Fish Physiology and Biochemistry vol. 14 no. 6 pp 471-480 (1995) Kugler Publications, Amsterdam/New York
Effects of melatonin, p-chlorophenylalanine, and a-methylparatyrosine on plasma gonadotropin level and ovarian activity in the catfish, Heteropneustes fossilis: A study correlating changes in hypothalamic monoamines 1 B. Senthilkumaran and K.P. Joy* Centre of Advanced Study in Zoology, BanarasHindu University, Varanasi-221 005, India Accepted: May 8, 1995 Keywords: melatonin, a-methylparatyrosine, parachloropenylalanine, hypothalamus, monoamine, gonadotropin, ovarian function, estradiol
Abstract The effects of (ip, 10 injections over 20 days) of melatonin (75 Plg 100 g-1 BW), the serotonin (5-HT)synthesis blocker, para-cholorophenylalanine (p-CPA, 10 mg 100g- ' BW) and the catecholamine-synthesis blocker, a-methylparatyrosine (a-MPT, 10 mg 100 g-l BW) on gonadotropin (GTH) secretion and ovarian activity were studied in Heteropneustesfossilisduring late preparatory to early prespawning (April-May). The treatments resulted in significant reductions of plasma GTH and estradiol-17[ levels, the gonadosomatic index, frequency distribution of vitellogenic and postvitellogenic oocytes, and ovarian and serum 32p_ labelled alkali-labile phosphoprotein (a marker of vitellogenic activity). Most of the oocytes were nonvitellogenic or had undergone atretic changes. The hepatic 3 2 p-phosphoprotein content increased significantly over the saline control value. The effects were similar and pronounced in the p-CPA and melatonin-treated groups but were moderate in the a-MPT-treated group. Hypothalamic 5-HT content and turnover were significantly inhibited in the p-CPA and melatonin-treated groups but the content and turnover of catecholamines were not. The a-MPT treatment decreased significantly the content and turnover of dopamine (DA), noradrenaline (NA), and adrenaline (A) but did not influence the 5-HT content or turnover. These results suggest that 5-HT, NA and A are stimulatory to GTH secretion and that melatonin may act on the serotonergic system to inhibit the pituitary-gonadal axis.
Introduction Central monoamines (MA) play an important role in the regulation of gonadotropin (GTH) secretion and modulate basal and/or gonadotropin releasing hormone (GnRH)-induced GTH release in goldfish (Peter et al. 1991), African catfish (de Leeuw et al. 1987), Atlantic croaker (Khan and Thomas 1994), and Heteropneustesfossilis (Senthilkumaran and * to
Joy 1994a, 1995). These studies have demonstrated that serotonin (5-HT) and noradrenaline (NA) are stimulatory, and dopamine (DA) is inhibitory to GTH secretion. The central MA-ergic system is suggested to act as a common pathway for mediation of both extrinsic (photoperiod and temperature) and intrinsic (steroid feedback) factors in the neuroendocrine control of GTH secretion (de Vlaming and Olcese 1981; Khan and Joy 1990; Senthilkuma,
whom correspondence should be addressed. I A part of the results was presented at the International Workshop on Pineal gland: Its molecular signals and published as an abstract in Neuroendocrinol. Lett. 14: 399 pp., 1992.
472 ran and Joy 1994a,b, 1995). The hypothalamic serotonergic system is considered an important component of photosexual mechanism in teleosts (de Vlaming and Olcese 1981; Joy and Khan 1991). Pinealectomy and melatonin treatment are known to influence hypothalamic 5-HT in mammals (Kachi 1987; Glass 1988) and teleosts (de Vlaming and Olcese 1981; Joy and Khan 1991). Most investigations in vertebrates show that the endocrine role of the pineal organ is mediated through melatonin, the secretion of which is influenced by changes in external photoperiod (Reiter 1982; Ralph 1983). The teleost pineal organ secretes melatonin with a nocturnal surge (Gern et al. 1978; Falcon et al. 1987). The administration of melatonin is reported to have varied effects on gonadal activity depending on season, photoperiod, temperature, and the dose administered (see de Vlaming and Olcese 1981; Joy and Khan 1991). A previous morphological study in the catfish, H. fossilis has indicated an inhibitory effect of melatonin on the gonadal activity (Joy and Agha 1991); however, although reports describing effects of pinealectomy and melatonin treatment on gonads of teleosts are numerous, studies elucidating the neuroendocrine mechanisms associated with these morphological effects are scarce. Previous investigations in teleosts have reported short-term effects of MA-synthesis blockers or inhibitors on pituitary or blood levels of GTH (Peter et al. 1991) but were not extended to monitor their long-term effects on gonadal activity. Such longterm studies will be useful to understand the neuroendocrine mechanisms controlling seasonal reproduction. In H. fossilis, hypothalamic 5-HT and catecholamines (CA except DA) levels are high in the gonadal recrudescent phase and low in the quiescent phase (Senthilkumaran and Joy 1993, 1995). However, the significance of these high levels to gonadal activity have not been examined in any teleost. In the present study, therefore, both 5-HT and CA activities were blocked by parachlorophenylalanine (p-CPA) and a-methylparatyrosine (a-MPT), their respective synthesis blockers, at the peak period of gonadal activity and changes in GTH and ovarian activity were monitored to understand the specific role of the MA sys-
tem on the pituitary-gonadal axis. Secondly, effects of administration of melatonin on GTH secretion and ovarian activity in the catfish were also studied and correlated with changes in hypothalamic monoamines, such as 5-HT, DA, NA and adrenaline (A) to elucidate the possible mechanism of action of melatonin on the hypothalamo-hypophyseal-gonadal system.
Materials and methods The reproductive cycle of H. fossilis, an airbreathing catfish, can be divided into four phases (Senthilkumaran and Joy 1993). Adult female catfish (35-45g) were collected from local fishermen in Varanasi during late preparatory phase (April 10, 1992). They were treated with benzanthine penicillin (16,000 IU 1-1) daily for three days to prevent skin infection and were maintained in aquarium tanks under ambient photoperiod and temperature conditions (13.21L: 10.39D; 24 + 2C). They were acclimated for 14d and fed goat liver daily.
Experimental design After acclimation, the fish were divided into 5 groups of 50 each. Group I served as initial control and the fish were killed at the beginning of the experiment (last week of April). Group II was injected with p-CPA (Sigma) (10 mg 100 g-1 BW), Group III with a-MPT (Sigma) (10 mg 100 g-1 BW), and Group IV with melatonin (Sigma) (75 g 100 g-1 BW). The doses of p-CPA and a-MPT used in the study were low compared to those used in rats for depletion of the MA (Koe and Weissman 1966; Ojeda and McCann 1973). The compounds were dissolved in acidic saline (dissolved in half the required volume of saline (0.65% NaCI) at pH 10 with 5N NaOH, rapid precipitation by acidifying to pH 1.5 with 5N HCI and diluted with remaining amount of saline to give a final pH of 1.8) and were given 10 intraperitoneal injections (0.1 ml per injection) in the evening of every second day. The Group 5 fish were injected with the same volume of the vehicle (acidified saline) in a similar manner. Two fish
473 died in the p-CPA group due to lesions in the injection sites. Five fish in each group were injected with 3 2 p-labelled NaH PO (Sp. Act. 5 mCI mMol- 1, 2 4 BARC, Bombay), 24h before sacrificing. Ten fish in each group were injected with saline (0.65% NaCl)/pargyline (75 mg kg-' BW), 6h before sacrificing, for the estimation 5-HT content and turnover (Senthilkumaran and Joy 1993), respectively. Five fish in each group was injected with saline/aMPT (250 mg g-1 BW), 2h before sacrificing, for the estimation of CA content and turnover (Senthilkumaran and Joy 1995), respectively. On completion of the experiments, the fish were weighed, blood was collected in heparinized tubes by puncturing the caudal vasculature, plasma was separated by centrifuging at 800 x g for 20 min at 4°C and stored at -20 0 C until assayed for hormones. The fish were killed by decapitation and the brain was dissected out quickly on ice. The hypothalami were separated from brain samples as per the method explained previously (Senthilkumaran and Joy 1993) and were utilized for the estimation of 5-HT and CA. The ovaries were dissected out, weighed and fixed in Bouin's fluid for histology. Blood, liver and ovary of the tracer-injected fish were collected for determination of 3 2 p-labelled phosphoprotein as an indirect method to estimate vitellogenic activity. Serum was separated from the blood by allowing it to clot at room temperature and centrifuged at 800 x g for 20 min at 40 C.
Study parameters Gonadosomatic index (GSI) was expressed as the weight of the ovary per 100 g BW of fish. The Bouin-fixed ovaries were processed for paraffin embedding and 7.5 Im transverse sections were stained with Ehrlich's hematoxylin and eosin. For the frequency distribution study, the oocytes were identified as nonvitellogenic (stage I), vitellogenic (stage II and III) and postvitellogenic (stage IV), as described previously (Joy and Khan 1991). The atretic oocytes were not enumerated separately. Twenty fields of different regions of the ovary (anterior, middle and posterior) were examined for counting and staging the oocytes. The percentage
of oocytes in each stage was then calculated for each field out of the total number of oocytes present; the mean of the 20 fields for each category was calculated. The uptake of 3 2 p in the liver, ovary and serum was estimated according to the method of Nath and Sundararaj (1981). The estimate of the phosphoprotein provides an indirect assessment of vitellogenin, the yolk precursor protein. The tissues from the 3 2 P-injected fish were homogenized in 5 ml of cold 0.5N perchloric acid (PCA) and the homogenate was centrifuged at 1000 x g for 15 min. The tissue precipitates were resuspended in 5 ml of PCA. The serum samples were directly suspended in 5 ml of PCA. The tubes were incubated at 37°C for lh to remove RNA. The incubates were centrifuged and incubated at 75-80°C for 20 min to remove DNA. The mixture was cooled and again centrifuged. The precipitate was dissolved in 1 ml of 2N NaOH and incubated for 15 min at 100 0C. The solution was cooled and neutralized with HCI. The presence of inorganic phosphorus (alkali-labile) was determined by a rapid extraction after adding 5 ml of toluene-isobutyl alcohol (1:1) mixture followed by 1 ml each of silicotungstic and molybdate reagents. One ml aliquots of isobutyl alcohol-toluene layer were added to 10 ml of scintillation fluid [ReadysolvTMEP (Universal), Beckman]. The radioactivity was expressed as CPM ml-' serum or g-1 tissue. Plasma estradiol-170 (E2 ) and GTH (maturational GTH-II) levels were measured by the RIA methods described for this species (Senthilkumaran and Joy 1994a). The E2 antiserum was a generous gift from Professor G.D. Niswender, Colorado State University, USA. The lower sensitivity limit of the E2 assay was 10 pg ml-'. The coefficients of intra- and inter assay variations for E2 were 1.2 and 2.3%, respectively. The purified catfish GTH and the antiserum were obtained from Professor H.J.Th. Goos, University of Utrecht, The Netherlands as generous gifts. The lower and upper sensitivity limits of the assay were 0.8 and 12.5 ng mlrespectively. Intra- and inter assay coefficients of variation for the assay were found to be 5.1 and 9.1%, respectively. Other details of both the assays including validation were described earlier (Senthilkumaran and Joy 1994a).
474 ,,
DGSI
GTH
T E2
I
I E c
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I .n
*
o
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vU, CD
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Initial Control
Saline Control
o-MPT
p-CPA
Melatonin
1 Fig. 1. Effects of administration of p-CPA (10 mg 100 g-l BW), a-MPT (10 mg 100 g-l BW), and melatonin (75 Igg 100 g- BW) on gonadosomatic index (GSI), plasma estradiol-1713 (E2 ) and gonadotropin (GTH) levels in H. fossilis during late preparatoryprespawning phases (mean + SEM; Group size, n = 5). *p < 0.05; **p < 0.01; ***p < 0.001; a, compared with the initial control group; b, compared with the saline control group (Student's t test).
Hypothalamic 5-HT content was estimated by the method of Snyder et al. (1965) and was expressed as ,ug g-1 tissue. 5-HT turnover (activity) was calculated as the percentage increase of 5-HT after pargyline treatment over that of the saline control. The details of the 5-HT assay were described previously (Senthilkumaran and Joy 1994a). The CA content was measured by the radioenzymatic method of Ben-Jonathan and Porter (1976) as modified by them later (personal communication, 1981), and expressed as ng mg- 1 protein. The assay sensitivity was 32 pg mg- ' protein. Other details of the assay including validation were described previously (Senthilkumaran and Joy 1995). The protein content in each aliquot was estimated by the method of Lowry et al. (1951), using crystalline bovine serum albumin (BSA, fraction V, Sigma) as the standard.
Statisticalanalysis All data were expressed as means SEM. Student's t test was used for comparison between the experimental and control groups.
Results A comparison of the data of the initial and saline control groups on various parameters showed that gonadal recrudescence continued normally during the period of the experiments. The GSI, and levels of plasma GTH and E2 in the saline control group were significantly elevated compared to that of the initial control group (Fig. 1). The percentage of non-vitellogenic (Non-VT) oocytes decreased from 37.3 to 13.7, while that of post-vitellogenic (post-
475 Table 1. Effects of administration of p-CPA (10 mg 100g - 1 BW), a-MPT (10 mg 100 g-' BW), and melatonin (75 Btg 100 g-1 BW) on the frequency distribution of oocytes and 3 2 P-labelled phosphoprotein content in the liver, serum, and ovary of H. fossilis during late preparatory-prespawning phases (Mean + SEM; group size, n = 5) Groups
Initial Control Saline Control a-MPT
p-CPA
Melatonin
32
Frequency distribution of oocytes (0)
P-labelled phosphoprotein
Non-VT St. I
VT St. II&III
Post-VT St. IV
Liver (cpm mg- l)
Serum (cpm ml- l)
37.3 ±2.1 ***a 13.7 ± 1.8 ***b 54.4 + 2.0 ***b 88.6 ±0.8 ***b 86.1 +1.1
27.6 +2.0 NS 32.9 +2.1 ***b 20.2 + 1.2 ***b 7.4 +0.5 ***b 8.6 +0.9
35.1 + 2.4 ***a 53.5 + 2.9 ***b 25.4 + 1.6 ***b 4.1 ±0.7 ***b 5.3 +0.6
1556.3 104.5 ***a 4450.0 ±281.4 ***b 6657.1 ± 194.4 ***b 7748.5 +96.1 ***b 6320.0 +123.7
2970.0 +160.7 ***a 4133.3 +262.0 ***b 983.3 +34.2 ***b 1316.7 + 105.7 ***b 1083.3 ± 90.4
Ovary (cpm mg- 1) 1153.3 +66.8 ***a 2225.3 +59.7 ***b 1236.6 +61.4 ***b 1617.5 +64.2 ***b 1416.7 +85.7
VT, vitellogenic oocytes; St., stage; NS, nonsignificant; ***p < 0.001; a, compared with initial control; b, compared with saline control.
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Content
Turnover
Ole o
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Initial Control
Saline Control
o(-MPT
p-CPA
Melatonin
Fig. 2. Effects of administration of p-CPA, a-MPT and melatonin on hypothalamic serotonin (5-HT) content and turnover in H. fossilis during late preparatory-prespawning phases (mean + SEM, group size n = 5). Other details are as in Figure 1 (Student's t test).
VT) oocytes increased from 35.1 in the initial control group to 53.5% in the saline control group (Table 1). There was no significant change in the frequency distribution of the vitellogenic (VT) oocytes. Concomitantly, 3 2 P-labelled phosphopro-
tein contents in the liver, serum and ovary registered significant increases. In the hypothalamus, the turnover of 5-HT (Fig. 2) increased from 19.3 to 30.6% without any change in 5-HT content. The NA and A content increased significantly while the
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Fig. 3. Effects of administration p-CPA, a-MPT and melatonin on hypothalamic catecholamine (CA) contents (DA dopamine; NA noradrenaline; A adrenaline) in H. fossilis during late preparatory-prespawning phases (mean ± SEM; group size n = 5). Other details are as in Figure 1 (Student's t test).
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Effects of melatonin, p-chlorophenylalanine, and a-methylparatyrosine on plasma gonadotropin level and ovarian activity in the catfish, Heteropneustes fossilis: A study correlating changes in hypothalamic monoamines 1 B. Senthilkumaran and K.P. Joy* Centre of Advanced Study in Zoology, BanarasHindu University, Varanasi-221 005, India Accepted: May 8, 1995 Keywords: melatonin, a-methylparatyrosine, parachloropenylalanine, hypothalamus, monoamine, gonadotropin, ovarian function, estradiol
Abstract The effects of (ip, 10 injections over 20 days) of melatonin (75 Plg 100 g-1 BW), the serotonin (5-HT)synthesis blocker, para-cholorophenylalanine (p-CPA, 10 mg 100g- ' BW) and the catecholamine-synthesis blocker, a-methylparatyrosine (a-MPT, 10 mg 100 g-l BW) on gonadotropin (GTH) secretion and ovarian activity were studied in Heteropneustesfossilisduring late preparatory to early prespawning (April-May). The treatments resulted in significant reductions of plasma GTH and estradiol-17[ levels, the gonadosomatic index, frequency distribution of vitellogenic and postvitellogenic oocytes, and ovarian and serum 32p_ labelled alkali-labile phosphoprotein (a marker of vitellogenic activity). Most of the oocytes were nonvitellogenic or had undergone atretic changes. The hepatic 3 2 p-phosphoprotein content increased significantly over the saline control value. The effects were similar and pronounced in the p-CPA and melatonin-treated groups but were moderate in the a-MPT-treated group. Hypothalamic 5-HT content and turnover were significantly inhibited in the p-CPA and melatonin-treated groups but the content and turnover of catecholamines were not. The a-MPT treatment decreased significantly the content and turnover of dopamine (DA), noradrenaline (NA), and adrenaline (A) but did not influence the 5-HT content or turnover. These results suggest that 5-HT, NA and A are stimulatory to GTH secretion and that melatonin may act on the serotonergic system to inhibit the pituitary-gonadal axis.
Introduction Central monoamines (MA) play an important role in the regulation of gonadotropin (GTH) secretion and modulate basal and/or gonadotropin releasing hormone (GnRH)-induced GTH release in goldfish (Peter et al. 1991), African catfish (de Leeuw et al. 1987), Atlantic croaker (Khan and Thomas 1994), and Heteropneustesfossilis (Senthilkumaran and * to
Joy 1994a, 1995). These studies have demonstrated that serotonin (5-HT) and noradrenaline (NA) are stimulatory, and dopamine (DA) is inhibitory to GTH secretion. The central MA-ergic system is suggested to act as a common pathway for mediation of both extrinsic (photoperiod and temperature) and intrinsic (steroid feedback) factors in the neuroendocrine control of GTH secretion (de Vlaming and Olcese 1981; Khan and Joy 1990; Senthilkuma,
whom correspondence should be addressed. I A part of the results was presented at the International Workshop on Pineal gland: Its molecular signals and published as an abstract in Neuroendocrinol. Lett. 14: 399 pp., 1992.
472 ran and Joy 1994a,b, 1995). The hypothalamic serotonergic system is considered an important component of photosexual mechanism in teleosts (de Vlaming and Olcese 1981; Joy and Khan 1991). Pinealectomy and melatonin treatment are known to influence hypothalamic 5-HT in mammals (Kachi 1987; Glass 1988) and teleosts (de Vlaming and Olcese 1981; Joy and Khan 1991). Most investigations in vertebrates show that the endocrine role of the pineal organ is mediated through melatonin, the secretion of which is influenced by changes in external photoperiod (Reiter 1982; Ralph 1983). The teleost pineal organ secretes melatonin with a nocturnal surge (Gern et al. 1978; Falcon et al. 1987). The administration of melatonin is reported to have varied effects on gonadal activity depending on season, photoperiod, temperature, and the dose administered (see de Vlaming and Olcese 1981; Joy and Khan 1991). A previous morphological study in the catfish, H. fossilis has indicated an inhibitory effect of melatonin on the gonadal activity (Joy and Agha 1991); however, although reports describing effects of pinealectomy and melatonin treatment on gonads of teleosts are numerous, studies elucidating the neuroendocrine mechanisms associated with these morphological effects are scarce. Previous investigations in teleosts have reported short-term effects of MA-synthesis blockers or inhibitors on pituitary or blood levels of GTH (Peter et al. 1991) but were not extended to monitor their long-term effects on gonadal activity. Such longterm studies will be useful to understand the neuroendocrine mechanisms controlling seasonal reproduction. In H. fossilis, hypothalamic 5-HT and catecholamines (CA except DA) levels are high in the gonadal recrudescent phase and low in the quiescent phase (Senthilkumaran and Joy 1993, 1995). However, the significance of these high levels to gonadal activity have not been examined in any teleost. In the present study, therefore, both 5-HT and CA activities were blocked by parachlorophenylalanine (p-CPA) and a-methylparatyrosine (a-MPT), their respective synthesis blockers, at the peak period of gonadal activity and changes in GTH and ovarian activity were monitored to understand the specific role of the MA sys-
tem on the pituitary-gonadal axis. Secondly, effects of administration of melatonin on GTH secretion and ovarian activity in the catfish were also studied and correlated with changes in hypothalamic monoamines, such as 5-HT, DA, NA and adrenaline (A) to elucidate the possible mechanism of action of melatonin on the hypothalamo-hypophyseal-gonadal system.
Materials and methods The reproductive cycle of H. fossilis, an airbreathing catfish, can be divided into four phases (Senthilkumaran and Joy 1993). Adult female catfish (35-45g) were collected from local fishermen in Varanasi during late preparatory phase (April 10, 1992). They were treated with benzanthine penicillin (16,000 IU 1-1) daily for three days to prevent skin infection and were maintained in aquarium tanks under ambient photoperiod and temperature conditions (13.21L: 10.39D; 24 + 2C). They were acclimated for 14d and fed goat liver daily.
Experimental design After acclimation, the fish were divided into 5 groups of 50 each. Group I served as initial control and the fish were killed at the beginning of the experiment (last week of April). Group II was injected with p-CPA (Sigma) (10 mg 100 g-1 BW), Group III with a-MPT (Sigma) (10 mg 100 g-1 BW), and Group IV with melatonin (Sigma) (75 g 100 g-1 BW). The doses of p-CPA and a-MPT used in the study were low compared to those used in rats for depletion of the MA (Koe and Weissman 1966; Ojeda and McCann 1973). The compounds were dissolved in acidic saline (dissolved in half the required volume of saline (0.65% NaCI) at pH 10 with 5N NaOH, rapid precipitation by acidifying to pH 1.5 with 5N HCI and diluted with remaining amount of saline to give a final pH of 1.8) and were given 10 intraperitoneal injections (0.1 ml per injection) in the evening of every second day. The Group 5 fish were injected with the same volume of the vehicle (acidified saline) in a similar manner. Two fish
473 died in the p-CPA group due to lesions in the injection sites. Five fish in each group were injected with 3 2 p-labelled NaH PO (Sp. Act. 5 mCI mMol- 1, 2 4 BARC, Bombay), 24h before sacrificing. Ten fish in each group were injected with saline (0.65% NaCl)/pargyline (75 mg kg-' BW), 6h before sacrificing, for the estimation 5-HT content and turnover (Senthilkumaran and Joy 1993), respectively. Five fish in each group was injected with saline/aMPT (250 mg g-1 BW), 2h before sacrificing, for the estimation of CA content and turnover (Senthilkumaran and Joy 1995), respectively. On completion of the experiments, the fish were weighed, blood was collected in heparinized tubes by puncturing the caudal vasculature, plasma was separated by centrifuging at 800 x g for 20 min at 4°C and stored at -20 0 C until assayed for hormones. The fish were killed by decapitation and the brain was dissected out quickly on ice. The hypothalami were separated from brain samples as per the method explained previously (Senthilkumaran and Joy 1993) and were utilized for the estimation of 5-HT and CA. The ovaries were dissected out, weighed and fixed in Bouin's fluid for histology. Blood, liver and ovary of the tracer-injected fish were collected for determination of 3 2 p-labelled phosphoprotein as an indirect method to estimate vitellogenic activity. Serum was separated from the blood by allowing it to clot at room temperature and centrifuged at 800 x g for 20 min at 40 C.
Study parameters Gonadosomatic index (GSI) was expressed as the weight of the ovary per 100 g BW of fish. The Bouin-fixed ovaries were processed for paraffin embedding and 7.5 Im transverse sections were stained with Ehrlich's hematoxylin and eosin. For the frequency distribution study, the oocytes were identified as nonvitellogenic (stage I), vitellogenic (stage II and III) and postvitellogenic (stage IV), as described previously (Joy and Khan 1991). The atretic oocytes were not enumerated separately. Twenty fields of different regions of the ovary (anterior, middle and posterior) were examined for counting and staging the oocytes. The percentage
of oocytes in each stage was then calculated for each field out of the total number of oocytes present; the mean of the 20 fields for each category was calculated. The uptake of 3 2 p in the liver, ovary and serum was estimated according to the method of Nath and Sundararaj (1981). The estimate of the phosphoprotein provides an indirect assessment of vitellogenin, the yolk precursor protein. The tissues from the 3 2 P-injected fish were homogenized in 5 ml of cold 0.5N perchloric acid (PCA) and the homogenate was centrifuged at 1000 x g for 15 min. The tissue precipitates were resuspended in 5 ml of PCA. The serum samples were directly suspended in 5 ml of PCA. The tubes were incubated at 37°C for lh to remove RNA. The incubates were centrifuged and incubated at 75-80°C for 20 min to remove DNA. The mixture was cooled and again centrifuged. The precipitate was dissolved in 1 ml of 2N NaOH and incubated for 15 min at 100 0C. The solution was cooled and neutralized with HCI. The presence of inorganic phosphorus (alkali-labile) was determined by a rapid extraction after adding 5 ml of toluene-isobutyl alcohol (1:1) mixture followed by 1 ml each of silicotungstic and molybdate reagents. One ml aliquots of isobutyl alcohol-toluene layer were added to 10 ml of scintillation fluid [ReadysolvTMEP (Universal), Beckman]. The radioactivity was expressed as CPM ml-' serum or g-1 tissue. Plasma estradiol-170 (E2 ) and GTH (maturational GTH-II) levels were measured by the RIA methods described for this species (Senthilkumaran and Joy 1994a). The E2 antiserum was a generous gift from Professor G.D. Niswender, Colorado State University, USA. The lower sensitivity limit of the E2 assay was 10 pg ml-'. The coefficients of intra- and inter assay variations for E2 were 1.2 and 2.3%, respectively. The purified catfish GTH and the antiserum were obtained from Professor H.J.Th. Goos, University of Utrecht, The Netherlands as generous gifts. The lower and upper sensitivity limits of the assay were 0.8 and 12.5 ng mlrespectively. Intra- and inter assay coefficients of variation for the assay were found to be 5.1 and 9.1%, respectively. Other details of both the assays including validation were described earlier (Senthilkumaran and Joy 1994a).
474 ,,
DGSI
GTH
T E2
I
I E c
1*
I .n
*
o
-0
E 0o
vU, CD
2
¢
)
Initial Control
Saline Control
o-MPT
p-CPA
Melatonin
1 Fig. 1. Effects of administration of p-CPA (10 mg 100 g-l BW), a-MPT (10 mg 100 g-l BW), and melatonin (75 Igg 100 g- BW) on gonadosomatic index (GSI), plasma estradiol-1713 (E2 ) and gonadotropin (GTH) levels in H. fossilis during late preparatoryprespawning phases (mean + SEM; Group size, n = 5). *p < 0.05; **p < 0.01; ***p < 0.001; a, compared with the initial control group; b, compared with the saline control group (Student's t test).
Hypothalamic 5-HT content was estimated by the method of Snyder et al. (1965) and was expressed as ,ug g-1 tissue. 5-HT turnover (activity) was calculated as the percentage increase of 5-HT after pargyline treatment over that of the saline control. The details of the 5-HT assay were described previously (Senthilkumaran and Joy 1994a). The CA content was measured by the radioenzymatic method of Ben-Jonathan and Porter (1976) as modified by them later (personal communication, 1981), and expressed as ng mg- 1 protein. The assay sensitivity was 32 pg mg- ' protein. Other details of the assay including validation were described previously (Senthilkumaran and Joy 1995). The protein content in each aliquot was estimated by the method of Lowry et al. (1951), using crystalline bovine serum albumin (BSA, fraction V, Sigma) as the standard.
Statisticalanalysis All data were expressed as means SEM. Student's t test was used for comparison between the experimental and control groups.
Results A comparison of the data of the initial and saline control groups on various parameters showed that gonadal recrudescence continued normally during the period of the experiments. The GSI, and levels of plasma GTH and E2 in the saline control group were significantly elevated compared to that of the initial control group (Fig. 1). The percentage of non-vitellogenic (Non-VT) oocytes decreased from 37.3 to 13.7, while that of post-vitellogenic (post-
475 Table 1. Effects of administration of p-CPA (10 mg 100g - 1 BW), a-MPT (10 mg 100 g-' BW), and melatonin (75 Btg 100 g-1 BW) on the frequency distribution of oocytes and 3 2 P-labelled phosphoprotein content in the liver, serum, and ovary of H. fossilis during late preparatory-prespawning phases (Mean + SEM; group size, n = 5) Groups
Initial Control Saline Control a-MPT
p-CPA
Melatonin
32
Frequency distribution of oocytes (0)
P-labelled phosphoprotein
Non-VT St. I
VT St. II&III
Post-VT St. IV
Liver (cpm mg- l)
Serum (cpm ml- l)
37.3 ±2.1 ***a 13.7 ± 1.8 ***b 54.4 + 2.0 ***b 88.6 ±0.8 ***b 86.1 +1.1
27.6 +2.0 NS 32.9 +2.1 ***b 20.2 + 1.2 ***b 7.4 +0.5 ***b 8.6 +0.9
35.1 + 2.4 ***a 53.5 + 2.9 ***b 25.4 + 1.6 ***b 4.1 ±0.7 ***b 5.3 +0.6
1556.3 104.5 ***a 4450.0 ±281.4 ***b 6657.1 ± 194.4 ***b 7748.5 +96.1 ***b 6320.0 +123.7
2970.0 +160.7 ***a 4133.3 +262.0 ***b 983.3 +34.2 ***b 1316.7 + 105.7 ***b 1083.3 ± 90.4
Ovary (cpm mg- 1) 1153.3 +66.8 ***a 2225.3 +59.7 ***b 1236.6 +61.4 ***b 1617.5 +64.2 ***b 1416.7 +85.7
VT, vitellogenic oocytes; St., stage; NS, nonsignificant; ***p < 0.001; a, compared with initial control; b, compared with saline control.
*I
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Content
Turnover
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o(-MPT
p-CPA
Melatonin
Fig. 2. Effects of administration of p-CPA, a-MPT and melatonin on hypothalamic serotonin (5-HT) content and turnover in H. fossilis during late preparatory-prespawning phases (mean + SEM, group size n = 5). Other details are as in Figure 1 (Student's t test).
VT) oocytes increased from 35.1 in the initial control group to 53.5% in the saline control group (Table 1). There was no significant change in the frequency distribution of the vitellogenic (VT) oocytes. Concomitantly, 3 2 P-labelled phosphopro-
tein contents in the liver, serum and ovary registered significant increases. In the hypothalamus, the turnover of 5-HT (Fig. 2) increased from 19.3 to 30.6% without any change in 5-HT content. The NA and A content increased significantly while the
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a(- MPT
p -CPA
Melatonin
Fig. 3. Effects of administration p-CPA, a-MPT and melatonin on hypothalamic catecholamine (CA) contents (DA dopamine; NA noradrenaline; A adrenaline) in H. fossilis during late preparatory-prespawning phases (mean ± SEM; group size n = 5). Other details are as in Figure 1 (Student's t test).
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