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Effect of Peptide Histidine Isoleucine on In Vitro and In Vivo Prolactin Secretion in the Turkey
Effect of Peptide Histidine Isoleucine on In Vitro and In Vivo Prolactin Secretion in the Turkey J. A. PROUDMAN and H. OPEL USDAJARS-Avian Physiology Laboratory, Beltsville, Maryland 20705 (Received for publication October 17, 1989)
1990 Poultry Science 69:1209-1214 INTRODUCTION
The neuroendocrine regulation of prolactin (PRL) secretion in the turkey is complex and poorly understood (reviewed by El Halawani et ah, 1988). In mammalian species, most notably the rat, PRL release is strongly stimulated by several neurohormones, including vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), thyrotropin-releasing hormone (TRH), and neurophysin. Of these peptides, VIP is widely viewed as an authentic prolactin-releasing factor (PRF) in the rat (Shimatsu et ah, 1985), while both TRH and PHI are equipotent to VIP in stimulating PRL release in vitro and in vitro (Kaji et ah, 1984). Proudman and Opel (1988) and Opel and Proudman (1988) have recently shown that VIP is a potent stimulus for PRL secretion in the turkey. Peptide histidine isoleucine is a peptide composed of 27 amino acids and is found in the brain and the gut of
Sigma Chemical Co., St. Louis, MO 63178.
numerous species. It has a high (>50%) homology to VIP (Tatemoto, 1984). A preliminary report by Fehrer et ah (1987) has suggested that this peptide may also release PRL in the turkey. The present study was designed to determine if PHI functions as a PRF in the turkey. MATERIALS AND METHODS
hi Vitro Studies Three replicate preparations of pituitary cells were used; two were from pituitaries collected from large white laying turkey hens and one from medium white laying hens. For each preparation, the cells were dispersed enzymatically using collagenase and pancreatin and cultured for 4 days in monolayer flasks as described by Proudman and Opel (1988). On the 5th day of culturing, the cells were treated with either synthetic porcine VIP1 or synthetic porcine PHI1 at dosages ranging from 10""6 M to 10-11 M. Untreated flasks served as controls. Five flasks were treated at each dose. After 4 h of
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ABSTRACT Studies were conducted both in vitro, using monolayer cultures of turkey pituitary cells, and in vivo, using ovariectomized turkey hens, steroid-primed ovariectomized hens, and immature toms to compare the effectiveness of peptide histidine isoleucine (PHI) with that of vasoactive intestinal peptide (VIP) in stimulating prolactin (PRL) secretion. Vasoactive intestinal peptide, a putative PRL-releasmg factor (PRF) in the turkey, was approximately 1000-fold more effective than PHI in stimulating PRL secretion in vitro. Prolactin secretion was significantly enhanced by the exposure of pituitary cells to 10~7 M PHI and a similar stimulation was observed with 10~ 10 M VTP. Injection of carmulated, unrestrained turkeys with PHI at doses up to 100 ug per kg of BW caused no significant change in circulating PRL concentrations, but injection of 10 fig of VTP per kg resulted in a 7 to 22-fold increase in plasma PRL concentration within 10 to 30 min following injectioa These results demonstrate a marked difference between the turkey and the rat in their response to a PRLstimulating neuropeptide. In contrast to what was observed in the turkey, PHI is a strong PRF in the rat, with a potency equal to or greater man that of VTP. Earlier studies have shown that thyrotropin-releasing hormone, another strong PRF in mammals, has little consistent PRF activity in the turkey. Thus, die present studies add additional evidence of major differences between mammals and birds in the control of PRL secretion by hypothalamic neuropeptides. (Key words: prolactin, peptide histidine isoleucine, turkey, vasoactive intestinal peptide, prolactin-releasing factor)
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PROUDMAN AND OPEL
incubation at 37 C, die medium was poured into tubes and stored at -20 C until the PRL concentration was determined by homologous radioimmunoassay (Proudman and Opel, 1981).
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In Experiment 3, ovariectomized hens were primed with a 3- wk time-release pellet implant2 of either progesterone (50 or 100 mg), estradiol (50 or 75 mg), or both progesterone (50 mg) and estradiol (25 or 50 mg) inserted subcutaneously in the breast area. The doses of steroid used were In Vivo Studies based on those used by Saeed and El Halawani Five experiments were conducted to deter- (1986) to prime ovariectomized turkey hens mine the effects of intravenous injections of a prior to stimulation of PRL release. After 20 wide range of doses of PHI or of a known days of steroid priming, PHI was administered at effective dose of VIP on PRL secretion in doses of 5 or 10 jig per kg of BW. Because an turkeys in various physiological conditions. All infinite combination of doses of priming steroids birds were cannulated prior to experimentation and PHI was possible, a limited number of birds and were unrestrained in individual cages during (1 to 3) were tested at each of many treatment blood collection as previously described (Opel combinations to screen for an effective combiand Proudman, 1988). Samples were collected nation. Blood was collected as in Experiment 2, into heparinized syringes, centrifuged, and the except that the saline injection protocol was plasma was stored at -70 C prior to the omitted. measurement of PRL. Experiment 1 was conExperiment 4 tested the response of cannuducted using mature, ovariectomized, Diamond lated, unrestrained, Diamond Hybrid large Hybrid large white turkey hens. Hens were white toms treated with PHI or VIP at 14 wk of ovariectomized at 12 to 14 wk of age and raised age. The PHI was administered at doses of 5,10, in floor pens under a 6 h light (L): 18 h dark (D) 20, 50, or 100 Jig per kg of BW. The VIP was photoperiod from 18 to 28 wk of age. Hens were administered at doses of 10 or 20 ug per kg of then placed in individual laying cages within a BW (n = 1 or 2 per dose). Samples were battery room, given a stimulatory photoperiod collected prior to injection and 5,10,15,30,60, (14L:10D), and sampled between 30 and 38 wk and 90 min postinjection. of age. The 2-day injection and sampling Experiment 5 was conducted to test the protocol used was similar to that used previously possibility that PHI may have been lost due to (Opel and Proudman, 1988). Briefly, on Day 1 a adsorption of the peptide to the plastic containpreinjection blood sample (.5 mL) was drawn ers used for preparing and injecting the solufrom six birds, and a bolus of saline vehicle was tions. Mature, ovariectomized, Diamond Hybrid injected. Additional blood samples were col- medium white hens were treated with a wide lected at 5, 10, 15, 30,45, 60, 90, and 120 min range of doses of PHI (5 to 100 |ig/kg) or 10 jig following injection. On the 2nd day, hens were of VTP per kg (n = 1 to 3 per dose). The surfaces randomly selected to receive 2.5,5, or 10 u.g of of all cannulas, tubes, and syringes were PHI per kg of BW administered in 1 mL of pretreated with Silane to preclude peptide saline, and the sampling protocol used on Day 1 adsorption. Samples were collected at the was repeated. The entire protocol was repeated intervals used in Experiment 4. on a second group of six hens so that each dose was administered to a total of four birds. One hen was determined at necropsy to be incom- Statistical Analysis pletely ovariectomized and was deleted from the The results of the in vitro experiments did not experiment. differ between preparations of cells from large Experiment 2 was similar to Experiment 1, and medium white hens. Therefore, the data except that higher doses of PHI were tested (25, were combined to provide three independent 50, and 100 u.g per kg of BW), and postinfection replicates (n = 3) for statistical analysis. samples were collected at 10,20,30, and 60 min. Regression analysis was used to establish a In addition, three hens were treated with VIP at a significant dose effect (P<.05) for each treatdose of 10 u.g per kg of BW to confirm diat the ment. Data were then analyzed by ANOVA and hens were capable of responding to a known a two-sided Dunnett's procedure was applied to PRF. compare treatment means with die control. Except as noted below, the results on in vivo experiments were analyzed by a repeated'innovative Research of America, Toledo, OH 43606. measures ANOVA to establish differences over
PROLACTIN SECRETION IN THE TURKEY
FIGURE 1. Effect of various dosages of peptide histidine isoleucine (PHI) and vasoactive intestinal peptide (VTP) on prolactin secretion by monolayer cultures of turkey anterior pituitary cells. Treatment x + SEM with an asterisk differ significantly (P<05) from untreated controls. Each x represents three cell preparations (n = 3) with five replicates each.
treatment (dose of peptide) and time for each peptide. Where treatment by time interactions were present, Dunnett's procedure was used to compare means from a given sampling time to the zero time (P<05) as described by Opel and Proudman (1988). RESULTS
The effect of PHI and VIP on PRL secretion by dispersed turkey pituitary cells is shown in Figure 1, Vasoactive intestinal peptide significantly stimulated PRL secretion at a dose of 10 -10 M, with maximal stimulation observed from 10"8 to 10 -6 M VIP. Peptide histidine isoleucine significantly stimulated PRL secretion only at doses of 10 -7 and 10 -6 M. The response of cannulated, unrestrained, ovariectomized turkey hens to PHI and VIP in Experiments 1, 2, and 5 is presented in Table 1. Neither saline injection (data not shown) nor administration of PHI at doses ranging from 2.5 to 100 (ig per kg of BW produced any significant change in circulating PRL levels. The procedure utilized to preclude adsorption of PHI to plastic utensils (Experiment 5) did not influence these results. In contrast, VTP elicited a significant increase in concentrations
of PRL in plasma within 10 min of administration. Peak PRL concentrations were observed at 10 to 20 min following VIP injection in Experiment 2 and at 30 min after injection in Experiment 5. The increase in PRL following VIP administration ranged from 7 to 12-fold that of the basal levels. Priming of ovariectomized hens with steroids prior to treatment with PHI (Experiment 3) did not enhance the PRL response to PHI (Table 2). Because some steroid-PHI combinations were represented by only a single bird, regression analysis was used to test for PRL responses over time for each treatment combination. Although this analysis revealed significant increases in PRL over time in several instances, these increases in no case equaled that observed in one of the control groups. The reason for the increase in PRL over time in some steroid-primed hens (controls and PHI-treated) was unclear. We observed no behavioral indications of stress in these birds and observed no similar increase was observed in other cannulated birds used in these experiments. After combining PHI doses, there was still no significant change in PRL following PHI treatment in any of the steroidprimed groups. Immature torn turkeys (Experiment 4) also failed to respond to PHI administration at a wide range of doses (Table 3). These birds responded significantly to VIP as shown by regression analysis of PRL levels at each dose of VIP. Both 10 and 20 ug of VTP per kg elicited a maximum increase in plasma PRL at 15 min following injection. DISCUSSION
The present evidence that PHI has no effect on in vivo PRL secretion in the turkey and that PHI is approximately 1,000 times less effective than VD? in vitro is in sharp contrast to reports that PHI is a putative PRF in the rat (Werner et al, 1983; Kaji et al, 1984; Ohta et al, 1985). Administration of 1 \ig of PHI to a 300 g rat significantly increases plasma PRL within 5 min, and equimolar doses of PHI, VTP, and TRH given i.v. produce an equivalent rise in plasma PRL (Kaji et al., 1984). Peptide histidine isoleucine also stimulates PRL secretion from superfused rat pituitary cells (Kaji et al, 1984; Ohta et al, 1985), monolayer cultures of pituitary cells, and hemipituitaries (Werner et al, 1983). In these in vitro studies
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CONCENTRATION (Ml
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12.55 13.34 12.00 103.93* 10 7.90 16.75 13.15 6.40 12.65 8.75 27.65*
11.27 13.11 11.60 111.40* 5
7.95 16.10 13.53 6.00 10.80 8.08 8.65
1153 13.88 11.65 14.58 0
8.80 15.60 13.63 7.10 1058 7.45 9.40
10 3.94 4.82 356 20
3.63 5.55 3.86 10
3.59 4.85 2.93 0
•Significantly different from the 0 min x (P<05).
Standard error of the least squares x.
Experiment 5 5 ug PHI 10 ug PHI 20 Ug PHI 40 ug PHI 50 ug PHI 100 ug PHI 10 Ug VIP
Experiment 2 25 ug PHI 50 ugPHI 100 ug PHI 10 Ug VIP
Experiment 1 2.5 ug PHI 5 HgPHI 10 Mg PHI
Dose (per kg of BW)
1.02 .86 .31 9.54 60 7.05 22.45 14.90 6.00 10.43 7.80 50.60*
12.01 1354 11.80 44.88 30 6.60 16.90 1555 650 1050 7.15 115.10*
11.58 13.47 11.65 66.43 15 7.40 15.95 15.45 6.05 10.15 850 79.35*
45 3.65 557 3.35 SE
30 3.35 4.45 3.05 60
3.39 4.95 3.43 30
15
Minutes after injection 3 5 3
3
1
6 1 13
TABLE 1. Mean plasma prolactin levels (nglmL) in ovariectomized turkey hens given peptide or vasoactive intestinal peptide (VIP) in Experiments 1, 2, and 5
d from http://ps.oxfordjournals.org/ at Michigan State University on April 3, 2015
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PROLACTIN SECRETION IN THE TURKEY
TABLE 2. Mean plasma prolactin levels (ng/mL) in steroid-primed, ovariectomized turkey hens given peptide histidine isoleucine (PHI) or vasoactive intestinal peptide (VIP) (Experiment 3) Minutes after injection
Dose of PHI (Mg/kg of BW) n
Steroid treatment1
Progesterone (50 mg) plus estradiol (25 mg) Progesterone (50 mg) plus estradiol (50 mg)
10
20
30
60
SE 2
0 5 5 10
2 2 1 3
11.20 9.08 11.15 10.40
15.08 12.00 12.45 11.93
22.52 11.40 14.00 13.42
25 JO 12.85 16.65 16.22
31.68 17.00 20.25 18.25
2.11* 1.89 3.62* 1.18*
0 5 10 5 10
1 1 1 2 2
15.75 10.40 8.30 12.05 16.48
14.50 10.90 8.05 13.18 19.10
16.50 11.60 8.65 13.22 17.30
14.85 11.95 8.70 13.50 19.65
16.25 12.40 8.10 13.42 21.60
.87 .80* .30 .89 36*
5 10
1 2
10.40 19.35
10.30 18.90
10.80 21.58
10.85 23.00
12.60 24.90
.93 .16*
5 10
1 1
20.90 12.85
20.15 12.00
22.50 14.35
25.70 14.60
25.25 15.90
2.50* 1.53*
'Total steroid dose in 3-wk time-release implants. Body weight was 7.6 to 10.4 kg. Standard error of the least squares x. *Row x change significantly with time (P<05).
of the rat, PHI was shown to be more effective than VIP in stimulating PRL secretion. We have administered a wide range of doses of PHI to turkeys in various physiological states and under conditions in which we have demonstrated herein and previously that VIP is highly effective in stimulating PRL secretion. Clearly, PHI has no effect on PRL secretion in ovariectomized turkey hens, immature toms, and steroid-primed ovariecto-
mized hens at the steroid-PHI treatment combinations that were tested. Peptide histidine isoleucine was not given to laying or incubating hens. Thus, although the possibility that PHI may affect PRL secretion in vivo under other conditions cannot be precluded by the present study, the results strongly suggest that PHI is unlikely to function as a physiological PRF in the turkey. This finding, together with previous evidence (El Halawani et al.,
TABLE 3. Mean plasma prolactin levels (ng/mL) in i'mmature torn turkeys given peptide histidine isoleucine (PHI) or vasoactive intestinal I peptide (VIP) (Experiment 4) Minutes after injection
Dnsfi
(per kg of BW)
n
0
5
10
15
30
60
90
SE1
PHI •5 Mg 5 Mg
10 jig 20 Mg 50 Mg 100 Mg
VTP 10 Mg 20 Mg
2.45 3.40 4.12 2.82 5.18 3.92
2.70 3.45 3.68 2.68 4.48 3.08
2.40 3.10 3.70 2.68 5.18 3.90
2.05 3.55 3.92 2.58 5.55 4.12
2.45 4.35 3.75 2.82 5.68 4.02
2.25 3.75 3.58 2.58 5.05 3.95
2.60 3.05 4.45 2.60 4.88 338
22 .44 .79 .17 SI .34
3.75 3.12
11.60 9.30
22.50 17.90
82.90 67.85
55.40 49.15
23.60 29.10
3.10 11.20
28.98* 24.63*
Standard error of the least squares x. *Row x change significantly with time (P<05).
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Progesterone 50 mg 50 mg 100 mg 100 mg Estradiol 50 mg 50 mg 50 mg 75 mg 75 mg
0
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PROUDMAN AND OPEL
1988; Proudman and Opel, 1988) that TRH (a highly effective PRF in mammals) is also not a PRF in the turkey, demonstrates major differences in the neuroendocrine regulation of PRL secretion between an avian specie (the turkey) and the mammalian species studied to date.
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prolactin release in the rat. Life. Sci. 35:641-647. Ohta, H., Y. Kato, K. Tojo, A. Shimatsu, T. Inoue, Y. Kabayama, and H. Imura, 1985. Further evidence that peptide histidine isoleucine (PHI) may function as a prolactin releasing factor in rats. Peptides 6:709-712. OpeLH., and J. A. Proudman, 1988. Stimulation of prolactin release in turkeys by vasoactive intestinal peptide. Proc. Soc. Exp. Biol. Med. 187:455-460. Proudman, J. A., and H. Opel, 1981. Turkey prolactin: validation of a radioimmunoassay and measurement of ACKNOWLEDGMENTS changes associated with broodiness. Biol. Reprod. 25: 573-580. The authors gratefully acknowledge the Proudman, J. A., and H. Opel, 1988. Stunulation of prolactin technical assistance of Gaynelle Campbell and secretion from turkey anterior pituitary cells in culture. Proc. Soc. Exp. Biol. Med. 187:448-*54. Beverly Russell. Saeed, W., and M. E. El Halawani, 1986. Modulation of the prolactin response to thyrotropin releasing hormone by ovarian steroids in ovariectomized turkeys (Meleagris REFERENCES gattopavo). Gen. Comp. Endocrinol. 62:129-136. El Halawani, M. E., S. Fehrer, B. M. Hargis, and T. E. Porter, Shimatsu, A., Y. Kato, H. Ohta, K. Tojo, Y. Kabayama, T. Inoue, and H. Imura, 1985. Involvement of vasoactive 1988. Incubation behavior in the domestic turkey: intestinal polypeptide in serotonergic stimulation of physiological correlates. CRC Crit. Rev. Poult Biol. 1: prolactin secretion in rats. Pages 74-78 in: Prolactin. 285-314. Basic and Clinical Correlates. R. M. MacLeod, M. O. Fehrer, S. C, J. L. Silsby, and M. E. El Halawani, 1987. Thorner, U. Scapagnini, ed. Springer Verlag, New Capacity of various neuropeptides to induce prolactin York, NY. (PRL) or luteinizing hormone (LH) release by dissoci- Tatemoto, K., 1984. PHI-A new brain-gut peptide. Peptides ated turkey anterior pituitary cells. Poultry Sci. 5:151-154. 66(Suppl. 1):98. (Abstr.) Werner, S., A.-L. Hurting, T. Hokfelt, P. Eneroth, K. Kaji, H., K. Chihara, H. Abe, N. Minamitani, H. Kodama, T. Tatemoto, V. Mutt, L. Maroder, and E. Wunsch, 1983. Kita, T. Fujita, and K. Tatemoto, 1984. Stimulatory Effect of the peptide PHI-27 on prolactin release in effect of peptide histidine isoleucine amide 1-27 on vitro. Neuroendocrinology 37:476-478.
1990 Poultry Science 69:1209-1214 INTRODUCTION
The neuroendocrine regulation of prolactin (PRL) secretion in the turkey is complex and poorly understood (reviewed by El Halawani et ah, 1988). In mammalian species, most notably the rat, PRL release is strongly stimulated by several neurohormones, including vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), thyrotropin-releasing hormone (TRH), and neurophysin. Of these peptides, VIP is widely viewed as an authentic prolactin-releasing factor (PRF) in the rat (Shimatsu et ah, 1985), while both TRH and PHI are equipotent to VIP in stimulating PRL release in vitro and in vitro (Kaji et ah, 1984). Proudman and Opel (1988) and Opel and Proudman (1988) have recently shown that VIP is a potent stimulus for PRL secretion in the turkey. Peptide histidine isoleucine is a peptide composed of 27 amino acids and is found in the brain and the gut of
Sigma Chemical Co., St. Louis, MO 63178.
numerous species. It has a high (>50%) homology to VIP (Tatemoto, 1984). A preliminary report by Fehrer et ah (1987) has suggested that this peptide may also release PRL in the turkey. The present study was designed to determine if PHI functions as a PRF in the turkey. MATERIALS AND METHODS
hi Vitro Studies Three replicate preparations of pituitary cells were used; two were from pituitaries collected from large white laying turkey hens and one from medium white laying hens. For each preparation, the cells were dispersed enzymatically using collagenase and pancreatin and cultured for 4 days in monolayer flasks as described by Proudman and Opel (1988). On the 5th day of culturing, the cells were treated with either synthetic porcine VIP1 or synthetic porcine PHI1 at dosages ranging from 10""6 M to 10-11 M. Untreated flasks served as controls. Five flasks were treated at each dose. After 4 h of
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Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on April 3, 2015
ABSTRACT Studies were conducted both in vitro, using monolayer cultures of turkey pituitary cells, and in vivo, using ovariectomized turkey hens, steroid-primed ovariectomized hens, and immature toms to compare the effectiveness of peptide histidine isoleucine (PHI) with that of vasoactive intestinal peptide (VIP) in stimulating prolactin (PRL) secretion. Vasoactive intestinal peptide, a putative PRL-releasmg factor (PRF) in the turkey, was approximately 1000-fold more effective than PHI in stimulating PRL secretion in vitro. Prolactin secretion was significantly enhanced by the exposure of pituitary cells to 10~7 M PHI and a similar stimulation was observed with 10~ 10 M VTP. Injection of carmulated, unrestrained turkeys with PHI at doses up to 100 ug per kg of BW caused no significant change in circulating PRL concentrations, but injection of 10 fig of VTP per kg resulted in a 7 to 22-fold increase in plasma PRL concentration within 10 to 30 min following injectioa These results demonstrate a marked difference between the turkey and the rat in their response to a PRLstimulating neuropeptide. In contrast to what was observed in the turkey, PHI is a strong PRF in the rat, with a potency equal to or greater man that of VTP. Earlier studies have shown that thyrotropin-releasing hormone, another strong PRF in mammals, has little consistent PRF activity in the turkey. Thus, die present studies add additional evidence of major differences between mammals and birds in the control of PRL secretion by hypothalamic neuropeptides. (Key words: prolactin, peptide histidine isoleucine, turkey, vasoactive intestinal peptide, prolactin-releasing factor)
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PROUDMAN AND OPEL
incubation at 37 C, die medium was poured into tubes and stored at -20 C until the PRL concentration was determined by homologous radioimmunoassay (Proudman and Opel, 1981).
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on April 3, 2015
In Experiment 3, ovariectomized hens were primed with a 3- wk time-release pellet implant2 of either progesterone (50 or 100 mg), estradiol (50 or 75 mg), or both progesterone (50 mg) and estradiol (25 or 50 mg) inserted subcutaneously in the breast area. The doses of steroid used were In Vivo Studies based on those used by Saeed and El Halawani Five experiments were conducted to deter- (1986) to prime ovariectomized turkey hens mine the effects of intravenous injections of a prior to stimulation of PRL release. After 20 wide range of doses of PHI or of a known days of steroid priming, PHI was administered at effective dose of VIP on PRL secretion in doses of 5 or 10 jig per kg of BW. Because an turkeys in various physiological conditions. All infinite combination of doses of priming steroids birds were cannulated prior to experimentation and PHI was possible, a limited number of birds and were unrestrained in individual cages during (1 to 3) were tested at each of many treatment blood collection as previously described (Opel combinations to screen for an effective combiand Proudman, 1988). Samples were collected nation. Blood was collected as in Experiment 2, into heparinized syringes, centrifuged, and the except that the saline injection protocol was plasma was stored at -70 C prior to the omitted. measurement of PRL. Experiment 1 was conExperiment 4 tested the response of cannuducted using mature, ovariectomized, Diamond lated, unrestrained, Diamond Hybrid large Hybrid large white turkey hens. Hens were white toms treated with PHI or VIP at 14 wk of ovariectomized at 12 to 14 wk of age and raised age. The PHI was administered at doses of 5,10, in floor pens under a 6 h light (L): 18 h dark (D) 20, 50, or 100 Jig per kg of BW. The VIP was photoperiod from 18 to 28 wk of age. Hens were administered at doses of 10 or 20 ug per kg of then placed in individual laying cages within a BW (n = 1 or 2 per dose). Samples were battery room, given a stimulatory photoperiod collected prior to injection and 5,10,15,30,60, (14L:10D), and sampled between 30 and 38 wk and 90 min postinjection. of age. The 2-day injection and sampling Experiment 5 was conducted to test the protocol used was similar to that used previously possibility that PHI may have been lost due to (Opel and Proudman, 1988). Briefly, on Day 1 a adsorption of the peptide to the plastic containpreinjection blood sample (.5 mL) was drawn ers used for preparing and injecting the solufrom six birds, and a bolus of saline vehicle was tions. Mature, ovariectomized, Diamond Hybrid injected. Additional blood samples were col- medium white hens were treated with a wide lected at 5, 10, 15, 30,45, 60, 90, and 120 min range of doses of PHI (5 to 100 |ig/kg) or 10 jig following injection. On the 2nd day, hens were of VTP per kg (n = 1 to 3 per dose). The surfaces randomly selected to receive 2.5,5, or 10 u.g of of all cannulas, tubes, and syringes were PHI per kg of BW administered in 1 mL of pretreated with Silane to preclude peptide saline, and the sampling protocol used on Day 1 adsorption. Samples were collected at the was repeated. The entire protocol was repeated intervals used in Experiment 4. on a second group of six hens so that each dose was administered to a total of four birds. One hen was determined at necropsy to be incom- Statistical Analysis pletely ovariectomized and was deleted from the The results of the in vitro experiments did not experiment. differ between preparations of cells from large Experiment 2 was similar to Experiment 1, and medium white hens. Therefore, the data except that higher doses of PHI were tested (25, were combined to provide three independent 50, and 100 u.g per kg of BW), and postinfection replicates (n = 3) for statistical analysis. samples were collected at 10,20,30, and 60 min. Regression analysis was used to establish a In addition, three hens were treated with VIP at a significant dose effect (P<.05) for each treatdose of 10 u.g per kg of BW to confirm diat the ment. Data were then analyzed by ANOVA and hens were capable of responding to a known a two-sided Dunnett's procedure was applied to PRF. compare treatment means with die control. Except as noted below, the results on in vivo experiments were analyzed by a repeated'innovative Research of America, Toledo, OH 43606. measures ANOVA to establish differences over
PROLACTIN SECRETION IN THE TURKEY
FIGURE 1. Effect of various dosages of peptide histidine isoleucine (PHI) and vasoactive intestinal peptide (VTP) on prolactin secretion by monolayer cultures of turkey anterior pituitary cells. Treatment x + SEM with an asterisk differ significantly (P<05) from untreated controls. Each x represents three cell preparations (n = 3) with five replicates each.
treatment (dose of peptide) and time for each peptide. Where treatment by time interactions were present, Dunnett's procedure was used to compare means from a given sampling time to the zero time (P<05) as described by Opel and Proudman (1988). RESULTS
The effect of PHI and VIP on PRL secretion by dispersed turkey pituitary cells is shown in Figure 1, Vasoactive intestinal peptide significantly stimulated PRL secretion at a dose of 10 -10 M, with maximal stimulation observed from 10"8 to 10 -6 M VIP. Peptide histidine isoleucine significantly stimulated PRL secretion only at doses of 10 -7 and 10 -6 M. The response of cannulated, unrestrained, ovariectomized turkey hens to PHI and VIP in Experiments 1, 2, and 5 is presented in Table 1. Neither saline injection (data not shown) nor administration of PHI at doses ranging from 2.5 to 100 (ig per kg of BW produced any significant change in circulating PRL levels. The procedure utilized to preclude adsorption of PHI to plastic utensils (Experiment 5) did not influence these results. In contrast, VTP elicited a significant increase in concentrations
of PRL in plasma within 10 min of administration. Peak PRL concentrations were observed at 10 to 20 min following VIP injection in Experiment 2 and at 30 min after injection in Experiment 5. The increase in PRL following VIP administration ranged from 7 to 12-fold that of the basal levels. Priming of ovariectomized hens with steroids prior to treatment with PHI (Experiment 3) did not enhance the PRL response to PHI (Table 2). Because some steroid-PHI combinations were represented by only a single bird, regression analysis was used to test for PRL responses over time for each treatment combination. Although this analysis revealed significant increases in PRL over time in several instances, these increases in no case equaled that observed in one of the control groups. The reason for the increase in PRL over time in some steroid-primed hens (controls and PHI-treated) was unclear. We observed no behavioral indications of stress in these birds and observed no similar increase was observed in other cannulated birds used in these experiments. After combining PHI doses, there was still no significant change in PRL following PHI treatment in any of the steroidprimed groups. Immature torn turkeys (Experiment 4) also failed to respond to PHI administration at a wide range of doses (Table 3). These birds responded significantly to VIP as shown by regression analysis of PRL levels at each dose of VIP. Both 10 and 20 ug of VTP per kg elicited a maximum increase in plasma PRL at 15 min following injection. DISCUSSION
The present evidence that PHI has no effect on in vivo PRL secretion in the turkey and that PHI is approximately 1,000 times less effective than VD? in vitro is in sharp contrast to reports that PHI is a putative PRF in the rat (Werner et al, 1983; Kaji et al, 1984; Ohta et al, 1985). Administration of 1 \ig of PHI to a 300 g rat significantly increases plasma PRL within 5 min, and equimolar doses of PHI, VTP, and TRH given i.v. produce an equivalent rise in plasma PRL (Kaji et al., 1984). Peptide histidine isoleucine also stimulates PRL secretion from superfused rat pituitary cells (Kaji et al, 1984; Ohta et al, 1985), monolayer cultures of pituitary cells, and hemipituitaries (Werner et al, 1983). In these in vitro studies
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on April 3, 2015
CONCENTRATION (Ml
1211
12.55 13.34 12.00 103.93* 10 7.90 16.75 13.15 6.40 12.65 8.75 27.65*
11.27 13.11 11.60 111.40* 5
7.95 16.10 13.53 6.00 10.80 8.08 8.65
1153 13.88 11.65 14.58 0
8.80 15.60 13.63 7.10 1058 7.45 9.40
10 3.94 4.82 356 20
3.63 5.55 3.86 10
3.59 4.85 2.93 0
•Significantly different from the 0 min x (P<05).
Standard error of the least squares x.
Experiment 5 5 ug PHI 10 ug PHI 20 Ug PHI 40 ug PHI 50 ug PHI 100 ug PHI 10 Ug VIP
Experiment 2 25 ug PHI 50 ugPHI 100 ug PHI 10 Ug VIP
Experiment 1 2.5 ug PHI 5 HgPHI 10 Mg PHI
Dose (per kg of BW)
1.02 .86 .31 9.54 60 7.05 22.45 14.90 6.00 10.43 7.80 50.60*
12.01 1354 11.80 44.88 30 6.60 16.90 1555 650 1050 7.15 115.10*
11.58 13.47 11.65 66.43 15 7.40 15.95 15.45 6.05 10.15 850 79.35*
45 3.65 557 3.35 SE
30 3.35 4.45 3.05 60
3.39 4.95 3.43 30
15
Minutes after injection 3 5 3
3
1
6 1 13
TABLE 1. Mean plasma prolactin levels (nglmL) in ovariectomized turkey hens given peptide or vasoactive intestinal peptide (VIP) in Experiments 1, 2, and 5
d from http://ps.oxfordjournals.org/ at Michigan State University on April 3, 2015
1213
PROLACTIN SECRETION IN THE TURKEY
TABLE 2. Mean plasma prolactin levels (ng/mL) in steroid-primed, ovariectomized turkey hens given peptide histidine isoleucine (PHI) or vasoactive intestinal peptide (VIP) (Experiment 3) Minutes after injection
Dose of PHI (Mg/kg of BW) n
Steroid treatment1
Progesterone (50 mg) plus estradiol (25 mg) Progesterone (50 mg) plus estradiol (50 mg)
10
20
30
60
SE 2
0 5 5 10
2 2 1 3
11.20 9.08 11.15 10.40
15.08 12.00 12.45 11.93
22.52 11.40 14.00 13.42
25 JO 12.85 16.65 16.22
31.68 17.00 20.25 18.25
2.11* 1.89 3.62* 1.18*
0 5 10 5 10
1 1 1 2 2
15.75 10.40 8.30 12.05 16.48
14.50 10.90 8.05 13.18 19.10
16.50 11.60 8.65 13.22 17.30
14.85 11.95 8.70 13.50 19.65
16.25 12.40 8.10 13.42 21.60
.87 .80* .30 .89 36*
5 10
1 2
10.40 19.35
10.30 18.90
10.80 21.58
10.85 23.00
12.60 24.90
.93 .16*
5 10
1 1
20.90 12.85
20.15 12.00
22.50 14.35
25.70 14.60
25.25 15.90
2.50* 1.53*
'Total steroid dose in 3-wk time-release implants. Body weight was 7.6 to 10.4 kg. Standard error of the least squares x. *Row x change significantly with time (P<05).
of the rat, PHI was shown to be more effective than VIP in stimulating PRL secretion. We have administered a wide range of doses of PHI to turkeys in various physiological states and under conditions in which we have demonstrated herein and previously that VIP is highly effective in stimulating PRL secretion. Clearly, PHI has no effect on PRL secretion in ovariectomized turkey hens, immature toms, and steroid-primed ovariecto-
mized hens at the steroid-PHI treatment combinations that were tested. Peptide histidine isoleucine was not given to laying or incubating hens. Thus, although the possibility that PHI may affect PRL secretion in vivo under other conditions cannot be precluded by the present study, the results strongly suggest that PHI is unlikely to function as a physiological PRF in the turkey. This finding, together with previous evidence (El Halawani et al.,
TABLE 3. Mean plasma prolactin levels (ng/mL) in i'mmature torn turkeys given peptide histidine isoleucine (PHI) or vasoactive intestinal I peptide (VIP) (Experiment 4) Minutes after injection
Dnsfi
(per kg of BW)
n
0
5
10
15
30
60
90
SE1
PHI •5 Mg 5 Mg
10 jig 20 Mg 50 Mg 100 Mg
VTP 10 Mg 20 Mg
2.45 3.40 4.12 2.82 5.18 3.92
2.70 3.45 3.68 2.68 4.48 3.08
2.40 3.10 3.70 2.68 5.18 3.90
2.05 3.55 3.92 2.58 5.55 4.12
2.45 4.35 3.75 2.82 5.68 4.02
2.25 3.75 3.58 2.58 5.05 3.95
2.60 3.05 4.45 2.60 4.88 338
22 .44 .79 .17 SI .34
3.75 3.12
11.60 9.30
22.50 17.90
82.90 67.85
55.40 49.15
23.60 29.10
3.10 11.20
28.98* 24.63*
Standard error of the least squares x. *Row x change significantly with time (P<05).
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on April 3, 2015
Progesterone 50 mg 50 mg 100 mg 100 mg Estradiol 50 mg 50 mg 50 mg 75 mg 75 mg
0
1214
PROUDMAN AND OPEL
1988; Proudman and Opel, 1988) that TRH (a highly effective PRF in mammals) is also not a PRF in the turkey, demonstrates major differences in the neuroendocrine regulation of PRL secretion between an avian specie (the turkey) and the mammalian species studied to date.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on April 3, 2015
prolactin release in the rat. Life. Sci. 35:641-647. Ohta, H., Y. Kato, K. Tojo, A. Shimatsu, T. Inoue, Y. Kabayama, and H. Imura, 1985. Further evidence that peptide histidine isoleucine (PHI) may function as a prolactin releasing factor in rats. Peptides 6:709-712. OpeLH., and J. A. Proudman, 1988. Stimulation of prolactin release in turkeys by vasoactive intestinal peptide. Proc. Soc. Exp. Biol. Med. 187:455-460. Proudman, J. A., and H. Opel, 1981. Turkey prolactin: validation of a radioimmunoassay and measurement of ACKNOWLEDGMENTS changes associated with broodiness. Biol. Reprod. 25: 573-580. The authors gratefully acknowledge the Proudman, J. A., and H. Opel, 1988. Stunulation of prolactin technical assistance of Gaynelle Campbell and secretion from turkey anterior pituitary cells in culture. Proc. Soc. Exp. Biol. Med. 187:448-*54. Beverly Russell. Saeed, W., and M. E. El Halawani, 1986. Modulation of the prolactin response to thyrotropin releasing hormone by ovarian steroids in ovariectomized turkeys (Meleagris REFERENCES gattopavo). Gen. Comp. Endocrinol. 62:129-136. El Halawani, M. E., S. Fehrer, B. M. Hargis, and T. E. Porter, Shimatsu, A., Y. Kato, H. Ohta, K. Tojo, Y. Kabayama, T. Inoue, and H. Imura, 1985. Involvement of vasoactive 1988. Incubation behavior in the domestic turkey: intestinal polypeptide in serotonergic stimulation of physiological correlates. CRC Crit. Rev. Poult Biol. 1: prolactin secretion in rats. Pages 74-78 in: Prolactin. 285-314. Basic and Clinical Correlates. R. M. MacLeod, M. O. Fehrer, S. C, J. L. Silsby, and M. E. El Halawani, 1987. Thorner, U. Scapagnini, ed. Springer Verlag, New Capacity of various neuropeptides to induce prolactin York, NY. (PRL) or luteinizing hormone (LH) release by dissoci- Tatemoto, K., 1984. PHI-A new brain-gut peptide. Peptides ated turkey anterior pituitary cells. Poultry Sci. 5:151-154. 66(Suppl. 1):98. (Abstr.) Werner, S., A.-L. Hurting, T. Hokfelt, P. Eneroth, K. Kaji, H., K. Chihara, H. Abe, N. Minamitani, H. Kodama, T. Tatemoto, V. Mutt, L. Maroder, and E. Wunsch, 1983. Kita, T. Fujita, and K. Tatemoto, 1984. Stimulatory Effect of the peptide PHI-27 on prolactin release in effect of peptide histidine isoleucine amide 1-27 on vitro. Neuroendocrinology 37:476-478.