J Huazhong Univ Sci TechnolMed Sci

DOI 10.1007/s11596-014-1346-5 34(5):745-749,2014

J Huazhong Univ Sci TechnolMed Sci 34(5):2014 745

Expression of Attractin in Male Reproductive Tract of Human and

Mice and Its Correlation with Male Reproduction*
Dan CHENG ( )1, 2, Yu MING ( )1, Jie LI ( )2, Yan CHI ( )1, Hong-gang LI ( )1, 2,
Yu-jie ZOU ()2, Cheng-liang XIONG ()1#
1
Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030,
China
2
Reproductive Medical Center, Renmin Hospital of Wuhan University, Wuhan 430060, China

Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014

Summary: The expression of Attractin mRNA and protein in testis and semen of human and male
mice was investigated. Human testis and semen samples were all collected from Reproductive Center
of Renmin Hospital, Wuhan University in December, 2012. Testis samples were collected from 7
cases of obstructive azoospermias when they were subjected to diagnosed testis biopsy, and 30 nor-
mal human semen samples were obtained from those cases of semen analysis. Adult mice testis tis-
sues were obtained from 10 2-month-old male BALB/c mice, and 60 male mice at different ages were
classified into 10 groups (day 1, 5, 10, 15, 21, 28, 35, 42, 56, and 120 respectively, n=6 each). The
expression of Attractin mRNA and protein in testis was detected by RT-PCR and Western blotting re-
spectively. Human semen samples were centrifuged into sperm plasma (SP) and sperm extract (SE),
and mice sperm samples were collected from the epididymis of 10 adult male BALB/c mice. Western
blotting was used to determine the Attractin protein expression level. Attractin mRNA and protein
were expressed in the testis of both patients with obstructive azoospermias and adult Bcl/B mice.
Quantitative RT-PCR revealed that no Attractin mRNA was detectable in day 1 male BALB/c mice
group. The Attractin mRNA and protein levels were low on the day 10, and increased with age until
day 56. On the day 120, the expression levels of Attractin were decreased. As for human semen sam-
ples, Attractin protein was expressed in both SP and SE, but didnt exist in samples from the epidi-
dymis of male BALB/c mice. It was suggested that Attractin acted as a novel active substance and
was involved in male reproduction in both human and BALB/c mice, but it exerted a different ex-
pression profile in different mammal species.
Key words: Attractin; male reproduction; spermatogenesis

Attractin, which is identified as the product of
the murine Mahogany gene (Mahoganin)[1], is located
on chromosome 20, P13 in human[2]. It contains 29
exons, and its mRNA is about 9 kb. Previous re-
searches were focused on its control of pigmentation
and energy metabolism, while it was found that At-
tractin may also play roles in male reproduction.
Wjst[3] reported that Attractin was located at up-
stream of the domain of a disintegrin and metallopro-
teinase (ADAM), and one of the most important mem-
bers in ADAM is fertilin[4], which is closely related
with male reproduction. It was also proven by Rad-
hakrishnan[5] that Attractin can act in male reproduc-
tion as the ligand of human sperm associated antigen
11 B isoform D (SPAG11B/D).
Dan CHENG, E-mail: chengdanny@126.com
#
Corresponding author, E-mail: clxiong951@sina.com
*
This project was supported by the National Natural Science
Foundation of China (No. 30570769); Funds of Health and
Family Planning Commission of Hubei Province (No.
JS2013009) and the Fundamental Research Funds for Cen-
tral Universities (No. 2042014kf0120).
Previous observations from our laboratory
showed that the fertility of Mahoganin gene knock-out
animal was decreased, presented as the decrease of pi-
tuitary hormones FSH and LH which regulate sper-
matogenesis, degeneration in reproductive tract in
which sponge occurs in the hypothalamus and pitui-
tary while vacuolization in the testis, and the dysfunc-
tion of sperm[5-8]. Attractin and Attractin like-1 double
knockout male mice were infertile[9,10].
Both immunohistochemistry and in situ hybridi-
zation proved that Attractin mRNA is widely ex-
pressed in the high grade center of reproductive axis
such as cerebral cortex and hypothalamus[11,12]. Nev-
ertheless, the existence of Attractin in the testis and
semen of human and mice has not been delicately in-
vestigated previously, partly by the restrictions of eth-
ics. This study compared the expression of Attractin
mRNA and protein in the reproductive tract in human
and mice, and it was found that Attractin mRNA and
protein were both expressed in the testis of human and
Bcl/B mice, but Attractin protein was only expressed
in human semen and didnt exist in sperm samples
from the epididymis of male BALB/c mice. We also
746
provided the expression profiles of Attractin tran-
scripts in the mouse testis during post-natal develop-
ment and described the close relationship of Attractin
with spermatogenesis. Our data proved that Attractin
may be involved in male reproduction and may play
multi-link roles in different species.
1 MATERIALS AND METHODS
1.1 Animals
The BALB/c male mice at different ages were
obtained from the Center of Animal Experiments of
Wuhan University (China) for RT-PCR and Western
blotting. Ten of 2-month-old male BALB/c mice were
collected as matured adult group, and 60 male mice at
different ages were classified into 10 groups (day 1,
5,10, 15, 21, 28, 35, 42, 56, and 120 respectively, n=6
each).
All mice were sacrificed by CO2 inhalation and
the testis were obtained and immediately cryopre-
served in liquid nitrogen for subsequent experiments.
The seminal vesicles of 10 matured mice were col-
lected before sacrifice and were put into the Preheat
medium of M16 (0.5 mL) (Sigma-Aldrich, USA) for
incubation at 37C for 30 min, waiting for the hatch-
ing of sperm, then the suspensions were cryopreserved
at 80C for testing. All procedures involving mice
were performed according to the NIH Guiding Princi-
ples in the Care and Use of Animals.
1.2 Human Samples
Seven of human testis samples were obtained from
those patients undergoing diagnostic testicular sperm as-
piration (TESA) with written consent in December 2012,
according to procedures approved by the ethics board of
Renmin Hospital of Wuhan University (China). All cases
were diagnosed as having obstructive azoospermatism
Table 1 PCR primer sequences, amplification fragments and conditions
Genes Primer sequences (5'3')
Human attractin Sense: GGT TAC GGC CAT AGC AGT GT
Antisense: TAA CGG AAA AAT CGGCTG TC
Human -actin Sense: CAC GAT GGA GGG GCC GGA CTC
ATC
Antisense: TAA AGA CCT CTA TGC CAA CAC
AGT
Mouse attractin Sense: TGT CTC CCA TCT CCT TCA GCC
Antisense: CTG TGT CTT GCC CTT CTG TAG
Mouse -actin Sense: ATC GCT GCG CTG GTC GTC
Antisense: GCT CTG GC CTC GTC ACC
1.5 Real-time Quantitative PCR
Real-time fluorescence quantitative PCR (Q-PCR)
was performed with an Mx3000P thermocycler (Strata-
gene, USA) to determine the Attractin mRNA levels of
postnatal BALB/c male mouse testis by using SYBR
GREEN 1 (Invitrogen, Switzerland) fluorescence dye.
Primer sequences were also noted in table1. Data were
analyzed according to the standard curve and values of
Attractin were normalized to endogenous control,
-actin.
J Huazhong Univ Sci TechnolMed Sci 34(5):2014
(OA) and normal testicular organization structure was
seen by hematoxylin-eosin (HE) staining. All samples
were cryopreserved in liquid nitrogen for subsequent
experiments.
The semen samples were collected from those
undergoing sperm analysis in outpatient service in
Renmin Hospital of Wuhan University in December
2012 and judged as normal according to WHO the
fourth edition standard. The semen samples from 30
cases were centrifuged at 1500 r/min for 15 min after
liquefaction. Seminal plasma (SP) was obtained from
the supernatant fluid, and sperm extraction (SE) from
the sublayer. Then all samples were labeled and cryo-
preserved at 80oC for further experiments.
Our study was approved by the ethical committee.
All the experiments were performed in the lab of Family
Planning Research Institute, Tongji Medical College,
Huazhong University of Science and Technology (China)
from Jan. 1, to June 28, 2012.
1.3 Chemicals
Chemicals were purchased from Sigma Chemical,
BioRad, Qiagen, and Invitrogen Life Technologies
(USA), unless specified. Human Attractin antibody
(1:200, Cat#sc-9328) was purchased from Santa Cruz
Biotechnology (USA).
1.4 Reverse Transcription-Polymerase Chain Reac-
tion (RT-PCR)
The testis samples were taken out from liquid ni-
trogen and total RNA was isolated with Trizol reagent
(Invitrogen, USA) according to the manufacturers in-
structions. A total of 1 mg RNA for each sample was re-
verse-transcribed, and -actin was used in parallel for
each run as internal control. The PCR amplification was
performed as previously described in detail. The primers
used for Attractin were selected from the Harvard Primer
Bank and primer sequences were listed in table 1.
Fragments (bp) Melting temperature (C)
167 85
184 87
269 85
168 92
1.6 Tissue and Sperm Protein Extracts, SDS-PAGE,
and Western Immunoblotting
Liquid nitrogen frozen testes were homogenized on
ice and sonicated briefly and centrifuged for 10 min at
14000 g. Protein in the supernatant was quantified by
BCA Protein Assay Kit (Pierce, USA). Proteins were
then electrophoretically transferred onto PVDF mem-
branes at 4C, 30 V for 100 min. Western Breeze kit (In-
vitrogen, USA) was applied for following immunodetec-
tion of Western blots, conforming to the protocols. For
J Huazhong Univ Sci TechnolMed Sci 34(5):2014 747

protein loading control, GAPDH primary antibody was lecular weight of 160 kD (fig. 3A), which was similar
used. to that in the brain tissues of mice. There was also
1.7 Statistical Analysis positive blotting in the testis of OA patients (fig. 3B),
The data were presented as s. Statistical suggesting Attractin protein exists in the testis of both
analyses were performed using the SPSS software ver- human and Bcl/B mice.
sion 12.0 (SPSS In., USA). The changes of Attractin
mRNA expression during post-natal development were
analyzed by one-way ANOVA. All statistical tests
were two-tailed and P<0.01 was considered to be sta-
tistically significant.

2 RESULTS

2.1 Attractin mRNA Expression in Testis of Adult

Men and BALB/c Male Mice
RT-PCR showed that there were expected 269-bp
specific bands of Attractin mRNA in the testis of
BALB/c male mice and 187-bp in the testis of OA pa-
tients (fig. 1). It was revealed that Attractin mRNA
was expressed in the testis of both BALB/c male mice
and adult men.
2.2 Attractin mRNA Expression in BALB/c Mouse
Testis at Different Postnatal Days
Real-time Q-PCR was performed to test Attractin
mRNA expression in the testis of BALB/c mice at dif-
ferent postnatal days. It was revealed that the Attractin
mRNA was not detectable in the testis of mice at postna-
tal day 1, and weak at postnatal day 5, then increased
gradually with age and reached the peak at postnatal day
35, and then decreased (fig 2A and 2B).
2.3 Attractin Protein in the Testis of Adult Men and
Male Mice
Western blotting showed that there was positive
blotting in the testis of elder BALB/c mice, with mo-
Fig. 1 The expression of Attractin mRNA in the testis of OA
patients and BALB/c male mice detected by RT-PCR
Attractin mRNA was 269 bp in Bcl/B mice, and 187 bp
in human testis. -actin (168 bp) was used as a loading
control. M: DNA marker; N: negative control; hT: hu-
man testis; mT: mouse testis
2.4 Attractin Protein in BALB/c Mouse Testis at
Different Postnatal Days
Attractin positive blotting was very weak in the
testis of postnatal day 1 and 5 mice, then increased
gradually with postnatal days, and retained a high
level after maturatione, which has the similar trend
with Attractin mRNA (fig. 4A and 4B).

Fig. 2 The expression of Attractin mRNA in the testis of BALB/c male mice during different postnatal days identified by Q-PCR
A: Attractin mRNA was 269 bp, and -actin (168 bp) was used as a loading control; B: Expression of Attractin mRNA nor-
malized to -actin. The expression levels of Attractin mRNA were expressed as a ratio relative to those of the matured mice at
2 months. Results are averaged from three independent experiments in all groups.

Fig. 3 The expression of Attractin protein in the testis of BALB/c male mice and OA patients detected by Western blotting
A: the positive blotting of mouse Attractin (160 kD). mB, mice brain tissues; mT, mice testis. GAPDH as the control. B: the
positive blotting of human Attractin (175 kD). Con: control; hT: the testis of OA patients
748 J Huazhong Univ Sci TechnolMed Sci 34(5):2014

Fig. 4 The expression of Attractin protein expression in the testis of BALB/c male mice during different postnatal days.
A: the blotting of Attractin in the testis of mice at different postnatal days; B: the relative expression of Attractin. The ratio
in the testis of mice at postnatal day 120 is 1.0. A: absorbance

2.5 Attractin Protein in Semen of Adult Men and hatched from the seminal vesicle of BALB/c mice (fig.
Male Mice 5A), but the Attractin protein expression could be de-
Western blotting showed that there was unde- tected in both SP and SE of human semen (fig. 5B).
tectable Attractin protein in the suspension which was

Fig. 5 The expression of Attractin protein in semen of adult BALB/c mice and human
A: the expected positive blotting is 160 kD. mS, the suspension which was hatched from the seminal vesicle of BALB/c
mice; mT, mice testis. B: the expected positive blotting is 175 kD. GAPDH was used as the control. Con: control; hSP: hu-
man seminal plasma; hSE, human sperm extraction

3 DISCUSSION implied that Attractin acted in male reproduction through

Attractin, which was first reported in 1960 as a
mutator of Mahogany, is involved in many physio-
logical and pathological events such as the regulation
of immune system, weight control, pigmentation,
myelin formation and tumor susceptibility[13, 14]. It was
reported that knockout of Mahogany may result in the
defect of male reproduction in mice[9].
Radhakrishnan reported that Attractin could act
in regulation of male reproduction through interactions
with SPAG11B/D[5]. The association between
SPAG11B/D and Attractin was confirmed by
co-immunoprecipitation experiments. Tokuda[15] re-
ported that Attractin was widely expressed in higher
center of reproductive axisthe hypothalamus and
pituitary both in mice and humans, which also implied
it may act in male reproduction.
Our group has previously testified that Attractin
mRNA existed in the hypothalamus, pituitary, testis, epi-
didymis and seminal vesicle of rats. In 2004 it was firstly
reported by our laboratory that Attractin existed in leydig
and convoluted tubule of rats, and its expression changed
obviously in accordance with the first wave of sper-
matogenesis[6]. This experiment revealed that both At-
tractin mRNA and protein existed in the testis of human
and mice, and its expression changed obviously in ac-
cordance with the first wave of spermatogenesis, which
its specific regulation in spermatogenesis.
As the function unit of male reproduction, semen is
composed by SP and SE. Sperms are produced by the
male gonad-testis and are directly related to the male fer-
tility. SP is secreted by the prostate, seminal vesicle and
bulbourethral glands, which contains water, fructose,
protein and fat, besides a variety of enzymes and inor-
ganic salt, and provides nutrients to the sperm[16,17]. It
was revealed by our research that Attractin protein
couldnt be detected in the semen of mice but in human
which may be attributed to the species specificity.
As a soluble human plasma protein, human At-
tractin exerts two different formations: membrane type
which is similar to the mice Attractin; secretory gly-
coprotein. Secretory Attractin is released by active T
immune cells[18], can facilitate initial immune cell
clustering and, through a putative dipeptidyl peptidase
(DPP) activity similar to that of CD26, has the
potential to regulate chemotactic activity of chemoki-
nes[19]. Different types of Attractin protein may be due
to the selective shear during gene transcription[20], and
may have different distribution profiles.
It was proved by knock-out animal model that in
the absence of Attractin, the semen quality was de-
creased dramatically, the swimming-up speed of sperm
slowed down obviously and the fertilization rate of
sperm in vitro decreased evidently. The existence of
J Huazhong Univ Sci TechnolMed Sci 34(5):2014
Attractin protein in human semen implied that the re-
flection of Attractin on male reproduction was not only
in spermatogenesis but also in its functional
unitsemen. Attractin has higher content in SP than in
SE, which would be attributed to its secretory form
and different forms of Attraction may play a different
role in male reproduction.
The limitations of this research are that it con-
fined to the distribution profiles of Attractin in the tes-
tis and semen of human and BALB/c male mice, but
lacked the study on specific steps which act on male
reproduction. Muto[21] reported that Attractin had a
pivotal role in cell survival under oxidative stress, but
Sun[22] revealed that it may be restricted with mito-
chondrial dysfunction. Further research will be fo-
cused on the mechanism of Attractin gene on male re-
production.
Acknowledgements
We are grateful to Dr. Jing Yang and Dr. Wangming
Xu in the Reproductive Center of Renmin Hospital of
Wuhan University for providing us with the human sam-
ples of testis and semen.
Conflict of Interest Statement
The authors declare that there is no conflict of interest
with any financial organization or corporation or individual that
can inappropriately influence this work.
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