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RELATIONSHIPS BETWEEN THE LUTEINIZING HORMONE. RESPONSE TO GONADOTROPIN RELEASING HOR-. MONE AND ENDOGENOUS STEROIDS 1 , ...
RELATIONSHIPS BETWEEN THE LUTEINIZING HORMONE RESPONSE TO GONADOTROPIN RELEASING HORMONE A N D E N D O G E N O U S S T E R O I D S 1 ,2 Josef Zolman, Edward M. Convey and ~lackT:l. Britt

Michigan State University, East Lansing 48824 Summary Twenty-four yeading heifers, exhibiting regular estrous cycles, were assigned at random to groups of four and received 5, 40 or 320 #g of synthetic gonadotropin releasing hormone (GnRH) via jugular cannula on day 14 of the estrous cycle or on the day following corpus luteum regression which occurred on the average on day 20 of the cycle. Serum estradiol g/ml), estrone (pg/ml) and progesterone g/ml) prior to GnRH treatment averaged 5.1, 6.9 and 3.9 on day 15 and 10.8, 7.5 and 0.9 on the day following luteal regression and differences between days were significant (P < 0.01) for estradiol and progesterone. Peak serum LH (ng/ml) after 5,40 and 320/.tg GnRH was 1.8, 7.7 and 13.9 on day 15 and 1.0, 13.6 and 8.3 on day 20 but differences between days of the cycle were not significant. However analysis revealed a significant (P < .01) relationship between serum estradiol and estrone concentration at the time GnRH was administered, and the magnitude of LH release in response to GnRH. Serum estradiol measured at 4 hr. post-GnRH was increased on day 15 but decreased on day 20 of the cycle (P < .02), while serum estrone and progesterone remained unchanged (P < .05). It appears that the LH response to GnRH is related to serum estrogen concentration.

~n

Introduction

Synthetic gonadotropin releasing hormone (GnRH) causes a dose-response increase in

1Published with the approval of the Michigan Agriculture Experiment Station as Journal Article No. 6567. Supported in part by NIH Grant No. AM 15899. 2 Animal Reproduction Laboratory, Department of Dairy Science.

serum luteinizing hormone (LH) concentration in heifers when administered during the luteal phase of the estrous cycle (Zolman et al., 1973). But the magnitude of LH released in response to a dose of GnRH which was determined to cause maximum LH _r_elease during the luteal phase of the cycle was only half that which has been reported from this laboratory to characterize the preovulatory surge of LH in heifers (Swanson and Hafs, 1971). If the quantity of LH released was determined only by the quantity of releasing hormone acting on the pituitary, then an LH surge induced by GnRH during the luteal phase of the estrous cycle should be comparable in magnitude to the normal preovulatory surge. Since this was not the case, other factors appear to be involved. The possibility that ovarian steroids may modify the responsiveness of the pituitary to exogenous GnRH has been considered by others. Thus Reeves, Arimura and Schally (1971a) demonstrated that LH release in response to GnRH in ewes was greatest during an 8-hr. period on day I of the estrous cycle relative to any other time tested. In ewes, these results provide indirect evidence that pituitary sensitivity to GnRH may be enhanced by removal of a progesterone block or by estrogen stimulation. In addition, pretreatment of ewes with 250 to 500/zg of estradiol benzoate 20 hr. before GnRH administration increased LH release relative to control ewes (Reeves et al., 1971b). Whether changes in endogenous ovarian steroids influence sensitivity to exogenous GnRH has not been determined. The present study was undertaken to determine whether the magnitude of LH released in response to various doses of GnRH changes with shifts in ovarian steroid concentrations that occur normally during the bovine estrous cycle (Wettemann et al., 1972).

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JOURNAL OF ANIMAL SCIENCE, vol. 39, no. 2, 1974

356

ZOLMAN ET AL. Materials and M e t h o d s

Twenty-four Holstein heifers were assigned at random to groups of four to receive 5, 40 or 320/lg of synthetic GnRI-I a on day 15 of the estrous cycle (estrus = day 0) or on the day following corpus luteum regression as determined by rectal palpation (~ = 20 days). Heifers were 12 to 16 months old at the time of treatment and were observed to have at least two regular consecutive estrous cycles prior to this experiment. Heifers were palpated per rectum daily beginning 4 days before expected estrus to detect corpus luteum regression and ovulation. This procedure was followed during both pretreatment and experimental estrous cycles. Each heifer assigned to be treated following luteal regression was palpated per rectum twice daily beginning 4 days before expected estrus and treatment was administered within 12 hr. after the corpus luteum regressed to approximately 1.0 cm diameter. On the average this occurred on day 20 of the estro~:s cycle and this group will subsequently be referred to as having been treated on day 20. Rectal palpation revealed that no heifer in this group ovulated prior to GnRH administration. Twenty-four hours prior to each experiment a cannula (Vinyl Tubing, Clay Adams, Inc., New York City) was inserted into the left jugular vein of each animal. Synthetic GnRH in 10 ml .isotonic saline was administered intravenously as a single injection via the jugular cannulae. Blood samples (10 ml) were collected at 60, 30 and 10 min. prior to GnRH treatment and following GnRH treatment at 2-min. intervals for 30 rain., at 45 min. and at half-hr. intervals from 60 to 180 minutes. In addition blood samples (50 ml) were withdrawn at time 1 min. before GnRH and at 4 hr. after GnRH treatment for quantitation of serum steroid concentrations. Serum samples were stored at - 2 0 ~ until assayed for LH (Oxender, Hafs and Edgerton, 1972), estradiol, estrone (Smith et al., 1973) and progesterone (Kittok, Britt and Convey, 1973) by radioimmunoassays. Variance was analysed to evaluate the significance of dose of GnRH and day of treatment on serum hormone concentrations. Relationships between ovarian steroid concentrations in serum and LH response to GnRH

TABLE 1. SERUM ESTRADIOL, ESTRONE AND PROGESTERONE ON DAY 15 AND 20 OF THE ESTROUS CYCLE IN HOLSTEIN HEIFERS PRIOR TO GONADOTROPINRELEASING HORMONEa D a y o f the estrous c y c l e Hormone Estradiol ( p g / m l ) Estrone ( p g / m l ) Progesterone ( n g / m l )

15 5.1 -+ 0.7 6.9 + 1.3 3.9 + 0.5

20 b 1 0 . 8 + 2.3 c 7.5 + 0.9 0.4 + 0.1 c

a v a l u e s axe m e a n s + s t a n d a x d e r r o r , n = 4. b D a y f o l l o w i n g l u t e a l r e g r e s s i o n , x = 2 0 days.

c s i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o m p a r a b l e value o n day (P < . 0 1 ) .

were also investigated. Variables which were related to the magnitude of LH release in response to GnRH were identified, using the stepwise addition method of Draper and Smith (1966), for inclusion in a covariance analysis to adjust the data on LH release. The 90% confidence level was required for inclusion of variables in the analysis of covariance. Results

Serum estradiol averaged 10.8 pg/ml immediately prior to GnRH treatment (time 0) on day 20 of the estrous cycle and was greater (P < 0.01) than the comparable average on day 15 (5.1 pg/ml; table 1). Mean estrone concentrations were 6.9 and 7.5 pg/ml in serum collected on day 15 and 20 of the estrous cycle, respectively, and differences between means were not significant (P > .05). However, serum progesterone concentration decreased (P < .01) from 3.9 ng/ml on day 15 of the estrous cycle to 0.4 on the day following regression of the corpus luteum. Serum LH concentrations prior to GnRH treatment were 0.6 and 0.8 ng/ml on day 15 and 20 of the estrous cycle and differences between means were not significant (P > .05). GnRH at doses of 5, 40 or 320/ag increased serum LH concentrations to peaks of 1.8, 7.7 and 13.9 ng/ml, respectively, in heifers treated on day 15 of the estrous cycle and to peaks of 1.0, 13.6 and 8.8 ng/ml in serum of heifers treated on day 20 of the cycle (table 2). Variation in peak serum luteinizing hormone concentration due to dose of GnRH was significant SKindly supplied by Dr. R. Rippel, Abbot (P < .01) but the effect of day of estrous cycle Laboratories, North Chicago, Illinois. The GnRH when treatments were administered was not supplied us by Abbot Laboratories was equivalent in (P > .05). Interaction of the main effects (dose potency to natural porcine GnRH (AVS LH-RH of GnRH • day of estrous cycle) was signifi77-33, fractions 215-269) and was 98 to 100% pure cant (P < .05) indicating a failure of each dose decapeptide diacetate tetrahydrate.

357

LH RESPONSE TO GnRH AND ENDOGENOUS STEROIDS

the estrous cycle estradiol was decreased by 4 hr. post-GnRH in seven of eight heifers receiving 5 or 40 pg GnRH, but increased in all heifers receiving 320 pg GnRH. Overall analysis of variance indicated a marDay of the estrous cycle ginal (P < .08) effect of GnRH treatment on GnRHb 15 20 c serum progesterone concentration at 4 hr. post-treatment (table 4). However, the mean in(ug) crease in serum progesterone at 4 hr. after 40 or 5 1.8• 1.0• 40 7.7• 13.6• 320 pg GnRH on day 15 was greater than the 320 13.9• 8.8• means of all other groups (P < .05). ~d 7.9• 7.8• The change in estrone from time zero to 4 hr. post-GnRH is shown in table 5. Animal variaValues are m e a n s • s t a n d a r d e r r o r , n = 4. b A d m i n i s t e r e d iv. ation was large and changes in serum estrone CDay f o l l o w i n g l u t e a l r e g r e s s i o n ; ~ = 2 0 days. were not related to day of the estrous cycle d Overall m e a n . when treatment took place or to,dose of GnRH (P :> .05). Because differences in concentration of seof GnRH to cause a similar release of LH on both treatment days. This interaction may be rum estrogens between day 15 and day 20 of explained on the basis of differences in serum the cycle were not marked, art attempt was ovarian steroids prior to GnRH treatment and made to clarify the relationship between serum will be discussed below. Invariably, serum LH steroid concentrations and magnitude of LH reconcentration reached m~/ximum levels within lease in response to GnRH using covariance 30 min of GnRH administration. During the analysis. Variables were identified from among 4-hr. interval following GnRH administration the following for inclusion in the covariance serum LH concentration returned to with- analysis: GnRH dose, day, GnRH dose x day, and pretreatment, post-treatment or change in + 0.3 ng/ml of the pretreatment baseline. Estradiol concentration in serum collected 4 (A = hormone concentration at 4 hr. after hr. after GnRH treatment was, on the average, GnRH minus hormone concentration at time 0) higher (P < .92) on day 15 but lower (P < .05) in serum steroid hormone concentration. Vario n day 20 of the estrous cycle relative to the ables shown in table 6 include only those that comparable pretreatment average (table 3). Al- are related to the magnitude of LH released in though the change-in-serum estradiol concentra- response to GnRH ( P < .10). The greatest tion on day 15 was positive there was a great determinant to magnitude of LH released i,~ deal of within group variation in magnitude of response to GnRH was the quantity of GnRH increase as evidenced by the large standard er- used. However, serum estradiol and estrone rors associated with the means. On day 20 of concentrations at the time GnRH was adminT A B L E 2. P E A K C O N C E N T R A T I O N O F L U T E I N I Z ING HORMONE IN SERUM OF HEIFERS ADMINISTERED GONADOTROPIN RELEASING H O R M O N E ( G n R H ) O N D A Y S 15 A N D 20 OF THE ESTROUS CYCLEa

T A B L E 3. C H A N G E IN SI':RUM E S T R A D I O L CONCENTRATION FOLLOWING GnRH A D M I N I S T R A T I O N IN H E I F E R S a

GnRH b (ug) 5 40 320 ~c

Day of the estrous cycle 15 20 b pg/ml 0.9 + 1.1 --6.4 + 4.6 8.4 • 7.5 --7.3 + 3.8 12.5 • 7.7 4.4 -+ 1.9 7.3 + 3.6d - 3 . 1 + 2.5 e

a V a l u e s are m e a n s • s t a n d a r d e r r o r , n = 4. C a l c u l a t e d as s e r u m e s t r a d i o ] c o n c e n t r a t i o n a t 4 hr. after G n R H t r e a t m e n t m i n u s s e r u m e s t r a d i o l c o n c e n t r a t i o n at t i m e z e r o . G n R H a d m i n i s t e r e d iv. b D a y of t h e e s t r o u s c y c l e f o l l o w i n g l u t e a l regression. COverall m e a n . d S i g n i f l c a n t l y d i f f e r e n t f r o m zero (P < . 0 2 ) . e S i g n i f l c a n t l y d i f f e r e n t f r o m z e r o (P < . 0 5 ) .

T A B L E 4. C H A N G E IN S E R U M P R O G E S T E R O N E CONCENTRATION FOLLOWING GnRH A D M I N I S T R A T I O N IN H E I F E R S a

GnRH b (~g) 5 40 320 id

Day of the estrous cycle 15 20c ng/ml -.8• 3.9• 2.7• 1.9•

e 1.2 e

0.2• 0.5• 0.8• 0.5•

a V a l u e s are m e a n s + s t a n d a r d e r r o r , n = 4. C a l c u l a t e d as s e r u m p r o g e s t e r o n e c o n c e n t r a t i o n at 4 hr. a f t e r G n R H t r e a t m e n t m i n u s s e r u m p r o g e s t e r o n e c o n c e n t r a t i o n at t i m e z e r o . b A d m i n i s t e r e d iv. CDay f o l l o w i n g h i t e a l r e g r e s s i o n ; x = 2 0 days. d Overall m e a n . e s i g n i f l c a n t l y h i g h e r t h a n 5 # g r e s p o n s e (P < . 0 5 ) .

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ZOLMAN ET AL. TABLE 5. CHANGE IN SERUM ESTRONE CONCENTRATION FOLLOWING GnRH ADMINISTRATION IN HEIFERS a D a y o f the estrous c y c l e

GnRH b

15

20c

(/Jg)

pg/ml - . 1 • 0.4 13.3 • 15.6 4.3 • 6.6 5.8 • 5.4

5

40 320 ~d

11.3 -.1 3.6 4.9

+- 7.0 • 2.1 + 3.6 • 2.8

means + standard error, n = 4. estrone concentration at 4 hr. after GnRH treatment minus serum estrone concentration at time zero. aValues Calculated

are

as s e r u m

bAdminist ered iv.

CDay following

luteal regression; x = 20 days.

doverall mean.

release. Evidence that ovarian steroids may function in this capacity has recently been reviewed (Convey, 1973). Day 15 and the day following corpus luteum regression were chosen as treatment days in this experiment to take advantage of physiological shifts in ovarian steroids in investigating their role in modifying pituitary sensitivity to GnRH. Estradiol and progesterone concentrations on istered were positively related to magnitude of the LH released by GnRH. In this regard it is important to note that serum progesterone at time zero was not related to the magnitude of LH response to GnRH. Estrone concentrations in serum collected at 4 hr. after treatment and A serum estradiol concentration were also significantly related to peak concentration of serum LH after GnRH treatment. Discussion In a previous study Zolman etal. (1973) observed an increase in magnitude of LH released TABLE 6. ANALYSIS OF COVARIANCE OF PEAK SERUM LUTEINIZING HORMONE IN RESPONSE TO GONADOTROPIN RELEASING HORMONE (GnRH) ADMINISTERED ON DAYS 15 AND 20 OF THE ESTROUS CYCLE Variable GnRH

Estradiol-prea Estrone-pre Estrone-post b EstradiolA Error

df

M.S.

F

2

0.041

13.59

1 1 1 1 11

0.020 0.016 0.022 0.011 0.003

6.72 5.29 7.36 3.49

SIG 0.0005 0.019 0.034 0.015 0.079

9 a p r e - s a m p l e s w e r e t a k e n at t i m e 0 . bpost-samples w e r e t a k e n at 4 h r a f t e r G n R H . CA = s t e r o i d c o n c e n t r a t i o n m s e r u m a t 4 hr. a f t e r GnRH minus the comparable concentration at time 0.

with increasing dose of GnRH in heifers treated during the luteal phase of the estrous cycle. Although the LH response was maximal after 80 pg GnRH it was only about 50% that characterizing the preovulatory LH peak in heifers previously reported (Swanson and Hafs, 1971). Therefore it is a possibility that factors other than the quantity of releasing hormone reaching the pituitary played a role in modifying LH each treatment day at the time of GnRH treatment in this study are similar to comparable values previously reported from this laboratory (Wettemarm et al., 1972). Similarly, serum estrone concentrations on day 15 in this study are comparable to values previously reported for serum collected from cows during the luteal phase of the estrous cycle (Kittok et al., 1973). Failure to detect significant variation in the .magnitude of LH release in response to GnRH due to days of the estrous cycle tested suggests that factors influencing pituitary sensitivity to GnRH may vary independent of day o f the estrous cycle. The significant interaction between dose of GnRH and day of the estrous cycle may result from differences in ovarian steroid concentration in serum at the time GnRH was administered. This view is supported by failure of the interaction to remain an important variable when serum steroid hormones were included in the analysis. Covariance analysis revealed that serum estradiol and estrone, in addition to dose of GnRH were related to magnitude of the LH surge. Although covariance analysis does not determine cause and effect, these data support those of others who have implicated changes in estrogen concentration as a factor in modifying pituitary sensitivity to GnRH. Reeves et al. (1971a) rePorted increased pituitary sensitivity to GnRH in ewes during an 8-hr. period on day 1 of the estrous cycle. Although serum estradiol was increased relative to day 15 of the estrous cycle, and serum progesterone was markedly decreased in these heifers there was no increase in pituitary sensitivity to GnRH, as measured by LH release, comparable to that seen in ewes. Perhaps, development of maximum pituitary sensitivity to GnRH after estrogen stimulation or progesterone withdrawal requires time to develop and enhanced sensitivity may be more obvious nearer estrus. Of particular interest here is the failure of serum progesterone to influence LH response to the decapeptide. If there was a progesterone block on LH release by GnRH then the marked decrease in progesterone that followed corpus luteum regression in these heifers would be ex-

LH RESPONSE TO GnRH AND ENDOGENOUS STEROIDS

359

pected to constitute removal of this block. Yet, o f LH in the bovine (Carlson, Norimoto and serum progesterone concentration was not sig- Hansel, 1971 ). nificantly related to LH release in the analysis o f covariance. But the possibility that changes in pituitary sensitivity to GnRH following proLiterature Cited gesterone withdrawal may be time dependent cannot be excluded on the basis Of these data. Carlson, J. C., K. Norimoto and W. Hansel. 1971. Effects of LH on peripheral progesterone concenThis suggestion is supported by a previous obtrations in intact and hysterectomized heifers. servation (Kittok etal., 1973) of a twofold Endocrinol. 89:1530. greater LH response to GnRH in cows with Convey, E. M. 1973. Neuroendocrine relationships in farm animals: A review. J. Anim. Sci. 37:745. ovarian follicular cysts compared with hermates treated during the luteal phase of the estrous Debeljuk, L., A. Arimura and A. V. Schally. 1972. Effect of estradiol and progesterone on the LH cycle. Differences in pretreatment serum estrorelease induced by LH-releasing hormone (LH-RH) gens in the two groups o f cows were not signifiin intact diestrous rats and anestrous ewes. Proc. Soc. Exp. Biol. Med. 139:774. cant, but serum progesterone in cows with follicular cysts was less than 1.5 ng/ml for more Draper, N. R. and H. Smith. 1966. Applied regression analysis. John Wiley & Sons, Inc., New York. than 24 hr. prior to treatment compared to Kittok, R. J., J. H. Britt and E. M. Convey. 1973. values o f 5 to 6 ng/ml for cows treated during Endocrine response after GnRH in luteal phase cows and cows with ovarian follicular cysts. J. the luteal phase. Additionally, Debeljuk, Anim. Sci. 37:985. Arimura and Schally (1972) reported proges9 Oxender, W. D., H. D. Hafs and L. A. Edgerton. 1972. terone_ injections inhibited LH release after Serum growth hormone, LH and prolacfin in the GnRH in rats and completely masked the pregnant cow. J. Anim. Sci. 35:51. increased responsiveness of the pituitary to Reeves, J. J., A. Arimura and A. V. SchaUy. 1971a. Pituitary responsiveness to purified luteinizing GnRH induced with estradiol. hormone-releasing hormone (LH-RH) at various Since follicle stimulating hormone (FSH) is stages of the estrous cycle in sheep. J. Anim. SeL released in bulls" by GnRH (Zolman and 32:123. Convey, 1973), the increase in serum estradiol Reeves, J. J., A. Arimura and A. V. Schally. 1971b. Changes in pituitary responsiveness to luteinizing in heifers treated on day 15 o f the estrous cycle hormone-releasing hormone (LH-RH) in anestrous may have resulted from FSH stimulation of esewes pretreated with estradiol benzoate. Biol. trogen synthesis perhaps via increased follicular Reprod. 4:88. growth. Why gonadotropin released b y GnRH Smith, V. G., L. A. Edgerton, H. D. Hafs and E. M. Convey. 1973. Bovine serum estrogens, progestins should cause a decrease in serum estradiol in and glucocorticoids during late pregnancy, parturisome heifers but not others on the day foltion and early lactation. J. Anim. Sci. 36:391. lowing luteal regression is not clear at this time, Swanson, L. V. and H. D. Hafs. 1971. LH and prolactin in blood serum from estrus to ovulation but perhaps is related to the steroidogenic in Holstein heifers. J. Anim. Sci. 33:1038. capacity of the ovarian follicles. Wettemann, R. P., H. D. Hafs, L. A. Edgerton and L. Progesterone was increased in those heifers V. Swanson. 1972. Estradiol and progesterone in that had a functional corpus luteum and blood serum during the b~vine estrous cycle. J. Anim. Sci. 34:1020. received sufficient GnRH to cause a rise in serum LH greater than 1 ng/ml. This agrees Zolman, J., E. M. Convey, J. H. Britt and H. D. Hafs. 1973. Release of bovine luteinizing hormone by with previous results (Kittok et al., 1973) of purified porcine and synthetic gonadotropin releasing hormone. Proc. Soc. Exp. Biol. Med. increased progesterone in serum o f cows treated 142:189. with GnRH during the luteal phase o f the Zolman, J. and E. M. Convey. 1973. GnRH: Effect on estrous _cycle. Increased progesterone after an .... serum FSH and androgens in bulls. J. Anim. Sci. LH surge is consistent with the luteotropic role 37:344 (Abstr.).