STRESS AND THE HYPOTHALAMUS-PITUITARY GONADAL AXIS IN THE CYCLIC RAT

Transcription

1 STRESS AND THE HYPOTHALAMUS-PITUITARY GONADAL AXIS IN THE CYCLIC RAT

2 Prmtr: Prf. Dr. V.M. Wiegant Hgleraar in de Neurendcrinlgie C-prmtr: Dr. J.A.M. Mattheij Universitair Hfddcent bij het departement Dierwetenschappen, leerstelgrep Fysilgie van Mens en Dier

3 titini ),r^n STRESS AND THE HYPOTHALAMUS-PITUITARY- GONADAL AXIS IN THE CYCLIC RAT Marjlijn M. Rzendaal Prefschrift ter verkrijging van de graad van dctr p gezag van de rectr magnificus, van de Landbuwuniversiteit Wageningen, Dr. C.M. Karssen, in het penbaar te verdedigen p dinsdag 25 nvember 1997 des namiddags te vier uur in de Aula

4 Omslag: Esther Leuvenink CIP-DATA KONINKLIJKE BIBLIOTHEEK, DEN HAAG Marjlijn M. Rzendaal Stress and hypthalamus-pituitary-gnadal axis in the cyclic rat Marjlijn Rzendaal Thesis University Wageningen. - With réf. - With summary in Dutch. ISBN X Subject headings: stress / hypthalamus-pituitary-gnadal axis / cyclic rats BIBLIOTHEEK LANDBOUWUNIVERSITEIT WAGENINGEN

5 /VJKi'^0 ) &3H^ Stellingen 1. Stress kan de pre-vulatire LH-piek verstren. (dit prefschrift) 2. Er is relatief weinig LH ndig m Graafse fllikels te laten vuleren. (dit prefschrift) 3. Onder stress-mstandigheden is de pre-vulatire LH-piek afhankelijk van Vaspressine. (dit prefschrift) 4. CRH, endgene Opiiden en GABA spelen geen belangrijke rl bij de invled van stress p de pre-vulatire LH-piek. (dit prefschrift) 5. Substitutie van estradil en prgestern na variëctmie maakt van een rat ng geen ged mdel vr nderzek naar de invled van stress p de pre-vulatire LH-piek. (Salisbury et al. (1980), Endcrinlgy 101: ) (Kub et al. (1983), Endcrinl. Jpn. 30: ) 6. Het ntstaan van "luteinized unruptured fllicles" kan niet verklaard wrden als verrzaakt dr een te lage LH-afgifte alleen. (Mattheij & Swarts (1995), Eur. J. Endcrinl. 132:91-96) 7. Alleen een grte bedreiging van hun welzijn zal dieren ervan weerhuden m zich vrt te planten. (Mberg G.P. (1985). In: Animal stress, The Williams & Wilkins Cmpany, Baltimre, biz 245) 8. Bij het induceren van vulatie in de vruwelijke meerval zijn fermnen betrkken. (prefschrift J.W. Resink (1988), Utrecht) 9. Mannelijke watervlien nstaan pas als het vr vruwelijke meilijk wrdt m te verleven.

6 10. De vrgeschiedenis van het prefdier is van invled p de uitkmsten van de dierpref. 11. Hersenen zijn gecmpliceerder als je denkt. 12. Omdat "verwacht rendement" en "risic" subjectieve begrippen zijn, zullen de beurskersen altijd beïnvled wrden dr sentimenten. 13. Een geaccepteerde hypthese blijft een hypthese en laat dus vele vragen nbeantwrd. 14. Wachtgeld betekent sms "wacht p je geld" De besluitvrming in de Tweede Kamer wrdt versluierd dr het gebruik van vr leken nduidelijke afkrtingen. 16. Hewel het geslacht van een ngebren baby drgaans niet bekend is wrdt ze in het spraakgebruik altijd aangeduid als "hij". Marjlijn M. Rzendaal Stress and the hypthalamus-pituitary-gnadal axis in the cyclic rat Wageningen, 20 nvember 1997

7 Cntents page Chapter 1 General intrductin 1 Chapter 2 Effect f restraint stress n the prevulatry LH prfile and vulatin in the rat 35 Chapter 3 Effect f CRH n the prevulatry LH and FSH surge in the cyclic rat: a rle fr arginine vaspressin? 51 Chapter 4 Evidence fr a rle f arginine vaspressin and nt f crtictrpin releasing hrmne in the restraintinduced inhibitin f the LH surge in the cyclic rat 69 Chapter 5 Inhibitin f the LH surge in cyclic rats by stress is nt mediated by piids 85 Chapter 6 Inhibitin f the LH surge by restraint stress in cyclic rats: studies n the rle f GABA A and GABA B receptrs 99 Chapter 7 Summary and cnclusins 113 Glssary Samenvatting Dankwrd Curriculum vitae

8 Chapter 1 General intrductin 1 Intrductin The cncept f hmestasis was first frmulated by Bernard (1). He recgnized the interactin between the external envirnment ("milieu extérieur") that surrunds the rganism and its internal envirnment ("milieu intérieur"). An rganism is cnstantly subjected t changes in the external envirnment. By maintaining the internal envirnment within fixed limits, rganisms have evlved t becme mre independent frm changes in the uter wrld. This maintenance f a relative stability f the internal envirnment was termed hmestasis by Cannn (2). Fluctuatins in the internal envirnment are tlerated but are kept within limits. When external influences alter a physilgical variable (e.g. temperature, bld pressure) s that a deviatin frm a physilgical setpint is detected, feedback mechanisms will be activated, that re-adjust the variable twards the physilgical setpint. Activatin f feedback systems in rder t reinstate hmestasis is termed "adaptatin". In a cnstantly changing external envirnment, maintenance f a relative stability f the internal envirnment (hmestasis) is necessary fr an rganism t survive. An envirnmental cnditin r factr that threatens r disturbs hmestasis is usually referred t as a stressr. Activatin f behaviural, autnmic and endcrine mechanisms, the stress respnse, aims at prtecting the rganism against (ptential) damaging cnditins. 2 Stress Stress is an ubiquitus feature f life and the cncept f stress has been widely and frequently discussed. After many decades f stress research, an exact definitin f the phenmenn stress is still nt established. Fr an animal t survive, it must maintain its vital variables within preset limits. Thus, specific, rutinely perating hmestatic mechanisms, cnsisting f behaviural and physilgical prgrammes, cntrl the vital functins and prtect hmestasis. Any cnditin that culd lead t a change in such functins beynd the capacity 1

9 f these mechanisms - and thus threatens hmestatic cntrl - triggers a general defence mechanism, the stress respnse, that includes behaviral, autnmic and neurendcrine adaptatins aiming at remval f the threat and reestablishment f hmestasis. Whether r nt a stimulus represents a significant threat t cntrl (and is a stressr), is assessed in the central nervus system (CNS). This nt nly depends n qualities f the stimulus cnditin itself (e.g. intensity, frequency, duratin), but als n characteristics f the animal such as genetic cnstitutin (e.g. species, selectin line, gender), pre- and pstnatal develpment, prir experiences and skills (e.g. histry f the individual, learning, memry), and the physilgical (nrmal r pathlgical) and psychlgical state f the animal at that particular mment (Fig. 1). CNS Stimulus Perceptin f stressr Organizatinf bilgical defence - experience - genetic cnstitutin - physilgical state - psychlgical state /\ Bilgical respnse behaviural autnmic neurendcrine STRESS "RESPONSE V Change in bilgical functin Fig. 1. Schematic representatin f the respnse t a ptential stressful event and examples f factrs that influence this respnse (adapted frm Mberg [3D The stress respnse cmes in several mdes. Active behaviur can be used t apprach and fight r t flee frm the stressr (fight-flight respnse). When an active respnse is nt pssible r available, behaviural inactivity is displayed.

10 Chapter 1 Each mde is parallelled by a different set f physilgical reactins, that basically thught t cncern mbilizatin and redistributin f energy t allw r prepare fr the behaviural respnse t be generated (4). Thus, energy is mbilized frm stres, and strage f energy is blcked. Anablic prcesses are deferred, and physilgical functins which nrmally cst energy and are nt needed t survive the stressful situatin, are suppressed (such as digestin, grwth and repair, immunity, and reprductin). Thus, the stress respnse cmes with csts fr the rganism, and during prlnged stress it can therefre becme as damaging as the stressr itself. If the stress experience lasts t lng r the stress respnse can nt be apprpriately terminated at the end f stress, stress-related disturbances r diseases can emerge. These disturbances actually cncern physilgical systems that are affected during the stress respnse. It is generally thught, that they arise as a cnsequence f bichemical/cellular changes induced by mediatrs f the stress respnse (hrmnes, neurpeptides, neurtransmitters). The neurendcrine respnse t stress has been the subject f extensive research. A majr fcus f these studies has been n the hypthalamus-pituitary-adrenal (HPA) axis. With the nset f stress, crtictrpin-releasing hrmne (CRH) is secreted frm the hypthalamus int the hypphysial prtal circulatin (see review 5). CRH then triggers the release f adrencrtictrpic hrmne (ACTH) frm the pituitary, which in turn triggers the release f gluccrticids frm the adrenal crtex (6-9). Activatin f the HPA axis blcks energy strage, helps in mbilizing energy frm strage sites, and inhibits anablic prcesses such as grwth, immunity and reprductin. Therefre, a relatinship is suggested between hrmnes f the HPA axis (which are released during stress) and thse f the hypthalamic-pituitary-gnadal (HPG) axis (which are als influenced by stress). 3 The reprductive cycle f the female rat 3.1 Ovarian fllicular cycle In the adult labratry rat, the varian cycle cntinues thrughut the year and has a length f 4 r 5 days. The stages f the estrus cycle in the rat can be determined by micrscpical rating f the cell types that appear in the vaginal smear (see review 10). Pr-estrus is characterized by a predminance f

11 nucleated epithelial cells. On estrus, the dminant cell is the nnnucleated, crnified squamus epithelial cell. During metestrus, leuccytes appear alng with a significant number f crnified squamus epithelial cells. The stage f diestrus is characterized by a predminance f small leuccytes interspersed by a few nucleated epithelial r crnified squamus epithelial cells. At birth the varies cntain apprximately cytes. The cytes have entered int the prphase f meisis I which is prgressed t the dictyate r "resting stage" 4 days after birth (11). Only a small fractin f these viable cytes will develp int mature va. The cyte is surrunded by a single layer f flattened epithelium-derived granulsa cells; the cmbined structure is called a primrdial fllicle. After transfrmatin f the fllicular epithelial cells int a single layer f cubidal cells, the cmpsite structure is referred t as a primary fllicle. During each estrus cycle a randm grup f primary fllicles develps int secndary fllicles; a fully develped mature fllicle is knwn as a graafian fllicle. After rupture f the mature fllicle and liberatin f the vum during vulatin, the cells f the fllicle are luteinized which leads t frmatin f the crpus luteum. 3.2 Hrmnal regulatin f the varian cycle (see reviews 10,12) Luteinizing hrmne and fllicle stimulating hrmne The varies perfrm tw basic functins: prductin and release f gametes and prductin and secretin f sterids. Bth functins are under cntrl f gnadtrpic hrmnes i.e. luteinizing hrmne (LH) and fllicle stimulating hrmne (FSH). Hypthalamic release f the gnadtrpin releasing hrmne (GnRH) induces the secretin f these gnadtrpins frm the anterir pituitary int the systemic circulatin. The secretin f LH and FSH during the varian cycle f the rat is maintained at lw levels n metestrus, diestrus, and the mrning f pr-estrus (Fig. 2). This basal gnadtrpin secretin is regulated by gnadal sterids (estrgen, prgesterne and teststerne) thrugh negative feedback actin n hypthalamus and pituitary levels. On pr-estrus this negative feedback actin cnverts int a psitive feedback by rising cncentratins f circulating estrgens, which are secreted by ripening fllicles, and subsequently a surge f LH and FSH is induced (Fig. 2). The LH surge induces resumptin f meisis f the cyte, triggers vulatin n the ensuing mrning, and induces

12 Chapter 1 transfrmatin f the vulated fllicle int a crpus luteum (luteinizatin). Preccius fllicle luteinizatin can lead t luteinized unruptured fllicles (LUF). Mattheij et al. (14,15) shwed that an injectin f a small amunt f LH in the mrning f pr-estrus causes LUF. The FSH surge is f influence upn the develpment f graafian fllicles f the fllwing estrus cycle. It has been knwn fr many years that the prevulatry rise f estrgen tgether with a daily signal generated by the bilgical clck are the primary determinants f GnRH secretin and the subsequent LH and FSH surge (16). It has been clearly established that the prevulatry surge f LH and FSH in intact rats ccurs at a fixed time relative t the light-dark cycle. The nset f the prestrus LH and FSH surge can be predicted frm the knwn light-dark schedule i.e. apprximately half f the light perid plus tw hurs. OESTRUS METOESTRUS DIOESTRUS PRO-OESTRUS OESTRUS Fig. 2. Schematic diagram f the hrmne prfiles f LH ( ), FSH ( ), estradil (. ) and prgesterne ( ) during the estrus cycle f the rat. Black bar represents the dark perid f the lighting cycle, (adapted frm Smith et al. [13]) Gnadtrpin-releasing hrmne GnRH prvides a humral link between the brain and the reprductive system. GnRH neurns in the rat are lcated in the diagnal band f Brca (DBB), the medial nucleus f the septum, the medial preptic area (MPOA), and the anterir hypthalamic area (AHA), and als in the retrchiasmatic area f the medial hypthalamus (see reviews 10,17). In the rat, tracer studies shwed that vir-

13 tually (>90%) all GnRH neurns prject t the median eminence (ME) irrespective f sex, age r gnadal status f the animal (18). The GnRH neurns that prject t the ME are cncentrated basally in the midline regins frm the MPOA rstrally and thrugh the retrchiasmatic area caudally. GnRH is released in a pulsatile manner frm the ME int the prtal system and then transprted t the anterir pituitary, where it stimulates release f LH and FSH. GnRH neurns are regulated by estrgens (and prgesterne) but because these GnRH neurns d nt have estrgen receptrs (19), it is believed that they are cntrlled by interneurns cntaining estrgen receptrs. At pr-estrus there is a shift frm a negative t a psitive feedback actin f estrgen n the HPG axis (see reviews 10,12,20). A steady rise f estrgens n prestrus entrains the bilgical clck with neursecretry events in peptidergic systems that participate in the inductin f the LH and FSH surge. This psitive feedback actin f estrgens requires the synergistic participatin f prgesterne (21). Increments f prgesterne facilitate the neural event leading t hypersecretin f GnRH frm the ME nerve terminals. Additinally, the increased levels f prgesterne als prevent the recurrence f a LH and FSH surge the fllwing day. It was suggested that n pr-estrus the bilgical clck perates in tw stages t induce tw distinct neursecretry events (22). Initially, the clck disrupts the inhibitry influence f hypthalamic signals (including piids) n the mrning f pr-estrus. A gradual restraint n the hypthalamic inhibitry systems may stimulate an increase in the rate f psttranslatinal prcessing leading t accumulatin f GnRH in ME nerve terminals. The evidence that preventin f this disinhibitin interrupted the accumulatin f GnRH in the medial basal hypthalamus (MBH) is in accrd with this suggestin (23). Subsequently, the clck may act again t augment neurchemical signalling fr accelerated release f GnRH int the prtal system. The peptidergic pathways that prpagate and transmit impulses fr the GnRH surge reside in the suprachiasmatic nucleus (SCN) - medial preptic nucleus (MPN) - arcuate nucleus (ARC) - MPOA - ME neural cmplex. The timely initiatin f the impulses is entrained t the phtperidic input reaching the SCN (bilgical clck). An increase in ME levels f GnRH is the mst cnsistent earliest event, which ccurs 1-2 h befre the nset f the LH surge n prestrus (24). This is bserved in adult cyclic (25-27) and prepubertal rats (28).

14 Chapter 1 The ccurrence f this ME GnRH respnse is clsely tied t the nset f a rise in prtal GnRH levels. After stimulatin f the MPOA n pr-estrus the GnRH cntent f the ME is decreased (29), and GnRH release int the prtal system is increased cmpared t ther days f the cycle (30). On pr-estrus there is an increased pulsatile release f GnRH but als the anterir pituitary is sensitized t the actins f GnRH (31-33). The rapid increase in pituitary respnsiveness t GnRH that ccurs n pr-estrus has been attributed t the acute self-priming actin f GnRH, in cmbinatin with a psitive effect f estrgen at the level f the pituitary. Indeed, expsure f the pituitary t GnRH sensitizes the pituitary t further injectins f GnRH (34). The sensitivity is als augmented by direct actins f prgesterne (35,36) and there is evidence that the neurmediatrs galanin (37), pituitary adenylate cyclase activating peptide (38) and neurpeptide Y (39,40) enhance the respnsiveness f gnadtrpes t stimulatin f GnRH. There is n evidence that the GnRH pulse frequency is increased during the initiatin f the LH surge but the increase in GnRH release appears t take the frm f pulses f increased amplitude (see review 41). Opiids may be invlved in the increase f the amplitude f GnRH release that results frm the psitive feedback actin f sterids (42). A majr difficulty when studying the GnRH neurnal system is the distributin f GnRH neurns and the high number f factrs (e.g. classical neurtransmitters, neurpeptides, sterids, etc.) that have been reprted t mdulate directly r indirectly GnRH neurns at the level f the cell bdies r at their axnal endings in the ME (Fig. 3). There is extensive literature n the ptential rle f varius neurmediatrs (neurtransmitters and neurpeptides) in the regulatin f LH release (see reviews 20,44). Neurtransmitters invlved in the regulatin f LH release are fr instance nrepinephrine, epinephrine, dpamine, sertnin, histamine, gamma aminbutyric acid, and glutamate. Als neurpeptides are invlved in the regulatin f LH secretin, i.e. neurpeptide Y, galanin, xytcin, vaspressin, vasactive intestinal plypeptide, CRH and endgenus piids. The mdulatin f GnRH release depends n cmplex interactins between several f these factrs within a hypthalamic netwrk. Depending n the sterid envirnment, these neurmediatrs may play facilitry r inhibitry rles in GnRH secretin.

15 Fig. 3. Summary f factrs which mdulate directly r indirectly GnRH neurnal activity frm either cell bdies r nerve terminals. Slid arrws, stimulatry effect; dashed arrws inhibitry effect. AVP, arginine vaspressin; BEND, ß-endrphin; CRH, crtictrpin-releasing hrmne; DA, dpamine; E, epinephrine; EOP, endgenus piid peptides; GABA, gamma aminbutyric acid; GAL, galanin; 5-HT, sertnin; NE, nrepinephrine; NPY, neurpeptide Y; OT, xytcin; VIP, vasactive intestinal peptide, (mdified after Rivest and Rivier [43]) 4 Stress and reprductin A well knwn cnsequence f stress is disruptin f fertility. In animals, ne f the earliest studies discussing the relatinship between stress and reprductin, was based n the effects f vercrwding in bth natural and captive animal ppulatins (45). Undubtedly reprductin in male and female is vulnerable t the effects f stress. A primary mechanism by which stress can disrupt reprductin is by altering the cntrl f gnadtrpin secretin (Fig. 3). In mst studies an inhibiting effect f stress n gnadtrpin secretin has been fund

16 Chapter 1 (table 1) but shrtly after applicatin f a stressr a small transient rise f basal LH release can be induced (table 1a,b: 46-49). During the estrus cycle f the female rat stressrs appear t have their greatest impact just prir t vulatin, when precise neurendcrine cntrl is mst essential fr reprductive success. Hwever, the exact mechanism thrugh which stress alters reprductive functin remains largely unknwn. Many investigatrs have studied the effects f varius stressrs n LH and t a lesser extent FSH secretin in the rat (table 1a,b). Mst f the studies have been perfrmed with male rats r with variectmized r variectmized steridprimed female rats. Only a few studies have used intact cyclic female rats t investigated the influence f stress (table 1a: 50,51) n the surge f gnadtrpins. The effect f administratin f ptential stress-related neurmediatrs in the intact cyclic rat has been studied mre detailed (table 2a,b). Stress can influence reprductive functins at the three levels f the HPG axis; at the hypthalamus t alter GnRH secretin, at the pituitary t interfere with GnRH-induced gnadtrpin release, and at the gnads t alter the stimulatry effect f gnadtrpins n sterid secretin. The pathways via which stress influences reprductive functins have been subject f intense research fr many years. Investigatin f this relatinship is cmplicated by the variety f respnses an animal can display under stress and the cmplex regulatin f the prcesses f reprductin. Selye (52) was the first wh suggested a pssible relatinship between hrmnes and neurpeptides f the HPA axis, which are liberated during stress, and thse invlved in the HPG axis. Apart frm gluccrticids, indeed, CRH, the piid ß-endrphin (ßEND) and vaspressin (AVP) may play a rle in the effect f stress n reprductive functins (table 3, and sectins belw). Additinally, administratin f CRH, AVP and ßEND can inhibit the release f gnadtrpins and vulatin (table 2a,b). Besides these hrmnes and peptides, varius ther factrs may be invlved in stress-induced inhibitin f reprductive functins. Amng these, the majr inhibitry neurtransmitter f the brain, gamma aminbutyric acid (GABA) is a likely candidate. GABA can alter the activity f the HPA and HPG axis; GABA metablism and binding and functin f its receptrs are influenced by several stressrs (see review 53 and table 3).

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22 Chapter Crtictrpin-releasing hrmne In 1955, the first cnvincing demnstratins were prvided f the existence f a factr derived frm the hypthalamus that culd elicit ACTH secretin frm the pituitary (54, 55). This factr was named crtictrpin-releasing factr (CRF) because f its ability t stimulate secretin f ACTH. In 1981, Vale et al. (7) succeeded in determining the amin acid sequence f vine CRF and since then this factr is als indicated as crtictrpin-releasing hrmne (CRH). CRH is a 41-amin acid hrmne derived frm a 196-amin acid precursr. The amin acid sequence f this hrmne has been determined in sheep, man, rat, pig, gat and cattle; in all it shws a high hmlgy in structure (56). In the hypthalamus, it is synthesized in parvcellular neurns f the paraventricular nucleus (PVN) where it is clcalized with a variety f peptides, e.g. xytcin and vaspressin (8,57-60). Paraventricular neurns prject t the ME, and t the pituitary. In additin t its hypthalamic lcalizatin, CRH-cntaining cell bdies and binding sites have been identified in many extra-hypthalamic sites including the necrtex, areas f the limbic system invlved in emtin and stress respnses, such as the amygdala, nucleus accumbens, and hippcampus and regins invlved in the regulatin f the autnmic nervus system (lcus ceruleus, nucleus f the slitary tract, lfactry bulb, cerebral crtex and the spinal crd) (5,59-62). The wide distributin f CRH and its binding sites in the CNS has implicated the hrmne in the regulatin f endcrine, autnmic and behaviural prcesses. CRH is the majr endgenus crtictrpin releasing hrmne, althugh a number f ther factrs (e.g. AVP, xytcin (OXT) and endgenus piids) participate in the regulatin f ACTH release (62-64). ACTH subsequently elicits the secretin f gluccrticids frm the adrenal crtex which exert a negative feedback n hypthalamus and pituitary sites (66,67). Activatin f the HPA axis is cnsidered t be characteristic f the stress respnse and serves t cunteract disturbances f hmestasis. In additin t its ability t activate the HPA axis, central CRH may functin as neurtransmitter in the brain. Indeed, central administratin f CRH has indicated that the hrmne has a variety f endcrine, physilgical, neurchemical and behaviural activities that are nt shared with ACTH and crticsterne. It has been recgnized that a lt f the effects f CRH resembled thse bserved during stress, suggesting that CRH may be an endgenus mediatr f such 15

23 respnses (table 4). The bservatin that intracerebrventricular (icv) injectin f CRH suppresses the activity f the HPG axis in castrated male, and in intact and variectmized female rats (table 2a: 69-72) indicates that CRH plays a rle in stress-induced inhibitin f reprductin. In the pr-estrus intact female rat, central administratin f CRH attenuates LH secretin by inhibiting GnRH release int the hypphysial prtal system (70). During stress cnditins, central administratin f the CRH antagnist a-helical CRH prevented the inhibitin f LH release induced by ftshcks in castrated male rats (73). Table 4: Respnses fllwing CRH administratin that resemble thse bserved during stress (adapted frm Dunn and Berridge (67)) Endcrine Physilgical Electrphysilgical Neurchemical Behaviural decreases LH (GnRH) secretin decreases grwth hrmne (grwth-releasing hrmne) secretin initiates the hypthalamic-pituitary-adrenal respnse increases large bwel transit and fecal excretin decreases gastric acid secretin, decreases gastric emptying and small intestinal transit activates the EEG;seizures at higher dses increases the activity f cerebral nradrenergic and dpaminergic neurns decreases sexual behaviur in males and females increases grming decreases feeding increases lcmtr activity decreases respnding in a cnflict test decreases scial interactin enhances ftshck-induced freezing The rigin f CRH neurns innervating GnRH neurns remains unclear (43). Als the mechanisms thrugh which endgenus CRH can influence GnRH release are nt fully understd. MacLusky et al. (74) demnstrated a direct anatmical cnnectin between CRH axn terminals and dendrites f GnRH- 16

24 Chapter 1 secreting neurns. Hwever, the mechanisms thrugh which CRH influences GnRH release are likely t invlve the activatin f ther pathways, such as thse dependent n vaspressin and/r endgenus piids (69,75-77). 4.2 Vaspressin In 1953, Du Vigneaud et al. islated tw hrmnes frm the pituitary, AVP (arginine vaspressin) and OXT (78,79). AVP and OXT are peptides made up f nine amin acids. The sequence f AVP differs in nly tw psitins f OXT. These differences are sufficient t markedly alter the bilgical activities, althugh AVP and OXT can act n each thers' receptrs due t chemical similarity in structure. AVP is synthesized in the supraptic nucleus (SON) and PVN. Tw distinct ppulatins f neurns supply AVP t the anterir pituitary: parvicellular neurns frm the PVN which terminate in the ME, and magncellular neurns frm the PVN and SON which terminate in the psterir pituitary (80). In additin, AVP is als distributed in extrahypthalamic structures (81-83). A well-knwn effect f AVP n the pituitary is the stimulatin f ACTH release and the ptentiatin f CRH effects. AVP mdulates the effect f CRH n ACTH secretin and appears t take part in mediating the ACTH respnse t stress (see review 84). Hwever, little attentin has been paid n a rle f AVP in stress-induced alteratins f the HPG axis. Several studies suggest that the release f CRH and AVP in the prtal system is increased during stress (85-88) but nly a few studies are fcused n a pssible rle fr AVP in cntrl f LH secretin during stress. In variectmized (OVX) rats, basal LH tended t increase fllwing ether stress. LH levels were significantly lwer, hwever, in rats pretreated with an AVP-antiserum than in rats pretreated with nrmal rabbit serum (49). In AVP deficient male Brattlebr rats, histamine stress had n inhibiting effect n LH whereas in cntrl parent strain rats (Lng Evans) basal LH was suppressed (89). In the OVX rhesus mnkey, vaspressin antagnism prevented hypglycemia-induced LH suppressin (90). In additin, administratin f high dses f AVP caused suppressin f the LH surge and vulatin in intact and OVX sterid-primed female rats (table 2a: 91-93). The actins f AVP, like thse f CRH, are prbably exerted at the level f the hypthalamus because AVP des nt affect LH secretin frm islated pituitary tissue in vitr (49). Anatmical studies indicate that CRH neurns, which als cntain AVP (94), lie in clse appsitin with ß-endrphinergic neurns 17

25 within the hypthalamus (95). Mrever, AVP, like CRH (96,97), has been shwn t stimulate the release f ßEND frm hypthalamic tissue in vitr (98) and in viv (76). Burns et al. (77) demnstrate that CRH acts via AVP t release ßEND frm hypthalamic slices f male rats in vitr, and there is evidence that endgenus piid peptides can mediate the CRH-induced inhibitin f LH secretin (69, 70, 99, 100). 4.3 Endgenus piid peptides The first endgenus piid peptides (EOP), the enkephalins, were islated frm brain tissue and sequenced in 1975 (101). At present three families f EOP are knwn. Each family is derived frm a separate precursr: ßEND is derived frm pr-pimelancrtin (POMC), enkephalins are derived frm prenkephalin and dynrphins are derived frm prdynrphin. Peptides derived frm POMC include the piid ßEND and the nn-piid hrmnes ACTH and c-melancyte stimulating hrmne (a-msh). All three families f EOP have been identified in the brain, each with a characteristic distributin within particular neural netwrks (see reviews 20,44). The multiple piid effects are mediated by multiple piid receptrs. Opiid receptrs have been subdivided int three "main" classes: p, ô and K receptrs ( ). Each f the three piid peptide precursrs generates predminantly ligands t ne f the three piid receptr types. Pr-enkephalin derived enkephalins bind preferentially t the 5-receptrs, while pr-dynrphin derived piids are all K-ligands, and POMC derived piids bind t the //-receptrs. This categrizatin was initially based n distinct behaviural syndrmes crrelated t different classes f piate drugs. EOP and their receptrs have als been lcalized in the hypthalamus suggesting that EOP may play a rle in reprductive endcrinlgy. Research in this area during the last years has revealed many aspects f the rle f EOP in regulatin f reprductin which is reviewed by several authrs, (see reviews 20,41,44). In summary, it has been demnstrated that EOP neurns tnically inhibit secretin f LH in the rat thrugh intrahypthalamic inhibitin f GnRH release, except fr a brief interval during the prestrus LH surge in the female r during the perid f a prgesterne-induced LH surge in the estrgen-primed OVX rat. Using selective p piid agnists and antagnists it was demnstrated that mainly the /y-receptr is invlved in the inhibitin f hypthalamic GnRH secretin. Administratin f the piid receptr 18

26 Chapter 1 antagnist nalxne stimulates LH release and can reverse the exgenus piid induced suppressin f LH secretin ( ). A prevailing thery hlds that the tnic EOP inhibitin f GnRH release may be imprtant in transmissin f the negative feedback f gnadal sterids. The activatin f the EOP system is, at least partly, dependent n gnadal sterids. Opiid agnists usually inhibit, and piid antagnists stimulate LH secretin, but they fail t evke an LH respnse in lng-term OVX rats; the respnse is restred after substitutin with estradil, r with the cmbinatin f estradil and prgesterne. The primary actin f EOP in the rat appears t invlve suppressin f the amplitude f GnRH release while gnadal sterids decelerate the rate f pulse generatin. Therefre, it seems that piids may play an imprtant rle in GnRH release that results frm the psitive feedback f gnadal sterids (41,42). As GnRH neurns d nt cntain sterid receptrs, the actin f sterids must be mediated by ther neurns. Opiids appear t mdulate the regulatin f GnRH directly (see reviews 41,44) at the hypthalamic level and/r by multiple interactins with ther central regulatry mechanisms. Within the hypthalamus, piids can affect the actin f dpamine, nradrenaline, adrenaline, sertnin, GABA, neurpeptide Y, CRH, excitatry amin acids, etc. Several studies have suggested a rle fr EOP in the effects f stress n reprductive parameters. Experimental evidence exists that stress-related inhibitin f LH secretin is mainly mediated by // piid receptrs. Pretreatment with nalxne r naltrexne (piid receptr antagnists) which have a high affinity fr ^-receptrs, reverses inhibitry effects n LH by varius stressrs in mainly male rats and variectmized female rats (table 1a,b: 49,51, ). In the intact female rat, it was demnstrated that ftshck stress alters reprductive functin by inhibiting LH secretin thrugh mediatin f EOP (51). In additin, as mentined abve, piids may be invlved in the mediatin f the inhibitry actins f stress-ipduced CRH and AVP release n GnRH secretin. 4.4 Gamma aminbutyric acid GABA was discvered in the nervus system in 1950 (112). GABA is synthesized in neural tissues thrugh decarbxylatin f L-glutamic acid. The reactin is catalyzed by L-glutamic acid decarbxylase (GAD). GABA is degraded in the CNS by GABA-a-xglutarate transaminase (GABA-T). First evidence f the general inhibitry rle f GABA was btained frm the bservatin that the 19

27 enzyme GAD was fund in high cncentratin in inhibitry axns f the crayfish stretch receptr system, and nt in excitatry axns (113). Later GABA was als lcalized in areas f the vertebrate nervus system with knwn inhibitry functins. Frm bichemical and electrphysilgical studies, GABA has been shwn t be widely distributed thrughut the mammalian nervus system (see review 114). Neurchemical changes affecting the GABAergic system are judged by measurements f i.e. GABA cncentratins, GABA turnver, r the activity f the enzyme GAD. There are at least tw kinds f receptr classes, GABA A (chlride channelactivating) and GABA B receptrs (guansine triphsphate cupled, als an autreceptr t cntrl the release f GABA itself frm nerve endings) (115,116). The primary effect f central GABA B receptr activatin is a diminutin in membrane K + and Ca + + cnductance. The GABA A receptr is a cmplex entity; in additin t the GABA recgnitin site it cnsists several ther binding sites: a benzdiazepine receptr, receptr sites fr cnvulsant and anticnvulsant drugs such as Picrtxin and barbiturates, and a receptr binding site fr sterids (see reviews 53,115,116). Nt all GABA receptr cmplexes cntain all the cmpnents f the receptr cmplex as described abve. GABA appears t have a significant influence n pituitary hrmne secretin, primarily thrugh direct hypthalamic effects. It has been prpsed that GABA neurns in the MPOA mediate the negative feedback actin f estrgen n GnRH (117,118). GAD-cntaining neurns were shwn t have estrgen receptrs (118) and t synapse n GnRH-cntaining neurns (119). Additinally, GABA release in the preptic hypthalamic regin decreased cincident with the estrgen-induced LH surge in OVX rats (117,120,121). Central administratin f GABA in the intact female rat suppressed the LH surge and vulatin. Administratin f the GABA A agnist musciml r the GABA B agnist baclfen in the sterid-primed female rat caused als a suppressin f the sterid-induced LH surge (table 2b: ). GABA inhibits the activity f the HPA axis, and therefre influences the stress respnse (see review 53). Many studies have been dne n the effect f a variety f stressrs n GABAergic functins in several brain regins. Fr instance, in the hypthalamus f male rats, pain reduced GABA levels whereas 5 min f restraint did nt alter GABA levels. Three hurs f immbilizatin stress r 30 sec/day f ether stress (during 20 days) induced increased hypthalamic GABA levels (table 3: ). The effect f stress n the GABA cntent in 20

28 Chapter 1 the hypthalamus seems t depend n the type f stressr applied and the stress prtcl. Varius stressrs can als induce alteratins in GABA A receptr binding and functin, especially at the benzdiazepine binding site, which have been the subject f several studies (see review 53). Hwever, little attentin has been paid t the invlvement f GABA as inhibitry neurtransmitter in the altered gnadtrpin secretin during stress. In wmen with stress-related anvulatin, treatment with alprzalam, a benzdiazepine, restred LH pulsatility, and increased mean LH pulse frequency and amplitude (132). In the latter, activatin f the GABA receptr seemed t stimulate GnRH release by inhibitin f the stress-induced CRH release. T ur knwledge n studies have been dne n the rle f GABA as a mediatr f the stress-induced inhibitin f GnRH release in the female rat. 5 Outline f this thesis Stress f physilgical r emtinal rigin can influence the release f GnRH and subsequently that f LH and FSH. In the intact female rat, the LH surge n the day f pr-estrus induces meitic resumptin f the cyte, triggers vulatin and induces luteinizatin f the fllicle. An inadequate LH surge may induce premature luteinizatin in graafian fllicles and/r subsequently prevent vulatin. Very little research has been dne with respect t the effect f stress n the pr-estrus LH and FSH surge and subsequent effects n varian functin in the intact female rat. It has been reprted that ftshck stress results in an inhibitin f the pr-estrus LH surge and vulatin (51) and that restraint (immbilizatin) stress n pr-estrus inhibits vulatin (50,111). In view f the scarce literature n the effect f stress n reprductin in the intact cyclic female rat, we first investigated in this thesis the effect f different perids f restraint stress n pre-vulatry surge prfiles f gnadtrpins in the 5-day cyclic female rat and cncmitant effects n varian histlgy (Chapter 2). Additinally, experiments were perfrmed t gain mre insight in the mechanism underlying the restraint-induced inhibitin f the surge f gnadtrpins. Therefre, the rle f several neurpeptides knwn t be released in the brain during cnditins f stress was investigated. First, the effect f central administratin f exgenus CRH in pr-estrus rats was studied (Chapter 3). lev injectins f CRH were given just befre the presumed nset f the LH surge. 21

29 Als the effect was studied f infusins with CRH which were started befre the presumed nset f the LH surge and cntinued until the beginning f the dark perid thereby cvering mst f the perid during which the surge appears. Because a rle fr AVP in mediating the effect f CRH n LH secretin is suggested, rats were pretreated with an AVP-antiserum befre administratin f CRH. Subsequently, the rle f CRH and AVP in the restraint-induced suppressin f the pr-estrus LH surge was studied (Chapter 4). Therefre, restrained rats were pretreated with the CRH antagnist, a-helical CRH r with AVP-antiserum. A rle fr endgenus piids as mediatr in the inhibitry effect f stress n basal and pr-estrus LH release has als been suggested. In Chapter 5, we studied the effect f pretreatment with the piid antagnist, nalxne and its lnger acting analg naltrexne n the inhibitry effect f restraint n the LH surge. Finally, GABA appears t be invlved as inhibitry neurtransmitter in the regulatin f GnRH release. During the applicatin f several stressrs the GABAergic system is altered. Since an interactin between the HPG axis and GABAergic mdulatin f the stress respnse have been hardly investigated, a pssible invlvement f GABA in the restraint-induced inhibitin f the LH surge was studied by pretreatment with a GABA A and GABA B antagnist (Chapter 6). References 1. Bernard C. (1878) Leçns sur les phénmènes de la vie cmmuns aux animaux et aux végétaux. J.B. Bailliere, Paris. 2. Cannn W. (1936) The wisdm f the bdy. W.W. Nrtn, New Yrk. 3. Mberg G.P. Bilgical respnse t stress: key t assessment f animal wellbeing? In: Animal stress. Mberg G.P. (ed). The Williams & Wilkins Cmpany, Baltimre, pp Saplsky R.M. (1992) Stress, the aging brain, and the mechanisms f neurn death. A Bradfrd Bk, The MITT Press, Cambridge. 5. Dunn A.J., Berridge C.W. (1990) Physilgical and behaviural respnses t crtictrpin-releasing factr administratin: is CRF a mediatr f anxiety r stress respnses? Brain Res. Rev. 15:

30 Chapter 1 6. Rivier C, Brwnstein M., Spiess J., Rivier J., Vale W. (1982) In viv crtictrpin-releasing factr-induced secretin f adrencrtictrpin, ß- endrphin and crticsterne. Endcrinlgy 110: Vale W., Spiess J., Rivier C, Rivier J. (1981) Characterizatin f a 41-residue vine hypthalamic peptide that stimulates secretin f crtictrpin and ß- endrphin. Science 213: Antni F. (1986) Hypthalamic cntrl f adrencrtictrpin secretin: advances since the discvery f 41-residue crtictrpin-releasing factr. Endcr. Rev. 7: Rivier C, Pltsky P.M. (1986) Mediatin by crtictrpin releasing factr (CRF) n adenhypphyseal hrmne secretin. Ann. Rev. Physil. 48: Freeman M.E. (1994) The neurendcrine cntrl f the varian cycle f the rat. In: The Physilgy f Reprductin, Knbil E. and Neill J.D. (eds.), Raven Press New Yrk, pp Mandle A.M. (1963) Prevulatry changes in the cyte f the adult rat. Prc. R. Sc. Lnd. [Bil.] 158: Fink G. (1988) Gnadtrpin secretin and its cntrl. In: The Physilgy f reprductin, Knbil E. and Neill J.D. (eds.). Raven Press New Yrk, pp Smith M.S., Freeman M.E., Neill J.D. (1975) The cntrl f prgesterne secretin during the estrus cycle and early pseudpregnancy in the rat: prlactin, gnadtrpin and sterid levels assciated with the recue f the crpus luteum f pseudpregnancy. Endcrinlgy 96: Mattheij J.A.M., Swarts J.J.M., Van der Heijden A.J.H., Van Helvrt A.L.B., Küsters I.C. (1993) Advancement f meitic resumptin in graafian fllicles reduces fertility in the rat. Gynecl. Obstet. Invest. 36: Mattheij J.A.M., Swarts J.J.M. (1993) Inductin f luteinized unruptured fllicles after injectin f a small amunt f LH in the rat. J. Reprd. Pert. Abstract Series 12 (abstract 63). 16. Everett J.W., Sawyer C.H., Markee J.E. (1949) A neurgenic timing factr in cntrl f the vulatry discharge f luteinizing hrmne in the cyclic rat. Endcrinlgy 44: Silverman A.J., Livine I., Witkin J.W. (1994) The gnadtrphin-releasing hrmne (GnRH) neurnal systems: immuncytchemistry and in situ hybridizatin. In: The Physilgy f Reprductin, Knbil E., Neill J. (eds.). Raven Press New Yrk, pp Witkin J.W. (1990) Access f luteinizing hrmne-releasing hrmne neurns t the vasculature in the rat. Neurscience 37:

31 19. Shivers B.D., Harlan R.E., Mrell J.I., Pfaff D.W. (1983) Absence f estradil cncentratin in cell nuclei f LHRH-immunreactive neurnes. Nature 304: Kalra S.P. (1993) Mandatry neurpeptide-sterid signalling fr the prevulatry luteinizing hrmne-releasing hrmne discharge. Endcr. Rev. 14: Freeman M.C., Dupke K.C., Crteau CM. (1976) Extinctin f estrgen-induced daily signal fr LH release in the rat: a rle fr the prestrus surge f prgesterne. Endcrinlgy 99: Kalra S.P. (1986) Neurnal circuitry invlved in the cntrl f LHRH secretin: a mdel fr prevulatry LH release. In: Frntiers in Neurendcrinlgy. Martini L., Ganng W.F. (eds). Raven Press New Yrk, 9: Kalra S.P., Simpkins J.W. (1981) Evidence fr nradrenergic mediatin f piids effects n LH secretin. Endcrinlgy 109: Kalra S.P., Kalra P.S. (1983) Neural regulatin f luteinizing hrmne secretin in the rat. Endcr. Rev. 4: Kalra S.P., Kalra P.S. (1979) Tempral changes in the hypthalamic and serum luteinizing hrmne-releasing hrmne (LH-RH) levels and the circulating varian sterids during the rat estrus cycle. Acta Endcrinl. (Cpenh.) 85: Kalra S.P., Krulich L., McCann S.M. (1973) Changes in gnadtrpin releasing factr cntent in the rat hypthalamus fllwing electrchemical stimulatin f the anterir hypthalamic area and during the estrus cycle. Neurendcrinlgy 12: Wise P.M., Ranee N., Selmanff M., Barraclugh C.A. (1981) Changes in radiimmunassayable luteinizing hrmne-releasing hrmne in discrete brain areas f the rat at varius times at prestrus, diestrus day 1 and after phénbarbital administratin. Endcrinlgy 108: Advis J.P., Krause J.U.E., McKelvy J.F. (1982) Luteinizing hrmne-releasing hrmne peptidase activities in discrete hypthalamic regins and anterir pituitary f the rat: apparent regulatin during the prepubertal perid and first estrus cycle at puberty. Endcrinlgy 110: Pkwska J., Jutisz M. (1979) Lcal changes in immunreactive gnadtrphin releasing hrmne in the rat median eminence during the estrus cycle, crrelatin with the pituitary luteinizing hrmne. Neurendcrinlgy 28: Fink G., Aiyer M.S. (1974) Gnadtrphin secretin after electrical stimulatin f the preptic area during the estrus cycle f the rat. J. Endcrinl. 62: Aiyer M.S., Fink G., Greig F. (1974) Changes in the sensitivity f the pituitary gland t luteinizing hrmne releasing factr during the estrus cycle f the rat. J. Endcrinl. 60:

32 Chapter Sarkar D.K., Fink G. (1980) Luteinizing hrmne releasing factr in pituitary stalk plasma frm lng-term variectmized rats: effects f sterids. J. Endcr. 86: Fink G., Rsie R., Thmsn E. (1991) Sterid actins n hypthalamic neurns with special reference t estrgen cntrl f luteinizing hrmne-releasing hrmne bisynthesis and release. In: Vlume transmissin in the brain: Nvel mechanisms fr neurnal transmissin. Fuxe K. and Agnati L.F. (eds.), Raven Press New Yrk, pp Aiyer M.S., Chiappa S.A., Fink G. (1974) A priming effect f luteinizing hrmne releasing factr n the anterir pituitary gland in the female rat. J. Endcrinl. 62: Aiyer M.S., Fink G. (1974) The rle f sex sterid hrmnes in mdulating the respnsiveness f the anterir pituitary gland t luteinizing hrmne releasing factr in the female rat. J. Endcrinl. 62: Attardi B., Happe H.K. (1986) Mdulatin f the estrgen-induced luteinizing hrmne surge in the rat by prgesterne r antiestrgens: effects n pituitary gnadtrpin-releasing hrmne receptrs. Endcrinlgy 119: Lpez F.J., Merchentaler I., Ching M., Witsniewski M.G., Negr-Villar A. (1991) Galanin: a hypthalamic-hypphysitrpic hrmne mdulating reprductive functins. Prc. Natl. Acad. Sei. USA 88: Culler M.D., Pashai! CS. (1991) Pituitary adenylate cyclase-activating plypeptide (PACAP) ptentiates the gnadtrphin-releasing activity f luteinizing hrmne-releasing hrmne. Endcrinlgy 129: Crwley W.R., Hassid A., Kalra S.P. (1987) Neurpeptide Y enhances the release f luteinizing hrmne (LH) induced by LH-releasing hrmne. Endcrinlgy 120: Suttn S.W., Tyama T.T., Ott S., Pltsky P.M. (1988) Evidence that neurpeptide Y (NPY) released int the hypphysial-prtal circulatin participates in priming gnadtrphs t the effects f gnadtrpin-releasing hrmne (GnRH). Endcrinlgy 123: Levine J.E., Bauer-Dantin A.C., Besecke L.M., Cnaghan L.A., Legan J., Meredith J.M., Strble F.J., Urban J.H., Vgelsng K.M., Wlfe A.M. (1991) Neurendcrine regulatin f the luteinizing hrmne-releasing hrmne pulse generatr in the rat. Rec. Prg. Hrm. Res. 47: Levine J.E., Ramirez V.D. (1980) In viv release f luteinizing hrmne-releasing hrmne estimated with push-pull cannulae frm the medibasal hypthalami f variectmized sterid primed rats. Endcrinlgy 107: