Effects of ovarian hormones on levels of luteinizing hormone in plasma and on serotonin concentrations in discrete brain nuclei

Bruin Rrs~crrc~h Bulletin, Vol. 4, pp. 571-574. Printed in the U.S.A.

Effects of Ovarian Hormones on Levels of Luteinizing Hormone in Plasma and on Serotonin Concentrations in Discrete Brain Nuclei WILLIAM

R. CROWLEY,

THOMAS

L. O’DONOHUE*,

ERIC A. MUTH””

AND DAVID

M. JACOBOWITZ

Laborcrtovy of Clinical Science, Nationul Institute of Mental Health Bethesda MD 20014 ~Dep~lrtinent of Pharmacology, Howard University School of Medicine Wushjngton DC and **Department of Pharmacology, George Washington University School qf Medicine Washington DC 20059 (Received

9 December

1978)

CROWLEY, W. R., T. L. O’DONOHUE,

E. A. MUTH AND D. M. JACOBOWITZ. Effects rfol~arian hormcmrs on in discrete brain nuclei. BRAIN RES. BULL. 4(4) 571-574, 1979.-Levels of serotonin were measured in microdissected, individual brain nuclei in ovariectomized rats after treatment with ovarian hormones. Regions sampled included nuclei in the forebrain, rostral and medial hypothalamus, and midbrain tegmentum. Estradiol benzoate decreased levels of luteinizing hormone in plasma but did not affect serotonin levels in any region. Progesterone alone elevated serotonin content in the nucleus tractus diagonalis and ventral tegmental area. The combined estrogen plus progesterone regime produced a surge in plasma luteinizing hormone and also markedly elevated serotonin in the median eminence. These results may be of significance for ovarian hormonal regulation of gonadotropin secretion and reproductive behavior. ire&

ofluteinizing

Estradiol

hormone in plasma and on serotonin concentrations

Luteinizing hormone

Progesterone

SEVERAL studies suggest an important inhibitory role for central serotonergic (S-HT) neurons in the control of luteinizing hormone (LH) release and mating behavior in rats. For example, intraventricular administration of 5-HT depresses basal LH secretion in male rats [lo] and blocks Pregnant Mare’s Seem-induced ovulation in immature female rats 1231. These inhibitory effects have been localized to the medial basal hypothalamus [6,121. A .5-HT system also appears to modulate the circadian release of LH in estrogen-treated rats [71. Serotonergic agonists suppress, while antagonists facilitate the hormonal induction of sexual receptivity in female rats 14,161, and these effects have been localized to the preoptic area and medial basal hypothal~us 15,221. However, few studies have attempted regional analyses of 5-HT after hormone treatments that have physiological significance for control of reproductive processes [4,17]. In the present study, the ovarian hormone regime described by Kalra and McCann [91 was employed in order to lower or elevate plasma LH and to correlate such changes with S-HT levels in microdissected brain nuclei. Any changes in 5-HT levefs detected by this approach could serve to identify sites of particularly active endocrine-S-HT interaction.

Copyright

D 1979 ANKHO

International

Serotonin

METHOD

Animals Bilaterally ovariectomized Sprague-Dawley albino female rats, 60 days of age, were obtained from Zivic-Miller Laboratories (Allison Park, PA). Animals were housed 5 per cage and maintained on a 12: 12 hr light/dark cycle (lights on from 0600 to 1800) and ad lib food and tap water.

Hormone Treatments Four weeks after ovariectomy, all animals received one of four hormone treatments. At 0 hr, either estradiol benzoate (EB, 5 pgirat, SC in 0.1 ml sesame oil) or oil vehicle was administered. At 48 hr, animals received either progesterone (1.5 mgirat, SC in 0.3 ml sesame oil) or oil and were killed by decapitation 6 hr later. Thus, four hormone groups were generated (n=7-1 l/group): oil vehicle oniy, EB only, progesterone only, or EB and progesterone. All hormone injections were administered between 0700-1000 hr, and animals were killed between 1300 and 1600 hr.

Inc.-0361-9230/79/~0571-#$~.90/0

TABLE MICRODISSECTION

Region

OF‘ BRAIN

(‘annula Size (mm)

4

0.50

4 z 3 2 4 4 4 3 4 2

0.50 II.75 0.30 0.75 0.5n 0.50 11.50 0.50 Cl.SO 0.75

and Palkovits [ 81

.5-HT (P4EAN

i

SEM IN PG/pG

PROTEIN)

OVARJAN HORMONE TREATMENT

Region N. Tractus Diagonalis N. Interstitialis Striae Terminalis Medial Preoptic N. Suprachiasmatic N. Anterior Hypothalamic N. Supraoptic N. Paraventricular N. Dorsomedial N. Median Eminence Ventral Tegmental Area Interpeduncular N.

2 IN MICRODISSECTED IN OVARIECTOMIZED

Estradiol Benzoate (EB)

Vehicle (V) 10.9 1.2 1.6 11.2 5.6 5.8 5.7 5.6 10.5 9.9 12.8

? i * f .i + t + i i f

A8620, 78’10 A6860 A6860 .A636(1: i>O6(l AS660 A6060, i7XO A.5660. <.UO 14620. 4380 A4620. 438(1 A 1800, I400 A 1800. I Mlf,

tTissue from 1 animals were pooled.

TABLE LEVELS OF

NUC’LEI

No. Punches per Brain

N. Tractus Diagonalis N. Interstitialis Striae Terminalis Medial Preoptic N. Suprachiasmatic N.1 Anterior Hypothalamic N. Supraoptic N. Paraventricular N. Dorsomedial N. Median Eminence Ventral Tegmental Area Interpeduncular N. *Based on Jacobowitz

1

1.1 0.5 0.7 1.5 0.4 0.4 0.4 0.4 2.0 1.2 3.1

13.5 5.9 6.6 10.8 6.4 6.2 5.9 5.5 10.2 7.7 15.0

+ 1.2 i 0.2 1. 0.s i 1.9 t 0.5 i 0.7 + 0.3 i 0.5 + 3.3 i 1.8 i 2.6

BRAIN RATS

NtIC’LEI

EB + Progesteione 14.8 6.2 6.9 9.6 6.8 6.1 6.0 5.6 24.7 11.5 9.1

+ 2.6 + 0.5 1 0.5

! 1.2 f 0.8 ’ 0.4 + 0.2 i 0.5 t 6.8+ ! 1.8 ? 2.0

AF’I’FR

Prc)gesteronc 16.0 c 1.3” ‘.l ? 0.6 5.9 i 0.7 12.8 i 0.9 7.5 1.3 h.3 0.5 6.5 2. 0,s 6.0 r 0.6 0.5 T:2.0 14.4 r 2.2;r 12.4 ?. 1.7

tEB + P vs all others, p
lMic,rodissPc,fior1 cd

Biod~crnicd A.ssci~.s

Trunk blood was collected and plasma levels of LH were determined by radioimmunoassay with NIAMDD reagents and procedures. LH-RP-I served-as the reference standard. Only those animals exhibiting a clearcut decrease in LH after EB or a surge of LH after EB plus progesterone compared to controls were used for further study. Brains were removed rapidly and frozen with dry ice. Serial 300 Frn sections were cut in the frontal plane in a cryostat at -7°C and frozen onto chilled microscope slides. Eleven brain nuclei were removed with stainless steel cannulae as described previously [ 191. Table I presents further details of the microdissection procedure. Tissue punches were expelled into 30 ~1 of pH-adjusted 0.1 N HCI and sonicated for 3 sec. A 5 ~1 aliquot was taken for protein analysis [ 131, and a 10 ~1 aliquot was assayed for 5-HT by the method of !%aavedra t’f tr/. (211.

Rt:Sl~I.IS Lltl~‘illi:illL’ Hol7tlofrc As demonstrated previously [9l. treatment of long term ovariectomized females with EB alone significantly decreased plasma LH levels (vehicle mean z SEM. 411 k 32 ngiml, EB 192 2 39 ng:ml: /7<0.05 based on one way analysis of variance plus Duncan’s Multiple Range Test). Progesterone alone had no significant effect on LH (352 t 30 r&ml), while its administration to EB-primed animals produced a marked surge in LH 6 hr later (loo0 + 266 @ml: p
Table 2 presents 5-H?‘ levels in individual brain nuclei for the four treatment groups. EB alone failed to affect 5-HT

OVARIAN

HORMONES,

LH AND BRAIN SEROTONIN

levels in any area. However, progesterone alone significantly elevated 5-HT in the nucleus tractus diagonalis and ventral tegmental area, an effect that was not seen in EBprimed animals. The combined EB plus progesterone treatment markedly elevated 5-HT levels in the median eminence Q
The present study demonstrates an interaction between ovarian hormones and 5-HT in several discrete areas of the rat brain. In the median eminence, _5-HT levels were elevated after combined estrogen plus progesterone treatment that also produced a surge in LH. Previous studies have associated the medial-basal hypothalamic region with the inhibitory action of 5-HT on gonadotropin release [6,12], and it is possible that the increase of S-HT in the median eminence observed in the present study reflects this inhibition. However, further studies of changes in 5-HT turnover or metabolites will be required in order to clarify the functional significance of this increase. Interestingly, progesterone alone, which was without significant effect on gonadotropin levels, elevated 5-HT content in the ventral tegmental area and nucleus tractus diagonalis. These increases did not occur if animals were pretreated with estrogen. The ventral tegmentum has been identi~ed as a site for avid progesterone uptake [ 141 and for progesterone indltction of sexual receptivity in estrogen-

573 primed rats [15,20] and the present results suggest a site of progesterone-5-HT interaction in mediation of mating behavior. Further work is required to clarify the significance of the progesterone-induced increase of S-HT in the nucleus tractus diagonalis. This region has been identified as a site for the stimulatory feedback effects of progesterone on gonadotropin release [ll]. Moreover, in previous work, we have demonstrated that the nucleus tractus diagonalis exhibits cyclic changes in dopamine content over the estrous cycle [ 11, changes in dopamine turnover after ovarian hormones [31, sex differences in dopamine that are apparently under the control of neonatal androgen [2) and gonadal hormoneinduced alterations in the activity of choline acetyltransferase (1XJ. Thus, this region appears to be a site for complex interactions among several neurochemical systems in response to gonadal steroids.

ACKNOWLEDGEMENTS

We thank Ms. V. Stranov for technical assistance and Ms. P. Norton for secretarial aid. Reagents for LH radioimmunoassay were generously provided by the NIAMDD Rat Pituitary Hormone Distribution Program and Dr. A. F. Parlow. W.R.C. was supported by National Research Service Award Postdoctoral Fellowship No. 5F32 MH 07131-02 from NJMH. Present address: Department of Pharmacology, University of Tennessee Center for the Health Sciences, Memphis. Tennessee. T.L.O’D. was supported by 5TO2-GMO5~ MARC from NIGMS, NIH.

REFERENCES I. Crowley, W. R., T. L. O’Donohue and D. M. Jacobowitz. Changes in catecholamine content in discrete brain nuclei during the estrous cycle of the rat. Bruin Rrs. 147: 31.5-326, 1978. 2. Crowley, W. R., T. L. O’Donohue and D. M. Jacobowitz. Sex differences in catecholamine content in discrete brain nuclei of the rat: Effects of neonatal castration or testosterone treatment. Actcr Entfo~r. 89: 20-28, 1978. 3. Crowley. W. R., T. L. O’Donohue, H. Wachslicht and D. M. Jacobowitz. Effects of estrogen and progesterone on plasma gonadotropins and on catecholamine levels and turnover in discrete brain regions of ovariectomized rats. Bruin Rcs. 154: 345-357, 1978. 4. Eve&t. B. J., K. Fuxe, T. Hiikfelt and G. Jonsson. Role of monoamines in the control by hormones of sexual receptivity in the female rat. .l. camp. ph.~~io/.Psycho/. 89: 5X-572, 1975. 5. Foreman, M. M. and R. L. Moss. Role of hypothalamic serotonergic receptors in the control of lordosis behavior in the female rat. Hormonc~s Bahm~. 10:97-106, 1978. 6. Galio, R. V. and G. P. Moberg. Serotonin mediated inhibition of episodic luteinizing hormone release during electrical stimulation of the arcuate nucleus in ovariectomized rats. Entttw-hto~y loo: 945-954, 1977. 7. Hery, M., E. La Plante and C. Kordon. Participation of serotonin in the phasic release of LH. 1. Evidence from pharmacological experiments. Endocrinology 99: 496503, 1976. 8. Jacobowitz. D. M. and M. Palkovits. Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the brain. I. Forebrain (telencephalon, diencephalon~. J. corny. (Vrtrvtt(. 157: 13-28, 1974. 9. Kalra, P. S. and S. M. McCann. Involvement of catecholamines in feedback mechanisms. frog. Brczin Rev. 39: 185-198, 1973.

IO. Kamberi, 1. A., R. S. Mica1 and J. C. Porter. Effect of anterior pituitary perfusion and intraventricular injection of catecholamines and indoleamines on LH release. Endocrinokxv 87: l-12, 1970. 1I. Kawakami, M., E. Yoshioka, M. Kondo, J. Arita and S. Vissessuvan. Data on the sites of stimulatory feedback action of gonadal steroids indispensable for luteinizing hormone release in the rat. Endocrinology 102: 791-799, 1978. 12. Kordon, C. Effects of selective experimental changes in regional hypothalamic monoamine levels on superovulation in the immature rat. NezrroetzdocrinoloR 4: 129- 138, 1969. 13. Lowrv. 0. H.. M. Rosebroueh. A. Farr and R. Randall. Protein measurement with the Folin~h~nol reagent. .I. bid. Chtw. 193: 265-275, 19.51. 14. Luttge, W. G., R. B. Chronister and N. R. Hall. Accumulation of YH-progestins in limbic, diencephalic, and mesencephalic regions of mouse brain. L$e Sci. 12: 419-424, 1973. 1.5. Luttge, W. G. and J. R. Hughes. Intracerebral implantation of progesterone: Re-examination of the brain sites responsible for facilitation of sexual receptivity in estrogen-p~med ovariectomized rats. Physiol. Befwzv. 17: 771-775, 1976. 16. Meyerson, B. J. Central nervous monoamines and hormone induced estrus behaviour in the spayed rat. Acta ph~siol. scmd. 63: Suppl. 241: l-32, 1964. 17. Munaro, N. I. The effect of ovarian steroids on hypothalamic S-hydroxytryptamine neuronal activity. Nelrvoc~n;jo~~i,z~~/~~~~ 26: 27&276, 1978. 18. Muth, E. A., W. R. Crowley, T. L. O’Donohue and D. M. Jacobowitz. The effect of gonadal steroids on the activity of choline acetyltransferase in discrete brain regions of gonadectomized rats. Sot,. Ncrrrosci. Abstr. No. II 17, p. 360, 1978.

19. Palkovits, M. Isolated removal of hypothalamic nuclei for neuroendocrinological and neurochemical studies. In: Am~omicd Nfnnteltdotrino/f~.~~. edited by W. E. Stmnpf and 1,. D. Grant. Basel: Karger, 1975. 20. Ross, J., C. Claybaugh, L. G. Clemens and R. A. Gorski. Short latency induction of estrous behavior with intracerebral gonadal hormones in ovariectomized rats. .Gr/oc~rino/o~~ 89: 32-38. 1971.

71. Saavedra, .I. M.. M. BrownsteIn and J. Axeirocl. A qxx~fi< :mtl sensitive enzymatic-isotopic microassay for serotonin in tissties. .I, Phtrr0rtic.. <~xp_Tli~~. 186: 5OP-5 1. 197.3. 22. Ward, I. L.. W. R. Crowley. F. P. TernIan and D. L. %rgulei; Monoaminergic mediation of fern& se.xt~albehavxtr-. .f. <.+ur,,n. ph~sir~l. Psvc+of. 81: 53-6 t 1 1975. 23. Zolovick, A. and A. P. Labhsetwa:. ~:v~dence t’or the theory ot dual hypothalamic control of ovul:ttictn. .Ytrt;l/.c, 215: 1% 157. 1973.