evidence that long-term estrogen treatment disrupts opioid involvement in the induction of pituitary...

BrainResearch, 583 (1992) 183-188© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

BRES 17882

183

Evidence that long-term estrogen treatment disrupts opioid involvementin the induction of pituitary LH surge *

Miguel Fuentes **, Abhiram Sahu and Satya P. Kalra

Departmentof Obstetrics and Gynecology, University of Florida College ofMedicine, Gainesville, FL 32610(USA)

(Accepted 11 February 1992)

Key words: Estrogen; Luteinizing hormone surge; Abolition; Opioid; Naloxone

Recent evidence suggests that a decrease in the inhibitory opioid influence is a necessary hypothalamic neural event in the preovulatory andovarian steroid-induced luteinizing hormone (LH) surge. Whether shifts in ovarian steroidal milieu disrupts this neural event is not known.Therefore, the effects of short-term (3 days) and long-term (13 or 17 days) estradiol 17f3 (E z) exposure on spontaneous, naloxone (NAL) andprogesterone (P)-induced LH surges were assessed in ovariectomized (ovx)rats. Two weeks after ovariectomy, rats received subcutaneous Silasticcapsules filled with crystalline Ez. In rats exposed to Ez for 3 days and infused with saline intravenously between 11.00-14.00 h, plasma LH rosesignificantlyat 17.00 h. NAL infusion between 11.00-14.00 h in these rats to decrease the inhibitory opioid influence, advanced both the onset ofLH rise and amplified the secretion of LH in the afternoon. Continuation of Ez exposure for 13 days produced no deleterious effects on eitherthe spontaneous or NAL-induced augmentation in LH responses. However, uninterrupted Ez exposure for 17 days abolished the spontaneousafternoon LH rise and drastically diminished the ability of NAL to advance and amplify the LH response. In the next experiment, we evaluatedthe effect of P injection (2 mgyrat) at 11.00h on the afternoon LH release in rats similarly exposed to Ez for either 3 or 17 days. In rats exposedto Ez for 3 days, the P-induced LH release was significantly greater than that observed after saline or NAL infusion. In marked contrast to theabolition of spontaneous and diminution of NAL-induced LH response in rats exposed to Ez for 17 days, P injection elicited an undiminishedLH response. These findings show that the neural circuitry involved in elicitation of LH surge in ovx rats fails to operate after uninterrupted Ezexposure for longer than 13 days, but it can be readily reactivated by P and not by opiate receptor antagonism with NAL infusion. Theseobservations demonstrate that expression of the hypothalamic opioid link necessary for induction of LH surge is disrupted by prolonged Ezexposure.

INTRODUCTION

Estradiol 17f3 (Ez) and progesterone (P) are the twoimportant ovarian signals responsible for induction ofpreovulatory discharge of hypothalamic luteinizing hor­mone releasing hormone (LHRH) and pituitary LHduring the critical period on the afternoon of pro­estrus9,ZO,Z3,39. Additionally, several observations attestto the view that these steroids facilitate different neu­roendocrine events in activation of the LHRH-LH axison proestrus. Characteristically, the role of Ez is tofacilitate the occurrence of hypothalamic sequelae nec­essary for the initiation of LHRH surge by the neuralclock, and the role of P is to synchronize and enhancethe magnitude and duration of LHRH and LH

surges14,16 - 18,20,Z6,31,39,4Z. The central feedback action of

Ez and P during the estrous cycle is known to occur ina strict temporally related sequence in conjunctionwith the daily operation of the neural clock9,zo,z3.

Therefore, a change in the timing, strength and dura­tion of action of anyone of these ovarian signalsresults in loss of the LH surge and anovulatory condi­tion prevails8

,9,22 . For instance, inappropriate Ez mi­lieu is believed to be responsible for cessation of pre­ovulatory LH surge and anovulatory and persistentestrous conditions in aging rats7.9,30. In intact youngrats, either daily Ez injections or a single injection oflong-acting estradiol valerate produces a persistent es­trous syndrome3,9,3z , Similarly, continuous E z exposureto acutely ovariectomized (ovx) rats was shown to per-

* Presented at the 73rd annual meeting of the Endocrine Society, Washington, DC, June 19-22, 1991.** Present address: Department of Pediatrics, Sacred Heart Hospital, Pensacola, FL 32504, USA.Correspondence: S.P. Kalra, Department of Obstetrics and Gynecology, University of Florida College of Medicine, Box 100294 JHMHCGainesville, FL 32610, USA. Fax: (l) (904) 392-6994,

184

mit daily afternoon surges only for a period of 10 days,thereafter these rats were rendered unresponsive tothe persistent E, feedback signal, as indicated by theabsence of LHRH hypersecretionf'-". How continuousE z exposure disrupts the operation of the hypothala­mic network responsible for elicitation of LHRH hy­persecretion has not yet been elucidated.

A sequence of neuroendocrine events that partici­pates in the ovarian steroid-dependent LH surge onproestrus and in ovx rats has been identified20,z3. Oneof the earliest neural events identified in a number ofspecies including the rat, is the decrease in inhibitoryinfluence of endogenousopioid peptides (EOP) in thehypothalamus1,z,34,35. Since ovarian steroids can regu­

late the activity of EOP neurons in the hypothalamusand preoptic area10,l1,Z4,33,40, it is possible that the

antecedent restraint on the inhibitory EOP tone in ratscontinuously exposed to E z fails to manifest in a timelyfashion. If this is the case, then we reasoned that itshould be possible to reinstate the LHRH- LH surgeexperimentally by restraining the inhibitory EOP tonewith the opioid receptor antagonist, naloxone(NAL)1,Z,31. On the other hand, if refractoriness to thestimulatory Ez feedback signal develops not in theEOP system itself, but somewhere in the sequencedistal to the opioid link in the hypothalamic neuralcircuitryZO,23,36, then NAL should fail to reinstate LH

surge. In order to evaluate whether the operation ofthe EOP link is impaired by continuous E z exposure,we studied the effects of NAL infusion on induction ofLH surge in ovx rats exposed to E z for varying lengthsof time - for 3 days (short-term) or for 13 or 17 days(long-term).

MATERIALS AND METHODS

Adult female rats (Charles River, Crl: CDBrl) were maintained inair-conditioned rooms under a controlled light-dark cycle (lights on05.00-17.00 h). Rats were bilaterally ovariectomized (ovx) whileunder tribromoethanol anesthesia (2%, 1 mlj100 g body wt., i.p.),Two weeks after ovariectomy, rats received either Silastic capsules(15 mm length, 1.57 mm i.d., 3.18 mm o.d., Dow Corning) filled withcrystalline E z (Steraloids Inc., Wilton, NH), as describedpreviouslyZ7,z8. This E z treatment has been shown to evoke daily LHsurges for up to 10 days in recently ovx rats Z7,Z8. Ez-filled capsuleswere soaked overnight in saline before subcutaneous implantationunder ether anesthesia.

Experiment 1Ovx rats implanted with Ez-filled capsules were divided into

three groups. .Group 1: short-term 3 day E2 exposure. Two days after Ez implan­

tation rats received intrajugular cannulae in the afternoon whileunder ether anesthesia. The following day at 10.00 h the jugularcannula was attached to a peristaltic pump for intravenous infusionof either saline (SAL) alone, or SAL containing NAL (2 mgjO.6mljh, naloxone hydrochloride, E.I. Dupont, Wilmington, DE) for 3h (11.00-14.00 h), as described previouslyl,Z,31. The selection of 2

mgjh (approximately 8 mgjkgjh) NAL infusion rate was based onthe observation of optimal LH stimulation'v (S. Kalra unpublished).Blood samples were withdrawn from the jugular cannula beforeinfusion at 11.00 h, during infusion at 12.00, 13.00 and 14.00 handafter infusion at 15.00, 16.00 and 17.00 h.

Group 2: long-term 13 days E2 exposure. In this experiment,intrajugular cannulae were placed 12 days after E z implantation.SAL or NAL infusions and blood sampling were performed the nextday as in Group 1.

Group 3: long-term 17 days E2 exposure. In this experiment,intrajugular cannulae were placed on day 16 post-implantation ofEz-filled capsules, and NAL or SAL infusions and blood samplingwere performed the next day as in Group 1. LH in plasma sampleswas determined by radioimmunoassay (RIA).

Experiment 2In this experiment, the effect of P injection on LH release was

studied in rats exposed to short-term (3 days) and long-term (17days) Ez. The experimental design was the same as for Groups 1 and3 in Experiment 1, except that P in sesame oil (2 mgjratjO.l ml oil)or oil alone was injected s.c. at 11.00 h on day 3 or 17 and these ratswere infused with SAL between 11.00-14.00 h. Blood samples werewithdrawn at hourly intervals between 11.00-17.00 h. LH in plasmasamples was determined by RIA.

Plasma LH levels were analyzed with the help of RIA kitsprovided by Dr. A.F. Parlow and the National Hormone and Pitu­itary Agency, NIDDK. Plasma LH levels were expressed in terms ofLH-RP-2. The assay sensitivity was 5 pgjtube and intra-assay coeffi­cient of variation was 13.1. The data were analyzed by two-wayanalysis of variance, followed by Duncan's new multiple range test,using a MacIntosh Plus computer. Also, LH response was calculatedas area under the curve for the entire experimental period and afterP or oil injectiorr' '. Student's t-test was used to compare the areaunder the curve response between the two treatment groups.

RESULTS

Effect of NAL infusion on LH release in rats aftershort-term (3 days) or long-term (13 or 17 days) E2

exposureAs shown in Fig. lA, exposure of ovx rats to E z for

3 days stimulated LH hypersecretion that began at15.00 h to reach significant levels at 17.00 h (P < 0.05).The mean plasma LH levels in these rats at 11.00 hwere 0.6 ± 0.08 ngyrnl and rose to 2.1 ± 0.5 ngyml at17.00 h when the experiment was terminated. NALinfusion advanced both the onset of LH rise and ampli­fied the LH surge in the afternoon. The peak values at17.00 h (3.7 ± 0.4 ngyrnl) in NAL-infused rats werehigher than those in SAL-infused control rats at thistime (P < 0.05), and the amount of LH secretion asdetermined by area under the curve analysis was four­times higher in NAL-infused as compared to SAL-in­fused control rats (P < 0.05, Fig. 2). Furthermore, ex­tension of E z exposure from 3 to 13 days produced noimpact on either the spontaneous or NAL-induced LHresponse (Figs. lB and 2). The time-course and magni­tude of LH rise in SAL- and NAL-infusion was similarto that seen in rats exposed to Ez for 3 days (Figs. 1Aand 2).

A --0-- Saline

- Naloxone6

B16

E2 =17 days12

*E *OJ 8.s. P or oil

:r: ( 5 )...J

+4

( 5 )0900 1100 1300 1500 1700

Effect of P on LH release in rats exposed to E2 for 3 or17 days

In contrast, the ability of P to evoke LH surge wasundiminished after long-term E 2 exposure. As is evi­dent in Figs. 3 and 4, P injection was more effectivethan NAL infusion in eliciting LH release in two ways.

First, in short-term E 2-exposed rats, stimulation of LHrelease by P was greater than that produced by NAL

I Sallnfuslon TIME (hrs)

Fig. 3. Effects of E z exposure for 3 days (A) or 17 days (B) on plasmaLH after oil or progesterone (P) injection at 11.00 h. These rats wereinfused with saline between 11.00-14.00 h. * P < 0.05 vs. 11.00 h

values.

in SAL-infused rats or as determined by area underthe curve (Fig. 2), was not significantly different be­tween SAL- and NAL-infused rats. Furthermore, acomparison of NAL-induced LH response among thethree treatment groups showed that (1) plasma LH at17.00 h was at or near peak levels in rats exposed to E 2

for 3 or 13 days, but returned to low values of 11.00 hin rats exposed to E 2 for 17 days, and (2) the LHresponse during the entire period of NAL infusion wasmarkedly reduced in rats exposed to E2 for 17 days ascompared to that observed after E 2 exposure for 3 or13 days (Fig. 2).

1800

(5 )

(6 )

1800

( 5)

( 5)

(6)

(6)

Gl Salineria Naloxone

17

1600

1600

1600

TIME (hrs)

a

13

1400

1400

1400

Infusion

1200

1200

1200

E2 =13 days

B

E2 =3 days

c

4

o+-~::=;'=~~==:---:-~---.-~1000

o-l--;"---,,-~-"":;:--~-.-----.--­1000

~...J

16Gl

e:::lu 12Gl=... 8Gl"0C:::l

III 4eIII

03

~

Etil E2 =17 days.s~...J

Days of E2 treatment

Fig. 2. Plasma LH response as determined by area under the curveanalysis after saline or naloxone infusion in rats exposed to 17{3­estradiol (E z) for 3, 13 or 17 days. Similar superscripts indicate no

significant difference.

Fig. 1. Effects of Ez exposure for 3 days (A), 13 days (B) or 17 days(C) on saline- or naloxone-induced plasma LH levels in ovariec­tomized rats. * P < 0.05 vs. 11.00 h values; numbers in parentheses

indicate the number of rats.

On the other hand, extension of E 2 exposure from13 to 17 days abolished the spontaneous rise in plasmaLH in the afternoon (Fig. IC). Although NAL infusionelicited a small increase in plasma LH at 15.00 and16.00 h as compared to values at 11.00 h (Fig. LC), theLH response, as compared to that at 15.00 and 16.00 h

186

• Oil

'" ~ Progesteronet> 30>:;u

t>20-=

Ci"l::l 10

'"t>i:;

03 17

E2 treatment, days

Fig. 4. Plasma LH response as determined by area under the curveanalysis in response to progesterone injection at 11.00 h in rats

exposed to Ez for 3 or 17 days. * P < 0.05vs. oil controls.

infusion (Figs. 2 and 4). Second, despite the fact thatspontaneous LH surge failed to occur and NAL infu­sion was largely ineffective in enhancing the LH re­sponse, P injection readily provoked LH release in ratsexposed to E 2 for 17 days in amounts equivalent tothat in rats exposed to E 2 for 3 days (Fig. 4).

DISCUSSION

These studies, designed to evaluate the effects ofcontinuous E 2 exposure to ovx rats on spontaneousand NAL-induced LH hypersecretion in the afternoonreveal several new findings. First, although the E 2

implants employed in this study raise plasma E 2 levels2-3-fold higher than the range found on the morningof proestrus of cycling female rats14,18,27,28 , the LHsurge stimulated by these supraphysiological E 2 levelswas similar in timing and magnitude to that induced byphysiological E 2 levels". Furthermore, consistent withprevious observations12,13,16,17,31,42, the magnitude of LHrise by either of these two E2 regimens was far lessthan that seen normally on the afternoon ofproestrus14,16,17,21. Thus, there is a general consensusthat E2 is an inadequate ovarian feedback signal forinduction of the preovulatory-type LH surge in ovxrats.

Second, Legan and Karsclr" reported that ratsovariectomized on diestrus and then immediately ex­posed to continuous E 2 , displayed daily LH surges forup to 10 days. Our studies show that in long-term ovxrats, similar supraphysiological E 2 exposure for as longas 13 days produced no discernible alteration of LHhypersecretion in the afternoon. However, prolonga­tion of E z exposure for an additional 4 days abolished

the afternoon LH rise. Clearly then, there is a tempo­ral window of vulnerability to continuous E2 exposurebetween 13 and 17 days in long-term ovx rats. Wesuspect that during this interval either one or morethan one loci in the hypothalamic neural circuitry in-

volved in elicitation of LH hypersecretion20,23,36 , fails tooperate. Third, we observed that disappearance ofspontaneous LH rise was concurrent with a markeddiminution in the ability of NAL to amplify the magni­tude and duration of LH rise in the afternoon. Fourth,the robust LH responses seen after P injection inshort- and long-term E2-exposed rats clearly demon­strated that abolition of spontaneous LH surge andineffectiveness of NAL in long-term E 2-exposed ratswas neither due to the inability of LHRH neurons toproduce and release neurohormone nor due to unre­sponsiveness of pituitary gonadotrophs to release LHin response to endogenous LHRH surge. Then thequestion is how and where does the sustained E2 signalact to disrupt the spontaneous and NAL-inducedLHRH hypersecretion25,36.

Figure 5 depicts the principal features of the dynam­ics of the neural circuitry that includes participation ofendogenous opioid peptides (EOP) in initiation of LHsurge on proestrus and that induced by ovarian steroidsin ovx rats1,2,20,23,36. According to the available evi­dence'r', the neural clock curtails the inhibitory EOPinfluence locally in the hypothalamus and preopticarea to allow increased output of excitatory peptidergicand aminergic signals, which in turn activate LHRHsecretion to increase the release of pituitary LH. Ac­cordingly, there are several loci where an uninter­rupted E 2 feedback signal for 17 days may act toabolish the LH surge. It is possible that the clock itselfmay not operate in a timely fashion to curtail theinhibitory EOP influence. A more likely possibility isthat the progression and transduction of message fromthe neural clock may be interrupted either at the levelof the EOP link or at loci downstream along thetransmission line to the final destination, LHRH neu­rons. The observation that NAL infusion was renderedlargely ineffective in these rats suggests a deficiencyeither in EOP ligand and/or interference of ligandbinding to the postjunctional sites in the preoptic areaand basal hypothalamus4,19,2o,29. Under these deficientconditions, it is reasonable to expect that the expres­sion of antagonistic effects of NAL may not manifest.The possibility that infusion of doses of NAL higherthan that employed in this study (approximately 8mg/kg/h) may reinstate LH surge is unlikely becausethese pharmacological doses have been found to maxi­mally antagonize the central opiate receptors. More­over, Desjardin et a1.5 reported that eight weeks of E 2

exposure to intact female rats neither affected thehypothalamic f3-endorphin levels nor altered the distri­bution and density of 0, J.t and K opiate receptorbinding sites in hypothalamic regions implicated in theinduction of LH surge. On the other hand, ovx rats

NEURALCLOCK

~

INHIBITORY SIGNALS

(EOP)

.>PROG. P NAL

-, II/II,

EXCITATORYSIGNALS

~

LHRHNEURONS

~

!LH

Fig. 5. A diagrammatic representation of the principal features ofthe neural circuitry in regulation of LH surge on proestrus and thatinduced by ovarian steroids in ovariectomized rats. Several lines ofevidence suggest that a neural clock-dependent restraint on theinhibitory influence of EOP in the hypothalamus allows increasedoutput of excitatory signals (e.g, neuropeptide Y and adrenergictransmitters) which in turn accelerate the rate of LHRH secretioninto the hypophysial portal system necessary for LH surge from thepituitary gonadotrophs. The curtailment (= =» of inhibitory EOPinfluence can be reproduced by infusion of the opiate receptorantagonist, naloxone. Our results show that in rats treated with E 2for 17 days the antagonistic effects of naloxone, which normally leadto increased output of excitatory signals, were absent. On the otherhand, progesterone (PROG) injection overcame the disruptive ef­fects of long-term E 2 treatment, perhaps by reactivation of one ormore loci in the transmission line to LHRH neurons (for details,

see text).

treated with physiological or supraphysiological levelsof estrogen for 3 weeks or longer reduced hypothala­mic {3-endorphin levels", increased [3H]NAL bindingin the hypothalamus" and selectively decreased thenumber of {3-endorphin immunopositive neurons inthe arcuate nucleus". Thus, although suggestive of amarked effect on the EOP system, it is difficult to drawany definitive conclusions from these studies onwhether the tonic release of opioids or ligand bindingto opiate receptors in relevant sites in the preopticarea and basal hypothalamus is suppressed after pro­longed exposure to E 2, a possibility raised by ourstudies.

Moreover, one cannot discount the fact that 17 daysof uninterrupted E2 exposure may also independently

187

disrupt the neurosecretory function of several interven­ing E2 target neuronal systems such as neuropeptide Yand adrenergic transmitters (Fig. 5)2,20,23,24,28,37,38. How-

ever, the fact that P injection readily provoked a robustLH surge indicates that these intervening neural cir­cuits are operational if driven appropriately. This ob­servation also reveals that the facilitatory action of Pmay be more broad-based, encompassing several P-re­sponsive networks, including the EOP system itself 11,33

and those located distally in the transmission line toLHRH neurons 37.38. Thus, P may either transientlyovercome the suppressive effects of persistent E2 atthe EOP link, and thereby re-initiate the sequence ofevents leading to elicitation of LH surge, or bypass theEOP link to facilitate the increase in the rate of LHRHand LH release in the afternoon. Further studies areunderway to precisely identify the neural loci of Paction in rats treated chronically with E2.

In summary, these studies show that there is acoincidental loss of spontaneous daily afternoon in­crease in the release of pituitary LH and developmentof refractoriness to NAL to augment the LH responsein rats exposed to E2 for longer than 13 days. How­ever, the neuroendocrine circuitry responsible for LHhypersecretion remains fully responsive to P in theserats. These results imply that the expression of theEOP link in this neural circuitry is adversely affectedby long-term E2 exposure.

Acknowledgements. Supported by a grant from the National Insti­tutes of Health (HD 08634). Thanks are due to Ms. Sally McDonellfor secretarial assistance.

REFERENCES

1 Allen, L.G., Hahn, E., Caton, D. and Kalra, S.P., Evidence that adecrease in opioid tone on proestrus changes the episodic patternof luteinizing hormone (LH) secretion: implications in the pre­ovulatory LH hypersecretion, Endocrinology, 122 (1988) 1004­1013.

2 Allen, L.G. and Kalra, S.P., Evidence that a decrease in opioidtone may evoke preovulatory luteinizing hormone release in therat, Endocrinology, 118 (1986) 2375-2381.

3 Brawer, 1.R., Naftolin, E, Martin, J. and Sonneschein, C, Effectsof a single injection of estradiol valerate on the hypothalamicarcuate nucleus and on reproductive function in the female rat,Endocrinology, 103 (1978) 501-512.

4 Chen, W.-P., Witkin, I.W. and Silverman, AI., /:I-Endorphin andgonadotropin releasing hormone synaptic input to gonadotropinreleasing hormone neurosecretory cells in the male rat, 1. CompoNeurol., 286 (1989) 85-95.

5 Desjardins, G., Baudet, A and Brawer, J.R., Alterations inopioid parameters in the hypothalamus of rats with estradiol-in­duced polycystic ovarian disease, Endocrinology, 127 (1990)2969-2979.

6 Desjardins, G.c., Brawer, 1.R. and Baudet, A, Selelctive loss of/:I-endorphin neurons and compensatory increase in I.L opioidreceptors in the hypothalamus of estradiol-valerate-treated rats,(Abst.) 21st Annu. Mtg. Soc. Neurosci., (1991) Nov 10-15, NewOrleans, LA.

188

7 Everett, J.W., Spontaneous persistent estrus in a strain of albinorat , Endocrinology, 25 (1939) 127-129.

8 Everett, J.W., Further studies on the relat ionship of progesteroneto ovulation and luteinization in the pers istent estrous rat , En­docrinology, 32 (1943) 285-292.

9 Everett, J.W., Neurobiology of Reproduction in the Female Rat,Springer-Verlag, New York, 1986.

10 Ferin, M., Van Vugt, D. and Wardl aw, S., The hypothalamiccontrol of the menstrual cycle and the role of endogenous opioidpeptides, Rec. Progr. Horm. Res., 40 (1983) 441-480.

11 Fox, S.R., Harlan, RE., Shivers, B.D. and Pfaff, D.W., Chemicalcharacterization of neuroendocrine targets for progesterone infemale rat brain and pituitary, Neuroendocrinology, 51 (1990)276-283.

12 Hiemke, C. and Ghraf, R, Re-establishment of stimulatory estra­diol effects on luteinizing hormone secretion in long-termovariectomized rats, Brain Res" 294 (1984) 182-185.

13 Hiemke, c., Schmid, A., Buttner, D. and Ghraf, R., Improvedmodel for the induction of proestrous-like gonadotropin surges inthe long-term ovariectomized rats, J. Ster. Biochem., 28 (1987)357-359.

14 Kalra, P.S. and Kalra, S.P., Temporal changes in the hypothala ­mic and serum luteinizing hormone-releasing hormone (LH-RH)levels and the circulating ovarian steroids during the rat oestrouscycle, Acta Endocrinol. , 85 (1977) 449-455.

15 Kalra, P.S. and Kalra , S.P., The effect of hyperprolactinemiaproduced by transplantable pituitary MtTW15 tumor cells inmale rats on hypothalamic LHRH release in vitro: effects ofnaloxone and K+, Endocrinology , 121 (1987) 310-315.

16 Kalra, P.S., Kalra, S.P., Krulich, L., Fawcett, CP, McCann , S.M.,Involvement of norepinephrine in transmission of the stimulatoryinfluence of progesterone on gonadotropin release, Endocrinol­ogy, 90 (1972) 1168-1176.

17 Kalra , P.S. and McCann, S.M., Involvement of catecholamines infeedback mechanisms, Progr. Brain Res., 39 (1973) 185-198.

18 Kalra , S.P., Observation on facilitation of the preovulatory rise ofLH by estrogen, Endocrinology , 96 (1975) 23-28.

19 Kalra , S.P., Neural loci involved in naloxone-induced luteinizinghormone release: effects of a norepinephrine synthesis inhibitor,Endocrinology, 109 (1981) 1805-1810.

20 Kalra , S.P., Neural circuitry involved in control of LHRH secre­tion: a model for preovulatory LH release. In: L. Martini andW.F. Ganong (Eds.), Frontiers in Neuroendocrinology, Vol. 9,Raven , New York, 1986, pp. 31-75.

21 Kalra, S.P., Ajika, K., Krulich, L., Fawcett , c.r., Quijada, M. andMcCann , S.M., Effects of hypothalamic and preoptic electro­chemical stimulation on gonadotropins and prolactin release inproestrous rats , Endocrinology, 8 (1971) 1150-1158.

22 Kalra, S.P. and Kalra , P.S., Temporal interrelationships amongcirculating levels of estradiol, progesterone and LH during the ratestrous cycle: effects of exogenous progesterone, Endocrinology ,95 (1974) 1711-1718.

23 Kalra , S.P. and Kalra, P.S., Neural regulation of luteinizinghormone secretion in the rat , Endocr. Reu., 4 (1983) 311-351.

24 Kalra , S.P. and Kalra , P.S., Steroid-peptide interaction in theendocrine brain: reproduction. In: M. Motta (Ed.), Brain En­docrinology, Raven, New York, 1991, pp 177-216.

25 Leadem, C.A, Crowley, W.R., Simpkins, J.W. and Kalra, S.P.,Effects of naloxone on catecholamine and LHRH release fromthe peri fused hypothalamus of the steroid-primed rat, Neuroen­docrinology, 40 (1985) 497-500.

26 Leadem, CA. and Kalra, S.P., Stimulation with estrogen andprogesterone of luteinizing hormone (LH)-releasing hormone re­lease from perifused adult female rat hypothalami: correlationwith the LH surge , Endocrinology , 114 (1984) 51-56.

27 Legan, S., Coon, GA. and Karsch, FJ., Role of estrogen asinitiator of daily LH surges in the ovariectomized rat , Endocrinol­ogy, 96 (1975) 50-56.

28 Legan, SJ. and Karsch, F.J ., A daily signal for the LH surge inthe rat , Endocrinology , 96 (1975) 57-62.

29 Leranth, C, MacLusky, N.J., Shanabrough, M. and Naftolin , F.,Immunohistochemical evidence for synaptic connections betweenproopiomelanocortin-immunoreactive axons and LHRH neuronsby the preoptic area of the rat , Brain Res., 449 (1988) 167-176.

30 Lu, J.K.R, LaPolt, P.S., Nass, T.E., Matt , D.W. and Judd, RL.,Relation of circulating estradiol and progesterone to go­nadotropin secretion and estrous cyclicity in aging female rats ,Endocrinology, 116 (1985) 1953-1 959.

31 Masotto, C; Sahu, A, Dube, M.G. and Kalra, S.P., A decrease inopioid tone amplifies the luteinizing hormone surge in estrogen­treated ovariectomized rats : comparisons with progesterone ef­fects, Endocrinology, 126 (1990) 18-25.

32 McCarthy, G.F. and Brawer, J.W., Induction of Stein-Levan­thaI-like polycystic ovaries (PCO) in the rat : a new model forcystic ovarian disease , Anat. Rec., 228 (1990) 137-144.

33 Morrell , J. , McGinty, F. and Pfaff, D.W., A subset of (j-en ­dorphin- or dynorphin-containing neurons in the medial basalhypothalamus accumulates estradiol, Neuroendocrinology , 41(1985) 417-426.

34 Quirick, P.D., Woller, M.J., Shapiro, S.S. and Terasawa, E. , Roleof endogenous opiates in the control of positive feedback effectsof ovarian steroids on the LH surge in rhesus monkeys, (Abst)22nd Annu. Mtg. Soc. Study Reprod., Columbia, MO, 1989, p. 168.

35 Rossmanith, W.G., Mortola, J.F. and Yen, S.S.C., Role of en­dogenous opioid pep tides in the initiation of the midcycleluteinizing hormone surge in normal cycling women, J. Clin.Endocrinol. Metab., 67 (1988) 695-700.

36 Sahu, A , Crowley, W.R. and Kalra , S.P., An opioid-neuropeptideY transmission line to luteinizing hormone (LH) releasing hor­mone neurons: a role in the induction of LH surge , Endocrinol­ogy, 126 (1990) 876-883.

37 Sar, M., Sahu, A, Crowley, W.R and Kalra, S.P., Localization ofneuropeptide Y (NPY) immunoreactivity in estradiol concentrat­ing cells in the hypothalamus, Endocrinology, 127 (1990) 2752­2756.

38 Sar, M. and Stumpf, W.E., Central noradrenergic neurons con­centrate [3Hloestradiol, Nature, 289 (1981) 500-502.

39 Sarkar, D.K., Chiappa, S.A and Fink, G., Gonadotropin-releas­ing hormone surge in pro-oestrous rats, Nature, 264 (1976) 461­463.

40 Wardlaw, S.c., Thoron, L. and Frantz, A.G., Effects of sexsteroids on brain f3-endorphin, Brain Res., 245 (1982) 327-331.

41 Wilkins, M., Bhanot, R. , Wilkinson, D. and Brawer, l.R., Pro­longed estrogen treatment induces changes in opiate, benzodi­azepine and f3-adrenergic binding sites in female rat hypothala­mus, Brain Res. Bull., 11 (1983) 279-281.

42 Wise, P.M., Camp-Grossman, P. and Barraclough, C.A, Effectsof estradiol and progesterone on plasma gonadotropins, prolactinand LHRH in specific brain areas of ovariectomized rats, Biol.Reprod., 24 (1981) 820-830.