Hyperprolactinemia does not promote testicular recrudescence in photoregressed Siberian hamsters

Physwlogy&Behavior,Vol 54, pp. 175-178, 1993

0031-9384/93$6 00 + .00 Copyright© 1993PergamonPressLtd

Pnntedm the USA

Hyperprolactinemia Does Not Promote Testicular Recrudescence in Photoregressed Siberian Hamsters R I C H A R D D. W H I T T E N , T I M O T H Y G. Y O U N G S T R O M

A N D T I M O T H Y J. B A R T N E S S l

Departments of Psychology and of Biology, Neurobtology and Behavior Program, Georgia State University, Atlanta, GA 30303 Received 9 September 1992 WHITTEN, R. D., T. G. YOUNGSTROM AND T. J. BARTNESS.Hyperprolactmemladoesnotpromotetestwularrecrudescence m photoregressedStbenan hamsters PHYSIOL BEHAV 54(1) 175-178, 1993.--Serum prolactin (PRL) concentrations, as well as body, epldldymal white adipose tissue (EWAT),and testes weights, decrease in Slbenan hamsters (Phodopussungorussungorus) followingshort-photoperiodexposure. Previously,we have shown that lesionsof the suprachiasmatlcnucleus of the hypothalamus (SCNx) block regressionof the testes and decreases in body weight and EWAT caused by short day-like,timed daffy subcutaneous melatonln infusions in pinealectomlzed Siberian hamsters and elevate dramatically serum PRL concentrations. We also have shown that SCNx, as well as lesions of the paraventncular nucleus of the hypothalamus (PVNx) and an area immediately ventral to the PVN (subPVN), promote accelerated tesUcular recrudescence, increases m EWAT and body weights, and increases m serum PRL concentrations, in short-day (SD)-housed, photoregressed Siberian hamsters. The stlmulaUon of the testes seen m these previous studies could have been due to the lesion-inducedincreasesin serum PRL concentrations. Therefore, the purpose of the present experiment was to test whether experimentallyreduced hyperprolactinemiacould stimulate testicularrecrudescence This was accomphshed by giving photoregressed, SD-housed Siberian hamsters chronic subcutaneous infusions of ovine PRL (oPRL) to mimic either long-day- or lesion-induced serum concentrations of hamster prolactin (hPRL). No increase m testes, body, or EWAT weights were observed following5 weeks of oPRL infusions in either group compared with their vehicle-infused counterparts. These data suggestthat hyperprolactmemiawas not solelyresponsiblefor the stimulaUonoftestlcular recrudescence m SCNx or PVNx photoregressed, or SCNx pinealectomizedhamsters recewing timed melatonln infusions seen previously. Photoperiod

Prolactm

Testes

S~benanhamster

SIBERIAN hamsters (Phodopus sungorus sungorus) display dramatic changes in body weight (19), fat content (5), pelage color (8), and reproductive status (10) following changes in the photoperiod. The photoperiodic signals received at the retina are transmitted via the retinohypothalamic tract to the suprachiasmatlc nucleus of the hypothalamus (SCN) (l l). The SCN neurons project to the paraventricular nucleus of the hypothalamus (PVN) and immediately ventral to the PVN (subPVN), whereas the PVN sends projections to the spinal cord, including the intermediolateral horn ( 12,13,15,20,21 ). Neurons of the mtermediolateral horn then project to the superior cervical ganglion (16) and ultimately to the pineal gland. The pineal gland secretes the hormone melatonin (MEL), which is involved in promoting seasonal changes in behavior and physiology (5,8, I0). We have been interested in determining the melatonin target sites that mediate the effects of the photoperiod on energy balance and reproductive status. Previously, we have shown that SCN lesions (SCNx) block regression of the testes and decreases m body weight and fat caused by long-duration, short-day (SD)-

like, timed daily subcutaneous melatonin infusions in pinealectomized Sibcrlan hamsters (4). Associated with these SCN lesions were dramatically elevated serum PRL concentrations. One interpretation of these findings is that SCNx block the effects of SD-type melatonin infusions on these responses. Alternatively, the blockade of the SD-type responses could have been due to hyperprolactinemia-induced stimulation of the testes (3) and, less likely, body weight (fat) (6). This latter alternative interpretation appeared to be strengthened by our recent findings that SCNx, PVNx, and subPVNx all promote accelerated testicular recrudescence and increases in fat pad and body weights in SDhoused, photoregressed Siberian hamsters, and, moreover, increase dramatically serum PRL concentrations (14). Therefore, all three lesions may be affecting a normally active gonadotropin and/or PRL inhibitory system, and thus increases in these hormones may stimulate testicular regrowth. Indeed, at least for the PVN or subPVN lesion, some evidence supports the possible role of the PVN in the mediation ofgonadotropin or PRL release (1,2,12,13,15,17). The purpose of the present experiment was

J Requests for reprints should be addressed to Timothy J. Bartness.

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to test whether hyperprolactlnemla could stimulate test~cular and body weight (fat) recrudescence. This was accomplished by gaving photoregressed, SD-housed Sibenan hamsters chromc subcutaneous infusions of ovme PRL (oPRL) to mimic either long-day- or lesion-induced serum concentrations of hamster prolactln (hPRL) METHOD

Ammal, s and Housing Adult, male Siberian hamsters (n = 61) were group housed and raised m long days (16 h hght, 8 h darkness; lights on 0300 h) from birth. One cage of animals (n = 9) remained m long days as a control group, while other cages were transferred to SDs (L:D 8:16: lights on 0700 h) for 14 weeks. Hamsters had ad lib access to Punna Rodent Chow (#5001) and tap water throughout the experiment. At 14 weeks, animals housed in SDs continued m the experiment if they had a pelage color rating of either 3 or 4 [short day, winter-white color = 4, long day, graybrown color = 1; (9)] and/or had lost at least 6% of their ongmal body weight. Ammals were divided into groups that were balanced for body weight loss. At this time, subcutaneous catheters were implanted in all SD-housed ammals and a small patch of fur was plucked ( ~ 0 25-0.50" diameter patch) from the right hindquarter. The latter treatment was done to permit an rodependent indication of the bioactwity of the refused oPRL on a PRL-sensitwe endpomt other than the testes. We (6) and others (9) have shown that SD-housed hamsters exhibiting a whitewinter pelage and infused with oPRL will regrow dark summertype hair m the region that is plucked, whereas vehicle-refused controls grow back white fur. Body weights were recorded weekly. Hamsters were lightly anesthetized at week 4 with methoxyflurane to permit blood sampling from the orbital plexus for subsequent measurements of serum PRL concentrations. At the end of the experiment (week 5), the animals were sacnficed by decapltaUon. Trunk blood was collected for oPRL and hPRL measurement, and testes and EWAT pads were removed, blotted dry, and weighed.

Chromc Subcutaneous oPRL lnfitston~ In order to elevate serum PRL concentrations to those of long-day-housed, and SCNx, PVNx, or subPVNx animals, SDexposed hamsters were fitted with a subcutaneous catheter for chronic infusmns of oPRL as described previously (3). Catheters were implanted under methoxyflurane anesthesia. The vehicle group (n = 10) was infused chronically with 0.01 M sodium bicarbonate (0.4 ml/day) as the vehicle control. The 20-/ag group (n = 11) received 20 #g of oPRL/day in an attempt to mimic endogenous hPRL serum concentraUons of long-dayexposed ammals, whereas the 100-#g group (n = 9) received 100 tag of oPRL/day to mimic the elevated levels produced by SCNx, PVNx, and subPVNx (8). We recognize that oPRL Is not the native form of PRL in Siberian hamsters; however, previous work has demonstrated that the hair pigmentation effects of PRL can be duplicated by oPRL infusions. Thus, at least for this response, oPRL seems to have a similar bioactivity to that of hPRL (9).

and Antlsera Center (Harbor-UCLA Medical Center) 1hc k)l employs antibody AFP-7472988 (raised m rat) and antigen AFt'10302E for lodmat~on and hPRL reference standards (14). Lnmts of assay sensitivity for a 30-M sample were 0 54 and 144() ng/ml The oPRL levels also were measured wa RIA using a k~t supplied by the Pituitary Hormones and Antisera Center (Harbor-UCLA Medical Center) Antibody AFP-973269 (raised tn rabbit) and antigen AFP-7150B were used tbr ~odmatlon and oPRL reference standards, respectively Multiple ddut~ons of sera were assayed The results obtained from assaying 10-tal samples of a 1 4 dilution will be reported. In those instances where the level of oPRL was above or below the range of detectablhty, the appropriate corresponding maximum or mm~mum detectability was assigned to the sample In this case, assay sensitivity was 10.52 and 209 4 ng/ml.

Start s'twal A hall'.St s All data were analyzed by analys~s of vanance and, where apphcable, post hoc comparisons were made using StudentNewman-Keuls (SNK) (18) Body weight change data were analyzed by t-tests Results for all statistical tests were considered statistically significant lfp < 0.05 Exact probabilities and SNK values are not reported, to s~mplify the presentation of the results RESUI TS

Ovlne PRL serum concentrations in the 20-tag and 100-#g groups were elevated three- to fourfold compared with long-day controls, F(3, 74) = 20.15, all p < 0.05 (Fig. 1, top) Serum hPRL concentrations were nearly nondetectable Jn the three SD-housed groups and thus significantly lower than hPRL concentrations for the long-day controls, F(3, 77) = 17.59, all p < 0.05 (Fig. 1, bottom). Both the 20-~g and 100-tag groups had approximately tenfold increases in serum oPRL compared with long-day-housed serum hPRL concentrations [20 tag vs. LD, t(22 64) = 6.68, p < 0.05; 100 ~g vs. LD, t(17.72) = 5.22, p < 0.05: Fig 1] All three SD-housed groups had significantly lower body weights than the LD controls [week 1, F(3, 35) = 8.79, p < 0.05; vehicle vs. LD, t(l I 7) = 3.74, p < 0.05; 20 tag vs LD, t(10.64) = 3 73, p < 0 05; 100 tag vs LD, t(16) = 3.28, p < 0.05; week 5, F(3, 35) = 11.86; vehicle vs LD, t(10.15) = 4.02, p < 0.05: 20 #g vs. LD, t(18) = 4.00, p < 0.05; 100 tzg vs. LD, t(11.13) = 3 23, p < 0.05; Fig. 2] and were not different from one another (all p > 0.05). Body weights of all groups were not different between week 1 and week 5 (all p > 0.05; Fig. 2). The oPRL infusions did not increase paired testes or EWAT wetghts compared to the vehicle-refused group (all p > 0.05; Fig.3). All SD-housed groups had regressed testes and decreased EWAT weights compared with long-day controls testes, F(3,35) = 98 36, p < 0.05: EWAT, /:(3,35) = 26 11, all p < 0.05, Fig. 3] After 5 weeks of infusion, all hamsters m the 20-tag and 100tag groups grew dark hair (long-day-like, stage l) in the region where the fur was plucked, whereas the rest of their pelage remained white (short-day-hke, stage 4). Regrown hair m all vehicle-infused animals was white (stage 4)

Hormone Assay, DISCUSSION

Blood collection tubes were stored for 24 h at 4°C, then centrifuged at 2000 rpm for 20 min to obtain serum samples. The serum was stored at - 2 0 ° C untd assayed. Serum hPRL concentrations were determined by radloimmunoassay (RIA) using a kit supphed by the Pituitary Hormones

In a prewous study, elevated serum PRL concentrations were observed along with the blockade of testicular regression and decreases m body weight and fat m long-day-housed, pinealectomlzed SCNx Sibenan hamsters receiving long-duration, SD-

PROLACTIN AND RECRUDESCENCE

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FIG. 2 Mean __SEM body weghts followLngthe fast week of the experiment (Week 1) or the fifth week (Week 5). See caplaon ofF'~ I for group acronyms and treatments. * p < 0.05 vs. respeetaveweek for all other groups vated serum PRL was not solely responsible for SCNx-, PVNx-, and subPVNx-induced recrudescence seen previously (14). The results also suggest hyperprolactinemm was not responsible for the blockade of testicular regression in SCNx MEL-

15

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907 •-~ 06 0.5 Vehicle

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FIG. 1 Mean serum owne prolactin (oPRL, top) or hamster prolactln (hPRL, bottom) concentrations for SD-housed hamsters receiving eqmvolemic continuous SC infusions of the vehicle (Vehicle) or oPRL at 20 #g or 100/zg/day (20-/~g and 100-/~g groups, respectively). * p < 0.05 vs. all other groups. SD-housed ammals infused with 20 ttg or 100 #g of oPRL per day. Mimmum and maximum detectability of the oPRL radioimmunoassay was 10.52 ng/ml and 209 4 ng/ml, respectively. Minimum and maximum detectability of the hPRL radioimmunoassay was 0.54 ng/ml and 144.0 ng/ml, respectively. Note the tenfold scale difference of the ordinate between the top and bottom graphs

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0.4 03 02 0.1

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20 ~g

100/~g LD Control

10 TESTES 09

hke subcutaneous melatonin infusions (4). In addition, in another experiment, photoregressed, SD-housed SCNx, PVNx, and subPVNx Siberian hamsters had similar elevations in serum PRL concentrations to those reported in the present experiment (4), and exhibited testlcular and body weight (fat) recrudescence (14). Therefore, in the present experiment, we chronically infused o P R L subcutaneously to mimic lesion-induced serum concentrations of h P R L to determine whether hyperprolactinemia has a stimulatory effect on testes and/or body weight (fat) recrudescence. We also used the pigmentation o f regrown fur as an indicator o f the bioactivity o f the o P R L infusions. In spite of the ability o f o P R L to induce pigmentation of regrown fur to a long-day-like color, the testes, body, and fat pad weights in both oPRL-infused groups did not recrudesce. The inability of experimentally induced hyperprolactinemia to promote testicular and body weight (fat) recrudescence suggests ele-

07 /

0 08~

.

Vehicle

20 ~g

100 ~g LD Control

FIG 3. Mean + SEM paired epididymal white adipose tissue (EWAT) and testes wet weights at the conclusion of the expenment. See caption of Fig. 1 for description of group acronyms and treatments *, p < 0.05 vs. all other groups.

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refused animals Moreover, m the present experiment, E W A T a n d body weights were unaffected by hyperprolactmemm, a finding consistent with our previous study suggesting that P R L does not play a p r e d o m i n a n t role in the p h o t o p e n o d l c control of these responses (6) It might be argued that o P R L a n d h P R L are so biologically &ss~milar that comparably produced results are invahd, or that elevated serum P R L inhibits rather t h a n s h m u l a t e s tesucular growth. However, the results from the regrowth of p~gmented pelage suggest that oPRL was b~oactwe, at least for th~s response In addmon, elevated serum o P R L concentrations did not further reduce testes weight c o m p a r e d to the vehicle group. Th~s would suggest that hyperprolactinemm does not have an mhlbxtory effect

AND BARINESS

on the testes, although, g~ven that the testes weights ol the experimental groups are not slgmficantly &fferent from the SD vehicle-refused group, a floor effect preventing detection of such m h t b i t l o n is possible Contrary to results obtained using Syrian hamsters (3), the results of this study show that hyperprolactinemla does not stimulate the testes m th~s species, n o r does it suggest a st~mulatoD , role for P R L m the regulation of body weight a n d fat, as has been suggested in Syrian hamsters (7) ACKNOWLEDGEMENTS This research was supported by NIH RO-I DK35254 and NIMH Research Scientist Development Award KO2-00841

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