Progesterone and 17α-hydroxyprogesterone advance the estrogen-induced bioassayable luteinizing hormone surge in castrate monkeys*

Vol. 43. No.2. February 1985 Printed in U.8A.

FERTILITY AND STERILITY Copyright © 1985 The American Fertility Society

Progesterone and 17n-hydroxyprogesterone advance the estrogeninduced bioassayable luteinizing hormone surge in castrate monkeys *

Robert S. Schenken, M.D.t:J: Robert F. Williams, Ph.D.§ Bryan D. Cowan, M.D.§ Gary D. Hodgen, Ph.D.§11 The University of Texas Health Science Center at San Antonio, San Antonio, Texas, and Pregnancy Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland

Recent studies have demonstrated marked disparities between bioassayable (BIO) versus radioimmunoassayable concentrations of luteinizing hormone (LH) at midcycle. Other studies in intact women and monkeys have indicated that progesterone (P) may facilitate estrogen induction of the preovulatory gonadotropin surges. We have combined the study of these observations as follows: long-term castrate female monkeys were given estradiol benzoate with and without subsequent P or 17a-hydroxyprogesterone (17-0HP) injections. Plasma was collected during a selected 24-hour interval via a chronic indwelling femoral vein cannula. Initially, estrogen-negative feedback decreased the pulsatility and circulating levels of gonadotropins. P and 17-0HP treatment (after estradiol) hastened initiation of the BIO-LH surge by up to 8 hours. Although estrogen-positive feedback alone enhanced the biologic activity of LH, P and 17-0HP expedited the midcycle-like BIO-LH surges. Fertil Steril43:301, 1985

Received September 11,1984; revised and accepted October 26,1984. *Presented in part at the Thirty-Eighth Annual Meeting of The American Fertility Society, March 20 to 24, 1982, Las Vegas, Nevada. tDepartment of Obstetrics and Gynecology, Division ofHuman Reproduction. tReprint requests: Robert S. Schenken, M.D., Department of Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284. §Pregnancy Research Branch, National Institute of Child Health and Human Development, National Institutes of Health. IIPresent address: The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Lewis Hall, Room 2049, Eastern Virginia Medical School, Norfolk, Virginia 23501. Vol. 43, No.2, February 1985

Previous reports have demonstrated marked disparities between radioimmunoassayable (RIA) luteinizing hormone (LH) and bioassayable (BIO) LH in humans and monkeys under certain physiologic conditions. i-4 Study of this discordance during the follicular phase of the primate menstrual cycle has shown that the pulsatile secretion of BIO-LH undergoes a peri ovulatory transition that is temporally and quantitatively distinct from that of RIA-LH. 5 • 6 Typically, BIO-LH begins to rise - 6 hours before the surge mode of RIA-LH and RIA-follicle-stimulating hormone (FSH); further, the BIO/RIA-LH ratio often rose from near 1:1 to as much as 10:1 during the preovulatory gonadotropin surge. Moreover, exogenous treatment of castrate monkeys with estra-

Schenken et a1. Progestins and the E:zB-induced BIO-LH surge

301

diol benzoate (E 2B) induces gonadotropin surges indistinguishable from those seen in intact monkeys at midcycle, which demonstrates that estrogen"positive feedback alone can enhance the biologic activity of LH, possibly by reversal of the negative feedback state. 5 Numerous studies have discussed the supposed augmentative effects ofprogestins on estrogen-induced gonadotropin surges. 7-11 However, these observations were based exclusively on immunologic LH assays. In view of the recent evidence indicating an independent course of the BIO-LH surge in intact monkeys,5 we have studied progesterone (P) and 17a-hydroxyprogesterone (17OHP) potentiation of the estrogen-induced BIOLH surge, using castrate female monkeys. MATERIALS AND METHODS

Long-term castrate adult female rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) monkeys (n = 24) having elevated peripheral serum gonadotropin concentrations were chosen for study. These primates were maintained as described previously and provided with artificial light from 6:00 A.M. until 6:00 p.M.12 Monkeys were fitted with a novel vest and mobile tether, which permitted placement of a chronic indwelling femoral vein cannula and blood collection without anesthesia; details of these procedures have been described elsewhere. 13 Monkeys were given an intramuscular injection of E2B (50 fLg/kg) at To. Six monkeys served as estrogen-treated controls and received no further treatment; nine monkeys were given intramuscular injections ofP (35 fLg/kg) in oil at 8 (n = 3), 12 (n = 3), or 16 (n = 3) hours after E2B. Another nine monkeys were given intramuscular injections of 17 -OHP (35 fLglkg) in oil at 8 (n = 3), 12 (n = 3), or 16 (n = 3) hours after E2B. It was our intent that these estrogen and progestin treatments simulate the preovulatory elevation of these steroids in intact monkeys. Blood was collected every 15 minutes throughout a 24-hour interval, preselected to include onset of the induced gonadotropin surges. In addition, during the subsequent 24 hours, plasma was collected every 6 hours; thus, up to 100 samples were taken from each monkey. Plasma was harvested and frozen at - 4°C; red blood cells were resuspended in 0.9% saline and returned to the animals. For all plasma specimens, RIAs of FSH and LH were performed,14 as 302

36

-

34

32

BIO-LH RIA-LH RIA-FSH

30 28 26 24 22

20

I'giml ~: 14 12 10

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 3B 39 40

TIME (hoursl

Figure 1 Plasma concentration of LH and FSH determined by RIA and of LH determined by in vitro bioassay at 15-minute intervals over 24 hours, then every 6 hours on the subsequent day. Intramuscular injection of E2B (50 fJ-g/kg) at To.

well as an in vitro bioassay for LH.15 Plasma samples were pooled over 4-hour intervals before RIA of 17f3-estradiol (E 2), P, and 17-0HPY Tests for differences between group means were determined by the F statistic and the t-test. 16 However, the frequency an.d number of observations made here prohibit sophisticated statistical evaluation of the pulse amplitude and frequency characteristics of LH and FSH secretion at the onset of the gonadotropin surge mode. 17 Accordingly, the onset of the surge mode of secretion was determined arbitrarily by retrospective visualization of the ascending course of LH or FSH in circulation, as assessed by each assay system. We defined the onset of the surge as the time when the first plasma concentration exceeded the mean + 2 standard deviations of the preceding three values for BIO_LH.18 RESULTS ESTRADIOL ALONE

The monkeys in this group received E2B at To; blood collections were initiated either immediately or at T 16' Mean plasma E2 levels were in the range of 350 to 400 pg/ml during the interval of study. Throughout, plasma P and 17-0HP remained at or below the limits of detection « 0.1 ng/mI). Typically, plasma levels of pituitary gonadotropins measured by RIA and BIO were maximally suppressed by T 16; concurrently, the characteristic pulsatility of FSH and LH release, noted in

Schenken et al. Progestins and the E 2 B-induced BIO-LH surge

Fertility and Sterility

Table 1. Advancement of the Estrogen-Induced BIO-LH Surge by P (Hours [Mean ± SE] from Estrogen Injection to Onset of the BIO-LH Surge) E.B alone (rJ = 6)

26.2 ± 3.2

P (8 hours)

18.3 ±

E.B + pa (rJ = 3) P (12 hours)

2.3 b

21.5 ± 3.1

P (16 hours)

19.1 ±

.

0.7 b

I----------~----------I

I

19.8 ± 2.0c ap given 8, 12, or 16 hours after estrogen. bSignificantly different by comparison with estrogen treatment alone using the F statistic (P < 0.05). CSignificantly different by comparison with estrogen treatment alone using the t-test (P < 0.05).

the pretreatment state, became suppressed. In this group, the mean onset of the BIO-LH surge was 26.2 ± 3.2 (standard error [SE]) hours after estrogen treatment. Data from one representative monkey are presented in Figure 1. Uniformly, ascending limbs of these BIO-LH surges were established - 6 hours before initiation of the surges of LH and FSH by RIA. ESTRADIOL BENZOATE IN COMBINATION WITH PROGESTERONE

Three monkeys in this group received E2B at To and P at Ts. Plasma E2 levels were - 350 pg/ml from To to T 20, declining to - 200 pg/ml after 20 hours. P rose from the limits of detection « 0.1 ng/mD to near 0.4 ng/ml between Ts and T 12 , whereas plasma 17-0HP levels remained < 0.2 ng/ml. As expected, BIO-LH and RIA-LH were promptly suppressed after E2B administration, reaching a nadir by T s. Suppression of FSH in circulation was less dramatic. During this nega38 34

32 30

E,

+

p

-

tive feedback interval, the amplitudes and frequencies of FSH and LH pulses were markedly diminished in all assay systems. Onset of the BIO-LH surges was evident 18.3 ± 2.3 (SE) hours after E2B administration (Table 1). Uncharacteristically, one monkey manifested a near simultaneous rise in BIO-LH and RIA-LH (Fig. 2), whereas the other monkeys showed a distinctly earlier onset of the BIO-LH surge. Among monkeys receivihg E2B at To and P at T12, plasma E2 levels were near 400 pg/ml from To to T20, falling to - 250 pg/ml thereafter. Plasma P increased to near 0.6 ng/ml from T12 to T 20 . Throughout the sampling interval, plasma 17OHP levels were < 0.2 ng/ml. As in the other groups, E2B treatment caused a prompt suppression of pulsatile gonadotropin secretion, although the E 2-negative feedback effect on FSH was less dramatic. Interestingly, in one monkey (Fig. 3), the pulsatile character of BIO-LH secretion was reestablished briefly, coincident with the time of P administration at T 12 . Subsequently, BIO-LH in plasma returned to the baseline before transition to the midcycle-like surge near T l9 . One monkey in this group failed to demonstrate a BIO-LH surge onset until T 2S . Thus, the mean onset of the BIO-LH surge in this group was not significantly advanced as compared with that of monkeys treated with E2B alone (Table 1). Monkeys receiving P 16 hours after E2B demonstrated plasma E 2 levels of - 450 pg/ml from To to T20 , which later declined to - 300 pg/ml. Circulating P and 17-0HP levels were initially < 0.1 ng/ml; then, P levels increased to 0.6 ng/ml from TIS to T24 . Note that in one monkey (Fig. 4), BIOLH and.RIA-LH values were already significantly

BIO-LH RIA-LH RIA-FSH

+

28 26

24 22

12

-Wl~O~I~2~3~4~5~6~7~8~9~10~11~1~2~13~1~4~15~1~6~17~1~8wI9~~~2~122~n~~~~~ TIME (hours)

Vol. 43, No.2, February 1985

Figure 2 Plasma concentration of LH and FSH determined by RIA and of LH determined by in vitro bioassay at 15-minute intervals over 24 hours, then every 6 hours on the subsequent day. Intramuscular injection of E2B (50 ILg/kg) at To and P (35 ILglkg) in oil at +8 hours.

Schenken et al. Progestins and the E~-induced BIO-LH surge

303

36 34

p

E,

32

+

+

30

28 28

_ _ -

BIO-LH RIA-LH RIA-FSH

Figure 3 Plasma concentration of LH and FSH determined by RIA and of LH determined by in vitro bioassay at 15-minute intervals over 24 hours, then every 6 hours on the subsequent day. Intramuscular injection of E2B (50 J.Lglkg) at To and P (35 J.Lglkg) in oil at +12 hours.

"g/ml

-1 0 1 2 3 4 5 6 7 8 9

suppressed by T4, and the nadirs were reached by T 14. In this monkey, transition from the tonic to the surge mode of BIO-LH occurred near T 20 . No concomitant elevations of RIA-LH or FSH were detected during the sampling period. Table 1 summarizes the advancement of the estrogen-induced BIO-LH surge by P given 8, 12, and 16 hours after E2B. ESTRADIOL BENZOATE IN COMBINATION WITH 17a-HYDROXYPROGESTERONE

Three monkeys in this group received E 2B, followed by 17-0HP at Ts. Plasma E2 levels ranged from 350 to 450 pg/ml from T4 to T20 and declined to - 250 pg/ml thereafter. Plasma P levels remained undetectable during the sampling inter>50 36

__ RIA-LH

32

_RIA-FSH

30 28

26

/0

E,

I

2' 22

,.

20 ~g/ml

'8

j 1 0

1 2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

>90

>90

>50

rY~

. ....-BIO-LH

34

val, but 17-OHP increased to 0.8 to 1.0 ng/ml from Ts to T20 . Plasma gonadotropins were maximally suppressed when blood sampling was initiated at T4. The onset of the BIO-LH surge occurred 19.2 ± 1.0 (SE) hours after E2B injection (Table 2). In one monkey, a dramatic increase in BIO-LH was noted at T12, and the level exceeded 30 j.Lg/ml by T21 (Fig. 5). Monkeys receiving 17-0HP 12 hours after E2B demonstrated plasma E2 levels of - 450 pg/ml from T4 to T20 ; thereafter, levels fell to - 200 pg/ ml. Plasma P levels remained < 0.2 ng/ml, while plasma 17-0HP levels increased to 1.1 to 1.3 ng/ ml from T 12 to T 24. Interestingly, in one monkey a dramatic increase in the BIO-LH pulse amplitude was detected immediately upon 17-OHP injection (Fig. 6). However, BIO-LH levels gradually re60 50 40 30 28 26

"I

17QHP

......... BIO-LH _RIA_LH -RIA-FSH

~~

I >2.

2. 22

~"-

,.

20

'8

t~

~*'4k

~~~~~~~~~~::~~~~~~~~~~~~

-2 -1 0 4 5 6

7 8 9 10 11 12 13 ,,( 15 18 17 18 19 20 21 22 23 24 25 26 27 28 36 42 48 54

TIME (hours)

TIME

Figure 4 Plasma concentration of LH and FSH determined by RIA and of LH determined by in vitro bioassay at 15-minute intervals over 24 hours, then every 6 hours on the subsequent day. Intramuscular injection of E2B (50 J.Lglkg) at To and P (35 J.Lg/kg) in oil at + 16 hours. 304

Figure 5 Plasma concentration of LH and FSH determined by RIA and of LH determined by in vitro bioassay at 15-minute intervals over 24 hours, then every 6 hours on the subsequent day. Intramuscular injection of E2B (50 J.Lglkg) at To and 17 -OHP (35 J.Lglkg) in oil at + 8 hours. .

Schenken et aI. Progestins and the E;zB-induced BIO-LH surge

Fertility and Sterility

38>50 E2

........ 810- LH ...... RIA-LH ........ RIA-FSH

170HP

34

32 30

28 28

~

"

r-~

>25

Jr /

2' 22

-

>50>80>80

I.,. ,.

20

12

I 2

-~2~-1~0~'~5~.~7~.~9~10~1~11~2~13~1'~15~1.~1~71~.~'9~~~21~2~22~32~'~25~~~27~28~.2~'~ Time (hours)

Figure 6 Plasma concentration of LH and FSH determined by RIA and of LH determined by in vitro bioassay at 15-minute intervals over 24 hours, then every 6 hours on the subsequent day. Intramuscular injection of E2B (50 ....g/kg) at To and 17 -OHP (35 ....glkg) in oil at + 12 hours.

turned to baseline at T 14 before demonstrating a sustained surge onset at T 17 . In this group, the BIO-LH surge was evident 18.5 ± 3.0 (SE) hours after E2B injection (Table 2). Among monkeys receiving 17-0HP injection 16 hours after E 2B, E2 levels were 400 to 450 pg/ml from T4 to T24 and then decreased to - 250 pg/ml. Similar to those in other 17-0HP-treated monkeys, P levels remained < 0.2 ng/ml, and plasma 17-0HP levels rose to 1.1 to 1.4 ng/ml from T 20 to T24 • Onset ofthe BIO-LH surge in this group was apparent 18.8 ± 1.5 (SE) hours after E2B (Table 2).

DISCUSSION

It has been reported that in primates estrogen induces the preovulatory gonadotropin surges by acting directly upon the pituitary.19 Furthermore, recent evidence has shown that estrogenpositive feedback may cause a relative increase in the biologic activity of LH.5 Here we have studied these events in unison by sequential estrogen and progestin treatments of castrate monkeys. Whereas E2B alone provokes a transient inhibition of pituitary gonadotropin secretion, typically the BIO~LH surge is initiated 26 hours later. Note that these BIO-LH surges occurred up to 6 hours before the surge of LH and FSH measured by RIA; a few exceptions were found. Clearly, among monkeys receiving P 8,12, or 16 hours after exogenous estrogen, the onset of the BIO-LH surge was advanced up to 8 hours in eight of nine monkeys and occurred before the Vol. 43, No.2, February 1985

onset of the immunoassayable gonadotropin surges. The lone exception was a monkey receiving P 12 hours after E2B and manifesting a latent rise in all gonadotropin measurements. All monkeys receiving 17-0HP after exogenous estrogen demonstrated an earlier onset of the BIO-LH surge as compared with E2B treatment alone. Not unexpectedly, we were unable to discern a differential effect of the interval from estrogen injection to P or 17-OHP therapy in the'se small groups. Our interpretation of the physiologic importance of this facilitory effect of P and 17 -OHP on the LH surge, especially its BIO components, must be regarded as speculative in view of the continuing debate over the periovulatory time course of progestin elevations and LH changes. 18. 20-25 Until recently, many reports failed to acknowledge an increase in progestins at or before the onset of the LH surge. However, recent studies have shown that serum P and 17-0HP begin to increase - 12 hours before a detectable rise in RIA-LH in women and - 6 to 12 hours before a midcycle increment in RIA-LH or BIOLH in monkeys.6.l8 Collectively, this growing body of evidence illustrates the probable physiologic significance of these small increments in progestins in staging the ovulatory event. Apparently, in the late follicular phase, the BIO-LH surge may be regulated temporally by the secretion of P and/or 17-0HP from the near-ovulatory dominant follicle. 18. 19 We must consider whether the preovulatory follicle, in the presence of a high-estrogen milieu, schedules (hastens) the final stages of ovum maturation and release through the augmentative action of one or more of these progestins. Whereas these castrate monkeys have proven useful in defining the augmentative effects of proTable 2. Advancement of the Estrogen-Induced BIO-LH Surge by 17-0HP (Hours [Mean ± SE] from Estrogen Injection to Onset of the BIO-LH Surge) E.B alone _ _--::c=,--E=-.B_+_l~7::_-0_::H:::pa=_(n-=-3)__:_:=_==-(n = 6) 17-0HP 17-0HP 17-0HP (8 hours) (12 hours) (16 hours)

26.2 ± 3.2

19.2 ± l.Ob

18.5 ± 3.0 b

18.8 ± l.5 b

I----------~--------I

I

18.7 ± l.8e

a17-0HP given 8,12, or 16 hours after estrogen. bSignificantly different by comparison with estrogen treatment alone using the F statistic (P < 0.05). eSignificantly different by comparison with estrogen treatment alone using the t-test (P < 0.05).

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gestins during estrogen-induced gonadotropin surges, this model cannot account for all the interactive processes of the intact state. These limitations must be realized in interpreting the results presented. An additional limitation of this study is the inability to apply refined statistical assessments of the changes in endocrine signaling which accompany the transition from negative to positive feedback by estrogens on gonadotropin secretion, with or without progestin treatment. Indeed, an experimental design adequate to determine changes in pulse frequency would require a sampling frequency at least six times higher than the signal (pulse) under scrutinyP For example, if LH is secreted with a circhoral rhythmicity,5 the sampling frequency must be no less than six per hour. Obvious constraints (blood volume, time course of the biologic event, technical effort, cost, etc.) make such intensive protocols impractical. In summary, we confirm and extend claims that estrogen-positive feedback alone can induce midcycle-like surges of LH and FSH in long-term castrate monkeys, including enhancement of BIO-LH activity. Furthermore, P and 17-0HP expedite the onset of the gonadotropin surges, most notably that of BIO-LH. Indeed, P and/or 17-OHP may be an ovarian signal synchronizing the release of biologically active LH with final maturation of the dominant follicle and provision of a fertilizable ovum. REFERENCES 1. Dufau ML, Beitins IZ, McArthur JW, Catt KJ: Effects of luteinizing hormone releasing hormone (LHRH) upon bioactive and immunoreactive serum LH levels in normal subjects. J Clin Endocrinol Metab 43:658, 1976 2. Dufau ML, Hodgen GD, Goodman AL, Catt KJ: Bioassay of circulating luteinizing hormone in the rhesus monkey: comparison with radioimmunoassay during physiological changes. Endocrinology 100:1557, 1977 3. Neill JD, Daily RA, Tsou RL, Reichert LE Jr: Immunoreactive LH-like substances in serum of hypophysectomized and prepubertal monkeys: inactive in an in vitro LH bioassay. Endocrinology 100:856, 1977 4. Dufau ML, Veldhuis JD, Fraioli F, Johnson ML, Beitins IZ: Mode of secretion of bioactive luteinizing hormone in man. J Clin Endocrinol Metab 57:993, 1983 5. Marut EL, Williams RF, Cowan BD, Lynch A, Lerner SP, Hodgen GD: Pulsatile pituitary gonadotropin secretion during maturation of the dominant follicle in monkeys: estrogen positive feedback enhances the biological activity of LH. Endocrinology 109:2270, 1981 6. Schenken RS, Werlin LB, Williams RF, Hodgen GD: Periovulatory hormonal dynamics: roles of ovarian steroids in initiating the immunoassayable and bioassayable LH

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23. Landgren BM, Aedo AR, Nunes M, Cekan SZ, Diczfalusy E: Studies on the pattern of circulating steroids in the menstrual cycle. Acta Endocrinol (Copenh) 84:620, 1977 24. Laborde N, Carril M, CheviakoffS, Croxatto HD, Penroze E, Rosner JM: The secretion of progesterone during the,

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periovulatory period in women with certified ovulations. J Clin Endocrinol Metab 43:1157,1976 25. Uemura S, Tsuchihashi S: Changes in serum 17a-hydroxyprogesterone levels during the peri ovulatory phase in women. Endocrinol Jpn 25:295, 1978

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