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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 Previous observations from our laboratory
the murine Mahogany gene (Mahoganin)[1], is located showed that the fertility of Mahoganin gene knock-out
on chromosome 20, P13 in human[2]. It contains 29 animal was decreased, presented as the decrease of pi-
exons, and its mRNA is about 9 kb. Previous re- tuitary hormones FSH and LH which regulate sper-
searches were focused on its control of pigmentation matogenesis, degeneration in reproductive tract in
and energy metabolism, while it was found that At- which sponge occurs in the hypothalamus and pitui-
tractin may also play roles in male reproduction. tary while vacuolization in the testis, and the dysfunc-
Wjst[3] reported that Attractin was located at up- tion of sperm[5-8]. Attractin and Attractin like-1 double
stream of the domain of a disintegrin and metallopro- knockout male mice were infertile[9,10].
teinase (ADAM), and one of the most important mem- Both immunohistochemistry and in situ hybridi-
bers in ADAM is fertilin[4], which is closely related zation proved that Attractin mRNA is widely ex-
with male reproduction. It was also proven by Rad- pressed in the high grade center of reproductive axis
hakrishnan[5] that Attractin can act in male reproduc- such as cerebral cortex and hypothalamus[11,12]. Nev-
tion as the ligand of human sperm associated antigen ertheless, the existence of Attractin in the testis and
11 B isoform D (SPAG11B/D). 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
Dan CHENG, E-mail: chengdanny@126.com mRNA and protein in the reproductive tract in human
#
Corresponding author, E-mail: clxiong951@sina.com and mice, and it was found that Attractin mRNA and
*
This project was supported by the National Natural Science protein were both expressed in the testis of human and
Foundation of China (No. 30570769); Funds of Health and Bcl/B mice, but Attractin protein was only expressed
Family Planning Commission of Hubei Province (No. in human semen and didnt exist in sperm samples
JS2013009) and the Fundamental Research Funds for Cen- from the epididymis of male BALB/c mice. We also
tral Universities (No. 2042014kf0120).
746 J Huazhong Univ Sci TechnolMed Sci 34(5):2014

provided the expression profiles of Attractin tran- (OA) and normal testicular organization structure was
scripts in the mouse testis during post-natal develop- seen by hematoxylin-eosin (HE) staining. All samples
ment and described the close relationship of Attractin were cryopreserved in liquid nitrogen for subsequent
with spermatogenesis. Our data proved that Attractin experiments.
may be involved in male reproduction and may play The semen samples were collected from those
multi-link roles in different species. undergoing sperm analysis in outpatient service in
Renmin Hospital of Wuhan University in December
1 MATERIALS AND METHODS 2012 and judged as normal according to WHO the
fourth edition standard. The semen samples from 30
1.1 Animals cases were centrifuged at 1500 r/min for 15 min after
The BALB/c male mice at different ages were liquefaction. Seminal plasma (SP) was obtained from
obtained from the Center of Animal Experiments of the supernatant fluid, and sperm extraction (SE) from
Wuhan University (China) for RT-PCR and Western the sublayer. Then all samples were labeled and cryo-
blotting. Ten of 2-month-old male BALB/c mice were preserved at 80oC for further experiments.
collected as matured adult group, and 60 male mice at Our study was approved by the ethical committee.
different ages were classified into 10 groups (day 1, All the experiments were performed in the lab of Family
5,10, 15, 21, 28, 35, 42, 56, and 120 respectively, n=6 Planning Research Institute, Tongji Medical College,
each). Huazhong University of Science and Technology (China)
All mice were sacrificed by CO2 inhalation and from Jan. 1, to June 28, 2012.
the testis were obtained and immediately cryopre- 1.3 Chemicals
served in liquid nitrogen for subsequent experiments. Chemicals were purchased from Sigma Chemical,
The seminal vesicles of 10 matured mice were col- BioRad, Qiagen, and Invitrogen Life Technologies
lected before sacrifice and were put into the Preheat (USA), unless specified. Human Attractin antibody
medium of M16 (0.5 mL) (Sigma-Aldrich, USA) for (1:200, Cat#sc-9328) was purchased from Santa Cruz
incubation at 37C for 30 min, waiting for the hatch- Biotechnology (USA).
ing of sperm, then the suspensions were cryopreserved 1.4 Reverse Transcription-Polymerase Chain Reac-
at 80C for testing. All procedures involving mice tion (RT-PCR)
were performed according to the NIH Guiding Princi- The testis samples were taken out from liquid ni-
ples in the Care and Use of Animals. trogen and total RNA was isolated with Trizol reagent
1.2 Human Samples (Invitrogen, USA) according to the manufacturers in-
Seven of human testis samples were obtained from structions. A total of 1 mg RNA for each sample was re-
those patients undergoing diagnostic testicular sperm as- verse-transcribed, and -actin was used in parallel for
piration (TESA) with written consent in December 2012, each run as internal control. The PCR amplification was
according to procedures approved by the ethics board of performed as previously described in detail. The primers
Renmin Hospital of Wuhan University (China). All cases used for Attractin were selected from the Harvard Primer
were diagnosed as having obstructive azoospermatism Bank and primer sequences were listed in table 1.

Table 1 PCR primer sequences, amplification fragments and conditions


Genes Primer sequences (5'3') Fragments (bp) Melting temperature (C)
Human attractin Sense: GGT TAC GGC CAT AGC AGT GT 167 85
Antisense: TAA CGG AAA AAT CGGCTG TC
Human -actin Sense: CAC GAT GGA GGG GCC GGA CTC 184 87
ATC
Antisense: TAA AGA CCT CTA TGC CAA CAC
AGT

Mouse attractin Sense: TGT CTC CCA TCT CCT TCA GCC 269 85
Antisense: CTG TGT CTT GCC CTT CTG TAG
Mouse -actin Sense: ATC GCT GCG CTG GTC GTC 168 92
Antisense: GCT CTG GC CTC GTC ACC

1.5 Real-time Quantitative PCR 1.6 Tissue and Sperm Protein Extracts, SDS-PAGE,
Real-time fluorescence quantitative PCR (Q-PCR) and Western Immunoblotting
was performed with an Mx3000P thermocycler (Strata- Liquid nitrogen frozen testes were homogenized on
gene, USA) to determine the Attractin mRNA levels of ice and sonicated briefly and centrifuged for 10 min at
postnatal BALB/c male mouse testis by using SYBR 14000 g. Protein in the supernatant was quantified by
GREEN 1 (Invitrogen, Switzerland) fluorescence dye. BCA Protein Assay Kit (Pierce, USA). Proteins were
Primer sequences were also noted in table1. Data were then electrophoretically transferred onto PVDF mem-
analyzed according to the standard curve and values of branes at 4C, 30 V for 100 min. Western Breeze kit (In-
Attractin were normalized to endogenous control, vitrogen, USA) was applied for following immunodetec-
-actin. 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 Fig. 1 The expression of Attractin mRNA in the testis of OA
and adult men. patients and BALB/c male mice detected by RT-PCR
2.2 Attractin mRNA Expression in BALB/c Mouse Attractin mRNA was 269 bp in Bcl/B mice, and 187 bp
Testis at Different Postnatal Days in human testis. -actin (168 bp) was used as a loading
Real-time Q-PCR was performed to test Attractin control. M: DNA marker; N: negative control; hT: hu-
mRNA expression in the testis of BALB/c mice at dif- man testis; mT: mouse testis
ferent postnatal days. It was revealed that the Attractin
mRNA was not detectable in the testis of mice at postna- 2.4 Attractin Protein in BALB/c Mouse Testis at
tal day 1, and weak at postnatal day 5, then increased Different Postnatal Days
gradually with age and reached the peak at postnatal day Attractin positive blotting was very weak in the
35, and then decreased (fig 2A and 2B). testis of postnatal day 1 and 5 mice, then increased
2.3 Attractin Protein in the Testis of Adult Men and gradually with postnatal days, and retained a high
Male Mice level after maturatione, which has the similar trend
Western blotting showed that there was positive with Attractin mRNA (fig. 4A and 4B).
blotting in the testis of elder BALB/c mice, with mo-

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


its specific regulation in spermatogenesis.
Attractin, which was first reported in 1960 as a As the function unit of male reproduction, semen is
mutator of Mahogany, is involved in many physio- composed by SP and SE. Sperms are produced by the
logical and pathological events such as the regulation male gonad-testis and are directly related to the male fer-
of immune system, weight control, pigmentation, tility. SP is secreted by the prostate, seminal vesicle and
myelin formation and tumor susceptibility[13, 14]. It was bulbourethral glands, which contains water, fructose,
reported that knockout of Mahogany may result in the protein and fat, besides a variety of enzymes and inor-
defect of male reproduction in mice[9]. ganic salt, and provides nutrients to the sperm[16,17]. It
Radhakrishnan reported that Attractin could act was revealed by our research that Attractin protein
in regulation of male reproduction through interactions couldnt be detected in the semen of mice but in human
with SPAG11B/D[5]. The association between which may be attributed to the species specificity.
SPAG11B/D and Attractin was confirmed by As a soluble human plasma protein, human At-
co-immunoprecipitation experiments. Tokuda[15] re- tractin exerts two different formations: membrane type
ported that Attractin was widely expressed in higher which is similar to the mice Attractin; secretory gly-
center of reproductive axisthe hypothalamus and coprotein. Secretory Attractin is released by active T
pituitary both in mice and humans, which also implied immune cells[18], can facilitate initial immune cell
it may act in male reproduction. clustering and, through a putative dipeptidyl peptidase
Our group has previously testified that Attractin (DPP) activity similar to that of CD26, has the
mRNA existed in the hypothalamus, pituitary, testis, epi- potential to regulate chemotactic activity of chemoki-
didymis and seminal vesicle of rats. In 2004 it was firstly nes[19]. Different types of Attractin protein may be due
reported by our laboratory that Attractin existed in leydig to the selective shear during gene transcription[20], and
and convoluted tubule of rats, and its expression changed may have different distribution profiles.
obviously in accordance with the first wave of sper- It was proved by knock-out animal model that in
matogenesis[6]. This experiment revealed that both At- the absence of Attractin, the semen quality was de-
tractin mRNA and protein existed in the testis of human creased dramatically, the swimming-up speed of sperm
and mice, and its expression changed obviously in ac- slowed down obviously and the fertilization rate of
cordance with the first wave of spermatogenesis, which sperm in vitro decreased evidently. The existence of
J Huazhong Univ Sci TechnolMed Sci 34(5):2014 749

Attractin protein in human semen implied that the re- tive hormone of male mice after knocking out Attractin
flection of Attractin on male reproduction was not only gene or Mahoganoind gene. Zhong Guo Ji Hua Sheng Yu
in spermatogenesis but also in its functional Xue Za Zhi (Chinese), 2007,15(12):729-731
unitsemen. Attractin has higher content in SP than in 9 Cheng D, Xiong C, Li J, et al. The effect of Mahogunin
SE, which would be attributed to its secretory form gene mutant on reproduction in male mice: a new sight
and different forms of Attraction may play a different for infertility? Andrologia, 2014,46(2):98-105
role in male reproduction. 10 Walker WP, Aradhya S, Hu CL, et al. Genetic analysis of
The limitations of this research are that it con- attractin homologs. Genesis, 2007,45(12):744-756
fined to the distribution profiles of Attractin in the tes- 11 Gohma H, Kuramoto T, Matalon R, et al. Absence-like
tis and semen of human and BALB/c male mice, but and tonic seizures in aspartoacylase/attractin dou-
lacked the study on specific steps which act on male ble-mutant mice. Exp Anim, 2007,56(2):161-165
reproduction. Muto[21] reported that Attractin had a 12 Paz J, Yao H, Lim HS, et al. The neuroprotective role of
pivotal role in cell survival under oxidative stress, but attractin in neurodegeneration. Neurobiol Aging,
Sun[22] revealed that it may be restricted with mito- 2007,28(9):1446-1456
chondrial dysfunction. Further research will be fo- 13 Cota CD, Liu RR, Sumberac TM, et al. Genetic and phe-
cused on the mechanism of Attractin gene on male re- notypic studies of the dark-like mutant mouse. Genesis,
production. 2008,46(10):562-573
14 Fernandez-Fernandez R, Martini AC, Navarro VM, et al.
Acknowledgements Novel signals for the integration of energy balance and
We are grateful to Dr. Jing Yang and Dr. Wangming reproduction. Mol Cell Endocrinol, 2006,254-255:
Xu in the Reproductive Center of Renmin Hospital of 127-132
Wuhan University for providing us with the human sam- 15 Tokuda S, Kuramoto T, Serikawa T. PCR-based genotyp-
ples of testis and semen. ing of the rat Atrn (mv) mutation. Exp Anim,
2004,53(1):73-76
Conflict of Interest Statement 16 Pant N, Pant AB, Chaturvedi PK, et al. Semen quality of
The authors declare that there is no conflict of interest environmentally exposed human population: the toxico-
with any financial organization or corporation or individual that logical consequence. Environ Sci Pollut Res Int,
can inappropriately influence this work. 2013,20(11):8274-8281
17 Li HG, Ding XF, Liao AH, et al. Expression of CatSper
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