https://www.ebi.ac.uk/gxa). Human

CYLC1

mRNA was specifically expressed

in testes. (

) According to the Single Cell Expression dataset

(https://www.ebi.ac.uk/gxa/sc), human

CYLC1

mRNA was restricted to

early/late spermatids within the testis.

Figure 1

—figure supplement 2.

Generation of cylicin-1 antiserum. Flow chart

showing major steps to generate the cylicin-1 antiserum. First, the N-terminus

of mouse CYLC1 was cloned into the pET-N-His-C-His vector and transfected

into the ER2566 E. coli strain. CYLC1-N protein expression was induced by

IPTG. Second, anti-His beads were added to the E.coli lysates. After incubation

and washing, the CYLC1-N protein was eluted with imidazole. Third,

recombinant CYLC1-

N protein was emulsified with Freund’s complete adjuvant

and administered subcutaneously into New Zealand white rabbits four times

with an interval of 2 weeks. Fourth, one week after the last immunization, blood

was collected, and the serum was separated. Fifth, the cylicin-1 antiserum was

verified by Western blotting and immunofluorescence staining. Cylicin-1

antiserum specifically recognized the cylicin-1 protein (~72 kDa) in the protein

lysates of CYLC1-overexpressing HEK293T cells and testis tissues.

Immunofluorescence staining of mouse sperm indicated that cylicin-1 was

specifically localized to the postacrosomal region (PAR) of heads and that its

expression was absent in the sperm of

Cylc1

KO mice. Scale bar, 5 μm.

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Figure 1

—figure supplement 3.

Construction report of

Cylc1

-KO mice. (

)

Wild-type genomic sequence of the mouse

Cylc1

gene. Two gRNAs are

marked in blue, and exons are marked in red. The sequence between the two

yellow boxes was deleted. (

) Sanger sequencing confirmed that a 3651 bp

region containing exons 3 and 4 of the

Cylc1

gene was successfully deleted.

Figure 1

—figure supplement 4.

Analysis of the reproductive system of

Cylc1

KO male mice.

(

)

There was no obvious difference in testis size or weight

between adult wild-type (WT) mice and

Cylc1

-KO mice. Data are presented as

the mean ± SEM (

=3 each

); Student’s

test; ns, no significance. t, testis; epi,

epididymis. (

) Histological morphology of testis and cauda epididymis from

WT mice and

Cylc1

-KO mice by hematoxylin-eosin (H&E) staining. SPC,

spermatocytes; RS, round spermatids; ES, elongating/elongated spermatids.

Scale bars,

50 μm.

Figure 2

—figure supplement 1.

Normal development of manchette in

spermatids of

Cylc1

-KO mice.

(

) The manchette of spermatids from WT mice

and

Cylc1

-KO mice was observed by staining using Tubulin-Tracker Red dye.

Nuclei were counterstained with DAPI. Double-headed arrows indicate the

position of the perinuclear ring. Scale bars, 2 µm. (

) Transmission electron

microscopy (TEM) of a spermatid of

Cylc1

-KO mice showing the generally well-

formed manchette structure. Scale bar, 200 nm.

Figure 3

—figure supplement 1.

CYLC1 interacts with itself and several other

PT proteins. (

)

FLAG- and/or Myc-tagged CYLC1 was transfected into

HEK293T cells. Protein lysates were immunoprecipitated with anti-Myc

antibody and then detected with anti-

FLAG antibody by SDS‒PAGE. (

B-E

)

Myc-tagged CYLC1 was transfected into HEK293T cells with or without FLAG-

tagged ACTRT1 (b), ACTRT2 (c), CAPZA3 (d), or CCIN (e). Protein lysates

were immunoprecipitated with anti-FLAG antibody and then detected with anti-

Myc antibody by SDS‒PAGE.

Figure 3

—figure supplement 2.

Expression of PT proteins in sperm samples

of WT mice and

Cylc1

-KO mice.

(

)

Protein contents of PT proteins, such as

CYLC1, ACTRT1, ACTRT2, ACTL7A, CCIN, CAPZA3, CAPZB, CYLC2, and

PLCζ, were analysed by Western blotting in sperm protein lysates

from WT

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mice and

Cylc1

-KO mice.

β-actin served as an internal control. (

)

Bar graphs

representing band intensities, which were analysed by ImageJ software. Data

represent the mean ± SEM of three biological replicates.

Student’s

test, ns:

not significant.

Figure 6

—figure supplement 1.

Analysis of the reproductive system of

Cylc1

mutant

mice. (

)

The testis size and weight were similar between wild-type (WT)

mice and

Cylc1

-mutant mice. Data are presented as the mean ± SEM (

each); Student’s

test; ns, no significance. t, testis; epi, epididymis. (

)

Histological morphology of testis and cauda epididymis from WT mice and

Cylc1

-mutant mice by hematoxylin-eosin (H&E) staining. SPC, spermatocytes;

RS, round spermatids; ES, elongating/elongated spermatids. Scale bars,

50 μm.

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References

Aarabi M

, Balakier H, Bashar S, Moskovtsev SI, Sutovsky P, Librach CL, Oko

R. 2004. Sperm-derived WW domain-binding protein, PAWP, elicits

calcium oscillations and oocyte activation in humans and mice.

FASEB

:4434

–4440.

DOI: https://doi.org/10.1096/fj.14-256495

PMID: 24970390

Adzhubei I

, Jordan DM, Sunyaev SR. 2013. Predicting functional effect of

human missense mutations using PolyPhen-2.

Cur Protoc Hum Genet

Chapter 7

:Unit7 20.

DOI: https://doi.org/10.1002/0471142905.hg0720s76

PMID: 23315928

Aul RB

, Oko RJ. 2002. The major subacrosomal occupant of bull spermatozoa

is a novel histone H2B variant associated with the forming acrosome

during spermiogenesis.

Dev Biol

242

:376

–387.

DOI: https://doi.org/10.1006/dbio.2001.0427

PMID: 11892742

Baker MA

, Hetherington L, Weinberg A, Velkov T. 2014. Phosphopeptide

analysis of rodent epididymal spermatozoa.

J Vis Exp

:51546.

DOI: https://doi.org/10.3791/51546

PMID: 25591077

Barratt CLR

, Björndahl L, De Jonge CJ, Lamb DJ, Osorio Martini F, McLachlan

R, Oates RD, van der Poel S, St John B, Sigman M, Sokol R, Tournaye

H. 2017. The diagnosis of male infertility: an analysis of the evidence to

support the development of global WHO guidance-challenges and future

research opportunities.

Hum Reprod Update

:660

–680.

DOI: https://doi.org/10.1093/humupd/dmx021

PMID: 28981651

Bulow M von

, Rackwitz HR, Zimbelmann R, Franke WW. 1997. CP beta3, a

novel isoform of an actin-binding protein, is a component of the

cytoskeletal calyx of the mammalian sperm head.

Exp Cell Res

233

:216

–224.

DOI: https://doi.org/10.1006/excr.1997.3564

PMID: 9184090

Castaneda JM

, Shimada K, Satouh Y, Yu Z, Devlin DJ, Ikawa M, Matzuk MM.

2021. FAM209 associates with DPY19L2, and is required for sperm

acrosome biogenesis and fertility in mice.

J Cell Sci

134

:jcs259206.

DOI: https://doi.org/10.1242/jcs.259206

PMID: 34471926

Chen P

, Saiyin H, Shi R, Liu B, Han X, Gao Y, Ye X, Zhang X, Sun Y. 2021.

CC-BY 4.0 International license

It is made available under a

perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in

(which was not certified by peer review)

preprint

The copyright holder for this

this version posted January 12, 2024.

;

https://doi.org/10.1101/2024.01.11.24301040

doi:

medRxiv preprint

Loss of SPACA1 function causes autosomal recessive globozoospermia

by damaging the acrosome-acroplaxome complex.

Hum Reprod

:2587

–2596.

DOI: https://doi.org/10.1093/humrep/deab144

PMID: 34172998

Dai J

, Zhang T, Guo J, Zhou Q, Gu Y, Zhang J, Hu L, Zong Y, Song J, Zhang

S, Dai C, Gong F, Lu G, Zheng W, Lin G. 2021. Homozygous pathogenic

variants in ACTL9 cause fertilization failure and male infertility in humans

and mice.

Am J Hum Genet

108

:469

–481.

DOI: https://doi.org/10.1016/j.ajhg.2021.02.004

PMID: 33626338

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. 2022.

Mutations in CCIN cause teratozoospermia and male infertility.

Sci Bull

:2122

–2123.

DOI: https://doi.org/10.1016/j.scib.2022.09.026

PMID: 36546111

Fujihara Y

, Satouh Y, Inoue N, Isotani A, Ikawa M, Okabe M. 2012. SPACA1-

deficient male mice are infertile with abnormally shaped sperm heads

reminiscent of globozoospermia.

Development

139

:3583

–3589.

DOI: https://doi.org/10.1242/dev.081778

PMID: 22949614

Gnoth C

, Godehardt E, Frank-Herrmann P, Friol K, Tigges J, Freundl G. 2005.

Definition and prevalence of subfertility and infertility.

Hum Reprod

:1144

–1147.

DOI: https://doi.org/10.1093/humrep/deh870

PMID: 15802321

Gonzales PR

, Andersen EF, Brown TR, Horner VL, Horwitz J, Rehder CW,

Rudy NL, Robin NH, Thorland EC, On Behalf Of The Acmg Laboratory

Quality Assurance Committee. 2022. Interpretation and reporting of

large regions of homozygosity and suspected consanguinity/uniparental

disomy, 2021 revision: A technical standard of the American College of

Medical Genetics and Genomics (ACMG).

Genet Med

:255

–261.

DOI: https://doi.org/10.1016/j.gim.2021.10.004

PMID: 34906464

Gudmundsson S

, Singer-Berk M, Watts NA, Phu W, Goodrich JK,

Solomonson M; Genome Aggregation Database Consortium; Rehm HL,

MacArthur DG, O'Donnell-Luria A. 2022. Variant interpretation using

population databases: Lessons from gnomAD.

Hum Mutat

:1012

–

CC-BY 4.0 International license

It is made available under a

perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in

(which was not certified by peer review)

preprint

The copyright holder for this

this version posted January 12, 2024.

;

https://doi.org/10.1101/2024.01.11.24301040

doi:

medRxiv preprint

1030.

DOI: https://doi.org/10.1002/humu.24309

PMID: 34859531

Hachem A

, Godwin J, Ruas M,

Lee HC, Ferrer Buitrago M, Ardestani G,

Bassett A, Fox S, Navarrete F, de Sutter P, Heindryckx B, Fissore R,

Parrington J. 2017. PLCzeta is the physiological trigger of the Ca

(2+)

oscillations that induce embryogenesis in mammals but conception can

occur in its absence.

Development

144

:2914

–2924.

DOI: https://doi.org/10.1242/dev.150227

PMID: 28694258

Hao J

, Chen M, Ji S, Wang X, Wang Y, Huang X, Yang L, Wang Y, Cui X, Lv L,

Liu Y, Gao F. 2014. Equatorin is not essential for acrosome biogenesis

but is required for the acrosome reaction.

Biochem Biophys Res

Commun

444

:537

–542.

DOI: https://doi.org/10.1016/j.bbrc.2014.01.080

PMID: 24480441

Hamilton LE

, Acteau G, Xu W, Sutovsky P, Oko R. 2017. The developmental

origin and compartmentalization of glutathione-s-transferase omega 2

isoforms in the perinuclear theca of eutherian spermatozoa.

Biol Reprod

:612

–621.

DOI: https://doi.org/10.1093/biolre/iox122

PMID: 29036365

Heid H

, Figge U, Winter S, Kuhn C, Zimbelmann R, Franke W. 2002. Novel

actin-related proteins Arp-T1 and Arp-T2 as components of the

cytoskeletal calyx of the mammalian sperm head.

Exp Cell Res

279

:177

–187.

DOI: https://doi.org/10.1006/excr.2002.5603

PMID: 12243744

Herrada G

, Wolgemuth DJ. 1997. The mouse transcription factor Stat4 is

expressed in haploid male germ cells and is present in the perinuclear

theca of spermatozoa.

J Cell Sci

110

:1543

–1553.

DOI: https://doi.org/10.1242/jcs.110.14.1543

PMID: 9247188

Hess H

, Heid H, Franke WW. 1993. Molecular characterization of mammalian

cylicin, a basic protein of the sperm head cytoskeleton. J Cell Biol

122

:1043

–1052.

DOI: https://doi.org/10.1083/jcb.122.5.1043

PMID: 8354692

Koscinski I

, Elinati E, Fossard C, Redin C, Muller J, Velez de la Calle J, Schmitt

F, Ben Khelifa M, Ray PF, Kilani Z, Barratt CL, Viville S. 2011. DPY19L2

CC-BY 4.0 International license

It is made available under a

perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in

(which was not certified by peer review)

preprint

The copyright holder for this

this version posted January 12, 2024.

;

https://doi.org/10.1101/2024.01.11.24301040

doi:

medRxiv preprint

deletion as a major cause of globozoospermia.

Am J Hum Genet

:344

–350.

DOI: https://doi.org/10.1016/j.ajhg.2011.01.018

PMID: 21397063

Li H

, Durbin R. 2010. Fast and accurate long-read alignment with Burrows-

Wheeler transform.

Bioinformatics

:589

–595.

DOI: https://doi.org/10.1093/bioinformatics/btp698

PMID: 20080505

Liu G

, Shi QW, Lu GX. 2010. A newly discovered mutation in PICK1 in a human

with globozoospermia.

Asian J Androl

:556

–560.

DOI: https://doi.org/10.1038/aja.2010.47

PMID: 20562896

Longo FJ

, Krohne G, Franke WW. 1987. Basic proteins of the perinuclear theca

of mammalian spermatozoa and spermatids: a novel class of

cytoskeletal elements.

J Cell Biol

105

:1105

–1120.

DOI: https://doi.org/10.1083/jcb.105.3.1105

PMID: 3308904

McKenna A

, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A,

Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA. 2010. The

Genome Analysis Toolkit: a MapReduce framework for analyzing next-

generation DNA sequencing data.

Genome Res

:1297

–1303.

DOI: https://doi.org/10.1101/gr.107524.110

PMID: 20644199

Ng PC

, Henikoff S. 2003. SIFT: Predicting amino acid changes that affect

protein function.

Nucleic Acids Res

:3812

–3814.

DOI: https://doi.org/10.1093/nar/gkg509

PMID: 12824425

Oko R

, Morales CR. 1994. A novel testicular protein, with sequence similarities

to a family of lipid binding proteins, is a major component of the rat sperm

perinuclear theca.

Dev Biol

166

:235

–245.

DOI: https://doi.org/10.1006/dbio.1994.1310

PMID: 7958448

Oko R

, Sutovsky P. 2009. Biogenesis of sperm perinuclear theca and its role in

sperm functional competence and fertilization.

J Reprod Immunol

–

DOI: https://doi.org/10.1016/j.jri.2009.05.008

PMID: 19883945

Rentzsch P

, Witten D, Cooper GM, Shendure J, Kircher M. 2019. CADD:

predicting the deleteriousness of variants throughout the human genome.

Nucleic Acids Res

:D886

–D894.

DOI: https://doi.org/10.1093/nar/gky1016

PMID: 30371827

CC-BY 4.0 International license

It is made available under a

perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in

(which was not certified by peer review)

preprint

The copyright holder for this

this version posted January 12, 2024.

;

https://doi.org/10.1101/2024.01.11.24301040

doi:

medRxiv preprint

Schneider S

, Kovacevic A, Mayer M, Dicke AK, Arévalo L, Koser SA, Hansen

JN, Young S, Brenker C, Kliesch S, Wachten D, Kirfel G, Struenker T,

Tüttelmann F, Schorle H. 2023. Cylicins are a structural component of

the sperm calyx being indispensable for male fertility in mice and human.

Elife

:RP86100.

DOI: https://doi.org/10.7554/eLife.86100

PMID: 38013430

Singh P

, Schimenti J. 2015. The genetics of human infertility by functional

interrogation of SNPs in mice.

Proc Natl Acad Sci U S A

112

:10431

–

10436.

DOI: https://doi.org/10.1073/pnas.1506974112

PMID: 26240362

Wang K

, Li M, Hakonarson H. 2010. ANNOVAR: functional annotation of

genetic variants from high-throughput sequencing data.

Nucleic Acids

Res

:e164.

DOI: https://doi.org/10.1093/nar/gkq603

PMID: 20601685

Xin A

, Qu R, Chen G, Zhang L, Chen J, Tao C, Fu J, Tang J, Ru Y, Chen Y,

Peng X, Shi H, Zhang F, Sun X. 2020. Disruption in ACTL7A causes

acrosomal ultrastructural defects in human and mouse sperm as a novel

male factor inducing early embryonic arrest.

Sci Adv

:eaaz4796.

DOI: https://doi.org/10.1126/sciadv.aaz4796

PMID: 32923619

Yatsenko AN

, O'Neil DS, Roy A, Arias-Mendoza PA, Chen R, Murthy LJ, Lamb

DJ, Matzuk MM. 2012. Association of mutations in the zona pellucida

binding protein 1 (ZPBP1) gene with abnormal sperm head morphology

in infertile men.

Mol Hum Reprod

:14

–21.

DOI: https://doi.org/10.1093/molehr/gar057

PMID: 21911476

Zhang XZ

, Wei LL, Jin HJ, Zhang XH, Chen SR. 2022a.The perinuclear theca

protein Calicin helps shape the sperm head and maintain the nuclear

structure in mice.

Cell Rep

:111049.

DOI: https://doi.org/10.1016/j.celrep.2022.111049

PMID: 35793634

Zhang XZ

, Wei LL, Zhang XH, Jin HJ, Chen SR. 2022b. Loss of perinuclear

theca ACTRT1 causes acrosome detachment and severe male

subfertility in mice.

Development

149

:dev.200489.

DOI: https://doi.org/10.1242/dev.200489

PMID: 35616329

CC-BY 4.0 International license

It is made available under a

perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in

(which was not certified by peer review)

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Primers for mouse genotyping.

Cylc1

-KO mice (based on band size):

PCR No.

Primer No.

Sequence (5’→3’)

Band size

PCR (1)

GAAGTCAGGACTGTAACTCAAGCAG

WT: 4222 bp

KO: 571 bp

GCATCTTCTGAACCATTGTACCC

PCR (2)

CCTGATATAGCTGTCTCTTGTGAGG

WT: 266 bp

KO: 0 bp

AGTTTGCAAGACCAATGGGC

Cylc1

-mutant mice (based on sequencing):

PCR No.

Primer No.

Sequence (5’→3’)

Band size

PCR

AAATCAAAACACGATAAAA

508 bp

AATCACTGAAACTCACTAT

Figure 1

—source data 1

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Antibodies used in this study.

Rabbit polyclonal anti-CYLC1

In this study

N/A

Rabbit polyclonal anti-FAM209

In this study

N/A

Rabbit polyclonal anti-ACTL7A

Proteintech

Cat#17355-1-AP; RRID:AB_2273677

Rabbit polyclonal anti-SPACA1

Abcam

Cat#

ab191843

Rabbit polyclonal anti-ACTRT1

Invitrogen

Cat#

PA5-31691

; RRID:AB_2549164

Rabbit polyclonal anti-ACTRT2

Proteintech

Cat#

16992-1-AP

; RRID:AB_2878338

Guinea pig polyclonal anti-CCIN

Progen

Cat#GP-SH3

Guinea pig polyclonal anti-CAPZA3

Progen

Cat#GP-SH4

Rabbit polyclonal anti-CAPZB

Proteintech

Cat#

25043-1-AP

;

RRID:AB_2879867

Rabbit polyclonal anti-CYLC2

Proteintech

Cat#

18729-1-AP

;

RRID:AB_2276962

Rabbit polyclonal anti-

PLCζ

Abcam

Cat#

ab181816

;

RRID:AB_1071092

Mouse monoclonal anti-acetylated

Tubulin

Proteintech

at#66200-1-Ig;

RRID:AB_2722562

Mouse monoclonal anti-beta Actin

Abcam

Cat#ab8226; RRID:AB_306371

PNA-FITC

Sigma-Aldrich

Cat# L7381

Actin-Tracker Red

Beyotime

Cat#C2203

Mouse polyclonal anti-DYKDDDDK-

tag

Abmart

Cat#M20008; RRID:AB_2713960

Mouse polyclonal anti-Myc-tag

Abmart

Cat#M20002; RRID:AB_2861172

Alexa Fluor 555-labeled anti-Mouse

IgG

Beyotime

Cat#A0428; RRID:AB_2893435

Alexa Fluor 555-labeled anti-Rabbit

IgG

Beyotime

Cat#A0453; RRID:AB_2890132

Goat anti-Guinea pig IgG-HRP

Solarbio

Cat#K0040G-HRP

Goat anti-Rabbit IgG-HRP

Abmart

Cat#M21002; RRID:AB_2713951

Goat anti-Mouse IgG HRP

Abmart

Cat#M21001; RRID:AB_2713950

Figure 3

—source data 1

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WES reveals variants in known sperm head deformity-associated genes in infertile
men.

Gene

Mutated sites

gnomAD

Read

depth

DPY19L2

heter: c.814C>A/p.Leu272Met

0.000007422

heter: c.830C>A/p.Thr277Lys

—

homo: c.1213_1217del/p.T405fs

—

ZPBP

heter: c.427T>C/p.Tyr143His

0.00005576

heter: c.430T>C/p.Tyr144His

0.00005576

heter: c.74G>C/p.Arg25Pro

0.001676

PICK1

heter: c.839T>C/p.Leu280Pro

—

heter: c.851T>C/p.Leu284Pro

—

126

heter: c.905A>C/p.Gln302Pro

—

156

heter: c.839T>C/p.Leu280Pro

—

heter: c.874T>C/p.Tyr292His

—

heter

：

c.839T>A/p.Leu280Gln

—

heter: c.851T>A/p.Leu284His

—

100

heter: c.874T>A/p.Tyr292Asn

—

114

heter: c.887T>A/p.Leu296Gln

—

123

ACTL7A

homo: c.659G>C/p.Gly220Ala

0.000006572

336

heter: c.871A>T/p.Lys291*

—

230

heter: c.1035_1036delGT/p.Cys346fs

—

225

ACTL9

heter: c.553_556delGTCT/p.Val185fs*42

—

146

heter: c.1058G>A/p.Arg353His

—

220

CCIN

homo: c.125A>T/p.His42Leu

0.000006571

homo: c.125A>T/p.His42Leu

0.000006571

heter: c.1294C>T/p.Arg432Trp

0.000006568

heter: c.1341C>A/p.Cys447*

—

gnomAD v.3.1.1 (http://www.gnomad-sg.org/).

—, not available. We did not observed the

homozygous, double heterozygous, or hemizygous variants (a minor allele frequency of ≤
1% in public databases) of 40 sperm head deformity-associated genes, including

SPACA1

GOPC

CSNK2A2

TNP2

FAM209

VPS13B

GALNT3

SPAG6

HSP90B1

SPATA46

TMF1

SIRT1

NHE8

LMNA

PPP1CC

ACTRT1

ACTRT2

ACTRT3

CAPZA3

PLCZ1

PARP11

ATG5

ATG7

PIWIL4

CC2D1B

CCNB3

KIAA1210

CHPT1

SEPTIN12

PDCL2

PCSK4

HIPK4

SUN3

SUN4

ACRBP

KPNA4

SPAG17

AXDND1

RIMBP3

and

CEP70

Figure 4

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ICSI experiments using sperm from WT mice or

Cylc1

-KO mice.

Sperm from WT mice

Sperm from

Cylc1

-KO mice

No. oocytes for injection

No. 2-cell embryos

Rate of 2-cell embryos

86.76%

88.33%

85.33%

82.26%

84.06%

86.15%

No. blastocysts

Rate of blastocysts

61.02%

66.04%

64.06%

62.75%

58.62%

62.50%

Table 3

—source data 1

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