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Circulating concentrations of 17-estradiol influence pattern of LH in circulation of cows
DOMESTICANIMALENDOCRINOLOGY
Vol. 8(4):463-469,1991
CIRCULATING CONCENTRATIONS OF 17-ESTRADIOL INFLUENCE PATTERN OF LH IN CIRCULATION OF COWS 1 J.E. Kinder*z, M. Garcia-Winder *,a, K. Imakawa.4, M.L. Day*,s D.D. Zalesky .6, M.L. D'Occhio *,**,7, T.T. Stumpf*,8 R.J. Kittok* and B.D. Schanbacher **,9 *Department of Animal Science University of Nebraska Lincoln, Nebraska 68583 **USDA, Agricultural Research Service Roman L. Hruska U.S. Meat Animal Research Center Clay Center, Nebraska 6893371° Received July 21, 1989
ABSTRACT The objective of the research was to determine the relationship between circulating 17[3-estradiol (E2) and secretion of luteinizing hormone (LH) in cows. A second objective was to determine if response to E 2 was influenced by interval between ovariectomy and the start of E2 treatment. Thirtyone nulliparous cows 3 yr of age were randomly assigned to a 2 x 4 factorial arrangement of treatments. Sixteen cows were ovariectomized at 18 mo of age (long term), and the other 15 cows were ovariectomized at 36 mo of age (short term). At the time of ovariectomy of cows in the short term group, 11 cows in the short term group and 12 cows in the long term group were implanted subcutaneously with 1, 2 or 4 polydimethylsiloxane capsules containing E 2. The other eight cows served as non-implanted controls (n=4-short term, n=4-1ong term). All cows were fitted with jugular vein catheters on day 29 of treatment, and on day 30 blood samples were collected at 12-rain intervals for 6 hr. At the end of 6 hr, luteinizing hormone-releasing hormone (LHRH) was administered and blood sampling continued at 12-rain intervals for an additional hour. Serum was analyzed for LH and E 2. Variables of LH secretion analyzed were mean concentration, frequency of pulses, amplitude of pulses and maximum concentration after LHRH. There were no significant interactions for any of the variables of LH among cows ovariectomized for the long and short term. There was a significant linear increase in mean concentration of LH with increased circulating concentration of E 2. Frequency of LH pulses was not affected by circulating concentration of E2. As circulating concentration of E 2 increased, amplitude of LH pulses increased and response to LHRH increased - resulting in an increase in mean LH. Interval from time of ovariectomy to the start of E2 treatment only had a minor influence on mean concentration of LH and profile of LH concentrations in circulation. INTRODUCTION Secretion o f LH from the anterior pituitary of the bovine female is under the influence of ovarian steroids (1), and is further modulated by age, (2,3), nutrition (4,5) and photoperiod (6,7,8). Heifers ovariectomized during prepuberty that receive E 2 have lower concentrations of LH than ovariectomized heifers not treated with E2; however, after age-matched intact heifers attain puberty, concentrations of LH in ovariectomized heifers implanted with E 2 are higher than in ovariectomized heifers not receiving E 2 (2,3). Similarly, cows that are o v a r i e c t o m i z e d as a d u l t s and i m p l a n t e d with E 2 have h i g h e r c i r c u l a t i n g L H than ovariectomized cows not receiving E 2 (9). Increased concentrations of LH in circulation of ovariectomized cows implanted with E2 are associated with a higher amplitude o f L H pulses (7,8,9). In contrast, E 2 suppressed amplitude o f LH pulses in adult ovariectomized ewes and also reduced magnitude o f the Copyright © 1991 Butterworth-Heinemann
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increase in tonic secretion of LH after progesterone withdrawal (10). Dose-response relationships between circulating concentrations of steroids and patterns of LH in circulation that occur in males (11) may also occur in females. The male bovine gonadectomized for the long term before treatment with testosterone fails to respond to exogenous testosterone by having changed patterns of LH in circulation (11). An understanding of these relationships in bovine females is important to properly interpret results of studies utilizing hormone replacement, and appreciate the physiological role of E 2. The specific aims of the present research were 1) to determine the relationship between circulating E 2 and secretion of LH in cows and 2) to determine if responses to E 2 are influenced by the interval between ovariectomy and commencement of E 2 treatment.
MATERIALS AND METHODS Animals and Experimental Protocol. Thirty-one Angus-Hereford crossbred and straightbred Hereford nulliparous cows 18 mo of age were randomly assigned by breed groups to a 2 x 4 factorial arrangement of treatments. Sixteen cows were ovariectomized at 18 mo of age, and no further treatments were administered for the following 18 mo (long term). Eighteen months following ovariectomy of cows in the long term group, estrus was synchronized in another group (short term, 36 mo of age) of cows (n=15) by using prostaglandin F2a (Lutalyse, Upjohn Co., Kalamazoo, MI), and cows were ovariectomized on the twelfth d of the estrous cycle. At the time of ovariectomy of cows in the short term group, 11 cows were implanted subcutaneously, caudal to the shoulder blade with 1,2 or 4 polydimethylsiloxane capsules (3.35 mm id x 4.65 mm od x 135 mm) containing crystalline E 2. Twelve cows ovariectomized for the long term were implanted with 1,2 or 4 implants (n=4/group) containing E 2 at the same time as cows ovariectomized for the short term were implanted with E 2. The other eight cows served as non-implanted controls (short term n=4, long term n=4) for their respective groups. The jugular vein was catheterized 29 d after insertion of the implants containing E2 and on the next day serial blood samples (10 ml) were collected at 12-min intervals for 6 hr. Immediately following the 6 hr of blood collection, 1 ~tg of LH-releasing hormone (LHRH) was administered intravenously and blood samples were collected at 12-min intervals for an additional 1 hr. Concentrations of LH were determined by radioimmunoassay (12,13). Intra- and interassay coefficients of variation were 2.5 and 11.6%, respectively. Sensitivity of the assay was 130 pg/ml. Concentrations of E 2 were determined in a single assay (14). Samples for quantifying E 2 were collected at the start, middle and end (preceding LHRH) of the 6 hr sampling period. Statistical Analyses. The variables of LH secretion evaluated were mean concentrations, frequency of pulses, amplitude of pulses and maximum concentrations subsequent to administration of LHRH. Mean concentration of LH for individual cows was the average concentration in the 30 samples collected before administration of LHRH. In addition, LH pulses were identified and amplitude of LH pulses was calculated (15). Frequency of LH pulses was expressed as the number of pulses detected during the 6 hr of blood collection before administration of LHRH. Amplitude of LH pulses was expressed as the average amplitude of all pulses detected during this 6 hr period of blood collection for an individual COW.
A general linear model's procedure was used to analyze the main effects of treatment (short or long term). The sums of squares for circulating concentration of E2 were divided into partial sums of squares to study linear and quadratic effects of E 2 on LH (16). Interactions of the regression coefficients between treatment groups were tested to determine if treatment (short or long term) affected response to E 2. Linear and quadratic regression coefficients for mean concentrations of E 2 and LH in circulation, frequency of LH pulses, amplitude of LH pulses and maximum concentration of LH after administration of LHRH were determined as circulating concentrations of E e increased. The concentration of LH
E S T R A D I O L M O D U L A T I O N O F LH
TABLE 1. MEANCONCENTRATIONSOFLH,
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FREQUENCYOF LH
PULSESANDAMPLITUDEOFLH PULSESIN BLOOD
SERUM OF Cows RECEIVINGDIFFERINGNUMBERS OF 17~-ESTRADIOL(E2) IMPLANTSTHATWERE OVARIECTOMIZED
FORA SNORT(ST) ORLONGTERM(LT) ATTHETIMEOFADMINISTRATIONOFE2a. Number of implants"
0 1 2 ,4
Mean concentration E2 (pg/ml)'
Mean concentration LH (ng/ml)a'e
Frequency of LH pulses (pulses/6 hr) f
Amplitude of LH pulses (ng)g
LT 1.5 2.7 3.9 8.6
LT 2.15 2.81 3.91 3.53
LT 5.3 7.0 6.8 5.5
LT .75 1.17 2.77 2.69
ST 2.1 3.4 4.7 6.0
ST 2.35 3.38 3.60 2.95
ST 7.5 6.0 5.5 3.7
Changes in LH after LHRH h
ST .98 2.50 3.24 3.65
LT 5.88 8.52 5.92 12.37
ST 5.91 7.41 4.87 11.73
'Cows ovariectomized for the short term were ovariectomized and implanted with E 2 on day 12 of the estrous cycle (day 0=estrus). Cows ovariectomized for the long term were ovariectomized 18 mo before being implanted with E r bCows were implanted with E., subcutaneously caudal to the shoulder blade• ePooled SEM = 0.62. dMean concentration of LH during the 6-hr period of blood sampling 30 d after E 2 was administered. ~'r'g'hPooledSEM = 0.36, 0.89, 0.52 and 0.56 for mean concentration of LH, frequency of LH pulses, amplitude of LH pulses and changes in LH after LHRH, respectively.
i m m e d i a t e l y before administration o f L H R H was subtracted from m a x i m u m concentration o f L H after administration o f L H R H to evaluate responsiveness o f the pituitary to L H R H . RESULTS N o difference was d e t e c t e d in m e a n concentrations o f L H (Table 1) a m o n g c o w s that w e r e o v a r i e c t o m i z e d for the short or long term. N o interaction for m e a n concentration o f L H in response to E 2 was detected a m o n g cows in the two treatment groups. W h e n data for c o w s f r o m the two treatment groups were combined, there was a linear increase (P<0.001) o f m e a n concentration o f L H as concentration o f E 2 in circulation increased (Figure 1). N o
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Serum Estradiol (pg/ml) Figure 1. Linear regression (P<0.001) for mean concentrations of LH in cows ovariectomized for a long and short lerm that received 0, 1, 2 or 4 implants of E_,. There was no interaction between the two groups and no effect of treatment, thus data from cows of the two groups were combined for regression analysis.
466
KINDER ET AL.
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Serum Estradiol (pg/ml) Figure 2. Linear regressions (P<0.001) of LH pulse amplitude in cows that were ovariectomized for a long or short term that received 0, 1, 2 or 4 implants of E,. There was no interaction between the two groups, but there was an effect (P<0.05) of treatment.
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Serum Estradiol (pg/ml) Figure 3. Linear regression (P<0.002) of maximum LH after administration of LHRH in cows ovariectomized for a long and short term that received 0, I, 2 or 4 implants of E 2. There was no interaction between the two groups and no effect of treatment, thus data from cows of the two groups were combined for regression analysis.
ESTRADIOL MODULATION OF LH
467
significant difference in frequency of LH pulses (Table 1) existed between the short and long term groups. There was no effect (P>0.10) of circulating concentration of E 2 on frequency of LH pulses. Amplitude of LH pulses was lower (P<0.05) in cows that were ovariectomized for the short term as compared to cows ovariectomized for the long term (Table 1). There was no interaction between the long and short term groups for amplitude of LH pulses. When cows from the two groups were combined there was a linear increase (P<0.001) in amplitude of LH pulses as circulating concentrations of E2 increased (Figure 2). Response of the pituitary to LHRH was not different between cows ovariectomized for short or long term. There was no interaction in response to LHRH between the two treatment groups. Peak response to LHRH increased (P<0.003) linearly as circulating concentration of E 2 increased (Figure 3). DISCUSSION Estradiol is a potent suppresser of secretion of LH in prepubertal heifers (2), ewe lambs (17) and in postpubertal heifers if E 2 and progesterone are both present (18,19). In addition, E 2 is a potent suppresser of LH secretion in the heifer (4,5) and ewe lamb (20) fed diets containing low levels of energy. Estradiol suppresses secretion of LH in adult ovariectomized ewes during both the breeding and nonbreeding season (10,15) but enhances secretion of LH in ovariectomized cows during all seasons of the year (8,21). In the present study, variables of LH secretion were similar between cows ovariectomized for the short or long term. Therefore, cows respond to E2 replacement by modulating secretion of LH regardless of whether they have been ovariectomized for the long or short term at the time of administration of E 2. Amplitude of LH pulses increases as circulating concentration of E2 increases. This occurs when frequency of LH pulses is not affected by circulating concentration of E 2. In the ovariectomized ewe, E 2 suppresses amplitude of LH pulses but has no influence on frequency of LH pulses (10). Differences between the ewe and cow might result from differing effects of E 2 o n the hypothalamo-pituitary axis between the species. During the follicular phase of the estrous cycle in the cow, increasing concentrations of E2 enhance the amplitude of LH pulses (9). Presumably this is one factor involved in the follicular phase rise in secretion of LH during the estrous cycle before the preovulatory surge of LH. When progesterone is present, E 2 administered at the lowest dose (1 E 2 implant - present study) is a potent suppresser of LH in circulation (19). Thus, it appears that E 2 loses the ability to suppress secretion of LH when progesterone declines with the regression of the corpus luteum in the bovine female. Concentrations of E 2 similar to concentrations detected during late pregnancy in the bovine female suppress LH secretion to levels below that of ovariectomized cows not receiving E 2 (22). Estradiol increases the sensitivity of the anterior pituitary to exogenous LHRH in sheep (23), rats (24), cows (25, 26) and monkeys (27). Concentrations of LH after a single dose of LHRH vary during different stages of the estrous cycle in sheep (21), rats (22), cattle (26) and women (27); the greatest LHRH-induced release of LH occurs just before the preovulatory surge of LH (period of increased endogenous secretion of E2). Estradiol increases the LHRH induced release of LH from pituitary cells of rats (30) and cows (31) in vitro. Receptor populations for LHRH in the pituitary are elevated at proestrus in the rat (32). In addition, nuclear receptors for E 2 are higher in pituitary glands collected during the follicular phase than during the luteal phase of the ovine estrous cycle (33). Estradiol increases the number of pituitary membrane receptors for LHRH in the gonadotrophs of ovariectomized sheep (34,35), rats (36) and cows (37). In the present study E2 may have increased the number of receptors for LHRH, thus sensitizing the pituitary to LHRH and promoting a greater release of LH.
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T h e e f f e c t o f E 2 o n p a t t e r n o f L H in c i r c u l a t i o n at t h e l o w d o s e s u s e d in t h e p r e s e n t res e a r c h w a s to e n h a n c e s e c r e t i o n o f L H . T h i s o c c u r r e d a s a r e s u l t o f i n c r e a s e d a m p l i t u d e o f LH pulses when E 2 was present. ACKNOWLEDGEMENTS/FOOTNOTES We thank Laura Rife for her patience in preparation of this manuscript; Karl Moline, Bob Broweleit and Jeff Bergman for management of the experimental animals; Dr. Jerry Reeves and Dr. Norman Mason, respectively, for providing the LH and E~ antisera and Dr. Leo Reichert, Jr. for the purified LH for radioiodination. tPublished as paper no. 8709, Journal Series Nebraska Agricultural Research Division. 2From whom reprints should be requested. 3Present Address: Colegio de Postgraduados, Texcocco, Mexico. 4Present Address: Department of Obstetrics & Gynecology, University of Kansas, School of Medicine, Wichita, KS. 5Present Address: Department of Animal Science, Ohio State University, Columbus, OH 4321(t. 6Present address: Louisiana Cooperative Extension Service, Louisiana State University, Baton Rouge, LA 70803. 7Present Address: CSIRO, Division of Tropical Animal Production, Tropical Cattle Research Centre, Queensland Australia. SPresent address: Department of Animal Science, University of Missouri, Columbia, MO 65211. +Present address: Route AI, Box 4A, Clay Center, NE 68933. mMention of a trade name, proprietary product or specific equipment does not constitute a guarantee or warrenty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.
REFERENCES 1. Kinder JE, Day ML, Kittok RJ. Endocrine regulation of puberty in cows and ewes. J Reprod Fertil [Suppl 34]:167-186, 1987. 2. Day ML, Imakawa K, Garcia-Winder M, Zalesky DD, Schanbacher BD, Kittok R J, Kinder JE. Endocrine mechanisms of puberty in heifers. Estradiol negative feed-back regulation of luteinizing hormone secretion. Biol Reprod 31:332-341, 1984. 3. Day ML, Imakawa K, Garcia-Winder M, Kittok RJ, Schanbacher BD, Kinder JE+ Influence of prepubertal ovariectomy and estradiol replacement therapy on secretion of luteinizing hormone before and after pubertal age in heifers. Domest Anim Endocrinol 3:17-25, 1986. 4. Imakawa K, Day ML, Garcia-Winder M, Zalesky DD, Kittok RJ+ Schanbacher BD, Kinder JE. Endocrine changes during restoration of estrous cycles following induction of anestrus by restricted nutrient intake in beef heifers. J Anim Sci 63:565-571, 1986. 5. Imakawa K, Day ML, Zalesky DD, Garcia-Winder M, Kittok RJ, Kinder JE. Regulation of pulsatile LH secretion by ovarian steroids in the heifer. J Anim Sci 63:162-168, 1986. 6. Critser JK, Miller KF, Gunsett FC, Ginther OJ. Seasonal LH profile in ovariectomized cattle. Theriogenology 19:181-191, 1983. 7. Day ML, Imakawa K, Pennel PL, Zalesky DD, Clutter AC, Kittok RJ, Kinder JE. Influence of season and estradiol on secretion of LH in ovariectomized cows. Biol Reprod 62:1641-1648, 1986. 8. Stumpf 1~, Day ML, Wolfe PL, Wolfe MW, Clutter AC, Kittok RJ, Kinder JE. Feedback of 17[~-estradiol on secretion of luteinizing hormone during different seasons of the year. J Anim Sci 66:447-451, 1988. 9. Stumpf TT, Day ML, Stotts JA, Wolfe MW+ Pennel PL, Kittok RJ, Kinder JE. Effect of estradiol on luteinizing hormone secretion during the follicular phase of the bovine estrous cycle. Biol Reprod 41:91 97, 1989. 10. Goodman RL, Karsch FJ. Pulsatile secretion of luteinizing hormone: Differential suppression of ovarian steroids. Endocrinology 107:1286-1290, 1980. 11. D+Occhio MJ, Kinder JE, Schanbacher BD. Patterns of LH secretion in castrated bulls (steers) during intravenous infusion of androgenic and estrogenic steroids: Pituitary response to exogenous luteinizing hormone releasing hormone. Biol Reprod 26:249-257, 1982. 12. Goiter TD, Reeves JJ, O'Mary CC, Arimura A, Schally AV. Serum LH levels in bulls treated with synthetic luteinizing hormone- releasing hormone/follicle stimulating hormone-releasing hormone (LH-RH/ FSH-RH). J Anim Sci 37:123-127, 1973. 13. Garcia-Winder M, Imakawa K, Day ML, Zalesky DD, Kittok RJ, Kinder JE. Effects of suckling and low doses of estradiol on luteinizing hormone secretion during the postpartum period in beef cows. Domest Anim Endocrinol 3:79-86, 1986. 14. D'Occhio MJ, Schanbacher BD, Kinder JE. Relationship between serum testosterone concentration and patterns of LH secretion in male sheep. Endocrinology 110:1547-1554, 1982. 15. Goodman RL, Legan SJ, Ryan KD, Foster DL, Karsch FJ. Two effects of estradiol that normally contribute to the control on tonic LH secretion in the ewe. Biol Reprod 23:415422, 198(I.
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16. SAS. SAS Users Guide. Cary NC, SAS Institute, Inc, 1985. 17. Foster DL, Ryan KD. Endocrine mechanisms governing transition into adulthood: A marked decrease in inhibitory feedback action of estradiol on tonic secretion of luteinizing hormone in the lamb during puberty. Endocrinology 105:896--904, 1979. 18. Beck TW, Smith VG, Seguin BE, Convey EM. Bovine serum LH, GH and prolactin following chronic implantation of ovarian steroids and subsequent ovariectomy. J Anim Sci 42:461-468, 1976. 19. Stumpf TF, MS Roberson, DL Hamemik, RJ Kittok. Inhibitory influence of progesterone on luteinizing hormone pulse frequency is amplified by low levels of 17~-estradiol during the bovine estrous cycle. Biol Reprod 38:[Suppl. 11123, 1988. 20. Foster DL, Olster DH. Effect of restricted nutrition on puberty in the lamb: Patterns of tonic luteinizing hormone (LH) secretion and competency of the LH surge system. Endocrinology 116:375-381, 1985. 21. Critser JK, Miller KF, Gunsett FC and Ginther OJ. Seasonal LH profile in ovariectomized cattle. Theriogenology 1912]:181-191, 1983. 22. Wolfe MW, Stumpf TT, Kinder JE. Estradiol feedback on secretion of luteinizing hormone in the bovine male. Biol Reprod 38:[Suppl. I]27, 1988. 23. Reeves JJ, Arimura A, Schally AV. Pituitary responsiveness to purified luteinizing hormone-releasing hormone (LH-RH) at various stages of the estrous cycle in sheep. J Anita Sci 32:123-126, 1971. 24. Aiyer MS, Fink G, Greig F. Changes in the sensitivity of the pituitary gland to luteinizing hormone releasing factor during the oestrus cycle of the rat. J Endocrinol 60:47-64, 1974. 25. Kaltenbach CC, Dunn TG, Kiser TE, Corah LR, Akbar AM, Niswender GD. Release of FSH and LH in beef heifers by synthetic gonadotropin releasing-hormone. J Anim Sci 38:357-362, 1974. 26. Kesner JS, Convey EM, Anderson CR. Evidence that estradiol induces the preovulatory LH surge in cattle by increasing pituitary sensitivity to LHRH and then increasing LHRH release. Endocrinology 108:13861391, 1981. 27. Knobil E, Plant TM, Wildt L, Belchetz PE, Marshall G. Neuroendocrine control of the Rhesus monkey menstrual cycle: Permissive role of the hypothalamic gonadotropin-releasing hormone (GnRH). Science 207:1371-1373, 1980. 28. Convey EM, Beal WE, Sequin BE, Tannen K J, Lin YC. Gonadotropin- releasing hormone induced luteinizing hormone release after prostaglandin F2~ in heifers. Proc Soc Exp Biol Med 151:84-88, 1976. 29. Yen SS, Vandeberg G, Rebar R, Ehara Y. Variation of pituitary responsiveness to synthetic LRH during different phases of the menstrual cycle. J Clin Endocrinol Metab 35:931-934, 1972. 30. Drouin J, Lagace L, Labrie F. Estradiol-induced increase of the LH responsiveness to LH releasinghormone (LHRH) in rat anterior pituitary cells in culture. Endocrinology 99:1477-1481, 1976. 31. Padmanabhan V, Kesner JS, Convey EM. Effects of estradiol on basal and luteinizing hormone releasing hormone (LHRH)- induced release of luteinizing hormone (LH) from bovine pituitary cells in culture. Biol Reprod 18:608-613, 1978. 32. Clayton RN, Solano AR, Garcia-Vela A, Dufau ML, Catt KJ. Regulation of pituitary receptors for gonadotropin-releasing hormone during the rat estrous cycle. Endocrinology 107:699-706, 1980. 33. Clarke IJ, Burman K, Funder JW, Findlay JK. Estrogen receptors in the neuroendocrine tissues of the ewe in relation to breed, season and stage of the estrous cycle. Biol Reprod 24:323-331,1981. 34. Moss GE, Crowder ME, Nett TM. GnRH-receptor interaction. VI. Effect of progesterone and estradiol on hypophyseal receptors for GnRH, and serum and hypophyseal concentrations of gonadotropins in ovariectomized ewes. Biol Reprod 25:938-944, 1981. 35. Crowder ME, Nett TM. Pituitary content of gonadotropins and receptors for gonadotropin-releasing hormone (GnRH) and hypothalamic content of GnRH during the periovulatory period of the ewe. Endocrinology 114:234-239, 1984. 36. Marchetti B, Reeves JJ, Pelletier G, Labrie F. Modulation of pituitary luteinizing hormone-releasing hormone receptors by sex steroids and luteinizing hormone-releasing hormone in the rat. Biol Reprod 27:133-145, 1982. 37. Schoenemann HM, Humphrey WD, Crowder ME, Nett TM, Reeves JJ. Pituitary luteinizing hormonereleasing hormone receptors in ovariectomized cows after challenge with ovarian steroids. Biol Reprod 32:574-583, 1985.
Vol. 8(4):463-469,1991
CIRCULATING CONCENTRATIONS OF 17-ESTRADIOL INFLUENCE PATTERN OF LH IN CIRCULATION OF COWS 1 J.E. Kinder*z, M. Garcia-Winder *,a, K. Imakawa.4, M.L. Day*,s D.D. Zalesky .6, M.L. D'Occhio *,**,7, T.T. Stumpf*,8 R.J. Kittok* and B.D. Schanbacher **,9 *Department of Animal Science University of Nebraska Lincoln, Nebraska 68583 **USDA, Agricultural Research Service Roman L. Hruska U.S. Meat Animal Research Center Clay Center, Nebraska 6893371° Received July 21, 1989
ABSTRACT The objective of the research was to determine the relationship between circulating 17[3-estradiol (E2) and secretion of luteinizing hormone (LH) in cows. A second objective was to determine if response to E 2 was influenced by interval between ovariectomy and the start of E2 treatment. Thirtyone nulliparous cows 3 yr of age were randomly assigned to a 2 x 4 factorial arrangement of treatments. Sixteen cows were ovariectomized at 18 mo of age (long term), and the other 15 cows were ovariectomized at 36 mo of age (short term). At the time of ovariectomy of cows in the short term group, 11 cows in the short term group and 12 cows in the long term group were implanted subcutaneously with 1, 2 or 4 polydimethylsiloxane capsules containing E 2. The other eight cows served as non-implanted controls (n=4-short term, n=4-1ong term). All cows were fitted with jugular vein catheters on day 29 of treatment, and on day 30 blood samples were collected at 12-rain intervals for 6 hr. At the end of 6 hr, luteinizing hormone-releasing hormone (LHRH) was administered and blood sampling continued at 12-rain intervals for an additional hour. Serum was analyzed for LH and E 2. Variables of LH secretion analyzed were mean concentration, frequency of pulses, amplitude of pulses and maximum concentration after LHRH. There were no significant interactions for any of the variables of LH among cows ovariectomized for the long and short term. There was a significant linear increase in mean concentration of LH with increased circulating concentration of E 2. Frequency of LH pulses was not affected by circulating concentration of E2. As circulating concentration of E 2 increased, amplitude of LH pulses increased and response to LHRH increased - resulting in an increase in mean LH. Interval from time of ovariectomy to the start of E2 treatment only had a minor influence on mean concentration of LH and profile of LH concentrations in circulation. INTRODUCTION Secretion o f LH from the anterior pituitary of the bovine female is under the influence of ovarian steroids (1), and is further modulated by age, (2,3), nutrition (4,5) and photoperiod (6,7,8). Heifers ovariectomized during prepuberty that receive E 2 have lower concentrations of LH than ovariectomized heifers not treated with E2; however, after age-matched intact heifers attain puberty, concentrations of LH in ovariectomized heifers implanted with E 2 are higher than in ovariectomized heifers not receiving E 2 (2,3). Similarly, cows that are o v a r i e c t o m i z e d as a d u l t s and i m p l a n t e d with E 2 have h i g h e r c i r c u l a t i n g L H than ovariectomized cows not receiving E 2 (9). Increased concentrations of LH in circulation of ovariectomized cows implanted with E2 are associated with a higher amplitude o f L H pulses (7,8,9). In contrast, E 2 suppressed amplitude o f LH pulses in adult ovariectomized ewes and also reduced magnitude o f the Copyright © 1991 Butterworth-Heinemann
463
0739-7240/91/$3.00
464
KINDER ET AL.
increase in tonic secretion of LH after progesterone withdrawal (10). Dose-response relationships between circulating concentrations of steroids and patterns of LH in circulation that occur in males (11) may also occur in females. The male bovine gonadectomized for the long term before treatment with testosterone fails to respond to exogenous testosterone by having changed patterns of LH in circulation (11). An understanding of these relationships in bovine females is important to properly interpret results of studies utilizing hormone replacement, and appreciate the physiological role of E 2. The specific aims of the present research were 1) to determine the relationship between circulating E 2 and secretion of LH in cows and 2) to determine if responses to E 2 are influenced by the interval between ovariectomy and commencement of E 2 treatment.
MATERIALS AND METHODS Animals and Experimental Protocol. Thirty-one Angus-Hereford crossbred and straightbred Hereford nulliparous cows 18 mo of age were randomly assigned by breed groups to a 2 x 4 factorial arrangement of treatments. Sixteen cows were ovariectomized at 18 mo of age, and no further treatments were administered for the following 18 mo (long term). Eighteen months following ovariectomy of cows in the long term group, estrus was synchronized in another group (short term, 36 mo of age) of cows (n=15) by using prostaglandin F2a (Lutalyse, Upjohn Co., Kalamazoo, MI), and cows were ovariectomized on the twelfth d of the estrous cycle. At the time of ovariectomy of cows in the short term group, 11 cows were implanted subcutaneously, caudal to the shoulder blade with 1,2 or 4 polydimethylsiloxane capsules (3.35 mm id x 4.65 mm od x 135 mm) containing crystalline E 2. Twelve cows ovariectomized for the long term were implanted with 1,2 or 4 implants (n=4/group) containing E 2 at the same time as cows ovariectomized for the short term were implanted with E 2. The other eight cows served as non-implanted controls (short term n=4, long term n=4) for their respective groups. The jugular vein was catheterized 29 d after insertion of the implants containing E2 and on the next day serial blood samples (10 ml) were collected at 12-min intervals for 6 hr. Immediately following the 6 hr of blood collection, 1 ~tg of LH-releasing hormone (LHRH) was administered intravenously and blood samples were collected at 12-min intervals for an additional 1 hr. Concentrations of LH were determined by radioimmunoassay (12,13). Intra- and interassay coefficients of variation were 2.5 and 11.6%, respectively. Sensitivity of the assay was 130 pg/ml. Concentrations of E 2 were determined in a single assay (14). Samples for quantifying E 2 were collected at the start, middle and end (preceding LHRH) of the 6 hr sampling period. Statistical Analyses. The variables of LH secretion evaluated were mean concentrations, frequency of pulses, amplitude of pulses and maximum concentrations subsequent to administration of LHRH. Mean concentration of LH for individual cows was the average concentration in the 30 samples collected before administration of LHRH. In addition, LH pulses were identified and amplitude of LH pulses was calculated (15). Frequency of LH pulses was expressed as the number of pulses detected during the 6 hr of blood collection before administration of LHRH. Amplitude of LH pulses was expressed as the average amplitude of all pulses detected during this 6 hr period of blood collection for an individual COW.
A general linear model's procedure was used to analyze the main effects of treatment (short or long term). The sums of squares for circulating concentration of E2 were divided into partial sums of squares to study linear and quadratic effects of E 2 on LH (16). Interactions of the regression coefficients between treatment groups were tested to determine if treatment (short or long term) affected response to E 2. Linear and quadratic regression coefficients for mean concentrations of E 2 and LH in circulation, frequency of LH pulses, amplitude of LH pulses and maximum concentration of LH after administration of LHRH were determined as circulating concentrations of E e increased. The concentration of LH
E S T R A D I O L M O D U L A T I O N O F LH
TABLE 1. MEANCONCENTRATIONSOFLH,
465
FREQUENCYOF LH
PULSESANDAMPLITUDEOFLH PULSESIN BLOOD
SERUM OF Cows RECEIVINGDIFFERINGNUMBERS OF 17~-ESTRADIOL(E2) IMPLANTSTHATWERE OVARIECTOMIZED
FORA SNORT(ST) ORLONGTERM(LT) ATTHETIMEOFADMINISTRATIONOFE2a. Number of implants"
0 1 2 ,4
Mean concentration E2 (pg/ml)'
Mean concentration LH (ng/ml)a'e
Frequency of LH pulses (pulses/6 hr) f
Amplitude of LH pulses (ng)g
LT 1.5 2.7 3.9 8.6
LT 2.15 2.81 3.91 3.53
LT 5.3 7.0 6.8 5.5
LT .75 1.17 2.77 2.69
ST 2.1 3.4 4.7 6.0
ST 2.35 3.38 3.60 2.95
ST 7.5 6.0 5.5 3.7
Changes in LH after LHRH h
ST .98 2.50 3.24 3.65
LT 5.88 8.52 5.92 12.37
ST 5.91 7.41 4.87 11.73
'Cows ovariectomized for the short term were ovariectomized and implanted with E 2 on day 12 of the estrous cycle (day 0=estrus). Cows ovariectomized for the long term were ovariectomized 18 mo before being implanted with E r bCows were implanted with E., subcutaneously caudal to the shoulder blade• ePooled SEM = 0.62. dMean concentration of LH during the 6-hr period of blood sampling 30 d after E 2 was administered. ~'r'g'hPooledSEM = 0.36, 0.89, 0.52 and 0.56 for mean concentration of LH, frequency of LH pulses, amplitude of LH pulses and changes in LH after LHRH, respectively.
i m m e d i a t e l y before administration o f L H R H was subtracted from m a x i m u m concentration o f L H after administration o f L H R H to evaluate responsiveness o f the pituitary to L H R H . RESULTS N o difference was d e t e c t e d in m e a n concentrations o f L H (Table 1) a m o n g c o w s that w e r e o v a r i e c t o m i z e d for the short or long term. N o interaction for m e a n concentration o f L H in response to E 2 was detected a m o n g cows in the two treatment groups. W h e n data for c o w s f r o m the two treatment groups were combined, there was a linear increase (P<0.001) o f m e a n concentration o f L H as concentration o f E 2 in circulation increased (Figure 1). N o
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Serum Estradiol (pg/ml) Figure 1. Linear regression (P<0.001) for mean concentrations of LH in cows ovariectomized for a long and short lerm that received 0, 1, 2 or 4 implants of E_,. There was no interaction between the two groups and no effect of treatment, thus data from cows of the two groups were combined for regression analysis.
466
KINDER ET AL.
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Serum Estradiol (pg/ml) Figure 2. Linear regressions (P<0.001) of LH pulse amplitude in cows that were ovariectomized for a long or short term that received 0, 1, 2 or 4 implants of E,. There was no interaction between the two groups, but there was an effect (P<0.05) of treatment.
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Serum Estradiol (pg/ml) Figure 3. Linear regression (P<0.002) of maximum LH after administration of LHRH in cows ovariectomized for a long and short term that received 0, I, 2 or 4 implants of E 2. There was no interaction between the two groups and no effect of treatment, thus data from cows of the two groups were combined for regression analysis.
ESTRADIOL MODULATION OF LH
467
significant difference in frequency of LH pulses (Table 1) existed between the short and long term groups. There was no effect (P>0.10) of circulating concentration of E 2 on frequency of LH pulses. Amplitude of LH pulses was lower (P<0.05) in cows that were ovariectomized for the short term as compared to cows ovariectomized for the long term (Table 1). There was no interaction between the long and short term groups for amplitude of LH pulses. When cows from the two groups were combined there was a linear increase (P<0.001) in amplitude of LH pulses as circulating concentrations of E2 increased (Figure 2). Response of the pituitary to LHRH was not different between cows ovariectomized for short or long term. There was no interaction in response to LHRH between the two treatment groups. Peak response to LHRH increased (P<0.003) linearly as circulating concentration of E 2 increased (Figure 3). DISCUSSION Estradiol is a potent suppresser of secretion of LH in prepubertal heifers (2), ewe lambs (17) and in postpubertal heifers if E 2 and progesterone are both present (18,19). In addition, E 2 is a potent suppresser of LH secretion in the heifer (4,5) and ewe lamb (20) fed diets containing low levels of energy. Estradiol suppresses secretion of LH in adult ovariectomized ewes during both the breeding and nonbreeding season (10,15) but enhances secretion of LH in ovariectomized cows during all seasons of the year (8,21). In the present study, variables of LH secretion were similar between cows ovariectomized for the short or long term. Therefore, cows respond to E2 replacement by modulating secretion of LH regardless of whether they have been ovariectomized for the long or short term at the time of administration of E 2. Amplitude of LH pulses increases as circulating concentration of E2 increases. This occurs when frequency of LH pulses is not affected by circulating concentration of E 2. In the ovariectomized ewe, E 2 suppresses amplitude of LH pulses but has no influence on frequency of LH pulses (10). Differences between the ewe and cow might result from differing effects of E 2 o n the hypothalamo-pituitary axis between the species. During the follicular phase of the estrous cycle in the cow, increasing concentrations of E2 enhance the amplitude of LH pulses (9). Presumably this is one factor involved in the follicular phase rise in secretion of LH during the estrous cycle before the preovulatory surge of LH. When progesterone is present, E 2 administered at the lowest dose (1 E 2 implant - present study) is a potent suppresser of LH in circulation (19). Thus, it appears that E 2 loses the ability to suppress secretion of LH when progesterone declines with the regression of the corpus luteum in the bovine female. Concentrations of E 2 similar to concentrations detected during late pregnancy in the bovine female suppress LH secretion to levels below that of ovariectomized cows not receiving E 2 (22). Estradiol increases the sensitivity of the anterior pituitary to exogenous LHRH in sheep (23), rats (24), cows (25, 26) and monkeys (27). Concentrations of LH after a single dose of LHRH vary during different stages of the estrous cycle in sheep (21), rats (22), cattle (26) and women (27); the greatest LHRH-induced release of LH occurs just before the preovulatory surge of LH (period of increased endogenous secretion of E2). Estradiol increases the LHRH induced release of LH from pituitary cells of rats (30) and cows (31) in vitro. Receptor populations for LHRH in the pituitary are elevated at proestrus in the rat (32). In addition, nuclear receptors for E 2 are higher in pituitary glands collected during the follicular phase than during the luteal phase of the ovine estrous cycle (33). Estradiol increases the number of pituitary membrane receptors for LHRH in the gonadotrophs of ovariectomized sheep (34,35), rats (36) and cows (37). In the present study E2 may have increased the number of receptors for LHRH, thus sensitizing the pituitary to LHRH and promoting a greater release of LH.
468
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T h e e f f e c t o f E 2 o n p a t t e r n o f L H in c i r c u l a t i o n at t h e l o w d o s e s u s e d in t h e p r e s e n t res e a r c h w a s to e n h a n c e s e c r e t i o n o f L H . T h i s o c c u r r e d a s a r e s u l t o f i n c r e a s e d a m p l i t u d e o f LH pulses when E 2 was present. ACKNOWLEDGEMENTS/FOOTNOTES We thank Laura Rife for her patience in preparation of this manuscript; Karl Moline, Bob Broweleit and Jeff Bergman for management of the experimental animals; Dr. Jerry Reeves and Dr. Norman Mason, respectively, for providing the LH and E~ antisera and Dr. Leo Reichert, Jr. for the purified LH for radioiodination. tPublished as paper no. 8709, Journal Series Nebraska Agricultural Research Division. 2From whom reprints should be requested. 3Present Address: Colegio de Postgraduados, Texcocco, Mexico. 4Present Address: Department of Obstetrics & Gynecology, University of Kansas, School of Medicine, Wichita, KS. 5Present Address: Department of Animal Science, Ohio State University, Columbus, OH 4321(t. 6Present address: Louisiana Cooperative Extension Service, Louisiana State University, Baton Rouge, LA 70803. 7Present Address: CSIRO, Division of Tropical Animal Production, Tropical Cattle Research Centre, Queensland Australia. SPresent address: Department of Animal Science, University of Missouri, Columbia, MO 65211. +Present address: Route AI, Box 4A, Clay Center, NE 68933. mMention of a trade name, proprietary product or specific equipment does not constitute a guarantee or warrenty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.
REFERENCES 1. Kinder JE, Day ML, Kittok RJ. Endocrine regulation of puberty in cows and ewes. J Reprod Fertil [Suppl 34]:167-186, 1987. 2. Day ML, Imakawa K, Garcia-Winder M, Zalesky DD, Schanbacher BD, Kittok R J, Kinder JE. Endocrine mechanisms of puberty in heifers. Estradiol negative feed-back regulation of luteinizing hormone secretion. Biol Reprod 31:332-341, 1984. 3. Day ML, Imakawa K, Garcia-Winder M, Kittok RJ, Schanbacher BD, Kinder JE+ Influence of prepubertal ovariectomy and estradiol replacement therapy on secretion of luteinizing hormone before and after pubertal age in heifers. Domest Anim Endocrinol 3:17-25, 1986. 4. Imakawa K, Day ML, Garcia-Winder M, Zalesky DD, Kittok RJ+ Schanbacher BD, Kinder JE. Endocrine changes during restoration of estrous cycles following induction of anestrus by restricted nutrient intake in beef heifers. J Anim Sci 63:565-571, 1986. 5. Imakawa K, Day ML, Zalesky DD, Garcia-Winder M, Kittok RJ, Kinder JE. Regulation of pulsatile LH secretion by ovarian steroids in the heifer. J Anim Sci 63:162-168, 1986. 6. Critser JK, Miller KF, Gunsett FC, Ginther OJ. Seasonal LH profile in ovariectomized cattle. Theriogenology 19:181-191, 1983. 7. Day ML, Imakawa K, Pennel PL, Zalesky DD, Clutter AC, Kittok RJ, Kinder JE. Influence of season and estradiol on secretion of LH in ovariectomized cows. Biol Reprod 62:1641-1648, 1986. 8. Stumpf 1~, Day ML, Wolfe PL, Wolfe MW, Clutter AC, Kittok RJ, Kinder JE. Feedback of 17[~-estradiol on secretion of luteinizing hormone during different seasons of the year. J Anim Sci 66:447-451, 1988. 9. Stumpf TT, Day ML, Stotts JA, Wolfe MW+ Pennel PL, Kittok RJ, Kinder JE. Effect of estradiol on luteinizing hormone secretion during the follicular phase of the bovine estrous cycle. Biol Reprod 41:91 97, 1989. 10. Goodman RL, Karsch FJ. Pulsatile secretion of luteinizing hormone: Differential suppression of ovarian steroids. Endocrinology 107:1286-1290, 1980. 11. D+Occhio MJ, Kinder JE, Schanbacher BD. Patterns of LH secretion in castrated bulls (steers) during intravenous infusion of androgenic and estrogenic steroids: Pituitary response to exogenous luteinizing hormone releasing hormone. Biol Reprod 26:249-257, 1982. 12. Goiter TD, Reeves JJ, O'Mary CC, Arimura A, Schally AV. Serum LH levels in bulls treated with synthetic luteinizing hormone- releasing hormone/follicle stimulating hormone-releasing hormone (LH-RH/ FSH-RH). J Anim Sci 37:123-127, 1973. 13. Garcia-Winder M, Imakawa K, Day ML, Zalesky DD, Kittok RJ, Kinder JE. Effects of suckling and low doses of estradiol on luteinizing hormone secretion during the postpartum period in beef cows. Domest Anim Endocrinol 3:79-86, 1986. 14. D'Occhio MJ, Schanbacher BD, Kinder JE. Relationship between serum testosterone concentration and patterns of LH secretion in male sheep. Endocrinology 110:1547-1554, 1982. 15. Goodman RL, Legan SJ, Ryan KD, Foster DL, Karsch FJ. Two effects of estradiol that normally contribute to the control on tonic LH secretion in the ewe. Biol Reprod 23:415422, 198(I.
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16. SAS. SAS Users Guide. Cary NC, SAS Institute, Inc, 1985. 17. Foster DL, Ryan KD. Endocrine mechanisms governing transition into adulthood: A marked decrease in inhibitory feedback action of estradiol on tonic secretion of luteinizing hormone in the lamb during puberty. Endocrinology 105:896--904, 1979. 18. Beck TW, Smith VG, Seguin BE, Convey EM. Bovine serum LH, GH and prolactin following chronic implantation of ovarian steroids and subsequent ovariectomy. J Anim Sci 42:461-468, 1976. 19. Stumpf TF, MS Roberson, DL Hamemik, RJ Kittok. Inhibitory influence of progesterone on luteinizing hormone pulse frequency is amplified by low levels of 17~-estradiol during the bovine estrous cycle. Biol Reprod 38:[Suppl. 11123, 1988. 20. Foster DL, Olster DH. Effect of restricted nutrition on puberty in the lamb: Patterns of tonic luteinizing hormone (LH) secretion and competency of the LH surge system. Endocrinology 116:375-381, 1985. 21. Critser JK, Miller KF, Gunsett FC and Ginther OJ. Seasonal LH profile in ovariectomized cattle. Theriogenology 1912]:181-191, 1983. 22. Wolfe MW, Stumpf TT, Kinder JE. Estradiol feedback on secretion of luteinizing hormone in the bovine male. Biol Reprod 38:[Suppl. I]27, 1988. 23. Reeves JJ, Arimura A, Schally AV. Pituitary responsiveness to purified luteinizing hormone-releasing hormone (LH-RH) at various stages of the estrous cycle in sheep. J Anita Sci 32:123-126, 1971. 24. Aiyer MS, Fink G, Greig F. Changes in the sensitivity of the pituitary gland to luteinizing hormone releasing factor during the oestrus cycle of the rat. J Endocrinol 60:47-64, 1974. 25. Kaltenbach CC, Dunn TG, Kiser TE, Corah LR, Akbar AM, Niswender GD. Release of FSH and LH in beef heifers by synthetic gonadotropin releasing-hormone. J Anim Sci 38:357-362, 1974. 26. Kesner JS, Convey EM, Anderson CR. Evidence that estradiol induces the preovulatory LH surge in cattle by increasing pituitary sensitivity to LHRH and then increasing LHRH release. Endocrinology 108:13861391, 1981. 27. Knobil E, Plant TM, Wildt L, Belchetz PE, Marshall G. Neuroendocrine control of the Rhesus monkey menstrual cycle: Permissive role of the hypothalamic gonadotropin-releasing hormone (GnRH). Science 207:1371-1373, 1980. 28. Convey EM, Beal WE, Sequin BE, Tannen K J, Lin YC. Gonadotropin- releasing hormone induced luteinizing hormone release after prostaglandin F2~ in heifers. Proc Soc Exp Biol Med 151:84-88, 1976. 29. Yen SS, Vandeberg G, Rebar R, Ehara Y. Variation of pituitary responsiveness to synthetic LRH during different phases of the menstrual cycle. J Clin Endocrinol Metab 35:931-934, 1972. 30. Drouin J, Lagace L, Labrie F. Estradiol-induced increase of the LH responsiveness to LH releasinghormone (LHRH) in rat anterior pituitary cells in culture. Endocrinology 99:1477-1481, 1976. 31. Padmanabhan V, Kesner JS, Convey EM. Effects of estradiol on basal and luteinizing hormone releasing hormone (LHRH)- induced release of luteinizing hormone (LH) from bovine pituitary cells in culture. Biol Reprod 18:608-613, 1978. 32. Clayton RN, Solano AR, Garcia-Vela A, Dufau ML, Catt KJ. Regulation of pituitary receptors for gonadotropin-releasing hormone during the rat estrous cycle. Endocrinology 107:699-706, 1980. 33. Clarke IJ, Burman K, Funder JW, Findlay JK. Estrogen receptors in the neuroendocrine tissues of the ewe in relation to breed, season and stage of the estrous cycle. Biol Reprod 24:323-331,1981. 34. Moss GE, Crowder ME, Nett TM. GnRH-receptor interaction. VI. Effect of progesterone and estradiol on hypophyseal receptors for GnRH, and serum and hypophyseal concentrations of gonadotropins in ovariectomized ewes. Biol Reprod 25:938-944, 1981. 35. Crowder ME, Nett TM. Pituitary content of gonadotropins and receptors for gonadotropin-releasing hormone (GnRH) and hypothalamic content of GnRH during the periovulatory period of the ewe. Endocrinology 114:234-239, 1984. 36. Marchetti B, Reeves JJ, Pelletier G, Labrie F. Modulation of pituitary luteinizing hormone-releasing hormone receptors by sex steroids and luteinizing hormone-releasing hormone in the rat. Biol Reprod 27:133-145, 1982. 37. Schoenemann HM, Humphrey WD, Crowder ME, Nett TM, Reeves JJ. Pituitary luteinizing hormonereleasing hormone receptors in ovariectomized cows after challenge with ovarian steroids. Biol Reprod 32:574-583, 1985.