Bone 44 (2009) 691698

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Bone
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Testosterone, but not IGF-1, LH, prolactin or cortisol, may serve as antler-stimulating hormone in red deer stags (Cervus elaphus)
Ludk Barto a,, Dieter Schams b, George A. Bubenik c
a b c

Department of Ethology, Institute of Animal Science, Ptelstv 815, 106 00 Praha 10-Uhnves, Czech Republic Institute of Physiology, University of Munich-Weihenstephan, Freising-Weihenstephan, Germany University of Guelph, Guelph, Ontario, Canada

a r t i c l e

i n f o

a b s t r a c t
The role of androgens and insulin-like growth factor 1 (IGF-1) in antler growth has been disputed. We predicted that the secretory of IGF-1 may be associated with an acceleration of body growth rather than with antler growth. Furthermore we anticipated a relationship between the increase of testosterone and the progress of antler growth. If IGF-1 is involved in the stimulation of antler growth, this should be more obvious in young than in mature stags. Eight two-year-old red deer stags (Cervus elaphus), and twelve adult red deer stags were blood sampled and the length of their velvet antlers was measured in one-week intervals during the period of antler growth. Concentrations of testosterone, cortisol, IGF-1, luteinizing hormone (LH), and prolactin were determined in plasma by enzyme immunoassay or radioimmunoassay. Antler growth per day was primarily dependent on changes in testosterone concentration per day in both groups of stags. As expected, only in two-year-old stags we detected a possible role of IGF-1 in the antler growth regulation, but that was not in agreement with previously published studies. Nevertheless, this effect was still utilized in interaction with testosterone. In addition to total antler length, only concentrations of testosterone and LH were signicantly higher in adult males in comparison to two-year-old males. Our present results lead us to conclude that it is not IGF-1 but testosterone which is responsible for the intensity of antler growth in subadult and adult red deer stags. 2008 Elsevier Inc. All rights reserved.

Article history: Received 1 October 2008 Revised 12 November 2008 Accepted 5 December 2008 Available online 16 December 2008 Edited by: T. Matsumoto Keywords: Antler growth Testosterone IGF-1 Red deer Cervus elaphus

Introduction The regeneration and growth of deer antlers has been proposed as a model for biomedical research in several publications [1,2]. As shown previously [3,4], understanding of the processes in antlers may be helpful in human osteological research. Role of steroids, testosterone and estrogen in particular, in bone biology seems to be of special importance in elderly people. Generally, testosterone promotes secondary sexual characters across many taxa [5]. The observed evolutionary conservation in the regulation of traits induced by testosterone in male vertebrates suggests that this system evolves as a complex, with the linkage between testosterone and male traits being inseparable [6]. In this respect, some authors suggest, that antlers, as typical male secondary sexual characters, should be controlled otherwise. When Suttie et al. [7, 8] compared seasonal variations of hormones with the progress of antler growth in red deer, Cervus

Sources of support: Funded by grants from the Czech Science Foundation (grant number 523/08/0808) and Ministry of Agriculture of the Czech Republic (grant numbers MZe 0002701402 and MZe 0002701404). All authors have no conicts of interest. Corresponding author. Fax: +420 267 710 779. E-mail addresses: bartos@vuzv.cz (L. Barto), schams@wzw.tum.de (D. Schams), gbubenik@uoguelph.ca (G.A. Bubenik). 8756-3282/$ see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2008.12.004

elaphus, they concluded that insulin-like growth factor 1 (IGF-1) is an antler-stimulating hormone. This hypothesis replaced an earlier notion which suggests that the antler-stimulating hormones are androgens, particularly testosterone or its derivatives [912]. To further support the role of IGF-1 as the antler-stimulating hormone, a number of in vivo and in vitro studies have been performed [1317]. A similar increase of IGF-1 concentrations corresponding to the progress of antler growth was also reported in roe deer, Capreolus capreolus [18] and pudu, Pudu puda [19]. Nevertheless, in a later study we have demonstrated that in pudu the increase of IGF-1 is found only in the dominant males but not in the subordinate ones [20]. Data on male and female reindeer, Rangifer tarandus [21,22] and on castrated fallow deer, Dama dama [23] did not conrm Suttie et al.'s [7] conclusions either. In another study with yearling red deer stags, Suttie et al. [24] reported that IGF-1 correlated positively with antler growth rate, but the pattern of the hormone shown in their Fig. 1 raises some questions. The most pronounced discrepancy was apparent during the last two months of the antler growth period presented in the same gure in that study [24], when IGF-1 concentrations were sharply decreasing, while antlers were still growing. Similar discrepancy may be found also in a study on reindeer calves [21]. Thus, the role of androgens and IGF-1 in antler growth remains disputed. IGF-1 is generally an important component of the growth process and a

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Fig. 1. Total antler length (means S.E.) of two-year-old and adult red deer males.

certain role in antler growth seems very plausible [25]. However, the role of IGF-1 as the principle hormone responsible for controlling antler growth is controversial. Otherwise all reports examining IGF-1 time course during the period of antler growth should be consistent. One of the reasons for the inconsistency in the reports may be the age of the subjects. In their rst experiment Suttie et al. [7] used yearling stags. Also all other studies providing information on function and regulation of IGF-1 in red deer have been made on calves [24,2630]. Yearling red deer are not yet mature and their antler growth occurs at the time of the accelerated body growth [31,32]. IGF-1 correlates strongly with body growth [33]. Hence, we can hypothesize that the IGF-1 pattern may be basically associated with the accelerated body growth rather than with the antler growth. As such, an effect of the hormones on the antler growth should be tested in principle in mature males with nished body growth and not in still growing yearlings. In addition increasing IGF-1 concentrations associated with body growth spurt in yearlings could mimic association with antler enlargement spurting at the same time. Therefore, we expected to nd differences in the IGF-1 time courses of young and mature stags as they relate to antler growth. Furthermore we anticipated a relationship between the increase in testosterone and the progress of antler growth. If IGF-1 is involved in the stimulation of antler growth, this should be more obvious in young, than in mature stags. Finally, taking into account an increase in the antler length with age [34], an increase in antler size should be associated with a comparable increase in concentrations of hormones responsible for antler growth. Material and methods Animals and procedures Eight two-year-old red deer stags (Cervus elaphus) and twelve stags, age 4 to 9 years (adults), were kept in a paddock at the animal facility of the Institute of Animal Science (former Research Institute of Animal Production). The deer were kept in a group outdoors and were fed on grass supplemented with barley (0.75 kg/animal/day) and hay. Always at the same time of day they were handled in a crush and blood sampled and the length of their velvet antlers was measured using a exible measuring tape. Deer were sampled in one week intervals ranging from the time prior the antler casting to the end of antler growth and velvet shedding. Left and right antlers were measured and mean values of the antler lengths from both sides entered the analysis. Blood samples were collected using VENOSAFE blood collection system (TERUMO Europe N. V.).

Hormone analyses Concentrations of testosterone, cortisol, IGF-1, and luteinizing hormone (LH) were determined in plasma by enzyme immunoassay (EIA) or radioimmunoassay (RIA). For each hormone, all samples were assayed at the same time. Testosterone was measured by EIA [35] with a double antibody technique after extraction of 1 ml plasma with 3 ml tertiary butylmethylether/petrolether 30/70 (v/v). After freezing, the solvent fraction was decanted and evaporated in a water bath at 60 C. The extract was dissolved in 100 l assay buffer, and 20 l/tube were analyzed. The assay used a polyclonal antibody raised in a rabbit against testosterone-11-hemisuccinate-BSA, and the label was testosterone-3-carboxy methyl-oxime-horseradish peroxidase. Cross reactivities to other steroids were as follows: testosterone 100%, 5adihydrotestosterone 10%, androstendione 2%, oestradiol b 0.1% and progesterone b 0.1%. The standard curve ranged from 0.225 pg/tube, corresponding to a sensitivity of 10 pg/ml plasma or 1 pg/ml plasma after concentration (dissolving in 100 l buffer only). The ED50 of the standard curve was 2 pg/tube. The intra- and inter-assay coefcients of variation were an average 6.7% and 10.5% respectively. IGF-1 was determined by RIA after acidethanol extraction of 50 l of blood plasma [18]. Recombinant human IGF-1 (Lilly Company, Saint Louis, USA) was used for iodination according to the lactoperoxidase method and as standard. Separation of bound and free hormone was accomplished by a combination of second-antibody technique with 4% polyethylene glycol. The assay was validated for red deer plasma by recovery studies (IGF-1 added to plasma samples before extraction) and was an average 89% and complete parallel displacement was observed between extracted samples and the standard curve. Intraand inter-assay CVs were b 10% and b 16%, respectively. Cortisol (4-pregnene-11-17-21-triol-3, 20-dione, Steraloids, Wilton, N. H., USA) concentrations were measured with a competitive EIA [36]. The antibody was raised in a rabbit against cortisol-21-hemisuccinateBSA. Cross reactivities were: cortisol 100%, cortisone 8%, corticosterone 9.5%, prednisolone 18%. Blood plasma was extracted with a 10 fold volume of tert-butyl methyl ether. After freezing at 60 C, extracts were decanted, evaporated and reconstituted. Interassay CV was between 7 and 14%. LH was determined by a homologous bovine assay with no cross reactivity to other pituitary hormones [37,38]. The reference preparation was a pure bovine pituitary extract prepared in our laboratory (LH-DSA with a biological activity of 1.0 times NIH-LH-S1). The assay was validated for fallow deer plasma by recovery studies of 4 different concentrations added to plasma. Average recovery was 96.4 6.4%.

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Fig. 2. Concentrations of testosterone, IGF-1, LH, cortisol, and prolactin (means S.E.) in the period from the day of antler casting to the end of antler growth.

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Dilution curves of fallow deer plasma ran parallel to bovine standard. The sensitivity was 0.05 ng/tube; intraassay CV averaged 8.2%, and interassay CV was 10.414%. Prolactin was determined by a homologous bovine RIA [37]. The antiserum produced in rabbits showed no cross-reactivity to other pituitary hormones. The sensitivity was 50 pg/ml. The assay was validated for the deer plasma by recovery experiments (recovery range 95%102% in deer plasma) and dilution curves, which run parallel to the standard. The pituitary bovine reference preparation (USDA-P-B4) was used. The intra-assay CV was 4.08.4% and the interassay CV was in the range of 8.212.4%. Statistics The analysis of the relationship between antler growth and hormone concentrations was done in three steps. First, we plotted raw data for two-years-old and adult males against the time course and compared visually possible relationship, as has been done previously by Suttie et al. [7]. In the second step we analyzed separately data for two-year-old and adult males to see if there is any difference in factors involved. As in our previous studies [23,39], in each male, areas under the curve were calculated for antler growth per day, testosterone, IGF-1, LH, cortisol and prolactin. To accomplish that, the interval between sampling sessions (in days) was multiplied by the mean value of each hormone calculated from the two successive sampling sessions. All data were analyzed with the aid of SAS System (SAS, version 9). The statistical analysis, using a generalized linear mixed model (GLMM) procedure (PROC MIXED), was performed only for samples showing detectable antler growth, i.e., at least one centimeter elongation since previous measurement. We analyzed associations between an increase in antler growth per day as the dependent variable and change per day in the area under the curve of hormonal concentration. For these variables we found an appropriate power transformation of the non-normally distributed observations/data by computing BoxCox family of power transformations [40] using PROC TRANSREG. To account for the repeated measures on the same individuals, the analysis was performed with individual male as a random factor in GLMM. Fixed effects were continuous variables change in area under the curve of testosterone, IGF-1, LH, cortisol and prolactin, body weight, age (from 4 to 9 years, used only in analyzing the adult males) and the order within the group in which the male was sampled, and a categorical variable month of collecting the samples (April, May, June, July, August and September). The signicance of each xed effect in the GLMM was assessed by the F-test, on sequential dropping of the least signicant effect, starting with a full model. When tting the best GLMM model, we followed the Fit Statistics table. Of the criteria available, we chose Bayesian Information Criterion (BIC) [41] which tends to favor less complex model. Associations between an increase in antler growth per day and change in the area under the curve of hormonal concentrations per day and age were estimated by tting a random coefcient model using PROC MIXED as described by Tao et al. [42]. With this random coefcient model we calculated predicted increase in antler growth per day and plotted them against change per day in the area under the curve of hormonal concentrations and/or age with predicted regression lines for each site of the sample collection obtained in Solution for xed effect table in the output of the analysis. The regression lines are based on partial tests. They measure the contribution of a regressor in the presence of all other regressor variables in the model. In the third step, we analyzed data of two-year-old and adult males together. We constructed a GLMM model for dependent variable total antler length, concentrations of testosterone, IGF-1, cortisol, LH or prolactin with only two xed factors: group (two-year-olds, adults) and month of collecting the samples (April, May, June, July, August and September). Also in this case, to account for the repeated measures on the same individuals, the analysis was performed with individual

male as a random factor in GLMM. In unbalanced designs with more than one effect, the arithmetic mean for a group may not accurately reect response for that group, since it does not take other effects into account. Therefore, we used Least-squares means, which are in effect within-group means appropriately adjusted for the other effects in the model (further referred as adjusted means). Results Antler growth and pattern of hormone concentrations Antler growth of two-year-old stags started in May and ceased at the beginning of August, while that of adult stags started in mid April and ceased in mid August (Fig. 1). Fig. 2 shows the pattern of testosterone, IGF-1, LH, cortisol, and prolactin concentrations of twoyear-old and adult stags during the period of antler growth. In contrast to all other hormones, only testosterone concentrations showed more or less consistent increase during the time of antler growth in both categories of the stags. A decline in testosterone concentrations observed at the end and/or just after the antler elongation was

Fig. 3. Top antler growth of two-year-old males (BoxCox transformed predicted values of the increase in antler length per day in cm) plotted against increase in testosterone concentrations (BoxCox transformed changes in area under the curve of testosterone per day) and bottom antler growth of two-year-old males (BoxCox transformed predicted values of the increase in antler length per day in cm) plotted against increase in IGF-1 concentrations (BoxCox transformed changes in area under the curve of IGF-1 per day). Regression lines were calculated according to equation Predicted value = 0.295 + 184.57 Testosterone 0.336 IGF-1 165.39 Testosterone IGF-1 for both testosterone (top, P b 0.03) and IGF-1 (bottom, NS).

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Fig. 4. Antler growth of two-year-old males (BoxCox transformed predicted values of the increase in antler length per day in cm) plotted against interaction between increase in testosterone concentrations (BoxCox transformed changes in area under the curve of testosterone per day) and increase in IGF-1 concentrations (BoxCox transformed changes in area under the curve of IGF-1 per day).

completed, was followed by another steady increase of testosterone concentrations (not shown here). Relationship between antler growth and hormones in two-year-olds The GLMM model showed a signicant effect of changes in testosterone concentration per day (GLMM, F(1, 18) = 5.57, P b 0.03), non-signicant changes in IGF-1 concentration per day (GLMM, F(1, 18) = 1.84, NS) and again, a signicant effect of the interaction between the changes in testosterone and IGF-1 concentrations per day (GLMM, F(3, 42) = 6.64, P b 0.02). Antler growth (an increase of antler length per day) was positively dependent on changes in testosterone concentration per day (Fig. 3 top). The potentially negative dependency of antler growth on increase in IGF-1 shown in Fig. 3 (bottom) was not signicant. Fig. 4 displays the relationship between antler growth and

the interaction between testosterone and IGF-1 concentrations demonstrating that antler growth was slowest with the highest concentrations of the two hormones when effect of testosterone as a single factor was statistically signicant. There was no other signicant GLMM model which would contain testosterone and any other single hormone. Relationship between antler growth and hormones in adults In adults, we tted the nal GLMM model containing xed factors changes in testosterone concentrations, age and the month when samples were taken. Antler growth per day was dependent on changes in testosterone concentration per day (Fig. 5, F(1, 42) = 15.94, P b 0.001), the month when samples were taken (F(4, 42) = 11.06, P b 0.001) and the interaction between age of the male and changes in testosterone concentration per day (Fig. 6, F(3, 42) = 9.56, P b 0.001). Antler growth was not dependent on any other hormone involved, either alone or in interaction with other factors. Comparison between two-year-old and adult males in antler length and hormonal concentrations Fig. 7 shows adjusted means (S.E.) of total antler length (F(1, 20) = 22.78, P b 0.001) and overall concentrations of testosterone (F(1, 20) = 15.58, P b 0.001), IGF-1 (F(1, 20) = 0.10, NS), LH (F(1, 20) = 11.19, P b 0.001), cortisol (F(1, 20) = 0.29, NS) and prolactin (F(1, 20) = 0.76, NS). Apart from the total antler length, only concentrations of testosterone and LH, but not of IGF-1 or prolactin, were signicantly higher in adult males than in the two-year-old males. Discussion In both age categories it was basically the change in testosterone concentrations which was positively associated with antler growth. This is in full agreement with our previous results on castrated fallow deer [23,39]. In Iberian red deer the most precocious males in antler growth also showed higher levels of testosterone during the rst and second year of age [43]. Moreover, it is also coherent with studies on mice and humans. Androgen receptor activation dominated normal trabecular bone development and cortical bone modeling in male

Fig. 5. Antler growth of adult males (BoxCox transformed predicted values of the increase in antler length per day in cm) plotted against increase in testosterone concentrations (BoxCox transformed changes in area under the curve of testosterone per day).

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Fig. 6. Antler growth of adult males (BoxCox transformed predicted values of the increase in antler length per day in cm) plotted against interaction between increase in testosterone concentrations (BoxCox transformed changes in area under the curve of testosterone per day) and age of the males (years).

mice [44,45]. A steep rise in testosterone levels in boys and girls precedes the pubertal increase in bone mass [46]. Supplemental treatment with testosterone increased bone density in hypogonadal and eugonada1 men [47,48]. As expected, only in two-year-old stags we detected a possible role of IGF-1 in the antler growth regulation, although far from the extent reported earlier [7,18]. The effect of IGF-1 was still utilized in interaction with testosterone. This was not consistent with earlier reports on red and roe deer [7,18]. In contrast, we found almost an opposite trend to those earlier reports. Our GLMM model for twoyear-old stags contained two hormones while that for the adults contained testosterone only. This may suggest some support for our prediction that the IGF-1 patterns in young animals are basically associated with the accelerated body growth rather than with the antler growth. (Still this might be clearer in calves which were not available in our study.) Future experiments dealing with antler growth physiology should be done with adult rather than young animals. It should also be pointed out that, whereas the levels of LH and testosterone were much higher in adult stags over all (Fig. 7), peak seasonal levels of both hormones in both groups were much closer (Fig. 2). Of the other hormones analyzed, only cortisol seemed to increase in the two-year-old stags. This may reect their increasing

stress while sharing the paddock with adult stags whose seasonal increase of aggression could be expected. It agrees with the data of Bubenik and Leatherland [49] who reported peak seasonal concentrations of cortisol in white-tailed bucks shortly before and during the rutting season. In adults, the increase in testosterone concentrations and the intensity of antler growth tended to increase with age while no signicant dependency was established between the rate of antler growth and the changes in concentrations of any other hormone investigated. This may reect a general trend of an increase of antler length in red deer stags even after ceasing somatic development [50]. Antlers were signicantly larger in our adult stags in comparison with two-year-olds as were the concentrations of testosterone and LH. This ts very well with our expectations. In cervids LH concentrations usually precede those of testosterone [23,38,5157]. LH concentrations thus stimulate an increase of testosterone which in this study appeared to be the principal hormone associated with antler growth. The extent of the increase in antler size in adults in comparison to two-years-olds was apparently comparable to the increase in concentrations of testosterone. The fact that no variation in seasonal concentrations of IGF-1, cortisol and prolactin was detected between two-year-olds and adults further supports the idea that these hormones are not involved in the principal regulation of antler growth.

Fig. 7. Two-year-old and adult males' adjusted means ( S.E.) of total antler length and over all concentrations of testosterone, IGF-1, LH, cortisol, and prolactin.

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As mentioned earlier, IGF-1 was associated with antler growth in some studies [7,18,19] but not in others including the present results [20,22,23]. In contrary to these data, antler growth was invariably associated with an increase of testosterone concentrations in all studies across many cervid species, such as red deer [7], roe deer [5860], fallow deer [23,39,61,62], white-tailed deer, Odocoileus virginianus [6366], Columbian black-tailed deer, Odocoileus hemionus columbianus [67], axis deer, Axis axis [55,68], pudu [19], Eld's deer, Cervus eldi thamin [69], reindeer [21], and rusa deer, Cervus (Rusa) timorensis [70]. It is obvious that the sharp increase in testosterone concentration which causes the mineralization of antlers and the shedding of velvet occurs only after the cessation of antler growth and the completion of antler bone development. The conclusion that a low concentration of testosterone and not IGF-1 serves as a permissive antler-stimulating hormone however does not challenge the fact that higher levels of testosterone stop all bone growth and induce antler bone mineralization, as observed across various deer taxa [12]. This ts very well with the earlier study of Brown et al. [10] who found positive correlations between increasing serum androgen concentrations and increasing antler mass in whitetailed bucks. They concluded that low concentrations of testosterone can stimulate bone growth, while a higher level will cause inhibition [10]. Recent in vitro studies support the notion that androgens (such as testosterone, DHT or DHEA) excert dose-dependent mitogenic effect on antler cells derived from the proliferation zone of the antler tip. However, the androgen effect may also depend on other serum factors such as anabolic hormones and serum-containing culture medium [71]. Testosterone has also a direct stimulatory effect on the growth of the mouse male-derived mandibular condyle. Overtreatment with testosterone (regarding both dosage and duration) causes enhanced calcication at the expense of chondrogenic activity, resulting in reduced overall condylar growth [72]. Recently, low but signicant levels of testosterone and estradiol were detected in growing antler bone of white-tailed deer [73] thus indicating a role of sexual steroids in antler development. A growth promoting effect of steroids can be brought about by increasing the production of IGF-1, which then acts as an autocrine, paracrine or endocrine growth factor [23,74]. In a number of studies and various taxa it has been reported, that testosterone stimulates growth and local production of IGF-I [72,73,75,76]. A lack of statistical difference between over all levels of IGF-1 in subadult and adult stags in the present study contrasts sharply with the signicant differences found for LH, testosterone and the rate of antler elongation (Fig. 7). Furthermore the time course of IGF-1 in both groups is rather at, unlike a rising trend of testosterone concentrations. Our present results thus lead us to conclude that it is testosterone (possibly in an interaction with other steroids) and not IGF-1 which is responsible primarily for the intensity of antler growth in subadult and adult red deer stags. Acknowledgments The authors gratefully acknowledge the assistance of Ji iler, Sobslav Losos and Petr Janovsk in managing the deer as well as the consultations on statistic with Petr imeek. We wish to thank two anonymous referees for their helpful comments. This study was supported by the Czech Science Foundation (grant number 523/08/ 0808) and Ministry of Agriculture of the Czech Republic (grant numbers MZe 0002701402 and MZe 0002701404). References
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