Effects of Methylparathion on the Reproductive System in Male Frogs, Hoplobaprachus rugulosus

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1 Effects of Methylparathion on the Reproductive System in Male Frogs, Hoplobaprachus rugulosus Artchariya Chaiyarat, 1 Prakong Tangpraprutgul, 1,2 Putsatee Pariyanonth 1 and Kingkaew Wattanasirmkit 1,2 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 C until undergoing a testosterone radioimmunoassay. Testes were fixed in Bouin, s solution for histological study. Plasma testosterone levels decreased 10.50%, 15.94%, %, 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

2 Artchariya Chaiyarat, Prakong Tangpraprutgul, Putsatee Pariyanonth and....kingkaew Wattanasirmkit ผลของเมท ลพาราไทออนต อระบบส บพ นธ กบนาเพศผ Hoplobaprachus rugulosus อ จฉร ยา ไชยร ตน ประคอง ต งประพฤทธ ก ล ผ สต ปร ยานนท และ ก งแก ว ว ฒนเสร มก จ (2546) วารสารว จ ยว ทยาศาสตร จ ฬาลงกรณ มหาว ทยาล ย 28 (Special Issue I) การศ กษาผลของเมท ลพาราไทออนต อระบบส บพ นธ ของกบนาเพศผ โดยด ผล ท ม ต อปร มาณฮอร โมนเทสโทสเตอโรนในพลาสมาและต อเน อเย อของอ ณฑะ กบนาเพศผ Hoplobaprachus rugulosus อาย 2, 4, 6, 8, 10 และ 12 เด อน โดยใช เมท ลพาราไทออนท ความเข มข น 20 ppm ต ดต อก นเป นเวลา 60 ว น แล วเก บ ต วอย างเล อดของกบนาเพ อน าไปป นเอาพลาสมาไปเก บไว ท อ ณหภ ม -20 องศา เซลเซ ยส จนกว าจะน าไปว ดปร มาณฮอร โมนเทสโทสเตอโรนโดยว ธ เรด โออ มม ว- โนเอสเสย ท าการเก บต วอย างอ ณฑะของกบนาโดยต ดออกมาท งสองข างแล วแช ลง ใน Bouin's solution ท นท เพ อน าไปศ กษาทางด านเน อเย อต อไป ผลการศ กษา พบว าเมท ลพาราไทออนม ผลท าให ปร มาณของฮอร โมนเทสโทสเตอโรนใน พลาสมาของกบนาลดลงอย างม น ยส าค ญทางสถ ต (p 0.05) เม อเปร ยบเท ยบก บ กล มควบค มยกเว นในกบนาเพศผ อาย 4 เด อน โดยพบว าในกบนาอาย 6, 8, 10, 12 และ14 เด อนม ปร มาณฮอร โมนเทสโทสเตอโรนลดลง 15.94%, 17.68%, 29.05%, 19.18% และ 27.12% ตามล าด บ จากการศ กษาเน อเย อของอ ณฑะกบนาพบว า เม ท ลพารา-ไทออนม ผลท าลายเซลล ล ยด กโดยตรงและท าให เก ดการเปล ยนแปลงใน ล กษณะท ม การรวมกล มของโครมาต นรอบๆ เย อห มน วเคล ยส น วเคล ยสอ ดก น แน นเป นก อนต ดส ท บอย กลางเซลล ม การบวมของเซลล ม เย อห มน วเคล ยสฉ กขาด และเก ดการสลายต วของน วเคล ยสและม การท าลายของเน อเย อท อย ระหว างท อเซ ม น เฟอร ส ผลการทดลองคร งน สร ปได ว าผลของเมท ล พาราไทออนต อระบบ ส บพ นธ ของกบนาเพศผ โดยเฉพาะท ท าให ปร มาณของฮอร โมนเทสโทสเตอโรนใน พลาสมาของกบนาเพศผ ลดลง เก ดจากการท เมท ล พาราไทออนท าลายล ยด กส เซลล โดยตรง ค าส าค ญ เมท ลพาราไทออน ระบบส บพ นธ เทสโทสเตอโรน กบนาเพศผ 30 J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)

3 Effects of Methylparathion on the Reproductive System in Male Frogs, Hoplobaprachus rugulosus. INTRODUCTION The native Thai frog, Hoplobaprachus rugulosus, is distributed all over the country. (1) 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 (2) breeding, both in nature and in farms. 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 (4) the reproductive system in fish, 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 m 3 ) 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 C 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 (2) September. At the end of the experiment, trunk blood was collected in heparinized tubes. Plasma samples were stored at C 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

4 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 (7) was conducted as previously described 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 4 0 C for 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 Testosterone ( pmol / L ) pmol/l Control Treated 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)

5 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 GSI% Age (Months) Control Treated 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

6 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)

7 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

8 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)

9 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

10 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 (9) reported by Somkoti and Chapin, (10) 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 (13) leydig cells. 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, 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 Royal Development Study Center and the National Research Center for Environmental and Hazardous Waste 38 J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)

11 Effects of Methylparathion on the Reproductive System in Male Frogs, Hoplobaprachus rugulosus.. Management (NRC-EHWM) for their support of this work. REFERENCES 1. Tayler, E.H. (1962) The amphibian fauna in Thailand, The university of Kansus bulletin. 8, Tangpraprutgul, P., Pariyanonth, P., and Chaitiamwong, R. (1996) Seasonal changes in plasma gonadal steroids in Rana tigerina rugulosa and Rana catesbeiana, Thai J. Physiological Science. 9(1), Sretarugsa, P., Nakiem, V., Sobhon, P., Chavadej, J., Kruatrachue, M., and Upatham, E.S. (1997) Structure of the testis of Rana tigerina and its changes during development and seasonal variation, J. Sci. Soc. Thailand. 23, Harnchariyakul, P. (1992) Acute toxicity and sublethal effect of methylparathion in giant perch (Lates calcarifer). Doctoral dissertation, Chulalongkorn University Gupta, R.C., Thornburg, J.E., Stedman, D.B., and Weisch, F. (1984) Effect of subchronic administration of methyl parathion on in vivo protein synthesis in pregnant rat and their conceptuses, Toxicol. Appl. Pharmaco. 73(3), Mathew, G., Vijayalaxmi, K.K., and Abdul, R.M. (1992) Methyl parathion induced sperm shape abnormalities in mouse, Mut.Res 280(3), Tangpraprutgul, P., and Pariyanonth, P. (1999) The influence of transportation on plasma gonadal steroid concentrations in adult frogs, Rana tigerina rugulosa and Rana catesbeiana, Mole. and Comp. Endocrinol Samanya, M. (1996) Effect of mevinphos and methylparathion on testosterone secretion in male rats, M. Sc. Thesis, Chulalongkorn University Somkuti, S.G. (1987) Reproductive tract esions resulting from subchronic administration (63days) of tri- o - cresylphosphate in male rats, Toxicol. Appl. Pharmacol. 89, Chapin, R.E., Phelps, J.L., Somkuti, S.G., Heindel, J.J., and Burka, L.T. (1990) The interaction of sertoli and leydig cells in the testicular toxicity of tri-o-cresyl phosphate, Toxicol. Appl. Pharmacol 104, Rattner, B.A., and Michael, S.D. (1985) Oganophosphorus insecticide induce decrease in plasma luteinizing hormone concentration in white - footed mice Toxicol. Lett. 24, Smallridge, R.C., Carr, F.E., and Fein, H.G. (1991) Diisopropyl fluorphosphate (DFP) reduced serum prolactin thyrotropin, luteinizing hormone and growth hormone and increases adrenocorticotropin and corticosterone in rats : involvement of dopaminergic and somatostatinergic as well as cholinergic pathway Toxicol. Appl. Pharmacol. 108, Song, K-H., Lee, K., Choi, H-S. (2002) Endocrine disrupter bisphenol A induces orphan nuclear receptor Nur77 gene expression and steroidogenesis in mouse testicular leydig cells, Endocrinology. 143 (6), McCreery, B.R., and Litcht, P. (1983) Induced ovulation and changes in pituitary responsiveness to continuous infusion of gonadotropin releasing hormone during the ovarian cycle in the bullfrog, Rana catesbeiana Biol. Reprod 29, Chaitiamwong, R. (1995) Annual changes in testicular and plasma testosterone levels in Rana tigerina and Rana catesbeiana, M. Sc. Thesis,Chulalongkorn University Matthew, P.H., Robert, L.S., and Larry L.E. (1992) Leydig cell renewall in testes of breeding animals, J. Exp. Zool. 261, Received: July 4, 2003 Accepted: August 22, 2003 J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003) 39

12 Artchariya Chaiyarat, Prakong Tangpraprutgul, Putsatee Pariyanonth and kingkaew Wattanasirmkit 40 J. Sci. Res. Chula. Univ., Vol.28, Special Issue I (NRC-EHWM), (2003)