Effects of Methylparathion on the
Reproductive System in Male
Frogs, Hoplobaprachus rugulosus
Artchariya Chaiyarat,1 Prakong Tangpraprutgul,1,2
Putsatee Pariyanonth1 and Kingkaew Wattanasirmkit1,2
The effect of methylparathion (MPT), a potent insecticide, on the
reproductive system of the male frog Hoplobaprachus rugulosus was
investigated. Male frogs aged 2, 4, 6, 8, 10 and 12 months old were exposed
to 20 ppm of MPT for 60 days. At the end of the experiment, animals were
sacrificed. Plasma samples were collected and kept at -200C until undergoing
a testosterone radioimmunoassay. Testes were fixed in Bouin, s solution for
histological study. Plasma testosterone levels decreased 10.50%, 15.94%,
17.68 %, 29.05%, 19.18% and 27.12% in frogs autopsied at the ages of 4, 6, 8,
10, 12 and 14 months, respectively. Microscopy studies showed that MPT
directly injured leydig cells and affected their cell structure. The histological
alterations of leydig cells in all exposed frogs included hydropic swelling,
pyknotic nuclei, karyolysis, nuclear membrane disruption and vacuolated
interstitial tissue. The gonadosomatic index percentage (GSI%) showed a
correlation with the plasma testosterone levels in the frog at the ages of 10, 12
and 14 months old. It is concluded that the effect of MPT on the reproductive
system, especially on lowering of testosterone levels, may be exerted directly
on the leydig cell functions. The mature male frog seems to be more sensitive
to decreasing sexual ability due to MPT than the immature frog.
Key word: methylparathion, reproductive system, testosterone, male frogs,
Hoplobaprachus rugulosus.
1
Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330,
Thailand.
2
National Research Center for Environmental and Hazardous Waste Management
(NRC – EHWM), Chulalongkorn University, Bangkok 10330, Thailand.
J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)
29
Artchariya Chaiyarat, Prakong Tangpraprutgul, Putsatee Pariyanonth and
……………………………………………….……………………………...Kingkaew Wattanasirmkit
ผลของเมทิลพาราไทออนตอระบบสืบพันธุกบนาเพศผู
Hoplobaprachus rugulosus
อัจฉริยา ไชยรัตน ประคอง ตังประพฤทธิ์กุล ผุสตี ปริยานนท และ
กิ่งแกว วัฒนเสริมกิจ (2546)
วารสารวิจัยวิทยาศาสตร จุฬาลงกรณมหาวิทยาลัย 28 (Special Issue I)
การศึกษาผลของเมทิลพาราไทออนตอระบบสืบพันธุของกบนาเพศผูโดยดูผล
ที่มีต อปริ มาณฮอรโมนเทสโทสเตอโรนในพลาสมาและต อเนื้อเยื่ อของอั ณฑะ
กบนาเพศผู Hoplobaprachus rugulosus อายุ 2, 4, 6, 8, 10 และ 12 เดือน โดยใช
เมทิลพาราไทออนที่ความเขมขน 20 ppm ติดตอกันเปนเวลา 60 วัน แลวเก็บ
ตัวอยางเลือดของกบนาเพื่อนําไปปนเอาพลาสมาไปเก็บไวที่อุณหภูมิ -20 องศา
เซลเซียส จนกวาจะนําไปวัดปริมาณฮอรโมนเทสโทสเตอโรนโดยวิธี เรดิโออิมมิวโนเอสเสย ทําการเก็บตัวอยางอัณฑะของกบนาโดยตัดออกมาทั้งสองขางแลวแชลง
ใน Bouin's solution ทันที เพื่อนําไปศึกษาทางดานเนื้อเยื่อตอไป ผลการศึกษา
พบว า เมทิ ล พาราไทออนมี ผ ลทํ า ให ป ริ ม าณของฮอร โ มนเทสโทสเตอโรนใน
พลาสมาของกบนาลดลงอยางมีนัยสําคัญทางสถิติ (p≤ 0.05) เมื่อเปรียบเทียบกับ
กลุมควบคุมยกเวนในกบนาเพศผูอายุ 4 เดือน โดยพบวาในกบนาอายุ 6, 8, 10, 12
และ14 เดือนมีปริมาณฮอรโมนเทสโทสเตอโรนลดลง 15.94%, 17.68%, 29.05%,
19.18% และ 27.12% ตามลําดับ จากการศึกษาเนื้อเยื่อของอัณฑะกบนาพบวา เม
ทิลพารา-ไทออนมีผลทําลายเซลลลัยดิกโดยตรงและทําใหเกิดการเปลี่ยนแปลงใน
ลักษณะที่มีการรวมกลุมของโครมาตินรอบๆ เยื่อหุมนิวเคลียส นิวเคลียสอัดกัน
แนนเปนกอนติดสีทึบอยูกลางเซลล มีการบวมของเซลล มีเยื่อหุมนิวเคลียสฉีกขาด
และเกิดการสลายตัวของนิวเคลียสและมีการทําลายของเนื้อเยื่อที่อยูระหวางทอเซ
มินิเฟอรัส ผลการทดลองครั้งนี้สรุปไดวาผลของเมทิล พาราไทออนตอระบบ
สืบพันธุของกบนาเพศผูโดยเฉพาะที่ทําใหปริมาณของฮอรโมนเทสโทสเตอโรนใน
พลาสมาของกบนาเพศผูลดลง เกิดจากการที่เมทิล พาราไทออนทําลายลัยดิกสเซลล
โดยตรง
คําสําคัญ เมทิลพาราไทออน ระบบสืบพันธุ เทสโทสเตอโรน กบนาเพศผู
30
J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)
Effects of Methylparathion on the Reproductive System in Male Frogs,
Hoplobaprachus rugulosus……………….……………………………………………………………
INTRODUCTION
The
native
Thai
frog,
Hoplobaprachus
rugulosus,
is
(1)
distributed all over the country. Like
other amphibians, the frogs spend most
of their lives in aquatic habitats but can
also hunt for food and shelter on
dry ground. They exhibited seasonal
breeding, both in nature and in farms.(2)
The spermatogenesis was completed at
the age of 6 months in the male frog.(3)
It has been observed throughout the
country that the population of the
frog has dramatically declined. The
declining of the frog population may be
due to various reasons such as a lack of
the proper habitat, since it has been
invaded by the expansion of agriculture.
Other reasons for the declining frog
population may be exposure to toxic
chemical pollutants in the environment,
since frogs can easily absorb pollutants
though their wet skin. Pesticides are
believed to be among the most potent
toxic chemicals in the environment.
Organophosphate groups are widely
used as insecticides in Thailand.
Methylparathion (MPT) is one of
the organophosphates that has been
intensively used as a potent insecticide
in Thailand. MTP was reported to affect
the reproductive system in fish,(4) in
rats(5) and in mice.(6) It is of interest to
determine the effects of MTP on
the reproductive system in frogs to
determine whether the declining frog
population is due to contamination by
insecticides in the environment. We
therefore aimed to study the effect of
MPT on the reproductive system,
emphasizing the synthesis and secretion
of testosterone and sexual ability
of male frogs at different ages.
The results obtained may indicate the
reason for the declining frog population
in the natural habitat, since frogs are
more easily affected by any alteration of
the environment.
MATERIALS AND METHODS
Animals
Male frogs, Hoplobaprachus
rugulosus, were used. They were bred
and reared in a concrete pond (2.0 x 2.5
x 1.0 m3) with a constant water level of
10 cm depth which was changed every
two days. They were fed daily (5%
body weight/kg) with frog chow and
kept in a natural environment at a farm.
The average ambient temperature
ranged from 25.0 - 29.60C and the
relative humidity was about 69 - 81%
throughout the year. The animals were
exposed to a natural daily light and dark
cycle, both for approximately 12 hours,
in Petchaburi Province at the Royal
Development Study Center.
Male frogs aged 2, 4, 6, 8, 10 and
12 months were used. Each age group
was subdivided into experimental and
control groups of 20 animals each. The
experimental group was exposed to 20
ppm methylparathion for 2 months.
The solution of methylparathion was
changed every two days until the end
of experiment. The experiment was
designed to end during the months of
high plasma testosterone level: April –
September.(2) At the end of the
experiment, trunk blood was collected
in heparinized tubes. Plasma samples
were stored at -200C for the hormone
determination. Testes were excised,
weighed and fixed in Bouin,s fixation
for the histological study.
Hormone assay
Testosterone was determined by
radioimmunoassay. The testosterone
J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)
31
Artchariya Chaiyarat, Prakong Tangpraprutgul, Putsatee Pariyanonth and
……………………………………………….……………………………...Kingkaew Wattanasirmkit
antibody was kindly supplied by
Dr. Fortune Kohen of the Weizmann
Institute of Science, Israel. The assay
was conducted as previously described (7)
as follows: duplicates of 200 μl of
plasma from each sample were extraced
in 5 ml diethyl ether. The samples were
dried and later were resuspended in 500
μl phosphate buffer saline (0.05 M, pH
7.4). Then 100 μl of hormone tracer
and 100 μl of antiserum were added.
The mixture was incubated at 40C for
18-20 hours. Free forms of hormones
were separated by dextran-coated
charcoal, where the bound form was
transferred into scintillation counting
fluid and counted in a beta counter.
RESULTS
Effects of MPT on plasma testosterone
levels
Figure 1 showed that the plasma
testosterone levels in frogs exposed to
methylparathion (MPT) for two months
decreased by 10.50%, 15.94%, 17.68%,
29.05%, 19.18% and 27.12% in frogs
autopsied at the ages of 4, 6, 8, 10, 12
and 14 months, respectively. Plasma
testosterone levels in treated frogs
autopsied at the ages of 6, 8, 10, 12 and
14 months were significantly lower than
those of the control group (p ≤ 0.05).
No difference were observed in frogs
autopsied at 4 months old.
4500
4000
3500
pmol/L
Testosterone ( pmol / L )
5000
3000
Control
2500
Treated
2000
1500
1000
500
0
4
6
8
10
12
14
Age (Months)
Figure 1. Plasma testosterone levels in frogs exposed to MPT for 2 months,
autopsied at ages of 4, 6, 8, 10, 12 and 14 months (open bar =
control group, dark bar = treated group,
= significant
difference at p≤ 0.05).
32
J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)
Effects of Methylparathion on the Reproductive System in Male Frogs,
Hoplobaprachus rugulosus……………….………………….…………………………………………
Effects of MPT on gonadosomatic
index percentage (GSI%) in male frogs
A higher value of the
gonadosomatic index percentage
(GSI%) or gonadal weight as a
percentage of body mass indicates
greater sexual ability of the frog. Figure
2 showed that GSI% in frogs exposed to
MPT for 2 months was significantly
lower than those of control groups when
autopsied at ages of 10, 12 and 14
months (p ≤ 0.05). No difference was
observed in frogs autopsied at the age of
8 months. GSI% in exposed frogs
autopsied at ages of 4 and 6 months old
were higher than those of the control.
.
240
210
180
Control
150
GSI%
120
Treated
90
60
30
0
4
6
8
10
Age (Months)
12
14
Figure 2. GSI % in frogs exposed to MPT for 2 months, autopsied at the ages
of 4, 6, 8, 10, 12 and 14 months (open bar = control group, dark
bar = treated group,
= significant difference at p ≤ 0.05).
Effects of MPT on leydig cell histology
Microscopic study of the testes
showed that the leydig cells of the MPT
exposed frogs were altered in the
same manner in all groups of frogs
studied. The histological alterations
observed in the leydig cells included of
hydropic swelling (Figure 4), pyknotic
nuclei (Figure 5), perinuclearchromatin
clumping nuclei (Figure 6), karyolysis
(Figure 7), and nuclear membrane
disruption (Figure 8), compared with
the leydig cells of the control frogs
(Figure 3)
J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)
33
Artchariya Chaiyarat, Prakong Tangpraprutgul, Putsatee Pariyanonth and
…...…………….…………………………….……………………………...Kingkaew Wattanasirmkit
Control frog
Figure 3. A representative image of normal leydig cells of control
frogs autopsied at the age of 10 months. Some healthy
nuclei of the cells are indicated (
).
Scale Bar = 10 μm. Stained by haematoxylin & eosin.
Treated frog
Hydropic
swelling
34
J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)
Effects of Methylparathion on the Reproductive System in Male Frogs,
Hoplobaprachus rugulosus……………….………………….…………………………………………
Figure 4. A representative image of hydropic swelling (E) of leydig cells
in the testes of MPT exposed frogs autopsied at the age of
8 months.
Scale Bar = 10 μm. Stained by haematoxylin & eosin.
J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)
35
Artchariya Chaiyarat, Prakong Tangpraprutgul, Putsatee Pariyanonth and
…...…………….…………………………….……………………………...Kingkaew Wattanasirmkit
Figure 5. A representative image of pyknotic nuclei (t) of the leydig cells in
the testes of MPT exposed frogs autopsied at the age of 12
months.
Scale Bar = 10 μm. Stained by haematoxylin & eosin.
R
Figure 6. A representative image of a perinuclearchromatin clumping nucleus
(R) of a leydig cell in the testes of MPT exposed frogs autopsied at
the age of 6 months.
Scale Bar = 10 μm. Stained by haematoxylin & eosin.
36
J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)
Effects of Methylparathion on the Reproductive System in Male Frogs,
Hoplobaprachus rugulosus……………….………………….…………………………………………
Figure 7. A representative image of karyolysis ( ∗ ) of leydig cells in the testes
of MPT exposed frogs autopsied at the age of 14 months.
Scale Bar = 10 μm. Stained by haematoxylin & eosin.
Figure 8. A representative image of nuclear membrane disruption (S) of leydig
cells in the testes of MPT exposed frogs autopsied at the age of 10
months.
Scale Bar = 10 μm. Stained by haematoxylin & eosin.
J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)
37
Artchariya Chaiyarat, Prakong Tangpraprutgul, Putsatee Pariyanonth and
…...…………….…………………………….……………………………...Kingkaew Wattanasirmkit
DISCUSSION
Methylparathion (MPT) has been
demonstrated to alter the testosterone
secretion in frogs. MPT reduced the
testosterone secretion more dramatically
in frogs that were exposed to the MPT
at the ages of 6, 8, 10 and 12 months
than those exposed at the ages of 2 and
4 months. The results obtained were
similar to those found in rats by
Samanya,(8) who showed that MPT
reduced plasma testosterone levels in
adult rats but not in immature rats.
The results are also similar to those
reported by Somkoti(9) and Chapin, ( 1 0 )
in which another organophosphate tri-ocresyl phosphate, reduced spermatogenesis
and testosterone secretion in adult rats.
The mechanism of MPT to reduce
testosterone secretion is still unclear.
MPT may exert its effect via reduction
of LH secretion, resulting in reduction
of testosterone secretion.(11, 12) The
mechanism of action of MPT to
reduce testosterone secretion in
frog testicular leydig cells is still not
known. However it has been reported
that a potent endocrine disrupter,
bisphenol A, induced orphan nuclear
receptors and subsequently altered the
steroidogenesis in mouse testicular
leydig cells.(13)
The higher value of the
gonadosomatic index percentage (GSI%)
or gonadal weight as percentage of body
mass indicates greater sexual ability in
frogs of both sexes.(14) GSI% increased
in older frogs (Figure 2), showing a
direct correlation between testicular
weight and testosterone production.(15)
MPT reduced GSI% in frogs at ages of
10, 12 and 14 months but the effect was
not observed in younger frogs. The
GSI% in the treated frogs at ages of
4 and 6 months, which seemed slightly
higher than those in the control group,
38
could not be conclusively confirmed.
The results obtained may be due to
uncertain activity of the testes in the
immature frogs. However the GSI%
values obtained from mature frogs were
concomitant with testosterone secretion
affected by MPT. Therefore it seems
that the mature frogs are more sensitive
to MPT in terms of decreasing sexual
ability than the immature ones.
Microscopic study of the leydig
cells revealed MPT injured leydig
cells in all ages of frogs studied.
Common alterations of the cell included
hydropic swelling, pyknotic nuclei,
peri- nuclearchromatin-clumping nuclei,
karyolysis and membrane disruption.
These alterations of the cell may affect
the synthesis as well as secretion of
testosterone from MPT injured cells,
resulting in lowering of plasma
testosterone levels.
It is therefore concluded from this
study that mature frogs are more
sensitive to MPT altering their
reproductive functions than immature
ones. The effects of MPT on reducing
plasma testosterone levels may be
exerted directly on the synthesis and/or
secretion of testosterone from the leydig
cells.
Results obtained from the study
indicated that any pesticides or
toxic chemicals contaminating the
environment might affect the
reproductive system in frogs, e. g., by
lowering their sexual ability which may
result in a declining frog population in
the natural habitat.
ACKNOWLEDGMENTS
We
thank
the
Roy al
Development Study Center and the
National Research Center for
Environmental and Hazardous Waste
J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)
Effects of Methylparathion on the Reproductive System in Male Frogs,
Hoplobaprachus rugulosus……………….………………….…………………………………………
Management (NRC-EHWM) for their
support of this work.
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Received: July 4, 2003
Accepted: August 22, 2003
J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)
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Artchariya Chaiyarat, Prakong Tangpraprutgul, Putsatee Pariyanonth and
…...…………….…………………………….……………………………...Kingkaew Wattanasirmkit
40
J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)