JEZ 724 THE JOURNAL OF EXPERIMENTAL ZOOLOGY 277:99–105 (1997) Effect of the Photoperiod in Modulating the Androgenic Control of 1-Alkyl-2,3-Diacylglycerol Composition in the Harderian Gland of the Golden Hamster, Mesocricetus auratus GERALD R. BUZZELL,1* AZUMI HIDA,2 STANLEY FU, 1 AND YOUSUKE SEYAMA2 1 Department of Anatomy and Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada 2 Department of Physiological Chemistry and Nutrition, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan ABSTRACT The golden hamster Harderian gland produces a lipid secretion consisting mainly of 1-alkyl-2,3-diacylglycerol. We investigated the composition of alkyl and acyl groups in male and female hamster Harderian alkyldiacylglycerol, in animals kept in long and short photoperiods. Female hamsters in long days have alkyl groups with long saturated straight chains (C18:0 and C20:0) and methyl-branched chains (even and odd chain length iso-branched and odd chain length anteiso-branched chains). Acyl groups in females in long days are mostly long straight chains (C16:0) and methyl-branched chains. In females, short photoperiods led to reductions in the proportions of methyl-branched chains and changes in the proportions of straight chain alkyl and acyl groups; these changes were prevented by pinealectomy. Male hamsters with intact gonads, maintained in long days, had no methyl-branched chain alkyl or acyl groups; saturated straight chains were generally shorter than those of females and the odd chain length saturated C15:0 acyl group was common. Short photoperiods did not significantly alter the composition of male alkyldiacylglycerol. Castrated male hamsters in long days showed a distinctively female phenotype, with long straight chains and methyl-branched alkyl and acyl groups. Castrated males in short days showed a mixture of male and female characteristics: shorter straight chain alkyl and acyl groups, a total absence of methyl-branched alkyl groups, and the presence of methyl-branched acyl groups. These results and those of other studies suggest that testosterone controls the enzymes isovaleryl acyl-CoA dehydrogenase and 2-methyl branched-chain acyl-CoA dehydrogenase; in the absence of these enzymes, the primers for the synthesis of methyl-branched chain fatty acids are produced. Our results indicate that this control is modulated by short photoperiods (perhaps due to reduced prolactin levels). It is also suggested that characteristics of male-type alkyldiacylglycerol are better adapted to conditions of autumn and winter than are those of female-type alkyldiacylglycerol. J. Exp. Zool. 277:99–105, 1997. © 1997 Wiley-Liss, Inc. The rodent Harderian gland secretes a lipid material to the medial portion of the eyeball. In the golden (Syrian) hamster, Mesocricetus auratus, and in several other species including the guinea pig, most of this lipid is unusual in that it has the composition of 1-alkyl-2,3-diacylglycerol (ADG) (Yamazaki et al., ’81; Murawski et al., ’91; Park et al., ’93; Seyama et al., ’95) (Fig. 1). The function of Harderian ADGs is unknown, though it has been suggested that they are involved in thermoregulation, lubricate the nictitating membrane, have bacteriocidal properties, or are solvents for biologically active materials (Seyama et al., ’92). All the evidence for these suggestions is circumstantial. © 1997 WILEY-LISS, INC. The Harderian gland of golden hamsters is also characterized by marked sexual dimorphisms (Buzzell, ’96) and this also applies to their lipid. Secretory lipid droplets in both sexes are membrane-bounded and are released by exocytosis (López et al., ’93). Male lipid droplets are of two sorts, distinguished by size, being small (type 1) or large (type 2), whereas those of females are entirely small (type 1) (Buzzell, ’96; Buzzell et al., *Correspondence to: Dr. G.R. Buzzell, Department of Anatomy and Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Received 13 March 1996; Revision accepted 12 September 1996 100 G.R. BUZZELL ET AL. females with testosterone has the opposite effects (Buzzell, ’96; Buzzell et al., ’95; Seyama et al., ’96). However, androgens are not the entire story: both male and female hamsters subjected to short photoperiods exhibit an increase in type 2 lipid droplets, and castrated males in short days also do not show the expected decrease in type 2 lipid droplets, such as is seen in castrated males in long days (Buzzell, ’96; Buzzell et al., ’95). This suggests that the effects of low androgens are modulated by other factors following exposure to short days. In this study, we examine the composition of Harderian ADG in golden hamsters exposed to short days to determine whether this treatment also modulates the biochemical makeup of the lipid droplets. MATERIALS AND METHODS Animals and experiments Fig. 1. Structures of (a) 1-alkyl-2,3-diacylglycerol (ADG) and (b) fatty acids in the golden hamster Harderian gland. a: R1– is an alkyl residue, R2CO- and R3CO- are acyl residues. b: Fatty acids form ester linkages at the number 2 and 3 positions of glycerol, to make up the acyl portion of ADG. Fatty alcohols, formed from the corresponding fatty acid, form ether linkages at the number 1 position of glycerol, to make up the alkyl portion of ADG. Methyl branching resides at the (n-1) position in iso-branched chain fatty acids and the (n-2) position in anteiso-branched chain fatty acids. Abbreviations: ai CAS FA INT is LD PINX SD anteiso-branched castrated fatty acid intact iso-branched long days (14 h:10 h, L:D) pinealectomized short days (8 h:16 h, L:D) ’95). ADGs of males consist of saturated straightchain alkyl and acyl groups, whereas those of females contain saturated straight-chains as well as iso- and anteiso-branched chain alkyl and acyl groups (Fig. 1b) (Seyama et al., ’95). The primary control of lipid droplet type and of ADG composition is hormonal, specifically androgenic. Thus, orchidectomy leads to a loss of type 2 lipid droplets and the production of branchedchain alkyl and acyl groups whereas treatment of Golden hamsters (Mesocricetus auratus) were obtained from Charles River Canada (St. Constant, Québec) and maintained in a vivarium at constant temperatures (20° ± 2°C), with food and water available ad libitum. They were placed in long photoperiods (14 h light/day, lights on at 06:00 h) or short photoperiods (8 h light/day, lights on at 08:00 h), as outlined below. Female control hamsters in long days were untreated (n = 6); data from this group have been reported in a previous study (Seyama et al., ’96). Experimental groups run at the same time as the controls were maintained in short days; they were intact (n = 8) or pinealectomized (n = 5). Female hamsters were monitored during the estrous cycle and were sampled with 7 of the intact animals in short days were anestrus, 9–9 1/2 weeks following the beginning of the experiment. They were sampled over a 4-day period, when all animals were either in diestrus I or would have been in diestrus I, had they not been anestrus (Buzzell et al., ’95; Seyama et al., ’96). Male hamsters in long days were intact (n = 6) or castrated (n = 6) as controls. Data from these groups have been reported in a previous paper (Seyama et al., ’96). Experimental groups run with the controls were maintained in short days; they were intact (n = 8), pinealectomized (n = 5), or castrated (n = 6). Male hamsters were kept in these conditions for 8 1/2 weeks and then sampled. All procedures using animals were approved by the University of Alberta Health Sciences Animal Welfare Committee. Castration of male hamsters was done via the scrotal route. Pinealectomy of ALKYLDIACYLGLYCEROL IN HARDERIAN GLANDS males or females was by the method of Hoffman and Reiter (1965). Animals were anesthetized for surgery by Rompun (Chemagro Ltd., Etobicote, Ontario) (20 mg/kg) and Ketamine (M.T.C. Pharmaceuticals, Cambridge, Ontario) (100 mg/kg). Animals were anesthetized with Rompun and Ketamine before being decapitated at sampling. Harderian glands were quickly dissected from the orbits and frozen on dry ice. They were kept at –70°C until subjected to extraction of their lipid. In addition, pieces of the glands were processed for histology and serum was analyzed for hormone levels; these results have been reported previously (Buzzell et al., ’95). Extraction of lipids Crude lipid extracts were made from glands homogenized in chloroform/methanol (2:1, v/v), left overnight at room temperature, vortexed, and filtered. The liquid phase was evaporated under nitrogen. The experiments and the crude lipid extraction were done in Edmonton. The tubes with crude lipid extracts were then sent via air mail to Tokyo where they arrived 5 days later. Preparation and analyses of alkyldiacylglycerol Lipid biochemistry was done in Tokyo. Detailed methods used are reported elsewhere (Seyama et al., ’96). Briefly, alkyldiacylglycerol (ADG) was prepared from crude lipid extracts by elution from Silica SEP-PAC columns by hexane/benzene (4:6, v:v). Acyl groups were extracted as fatty acid methyl esters by standard techniques. Alkyl groups were extracted from residue, as isopropylidine derivatives. Fatty acid methyl esters and isopropylidine derivatives were measured by gasliquid chromatography (GLC) (Seyama et al., ’96). 101 Student-Neuman-Keuls (SNK) test was done to determine which group(s) contributed to the observed differences. Castrated males in long days were compared with castrated males in short days by Student’s t-test. In all cases, significant differences were accepted with P < 0.05. RESULTS Female hamsters Alkyl groups Short photoperiods led to significant increases in the proportions of the straight-chain alkyl groups 14:0, 16:0, and 18:0 and a reduction in 20:0 in female hamsters. The total proportion of branched-chain alkyl groups was reduced from 52 to 23% in short days. Amongst the branched-chain alkyl groups, iso-branched C17, C19, and C21, as well as anteiso-branched C17 and C19, were reduced. Pinealectomy prevented the changes in straight-chain 14:0, 16:0, and 20:0; it reduced or prevented the decreases in the branched-chain alkyl groups (Fig. 2). Acyl groups In female hamsters, short photoperiods led to significant reductions in the proportions of the straight-chain acyl groups 16:0 and 20:0 and increases in 12:0, 17:0, 18:0, and especially 14:0; pinealectomy prevented all these changes except Data management Concentrations of the individual fatty acid methyl esters and isopropylidine derivatives (with 12 or more carbon atoms) in each gland were determined from the GLC plots, as proportions of the total fatty acid methyl esters and of the total isopropylidine derivatives for that gland. From these data, the means ± SEM of each alkyl and acyl group in that gland were calculated. Data were compared as follows: Females and males with intact gonads (long days, short days, pinealectomized/short days) were compared by analysis of variance (ANOVA); where the ANOVA showed significant between-group differences, a Fig. 2. Alkyl groups in female golden hamster ADGs, expressed as the percentages of total alkyl groups for each experimental condition. *P < 0.05 vs. INT-LD (ANOVA, SNK). 102 G.R. BUZZELL ET AL. those to 17:0 and 20:0. The total proportion of branched-chain acyl groups was reduced from 25 to 18% in short days. Changes to branched-chain acyl groups included increases to iso- and anteisobranched C15 and reductions to iso-branched C17, C18, and C19, and to anteiso-branched C19. Pinealectomy had little effect on these branchedchain fatty acids (Fig. 3). Male hamsters with intact gonads Alkyl groups Neither short days nor pinealectomy in short days had any effect on the alkyl group composition of Harderian ADG in male hamsters (Fig. 4). No branched-chain alkyl groups were present in any experimental condition. Acyl groups Acyl group composition of ADGs in male hamsters kept in short days, with or without their pineal glands, was essentially identical to that of control animals kept in long days (Fig. 5). Castrated male hamsters Alkyl groups Fig. 4. Alkyl groups in ADGs of male golden hamsters with intact gonads, expressed as the percentages of total alkyl groups for each experimental condition. Castrated male hamsters maintained in short days showed significantly increased proportions of straight-chain alkyl groups 14:0, 16:0, and 18:0 and Fig. 3. Acyl groups in female golden hamster ADGs, expressed as the percentages of total acyl groups for each experimental condition. *P < 0.05 vs. INT-LD (ANOVA, SNK). Fig. 5. Acyl groups in ADGs of male golden hamsters with intact gonads, expressed as the percentages of total acyl groups for each experimental condition. *P < 0.05 vs. INTLD (ANOVA, SNK). ALKYLDIACYLGLYCEROL IN HARDERIAN GLANDS 103 a reduction in 20:0. These animals showed a total absence of branched-chain alkyl groups (Fig. 6). Acyl groups Short photoperiods led to significant increases in the straight-chain acyl groups 12:0, 14:0, and 18:0 and a reduction in 16:0 in castrated male hamsters. There were also significant increases in iso- and anteiso-branched C15 and reductions in iso-branched C17, C18, and C19 and anteisobranched C17 from those of long day castrated controls (Fig.7). DISCUSSION 1-Alkyl-2,3-diacylglycerol (ADG) (Fig. 1) is the major secretory lipid in the Harderian gland of the golden hamster. The composition of ADG in this animal shows marked sexual differences in hamsters kept in long photoperiods. In these conditions, female ADGs contain both straight- and branched-chain alkyl and acyl groups whereas those of males have entirely straight chains. Unsaturated alkyl or acyl groups are not found in either sex (Seyama et al., ’95). Branched-chain alkyl and acyl groups were characteristic of ADGs from female hamsters and from castrated males maintained in long days. The enzyme isovaleryl-CoA dehydrogenase promotes the catabolism of the methyl-branched amino acid, leucine; in its absence, leucine forms isovalerylCoA which can act as a primer for odd chain length iso-branched fatty acids. Another enzyme, 2-methyl branched-chain acyl-CoA dehydrogenase, Fig. 6. Alkyl groups in ADGs of castrated male golden hamsters, expressed as the percentages of total alkyl groups for each experimental condition. *P < 0.05 vs. CAS-LD (t-test). Fig. 7. Acyl groups in ADGs of castrated male golden hamsters, expressed as the percentages of total acyl groups for each experimental condition. *P < 0.05 vs. CAS-LD (t-test). promotes the catabolism of valine and isoleucine; in its absence, valine forms isobutyryl-CoA and isoleucine forms 2-methylbutyryl-CoA, primers for even chain length iso-branched and odd chain length anteiso-branched fatty acids, respectively (Seyama et al., ’95). Previous studies suggest that testosterone stimulates the activity of these enzymes; when testosterone levels were low (castrated males and females), methyl-branched alkyl and acyl groups were present whereas when testosterone levels were high (males and females given testosterone), methyl-branched chains were absent (Seyama et al., ’95, ’96). The present study suggests that this effect is modulated in short photoperiods. Long days are stimulatory for many features of the physiology of golden hamsters, and are generally regarded as the “control” condition in experiments. In these conditions, animals are reproductively competent. Serum levels of gonadotrophins, prolactin, sex steroids, thyroxin, and other hormones are high; gonads are active; mating occurs; and, since (in nature) long days occur during the spring and summer, survival of the offspring is optimal (Reiter, ’80). Short photoperiods are features of the autumn and winter. Golden hamsters maintained in short days become reproductively quiescent. Serum levels of gonadotrophins, prolactin, sex steroids, thyroxin, and other hormones are low; testes, uteri, and accessory sex glands atrophy; and estrous cycles cease. These effects probably prevent reproduction from occurring during winter, when survival of the offspring would be doubtful. Short 104 G.R. BUZZELL ET AL. photoperiod-mediated changes in the physiology of golden hamsters are controlled by the pineal gland and its hormone melatonin; pinealectomy prevents these effects (Reiter, ’80). Despite the low testosterone levels in male hamsters maintained in short days (0.3 ± 0.1 vs. 2.9 ± 0.4 ng/ml in long days; Buzzell et al., ’95), the compositions of both alkyl and acyl groups in these conditions were identical to those of long day males and quite different from those of castrated male hamsters in long days. Significantly, no methyl-branched alkyl or acyl groups were present in ADGs of male hamsters with intact gonads, regardless of the photoperiod, suggesting that Harderian isovaleryl-CoA dehydrogenase and 2methyl branched-chain acyl-CoA dehydrogenase are active in short days, despite the low testosterone levels. In females, changes which occur in short days generally decrease the proportions of branchedchains and increase the proportions of shorter straight-chain alkyl and acyl groups, trends which could be described as masculinizing. Pinealectomy prevented most of these masculinizing effects. Morphometric analysis of lipid droplet types in these same animals also showed short photoperiods leading to masculine phenotypes in both sexes (Buzzell et al., ’95). Probably the most interesting effects noted were those of short days in castrated male hamsters. We expected, from our morphometric studies, that Harderian glands in these experimental conditions would show a male pattern, rather than a female one, i.e., that short days would prevent the feminizing effects of orchidectomy (Buzzell, ’96; Buzzell et al., ’95). What we found was a mixture of masculine and feminine characteristics. In this group, the straight chain alkyl and acyl groups showed a pattern which was mainly masculine. Also masculine was the absence of branched chain alkyl groups; however, the presence, under these conditions, of branched chain acyl groups was a feminine characteristic. This was the only instance in which branched chain alkyl groups were absent when branched chain acyl groups were present. The low testosterone levels and presence of methyl-branched acyl groups in these glands suggest that isovaleryl-CoA dehydrogenase and 2-methyl branched-chain acylCoA dehydrogenase are not active in these conditions and that the methyl-branched amino acids produce methyl-branched primers for fatty acid synthesis. The question of why these methylbranched fatty acids do not produce methyl- branched fatty alcohols for formation of ether linkages (alkyl groups) of ADGs must await further study. Several other dimorphic characteristics of golden hamster Harderian glands, which are controlled by androgens, are also modulated in response to short photoperiods. Since circulating testosterone levels drop in male hamsters on short days, it was surprising, at first, that these changes are not always similar to those seen after orchidectomy (and may, in fact, prevent the effects of low testosterone levels) (Hoffman, ’71; Buzzell and MenendezPelaez, ’92; Buzzell, ’96; Buzzell et al., ’94, ’95; Nadakavukaren and Lin, ’83; Menendez-Pelaez et al., ’88). Sometimes the effect of short days is to maintain the status quo, despite the decrease in androgen levels; we have suggested that the short day–induced decrease in serum prolactin levels prevents the porphyrinogenesis which otherwise follows low testosterone levels (Buzzell, ’96; Buzzell et al., ’92, ’94). A similar mechanism may operate here, but more studies are needed to establish this. The effects of short photoperiods on Harderian lipid in both sexes is to produce a more masculine phenotype. Because short photoperiods are characteristic of autumn and winter, it seems likely that the male type of ADG is, in some way, well suited to function in cold ambient temperatures. Because we do not know the precise function of Harderian ADG, clarification of this matter must also await further studies. ACKNOWLEDGMENTS These studies were supported by grants from NSERC Canada (G.R.B.), a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (Y.S.), and a grant from the Cosmetology Research Foundation (Y.S.). We thank Kirsten Oates and Shuichiro Hayashi for their assistance. LITERATURE CITED Buzzell, G.R. (1996) Sexual dimorphism in the Harderian gland of the Syrian hamster is controlled and maintained by hormones, despite seasonal fluctuations in hormone levels: Functional implications. Microsc. Res. Tech., 33:133–138. Buzzell, G.R., and A. 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