Effect of restraint stress on the preovulatory luteinizing hormone

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1 Effect f restraint stress n the prevulatry luteinizing hrmne prfile and vulatin in the rat Marjlijn M Rzendaal, Hans JM Swarts, Victr M Wiegant and Jhn AM Mattheij :; Department f Human and Animal Physilgy. Agricultural University. Wageningen, The Netherlands; Rudlf Magnus Institute, Department f Medical Pharmaclgy, University f Utrecht, The Netherlands Rzendaal MM, Swarts HJM, Wiegant VM, Mattheij JAM. Effect f restraint stress n the prevulatry luteinizing hrmne prfile and vulatin in the rat. Eur J Endcrinl 995;33:347\p=n-\53. ISSN 84\p=n-\ 4643 Plasma prfiles f luteinizing hrmne (LH) and fllicle-stimulating hrmne (FSH) were measured during restraint stress n the day f pr-estrus; these prfiles were cnsidered in relatin t vulatin rate n the next day. Rats bearing a permanent jugular vein cannula were subjected t restraint, which was started, r 2 h befre the presumed nset f the LH surge and ended just befre the beginning f the dark perid. Expsure t restraint resulted in a suppressin f the secretin f bth gnadtrphins n the day f pr-estrus. Suppressin f the LH surge was virtually cmplete (plasma LH \m=le\.2 ng/ml) in 5 ut f 32 stressed rats, and the varies f these rats cntained graafian fllicles with cytes in germinal vesicle stage. In these rats, the LH surge did nt ccur 24 h later. In the remaining 7 rats, restraint resulted in a cnsiderable suppressin f the LH surge. Of these rats, five had an vulatin rate f % and fur vulated partially. In unruptured fllicles f the latter, the cyte had nt resumed meisis and the fllicle wall was nt luteinized. In the remaining eight rats with a reduced LH surge, vulatins had nt ccurred and graafian fllicles were unaffected. The results f this study indicate that during pr-estrus restraint stress suppresses and des nt delay the release f prevulatry gnadtrphins. Partial suppressin f LH by restraint des nt result in inductin f meitic resumptin withut subsequent vulatin r in luteinized unruptured fllicles. JAM Mattheij, Department f Human and Animal Physilgy, Agricultural University, Haarweg, 679 PJ, Wageningen, The Netherlands Stress f physical r emtinal rigin may interfere with reprductive functins (, 2). Stress can influence the release f gnadtrphin-releasing hrmne (GnRH) (3) and als f luteinizing hrmne (LH) and flliclestimulating hrmne (FSH). Mst studies have been perfrmed in male r in variectmized female animals (4-6). Little research has been dne int the effect f stress n the pr-estrus LH and FSH surge in intact female rats. In adult cyclic rats, unpredictable ftshcks applied n pr-estrus caused a partial inhibi tin f the LH surge and f subsequent vulatin (7). After expsure t immbilizatin stress n the day f pr-estrus, vulatin was blcked and delayed fr day, implying als a delay f the LH surge by day (8). In the intact female rat, the LH surge n the day f pr-estrus triggers vulatin. Luteinizing hrmne als induces meitic resumptin f the cyte and can induce premature luteinizatin in graafian fllicles. In graafian fllicles, meitic resumptin and luteinizatin have been shwn t start after a dse f LH smaller than needed fr vulatin (9, ). If restraint stress partially suppresses the LH surge, it may cnceivably induce either meitic resumptin withut subsequent vula tin r luteinized unruptured fllicles (LUF). In the present study, we investigated the effect f restraint stress n prevulatry surge prfiles f gnadtrphins in rats. Different starting times f restraint n prestrus were chsen with the aim f btaining a variety f mderately t strngly reduced LH surge prfiles. In these rats, we wanted t investigate whether a partially suppressed LH surge might induce meitic resumptin and/r luteinizatin withut vulatin. T that end, the varies were cllected fr histlgy the day after restraint. Occurrence f vulatin, meitic resumptin withut subsequent vulatin and LUF were studied in relatin t the LH prfiles. Materials and methds Rats, husing and surgery The experiments were perfrmed with female Fl hybrids (6-8 mnths f age, 2-25 g bdy weight) f tw Wistar substrains (U-inbred males and R-inbred females) frm the University breeding clny. They were maintained in a cntrlled temperature envirn ment (22 ± C) with lights n frm. t 4. h. With this light regimen these rats have 5-day cycles and the nset f the gnadtrphin surge is at arund 9. h, i.e. 2 h after the middle f the light perid (). Dwnladed frm Biscientifica.cm at /5/28 4:7:33PM

2 The rats were hused individually and received standard fd pellets and tapwater ad libitum. A dim light was left n during the dark perid t facilitate bld sampling. Rats were prvided with a jugular vein cannula accrding t the methd f Steffens (), with sme mdificatins (3) under ketamine (4-6 mg/kg intraperitneal) and xylazine (2µ sc f a 2% slutin diluted with tw vlumes f saline) anaes thesia. Starting day after surgery, the rats were handled daily t minimize stress during bld sampling. Oestrus cyclicity was assessed each day by inspectin f vaginal smears and bservatin f lrdsis behaviur induced after intrductin f a male rat fr a brief perid in the cage. Rats that exhibited at least tw cnsecutive 5-day cycles fllwing surgery were used fr experimentatin. Restraint prcedure After being cnnected t a bld sampling cannula, individual rats were placed in a perspex cylinder f 4.8 cm inner diameter. One end f the cylinder was cne-shaped and prvided with perfratins t facilitate air supply t the rat. The ther end f the cylinder cntained utlet drains fr urine and an pening fr the tail f the rat. A slit (.7 cm wide) running alng the length f the cylinder enabled fixatin f a partitinwall t adjust the interir length f the cylinder t the size f the rat. The bld sampling cannula was exterirized via the slit. Experimental prtcl The experiments were cnducted after apprval by the University Cmmittee n Animal Care and Use (DEC). Rats (tw t five rats at a time) were transferred t a nvel experimental rm and immediately placed in a restraint cylinder. The restraint was started at, r 2 h befre the nset f the LH surge, which was 9. h. The rats anticipated t begin at apprximately were released and returned t their hme cage shrtly befre the nset f the dark perid at 4. h. After a recvery perid f 2h, estrus behaviur (lrdsis) was assessed by allwing a male int the cage. Bld samples were taken hurly frm 9. h up t 7. h and als n the next day at.,. and 3. h. After the 3. h sample the rats were anaesthetized by ether and killed by cervical dislcatin. Ovaries were cllected fr histlgical examinatin. Cntrls were cannulated rats in pr-estrus, f which bld samples were taken hurly in their hme cage frm 9. t 7. h and they were killed the next day at 3.h. Radiimmunassay Bld samples (5µ) were cllected in ice-cled heparinized tubes. After centrifugatin, plasma samples (6 µ ) were diluted with three vlumes f phsphatebuffered saline (ph 7.5) cntaining.% bvine serum albumin (Sigma RIA-grade) and stred at 2 C. Levels f LH and FSH were measured using kits prvided by the NIDDK. Rat LH-RP-2 and rfsh-rp-2 diluted in serum f hypphysectmized rats were used as reference materi als. The secnd antibdy was dnkey anti-rabbit (Saceel, Wellcme Reagents, Beckenham, UK). Determina tins were perfrmed in triplicate. Quality cntrl sera with lw, medium and high LH r FSH cncentratins were included in each RIA. Assay sensitivity at 9% B/ B was.5 ng/tube fr LH and.4 ng/tube fr FSH. In the diluted plasma samples, LH cncentratins lwer than.2 ng/ml and FSH cncentratins lwer than.6 ng/ml were cnsidered as baseline levels. The intraassay cefficients f variatin fr LH and FSH were 4.3% and 5.7%, respectively. Histlgy Ovaries were fixed in Buin's slutin and embedded in paraffin wax. Serial sectins (8- pm) were stained with hematxylin (Gurr, UK) and esin and munted in DePeX (Gurr, UK). In bth varies f each rat, graafian fllicles with r withut meitic resumptin, luteinized unruptured fllicles and ruptured fllicles were cunted; the latter were cmpact structures cntaining numer us dark-staining nuclei and these structures culd easily be distinguished frm crpra lutea f preceding cycles (fr details, see Ref. 9). Statistical analysis Peak values f LH and FSH were based n the value frm zer t maximum. T cmpare the respnse curves f LH and FSH t restraint stress, the individual data f the surge between 9. and 7. h were expressed as the area under the curve (AUC). The AUC is the integrated area between the baseline and the LH/FSH respnse abve the baseline. The AUC f LH and FSH, the peak values f LH and FSH and the vulatin rate were analysed by the Kruskal-Wallis test (ne-way analysis f variance), with nn-parametric cmparisns made using the Mann-Whitney U-test when trends were fund t be significant. Fr statistical analyses the Statistical Prgram System fr Scial Sciences (SPSS/ PC+ V2., SPSS, Inc., Chicag, IL) was used. Significance was defined at the.5 level. Values are reprted as means ± sem. Results Figure illustrates the effect f restraint n plasma LH during pr-estrus. In individual cntrl rats, the peak value f LH ranged between 7.5 and.5 ng/ml and was reached between. and 4. h. In restraint rats, the highest plasma LH levels ranged between basal and 4.3 ng/ml. Expsure t restraint resulted in a Dwnladed frm Biscientifica.cm at /5/28 4:7:33PM

3 3 t ( 9: : : : 3: 4: Time f day (h) 5: 6: 7: Fig.. Prfiles f rat plasma LH during pr-estrus: (D) cntrls ( = 8): (O) restraint h ( = ); ( ) restraint 8.-4.h ( = ): ( ) restraint 7.-4.h ( = ). Black hrizntal bar represents dark perid; data are given as means ± sem. significant inhibitin f the LH respnse (peak value and AUC; bth <. cmpared t cntrls) in all experimental grups. N significant verall differences were bserved between the three grups with different starting times f restraint. A cnsiderable variatin f individual LH prfiles was bserved in all restraint grups. Restraint cmpletely blcked the LH surge (plasma LH ^.2 ng/ml) in 5 ut f 32 rats. In rats, maximum LH levels varied between.5 and.7 ng/ml. Maximum LH levels between 2. and 4.3 ng/ml were reached in seven rats. Figure 2 illustrates LH prfiles f six individual rats in which restraint did nt cmpletely suppress the LH surge. In stressed rats, suppressed maximum LH levels were reached at any time pint between 9. and 6. h. The rat in Fig. 2b shws an advanced nset f the LH surge. In all restraint-stressed rats the LH levels were lw (.2-.4 ng/ml) n the next day at.,. and 3. h. This indicates that the restraint had nt delayed the surge f LH by 24 h. As shwn in Fig. 3, in cntrl rats plasma FSH increased gradually n the day f pr-estrus and restraint significantly suppressed this gradual FSH rise (p<.). The FSH level was 2.9 ±. ng/ml at.,. and 3.h the day after pr-estrus. N crrelatin was bserved between the peak and AUC values f LH and FSH. In Table the data n the varies, peak LH cncentratin in the plasma, relative amunt f LH released during LH surge (AUC) and vulatin rate are given fr cntrls and stressed rats. Data fr FSH are nt given because n crrelatin was seen between vulatin rate and peak r AUC values f FSH. In cntrl rats, virtually % vulatin had ccurred. Ovulatin rate was significantly reduced in the restraint grups (p <.5). N significant differ ences were fund between the three restraint grups with regard t vulatin rate, s they were cnsidered as ne grup. In the 5 rats that had LH levels <.2 ng/ml, the graafian fllicles were unaffected, i.e. they cntained an cyte with germinal vesicle and shwed n luteinizatin. In seven rats with a maximum LH level between.5 and.7 ng/ml and an AUC between.4 and 3., the graafian fllicles were als apparently unaffected. Exceptins were tw rats with a maximum LH level f.5 and.3 ng/ml and an AUC f.8 and 3. in which, respectively, % and 7% f the graafian fllicles had vulated. Rats with a peak LH level > 2 ng/ml r an AUC f ^4. generally shwed a high vulatin rate, with the exceptin f ne rat with a peak level f 2.8 ng/ml and an AUC f 4. that had nt vulated. In rats with partial vulatin, mst f the unruptured graafian fllicles were unaffected. Very few luteinized unruptured fllicles were bserved; they cntained an cyte in metaphase II stage. A ntewrthy trend was that the number f rats that had a relatively high LH peak, AUC and vulatin rate Dwnladed frm Biscientifica.cm at /5/28 4:7:33PM

4 (a) (b) (? Q Q 9= -- -T -O--p, ' O- --- ~l ~ zcx Q en c (C) (d) ( 2 E ^. 6-\» r (e) " - ( == = *=^ " 9: : : : 3: 4: 5: 6: 7: 9: : : : 3: 4: 5: 8: 7: Time f day (h) Time f day (h) Fig. 2. Release pattern f LH in six individual rats f which restraint suppressed the LH surge partially: (a, b) 5-h restraint; (c, d) 6-h restraint; (e, f ) 7-h restraint. Black hrizntal bar represents dark perid. was greater in the 6-h and 7-h restraint grups than in the 5-h restraint grup. On pr-estrus all cntrl rats and 7% f the stressed rats shwed lrdsis when a male was intrduced in their hme cage at 6.h. Discussin In male and variectmized female rats LH secretin is suppressed and the FSH secretin has been fund nt t be affected by varius Stressrs (5, 4-7). In the present study, expsure f cyclic rats t restraint n the day f pr-estrus resulted in a strng inhibitin f bth the LH and the FSH surge. Hülse and Cleman (7) bserved a partial suppressin f the LH surge in cyclic rats subjected t inescapable ftshcks fr 3 h during the surge n pr-estrus. T the authrs' knwledge, a suppressive effect f stress n the FSH surge f cyclic rats has nt been reprted befre. A reduced FSH surge may cnceivably cause a decrease f the number f vulatins in the next cycle. Transferring the rats t a nvel experimental rm befre restraint may be an additinal stressr. With respect t restraint stress the effect f nvel envirn ment n the LH surge is prbably small because we bserved n effect f mving the rats t a nvel experimental rm in pilt studies. Restraint ttally blcked the LH surge in 5 rats. These rats did nt have elevated LH and FSH levels the next day, indicating that the restraint had nt delayed the surge f gnadtrphins by ne day. The cmplete absence f vulatins and unruptured fllicles cntain ing an cyte in metaphase indicates that in these rats n substantial gnadtrphin secretin had ccurred between the perid f bld sampling n pr-estrus and the sampling perid n the next day. This crrbrates the view that in the rat the prevulatry surge f LH ccurs in a fixed perid f the day relative t the light/dark cycle (8). In cntrast t ur results, Ynetani et al. (8) reprted that frced immbilizatin fr 4 h starting at the beginning f the LH surge delayed the LH surge by day. These discrepant results may be due t a strain difference r may reflect a difference between the stress mdel used, i.e. restraint in a cylinder versus tying up in a supine psitin. The prevulatry LH surge is blcked and delayed by 24 h in 4- and 5-day cyclic rats after injectin f Nembutal n the day f pr-estrus (9, 2). After injectin f Nembutal n pr-estrus, LH and FSH are suppressed but estradil levels remain Dwnladed frm Biscientifica.cm at /5/28 4:7:33PM

5 Table. Effect f restraint applied during pr-estrus n plasma LH and subsequent vulatin in the rat.a Plasma LH during "surge time" Peak height (ng/ml) Area under curve (arbitrary units) Number f Number f Number f luteinized Number f graafian ruptured unruptured Ovulatin large fllicles fllicles fllicles fllicles rate (GF + RF + LUF) (GF) (RF) (LUF) (%) Cntrl.4 ± ±.6.4 ±.4.8 ±.4.9±.6 96 ±3.4 Restraint 5 h (9.-4. h) (a) (b).2 ± ±.: 4 3.2±.5 Restraint 6 h (8.-4. h) 4 (c) Kd).2 ± ± ± Restraint 7 h (7.-4. h) 6 (e) (f).2 ± ±. 3.5 ±. ameans ± sem are given fr cntrl rats and fr stressed rats with cmpletely suppressed LH surge; individual data are given fr rats with partially suppressed LH surge. "Surge time" is the perid f the day f pr-estrus at which the LH surge was presumed t ccur. Ovulatin rate is the number f RF versus the number f large fllicles relatively high; apparently, estradil remains suffi ciently high t evke an LH surge n the day after prestrus (2). In the present study, the absence f an LH surge n the day after restraint is hard t explain. It may have been caused by restraint-stimulated crticsterne secretin (22); increased crticsterne may inhibit FSH-induced armatase activity and cnsequently estrgen prductin in granulsa cells (23). Hw ever, the crticsterne increase during restraint is transient (24); it is unlikely that a relative brief rise f crticsterne caused a lasting suppressin f estrgen synthesis. Stress-induced suppressin f gnadtr phins might have caused atresia f fllicles and s a decrease f estrgen synthetic capacity. This is als unlikely because after Nembutal-induced suppressin f gnadtrphins an LH and FSH surge ccurred the next day (2). Ovulatin is initiated by a gnadtrphin surge n the day f pr-estrus; the magnitude f the LH surge is cnsiderably larger than needed t cause full vulatin. Greig and Weisz (25) reprted that apprximately 4% f the peak LH value is sufficient t induce vulatin, which agrees with the results f the present study. In the present study, it appears that als apprximately 4% f the cntrl AUC value is sufficient t trigger vulatin. The LH surge induces meitic resumptin f the cyte in graafian fllicles. In Nembutal-anaesthesized rats, a dse-related effect f LH n meitic resumptin, luteinizatin and vulatin was reprted (9, ): injectin f µg f LH caused meitic resumptin but n vulatin; 2 µg f LH caused vulatin f sme fllicles and meitic resumptin plus luteinizatin but n vulatin f the ther graafian fllicles; 4 µg f LH caused mre vulatins. Based n these data we expected that n vulatins but meitic resumptin with r withut luteinizatin shuld have been induced in the rat with a small rise f LH during pr-estrus. Hwever, the present data shw that after a cnsiderably reduced LH surge either all graafian fllicles vulated r the graafian fllicles appeared unaffected; they cntained an cyte in germinal vesicle stage and were nt luteinized. The explanatin f the discrepancy between Mattheij et al. (9, ) and the present results may be that in the frmer studies injectin f LH caused a steep and shrt-lasting (less than 6 min) increase f LH in the plasma, whereas in the present study the rats with a partially suppressed LH surge had a slightly increased LH level fr several hurs. The discrepancy may als be due t the fact that Mattheij et al. (9, ) injected LH 8 h befre the Dwnladed frm Biscientifica.cm at /5/28 4:7:33PM

6 c 6- u. (O E W _CO Q. T T 9: : : : 3: 4: Time f day (h) 5: 6: 7: Fig. 3. Prfiles f rat plasma FSH during pr-estrus: (D) cntrls ( = 8); (O) restraint 9.-4.h ( = ); ( ) restraint h ( = ); ( ) restraint 7.-4.h ( = ). Black hrizntal bar represents dark perid; data are given as means ± sem. presumed nset f the LH surge. Cnceivably, at that time fewer LH receptrs were present in the granulsa layer f the graafian fllicles than at the beginning f the presumed nset f the LH surge (26). In the present study, 7% f the stressed rats shwed lrdsis 2 h after the end f restraint while they had a reduced r ttally suppressed LH surge. In the cyclic rat, estradil and prgesterne (27, 28) tgether with GnRH (29) initiate and intensify estrus behaviur. In 5 rats, restraint cmpletely suppressed the LH surge. S an increase f the GnRH secretin in the hypphy seal prtal system and a cnsequent increase f varian prgesterne presumably did nt ccur. Yet 3 f these rats shwed lrdsis. The elevated plasma estrgen n pr-estrus tgether with a stress-induced increase f the prductin f adrenal prgesterne (27) r ther adrenal sterids (3) may have induced lrdsis in these rats. We cnclude that restraint stress inhibited and did nt delay gnadtrphin secretin n pr-estrus. Partial inhibitin f LH secretin by restraint is nt fllwed by inductin f meitic resumptin withut subsequent vulatin r by luteinized unruptured fllicles. Acknwledgments. The authrs thank NIH and NIDDK (Bethesda, MD, USA) fr prviding the materials fr the LH and FSH radiimmu nassays. They thank JG Leber (Natinal Institute f Public and Envirnmental Health, Bilthven, The Netherlands) fr prviding bld plasma frm hypphysectmized rats and Dr FPT Hamers (Utrecht University) fr kindly prviding us with sftware fr determining the AUC. References. Chattertn RT. The rle f stress in the female reprductin: animal and human cnsideratins. Int J Fértil 99:35: Rivier C, Rivest S. Effect f stress n the activity f the hypthalamic-pituitary-gnadal axis: peripheral and central mechanisms. Bil Reprd 99;45: Petraglia F, Suttn S, Vale W, Pltsky P. Crtictrpin-releasing factr decreases plasma luteinizing hrmne levels in female rats by inhibiting gnadtrpin-releasing hrmne release int hypphysial-prtal circulatin. Endcrinlgy 987:: Higuchi T, Hnda K, Negr H. Influence f estrgen and nradrenergic afferent neurns n the respnse f LH and xytcin t immbilizatin stress. J Endcrinl 986:: Briski KP, Sylvester PW. Effects f specific acute Stressrs n luteinizing hrmne release in variectmized and variectmized estrgen-treated female rats. Neurendcrinlgy 988:47: Armari A, Marti O, Gavalda A, López-Calderón A. Evidence fr the invlvement f sertnin in acute stress-induced release f luteinizing hrmne in the male rat. Brain Res Bull 993:3: Hülse GK, Cleman GJ. The rle f endgenus piids in the blckade f reprductin functin in the rat fllwing expsure t acute stress. Pharm Bichem Behav 983:9: Ynetani S, Jjima M, Suzuki Y. Blckade f vulatin in rats by frced immbilizatin fr surgical treatment. Endcrinl Jpn 974;2:6-8 Dwnladed frm Biscientifica.cm at /5/28 4:7:33PM

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