Effects of manipulating catecholamines on the incidence of the preovulatory surge of luteinizing hormone and ovulation in the rat: Evidence for a necessary involvement of hypothalamic adrenaline in the normal or ‘midnight’ surge

Neuroscience Vol. 10, No. I. pp. 187-206, Printed in Great Britain

0306-4522/83 $3.00+0.00 Pergamon Press Ltd 0 1983 IBRO

1983

EFFECTS OF MANIPULATING CATECHOLAMINES ON THE INCIDENCE OF THE PREOVULATORY SURGE OF LUTEINIZING HORMONE AND OVULATION IN THE RAT: EVIDENCE FOR A NECESSARY INVOLVEMENT OF HYPOTHALAMIC ADRENALINE IN THE NORMAL OR ‘MIDNIGHT’ SURGE C. W. COEN* and M. C. CooMnst Department of Human Anatomy, South Parks Road, Oxford, OX1 3QX, U.K. Abstract-The preovulatory surge of luteinizing hormone reaches a maximum at 18.00 h on the day of pro-oestrus in female rats maintained with regular lighting from 06.00 to 20.00 h. This surge is initiated by a discharge of luteinizing hormone-releasing hormone into hypophysial portal blood. In this study, drugs which affect catecholamine-mediated neurotransmission were administered on the day of prooestrus and the effects on serum concentrations of luteinizing hormone and on subsequent ovulation were observed. a-Methyl-p-tyrosine, diethyldithiocarbamate and SKF 64139 inhibit catecholamine synthesis at the level of tyrosine hydroxylase, dopamine /I-hydroxylase and phenylethanolamine N-methyltransferase, respectively. Although a-methyl-p-tyrosine suppressed ovulation, it had a negligible effect on the incidence of the preovulatory surge. In contrast, the various treatments with diethyldithiocarbamate and SKF 64139 resulted in a minimal occurence of the 18.00 h surge; at relatively low doses, however, these drugs frequently elicited a surge at 22.00 or 24.00 h which invariably resulted in ovulation. The failure of the surge after diethyldithiocarbamate or SKF 64139 was not associated with a loss of pituitary sensitivity to luteinizing hormone-releasing hormone. In terms of the hypothalamic concentration of dopamine, noradrenaline, adrenaline and S-hydroxytryptamine at 18.00 h on pro-oestrus, the only common effect of diethyldithiocarbamate and SKF 64139, given in a dose which blocks the surge, was a severe depletion of adrenaline; a-methyl-p-tyrosine failed to produce this effect despite inducing a marked depression of dopamine and a moderate loss of noradrenaline. Neither the increase in hypothalamic dopamine after diethyldithiocarbamate, nor the a2 receptor blocking properties of SKF 64139 appear to be relevant in this context since injections of L-dopa or piperoxane, an a2 receptor antagonist, were without effect on the surge or ovulation. The failure of the surge after prazosin, an a, receptor antagonist, indicates that the function of adrenaline may be mediated postsynaptically by a, receptors. Clonidine, an a2 receptor agonist which reduces the turnover rate of hypothalamic adrenaline, had effects on the surge and ovulation which were comparable to those of diethyldithiocarbamate and SKF 64139, the relatively low doses causing some of the surges to occur at 24.00 instead of 18.00 h and higher doses suppressing the surge at both times and thus preventing ovulation. The hypotensive effects of clonidine are unlikely to be of significance in this respect since mecamylamine, a ganglionic blocking hypotensive agent, did not disturb the surge or ovulation. Piperoxane, an GL~ receptor antagonist which enhances the turnover rate of hypothalamic adrenaline, counteracted the effect of clonidine on the timing of the surge. The results of this study are consistent with a necessary role for adrenaline in the production of the spontaneous preovulatory luteinizing hormone surge. Evidence specifically implicating dopamine or noradrenaline in this process was not obtained. The results emphasize the importance of the sequential study of serum concentrations of luteinizing hormone and ovulation in individual animals and of recognizing the possibility of a delayed “midnight” surge. This study also indicates that tyrosine hydroxylase inhibition may fail to produce the functional deficits which follow the inhibition of enzymes involved in the subsequent biosynthetic pathway for adrenaline.

*Present address: Section of Neurosurgery, Yale University School of Medicine, New Haven, Connecticut 06510, U.S.A. tPresent address: St. Mary’s Hospital Medical School, Norfolk Place, London, W2 IPG, U.K. Abbreviations: aMPT, a-methyl-p-tyrosine; DA, dopamine; DBH, dopamine P-hydroxylase; DDC, diethyldithiocarbamate; DOPA, dihydroxyphenylalanine; DOPS, dihydroxyphenylserine; S-HT, 5-hydroxytryptamine (serotonin); LH, luteinizing hormone; LHRH, luteinizing hormone-releasing hormone; NA, noradrenaline; 6-OHDA, 6-hydroxydopamine; PNMT, phenylethanolamine N-methyltransferase. 18

An involvement of catecholamines in the occurrence of the spontaneous preovulatory surge of luteinizing hormone (LH) was proposed by Sawyer, Everett and

Markee in 1949 when they reported that rats treated with dibenamine (an a-adrenergic receptor blocking During the subsequent drug) failed to ovulate. 48,49~‘13 two decades comparable effects on spontaneous ovulation were found after administrationof SKF 501’r4 or dibenzyline93 (both a-adrenergic antagonists), chlorpromazine (a drug with catecholamine receptor blocking properties),3 reserpine (a monoamine de-

188

C. W. Coen and M. C. Coombs

pleting agent),3s”,34,9’ a -methyl-p-tyrosine (a MPT) (an inhibitor of cat~holamin~ synthesis)‘~,79.E4or a-methyl-dopa (a drug which disrupts catecholaminergic transmission).34,7v It was also reported that ovulation can be restored when the catecholaminedepleting effect of reserpine or lr MPT is counteracted by treatment with L-dopa”.79 or, in the case of reserpine, with monoamine oxidase inhibitors.34,9’ None of these studies presented direct evidence of an effect of the various treatments on the serum concentration of LH itself; it was generally assumed that a failure of ovulation indicated a failure of the LH surge. Nevertheless, several reports stated that the induction of ovulation by exogenous LH or human chorionic gonadotrophin could be inhibited by treatment with chlorpromazine,‘00 reserpine”,5’,69,‘00or dibenamine;“x’ furthermore, the evidence presented against an ovarian site of action of these drugs was derived from studies’4*“3 in which the gonadotrophins were administered at a high dose normally resulting in superovulation. 13’Peripheral actions may therefore have contributed to the reported capacity of reserpine to suppress ovulation following electrical stimulation of the preoptic area,“’ the site of cell bodies containing LH-releasing hormone (LHRH) in the rat;“* moreover, the reversal of this effect by inhibiting monoamine oxidase ‘*’ does not necessarily imply a change of function within the hypothalamus since a comparable treatment has been reported to restore the reserpine-impaired responsiveness of the ovary to exogenous gonadotrophins.51 Despite the lack of evidence involving serum LH concentrations, it was generally assumed by 1970R9 that catecholamine-containing neurons in the central nervous system of the rat participate in the production of the LH surge. It is therefore remarkable that following the introduction of the radioimmunoassay for LH relatively little research has been undertaken on catecholamine involvement in LH release specifically in the context of the spontaneous surge. Considerable attention has, however, been given to the effects of manipulating catecholaminergic transmission on basal serum LH concentrations in intact males2~77~7s~95~‘i8 or females at times other than that of the LH surge,“’ or on the elevated LH levels found after gonadectomy; some of the experiments recognized the marked pulses of LH which occur in the latter condition.7~4s~7~5s~LX~X3~13.1 but others failed to do SO,~~~~~‘~~~‘~~~~“~.‘~” and others involved a suppression of those levels by steroids.5’.76~X’~X’~“X~‘3” The evidence from these disparate experimental conditions suggesting that dopamine (DA) may inhibit LH release after gonadectomy6~7~45~46*ss~58 and yet stimulate it in the presence of the gonads or gonadal steroids, “J’*J~~ is offset by studies which have failed to demonstrate any conclusive effect on LH secretion after manipulating dopaminergic transmission in either the former~46.97.9x.“x.‘~oor the latter5’.K’.Y’circumstances. Many of the results concerning noradrenaline (NA) have been taken as evi-

dence for its involvement in the stimulation of LH release ,2~26~7b.R’~irli some of the experiments indicating that this may depend upon the presence of gonadal steroids:55.*3.‘30nevertheless, several reports fail to demonstrate such a stimulatory role for NA,“4.77.“X and others suggest that it performs a function in the pulsatile regulation of LH which is merely permissive45,47,5X.133 or even inhibitory.55.“’ None of these studies was designed specifically to elucidate the neuronal mechanisms underlying the spontaneous preovulatory surge. Each of the instances of experimentally reduced LH levels may have reflected an interference with the pulsatile release mechanism; furthermore, since rats possess a vestigial capacity for reflex ovulation,” the first neuroendocrine process for which a necessary involvement of catecholamines was proposed,“’ each of the instances of enhanced LH release may have been the result of an activation of this reflex or an overdriving of the system controlling LH pulses, neither effect necessarily involving catecholaminergic systems contributing to the normal occurrence of the surge. These possibilities also apply to the restoration of ovulation by adrenergic or dopaminergic agonists after treatment with nembutal or reserpine24s’04.‘07 or after various experimental procedures resulting in chronic anovulation.~2s The case for a necessary involvement of catecholamines in the occurrence of the spontaneous prooestrous LH surge rests upon a few studies which have indicated that the surge may be blocked by acute treatment with 6-hydroxydopamine (6-OHDA),“” a neurotoxin for catecholamine-containing neurons, with the DA receptor antagonists haloperido14~ or pimozides.‘3 or with inhibitors of catecholamine synthesis at the level of tyrosine hydroxylase75 or dopamine fi-hydroxylase (DBH).“.“? Nevertheless, only three of these studies5.75.90 involved more than one sample of blood from each animal and included an indication of LH secretion prior to the treatment; in the other studies a depression in the amplitude or a change in the timing of the surge could have occurred without detection. These possibilities also apply in an extensive but, for technical reasons, necessarily non-sequential study which indicated an active involvement of NA and two separate and opposing roles for DA in the induction of an LHRH surge in hypophysial portal blood in immature rats.“’ The failure of the pro-oestrous LH surge after acute treatment with 6-OHDA is difficult to interpret since this effect has been found only when the B-OHDA is administered on the day of the presumptive surge,” normal oestrous cycles and preovulatory surges resuming within a few days of the treatment despite a severe depletion of hypothalamic NA;“J0*96 the acute neurochemical consequences of 6-OHDA treatment are unclear but in addition to the initial processes leading to a degeneration of catecholamine-containing neurons they may involve various non-specific2’ and stimulatory effects.** Similar

Hypothalamic adrenaline and the luteinizing hormone surge

problems of specificity are associated with the use of haloperidol or pimozide given that the former has adrenergic receptor blocking properties’ and a direct inhibitory effect on LH release from the anterior pituitary gland4’ and both enhance the rate of turnover of NA.’ 36 Close inspection of the data of Kalra and McCannr5 concerning the prevention of the prooestrous LH surge and ovulation by crMPT, an inhibitor of tyrosine hydroxylase, or by DDC, an inhibitor of DBH, actually indicates that none of the treatments involving EMPT alone was completely successful and that ovulation could occur despite the actions of DDC. Although these drugs or 6-OHDA can prevent the progesterone-induced surge in oestrogen-primed ovariectomized rats,73,‘20the physiological validity of this method of induction should be questioned since spontaneous ovulation is unaffected by the presence of antibodies to progesterone;” furthermore, the surge occurs without being preceded by the pro-oestrous rise in progesterone release12’ (which it stimulates”) and in response to oestrogen alone following adrenalectomy and ovariectomy.33,xx The progesterone-induced surge blocked by ctMPT or DDC may be restored by treatment with dihydroxyphenylserine (DOPS), a precursor which is decarboylated to form NA, but not with r_-dopa, the precursor of DA.” In prooestrous rats, however, the use of these precursors in addition to ctMPT or DDC seems to be generally incompatible with the occurrence of the surge and ovulation.” Clear sequential information directly concerning the involvement of the three catecholamines, DA, NA and adrenaline, in the production of the spontaneous pro-oestrous LH surge and subsequent ovulation is therefore required. The present study was undertaken systematically to investigate some of the consequences of administering drugs with various effects on catecholamine-containing neurons. Given the risk of inducing pseudopregnancy by the suppression of catecholaminergic transmission prior to the day of pro-oestrus4,35 and the possibility of druginduced changes in the timing of the surge or direct deleterious effects on the process of ovulation, drugs were administered to pro-oestrous rats, blood samples were taken before and during the presumptive surge, and at additional times, and oviducts were routinely examined for ova, but not until the afternoon of the day following the treatment. Since it has been our experience that ovulation per se may be influenced by the route of administration of a drug,28 subcutaneous and intraperitoneal routes were used in testing the effects of certain compounds. A preliminary report of some of the results has been presented previously.32 EXPERIMENTAL PROCEDURES

Animals Female Wistar rats (Charles River U.K., Margate) weigh-

189

ing between 250 and 300g were maintained under regular lighting conditions (lights on from 06.00 to 20.00 h). Diet FFG (Dixon & Sons Ltd., Ware) and water were available ad libitum. Only animals exhibiting at least two consecutive Cday oestrous cycles were selected for study. Administration of compounds a-Methyl-n-tvrosine methyl ester hydrochloride (aMPT; Sigma Chemical Co., Poole), sodium diethyldithiocarbamate (DDC: Siema). SKF 64139 (Smith. Kline & French. Welwyn Garde; City), L-3,4_dihydroxyphenylalanine (L: dopa; Sigma), piperoxane hydrochloride (RhBne-PoulencSante, Paris), prazosin hydrochloride (Pfizer, Sandwich), clonidine (Cataores: Boehringer Ingelheim. Bracknell) and mecamylamine ISigma) were administered, as indicated in the Results, in propylene glycol (BDH Chemicals, Poole), for subcutaneous (s.c.) injection and for some of the intraperitoneal (i.p.) treatments, or in 0.9% saline. The releasing hormone for LH (LHRH; ICI. Pharmaceuticals Division, Macclesfield) was dissolved in 0.9% saline and injected via an atria1 cannula. The doses and times of administration are indicated in the Results. All compounds were injected in a volume of 2.0ml/kg body weight with the exception of clonidine which was administered in 1.Oml/kg body weight. Collection and analysis of data

As described previously,30 blood samples were taken by venipuncture of the external jugular under light ether anaesthesia (animal anaesthetized < 1.5 min) or, when indicated, by means of an atria1 cannula, the animal being conscious and unrestrained. After centrifugation serum samples were stored at -20°C. Serum concentrations of LH were assayed by a double-antibody radioimmunoassay using ovine LH (S13, National Institutes of Health; biological potency 0.95 x NIH-LH-Sl) as the reference preparation.‘23 The radioimmunoassay described by Turnbull et al.‘*’ was used to measure serum concentrations of progesterone. In order to examine the effects of various pharmacological treatments on the occurrence of the prooestrous surge of LH and subsequent ovulation a blood sample was taken immediately before treatment and also at 18.00 h, the time of the peak of the surge which, under these lighting conditions, begins at approximately 16.30 h; additional times at which blood samples were taken are indicated in the Results. The 350 pro-oestrous rats used in this part of the study showed an increment in the level of LH between the initial time of bleeding and 18.00 h which was either > 11.3 ng/ml (n = 178) and classified as a surge (the lowest of these increments occurring in a vehicle-treated animal and being compatible with full ovulation) or ~3.5 ng/ml (n = 161); there were 11 exceptions in which the increment was 4.8-9.5 ng/ml, 55% of these cases resulting in ovulation including that with the lowest increment in this group. Given the variation which occurs between individuals in the timing of the peak of the LH surge and the observation that only approximately 14% of the peak value attained is required for ovulation per se,63 these exceptions have, for the purposes of this study, been classified as surges; they are, nevertheless, specifically

190

C.

W. Coen and M. C. Coombs

identified in the Results. Oviducts were examined for ova between 14.00 and 17.00 h on the day after the presumptive LH surge. Full ovulation was recorded when a total of 8 or more ova were observed. In order to measure the concentration of amines in the hypothalamus, brains were removed rapidly and transverse sections were made which were sufficiently rostra1 to the optic chiasma to include the preoptic area and which, at the caudal level, bisected the mammillary bodies. The lateral hypothalamic sulci formed the lateral borders of the dissection. The dorsal surface of the wedge of tissue passed from the base of the anterior commissure to the ventral limit of the caudal transverse section. Tissue samples were stored in liquid nitrogen until they were assayed. The hypothalamic content of the catecholamines and 5-hydroxytryptamine (5-HT) was measured by radioenzymatic assays modified from those of Saller and Zigmond’09 and Boireau et aL9 respectively. The tissue was homogenized in 800 ~1 0.1 M hydrochloric acid containing 1 mM diethylenetriamine-pentaacetic acid (Sigma) and 0.1% sodium metabisulphite. An aliquot of homogenate was removed for protein estimation.86 The remaining homogenate was centrifuged at 10,OOOgfor 30 min at 4°C and 10 ~1 aliquots of supernatant were used for estimation of the amines. The catecholamine assay has a sensitivity (defined as twice blank) of 4.2 pg DA, 12.9 pg NA and 5.4 pg adrenaline, with an intra-assay variation for all three catecholamines of less than 5%. Corrections were made in the data for a 0.46% crossover of NA and a 0.70% crossover of DA into the counts associated with adrenaline. The assay for 5-HT has a sensitivity of 10.4 pg with an intra-assay variation of 3.3%. The samples were run in duplicate in a single assay for catecholamines and for 5-HT. All amine concentrations are expressed in units of pg amine per pg protein. Statistical significance was evaluated by Student’s t-test or Fisher’s exact test. RESULTS

Effects of‘catecholamine synthesis inhibitors on the LH surge at 18.00 h on pro-oestrus and on subsequent ovulation

Rats were injected with EMPT, an inhibitor of tyrosine hydroxylase,“4 either S.C. or i.p., between 10.00 and 14.00 h on the day of pro-oestrus at a dose ranging from 300 to 400 mg/kg (Table 1). Treatment with 3OOmg aMPT/kg S.C.was without effect on the occurrence of the LH surge at 18.00 h and on subsequent ovulation (n = 12). The same dose of ctMPT administered i.p. and greater doses given either S.C. or i.p. (n = 52) were associated with a failure of the surge at 18.00 h in only 8% of the cases; nevertheless, ovulation was suppressed (presumably by a direct effect on the ovaries) with 48% of the animals failing to ovulate and 27% producing less than 8 ova. After treatment with either of the vehicles by either route of administration (n = 29) only 7”/, of the rats failed

to produce the 18.00 h surge and full ovulation occurred without exception. Treatment with DDC, an inhibitor of DBH,59 between 10.00 and 14.00 h on the day of pro-oestrus at either 600 or 650 mg/kg (Table 2) was compatible with a surge of LH at 18.00 h in only 11% of the rats (n = 46). Nevertheless, full ovulation took place in 61% of the animals and a further 7% produced at least 6 ova. There was minimal evidence to suggest that the route of administration of this drug might directly affect the process of ovulation; the one animal failing to ovulate after producing a normal LH surge had received the DDC by i.p. injection. When rats were treated with SKF 64139, an inhibitor of phenylethanolamine N-methyltransferase (PNMT),“’ between 10.00 and 14.00 h on the day of pro-oestrus, a dose of 50 mg/kg (n = 20) was associated with a normal occurrence of the LH surge in 75% of the animals and yet full ovulation took place in 90% of the animals and a further 5% produced 7 ova (Table 2). None of the animals treated with SKF 64139 at a dose of 100 mg/kg (n = 11) displayed the normal LH surge but 18% of them produced a full complement of ova (Table 2). In order further to investigate the effects of the inhibitors of catecholamine synthesis on the preovulatory LH surge and ovulation, rats were treated with aMPT, DDC or SKF 64139 on two occasions between 12.00 and 17.00 h on the day of pro-oestrus (Table 3). None of the animals given 400mg rMPT/kg twice (n = 12) ovulated, but 75% of them produced the 18.00 h surge, albeit with an apparently depressed amplitude in 33Ygof the cases. None of the animals receiving two injections of 600 or 650mg DDCjkg (n = 32) or of 50 mg SKF 64139/kg (n = 27) produced a surge at 18.00 h and yet, respectively, 19 and 22:/, of them produced a full complement of ova with an additional 6”,, of those treated with DDC producing at least one ovum. Following two treatments with either of the vehicles by either route of administration (n = 16) only 12”;)of the rats failed to produce the 18.00 h surge and full ovulation occurred without exception. Occurrence qf’a preovulatory LH surge during the dark phase between pro-oestrus and oestrus following inhibition of noradrenaline and/or adrenaline svnthesis

Pro-oestrous rats were treated i.p. with DDC, at a dose of 650 mg/kg at 13.00 h (n = 16) or 600 mg/kg at 13.00 and 16.00 h (n = 12), or with SKF 64139, at a dose of 100 mg/kg at either 12.00, 13.00 or 14.00 h (n = 11) or 50 mg/kg at 13.00 and 16.00 h (n = 8). Blood samples were taken at times in addition to that immediately preceding the treatment and 18.00 h (Fig. I). None of the animals produced a surge of LH at the normal time of 18.00 h; nevertheless, in some rats a surge was observed at 22.00 or 24.00 h (none of the animals being bled at both of these times) and this invariably resulted in tubal ova (10.7 k 3.0 [S.D.], n = 13) between 14.00 and 17.00 h on the following

25

4 4 4 4 4 4 4 4 4

12.00 13.00 14.00 12.00 13.00 14.00 12.00 13.00 14.00 10.00 12.00 13.00 14.00 10.00 12.00 13.00 14.00

300 mg aMPT/kg in propylene gtycol i.p.

350 mg a MPTjkg in propylene glycol se.

350 mg aMPT/kg in propylene glycol i.p.

400 mg aMPT/kg in 0.9% saline i.p.

Either vehicle S.C.or i.p.

12.0 f 2.4 II 13.2 k 3.4 12.6 & 3.4 14.0 + 2.3 14.0 + 2.8

86 100 86.t 100

13 9

100 25

25 25

1.5 9

14 14

2s 25

6 3

4.0 rf: 0.0

1.3 & 0.6

15

50

3.5k2.1

2 4.5 f 0.7 5

50

25 sot 25 25

6

Reduced ovulation LH surge Basal LH % No. of ova % No. of ova

1 f

50 7s

100

25 75 25

25t

75

:it

%

25

50

%

No ovulation LH surge Basal LH

*Oviducts were examined for ova between 14.00 and 17.00 h on the day after treatment. Full ovulation was recorded when 8 or more ova were observed. tAnimals showed an increment in LH levels between the time immediately preceding treatment and 18.00 h on “pro-oestrus” which was > 13.0 ngjml (registered as “LH surge”) or cl.7 ngjml (registered as “basal LH”) with 7 exceptions in the groups marked+ in which the increment was 4.&8.8ng/ml.

7 8 7 7

4 4 4 4

14

25

4 4

12.313 + 3.5

12

100 loot

4

12.00 13.qo 14.00

300 mg aMPT/kg in propylene giycol S.C.

::t

12.5 & 3.7 11.5 + 1.7 11.8 * 1.5

100

No.

Time (h)

Treatment

Full ovulation* Basal LHf LH surge$ % No. of ova % No. of ova

Table 1. Incidence of ovulation (with mean _t S.D. no. of ova) and of the surge in the serum concentration of htteinizing hormone (LH) at 18.00 h on the day of pro-oestrus following S.C. or i.p. treatment with a-methyl-p-tyrosine (cLMPT) or vehicle at various times on that day

rc 2

No. 4 4 4

Time (h) 12.00 13.00 14.00 12.00 13.00 14.00

600 mg DDC/kg in propylene glycol S.C.

600 mg DDCjkg in propylene glycol i.p.

650 mg DDCjkg in 0.9% saline i.p.

10 10.5 f 2.1

10 13

2.5 33

2s 33

17

12.5

25 25

7

6

7 7

Reduced ovulation Basal LH No. of ova %

25

67

15 100

25

25 2.5 12.5 50

25 25 25

50 75

No ovulation LH surge Basal LH “/, %

*Oviducts were examined for ova between 14.00 and 17.00 h on the day after treatment. Full ovulation was recorded when 8 or more ova were observed. $Animals showed an increment in LH levels between the time immediately preceding treatment and 18.00 h on “pro-oestrus” which was > I 1.3 ngjml (registered as “LH surge”) or < I A ng/ml (registered as “basal LH”) with 1 exception in the group marked t in which the increment was 6.2 ng/ml.

4 4 3

12.00 13.00 14.00

1.4 2. I 1.1 1.3

100 mg SKF 64139/kg in 0.9% saline i.p.

12.0+ 10.5 + 10.8 + 12.3 +

50 67 83 100

4 6 6 4

10.00 12.00 13.00 14.00

9.2 0.0 2.1 0.0

50mg SKF 64139/kg in 0.9% saline i.p.

19.5 + 12.0 f 10.8 f 12.0 *

50 50 75 50

4 4 8 6

10.00 12.00 13.00 14.00

I5 9

12.3 f 2.9 12.0 rt 3.6 10.5 f 0.7

75 15 50

4 4 4 25t 25

ll.Ok2.6

75

Treatment

Fuli ovulation* LH surgef Basal LHJ No. of ova 3; No. of ova % 50 9.5 + 0.7

Table 2. Incidence of ovulation (with mean + S.D. no. of ova) and of the surge in the serum concentration of luteini~ng hormone (LH) at 18.00 h on the day of pro-oestrus following se. or i.p. treatment with diethyldithiocarbamate (DDC) or SKF 64139 at various times on that day

6 6 4

3 I2 4

4

4

4 4 4

12

4

4

4 4 4

No.

I00 83 75

13.3 + I.5 ll.O+2.5 10.7 + 2.1

I7 2s

33 25

25

25

2.5

25

50

I3 IO

IO 13.0 f 3.5

I5

II

8

12.3 f 3.5

14.0 + 2.8

25

8

I

2

Reduced ovulation Basal LH No. of ova %

751 50 loot

67 75 I00

1.5

75

75 I00 15

67

50

I00

25 50

No ovulation Basal LH LH surge % %

following

*Oviducts were examined for ova between 14.00 and 17.00 h on the day after treatment. Full ovulation was recorded when 8 or more ova were observed. IAnimals showed an increment in LH levels between the time immediately preceding treatment and 18.00 h on “pro-oestrus” which was > 23.8 ng/mi (registered as ‘“LH surge”) or (3.2 “g/ml (registered as “Basal LH”) with 3 exceptions in the groups marked t in which the increment was 6.9-9.5 ng/mI.

Vehicles i-p. or se. (pooled data) 12.00 & 15.00 13.00 & 16.00 14.00 & 17.00

13.00 & 15.00 13.00 & 16.00 14.00 & 17.00

50

mg SKF 64139/kg in 0.9% saline i.p.

13.00 & 16.00 13.00 & 16.00

50 mg SKF 64139/kg in propylene glycol i.p.

12.OQ& 15.00 13.00 & 16.00 14.00 & 17.00

650 mg DDC/kg in 0.9% saline i.p.

50mg SKF 64139/kg in propylene glycol se.

13.00 &. 16.00 13.00 & 16.00

6OOmg DDCjkg in propylene glycol i.p.

600 mg DDCjkg in 0.9% saline i.p.

13.00 & 16.00

Time (h)

600 mg DDCjkg in propylene glycol S.C.

- --_ 12.00 & 15.00 13.00 & 16.00 14.00 & 17.00

Treatment

400mg aMPT/kg in 0.9% saline i.p.

__I

Full ovulation* LH surge: Basal LHf No. of ova % No. of ova % --

Table 3. Incidence of ovulation (with mean + SD. no. of ova) and of the surge in the serum concentration of luteinizing hormone (LH) at 18.00 h on the day of pro-oestrus S.C. or i.p. treatment with either u-methyl-p-tyrosine (ctMPT), diethyldithiocarbamate (DDC), SKF 64139 or vehicle at two times on that day

194

C. W. Coen and M. C. Coombs

0

650mg DDC /kg13.OOh 600mg DDC Ikg1300& 16eOOh

3

25

15

10 z l-7 5; s k z F

5 4 i 30

Ezl1OOmgSKF64139/kg12.00,13DOor14.OOh(pooled) 50mg SKF64139 /kg13.00& 16.OOh

20

15

10 5

PreTreatment 14-00

16*DD

15*DO

18.00

200l

22.00

24.00 02.00

17.00 Time I h IPro-oestrus

Fig. I. Mean (+S.D.) serum luteinizing hormone (LH) concentratton, at times indicated, expressed as ng NIH-LH-Sl3/ml, in pro-oestrous rats (number indicated) given 650 mg diethyldithiocarbamate (DDC)/kg at 13.00 h (open bars), 600 mg DDC/kg at 13.00 and 16.00 h (cross-hatched bars), 100 mg SKF 64139/kg at either 12.00, 13.00 or 14.00 h (pooled data) (hatched bars) or 50 mg SKF 64139/kg at 13.00 and 16.00 h (solid bars). Compounds were administered in 0.9% saline. Blood samples were taken immediately before the (first) treatment and 2-3 times subsequently. Whenever there was a surge in the concentrationof LH in some animals but maintenance of a basal level in others, the bars have been split. The single instance of an elevated level of LH before 22.00 h (t) re p resents an early stage of the surge since the level had risen four-fold by 24.00 h in this animal.

day. The double injection of DDC and the higher dose of SKF 64139 were the most effective of the treatments in preventing the surge at each of the times investigated, a surge occurring in 17 and 9%, respectively, of the animals at 24.00 h. Of the cases in which a surge of LH failed to occur at 18.00, 22.00 or 24.00 h (n = 34) only 9% were subsequently found to have ovulated (9.0 f 6.1 [SD.] ova, n = 3). In a concurrent study, pro-oestrous rats injected i.p. with

0.9% saline at 13.00 and 16.00 h (n = 8) consistently produced a surge of LH at 18.00 h but only basal LH levels at 24.00 h; each of these animals generated a full complement of ova (11.7 + 1.5 [S.D.]). Responsiveness qf the anterior pituitary gland to LHRH following inhibition of noradrenaline and/or adrenaline synthesis The

failure

to

prevent

the

surge

with

aMPT

Hypothalamic adrenaline and the luteinizing hormone surge

195

Vehicle lO+OOhPro-oestrus (n-5 1 65Omg DDClkglO%Oh Pro-oestrus (n=3 1

1OOmg SKF 64139Ikg 104OhPro-oestrus ( n=3 1 50ngLHRHllOOg

20

124OhPro-oestrus

16

20

30

40

60

Time Imin) Fig. 2. Mean (+ S.E.M.) serum luteini~ng hormone (LH) concentration expressed as ng NIH-LH-S13/ ml in pro-oestrous rats treated i.p. at 10.00 h with vehicle (0.9% saline) (solid bars, n = 5), 65Omg diethyIdithio~rbamate (DDC)/kg (open bars, n = 3) or IO0 mg SKF 64139/kg (hatched bars, n = 3) and bled through an atria1 cannula immediately before and for 60min after receiving 50ng LH-releasing hormone (LHRH)/lOO g at 12.00 h.

(Tables

more comprehensive is insynthesis than DDC or SKF

l-3), theoretically

hibiting catecholamine

64139, indicated that the latter drugs might have an acute deleterious effect on pituitary sensitivity to LHRH. This possibility was therefore examined. Rats were cannulated at least four days before experimental treatment. At 10.00 h on the day of pro-oestrus they were injected i.p. with vehicle

(n = 5), DDC (45Omg/kg, n = 3) or SKF 64139 (100 mg/kg, n = 3). Blood samples were taken immediately before and continuing for 60 min after the administration of 50 ng LHRHjlOO g body weight at 12.00 h. There were no significant differences in the serum concentrations of LH between the groups in response to LHRH treatment (Fig. 2). Effects of catecholamine synthesis inhibitors on the hypothalamic concentration of amines

Rats were treated i.p. with aMPT, DDC or SKF 64139 at 12.00 h on the day of pro-oestrus at the highest dose used in studying the effect of a single injection of these compounds on the LH surge and ovulation (Tables 1 and 2) (i.e. 400mg crMPT/kg, 650 mg DDCjkg and 100 mg SKF 64139/kg, each administered in 0.9% saline). The hypothalamic concentration of DA, NA, adrenaline and 5-HT at 18.00 h following these treatments was measured (Fig. 3). Injection of clMPT (n = 5), at a dose which failed to affect the 18.00 h LH surge (Table l),

resulted in a severe depression of DA, a moderate depletion of NA but no significant change in the concentration of adrenaline; the level of 5-HT was unaffected. Administration of DDC (n = 5), at a dose which inhibited the 18.00 h surge (Table 2), led to a rise in the concentration of DA and a depression in that of NA and adrenaline; the level of S-HT was unaffected. Treatment with SKF 64139 (n = 5) at a dose which consistently blocked the 18.00 h surge failed to affect the concentration of DA and NA but resulted in a severe depletion of adrenaline; the level of 5-HT was mildly elevated. Effects of other drugs on the LH surge either at 18.00 h on pro-oestrus or during the dark phase between pro-oestrus and oestrus and on subsequent ovulation

Of the three inhibitors of catecholamine synthesis considered thus far only DDC and SKF 64139 consistently blocked the occurrence of the normal preovulatory LH surge. The only common effect of these two drugs detected when the con~ntration of hypothalamic amines was measured was a severe depletion of adrenaline. It should not, however, be assumed that the blockade of the 18.00 h surge following treatment with DDC or SKF 64139 and the change in its timing following the relatively low doses of these drugs necessarily involve a common effect on adrenaline. Alternative processes possibly underlying the disruption of the surge are suggested by the rise

196

C. W. Coen and M. C. Coombs 17

Vehicle 12.Wh Prwestrur 4Wmg d4PTlkg 12. CKlh Pro-oertrus 650mg ODC/kg 12.KIh Pro-wrtrus ,@,mg 5Kf 6 IWIg

*Ro.c5

I*

ND. 01

12. ooh Pro-oestrus

#rPcO.

ml

-

Y. Vehicle

Fig. 3. Mean (+S.E.M.) concentration (pg/pg protein) of dopamine (DA), noradrenaline (NA), adrenaline (A) and 5-hydroxytryptamine (5-HT) in hypothalami of pro-oestrous rats treated i.p. with vehicle (0.9% saline) (open bars, n = 5), 400mg a-methyl-p-tyrosine (aMPT)/kg (hatched bars, n = 5), 650 mg diethyldithiocarbamate (DDC)/kg (solid bars, n = 5) or 1OOmg SKF 64139/kg (cross-hatched bars, n = 5) at 12.00 h and killed at 18.00 h.

in the level of hypothalamic DA following DDC (Fig. 3), an effect which may be related inter aliu to an increase in tyrosine concentration” and tyrosine hydroxylase activity,66 and by the ~1~adrenergic receptor blocking properties of SKF 64139.6’ Nevertheless, a single or double injection of L-dopa (200 mg/kg) between 10.00 and 17.00 h on pro-oestrus, this precursor producing a considerable increase in the hypothalamic content of DA without enhancing that of NA,57.70 failed to affect the normal occurrence of the surge in 90% of the animals (n = 29) (Table 4). Similarly, it appears to be unlikely that CI*receptor blockade by SKF 64139 accounts for the effect of this drug on the LH surge since piperoxane, an tlr receptor antagonist,82 did not alter the surge and ovulation (n = 8) when administered (50 mg/kg) twice between 12.00 and 17.00 h on pro-oestrus (Table 4). Treatment with prazosin (5 mg/kg), an c(, adrenergic receptor antagonist92.‘28 at 14.00 and 17.00 h on prooestrus did, however, block the 18.00 h surge and subsequent ovulation (Table 4), suggesting that the function of adrenaline may be mediated by postsynaptic CI, receptors. Clonidine, an u2 receptor agonist, suppresses the turnover rate of hypothalamic adrenaline by a putatively presynaptic actiom5*,“’ the effects of this

drug on the pro-oestrous surge and ovulation were therefore examined (Table 5 and Fig. 4). The consequences of clonidine treatment between 12.00 and 17.00 h on pro-oestrus at a dose of 20 or 100 pgg/kg, the higher dose given twice to some animals, were comparable to those of DDC or SKF 64139, the relatively low or single higher dose of the drug having a negligible effect on the normal occurrence of ovulation but changing the timing of the surge to 24.00 h in some of the animals; double treatment with clonidine (n = 8) consistently prevented the surge at 18.00 h and permitted an occurrence of the 24.00 h surge and subsequent ovulation in only 37% of the cases. A surge of LH was invariably detected at either 18.00 or 24.00 h in each of the animals that ovulated. The hypotensive effects of clonidine” are unlikely to be of significance per se in this respect, since mecamylamine, a ganglionic blocking hypotensive agent,62 was without effect on the surge and ovulation when administered (3 mg/kg) at 14.00 and 17.00 h on pro-oestrus (Table 5). Treatment with piperoxane (50 mg/kg), an x2 receptor antagonist which enhances the rate of turnover of hypothalamic adrenaline,” 10 min before an injection of clonidine (100 pg/kg at 12.00, 13.00 or 14.00 h) counteracted the effect of the latter drug (P < 0.05) the surge occurring at the normal time in 63% of the animals (n = 16) (Table 5 and Fig. 4). Effects of inhibitors of catecholumine synthesis on the serum concentration qf progesterone on dioestrus Since treatment with an inhibitor of DBH on the day preceding pro-oestrus can prevent the LH surge, it has been suggested that an increase in noradrenergic activity on dioestrus may play a significant role in the production of the surge.13* A premature rise in the secretion of progesterone would, however, provide an alternative explanation; in order to investigate this possibility and elucidate the conditions in which such a stimulation may occur, the effect on serum progesterone levels of the three catecholamine synthesis inhibitors used in the present study were examined. Dioestrous rats were given vehicle (O.Su/:, saline) (n = 5) 400mg stMPT/kg (n = 4) 600 mg DDCjkg (n = 4) or 100 mg SKF 64139/kg (n = 4) at 12.00 h and terminal blood samples were rapidly collected 6 h later. Treatment with aMPT or DDC resulted in an increased secretion of progesterone (P < 0.05 and < 0.001, respectively) consistent with an induction of pseudopregnancy;12’ SKF 64139 did not, however, produce such an effect (Fig. 5). DlSCUSSlON

The presence of adrenaline within the hypothalamus was first reported by Vogt;“’ it has been subsequently established that the hypothalamic distribution of this amine’29 corresponds closely to that of PNMT (identified either biochemically’(‘* or immunohistochemically”), the enzyme responsible for

12.00 & 15.00 14.00 & 17.00

14.00 & 17.00

50 mg Piperoxane/kg

5 mg Prazosin/kg

5

4 4

4 4 4

4 4 5 4

No.

11.5 & 2.6 12.2 * 1.0 11.3 k 3.2 9.7 f 0.9 11.2+2.1

100 100

1.0 1.0 0.8 1.8

100 100 75

+ * + f

10.0 13.3 13.0 10.0

75 100 80 100

%

20

12

Basal LHJ No. of ova

25

6

Reduced ovulation LH surge No. of ova %

100

25

%

No ovulation Basal LH

h on the day after treatment. Full ovulatton was recorded when 8 or more ova were observed. *Oviducts were examined for ova between 14.00 and 1 which was > 18.7 ng/ml (registered SAnimals showed an increment in LH levels between the time immediately preceding treatment and 18.00 h on “pro-oestrus” as “LH-surge”) or < 2.4 ng/ml (registered as “basal LH”).

12.00 & 15.00 13.00 & 16.00 14.00 & 17.00

200 mg L-dopa/kg

Time (h)

10.00 12.00 13.00 14.00

Treatment

200 mg L-dopa/kg

Full ovulation* LH surgej. No. of ova %

Table 4. Incidence of ovulation (with mean + S.D. no. of ova) and of the surge in the serum concentration of luteinizing hormone (LH) at 18.00 h on the day of pro-oestrus following i.p. administration of L-dihydroxyphenylalanine (L-dopa), piperoxane or prazosin in 0.9% saline at various times on that day

12.00 13.00 14.00 4 6 6

4 4 17 50

50

50 2s

9.0* 1.4 8 10.0 f 2.0

13.0 & 2.8 8

33 17

25

50 2.5 75

ll.Orf:4.2 I1

15

9.5 t_ 0.7 14 12.7 + 3.8

50 17 17

0’ ,*

4.0 + 1.4 3 I

33

50

50

“/,

5.5 r?_0.7

5.0 rt: 1.4

6.0 & I .4

Reduced ovulation LH surge Basal LH No. of ova

17

50 75

25

No ovulation Basal LH

3 mg mecamylamine/kg

ll.Oi 1.6 100 14.00 & 17.00 4 ~-- .--*Oviducts were examined for ova between 14.00-17.00 h on the day after treatment. Full ovulation was recorded when 8 or more ova were observed. ZAnimals showed an increment in LH levels between the time immediately preceding treatment and 18.00 h on “pro-oestrus” which was r t 2.1 ngjml (regrstered as “LN surge”) or ~3.5 ng/ml (registered as “basal LH”).

100pg clonidineikg with 50 mg piperoxane/kg at - IO min

12.00 & 15.00 14.00 8L 17.00

100 pg cionidine/kg

4 4 4

3 4 4

12.00 13.00 14.00

12.00 13.00 14.00

No.

Time (h)

100 kg clonidine/kg

Treatment ____.--~ 20 pg clonidinejkg

Full ovulation* LH surge$ Basal LIJS No. of ova Y; No. of ova “/, 61 11.0&2.8 33 13 15 12.0 + 2.0 25 12 50 10.5 * 2. I 50 12.5 k2.1

Table 5. Incidence of ovulation (with mean + S.D. no. of ova) and of the surge in the serum concentration of luteinizing hormone (LH) at 18.00 h on the day of pro-oestrus following i.n. iniection of clonidine, with or without piperoxane, or mecamylamine. each administered in 0.92, saline at various times on that day

Hypothalamic

adrenaline

and the luteinizing

199

hormone surge

q 2O~gClonidinelkg 12Gl, 13%l or 14GJh (Pooled) l 100 pg Clonidinelkg 12~00,13~00 or 14.DOh (Pooled) 17

1OOpg Clonidinelkg

12~00&15~W or 14~00&1~00h

100 pg Clonidinelkg

12*00,1300

3

IPooled)

or 14.W with 50mg Piperox;;elkg

at-10min

(Pooled)

Time (h) Pro-oestrus Fig. 4. Mean (+S.D.) serum luteinizing hormone (LH) concentration, at times indicated, expressed as ng NIH-LH-S13/ml, in pro-oestrous rats given 20 pg clonidine/kg at either 12.00, 13.00 or 14.00 h (pooled data) (hatched bars, n = 1l), 100 pg clonidine/kg at either 12.00, 13.00 or 14.00 h (pooled data) (solid bars, n = 12) 100 pg clonidine/kg at either 12.00 and 15.00 h or 14.00 and 17.00 h (pooled data) {cross-hatched bars, n = 8) or 100 pg clonidineikg at 12.00, 13.00 or 14.00 h with a pretreatment of 50 mg piperoxane/kg 10min earlier (pooled data) (open bars, n = 16). Compounds were administered i.p. in 0.9% saline. Whenever there was a surge in the concentration of LH in some animals but maintenance of a basal level in others, the bars have been split and the number of animals exhibiting the respective responses is shown. Each of the groups marked * contains 2 animals which had produced a surge of LH at 18.00 h but still showed a level > 10.0 ng/mI at 24.00 h.

its biosynthesis. The proposition that adrenaline functions as a neurotransmitter in the brain is corroborated by reports indicating that it can stimulate adenylate cyclase activity in PNMT-containing regions’35 and that endogenous hypothalamic adrenaline can be released by various agents in vitro” and in vivo, the latter being dependent upon the integrity of projections from cell bodies loacted caudal to the hypothalamus.43

There is considerable evidence for an involvement of adrenaline within the hypothalamus in cardiovascular regulation.54 The results of the present study are consistent with the proposition that hypothalamic adrenaline also plays a necessary role in the release of LHRH underlying the preovulatory LH surge in rats. This surge peaks at lg.00 h (beginning at approximately 16.30 h) on pro-oestrus and can be blocked by acute treatment with DDC (Tables 2 and 3, Fig. 1), an inhibitor of DBH” or with SKF 64139 (Tables 2 and 3, Fig. l), an inhibitor of PNMT,‘r2 these drugs causing a depletion of hypothalamic NA together with adrenaline, or adrenaline afone, respectively

(Fig. 3). The release of LH in response to LHRH is not affected by treatment with DDC or SKF 64139 (Fig. 2). Additional effects of these drugs such as the rise in hypothalamic DA following DDC (Fig. 3) and the cz2adrenergic receptor blocking properties of SKF 64139’j’ appear to be irrelevant to the prevention of the surge since injections of L-dopa or piperoxane, an a2 receptor antagonist,82 were without effect on the surge or ovulation (Table 4). Despite the points raised in the Introduction con~rning the physiolo~cal validity of inducing a surge of LH with progesterone, the recent report indicating that SKF 64139 can block such a surge3g is at least in accord with the findings of the present study. The failure of the pro-oestrous surge after treatment with prazosin, an LY,adrenergic receptor antagonist,g2~‘28(Table 4) suggests that the contribution of adrenaline to the production of the surge may be mediated by postsynaptic CI, receptors. It remains to be determined whether these receptors are directly associated with the LHRH-containing neurons of the preoptic area. The normal pro-oestrous surge can also be prevented by treatment with clonidine (fable 5, Fig. 4), a drug which reduces the turnover rate of hypo-

C. W. Coen and M. C. Coombs

200

q

Vehicle 12Wh Dioestrus 400mgWPT

I kg 12alh

Dioestrur

6CQmgDDC I kg 12Olh Dioestrus

1oOmgSKFMl39 i kg 12mh Dioestrus

-i

25 -

z s $

20 -

% p15

-

18. CQh Dioestrus

Fig. 5. Mean (+S.E.M.) serum progesterone concentration at 18.00 h in dioestrous rats treated i.p. at 12.00 h with vehicle (0.9% saline) (open bar, n = 5), 400 mg a-methyl-ptyrosine (a fiPT)/kg) (hatched bar, n = 4), 600 mg dieihildithiocarbamate (DDC)/kg (solid bar, n = 4) or 100 mg SKF 64139/kg (cross-hatched bar, n = 4). Significance is indicated with respect to the vehicle-treated group.

thaiamic adrenaline by a putatively presynaptic action.52z”7 this effect may be counteracted by treatment with’ piperoxane (Table 5, Fig. 4), a drug which blocks the a2 receptors activated by clonidine and enhances the turnover rate of hypothalamic adrenaline.52 The hypotensive effects of clonidine6* are unlikely to be of significance in the prevention of the surge since treatment with mecamylamine, a ganglionic blocking hypotensive agent,62 did not disturb the surge or ovulation (Table 5). It is possible that the experimental suppression of adrenaline-mediated transmission may trigger a mechanism which actively prevents the LH surge. This possibility is not, however, consistent with the evidence indicating that LH release is stimulated by adrenaline when the latter is administered intraventricularly in the presence of ovarian steroids.55.‘07.‘30The failure of the LH surge following the treatments used in this study may, of course, be mediated by various actions other than those directed against adrenaline. Nevertheless, further circumstantial evidence implicating adrenaline in this function is provided by the discovery that the normal occurrence of the pro-oestrous surge after treatment with aMPT (Tables 1 and 3), an inhibitor of tyrosine by an undiminished hydroxylase, ‘34 is accompanied concentration of hypothalamic adrenaline despite a marked depression of DA and a moderate loss of NA

(Fig. 3). This absence of effect on the surge is at variance with the conclusions of an earlier study’j in which, however, relatively few animals were given ctMPT alone and, according to the data, several of them certainly surged; furthermore, in the light of the present results, an explanation of the reported inhibitory effect of aMPT on the release of LH following electrochemical stimulation of the preoptic area74 is not apparent. It could be argued that the normal occurrence of the LH surge in the presence of %MPT is related to the concurrent removal of DA-mediated inhibition and NA-mediated stimulation, these effects neutralizing each other. Such a simple relationship of function seems implausible. An alternative explanation of the absence of effect on the surge is provided by the apparent resistance of hypothalamic adrenaline to the depleting effects of tyrosine hydroxylase inhibition (Fig. 3), an effect which has been mentioned elsewhere.53 It remains to be determined whether this failure is the consequence of processes comparable to those by which NA-containing neurons are less responsive to the effects of aMPT than those containing DA.‘24.‘34 These effects could be related to the existence of multiple forms of tyrosine hydroxylase which differ in their distribution between the neuronal systems synthesizing DA and NAioS and possibly adrenaline. Furthermore, there is evidence indicating that stored DA is available for the synthesis of NA after tyrosine hydroxylase inhibition;b”.‘24 the possibility of a comparable storage of DA or NA in adrenaline-containing neurons remains open and such an effect might be complemented by a continued uptake of NA released in the vicinity of adrenaline-containing terminals after treatment with crMPT. The failure to depress adrenaline may, of course, be related to specific aspects of the present experimental conditions such as the duration of exposure to aMPT and the stimulation of PNMT activity by oestrogen.” Even when administered at a dose of 400 mg/kg, aMPT does not completely block catecholamine synthesis from tritiated tyrosine, NA being significantly less affected than DA;‘34 furthermore, inhibition of tyrosine hydroxylase would not necessarily preclude the synthesis of NA and adrenaline from tyrosine via tyramine and octopamine.” It should be recognized that r-methylated catecholamines may have been detected but not identified as such in our catecholamine assay; such compounds were not, however, observed in the brain following the injection of 400 mg ctMPT/kg in a study involving similar assay procedures.” Failure of’ ovulation despite a normal LH surge Although aMPT treatment failed to affect the LH surge, it did cause a serious disruption of ovulation (Tables 1 and 3). This effect demonstrates the invalidity of assuming that a failure of ovulation indicates a failure of the surge. a supposition which prevailed, as described in the Introduction, during the

Hypothalamic ad~naline and the iutei~i~ng hormone surge first two decadesof research in this field. The exact nature of the failure induced by clMPT at the level of the ovaries remains to be determined; the effect should, however, be considered in relation to recent observations concerning ovarian inneffation1E,s6 and to the finding that in some species the ovulatory action of LH may depend upon a mechanism involving a-adrenergic receptors.94

201

procedure the validity of which has been questioned in the Introduction; the results implicated the same wide range of sites mentioned in connection with the pro-oestrous surge. In these studies the rates of turnover of DA and NA were estimated according to the degree of depletion of the amine following inhibition of tyrosine hydroxylase;‘* it should therefore be noted that this procedure inhibits the synthesis of DA more Ovuiation despite a failure of the normal LH surge effectively than that of NA’24*‘34and also has a marked stimulatory effect on the release of proThe failure of the 18.00 h LH surge after DBH lactin,44 a~en~orticotrophic ho~one,‘16 corticoinhibition with DDC (Tables 2 and 3) is in agreement sterone’16 and progesterone (Fig. 5). Furthermore, with previous studies in which Dl3H inhibitors have been administered before the pro-oestrous surge;75s’32 since studies of neurotransmitter turnover rates provide only correlative information, any of the spontanevertheless, an examination of oviducts on the afterneous changes in the endocrine system occurring in noon of the day following that of the presumptive association with the LH surge may be pertinent; these surge has led to the discovery of an anomalous group of animals which ovulate without a surge of LH at include the surge of prolactin, follicle stimulating hormone and progesterone,‘2’ an increase in the the conventional time of 18.00 h (Tables 2 and 3). The amplitude of the circadian release of adresame phenomenon was apparent in some animals treated with the relatively low doses of SKF 64139 nocorticotrophic hormone” and corticosterone,” a biphasic pattern in the secretion of thyroid stimu(Tables 2 and 3) or clonidine (Table 5). When blood samples were taken at additional times, 90% of the lating hormone’s and thyroxine” and a fall in the serum concentration of oestradiol either beforeI or animals treated with DDC, SKF 64139 or clonidine and subsequently ovulating, despite a failure of the during’*’ the LH surge. In several of the reports concerning noradrenergic 18.00 h LH surge (n = 31), were found to be producing a surge at 22.00 or 24.00 h (Figs 1 and 4). It is activity associated with the LH surge, comparisons unclear whether these abnormally timed surges reflect have been made merely between the rate of turnover a shift in a timing mechanism or a change of state in at a particular time on the day of the presumptive which an underlying capacity to produce a surge in surge and that observed at the same time on a day the middle of the dark phase is given expression. The without such an ~urrence.37,38,~,83 These studies latter possibility is suggested by the observation of a consequently fail to identify a change in activity during the day of the surge and leave open the similarly timed second surge of LHRH in hypophysial portal blood, which, in the event of a normal LH possibility that significant differences may reflect less abrupt endocrine changes such as those occurring in, surge at 18.00 h, fails to produce a corresponding discharge of LH, but may be involved in regulating for example, the serum concentration of oestrogen. the release of follicle stimulating hormone.‘w The Furthe~ore, many of the studies have failed to occurrence of a “midnight” surge after treatment consider the concentration of the amine at the time with relatively low doses of the drugs, may reflect a at which the synthesis inhibitor is administered as the sufficient recovery of adrenaline- mediated transinitial value from which the decline is calcumission to fulfil the requirements of the oscillator that lated37~38*4’~68*“9 and have assumed that the concengoverns the timing of this event. tration observed in a vehicle-treated group killed at the same time as the drug-treated group is an adernvo~vement of noradrena~ine in the LH surge quate substitute. This practice contravenes the prinThe present results suggest that the contribution of ciple of estimating the rate of turnover as a function NA to the preovulatory LH surge may be limited to of the degree of depletion following a cessation of its role in the biosynthetic pathway for adrenaline. It synthesis;” it may also lead to inappropriately high is therefore important to examine the case for a direct “initial” levels of catecholamines given that tyrosine noradrener~c involvement in this process as hydroxylase activity has been reported to rise in presented in several studies reporting an increased conjunction with the LH surge.** rate of turnover of hypothalamic NA on the day of Only Barraclough and his associates have prooestrus. 4’*M1~s5~‘o’~‘03 Various sites have been re- presented data on the rate of NA turnover at ported to show this increased noradrenergic activity various times on the day of a presumptive LH surge including the medial preoptic area,6*85~‘o’~‘03 the su- using the true initial concentration in their calcuprachiasmatic nuclei,‘0t*‘03the arcuate nuclei’03 and lations.‘“‘~‘oz~‘03~‘36 Of their two studies concerned with the median e~nence~‘~85~‘o’~‘03 The turnover rate of the day of pro-oestrus, one”’ indicates a marked hypothalamic NA has also been examined in associ- elevation in noradrenergic activity in the medial ation with the LH surge induced in overiectomized preoptic area and suprachiasmatic nuclei at the time rats by oestrogen alone’o2*‘36or by progesterone fol- of the LH surge. This level of activity in the medial lowing a priming dose of oestrogen,37*38~68~“9~‘3” a preoptic area is not, however, altered when the LH

C. W. Coen and M. C. Coombs

202

surge is blocked by the central actions of phenobarbital.“’ Furthermore, although their other paper on pro-oestrous rats lo3includes the same data for the two times of day prior to the LH surge, the different results presented in association with the surge indicate that the level of noradrenergic activity in the medial preoptic area and suprachiasmatic nuclei at the latter time is not significantly higher than that observed three hours earlier, thus suggesting that the changes in these parameters during pro-oestrus are only gradual and not comparable to the abrupt discharge of LH. Similarly, the increase in the rate of NA turnover in the median eminence is found three hours before the LH surge.‘0’,‘03 An apparently abrupt increase in noradrenergic activity at the time of the surge is, however, observed in the arcuate nuclei,lo3 but the importance of this enhanced activity is unctear since it is corroborated in only oneb3’ of their two studies concerned with the LH surge in oestrogen-treated ovariectomized rats.‘02.‘3h A significant contribution to the production of the LH surge involving an increase in noradrenergic activity in the mediobasal hypothalamus on dioestrus, the day preceding pro-oestrus, has been proposed.“’ Treatment with inhibitors of tyrosine hydroxylase or DBH on dioestrus can indeed prevent the pre-

ovulatory surge and ovulation.‘*‘~“’ These results may, however, be explained without invoking a necessary noradrenergic function on the day preceding the surge since the inhibition of these enzymes (but not PNMT) on dioestrus results in a considerable increase in the serum level of progesterone (Fig. .5), an effect which can prevent ovulation when it occurs prior to the rise in the secretion of oestrogen.” CarechoIami~e pathways and the

LH

surge

The rapid resumption of apparently normal LH surges and sexual cycles after an administration of 6-OHDA64,W,96may be related to the compensatory increase in hypothalamic PNMT activity which occurs within a few days of its acute depression following such treatment. I9 Similarly, transections of the ascending noradrenergic tracts have been reported to cause only a brief disruption of these processes;‘s recent evidence suggests that these tracts do not, however, include the majority of the adrenalinecontaining projections from the medulla oblongata to the hypothalamus. 99Although the site at which adrenaline may participate in the production of the LH surge remains to be determined, it is unlikely to be outside the blood-brain barrier in the median emi-

nence since there is evidence suggesting that a systemic infusion of adrenaline on the day of pro-oestrus can block the LH surge by suppressing LHRH release.8 Our recent research indicates that at the time of the LH surge there is an increase in the rate of turnover of adrenaline in the preoptic area, the site of the LHRH cell bodies projecting to the hypophysial portal vessesls.32” Involvement

of serotonin in the LH surge

Several studies have implicated serotonin in the hypothalamic regulation of the surge by showing that the latter can be blocked by p-chlorophenyfaianine. an inhibitor of serotonin synthesis, and that this effect can be counteracted when the hypothalamic concentration of serotonin is restored by 5hydroxytryptophan, its immediate precursor.27-31,65These studies actually provide further circumstantial evidence for the involvement of adrenaline in the surge since we have recently demonstrated that p-chlorophenylalanine treatment suppresses PNMT activity and that the depleted concentration of hypothalamic adrenaline is restored by an injection of S-hydroxytryptcphan; furthermore, treatment with a neurotoxin specific to serotonin-containing neurons is compatible with the normal occurrence of the surge.27

The present study was designed to examine whether cat~holamines play a necessary role in the production of rhe preovulatory LH surge. The evidence indicates that such a role exists and that adrenaline is the catecholamine by which it is mediated. The results emphasize the importance of the sequential study of the serum concentration of LH and ovulation in individual animals and of examining oviducts at a time which recognizes the possibility of a delayed “midnight” surge. Arlolol~lrd~nnrnts--We

search

Council

are grateful to the Medical Re-

for a Fellowship to C.W.C.. a Studentship

to M.C.C. and a grant (G 979/903) to Dr P. C. B. MacKinnon, whose support throughout this study and assistance with ovum counting was in~~lllable. and to Smith, Kline and French for SKF 64139, to RhBne-PoulencSant& for piperoxane. to Pfizer for prazosin, to BoehringerIngelheim for clonidine, to Professor G. D. Niswender for anti-ovine LH serum no. IS. to Professor L. E. Reichert Jr for purified LH (LER-1056.C2). to the Endocrinology Section of the National Institute of Arthritis, Metabolism and Digestive Diseases for the standard preparation of ovine LH (NIH-LH-SI3), to Mr R. Laynes for the measurement of serum LH concentrations, and to Dr C. Jones for the measurement of serum progesterone concentrations.

REFERENCES 1. Andin N.-E., Butcher S. G., Corrodi H., Fuxe K. and Ungerstedt U. (1970) Receptor activity and turnover of dopamine and noradrenaline after neuroleptics. Eur. J. Pharmuc. 11, 303-314. 2. Bacha J. C. and Donoso A. 0. (1974) Enhanced luteinizing hormone release after noradrenaline treatment in &hydroxydopamine-treated rats. J. En&r. 62, 169-170. 3. Barraclough C. A. and Sawyer C. H. (1957) Blockade of the release of pituitary ovulating hormone in the rat by chlorpromazine and reserpine: possible mechanisms of action. Endocrinology 61, 341-35 I. 4. Barraclough C. A. and Sawyer C. H. (1959) Induction of pseudopregnancy in the rat by reserpine and chlorpromazine. Endocrinology

65, 563-57 I.

Hypothalamic

adrenaline

and the luteinizing

hormone

surge

203

5. Beattie C. W., Gluckman M. I. and Corbin A. (1976) A comparison of y-butyrolactone and pimozide on serum gonadotrophins and ovulation in the rat. Proc. Sot. exp. Biol. Med. 153, 147-150. 6. Beck W., Hancke J. L. and Wuttke W. (1978) Increased sensitivity of dopaminergic inhibition of luteinizing hormone release in immature and castrated female rats. Endocrinology 102, 837-843. 7. Beck W. and Wuttke W. (1977) Desensitization of the dopaminergic inhibition of pituitary luteinizing hormone release by prolactin in ovariectomized rats. J. Endocr. 74, 67-74. 8. Blake C. A. (1976) Effects of intravenous infusion of catecholamines on rat plasma luteinizing hormone and prolactin concentrations. Endocrinology 98, 99-104. 9. Boireau A., Temaux J. P., Bourgoin S., Hery F., Glowinski J. and Hamon M. (1976) Determination of picogram levels of 5-HT in biological fluids. J. Neurochem. 26, 201-204. 10. Brodie B. B., Costa E., Dlabac A., Neff N. H. and Smookler H. H. (1966) Application of steady state kinetics to the estimation of synthesis rate and turnover time of tissue catecholamines. J. Pharmac. exp. 7’hir. 154, 493-498. 11. Brown P. S. (1966) The effect of reserpine, 5hydroxytryptamine and other drugs on induced ovulation in immature mice. J. Endocr. 35, 161-168. 12. Brown P. S. (1967) Antigonadotrophic effects of a-methyltyrosine, methysergide and reserpine. Nature, Lond. 214, 1268-1269. 13. Brown-Grant K. (1969) The effects of progesterone and of pentobarbitone administered at the dioestrous stage on the ovarian cycle of the rat. J. Endocr. 43, 539-552. 14. Brown-Grant K., Davidson J. M. and Greig F. (1973) Induced ovulation in albino rats exposed to constant light. J. Endocr. 51, 7-22. 15. Brown-Grant K., Dutton A. and ter Haar M. B. (1977) Variations in plasma thyrotrophin concentration during the rat oestrous cycle. J. Endocr. 72, 33P-34P. 16. Brown-Grant K., Exley D. and Naftolin F. (1970) Peripheral plasma oestradiol and luteinizing hormone concentrations during the oestrous cycle of the rat. J. Endocr. 48, 295-296. 17. Buckingham J. C., Diihler K.-D. and Wilson C. A. (1978) Activity of the pituitary-adrenocortical system and thyroid gland during the oestrous cycle of the rat. J. Endocr. 78, 359-366. 18. Burden H. W., Lawrence I. E., Louis T. M. and Hodson C. A. (1981) Effects of abdominal vagotomy on the estrous cycle of the rat and the induction of pseudopregnancy. Neuroendocrinology 33, 218-222. 19. Burgess S. K., Eiden L. E. and Tessel R. E. (1980) 6-hydroxydopamine differentially affects PNMT activity and epinephrine content in rat hypothalamus: evidence for compensatory enhancement in neuronal function. Sot. Neurosci. Abs. 6, 441. 20. Burgess S. K. and Tessel R. E. (1980) Epinephrine is released from hypothalamus by depolarizing agents and amphetamine. Brain Res. 194, 259-262. 21. Butcher L. L., Eastgate S. M. and Hodge G. K. (1974) Evidence that punctate intracerebral administration of 6-hydroxydopamine fails to produce selective neuronal degeneration. Naunyn-Schmiedebergs Arch. Pharmac. 258, 3 l-70. 22. Carr L. A. and Voogt J. L. (1980) Catecholamine synthesizing enzymes in the hypothalamus during the estrous cycle. Brain Res. l%, 4377445. 23. Choudhury S. A. R., Sharpe R. M. and Brown P. S. (1974) The effect of pimozide, a dopamine antagonist, on pituitary gonadotrophin function in the rat. J. Reprod. fert. 39, 275-283. 24. Clemens J. A., Tinsley F. C. and Fuller R. W. (1977) Evidence for a dopaminergic component in the series of neural events that lead to the pro-oestrous surge of LH. kcfa Endocr. 85, 18-24. _ 25. Clifton D. K. and Sawver C. H. (1979) LH release and ovulation in the rat followine deuletion of hvoothalamic ,norepinephrine: chronic’versus acute effects. Neuroendocrinology 28, 442449. ’ 26. Cocchi D., Fraschini F., Jalanbo H. and Miiller E. E. (1974) Role of brain catecholamines in the postcastration rise in plasma LH of prepubertal rats. Endocrinology 95, 1649-1657. 27. Coen C. W., Coombs M. C., Wilson P. M. J., Clement E. M. and MacKinnon P. C. B. (1983) Possible resolution of a paradox concerning the use of p-chlorophenylalanine and 5hydroxytryptophan: evidence for a mode of action involving adrenaline in manipulating the surge of luteinizing hormone in rats. Neuroscience 8, 583-591. 28. Coen C. W., Franklin M., Laynes R. W. and MacKinnon P. C. B. (1980) Effects of manipulating serotonin on the incidence of ovulation in the rat. J. Endocr. 87, 195-201. 29. Coen C. W. and MacKinnon P. C. B. (1976) Serotonin involvement in oestrogen-induced luteinizing hormone release in ovariectomized rats. J. Endocr. 71, 49P-50P. 30. Coen C. W. and MacKinnon P. C. B. (1979) Serotonin involvement in the control of phasic luteinizing hormone release in the rat: evidence for a critical period. J. Endocr. 82, 105-113. 3 1. Coen C. W. and MacKinnon P. C. B. (1980) Lesions of the suprachiasmatic nuclei and the serotonin-dependent release of luteinizing hormone in the rat: effects on drinking rhythmicity and on the consequences of preoptic area stimulation. J. Endocr. 84, 231-236. 32. Coen C. W. and MacKinnon P. C. B. (1981) The effects of blocking catecholamine synthesis on the incidence of the LH surge and ovulation in the rat. Neuroscience Letters Suppl. 7, S252. 32a. Coombs M. C. and Coen C. W. (1983) Adrenaline turnover rates in the medial preoptic area and mediobasal hypothalamus in relation to the release of luteinizing hormone in female rats. Neuroscience 10, 207-210. 33. Coon G. A., Legan S. J. and Karsch F. J. (1975) Persistence of a dailv surge of luteinizitm hormone (LH) followine adrenalectomy in estrogen-treated ovariectbmizdd rats. Fedn Proc. Fedh A% Sots exp. EoZ. 34, 287: ’ 34. Coppola J. A., Leonardi R. G. and Lippmann W. (1966) Ovulatory failure in rats after treatment with brain norepinephrine depletors. Endocrinology 78, 225-228. 35. Coppola J. A., Leonardi R. G., Lippmann W., Perrine J. W. and Ringler I. (1965) Induction of pseudopregnancy in rats by depletors of endogenous catecholamines. Endocrinology 77, 485490. 36. Corrodi H., Fuxe K. and Hiikfelt T. (1967) The effect of neuroleptics on the activity of central catecholamine neurones. Ltfe Sci. 6, 767-774. 37. Crowley W. R. (1982) Effects of ovarian hormones on norepinephrine and dopamine turnover in individual hypothalamic and extrahypothalamic nuclei. Neuroendocrinology 34, 381-386. 38. Crowley W. R., O’Donohue T. L., Wachslicht H. and Jacobowitz D. M. (1978) Effects of estrogen and progesterone

204

39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52.

53.

54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68.

69. 70. 71. 72.

C. W. Coen and M. C. Coombs on plasma gonadotropins and catecholamine levels and turnover in discrete brain regions of ovariectomized rats. Brain Res. 154, 345-357. Crowley W. R. and Terry L. C. (1981) Effects of an epinephrine synthesis inhibitor, SKF 64139, on the secretion of luteinizing hormone in ovariectomized female rats. Brain Res. 204, 231-235. Deanesly R. (1966)The effects of reserpine on ovulation and on the corpus luteum of the guinea-pig. J. Reprod. Fert. 11, 429-438. Demarest K. T., Johnston C. A. and Moore K. E. (1981) Biochemical indices of catecholamine neuronal activity in the median eminence during the estrous cycle of the rat. Neuroendocrinology 32, 2427. Dickerman S., Kledzik G., Gelato M., Chen H. J. and Meites J. (1974) Effects of haloperidol on serum and pituitary prolactin, LH and FSH and hypothalamic PIF and LRF. Neuroendocrinology 15, 1620. Diet1 H., Sinha J. N. and Phihppu A. (1981) Presynaptic regulation of the release of catecholamines in the cat hypothalamus. Bruin Res. 208, 213-218. Donoso A. 0.. Bishop W., Fawcett C. P.. Krulich L. and McCann S. M. (1971) EITects of drugs that modify brain monoamine concentrations on plasma gonadotropin and prolactin levels in the rat. Endocrinology 89, 774-784. Drouva S. V. and Gallo R. V. (1976) Catecholamine involvement in episodic luteinizine. hormone release in adult ovariectomized rats. Endocrinology !I$ 651-658. Drouva S. V. and Gallo R. V. (1977) Further evidence for inhibition of episodic luteinizing hormone release in ovariectomized rats by stimulation of dopamine receptors. Endocrinology 100, 792-798. Estes K. S., Simpkins J. W. and Kalra S. P. (1982) Resumption with clonidine of pulsatile LH release following acute norepinephrine depletion in ovariectomized rats. Neuroendocrinology 35, 5662. Everett J. W. and Sawyer C. H. (1949) A neural timing factor in the mechanism by which progesterone advances ovulation in the cyclic rat. Endocrinology 45, 581-595. Everett J. W., Sawyer C. H. and Markee J. E. (1949) A neurogenic timing factor in control of the ovulatory discharge of luteinizing hormone in the cyclic rat. Endocrinology 44, 234250. _ Ferin M.. Temoone A.. Zimmerinz P. E. and Vande Wiele R. L. (1969) Effects of antibodies to 17-0-estradiol and progesterone on the estrous cycle of the rat. Endocrinology 85, IO%l~78. France E. S. (1970) Reversal by pargyhne of reserpine block of induced ovulationdirect ovarian effects. Neuroendocrinology 6, 77-89. Fuller R. W., Perry K. W., Hemrick S. K. and Molloy B. B. (1979) Lowering of brain epinephrine by inhibition of norepinephrine N-methyltransferase in rats. In Catecholamines: Basic and Clinical Frontiers (eds Usdin E., Kopin I. J. and Barchas J.) pp. 186188. Pergamon Press, New York. Fuxe K., Andersson K., Agnati L. F., Ferland L., Hokfelt T., Eneroth P., Gustafsson J.-A. and Skett P. (1979) Central catecholamine systems and neuroendocrine regulation. Controllers of anterior pituitary secretion. In Cutecholumines: Basic and Clinical Fronfiers (eds Usdin E., Kopin I. J. and Barchas J.) pp. 1187-1203. Pergamon Press, New York. Fuxe K., Goldstein M., Hiikfelt B. and Hiikfelt T. (1980) Central Adrenaline Neurons. Basic Aspects and Their Role in Cardiovascular Functions. Pergamon Press, Oxford. Gallo R. V. and Drouva S. V. (1979) Effects of intraventricular infusion of catecholamines on luteinizing hormone release in ovariectomized and ovariectomized, steroid-primed rats. Neuroendocrinology 29, 149-162. Gerendai I. and Hal&z B. (1981) Participation of a pure neuronal mechanism in the control of gonadal functions. Andrologia 13, 275-282. Glowinski J. and lversen L. L. (1966) Regional studies of catecholamines in the rat brain--I. The disposition of ‘H norepinephrine, ‘H dopamine and ‘H dopa in various regions of the brain. J. Neurochem. 13, 655-669. Gnodde H. P. and Schuiling G. A. (1976) Involvement of catecholaminergic and cholinergic mechanisms in the pulsatile release of LH in the long-term ovariectomized rat. Neuroendocrinology 20, 212-223. Goldstein M. B., Anagnoste B., Lauber E. and McKereghan M. R. (1964) Inhibition of dopamine-p-hydroxylase. Life Sci. 3, 763-767. Goldstein M. and Nakajima K. (1967) The effect of disulfiram on catecholamine levels in the brain. J. Pharmac. exp. ThPr. 157, 96-102. Goldstein M., Saito M., Lew J. Y., Hieble J. P. and Pendleton R. G. (1980) The blockade of a,-adrenoceptors by the PNMT inhibitor SKF 64139. Eur. J. Pharmnc. 67, 305-308. Gilman A. G., Goodman L. S. and Gilman A. (1980) The Pharmacological Basis of Therapeutics (6th edition). MacMillan Publishing Co., New York. Greig F. and Weisz J. (1973) Preovulatory levels of luteinizing hormone, the critical period and ovulation in rats. J. Endocr. 57, 235-245. Hancke J. L. and Wuttke W. (1979) Effects of chemical lesion of the ventral noradrenergic bundle or the medial preoptic area on preovulatory LH release in rats. Expl. Bruin Res 35, 127-134. Hkry M., Laplante E. and Kordon C. (1976) Participation of serotonin in the phasic release of LH--I. Evidence from pharmacological experiments. Endocrinology 99, 496503. Heubusch P. and DiStefano V. (1978) Activation of brain tyrosine hydroxylase in rats exposed to carbon disuhide and sodium diethyldithiocarbamate. Toxicol. Appl. Pharmac. 46, 143-149. Hokfelt T., Fuxe K., Goldstein M. and Johansson 0. (1974) Immunohistochemical evidence for the existence of adrenaline neurons in rat brain. Brain Res. 66, 235-251. Honma K. and Wuttke W. (1980) Norepinephrine and dopamine turnover rates in the medial preoptic area and the mediobasal hypothalamus of the rat brain after various endocrinological manipulations. Endocrinology 106, 184881853. Hopkins T. F. and Pincus G. (1963) Effects of reserpine on gonadotropin-induced ovulation in immature rats. Endocrinology 73, 775-780. Hyyppa M., Lehtinen P. and Rinne U. K. (1971) Effect of L-dopa on the hypothalamic, pineal and striatal monoamines and on the sexual behaviour of the rat. Bruin Res. 30, 265-272. Ichikawa S., Morioka H. and Sawada T. (1972) Acute effect of gonadotrophins on the secretion of progestins by the rat ovary. Endocrinology 90, 13561362. Iversen L. L. (1970) Metabolism of catecholamines. In Handbook of Neurochemisfry (ed. Lajtha A.) Vol. 4, pp. 197-220. Plenum Press, New York.

Hypothalamic adrenaline and the luteinizing hormone surge

205

73. Kalra P. S., Kalra S. P., Krulich L., Fawcett C. P. and McCann S. M. (1972) Involvement of norepinephrine in transmission of the stimulatory influence of progesterone on gonadotropin release. Endocrinology 90, 1168-1176. 14. Kalra S. P. and McCann S. M. (1973) Effect of drugs modifying catecholamine synthesis on LH release induced by preoptic stimulation in the rat. Endocrinology 93, 356362. 15. Kalra S. P. and McCann S. M. (1974) Effects of drugs modifying catecholamine synthesis on plasma LH and ovulation in the rat. Neuroendocrinology 15, 19-91. 16. Kalra S. P. and Simpkins J. W. (1981) Evidence for noradrenergic mediation of opioid effects on luteinizing hormone secretion. Endocrinology 109, 776782. 1% Kamberi I. A., Mica1 R. S. and Porter J. C. (1970) Effect of anterior pituitary perfusion and intraventricular injection of catecholamines and indoleamines on LH release. Endocrinology 87, 1-12. 78. Kitchen J. H. (1974) Effects of intracerebral injections of 6-hydroxydopamine on LH and FSH release in male rats. Neuroendocrinology 15, 24&244. 79. Kordon C. and Glowinski J. (1969) Selective inhibition of superovulation by blockade of dopamine synthesis during the “critical period” in the immature rat. Endocrinology 85, 924-931. 80. Kostrzewa R. M. and Jacobowitz D. M. (1974) Pharmacological actions of 6-hydroxydopamine. Pharmac. Rev. 26, 199-288. 81. Krieg R. J. and Sawyer C. H. (1976) Effects of intraventricular catecholamines on luteinizing hormone release in ovariectomized-steroid-primed rats. Endocrinology 99, 411-419. 82. Langer S. Z. (1979) Presynaptic adrenoceptors and regulation of release. In The Release of Catecholamines from Adrenergic Neurons (ed. Paton D. M.) pp. 5985. Pergamon Press, New York. 83. Leung P. C. K., Arendash G. W., Whitmoyer D. I., Gorski R. A. and Sawyer C. H. (1982) Differential effects of central adrenoceptor agonists on luteinizing hormone release. Nemoendocrinology 34, 207-214. 84. Lippmann W. (1968) Relationship between hypothalamic norepinephrine and serotonin and gonadotrophin secretion in the hamster. Nature, Land 218, 173-174. 85. Liifstriim A. (1977) Catecholamine turnover alterations in discrete areas of the median eminence of the 4 and S-day cyclic rat. Brain Res. 120, 113-131. 86. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. 8% Magos L. and Jarvis J. A. (1970) Effects of diethyldithiocarbamate and carbon disulphide on brain tyrosine. J. Phartn. Pharmac. 22, 936938. 88. Mann D. R., Korowitz C. D., Macfarland L. A. and Cost M. G. (1976) Interactions of the light-dark cycle, adrenal

glands and time of steroid administration in determining the temporal sequence of LH and prolactin release in female rats. Endocrinology 99, 1252-1262. 89. Martini L. and Meites J. (1970) Neurochemical Aspects of Hypothalamic Function. Academic Press, New York. 90. Martinovic J. V. and McCann S. M. (1977) Effect of lesions in the ventral noradrenergic tract produced by microinjection of 6-hydroxydopamine on gonadotrophin release in the rat. Endocrinology 100, 12061213. 91. Meyerson B. J. and Sawyer C. H. (1968) Monoamines and ovulation in the rat. Endocrinology 83, 17&176. alpha 92. Miach P. J., Dausse J.-P. and Meyer P. (1979) Biochemical evidence for “pre-” and “postsynaptic” adrenoceptors in rat brain. In Catecholamines: Basic and Clinical Fronfiers (eds Usdin E., Kopin I. J. and Barchas J.) pp. 571-573. Pergamon Press, New York. 93. Moore W. W. (1961) Failure of adrenergic and cholinergic blocking agents to block ovulation in the rat. Am. J. Physiol. 200, 1293-1295. 94 Moudgal R. P. and Razdan M. N. (1981) Induction of ovulation in vitro by LH and catecholamines in hens is mediated by cc-adrenergic receptors. Nature, Lond. 293, 738-739. 95 Mueller G. P., Simpkins J., Meites J. and Moore K. E. (1976) Differential effects of dopamine agonists and haloperidol

on release of prolactin, thyroid stimulating hormone, growth hormone and luteinizing hormone in rats. Neuroendocrinology 20, 121-135. 96. Nicholson G., Greeley G., Humm J., Youngblood

W. and Kizer J. S. (1978) Lack of effect of noradrenergic denervation of the hypothalamus and medial preoptic area on the feedback regulation of gonadotropin secretion and the estrous cycle of the rat. Endocrinology 193, 559-566. 97. Ojeda S. R., Harms P. G. and McCann S. M. (1974) Effect of blockade of dopaminergic receptors on prolactin and LH release: median eminence and pituitary sites of action. Endocrinology 94, 1650-1657. 98. Ojeda S. R. and McCann S. M. (1973) Evidence for participation of a catecholaminergic mechanism in the post-castration rise in plasma gonadotropins. Neuroendocrinology 12, 295-315. 99. Palkovits M., Mezey E., Zaborsky L., Feminger A., Versteeg D. H. G., Wijnen H. J. L. M., De Jong W., Fekete M. I. K., Herman J. P. and Kanyicska B. (1980) Adrenergic innervation of the rat hypothalamus. Neuroscience Letters 18, 2377243. 100. Purshottam N., Mason M. M. and Pincus G. (1961) Induced ovulation in the mouse and the measurement of its inhibition. Fert. Steril. 12, 346352. 101. Rance N. and Barraclough C. A. (1981) Effects of phenobarbital on hypothalamic LHRH and catecholamine turnover rates in proestrous rats. Proc. Sot. exp. Biol. Med. 166, 425431. 102. Rance N., Wise P. M. and Barraclough C. A. (1981) Negative feedback effects of progesterone correlated with changes in hypothalamic norepinephrine and dopamine turnover rates, median eminence luteinizing hormone-releasing hormone, and peripheral plasma gonadotrophins. Endocrinology 108, 21942199. 103. Rance N., Wise P. M., Selmanoff M. K. and Barraclough C. A. (1981) Catecholamine turnover rates in discrete hypothalamic gonadotropins

areas and associated changes in median eminence luteinizing hormone-releasing hormone and serum on proestrus and diestrous day 1. Endocrinology 108, 179>1802. 104. Raziano J., Cowchock S., Ferin M. and Vande Wiele R. L. (1971) Estrogen-dependency of monoamine-induced ovulation. Endocrinology 88, 15161518. 105. Reis D. J., ROSSR. A., Pickel V. M., Lewander T. and Joh T. H. (1975) Some differences in tyrosine hydroxylase in central noradrenergic and dopaminergic neurons. In Chemical Tools in Catecholamine Research (eds Almgren O., Carlsson A. and Engel J.) Vol. 2, pp. 53-60. North-Holland Publishing Company, Amsterdam. 106. Rethelyi M., Vigh S., Set616 G., Merchenthaler I., Flerkb B. and Petrusz P. (1981) The luteinizing hormone releasing

206

C. W. Coen and M. C. Coombs

hormone-containing pathways and their co-termination with tanycyte processes in and around the median eminence and in the pituitary stalk of the rat. Acta morph. Acad. Sci. hung. 29, 259-283. 107. Rubinstein L. and Sawyer C. H. (1970) Role of catecholamines in stimulating release of pituitary ovulating hormone(s) in rats. Endocrinology 86, 988-995. 108, Saavedra J. M., Palkovits M., Brownstein M. J. and Axehod J. (1974) Localization of phenylethanolamine ~-methyltransfer~e in rat brain nuclei. Nature, Lond. 248, 695-696. 109. Sailer C. F. and Zigmond M. J. (1978) A radioenzymatic assay for catecholamines and ~hydroxyphenyla~tic acid. Life sci. 23, 1117-l 130.

110.Sarkar D. K., Chiappa S. A., Fink G. and Sherwood N. M. (1976) Gonadotropin-releasing

hormone surge in pro-oestrous rats. Nature, Lond. 264, 461-463. 111. Sarkar D. K. and Fink G. (1981) Gonadotropin-releasing hormone surge: possible modulation through postsynaptic a-adrenoreceptors and two pharmacologically distinct dopamine receptors. Endocrinology 108, 862-867. 112. Sauter A. M., Lew J. Y., Baba Y. and Goldstein M. (1977) Effect of phenylethanolamine ~-methyitransferase and dopamine-~-hydroxyla~ inhibition on epinephrine levels in the brain. Life Sci. 21, 261-266. 113. Sawyer C. H., Everett J. W. and Markee J. E. (1949) A neural factor in the mechanism by which estrogen induces the release of luteinizing hormone in the rat. Endocrinology 44, 218-233. 114. Sawyer C. H., Markee J. E. and Everett J. W. (1950) Further experiments on blocking pituitary activation in the rabbit and the rat. J. expl 2001. 113, 659-682. 115. Sawyer C. H., Markee J. E. and Hollinshead W. H. (1947) Inhibition of ovulation in the rabbit by the adrenergic-blocking agent Dibenamine. Endocrinology 41, 395402. 116. Scapagnini U., Annunziato L., Lombardi G., Oliver C. and Preziosi P. (1975) Time-course of the effect of ~-methyl-F-tyrosine on ACTH secretion. ~euroendocr~no~o~y 18, 2722276. 117. Scatton B.. Pelavo F.. Dubocovich M. L., Lancer S. 2. and Barthohni G. (1979) Effects of clonidine on the cerebral adrenaline turnover and the adrenaline release-in nucleus tractus solitarii ‘of the rat. In Presynapfic Receptors (eds Langer S. Z., Starke K. and Dubocovich M. L.) pp. 231.-236. Pergamon Press, New York. 118. Schneider H. P. G. and McCann S. M. (1970) Mono- and indolamines and control of LH secretion. Endocrinology 86, 1127-1133. 119. Simpkins J. W., Huang H. H., Advis J. P. and Meites J. (1979) Changes in hypothalamic NE and DA turnover resulting from steroid-indu~ LH and prolactin surges in o~ri~tomized rats. Bioi. Reprod. 20, 625-632. 120. Simpkins J. W. and Kalra S. P. (1979) Blockade of progesterone-induced increase in hypothalamic luteinizing hormone-releasing hormone levels and serum gonadotropins by intrahypothalamic implantation of 6-hydroxydopamine. Brain Res. 170, 475484. 121. Smith M. S., Freeman M. E. and Neil1 J. D. (1975) The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin, gonadotrophin and steroid levels associated with rescue of the corpus luteum of pseudopr&nancy. Endocrinology %, 219-226. 122.Terasawa E.. Bridson W. E.. Davennort J. W. and Gov R. W. (1975) Role of brain monoamines in release of gonadotropin before proestrus in the‘cyclic rat. ~e~~~~~~~crjno~og~18, ‘345358. 123. Ter Haar M. B. and MacKinnon P. C. B. (1973) Changes in serum gonadotrophin levels. and in protein levels and in-viva incorporation of % methionine into protein of discrete brain areas and the anterior pituitary of the rat during the oestrous cycle. J. Endocr. 58, 563-576. 124. Thierry A. M., Blanc G. and Glowinski J. (1971) Dopamine-norepinephrine: another regulatory step of norepinephrine synthesis in central noradrenergic neurons. Eur. J. Pharmac. 14, 303-307. 125. Tima L. and FlerkB B. (1974) Ovulation induced by norepinephrine in rats made anovulatory by various experimental procedures. ~eur~endocrino~ogy 15, 346-354. 126. Tizabi Y., Massari V. J. and Jacobovitz D. M. (1979) Study of a possible interference of a-methyl-para-tyrosine pretreatment on the radioenzymatic assay of catecholamines in the rat brain. J. Neurochem. 33, 959-961. 127. Turnbull A. C., Patten P. T., Flint A. P. F., Keirse M. J. N. C., Jeremy J. Y. and Anderson A. B. M. (1974) Significant fall in progesterone and rise in oestradiol levels in human peripheral plasma before onset of labour. Lancer 1, 101-104. 128. UPrichard D. C., Charness M. E., Robertson D. and Snyder S. H. (1978) Prazosin: differential affinities for two populations of a-noradrenergic receptor binding sites. Eur. J. Pharmac. 50, 87-89. 129. Van der Gugten J., Paikovits M., Wijnen H. L. J. M. and Versteeg D. H. G. (1976) Regional distribution of adrenaline in rat brain. Brain Res. 107, 171-175. 130. Vijayan E. and McCann S. M. (1978) Re-evaluation of the rote of catecholamines in control of gonadotropin and prolactin release. Neuroendocrinology 25, 15(t165. 131. Vogt M. (1954) The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs. J. Physiol., Lond. 123, 451.-481. 132. Voogt J. L. and Carr L. A. (1981) Inhibition of LH and prolactin release in the cycling rat following inhibition of dopamine-fi-hydroxylase. Brain Res. 209, 411419. 133. Weick R. F. (1978) Acute effecis of adrenergic receptor blocking drugs and neuroleptic agents on pulsatile discharges of iuteinizing hormone in the ovariectomized rat. -~euroend~cr~n*~~~y 26, 108---lI?. _ 134. Wider%% E. and Lewander T. (1978) Inhibition of the in uirjo biosynthesis and changes of catecholamine levels in rat brain after a-methyl-p-tyrosine: time- and dose-response relationships. Naunyn-Sch;niedehergs Arch. Pharmac. 304, 111-123. 135. Wilkening D., Dvorkin B., Makman M. H., Lew J. Y., Matsumoto J., Baba Y., Goldstein M. and Fuxe K. (1980)

Catecholamine-stimulated cyclic AMP formation in phenylethanolamine N-methyhransferase containing brain stem nuclei of normal rats and of rats with spontaneous genetic hypertension. Brain Res. 186, 133-143. 136. Wise P. M., Rance N. and Barraclough C. A. (1981) Effects of estradiol and progesterone on catecholamine turnover rates in discrete hypothalamic regions in ovariectomized rats. ~ndocr~no~~g~ lb, 2 1862193. 137. Zarrow M. X.. Caldwell A. L., Hafez E. S. E. and Pincus G. (1958) Superovulation in the immature rat as a possible assay for LH and HCG. Endocrinology 63, 748-758. (Accepted 30 January 1983)