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Serum prolactin response to thyrotropin releasing hormone in estrogen treated hypophysial stalk-transected gilts
DOMESTIC ANIMAL ENDOCRINOLOGY
Vol. 3(4):289-294,1986
SERUM PROLACTIN RESPONSE TO THYROTROPIN RELEASING HORMONE IN ESTROGEN TREATED HYPOPHYSlAL STALK-TRANSECTED GILTS Robert R. Kraeling* and George B. Rampacek** *Animal Physiology Unit, Richard B. Russell Agricultural Research Center ARS, USDA, Athens, GA 30613 and **Animal and Dairy Science Department, University of Georgia Athens, Georgia 30602 Received March 10, 1986
ABSTRACT Twelve crossbred gilts, 169 + 3 days of age and 72.8 + 3.4 kg body weight, were hypophysial stalk-transected (I-IST)I or sham hypophysial stalk-transected (S-HST). Gilts were ovariectomized 6 days later and assigned to four treatments of 3 gilts each in a 2 x 2 factorial arrangement. One-half of the HST and S-HSTgilts received 5 mg estradiolbenzoate (EB) or corn oil vehicle tm at 0800 hr daily for 5 days beginning 64 _+ 3 days after HST or S-HST. Blood was collected by jugular vein cannula at 0830 and 0900 hr the day after the last injection of EB or oil. Immediately after the 0900 hr sample, 200 ttg thyrotropin releasing hormone (TRH) were injected (iv). Mean basal serum prolactin (PRL) concentration was similar for HST (10.3 + 1.0 ng/ml) and S-HST (12.3 _+ 1.7 ng/ml) gilts, however mean basal serum PRL concentration was greater (P<.05) for EB-treated gilts (13.7 -+ 1.3 ng/ml) than for oil-treated gilts (8.8 -+ .5 ng/ml). Mean serum PRL concentration of all gilts increased within 10 rain and returned to approximately 20 ng/ml by 150 rain after TRH. Maximum serum PRL concentrations at 10 min after TRH were greater (P<.01) for S-HST (255.9 -+ 29.6 ng/ml) than HST gilts (83.4 - 18.8 ng/ml), but were not different for EB (198.0 + 50.6 ng/ml) and oil-treated gilts (141.4 + 36.3 ng/ml). Area under the serum PRL response curve after TRH was greater (P<.005) for S-HST than HST gilts and for EB than oil-treated gilts (P<.05). These results do not eliminate the possible influence of estrogen on PRL secretion at the hypothalamus, but do indicate that estrogen directly stimulated the anterior pituitary gland to secrete PRL. INTRODUCTION The t e m p o r a l relationships b e t w e e n b l o o d estrogen, luteinizing h o r m o n e (LH) and prolactin (PRL) c o n c e n t r a t i o n s during the periestrous p e r i o d in the gilt and p o s t p a r t u m sow have b e e n described by Guthrie et al (1), HHenricks et al (2), Berets et al (3), Dusza and Krzymowska (4), Brinkley (5), and Van de Wiel et al (6). Mean b l o o d estrogen concentrations were basal until proestrus w h e n a three- to fourfold increase occurred. A decline in b l o o d estrogen c o n c e n t r a t i o n began p r i o r to the p r e o v u l a t o r y LH surge and the p r e o v u l a t o r y LH surge o c c u r r e d w i t h i n 24 hr f o l l o w i n g the onset o f behavioral estrus. Two surges o f b l o o d PRL c o n c e n t r a t i o n s generally o c c u r r e d during the periestrous p e r i o d in gilts; one surge began near the end o f luteal regression and o t h e r just before the p r e o v u l a t o r y LH surge. The cause and effect relationship b e t w e e n estrogen and PRL secretion has not b e e n d e t e r m i n e d in the pig. However, Bevers et al (3) and Stevenson et al (7) suggested that estrogen not only stimulates the p r e o v u l a t o r y LH surge, but also stimulates the periestrous surge of PRL. In addition, H o o v e r et al (8) and Stevenson et al (7) r e p o r t e d that e x o g e n o u s estrogen caused an increase in plasma PRL c o n c e n t r a t i o n in ovar-
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1986 by DOMENDO, INC.
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0739-7240/86/$3.00
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i e c t o m i z e d sows. The mechanism by w h i c h estrogen might stimulate PRL secretion in the pig has not b e e n investigated. Therefore, the objective of this e x p e r i m e n t was to d e t e r m i n e if estrogen stimulates PRL secretion in the pig by direct action on the anterior pituitary gland. MATERIALS A N D METHODS
Twelve p r e p u b e r a l crossbred gilts, 169 _+ 3 days of age and 72.8 + 3.4 kg b o d y w e i g h t w e r e hypophysial stalk-transected (HST) or sham hypophysial stalk-transected (S-HST) using the surgical a p p r o a c h to h y p o p h y s e c t o m y of du Mesnil du Buisson et al (9) as modified by Kraeling (10). A Teflon disc, 5 mm in diameter and .5 m m thick, was p l a c e d b e t w e e n the severed ends of the stalk to p r e v e n t vascular regeneration from the hypothalamus to the anterior pituitary gland. The same surgical p r o c e d u r e s w e r e p e r f o r m e d on the S-HST gilts as the HST gilts, e x c e p t the hypophysial stalk was not severed. All pigs r e c e i v e d 50 mg cortisone acetate (im) immediately before and after surgery. All gilts w e r e fed a 14% protein corn-soybean meal diet and mineral mix was added to the feed of HST gilts daily Gilts w e r e o v a r i e c t o m i z e d 6 days after HST or S-HST. At 60 _+ 3 days after HST or S-HST, a cannula was p l a c e d nonsurgically into a jugular vein (11) and the gilts w e r e assigned to four treatment groups of 3 gilts each in a 2 )< 2 factorial arrangement. Half of the HST and S-HST gilts received 5 mg estradiol benzoate (EB) in corn oil or corn oil vehicle injected (im) at 0 8 0 0 hr daily for 5 days beginning 4 days after cannulation. Blood samples w e r e c o l l e c t e d at 0 8 3 0 and 0 9 0 0 hr the day after the last injection of EB or corn oil. Immediately after the 0 9 0 0 hr sample, 200 ~tg of thyrotropin releasing h o r m o n e (TRH) w e r e injected (iv) to d e t e r m i n e the capacity of the anterior pituitary gland to release PRL. Blood was c o l l e c t e d at 10 min intervals for 1 hr and every 30 min for the next 1.5 hr. Serum was stored at - - 2 0 C until quantitated for PRL by a d o u b l e - a n t i b o d y radioimmunoassay (RIA) described by Kraeling et al. (11) The intraassay coefficient of variation was 16.3%. All samples were quantitated in the same assay. Body w e i g h t gain was r e c o r d e d 59 + 3 days after HST or S-HST. At n e c r o p s y (86 +_ 7 days after HST or S-HST), w h o l e pituitary, thyroid and adrenal gland weights w e r e recorded. Sections of pituitary glands w e r e cut at 10 ~tm and stained with hematoxylin-eosin for histological examination. These data w e r e subjected to a one-way analysis of variance by the p r o c e d u r e s of the Statistical Analysis System (12). The area u n d e r the serum PRL response curve after TRH was estimated by an integration of the data using a mean of the 0 8 3 0 and 0 9 0 0 hr serum PRL concentrations at baseline. Area u n d e r the PRL response curve was expressed as ng PRL'mI-I'150 min -1. These data w e r e subjected to a two-way analysis of variance by SAS. RESULTS Examination of the hypothalamo-hypophysial region of the HST gilts at n e c r o p s y revealed that the hypophysial stalk was severed c o m p l e t e l y and that the Teflon barrier was in the p r o p e r position to p r e v e n t vascular regeneration of the hypophysial stalk. Body weight gain, and w h o l e pituitary and adrenal weights w e r e greater ( P < . 0 5 ) for S-HST than for HST gilts, whereas thyroid w e i g h t was similar for S-HST and HST gilts (Table 1). TABLE 1. BODYWEIGHT GAIN AND ENDOCRINE GLANDWEIGHTS (MEAN + SE) OF GILTS AFTER HYPOPHYSIAL STALK-TRANSECTION(HST) AND SHAM HST (S-HST). Body wt. Whole pituitary Thyroid Total Adrenal Treatment gain, kg weight, mR weight, g weight, g S-HST 21.5 +- 1.5" 322.0 _+ 9.9" 8.3 + .3 6.9 -+ 1.0" HST 13.2 -+ 2.2 106.8 + 13.5 7.3 + .7 3.7 -+ .5
•Greater (P<.05) than HST.
P R L IN E B - T R E A T E D , S T A L K - T R A N S E C T E D
GILTS
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Fig. 1. Serum prolactin (PRL) concentrations in response to thyrotropin releasing hormone (TRFI) in hypophysial stalk-transected (HST) and sham HST (S-HST) gilts treated with estrogen (E2) or oil. (Serum PRL concentration at 0 rain is the average of the - - 3 0 and 0 min samples). Time of TRH injection = 0 minutes.
Gross examination of the pituitary of FIST gilts revealed variable quantities of surviving tissue in the peripheral regions of the adenohypophysis. Histological examination of the adenohypophysis revealed the presence of acidophils and basophils within the viable tissue of the HST gilts. Serum PRL concentrations after TRH are presented in Figure 1 and areas under the PRL response curves are presented in Table 2. Surgery (HST vs SFIST) x treatment (EB vs oil) interactions were not significant for either parameter. Mean basal (-- 30 and 0 min before TRH) serum PRL concentrations were similar for the HST (10.3 - 1.0 ng/ml) and S-HST (12.3 -+ 1.7 ng/ml) gilts, however, the mean basal serum PRL concentrations were greater (P<.05) for the EB-treated gilts (13.7 - 1.3 ng/ml) than for the oil.treated gilts (8.8 -+ .5 ng/ml). Mean serum PRL concentration increased significantly within 10 rain and returned to approximately 20 ng/ml by 150 min after TRH in all four groups. Maximum serum PRL concentrations observed at 10 rain after TRH were greater ( P < . 0 1 ) for the S-HST ( 2 5 5 . 9 -+ 29.6 ng/ml) than HST gilts TABLE 2. S~RUM PROLACrIN (PRL) RESPONSE"TO THYROTROPIN RELEASINGHORMONE IN ESTROGEN (EB) TRI~TED HYPOPHYSIALSTALK-TRANSECTIONED,(HST), AND SHAM OPERATED (S-HST) GILTS Area under the curve a¢ Treatment No. of Gilts n~ PRL ml "1 150rain" S-HST -t- oil 3 703.9 S.HST + EB 3 1,389.9 HST -k oil 3 166.9 HST -4- EB 3 337.7 •Expressed as area under the response curve (ng PRL ml -~ 1 5 0 rain-l). bPooled S.E. = 1 1 4 . 0 ng ml" 1 5 0 min". =Main effects of HST vs S.HST and EB vs oil are significant.
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(83.4 + 18.8 n g / m l ) , b u t s e r u m PRL c o n c e n t r a t i o n s at 10 rain after TRH w e r e not different for the EB ( 1 9 8 . 0 _+ 50.6 n g / m l ) and oil treated gilts (141.4 _+ 36.3 n g / m l ) . Area u n d e r the s e r u m PRL r e s p o n s e c u r v e (Table 2) after TRH was greater ( P < . 0 0 5 ) for S-HST than HST gilts and for EB than oil-treated gilts
(P<.05). DISCUSSION Although there w e r e extensive areas of necrosis in anterior pituitary glands of HST gilts, there w e r e also extensive areas of surviving tissue o b s e r v e d u p o n histological e x a m i n a t i o n . Mean s e r u m PRL c o n c e n t r a t i o n s at 0 8 3 0 and 0 9 0 0 hr (Figure 1) indicate that basal PRL secretion was similar for the HST and SHST gilts. Anderson et al (13) r e p o r t e d that s e r u m PRL c o n c e n t r a t i o n s r e m a i n e d elevated in HST gilts c o m p a r e d to S-HST gilts to 192 hr after surgery and therefore, c o n c l u d e d that basal PRL s e c r e t i o n in the pig is u n d e r tonic inhibition by the h y p o t h a l a m u s . In o u r study, pituitary gland w e i g h t was 65% less in gilts 86 + 7 days after HST c o m p a r e d to S-HST gilts w h e r e a s in the study r e p o r t e d b y Anderson et al (13) pituitary gland w e i g h t was 32% less in gilts 10 days after HST c o m p a r e d to S-HST gilts. Another factor c o n t r i b u t i n g to the i n c o n s i s t e n c y b e t w e e n o u r data and that of Anderson and c o w o r k e r s (13) m a y be the difference in the interval b e t w e e n surgery and b l o o d sampling. Perhaps s e r u m PRL c o n c e n t r a t i o n s in HST gilts d e c r e a s e d w i t h t i m e after surgery. The pattern of PRL release in r e s p o n s e to TRH was similar to that r e p o r t e d b y Van L a n d e g h e m and Van de Weil ( 1 4 ) , Bevers and W i l l e m s e (15) and Kraeling et al ( 1 6 ) . Based on basal s e r u m PRL c o n c e n t r a t i o n s and the s e r u m PRL r e s p o n s e after TRH, e x p r e s s e d as area u n d e r the curve, w e c o n c l u d e that estrogen s t i m u l a t e d basal PRL s e c r e t i o n and the capacity of the pituitary gland to release PRL in r e s p o n s e to a s e c r e t a g o g u e such as TRH. Estrogen stimulation of PRL s e c r e t i o n also was d e m o n s t r a t e d in v i v o in the rat ( 1 7 , 1 8 , 1 9 , 2 0 ) , s h e e p ( 2 1 , 2 2 ) and p r i m a t e ( 2 3 ) . In addition, in v i t r o i n c u b a t i o n of anterior pituitary tissue in the p r e s e n c e of estrogen resulted in s t i m u l a t i o n of PRL synthesis and release in rats ( 2 4 , 2 5 , 2 6 , 2 7 , 2 8 , 2 9 ) , s h e e p ( 3 0 , 3 1 ) and cattle (32). These results do not eliminate the possible influence of estrogen at the h y p o t h a l a m i c level, b u t indicate that estrogen directly s t i m u l a t e d the anterior pituitary gland to secrete PRL. These results s u p p o r t the c o n c e p t suggested by Bevers et al (3) and Stevenson et al (7) that estrogen is the trigger w h i c h causes the p e r i e s t r o u s surge in s e r u m PRL c o n c e n t r a t i o n s in the pig and at least part of the m e c h a n i s m involved is a direct action of estrogen on the anterior pituitary gland. ACKNOWLEDGMENTS The authors gratefully acknowledge Dr. Douglas J. Bolt, USDA, Beltsville, MD, for supplying the purified PRL used in the RIA's. The assistance of Mr. Ruel L. Wilson, USDA, with the statistical analysis and the technical assistance of Ms. Elizabeth E. PriceTaras, Mr. Bennett Johnson, Ms. Nancy A. Fiorello-Stocks, Ms. Mary A. Hart are greatly appreciated. REFERENCES 1. Guthrie HD, Henricks DM, Handlin DL. Plasma estrogen, progesterone and luteinizing hormone prior to estrus and during early pregnancy in pigs. Endocrinology 91:675-679, 1972. 2. Henricks DM, Guthrie HD, Handlin DL. Plasma estrogen, progesterone and luteinizing hormone levels during the estrous cycle in pigs. Biol Reprod 6:210-218, 1972.
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3. Bevers MM, Willemse AH, Kruip ThAM. Plasma prolactin levels in the sow during lactation and the postweaning period as measured by radioimmunoassay. Biol Reprod 19:628-634, 1978. 4. Dusza L, Krzymowska H. Plasma prolactin concentrations during the oestrous cycle of sows. J Reprod Fert 57:511-514, 1979. 5. Brinkley HJ. Endocrine signaling and female reproduction. Biol Reprod 24:22-43, 1981. 6. Van de Wiel DFM, Erkins J, Koops W, Vos E, Van Landeghem AAJ. Periestrous and midluteal time courses of circulating LH, FSH, prolactin, estradiol-17[3 and progesterone in the domestic pig. Biol Reprod 24:223-233, 1981. 7. StevensonJS, Cox NM, Britt JH. Role of the ovary in controlling luteinizing hormone, follicle stimulating hormone, and prolactin secretion during and after lactation in pigs. Biol Reprod 24:341-353, 1981. 8. Hoover DJ, Brinkley HJ, Rayford PL, Young EP. Effect of injected progesterone, estradiol and estrone on serum LH, FSH and PRL in ovariectomized (OVX)o sows. J Anim Sci 45(Suppl 1):171, 1977. 9. Du Mesnil Du Buisson F, Leglise PC, Chodkiewicz JP. Technique de l'hypophysectomie par vole transfrontale sus-orbitaire chez le porc. Ann Biol Anim Biochim Biophys 4:229-237, 1964. 10. Kraeling RR. A modified supra-obital approach to hypophysectomy in the pig. Amer J Vet Res 34:283-285, 1973. 11. Kraeling RR, Rampacek GB, Cox NM, Kiser TE. Prolactin and luteinizing hormone secretion after bromocryptine (CB-154) treatment in lactating sows and ovariectomized gilts. J Anim Sci 54:1212-1220, 1982. 12. SAS. SAS User's Guide. Statistical Analysis System Institute, Inc, Cary, NC, 1979. 13. Anderson LL, Berardinelli JG, Malven PV, Ford JJ. Prolactin secretion after hypophysial stalk section in pigs. Endocrinology 111:380-384, 1982. 14. Van Landeghem AAJ, Van de Wiel DFM. Radioimmunoassay for porcine prolactin plasma levels during lactation, suckling and weaning and after TRH administration. Acta Endocrinol 88:653-667, 1978. 15. Bevers MM, Willemse AH. Effect of synthetic TRH on prolactin release in the pig. Theriogenology 18:303-309, 1982. 16. Kraeling RR, Rampacek GB, Mabry JW, Cunningham FL, Pinkert CA. Serum concentrations of pituitary and adrenal hormones in female pigs exposed to two photoperiods. J Anim Sci 57:1243-1250, 1983. 17. Meites J, Lu KH, Wuttke W, Welsch CW, Nagasawa H, Quadri SK. Recent studies on functions and control of prolactin secretion in rats. Rec Progr Hormone Res 28:471-516, 1972. 18. Caligaris L, Astrada JJ, Taleisnik S. Oestrogen and progesterone influence on the release of prolactin in ovariectomized rats. J Endocrinol 60:205-215, 1974. 19. Lawson DM. Evidence for a rapid in v i v o effect of estradiol-17[3 on prolactin secretion in ovariectomized rats. Endocrine Res Comm 6:135-148, 1979. 20. Johnson MD, Crowley WR. Acute effects of estradiol on circulating luteinizing hormone and prolactin concentrations and on serotonin turnover in individual brain nuclei. Endocrinology 113:1935-1941, 1983. 21. Fell LR, Beck C, Brown JM, Cumming IA, Goding JR. Radioimmunoassay for ovine prolactin. The secretion of prolactin as affected by milking, oestradiol administration and onset of partur.ition. J Reprod Fertil 28:133-134, 1972. 22. Shupnik MA, Baxter LA, French LR, Gorski J. In vivo effects of estrogen on ovine pituitary prolactin and growth hormone biosynthesis and messenger ribonucleic acid translation. Endocrinology 104:729-735, 1979. 23. Milmore JE. Influence of ovarian hormones on prolactin release in the Rhesus monkey. Biol Reprod 19:593-596, 1978. 24. Nicoll CS, Meites J. Estrogen stimulation of prolactin production by rat adenohypophysis in vitro. Endocrinology 70:272-277, 1962. 25. Haug E, Gautvik KM. Effects of sex steroids on prolactin secreting rat pituitary cells in culture. Endocrinology 99:1428-1489, 1976. 26. Jaques S Jr, Gala RR. The influence of oestrogen administration on in v i t r o prolactin release. Acta Endocrinol 92:437-447, 1979.
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27. West P, Dannies PS. Effects of estradiol on prolactin production and dehydroergocryptine-induced inhibition of prolactin production in primary cultures of rat pituitary cells. Endocrinology 106:1108-1113, 1980. 28. Zyzek E, Dufy-Barbe L, Dufy B, Vincent JD. Short-term effects of estrogen on release of prolactin by pituitary cells in culture. Biochem and Biophys Res Comm 102:1151 1157, 1981. 29. Lieberman ME, Maurer RA, Claude P, GorskiJ. Prolactin synthesis in primary cultures of pituitary cells: Regulation by estradiol. Molec and Cell Endocrinol 25:277-294, 1982. 30. Vician L, Shupnik MA, Gorski J. Effects of estrogen on primary ovine pituitary cell cultures-stimulation of prolactin secretion, synthesis, and preprolactin messenger ribonucleic acid activity. Endocrinology 104:736-743, 1979. 31. Peclaris GM. Effects of estradiol and progesterone on PRL release for dispersed pituitary cells of immature female lambs. Endocrinologia Experimentalis 17:5358, 1983. 32. Padmanabhan V, Convey EM. Estradiol-17-~ stimulates basal and thyrotropin releasing hormone induced prolactin secretion by bovine pituitary cells in primary culture. Molec and Cell Endocrinol 14:103-112, 1979.
Vol. 3(4):289-294,1986
SERUM PROLACTIN RESPONSE TO THYROTROPIN RELEASING HORMONE IN ESTROGEN TREATED HYPOPHYSlAL STALK-TRANSECTED GILTS Robert R. Kraeling* and George B. Rampacek** *Animal Physiology Unit, Richard B. Russell Agricultural Research Center ARS, USDA, Athens, GA 30613 and **Animal and Dairy Science Department, University of Georgia Athens, Georgia 30602 Received March 10, 1986
ABSTRACT Twelve crossbred gilts, 169 + 3 days of age and 72.8 + 3.4 kg body weight, were hypophysial stalk-transected (I-IST)I or sham hypophysial stalk-transected (S-HST). Gilts were ovariectomized 6 days later and assigned to four treatments of 3 gilts each in a 2 x 2 factorial arrangement. One-half of the HST and S-HSTgilts received 5 mg estradiolbenzoate (EB) or corn oil vehicle tm at 0800 hr daily for 5 days beginning 64 _+ 3 days after HST or S-HST. Blood was collected by jugular vein cannula at 0830 and 0900 hr the day after the last injection of EB or oil. Immediately after the 0900 hr sample, 200 ttg thyrotropin releasing hormone (TRH) were injected (iv). Mean basal serum prolactin (PRL) concentration was similar for HST (10.3 + 1.0 ng/ml) and S-HST (12.3 _+ 1.7 ng/ml) gilts, however mean basal serum PRL concentration was greater (P<.05) for EB-treated gilts (13.7 -+ 1.3 ng/ml) than for oil-treated gilts (8.8 -+ .5 ng/ml). Mean serum PRL concentration of all gilts increased within 10 rain and returned to approximately 20 ng/ml by 150 rain after TRH. Maximum serum PRL concentrations at 10 min after TRH were greater (P<.01) for S-HST (255.9 -+ 29.6 ng/ml) than HST gilts (83.4 - 18.8 ng/ml), but were not different for EB (198.0 + 50.6 ng/ml) and oil-treated gilts (141.4 + 36.3 ng/ml). Area under the serum PRL response curve after TRH was greater (P<.005) for S-HST than HST gilts and for EB than oil-treated gilts (P<.05). These results do not eliminate the possible influence of estrogen on PRL secretion at the hypothalamus, but do indicate that estrogen directly stimulated the anterior pituitary gland to secrete PRL. INTRODUCTION The t e m p o r a l relationships b e t w e e n b l o o d estrogen, luteinizing h o r m o n e (LH) and prolactin (PRL) c o n c e n t r a t i o n s during the periestrous p e r i o d in the gilt and p o s t p a r t u m sow have b e e n described by Guthrie et al (1), HHenricks et al (2), Berets et al (3), Dusza and Krzymowska (4), Brinkley (5), and Van de Wiel et al (6). Mean b l o o d estrogen concentrations were basal until proestrus w h e n a three- to fourfold increase occurred. A decline in b l o o d estrogen c o n c e n t r a t i o n began p r i o r to the p r e o v u l a t o r y LH surge and the p r e o v u l a t o r y LH surge o c c u r r e d w i t h i n 24 hr f o l l o w i n g the onset o f behavioral estrus. Two surges o f b l o o d PRL c o n c e n t r a t i o n s generally o c c u r r e d during the periestrous p e r i o d in gilts; one surge began near the end o f luteal regression and o t h e r just before the p r e o v u l a t o r y LH surge. The cause and effect relationship b e t w e e n estrogen and PRL secretion has not b e e n d e t e r m i n e d in the pig. However, Bevers et al (3) and Stevenson et al (7) suggested that estrogen not only stimulates the p r e o v u l a t o r y LH surge, but also stimulates the periestrous surge of PRL. In addition, H o o v e r et al (8) and Stevenson et al (7) r e p o r t e d that e x o g e n o u s estrogen caused an increase in plasma PRL c o n c e n t r a t i o n in ovar-
Copyright©
1986 by DOMENDO, INC.
289
0739-7240/86/$3.00
290
KRAELING AND RAMPACEK
i e c t o m i z e d sows. The mechanism by w h i c h estrogen might stimulate PRL secretion in the pig has not b e e n investigated. Therefore, the objective of this e x p e r i m e n t was to d e t e r m i n e if estrogen stimulates PRL secretion in the pig by direct action on the anterior pituitary gland. MATERIALS A N D METHODS
Twelve p r e p u b e r a l crossbred gilts, 169 _+ 3 days of age and 72.8 + 3.4 kg b o d y w e i g h t w e r e hypophysial stalk-transected (HST) or sham hypophysial stalk-transected (S-HST) using the surgical a p p r o a c h to h y p o p h y s e c t o m y of du Mesnil du Buisson et al (9) as modified by Kraeling (10). A Teflon disc, 5 mm in diameter and .5 m m thick, was p l a c e d b e t w e e n the severed ends of the stalk to p r e v e n t vascular regeneration from the hypothalamus to the anterior pituitary gland. The same surgical p r o c e d u r e s w e r e p e r f o r m e d on the S-HST gilts as the HST gilts, e x c e p t the hypophysial stalk was not severed. All pigs r e c e i v e d 50 mg cortisone acetate (im) immediately before and after surgery. All gilts w e r e fed a 14% protein corn-soybean meal diet and mineral mix was added to the feed of HST gilts daily Gilts w e r e o v a r i e c t o m i z e d 6 days after HST or S-HST. At 60 _+ 3 days after HST or S-HST, a cannula was p l a c e d nonsurgically into a jugular vein (11) and the gilts w e r e assigned to four treatment groups of 3 gilts each in a 2 )< 2 factorial arrangement. Half of the HST and S-HST gilts received 5 mg estradiol benzoate (EB) in corn oil or corn oil vehicle injected (im) at 0 8 0 0 hr daily for 5 days beginning 4 days after cannulation. Blood samples w e r e c o l l e c t e d at 0 8 3 0 and 0 9 0 0 hr the day after the last injection of EB or corn oil. Immediately after the 0 9 0 0 hr sample, 200 ~tg of thyrotropin releasing h o r m o n e (TRH) w e r e injected (iv) to d e t e r m i n e the capacity of the anterior pituitary gland to release PRL. Blood was c o l l e c t e d at 10 min intervals for 1 hr and every 30 min for the next 1.5 hr. Serum was stored at - - 2 0 C until quantitated for PRL by a d o u b l e - a n t i b o d y radioimmunoassay (RIA) described by Kraeling et al. (11) The intraassay coefficient of variation was 16.3%. All samples were quantitated in the same assay. Body w e i g h t gain was r e c o r d e d 59 + 3 days after HST or S-HST. At n e c r o p s y (86 +_ 7 days after HST or S-HST), w h o l e pituitary, thyroid and adrenal gland weights w e r e recorded. Sections of pituitary glands w e r e cut at 10 ~tm and stained with hematoxylin-eosin for histological examination. These data w e r e subjected to a one-way analysis of variance by the p r o c e d u r e s of the Statistical Analysis System (12). The area u n d e r the serum PRL response curve after TRH was estimated by an integration of the data using a mean of the 0 8 3 0 and 0 9 0 0 hr serum PRL concentrations at baseline. Area u n d e r the PRL response curve was expressed as ng PRL'mI-I'150 min -1. These data w e r e subjected to a two-way analysis of variance by SAS. RESULTS Examination of the hypothalamo-hypophysial region of the HST gilts at n e c r o p s y revealed that the hypophysial stalk was severed c o m p l e t e l y and that the Teflon barrier was in the p r o p e r position to p r e v e n t vascular regeneration of the hypophysial stalk. Body weight gain, and w h o l e pituitary and adrenal weights w e r e greater ( P < . 0 5 ) for S-HST than for HST gilts, whereas thyroid w e i g h t was similar for S-HST and HST gilts (Table 1). TABLE 1. BODYWEIGHT GAIN AND ENDOCRINE GLANDWEIGHTS (MEAN + SE) OF GILTS AFTER HYPOPHYSIAL STALK-TRANSECTION(HST) AND SHAM HST (S-HST). Body wt. Whole pituitary Thyroid Total Adrenal Treatment gain, kg weight, mR weight, g weight, g S-HST 21.5 +- 1.5" 322.0 _+ 9.9" 8.3 + .3 6.9 -+ 1.0" HST 13.2 -+ 2.2 106.8 + 13.5 7.3 + .7 3.7 -+ .5
•Greater (P<.05) than HST.
P R L IN E B - T R E A T E D , S T A L K - T R A N S E C T E D
GILTS
291
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Fig. 1. Serum prolactin (PRL) concentrations in response to thyrotropin releasing hormone (TRFI) in hypophysial stalk-transected (HST) and sham HST (S-HST) gilts treated with estrogen (E2) or oil. (Serum PRL concentration at 0 rain is the average of the - - 3 0 and 0 min samples). Time of TRH injection = 0 minutes.
Gross examination of the pituitary of FIST gilts revealed variable quantities of surviving tissue in the peripheral regions of the adenohypophysis. Histological examination of the adenohypophysis revealed the presence of acidophils and basophils within the viable tissue of the HST gilts. Serum PRL concentrations after TRH are presented in Figure 1 and areas under the PRL response curves are presented in Table 2. Surgery (HST vs SFIST) x treatment (EB vs oil) interactions were not significant for either parameter. Mean basal (-- 30 and 0 min before TRH) serum PRL concentrations were similar for the HST (10.3 - 1.0 ng/ml) and S-HST (12.3 -+ 1.7 ng/ml) gilts, however, the mean basal serum PRL concentrations were greater (P<.05) for the EB-treated gilts (13.7 - 1.3 ng/ml) than for the oil.treated gilts (8.8 -+ .5 ng/ml). Mean serum PRL concentration increased significantly within 10 rain and returned to approximately 20 ng/ml by 150 min after TRH in all four groups. Maximum serum PRL concentrations observed at 10 rain after TRH were greater ( P < . 0 1 ) for the S-HST ( 2 5 5 . 9 -+ 29.6 ng/ml) than HST gilts TABLE 2. S~RUM PROLACrIN (PRL) RESPONSE"TO THYROTROPIN RELEASINGHORMONE IN ESTROGEN (EB) TRI~TED HYPOPHYSIALSTALK-TRANSECTIONED,(HST), AND SHAM OPERATED (S-HST) GILTS Area under the curve a¢ Treatment No. of Gilts n~ PRL ml "1 150rain" S-HST -t- oil 3 703.9 S.HST + EB 3 1,389.9 HST -k oil 3 166.9 HST -4- EB 3 337.7 •Expressed as area under the response curve (ng PRL ml -~ 1 5 0 rain-l). bPooled S.E. = 1 1 4 . 0 ng ml" 1 5 0 min". =Main effects of HST vs S.HST and EB vs oil are significant.
292
KRAELING AND RAMPACEK
(83.4 + 18.8 n g / m l ) , b u t s e r u m PRL c o n c e n t r a t i o n s at 10 rain after TRH w e r e not different for the EB ( 1 9 8 . 0 _+ 50.6 n g / m l ) and oil treated gilts (141.4 _+ 36.3 n g / m l ) . Area u n d e r the s e r u m PRL r e s p o n s e c u r v e (Table 2) after TRH was greater ( P < . 0 0 5 ) for S-HST than HST gilts and for EB than oil-treated gilts
(P<.05). DISCUSSION Although there w e r e extensive areas of necrosis in anterior pituitary glands of HST gilts, there w e r e also extensive areas of surviving tissue o b s e r v e d u p o n histological e x a m i n a t i o n . Mean s e r u m PRL c o n c e n t r a t i o n s at 0 8 3 0 and 0 9 0 0 hr (Figure 1) indicate that basal PRL secretion was similar for the HST and SHST gilts. Anderson et al (13) r e p o r t e d that s e r u m PRL c o n c e n t r a t i o n s r e m a i n e d elevated in HST gilts c o m p a r e d to S-HST gilts to 192 hr after surgery and therefore, c o n c l u d e d that basal PRL s e c r e t i o n in the pig is u n d e r tonic inhibition by the h y p o t h a l a m u s . In o u r study, pituitary gland w e i g h t was 65% less in gilts 86 + 7 days after HST c o m p a r e d to S-HST gilts w h e r e a s in the study r e p o r t e d b y Anderson et al (13) pituitary gland w e i g h t was 32% less in gilts 10 days after HST c o m p a r e d to S-HST gilts. Another factor c o n t r i b u t i n g to the i n c o n s i s t e n c y b e t w e e n o u r data and that of Anderson and c o w o r k e r s (13) m a y be the difference in the interval b e t w e e n surgery and b l o o d sampling. Perhaps s e r u m PRL c o n c e n t r a t i o n s in HST gilts d e c r e a s e d w i t h t i m e after surgery. The pattern of PRL release in r e s p o n s e to TRH was similar to that r e p o r t e d b y Van L a n d e g h e m and Van de Weil ( 1 4 ) , Bevers and W i l l e m s e (15) and Kraeling et al ( 1 6 ) . Based on basal s e r u m PRL c o n c e n t r a t i o n s and the s e r u m PRL r e s p o n s e after TRH, e x p r e s s e d as area u n d e r the curve, w e c o n c l u d e that estrogen s t i m u l a t e d basal PRL s e c r e t i o n and the capacity of the pituitary gland to release PRL in r e s p o n s e to a s e c r e t a g o g u e such as TRH. Estrogen stimulation of PRL s e c r e t i o n also was d e m o n s t r a t e d in v i v o in the rat ( 1 7 , 1 8 , 1 9 , 2 0 ) , s h e e p ( 2 1 , 2 2 ) and p r i m a t e ( 2 3 ) . In addition, in v i t r o i n c u b a t i o n of anterior pituitary tissue in the p r e s e n c e of estrogen resulted in s t i m u l a t i o n of PRL synthesis and release in rats ( 2 4 , 2 5 , 2 6 , 2 7 , 2 8 , 2 9 ) , s h e e p ( 3 0 , 3 1 ) and cattle (32). These results do not eliminate the possible influence of estrogen at the h y p o t h a l a m i c level, b u t indicate that estrogen directly s t i m u l a t e d the anterior pituitary gland to secrete PRL. These results s u p p o r t the c o n c e p t suggested by Bevers et al (3) and Stevenson et al (7) that estrogen is the trigger w h i c h causes the p e r i e s t r o u s surge in s e r u m PRL c o n c e n t r a t i o n s in the pig and at least part of the m e c h a n i s m involved is a direct action of estrogen on the anterior pituitary gland. ACKNOWLEDGMENTS The authors gratefully acknowledge Dr. Douglas J. Bolt, USDA, Beltsville, MD, for supplying the purified PRL used in the RIA's. The assistance of Mr. Ruel L. Wilson, USDA, with the statistical analysis and the technical assistance of Ms. Elizabeth E. PriceTaras, Mr. Bennett Johnson, Ms. Nancy A. Fiorello-Stocks, Ms. Mary A. Hart are greatly appreciated. REFERENCES 1. Guthrie HD, Henricks DM, Handlin DL. Plasma estrogen, progesterone and luteinizing hormone prior to estrus and during early pregnancy in pigs. Endocrinology 91:675-679, 1972. 2. Henricks DM, Guthrie HD, Handlin DL. Plasma estrogen, progesterone and luteinizing hormone levels during the estrous cycle in pigs. Biol Reprod 6:210-218, 1972.
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3. Bevers MM, Willemse AH, Kruip ThAM. Plasma prolactin levels in the sow during lactation and the postweaning period as measured by radioimmunoassay. Biol Reprod 19:628-634, 1978. 4. Dusza L, Krzymowska H. Plasma prolactin concentrations during the oestrous cycle of sows. J Reprod Fert 57:511-514, 1979. 5. Brinkley HJ. Endocrine signaling and female reproduction. Biol Reprod 24:22-43, 1981. 6. Van de Wiel DFM, Erkins J, Koops W, Vos E, Van Landeghem AAJ. Periestrous and midluteal time courses of circulating LH, FSH, prolactin, estradiol-17[3 and progesterone in the domestic pig. Biol Reprod 24:223-233, 1981. 7. StevensonJS, Cox NM, Britt JH. Role of the ovary in controlling luteinizing hormone, follicle stimulating hormone, and prolactin secretion during and after lactation in pigs. Biol Reprod 24:341-353, 1981. 8. Hoover DJ, Brinkley HJ, Rayford PL, Young EP. Effect of injected progesterone, estradiol and estrone on serum LH, FSH and PRL in ovariectomized (OVX)o sows. J Anim Sci 45(Suppl 1):171, 1977. 9. Du Mesnil Du Buisson F, Leglise PC, Chodkiewicz JP. Technique de l'hypophysectomie par vole transfrontale sus-orbitaire chez le porc. Ann Biol Anim Biochim Biophys 4:229-237, 1964. 10. Kraeling RR. A modified supra-obital approach to hypophysectomy in the pig. Amer J Vet Res 34:283-285, 1973. 11. Kraeling RR, Rampacek GB, Cox NM, Kiser TE. Prolactin and luteinizing hormone secretion after bromocryptine (CB-154) treatment in lactating sows and ovariectomized gilts. J Anim Sci 54:1212-1220, 1982. 12. SAS. SAS User's Guide. Statistical Analysis System Institute, Inc, Cary, NC, 1979. 13. Anderson LL, Berardinelli JG, Malven PV, Ford JJ. Prolactin secretion after hypophysial stalk section in pigs. Endocrinology 111:380-384, 1982. 14. Van Landeghem AAJ, Van de Wiel DFM. Radioimmunoassay for porcine prolactin plasma levels during lactation, suckling and weaning and after TRH administration. Acta Endocrinol 88:653-667, 1978. 15. Bevers MM, Willemse AH. Effect of synthetic TRH on prolactin release in the pig. Theriogenology 18:303-309, 1982. 16. Kraeling RR, Rampacek GB, Mabry JW, Cunningham FL, Pinkert CA. Serum concentrations of pituitary and adrenal hormones in female pigs exposed to two photoperiods. J Anim Sci 57:1243-1250, 1983. 17. Meites J, Lu KH, Wuttke W, Welsch CW, Nagasawa H, Quadri SK. Recent studies on functions and control of prolactin secretion in rats. Rec Progr Hormone Res 28:471-516, 1972. 18. Caligaris L, Astrada JJ, Taleisnik S. Oestrogen and progesterone influence on the release of prolactin in ovariectomized rats. J Endocrinol 60:205-215, 1974. 19. Lawson DM. Evidence for a rapid in v i v o effect of estradiol-17[3 on prolactin secretion in ovariectomized rats. Endocrine Res Comm 6:135-148, 1979. 20. Johnson MD, Crowley WR. Acute effects of estradiol on circulating luteinizing hormone and prolactin concentrations and on serotonin turnover in individual brain nuclei. Endocrinology 113:1935-1941, 1983. 21. Fell LR, Beck C, Brown JM, Cumming IA, Goding JR. Radioimmunoassay for ovine prolactin. The secretion of prolactin as affected by milking, oestradiol administration and onset of partur.ition. J Reprod Fertil 28:133-134, 1972. 22. Shupnik MA, Baxter LA, French LR, Gorski J. In vivo effects of estrogen on ovine pituitary prolactin and growth hormone biosynthesis and messenger ribonucleic acid translation. Endocrinology 104:729-735, 1979. 23. Milmore JE. Influence of ovarian hormones on prolactin release in the Rhesus monkey. Biol Reprod 19:593-596, 1978. 24. Nicoll CS, Meites J. Estrogen stimulation of prolactin production by rat adenohypophysis in vitro. Endocrinology 70:272-277, 1962. 25. Haug E, Gautvik KM. Effects of sex steroids on prolactin secreting rat pituitary cells in culture. Endocrinology 99:1428-1489, 1976. 26. Jaques S Jr, Gala RR. The influence of oestrogen administration on in v i t r o prolactin release. Acta Endocrinol 92:437-447, 1979.
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27. West P, Dannies PS. Effects of estradiol on prolactin production and dehydroergocryptine-induced inhibition of prolactin production in primary cultures of rat pituitary cells. Endocrinology 106:1108-1113, 1980. 28. Zyzek E, Dufy-Barbe L, Dufy B, Vincent JD. Short-term effects of estrogen on release of prolactin by pituitary cells in culture. Biochem and Biophys Res Comm 102:1151 1157, 1981. 29. Lieberman ME, Maurer RA, Claude P, GorskiJ. Prolactin synthesis in primary cultures of pituitary cells: Regulation by estradiol. Molec and Cell Endocrinol 25:277-294, 1982. 30. Vician L, Shupnik MA, Gorski J. Effects of estrogen on primary ovine pituitary cell cultures-stimulation of prolactin secretion, synthesis, and preprolactin messenger ribonucleic acid activity. Endocrinology 104:736-743, 1979. 31. Peclaris GM. Effects of estradiol and progesterone on PRL release for dispersed pituitary cells of immature female lambs. Endocrinologia Experimentalis 17:5358, 1983. 32. Padmanabhan V, Convey EM. Estradiol-17-~ stimulates basal and thyrotropin releasing hormone induced prolactin secretion by bovine pituitary cells in primary culture. Molec and Cell Endocrinol 14:103-112, 1979.