Role of the Dopaminergic System in Luteinizing Hormone Release and Ovulation in the Hen1

Role of the Dopaminergic System in Luteinizing Hormone Release and Ovulation in the Hen1 PATRICIA A. JOHNSON, A. VAN TIENHOVEN, and J. FRIEDLANDER Department of Poultry and Avian Sciences, Cornell University, Ithaca, New York 14853 (Received for publication March 25, 1981)

1981 Poultry Science 60:2551-2556

INTRODUCTION Fuxe and Ljunggren (1965) first demonstrated a monoaminergic system in the upper brain stem of pigeons by histochemical methods. Several groups of investigators subsequently examined the role of the monoaminergic system relative to reproduction in a variety of avian species. Graber and Nalbandov (1972), measuring the hypothalamic content of norepinephrine (NE) and epinephrine (E) in the chicken under various endocrine conditions, found that when gonadotropin levels were elevated, as in castration, the NE content of the ventral hypothalamus was significandy increased over those values obtained when gonadotropin levels were low. Campbell and Wolfson (1974) reported a correlation in Japanese quail between an increased medial-basal hypothalamic NE content and an increased concentration of hypothalamic lutenizing hormone releasing factor (LRF). El Halawani and Burke (1975) concluded that NE was involved in photoperiodically stimulated testicular growth in quail. Additionally, in turkey hens, increases in brain NE and E were found associa-

1

Supported in part by NIH Grant HD12101.

ted with photoperiodically induced oviduct growth, and increased brain dopamine (DA) synthesis was associated with gonadal regression (El Halawani and Burke, 1976). Finally, increased serum concentrations of LH paralleled increases in brain NE and E in Japanese quail with recrudescing testes, and increases of DA content of the brain inhibited luteinizing hormone (LH) release and induced testicular regression (El Halawani et al., 1980a). Previous investigations in this laboratory (Wiseman, 1975) have examined the effect of exogenous DA and NE on ovulation in the chicken. Intraventricular injection of 10 /ig DA, administered approximately 14 hr prior to the first (C 1 ) ovulation of the sequence, blocked ovulation in 77.8% of the hens injected, whereas 10 jig of NE blocked in only 25%. At the higher dose of 20 /ig, DA blocked ovulation with an incidence of 55.6%, whereas NE blocked with an incidence of 57.1%. Hibbs et al. (1977) reported that intraventricular injections of DA at dosages above .1 /ig (that is, 1 to 20 /ig) in immature pullets caused a significant depression of plasma LH which was maximal 15 min after injection. The two experiments to be described in this paper were undertaken to examine further the role of the dopaminergic system in the ovulatory cycle of the hen.

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ABSTRACT Two experiments were conducted to examine further the role of the dopaminergic system in the ovulatory cycle of the hen. In Experiment 1, dopamine (DA; 8 Mg) was injected intraventricular^ 14 hr prior to the C, ovulation (to determine the efficacy of DA in causing premature ovulation) and the DA-receptor blocker, pimozide, implanted intraventicularly for the previous 24 hr was used to assess the specificity of the response. The luteinizing hormone (LH) radioimmunoassays from serial blood samples subsequent to the injection, indicated no effect of the DA treatment on either the time or the magnitude of the preovulatory LH surge. In Experiment 2, an intraventricular injection of DA (10 tig) was made 8 hr prior to the C1 ovulation (to examine the effect of DA on the normal preovulatory LH surge) and assay results from blood samples at short intervals immediately after the injection showed no effect of the treatment on plasma LH levels. These results, in addition to the finding that the pimozide implant caused no disruption of the ovulatory cycle, suggest that the dopaminergic system is not essential to the ovulatory process in the hen. (Key words: dopamine, ovulation, LH)

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JOHNSON ET AL. MATERIALS AND METHODS

Experiment

1. A 2 x 2 factorial experiment

TABLE 1. Effects of a pimozide implant in the third ventricle for 24 hr and/or an intraventricular injection of 8 txg dopamine (DA) on LH release and ovulation in White Leghorn hens (n = 6 hens for each treatment; all hens ovulated at the normally expected time) Treatment Implant

Injection (5 Ml)

Height of LH peak ± SEM (ng/ml)

Time c)f LH peak ± SEM1 (hr)

Cocoa butter Pimozide Pimozide Cocoa butter

Water 8MgDA Water 8MgDA

8.01 ± 8.49 ± 8.07 ± 8.18+

22:15 22:15 21:55 22:00

.68 .58 .69 1.39

± ± ± ±

.43 .39 .38 .38

Time of LH peak

Height of LH peak Source

df

F

Source

df

F

Analysis of variance Total Hens Treatment Error

23 5 3 15

5.02'* .11 NS .267*

Total Hens Treatment Error

23 5 3 15

3.05* .29 NS 2.47 2

1

Blood samples taken at 30 min intervals between 2015 to 2345 hr. Lights off 1800 to 0400 hr.

2

Value for error mean square.

*P<.05. **P<.01. NS = P>.05.

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Single Comb White Leghorn hens of the Cornell University C strain (Cole and Hutt, 1973) were used in their first year of production. Their sequence length was between 2 and 6 days with 1 day between sequences. The lighting schedule was 14L:10D (lights off 1800 to 0400 hr), and food and water were freely available. A chronic 22 gauge stainless steel guide cannula was stereotaxically implanted into the third ventricle of the hens (A 7.0, H +3.0, L 0) according to the procedure described by Scott and van Tienhoven (1974) and a 30 gauge cleanout wire was placed in the guide cannula to keep it patent. Birds were allowed to complete several regular sequences before they were used in an experiment. At the end of each experiment the hens were killed by an overdose of chloropent (Fort Dodge Laboratories, Fort Dodge, IA), and the heads perfused first with 1% saline and then 10% buffered formalin. After further fixation in formalin, the heads were placed in the stereotaxic instrument and dissected to determine whether the tip of the cannula was indeed in the third ventricle, and in all cases, it was.

was carried out in a semirandomized complete block design with 6 hens per treatment such that each hen received all four treatments. A specific DA receptor blocker, pimozide (Janssen Pharmaceutica, Beerse, Belgium), was mixed with cocoa butter (2 parts pimozide: 1 part cocoa butter) and implanted into the third ventricle to block endogenous DA receptors; cocoa butter implanted in the third ventricle was the control. Ten micrograms of DA-HCl (8 fig of free catecholamine) was the experimental intraventricular injection and distilled water was the vehicle control. Treatment A consisted of an intraventricular implant of cocoa butter followed 24 hr later by an intraventricular injection of distilled water. In treatment B, a pimozide implant was followed 24 hr later by an 8 fig DA injection. In treatment C, a pimozide implant was followed by an injection of distilled water, and treatment D consisted of a cocoa butter implant followed by an 8 fig DA injection. The fourth treatment was tested last in all cases as it was anticipated that the DA injection would cause the birds to go out of production following treatment (Wiseman, 1975). On the afternoon of the day prior to the last

RESEARCH NOTE

this response (Rowe-Murphy et al., 1980). Blood sampling was accomplished via a brachial vein cannula implanted at least several hours prior to bleeding (Johnson, 1981). Serial sampling (3 ml blood per sample with red blood cell [RBC] replacement at alternate sampling intervals) at 30 min intervals was done near a green light between 2015 and 2345 hr during the night following oviposition of the terminal egg of the sequence. This period of blood sampling began approximately 2 hr after the onset of darkness, as we had previously determined that it was likely to include the preovulatory LH surge of the Cj ovulation. The time of ovulation was determined by digital palpation on the following morning by an observer uninformed of the treatment. The LH was assayed by a homologous chicken LH radioimmunoassay as previously described (Johnson and van Tienhoven, 1980a). Experiment 2. Similar treatment and preparation of the animals were carried out as described in Experiment 1. In this experiment, a control and an experimental treatment were administered to each of 5 hens with random assignment to the first treatment. Intraventricular injection was made at 2000 hr (2 hr after lights off) on the terminal day of the sequence.

£ 01

c -3 I

• Control n:5 XDopamine 10ug n:5 0

15

30

45

60 minutes after injection

FIG. 1. The LH response to intraventricular injection of 10 Mg DA or vehicle in White Leghorn hens (n = 5). Vertical bars indicate standard error.

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day of the sequence, the hen was implanted intraventricular^ with either cocoa butter or pimozide. The implant consisted of a 30 gauge stainless steel tube packed at one end with cocoa butter or pimozide/cocoa butter (2:1 by weight) and placed within the chronic third ventricle guide cannula. At the same time on the following day (that is, on the last day of the sequence, approximately 14 hr prior to the first ovulation of the next sequence), the implant was removed and the hen received a 5 | i l intraventricular injection of either distilled water or 8 Aig of DA (as free catecholamine) dissolved in distilled water. All solutions were made immediately before injection and kept on ice until drawn into the syringe. Injections were made over a one minute period and the cleanout wire and cannula cap were subsequently replaced. The potency of both the DA and pimozide, as administered in these experiments, was tested in a preliminary trial according to previously published reports (Scott and van Tienhoven, 1974; Rowe-Murphy et al, 1980). In agreement with findings by Scott and van Tienhoven (1974), administration of the DA caused a depression of body temperature while intraventricular implantation of pimozide prevented

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JOHNSON ET AL.

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RESULTS AND DISCUSSION In Experiment 1 all hens ovulated normally. Table 1 shows the mean ± SEM of the height and the time of the preovulatory LH peak for

all treatments. Analysis of variance showed no effect of the treatments on either the height or the time of the preovulatory LH peak. However, significant differences were found among values for individual hens within treatments. Results from Experiment 2 revealed that 5 of 5 of the DA-treated hens ovulated at the normal time while 3 out of 5 of the control hens ovulated at the expected time. Two of the control hens showed blocked ovulation. One of these was assumed stressed since the brachial vein cannula had to be replaced during the sampling. The other blocked ovulation remains unexplained; however, it is known that occasionally a hen will have an interclutch interval of 2 days. Examination of the LH profile after the two treatments (Fig. 1) reveals no significant differences among the responses of the 5 birds for the 2 treatments. A paired t test was used for comparison of LH values between the baseline bleeding and 15 min after injection from the control- and the DA-treated hens. In contrast to the findings of Hibbs et al. (1977), no significant depression of LH was found after DA treatment. The slight decline in the LH values of both DA and control treatments during the first hour is attributed to the

• Control XDopamine

15

30

45

90

120

minutes after

150

180

n:3 10ug

n:3

270

injection

FIG. 2. The LH response to intraventricular injection of 10 ng DA or vehicle for those White Leghorn hens which ovulated normally on the following day (n = 3). Vertical bars indicate standard error.

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A 5 Ml quantity of either DA-HC1 (10 jig of free catecholamine) in saline or pH adjusted saline (pH = 5.8) was administered. Immediately before injection, a blood sample (1.5 ml) was drawn via a brachial vein cannula to determine baseline values of LH. After injection, serial bleedings (1.5 ml with no RBC replacement) were made at 15 min intervals for the first hour and then at 30 min intervals for 3.5 hr. The time of ovulation was determined on the following morning by digital palpation by an observer uninformed of the treatment, and the plasma was subsequently assayed for LH. Analysis of variance on both the height and the time of the preovulatory LH surge was used to evaluate the results from Experiment 1. A paired t test was used in Experiment 2 for comparison of LH values between the baseline bleeding and 15 min after injection from the control and the treated hens.

RESEARCH NOTE

Although Wiseman's study (1975) showed that intraventricular injection of DA at 10 and 20 ng dosages blocked ovulation in 78% and 56% of cases, respectively, his experiment did not include LH data, and in some cases, intraventricular injections were made immediately following implantation of the cannula, while the bird was anesthetized with Equi-thesin (Jensen Salsbery Laboratories); thus the possibility of a drug interaction cannot be excluded. Injections in the present study were made only after the birds had fully recovered from the stereotaxic surgery and had resumed laying normal sequences. Although our data in no way argue for or against a role of DA in the photoperiodic response or in gonadal regression, results of the two experiments reported herein indicate that intraventricular injection of DA does not acutely alter plasma concentrations of LH when injected 14 and 8 hr prior to the Ci ovulation. This was shown by the lack of a DA effect on the magnitude or time of the preovulatory LH surge and by the lack of an acute effect on basal LH values. These results are in contrast to those reported by Hibbs et al. (1977). Experiments in which we injected 100 Hg of DA in 4 hens at 8 hr prior to the expected Ci ovulation showed no effect of the DA on ovulation in any of the hens. We, therefore,

judge that DA in a wide range of doses has no effect on ovulation of the C t follicle of hens. Finally, the study by Hibbs et al. (1977) was accomplished in immature pullets. The difference in hypothalamic/pituitary sensitivity to exogenous catecholamines between the immature reproductive state and the laying state, as in the present study, has not been determined and may explain the difference in response. In addition, maturational changes in the rate of hypothalamic catecholamine turnover in response to castration have been demonstrated in turkeys (El Halawani et al, 1980b). ACKNOWLEDGMENTS We wish to thank P. A. J. Janssen of Janssen Pharmaceutica, N. V. Berse, Belgium, for the gift of pimozide; F. J. Cunningham for LH assay materials; and A. L. Johnson for his critical review of the manuscript.

REFERENCES Campbell, G. T., and A. Wolfson, 1974. Hypothalamic norepinephrine, luteinizing hormone releasing factor activity and reproduction in the Japanese quail, Cotumix cotumix japonica. Gen. Comp. Endocrinol. 23:302-310. Cole, R. K., and F. B. Hutt, 1973. Selection and heterosis in Cornell white Leghorns: a review, with special consideration of interstrain hybrids. Anim. Breeding Abstr. 41:103—118. El Halawani, M. E., and W. H. Burke, 1975. Role of catecholamines in photoperiodically-induced gonadal development in Cotumix quail. Biol. Reprod. 13:603-609. El Halawani, M. E., and W. H. Burke, 1976. Brain monoamine metabolism of turkey hens in various stages of their reproductive life cycle. Biol. Reprod. 15:254-259. El Halawani, M. E., W. H. Burke, and L. A. Ogren, 1980a. Involvement of catecholaminergic mechanisms in the photoperiodically induced rise in serum luteinizing hormone of Japanese quail (Corturnix cotumix japonica). Gen. Comp. Endocrinol. 41:14—21. El Halawani, M. E., W. H. Burke, and L. A. Ogren, 1980b. Age-dependent changes in hypothalamic catecholamine turnover rate following castration in turkeys. Gen. Comp. Endocrinol. 42:290— 296. Fuxe, K., and L. Ljunggren, 1965. Cellular localization of monoamines in the upper brain stem of the pigeon. J. Comp. Neurol. 125:355-382. Graber, J. W., and A. V. Nalbandov, 1972. Relationship of hypothalamic catecholamines and gonadotropin levels in the chicken. Neuroendocrinology 10:325-337. Hibbs, M., R. T. Gladwell, and F. J. Cunningham, 1977. Effect of the intraventricular administration of dopamine on plasma concentrations of

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stress of frequent sampling which has previously been reported (Wilson and Sharp, 1975; Johnson and van Tienhoven, 1980b). Figure 2 depicts the LH values from the 3 birds which ovulated after both DA and control treatments. These values are plotted as the LH values from the 2 control hens which did not ovulate may have depressed the LH profile in Figure 1 for the controls. In Experiment 1 (Table 1), there was no significant difference in LH values between the DA-treated and the control birds. In Experiment 2 (Fig. 1), examination of the LH profile shows that the average LH values of the DAtreated hens were increased earlier and for a longer time than the average of the control hens. From these results, we suggest that even if data from additional hens were included, a decrease in LH would not be expected. In addition, although DA was tested only at the doses of 8 (Experiment 1) and 10 (Experiment 2) Hg, the fact that the 24 hr implant of pimozide in the third ventricle did not disrupt the ovulatory cycle indicates that the dopaminergic system may not be essential in the ovulatory cycle of the hen.

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JOHNSON ET AL. P. E. Hillman, C. L. Wood, A. L. Johnson, and W. S. Schwark, 1980. Effects of 6-hydroxydopamine and pimozide on temperature maintenance by the chicken. Amer. J. Physiol. 239: R296-R302. Scott, N. R., and A. van Tienhoven, 1974. Biogenic amines and body temperature in the hen Gallus domesticus. Amer. J. Physiol. 227(6): 1 3 9 9 1405. Wilson, S. C , and P. J. Sharp, 1975. Episodic release of luteinizing hormone in the domestic fowl. J. Endocrinol. 64:77-86. Wiseman, F. C , 1975. Effects of the catecholamines, dopamine and norepinephrine, on ovulation when injected into the third ventricle of the fowl (Gallus domesticus). Masters thesis, Cornell University.

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luteinizing hormone in the domestic fowl. J. Endocrinol. 75:43 P. Johnson, A. L., 1981. Comparison of three serial blood sampling techniques on plasma hormone concentrations in the laying hen. Poultry Sci. 60:2322-2327. Johnson, A. L., and A. van Tienhoven, 1980a. Plasma concentrations of six steroids and LH during the ovulatory cycle of the hen, Gallus domesticus. Biol. Reprod. 23:386-393. Johnson, A. L., and A. van Tienhoven. 1980b. Hypothalamo-hypophyseal sensitivity to hormones in the hen. I. Plasma concentrations of LH, progesterone, and testosterone in response to central injections of progesterone and R5020. Biol. Reprod. 23:910-917. Rowe-Murphy, D. L., A. van Tienhoven, N. R. Scott,