BIOLOGY OF RPRODTION 37, 1-1 (1987) Pinelectomy or Superior ervicl Gnglionectomy Do Not Alter Reproduction in the Wolf (nis lupus) HRYL S. ASA, LYSSS S. SAL, MAR LTLLIR,3 DWARD D. PLOTKA, nd RI K. PTRSON5 Deprtment of Biochemistry1 niversity of Minnesot St. Pul, Minnesot 5511 A Medicl Reserch Service Minnepolis, Minnesot 551 7 Thed lrk Medicl enter3 Neenh, Wisconsin 5956 Mrshfield Medicl Reserch Foundtion Mrshfield, Wisconsin 59 nd Deprtment of A nto my,5 niversity of Minnesot Minnepolis, Minnesot 5555 ABSTRAT Twelve wolves (6 mle nd 6 femle) were used to study the role of the pinel in photo periodic medition of sesonl reproduction, ight wolves were pinelectomied (PNX) or shm -pinelectomied ( S-PNX) t 5 m oj ge, nd were superior cervicl gnglionectomied SGX) t 16 mo of ge ( mles nd femles per tret n:ent). All ttined puberty t the species-typicl time, during their second breeding seson, except SGX mles tht did not survive. Reproductive cycles of n dditionl mle tht ws SGX s n dult nd the PNX nd S-PNX wolves, followed for minimum of 3 yr, did not differ from ech other or from those of unoperted colony wolves on mesures /serum testosterone nd luteiniing hormone for mles, or of serum estrdiol nd progesterone for femles. Nor ws the rnge of dtes for ovultion different for treted vs. untreted femles. Surgicl trnsection of the olfctory trcts of 1 mle nd I femle PNX wolf, inducing nosmi to control for the possibility of pheromonlly synchronied cycles, lso filed to lter the sesonlity of these reproductive prmeters. These results do not conform to the model of pinel medition of sexul cycles for photo periodsensitive species. In spite of evidence for photo period influence, the wolf pprently relies on system other thn the pinel for sesonl control of reproduction. INTRODTION In studies of the effect of chnging dylength on sesonl reproduction, species cn be divided into two generl ctegories. The horse, ferret, nd most rodents re long-dy breeders; i.e., sexul recrudescence occurs during lengthening photoperiod in spring. In contrst, ruminnts such s deer, sheep, nd gots re short-dy breeders nd respond to the shorter dys of utumn. Wolves breed in mid- to lte Accepted Jnury 6, 1987. Received November 3, 1986, Reprint requests: heryl S. Ass, The Popultion ouncil, enter for Biomedicl Reserch, 13 York Avenue, New York, NY 11. winter (Mech, 197), time not only when dys re shortest but lso when the rte of chnge of dylength is reltively less. Although the time of estrus for wolves does not fit the prdigms of either the spring (long-dy) or fll (short-dy) breeder, there re severl resons to expect them to be photoperiod-sensitive. First, wolves, like other wild cnids, re sesonl breeders (wer, 1973). The effect is strongest t temperte nd polr ltitudes, but lso is seen in equtoril species, probbly becuse ncestors of the fmily nide evolved in North Americ (Kurtin, 1971; Stins, 1975). Second, trnsloction cross the equtor resulted in reversl of breeding seson by 6 mo for the fox nd the mned wolf (wer, 1973) nd 1 Downloded from https://cdemic.oup.com/biolreprod/rticle-bstrct/37/1/1/7655 on 13 Februry 18
WOLF PINALTOMY AND SPRIOR-RIAL-GANGLIONTOMY 15 the cpe hunting dog (unninghm, 195), the species for which dt re vilble. Third, the time of breeding for the wolf occurs lter t higher ltitudes (Mech, 197). These lines of evidence strongly suggest tht the wolf responds to photoperiodic cues, with the resulting predictble occurrence of estrus t the sme time ech yer. As shown in other mmmls, photic informtion is trnsmitted by the retino-hypothlmic trct to the suprchismtic nucleus nd thence to the pinel vi the superior cervicl gnglion (Reiter nd Hester, 1966). At the pinel, the signl is trnsduced into humorl messge, probbly meltonin (Turek nd mpbell, 1979). To ssess the role of this system in the sesonl breeding cycle of wolves, we surgiclly interrupted the pthwy t two points: Group 1 ws pinelectomied (PNX) nd Group ws superiorcervicl-gnglionectomied (SGX). In ddition, in third experiment, 1 mle nd 1 femle from the PNX group were mde nosmic to control to test the possibility tht olfctory informtion from intct wolves might be sufficient to prevent disruption of cyclicity in the PNX nd SGX nimls. Pinelectomy MATRIALS AND MTHODS Two mle nd two femle prepubertl wolves (5 mo old) were PNX in the fll of 1978 using the technique developed for white-tiled deer, with pproprite modifictions (Letellier et l., 1978). Briefly, the surgicl procedure entiled mking smll crniectomy in the midline, lterlly exposing the superior sgittl sinus. The dur ws cut nd the pinel pproched interhemisphericlly using gentle lterl retrction of the medil cudl pole of the ipsilterl hemisphere. Bipolr cogultion ws used to prevent bleeding when the communicting veins between the prietl nd occipitl lobes nd the superior sgittl sinus were cut. Slight bleeding ws controlled with bsorbble geltin sponges. The pinel glnd ws dissected wy nd recovered in one piece. The dur mter ws closed, the bone replced, nd the skin sutured. Four dditionl wolves, of like ge nd sex, were shm-operted (S-PNX). The wolves were returned to their two ntl pcks (1 PNX mle, 1 PNX femle, 1 S-PNX mle, nd 1 S-PNX femle per pck). Besides the operted nimls, the North Pck contined two dult mles nd two dult femles, plus three juveniles. The South Pck cornprised 3 dult mles, dult femles, nd 1 juvenile, in ddition to the operted wolves. ch pck ws kept in n outdoor,.16-h enclosure t pproximtely 5#{176} north ltitude. The nimls were provided with dog chow (Rlston-Purin o., St. Louis, MO) nd wter d libidum, plus eviscerted deer crcsses supplemented with vitmins nd minerls. Successful bltion ws confirmed by post-mortem exmintion of ll but 1 wolf, PNX mle tht ws still living. very second week, immobilition ws induced with mg ketmine H1 (Ketset; Bristol Lbs, Syrcuse, NY) nd 1 mg promine H1 (Sprine; Wyeth Lbs., Phildelphi, PA). Additionl ketmine ws dministered s required to mintin immobilition. ginl secretions were checked for the bloody dischrge chrcteristic of proestrus (Sel et!., 1979). Testis length nd dimeter were mesured with clipers, nd n index of testiculr sie ws clculted by summing the products of the length times dimeter for the two sides. Blood ws collected by venipuncture t 1 mm nd 9 mm fter injection of 1 ig luteiniing hormone-relesing hormone (LHRH; Sigm hemicl o., St. Louis, MO), for mesurement of luteiniing hormone (LH) nd testosterone, respectively. Serum ws froen until ssy. Testosterone ws ssyed s in McMillin et l. (197); LH, estrdiol, nd progesterone concentrtions were determined by the methods described in Sel et l. (1979). Sesonl cyclicity ws evluted by mesurement of LH nd testosterone for mles, nd by estrdiol nd progesterone for femles. Ovultion ws ssumed to hve occurred when progesterone concentrtions were greter thn ng/ml in t lest 3 successive smples. For the testosterone ssy, the coefficient of vrition () ws pproximtely 7 percent nd the sensitivity less thn 1 ng/1 ml. For the progesterone ssy, the ws 17 percent t 1 pg, nd the sensitivity ws 1 pg. For the estrdiol ssy, the ws percent t pg, nd the sensitivity ws pg. For the LH ssy, the ws 7 percent, nd the sensitivity ws. ng/ml. Superior ervicl Gnglionectomy Surgery ws performed on 1 dult mle in 198, nd in the following yer on 16-mo-old, prepubertl wolves ( mles, femles). The SGX nimls were kept in kennels ( X 6 m) nd fed s described bove. The nimls were nesthetied with ketmine nd promine s bove, nd endotrchil tubes were Downloded from https://cdemic.oup.com/biolreprod/rticle-bstrct/37/1/1/7655 on 13 Februry 18
16 ASA T AL. inserted. Gnglionectomy ws performed with the niml in supine position with hed fully extended nd immobilied. The initil incision extended from 1 cm lterl to the bse of the lrynx to the ngulr process of the jw. The externl jugulr vein ws dissected free nd retrcted from the field. Anterior strp muscles were visible nd were utilied s lndmrks. rotid rtery pulse ws detected immeditely lterl to the nterior strp muscles, nd the crotid sheth ws exposed by blunt dissection. During dissection, the externl mxillry vein nd lrge lymph nodes nterior to the crotid sheth were ligted nd removed. The vgosympthetic trunk ws identified immeditely posterior to the crotid rtery within the crotid sheth. The crotid rtery ws dissected free nd retrcted from the field. The vgosympthetic trunk ws isolted, dissected free, nd circled with umbilicl tpe for ese of mnipultion. The dissection of the trunk continued crnid, llowing identifiction of the following importnt lndmrk structures: (1) bifurction of the externl nd internl crotid rtery, () hypoglossl nerve, nd (3) posterior belly of the digstric muscle. Both the internl crotid rtery nd the hypoglossl nerve were exposed nd followed to the bse of the skull to provide positive identifiction of the SG nd vgl nodose gngli, which re locted in the petrobsilr fissure posterior-medil to the tympnic bull. In this region, the internl crotid rtery enters the crnium vi the petrobsl fissure, nd the hypoglossl nerve courses lterlly from point 1 cm posterior-medil to the fissure. In the fissure, the rtery courses between the two gngli tht re slightly inferior nd nterior to the SG. The epineureum of the vgosympthetic trunk sometimes surrounds both structures, mking them pper s one. In this cse, smll rtery prlleling the vgosympthetic trunk ws present in the epineureum. This rtery ws used s guideline to seprte the nodose nd SG. After the two gngli were isolted, the SG ws cut inferiorly nd superiorly nd the entire structure removed. The incision ws closed nd the process repeted on the opposing side. Successful SGX ws confirmed by the bilterl loss of sympthetic input to structures innervted by postgnglionic fibers derived from the SG (e.g., constricted pupils). S-PNX, nd 1 femle S-PNX) were mde nosmic in August 198, by trnsection of the olfctory peduncle s described in Peterson et l. (1981), procedure which blocks input from both the min nd ccessory olfctory trcts. The nimls were mintined s progesterone for the SGX femles were not different from those of representtive control femles (Fig. Olfctory Peduncle Trnsection Four wolves (1 mle PNX, 1 femle PNX, 1 mle 3). detiled Pinelectomy bove. RSLTS Throughout the four breeding sesons following pinelectomy, no differences in mesures of reproductive physiology were detected between PNX nd S-PNX or unoperted colony wolves. All PNX nd S-PNX wolves ttined puberty during the second breeding seson post-surgery (winter, 198), t the species-typicl ge, except S-PNX Femle 93 who ovulted t 1 rther thn yers of ge (Fig. ic). (Although the pttern of secretion ws similr, estrdiol vlues for Femle 93 were reltively higher due to lter ssy following pprent concentrtion during storge.) The inception of puberty nd the subsequent regulrity of sesonl cyclicity were evidenced in the femles by proestrous uterine bleeding nd ovultion, indicted by profiles of estdiol nd progesterone (Fig. l-d), nd in the mles by increses in serum testosterone nd luteiniing hormone (Fig. -d) nd testiculr sie. These curves of sesonl hormone chnges lso were like those of unoperted colony mles. ch of the PNX femles ws followed for totl of three cycles post-puberty; of the mles, one ws followed for five nd the other for six cycles, during which time no divergence from controls ws seen. Superior ervicl Gnglionectomy The younger SGX mles did not survive until the time of expected puberty, due to surgicl complictions. However, the SGX femles did ovulte for the first time during their second breeding seson. The pproximte dtes of these ovultions were between 1 Februry nd 3 Februry for Femle 111, nd between 3 Februry nd 9 Mrch for Femle 119. Ovultion dtes for other colony femles (post-pubertl: N=7) for tht seson were between 3 Februry nd 3 Februry. Profiles of estrogen nd Downloded from https://cdemic.oup.com/biolreprod/rticle-bstrct/37/1/1/7655 on 13 Februry 18
WOLF PINALTOMY AND SPRIOR-RIAL-GANGLIONTOMY 17 (,- +, ). ) ) -.. #{176}..... - (I) o o,. O7lSSd (1u/bd) 119 s3wn5,,.,o (lw/sd) I1P#{176} ) 3W S o #{176} (Iw/tu) SJe$$I6d W S - - II- I.-,. I ) ),,, ). l) AcTi IIT I -, - o e l) - (lw/sd) lolpojsb wnis (IW/bli) GOJOISObOJd WflJOS (i. I! o. I. ( (lw/sd) #{176}) 6#{176} d W S (,wfbd) 1191(311()5 Downloded from https://cdemic.oup.com/biolreprod/rticle-bstrct/37/1/1/7655 on 13 Februry 18
18 ASA T AL. -, - 1 F -,, -, -, -.. ( 1 ID ID 51 - I I (lw/so) WI WIIOS.5 1 _.. N - (19/6) 11 IDn)5. (IP/bO) )153J wn)5. (IP/b) 3u)ISlS1 1(15 N.! -, ( ). ), o = ) 1, -.#{176}., -, 1-1 O. #{176} #{19} 1 -, 5 -, 11. (Iw/b) H1I 1 S? 9 (lw/so) H1WI )S o I. (l. Q (19/6) (153j wflo$ O (19/OIl) 11)IllOOlSDj 1)5 Downloded from https://cdemic.oup.com/biolreprod/rticle-bstrct/37/1/1/7655 on 13 Februry 18
WOLF PINALTOMY AND SPRIOR-RIAL-GANGLIONTOMY 19 dtes of ovultion of pubertl PNX nd 1 of SGX femle wolves were within the rnge of those of I-J ) w (Li ) SONDIJFMAM MONTH SONDIJFMA FIG. 3. Serum concentrtions of estrdiol nd progesterone of superior-ceruicl gnglionectomied (SGX) nd representtive control femle wolves during one breeding seson. The SGX mle surgiclly ltered post-puberty ws followed for two sesons, during which time profiles of serum testosterone nd LH (Fig. ) nd chnges in the index of testis sie were similr to those of unoperted mles. Dt for SGX femles were vilble only through their pubertl yer, due to subsequent ssignment to nother experiment. Pinelectomy Plus Anosmi Olfctory-trct trnsection nd the resulting nosmi (see As et l., 1986, for procedures confirming successful bltion nd nosmi) did not ffect the sesonl cyclicity of either the PNX or S-PNX wolves (Figs. lb,d; b,d). DISSSION Neither PNX nor SGX ffected the onset of puberty or the sesonl breeding cycles of mle or femle wolves. This lck of effect ws suprising in light of the results of comprble experiments involving other photoperiod-sensitive species (see Lincoln nd Short, 198). All surgiclly ltered wolves reched puberty t the ge typicl of the species, in their second breeding seson (Mech, 197). nlike the ccelerted enlrgement nd regression of the testes of PNX, pubertl Soy rms (Lincoln, 198), no differences were detected between either PNX or SGX pubertl mle wolves nd shm or unoperted control groups in ptterns of serum testosterone nd LH, or indices of testis sie. Similrly, unoperted femles. The time of ovultion for the other SGX femle ws within wk of the lst ovultion of the intct, post-pubertl femles. However, it is not uncommon for young femle s first ovultion to occur lter thn those of the rest of the colony (unpublished observtions). (Li NI ) -J I- ) Ll I- LI- )( Ll - The ttenution of the testosterone peks observed for PNX Mle 8 nd S-PNX Mle 81 (Fig.,c) during the 1981-8 breeding seson is likely due to their seprtion from other members of their pck during December 1981, when they were moved to different enclosures. During their post-pubertl breeding sesons, mle nd femle PNX wolves continued to hve regulr cycles, in phse with the shm nd unoperted groups. In contrst, fter pinelectomy, ferrets were found to cycle synchronously with respect to ech other nd controls (Herbert et!., 1978). Although dt were collected from SGX femles for only one 3 I ONTROL d 8 ONTROL d85 I YAR FIG.. Sesonl chnges in the index of testiculr sie for superiorcervicl-gnglionectomied (SGX) nd control mles during three breeding sesons. (Lower vlues for Mle 85 re bsed on his single, descended testis.) Downloded from https://cdemic.oup.com/biolreprod/rticle-bstrct/37/1/1/7655 on 13 Februry 18
ASA T AL. cycle, the SGX mle wolf hd two sesons of norml testosterone, LH, nd testiculr cycles, which were synchronous with control mles. Olfctory-trct trnsection in two of the PNX wolves lso filed to lter their sesonl rhythms, demonstrting tht pheromonl cues were not mediting their reproductive synchrony. The results of the present series of experiments do not conform to the currently ccepted model, which predicts tht pinelectomy should lter the response of the neuroendocrine-gondl xis to photoperiod (Turek et l., 198). In ssessing the filure of pinelpthwy disruption to lter the sexul cycle of the wolf, we must consider in wht wys the wolf s cycle fits other spects of the model. First, it does seem tht wolves, nd likely other cnids, re photoperiodsensitive: 1) the wolf nd other cnids re sesonl breeders (wer, 1973), ) equtoril trnsloction of cnids for which there re dt results in reversl of breeding seson by 6 mo (unninghm, 195; wer, 1973), nd (3) the estrous periods of wolves occur lter t higher ltitudes (Mech, 197). vidence for ctegoriing the wolf s long- or short-dy breeder is not s cler. The pproximtely one-week estrus occurs during mid- to lte winter (depending on ltitude) when dys re lengthening, but t slower rte thn in the spring when the typicl long-dy breeders, like the horse, re entering their breeding seson. Furthermore, in the wolf, estrus culmintes n unusully lengthy series of events. strdiol levels begin to increse in the fll, but typiclly do not rech the threshold required for stimultion of proestrous bleeding until sometime fter the winter solstice (Sel et!., 1979; unpublished observtions). Similrly, mle testosterone nd LH levels begin their increse during utumn nd rech pek levels t bout the time of estrus (unpublished observtions). Thus, lthough estrus occurs during lenthening dys, the erly phses of sexul recrudescence occur when dys re becoming shorter. It is not known which of these photoperiodic phses is more importnt or dominnt, or if both ply eqully significnt roles in the yerly cycle. In discussing the effect of pinelectomy on photoperiod-sensitive species, Turek et l. (198) suggest tht dequte investigtion of pprent exceptions would eventully revel the predicted ltertion of neuroendocrine or gondl events. Further studies of this phenomenon in wolves or other cnids my indeed disclose such effects. However, preliminry experiment in which extended dylength (16L:8D) ws instituted on 16 November for the SGX femles nd 1 mle, plus 1 PNX mle, resulted in ovultion nd birth of pups 1 mo erlier thn for other colony nimls (unpublished observtions). These results not only confirm the bility of the wolf to respond to chnge in dylength, but lso indicte tht the pinel nd superior-cervicl gngli re not necessry for tht response. The filure of PNX nd SGX to disrupt the sesonl breeding cycles of the wolf does not preclude the involvement of meltonin in this phenomenon. In other species, meltonin hs been found in the retin nd Hrderin glnd, s well s in the pinel (Png et l., 1977), condition which my lso exist in the wolf. An bsence of meltonin, pprently the result of inbreeding in certin strins of mice (bihr et l., 1986), cnnot explin our results for the wolf. First, our colony hs been estblished nd crefully mintined s outbred, with originl founders from wolf popultions in Alsk, nd, nd Minnesot. Second, ssy of serum from unoperted wolves confirmed the presence of meltonin, lthough results were equivocl in regrd to diurnl rhythm (unpublished observtions). Studies re currently underwy to clrify ny role of meltonin in sesonl reproduction in the wolf. However, the uncertinty of meltonin influence does not chnge the fct tht PNX nd SGX did not lter the reproductive cycles in this photoperiod-sensitive species. AKNOWLDGMNTS The uthors wish to thnk N. Mnning, M. K. Twite, G.. Schmol- Icr, nd J. H. hmplin for hormone ssy nd computer dt entry, numerous technicins nd student volunteers for smple collection, Mrshfield Medicl Reserch Foundtion for finncil support, nd L. Knutsen nd R. Johnson, Minnesot Deprtment of Nturl Resources, for logisticl support of the wolf colony. RFRNS As S, Mech LD, Sel S, Plotk D, 1986. The effect of nosmi on reproduction in mle nd femle wolves (nis lupus). Behv Neurl Biol 6:7-8 unninghm DJ, 195. pe hunting dogs (Lycon pictus) in the grdens of the Royl Zoologicl Society of Irelnd. Proc R Soc dinb 5:83-8 bihr S, Mrks T, Hudson DJ, Menker M, 1986. Genetic control of meltonin synthesis in the pinel glnds of the mouse. Science 31: 91-93 wer RF, 1973. The rnivores. Ithc, New York: ornell niversity Press Herbert J, Stcey PM, Thorpe DH, 1978. Recurrent breeding sesons in pinelectomied or optic-nerve-sectioned ferrets. J ndocrinol 78:389-97 Kurtin B, 1971. The Age of Mmmls. New York: olumbi niversity Press Downloded from https://cdemic.oup.com/biolreprod/rticle-bstrct/37/1/1/7655 on 13 Februry 18
WOLF PINALTOMY AND SPRIOR-RIAL-GANGLIONTOMY 1 Letellier MA, Plotk D, Sel S, erme, Og JJ, 1978. A technique for pinelectomy in deer with notes on neurontomy. Am J et Res 39:1617- Lincoln GA, Short R, 198. Sesonl breeding: nture s contrceptive. Recent Prog Horm Res 36:1-5 McMillin JM, Sel S, Keenlyne KD, rickson AW, Jones J, 197. Annul testosterone rhythm in the dult white-tiled deer (Odocoileus virgininus borelis). ndocrinology 9:13- Mech D, 197. The Wolf: The cology nd Behvior of n ndngered Species. Grden ity, New York: Nturl History Press Png SF, Brown GM, Grot, hmbers JW, Rodmn RL, 1977. Determintion of N-cetylserotonin nd meltonin ctivities in the pinel glnd, retin, Hrderin glnd, brin nd serum of rts nd chickens. Neuroendocrinology 3:1-13 Peterson K, Letellier MA, Prsons JA, Plotk D, Mech LD, Sel S, 1981. Olfctory pedunculotomy-induced nosmi in the wolf (nislupus). Phys Behv 7:53-6 Reiter RJ, Hester RJ, 1966. Interreltionships of the pinel glnd, the superior cervicl gngli nd the photoperiod in the regultion of the endocrine systems of hmsters. ndocrinology 7:1168-7 Sel S, Plotk D, Pckrd JM, Mech LD, 1979. ndocrine correltes of reproduction in the wolf. I. Serum progesterone, estrdiol nd LH during the estrous cycle. Biol Reprod 1:157-66 Stins HJ, 1975. Distribution nd txonomy of the nide. In: Fox MW (ed), The Wild nids, Their Systemtics, Behviorl cology nd volution. New York: n Nostrnd Reinhold, pp. 3-6 Turek FW, mpbell S, 1979. Photoperiodic regultion of neuroendocrine-gondl ctivity. Biol Reprod :3-5 Turek FW, Swnn J, rnest DJ, 198. Role of the circdin system in reproductive phenomen. Recent Prog Horm Res :13-83 Downloded from https://cdemic.oup.com/biolreprod/rticle-bstrct/37/1/1/7655 on 13 Februry 18