Proc.Zool.Soc.India. 16 (1) : 47-53 : (2017) ISSN 0972 - 6683 : INDEXED AND ABSTRACTED 10 EFFECT OF P-CHLOROPHENYL ALANINE AND MELATONIN ON IMMUNE AND REPRODUCTIVE STATUS OF INDIAN PASSERIFORMES FINCH, LONCHURA PUNCTULATA SUNANDA DUBEY MITALI MEHROTRA AND S.N.P. YADAV "DEEN*" Dept. of Zoology, Udai Pratap College, Varanasi-221002 *Dept. of Zoology Magadh University, Bodhgaya Email: [email protected] Received - 11.11.2016 Accepted - 15.03.2017 ABSTRACT The effect of Melatonin (MEL) and p-chlorophenyl-alanine (PCPA), a β-adrenergic antagonist on immune and reproductive status was studied during reproductively active phase of Indian Passeriformes Finch, Lonchura punctulata. MEL treatment to these birds showed significant increase in the immune parameters such as spleen weight, blastogenic response of splenocytes, total leukocyte count and lymphocyte count, whereas PCPA treatment showed significant decrease in all these immune parameters. On the other hand the treatment of MEL and PCPA together to the birds showed an increase in immune parameters as compared with control group of birds. Histological observations of the spleen substantiate the data of spleen weight analysis as MEL treatment to the bird increased the splenocytes density in spleen, whereas PCPA treatment to the bird showed depletion of splenocytes in spleen. The results of the present study suggest immune-stimulatory and anti-gonadal effect of the neuro-hormone MEL in this avian species. Key words : Chlorophenyl Alanine, Melatonin, Immune and reproductive status, bird. INTRODUCTION The MEL signal controls the seasonal variation and sexual physiology of many animals (Guerrero and Reiter, 1992, Maestroni, 1993).There appeared some transitory and marginal studies on pineal effects, leading to the conclusion that the pineal gland may not be of primary importance in the control of avian reproduction (Ralph, 1981; Gwinner et al. 1981). Subsequent studies on Japanese quail (Simpson et al.1983), and on the turkey (Siopes and EI Halawani, 1986) have further reported that the pineal gland is not an important component of seasonal reproduction in birds. In contrast to these report, involvement of pineal gland in seasonal reproductive activity has been made in some tropical species of birds (Ramachandran et al. 1987, Ramachandran and Patel, 1986). Similar results have been also reported by Haldar and Ghosh (1990) in the Indian Jungle bush quail Perdicula asiatica. Seasonal peripheral MEL level and its response to the reproductive organs of the Jungle bush quail have shown the important role of MEL in the regulation of effect of seasonal photoperiod and its relation with gonad (Dubey and Haldar, 1997). Reproductive hormone plays an important role in the regulation of immune system in mammals (Bilbo and Nelson, 2001). Neutralizing or protective antibodies of the IgG class and IgM level show the most signicant sexual dimorphism and interaction between glucocorticoids and immune fuctions has been reported in relation to environmental stress (Nakono et al., 1987). Farner and Laniyonu (1984) studied the effects of PCPA on the sensitivity of rat intestine to agonists and on intestinal 5-hydroxytryptamine levels during Nippostrongylus brasiliensis infection, the treatment of infected rats with PCPA depleted the gut of 5-HT, and prevented the specic sub sensitivity to the amine. Further, PCPA suppresses immune status more in aged squirrels than in adult by reducing MEL level (Rai et al., 2009). MEL released by pineal is believed to be responsible for gonadal regression. By blocking MEL production at b-adrenergic receptor with b-adrenergic antagonist PCPA, the present investigations have been undertaken for the rst time for better understanding of the immunomodulatory role of pineal hormone MEL in relation to PCPA during reproductively active phase of tropical seasonally breeding Indian Passeriformes
(47) DUBEY ET AL. nch Lonchura punctulata. MATERIALS AND METHODS The birds were collected from the vicinity of Varanasi (Latitude 250 20' N, Longitude 830 00' E) during reproductively active phase. The birds were maintained in aviary exposed to ambient environmental conditions and were fed with grass seed and water ad libitum under laboratory conditions for two weeks. All experiments on animals were conducted in accordance with Institutional Practice and within the framework of Revised Animals (Scientic Procedures) Act of 2002 of Government of India on Animal Welfare MEL and PCPA from (SIGMA, St. Louis, Missouri, USA) were used for this study. MEL solution was made by dissolving it in few drops (20µl) of ethanol and then diluted with normal saline (0.9% NaCl) upto the desired concentration. PCPA was dissolved in 1 N NaOH and diluted with normal saline. Group No. of birds 1. Control 5 2. MEL treated 5 3. PCPA treated 5 4. PCPA + MEL treated 5 These birds were given daily injection of 20µg of MEL and 20µg of PCPA subcutaneously during evening hours (4.30 pm – 5.30 pm) for 30 consecutive days. The control birds were treated with saline. For the histological observation testes and spleen were immediately xed in bouin's uid and processed for Hematoxylin and Eosin staining. For total Leukocyte Count (TLC): Blood was diluted with standardized Turk's diluents in WBC Pipette, cells were counted in Haemocytometer under the microscope. Lymphocyte count was done by preparing blood smear on the glass slide and stained with the Leishman stain. For percent stimulation of spleen for splenocytes, the spleen was processed for blastogenic response (%SR). The lymphoid organ (Spleen) was dissected and a single cell suspension was prepared in chilled RPMI-1640. The cell viability was checked by 1.0% trypan blue exclusion method. In spleen cell suspension, the red cells were lysed with cold ammonium chloride tris buffer (tris hydroxyl methylene aminomethane, BDH, UK), 0.5% tris buffer and 0.84% NH4Cl mixed in 1:10 ratio and adjusted to pH 7.2 after washing twice with RPMI-1640. The cell suspension was adjusted to 106 cells/ml in RPMI-1640, containing sodium bicarbonate, antibodies (Penicillin 100 IU/ml, Streptomycin 100µg/ml, gentamycin 100µg/ml) and 10% foetal calf serum (Sigma, USA). For the study of blastogenic response the cell suspension was divided into aliquot of 2 ml each and the control tubes were cultured in the absence of mitogen, whereas the test cultures were stimulated with mitogen Con A at the concentration (5 µg/ml). CPM with mitogen x 100 % Stimulation Ratio = CPM without mitogen
All data were expressed as mean ± SEM. Student-Newman Kuel's test was used for comparisons. The difference were considered signicant when p<0.05. RESULTS MEL treatment signicantly reduced testes weight, whereas PCPA treatment showed non signicant increase. However, treatment of MEL along with PCPA resulted in reduced testes mass. Histological observation of testes during reproductive active phase of control birds showed enlarged seminiferous tubules lined with many layers of germ cells whereas MEL treatment reduced its functional activities as observed by shrinked and thin seminiferous tubules. MEL treatment to the birds increased the spleen weight and splenocytes density whereas, PCPA treatment, to the birds decreased the weight and splenocytes density in spleen. MEL treatment, along with PCPA also displayed an increased splenic cells function.
(48) PROC. ZOOL. SOC. INDIA MEL treatment alone signicantly increased the blastogenic response, percent stimulation ratio of splenocytes, total leukocyte count and lymphocyte count while PCPA treatment signicantly decreased the blastogenic response as well as percent stimulation ratio of splenocytes, total leukocyte and lymphocyte count when compared with control group of birds. Further, treatment of MEL along with PCPA enhanced these parameters. Discussion The result of present study on inuence of exogenous MEL administration is suggestive of an anti gonadal effect. Some previous studies have shown that MEL has no signicant effect on the gonadal growth of juvenile chickens (Singh and Turner, 1967) or juvenile quails (Saylor and Wolfson, 1968). However, some recent observation on tropical/subtropical species such as the lal Munia (Estrilda amandava) and the parakeet (Psittacula krameri) showed that exogenous MEL has an inhibitory effect on gonadal activity (Gupta et al. 1987, Maitra and Dey, 1992). Testicular regression and spermatogenic arrest with PCPA treatment were observed in Pigeons (Ramachandran et al., 1996). The effect of temperature on testicular cycle of wild birds Black headed munia and spotted Munia (Malabika, 2005) and the studies on photoperiodism and reproductive cycles in birds, state the role of natural light changes on avian species (Farner et al., 1953). Earlier studies revealed seasonal cyclicity of testicular volume of spotted Munia with greatest testicular volume found in the month of September (Chandola et al., 1976). Study of the seasonal cycle of female Indian jungle bush quail, Predicula astiaca suggested that plasma MEL and ovarian activity are reversely related (Ghosh et al., 1994, Haldar and Rai, 1997).The testicular changes indicate that MEL may have an inhibitory effect during reproductive active phase on the hypothalamo-hypophysial axis regulating gonad functions. MEL being the endocrine messenger of darkness, its endogenous synthesis depends on the photoperiod and shows seasonal variations. Similarly, it is reported that the pharmacological effects of MEL is season-dependent (Maestroni, 2001). The lymphocyte proliferation in response to mitogen Con A showed signicant enhancement of splenocytes proliferation in MEL treated groups. It seems that MEL may ameliorate the immuno- compromising effect of β-adrenergic receptor. Further, MEL receptors have been localized on circulating lymphocytes (Calvo et al., 1995) as well as on thymocytes and splenocytes (Martin-Cacao et al., 1993, RaiEI-Idrissi et al. 1995). Our study shows that MEL administration signicantly increased the spleen activity, while PCPA treatment to the bird signicantly reduced the spleen function. MEL treatment along with PCPA also showed increased activity of spleen, the direct effect of MEL on the regulation of the immune system suggested. It has been studied that testosterone treatment in young birds showed a strong negative effect on IgG production (Lerner et al., 1971). Chronic administration of MEL increased the splenocytes proliferation when stimulated with T-cell mitogen concanavalin A in male Syrian hamster (Champney et al., 1997). β- adrenergic receptors are also present on immune cells (Werb et al., 1978) and are capable of affecting immune function. Thus the inverse relationship between β-adrenergic receptor blocker, PCPA administration and pineal hormone MEL, which is known as anti-gonadal hormone might be playing an important role in the immune status of this seasonally breeding avian species. Hence, the neuro-hormone MEL may be a potent therapeutic agent in situations where, immune-proliferation is required, as no known toxicity of MEL is reported. ACKNOWLEDGEMENT Prof. C. Haldar, Dept. of Zoology, B.H.U., Varanasi is greatly acknowledged for her unabated cooperation for this work. REFERENCES Bilbo S, Nelson R. J. 2001. Sex steroid hormones enhance immune functions in male and female Siberian hamsters. Am. J. Physiol. Regul. Inte. Comp. Physiol. 280: R207-R213. Calvo, Juan R, Mohammed Rai-EI-ldrissi,D. Pozo,Juan Guerrero, M. 1995. Immunomodulatory role of melatonin: specic binding sites in human and rodent lymphoid cells Volume 18, Issue
(49) DUBEY ET AL. 3,Pages 119–126 Champney T.H.,Parado J, Youngblood T., Appel K. Mc Murray DN. 1997. Immune responsiveness of splenocyte after chronic daily melatonin administration in male Syrian hamsters. Immunol. Lett. 58: 95-100. Chandola A. and Thapliyal J.P. 1968. Endocrine regulation of Body weight in the spotted munia, Uroloncha punctulata The Condor. Vol.70,No.3ul0, pp.248-251. Chandola A. pavanaskar J. Thapliyal J.P. 1976. Decreasing photoperiods and the testicular cycle of a subtropical bird (Spotted Munia), Lonchura punctulata. Biological Rhythm research, 1744- 4179,7(2), 95-100. Dubey S and Haldar C. 1997. Environmental factors and annual haderian-pineal-gonadal interrelationship in Indian jungle bush quail, P. asiatiaca. General and comparative endocrinology, 106,17- 22. Farner,D.S., L.R.Mewaldt, and S.D Irving 1953. The roles of darkness and light in the photopreodic response of the testes of white crowned sparrows. Biol. Bull., 105:434-441. Farner,S.G. and Laniyonu,A.A. 1984. Effects of p-Chlorophenylanine on the sensitivity of rat intestine to agonists and on intestinal 5-hydroxytryptamine levels during Nippostrongylus barasiliensis infection. Br. J Pharmacol. 82 (4): 883-889. Ghosh,M., Dubey S., and Haldar C. 1994. Short and long photoperiodic responses of Harderian gland and Pineal gland in Jungle bush quail, P.astiaca. 2nd International symposium on the cell biology and physiology of the harderian gland, Sorrento, Italy. Guerrero J. M., Reiter R. J. 1992. A brief survey of pineal gland-immune system interrelation ships, Endocrinol. Res.18:91-113. Gupta,B.B.P, Haldar-Mishra C. Ghosh, M. and Thapliyal J.P.1987. Effect of melatonin on gonads, body weight and Luteinizing hormone (LH) dependent coloration of the Indian nch Lal munia, Estrilda amandava.Gen.comp.Endocr, 65,451-456.. Haldar C, Ghosh M 1990. Annual pineal and testicular cycle in the Indian jungle bush quail, Perdicula asiatica, with reference to the effect of pinealectomy, Gen.Comp.Endocrinol. 2:73-81. Lerner, K.G., Glick, B., McDuffe F.C. 1971. Role of the bursa of Fabricius in IgG and IgM production in the chicken. Evidence for the role of non bursal site in the development of humoral immunity . J. Immunol.107: 493-503. Maestroni G.J.M. 1993. The immunoneuroendocrine role of melatonin, J. Pineal Res. 14: 1-10. Maestroni G.J.M. 2001. The immunotherapeutic potential of melatonin. Expert Opin Investig. Drug.10:467-76. Malabika S. 2005. Effect of temperature on testicular cycle of wild birds black headed munia and spotted munia. J. Environ. Biol. (1): 135-139. Martin-Cacao A., Lopez- N.A., Reiter R.J. Calvo J.R., Guerrero J.M. 1993. Binding of 2(125) Melatonin by rat thymus membranes during postnatal development. Immunol. Lett. 36:59-64. Nakano K., Suzuki S. and O. C. 1987. Signicance of increased secretion of glucocorticoids in mice and rat injected with bacterial endotoxin. Brain. Behav. Immunol.1:159-172. Rai.S., Haldar C. and Singh R. 2009. Modulation of immunity in young, adult and aged squirrel, Funambulus pennant by melatonin and p-chlorophenylalanine. Immun Ageing. 6:5.1186/1742-49336-5. Ralph, C. L., 1981. The pineal and reproduction in birds. In The pineal gland, Vol.II(ed.R.J.Reiter) Boca, FL: CRC Press. Ramchandran A., Patel M., Patel C., 1996. Effects of pineal indoles and Parachlorophenylalanine on seasonal reproduction in the pigeon. J. Exp. Biol. 199-793-800. Ramchandran, A.V. and Patel, M.M. 1986. Seasonal histomorphological alterations of adrenal and thyroid
(50) PROC. ZOOL. SOC. INDIA in normal and pineaectomised domestic pigeons. Columba livia. Ind. J. exp. Biol. 24, 755- 759. Ramchandran, A.V., Patel, C.D., Asnani, M.V. and Shah, R.V. 1987. Seasonal histomorphological changes in gonad of normal and pinealectomized domestic pigeons. Columba livia. Ind. J. exp. Biol. 26,835—838. Reiter R.J. 1991. Melatonin in chemical expression in darkness. (In prep) Saylor,A. and Wolfson, A. 1968. Inuence of the pineal gland on gonadal maturation in the Japanese quail. Endocrinology 83, 1237-1246. Simpson, S.M. and Follett, B.K. 1981. Pineal and hypothalamic pacemakers: their role in regulating circadian rhythmicity in Japanese quail. J. Comp. Physiol. A. 144, 381-389. Simpson, S.M., Urbanski, H.F. and Robinson, J.E. 1983. The pineal gland and the photoperiodic control of luteinizing hormone secretion in intact and castrated Japanese quail. J. Endocr. 99, 281- 287. Singh, D.V. and Turner, C.W. 1967. Effect of melatonin upon thyroid hormone secretion rate and endocrine glands of chicks. Proc. Soc. exp. Biol. 125, 407-411. Siopes, T.D. and El halawani, M.E. 1986. Plasma prolactin and leteining hormone levels of pinealectomized and enucleated turkey hens. Gen. Comp. Endocr. 62,36-42. Werb Z., Foley R. and Munk A. 1978. Interaction of glucocorticoids with macrophages : Identication of glucocorticoids in monocytes and macrophages. J. Exp. Med. 147: 1684-1694.
Fig. : Showing the effect of different treatments on spleen weight, testes weight and total leucocyte counts in L. punctulata
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Fig 2 : Showing the effect of different treatments on lymphocyte counts and perecent stimulation in L. punctulata.
(52) PROC. ZOOL. SOC. INDIA Plate I (Fig. A-D) A. Histology of testes of control birds showing enlarged seminiferous tubules (ST) with round spermatids (RS) X 400. B. Histology of testes of MEL treated birds showing an arrest of spermatogenesis, tubular regression (TR) and intertubular space (ITS) X 400. C. Histology of testes of PCPA treated birds showing enlarged seminiferous tubules (ST) with round and elongated spermatids (ES) X 400. D. Histology of testes of MEL and PCPA treated birds showing regressed seminiferous tubules and interstitium (IC) X 400.
Plate 2 (Fig. A- D) A. Histology of spleen of control birds showing splenic lymphocytes (L) in periarterial region X 400. B. Histology of spleen of MEL treated birds showing more dense splenic lymphocyte (L) in periarterial region X 400. C. Histology of spleen of PCPA treated birds showing depletion of splenic lymphocytes (L) in periarterial region X 400. D. Histology of spleen of MEL and PCPA treated birds showing restoration of splenic lymphocytes (L) X 400.
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