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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.
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ALKYLDIACYLGLYCEROL IN HARDERIAN GLANDS
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