Injection of neuropeptide Y into the third cerebroventricle differentially influences pituitary secretion of luteinizing hormone and growth hormone in ovariectomized cows

DOMESTIC ANIMAL ENDOCRINOLOGY Vol. 16(3):159 –169, 1999

INJECTION OF NEUROPEPTIDE Y INTO THE THIRD CEREBROVENTRICLE DIFFERENTIALLY INFLUENCES PITUITARY SECRETION OF LUTEINIZING HORMONE AND GROWTH HORMONE IN OVARIECTOMIZED COWS M.G. Thomas,1 O.S. Gazal,2 G.L. Williams,3 R.L. Stanko,4 and D.H. Keisler5,6 1

Department of Animal and Range Sciences, New Mexico State University, Las Cruces, NM 88003 2 Department of Biological Sciences, St. Cloud State University, St. Cloud, MN 56301 3 Animal Reproduction Laboratory, Texas A&M University Agricultural Research Station, Beeville, TX 78102 4 Department of Animal and Wildlife Sciences, Texas A&M University-Kingsville, TX 78363 5 Department of Animal Science, University of Missouri, Columbia, MO 65211 Received September 22, 1998 Accepted January 11, 1999

Hypothalamic neurons that control the luteinizing hormone (LH) and growth hormone (GH) axes are localized in regions that also express neuropeptide Y (NPY). Increased hypothalamic expression of NPY due to diet restriction has been associated with suppressed secretion of LH and enhanced secretion of GH in numerous species. However, these physiological relationships have not been described in cattle. Thus, two studies were conducted to characterize these relationships using ovariectomized (Experiment 1) or ovariectomized estrogen-implanted (Experiment 2) cows. In Experiment 1, four well-nourished, ovariectomized cows received third cerebroventricular (TCV) injections of 50 and 500 mg of NPY in a split-plot design. Venous blood was collected at 10-min intervals from 24 hr (pre-injection control period) to 14 hr (postinjection treatment period) relative to TCV injection. NPY suppressed (P ¶ 0.04) tonic secretion of LH irrespective of dose and tended to stimulate (P ¶ 0.10) an increase in tonic secretion of GH. In Experiment 2, six ovariectomized cows that were well nourished and implanted with estradiol received TCV injections of 0, 50, or 500 mg of NPY in a replicated 3 3 3 Latin Square. Both doses of NPY suppressed (P , 0.06) mean concentration of LH relative to the 0-mg dose. The 50-mg dose of NPY tended (P , 0.10) to increase the amplitude of GH pulses. In conclusion, TCV injection of NPY suppressed pituitary secretion of LH and simultaneously tended to increase pituitary secretion of GH. © Elsevier Science Inc. 1999

INTRODUCTION Chronic undernutrition is commonly associated with impaired reproductive function. One mechanism by which undernutrition impairs reproductive function is through suppressed secretion of luteinizing hormone (LH) from the pituitary via suppression of the release of the hypothalamic peptide, luteinizing hormone releasing hormone (LHRH; (1– 4)). Concomitantly, as nutrient status of the animal declines, serum concentrations of growth hormone (GH) increase to mobilize energy stores (5–7). The mechanism(s) by which an animal’s nutrient status is perceived by the brain, and the way in which this information is relayed to hypothalamic neurons which regulate pituitary secretion of LH and GH, are not well defined. However, a neuromodulatory relationship between in© Elsevier Science Inc. 1999 655 Avenue of the Americas, New York, NY 10010

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creased cerebrospinal fluid concentrations of the orexigenic peptide, neuropeptide Y (NPY), during chronic undernutrition, and suppressed hypothalamic-pituitary secretion of LH, has been described in sheep, monkeys, rodents, and humans (8 –12). NPY receptors have been localized to arcuate and paraventricular nuclei of the hypothalamus in humans, rodents, and sheep (13–16). In cattle, as in all other species studied, the arcuate nucleus of the hypothalamus also contains neurons that secrete LHRH, growth hormone releasing hormone (GHRH), and somatostatin (17–18). Similar to reports in sheep, monkeys, rodents, and humans, a recent report from our laboratories demonstrated that infusion of NPY into the third ventricle of cattle suppressed the pulsatile release of LH (19). This effect was mediated in part through an inhibition of pulsatile LHRH release. Therefore, providing evidence to suggest that NPY could influence the neurons located in the hypothalamus and possibly serve as an interpretation signal of body condition or nutritional status to the growth and reproductive axes in ruminants. However, the simultaneous influence of NPY on LH and GH secretion in cattle has yet to be described. The objectives of experiments reported here were to expand knowledge of the effects of NPY on LH release, and to test the hypothesis that NPY simultaneously stimulates GH release in cattle. Studies were conducted in either ovariectomized (i.e., open-loop, Experiment 1) or ovariectomized, estrogen-implanted (i.e., closed-loop, Experiment 2) cows. MATERIALS AND METHODS Description of Animals. All animal-related procedures employed in this study were approved by the Institutional Agricultural Care and Use Committee of the Texas A&M University System (Protocol No. 246). The 10 Brahman 3 Hereford (F1) cows utilized in the following two experiments conducted in 1996 (Experiment 1) and 1997 (Experiment 2) were first acclimated to pens measuring 25 3 9 m and fed daily with hay and a concentrate supplement formulated to meet National Research Council recommendations for maintenance or lactation (20) and to maintain a body condition score of 5 to 6 (1 5 emaciated; 9 5 obese). Cows were ovariectomized by paralumbar laparotomy (21) and subsequently fitted with cerebroventricular cannulae surgically placed into the third ventricle (i.e., third cerebroventricule [TCV]) by the methods of Gazal et al. (19)). Location of cannulae were confirmed radiographically and by free flow of cerebrospinal fluid. Polyethylene tubing (0.58-mm inner diameter 3 0.97-mm outer diameter; Becton Dickinson) or silicone elastomer tubing (Silastic; 0.51-mm inner diameter 3 0.95-mm outer diameter; Konigsberg Instruments, Pasadena, CA) was inserted through the guide cannula and used to inject either control or treatment doses of NPY into the third ventricle. The four cows used in Experiment 1 averaged 9.8 6 2.2 years of age and were maintained as an open-loop model; no supplemental steroids were utilized to influence hypothalamic secretion of releasing hormones. The six cows utilized in Experiment 2 averaged 11.5 6 0.8 years of age and received at the time of ovariectomy, a subcutaneous (sc) Silastic® ear implant containing crystalline estradiol-17b (Sigma). Implants were designed to produce a physiological baseline of 2 to 6 pg/ml of plasma estradiol that yielded a closed-loop (negative feedback), ovariectomized model (21). Twenty-four hours before application of treatments in each experiment, jugular catheters (polyethylene tubing, inner diameter 1.4 mm; outer diameter 1.9 mm; Becton Dickinson) were placed into each cow for intensive blood sampling. Before initiating each sampling period, cows were placed in stanchions with locking headgates. Stanchions were used to facilitate TCV injection, frequent jugular blood collection, and maintenance of calm cows. Experimental Protocols. The effects of NPY on pituitary secretion of LH and GH were determined using four open-loop, ovariectomized cows in Experiment 1. Blood samples were collected at 10-min intervals from 24 hr to 14 hr relative to a 50- or 500-mg

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TCV injection of NPY in a split-plot design. The 4-hr blood collection period that preceded the TCV injection (pre-injection) was used as a control period for each cow and the 4-hr blood collection that followed each TCV-injection period (postinjection) was used as a treatment period. Each cow received the 50- and 500-mg dose of NPY. Cows were randomly selected to receive either the 50- or 500-mg dose of NPY on the 1st d of the experiment, and then 7 d later, each cow received the other respective dose of NPY needed to complete the experiment. The NPY (porcine NPY, Pennisula Laboratories) utilized in Experiments 1 and 2 was dissolved in 0.2 ml of vehicle (0.3% bovine serum albumin, 0.9% NaCl) as recommended by Pardridge (22). This experiment mimicked the study of the effects of NPY dose on LH release in ovariectomized ewes by McShane et al. (12) where vehicle, or the 0-mg dose, exhibited no effect on pituitary secretion of LH. This same relationship was also reported by Hileman et al. (23) and we confirmed this relationship in ovariectomized cattle in an additional preliminary study in which TCV injection of vehicle exhibited no detectable influence on serum concentration of LH (mean concentration 24 hr 5 12.4 6 1.3 ng/ml versus 14 hr 5 13.5 6 1.4 ng/ml; n 5 2) or GH (mean concentration 24 hr 5 10.9 6 4.7 ng/ml versus 14 hr 5 8.5 6 1.8 ng/ml; n 5 2). Thus, a 0-mg dose was not included in Experiment 1. In Experiment 2, six cows were randomized by dose to a replicated 3 3 3 Latin Square. Treatment doses were 0, 50, and 500 mg of NPY, respectively. During each of the three treatment periods, spaced at least 2 d apart, a single blood sample was collected from each cow immediately before injection of the treatment doses of NPY. After the injection, 10-ml blood samples were collected at 10-min intervals for 4 hr. Blood samples in each experiment were allowed to clot on ice and then centrifuged at 3,000 3 g for 30 min. Sera were collected and stored at 220°C until assayed for concentrations of LH and GH by radioimmunoassay. Hormone Assays. In Experiment 1, serum concentrations of LH were assayed in triplicate 100-ml samples by the methods of Zaied et al. (24), with the use of RAoLH TEA #35 heterologous ovine antiserum (25). The sensitivity of the assay was 0.05 ng/ml and the intra- and interassay coefficients of variation were 3.5% and 3.9%, respectively. Serum concentrations of GH were determined in triplicate 100-ml aliquots using the materials and procedures provided by the National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Pituitary Program (NIDDK-NHPP), and methods modified from the ovine GH assay described by Powell and Keisler (26). In brief, the following reagents were added to 12 mm 3 75 mm polypropylene culture tubes: 100 ml of 0.1% Gelatin-phosphate-buffered saline containing 0.1% Tween-20 (pH 5 7.2; PAB), 100 mL of AFPB55-monkey anti-bovine GH in PAB containing 0.01 m EDTA (1:500,000 final tube dilution), and 100 ml of serum or a standard solution of NIDDK-bGH (AFP11182B) in PAB ranging in mass from 0.05 to 2.5 ng/tube. Samples and standards were incubated at 4°C for 24 hr then 100 ml of [125I]-bovine GH (NIDDK-bGH AFP11182B), 15,000 dpm (specific activity 48 mCi/mg) were added to each tube and incubation continued for 24 hr at 4°C. Subsequently, the antigen-primary antibody complexes were precipitated following a 15 min, 22°C incubation using a preprecipitated goat-anti-monkey second antibody. Cold recovery was determined to be greater than 95% and sensitivity of the assay was determined to be 0.05 ng/ml. Intra- and interassay coefficients of variation were 5.4% and 9.8%, respectively. Samples that displaced [125I]-bovine GH at a level greater than 80% were diluted 1:100 with assay buffer and re-assayed. In Experiment 2, serum concentrations of LH were determined in 200-ml aliquots as described previously by McVey and Williams (27). Intra- and interassay coefficients of variation were 8.8% and 8.9%, respectively. Serum concentrations of GH were deter-

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TABLE 1. (EXPERIMENT 1,

OPEN-LOOP MODEL). TONIC SECRETION PATTERNS FOR SERUM

PRECEEDING AND FOLLOWING A

TCV

INJECTION OF

50

AND

500 mG

OF

NPY

LH

FOR THE

4

HR

IN OVARIECTOMIZED COWS

Variable

Preinjection

Postinjection

Probability

Mean LH (ng/ml) Pulse frequency/4 hr Pulse amplitude (ng/ml) Area under the curve

4.5 6 0.7 2.4 6 0.3 6.9 6 0.9 1001 6 165

1.8 6 0.7 1.3 6 0.3 3.2 6 1.1 645 6 154

0.01 0.04 0.01 0.01

Abbreviations: LH, luteinizing horomone; NPY, neuropeptide Y; TCV, third cerebroventricular. Samples were collected at 10-min intervals. Values are mean 6 SE.

mined in 100-ml aliquots by the methods described by Ryan et al. (28). Intra- and interassay coefficients of variation were 5.0% and 15.7%, respectively. Statistics. Mean concentration of LH and GH, number of pulses, amplitude of pulses, and the area under the curve for LH and GH were determined using the Cluster procedures of Veldhuis and Johnson (29) for each sampling period in Experiments 1 and 2. In Experiment 1, there was a 4-hr pre-injection period (i.e., control period) and a 4-hr postinjection period (i.e., treatment or dose period) relative to a 50- and 500-mg injection of NPY. Data from each of the periods were subjected to ANOVA procedures for a split-plot design using the General Linear Models procedure of SAS (30). The model included dose (i.e., 50 and 500 mg of NPY), cow (dose), period (i.e., pre-injection versus postinjection), and dose 3 period interaction as possible sources of variation. The effect of dose was tested with cow-within-dose used as an error term. In Experiment 2, effects of TCV injection of pNPY on serum concentrations of LH and GH were determined by ANOVA procedures for the replicated Latin-square design (30). The statistical model included cow, replicate, dose, and day as sources of variation. A rep effect was not detected (P , 0.70) in the analyses so it was eliminated from the model. When analyses revealed that treatment (i.e., dose) was a significant source of variation, means were separated using orthogonal contrast (i.e., 0-mg dose versus 50 or 500 mg of NPY) using the PDIFF procedure from SAS (30). RESULTS No dose (50 vs. 500 mg) or dose 3 period effects were detected (P , 0.20) in Experiment 1. However, the effect of period (pre-injection versus postinjection) was significant (P ¶ 0.04) for variables used to evaluate tonic secretion of LH. Thus, NPY decreased mean concentrations (P , 0.01), number of pulses (P , 0.04), amplitude of pulses (P , 0.01), and the area under the curve for serum LH (P , 0.01; Table 1). An effect (P ¶ 0.10) of period for the variables used to evaluate tonic secretion of GH was also detected in Experiment 1. Converse to the influence on secretion of LH, NPY tended to simultaneously increase mean concentration (P , 0.10), pulse amplitude (P , 0.07), and area under the curve for serum GH (P , 0.10; Table 2). Figures 1 and 2 illustrate representative profiles of serum concentrations of LH and GH in cows treated with 50 and 500 mg of NPY. TCV injection of 50- and 500-mg doses of NPY suppressed (P , 0.06) mean serum concentrations of LH relative to a 0-mg injection in Experiment 2 (Table 3). This occurred coincident with a tendency to suppress (P , 0.10) LH pulse amplitude. A tendency to suppress area under the LH curve was also detected (P , 0.10) for both doses of NPY. Simultaneously, the 50 mg dose of NPY tended to increase (P , 0.10) pulse amplitude and area under the curve for serum GH (Table 4). Figures 3 and 4 illustrate representative profiles of serum concentrations of LH and GH in cows treated with 0, 50, and 500 mg of NPY.

NPY SUPPRESSES LH AND ENHANCES GH TABLE 2. (EXPERIMENT 1,

163

OPEN-LOOP MODEL). TONIC SECRETION PATTERNS FOR SERUM

PRECEEDING AND AFTER A

TCV

INJECTION OF

50

AND

500 mG

OF

NPY

GH

FOR THE

4

HR

IN OVARIECTOMIZED COWS

Variable

Preinjection

Postinjection

Probability

Mean GH (ng/ml) Pulse frequency/4 hr Pulse amplitude (ng/ml) Area under the curve

5.4 6 0.9 2.1 6 0.3 8.8 6 1.9 1238 6 212.8

18.2 6 7.2 2.0 6 0.3 93.1 6 37.9 4562 6 1779

0.10 0.80 0.07 0.10

Abbreviations: GH, Growth hormone: NPY, neuropeptide Y; TCV, third cerebroventricular. Samples were collected at 10-min intervals. Values are mean 6 SE.

DISCUSSION NPY is a ubiquitously expressed by the central nervous system and is highly homologous across species (31–32). Multiple receptors and receptor subtypes of NPY have been defined in rodents, humans, sheep, and cattle (14 –16,33). In rodents and sheep, NPY and its receptors are localized in hypothalamic regions that contain neurons that synthesize and

Figure 1. (Experiment 1, open-loop model). Patterns of serum LH in two ovariectomized cows (ID#s 4300 and 4315) 4 hr before (pre-injection control period) and 4 h after (postinjection treatment period) receiving a 50- or 500-mg TCV injection of NPY. Serum samples were collected each 10 min for the 4-hr pre- and postinjection. Arrows indicate time of injection of NPY.

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Figure 2. (Experiment 1, open-loop model). Patterns of serum GH in two ovariectomized cows (ID#s 4300 and 4315) 4 hr before (pre-injection control period) and 4 hr after (postinjection treatment period) receiving a 50- or 500-mg TCV injection of NPY. Serum samples were collected each 10 min for 4 h pre- and postinjection. Arrows indicate time of injection of NPY.

secrete the releasing hormones for LH and GH (11,13,34 –36). These anatomic areas also contain the neurons for LHRH, GHRH, and somatostatin in cattle (17–18). Central administration of NPY, has been shown to stimulate feeding behavior in sheep, monkeys, rodents, and humans (8 –10,37). A relationship between NPY-induced feeding and impaired reproductive function was established by the discovery that hypothalamic TABLE 3. (EXPERIMENT 2, CLOSED-LOOP MODEL). TONIC SECRETION PATTERNS FOR SERUM LH FOR THE 4 HR AFTER A TCV INJECTION OF 0, 50, OR 500 mg OF NPY IN ESTRADIOL-IMPLANTED OVARIECTOMIZED COWS Dose of NPY Variable

0 mg

50 mg

500 mg

Mean LH (ng/ml) Pulse frequency/4 hr Pulse amplitude (ng/ml) Area under the curve

4.7 6 1.0 1.6 6 0.2 7.7 6 2.4 1162 6 241

2.9 6 0.5* 1.3 6 0.5 3.5 6 1.4** 674 6 107**

2.7 6 0.4* 0.8 6 0.4 2.1 6 0.9** 642 6 82**

Abbreviations: LH, luteinizing hormone; NPY, neuropeptide Y; TCV, third cerebroventricular. Doses were applied in a replicated 3 3 3 Latin Square (n 5 6). Values are mean 6 SE. * Differs from control (0 mg of NPY) P , 0.1. ** Differs from control (0 mg of NPY) P , 0.06.

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TABLE 4. (EXPERIMENT 2, CLOSED-LOOP MODEL). TONIC SECRETION PATTERNS FOR SERUM GH FOR THE 4 HR AFTER A TCV INJECTION OF 0, 50, OR 500 mG OF NPY IN ESTRADIOL-IMPLANTED OVARIECTOMIZED COWS Dose of NPY Variable

0 mg

50 mg

500 mg

Mean GH (ng/ml) Pulse frequency/4 hr Pulse amplitude (ng/ml) Area under the curve

4.8 6 0.7 2.2 6 0.2 6.3 6 0.8 1145 6 157

5.7 6 1.0 2.8 6 0.2* 8.6 6 1.8 1379.8 6 221*

4.7 6 1.1 2.6 6 0.4 6.2 6 1.4 1115 6 261

Abbreviations: GH, growth hormone; NPY, neuropeptide Y; TCV, third cerebroventricular. Doses were applied in a replicated 3 3 3 Latin Square (n 5 6). Values are mean 6 SE. * Differs from control (0 mg of NPY) P , 0.1.

concentrations of NPY, or NPY mRNA, were elevated in animals receiving limited nutrient intake (see Keisler and Lucy for review (38)). More specific to ruminants, an inverse relationship between cerebrospinal fluid concentrations of NPY and pituitary secretion of LH has been reported in ovariectomized ewes (11,12). Even though this relationship was not detected in intact ewes (39), the current experiments, in coordination with experiments where NPY was infused into the lateral cerebroventricles of ovariectomized ewes (12), provide evidence to suggest that NPY may influence the neurons that regulate the reproductive axis in ruminants. Previously, our laboratories reported that NPY suppressed tonic secretion of LH

Figure 3. (Experiment 2, closed-loop model). Patterns of serum LH in two estrogen-implanted ovariectomized cows (ID#s 4236 and 6232) that received a 0-, 50-, or 500-mg TCV injection of NPY in a Latin square. Serum samples were collected each 10 min for 4 hr postinjection. Arrows indicate time of injection of NPY.

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Figure 4. (Experiment 2, closed-loop model). Patterns of serum GH in two estrogen-implanted ovariectomized cows (ID#s 4236 and 6232) that received a 0-, 50-, or 500-mg TCV injection of NPY in a Latin square. Serum samples were collected each 10 min for 4 hr postinjection. Arrows indicate time of injection of NPY.

regardless of estrogen treatment in ovariectomized ewe lambs (12) and in estradiolimplanted, ovariectomized cows (19). The data in the current experiments do not clearly duplicate these observations. In Experiment 1, both doses of NPY—50 and 500 mg— dramatically suppressed tonic secretion of LH in the open-loop ovariectomized model. These responses were somewhat attenuated in Experiment 2 in estrogen-implanted, ovariectomized cows. However, these experiments were not designed to compare responses in ovariectomized (open-loop) cows to estrogen-implanted ovariectomized cows (closed-loop). Although the responses to NPY are not equivalent in the two experiments, the hypothalamus is known to be estrogen sensitive in numerous species (40 – 44) and further investigation is needed to define the interaction of estrogen with NPY in regulating secretion of LHRH in cattle. Gonadal steroids have been reported to enhance pituitary secretion of GH in both rodents and humans (45– 46). There is also evidence to suggest that this same relationship, particularly in regard to the effect of estrogens, may exist in ruminants (47– 49). In the current experiments, NPY appeared to stimulate an increase in serum concentrations of GH in ovariectomized (open-loop) cows, whereas only a slight increase in serum concentrations of GH was detected in estrogen-implanted, ovariectomized cows (note differences in the scale of the Y axes in Figures 2 and 4). The basis for these differences is unclear, although numerous influences should be considered. For example, age of the cows used in the two experiments differed somewhat and the effects of estrogen on the number and sensitivity of the neurons that regulate pituitary secretion of GH could be different in ruminants relative to the effects described in rodents (40 – 41). Moreover, high concentrations of NPY infused into the third ventricle could diffuse into the pituitary stalk and exert influences directly on the pituitary which is rich in NPY receptors and sensitive

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to estrogen (13,31–32,50). Whatever the case, these data provide evidence to suggest that NPY may be involved in stimulating pituitary secretion of GH which is typically enhanced in animals in poor body condition (5,28). Although knowledge of the relationship between NPY, LH, and GH is improved with these studies, the mechanism(s) which drive these relationships and account for the between animal variation observed within these relationships are not well defined. Further research is needed to define the interactions between NPY and the neurons that synthesize and secrete LHRH, GHRH, or somatostatin. Also, determining the metabolic cues that regulate hypothalamic synthesis of NPY may aid in understanding the mechanisms by which NPY influences pituitary secretion of LH and GH. This knowledge may provide a more detailed understanding of how nutritional status is perceived by the brain and provide an explanation for the variability in reproductive performance observed with suboptimal body condition in cattle. In conclusion, TCV injection of NPY decreased tonic secretion of LH and tended to increase serum concentrations of GH in ovariectomized cows. These data provide evidence supporting the hypothesis that NPY suppresses pituitary secretion of LH while simultaneously stimulating pituitary secretion of GH in cattle. ACKNOWLEDGMENTS/FOOTNOTES This work is a contribution from the Missouri Agricultural Experiment Station journal series (no. 12,554) and was supported in part by U.S. Department of Agriculture-NRICGP Grant no. 92–37203-8036 (D.H.K.), U.S. Department of Agriculture-National Research Initiative Competitive Grants Program Postdoctoral Fellowship Grant no. 95-37206-2119 (M.G.T.), U.S. Department of Agriculture-National Research Initiative Competitive Grants Program Grant no. 94-37203-0924 (G.L.W.), and the Texas Agricultural Experiment Station. The authors thank National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Pituitary Program and Dr. A.F. Parlow for bovine GH antigen and antisera, Dr. J.J. Reeves for LH antisera, and National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Pituitary Program for LH antigen. The authors also thank J.A. Daniel, B. Thedin, and P. Fajersson for their assistance with the TCV cannulations, and Dr. T. Ross for his assistance with statistics. 6 Address correspondence to: Dr. Duane H. Keisler, Department of Animal Sciences, 160 Animal Science Research Center, University of Missouri, Columbia, MO 65211. e-mail: [email protected].

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