GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
80,
138-145 (1990)
Enhanced Vasoactive Intestinal Peptide-Induced Prolactin Secretion from Anterior Pituitary Cells of Incubating Turkeys (Meleagris gallopavo)’ M. E. EL HALAWANI,
J. L. SILSBY, AND L.J.
MAURO
Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108 Accepted October 23, 1989 During incubation, female turkeys exhibit elevated circulating prolactin (PRL) which may be the result of enhanced pituitary responsiveness to vasoactive intestinal peptide (VIP). This hypothesis was tested by comparison of spontaneous and porcine VIP-induced PRL secretion from anterior pituitary cells of hens in various reproductive conditions. The effect of VIP and luteinizing hormone releasing hormone (LHRH), alone and in combination, on luteinizing hormone (LH) secretion was also examined. Incubation with pVIP (lo-” to 10v6 M) significantly stimulated PRL secretion at all incubation times tested (l-5 hr). This increase was greatest in cells from incubating hens, with those from laying, photorefractory, and quiescent (nonphotostimulated) hens secreting successively less PRL. These responses were obtained when spontaneous PRL secretions were compared. VIP induced approximately a similar 1.5fold increase in LH secretion, in all reproductive groups. Also, VIP enhanced LHRH-induced LH secretion (1.2- to 1.6-fold; P < 0.0001). It is concluded that PRL secretion in vitro by pituitary cells from turkey hens in various reproductive stages reflects the circulating levels of PRL at these stages. 8 1990Academic press, 1~.
Incubation behavior in the female turkey is associated with high concentrations of circulating prolactin (PRL) in relation to other stages of the reproductive cycle (for review, El Halawani et al., 1988a). The regulatory mechanisms responsible for this elevation in circulating PRL appear to involve changes in the hypothalamus as well as the anterior pituitary. The facilitation of in vivo PRL release by serotonin (5HT) and the increased 5-HT turnover in incubating hens indicates central PRL regulation (El Halawani and Burke 1976; El Halawani et al., 1980; Hargis and Burke, 1984). Changes in hypothalamic vasoactive intestinal peptide (VIP), a potent secretagogue of PRL both in vivo and in vitro (Proudman and Opel, 1988; Opel and Proudman, 1988;
r Minnesota Agricultural Experiment Station Scientific Journal Article 17,633. Supported by USDA Grant 88-37242-3845.
Knapp et al., 1988), may also contribute to changes in PRL release since the number of VIP immunoreactive (VIP-IR) neurons and fibers in the mediobasal hypothalamus increase in incubating turkey hens (Mauro et al., 1988). Regulation at the level of the pituitary is suggested by the findings that spontaneous as well as hypothalamic extract-induced PRL secretion is greatest from anterior pituitary cells of incubating hens as compared to cells from laying and photorefractory hens (El Halawani et al., 1988b). Considering these potential regulatory mechanisms, it is possible that increased circulating PRL concentrations in incubating female turkeys are in part the result of enhanced pituitary responsiveness to VIP, an apparent physiological prolactin-releasing peptide (Mauro et al., 1989; El Halawani et al., 1990). To test this hypothesis, anterior pituitary glands from hens at various stages of the reproductive cycle were 138
0016~6480190 $1.50 CopytQht 0 19!30 by Academic Press, Inc. All rights of reproduction in any form reserved.
139
VIP AND PRL SECRETION
dissociated and cultured, and the spontane- way analysis of variance using the SAS General Linear procedure (Helwig and Council, 1979). Statisous and VIP-induced PRL secretions were Model tical significance between treatment means was decompared. In addition, since VIP stimu- tected using Duncan’s multiple range test at a sign& lates LH secretion both in vitro (Fehrer et cance level of Q = 0.05. al., 1987) and in vivo (Macnamee et al., 1986), it was of interest to examine the efRESULTS fect of VIP and luteinizing hormoneExperiment I: Effect of VIP on PRL and releasing hormone (LHRH) alone and in LH secretion by anterior pituitary cells decombination on LH secretion. MATERIALS
AND METHODS
Hatchmate turkeys of the large white female line (Nicholas Turkey Farms, Sonoma, CA) were housed together to obtain groups required for each experiment (El Halawani et al., 1988b). The reproductive groups utilized in this study were (1) laying turkeys that had laid regularly up to the date of the experiment and showed one or two nest visits per day; (2) incubating birds that were on the nest six or seven times each day for at least 2 weeks without producing eggs; (3) photorefractory birds that had neither laid nor nested despite the 15L:9D lighting schedule and had hard, tight pubic bones and dry cloaca that are associated with ovarian quiescence; and (4) quiescent (nonphotostimulated) hens that were maintained under a 6L:18D lighting regime for at least 8 weeks. In Experiment 1, a balanced incomplete block design was used to compare pituitaries of hens from different reproductive stages. Comparisons were made between laying (n = 7) and incubating (n = 7), laying (n = 7) and photorefractory (n = 7), and incubating (n = 7) and photorefractory (n = 7) hens on 3 different days between hens of the same flock. Anterior pituitary cells were incubated for 3 hr in the presence of different dilutions (10-l’ to 10M6 M) of porcine VIP (PVIP) or with lo-’ M VIP at l-, 2-, 3-, 4-, and 5-hr increments of incubation. In Experiment 2, comparisons were made between laying hens (n = 7) and their semen-producing male hatchmates (n = 8). In Experiment 3, anterior pituitary cells obtained from quiescent, laying, and incubating hens and from semen-producing males (n = 7/group) were cultured with lo-’ M VIP, lo-* M LHRH, or a combination of the two. Cell cuhre. Dissociated anterior pituitary cells from the above turkeys were obtained using the procedure of Fehrer et al. (1985). The cells were incubated in glass culture tubes (four tubes/treatment). At the conclusion of each incubation period, the cell cultures were centrifuged and the supematant was collected and stored at - 20” until assayed. Radioimmunoassays for PRL and LH were conducted according to the methods of Proudman and Opel (1981) and Burke et al. (1979), respectively. Sratistical analysis. All data were analyzed by one-
rived from laying, incubating, and photorefractory turkeys. During the 3-hr incubation
period, the highest spontaneous secretion of PRL was by anterior pituitary cells from incubating hens (0.89 k 0.07 pg; Fig. 1) followed by cells from laying hens (0.32 + 0.02 kg) and those from photorefractory hens (0.17 + 0.01 pg). The VIP-stimulated PRL secretion was increased in a dose-related manner (P < O.OOOl),with maximal PRL secretion occurring at 10e8 A4 VIP. As with the spontaneous PRL secretion (Fig. l), VIP induced the highest PRL release in
7 7
,/p-.
I
J
z t 4
3.
g
2.
1.
o-
) 0
12
10 VIP (-log
6
6
MI
FIG. 1. Effect of increasing concentrations of porcine vasoactive intestinal peptide (VIP) on prolactin secretion by cultured pituitary cells from laying (solid line), incubating (large broken lines), and photorefractory (small broken lines) hens. Data points represent means + SEM of 8 to 10 determinations. In several cases, the SEM is within the data point drawn in the figure.
140
EL HALAWANI,
SILSBY, AND MAURO
cells from incubating hens (7.31 + 0.46 pg) as compared to cells from laying (3.17 -+ 0.37 Fg) and photorefractory (1.21 + 0.12 pg) hens. These responses to VIP represent 8.2-, 9.9-, and 7.1-fold increases in PRL by cells from incubating, laying, and photorefractory birds, respectively. The content of PRL in the medium increased (P < 0.0001) in a time-dependent manner regardless of the pituitary cell origin or the type of treatment except the spontaneous PRL secretion from cells of photorefractory hens (P = 0.1558; Fig. 2). Anterior pituitary cells from incubating hens had greater spontaneous and VIP (10e8 m-stimulated PRL secretion (P < 0.05) than cells from laying hens and these PRL releases by cells from laying hens were greater (P < 0.05) than those of pituitary cells from photorefractory hens at each incubation time. Anterior pituitary cells from laying hens SPONTANEOUS
had a greater (P < 0.05) spontaneous LH secretion than cells from incubating or photorefractory hens (Fig. 3). The presence of VIP (IO-* M) induced an increase (P < 0.05) in LH secretion from pituitary cells of incubating and photorefractory hens but not from cells of laying hens (P > 0.05) during the 5 hr of incubation. Experiment 2: Effect of VIP on PRL and LH secretion by anterior pituitary cells derived from semen-producing male and laying female turkeys. Anterior pituitary cells
from the males exhibited higher spontaneous and VIP (lo-* m-induced LH secretion than cells from laying hens (Fig. 4). After a 5-hr incubation, LH increased (P < 0.05) from the spontaneous value of 14.1 + 1.1 to 21.2 + 1.2 ng following VIP treatment of cells from laying hens. The corresponding values from male pituitary cells were 39.0 k 1.5 and 61.2 + 2.0 ng, respectively. Spontaneous and VIP-induced PRL secreVIP STIMULATED
SECRETION
INCUBATION
TIME
SECRETION
(hr)
FIG. 2. Spontaneous and porcine vasoactive intestinal peptide (lo-* m-induced prolactin secretion by pituitary cells from laying, incubating, and photorefractory hens cultured for varying lengths of time; otherwise as in Fig. 1.
VIP
35
AND
SPONTANEOUS
PRL
141
SECRETION VIP STIMULATED
SECRETION
SECRETION
30
25 3 8 8 8
20
z a 5
15
5
10
I
1
2
3
4
5
INCUBATION
2
1 TIME
3
4
5
(hr)
FIG. 3. Spontaneous and porcine vasoactive intestinal peptide (lOma M)-induced luteinizing hormone secretion by pituitary cells from laying, incubating, and photorefractory hens cultured for varying lengths of time; otherwise as in Fig. 1.
tion exhibited a similar pattern with the greatest response seen with cells from laying hens. Experiment 3: Effect of VIP (IO-* M), LHRH (IO-* M), or their combinations on PRL and LH secretions by anterior pituitary cells derived from nonphotostimulated, laying, and incubating hens andfrom semen-producing male turkeys. As in Experiment 1, anterior pituitary cells derived from incubating hens exhibited both greater (P < 0.05) spontaneous and VIP-stimulated PRL secretion than cells from laying hens. In Experiment 2, spontaneous and VIPstimulated PRL secretion by pituitary cells from laying hens was greater (P < 0.05) than those of pituitary cells from the males (Fig. 5). Anterior pituitary cells from quiescent hens had less VIP-stimulated PRL secretion (P < 0.05) compared to that of cells from any of the other groups of birds. No effect of LHRH was observed on PRL secretion. Coincubation of anterior pituitary cells with both LHRH and VIP at lo-* M each enhanced LH secretion compared to cultures exposed to either LHRH or VIP.
However, only pituitary cells from males responded to the coincubation with VIP and LHRH with greater net LH secretion (161.7 + 10.6 ng) than the combined net response to either peptide alone (VIP, 22.3 + 3.8 ng; LHRH, 76.5 ? 5.9 ng). Anterior pituitary cells from male turkeys had greater LHRH plus VIP-stimulated LH secretion (245.8 + 10.6 ng; P < 0.05) than cells from quiescent hens (138.9 2 7.6 ng) which in turn had greater LH secretions (P < 0.05) than cells from incubating (74.6 2 8.1 ng) or laying (60.0 2 5.4 ng) hens. DISCUSSION These results clearly show that VIP increases PRL secretion from anterior pituitary cells in vitro. The effect at all VIP concentrations is greatest on cells from incubating hens, followed in descending order by cell populations from laying, photorefractory, and nonphotostimulated hens. Whether this increase in the PRL-releasing capacity observed in the pituitary cell population of incubating hens results from an
142
EL
2
_
1
-
0
1:‘:
c____c__ 1
HALAWANI,
SILSBY,
1 I :
-C----C----* 2
INCUBATION
3
4
TIME
5
(hr)
FIG. 4. Spontaneous secretion of luteinizing hormone and prolactin by pituitary cells from laying female (solid line) and male turkeys (large broken lines) as well as the porcine vasoactive intestinal peptide (lo-* m-stimulated secretion of luteinizing hormone and prolactin (females, dotted large broken lines; males, small broken lines). Data points represent means -+ SEM of four to five determinations.
increase in the number of lactotrophs per pituitary, an increase in PRL production per lactotroph, or a combination of both remains to be determined. The observed in vitro PRL secretion at various reproductive states reflects the circulating levels of PRL at these states. Cells from incubating hens had the greatest secretion of PRL and their circulating PRL levels were also the greatest. Laying hens had lower circulating PRL levels than incubating hens, but had greater PRL levels than photorefractory or quiescent hens. Accordingly, anterior pituitary cells from laying hens had a smaller spontaneous and VIP-stimulated PRL secretion than such cells from incubating hens. In addition, pi-
AND
MAURO
tuitary cells from laying hens had greater spontaneous as well as VIP-stimulated PRL secretion than either photorefractory or quiescent hens. Similar results were obtained utilizing hypothalamic extract as a secretagogue of PRL (El Halawani et al., 1988b). On the other hand, VIP (10e7 M) did not effect PRL release when incubated with individual anterior pituitary glands from laying bantam hens in vitro (Macnamee et al., 1986). In a separate experiment, however, VIP was stimulatory to PRL secretion by anterior pituitary glands from incubating bantams (Macnamee et al., 1986). We propose that both hypothalamic inputs and the anterior pituitary itself may each contribute to the changes in circulating PRL levels observed in the various states of the reproductive cycle of the female turkey. VIP may be a prolactinreleasing neuropeptide in the turkey since hypothalamic VIP immunoneutralization and pituitary VIP receptor blockade significantly depressed the PRL response to hypothalamic extract in vitro (El Halawani er al., 1990). Moreover, changes in the number of VIP-like immunoreactive cells in the infundibular nuclear complex (INF) are correlated with the alterations in circulating PRL levels during the reproductive cycle of the turkey (Mauro et al., 1988). The increase in circulating PRL during incubation was accompanied by a dramatic increase in VIP-IR cells, whereas disruption of incubation behavior results in a decline in both circulating PRL levels and the number of VIP-IR cells (Mauro et al., 1989). Thus, the natural increases in circulating PRL during photostimulation, egg laying, and incubation may reflect changes in the synthesis and release of VIP and/or in anterior pituitary responsiveness to VIP. In the present study, pVIP induced approximately a 1.5fold increase in LH secretion from cultured anterior pituitary cells. Also, pVIP enhanced LHRH-induced LH secretion. Similarly, when in vitro cul-
VIP
AND
PRL
143
SECRETION
20. 3 z
16 -
8 3::
12.
‘a 3 I
6.
5 5 0 E
4. 2 1 30X
i
1 CELLS
ALONE I
LHRH
QUIESCENT
INCUBATING
LAYING REPRODUCTIVE
MALE
STATUS
FIG. 5. Effects of vasoactive intestinal peptide (lo-* AI), luteinizing hormone-releasing hormone (lo-* M), or their combinations on prolactin and luteinizing hormone secretions by cultured pituitary cells from quiescent (nonphotostimulated), laying, and incubating female turkeys and from male turkeys; otherwise as in Fig. 4.
ture of anterior pituitary cells from laying turkey hens was utilized, pVIP stimulated LH secretion (Fehrer et al., 1987). Macnamee et al. (1986) also observed an increase in circulating LH with VIP administration to bantam chickens. In the rat, intraventricular administration of VIP stimulated LH secretion (Vijayan et al., 1979), and VIP induced GnRH secretion in vitro by synaptosomes from median eminence tissue (Samson et al., 1981). In contrast to the latter results, Alexander et al. (1985) reported an inhibitory effect of intra-
ventricularly administered VIP on LH secretion. The physiological relevance of VIP in LH secretory regulation remains speculative, at best. The INF comprises a region of the brain containing high concentrations of neurons exhibiting VIP-like immunoreactivity (Mauro er al., 1988, 1989). Since the INF constitutes a link to the photoregulation of LH secretion and gonadal recrudescence by the presence of photoreceptors (Oliver and Bayle, 1976; Oliver et al., 1977; Ohta and Homma, 1987), it is possible that
144
EL HALAWANI,
SILSBY,
VIP may modulate photoperiodically induced gonadotropin secretion. The appearance of VIP-IR neurons in the INF following photostimulation (Mauro et al., 1989) and the colocalization of an opsin-like compound with VIP in the INF neurons (Silver et al., 1988) support such a hypothesis. REFERENCES Alexander, M. J., Cliion, D. K., and Steiner, R. A. (1985). Vasoactive intestinal polypeptide effects a central inhibition of pulsatile luteinizing hormone secretion in ovariectomized rats. Endocrinology 117, 2134-2139.
Burke, W. H., Licht, P., Papkoff, H., and Bona Gallo, A. (1979). Isolation and characterization of luteinizing hormone and follicle-stimulating hormone from pituitary glands of the turkey (Meleagris gallopavo).
Gen.
Comp.
Endocrinol.
37, 508-520.
El Halawani, M. E., and Burke, W. H. (1976). Brain monoamine metabolism of turkey hens in various stages of their reproductive life cycle. Biol. Reprod. 15, 254-259. El Halawani, M. E., Burke, W. H., and Dennison, P. T. (1980). Effects of p-chlorophenylalanine on the rise in serum prolactin associated with nesting in broody turkeys. Biol. Reprod. 23, 815-819. El Halawani, M. E., Fehrer, S. C., Hargis, B. M., and Porter, T E. (1988a). Incubation behavior in the domestic turkey: Physiological correlates. CRC Crit.
Rev.
Poult.
Biol.
l(4),
285-314.
El Halawani, M. E., Silsby, J. L., and Fehrer, S. C. (1988b). Basal and hypothalamic extract-induced luteinizing hormone and prolactin secretion by cultured anterior pituitary cells from female turkeys in various stages of the reproductive cycle. Gen.
Comp.
Endocrinol.
71, 45-54.
El Haiawani, M. E., Silsby, J. L., and Mauro, L. J. (1990). Vasoactive intestinal peptide is a hypothalamic prolactin-releasing neuropeptide in the turkey (Meleagris gallopavo). Gen. Comp. Endocrinol.
78, 66-73.
Fehrer, S. C., &by, J. L., Behnke, E. J., and El Halawani, M. E. (1985). Hypothalamic and serum factors influence on prolactin and luteinizing hormone release by the pituitary gland of the young turkey (Meleagris gallopavo). Gen. Comp. Endocrinol. 59, 73-81. Fehrer, S. C., Silsby, J. L., and El Halawani, M. E. (1987). Capacity of various neuropeptides to induce prolactin (PRL) or luteinizing hormone (LH) release by dissociated turkey hen anterior pituitary cells. Poult. Sci. 66, Suppl. 1, 98.
AND
MAURO
Hargis, B. M., and Burke, W. H. (1984). Prolactin and luteinizing hormone levels of prelaying, laying and postlaying turkey hens following central administration of serotonin and peripheral administration of quipazine maleate. Gen. Comp. Endocrinol. 55, 12-19. Helwig, J. T., and Council, K. A. (Eds.) (1979). “SAS User’s Guide.” SAS Institute, Cary, NC. Knapp, T. R., Fehrer, S. C., Silsby, J. L., Porter, T. E., Behnke, E. J., and El Halawani, M. E. (1988). Gonadal steroid modulation of basal and vasoactive intestinal polypeptide-stimulated prolactin release by turkey anterior pituitary cells. Gen.
Comp.
Endocrinol.
72,226-236.
Macnamee, M. C., Sharp, P. J., Lea, R. W., Sterling, R. J., and Harvey, S. (1986). Evidence that vasoactive intestinal polypeptide is a physiological prolactin-releasing factor in the bantam hen. Gen. Comp.
Endocrinol.
62, 470-478.
Mauro, L. J., Elde, R. P., and El Halawani, M. E. (1988). Evidence of a differential expression of hypothalamic VIP immunoreactivity (VIP-IR) associated with prolactin (PRL) levels in the domestic turkey hen. Endocrinology 122, Suppl. 1, 133. Mauro, L. J., Elde, R. P., Youngren, 0. M., Phillips, R. E., and El Halawani, M. E. (1989). Alterations in hypothalamic vasoactive intestinal peptide-like immunoreactivity are associated with reproduction and prolactin release in the female turkey. Endocrinology
125, 1795-1804.
Ohta, M., and Homma, K. (1987). Detection of neural connection to the infundibular complex by partial or complete hypothalamic deafferentation in male quail. Gen. Comp. Endocrinol. 68, 286-292. Oliver, J., and Bayle, J. D. (1976). The involvement of the preoptic-suprachiasmatic region in the photosexual reflex in quail: Effects of selective lesions and photostimulation. J. Physiol. (Paris) 72, 627637.
Oliver, J., Herbute, S., and Bayle, J. D. (1977). Testicular response to photostimulation by radioluminous implant in deafferented hypothalmus of quail. J. Physiol. (Paris) 73, 685-691. Opel, H., and Proudman, J. A. (1988). Stimulation of prolactin release in turkeys by vasoactive intestinal peptide. Proc. Sot. Exp. Biol. Med. 187,455460.
Proudman, J. A., and Opel, H. (1981). Turkey prolactin: Validation of a radioimmunoassay and measurement of changes associated with broodiness. Biol.
Reprod.
25, 573-580.
Proudman, J. A., and Opel, H. (1988). Stimulation of prolactin secretion from turkey anterior pituitary cells in culture. Proc. Sot. Exp. Biol. Med. 187, 448-454.
VIP
AND
PRL
Samson, W. K., Burton, K. P., Reeves, J. P., and McCann, S. M. (1981). Vasoactive intestinal peptide stimulates luteinizing hormone-releasing hormone release from median eminence synaptosomes. Regul. Pept. 2, 253. Silver, R., Witkovsky, P., Horvath, P., Alones, V., Bamstable, C. J., and Lehman, M. N. (1988). Coexpression of opsin- and VIP-like immunoreactiv-
SECRETION
145
ity in CSF-contacting neurons of the avian brain. Cell Tissue Res. 253, 189-198. Vijayan, E., Samson, W. K., Said, S. I., and McCann, S. M. (1979). Vasoactive intestinal peptide: Evidence for a hypothalamic site of action to release growth hormone, luteinizing hormone, and prolactin in conscious ovariectomized rats. Endocrinology 111, 2055.
AND
COMPARATIVE
ENDOCRINOLOGY
80,
138-145 (1990)
Enhanced Vasoactive Intestinal Peptide-Induced Prolactin Secretion from Anterior Pituitary Cells of Incubating Turkeys (Meleagris gallopavo)’ M. E. EL HALAWANI,
J. L. SILSBY, AND L.J.
MAURO
Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108 Accepted October 23, 1989 During incubation, female turkeys exhibit elevated circulating prolactin (PRL) which may be the result of enhanced pituitary responsiveness to vasoactive intestinal peptide (VIP). This hypothesis was tested by comparison of spontaneous and porcine VIP-induced PRL secretion from anterior pituitary cells of hens in various reproductive conditions. The effect of VIP and luteinizing hormone releasing hormone (LHRH), alone and in combination, on luteinizing hormone (LH) secretion was also examined. Incubation with pVIP (lo-” to 10v6 M) significantly stimulated PRL secretion at all incubation times tested (l-5 hr). This increase was greatest in cells from incubating hens, with those from laying, photorefractory, and quiescent (nonphotostimulated) hens secreting successively less PRL. These responses were obtained when spontaneous PRL secretions were compared. VIP induced approximately a similar 1.5fold increase in LH secretion, in all reproductive groups. Also, VIP enhanced LHRH-induced LH secretion (1.2- to 1.6-fold; P < 0.0001). It is concluded that PRL secretion in vitro by pituitary cells from turkey hens in various reproductive stages reflects the circulating levels of PRL at these stages. 8 1990Academic press, 1~.
Incubation behavior in the female turkey is associated with high concentrations of circulating prolactin (PRL) in relation to other stages of the reproductive cycle (for review, El Halawani et al., 1988a). The regulatory mechanisms responsible for this elevation in circulating PRL appear to involve changes in the hypothalamus as well as the anterior pituitary. The facilitation of in vivo PRL release by serotonin (5HT) and the increased 5-HT turnover in incubating hens indicates central PRL regulation (El Halawani and Burke 1976; El Halawani et al., 1980; Hargis and Burke, 1984). Changes in hypothalamic vasoactive intestinal peptide (VIP), a potent secretagogue of PRL both in vivo and in vitro (Proudman and Opel, 1988; Opel and Proudman, 1988;
r Minnesota Agricultural Experiment Station Scientific Journal Article 17,633. Supported by USDA Grant 88-37242-3845.
Knapp et al., 1988), may also contribute to changes in PRL release since the number of VIP immunoreactive (VIP-IR) neurons and fibers in the mediobasal hypothalamus increase in incubating turkey hens (Mauro et al., 1988). Regulation at the level of the pituitary is suggested by the findings that spontaneous as well as hypothalamic extract-induced PRL secretion is greatest from anterior pituitary cells of incubating hens as compared to cells from laying and photorefractory hens (El Halawani et al., 1988b). Considering these potential regulatory mechanisms, it is possible that increased circulating PRL concentrations in incubating female turkeys are in part the result of enhanced pituitary responsiveness to VIP, an apparent physiological prolactin-releasing peptide (Mauro et al., 1989; El Halawani et al., 1990). To test this hypothesis, anterior pituitary glands from hens at various stages of the reproductive cycle were 138
0016~6480190 $1.50 CopytQht 0 19!30 by Academic Press, Inc. All rights of reproduction in any form reserved.
139
VIP AND PRL SECRETION
dissociated and cultured, and the spontane- way analysis of variance using the SAS General Linear procedure (Helwig and Council, 1979). Statisous and VIP-induced PRL secretions were Model tical significance between treatment means was decompared. In addition, since VIP stimu- tected using Duncan’s multiple range test at a sign& lates LH secretion both in vitro (Fehrer et cance level of Q = 0.05. al., 1987) and in vivo (Macnamee et al., 1986), it was of interest to examine the efRESULTS fect of VIP and luteinizing hormoneExperiment I: Effect of VIP on PRL and releasing hormone (LHRH) alone and in LH secretion by anterior pituitary cells decombination on LH secretion. MATERIALS
AND METHODS
Hatchmate turkeys of the large white female line (Nicholas Turkey Farms, Sonoma, CA) were housed together to obtain groups required for each experiment (El Halawani et al., 1988b). The reproductive groups utilized in this study were (1) laying turkeys that had laid regularly up to the date of the experiment and showed one or two nest visits per day; (2) incubating birds that were on the nest six or seven times each day for at least 2 weeks without producing eggs; (3) photorefractory birds that had neither laid nor nested despite the 15L:9D lighting schedule and had hard, tight pubic bones and dry cloaca that are associated with ovarian quiescence; and (4) quiescent (nonphotostimulated) hens that were maintained under a 6L:18D lighting regime for at least 8 weeks. In Experiment 1, a balanced incomplete block design was used to compare pituitaries of hens from different reproductive stages. Comparisons were made between laying (n = 7) and incubating (n = 7), laying (n = 7) and photorefractory (n = 7), and incubating (n = 7) and photorefractory (n = 7) hens on 3 different days between hens of the same flock. Anterior pituitary cells were incubated for 3 hr in the presence of different dilutions (10-l’ to 10M6 M) of porcine VIP (PVIP) or with lo-’ M VIP at l-, 2-, 3-, 4-, and 5-hr increments of incubation. In Experiment 2, comparisons were made between laying hens (n = 7) and their semen-producing male hatchmates (n = 8). In Experiment 3, anterior pituitary cells obtained from quiescent, laying, and incubating hens and from semen-producing males (n = 7/group) were cultured with lo-’ M VIP, lo-* M LHRH, or a combination of the two. Cell cuhre. Dissociated anterior pituitary cells from the above turkeys were obtained using the procedure of Fehrer et al. (1985). The cells were incubated in glass culture tubes (four tubes/treatment). At the conclusion of each incubation period, the cell cultures were centrifuged and the supematant was collected and stored at - 20” until assayed. Radioimmunoassays for PRL and LH were conducted according to the methods of Proudman and Opel (1981) and Burke et al. (1979), respectively. Sratistical analysis. All data were analyzed by one-
rived from laying, incubating, and photorefractory turkeys. During the 3-hr incubation
period, the highest spontaneous secretion of PRL was by anterior pituitary cells from incubating hens (0.89 k 0.07 pg; Fig. 1) followed by cells from laying hens (0.32 + 0.02 kg) and those from photorefractory hens (0.17 + 0.01 pg). The VIP-stimulated PRL secretion was increased in a dose-related manner (P < O.OOOl),with maximal PRL secretion occurring at 10e8 A4 VIP. As with the spontaneous PRL secretion (Fig. l), VIP induced the highest PRL release in
7 7
,/p-.
I
J
z t 4
3.
g
2.
1.
o-
) 0
12
10 VIP (-log
6
6
MI
FIG. 1. Effect of increasing concentrations of porcine vasoactive intestinal peptide (VIP) on prolactin secretion by cultured pituitary cells from laying (solid line), incubating (large broken lines), and photorefractory (small broken lines) hens. Data points represent means + SEM of 8 to 10 determinations. In several cases, the SEM is within the data point drawn in the figure.
140
EL HALAWANI,
SILSBY, AND MAURO
cells from incubating hens (7.31 + 0.46 pg) as compared to cells from laying (3.17 -+ 0.37 Fg) and photorefractory (1.21 + 0.12 pg) hens. These responses to VIP represent 8.2-, 9.9-, and 7.1-fold increases in PRL by cells from incubating, laying, and photorefractory birds, respectively. The content of PRL in the medium increased (P < 0.0001) in a time-dependent manner regardless of the pituitary cell origin or the type of treatment except the spontaneous PRL secretion from cells of photorefractory hens (P = 0.1558; Fig. 2). Anterior pituitary cells from incubating hens had greater spontaneous and VIP (10e8 m-stimulated PRL secretion (P < 0.05) than cells from laying hens and these PRL releases by cells from laying hens were greater (P < 0.05) than those of pituitary cells from photorefractory hens at each incubation time. Anterior pituitary cells from laying hens SPONTANEOUS
had a greater (P < 0.05) spontaneous LH secretion than cells from incubating or photorefractory hens (Fig. 3). The presence of VIP (IO-* M) induced an increase (P < 0.05) in LH secretion from pituitary cells of incubating and photorefractory hens but not from cells of laying hens (P > 0.05) during the 5 hr of incubation. Experiment 2: Effect of VIP on PRL and LH secretion by anterior pituitary cells derived from semen-producing male and laying female turkeys. Anterior pituitary cells
from the males exhibited higher spontaneous and VIP (lo-* m-induced LH secretion than cells from laying hens (Fig. 4). After a 5-hr incubation, LH increased (P < 0.05) from the spontaneous value of 14.1 + 1.1 to 21.2 + 1.2 ng following VIP treatment of cells from laying hens. The corresponding values from male pituitary cells were 39.0 k 1.5 and 61.2 + 2.0 ng, respectively. Spontaneous and VIP-induced PRL secreVIP STIMULATED
SECRETION
INCUBATION
TIME
SECRETION
(hr)
FIG. 2. Spontaneous and porcine vasoactive intestinal peptide (lo-* m-induced prolactin secretion by pituitary cells from laying, incubating, and photorefractory hens cultured for varying lengths of time; otherwise as in Fig. 1.
VIP
35
AND
SPONTANEOUS
PRL
141
SECRETION VIP STIMULATED
SECRETION
SECRETION
30
25 3 8 8 8
20
z a 5
15
5
10
I
1
2
3
4
5
INCUBATION
2
1 TIME
3
4
5
(hr)
FIG. 3. Spontaneous and porcine vasoactive intestinal peptide (lOma M)-induced luteinizing hormone secretion by pituitary cells from laying, incubating, and photorefractory hens cultured for varying lengths of time; otherwise as in Fig. 1.
tion exhibited a similar pattern with the greatest response seen with cells from laying hens. Experiment 3: Effect of VIP (IO-* M), LHRH (IO-* M), or their combinations on PRL and LH secretions by anterior pituitary cells derived from nonphotostimulated, laying, and incubating hens andfrom semen-producing male turkeys. As in Experiment 1, anterior pituitary cells derived from incubating hens exhibited both greater (P < 0.05) spontaneous and VIP-stimulated PRL secretion than cells from laying hens. In Experiment 2, spontaneous and VIPstimulated PRL secretion by pituitary cells from laying hens was greater (P < 0.05) than those of pituitary cells from the males (Fig. 5). Anterior pituitary cells from quiescent hens had less VIP-stimulated PRL secretion (P < 0.05) compared to that of cells from any of the other groups of birds. No effect of LHRH was observed on PRL secretion. Coincubation of anterior pituitary cells with both LHRH and VIP at lo-* M each enhanced LH secretion compared to cultures exposed to either LHRH or VIP.
However, only pituitary cells from males responded to the coincubation with VIP and LHRH with greater net LH secretion (161.7 + 10.6 ng) than the combined net response to either peptide alone (VIP, 22.3 + 3.8 ng; LHRH, 76.5 ? 5.9 ng). Anterior pituitary cells from male turkeys had greater LHRH plus VIP-stimulated LH secretion (245.8 + 10.6 ng; P < 0.05) than cells from quiescent hens (138.9 2 7.6 ng) which in turn had greater LH secretions (P < 0.05) than cells from incubating (74.6 2 8.1 ng) or laying (60.0 2 5.4 ng) hens. DISCUSSION These results clearly show that VIP increases PRL secretion from anterior pituitary cells in vitro. The effect at all VIP concentrations is greatest on cells from incubating hens, followed in descending order by cell populations from laying, photorefractory, and nonphotostimulated hens. Whether this increase in the PRL-releasing capacity observed in the pituitary cell population of incubating hens results from an
142
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_
1
-
0
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c____c__ 1
HALAWANI,
SILSBY,
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-C----C----* 2
INCUBATION
3
4
TIME
5
(hr)
FIG. 4. Spontaneous secretion of luteinizing hormone and prolactin by pituitary cells from laying female (solid line) and male turkeys (large broken lines) as well as the porcine vasoactive intestinal peptide (lo-* m-stimulated secretion of luteinizing hormone and prolactin (females, dotted large broken lines; males, small broken lines). Data points represent means -+ SEM of four to five determinations.
increase in the number of lactotrophs per pituitary, an increase in PRL production per lactotroph, or a combination of both remains to be determined. The observed in vitro PRL secretion at various reproductive states reflects the circulating levels of PRL at these states. Cells from incubating hens had the greatest secretion of PRL and their circulating PRL levels were also the greatest. Laying hens had lower circulating PRL levels than incubating hens, but had greater PRL levels than photorefractory or quiescent hens. Accordingly, anterior pituitary cells from laying hens had a smaller spontaneous and VIP-stimulated PRL secretion than such cells from incubating hens. In addition, pi-
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MAURO
tuitary cells from laying hens had greater spontaneous as well as VIP-stimulated PRL secretion than either photorefractory or quiescent hens. Similar results were obtained utilizing hypothalamic extract as a secretagogue of PRL (El Halawani et al., 1988b). On the other hand, VIP (10e7 M) did not effect PRL release when incubated with individual anterior pituitary glands from laying bantam hens in vitro (Macnamee et al., 1986). In a separate experiment, however, VIP was stimulatory to PRL secretion by anterior pituitary glands from incubating bantams (Macnamee et al., 1986). We propose that both hypothalamic inputs and the anterior pituitary itself may each contribute to the changes in circulating PRL levels observed in the various states of the reproductive cycle of the female turkey. VIP may be a prolactinreleasing neuropeptide in the turkey since hypothalamic VIP immunoneutralization and pituitary VIP receptor blockade significantly depressed the PRL response to hypothalamic extract in vitro (El Halawani er al., 1990). Moreover, changes in the number of VIP-like immunoreactive cells in the infundibular nuclear complex (INF) are correlated with the alterations in circulating PRL levels during the reproductive cycle of the turkey (Mauro et al., 1988). The increase in circulating PRL during incubation was accompanied by a dramatic increase in VIP-IR cells, whereas disruption of incubation behavior results in a decline in both circulating PRL levels and the number of VIP-IR cells (Mauro et al., 1989). Thus, the natural increases in circulating PRL during photostimulation, egg laying, and incubation may reflect changes in the synthesis and release of VIP and/or in anterior pituitary responsiveness to VIP. In the present study, pVIP induced approximately a 1.5fold increase in LH secretion from cultured anterior pituitary cells. Also, pVIP enhanced LHRH-induced LH secretion. Similarly, when in vitro cul-
VIP
AND
PRL
143
SECRETION
20. 3 z
16 -
8 3::
12.
‘a 3 I
6.
5 5 0 E
4. 2 1 30X
i
1 CELLS
ALONE I
LHRH
QUIESCENT
INCUBATING
LAYING REPRODUCTIVE
MALE
STATUS
FIG. 5. Effects of vasoactive intestinal peptide (lo-* AI), luteinizing hormone-releasing hormone (lo-* M), or their combinations on prolactin and luteinizing hormone secretions by cultured pituitary cells from quiescent (nonphotostimulated), laying, and incubating female turkeys and from male turkeys; otherwise as in Fig. 4.
ture of anterior pituitary cells from laying turkey hens was utilized, pVIP stimulated LH secretion (Fehrer et al., 1987). Macnamee et al. (1986) also observed an increase in circulating LH with VIP administration to bantam chickens. In the rat, intraventricular administration of VIP stimulated LH secretion (Vijayan et al., 1979), and VIP induced GnRH secretion in vitro by synaptosomes from median eminence tissue (Samson et al., 1981). In contrast to the latter results, Alexander et al. (1985) reported an inhibitory effect of intra-
ventricularly administered VIP on LH secretion. The physiological relevance of VIP in LH secretory regulation remains speculative, at best. The INF comprises a region of the brain containing high concentrations of neurons exhibiting VIP-like immunoreactivity (Mauro er al., 1988, 1989). Since the INF constitutes a link to the photoregulation of LH secretion and gonadal recrudescence by the presence of photoreceptors (Oliver and Bayle, 1976; Oliver et al., 1977; Ohta and Homma, 1987), it is possible that
144
EL HALAWANI,
SILSBY,
VIP may modulate photoperiodically induced gonadotropin secretion. The appearance of VIP-IR neurons in the INF following photostimulation (Mauro et al., 1989) and the colocalization of an opsin-like compound with VIP in the INF neurons (Silver et al., 1988) support such a hypothesis. REFERENCES Alexander, M. J., Cliion, D. K., and Steiner, R. A. (1985). Vasoactive intestinal polypeptide effects a central inhibition of pulsatile luteinizing hormone secretion in ovariectomized rats. Endocrinology 117, 2134-2139.
Burke, W. H., Licht, P., Papkoff, H., and Bona Gallo, A. (1979). Isolation and characterization of luteinizing hormone and follicle-stimulating hormone from pituitary glands of the turkey (Meleagris gallopavo).
Gen.
Comp.
Endocrinol.
37, 508-520.
El Halawani, M. E., and Burke, W. H. (1976). Brain monoamine metabolism of turkey hens in various stages of their reproductive life cycle. Biol. Reprod. 15, 254-259. El Halawani, M. E., Burke, W. H., and Dennison, P. T. (1980). Effects of p-chlorophenylalanine on the rise in serum prolactin associated with nesting in broody turkeys. Biol. Reprod. 23, 815-819. El Halawani, M. E., Fehrer, S. C., Hargis, B. M., and Porter, T E. (1988a). Incubation behavior in the domestic turkey: Physiological correlates. CRC Crit.
Rev.
Poult.
Biol.
l(4),
285-314.
El Halawani, M. E., Silsby, J. L., and Fehrer, S. C. (1988b). Basal and hypothalamic extract-induced luteinizing hormone and prolactin secretion by cultured anterior pituitary cells from female turkeys in various stages of the reproductive cycle. Gen.
Comp.
Endocrinol.
71, 45-54.
El Haiawani, M. E., Silsby, J. L., and Mauro, L. J. (1990). Vasoactive intestinal peptide is a hypothalamic prolactin-releasing neuropeptide in the turkey (Meleagris gallopavo). Gen. Comp. Endocrinol.
78, 66-73.
Fehrer, S. C., &by, J. L., Behnke, E. J., and El Halawani, M. E. (1985). Hypothalamic and serum factors influence on prolactin and luteinizing hormone release by the pituitary gland of the young turkey (Meleagris gallopavo). Gen. Comp. Endocrinol. 59, 73-81. Fehrer, S. C., Silsby, J. L., and El Halawani, M. E. (1987). Capacity of various neuropeptides to induce prolactin (PRL) or luteinizing hormone (LH) release by dissociated turkey hen anterior pituitary cells. Poult. Sci. 66, Suppl. 1, 98.
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Hargis, B. M., and Burke, W. H. (1984). Prolactin and luteinizing hormone levels of prelaying, laying and postlaying turkey hens following central administration of serotonin and peripheral administration of quipazine maleate. Gen. Comp. Endocrinol. 55, 12-19. Helwig, J. T., and Council, K. A. (Eds.) (1979). “SAS User’s Guide.” SAS Institute, Cary, NC. Knapp, T. R., Fehrer, S. C., Silsby, J. L., Porter, T. E., Behnke, E. J., and El Halawani, M. E. (1988). Gonadal steroid modulation of basal and vasoactive intestinal polypeptide-stimulated prolactin release by turkey anterior pituitary cells. Gen.
Comp.
Endocrinol.
72,226-236.
Macnamee, M. C., Sharp, P. J., Lea, R. W., Sterling, R. J., and Harvey, S. (1986). Evidence that vasoactive intestinal polypeptide is a physiological prolactin-releasing factor in the bantam hen. Gen. Comp.
Endocrinol.
62, 470-478.
Mauro, L. J., Elde, R. P., and El Halawani, M. E. (1988). Evidence of a differential expression of hypothalamic VIP immunoreactivity (VIP-IR) associated with prolactin (PRL) levels in the domestic turkey hen. Endocrinology 122, Suppl. 1, 133. Mauro, L. J., Elde, R. P., Youngren, 0. M., Phillips, R. E., and El Halawani, M. E. (1989). Alterations in hypothalamic vasoactive intestinal peptide-like immunoreactivity are associated with reproduction and prolactin release in the female turkey. Endocrinology
125, 1795-1804.
Ohta, M., and Homma, K. (1987). Detection of neural connection to the infundibular complex by partial or complete hypothalamic deafferentation in male quail. Gen. Comp. Endocrinol. 68, 286-292. Oliver, J., and Bayle, J. D. (1976). The involvement of the preoptic-suprachiasmatic region in the photosexual reflex in quail: Effects of selective lesions and photostimulation. J. Physiol. (Paris) 72, 627637.
Oliver, J., Herbute, S., and Bayle, J. D. (1977). Testicular response to photostimulation by radioluminous implant in deafferented hypothalmus of quail. J. Physiol. (Paris) 73, 685-691. Opel, H., and Proudman, J. A. (1988). Stimulation of prolactin release in turkeys by vasoactive intestinal peptide. Proc. Sot. Exp. Biol. Med. 187,455460.
Proudman, J. A., and Opel, H. (1981). Turkey prolactin: Validation of a radioimmunoassay and measurement of changes associated with broodiness. Biol.
Reprod.
25, 573-580.
Proudman, J. A., and Opel, H. (1988). Stimulation of prolactin secretion from turkey anterior pituitary cells in culture. Proc. Sot. Exp. Biol. Med. 187, 448-454.
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Samson, W. K., Burton, K. P., Reeves, J. P., and McCann, S. M. (1981). Vasoactive intestinal peptide stimulates luteinizing hormone-releasing hormone release from median eminence synaptosomes. Regul. Pept. 2, 253. Silver, R., Witkovsky, P., Horvath, P., Alones, V., Bamstable, C. J., and Lehman, M. N. (1988). Coexpression of opsin- and VIP-like immunoreactiv-
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ity in CSF-contacting neurons of the avian brain. Cell Tissue Res. 253, 189-198. Vijayan, E., Samson, W. K., Said, S. I., and McCann, S. M. (1979). Vasoactive intestinal peptide: Evidence for a hypothalamic site of action to release growth hormone, luteinizing hormone, and prolactin in conscious ovariectomized rats. Endocrinology 111, 2055.
