Evidence of higher levels of testosterone during the velvet period in muntjac than in other cervids Chiu-Shuang Lin1, Kurtis Jai-Chyi Pei2, *, Paulus S. Wang3, Bing-Tsan Liu4




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Evidence of higher levels of testosterone during the velvet period in muntjac than in other cervids

Chiu-Shuang Lin1, Kurtis Jai-Chyi Pei2, *, Paulus S. Wang3, Bing-Tsan Liu4

1Graduate Institute of Bioresources, National Pingtung University of Science and Technology, Neipu, Pingtung 912, Taiwan

2 Institute of Wildlife Conservation, National Pingtung University of Science and Technology, Neipu, Pingtung 912, Taiwan

3Department of Physiology, National Yang-Ming University, Sec.2, Li-Nong St., Pei-Tou,Taipei 112, Taiwan

4Department of Animal Science, National Pingtung University of Science and Technology, Neipu, Pingtung 912, Taiwan
Running header: testosterone production by Leydig cells

Keywords: antler cycle, Cervus nippon, Rusa unicolor, deer reproduction physiology, Leydig cells, Muntiacus reevesi, in vitro testosterone production.


*To whom correspondence and reprint requests should be addressed. Tel: 886-8-7740285. Fax: 886-8-7740388. E-mail: kcjpei@mail.npust.edu.tw
Discipline: Mammal Reproduction Biology

ABSTRACT

Previous studies have shown that despite having a clear seasonal fluctuation in fecal testosterone concentration, the significantly lower testosterone levels found in velvet stags of the non-seasonal breeder muntjac (Muntiacus sp.) apparently did not stop their spermatogenesis as in other deer species. In the present study, in vitro cultivated Leydig cells isolated from adult stags of three native deer species of Taiwan were treated with androstenedione, with or without adding human chorionic gonadotropin (hCG). Results showed that, unlike the two seasonal breeders, sika deer (Cervus nippon) and sambar deer (Rusa unicolor), Leydig cells of velvet muntjac had no dramatic reduction in or even maintain the full capability of their testosterone productivity compared with the hard-antlered stage. The decrease in fecal testosterone level observed earlier in muntjac during the velvet period was probably due to a reduction of Leydig cells number. These results support the hypothesis that testosterone production in muntjac during its velvet period may never be low enough to trigger the quiescent phase of the reproduction cycle.


INTRODUCTION

It is well known that in male cervids, serum testosterone concentration in the blood fluctuate annually following seasonal changes in testicular activity, which then dictates, simultaneously, both the reproductive cycle, also known as annual re-puberty phenomenon, and the antler replacement cycle. Stags are fertile and behaviorally active in mating, together with the regaining of the pair of hard antlers, only during periods of high serum testosterone concentrations in autumn [1-14].

Male muntjacs (genus Muntiacus), however, despite demonstrating a regular annual testosterone and antler replacement cycle like other deer species, do not exhibit the annual re-puberty phenomenon and remain fertile throughout the year, even when they are in the velvet period, which from the beginning of antler regrowth until it is mineralized and cleaned [15-21]. As a result, births can occur every month of the year [22-27].

It was proposed that, unlike other deer species, the serum testosterone concentration threshold for initiating the casting and re-growing of the antler in muntjac may be set higher than that required to stop spermatogenesis [16]; in other words, the testosterone production in muntjac during their velvet period, although significantly lower than in the hard-antlered stage, may never be low enough to trigger the quiescent phase of the reproduction cycle. In this study, we used the in vitro approach [28] to test this hypothesis by comparing the testosterone productivity of the cultivated Leydig cells isolated from Reeves’ muntjac (Muntiacus reevesi) with that of the other two seasonal breeders, namely sika deer (Cervus nippon) and sambar deer (Rusa unicolor).


MATERIALS AND METHODS

For comparison purposes, comparative data in hard-antlered stage from our previous study [28] are also presented in this paper. All procedures (i.e. concentrations, formulas, durations of cell incubation, and testosterone assay) followed the same study.

Testicles of 1 adult male of each of the three species from deer farms in central Taiwan were castrated, 1 testicle in velvet period and another in hard-antlered stage, every year during 2006-2008. All individuals were over 2 years old and in normal condition. Direct observations in the deer farms showed that, besides sambar deer’s velvet period occurring in January-May, sika deer and muntjac’s were both in May-September (CSL, per. obs.). Velvet period samples were collected in the middle section of the velvet period (Sambar deer: February and March; sika deer and muntjac: July and August), while hard-antlered stage samples were collected during October-December.

After removal, the testicular Leydig cells were isolated and cultivated as described in Lin et al. [28]. Cell cultures of the Leydig cells purified from each individual were considered 1 sample, therefore sample sizes were 3 for all treatments in the present study.

For evaluating the ability of the testosterone production by the cultured Leydig cells, steroidogenic precursor androstenedione (102 nM) was added to Leydig cells suspensions of 105 cells /ml, in 35 mm culture dishes (Costar, Cambridge, MA) in medium 199 containing 1.4 g/l NaHCO3 and 10% BSA, and incubated at 34oC, 95% air and 5% CO2 for 3 days. Androstenedione has been shown to be the most effective steroidogenic precursors in inducing the testosterone production by the cultivated Leydig cells [28] (Lin et al. 2011). Two kinds of treatments were conducted, one with and one without human chorionic gonadotropin (hCG; 0.5 IU/ml) added. The hCG was used in this study to replace LH as the original stimulatory agent for testosterone biosynthesis. The culture medium was then centrifuged at 4oC and 200 g for 10 min and the supernatant was stored at -20oC until analyzed for testosterone by enzyme immunoassay (EIA) [28].

Testosterone concentrations were measured by IMMULATE 2000 (Siemens Medical Solutions Diagnostics Pte Ltd, 5210 Pacific Concourse Drive, Los Angeles, CA, USA) using a commercial kit (EIA L2KTW2, Simens Medical Solutions Diagnostics Pte Ltd) (The Quality of Siemens Medical Solutions Diagnostics Pte Ltd is certified with ISO 13485: 2003). The antiserum had the following cross-reactivities (provided by the company): 100% testosterone, 2.0% 5α-dihydrotestosterone, 0.5% 5α-androstan-3β, 17β-diol, 0.6% androstenedione, and 0% 5α-androstan-3,17-dione. Assay sensitivity was 0.2 ng/ml, and the inter- and intra- assay coefficients of variation were 5.1 and 7.2%, respectively. The IMMULITE 2000 assay is an automatic two-site sandwich immunoassay with chemiluminescent detection [28-29].

RESULTS

All treatments, except plain velvet Leydig cells of the sika and sambar deer, produced traceable testosterone after the 3-day incubation. Without Androstenedione, the control groups of plain Leydig cells or Leydig cells plus hCG only produced insignificant amount of the testosterone (average 0.7-1.3 nM/105 cells/3 days for velvet period; average 0.8-2.0 nM/105 cells/3 days for hard-antlered stage).



As expected, the supplementation of the Androstenedione in this study significantly increased the production of the testosterone for all 3 species in the hard-antlered stage (average 17.6-19.0 nM/105 cells/3 days for plain Leydig cells; average 23.8-44.4 nM/105 for Leydig cells plus hCG); however, only the muntjac’s velvet Leydig cells produced large amounts of testosterone as in the hard-antlered stage (average 13.0 nM/105 cells/3 days for plain Leydig cells; average 45.4 for Leydig cells plus hCG) (Fig. 1 and Fig. 2). Furthermore, the Leydig cells’ testosterone productivity in muntjac showed no differences between the velvet period and hard-antlered stages when treated with the hCG and Androstenedione (Mann-Whitney-Wilcoxon test, Z*=0.218, P>0.05).
DISCUSSION

The results of the present in vitro study suggest that, as predicted by the hypothesis, male muntjac sustained their ability to produce relatively high amounts of testosterone during their velvet period (Fig. 1 and Fig. 2), and, therefore, could maintain a year-round capability of generating fertile sperms. Apparently, the function of the muntjac testicle will not revert to a quiescent phase during the velvet period as in other deer species, a phenomenon which calls for further investigation at the cellular or molecular levels of their Leydig cells. The study also demonstrated the important role of the LH (i.e., hCG) in controlling the Leydig cell’s production of testosterone as in other deer species [14, 30].

Nevertheless, the high amount of, or even no reduction in, testosterone production during the velvet period, which is featured in the study, was not expected since our previous study did show significant annual fluctuations in the fecal testosterone level in male muntjac [17]. The concentration and number of Leydig cells used in this in vitro study were the same for all treatments; therefore, a possible explanation is that although the function of producing testosterone by the muntjac Leydig cell does not regress during the velvet period, the number of Leydig cells might decrease significantly, which caused the decrease in concentrations of fecal testosterone of muntjac during the velvet period than in the hard-antlered stage [17]. It has been shown that the intratesticular testosterone level was significantly related to the number of Leydig cells and the concentration of Leydig cells per testis was 53% greater during the breeding season in stallions [30].

Pei et al. [17] and the present study both show that although testosterone might play a critical role in both antlergenesis and spermatogenesis, the magnitude of its seasonal fluctuation in male muntjac seems to regulate only their antler cycle. Seasonal changes in testosterone production by male cervids are well known to be regulated by photoperiod through the hypothalamus-pituitary-gonad axis [30-38]. However, the surprisingly synchronous antler cycle in male muntjac over a broad geographical region, including zoos in Northern and Southern Hemispheres [39-45], suggests a different mechanism, other than photoperiod and testosterone-controlled, must be involved. It might be that the regular antler cycle in adult muntjac is endogenous, as proposed by Bedford and Marshall [46], Goss and Rosen [47], and Lincoln [48] for the deer of tropical and subtropical origins. If this is the case, both the antler and reproduction cycles in muntjac might not be dictated by photoperiod.

Interestingly, unlike other cervids, muntjacs do not stand on their hind feet and "box" when they are in the velvet stage [49-50]. The standing and "boxing" behavior of cervids not only serves as an antagonistic or threatening expression when they are antlerless, but also avoids damage to the sensitive tissue of the velvet. Sparring with the long pedicles covered with fresh, growing velvet in male muntjac usually results in abnormal hard antlers (KJCP, pers. obs.). Presumably, it has little value in fighting and displaying. This suggests that antler characteristics may not be as important to reproductive success in the muntjac as in other deer species.

ACKNOWLEDGMENTS

The authors would like to thank Mr. Hui-Wen Shaw, Mr. Chen-Shih Chen, Mr. Jung-In Chen and Mr. Kuen-Feng Lin for their help in sample collection. Thanks also to the Reproductive Physiology Laboratory of the Department of Animal Science, National Pingtung University of Science and Technology, Taiwan, for kindly providing facilities and technical supports for sample preparation. The Lezen Reference Laboratory (http://www.lezen.com.tw) conducted the EIA assays for testosterone measurements in the study. Funding was provided by the Forestry Bureau, Council of Agriculture, Taiwan (Projects: 96 Forest Management-02.1-Conservation-17).


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FIGURE CAPTIONS

Figure 1. Comparison of the average testosterone production (ng/ml/3 days) by cultivated Leydig cells (LC; 105 cells /ml) purified from sika deer, sambar deer and muntjac in hard-antlered stage (black bar) or velvet period (white bar). The top figure shows the result of plain LC and bottom figure shows the result of LC treated with androstenedione (102 nM). Sample size was 3 animals for all treatments and data are mean with 1 SD.



Figure 2. Comparison of the average testosterone production (ng/ml/3 days) by cultivated Leydig cells (LC; 105 cells /ml) purified from sambar deer, sika deer and muntjac in hard-antlered stage (black bar) or velvet period (white bar) with the human chorionic gonadotropin (hCG; 0.50 IU/ml) added. The top figure shows the result of plain LC and bottom figure shows the result of LC treated with androstenedione (102 nM). Sample size was 3 animals for all treatments and data are means with 1 SD.





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