Serum prolactin and dehydroepiandrosterone concentrations during the summer and winter hair growth cycles of mink (Mustela vison)

Comparative Biochemistry and Physiology Part A 121 (1998) 263–271

Serum prolactin and dehydroepiandrosterone concentrations during thesummer and winter hair growth cycles of mink (Mustela 6ison)

Jack Rose *, Michael Kennedy, Brad Johnston, Wade Foster

Department of Biological Sciences, Idaho State Uni6ersity, Campus Box 8007, Pocatello, ID 83209, USA

Received 26 May 1998; accepted 3 September 1998

Abstract

We investigated the relationship between serum concentrations of prolactin (PRL) and dehydroepiandrosterone (DHEA) duringinitiation and development of summer and winter hair growth (anagen) cycles in mink. In the spring, haloperidol (HAL) increasedPRL concentrations and induced summer anagen earlier than controls, whereas melatonin (MEL) inhibited PRL secretion andcompletely blocked summer anagen. In the fall, HAL increased PRL concentrations, inducing anagen at an earlier time thancontrols, although the resulting fur was abnormal being almost devoid of underhair fibers. Exogenous MEL during the fallreduced PRL concentrations, initiating winter anagen 4 weeks earlier than controls. Adrenalectomy (ADX) induced earlier onsetof summer and winter anagen and neutralized the inhibitory effects of HAL in the fall and MEL in the spring. No change inserum DHEA concentrations was observed during the onset of summer or winter anagen in any group although MEL increasedDHEA levels from 27 March through 5 June relative to HAL-treated mink. We conclude that changes in serum levels of DHEAand PRL are not requisite to onset of summer or winter anagen in mink. It is possible that metabolites of DHEA and/or PRLmay still affect other aspects of the hair growth cycle. © 1998 Elsevier Science Inc. All rights reserved.

Keywords: Adrenal; Dehydroepiandrosterone; DHEA; Fur; Hair; Haloperidol; Melatonin; Mink; Prolactin

1. Introduction

In the mink, increasing photoperiod in the springinitiates summer hair growth, whereas decreasing pho-toperiod in the fall induces growth of winter fur[14,39,40,53,56]. At the start of each hair growth cycle,epidermal stem cells within the outer root sheath of theresting (telogen) hair follicle are activated, and subse-quently divide and differentiate to reform the lowerportion of the hair follicle [16,31]. From this newlygrowing (anagen) hair follicle, a hair shaft develops,eventually dislodging the old hair from the previouscycle, resulting in the molt.

Part of the effects of photoperiod on induction ofsummer and winter anagen in mink are thought to bemediated through the pituitary hormone prolactin(PRL). Serum concentrations of PRL increase in thespring prior to summer anagen and decrease in the fallbefore onset of winter anagen [38,40,54,55]. Inhibitionof PRL secretion in the spring delays whereas exoge-nous PRL at this time advances the onset of summeranagen [2,39]. In contrast, inhibition of PRL secretionin the fall advances the onset of winter anagen[40,54,56]. The tissue on which PRL might act toinfluence anagen initiation is unknown, although PRLreceptors are present in the skin [57] and adrenal glands[52] of mink. Because bilateral adrenalectomy (ADX)of mink in early March or late June induces the onsetof summer and winter anagen up to 6 weeks earlierthan controls [49,51], it is possible that part of theeffects of PRL on fur growth are mediated through theadrenal glands and/or the skin.

* Corresponding author. Tel.: +1 208 2364261; fax: +1 2082364570; e-mail: [email protected]

1095-6433/98/$ - see front matter © 1998 Elsevier Science Inc. All rights reserved.PII S1095-6433(98)10127-7

J. Rose et al. / Comparati6e Biochemistry and Physiology, Part A 121 (1998) 263–271264

In primates, secretion of the adrenal steroid dehy-droepiandrosterone (DHEA) is elevated during hyper-prolactinemia and reduced during hypoprolactinemia[1,4,33,45,64]. Moreover, Higuchi et al. [22] demon-strated that, during hyperprolactinemia, DHEA andDHEA-S secretion were elevated, but not androstene-dione or cortisol. Interestingly, DHEA possesses littleintrinsic biological activity and must be converted tomore active metabolites in peripheral tissues to exertphysiological actions [26,28–30]. Skin, hair follicles andsebaceous glands contain the enzymes necessary tometabolize DHEA into active androgens (testosteroneand dihydrotestosterone) and estrogens (estradiol andD5-diol) as well as the sulfatase responsible for liberat-ing DHEA from DHEA-S [7,15,19,28,34,37,59]. Theproduction of biologically active hormones within thesame cells that respond to them is referred to as in-tracrinology [26–29], a phenomenon that could providefor local control of the hair growth cycle with little orno change in blood steroid concentrations. It is possiblethat anagen initiation in mink is regulated, in part, bythe intracrine formation of steroids in the skin, withDHEA serving as a substrate, under the influence ofPRL. Therefore, our objectives were to determine: (1)serum concentrations of DHEA in mink during thesummer and winter hair growth cycles, in animalsexhibiting elevated, suppressed and naturally changingserum PRL concentrations; (2) if inhibition of PRLsecretion with melatonin (MEL) and the resulting blockto summer anagen could be restored by ADX; and (3)if the onset of winter anagen could be inhibited byelevating endogenous PRL levels with the dopaminergicantagonist haloperidol (HAL).

2. Materials and methods

Animal care and research procedures were approvedby the Institutional Animal Care and Use Committeeof Idaho State University and complied with the Guidefor the Care and Use of Laboratory Animals.

2.1. Experiment 1

Seventy-two adult female dark mink were movedfrom outdoor ranch conditions to our indoor animalfacility on the Idaho State University campus duringthe last week of February. Animals were housed indi-vidually in wire mesh cages, fed a mixture of chickenand fish by-products, and received water ad libitum. Allmink were exposed to a photoperiod approximatingnatural changes in day length with room temperaturemaintained at 2593°C [51].

After a 1-week acclimatization period the animalswere assigned randomly to one of seven treatmentgroups (Table 1). On 13 March, mink in group one

(controls) each received an empty silastic (Dow Corn-ing, Midland, MI) implant (50 mm×3.35 mm ID×4.64 mm OD) inserted subcutaneously over theinterscapular region under ketamine hydrochlorideanesthesia (50 mg kg−1; Aveco Co., Fort Dodge, IA).On 14 March, animals in groups 3, 5 and 7 eachreceived two silastic implants each containing 25 mgMEL (Sigma Chemical Co., St. Louis, MO) insertedsubcutaneously over the interscapular region, to inhibitPRL secretion. On 15 March, mink in group 2 eachreceived a HAL implant (Innovative Research of Amer-ica, Sarasota, FL) containing 200 mg of the drug(60-day duration of release) to stimulate PRL secretion.Between 24 and 28 March, mink in groups 4–7 eachreceived a silastic implant containing 100 mg of deoxy-corticosterone (DOC; Sigma) in preparation for ADX.The administration of DOC, an adrenal steroid withmineralocorticoid activity, was essential in order tomaintain fluid and electrolyte balance following ADX.Between 29 and 30 March, mink in groups 4 and 5 wereadrenalectomized through a single mid-ventral incision,under isoflurane anesthesia (Ohmeda Caribe Inc., Lib-erty Corner, NJ) as previously described [49,51].

All mink had a 5 cm2 area of fur sheared from theright hip on 13 March. Guard hair measurements weresubsequently taken at weekly to biweekly intervals from13 March until 5 June 1995, as previously described[53]. The onset of anagen was estimated as the day onwhich hair growth could be measured above the surfaceof the epidermis. Although some investigators use in-creased skin pigmentation as an indicator of the onsetof anagen [2,39,40], we find this method to be lessconsistent than the visible appearance of hair shafts.For example, hair growth induced by HAL or DOCresults in a pelage that is less dense than normalbecause of a reduction in the number of underhair typefibers that develop. Because guard hair follicles developfirst, followed some 10–12 days later by underhairfollicles in mink [65], the increased skin pigmentationprior to eruption of hairs is not always obvious in minkwith reduced underhair development. Admittedly, ei-ther of these methods is still an approximation of theonset of anagen and histological analysis of skin sam-ples would be required to more accurately determinethe precise time of epidermal stem cell activation.

Table 1Experiment 1 design

NGroup Treatment

12 Control12 12 Haloperidol

12 Melatonin3114 ADX+DOC125 ADX+DOC+Melatonin

66 DOC77 Melatonin+DOC

J. Rose et al. / Comparati6e Biochemistry and Physiology, Part A 121 (1998) 263–271 265

Table 2Experiment 2 design

Group N Treatment

Control1212 12 Haloperidol

123 Melatonin8 ADX+DOC4

5 8 ADX+DOC+Haloperidol7 DOC6

tion 50 m l of 0.05 M phosphate buffered saline (PBS,pH 7.3–7.4) were added to the reaction tube contain-ing the cPRL (5 mg in 50 m l of 0.01 M NaHCO3, pH7.5), followed by 0.5 mCi Na125I (100 mCi ml−1;ICN Biomedicals, Irvine, CA) and 20 mg chloramine-T (Sigma) in 50 m l of 0.05 M phosphate buffer (pH7.5). After 15 s the reaction was stopped by adding80 mg of sodium metabisulfite (Sigma) in 100 m l of0.5 M PBS. The 125I-labeled cPRL was isolated fol-lowing elution of the entire reaction mixture througha column of Sephadex G-100 (Sigma).

Mink serum PRL concentrations were measured bya heterologous double antibody RIA [62]. To eachserum sample of 200 m l were added 100 m l of assaybuffer, followed by 100 m l of a 1:65000 dilution ofthe first antibody (guinea-pig anti cPRL;AFP1062091) and then 50 m l of 125I-labeled cPRL(20000 cpm). The tubes were gently vortexed, coveredand incubated at 4°C for 16–18 h. Subsequently, 200m l of assay buffer containing the second antibody(goat anti-guinea-pig IgG; Antibodies Inc., Davis,CA) at a 1:12 dilution were added to the tubes, vor-texed and incubated an additional 3–4 h. The reac-tion was then terminated by adding 2.0 ml of colddistilled water and centrifuging at 1500×g for 30min at 4°C. The resulting supernatant was decantedand the tubes allowed to dry in the inverted positionfor 2 h prior to counting the pellets.

To test for accuracy, 100 m l of mink serum (col-lected in November when endogenous serum PRLconcentrations are low) plus 200 m l of assay buffer orvarying amounts of reference standard cPRL (0.1–24ng in 200 m l of assay buffer) were assayed in parallelwith the reference standards alone.

In addition, a pool of mink serum collected fromHAL-treated animals (high PRL levels) was seriallydiluted and assayed for PRL concentrations to testfor parallelism with the assay standard curve.

The standard curve in the absence and presence ofmink serum exhibited a high degree of parallelism(r2=0.99 for both, with slopes of −2.309 for assaybuffer and −2.302 for mink serum) indicating thelack of non-specific interference from the mink sera.The recovery of varying amounts of cPRL added to aconstant volume of mink serum was highly correlated(r2=0.97). The slope for this relationship was only0.79, indicating that our assay detects less PRL thanis actually present. When a pool of mink serumknown to be high in PRL was serially diluted withassay buffer, the binding kinetics (r2=0.99; slope=−2.87) were very comparable to those obtained forthe assay standard curve (r2=0.99; slope= −2.43),also confirming little non-specific interference fromthe mink sera. The intra-assay coefficients of varia-tion were determined by pooling mink serum ob-tained on 10 April, from mink previously treated on13 March with HAL to increase PRL concentrations,

Blood samples were collected at approximately bi-weekly intervals (13 and 27 March, 10 and 24 April,8 and 22 May and 5 June) from mink in groups 1, 2and 3 by jugular venipuncture, under ketamine hy-drochloride anesthesia. Due to the high mortality pre-viously associated with ADX in mink [49,51], bloodsamples were only collected from these mink at thetime they were sacrificed (5 June).

2.2. Experiment 2

During the last week of June, 59 adult female minkwere moved to our indoor animal facility, housed asdescribed above and assigned at random to one of sixtreatment groups (Table 2). On 27 June, mink ingroup one each received an empty silastic implantinserted subcutaneously under ketamine hydrochlorideanesthesia and represented controls. Mink in groups 2and 5 each received a subcutaneous implant contain-ing 200 mg HAL, while mink in group 3 each re-ceived two subcutaneous silastic implants eachcontaining 25 mg MEL. Animals in groups 4–6 eachreceived an implant containing 100 mg DOC, on 6July. Between 9 and 10 July, mink in groups 4 and 5were adrenalectomized as described above. At thetime treatments were administered a 5 cm2 area of furwas sheared from the right hip and guard hair mea-surements were taken at weekly or biweekly intervalsfrom 12 July through 15 October. Blood sampleswere collected from mink in groups 1–3, on 12 and26 July, 14 August, 7 September and 8 October.Mink were sacrificed by anesthetizing with ketaminehydrochloride and then administering an overdose ofsodium pentobarbital. Blood samples were allowed toclot overnight at 4°C prior to harvesting and storageof serum at −80°C. Serum samples were subse-quently assayed for PRL and DHEA concentrationsby radioimmunoassay (RIA).

2.3. Prolactin RIA

Canine prolactin (cPRL; AFP2451B) was iodinatedby the chloramine-T procedure [18]. At each iodina-


MEL to reduce PRL concentrations and controls.Based on 12 determinations of each serum pool, intriplicate, the coefficients of variation were 4.28%(HAL), 10.49% (MEL) and 4.12% (control).

2.4. Dehydroepiandrosterone RIA

Dehydroepiandrosterone (DHEA) was measured by ahighly specific solid-phase RIA utilizing 125I-labeledDHEA (Diagnostic Products Corporation, Los Angeles,CA). The steroids were extracted from 1.0-ml serumsamples with diethyl ether (Fisher Scientific) and thedried extract was resuspended in 600 m l of assay kitbuffer. Each sample was measured in duplicate using 200m l of resuspended extract.

To test for accuracy, 1:1 dilutions of pooled minkserum (100 m l) and assay buffer (100 m l) containingvarious concentrations of DHEA ranging from 0.25 to15.0 ng were incubated in parallel with the standardcurve. In addition, a pool of mink serum was seriallydiluted to test for parallelism with the standard curveof the assay. Intra-assay variation was evaluated byrepeatedly (N=4) measuring the DHEA concentrationsin two pools of mink serum previously shown to exhibithigh and low DHEA concentrations in anotherlaboratory.

The standard curve in the absence and presence ofmink serum exhibited a high degree of parallelism(r2=0.999 for both, with slopes of −1.37 for assaybuffer and −1.49 for mink serum) indicating the lackof non-specific interference from the mink sera. Therecovery of varying amounts of DHEA added to aconstant volume (100 m l) of mink serum was highlycorrelated (r2=0.999; slope=0.99), indicating that theassay detects accurately the amount of DHEA presentand that extraction efficiency is very high. When poolsof mink serum, high and low in DHEA, were repeatedly(N=4) measured for DHEA levels, the intra-assaycoefficient of variation for the high DHEA serum poolwas 0.83% and for the low DHEA serum pool was4.77%.

2.5. Analysis of data

Only data obtained from mink that survived until thecompletion of the experiments were statistically ana-lyzed. All data obtained on serum PRL and DHEAconcentrations were transformed to natural logarithmsfor statistical analysis. Non-transformed data are uti-lized in graphs depicting the changes in serum hormoneconcentrations in control and treated mink over time.Differences between treatment groups were analyzed byone-way analysis of variance or unpaired Student’st-tests (Instat, San Diego, CA). All radioimmunoassaysfor each experiment were conducted in a single assay;thus, no interassay coefficients of variation are reported.

3. Results

3.1. Experiment 1

3.1.1. Fur growthControl mink exhibited summer anagen between the

last week of April and first week of May (Fig. 1a).Treatment with HAL induced summer anagen 2 weeksearlier than controls in approximately 40% of the ani-mals (PB0.05; Fig. 1a). On 17 April, 60% of the

Fig. 1. (a) Percent of mink in the actively growing (anagen) stage ofthe summer fur growth cycle from 13 March to 5 June. Mink weretreated with haloperidol (HAL) (, N=8), melatonin (MEL) (�,N=5), deoxycorticosterone (DOC) ( , N=6), bilaterally adrenalec-tomized (ADX)+DOC (", N=8), ADX+DOC+MEL (�, N=11), MEL+DOC (�, N=7) and controls (�, N=10). (b) Mean(9S.E.) serum prolactin concentrations in adult female mink from13 March to 5 June. Means were derived from triplicate determina-tions for each animal. (c) Mean (9S.E.) serum concentrations ofdehydroepiandrosterone (DHEA) from 13 March to 5 June. Meanswere derived from duplicate determinations for each animal.


HAL-treated mink exhibited hair growth whereas noneof the controls were in anagen (PB0.05). However, itwas not until early May that all HAL-treated mink werein anagen. All mink treated with MEL alone or incombination with DOC failed to exhibit summer anagenduring the entire course of the experiment. In contrast,mink treated with MEL and ADX displayed summeranagen at the same time as controls. Exogenous DOCresulted in summer anagen 2 weeks earlier than controlsin approximately 50% of the mink (PB0.05; Fig. 1a),whereas the remaining animals did not enter anagen untilafter controls. Mink that were treated with DOC andsubsequently subjected to ADX began summer anagenapproximately 2 weeks earlier than controls (PB0.05;Fig. 1a).

3.1.2. Serum PRLSerum PRL concentrations in control mink increased

from less than 10 ng ml−1 in mid-March to 60 ng ml−1

by late April (Fig. 1b) and remained elevated through 5June. HAL increased serum PRL concentrations from 10to 60 ng ml−1 on 10 April, 2 weeks earlier than controls(PB0.05); subsequently, hormone levels declined suchthat by mid-May blood levels were less than controls.Serum PRL levels in HAL-treated mink declined beforethe implants were expected to be depleted of the drug(mid-May). MEL prevented the increase in PRL levelsnormally observed in the spring, keeping PRL concentra-tions at less than 7 ng ml−1 from 13 March through 5June.

3.1.3. Serum DHEASerum DHEA concentrations in control mink de-

creased after 13 March, remained low until late April,then increased through the first week of May and thendeclined remaining low through 5 June (Fig. 1c). Treat-ment of mink with HAL resulted in DHEA levels thatwere not different than controls, except on 8 May, atwhich time they were lower (PB0.05). Exogenous MELresulted in DHEA concentrations that were significantlyhigher than HAL-treated animals from 27 March to 5June (PB0.05). On 10 April, DHEA levels in responseto MEL were approximately six times that of controls,and five times that of HAL-treated mink (PB0.05).Thereafter, DHEA concentrations in response to MELdecreased sharply to levels approximating controls by 24April, then continuously increased through 5 June.

3.2. Experiment 2

3.2.1. Fur growthMost (90%) control mink were in winter anagen by 20

September (Fig. 2a). All mink treated with MEL were inwinter anagen by 14 August, over 1 month earlier thancontrols (PB0.05). Mink treated with HAL (60%, N=6) exhibited anagen the same time as MEL-treated mink

Fig. 2. (a) Percent of mink in the actively growing (anagen) stage ofthe winter fur growth cycle from 12 July to 15 October. Mink weretreated with melatonin (MEL) (�, N=11), deoxycorticosterone(DOC) (�, N=5), bilaterally adrenalectomized (ADX)+DOC (",N=5), ADX+DOC+haloperidol (HAL) (�, N=5), or as controls(�, N=9). (b) Mean (9S.E.) serum prolactin concentrations inadult female mink from 12 July to 8 October. Animals were treatedwith haloperidol (, N=10), melatonin (�, N=11) and controls(�, N=9). Means were derived from triplicate determinations foreach animal. (c) Mean (9S.E.) serum dehydroepiandrosterone(DHEA) concentrations in mink from 12 July 1995 through 8 Octo-ber 1995. Animals were treated between 27 June and 6 July 1995 withhaloperidol (, N=10), melatonin (�, N=11) and controls (�,N=9). Means were derived from duplicate determinations for eachanimal.

(14 August), although the fur was not replaced in awave-like pattern, was patchy and almost devoid ofunderhair fibers and are therefore not graphically pre-sented. The remaining HAL-treated mink were in anagenon 8 October. Mink treated with HAL+ADX or ADXonly, exhibited winter anagen 5 weeks earlier thancontrols (PB0.001) and developed a normal densewinter pelage.


3.2.2. Serum PRLControl mink exhibited serum PRL levels of \40 ng

ml−1 from mid-July to mid-August, after which con-centrations declined to B15 ng ml−1 by 7 September(Fig. 2b). MEL reduced serum PRL levels to less than10 ng ml−1 between 14 August and 8 October. HALresulted in serum PRL concentrations that were higherthan controls from 12 July though 7 September (PB0.05), although a rapid decline was observed after 14August, analogous to controls. The HAL implants hada 60-day duration of release and should therefore havebeen depleted of the drug by 7 September, althoughPRL levels were still higher than controls until 8October.

3.2.3. Serum DHEASerum DHEA concentrations in control and HAL-

treated mink exhibited no significant change between 12July and 15 October, remaining at less than 5 ng ml−1

(Fig. 2c). In contrast, exogenous MEL resulted in acontinuous increase in serum concentrations of DHEAfrom basal levels of less than 2.090.342 ng ml−1 on 12July to over 14.094.466 ng ml−1 on 8 October (PB0.05). Serum DHEA levels in MEL-treated mink werehigher than controls between 14 August and 7 Septem-ber (PB0.05) and, although not significantly different,tended to be much higher on 8 October.

4. Discussion

The onset of summer anagen in control mink duringlate April was preceded by a large increase in serumPRL concentrations (Fig. 1a,b). Stimulating PRL secre-tion (HAL) advanced the time of onset, whereas inhibi-tion of PRL secretion (MEL) completely inhibitedsummer anagen. Although these findings are in agree-ment with those of others [2,39,41], showing that in-creasing serum PRL levels is correlated with onset ofsummer anagen, it remains to be demonstrated thatthere is a cause and effect between PRL and inductionof anagen in mink. For example, although exogenousMEL suppressed PRL secretion in male [2] and femalemink [39], and delayed summer anagen for as much as3 months, hair growth still occurred. Moreover, minkcontinue to exhibit summer and winter anagen whensubjected to artificial photoperiods that suppress PRLconcentrations to non-detectable levels [42]. In sheep,anagen precedes the seasonal rise in PRL secretion, andexposure to a short photoperiod that reduces PRLsecretion does not inhibit anagen [47].

Although the role of PRL in anagen induction isquestionable, the peptide may affect hair density. Insheep, elevated serum PRL levels are correlated with areduction in the number of anagen hair follicles,whereas reduced PRL levels are correlated with a

greater number of anagen follicles [3,10,47]. In thecashmere goat, exogenous PRL induced earlier growthof summer-type underhair but had no effect on the timeat which guard hairs were reactivated [12]. Because thetotal number of hair follicles is determined at birth [16],it is possible that low PRL levels permit a greaternumber of telogen follicles to become activated, enter-ing anagen.

ADX initiated summer anagen 2 weeks earlier thancontrols (PB0.05), whereas in a previous study [49] weobserved summer anagen 6 weeks earlier than controlsfollowing ADX. We suspect that, because the animalsin the present study were adrenalectomized at a latertime (29–30 March) compared to our previous study(2–11 March), there was a corresponding delay in onsetof anagen.

Serum PRL concentrations in ADX mink on 5 June(3 months after ADX) were 45.67910.236 ng ml−1

and were not different from control (58.19693.264 ngml−1) or DOC-treated animals (38.16696.301 ngml−1). In the rat, ADX produces a transient rise inserum PRL levels 12 days after surgery [5,32]. There-fore, it is possible that mink serum PRL levels wereelevated in the present study soon after ADX and thenreturned to basal levels 3 months later. However, in arecent study of the winter fur growth cycle, we coulddetect no difference in serum PRL levels between con-trol and ADX+DOC-treated mink starting as early as7 days after ADX (11 July) and continuing through 12November [24]. Although it remains to be determined ifADX increases PRL levels prior to summer anagen inmink, our data suggest that ADX-induced summeranagen is independent of changes in serum PRL con-centrations. Because the inhibition in PRL secretionand block in summer anagen in response to MEL werecompletely overcome by ADX (Fig. 1a,b) we concludethat the significance, if any, of increasing PRL levels inthe spring, might be to alter (decrease?) adrenal steroidsecretion and/or influence the metabolism of thesecreted steroids.

The stimulatory effect of DOC on induction of sum-mer anagen (Fig. 1a) is perplexing, yet is in agreementwith previous findings [49]. Although DOC has primar-ily mineralocorticoid actions [43], we cannot rule outthe possibility that the steroid influenced adrenocorti-cotropin (ACTH) secretion and therefore adrenal hor-mone production that contributed to the onset ofanagen. That DOC did not counteract the effects ofMEL (DOC+MEL) suggests that the anagen-inducingcapacity of DOC may require PRL.

Because serum concentrations of DHEA are higherin primates than rodents and other laboratory animals[26,29,30,43], it was of interest that mink serum DHEAconcentrations (1–15 ng ml−1; present study) werecomparable to those in primates [11,43,58]. Althoughwe could detect no change in serum DHEA concentra-


tions in mink during the telogen to anagen transition,there is evidence in primates to suggest that DHEA issecreted in a circadian pattern, with maximal concen-trations occurring during the morning hours [11,58]. Ifa circadian pattern of DHEA secretion exists in mink,then even a 1-h difference in time of blood collection ondifferent days might give misleading results with respectto circannual patterns of secretion. We believe that partof the variation in our data is the result of samplesbeing collected over a period of several hours.

Because DHEA levels were significantly higher inMEL- than HAL-treated mink suggests to us that PRLinhibits and/or MEL stimulates DHEA secretion (Fig.1c, Fig. 2c). That MEL might stimulate DHEA secre-tion in vivo in the mink is supported by the observa-tions that adrenal glands express MEL binding sites[6,21,48] and that MEL stimulates, in a dose-dependentmanner, ACTH induced production of DHEA fromcultured mouse adrenals [20]. The initial decline inDHEA levels on 27 March (similar to controls) inMEL-treated mink, may have occurred because MELimplants were administered on 14 March and bloodconcentrations of the hormone may not have becomeelevated sufficiently to be effective on the adrenalgland. Although PRL usually increases blood DHEAand DHEA-S levels in primates [1,64], we found nodifference in DHEA levels between control and HAL-treated mink during either the summer or winter furgrowth cycles, even though serum PRL levels weresignificantly different between the two treatments (Fig.1b,c, Fig. 2b,c). We hypothesize that part of the effectsof MEL on hair growth in mink may be mediateddirectly through the adrenal glands and the secretion ofDHEA.

During the winter fur growth cycle, control minkexhibited winter anagen during the second week ofSeptember after PRL levels had decreased to approxi-mately 25% of summer telogen values (Fig. 2a,b).Treatment with MEL caused serum PRL levels todecline to basal levels (B10 ng ml−1) by 14 August,and all mink were in winter anagen 4 weeks earlier thancontrols (PB0.05). These findings confirm previousresults [53,56] and suggest that a reduction in circulat-ing PRL levels is requisite to onset of winter anagen. Itwas surprising, therefore, that exogenous HAL whichincreased PRL secretion induced anagen in 50% of themink almost 4 weeks earlier than controls (PB0.05).The type of pelage in all mink treated with HAL alonewas less dense than winter or summer fur, exhibitingvery little underhair growth. In ADX+HAL-treatedmink, winter anagen began 4 weeks earlier than con-trols, analogous to ADX alone (Fig. 2a), and theresulting pelage was similar to controls with respect tohair length and density. Thus ADX neutralized theeffects of MEL (low PRL levels) in the spring and HAL(high PRL levels) in the fall. Interestingly, HAL has

been shown to stimulate ACTH secretion in rats [17]and dogs [25]. Because intradermal injections of ACTHstimulate hair growth in mice [46] and mink [50] it ispossible that part of the anagen-promoting effects ofHAL are mediated through pituitary ACTH secretion.It would appear that ADX-induced winter anagen isindependent of changes in serum PRL levels.

In an analogous manner to that observed during thesummer fur growth cycle, the role of PRL in develop-ment of the winter fur may be related to hair density. Inagreement with this hypothesis, exposure of mink to along photoperiod (16L:8D) increases PRL levels, anddelays the onset of winter anagen resulting in pelagewith reduced follicular density [40]. In contrast, follicu-lar density was increased 20% in these same mink whensimultaneously treated with bromocryptine to suppressPRL secretion.

Serum DHEA concentrations in control and HAL-treated mink exhibited no change during onset of win-ter anagen and were not different between the twotreatments during the entire study period (Fig. 2c). Incontrast, mink that were treated with MEL exhibited agradual increase in DHEA levels, which were higherthan controls between 14 August and 7 September(PB0.05), although no change was detected duringonset of anagen.

We conclude that changes in serum concentrations ofDHEA and PRL are not essential to the onset ofsummer or winter anagen in mink. However, this stilldoes not reject the hypotheses that metabolites ofDHEA and/or permissive actions of PRL are involvedin hair growth cycles. We suggest that guard hairfollicles and the smaller underhair follicles may responddifferently to the effects of PRL. High PRL levels in thespring may reduce activation of a subpopulation ofunderhair follicles resulting in the less dense summerfur, whereas low PRL levels during the fall may allowfor activation of a greater number of underhair folliclesresulting in the dense winter fur. In addition to themink [57], receptors for PRL are present in the skin ofsheep [8] and rats [44]. Moreover, PRL increases hairshaft elongation in isolated hair follicles of Red Deer[63] and goats [23], suggesting a direct action of thehormone on the hair follicle. Rats that are hyperpro-lactinemic exhibit increased skin mRNA expression andactivity of type IV 3b-hydroxysteroid dehydrogenase(3b-HSD), an enzyme that uses DHEA as a substrate[9]. In contrast, PRL has been shown to decrease3b-HSD activity (81% reduction) in ovarian tissue ofhypophysectomized rats [35–37] and therefore the con-version of DHEA to androstenedione. Thus, part of theeffects of PRL on hair growth could be mediatedthrough its affects on DHEA metabolism in the skinand/or hair follicle, even though serum levels of thesteroid remain unchanged. Finally, the effects of MELon hair growth may be mediated in part directly on the


skin. In support of this hypothesis, the skin of rodentssynthesizes MEL [61] and 3H-labeled MEL bindingsites [60]. However, Dicks et al. [13], using 125I-labeledMEL, failed to detect binding sites for the hormone inthe skin of sheep. Studies are currently in progress inour laboratory to determine if the skin of mink exhibits125I-labeled MEL binding sites.

Acknowledgements

This work was supported by The American MinkFarmers Research Foundation (Corvallis, OR), TheCanada Mink Breeders Association (Toronto, Canada)and a grant from the National Institutes of Health,NIH-IDeA/EPSCoR (1 P20 RR11833-01) to enhancecore research laboratories at Idaho Universities. Minkand feed were generously donated by Lee Moyle ofMoyle Fur Farms, Heyburn, ID. We thank Jim Peck,Jerry Van Tassel and Stan Burr for care of the animals.Dr Rodney Mead of the University of Idaho kindlyshared his PRL RIA procedure. We are especiallyindebted to Diagnostic Products for their assistance inhelping us to validate their DHEA RIA kits for minkserum. We also thank Bannock Regional Medical Cen-ter and Pocatello Regional Medical Center for theirkind donation of surgical supplies. The canine PRL forthe RIA, and first antibody (guinea-pig anti-caninePRL) was generously provided by Dr A.F. Parlow ofthe Pituitary Hormones and Antisera Center, Harbor-UCLA Medical Center. Funds to defray part of thecost of publishing this work were supplied by TheOffice of Research at Idaho State University.

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Documents

Serum prolactin and dehydroepiandrosterone concentrations during the summer and winter hair growth cycles of mink (Mustela vison)