ORIGINAL PAPER

Jpn J Reprod Endocrinol ⓒ2004 by
Japan Society of Reproductive Endocrinology

Novel SNP of the VCY2 Gene in Infertile Japanese Patientswith Sertoli Cell-only Phenotype
Jin CHOI1), Eitetsu KOH1), Masato FUKUSHIMA1), KenrouYAMAMOTO1), Yuji MAEDA1),
Atsumi YOSHIDA2) and Mikio NAMIKI1)
1) Department of Integrated Cancer Therapy and Urology, Kanazawa University Graduate School of Medical Science, Kanazawa 920-8641, Japan
2) Reproduction Center KIBA PARK CLINIC, Tokyo 135-0042, Japan

Abstract. Variable charged Y chromosome (VCY2) has been suggested to be involved in susceptibility to spermatogenic failure. Blood samples were collected from 126 proven fertile male volunteers and 271 infertile patients with nonobstructive azoospermia. We discovered three kinds of variant site in the 5 promoter region of the VCY2 gene located
417 upstream from exon 1. All Y chromosomes are in hemizygous condition. Therefore, the whole Y chromosome behaves as a haplotype. A homozygous A pattern was only observed at a rate of 5.1% (3/59) in Sertoli cell-only (SCO)
phenotype, but no variant was detected in idiopathic infertile or proven fertile subjects. Our results suggested that thealteration of total three copies of VCY2 within the AZFc region on the Y chromosome may markedly affect sperm production.

Key words: SNP, VCY2, BPY2, AZF, Male infertility

Introduction

Kuroda and Page s group reported the entire, intact sequence of the AZFc region [13,14]. The AZFc region con-

Y chromosomal microdeletion is involved in spermato-tains a complex of three palindromes (P1, P2, P3), and a

genesis impairment. The region required for spermatogen-conserved highly repetitive sequence. Total AZFc contains

esis is defined as the azoospermia factor (AZF) [1]. Genes

four copies of DAZ and two copies of both BPY2 and CDY1.

in the AZF regions are potential candidates for genes relat-

According to GenBank, at least 44 SNPs (single nucleotide

ed in spermatogenesis. Microdeletions in these genes arepolymorphisms) were found in the DAZ clusters, but there

divided into three main regions, AZFa, b, and c, according tohave been no previous reports of SNPs in the VCY2 gene

the histological phenotype and position [2]. Each AZF re-[15-18].

gion contains some genes involved in spermatogenesis.

The present study was performed to investigate geno-

Among cases with male infertility, microdeletions involving

type frequencies of VCY2 polymorphisms in 126 healthy

the AZFc gene family represent the most frequent finding

and 271 infertile male subjects.

[3-7]. The AZFc region is believed to play important roles
in normal spermatogenesis. Variable charged Y chromosome (VCY2, alias BPY2, GenBank accession no.
AC006366) is located within the AZFc region in fertile men

Subjects and methods
１）Subjects[8-12]. VCY2 is composed of nine exons spanning 21 kilo-

Blood samples were collected from Japanese subjects:

bases within the DAZ gene cluster. VCY2 encodes a product126 proven fertile male volunteers and 271 infertile patientsof 106 amino acids expressed specifically in the testis, andwith non-obstructive azoospermia. All patients underwent

is located on chromosome Yq in humans (OMIM 400013).

comprehensive examinations, including detailed history
taking, physical examination, at least two semen analyses,

Accepted: August 11, 2004
Correspondence: Eitetsu Koh, Department of Integrated Cancer Therapy and Urology, Kanazawa University Graduate Schoolof Medical Science, 13-1,Takara-cho, Kanazawa 920-8641, Japan
TEL: +81-76-265-2393
FAX: +81-76-222-6726
E-mail:kohei@med.kanazawa-u.ac.jp

endocrinology profile testing, karyotyping, and a molecular
test for Y-chromosome microdeletions. Severe oligozoospermia was defined as a sperm count of less than 1×106/
ml. Non-obstructive azoospermia was defined as germ cell
defects on testicular biopsy, elevated serum FSH level, or aJapanese Journal of Reproductive Endocrinology（2004）9：55-60

55

Jin CHOI et al.

Fig. 1. Targeting strategy
Protein-coding sequences, non-coding sequences, and 5 flanking regions are indicated as solid, striped, and open boxes, respectively.

Table 1. PCR primer,product length, polymorphic sites, and type of polymorphism
The
2 sequence from the completed human genome database (accession no. AC006366) was used as a reference for documenting thelocations of the PCR primers, with the first nucleotide of Exon 1 (position 122,361 in AC006366) designated as +1.

total testicular volume of less than 30 ml. Semen analysis

patients. Twenty-three subjects were not performed testic-

was performed according to the standard methods outlined

ular biopsy. Histological examination revealed the absenceby the World Health Organization [19].

of spermatogenesis (Sertoli cell-only) in 59 of these 108

A total of 271 infertile men presented with severe oligo-

subjects. The remaining 49 patients had various tissue

zoospermia or non-obstructive azoospermia. Of these 271

types, such as hypo-spermatogenesis or maturation arrest.

subjects, 140 showed at least one spermatozoon in the ejac-

This study was approved by the Ethical Committee ofulate. Testicular biopsy was performed in the remaining 108

Kanazawa University Hospital, and informed consent was

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Japanese Journal of Reproductive Endocrinology Vol. 9 2004

Novel SNP of the2

Fig. 2. Sequencing at variant site showed 1) homozygous (G/G), 2) heterozygous (G/A) and 3) homozygous (A/A) patterns

obtained from all volunteers.
２）Polymerase chain reaction (PCR)
Amplification genotyping was performed using allelespecific probes. Oligonucleotide primer and probe sets werecial Sciences statistical software (version 11.0). The Fisher s exact test was used to determine whether there wasany significant difference in genotype frequencies between
normal control and infertile male group. The value ofP<0.05 was taken to be statiscally significant.

designed based on gene sequences from the Gene Bank,
July 2003. VCY2 genomic structure and positions of prim-

Results and Discussion

ers are shown in Fig. 1. Primers for each locus are listed inTable 1. In each reaction, 19.2 µl of PCR Master Mix (Toyo-

We analyzed 271 infertile male patients with non-ob-

bo, Osaka, Japan) containing KOD DNA Polymerase,

structive azoospermia and 126 proven-fertile male volun-dNTPs, and MgCl2 were mixed with 10 nmol of each for-teers. No novel mutations of the VCY2 gene were identified

ward and reverse primer. DNA was allowed to stand at 94℃

in any of the exons. However, we discovered three kinds offor 1 min, followed by 30 cycles of amplification at 94℃ forvariant site in the 5 flanking region of the VCY2 gene lo-

30 s, 60℃ for 30 s, and 72℃ for 30 s, with a final extension

cated 417 upstream from exon 1 (Fig. 1). The sequences ofat 72℃ for 10 min and stored at 4℃. PCR was carried out

these three variants showed homozygous (G/G and A/A)

with a thermocycler (BIOMETRA, Goettingen, Germany).

and heterozygous (G/A) patterns (Fig. 2). All Y chromo-３）Restriction cleavage site
To detect a G > A variant at -417, we amplified 396-bp

somes are in hemizygous condition, and therefore thewhole Y chromosome behaves as a haplotype. These variants were mono-allelic on the Y chromosome.

fragments using oligonucleotide primers F2F/R. The prod-

The human DAZ gene cluster, which included the AZFc

ucts were digested with StyI, electrophoresed on 1.5% aga-

region, is located on the distal part of the long arm of the Yrose gels, and visualized under UV by staining with ethid-

chromosome and forms a huge repetitive region and palin-

ium bromide. The mismatch-containing primer introduced

drome complex [13, 14]. The genes in this region, such asa restriction site for StyI only in homogygous A, which was

DAZ, VCY2, and CDY
CDY, exist as multiple copies. VCY2 iscleaved into two fragments of 290 and 106 bp (Fig. 3a).

present as three copies as shown in Fig. 3. The distal two４）Nucleotide sequence analysis

genes are characterized by a palindrome structure in ahead-to-head orientation. The variants with homozygous A

PCR products were purified for sequencing using a Mi-

or G patterns contain three identical copies in the upstream

crocon PCR purification kit (Millipore, Bedford, MA, USA).

region, but we cannot determine which copies of VCY2 are

DNA sequencing was carried out on an ABI (Applied Bio-heterozygous.

systems, Foster City, CA) 377 DNA Sequencer using a BigDye Terminator v3.1 Cycle Sequencing Kit (ABI).
５）Statistical analysis
Data were analyzed using Statistical Package for the So-

To detect a G > A variant at -417, we performed PCRusing oligonucleotide primers F2F/R. The products after digestion with StyI were electrophoresed. The mismatchcontaining primer introduced a restriction site for StyI onlyin the homozygous (A) product, which was cleaved into

ORIGINAL PAPER

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Jin CHOI et al.

VCY2Fig. 3. Positions and orientations of three copies ofin the 5 flanking region of
2

VCY2

VCY2

2 on the Y chromosome and a novel SNP

Sty I

Fig. 4 Restriction fragment length polymorphism
a. The 396-bp PCR product was digested into two fragments.
b. PCR products of the 5 flanking region using primers F2F/R were digested with StyI.
1, Homozygous G type; 2, Heterozygous type; 3. Homozygous A type. P, PCR product; R, Restriction fragments

two fragments of 290 and 106 bp (Fig. 4a). There is no

the gene. A homozygous G/G pattern was observed at acleavage site for the homozygous (G) product (Fig. 4b).

rate of 91.5% (54/59) in the SCO phenotype, 97.2%

We examined the relationships between three VCY2 vari-(206/212) in idiopathic infertile, and 96.0% (121/126) inants and male infertility. As the Y chromosome is hemizy-

proven fertile subjects. A heterozygous G/A pattern wasgous and contains repetitive sequences, the 5 upstream se-observed at a rate of 3.4% in the SCO phenotype, 2.8% in

quence in the promoter region exists in all three copies of

idiopathic infertile, and 5.0% in proven fertile subjects. On

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Japanese Journal of Reproductive Endocrinology Vol. 9 2004

Novel SNP of theTable 2. Number of SNPS

the other hand, a homozygous A/A pattern was observed at
a rate of only 5.1% (3/59) in the SCO phenotype, but novariants were detected in idiopathic infertile or proven fertile subjects (Table 2). These polymorphisms were notfound in the normal population of unrelated healthy subjects
and idiopathic infertile without SCO, suggesting that thesevariants may be associated with the SCO phenotype. Therefore the alteration of all three copies of VCY2 (homozygous
A/A) within the AZFc region on the Y chromosome may
greatly affect sperm production.
The VCY2 -417G>A polymorphism is in the promoter
region, and it is therefore likely that this polymorphism mayalter the level of VCY2 expression. However, this polymorphic site had no consensus sequence for transcription factors by Promoter 2.0 Prediction Server (http://www.cbs.
dtu.dk/services/Promoter/). Further studies in larger numbers of subjects with idiopathic male infertility may be necessary to detect the influence of the VCY2 -417G>A polymorphism on the incidence of azoospermia. Further
analysis of the gene regulation system is necessary to elucidate the mechanism of spermatogenesis.
Although the precise function of VCY2 is not yet known,
this gene appears to have some links to DNA repair, theubiquitin system, and regulation of meiotic chromatin structure [20,21].
In summary, the present study provided the first evidence of a relationship between the common VCY2-417G>A polymorphism and male infertility. This finding
represents an important contribution to the molecular diagnosis of idiopathic azoospermia in Japan and may be usefulfor genetic counseling.

References
1. Tiepolo L, Zuffardi O (1976) Localization of factors controlling2

spermatogenesis in the nonfluorescent portion of the human Y
chromosome long arm. Hum Genet 34, 119-224.
2. Vogt PH, Edelmann A, Kirsch S, Henegariu O, Hirschmann P, Kiesewetter F, Kohn FM, Schill WB, Farah S, Ramos C, Hartmann M,
Hartschuh W, Meschede D, Behre HM, Castel A, Nieschlag E,
Weidner W, Grone HJ, Jung A, Engel W, Haidl G (1996) Human Y
chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet 5, 933-943.
3. Pryor JL, Kent-First M, Muallem A, Van Bergen AH, Nolten WE,
Meisner L, Roberts KP (1997) Microdeletions in the Y chromosome of infertile men. N Engl J Med 336, 534-539.
4. Simoni M, Gromoll J, Dworniczak B, Rolf C, Abshagen K, Kamischke A, Carani C, Meschede D, Behre HM, Horst J, Nieschlag
E (1997) Screening for deletions of the Y chromosome involving
the DAZ (Deleted in AZoospermia) gene in azoospermia and severe oligozoospermia. Fertil Steril 67, 542-547.
5. Nakashima M, Koh E, Namiki M, Yoshida A (2002) Multiplex sequence-tagged site PCR for efficient screening of microdeletions
in Y chromosome in infertile males with azoospermia or severe oligozoospermia. Arch Androl 48, 351-358.
6. Kent-First M, Muallem A, Shultz J, Pryor J, Roberts K, Nolten W,
Meisner L, Chandley A, Gouchy G, Jorgensen L, Havighurst T,
Grosch J (1999) Defining regions of the Y-chromosome responsible for male infertility and identification of a fourth AZF region(AZFd) by Y-chromosome microdeletion detection. Mol Reprod
Dev 53, 27-41.
7. Namiki, M (2000) Genetic aspects of infertility. World J Surg 24,
1176-1179.
8. Fernandes S, Paracchini S, Meyer LH, Floridia G, Tyler-Smith C,
Vogt PH (2004) A large AZFc deletion removes DAZ3/DAZ4 andnearby genes from men in Y haplogroup N. Am J Hum Genet 74,
180-187.
9. Saut N, Terriou P, Navarro A, Levy N, Mitchell MJ (2000) The human Y chromosome genes BPY2, CDY1 and DAZ are not essential
for sustained fertility. Mol Hum Reprod 6, 789-793.
10. Krausz C, Bussani-Mastellone C, Granchi S, McElreavey K, Scarselli G, Forti G (1999) Screening for microdeletions of Y chromosome genes in patients undergoing intracytoplasmic sperm injection. Hum Reprod 14, 1717-1721.
11. Wong J, Blanco P, Affara NA (1999) An exon map of the AZFc maleinfertility region of the human Y chromosome. Mamm Genome 10,57-61.
12. Kondo Y, Watanabe M, Nishigushi S, Shimda K, Yamanishi H, Kurokawa K, Yasunaga T, Koyama K (2002) Study of transcripts from
AC010088, a 199,485 bp fragment of the human Y chromosome located in the azoospermia factor region c. Reprod Biol 2, 59-72.
13. Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, Cordum HS, Hillier
L, Brown LG, Repping S, Pyntikova T, Ali J, Bieri T, Chinwalla A,
Delehaunty A, Delehaunty K, Du H, Fewell G, Fulton L, Fulton R,
Graves T, Hou SF, Latrielle P, Leonard S, Mardis E, Maupin R,
McPherson J, Miner T, Nash W, Nguyen C, Ozersky P, Pepin K,
Rock S, Rohlfing T, Scott K, Schultz B, Strong C, Tin-Wollam A,
Yang SP, Waterston RH, Wilson RK, Rozen S, Page DC (2003) The
male-specific region of the human Y chromosome is a mosaic ofdiscrete sequence classes. Nature 423, 825-837.
14. Kuroda-Kawaguchi T, Skaletsky H, Brown LG, Minx PJ, Cordum
HS, Waterston RH, Wilson RK, Silber S, Oates R, Rozen S, Page
DC (2001) The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men.

ORIGINAL PAPER

59

Jin CHOI et al.

Nat Genet 29, 279-286.
15. Ruiz LM, Szmulewicz MN, Herrera RJ (2002) Single-nucleotide
polymorphism in repetitive sequences of the human deleted inazoospermia complex. Electrophoresis 23, 1021-1024.
16. Szmulewicz MN, Ruiz LM, Reategui EP, Hussini S, Herrera RJ(2002) Single-nucleotide variant in multiple copies of a deleted inazoospermia (DAZ) sequence - a human Y chromosome quantitative polymorphism. Hum Hered 53, 8-17.
17. Wong EY, Tse JY, Yao KM, Tam PC, Yeung WS (2002) VCY2 protein interacts with the HECT domain of ubiquitin-protein ligase
E3A. Biochem Biophys Res Commun 296, 1104-1111.
18. Stuppia L, Gatta V, Fogh I, Gaspari AR, Grande R, Morizio E, Fantasia D, Pizzuti A, Calabrese G, Palka G (2000) Characterization of60

Japanese Journal of Reproductive Endocrinology Vol. 9 2004

novel genes in AZF regions. J Endocrinol Invest 23, 659-663.
19. World Health Organization (1992) Laboratory manual of the WHO
for the examination of human semen and semen-cervical mucus
interaction, Ed 3. Cambridge: Cambridge University Press 3–21
20. Wong EY, Tse JY, Yao KM, Lui VC, Tam PC, Yeung WS (2004)
Identification and characterization of human VCY2-interacting protein: VCY2IP-1, a microtubule-associated protein-like protein.
Biol Reprod 70, 775-784.
21. Tse JY, Wong EY, Cheung AN, O WS, Tam PC, Yeung WS (2003)
Specific expression of VCY2 in human male germ cells and its involvement in the pathogenesis of male infertility. Biol Reprod 69,
746-751.