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Effect of phenylethanolamine N-methyltransferase inhibition on serum concentrations of luteinizing hormone, follicle stimulating hormone and prolactin in pro-oestrous and ovariectomized rats
BrainResearch, 343(1985)383-387 Elsevier
383
BRE 21065
Effect of phenylethanolamine N-methyltransferase inhibition on serum concentrations of luteinizing hormone, follicle stimulating hormone and prolactin in pro-oestrous and ovariectomized rats CLIVE W. COEN 1, AGNES SIMONYI 2 and MARTON I. K. FEKETE 2
IDepartment of Anatomy and Human Biology, King's College, London University, London (U. K. ) and *-Institute of Experimental Medicine, Budapest (Hungary) (Accepted May 7th, 1985)
Key words: phenylethanolamine N-methyltransferase - - adrenaline - - luteinizing hormone - - follicle stimulating hormone - - prolactin - - ovulation
In a dose dependent manner, LY 87130, an inhibitor of adrenaline synthesis in the rat brain, either shifts the pro-oestrous surges of luteinizing hormone and follicle stimulating hormone from the late afternoon to the middle of the night or blocks any occurrence of the surges and prevents ovulation. Because the pulsatile release of LH is also blocked by LY 87130, it is proposed that the role of adrenaline in the production of the preovulatory surge is concerned with the regulation of the LH pulses which constitute the surge. A n involvement of adrenaline in the control of reflex ovulation in rabbits was first p r o p o s e d in 1947 (ref.16). It was subsequently r e p o r t e d that intraventricular adrenaline can restore ovulation in rats when the otherwise spontaneous p r e o v u l a t o r y surge of luteinizing h o r m o n e ( L H ) is blocked by barbiturate treatment~S; furthermore, intraventricular adrenaline was found to be m o r e p o t e n t than noradrenaline in restoring b a r b i t u r a t e - b l o c k e d ovulationlS and in stimulating L H release in steroid-primed ovariectomized rats 21. Nevertheless, evidence indicating a necessary involvement of adrenaline in the production of the spontaneous L H surge in rats was not available until 1981, when it was discovered that treatment with S K F 64139, an inhibitor of phenylethanolamine N-methyltransferase ( P N M T ) , the enzyme which catalyses the conversion of n o r a d r e n a line to adrenaline, blocks the pro-oestrous surge and subsequent ovulation 6 and also the surge induced by progesterone t r e a t m e n t following ovariectomy and a priming dose of oestrogen 9. In order further to elucidate the role of adrenaline in the control of spontaneous g o n a d o t r o p h i n secretion, we have examined the effect of LY 87130, an-
other P N M T inhibitor 12, on: (1) the pro-oestrous surges of L H , follicle stimulating h o r m o n e ( F S H ) and prolactin; (2) the incidence of ovulation; and (3) the pulsatile release of L H , a process most readily m o n i t o r e d after ovariectomy. The results have been previously p r e s e n t e d in abstract form 7. F e m a l e Wistar rats (150-200 g) were m a i n t a i n e d under regular lighting conditions (lights: 06.00-20.00 h). A t 13.00 h and, in some cases, at subsequent times on the day of pro-oestrus, animals received an intraperitoneal injection of the vehicle (2.0 ml water/kg) or LY 87130. T h e drug was administered at 20 or 40 mg/kg, doses previously r e p o r t e d to produce a m a r k e d depletion of central adrenaline12; the nadir, which represents a loss of 65 or 86% of the control value for the two respective doses, occurs about 6 h after t r e a t m e n t and is not a c c o m p a n i e d by a significant effect on the concentration of dopamine or noradrenaline 12. B l o o d samples (400 ktl) were taken under light ether anaesthesia ( < 1.5 min) immediately before the injection at 13.00 and again at 18.00 h, the a p p r o x i m a t e times of the p e a k of the LH surge. Because our recent studies on the effects of P N M T inhibition on the L H surge 3,6 indicate that a
Correspondence: C. W, Coen, Department of Anatomy and Human Biology, King's College, London University, Strand, London WC2R 2LS, U.K.
384 relatively low dose allows full ovulation to take place following a surge at 'midnight' instead of 18.00 h, an
tion). Statistical significance was evaluated by Student's t-test
additional blood sample was taken at 24.00 h. Oviducts were examined for ova between 14.00 and
Of the animals treated with the vehicle on the day of pro-oestrus, 91% produced a surge of LH at
17.00 h on the day after the presumptive L H surge. The rats used following ovariectomy were fitted with an indwelling cannula in the right atrium5 2 weeks after the former operation. O n the next day, blood samples (150 #1) were taken and replaced with 0.9% saline every 6 rain between 12.00 and 13.00 h.
18.00 h; such an occurrence was never observed in the animals given LY 87130 (Table I). Some of the animals in the latter group did, however, produce a surge at 24.00 h (Table I). The incidence of the 24.00 h surge was related to the dose and n u m b e r of treatments in which the LY 87130 was administered: thus,
The animals were given the vehicle or 20 mg LY 87130/kg immediately after the first period of sampling and again at 16.00 h. Further samples were taken every 6 rain between 17.00 and 18.30 h.
a single injection of 20 mg LY 87130/kg at 13.00 h resulted in an invariable occurrence of the surge at 24.00 h followed by either full or reduced ovulation, but the same dose given at 13.00, 16.00 and 19.'00 h led to a suppression of the surge at each of the possible times and a complete failure of ovulation (Table
Serum concentrations of LH, FSH and prolactin were assayed in duplicate in a double-antibody radioimmunoassay using ovine L H (NIH S13), rat FSH
I).
( N I A M D D RP-1) and rat prolactin ( N I A M D D RP-1) as the reference preparations. The results for each h o r m o n e in this study were obtained in a single
The 34 rats showed an increment in the levels of LH between 13.00 and 18.00 h on pro-oestrus which was either greater than 80.0 ng/ml (registered as a
assay; they were accepted only when the coefficient of variation of the duplicate was less than 5%. Pulsatile release of L H was analyzed according to the criteria of Gallo 14. The coefficient of variation of the concentrations of L H comprising the ascending and descending phase of each putative pulse was determined; a pulse was identified when this index was at
surge at 18.00 h; n = 10) or less than 4.9 ng/ml (n = 24) (Table I). In the latter case, the subsequent increment at 24.00 h was either greater than 26.9 ng/ml (registered as a surge at 24.00 h: n = 9) or less than 2.5 ng/ml (registered as no surge: n = 11); there were 4 exceptions with an increment of 7.0-11.0 ng/ml which, for the purpose of this study, have been
least 15% (1.5 x the intra-assay coefficient of varia-
classified as surges at 24.00 h (Table I). The 18.00 h
TABLE I Incidence o f ovulation (with mean +_ S.D. no. o f ova) and o f the surge in the serum concentration o f L H at either 18. O0 or 24. O0 h on the day o f pro-oestrus in rats given the vehicle or L Y 87130 at various times on that day Treatment
Vehicle Vehicle Vehicle 20 mg LY 87130/kg 40 mg LY 87130/kg 20 mg LY 87130/kg 20 mg LY 87130/kg
Time (h)
No.
13.00 4 13.00 and 16.00 4 13.00, 16.00 and 19.00 3 13.00 5 13.00 6 13.00and 16.00 7 13.00,16.00 and 19.00 5
Full owdation*
Reduced ovulation
No ovulation
LH surge-18. O0 h
LH surge- 24.00 h**
LH surge-24. O0 h
LH surge24. O0 h
No surge
%
No. o f ova
%
No. o f ova
%
No. o f ova
%
¢/c
100 75 100
8.7+0.9 9.3+1.1 8.7+0.6
25
14 40 17
4.5±2.1 2 29***
33 57 100
60 9.0± 1.0 50*** 10.7±2.9 14"** 14
* 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 > 80.0 ng/ml (registered as 'LH surge-18.00 h') or < 4.9 ng/ml. In the latter case the subsequent increment at 24.00 h was > 26.9 ng/ml (registered as 'LH surge-24.00 h ~) or < 2.5 ng/ml (registered as 'No surge') with 4 exceptions in the groups marked *** in which the increment was 7.0-11.0 ng/ml.
385 (n = 4) the increment in FSH was between 10.4 and
surge invariably resulted in full ovulation; the 'midnight' surge was followed by full ovulation, reduced
90.3 ng/ml. The various treatments with LY 87130
ovulation or no ovulation in, respectively 62, 23 and 15% of the cases, the latter group consisting of two of the 4 rats showing intermediate surges. There were
were without a significant effect on the pro-oestrous surge of prolactin (Table II). As indicated in Table III, ovariectomized rats
no instances of ovulation without the presence of a
given 20 mg LY 87130/kg at 13.00 and 16.00 h show a
surge at either 18.00 or 24.00 h. The m e a n increment for the full surge at 24.00 (45.54 + 6.04 ng/ml
suppression (P < 0.02) of the m e a n LH level to 36%
S.E.M., n = 9) was significantly lower (P < 0.001) than that observed at 18.00 h (96.70 _+ 4.28 ng/ml S.E.M., n = 10); the lowest increment (27.0 ng/ml) was, however, associated with a full c o m p l e m e n t of ova.
The occurrence of the pro-oestrous FSH surge was identical to that of the L H surge. W h e n e v e r a surge of LH occurred at either 18.00 or 24.00 h, the serum concentration of FSH exceeded that found in the previous blood sample by at least 129.4 ng/ml; in the absence of a surge of L H the increment in FSH levels was less than 22.3 ng/ml. In the case of the rats that produced an intermediary increment in L H at 24.00 h
of that observed before the treatment; no L H pulses were identified during the period monitored after the administration of the drug (Fig. 1). The ovariectomized rats given the vehicle instead of LY 87130 showed no significant changes either in the m e a n L H level or in the frequency or amplitude of the pulses (Table III, Fig. 1). There was insufficient serum to measure FSH and prolactin in all samples from the ovariectomized rats. In the case of FSH, at least 5 of the pre-treatment samples and 4 of the subsequent samples were assayed for each animal; for prolactin, the respective n u m b e r s were 9 and 11. While treatment with the vehicle failed to affect the m e a n FSH or prolactin level, the injections of LY 87130 resulted
TABLE lI Serum prolactin concentrations (mean + S.E.M.) in pro-oestrous rats given the vehicle or L Y 87130 at times and doses indicated in Table I Pro-oestrus 13. O0 h
18. O0 h
24. O0 h
Vehicle (Pooled data
(n = 11)
86.9 ± 9.12
326.1 ± 43.30
170.3 ± 37.62
LY 87130 (Pooled data
(n = 23)
77.4 ± 6.06
264.3 ± 32.45
173.8 _+28.15
TABLE III Mean (+_ S.E.M.) frequency and amplitude of L H pulses and mean (+ S.E.M.) serum concentrations of LH, FSH and prolactin in ovariectomized rats
Blood samples were taken every 6 min between 12.00 and 13.00 h and between 17.00 and 18.30 h. The animals were given the vehicle (n = 5) or 20 mg LY 87130/kg (n = 5) at 13.00 and 16.00 h. Before
LH pulse frequency (pulses/h) LH pulse amplitude (ng/ml) LH mean level (ng/ml) FSH mean level (ng/ml) Prolactin mean level (ng/ml)
Vehicle LY 87130 Vehicle LY 87130 Vehicle LY 87130 Vehicle LY 87130 Vehicle LY 87130
*P <0.05, **P <0.02, ***P <0.01 -vs pre-treatment level.
2.4 + 0.19 2.6 _+0.19 24.32 _+2.96 25.21 + 2.05 41.88 + 3.82 40.60 _+6.57 1602.8 _+229.4 1998.4 _+277.1 32.98 ± 2.79 33.26 + 1.40
After
2.32 _+0.11 21.47 + 2.72 43.39 _+4.93 14.51 + 0.48** 1464.0 _+ 198.1 1048.8 + 71.31" 36.58 _+4.11 68.09 _+6.02***
386 60
~
VEHICLEI l ~
~0 2O 60
VEHICLE
20
020 07LY870,kg :l= 20 mg LY87130/kg
z
20
60 ~0 20
~
v
~
Time (h)
Fig. 1. Serum LH concentrations in 5 representative ovariectomized rats. Blood samples were taken every 6 min between 12.00 and 13.00 h and between 17.00 and 18.30 h. The animals were given the vehicle or 20 mg LY 87130/kg at 13.00 and 16.00 h. in a suppression (P < 0.05) of the mean FSH level to 52% of that found previously and an elevation (P < 0.01) in the mean concentration of prolactin to 205% of the earlier level. LY 87130 is a potent inhibitor of brain PNMT 12, the enzyme which catalyses the conversion of noradrenaline to adrenaline. In a manner dependent upon the dose and number of treatments, LY 87130 either leads to an occurrence of the surge at 24.00 instead of 18.00 h or blocks the LH surge at both times and prevents ovulation (Table I); the incidence and timing of the FSH surge are affected in an identical fashion. The treatments with LY 87130 do not disturb the occurrence of the prolactin surge (Table II). We have previously found3,6 comparable effects on the incidence and timing of the LH surge after treatment with either SKF 64139 (another PNMT inhibitor), or diethyldithiocarbamate (an inhibitor of dopamine fl-hydroxylase (DBH) which depletes hypothalamic noradrenaline in addition to adrenaline) or clonidine (a drug which inter alia suppresses the turnover rate of hypothalamic adrenaline). Treatment with a-methyl-p-tyrosine (AMPT), an inhibitor of tyrosine hydroxylase which leads to a loss of hypotha-
lamic dopamine and noradrenaline without, under these conditions, a significant depletion of adrenaline, fails to affect the surge 3. Both LY 87130 and SKF 64139 have a2-adrenergic receptor blocking properties20; nevertheless, the possibility that such actions might be significant in the effect of these drugs on LH release seems unlikely since piperoxane, an a2-receptor antagonist, is without effect on the preovulatory surge3. The detailed characteristics of the 'midnight' surge and the possibility that it reflects a recovery of adrenaline-mediated transmission, after relatively low doses of drugs which compromise that function, remain to be investigated. The presence of a similarly timed second surge of LH-releasing hormone (LHRH) in hypophysial portal blood, which, in the event of a normal LH surge at 18.00 h, fails to produce a discharge of L H 19, may be of significance in the production of the 'midnight' surge. A pulsatile pattern in the release of LH is apparent in sequential blood samples taken from cannulated rats. The amplitude of the pulses is increased in the absence of oestrogen and attenuated during the oestrous cycle except at the time of the LH surge when the frequency and amplitude of the pulses are enhanced 13,14. The pulsatile release of LH in ovariectomized rats is blocked by treatment with LY 87130 at a dose which also prevents the pro-oestrous surge and ovulation (Tables I and II, Fig. 1). A comparable inhibition of LH pulses has also been observed after DBH inhibitionlL 15. Treatment with AMPT does not, however, affect the pulsesljijs; this result, like the failure of AMPT to affect the surge, may be related to the lack of effect on the concentration of hypothalamic adrenaline3. A previous report 10 indicating a lack of effect on LH pulses following treatment with the PNMT inhibitor, SKF 64139, is not incompatible with the present findings, since the dose of SKF 64139 used in that study is also insufficient to block the pro-oestrous LH surge 3. At present, no explanation can be offered for the capacity of LY 87130 to stimulate mean prolactin levels in ovariectomized rats without affecting the pro-oestrous surge of prolactin (Tables II and IlI). In conclusion, while recognizing that the interpretation of the effects of pharmacological manipulation requires extreme caution, we propose as a working hypothesis: (1) that the pulsatile release of LH is a
387
n e c e s s a r y c o n d i t i o n f o r t h e p r e o v u l a t o r y s u r g e ; (2)
t a i n i n g t e r m i n a l s in t h e p r e o p t i c a r e a a n d t h e r e l a -
t h a t t h e p u l s e s are d e p e n d e n t u p o n a d r e n a l i n e - c o n -
t i o n s h i p of t h o s e t e r m i n a l s to t h e L H R H - c o n t a i n i n g
t a i n i n g n e u r o n s ; a n d (3) t h a t t h e i n c r e a s e in t h e t u r n -
n e u r o n s in t h a t r e g i o n is c u r r e n t l y u n d e r i n v e s t i g a -
o v e r r a t e of p r e o p t i c a r e a a d r e n a l i n e at t h e t i m e o f
tion.
t h e surge8 m a y b e a s s o c i a t e d w i t h t h e i n c r e a s e in t h e f r e q u e n c y a n d a m p l i t u d e of t h e p u l s e s u n d e r l y i n g t h e
W e are g r a t e f u l to t h e R o y a l S o c i e t y a n d t h e H u n -
s u r g e 13. R e c e n t s t u d i e s h a v e i n d i c a t e d t h a t P N M T is
garian
p r e s e n t in cell b o d i e s n o t o n l y in t h e m e d u l l a b u t also
F e l l o w s h i p to C . W . C .
Academy
of
Sciences
for
a
Travelling
in t h e hypothalamus2,17; t h e o r i g i n o f t h e P N M T - c o n 1 Carr, L. A., Conway, P. M. and Voogt, J. L., Inhibition of brain catecholamine synthesis and release of prolactin and luteinizing hormone in the ovariectomized rat, J. Pharmacol. Exp. Ther., 192 (1975) 15-21. 2 Coen, C. W., Immunohistochemical localization of phenylethanolamine N-methyltransferase in the rat brain, Physiologist, 27 (1984) 223. 3 Coen, C. W. and Coombs, M. C., 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, 10 (1983) 187 -206. 4 Coen, C. W., Coombs, M. C., Wilson, P. M. J., Clement, E. M. and MacKinnon, P. C. B., Possible resolution of a paradox concerning the use of p-chlorophenylalanine and 5-hydroxytryptophan: evidence for a mode of action involving adrenaline in manipulating the surge of luteinizing hormone in rats, Neuroscience, 8 (1983) 583-591. 5 Coen, C. W. and MacKinnom P. C. B., Serotonin involvement in the control of phasic luteinizing hormone release in the rat: evidence for a critical period, J. Endocrinol., 82 (1979) 105-113. 6 Coem C. W. and MacKinnon, P. C. B., The effects of blocking catecholamine synthesis on the incidence of the LH surge and ovulation in the rat, Neurosci. Letts. Suppl., 7 (1981) 252. 7 Coen, C. W., Simonyi, A. and Fekete, M. I. K., Evidence for a necessary involvement of adrenaline in both the preovulatory surge and the pulsatile release of luteinizing hormone in rats, Seventh International Congress of Endocrino-
logy, Excerpta Medica International Congress Series, Vol. 652, Excerpta Medica, Amsterdam, 1984, p. 624. 8 Coombs, M. C. and Coen, C. W., Adrenaline turnover rates in the medial preoptic area and mediobasal hypothalamus in relation to the release of luteinizing hormone in female rats, Neuroscience, 10 (1983) 207-210. 9 Crowley, W. R. and Terry, L. C., Effects of an epinephrine synthesis inhibitor, SKF 64139, on the secretion of luteinizing hormone in ovariectomized female rats, Brain Research, 204 (1981) 231-235. 10 Crowley, W. R., Terry, L. C. and Johnson, M. D., Evidence for the involvement of central epinephrine systems in the regulation of luteinizing hormone, prolactin, and growth hormone release in female rats, Endocrinology, 110
(1982) 1102-1107. 11 Drouva, S. V. and Gallo, R. V., Catecholamine involvement in episodic luteinizing hormone release in adult ovariectomized rats, Endocrinology, 99 (1976) 651-658. 12 Fuller, R. W., Molloy, B. B., Hemrick, S. K. and Perry, K. W., Inhibition of rat brain norepinephrine n-methyltransferase by 2,3,4,5-tetrahydro-lH-indero (1,2-c)-pyridine hydrochloride (LY 87130), Brain Research, 190 (1980) 215-223. 13 Gallo, R. V., Pulsatile LH release during the ovulatory LH surge on proestrus in the rat, Biol. Reprod.. 24 (1981) 100-104. 14 Gallo, R. V., Pulsatile LH release during periods of low level LH secretion in the rat estrous cycle, Biol. Reprod., 24 (1981) 771-777. 15 Gnodde, H. P. and Schuiling, G. A., Involvement of catecholaminergic and cholinergic mechanisms in the pulsatile release of LH in the long term ovariectomized rat, Neuroendocrinology, 20 (1976) 212-223. 16 Markee, J. E., Sawyer, C. H. and Hollinshead, W. H., An adrenergic link in the ovulatory mechanism of the rabbit, Anat. Rec., 97 (1947) 398. 17 Ross, C. A., Ruggiero, D. A., Meeley, M. P., Park, D. H., Joh, T. H. and Reis, D. J., A new group of neurons in hypothalamus containing phenylethanolamine N-methyltransferase (PNMT) but not tyrosine hydroxylase, Brain Research, 306 (1984) 349-353. 18 Rubinstein, L. and Sawyer, C. H., Role of catecholamines in stimulating the release of pituitary ovulatory hormone(s) in rats, Endocrinology, 86 (1970) 988-995. 19 Sarkar, D. K., Chiappa, S. A., Fink, G. and Sherwood, N. M., Gonadotropin-releasing hormone surge in pro-oestrous rats, Nature (London), 264 (1976) 461-463. 20 Toomey, R. E., Horng, J. S., Hemrick-Euecke, S. K. and Fuller, R. W., a_,-Adrenoreceptor affinity of some inhibitors of norepinephrine N-methyltransferase, Life Sci., 29 (1981) 2467-2472. 21 Vijayan, E. and McCann, S. M., Re-evaluation of the role of catecholamines in control of gonadotropin and prolactin release, Neuroendocrinology, 25 (1978) 150-165. 22 Weick, R. F., Acute effects of adrenergic receptor blocking drugs and neuroleptic agents on pulsatile discharges of luteinizing hormone in the ovariectomized rat, Neuroendocrinology, 26 (1978) 108-117.
383
BRE 21065
Effect of phenylethanolamine N-methyltransferase inhibition on serum concentrations of luteinizing hormone, follicle stimulating hormone and prolactin in pro-oestrous and ovariectomized rats CLIVE W. COEN 1, AGNES SIMONYI 2 and MARTON I. K. FEKETE 2
IDepartment of Anatomy and Human Biology, King's College, London University, London (U. K. ) and *-Institute of Experimental Medicine, Budapest (Hungary) (Accepted May 7th, 1985)
Key words: phenylethanolamine N-methyltransferase - - adrenaline - - luteinizing hormone - - follicle stimulating hormone - - prolactin - - ovulation
In a dose dependent manner, LY 87130, an inhibitor of adrenaline synthesis in the rat brain, either shifts the pro-oestrous surges of luteinizing hormone and follicle stimulating hormone from the late afternoon to the middle of the night or blocks any occurrence of the surges and prevents ovulation. Because the pulsatile release of LH is also blocked by LY 87130, it is proposed that the role of adrenaline in the production of the preovulatory surge is concerned with the regulation of the LH pulses which constitute the surge. A n involvement of adrenaline in the control of reflex ovulation in rabbits was first p r o p o s e d in 1947 (ref.16). It was subsequently r e p o r t e d that intraventricular adrenaline can restore ovulation in rats when the otherwise spontaneous p r e o v u l a t o r y surge of luteinizing h o r m o n e ( L H ) is blocked by barbiturate treatment~S; furthermore, intraventricular adrenaline was found to be m o r e p o t e n t than noradrenaline in restoring b a r b i t u r a t e - b l o c k e d ovulationlS and in stimulating L H release in steroid-primed ovariectomized rats 21. Nevertheless, evidence indicating a necessary involvement of adrenaline in the production of the spontaneous L H surge in rats was not available until 1981, when it was discovered that treatment with S K F 64139, an inhibitor of phenylethanolamine N-methyltransferase ( P N M T ) , the enzyme which catalyses the conversion of n o r a d r e n a line to adrenaline, blocks the pro-oestrous surge and subsequent ovulation 6 and also the surge induced by progesterone t r e a t m e n t following ovariectomy and a priming dose of oestrogen 9. In order further to elucidate the role of adrenaline in the control of spontaneous g o n a d o t r o p h i n secretion, we have examined the effect of LY 87130, an-
other P N M T inhibitor 12, on: (1) the pro-oestrous surges of L H , follicle stimulating h o r m o n e ( F S H ) and prolactin; (2) the incidence of ovulation; and (3) the pulsatile release of L H , a process most readily m o n i t o r e d after ovariectomy. The results have been previously p r e s e n t e d in abstract form 7. F e m a l e Wistar rats (150-200 g) were m a i n t a i n e d under regular lighting conditions (lights: 06.00-20.00 h). A t 13.00 h and, in some cases, at subsequent times on the day of pro-oestrus, animals received an intraperitoneal injection of the vehicle (2.0 ml water/kg) or LY 87130. T h e drug was administered at 20 or 40 mg/kg, doses previously r e p o r t e d to produce a m a r k e d depletion of central adrenaline12; the nadir, which represents a loss of 65 or 86% of the control value for the two respective doses, occurs about 6 h after t r e a t m e n t and is not a c c o m p a n i e d by a significant effect on the concentration of dopamine or noradrenaline 12. B l o o d samples (400 ktl) were taken under light ether anaesthesia ( < 1.5 min) immediately before the injection at 13.00 and again at 18.00 h, the a p p r o x i m a t e times of the p e a k of the LH surge. Because our recent studies on the effects of P N M T inhibition on the L H surge 3,6 indicate that a
Correspondence: C. W, Coen, Department of Anatomy and Human Biology, King's College, London University, Strand, London WC2R 2LS, U.K.
384 relatively low dose allows full ovulation to take place following a surge at 'midnight' instead of 18.00 h, an
tion). Statistical significance was evaluated by Student's t-test
additional blood sample was taken at 24.00 h. Oviducts were examined for ova between 14.00 and
Of the animals treated with the vehicle on the day of pro-oestrus, 91% produced a surge of LH at
17.00 h on the day after the presumptive L H surge. The rats used following ovariectomy were fitted with an indwelling cannula in the right atrium5 2 weeks after the former operation. O n the next day, blood samples (150 #1) were taken and replaced with 0.9% saline every 6 rain between 12.00 and 13.00 h.
18.00 h; such an occurrence was never observed in the animals given LY 87130 (Table I). Some of the animals in the latter group did, however, produce a surge at 24.00 h (Table I). The incidence of the 24.00 h surge was related to the dose and n u m b e r of treatments in which the LY 87130 was administered: thus,
The animals were given the vehicle or 20 mg LY 87130/kg immediately after the first period of sampling and again at 16.00 h. Further samples were taken every 6 rain between 17.00 and 18.30 h.
a single injection of 20 mg LY 87130/kg at 13.00 h resulted in an invariable occurrence of the surge at 24.00 h followed by either full or reduced ovulation, but the same dose given at 13.00, 16.00 and 19.'00 h led to a suppression of the surge at each of the possible times and a complete failure of ovulation (Table
Serum concentrations of LH, FSH and prolactin were assayed in duplicate in a double-antibody radioimmunoassay using ovine L H (NIH S13), rat FSH
I).
( N I A M D D RP-1) and rat prolactin ( N I A M D D RP-1) as the reference preparations. The results for each h o r m o n e in this study were obtained in a single
The 34 rats showed an increment in the levels of LH between 13.00 and 18.00 h on pro-oestrus which was either greater than 80.0 ng/ml (registered as a
assay; they were accepted only when the coefficient of variation of the duplicate was less than 5%. Pulsatile release of L H was analyzed according to the criteria of Gallo 14. The coefficient of variation of the concentrations of L H comprising the ascending and descending phase of each putative pulse was determined; a pulse was identified when this index was at
surge at 18.00 h; n = 10) or less than 4.9 ng/ml (n = 24) (Table I). In the latter case, the subsequent increment at 24.00 h was either greater than 26.9 ng/ml (registered as a surge at 24.00 h: n = 9) or less than 2.5 ng/ml (registered as no surge: n = 11); there were 4 exceptions with an increment of 7.0-11.0 ng/ml which, for the purpose of this study, have been
least 15% (1.5 x the intra-assay coefficient of varia-
classified as surges at 24.00 h (Table I). The 18.00 h
TABLE I Incidence o f ovulation (with mean +_ S.D. no. o f ova) and o f the surge in the serum concentration o f L H at either 18. O0 or 24. O0 h on the day o f pro-oestrus in rats given the vehicle or L Y 87130 at various times on that day Treatment
Vehicle Vehicle Vehicle 20 mg LY 87130/kg 40 mg LY 87130/kg 20 mg LY 87130/kg 20 mg LY 87130/kg
Time (h)
No.
13.00 4 13.00 and 16.00 4 13.00, 16.00 and 19.00 3 13.00 5 13.00 6 13.00and 16.00 7 13.00,16.00 and 19.00 5
Full owdation*
Reduced ovulation
No ovulation
LH surge-18. O0 h
LH surge- 24.00 h**
LH surge-24. O0 h
LH surge24. O0 h
No surge
%
No. o f ova
%
No. o f ova
%
No. o f ova
%
¢/c
100 75 100
8.7+0.9 9.3+1.1 8.7+0.6
25
14 40 17
4.5±2.1 2 29***
33 57 100
60 9.0± 1.0 50*** 10.7±2.9 14"** 14
* 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 > 80.0 ng/ml (registered as 'LH surge-18.00 h') or < 4.9 ng/ml. In the latter case the subsequent increment at 24.00 h was > 26.9 ng/ml (registered as 'LH surge-24.00 h ~) or < 2.5 ng/ml (registered as 'No surge') with 4 exceptions in the groups marked *** in which the increment was 7.0-11.0 ng/ml.
385 (n = 4) the increment in FSH was between 10.4 and
surge invariably resulted in full ovulation; the 'midnight' surge was followed by full ovulation, reduced
90.3 ng/ml. The various treatments with LY 87130
ovulation or no ovulation in, respectively 62, 23 and 15% of the cases, the latter group consisting of two of the 4 rats showing intermediate surges. There were
were without a significant effect on the pro-oestrous surge of prolactin (Table II). As indicated in Table III, ovariectomized rats
no instances of ovulation without the presence of a
given 20 mg LY 87130/kg at 13.00 and 16.00 h show a
surge at either 18.00 or 24.00 h. The m e a n increment for the full surge at 24.00 (45.54 + 6.04 ng/ml
suppression (P < 0.02) of the m e a n LH level to 36%
S.E.M., n = 9) was significantly lower (P < 0.001) than that observed at 18.00 h (96.70 _+ 4.28 ng/ml S.E.M., n = 10); the lowest increment (27.0 ng/ml) was, however, associated with a full c o m p l e m e n t of ova.
The occurrence of the pro-oestrous FSH surge was identical to that of the L H surge. W h e n e v e r a surge of LH occurred at either 18.00 or 24.00 h, the serum concentration of FSH exceeded that found in the previous blood sample by at least 129.4 ng/ml; in the absence of a surge of L H the increment in FSH levels was less than 22.3 ng/ml. In the case of the rats that produced an intermediary increment in L H at 24.00 h
of that observed before the treatment; no L H pulses were identified during the period monitored after the administration of the drug (Fig. 1). The ovariectomized rats given the vehicle instead of LY 87130 showed no significant changes either in the m e a n L H level or in the frequency or amplitude of the pulses (Table III, Fig. 1). There was insufficient serum to measure FSH and prolactin in all samples from the ovariectomized rats. In the case of FSH, at least 5 of the pre-treatment samples and 4 of the subsequent samples were assayed for each animal; for prolactin, the respective n u m b e r s were 9 and 11. While treatment with the vehicle failed to affect the m e a n FSH or prolactin level, the injections of LY 87130 resulted
TABLE lI Serum prolactin concentrations (mean + S.E.M.) in pro-oestrous rats given the vehicle or L Y 87130 at times and doses indicated in Table I Pro-oestrus 13. O0 h
18. O0 h
24. O0 h
Vehicle (Pooled data
(n = 11)
86.9 ± 9.12
326.1 ± 43.30
170.3 ± 37.62
LY 87130 (Pooled data
(n = 23)
77.4 ± 6.06
264.3 ± 32.45
173.8 _+28.15
TABLE III Mean (+_ S.E.M.) frequency and amplitude of L H pulses and mean (+ S.E.M.) serum concentrations of LH, FSH and prolactin in ovariectomized rats
Blood samples were taken every 6 min between 12.00 and 13.00 h and between 17.00 and 18.30 h. The animals were given the vehicle (n = 5) or 20 mg LY 87130/kg (n = 5) at 13.00 and 16.00 h. Before
LH pulse frequency (pulses/h) LH pulse amplitude (ng/ml) LH mean level (ng/ml) FSH mean level (ng/ml) Prolactin mean level (ng/ml)
Vehicle LY 87130 Vehicle LY 87130 Vehicle LY 87130 Vehicle LY 87130 Vehicle LY 87130
*P <0.05, **P <0.02, ***P <0.01 -vs pre-treatment level.
2.4 + 0.19 2.6 _+0.19 24.32 _+2.96 25.21 + 2.05 41.88 + 3.82 40.60 _+6.57 1602.8 _+229.4 1998.4 _+277.1 32.98 ± 2.79 33.26 + 1.40
After
2.32 _+0.11 21.47 + 2.72 43.39 _+4.93 14.51 + 0.48** 1464.0 _+ 198.1 1048.8 + 71.31" 36.58 _+4.11 68.09 _+6.02***
386 60
~
VEHICLEI l ~
~0 2O 60
VEHICLE
20
020 07LY870,kg :l= 20 mg LY87130/kg
z
20
60 ~0 20
~
v
~
Time (h)
Fig. 1. Serum LH concentrations in 5 representative ovariectomized rats. Blood samples were taken every 6 min between 12.00 and 13.00 h and between 17.00 and 18.30 h. The animals were given the vehicle or 20 mg LY 87130/kg at 13.00 and 16.00 h. in a suppression (P < 0.05) of the mean FSH level to 52% of that found previously and an elevation (P < 0.01) in the mean concentration of prolactin to 205% of the earlier level. LY 87130 is a potent inhibitor of brain PNMT 12, the enzyme which catalyses the conversion of noradrenaline to adrenaline. In a manner dependent upon the dose and number of treatments, LY 87130 either leads to an occurrence of the surge at 24.00 instead of 18.00 h or blocks the LH surge at both times and prevents ovulation (Table I); the incidence and timing of the FSH surge are affected in an identical fashion. The treatments with LY 87130 do not disturb the occurrence of the prolactin surge (Table II). We have previously found3,6 comparable effects on the incidence and timing of the LH surge after treatment with either SKF 64139 (another PNMT inhibitor), or diethyldithiocarbamate (an inhibitor of dopamine fl-hydroxylase (DBH) which depletes hypothalamic noradrenaline in addition to adrenaline) or clonidine (a drug which inter alia suppresses the turnover rate of hypothalamic adrenaline). Treatment with a-methyl-p-tyrosine (AMPT), an inhibitor of tyrosine hydroxylase which leads to a loss of hypotha-
lamic dopamine and noradrenaline without, under these conditions, a significant depletion of adrenaline, fails to affect the surge 3. Both LY 87130 and SKF 64139 have a2-adrenergic receptor blocking properties20; nevertheless, the possibility that such actions might be significant in the effect of these drugs on LH release seems unlikely since piperoxane, an a2-receptor antagonist, is without effect on the preovulatory surge3. The detailed characteristics of the 'midnight' surge and the possibility that it reflects a recovery of adrenaline-mediated transmission, after relatively low doses of drugs which compromise that function, remain to be investigated. The presence of a similarly timed second surge of LH-releasing hormone (LHRH) in hypophysial portal blood, which, in the event of a normal LH surge at 18.00 h, fails to produce a discharge of L H 19, may be of significance in the production of the 'midnight' surge. A pulsatile pattern in the release of LH is apparent in sequential blood samples taken from cannulated rats. The amplitude of the pulses is increased in the absence of oestrogen and attenuated during the oestrous cycle except at the time of the LH surge when the frequency and amplitude of the pulses are enhanced 13,14. The pulsatile release of LH in ovariectomized rats is blocked by treatment with LY 87130 at a dose which also prevents the pro-oestrous surge and ovulation (Tables I and II, Fig. 1). A comparable inhibition of LH pulses has also been observed after DBH inhibitionlL 15. Treatment with AMPT does not, however, affect the pulsesljijs; this result, like the failure of AMPT to affect the surge, may be related to the lack of effect on the concentration of hypothalamic adrenaline3. A previous report 10 indicating a lack of effect on LH pulses following treatment with the PNMT inhibitor, SKF 64139, is not incompatible with the present findings, since the dose of SKF 64139 used in that study is also insufficient to block the pro-oestrous LH surge 3. At present, no explanation can be offered for the capacity of LY 87130 to stimulate mean prolactin levels in ovariectomized rats without affecting the pro-oestrous surge of prolactin (Tables II and IlI). In conclusion, while recognizing that the interpretation of the effects of pharmacological manipulation requires extreme caution, we propose as a working hypothesis: (1) that the pulsatile release of LH is a
387
n e c e s s a r y c o n d i t i o n f o r t h e p r e o v u l a t o r y s u r g e ; (2)
t a i n i n g t e r m i n a l s in t h e p r e o p t i c a r e a a n d t h e r e l a -
t h a t t h e p u l s e s are d e p e n d e n t u p o n a d r e n a l i n e - c o n -
t i o n s h i p of t h o s e t e r m i n a l s to t h e L H R H - c o n t a i n i n g
t a i n i n g n e u r o n s ; a n d (3) t h a t t h e i n c r e a s e in t h e t u r n -
n e u r o n s in t h a t r e g i o n is c u r r e n t l y u n d e r i n v e s t i g a -
o v e r r a t e of p r e o p t i c a r e a a d r e n a l i n e at t h e t i m e o f
tion.
t h e surge8 m a y b e a s s o c i a t e d w i t h t h e i n c r e a s e in t h e f r e q u e n c y a n d a m p l i t u d e of t h e p u l s e s u n d e r l y i n g t h e
W e are g r a t e f u l to t h e R o y a l S o c i e t y a n d t h e H u n -
s u r g e 13. R e c e n t s t u d i e s h a v e i n d i c a t e d t h a t P N M T is
garian
p r e s e n t in cell b o d i e s n o t o n l y in t h e m e d u l l a b u t also
F e l l o w s h i p to C . W . C .
Academy
of
Sciences
for
a
Travelling
in t h e hypothalamus2,17; t h e o r i g i n o f t h e P N M T - c o n 1 Carr, L. A., Conway, P. M. and Voogt, J. L., Inhibition of brain catecholamine synthesis and release of prolactin and luteinizing hormone in the ovariectomized rat, J. Pharmacol. Exp. Ther., 192 (1975) 15-21. 2 Coen, C. W., Immunohistochemical localization of phenylethanolamine N-methyltransferase in the rat brain, Physiologist, 27 (1984) 223. 3 Coen, C. W. and Coombs, M. C., 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, 10 (1983) 187 -206. 4 Coen, C. W., Coombs, M. C., Wilson, P. M. J., Clement, E. M. and MacKinnon, P. C. B., Possible resolution of a paradox concerning the use of p-chlorophenylalanine and 5-hydroxytryptophan: evidence for a mode of action involving adrenaline in manipulating the surge of luteinizing hormone in rats, Neuroscience, 8 (1983) 583-591. 5 Coen, C. W. and MacKinnom P. C. B., Serotonin involvement in the control of phasic luteinizing hormone release in the rat: evidence for a critical period, J. Endocrinol., 82 (1979) 105-113. 6 Coem C. W. and MacKinnon, P. C. B., The effects of blocking catecholamine synthesis on the incidence of the LH surge and ovulation in the rat, Neurosci. Letts. Suppl., 7 (1981) 252. 7 Coen, C. W., Simonyi, A. and Fekete, M. I. K., Evidence for a necessary involvement of adrenaline in both the preovulatory surge and the pulsatile release of luteinizing hormone in rats, Seventh International Congress of Endocrino-
logy, Excerpta Medica International Congress Series, Vol. 652, Excerpta Medica, Amsterdam, 1984, p. 624. 8 Coombs, M. C. and Coen, C. W., Adrenaline turnover rates in the medial preoptic area and mediobasal hypothalamus in relation to the release of luteinizing hormone in female rats, Neuroscience, 10 (1983) 207-210. 9 Crowley, W. R. and Terry, L. C., Effects of an epinephrine synthesis inhibitor, SKF 64139, on the secretion of luteinizing hormone in ovariectomized female rats, Brain Research, 204 (1981) 231-235. 10 Crowley, W. R., Terry, L. C. and Johnson, M. D., Evidence for the involvement of central epinephrine systems in the regulation of luteinizing hormone, prolactin, and growth hormone release in female rats, Endocrinology, 110
(1982) 1102-1107. 11 Drouva, S. V. and Gallo, R. V., Catecholamine involvement in episodic luteinizing hormone release in adult ovariectomized rats, Endocrinology, 99 (1976) 651-658. 12 Fuller, R. W., Molloy, B. B., Hemrick, S. K. and Perry, K. W., Inhibition of rat brain norepinephrine n-methyltransferase by 2,3,4,5-tetrahydro-lH-indero (1,2-c)-pyridine hydrochloride (LY 87130), Brain Research, 190 (1980) 215-223. 13 Gallo, R. V., Pulsatile LH release during the ovulatory LH surge on proestrus in the rat, Biol. Reprod.. 24 (1981) 100-104. 14 Gallo, R. V., Pulsatile LH release during periods of low level LH secretion in the rat estrous cycle, Biol. Reprod., 24 (1981) 771-777. 15 Gnodde, H. P. and Schuiling, G. A., Involvement of catecholaminergic and cholinergic mechanisms in the pulsatile release of LH in the long term ovariectomized rat, Neuroendocrinology, 20 (1976) 212-223. 16 Markee, J. E., Sawyer, C. H. and Hollinshead, W. H., An adrenergic link in the ovulatory mechanism of the rabbit, Anat. Rec., 97 (1947) 398. 17 Ross, C. A., Ruggiero, D. A., Meeley, M. P., Park, D. H., Joh, T. H. and Reis, D. J., A new group of neurons in hypothalamus containing phenylethanolamine N-methyltransferase (PNMT) but not tyrosine hydroxylase, Brain Research, 306 (1984) 349-353. 18 Rubinstein, L. and Sawyer, C. H., Role of catecholamines in stimulating the release of pituitary ovulatory hormone(s) in rats, Endocrinology, 86 (1970) 988-995. 19 Sarkar, D. K., Chiappa, S. A., Fink, G. and Sherwood, N. M., Gonadotropin-releasing hormone surge in pro-oestrous rats, Nature (London), 264 (1976) 461-463. 20 Toomey, R. E., Horng, J. S., Hemrick-Euecke, S. K. and Fuller, R. W., a_,-Adrenoreceptor affinity of some inhibitors of norepinephrine N-methyltransferase, Life Sci., 29 (1981) 2467-2472. 21 Vijayan, E. and McCann, S. M., Re-evaluation of the role of catecholamines in control of gonadotropin and prolactin release, Neuroendocrinology, 25 (1978) 150-165. 22 Weick, R. F., Acute effects of adrenergic receptor blocking drugs and neuroleptic agents on pulsatile discharges of luteinizing hormone in the ovariectomized rat, Neuroendocrinology, 26 (1978) 108-117.