Effects of Dopamine, Norepinephrine and Serotonin on Plasma Concentrations of Luteinizing Hormone and Prolactin in Ovariectomized and Anestrous Ewes

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1 BIOLOGY OF REPRODUCTION 27, (1982) Effects of Dopamine, Norepinephrine and Serotonin on Plasma Concentrations of Luteinizing Hormone and Prolactin in Ovariectomized and Anestrous Ewes D. R. DEAVER and R. A. DAILEY Division ofanimal and Veterinary Science West Virginia University Morgantown, West Virginia ABSTRACT Two experiments were conducted in ovariectomized and anestrous ewes to examine for effects of dopamine (DA), norepinephrine (NE) and serotonin (5-FIT) on basal concentrations of luteinizing hormone (LH) and prolactin (PrI) and the gonadotropin-releasing hormone (GnRH)-induced release of LH. In Experiment 1, ovariectomized ewes, during the breeding and anestrous seasons, were infused for 6 h intravenously with DA (0.66, 6.6 or 66 pg/kg per mm), NE or 5-HT (each at doses of 0.06, 0.66 or 6.6 pg/kg per mm). Effects of the biogenic amines on tonic concentrations and GnRH-induced release of LH were not different between seasons. Prior to GnRH, mean concentrations of LH in plasma were affected by DA, NE and 5-HT in a dose-dependent manner. The high dose of DA and NE decreased mean concentrations of LH, while the high dose of 5-FIT increased mean concentrations of LH during the first 2 h of infusion. Mean concentrations of LH in the 4 h following administration of CnRH were related to mean concentrations of LH prior to GnRH. After adjustment by analysis of covariance, there was a significant effect of the interaction of biogenic amine X dose on mean concentrations of LH following administration of CnRH. Dose-response patterns of LH to administration of GnRFI following infusion of biogenic arnines were similar to those observed prior to treatment with GnRH. While 5-FIT did not affect concentrations of PrI, DA and NE reduced concentrations of PrI. Dopamine was more effective in inhibiting secretion of PrI in fall than in spring. Changes in PrI and LH were not correlated. In Experiment 2, anestrous ewes were infused intravenously with DA (0.66 and 66 pg/kg per mm), NE or 5-HT (each at doses of 0.06 and 6.6 pg/kg per mm). Concentrations of LH were uniformly low prior to administration of CnRH. Following treatment with GnRH, NE inhibited and 5-HT potentiated the release of LH (P<0.01), while DA had no effect. Concentrations of PrI in plasma were inhibited by DA and NE in a dose-related manner (P<0.01). Intravenous infusion of 5-HT did not affect Prl. Again, there was no apparent relationship between changes in LH and PrI. INTRODUCTION The biogenic amines, dopamine (DA), norepinephrine (NE) and serotonin (5-HT) have been implicated in the neuroendocrine regulation of luteinizing hormone (LH) in sheep. Exact roles that these putative neurotransmitters play in controlling release of LH from the pituitary remain unclear. For example, intraventricular infusion of NE has been reported to inhibit secretion of LH in wethers (Riggs and Malven, 1974) but to enhance secretion of LH in anestrous ewes (Przekop et al., 1975). In support of the hypothesis that NE stimulates secretion of LH in ewes, Jackson Accepted May 6, Received December 10, l Reprint requests. (1977) reported that phenoxybenzamine (an a-adrenegic blocker), when given intramuscularly, depressed concentrations of LH in plasma. Similar controversy exists in regard to the effect of DA on secretion of LH. Intraventricular infusion of DA was reported to have no effect on secretion of LH in wethers (Riggs and Malven, 1974) and anestrous ewes (Przekop et al., 1975). In contrast, Jackson (1977) reported that the intramuscular injection of pimozide, a dopamine receptor antagonist, inhibited tonic and estradiol-induced secretion of LH in ovariectomized ewes. Recently Hill et a!. (1980) have reported that intramuscular injection of bromocryptine, a DA agonist, decreased LH in ovariectomized ewes. Based on these data, DA and NE may be involved in both stimulatory and inhibitory 624

2 EFFECTS OF BIOGENIC AMINES ON LH AND Prl IN EWES 625 pathways regulating the secretion of LH. The discrepancies among studies may be accounted for by differential stimulation (or inhibition) of a particular pathway(s) depending on the pharmacological agent, dose, route of administration and/or physiological status of the sheep. Few studies have reported effects of 5-HT on secretion of LH in sheep. Intraventricular administration of 5-I-IT suppressed tonic secretion of LH in wethers (Riggs and Malven, 1976) and blocked the preovulatory surge of LH in cycling ewes (Domanski et a!,, 1975). Concentrations of PrI and LH are correlated negatively during seasonal and lactational anestrus; however, preovulatory surges of both LH and PrI occur during estrus in the ewe. Comparing the effects of biogenic amines on secretion of Pri as well as LH may provide a better understanding of the neuroendocrine mechanisms regulating pituitary function in the ewe. While extensive literature is available pertaining to the effects of biogenic amines on secretion of Pr! in the rat, rhesus monkey and human (Weiner and Ganong, 1978), few studies have been reported for sheep. Bromocryptine (Niswender, 1974) and L-dihydroxyphenylalanine, the precursor of DA and NE, blocked release of Pr! while phenoxybenzamine increased (Davis and Borger, 1973) its release. The main objectives of the two experiments reported in this communication were to determine the effects of intravenously infused DA, NE and 5-HT on tonic secretion of LH and Prl and the GnRH-induced release of LH in ewes. Intravenous infusion of the biogenic amines was used to restrict the target sites within the hypothalamic-pituitary axis to those outside the blood-brain barrier. Since many agonists and antagonists of the biogenic amines cross the blood-brain barrier and may selectively affect different subgroups of biogenic amine receptors, the authentic amines were used in the present experiments. Since season is known to influence secretion of LH and Pr!, the influence of intravenous infusion of biogenic amines on secretion of LH and PrI in ovariectomized ewes was examined during the spring and fall. Ovariectomy eliminated possible interaction between season and ovarian secretion in mediating the effects of the biogenic amines. In the second experiment, effects of the biogenic amines on secretion of LH and Pr! in anestrous ewes were examined. In addition, since Dailey et a!. (1978) described a dose-dependent effect of DA on release of LH following GnRH treatment in rabbits, the effects of different doses of each biogenic amine on secretion of LH and PrI were studied in each experiment. General MATERIALS AND METHODS Mature ewes of mixed breeding were maintained on pasture. Approximately 16 h prior to initiation of the experimental treatments, one polyvinyl catheter was inserted into each jugular vein, flushed with heparinized sterile saline and anchored to the skin using 00 vetafil sutures. Two hours before initiation of infusion of biogenic amines or saline, ewes were placed in head stanchions and allowed access to hay and water. Ewes were infused (0.2 mi/mm) intravenously, via one jugular catheter, with one dose of one of the biogenic amines (described below) for a 6-h period. Two hours after initiation of the infusion, ewes were injected (intramuscularly) with 25 pg synthetic gonadotropin-releasing hormone (GnRH). A single 25 pg dose would be expected to induce a release of LH without depleting pituitary LH (Rippel et al., 1974). During the 6-h infusion period, 5 ml of blood were taken every 20 min, via the second jugular catheter, and placed into heparmnized tubes. Plasma was harvested and stored at -20#{176}C until assayed for LH (Fogwell et al., 1977) and PrI. Sensitivity of the LH assay was 50 pg/tube (NIH-S19). Samples prior to administration of GnRH were assayed in 100 p1, while samples following GnRH were assayed in 10 p1. Interand intra-assay coefficients of variation (CV) were 14 and 8%, respectively. The antiserum used to quantify concentrations of PrI was NIAMDD-anti-oPrl-1. This antiserum has been reported not to cross-react with ovine LI-I, growth hormone, thyroid-stimulating hormone or follicle-stimulating hormone (NIAMDD technical report 109 of the Pituitary Hormone and Antisera Center). NIAMDD-oPrl-1 was radiolabeled with 1251 using the method previously described for ovine LH (Fogwell et al., 1977). In this assay, approximately 50% of 1 ng 25I-oPrl-l was bound at a final tube dilution of 1:500,000 (total assay volume 500 p1) when incubated at room temperature for 18 h. Sensitivity of the Prl assay was 250 pg/tube (NIAMDD-oPrl-l-1). Samples collected in the fall were assayed in 100 p1, while samples collected in the spring were assayed at 20 p1. Inter- and intra-assay CV were 13 and 11%, respectively. Experiment I Seventy-two ewes, ovariectomized at least 8 weeks, were assigned randomly to be infused with DA (at doses of 0.66, 6.6 or 66 pg/kg per mm), NE or 5-HT (each at doses of 0.06, 0.66 or 6.6 pg/kg per mm). Ewes infused with these doses of DA, NE or 5-FIT displayed no behavioral changes or outward signs of physical distress. Four ewes in each group were infused when ewes in the intact flock maintained at the same facility were showing normal cyclic estrous behavior (October and November) and four ewes in each group were infused when the intact ewes were anestrous (March). In addition, 12 ewes were infused with saline and treated with 25 pg CnRH during each season.

3 626 DEAVER AND DAILEY Experiment 2 Anestrous ewes were assigned randomly to be infused with DA (at doses of 0.66 or 66 pg/kg per mm), NE or 5-HT (each at doses of 0.06 and 6.6 pg/kg per mm). The experiment was replicated, using a different group of ewes, during the month of May in two consecutive years (n=4 per group in year 1 and n=6 per group in year 2). In addition, 10 ewes were infused with saline and treated with 25 pg GnRH during each year. Seventy-two hours following administration of GnRH, all ewes were anesthetized with sodium pentobarbital and the ovaries were exteriorized via a midventral incision and observed for the presence of corpora Drugs Biogenic amine lutea. Dopamine (3-hydroxytyrammne HCI, H8502), NE (arterenol bitartrate, A9512) and 5-HT (5-hydroxytryptammne, H5755) were purchased from Sigma Chemical Co., St. Louis, MO. Biogenic amines were diluted with sterile 0.9% NaCl to appropriate concentrations immediately prior to infusion. During infusion all solutions were protected from exposure to light and kept in an ice-bath. Statistical Analyses In each experiment data for LI-I were analyzed using the method of least squares for a split-plot design with repeated measures in time (Fogwell et al., 1978). Due to the complexity of including a zero dose level into the analysis of experimental designs with a factorial arrangement of treatment, without a prior knowledge of the expected response curve (Kempthorne, 1967), in Experiment 1 the main effect of dose was determined independently for DA, NE and 5-FIT with saline included as a zero dose. Orthogonal polynomial contrasts were used to partition the sum of squares associated with the effect of dose into linear, quadratic and cubic components. Data were then re-analyzed to examine for interactions among the dose-response patterns of DA, NE and 5-UT by omitting the 0 dose and using the method of leastsquares for a 3 X 3 factorial arrangement of treat- TABLE 1. Effects of infusion of DA, NE and 5-HT on administration of GnRH in ovariectomized ewes.a,e Saline ments. In Experiment 1, concentrations of LH in plasma prior to and following administration of GnRH were analyzed separately. Analysis of covariance (Snedecor and Cochran, 1974) was used to determine if changes in LH during the first 2 h of infusion of biogenic amines were related to the subsequent release of LH following administration of GnRH. The method of least squares was used to analyze for changes in PrI at 0, 60, 120, 180, 240, and 360 mm of the infusion period. Due to the large variation in concentrations of PrI in plasma among ewes, these data were transformed to log10 prior to statistical analysis. The chi-square contingency test was used to examine for differences in frequency of ovulation among treatments in Experiment 2. A nonparametric, rank correlation test was used to determine if responses of LH and PrI to the infusions of biogenic amines were mean concentrations of LU (nglml) in plasma prior to Dose Low Mid High DAb NEC HTd associated. Experiment 1 RESULTS Concentrations of LH in plasma were not affected by season, nor were any season x treatment interactions significant. Therefore the data were pooled over seasons. Mean concentrations of LH during the first 2 h of infusion of biogenic amines are shown in Table 1. There were linear (P0.05) and quadratic (P<0.01) effects of dose of DA on concentrations of LH. The quadratic effect can be accounted for by the elevation of mean LH in ewes infused with the 0.66 pg/kg per mm dose of DA in comparison to mean LH in ewes infused only with saline. In contrast, only the linear effect of dose was significant (P=0.05) for NE with the 6.6 pg/kg per mm dose causing a decrease in mean concentrations of LH. Serotonin also affected mean LH in a doserelated manner with both linear and quadratic asaline N = 20 ewes; biogenic amines N = 8 ewes/dose. blmnear effect P0.05; quadratic effect P<0.05; error mean square = clinear effect P0.05; error mean square = dlinear effect P0.05; quadratic effect P<0.05; error mean square = ectholi linear vs. 5-UT linear P<0.01.

4 EFFECTS OF BIOGENIC AMINES ON LU AND PrI IN EWES 627 effects of dose (P <0.05). The quadratic component can be explained by the slight decrease in concentrations of mean LH in ewes infused with 0.06 and 0.66 pg/kg per mm of 5-HT and the elevated mean LH in ewes infused with 6.6 pg/kg per mm of 5-HT in comparison to mean LH in ewes infused with saline. When omitting the 0 dose and re-analyzing the experiment as a 3 X 3 factorial, there was an interaction (P<0.01) between dose of catecholamine and dose of 5-HT on mean LH. Concentrations of mean LH in plasma prior to and following administration of GnRU were related linearly (P<0.01). Least-squares mean concentrations of LH, adjusted by analysis of covariance for the mean LH prior to administration of GnRH, are shown in Table 2. Following administration of GnRH there were linear (P<0.05) and quadratic (P<O.01) effects of dose of DA. The dose of 0.66 pg/kg per mm of DA enhanced and higher doses of DA inhibited the release of LH following administration of GnRH when compared to the response of ewes infused with saline. Only the linear component of the dose effect was significant (P<O.05) for NE, with the dose of 6.6 pg/kg per mm of NE attenuating release of LH following GnRH. In ewes infused with 5-UT there was quadratic effect of dose (P=0.09). There was an interaction of biogenic amine x dose (P<0.01), accounted for by opposing slopes of the dose response of catecholeamines (DA and NE) and 5-HT. In addition to differences in mean LU, the interval from administration of GnRH to I so Ulnuts. Post GnRH FIG. 1. Effects of infusion of biogenic amines on patterns of LH release in ovariectomized ewes. The low, mid and high doses of each amine are represented by circle, triangle or square, respectively. The biogenic amine X time interaction is significant (P<0.001; error mean square=3868). TABLE 2. Effects of infusion of DA, NE and 5-UT on mean concentrations of LH (ng/ml) in plasma following administration of GnRH in ovariectomized ewes.a,e Biogenic amine DAb NEC 5HTd Saline Dose Low Mid High aleast square mean adjusted by analysis of covariance for mean LH prior to GnRH; N20 ewes; biogenic amines N=8 ewes/dose. blinear effect P<0.05; quadratic effect P<0.01; error mean square=7097. CLinear effect P<0.05; error mean square=6724. dquadratic effect P=O.09; error mean square=6294. cc hi vs. 5-UT linear P<0.01.

5 628 DEAVER AND DAILEY the peak of LH was longer in ewes infused with high doses of DA and NE than in ewes in the other groups (Fig. 1). There was an interaction between season, biogenic amine and dose of biogenic amine (P<0.01) on changes in concentrations of Pr! over time during the 6 h. The percentage changes in loglo Prl over time for all doses of DA and NE during the spring and fall are presented in Fig. 2 to illustrate the season x biogenic amine x dose x h interaction. In the fall, DA decreased Pr! at all three doses; however, the nadir achieved was maintained for the entire 6 h only by the high dose. In the spring, the low dose of DA only slightly depressed Pr!. Infusion of the mid and high doses of DA during the spring reduced Pr! to a greater extent, but these doses of DA were not as effective as during the fall. Norepinephrine decreased concentrations of Prl only at the mid and high doses. The apparent increase in Pr! in ewes infused with the low dose of NE is accounted for by high concentrations of Pr! in one ewe between the second and sixth h of the infusion. Concentrations of Pr! were not influenced by any dose of 5-UT during the spring or fall. Changes in LU and Pr! during the 6 h were not correlated. Experiment 2 Concentrations of LU before GnRU ranged between the sensitivity of the assay (0.5 ng/m!) and 1 ng/m! in all groups of anestrous ewes. In contrast to results in ovariectomized ewes in the first experiment, there was no significant effect of dose of biogenic amine on release of LU following GnRU. Mean concentrations of LH differed (P<0.05) among groups infused with saline, DA, NE or 5-FIT (30.1, 30.6, 21.4, 1 I Hour Inluuion FIG. 2. Effects of season (spring, open circle and fall, closed circle) and infusion of DA and NE on percentage change in log10 PrI in ovariectomized ewes. The season X biogenic amine X time interaction was significant (P<0.0O1; error mean square=0.058). For reference, percentage change in log10 Pr! in ewes (n= 18) infused with saline (dashed line, upper right) are shown. and 40.3 ng/ml, respectively). There was a biogenic amine x time interaction (P<0.01; Fig. 3). Intervals to the peak of the surge of LU were similar among all groups (approx. 120 mm). In ewes infused with saline, the pattern and magnitude of the release of LH were similar to those observed in ewes infused with DA. Ovulation rates as determined by the presence of new corpora lutea at laparotomy 3 days after GnRH were not different among the treatments. Uowever, corpora lutea were not observed in ewes infused with the high dose of TABLE 3. Effects of infusion of DA, NE and 5-UT on proportion of anestrous ewes ovulating in response to GnRH.5 Dose Biogenic amine DA NE 5-HT I. Saline Low 4/9 4/10 5/10 4/18 High 5/9 0/10 3/10 Totals 9/18b 4/20 8/20b 4/18b achi Square=5.67, d.f.=3, P=0.13. bin two ewes in each group the ovaries were adhered and were not examined.

6 EFFECTS OF BIOGENIC AMINES ON LU AND Pri IN EWES I M#{234}nutes Post GnRH FIG. 3. Effects of infusion of biogenic amines on patterns of LH release in anestrous ewes. Dopamine, NE and 5-HT are represented by triangle, square and circle, respectively. The biogenic amine X time interaction was significant (P<0.001; error mean square= 2t.). Fo reference, concentrations of LH following GnRH n ewes (cross) (n=18) infused with saline are shown. NE while the frequency of ovulation ranged from 25-50% in the other groups (Table 3). While there was no main effect of dose of biogenic amines on LH, there was an interaction of biogenic amine x dose x time on concentrations of Pr! (P<0.01; Fig. 4). Both doses of DA and NE decreased concentrations of Pr!. However, Pr! was not suppressed maximally for the entire 6 h at the low dose of either DA or NE. Concentrations of Pr! were not affected by either dose of 5-UT. As in Experiment 1, there was no correlation between the responses of LH and those of Pr! to the biogenic amines. DISCUSSION Effects of biogenic amines on secretion of LU in sheep are difficult to interpret because pharmacological agents, routes of administration and the physiological status of the sheep have varied among experiments. Several key findings from the present studies may clarify discrepancies concerning effects of DA, NE and 5-HT in modulating pituitary release of LU in ewes. These findings include: 1) in ovariectomized ewes a biogenic amine may increase or decrease LH depending on dose; 2) intravenous infusion of biogenic amines can affect pituitary responsiveness to luteinizing hormone releasing hormone (LHRH); 3) dose-related responses of LU to biogenic amines differ between ovariectomized and anestrous ewes; and 4) generally, catecholamines and 5-UT have opposite effects on LU. Because a biogenic amine can inhibit, stimulate or have no effect on secretion of LH, depending on the dose, it is not surprising that conflicting hypotheses have been advanced concerning how biogenic amines might regulate secretion of LU. The mechanisms by which different doses of biogenic amines affect secretion of LU are unclear. One possibility is that several classes of receptors exist with different affinities for purported agonists and antagonists, and activation (or inhibition) of these -J E a 3 Hour of Infusion FIG. 4. Effects of infusions of DA and NE on concentrations of log10 PrI in anestrous ewes. The low and high doses of each biogenic amine are depicted by square and triangle symbols, respectively. The biogenic amine X dose X time interaction was significant (P<0.0O1; error mean square=0.074). For reference, concentrations of log10, PrI in ewes infused with saline are shown (circle).

7 630 DEAVER AND DAILEY receptors might affect secretion of LU differently. For example, Kebabian and CaIne (1979) described different classes of DA receptors which varied in affinities for DA and agonists and antagonists of DA. Thus, the dose-related responses to DA observed in this study and the paradoxical effects of bromocryptine (Hill et al., 1980) and pimozide (Jackson, 1977) on secretion of LU might be mediated through different receptors. Further, since agonists and antagonists penetrate the blood-brain barrier more easily than biogenic amines (O!dendorf, 1971), sites might be activated that have opposing effects on secretion of LU. This is exemplified by the inhibitory effect of phenoxybenzamine (an -adrenergic blocker) reported by Jackson (1977) and the inhibitory effect of NE demonstrated in the present experiments on secretion of LU. Intravenous infusion of DA inhibited LURUinduced release of LH in the human (Leblanc et a!., 1976; Judd et al., 1978) and rabbit (Dailey et a!., 1978). The present experiments provide evidence that DA, NE and 5-UT modify pituitary responsiveness to LHRH in ewes. The mechanism by which exogenous biogenic amines affect the release of LH following administration of LURH is equivocal. Biogenic amines might act directly on the pituitary to increase or decrease responsiveness to LURH. In support of this concept, Dailey et a!. (1978) reported that DA inhibited LHRU-stimulated secretion of LH in stalk-sectioned and intact rabbits. While Martin et a!. (1977) reported that melatonin and 5-UT inhibited LHRU-induced release of LU from neonatal rat pituitaries in vitro, other attempts to show direct effects of biogenic amines have been unsuccessful (Schneider and McCann, 1969; Quijada et a!., 1973). Alternatively, as proposed by Judd et a!. (1978), biogenic amines might affect endogenous release of LURH from tuberoinfundibular neurons, thereby altering the self-priming effect of LHRH (Aiyer et al., 1974). The absence of stimu!atory effects on the low dose of DA and the high dose of 5-UT on tonic secretion of LU in anestrous ewes possibly reflects a failure of these biogenic amines to overcome inhibitory effects of estradiol-1 7j3 (Legan et a!., 1977). Norepinephrine inhibited and 5-UT potentiated LURU-induced release of LH, but no dose-response was seen. The response might be dependent on endogenous turnover rates of NE and 5-UT which might be higher in anestrous ewes than in ovariectomized ewes. This would allow the maxima! response to exogenous NE or 5-HT to be reached at a lower dose. Patterns of LU in plasma following administration of LURH were similar in anestrous ewes and in ovariectomized ewes infused with the high dose of either DA or NE. Anestrous ewes were less sensitive to LUreleasing hypothalamic extracts than cycling ewes (Domanski and Kochman, 1968) and have a lesser frequency of pulses of LH (Jackson and Davis, 1979). Perhaps, increases in turnover rates of catecholamines, NE in particular, may suppress endogenous release of LHRU, thus reducing the amount of releasable LH during anestrus. An interesting finding in these studies is that the slopes of dose-response curves, for both tonic and LHRH-induced release of LH, for DA and NE were opposite to those for 5-HT in ovariectomized ewes. In addition, NE and 5-UT exerted opposite effects on LURH-induced release of LU in anestrous ewes. These observations are consistent with those of Lippmann (1968) and Labhsetwar (1971) who proposed that in the hamster and rat, respectively, the ratio of catecho!amine/5-ht was critical in regulating secretion of LU. Based on the present results, the ratio of catecholamine/5-ht could have an important role in regulating secretion of LU in the ewe. Critical evaluation of the hypotheses presented awaits determination of turnover rates for biogenic amines, concentrations of the amines in hypophysial portal vessels and classes of receptors for biogenic amines located within the hypothalamic-pituitary axis of sheep. High concentrations of DA, NE and 5-UT are located within the stalk medium eminence of sheep (Wheaton et a!., 1975). However, content of biogenic amines in neurona! tissue is often a poor indicator of neuronal activity. Dopamine receptors have been located in the stalk median eminence of sheep (Cronin and Weiner, 1978); however, the class of receptor is unknown and information concerning receptors for other neurotransmitters is unavailable. In sheep, treatment with L-Dopa, a precursor of DA and NE, phenoxybenzamine (Davis and Borger, 1973) or bromocryptine (Niswender, 1974) inhibited secretion of Pr!. In the present studies, Pr! in plasma was decreased in ovariectomized and anestrous ewes by DA and NE. Dopamine was less effective in ovariectomized ewes in the spring than fall. Seasonal changes in Pr! have been reported for

8 EFFECTS OF BIOGENIC AMINES ON LU AND Pri IN EWES 631 the tam (Ravault, 1976) and ewe (Jackson and Davis, 1979). The finding in Experiment 1, that PrI in plasma of ovariectomized ewes was lower in the fall than in the spring, confirms the earlier observations of Parrot and Hi!! (1979) and Munro et a!. (1980) that the gonads of sheep are not required for the occurrence of seasonal changes in PrI. Based on the results of Experiment 1, seasonal changes in concentrations of Pr! in plasma of ovariectomized ewes might result in part from a changed sensitivity of the hypothalamic-pituitary axis to DA. Serotoninergic pathways have been reported to be involved in regulating the release of Pri in the rat, rhesus monkey and human (Weiner and Ganong, 1978). Failure to alter secretion of Pr! with intravenous infusion of 5-UT in the present studies is congruent with a hypothalamic and not pituitary site of action of 5-UT. In summary, intravenous administration of DA, NE and 5-UT altered plasma conceqtrations of LU, associated with tonic and LURHinduced secretion of LU, in ewes. Generally, catecho!amines and 5-UT had opposing dosedependent effects on secretion of LU. Concentrations of Pr! increased during the spring in the absence of the ovaries. Infusion of DA and NE, but not of 5-UT, inhibited secretion of Pr! in ewes. In ovariectomized ewes DA was less effective in the spring than in the fail. ACKNOWLEDGMENTS Published with the approval of the Director of the West Virginia Agricultural and Forestry Experiment Station as Scientific Paper No Supported by Hatch Project 224 (NE-72). The authors are indebted to Dr. R. H. Rippel, Abbott Laboratories, for the generous supply of GnRH, Dr. G. D. Niswender for the ovine LH antiserum (GDN #15), Dr. L. E. Reichert, Jr. for the purified ovine LH and the National Institute of Arthritis, Metabolism and Digestive Diseases for the ovine prolactin immunoreagents. We are grateful to Dr. W. V. Thayne for assistance with statistical analyses. REFERENCES Aiyer, M. S., Chiappa, S. A. and Fink, G. (1974). A priming effect of luteinizing hormone releasing factor on the anterior pituitary gland in the female rat. J. Endocrinol. 62: Cronin, M. J. and Weiner, R. I. (1978). Dopammnergic binding in neuroendocrine axis of the sheep. Fed. Proc. 37:226. Dailey, R. A., Tsou, R. C., Tindall, G. T. and Neill, J. D. (1978). Direct hypophysial inhibition of lutemnizing hormone release by dopamine in the rabbit. Life Sci. 22: Davis, S. L. and Borger, M. L. (1973). Hypothalamic catecholamine effects on plasma levels of prolactin and growth hormone in sheep. Endocrinology 92: Domanski, E. and Kochman, K. (1968). Induction of ovulation in sheep by intraadenohypophysial infusion of hypothalamic extracts. J. Endocrinol. 42: Domanski, E., Prezekop, F., Skubiszewski, B. and Wolinska, E. (1975). The effect and site of action of indoleamines on the hypothalamic centers involved in the control of LU release and ovulation in sheep. Neuroendocrmnology 17: Fogwell, R. L., Lewis, C. S., Butcher, R. L. and Inskeep, E. K. (1977). Effects of ovarian bisection on response to intrafollicular injection of PG F2a and on foilicular development in ewes. J. Anim. Sci. 45: Fogwell, R. L., Weems, C. W., Lewis, G. S., Butcher, R. L. and Inskeep, E. K. (1978). Secretion of steroids after induced luteal regression in beef heifers: Effects of PGF2a and removal of corpora lutea. J. Anim. Sci. 46: Hill, T. C., Alliston, C. W. and Malven, P. V. (1980). Plasma luteinizing hormone and prolactin in normothermic and hyperthermic ovariectomized ewes. Life Sci. 26: Jackson, G. L. (1977). Effect of adrenergic blocking drugs on secretion of tuteinizing hormone in the ovariectomized ewe. Biol. Reprod. 16: Jackson, G. L. and Davis, S. L. (1979). Comparison of luteinizing hormone and prolactin levels in cycling and anestrous ewes. Neuroendocrinoiogy 28: Judd, S. J., Rakoff, J. S. and Yen, S.S.C. (1978). Inhibition of gonadotropin and prolactin release by dopamine: Effect of endogenous estradiol levels. J. Clin. Endocrinol. Metab. 47: Kebabian, J. W. and CaIne, D. B. (1979). Multiple receptors for dopamine. Nature 277: Kempthorne, 0. (1967). The Design and Analysis of Experiments, 6th ptg. John Wiley and Sons, New York. Labhstwar, A. P. (1971). Effects of serotonin on spontaneous ovulation: A theory for the dual control of ovulation. Acta Endocrinol. 68: Leblanc, H., Lachelin, G.C.L., Abu-Fadil, S. and Yen, S.S.C. (1976). Effects of dopamine infusion on pituitary hormone secretion in humans. J. Clin. Endocrinol. Metab. 43: Legan, S. J., Karsh, F. J. and Douglas, D. L. (1977). The endocrine control of seasonal reproductive function in the ewes: A marked change in response to the negative feedback action of estradioi on lutemnizing hormone secretion. Endocrinology 101: Lippman, W. (1968). Relationship between hypothalmic norepinephrine and serotonin and gonadotrophin secretion in the hamster. Nature 218: Martin, J. E., Engel, J. N. and Klein, D. C. (1977). inhibition of the in vitro pituitary response to iuteinizing hormone by melatonin, serotonin, and 5-methoxytryptamine. Endocrinology 100: Munro, C. J., McNatty, K. P. and Renshaw, L. (1980).

9 632 DEAVER AND DAILEY Circa-annual rhythms of prolactin secretion in ewes and the effect of pinealectomy. J. Endocrinol. 84: Niswender, C. D. (1974). Influence of 2-Br-o-ergocryptine on serum levels of prolactin and the estrous cycle in sheep. Endocrinology 94: Oldendorf, W. U. (1971). Brain uptake of radiolabelled amino acids, anlines and hexoses after arterial injection. Am. J. Physioi. 221: Parrot, R. F. and Hills, F. (1979). Serum proiactin levels in castrated rams at various times of the year and during treatment with androgens or oestrogens. J. Endocrinol. 83: Preakop, F., Skubisiewski, B., Wolinska, E. and Domanski, E. (1975). The role of catecholamines in stimulating the release of pituitary hormones in sheep. Acts Physiol. Pol. 26: Quijada, M., Illner, P., Kruiich, L. and McCann, S. M. (1973). The effect of catecholamines on hormone release from anterior pituitaries and ventral hypothalami incubated in vitro. Neuroendocrinology 13: Ravault, J. P. (1976). Prolactin in the ram: Seasonal variations in the concentration of blood plasma from birth until three years old. Acta Endocrinol. 83: Riggs, B. L. and Malven, P. V. (1974). Effects of intraventricular infusion of serotonin, norepinephrine, and dopamine on spontaneous LU release in castrate male sheep. Biol. Reprod. 11: Rippel, R. H., Moyer, R. H., Johnson, E. S. and Mauer, R. E. (1974). Response of the ewe to synthetic gonadotropin releasing hormone. J. Anim. Sci. 38: Snedecor, C. W. and W. G. Cochran. (1974). Statistical Methods, 6th ed. Iowa State College Press, Ames. Weiner, R. I. and Ganong, W. F. (1978). Role of brain monoamines and histamine in regulation of anterior pituitary secretion. Physiol. Rev. 58: Wheaton, J. E., Martin, S. K. and Stormshak, F. (1975). Estrogen and L-dihydroxyphenylalanine induced changes in hypothalamic biogenic amine levels and serum LH in the ewe. J. Anim. Sci. 40: