PROSTAGLANDINS

INFLUENCE OF PROSTAGLANOINSAND THYROTROPIN RELEASING HORRONE(TRH) ON HORMONESECRETIOR AND GROWTHIN WETHER LAMBS S.L.

Davis,

M.S. Anfinson, J. Kllndt and D.L. Department of Animal Industries University of Idaho ~OSCW. Idaho 83843

Ohison

Abstract A series of experiments ware conducted in ewes and wether (castrate male) lambs to evaluate the influence of prostaglandlns on secretion of anabollc hormones and to determlne If repeated injections of prostaglandin (PG) Fsa would chronically Influence the secretion of these hormones and perhaps growth rate as well. A single intravenous injection of PGAl and PGBl (100 pglkg) exerted no significant (P > .lO) Influence on plasma concentrations of prolactln (PRL), growth hormone (GH) or thyrotropln (TSH) in awes. PGAI, but not an increase In the plasma concentration of insulin. InPGBl, stimulated fusion of PGFsa for 5.5 hr into awes resulted In increased (P < .05) plasma concantratlons of both GH and PRL while TSH and insulin ware not significantly influenced. Prostaglandin Fsa, when injected subcutaneously into wather lambs (10 mg twice weekly) stimulated (P < .05) plasma GH concentrations after the first injection, but not after 3 weeks of treatmsnt. Changes In plasma PRL or TSH ware not observed consistently in the lambs treated chronically with PGFsa or TRH. Prostaglandin Fsa, in the present studies, and PGEt in previously reported studies (I-3). has been damonstrated to be stimulatory to the secretion of PRL and GH. In contrast, PGAl and PGB1, which lack an llhydroxyl group, failed to influence the secretion of either PRL or GH. It would, therefore, appear that the Ii-hydroxyl group Is a structural requirement for prostaglandlns to influence the secretion of these two hormones in sheep. Treatment with thyrotropin releasing hormone (TRH), alone or in combination wlth PGFsa, significantly (P < .D5) increased grcwth rate (average datly gains) while PGFsa did not, despite the fact that both compounds exerted similar effects on plasma GH. ACKNOWLEDGEMENTS Idaho Agricultural Experimsnt Station Publication Nmnber 7644. in part, by a grant from NIH (HDD78301, These studies ware supported, State of Idaho Short Term Applied Research (STAR) funds and the Idaho Agriculture Experlment Station. Prostaglandins used in the present study ware generously supplied by Dr. James Lauderdale, Upjohn, Kalamazoo, Michigan. The authors would like to thank Dr. D.O. Everson and Mr. L.E. Christian for their help with statistical analysis. The authors are aiS grateful to Dr. J.G. Pierce, Dr. L.E. Reichert, Jr. and Dr. A.E. Wilhelmi for providing purlfled bovine TSH, ovine PRL and ovine GH used for radioiodlnatlons, and tha NIH Hormone Dlstributlon program for ovine hormones used for reference We also gratefully acknowledge Dr. R. Rlppel and Abbott standards. Laboratorles for supplying the TRH used In these studies.

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INTRODUCTION Previous studies (15) have indicated that some prostaglandins are StimujatOry to secretion of growth hormone (GH) and prolactin (PRL). Since both of these hormones have been shown to exert anabolic actions (6,7), It was of interest to determine if prostaglandin treatment would result in increased growth rate in lambs. Other evidence suggests that PGEl is inhibitory to Insulin secretion (5). No information has been reported concerning the effect of PGF2a on insulin secretion in sheep. The objectives of the present research, then, ware: to examine the influence of three prostaglandins--A], B1, and Fpu--on the secretion of GH, PRL, thyrotropin (TSH) and insulin in wether lambs; and to compare the influence of PGF2a with thyrotropin releasing hormone (TRH) on growth rate in wether lambs. he thods Study

1.

Influence

of PGAl and PGBl on Plasma Hormone Concentrations

Ten ewes (average body weight, 60 kg) were injected via indwelling jugular cannulae with PGAl at 100 pg/kg (n=5) or an equal volume of diluent (1.5 ml saline + 0.5 ml ethanol). Blood samples for hormone analysis were collected from the jugular cannulae at l5-minute intervals In a second experifor 1 hour pretreatment and 2 hours post treatment. ment 4 days later, the same animals were injected with either PGBl at 100 ug/kg or an equal volume of diluent (2.0 ml). The animals were not fasted during either of the studies. Blood samples were chilled on ice during collection, stored overnight at 4”C, then plasma was collected after centrifugation and frozen Treatment effects on plasma until assayed for the respective hormones. hormone concentrations were determlned by analysis of variance using a split plot design (8). Study 2.

Influence

of PGFso Infusion

on Plasma Hormone Cencentrations

Ten ewes (same animals used In the previous studies) were infused for 5.5 hours lntrajugularly wlth either 300 pg PGFpo tromethamine (THAH) salt/minute or an equal volume of diluent (.077 ml/min). Blood samples were obtained (from a cannula in the contralateral jugular) at 15-minute intervals during the 5.5 hour infusion period and were prepared as described in study 1. Analysis of variance using a split plot design (8) was used to exsmine treatment effects on plasma hormone concentrations. Study 3.

influence

of Subcutaneous

injection

of PGFso on Plasma Hormone

Concentrations Sixty wether lambs (initial weight approximately 30 kg) were given a single subcutaneous injection of one of the following treatments (15 lambs per treatment): 0, 1, 5 and 10 mg PGFao THAH salt dissolved in Blood samples were obtained by ethanol then suspended in mineral oil. jugular venipuncture 4, 10 and 24 hours after injection. The blood samples were collected and prepared as described in study 1. Statistical analysis of treatment effects on plasma hormone concentration was accomplished using a one-way analysis of variance. Study 4.

Influence

on Growth Rate

of Chronic

in Wether

Subcutaneous

Injection

of PGFsa and TRH

Lambs

The same 60 wether lambs used in study 3 were equally divided among 4 treatments (15 lambs per treatment) according to a split-plot design. Control (group 1) animals were treated with solvent solution (mineral oil) only. Group 2 received subcutaneous injections of PGFsa THAM salt (10 mg) at three day intervals; group 3, 1 mg of TRH at 3 day intervals; and group 4, 1 mg TRH + 10 mg PGFso THAI4 salt at 3 day intervals. The dose of TRH used in the present study has been previously shown to effectively stimulate Increased plasma concentrations of TSH and PRL Each group was divided into two replicates (8 and 7 animals/rep) (9).

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and group fad on an ad libitum basis. All groups received the same pelleted ration containing alfalfa, barley and soybean meal, supplemented with minerals and vitamin A. All lambs ware weighed twice monthly. Lambs were removed from the study and slaughtered when they reached 48 kg live body weight. Blood samples ware obtained by jugular venipuncture 8 hours post-injection on day 1 and day 21 of treatment for hormone analysis. To obtain estimates of feed consumption the feed was weighed as it was placed into self feeders. One feeder was placed between the two replicate pans for each treatment group. Therefore, it was not possible to estimate feed consumption par replicate, only on a par treatment basis. Treatment effects on growth rate (average daily gains) ware determined by analysis of variance using a split-plot design with initial body weight being used as a covariate in analysis of ADG. This design was used to test treatment effects for both 28-day and final ADG. A one-way analysis of variance was used to test treatment effects on plasma hormone concentrations.

radioeassay

- Plasma was assayed for PRL GH, TSH and Insulin systems developed praviiusly (10-13).

using

Results Study

levels PGB1, (fig. these prior

1.

Influence

of PGA, and PGB, on Plasma Hormone Concentration

Neither PGAl nor PGBl treatment exerted an influence on plasma of PRL. GH or TSH (fig. 1 and 2). Injection of PGA1, but not significantly (P < .05) increased plasma concentration of insulin 1 and 2). Variability in plasma insulin concentrations between two experiments may be due to differences in food consumption to and during the experiments.

FIGURE 1. Plasma concentrations of PRL, GH, TSH and insulin in awes injected with PCA, (e-e) or salineethanol (o-o). Time of PGAl injection 1s denoted on each figure by the dashed verticle line at time = 1 hr. Each point represents the mean + standard error of the mean (SEH)-of 5 observations.

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PROSTAGLANDINS

-3 I 4ooIL a

Study 2.

Influence

of PGF,a Infusion

FIGURE 2. Plasma PRL, GH, TSH and insulin in ewes injected with PGBl (o-o) or saline-ethanol (o-o). Time of PGBl injection is represented by the dashed vetticle line at t = Each point is the mean 1: SEM 1 hr. of 5 observations. on Plasma Hormone Concentrations

The continuous infusion of PGF2a (300 ug/min) resulted in a significant (P < .05) Increase in plasma GH (fig. 3) which remained above control GH levels for most of the infusion perlod. Similarly, PGF2a in plasma PRL within 30 stimulated a significant (P < .Ol) increase minutes after the start of the infuslon period (fig. 3). Plasma PRL levels in PGF2c treated ewes remained well above control levels over In contrast, PGF2a treatment was the entire infusion period (flg. 3). without effect on plasma levels of TSH and insulin (fig. 4). Study 3.

Influence

of Subcutaneous

Injection

of PGF,a on Plasma Hormone

Concentrations Subcutaneous injection of 10 mg PGF2a, but not 1 or 5 mg, resulted in a significant (P < .OS) increase in plasma GH IO hours post-injection These results led to the decision to inject 10 mg PGF2a in (table 1). Plasma concentrations of PRL were elevated above subsequent studies. controls (P < .lO) at 4 and 24 hours post-injection of 10 mg of PGF2a. Plasma TSH was not influenced by any of the PGF2a doses (table 1). Study 4.

Influence

on Growth Rate

of Chronic

in Wether

Subcutaneous

Injection

of PGF,(r and TRH

Lambs

of PGF2a resulted in a significant As observed in study 3, injection in plasma GH on the first day of treatment (table 2). (P d .05) increase Similarly, TRH and TRH plus PGF2a also stJmulated increased plasma GH on Plasma PRL concentrations were significantly the flrst day of treatment. < .lO) influenced by PGF2a (table 2) on day 1 of treatment and by TRH I; < .Ol) on day 20. Although there was a tendency for plasma TSH and PRL to be higher than in control lambs on both dates in all treated were not statlstically significant (P > .lO). groups, these differences

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PROSTAGLANDINS

Table 1.

Dose

PGw

Plasma concentrations of @I, TSH and PRL at varying time periods after subcutaneous injection of 0, 1. 5 and 10 mg PGF2a (mean + standard error of mean, SEM).

of (mg)

Hours after PGF 10

4

24

Plssms GH (r&g/ml) 0 1 5 10

15.9 + 13.5 5 14.1T 19.0 i

1.91 0.8 1.8 2.0

23.7 + 20.4 + 17.4 7 33.5 -

2.3 1.9 2.7 4.5**

16.6 + 1.8 16.0 7 2.2 14.2 7 1.1 14.6 f1.2

Plasma TSH (ng/ml) 0 1 5 10

3.9 3.4 3.7 3.5

+ T T 3

0.8 0.4 0.8 0.6

3.7 3.5 2.5 3.2

+ 7 7 5

0.6 0.4 0.2 0.5

2.3 + 2.17 3.7 + 2.8 3

0.4 0.5 0.6 0.4

195 + 192 + 194: 233 -

13.8 12.2 11.9 15.1*

Plssms PRL (ng/ml) 0 1 5 10

210 + 12.5' 200 T 13.5 2217 17.1 252 z11.6**

232 + 248 7 235 T 257 -

10.6 11.1 18.9 17.4

P C .lO 8s compared to controls. P < .05 ss compared to controls. These GH values are very high for wether lambs based upon previous experience. The samples were therefore asssyed for GH in three separate assays to ensure that the high values were not related to sassy problems. Such high concentrations in these wethers remain unexplained. Plasma PRL concentrations were quite high in all lambs. Plasma PRL could have been elevated due to stress of venipuncture, and/or perhaps due to season since these samples were obtained during June and July (14).

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PROSTAGLANDINS

Table 2.

Plasma concentrations of GH, TSH and PRL 8 hr after subcutaneous injection of PGFza, TRH or TRH + PGF2a (means ?SEM).

Days of Treatment 1 20

Treatment

Plasma GH (ng/ml) Control PGF2a TRH TRH + PGF2a

14.2 + 19.8 + 19.5 + 21.8 T -

1.2l 2.1** 2.4* 2.2***

13.7 + 13.6 + 15.6 + 14.2 -

1.7 1.2 2.6 1.7

Plasma TSH (ng/ml) Control PGF2a TRH TRH + PGFncc

4.3 3.8 4.4 5.5

+ T 7 3

0.5 0.6 0.6 0.8

4.0 4.9 5.6 4.0

+ T T 3

0.5 1.1 1.3 0.3

Plasma PRL (ng/ml) Control PGF2ci TRH TRH + PGFpcc

277 317 306 309

+ 2 + +

23.0' 17.0* 19.0 22.0

*

P < .lO as compared to control.

**

P < .05 as compared to control.

255 269 330 278

+ + T z

11.6 15.0 15.0*** 16.0

*** P < .Ol as compared to control. lr2 See footnote, table 1.

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&

r&n llbo Ii00 Till (IInS)

100

FIGURE 3. Plasma concentrations of CH (upper) and prolactin (lower) In ewes infused at a constant rate with saline or PGFza THAlj salt for 5.5 hours. Values are X + SEH of 5 animals.

Treatment with TRH was associated with 17.1% increase (P -z .05) in average daily gains after 28 days on feed and 8.7% increase (P < .lO) in final average daily gains as compared to the control group (table 3). Treatment with PCF2cr alone did not significantly (P > .lO) affect average daily gains over controls during either the first 28-day period or during the entire feeding period (table 3). Combined treatment with TRH and PGF1a produced ADG which were above controls (P < .DS), but which were not different from TRH alone over the entire period (table 3). Due to the experimental design it was not possible to examine treatment effects on feed/gain ratios statistically. It would appear, however, that the value of 5.1 in TRH treated lambs (28-days) was less than that of controls (6.1). Discussion Little information is available on the influence of prostaglandins. of anabolic hormones in with the exception of PGEL, on the secretion sheep. Warberg, -et al (15) have reported that PGA, and PCDl did not exert an effect on the secretion of LH, PRL or TSH in rats. Sacca, s fl (16,17) on the other hand, have observed that infusion of PGAl

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PROSTAGLANDINS

TABLE 3. sutwmY OF AVERAGE DAILY GAINS (ADG) AND FEED CONVERSION RATIOS (F/G) IN WETHER LAMBS INJECTED WITH PGF20 OR TRH

Treatment

Number of Lambs

28 Day ADG (kg/day)

F/G

Final ADG (kg/day)

F/G

Control PGF2a TRH TRH + PGF2a

’

Two lambs on this treatment were, therefore, removed. from urinary calculi.

a,b,c

1216

Means within scripts are P < .10 for

became ill Both of the

during the experiment and lambs subsequently died

columns which are followed by different letter supersignificantly different (P < .05 for 28 day ADG and final ADG).

JUNE 1977 VOL. 13 NO. 6

PROSTAGLANDINS

7111 Qss)

FIGURE 4. Plasma TGH (upper) and insulin (lower) concentration in ewes infused with PGFsa THAI4 salt (0-o) or sal ine (o-o). Values are ?+ gEH of 9 anikls;

lowered plasma insulin in rats and decreased the lnsul in response to glucose infusion while not Influencing basal plasma insulin In dogs. Our results in the sheep are in disagreement with these previous reports in that a single injection of PGAl slgnlficantly Increased plasms insulin The reasons for such differences remain speculative, but in the sheep. may be related to method of admInIstration (injectlon vs infusion) or species differences in energy metabolism and hormone secretlon (ruminants The Inability of PGBl injactlon to influence plasma vs nonruminants). insulin levels In sheep Is in agreement with the previous report that PGBl infusion failed to significantly alter plasma insulin In dogs. (18. The present observations on the influence of PGAl and PGB on GH secretion In sheep also disagree with the previously reported t 19) stimulation of GH release from ox-pituitaries Incubated --In vitro in the presence of PGAl (0.1 u) or PGBl (1 urn). Homver, the stlmulatory influence of PGA and PGBl was not observed In vitro in the absence of theophylline (19f suggesting the insensltivIGofltured anterior pituitary cells to PGAl or PGBl stimulation unless cyclic AMP phosphodiesterase is inhibited. This latter point may be one of the reasons why intravenous injection of PGAl or PGEl did not stimulate increased plasms GH concentrations in the present study. Of course, other points of difference include in and perhaps dose of prostaglandlns adminEtered. -vivo vs --In vitro experiments

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PROSTAGLANDINS

The presently reported inability of PGBl to influence either plasma PRL or TSH in sheep Is in agreement with the data of War-berg -et al (15) obtained from rats. Plasma concentrations of PRL and GH are reportedly elevated in cattle after PGFsa injectlon (4) which is in agreement with the results of the present infusion studies. The inabllity of PGFsa, PGAl and PGBI to influence plasma TSH concentrations in ruminants has not been previously documented. Warberg, 5 al., (15) have reported that the cis double bond in the 5, 6 position and the 11-OH group are essential for the LH releasing activity of prostaglandlns. Based upon present results, the ll-OH group also appears to be required for stimulation of PRL and GH secretion since, In its absence (PGA1 and PGBl have no hydroxyl group at the 11 posltlon), PRL and GH concentrations in the blood were not affected. The cis double bond In the 5, 6 position doas not appear to be required for stimulation of GH and PRL secretion In the sheep since both PGFsa (containing 5, 6 cis group) in the present study and PGE, (without the 5. 6 cis group) in previous studles (2.3) both stimulated the secretion of GH and PRL. Although TSH and PRL were not significantly changed by TRH in the present study, numerous studies have been reported demonstrating that TRH does stimulate increased PRL and TSH secretion in ruminant animals It is not too surprising that an increase in plasma PRL was (9.13,20). not observed in the present feeding trial studies after injection of TRH or PGFsa. First of all, plasma PRL concentrations have been shown to be quite variable in sheep, and this variability is known to increase when Stress associated with venipuncture blood samples are drawn by venipuncture. could easily have produced sufficient variability in plasma PRL concentrations to have masked the influence of TRH and PGFsa on PRL secretlon in the present studies. The lack of a significant influence of TRH on TSH secretion in the feeding trial Is more difficult to explain. However, it IS becoming increasingly more apparent that estimates of hormone concentrations from single blood samples follmlng treatment are, in many instances, inadequate to detect subtle changes in plasma hormone concentration. The inability to detect expected changes in plasma hormone concentrations following subcutaneous TRH or PGF2a inJectIon presents a serious problem in interpretation of the data. These data may suggest that TRH was not exerting its expected effects on the secretion of PRL and TSH. However. in view of the influence of TRH on growth rate in the present studies, and the fact that such an effect Is comparable to previous studies in sheep where the expected effects of TRH on PRL and TSH were evident (91, it seems more likely that the present sampling methods were inadequate to consistently detect hormonal changes associated with treatment.

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PROSTAGLANDINS

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Prostaglandin El and the release J.G. Nature, 228:179, 1970.

of

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Schofield, in vitro. --

2.

Hertelendy, F., A. Todd, K. Ehrhart and R. Blute. Studies on growth In vlvo effects of prostaglandin El. hormone secretion: IV. Prostaglandins 2:79. 1972.

3.

Davis, S.L. and M.S. Aufinson. Dose-response influence El and somatostatin on plasma levels of growth hormone, thyrotropin in sheep. J. Anim. Sci. 41:172, 1975.

4.

Louis, T.M., J.N. Stellflug, growth hormone, luteinising Fsa in heifers. Proc. Sot.

5.

Robertson, R.P., D.J. Inhibition of in viva Invest. 54:310, 1974.

of prostaglandln prolactin and

A.A. Tucker and E.D. Rafs. Plasma prolactln, hormone and glucocorticoids after prostaglandin Exp. Biol. Med. 147:128, 1974.

Gavareski, D. Porte, Jr. and E.L. Insulin secretion by prostaglandin

Bierman. El. J.

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Raben, M.S.. F. Matsusaki and P.R. Mintou. Growth promoting and metabolic effects of growth hormone. Metabolism 13:1102, 1964.

7.

Beck, J.C.. A. Gonda, H.A. Ramid, R.O. Morgan, D. Rubenstein and E.E. McGarry. Some metabolic changes induced by primate growth hormone and purified ovine prolactin. Metabolism 13:1108, 1964.

8.

Analysis of Gill, J.L. and H.D. Hafs. animals. J. Anti. Sci. 33:331, 1971.

9.

Davis, S.L., K.M. Hill, D.L. Ohlson and J.A. Jacobs. Influence of chronic thyrotropin-releasing hormone injections on secretion of . prolactin, thyrotropin and growth hormone and on growth rate in wether lambs. J. Anim. Sci. 42:1244. 1976.

repeated

measurements

of

10.

Davis, S.L., L.E. Reichert, Jr. and G.D. Niswender. of prolactln in sheep as measured by radiolmnunoassay. Reprod. 4:145, 1971.

Serum levels BiOl.

11.

Plasma levels of prolactin, growth hormone, and insulin Davis, S.L. in sheep following the infusion of arginine, leucine and phenylalanine. Endocrinol. 91:549, 1972.

12.

Borger, M.L. and S.L. (TSH) radioinrmunoassay 39:768, 1974.

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Inhibition Davis. S.L. and M.S. Anfinson. thyrotropin responses to thyrotropin-releasing J. Anti. Sci. 42:422. 1976.

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ovine thyrotropin Anim. Sci.

ovine prolactin and hormone by triiodothyronine.

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PROSTAGLANDINS

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Warberg, J., R.L. Eskay and J.C. of anterior pituitary hormones: Endocrinol. 98:1135. 1976.

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Sacca, L. , G. Perez, F. Rengo, I. Pascuccl and H. Condorelli. Reduction of circulating insulin levels during the infusion of different prostaglandins in the rat. Acta Endocrinol. 79:266,

Porter. Prostaglandln-induced release structure-activity relatlonshlps.

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Sacca, L., F. Rengo. l4. Chlariello and M. Condorelll. Glucose Intolerance and lmpalred insulin secretion by prostaglandln A1 in fasting anesthetized dogs. Endocrlnol. 92:31, 1973.

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Robertson, R.P.. and R.J. Guest. Effects of prostaglandln RI on basal and stimulated circulating levels of insulin, glucose, and free fatty acids. Prostaglandlns 8:231, 1974.

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Cooper, R.H., M. McPherson and J.G. Schofield. Tha effect prostaglandlns on ox pltultary content of Adensoslne 3’:5’ monophosphate and the release of growth hormone. glochem. 127:143, 1972.

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KcGuffey, R.K., J.W. Thanas and E.H. Convey. Growth, serum growth hormone, thyroxine, prolactln and insulin in calves after thyrotropln releasing hcmone or 3-methyl-thyrotropin releasing hormone. J. Anim. Scl. 44:422.

Received

1220

to

12/13/76

-

of - cyclic J.

Approved 5/2/77

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