CO13-7227/79/1044-0940$02.00/0 Endocrinology Copyright © 1979 by the Endocrine Society

Vol. 104, No. 4

Printed in U.S.A.

Further Evidence that Luteinizing Hormone-Releasing Hormone also Is Follicle-Stimulating Hormone-Releasing Hormone* PHYLLIS M. WISE, NAOMI RANCE,f GEORGE D. BARR4 AND CHARLES A. BARRACLOUGH Department of Physiology, School of Medicine, University of Maryland, Baltimore, Maryland 21201

ABSTRACT. While LHRH evokes the release of both LH and FSH under a wide spectrum of experimental conditions, this hormone has not been shown to induce the discharge of FSH unaccompanied by LH release. As a consequence, the possibility that a separate FSHRH exists has not been discounted. These experiments were designed to demonstrate that LHRH is capable of promoting marked increases in plasma FSH concomitant with physiologically insignificant changes in plasma LH and, thus, that LHRH is also FSHRH. Seven groups of cyclic female rats received phenobarbital at 1200 h proestrus to block spontaneous preovulatory LH and FSH surges. These various groups then were subdivided according to whether phosphate-buffered saline (PBS), LHRH, or both was infused (iv). The experimental protocol consisted of infusing material for an initial 30 min (priming period), stopping the infusion for 30 min (rest interval), and again infusing material for an additional 210 min (maintenance period). When PBS was infused during the priming and maintenance periods, plasma LH and FSH remained basal. In contrast, when a total of 25 ng LHRH was infused during the first 30 min, followed by the infusion of a total of 17.5 ng LHRH during the 210-min maintenance period, plasma FSH concentra-

I

S LHRH also FSHRH or does a separate FSHRH exist which has yet to be identified? This question has received considerable discussion; those who favor the single releasing hormone theory argue that despite numerous purification procedures of hypothalamic extracts, both highly purified hormone and synthetic LHRH induce the release of LH and FSH within a wide spectrum of experimental conditions (1-4). In certain situations, marked increases in plasma LH and slight rises in plasma FSH occur when an iv pulse injection of LHRH is given (5-7). However, if the pituitary gland is exposed to LHRH for prolonged periods, more pronounced releases of FSH accompany LH secretion (6-8). A criticism of

Received August 7,1978. Address all correspondence and requests for reprints to: Dr. Phyllis M. Wise, Department of Physiology, University of Maryland School of Medicine, 600 West Redwood Street, Baltimore, Maryland 21201. * This work was supported by USPHS Grant HD-02138. t Predoctoral fellow of the Reproductive Endocrine Training Program supported by USPHS Grant HD-00435. $ Present address: Department of Cell Biology, Baylor College of Medicine, Texas Medical Center, Houston, Texas 77030.

tions increased to levels comparable to those observed during the proestrous preovulatory FSH surge. In these animals, plasma LH increased to only 8% of proestrous values, which was insufficient to alter plasma estradiol and progesterone concentrations 60 min after the beginning of the initial LHRH infusion period. When the priming dose of LHRH was decreased by 50% (12.5 ng), the peak plasma FSH concentrations, which were induced by infusing 17.5 ng LHRH during the maintenance period, also were halved. Infusion of LHRH only during the priming or maintenance period did not appreciably affect basal plasma FSH concentrations. In Nembutal-blocked proestrous rats, low intensity electrical stimulation of the preoptic area was performed in an attempt to induce the endogenous release of LHRH in patterns and concentrations similar to those produced by our infusion protocol. Such stimuli induced the selective increase in plasma FSH. To our knowledge, these studies are the first to demonstrate that synthetic LHRH or medial preoptic area electrical stimulation can induce marked increase in plasma FSH without prior or simultaneous large increments in plasma LH; thus, they provide additional evidence that LHRH also is FSHRH. (Endocrinology 104: 940, 1979)

these studies is that the delayed rise in plasma FSH may be the result of the earlier increases in plasma LH. Such rises in LH may alter the steroid milieu and, thus, favor the secretion of FSH (9). However, previous studies have shown that the bolus injection of highly purified rat LH into Nembutal-blocked proestrous rats causes a rise in plasma FSH 3 h later, and this response is not due to a decline in plasma estrogen (10). LHRH also stimulates the secretion of LH and FSH in primary pituitary cell culture systems, although a change in the ratio of LH: FSH occurs if these monolayer cell cultures are preincubated with different steroid regimes (11). On the other hand, those investigators who propose that a separate FSHRH exists indicate that LHRH never has stimulated pituitary FSH release unaccompanied or preceded by the discharge of LH. Yet there are several physiological and experimental situations when the selective release of FSH occurs: 1) in cyclic rats, plasma FSH is elevated throughout estrous morning when LH concentrations are basal (12, 13); 2) plasma FSH levels rise within hours after ovariectomy before any change in 940

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LHRH INDUCES SELECTIVE FSH RELEASE plasma LH (14); and 3) electrochemical stimulation of the dorsal anterior hypothalamic area provokes the selective release of pituitary FSH but not LH (15). The hypothesis that a single releasing hormone exists would be considerably strengthened if experimental evidence could be presented that LHRH can induce the selective release of pituitary FSH in animal preparations competent to release both gonadotropins (viz the proestrous rat). We believe that the following studies provide such evidence.

941

120

mm 150 PBS

LHRH 17 5 ng

LHRH 17 5 ng

LHRH 17 5 ng

Materials and Methods Adult Sprague-Dawley female rats (ARS/Madison, WI), weighing 200-250 g, were housed in a temperature (22-24 C)and light (lights on 0400-1800 h)-controlled environment for 23 weeks before use. Estrous cyclicity was monitored by daily vaginal lavages and only those rats which exhibited at least two consecutive 4-day cycles were used in these studies. In these proestrous rats, the onset of the preovulatory LH and FSH surges occurred between 1300-1500 h proestrus. Infusion experiments Proestrous animals were anesthetized with ether between 0900-1100 h. The right external jugular vein was exposed, and a polyethylene cannula (od, 9.65 mm) was inserted to the level of the right atrium for subsequent infusion of hormone; a second cannula was placed in the femoral artery to collect blood. Both cannulae were coursed beneath the skin to the back and were cut short so that the rat could not disturb the cannulae. The entire surgical procedure was completed in 10-15 min. At 1215 h, animals were given phenobarbital in a dosage that does not affect the responsiveness of the pituitary gland to LHRH (75 mg/kg BW, ip) (6). Rats were then divided into eight experimental groups, as illustrated in Fig. 1. Phosphate-buffered saline (PBS) and/or LHRH (Beckman, lot D0420) dissolved in PBS was infused at a rate of 0.5 ml/h. There were two infusion periods. The first lasted 30 min and is termed the priming period; the second infusion was given for 210 min and is referred to as the maintenance period. The priming and maintenance periods were separated by a 30-min rest interval during which infusions were stopped. In groups 1-7, blood was collected (0.6 ml) before the beginning of the infusions and again at 30, 60,90, 120, 150, 210, and 270 min after the experiments were begun. The volume of blood removed was replaced by the combined injection and infusion of PBS (with or without LHRH). Group 1. These rats received only PBS (0.01 M, pH 7.4) during the priming and maintenance periods. Group 2. These rats received PBS during the priming period and LHRH was infused at a concentration of 10 ng/ml during the maintenance period. A total of 17.5 ng LHRH was given in this interval. Group 3. This group received a total of 25 ng LHRH (100 ng/ ml) during the priming period, followed by a total of 17.5 ng/ml LHRH during the maintenance period. Group 4. These animals received a total of 25 ng LHRH during the priming period and only PBS thereafter.

Blood Collections i 8.

| ELECTRICAL STIMULATION OF MPOA |

FIG. 1. Diagrammatic representation of the experimental protocol. Rats in groups 1-6 received PBS, LHRH, or a combination of both during the priming (30 min) and maintenance (210 min) periods. The two infusion periods were separated by a 30-min rest interval. Rats in group 6 were infused with highly purified rat LH (0.4 jug/h) for 90 min. Rats in group 7 received MPOA electrical stimulation for 2 h.

Group 5. Animals in this group received a total of 12.5 ng LHRH (50 ng/ml) during the priming period and a total of 17.5 ng LHRH during the maintenance period. Group 6. These rats received a total of 12.5 ng LHRH during the priming period and PBS thereafter. Group 7. This group of rats was infused with a total of 0.6 jug highly purified rat LH [NIAMDD-rat LH 1-4, which is approximately equal to 1.0 x NIH-LH-Sl(OAAD assay)] for 90 min (0.8 jug/ml at 0.5 ml/h). This amount of LH, when infused, produced peak plasma LH concentrations which were comparable to those which occurred after the LHRH infusion of rats in group 3. Group 8: effects of electrical stimulation of the medial preoptic area (MPOA) in pentobarbital-treated rats. Rats in this group received sodium pentobarbital (Nembutal) at 1200 h (31 mg/kg BW, ip), at 1500 h (10 mg/kg BW, ip), and at 1700 h (5 mg/kg BW, ip) on proestrus. Additional pentobarbital was given to produce prolonged deep anesthesia throughout the proestrous afternoon, as judged by leg withdrawal to foot pinch. These rats received external jugular cannulae, were placed in a stereotaxic instrument, and concentric bipolar stainless steel electrodes were oriented unilaterally into the MPOA. These electrodes consisted of a 27-gauge outer barrel and a 36-gauge inner stainless steel wire, which were insulated with epoxylite resin. Insulation was removed from 1.5 mm of the barrel and 0.2 mm of the center wire and these areas were separated by 0.4 mm of resin. Electrical stimulation was begun at 1300 h and terminated 120 min later. Current was supplied by two constant current stimulators which were triggered by two Grass model S-4 stimulators, and the stimuli were monitored by an oscilloscope. The stimulation parameters consisted of biphasic rectangular pulses of 10 jiiA peak to peak with a 1.0-msec pulse duration and a frequency of 50 Hz delivered at 15-sec on/off intervals for

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942

Endo • 1979 Vol 104 • No 4

WISE ET AL.

120 min. Blood (0.6 ml) was collected just before stimulation and at 60, 120, 180, 240, and 300 min after stimulation was begun. The animals then were sacrificed, and the brains were perfused with saturated potassium ferro-ferricyanide solution in 0.9% saline, followed by perfusion with 10% formalin. Sections of these brains were made to verify if the electrodes were correctly placed and if ferrous ion deposits or lesions occurred. In sham-control rats, electrodes were placed in the MPOA, but electrical current was not passed. RIAs Estradiol (E2). Plasma E2 levels were determined by RIA with an antibody (1:40,000 dilution) raised in sheep against 17/?-E2 using 6-O-carboxymethyloxime conjugated to bovine serum albumin. The specificity of this antibody to E2 has been previously determined (16). The isotope used was (2, 4, 6, 7[-3H]E2 (New England Nuclear) and the assay procedure was modified slightly from that previously reported (16). Briefly, determinations were run in duplicate on 500-jul plasma samples. After extraction with 2 ml benzene, the solvent phase was removed and evaporated with nitrogen gas. To the dried residue were added 100 /xl 0.1% gelatin in PBS, 100 jul antibody, and 3200 dpm [3H]E2, and this mixture was allowed to incubate at 4 C overnight. Antibody-bound steroid was separated from unbound hormone by dextran-coated charcoal and, after a 20-min incubation period, the mixture was centrifuged and decanted into scintillation vials. Scintillation cocktail (New England Nuclear, 950A) was added and radioactivity was measured in a Packard Tri-Carb scintillation counter. The assay was sensitive between 3-100 pg/tube, with 50% inhibition of binding occurring at 15 pg. Plasma blanks (4 ± 0.1 pg/ml) were subtracted from the assay values. The recovery was 95%, as determined by adding 10 pg E2 to plasma blanks. The internal standard was a plasma pool obtained from cyclic female rats. Using the method of Rodbard (17), the within-assay variability was calculated to be 8.5% and the between-assay variability was 10.8%. Progesterone. Progesterone was measured by RIA using an antibody raised in sheep against progesterone-11 bovine serum albumin (GDN 337) at a dilution of 1:25,000 and (1,2,6,7 -3H)progester6ne (New England Nuclear) as the isotope. The specificity of the antiserum has been reported previously (18) and the assay procedure was modified slightly from that described by Thorneycroft and Stone (18). Plasma samples of 25 or 50 jul were diluted to 1 ml with distilled water. Progesterone measurements were made on aliquots (100 and 200 jul) of these dilutions. After extraction with petroleum ether (2.0 ml), the solvent phase was removed and evaporated with nitrogen gas. Antibody (200 jul) and [3H]progesterone (32,000 dpm) were added to the dried residue and the assay tubes were incubated at 4 C overnight. After addition of dextran-coated charcoal and a 15-min incubation period, the mixture was centrifuged and the supernatant solution was counted, as described previously for the E2 assay. The sensitivity of this assay ranged between 10-1000 pg/tube, with 50% inhibition of binding at 100 pg. Plasma blanks were below the sensitivity of the assay (10 pg) and recoveries of progesterone in plasma blanks were 95%. The interassay variability was calculated to be 6.9% and the intraassay variability was 6.5%.

LH and FSH assays. Plasma LH concentrations were determined by RIA according to the ovine:ovine procedure of Niswender et al. (19). NIAMDD-rat LH-RP-1, which has a biological potency equivalent to 0.03 X NIH-LH-Sl, was used as a standard. In every animal, duplicate 10- and 25-jul aliquots and a single 50-jul aliquot of plasma were assayed. Serum pools obtained from castrated rats were measured in all assays with an interassay coefficient of variation of 6.0% and an intraassay coefficient of variation of 5.1%. Plasma FSH was assayed using rat RIA kit materials (FSH-I-3 and anti-FSH-8) and NIAMDD rat FSH-RP-1 as a standard which is 2.1 x NIH-FSH-S1 standard. Modifications of this assay procedure have been reported previously (15). Additional assay sensitivity was achieved by incubating the unknown samples and the standards with anti-FSH for 48 h and then with [125I]FSH for a second 48 h. At the end of the second incubation, antirabbit y-globulin was added and the assay was terminated 72 h later. We found that maximal precipitable counts per min were obtained when the antirabbit -/-globulin (Antibodies Inc., Davis, CA) was used at a 1:150 dilution. Duplicate 50-jul aliquots of plasma were assayed from every rat. The within-assay variation was 4.2% and the between-assay variation was 5.7%. Statistical analyses between and among experimental groups were performed using Duncan's multiple range test. Results While phenobarbital and pentobarbital were equally effective in blocking the proestrous LH surge (Figs. 2A and 4), these barbiturates had different effects on proestrous FSH release. Phenobarbital treatment completely prevented the proestrous afternoon rise in FSH and the infusion of PBS into these animals did not alter basal LH and FSH plasma concentrations when these values were compared to noninfused phenobarbital-treated proestrous rats (data not illustrated; Fig. 2A). In contrast, despite repetitive injections on proestrus, pentobarbital did not completely block the afternoon rise in FSH. Plasma FSH concentrations rose gradually and were significantly different from baseline levels at 240 min (P < 0.05; Fig. 4). It is important to note that all rats which exhibited concentrations of LH and FSH which were greater than the mean by more than 2 SDS in the initial blood collection (t = 0) were eliminated from all experimental groups. These animals apparently had already begun their spontaneous gonadotropin surges before the time that Nembutal or phenobarbital were administered, and thus intrinsic self-priming of the pituitary gland by LHRH presumably had occurred. As a consequence, when exogenous LHRH was infused into these animals or when the MPOA was stimulated, both LH and FSH surges occurred. If PBS was infused during the priming period and 17.5 ng LHRH were given for 210 min during the maintenance period, basal plasma LH and FSH levels were unaffected (Fig. 2B). In contrast, if a total of 25 ng LHRH was

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943

LHRH INDUCES SELECTIVE FSH RELEASE

FIG. 2. A, Plasma LH and FSH concentrations in phenobarbital-blocked rats during PBS infusions (n = 10). Vertical bars in this figure represent the SEM. B, Plasma LH and FSH responses to LHRH (17.5 ng) infused only during the maintenance period (n = 5). PBS was infused during the initial 30 min. C, Changes in plasma LH and FSH in rats receiving LHRH (25 ng) during the priming period and maintenance periods (17.5 ng; n = 7). While plasma LH increased to peak values of only 233 ± 28 ng/ml (normal proestrous LH peak is 3080 ng/ ml), plasma FSH rose to reach 657 ± 69 ng/ml, which is comparable to normal peak proestrous FSH concentrations (625 ng/ml). D, Plasma LH and FSH concentration changes in rats receiving LHRH (25 ng) only during the period (n = 5). E, Plasma LH and FSH responses in rats infused with LHRH (12.5 ng) during both the priming and maintenance periods (17.5 ng; n = 6). F, Plasma LH and FSH concentrations in rats receiving LHRH (12.5 ng) during the priming period only (n = 5).

— FSH •••

LH

o so «o »o 120 IBO

2TO 0

SO 60 90 120 IBO

270 0 SO 60 90 120 IBO

infused for the 30-min priming period, followed by 17.5 ng LHRH given, during the maintenance period, FSH plasma levels increased significantly by 90 min over PBSinfused controls (P < 0.01) and they eventually reached plasma concentrations normally attained on proestrous afternoon (659 ± 69 us. 625 ± 64 ng/ml; Fig. 2C). Plasma LH values were increased significantly in this group at 30 min and a second similar LH peak was observed at 90 min which was significantly elevated above the 60-min values. Thereafter, plasma LH declined slowly but remained significantly elevated above PBS-infused controls through 270 min. It should be emphasized that the peak LH concentrations reached in this experimental group (233 ± 28 ng/ml) represent only 8% of those values obtained during the normal spontaneous proestrous surge (3080 ± 263 ng/ml; Fig. 2C). When LHRH (total of 25 ng) is infused during the 30min priming period and only PBS is given during the 210-min maintenance period, a small delayed rise in FSH occurs which is significantly higher (P < 0.05) than basal concentrations at 210 min (Fig. 2D). The rise in plasma LH obtained in this group of rats during the initial infusion of LHRH was comparable to that obtained in group 3, although LH had returned to control concentrations by 120 min. When the LHRH dose was halved to 12.5 ng (group 5)

and infused during the priming period, followed by a maintenance dose of 17.5 ng LHRH, plasma FSH levels increased but only to approximately one half of those obtained in group 3 (Fig. 2E). Plasma concentrations of this gonadotropin rose gradually and became significantly different from PBS-infused controls at 120 min. In this group, plasma LH values remained significantly higher than PBS-infused controls throughout the infusion period (P < 0.05). Infusion of only the priming dose of 12.5 ng LHRH (group 6), followed by PBS administration during the maintenance period, failed to alter plasma FSH and produced only a transient rise in plasma LH which returned to basal control levels by 120 min (Fig. 2F). Since it was previously reported (10) that the elevation of plasma LH results in the subsequent rise in plasma FSH in Nembutal-blocked proestrous rats, we were concerned that the rise in plasma LH which occurred in rats receiving 25 ng LHRH during the priming period may have affected endogenous FSH secretion. Accordingly, we infused highly purified rat LH for 90 min (group 7) in concentrations which produced plasma LH levels similar to those obtained in rats in group 3. Such treatment caused only minimal FSH release, although levels of this hormone were significantly higher than PBS-infused controls at 210 and 270 min (Fig. 3).

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WISE ET AL.

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Rndo Vol 104

no evidence of lesion formation or deposition of ferrous ions.

LH 0 6 ng FSH LH

Discussion 2000

400

300 -

SH ng/

E

id

3 L

200 -

l000

500

100-

30

60

1979 No 4

90

120

150

210

270

These results clearly demonstrate that LHRH, when administered under certain restricted conditions, causes the selective release of pituitary FSH and has only minor effects on LH release. When we infused 25 ng LHRH over 30 min, allowed a 30-min rest interval, and then infused 17.5 ng LHRH for 210 min, plasma FSH concentrations increased 5-fold to levels normally attained during the spontaneous proestrous surge (Fig. 2C). Both the priming and maintenance components of this infusion schedule are required to evoke selective FSH secretion, for if LHRH is given during the maintenance period to TABLE 1. Plasma E2 and progesterone concentrations in rats treated with LHRH during the priming and/or maintenance periods or LH for 90 min

Minutes

FIG. 3. Plasma LH concentrations attained during infusion of purified rat LH (0.6 jug) for 90 min. Only minor changes in FSH secretion occurred in these rats (n = 5).

E2 (pg/ml)

1

47 47 47 47 47 47 47

4

5 6 7

270

60

0*

2 3

Plasma E2 and progesterone concentrations were measured in each experimental group at 0, 60, and 270 min. The small rises in plasma LH did not alter plasma levels of these steroids in any group at 60 min (Table 1). These observations are important, since changes in plasma steroids at this time (60 min) could have affected pituitary FSH secretion at later times in the experiment. Basal progesterone concentrations were higher in rats used in these experiments compared to animals which were decapitated at 1200 h proestrus (22 ± 3 vs. 7 ± 1 ng/ml), presumably as a consequence of the stress of ether anesthesia and surgical manipulation (Table 1). With the information obtained from groups 3 and 5, that LHRH can induce the secretion of pituitary FSH with only minor changes in LH release, we wished to determine if similar FSH and LH release patterns could be induced by activation of a brain region previously shown to induce marked preovulatory-like discharges of both gonadotropins (15). When low intensity electrical stimuli were presented to the MPOA over 120 min, plasma FSH concentrations gradually increased, were significantly elevated over sham-stimulated control values at 120 min, and reached peak levels of 458 ± 21 ng/ ml at 300 min (group 8; Fig. 4). In this preparation, plasma LH did not differ significantly from control concentrations at any of the times examined. Histological examination of the brains of animals receiving sham or electrical stimulation revealed that the electrodes were located in the dorsal MPOA. There was

Progesterone; (ng/ml)"

Group no. ±7 ±7 ±7 ±7 ±7 ±7 ±7

42 42 44 44

±8 ±8 ±4 ±4

40 ±2 40 ±2 38 ±8

0

41 ± 6 42 ± 4 50 ± 7 34 ± 7 46 ± 7 47 ± 8 39 ± 5

22 22 22 22 22 22 22

±3 ±3 ±3 ±3 ±3 ±3 ±3

60

270

20 ± 8 20 ± 8 18 ± 4 18 ± 4 23 ± 2 23 ± 2 15 ± 3

13 ± 3 15 ± 4 23 ± 3 12 ± 2 15 ± 4 15 ± 4 10 ± 6

" Plasma progesterone concentrations in rats decapitated at 1200 h without prior anesthesia or surgery (7 ± 1 ng/ml; n = 13). A Time of decapitation (minutes). INTERVAL OF ELECTRICAL OR' SHAM STIMULATION

500 T

. A .

. FSH-ES * FSH-SS . LH-ESorSS

400-

-2000

300-

M000

200-

•500

1200

I30C

1500

I40C

I6O0

hours

FIG. 4. LH and FSH responses after low intensity electrical stimulation of the MPOA for 2 h (n = 8) compared to sham-stimulated controls (n = 8).

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LHRH INDUCES SELECTIVE FSH RELEASE rats not previously primed with the decapeptide, plasma FSH concentrations remain basal (Fig. 2B). The rest interval, where no material is infused, may be important in permitting LHRH to mobilize pituitary FSH into the releasable pool so that it can be discharged in response to stimuli presented during the second 210-min infusion period. It also may allow for the clearance of the initially infused hormone from the peripheral circulation. In contrast, if LHRH is infused continuously for 3 h at rates administered during the priming period (50 ng/h), it promotes proestrous-like surges of both LH and FSH (6, 7, 20). Seemingly, the brief initial infusion period (30 min) of LHRH (25 ng) renders FSH gonadotrophs responsive to LHRH concentrations which normally are ineffective in evoking FSH release (17.5 ng; Fig. 2B). Yet this initial infusion period does not prime the LH gonadotrophs to a comparable degree. Further, the concentrations of LHRH presented to the pituitary gonadotrophs during the priming period also are critical, for it is this priming dose which dictates the magnitude of the FSH response to LHRH obtained during the second infusion period. Thus, if the priming dose of LHRH is reduced by 50% (from 25 to 12.5 ng), the subsequent plasma concentrations of FSH released after the infusion of LHRH during the maintenance period also are reduced by approximately 50% at 270 min (Fig. 2, compare C and E). Moreover, the higher the dosage of LHRH infused during the priming period, the more rapidly FSH rises in plasma during the second infusion period. These observations suggest that as the priming dose of LHRH is increased, responsiveness of the FSH gonadotroph also increases, perhaps via mobilization of a greater releasable pool of pituitary FSH. Varying the LHRH concentrations presented during the priming period also differentially affects the responsiveness of the LH gonadotroph to hormone infusions presented during the maintenance period. Thus, when 25 ng LHRH are infused during the priming period, a small LH plasma peak occurs. During the rest interval, plasma LH declines, but a comparable secondary plasma LH rise occurs in these rats during the second infusion period when only one tenth of the concentration of LHRH is infused. Seemingly, 25 ng LHRH are sufficient to produce some self-priming of the pituitary gland to the smaller concentrations of LHRH administered during the maintenance period. These data support the original observations of Aiyer et al. (21), who reported marked increases in responsiveness of pituitary LH gonadotrophs to a second pulse injection of LHRH given in an amount equal to that administered 60 min earlier. Previous studies have shown that the pulse injection of LH into Nembutal-blocked proestrous rats results in a rise in plasma FSH 3 h later (10). Since the infusion of 25 ng LHRH for 30 min elevated plasma LH (Fig. 2C), we reproduced these plasma gonadotropin concentra-

945

tions by infusing highly purified rat LH (Fig. 3) into phenobarbital-blocked proestrous rats. In these animals, only minimal plasma FSH increases occurred 210 and 270 min later. Therefore, it is doubtful that these small LHRH-induced increases in plasma LH are physiologically important, for not only is pituitary FSH release not affected, but plasma E2 and progesterone concentrations remain unaltered at 60 min. If changes in steroid milieu are important in the FSH response, one would expect to observe them by 60 min, since the first significant change in FSH is seen at 90 min in group 4. In the absence of any evidence of changing plasma steroid concentrations in these experimental animals, we believe that the selective rises we observed in plasma FSH are directly attributable to the action of LHRH on FSH gonadotrophs. Thus, proestrous pituitary glands can release LH, FSH, or both gonadotropins depending upon the LHRH signal input characteristics presented to the gonadotrophs. A brief pulse injection of high concentrations of LHRH to Nembutal-blocked proestrous rats elicits the selective release of pituitary LH, whereas glands exposed to low amounts of LHRH delivered over prolonged periods secrete primarily FSH. If high levels of the decapeptide are administered for prolonged periods, then both LH and FSH are released. Additional support for the concept that a single releasing hormone exists which can induce the release of either or both gonadotropins (LH and FSH) is provided by the results obtained after MPOA electrical stimulation. Previous reports from this laboratory have shown that electrochemical stimulation (ECS) of the MPOA induces the release of both LH and FSH (15), with the amount of LH secreted being directly proportional to the quantity of preoptic tissue activated (22). Further, MPOA-ECS causes pulsatile changes in medial basal hypothalamic LHRH concentrations (23) and a rise in portal plasma LHRH (24). With the information gained from the current LHRH infusion studies, we reasoned that we could affect the endogenous LHRH input signal to the pituitary gland by varying the stimulation parameters used to activate preoptic neurons. Electrical stimulation methodology was used to increase the duration and decrease the current strength and frequency at which such stimuli were presented to preoptic tissue. When such stimuli were delivered for a prolonged period to neural elements in the MPOA, the selective release of FSH occurred in Nembutal-blocked proestrous rats. Perhaps such stimuli produced temporal patterns and concentrations of LHRH in portal plasma which resembled those which occurred during our exogenous LHRH infusion schedule. These data further imply the dorsal anterior hypothalamic area (DAHA) ECS may activate only small numbers of LHRH-releasing cells to provide signal input of similar strength and duration to that seen by MPOAelectrical stimulation or by exogenous LHRH infusions.

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WISE ET AL.

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Thus, while small increments in peripheral plasma LH occur after DAHA-ECS, plasma FSH concentrations are markedly elevated. Indeed, Chiappa et al. (25) found that much less LHRH was released into portal plasma after DAHA vs. MPOA stimulation. The present data also aid in understanding why only FSH is elevated on estrous morning or why ovariectomy or LH pulse injections in Nembutal-blocked proestrous rats results in an increase in plasma FSH unaccompanied by LH. These other circumstances of selective FSH release may be reasonably explained if a substance similar to that obtained in porcine follicular fluid can be identified in peripheral circulation. This ovarian material (ovarian inhibin) selectively inhibits FSH secretion in proestrous and estrous rats (26). In this scheme, LHRH may be released on proestrous afternoon as pulses of high concentration (23) which promote the preovulatory rise in both LH and FSH. With the rise in plasma LH (and perhaps FSH), plasma E2 declines and this fall may be accompanied by a concomitant decrease in the ovarian inhibin material. Initially, as portal plasma LHRH levels decrease, peripheral plasma LH and FSH also decline. However, while plasma LH returns to basal levels, the FSH gonadotrophs, on being released from negative feedback inhibition, respond to low but constant amounts of endogenous LHRH by releasing FSH. As a consequence, a secondary rise in plasma FSH occurs which continues into estrous morning. Similarly, castration or LH pulse injections also reduce plasma E2 levels and may alter ovarian inhibin production. Thus, the FSH gonadotrophs are released from negative feedback inhibition and, in the presence of low concentrations of LHRH, FSH would increase in the peripheral circulation. In conclusion, we believe that these studies provide considerable support for the concept that LHRH is also FSHRH. By modifying the LHRH input signal, either LH or FSH secretion or both will occur. Perhaps different thresholds of responsiveness of LH vs. FSH gonadotrophs to LHRH exist. Alternatively, some investigators have concluded that both hormones (LH and FSH) are produced by one gonadotroph (27). If this is correct, then changing the concentrations and the duration of time that LHRH is presented to this cell may change its secretory characteristics from a cell which releases predominantly LH to one which secretes FSH. Acknowledgments We wish to thank Victoria Reck-Malleczewen and Joseph Connelly for their excellent technical assistance. Anti-E2, anti-LH (GDN-15), and purified ovine LH (LER-1056-2) used for RIA were kindly supplied by Drs. D. C. Collins, G. D. Niswender, and L. E. Reichert, respectively. RIA material for rat FSH and purified rat LH were kindly provided by the NIAMDD rat pituitary hormone distribution program.

References 1. Matsuo, H., Y. Baba, R. M. G. Nair, A. Arimura, and A. V. Schally, Structure of the porcine LH- and FSH-releasing hormone. I. The

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proposed amino acid sequence, Biochem Biophys Res Commun 43: 1334, 1971. 2. Matsuo, H., A. Arimura, R. M. G. Nair, and A. V. Schally, Synthesis of the porcine LH- and FSH-releasing hormone by the solid phase method, Biochem Biophys Res Commun 45: 822, 1971. 3. Schally, A. V., A. Arimura, A. J. Kastin, H. Matsuo, Y. Baba, T. W. Redding, R. M. G. Nair, L. Debeljuk, and W. F. White, The gonadotropin releasing hormone: a single hypothalamic polypeptide regulates the secretion of both LH and FSH, Science 173: 1036, 1971. 4. Arimura, A., H. Matsuo, Y. Baba, L. Debeljuk, J. Sandow, and A. V. Schally, Stimulation of LH by synthetic LHRH in vivo. I. Comparative study of natural and synthetic hormone, Endocrinology 90: 163, 1972. 5. Kastin, A. J., A. V. Schally, C. Gual, and A. Arimura, Release of LH and FSH after administration of synthetic LH-releasing hormone, J Clin Endocrinol Metab 34: 753, 1972. 6. Blake, C. A., Stimulation of the proestrous luteinizing hormone (LH) surge after infusion of LH-releasing hormone in phenobarbital-blocked rats, Endocrinology 98: 451, 1976. 7. Blake, C. A., Stimulation of the early phase of the proestrous follicle-stimulating hormone rise after infusion of luteinizing hormone-releasing hormone in phenobarbital-blocked rats, Endocrinology 98: 461, 1976. 8. Gonzalez-Barcena, D., A. J. Kastin, D. S. Schalch, J. A. Bermudez, D. Lee, A. Arimura, J. Ruelas, I. Zepeda, and A. V. Schally, Synthetic LH-releasing hormone (LH-RH) administered to normal men by different routes, J Clin Endocrinol Metab 37: 481, 1973. 9. Katz, Y., and D. T. Armstrong, Inhibition of ovarian estradiol-17/? secretion by luteinizing hormone in prepubertal, pregnant mare serum-treated rats, Endocrinology 99: 1442, 1976. 10. Chappel, S. C, and C. A. Barraclough, Further studies on the regulation of FSH secretion, Endocrinology 78: 41, 1976. 11. Lagace, L., F. Labrie, N. B. Schwartz, J. Lorenzen, and J. H. Dorrington, Similar selective inhibitory effect of ovarian follicular fluid and Sertoli cell culture medium on FSH secretion in vitro, Abstracts of The Endocrine Society, 1978 (Abstract 727). 12. Smith, M. S., M. E. Freeman, and J. D. Neill, The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rats: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy, Endocrinology 96: 219, 1975. 13. Daane, T. A., and A. F. Parlow, Periovulatory patterns of rat serum FSH and LH during the normal estrous cycle: effects of pentobarbital, Endocrinology 88: 653, 1971. 14. Brown-Grant, K., and F. Grieg, A comparison of changes in the peripheral plasma concentrations of luteinizing hormone and follicle-stimulating hormone in the rat, J Endocrinol 65: 389, 1975. 15. Chappel, S. C, and C. A. Barraclough, Hypothalamic regulation of pituitary FSH secretion, Endocrinology 98: 927, 1976. 16. Wright, K., D. C. Collins, and J. R. K. Preedy, Comparative specificity of antisera raised against estrone, estradiol-17/? and estriol using 6-0-carboxymethyloxime bovine serum albumin, Steroids 21: 755, 1973. 17. Rodbard, D., Statistical quality control and routine data processing for radioimmunoassays (RIA) and immunoradiometric assays (IRMA), Clin Chem 20: 1255, 1974. 18. Thorneycroft, I. H., and S. C. Stone, Radioimmunoassay of serum progesterone in women receiving oral contraceptive steroids, Contraception 5: 129, 1972. 19. Niswender, G. D., A. R. Midgley, S. E. Monroe, and L. E. Reichert, Radioimmunoassay for rat luteinizing hormone with anti-ovine LH serum and ovine LH 131I, Proc Soc Exp Biol Med 128: 807, 1968. 20. Wise, P. M., L. V. DePaolo, L. D. Anderson, C. P. Channing, and C. A. Barraclough, Evidence that the pituitary gland is the site of inhibitory action of porcine follicular fluid upon FSH secretion in the rat, In Channing, C. P., J. Marsh, and W. D. Sadler (eds.), Workshop on Ovarian Follicular and Corpus Luteum Function, Plenum Press, New York, in press. 21. Aiyer, M. S., S. A. Chiappa, and G. Fink, A priming effect of luteinizing hormone releasing factor on the anterior pituitary gland in the female rat, J Endocrinol 62: 573,1974. 22. Turgeon, J. L., and C. A. Barraclough, Temporal patterns of LH

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LHRH INDUCES SELECTIVE FSH RELEASE release following graded preoptic electrochemical stimulation in proestrous rats, Endocrinology 92: 755, 1973. 23. Barr, G. D., and C. A. Barraclough, Temporal changes in medial basal hypothalamic LH-RH correlated with plasma LH during the rat estrous cycle and following electrochemical stimulation of the medial preoptic area in pentobarbital-treated proestrous rats, Brain Res 148: 413, 1978. 24. Eskay, R. L., R. S. Mical, and J. C. Porter, Relationship between luteinizing hormone releasing hormone concentration in hypophysial portal blood and luteinizing hormone release in intact, castrated, and electrochemically-stimulated rats, Endocrinology 100: 263, 1977. 25. Chiappa, S. A., G. Fink, and N. M. Sherwood, Immunoreactive

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luteinizing hormone releasing factor (LRF) in pituitary stalk plasma from female rats: effects of stimulating diencephalon, hippocampus and amygdala, J Physiol 267: 625, 1977. 26. DePaolo, L. V., P. M. Wise, L. D. Anderson, C. A. Barraclough, and C. P. Channing, Suppression of the pituitary follicle-stimulating hormone secretion during proestrus and estrus in rats by porcine follicular fluid: possible site of action, Endocrinology 104: 402, 1979. 27. Soji, T., S. Saro, Y. Shishiba, M. Igarashi, T. Shioda, and F. Yoshimura, Chronic effect of TRH and LRH upon a series of basophils along with the serum and pituitary TSH, LH, and FSH concentration, Endocrinol Jap 24: 19, 1977.

Satellite Workshop of the Xllth Acta Endocrinologica Congress PROTEASES AND HORMONE ACTION The purpose of the workshop is to bring together workers in diverse areas where limited, controlled, partial proteolysis is known to activate the formation of products capable of physiological regulation. Examples of this type of control mechanism are to be found in steroid receptor systems, hormone activation, and blood coagulation. This is to be distinguished from the catabolic role of proteases leading to biological degradation and elimination, which will not be included. This workshop will be held on June 30, 1979, in the afternoon, immediately after the morning sessions of the Congress. Attendance is open and free to all interested scientists upon registration with the organizers. For further information, please contact: M. K. Agarwal INSERM U-36 17 Rue du Fer-a-Moulin, Paris 75005 France

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