ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 12,16 I- 165 ( 1986)

Effect of Commercial Formulation of Four Organophosphorus Insecticides on the LH-Induced Germinal Vesicle Breakdown

in the Oocytes of a Freshwater Teleost, Mystus vittatus (Bloch)-A Preliminary in Vitro Study

SHAMIM HAIDER ANDNETAJIUPADHYAYA

Department of Zoology, Banaras Hindu University, Varanasi-221005, India

Received March 23.1986

Effect of commercial formulation of four organophosphorus insecticides such as malathion, phosdrin (mevinphos), birlane (chlorfenvinphos), and gardona (tetrachlorvinphos) on LH-in- duced in vitrogerminal vesicle breakdown (GVBD) in the oocytes 0fMystu.r vittatus was investi- gated using three concentrations for each insecticide. All of these insecticides could significantly inhibit the LH-induced GVBD in all of their concentrations except two lower concentrations of birlane. A probable mechanism of inhibition of reproduction by these insecticides is discussed in the light of present findings. 0 1986 Academic p, IIIC.

INTRODUCTION

Aquatic ecosystems are gradually becoming contaminated by various insecticides used in crop protection. These insecticides are potentially toxic to fishes and the effects may be acute resulting in mass mortality, or chronic involving changes in survival, growth, and reproduction (Eaton, 1970; Lane and Scura, 1970; Konar, 198 la). Although the inhibition of reproduction in fishes has been reported in re- sponse to the exposure to sublethal concentration of organophosphorus insecticides (OPI) (Ghosh and Konar, 1976; Kapur et al., 1978; Saxena and Garg, 1978; Choud- hary et al., 198 1; Konar, 198 la,b; Mani and Saxena, 1985), the underlying mecha- nism for the inhibition is not known.

In our earlier study (Haider and Upadhyaya, 1985), we have also observed the loss of stage II and III oocytes accompanied by a significant fall in gonadosomatic index, cessation of vitellogenesis, and inactivation of steroidogenesis in gravid catfish, Mys- tus vittatus following 12 weeks chronic exposure to sublethal concentration of com- mercial formulations of four OPI. Since the maintenance of stage III oocytes, vitello- genesis, and steroidogenesis depends on the pituitary gonadotropic stimulation, its absence was thought to be the possible reason for these effects induced by OPI. Mani and Saxena (1985) also opined that low degree of recrudescence in the ovaries of fenitrothion (an OPI) treated Channa punctatus may be attributed to low titers of gonadotropin(s). However, the possibility of direct action of these OPI on affected organs cannot be ruled out, since it is known that OPI affect several enzymes other than acetylcholinesterase (Corbett, 1974; Eto, 1974; Cremlyn, 1978). Hence, to find out whether there exists any direct effect of these OPI on the ovaries, in the present investigation we have considered the oocyte maturation as the parameter.

In vertebrates, including fishes, meiotic activity in primary oocyte is arrested at the diplotene stage of first prophase which is followed by enormous enlargement of the

161 0147-6513186 $3.00 CopyrigJa 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

162 HAIDER AND UPADHYAYA

nucleus called germinal vesicle (GV) and cytoplasmic growth. At the end of cytoplas- mic growth phase, i.e., vitellogenesis, the meiotic activity of primary oocyte has to be resumed in order to render the oocytes fertilizable. Resumption of the meiotic activ- ity is known as oocyte maturation. In fishes gonadotropin acts on follicular layer of oocytes to produce the maturation inducing steroids (MIS) which in turn induces final maturation (Masui and Clarke, 1979). In M. vittatus we have observed that luteinizing hormone (LH) is the most potent in vitro inducer of oocyte maturation among all the mammalian pituitary hormones (details to be published elsewhere). In the present experiment, maturable oocytes of hf. vittatus are exposed to LH and commercial formulation of four OPI, viz., malathion, phosdrin, birlane, and gardona at various concentrations in vitro to explore the possibility of any direct effect of these insecticides on LH-induced oocyte maturation.

MATERIAL AND METHODS

Culture medium for in vitro oocyte maturation was prepared by dissolving 3.74 g NaCl, 0.32 g KCl, 0.16 g CaCl*, 0.1 g NaI-12P04-H20, 0.16 g MgS04-7Hz0, 0.8 g glucose, and 0.008 g phenol red in 1 liter of triple distilled water. The medium was filtered, autoclaved, and cooled. After adjusting its pH to 7.5 with sterilized 1 N so- dium bicarbonate, 0.2 g of streptomycin sulfate and 200,000 units of penicillin ben- zoate were added to it and stored in refrigerator below 10°C.

Bovine luteinizing hormone (NIH-LH-B9-Bovine) (bLH) obtained from National Institutes of Health, Bethesda, Maryland, was dissolved in sterilized distilled water to make the solution 1 mg/ml.

Malathion 50 EC, a commercial formulation manufactured by Pestochem, India, containing 50% (W/w) technical grade malathion (Sl,Zdi(ethoxy carbonyl) ethyl dimethyl phosphorothiolothionate) was purchased from a local market. The other three following commercial formulations of OPI were obtained from Shell Nether- land, Rotterdam: (1) Birlane or chlorfenvinphos (Zchloro- 1-(2,4dichlorophenyl) vi- nyl diethyl phosphate)-technical material 240 g/liter, EC A422 1, indent 9300/9039. (2) Gardona or tetrachlorvinphos (Zchloro-1-(2,4,5trichlorophenyl) vinyl dimethyl phosphate)-technical material 240 g/liter, EC A432 1, indent 9300/9038. (3) Phos- drin or mevinphos (2-methoxy carbonyl- 1 -methylvinyl dimethyl phosphate)-tech- nical material 240 g/liter, EC A4122, indent 9300/9036.

Stock and working solutions of all the OPI were prepared in ethanol. Considering our earlier in vivo investigation, in this preliminary in vitro study three concentrations of each OPI were used (see Table 1).

All glassware and dissecting instruments were properly cleaned and sterilized prior to initiating the experiment. Three milliliters of incubating medium were transferred to sterilized cavity blocks (each having a capacity of 15 ml) and LH (10 &ml) was added to it. Then OPI solution was added to it in such a way that there would be three concentrations for each OPI as mentioned in the Table. The final concentration of ethanol in each incubation was 1%. Control incubation received no OPI but etha- nol (0.0 1 ml/ml) and LH ( 10 &ml). Three replicate incubations were kept for each concentration.

Some gravid female M vittatus were obtained from a local supplier in the first week of July, 1984. Ovaries of these fishes contained maximum stage III oocytes (mean diameter 0.45 mm). Except for an average of 5.92 f 0.9% (mean f SE, n = 8), all of

INSECTICIDES AND OOCYTE MATURATION 163

TABLE 1

EFFECTS OF INSECTICIDES ON LH-INDUCED GERMINAL VESICLE BREAKDOWN IN Mystus vittatus @XYYTES

Treatment

Number of oocytes

Number of undergone Concentration oocytes exposed GVBD of insecticides

ppb W/V) I II III I II III GVBD

(W f SE”)

Malathion + LH lOkg/ml

Phosdrin + LH lOccg/ml

Birlane + LH IO&ml

Gardona +LH IO&ml

Control (LH 10 &ml)

1000 100 10

0.1 0.01 0.001

1.0 0.1 0.01

100 10 1.0

95 154 143 12 11 12 9.39 + 1.66* 172 109 106 18 11 10 9.99 f 0.30* 69 62 71 7 6 13 12.68 + 2.81**

122 134 69 21 20 12 16.50 f 0.79** 46 111 111 4 32 10 15.50 f 6.66*** 82 58 89 18 8 13 16.78 + 2.59**

42 91 134 10 12 11 15.06 + 4.60*** 79 57 129 9 26 23 24.94 2 10.5 b

114 92 90 27 44 22 31.98 + 7.92’

182 85 161 22 8 19 11.09 f 0.85* 102 85 93 9 12 14 12.66 f 1.94** 172 96 81 30 9 20 17.16 + 4.42***

56 133 95 23 72 51 49.63 + 4.28

’ Each value represents mean percentage + standard error of three replicate incubations (I, II, III, repli- cate incubations).

b Statistically not significant. *P<o.ool.

**P<o.o05. ***p<o.o1.

these oocytes have a centrally located GV and can undergo maturation when exposed to the appropriate stimulator under in vitro condition (personal observation). They were kept in a glass aquaria under natural photoperiod and fed with minced goat liver ad libitum for a week for acclimatization. Three fishes utilized in this experiment were decapitated. Their abdomens were cleaned by 90% ethanol and opened. Ovaries were removed and transferred to a petri dish containing 150 ml cool incubating me- dium. With a fme scissors the ovaries were cut into small pieces. Each one of these pieces were transferred to cavity blocks containing 3 ml of medium and LH with or without OPI in such a way that replicate Nos. I, 2, and 3 of all treatments would receive oocytes from first, second, and third fish, respectively. These cavity blocks were covered with their respective lids.

The entire experiment was conducted in an air conditioned laboratory where the temperature was maintained at 25 f 2°C. The culture was maintained for 24 hr. At the end of incubation the oocytes were separated from each other and observed under dissecting microscope for the incidence of germinal vesicle breakdown (GVBD) which is the first visible step toward oocyte maturation. The rate of GVBD was ex- pressed as the mean value of percentage of GVBD of three replicate incubations. The level of significance was evaluated by Student’s t test.

164 HAIDER AND UPADHYAYA

RESULTS

Oocytes of 6.96, 6.42, and 7.81% have already undergone GVBD in the ovaries of hrst, second, and third fish, respectively. The LH (10 &ml) treatment yielded a significant (P -=z 0.001) percentage of GVBD by 49.63 f 4.28 in these oocytes. When such incubations of oocytes containing LH were exposed to malathion, phosdrin, birlane, or gardona at all the three concentrations used in this experiment exhibited an appreciable change in the incidence of GVBD (Table 1).

When the oocytes were treated with malathion together with LH at the concentra- tions of 1000, 100, and 10 ppb, it resulted in a significant fall in the rate of GVBD by 9.39 f 1.66% (P < O.OOl), 9.99 + 0.3% (P < O.OOl), and 12.68 f 2.81% (P < O.OOS), respectively. Similar decrease in the GVBD percentage was also observed when the oocytes were exposed to phosdrin at the concentrations of 0.1, 0.01, and 0.001 ppb or gardona at the concentrations of 100, 10, and 1 ppb along with LH. In contrast, when the oocytes were exposed to birlane along with LH, the rate of GVBD was not significantly influenced at the concentrations of 0.1 and 0.0 1 ppb, while 1 ppb concentration of this OPI with LH decreased the rate of GVBD significantly (P < 0.0 1). None of the incubations produced ovulation.

DISCUSSION

Inhibition of reproduction in fishes after chronic exposure of various OPI has been reported by several investigators considering the parameters such as the activity of steroidogenic enzymes (Kapur et al., 1978, Haider and Upadhyaya, 1985), vitellogen- esis (Mani and Saxena, 1985; Haider and Upadhyaya, 1985), gonadosomatic index (Saxena and Garg, 1978; Choudhary et al., 1981; Mani and Saxena, 1985; Haider and Upadhyaya, 1985), maintenance of stage II and III oocytes (Saxena and Garg, 1978; Haider and Upadhyaya, 1985), fecundity (Ghosh and Konar, 1976; Konar, 198 lb), and ability to breed (Ghosh and Konar, 1976). Since, the majority of these events are controlled by pituitary gonadotropin(s) directly and/or via producing the female steroid hormone(s), their inhibition is attributed either to the disturbance in controlling hormone(s) (Mani and Saxena, 1985; Haider and Upadhyaya, 1985) or to the direct interference of OPI in the metabolism of concerned organ or to both (Haider and Upadhyaya, 1985).

The significant induction in the rate of GVBD found in the present experiment after the addition of malathion, phosdrin, gardona, or birlane to the incubating me- dium, despite the presence of LH in incubation, strengthen the view that there is a direct interference of OPI in the metabolism of oocytes.

In amago salmon, Oncorhynchus rhodurus, it is reported that gonadotropin-in- duced oocyte maturation involves stimulation of A5-3fi-hydroxysteroid dehydroge- nase (A5-3gHSD) to produce 17a,20@-dihydroxyprogesterone ( 17a,20&diOHprog), the natural MIS, which in turn induces the final maturation (Young et al., 1982). Further, they observed that when cyanoketone, an inhibitor of A5-3@-HSD was added to the incubations along with the salmon gonadotropin (SG-G loo), the oocyte matu- ration did not occur. Kapur et al. (1978) studied the effect of fenitrothion on repro- duction of Cyprinus carpio and observed that when fishes were exposed at 0.3 and 1.5 ppm, testicular and ovarian A’-3/3-HSD activity was reduced. Histochemical inhi- bition of the same enzyme was also reported in the ovaries of M. vittatus, when ex- posed to sublethal concentrations of malathion (2500 ppb), phosdrin (0.12 ppb), gar- dona (146.4 ppb), and birlane (3.02 ppb) (Haider and Upadhyaya, 1985). From all

INSECTICIDES AND OOCYTE MATURATION 165

the above studies it appears that the inhibition of LH-induced oocyte maturation may be due to the reduced activity of A’-3&HSD. Further study in this line is in progress.

CONCLUSION

The concentrations used in the present experiment were based on our earlier in vivo study (Haider and Upadhyaya, 1985). For the sake of methodological conve- nience, the concentration used in the above in vivo study was truncated to its nearest exponential value to be used as the highest concentration in this experiment for a given OPI. The ratio of higher and the next lower concentration was kept as 10. The inhibitory effect of malathion, phosdrin and gardona, though not birlane, at all concentrations used implies that a much lower concentration than that used in the exposure of the whole animal is sufficient to elicit the inhibition in in vitro GVBD.

It is felt that the in vitro oocyte maturation can serve as an alternative model for toxicity bioassay. We are continuing further study for its validation and perfection.

ACKNOWLEDGMENTS

This investi8ation was supported by Indian National Science Academy and Department of Environment grants. The gift of LH from NIH, U.S.A. is acknowledged.

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