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Testosterone-Dependent Primer Pheromone Production in the Sebaceous Gland of Male Goat¹

^a Laboratory of Veterinary Ethology, Department of Veterinary Medical Science, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113–8657, Japan

	ABSTRACT

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To test the hypothesis that the primer pheromone responsible for inducing the "male effect" is produced in the sebaceous gland androgen dependently, we examined the correlation between morphological changes of sebaceous glands and the pheromone activity in skin samples taken from castrated goats that had been treated with testosterone. Five castrated goats were implanted s.c. with testosterone capsules to maintain physiological levels of plasma testosterone for four weeks. Skin samples were obtained from the head region on Day 0 (the day of testosterone implant), Day 7, Day 14, Day 28 (the day of testosterone removal), Day 36, Day 42, and Day 56. Matched blood samples were also collected for measurement of testosterone concentration. The pheromone activity of the ether-extracts of the upper dermal layer containing sebaceous glands was assessed by its stimulatory effect on the hypothalamic GnRH pulse generator, which was monitored for changes of specific multiple unit activity (MUA) in ovariectomized estradiol-primed goats as described previously. The sebaceous gland enlarged during the testosterone treatment but reduced in size after testosterone removal. The pheromone activity first appeared in 2 out of 5 goats on Day 7 and in all the 5 goats by Day 28. Fourteen days after testosterone removal (Day 42), the pheromone activity was no longer detectable in any of the 5 goats. In short, the sebaceous gland size and the pheromone activity shifted almost in parallel. The present results provide strong support for the view that the primer pheromone is produced testosterone dependently in the sebaceous gland of the male goat.

	INTRODUCTION

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The "male effect" is well known in sheep [1, 2] and goats [3–5] as a good example of pheromone-induced activation of reproductive function in which seasonally anovulatory females resume ovarian cyclicity in response to the male pheromone. It has been shown that the olfactory signal emanating from the male hair stimulates the reproductive neuroendocrine system, and that the frequency of pulsatile LH secretion increases, leading to the normal sequence of preovulatory endocrine events [5, 6].

The GnRH pulse generator regulates the intermittent GnRH discharge into the pituitary portal circulation and thereby modulates the pulsatile secretion of LH [7, 8]. A method has been developed for monitoring the electrophysiological manifestation of this GnRH pulse generator activity from conscious and loosely restrained goats as the characteristic increases in the hypothalamic multiple-unit activity (MUA volleys) [9–12]. This technique provides us with the practical means for the real-time assessment of timed application of testing materials and their effects on the reproductive neuroendocrine system. These characteristics are particularly important for establishing a satisfactorily sensitive as well as specific bioassay system for assessing the primer pheromone activity [13]. In fact, exposure to male goat hair, which had been timed midway between succeeding MUA volleys, resulted in an occurrence of the MUA volley within a few minutes. On the other hand, hair samples obtained from castrated goats had no such stimulatory effect on the GnRH pulse generator, but testosterone replacement resulted in a restoration of the pheromone activity [13].

Although attempts have been made to purify the pheromone(s) responsible for the male effect [14, 15], chemical identification has not yet been successful. To achieve this objective, we need to know more about where and how the male pheromone is synthesized and released. This study was therefore aimed at revealing the time course of morphological change and pheromone-producing activity of the sebaceous gland, a putative pheromone-synthesizing organ, of the castrated male goat throughout 4 wk of testosterone treatment and the following 4-wk waning-off period by utilizing a novel neurophysiological bioassay system for the primer pheromone.

	MATERIALS AND METHODS

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Experimental Design

Five long-term (> 1 yr) castrated male goats (2–3 yr old) were obtained from the closed colony at the experimental station of the University of Tokyo. They were isolated from other male goats and housed under conditions of natural day length and temperature. Six testosterone capsules made of Silastic sheet (5 x 5 cm; Dow Corning Co., Ambridge, PA), each containing 1 g of crystalline testosterone (Wako Co., Osaka, Japan) were implanted s.c. for 28 days in each animal [16]. The sampling of skin and blood was conducted on Day 0 (immediately before the testosterone implantation), Day 7, Day 14, Day 28 (the day of testosterone removal), Day 35, Day 42, and Day 56. For sampling, the goat was anesthetized by ketamine HCl and xylazine HCl, and a square (1 cm x 1 cm) of skin was cut off by scalpel from the head region between the horns, where the sebaceous glands were shown to be well-developed [17]. The collected skin samples were stored at -80°C. For the testosterone assay, blood was collected on the same day, prior to the skin sampling. A blood sample (2 ml) was collected from the jugular vein with syringes containing heparin and centrifuged immediately after collection. The plasma was removed and stored at -20°C in a plastic tube until assayed for testosterone concentration.

Preparation of Skin Samples for Bioassay

Half of each skin sample was trimmed by removing the epidermal, subcutaneous layer, and the lower part of the dermal layer containing sweat glands, and only the upper dermal layer containing sebaceous glands was used for the bioassay to minimize interference by exogenous chemical compounds from the environment [18]. The samples were homogenized by ultrasonic homogenizer (Tomy Co., Tokyo, Japan), extracted by diethyl ether, filtered using filter paper No. 1 (Advantec TOYO Inc., Tokyo, Japan), and dehydrated using Na₂SO₄. The extracts were then dried under a stream of nitrogen gas at 40°C and stored at -20°C in a glass vial with a Teflon (DuPont, Wilmington, DE)-sealed cap that was filled with nitrogen gas to prevent oxidation.

On the day of bioassay, the sample was dissolved in diethyl ether again, in which a piece of gauze was soaked and dried again a few hours before the bioassay. The gauze was stored at -20°C in a sealed plastic bag filled with nitrogen until used.

MUA Recording and Bioassay for Pheromone Activity

Arrays of bilateral recording electrodes were stereotaxically placed in the medial basal hypothalamus of 3 long-term (> 2 yr) ovariectomized female goats [19]. The goats, from which clear MUA volleys had been consistently recorded [11, 20], were used for the bioassay of pheromone activity according to the procedure previously described by Hamada et al. [13] with a little modification.

In brief, the estradiol-primed goats were loosely tied to the stanchion in Faraday cage during MUA recording under controlled temperature (23°C), relative humidity (55%), and photoperiod (12L:12D). For the bioassay, the gauze containing the sample extract to be tested was placed 2–3 cm in front of the goat's nose for 3 min to allow olfactory, visual, and tactile investigation. When an MUA volley followed within a 3-min period of odor application, the sample was assessed to be pheromone positive. The bioassay was carried out 2–4 times a day in each of 3 female goats for 3–4 consecutive days, and the exposures to samples were separated by an interval sufficient in length (usually 2–3 h) for the frequency of MUA volleys to return to the same level as before exposure. Each female was tested with the samples from more than one male and the sequence of samples exposed was random, but the last sample was intentionally the predetermined positive control (male goat hair extract) for confirmation of normality of the bioassay.

Measurement of the Size of the Sebaceous Glands

The remaining halves of the skin samples were fixed in 10% paraformaldehyde and were cut on a freezing microtome at 15-µm thickness perpendicular to the skin surface. They were then stained with Sudan black B backgrounded by nuclear fast red. The sections were projected at a magnification of x100, and the outlines of 50 sebaceous glands were traced for each sample. The length of the major axis was measured for each gland, and mean values and standard errors (SE) were calculated for each experimental day. Analysis of variance and paired t-test were used for detection of significant differences.

Assay of Plasma Concentration of Testosterone

Plasma testosterone concentrations were measured by an enzyme-immunoassay using anti-testosterone serum (provided by Dr. A. Miyamoto, Obihiro University of Agriculture and Veterinary Medicine, Obihiro City, Japan), and HRP-testosterone (Medical System Service Co., Kanagawa, Japan) as described elsewhere [21]. The intra- and interassay coefficients of variation were 9.8% and 11.2%, respectively.

	RESULTS

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The effect of odor from five castrated goats (#1–5) on the intervals between MUA volleys in three female goats used for the bioassay is shown in Table 1. In most cases samples were applied 40 min (in some cases 35 min) after the onset of the preceding MUA volley. Representative positive data of the bioassay is shown in Figure 1, where the application induced advancement of the following MUA volley. This stimulatory effect, however, lasted only briefly, and the subsequent MUA volley interval returned to the preapplication level. The appearances of pheromone activity in the 5 castrated goats are shown in Figure 2 (upper). The pheromone activity was positive in 2 out of 5 goats on Day 7, soon after testosterone implantation. On Day 28, all of 5 goats turned positive; but after testosterone removal the pheromone activity vanished, and all individuals became negative again on Day 42. Four wk after the commencement of testosterone treatment all the castrated goats (Day 28) presented a male-like appearance, but they had returned to their pretreatment figures 4 wk after testosterone withdrawal (Day 56). Plasma concentrations of testosterone were maintained at physiologically high levels (0.8–15.8 ng/ml for the intact males in the same colony) during the period of testosterone implantation as shown in Figure 2 (middle).

View larger version (17K): [in this window] [in a new window]   FIG. 1. Changes in MUA in response to the application of the extract of the upper dermal layer in a representative ovariectomized goat carrying estradiol implant, shown as intervals (min) between MUA volleys. When the goat was exposed to the positive sample (vertical arrow) midway between volleys, the subsequent MUA volley was induced within a few minutes of the exposure.

View larger version (29K): [in this window] [in a new window]   FIG. 2. Proportion of pheromone positive samples obtained from the five castrated goats treated with testosterone between Day 0 and Day 56 (top). Plasma concentrations of testosterone between Day 0 and Day 56 (middle). The change of the major axis length of sebaceous glands between Day 0 and Day 56 (bottom). Each point represents the mean ± SE (n = 5). *P < 0.05 and **P < 0.01, by paired t-test, as compared with Day 0.

The sebaceous glands developed gradually during the course of the testosterone treatment (Days 0–28) and then atrophied during the follow-up period (Days 35–56) as shown in Figure 2 (bottom) and Figure 3. On Day 28 the size of the sebaceous glands eventually attained the normal size of mature intact males in the same colony (793.3 ± 32.26 µm), and this was due to increases in both the number and the volume of sebocytes. Consequently, the length of major axis of sebaceous gland was larger on Day 7 (P = 0.0297), Day 14 (P = 0.0013), Day 28 (P = 0.0001), Day 35 (P = 0.0004), and Day 42 (P = 0.0019) as compared with Day 0.

View larger version (114K): [in this window] [in a new window]   FIG. 3. Morphological changes of sebaceous glands in castrated goats treated with testosterone between Day 0 and Day 56. a) Day 0; b) Day 7; c) Day 14; d) Day 28; e) Day 35; f) Day 42; g) Day 56. Bar = 100 µm. Sebocytes in the sebaceous glands were small in size and in number on Day 0 (a, black arrow), developed on Day 28 (d, arrow head), then atrophied on Day 56 (g, white arrow)

	DISCUSSION

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In this study, primer pheromone activity appeared in upper dermal layer extracts obtained from castrated goats following testosterone treatment. This indicates that the primer pheromone responsible for the male effect is produced in the sebaceous gland under the control of testosterone. In previous attempts to purify this primer pheromone, the hair of male goats was used as a source of pheromone [13–15]. Now it has been revealed that sebaceous glands can be another and probably more suitable resource for the isolation of the pheromone.

Kealy et al. [22] isolated human sebaceous glands by shearing, but in this study we used the upper dermal skin layer for extraction because the glands at Day 0 were too small to shear off; nevertheless, we have confirmed that we could obtain results with the extracts from the sheared sebaceous gland that were similar to results from the skin of a mature male goat in our pilot study (data not shown).

In this study, primer pheromone activity first appeared on Day 7 and disappeared on Day 35 in some individuals. This change appears to be somewhat faster than the wax and wane of glandular development. Downing et al. [23] suggested that an average interval between synthesis and excretion of sebum was about 8 days. These results may suggest that pheromone production would have begun at an early stage of the sebum production process and that the size of the sebaceous gland enlarged gradually according to the flourish of sebum production. However, it is still unclear how testosterone acts on the sebaceous gland to induce pheromone production, and so further investigation, probably including the establishment of a primary culture of sebaceous gland cells, is needed to elucidate the mechanism of pheromone production as well as to identify pheromone molecules.

	ACKNOWLEDGMENTS

 
We would like to thank Dr. Sawazaki for providing animals from the closed colony of the University of Tokyo. This study was supported by "Research for the Future" Program, The Japan Society for the Promotion of Science (JSPS-RFTF 97L00904), Grants-in-aid, for Scientific research from the Ministry of Education, Science, Sports and Culture and CREST of the Japan Science and Technology Corporation.

	FOOTNOTES

 
First decision: 9 August 1999.

¹ This work was supported by "Research for the Future" Program, The Japan Society for the Promotion of Science (JSPS-RFTF 97L00904), Grants-in-aid for Scientific research from the Ministry of Education, Science, Sports and Culture and CREST of the Japan Science and Technology Corporation.

² Correspondence: Y. Mori, Laboratory of Veterinary Ethology, Department of Veterinary Medical Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. FAX: 81 3 5841 8190; aymori{at}mail.ecc.u-tokyo.ac.jp

Accepted: October 27, 1999.

Received: July 12, 1999.

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