Effect of apigenin on the reproductive system in male mice

doi:10.4236/health.2010.25065

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Vol.2, No.5, 435-440 (2010)

doi:10.4236/health.2010.25065

Health

Openly accessible at

Effect of apigenin on the reproductive system in male

mice

Hui Li, Hong-Bo Li, Ming Zhang, Fang Yan, Zhong-Xian Zhang, Zhi-Lan Li*

School of Public Health, Lanzhou University, Lanzhou, China; *Corresponding Author: lizhl@lzu.edu.cn

Received 23 November 2009; revised 7 January 2010; accepted 8 January 2010.

ABSTRACT

This study aimed to characterize the effect of

apigenin on the reproductive system in male

mice. Adult male mice were treated with intrap-

eritoneal injection of apigenin at the dose levels

of 5, 10, 15, 20 and 25 mg/kg.bw, 0.05% DMSO

and 0.9% normal saline daily for seven days.

Then, testis and epididymis sperms in sperm

motility, sperm morphology, the percentages of

ploidy cells and seminiferous epithelium cells at

the cell-circle phase, and the ratio of ploidy cells

were evaluated. The results showed that sperm

density significantly reduced in the 25 mg/kg

group compared with the solvent control group.

The abnormal sperms were mainly amorphous;

non-hook sperms took the second largest group;

and banana, double-tail and folded-tail sperms

were rare. Abnormal sperms were mainly in the

head sperm. Moreover, after intraperitoneal in-

jection of 5 mg/kg apigenin, the percentage of

1C population increased, and the percentage of

4C declined, leading to a significant increase of

the 1C:4C ratio, compared with the solvent and

negative control groups. The percentage of

seminiferous epithelium cells at the cell-circle

phase of G0/G1 exhibited a significant increase

in the 25 mg/kg group compared with the con-

trol groups. Taken together, that apigenin has

adverse effects on the reproductive system in

adult male mice is demonstrated.

Keywords: Apigenin; Male Mice;

Intraperitoneal Injection; Sperm Motility;

Sperm Morphology; Flow Cytometry

1. INTRODUCTION

In the early 1970s, several studies in the United States

first suggested a possible decline in human sperm con-

centration [1]. Since then, there has been increased

awareness of the possible effects of chemicals on male

fertility [2,3]. It is of paramount importance to assess

potential health risks associated with exposure to

chemical or physical agents since these agents may in-

terfere with the ability of individuals to produce normal

progeny. Apigenin is a common flavone present in diet:

it is not only in aromatic plants (camomilla, rosemary

and parsley), but also in celery, apple, honey, fennel and

wheat germ [4-6].

Although apigenin is less active than its homologous

isoflavone, genistein, much attention has been paid to its

endocrine properties and potential effects on fertility

recently. As a ligand of estrogen receptor [7,8], in vitro

apigenin has estrogenic activity on the growth of trans-

fected cells that are estrogen-dependent and have addi-

tive effects on 17-estradiol [6]. It has been shown that

apigenin reduces the endogenous level of estrogen re-

ceptors in mouse uterus [9], enhances the estrogenicity

of low-dose estradiol in immature rats [10], and has a

protective effect on skin tumorigenesis, a hormonal-

dependent cancer [11]. Moreover, anti-fertility proper-

ties of apigenin have been observed: apigenin is an ac-

tive constituent of Striga orobanchioides, a medicinal

plant with contraceptive properties [12]. Many potential

mechanisms have been proposed to explain these (anti-)

estrogenic and (anti-) carcinogenic properties including

interaction with estrogen receptors [6,13], modulation of

biosynthesis and metabolism of steroidhormones [14,15],

enhancement of gap-junction intercellular communica-

tion, and apoptosis induction [16,17].

While the effect of apigenin on the reproductive

system has been studied, to our best knowledge, the

adverse effects of apigenin on sperm motility, sperm

morphology and is still not very clear. Here these ef-

fects in male mice were investigated.

2. MATERIALS AND METHODS

2.1. Test Materials

This work was supported by the research foundation for the young an

the middle-aged scientist in Gansu Province, China (Grant No.

099RJYA003). Apigenin was obtained from Shanxi Hui Ke Plant Ex-

H. Li et al. / HEALTH 2 (2010) 435-440

436

ploitation Limited Company (China), whose purity was

98.00%. Dimethyl sulfoxide (DMSO) was purchased

from Shanxi Hua Mei Bioengineering Company (China).

All other chemicals were obtained from standard com-

mercial sources and were with the highest available

quality.

2.2. Animals

Eight-four healthy, adult male SPF mice of Kunming

strain, whose body weights ranged from 32 to 35 g, ap-

proximately seven weeks old, were obtained from Ex-

perimental Animal Center, Gansu College of Traditional

Chinese Medicine, Lanzhou, Gansu, China (Animal Cer-

tificate of Quality No: SCXK [Gan]: 2004-0006-0000

328). The use of these animals was approved by the

Chinese Association for Laboratory Animal Sciences.

They were housed in the GLP laboratory (Laboratory

Certificate of Quality No: SCXK [Gan]: 2004-0006-000

0328-0000120), where the temperature of 25°C, the

relative humidity of approximately 50%, and a photope-

riod of 12 h light: 12 h dark were maintained. All ani-

mals were housed on sawdust beds in cages and given

the standard diet and distilled water of ad libitum during

the whole study.

2.3. Chemicals and Experimental Design

A stock solution of 1.88 mg/ml apigenin was prepared

by dissolving 376 g apigenin in 1ml DMSO and 199 ml

0.9% normal saline (NS). The solvent was 0.05% DMSO,

and different concentrations of apigenin were prepared

after the dilution with 0.05% DMSO. According to their

body weights, male mice were randomly assigned to

seven groups (12 animals per group) including one sol-

vent group, one negative control group, and five experi-

mental groups. The solvent and negative control groups

were given 0.05% DMSO and 0.9% NS by intraperito-

neal injection, respectively. The five experimental

groups were given apigenin at the dose levels of 5, 10,

15, 20 and 25 mg/kg.bw once a day for seven consecu-

tive days. Solution concentrations were adjusted so that

a 30 g mouse received a solution of 0.4 ml. The body

weights were recorded since the start of dosing, then

once every two days, until the day of necropsy. The ac-

tual dose volume was adjusted according to the recorded

body weight. Male mice were killed after cervical dislo-

cation on the day following the last injection. On the day

of sacrifice, the body weight was recorded. Immediately

after sacrifice, both testes and epididymes were excised,

trimmed free fat, placed on a paper towel to remove any

liquid, and then weighted separately.

2.4. Sperm Motion Analysis

Sperm motion was analyzed with the WLJY-9000 sperm

quantity detection system (Beijing Wei Li New Century

Science and Technology Development Limited Company,

China).The left epididymes were excised and placed in a

pre-warmed peridish (37°C) consisting of 1 ml NS

(0.86% sodium chloride). The tissue was made three cuts

in the mid-to-distal region with a scalpel blade to release

spermatozoa into the medium, which was then placed in

a 37°C thermostatic water-bath box for 30 min prior to

the measurement of sperm motility. The suspension was

stirred, and 10 µl was placed on a clean counter board,

mounted with a special cover lip, and then observed with

four to six microscope fields. With the sperm quantity

detection system, the percentages of mobile sperms, cur-

vilinear velocity (VCL), straight-line velocity (VSL),

average path velocity (VAP), mean moving angle

(MAD), amplitude of lateral displacement (ALH), beat

cross frequency (BCF), linearity (LIN), straightness

(STR), wobble (WOB)and sperm density were calcu-

lated, respectively.

2.5. Sperm Morphology Analysis

The right epididymes were minced with an eye scissor in

3 ml NS (0.86% NaCl), repetitively beaten by pipette,

kept for 5 min and then filtered with four-layer lens pa-

per. The sperm morphology was assessed by smearing

the sperm suspension on a pre-cleaned slide with a drop

from the filtrate. Once air-dried, the samples were fixed

with methanol for 15 min, stained with 2% eosin for 1h,

rinsed thoroughly with distilled water and dried in air.

For each sample, 1,000 sperms from different fields

were observed under 40 × magnification with a light

microscope (Olympus, Japan), and according to the de-

scription of Huang et al. [18], classified as normal,

amorphous, non-hook, banana, double-tail and folded-tail.

2.6. Cell Cycle Analysis

The right testis was surgically removed, stripped of ad-

hering fat and connective tissues, and weighted. The

germ cells were released from seminiferous tubules in

cold phosphate buffered saline (PBS) at pH 7.4 by

mincing the testes with fine curved scissors. The cell

suspension was aspirated with PBS and filtered in a 200

mesh nylon mesh in order to remove tissue debris. The

suspension was centrifuged at 1,500 rpm for 5 min. The

supernatant was discarded, and the pellet was

re-suspended in 10 ml PBS, and then centrifuged at 800

rpm for 5 min. After rinsing three times as above, the

pellet was fixed in 75% ethanol and stored at 4°C. After

24 h, the fixed germ cells were washed with PBS to re-

move ethanol, and then the suspension was centrifuged

at 1,500 rpm for 5 min. The supernatant was discarded,

and the pellet was stained with Propidium iodide (PI) in

darkness for 30 min. The pellet was filtered in a 320

mesh nylon mesh to remove cell mass. The filtrates were

detected with flow cytometry (Epics. COULTER XL,

USA).

H. Li et al. / HEALTH 2 (2010) 435-440

437

2.7. Statistical Analyses

Statistical analyses were performed with SPSS (ver-

sion15.0). Body weights, organ indexes, sperm motility

parameters, the percentages of cell subpopulation were

analyzed by one-way ANOVA, and the obtained data

were expressed as mean ± SD. For parameters with sig-

nificant differences among groups, multiple comparison

tests were carried out. The percentages of sperm mor-

phology were analyzed by the χ2 test, and the data were

expressed as the constituent ratio. The parameters values

were compared at the 5% significance level.

3. RESULTS

3.1. Effect of Apigenin on Body and

Reproductive Organ Weights

The body weights of mice in seven groups increased at

different levels, but compared with the solvent and nega-

tive control groups, none of the experimental groups

showed statistically significant differences in body

weight, relative testes and epididymides weight (Table

1).

3.2. Effect of Apigenin on Sperm Motion and

Density

The mouse sperm densities in five experimental groups

reduced at different levels, and compared with the sol-

vent control group, there was a statistically significance

in the 25 mg/kg group (Table 2).

3.3. Effect of Apigenin on Sperm

Morphology

The mean percentage of abnormal sperms ranged from

2.69% to 3.16% in the experimental groups. The five

experimental groups did not significantly differ from

each other or the control groups. Furthermore, abnormal

sperms were mainly classified as amorphous; non-hook

sperm took the second largest group; banana, double-tail

and folded-tail sperms were rare, which were mainly

concentrated in the head sperms.

3.4. Effect of Apigenin on Cell Cycle

After injection of 5 mg/kg apigenin, the percentage of

1C population increased significantly compared with

the other groups; that of 4C decreased compared with

the negative control group; and a significant increase of

1C:4C ratio was observed compared with the control

groups.

Compared with the solvent and negative control

groups, the percentage of seminiferous epithelium cells

at the cell-circle phase of G0/G1 exhibited a significant

increase in the 25 mg/kg group.

4. DISCUSSION

The study on apigenin investigated whether apigenin had

any toxic effect on the reproductive system in adult male

mice. Intraperitoneal injection of apigenin for seven days

to mice led to no obvious change in their body weights.

The relative weights of testis and epididymis showed no

marked changes as well, when normalized with the

whole body weights.

Sperm motion is important for the sperm functional

capacity, and the assessment of sperm motion is very

useful for detecting or evaluating male reproductive tox-

icity [19]. For this purpose, both percentage of mobile

spermatozoa and characteristics of sperm movement

might provide key information. More quantitative and

qualitative evaluation of toxic effects on sperm motion

(e.g., motility) has been possible with the computer-

assisted sperm analysis (CASA) system. Besides the

conventional parameters, the system also describes

sperm kinematics movements [20-27]. In this study,the

results showed that compared with the solvent control

group, sperm density had a significantly reduction in the

25 mg/kg group was shown, which is consistent with the

results of Pu et al. [28]. On the other side, others pa-

rameters did not show significant variation in any ex-

perimental group. Taken together, our results suggested

that apigenin had some effects on sperm motion pa-

rameters in mice.

Sperm morphology is another important aspect in as-

sessing sperm quality as well as a key index to evaluate

reproductive toxicity and mutagenicity of exogenous

chemicals [29]. The results showed that abnormal

sperms were mainly concentrated in the head sperm,

which is consistent with the observation that the changes

of sperm morphology are mainly in the head.

In recent years, the flow cytometry analysis has grown

rapidly, which allows the recognition of several cell

types at various stages of spermagenesis [30,31]. In par-

ticular, after treatment with toxic agents, the flow cy-

tometry analysis of testicular tissue can be used to detect

variations in terms of the relative fractions of different

cell subpopulations, thus providing crucial evidence on

possible toxic effects [32]. In this study, the percentages

of different germ cell types in male mice as a function of

dose after the treatments with different doses of apigenin

were reported. Based on the DNA content, three

germ-cell peaks could be identified through flow cy-

tometry: 1C (round spermatids), 2C (spermatogonia) and

4C (primary spermatocytes). After intraperitoneal injec-

tion of 5 mg/kg apigenin, the percentage of 1C popula-

tion increased, the percentage of 4C decreased and so the

1C:4C ratio significantly increased, indicating that pri-

mary spermatocytes decreased but round spermatids

increased. This observation suggested that apigenin

stimulated the meiosis of spermatogonia, and the effect

H. Li et al. / HEALTH 2 (2010) 435-440

438

of apigenin in male mice occurred at the last stage of

spermagenia. Moreover, compared with the control gro-

ups, the sperm density and quality of this dose group did

not show any significant change.

Compared with the solvent and negative control

groups, the percentage of seminiferous epithelium cells

at the cell-circle phase exhibited a significant increase in

the 25 mg/kg group. This indicated that the dose of 25

mg/kg can slow the proliferation speed of germ cells,

and spermatogonia were blocked in G0/G1. This inhibi-

tion of spermatogonia is consistent with the decreased

sperm density in the group.

In conclusion, the results show that as a single agent,

apigenin can produce adverse effects on the reproductive

system in adult male mice at a dose of 25 mg/kg/day.

Thus, more efforts are required to elucidate the mecha-

nisms related the apigenin effects on sperm quality and

spermatogenesis.

Table 1. Body and organ weights (mean ± s.d.).

Apigenin (mg/kg/d)

5 10 15 20 25

0.05% DMS0 0.9% N.S

weight change (g) 3.86 ± 1.64 3.81 ± 2.62 4.53 ± 1.87 4.03 ± 1.25 4.15 ± 1.63 4.58 ± 1.37 4.42 ± 1.97

Relative organ weights (g/g.bw)

Testes 5.73 ± 0.74 5.42 ± 0.93 5.26 ± 0.54 5.51 ± 0.74 5.51 ± 0.76 6.22 ± 0.93 5.64 ± 0.78

Epididymides 2.00 ± 0.25 1.81 ± 0.21 1.87 ± 0.31 1.79 ± 0.28 1.87 ± 0.30 1.76 ± 0.31 1.87 ± 0.23

Table 2. Sperm motility in male mice treated with apigenin (n = 12/group; mean ± s.d.).

Apigenin (mg/kg/d)

5 10 15 20 25

0.05% DMS0 0.9% N.S

a-grade sperm 7.52 ± 5.97 6.08 ± 3.86 9.66 ± 5.86 9.42 ± 6.05 10.01 ± 4.73 9.88 ± 5.63 7.72 ± 3.26

b-grade sperm 14.19 ± 5.32 13.24 ± 6.77 10.48 ± 5.28 12.90 ± 5.78 16.44 ± 5.35 14.99 ± 4.90 14.22 ± 4.57

c-grade sperm 42.63 ± 15.77 46.36 ± 7.28 41.00 ± 11.6036.85 ± 12.3738.70 ± 17.02 40.93 ± 8.24 37.38 ± 8.07

d-grade sperm 35.66 ± 20.03 34.32 ± 13.01 38.85 ± 16.3640.72 ± 10.0734.85 ± 18.56 34.19 ± 13.21 40.72 ± 10.72

a + b sperm 20.71 ± 8.81 19.32 ± 8.81 19.37 ± 8.96 22.42 ± 9.11 26.45 ± 10.00 24.87 ± 8.90 21.9 ± 5.94

a + b + c sperm 64.34 ± 20.03 65.68 ± 13.01 61.10 ± 16.3759.27 ± 10.0765.15 ± 18.56 65.85 ± 13.21 59.28 ± 10.72

VCL(μm/s) 55.80 ± 10.11 51.94 ± 4.73 54.28 ± 9.01 55.21 ± 7.90 60.02 ± 6.68 55.52 ± 6.46 58.56 ± 8.51

VSL(μm/s) 17.19 ± 6.11 15.20 ± 4.60 18.14 ± 6.64 19.57 ± 5.62 20.22 ± 4.43 18.70 ± 5.31 18.61 ± 3.70

VA P ( μm/s) 24.27 ± 6.51 21.23 ± 4.39 24.31 ± 7.06 26.14 ± 4.98 26.74 ± 4.61 24.72 ± 5.19 25.15 ± 3.58

MAD(deg) 68.87 ± 8.76 71.40 ± 5.79 68.65 ± 8.08 69.03 ± 5.83 69.52 ± 6.93 69.00 ± 6.28 69.53 ± 6.26

ALH(μm) 1.87 ± 1.02 1.81 ± 0.81 1.83 ± 0.84 2.12 ± 0.80 2.64 ± 1.10 2.23 ± 0.92 2.57 ± 0.68

BCF(Hz) 10.54 ± 1.82 10.59 ± 3.44 9.38 ± 2.81 10.30 ± 1.37 9.95 ± 1.57 10.04 ± 1.58 9.82 ± 0.69

LIN 28.93 ± 6.10 28.17 ± 6.03 29.94 ± 6.12 32.75 ± 7.85 31.08 ± 6.49 31.47 ± 4.37 29.44 ± 4.00

WOB 44.57 ± 3.54 42.89 ± 3.73 46.60 ± 4.12 48.30 ± 4.73 46.17 ± 4.68 46.57 ± 2.30 45.03 ± 3.89

STR 63.33 ± 8.85 63.66 ± 9.57 63.42 ± 7.75 63.15 ± 9.31 68.27 ± 10.57 66.68 ± 6.76 66.96 ± 5.34

Sperm density

(× 106/ml) 1.25 ± 0.82 1.15 ± 0.38 1.51 ± 0.67 1.48 ± 0.85 0.96 ± 0.48★ 2.22 ± 1.50 1.92 ± 0.66

Note: ★significantly different from the solvent control group at P ＜ 0.05.

a: fast progressive motility

b: slow or dull progressive motility

c: non-progressive motility

d: immobility

a + b: progressive motility

a + b + c: total sperm motility

H. Li et al. / HEALTH 2 (2010) 435-440

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Openly accessible at

Table 3. Sperm morphology (n = 12/group; mean ± s.d.).

Apigenin (mg/kg/d)

5 10 15 20 25

0.05% DMS0 0.9% N.S

Total count 8000 10000 9000 7000 5000 9000 7000

Abnormal 215(2.69) 292(2.92) 264(2.93) 221(3.16) 156(3.12) 251(2.79) 165(2.36)

Amorphous 173(80.47) 232(79.45) 195(45.44) 185(83.37) 120(76.92) 205(81.67) 124(75.15)

Non-hook 36(16.74) 49(21.12) 66(25.00) 31(14.02) 34(21.79) 43(17.13) 35(21.21)

Banana 1(0.47) 1(3.13) 1(0.38) 3(1.36) 0(0.00) 0(0.00) 5(3.03)

Double-tail 5(2.33) 10(31.30) 2(0.76) 2(0.90) 1(0.64) 3(1.82) 1(0.61)

Folded-tail 0(0.00) 0(0.00) 0(0.00) 0(0.00) 1(0.64) 0(0.00) 0(0.00)

Table 4. Sperm motility parameters t (mean ± s.d.).

Apigenin (mg/kg/d)

5 10 15 20 25

0.05% DMS0 0.9% N.S

percentages of ploidy cells

1C 86.23 ± 2.60■ 77.50 ± 4.66 75.90 ± 2.12 76.68 ± 2.37 80.20 ± 1.15 77.13 ± 4.49 76.43 ± 5.12

2C 7.72 ± 1.07 11.35 ± 2.32 13.53 ± 0.05 11.69 ± 2.24 11.25 ± 0.66 10.86 ± 4.00 12.32 ± 2.75

4C 4.085 ± 0.46★ 7.281 ± 1.85 6.469 ± 2.44 5.270 ± 0.63 5.282 ± 1.56 6.048 ± 2.30 6.731 ± 1.78

AP 1.925 ± 1.66 1.060 ± 1.50 0.000 ± 0.00 3.833 ± 4.25 3.033 ± 1.91 2.125 ± 4.25 0.875 ± 1.75

percentages of seminiferous epithelium cells in cell circle phase

G0/G1 65.30 ± 2.01 54.10 ± 6.19 56.35 ± 4.74 60.67 ± 5.75 67.60 ± 5.84◆ 53.60 ± 6.22 55.93 ± 10.4

S-phase 0.00 ± 0.00 11.68 ± 8.24 17.20 ± 3.11 11.23 ± 10.15 1.33 ± 1.19 16.60 ± 6.99 14.13 ± 9.63

G2/M 34.70 ± 2.01 34.26 ± 2.24 26.50 ± 7.78 28.07 ± 7.17 31.07 ± 4.95 29.80 ± 2.69 29.93 ± 3.58

ratio of ploidy cells

1C:2C 11.39 ± 1.97 7.14 ± 1.93 5.61 ± 0.18 6.73 ± 1.32 7.14 ± 2.95 8.61 ± 5.50 6.51 ± 1.84

1C:4C 21.36 ± 2.94▲ 11.39 ± 3.72 12.70 ± 5.11 14.73 ± 2.36 16.09 ± 4.71 15.37 ± 9.38 12.40 ± 5.28

4C:2C 0.53 ± 0.05 0.63 ± 0.04 0.48 ± 0.18 0.46 ± 0.12 0.47 ± 0.11 0.56 ± 0.08 0.55 ± 0.14

Note: ■significantly different from others groups at P ＜ 0.05.

★significantly different from the NS control group at P ＜ 0.05.

▲significantly different from the NS and 0.05% DMSO control groups at P ＜ 0.05.

◆significantly different from the NS and 0.05% DMSO control

1C: haploidcell (round spermatids)

2C: diploidcell (spermatogonia)

4C: Tetraploidcell, (primary spermatocytes)

5. ACKNOWLEDGEMENTS

This work was supported by the research foundation for the young and

the middle-aged scientist in Gansu Province, China (Grant No.

099RJYA003). We thank lab members An Jing, Yu-Hui Dang, Chen Ya

and Shu-Yu Liu for their assistance. We also thank Dr. Si-Wu Fu and

Dr. Xing-Rong Liu for critical reading the manuscript.

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