Chem. Senses 24: 289-293,
1999
© Oxford University Press
Induction of Estrus in Grouped Female Mice (Mus domesticus) by Synthetic Analogues of Preputial Gland Constituents
Department of Chemistry, Indiana University, Bloomington, IN 47405, USA
Correspondence to be sent to: Milos V. Novotny, Department of Chemistry, Indiana University, Bloomington, IN 47405, USA. e-mail:novotny{at}indiana.edu
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Two major volatile constituents of the male mouse preputial gland, E,E-
-farnesene and
E-ß-farnesene, were examined for their role in inducing estrous cycles in grouped female
mice. The
results indicated that the mixture of the farnesenes was as effective as the homogenate of the
intact
preputial gland, while the extract of the castrate preputial tissue did not show a pronounced
response. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Primer pheromones play a critical role in the reproductive physiology of the house mouse (Mus domesticus) and other rodents (Whitten and Champlin, 1972;
Bronson,
1974, 1979; Bronson and MacMillan,
1983). Since the 1960s, urine has been investigated as the main source of the primer
pheromones that are chiefly responsible for estrus synchrony (Whitten, 1956),
puberty acceleration (Vandenbergh, 1969), puberty delay (Drickamer et al., 1978) and pregnancy block (Bruce, 1959). The subsequent
chemical characterization of some of these pheromones, together
with
biological testing of their synthetic analogues (Novotny et al., 1985a, b; Jemiolo et al., 1986; Jemiolo
and Novotny, 1993; M. Novotny et al., submitted for publication), has laid
a
foundation for a better understanding of the physiological and biochemical processes related to
chemical communication in M. domesticus.
Estrous cycles of the house mouse are closely related to caging conditions. While isolated
females
exhibit 4-5 day cycles, grouped females have been reported to develop longer and irregular
cycles
(Whitten, 1959). Induction of estrus in crowded females is one of the best
documented pheromonal phenomena. When the grouped females are exposed to a male, or male
urine,
the majority are stimulated into estrus with a high percentage occurring on the third day (Whitten, 1956). The male pheromones are androgendependent, as
castration depresses
the estrus acceleration capacity (Bruce, 1965). Since male urine alone
was
capable of inducing estrus in grouped female mice (Marsden and Bronson, 1964; Bronson and Whitten, 1968), the active substances must
originate
from either the internal metabolic products (originating from the liver or kidney metabolism) or,
alternatively, from the products of some androgen dependent sex accessary glands which are
located in
the path of voided urine. However, there has been much confusion concerning the source of the
male
chemosignals. Already in 1968, Bronson and Whitten (1968) reported
that the
androgen-dependent primer pheromone for estrus induction was present in the bladder urine.
Since the
bladder urine was found as potent as the voided urine in its biological effect, the authors
concluded that
the preputial gland was not the source of the pheromone. In contrast, other investigators (Gaunt, 1968; Chipman and Albrecht, 1974)
traced the
biologically active (estrus-accelerating) substances to the preputial gland. A more recent paper (Marchlewska-Koj et al., 1990) also implicates this gland as an
important source of the estrus-inducing chemosignal.
The development and metabolism of the preputial gland is androgen-dependent (Burdick and Gamon, 1941). The preputial glands were reported as the source of
androgendependent olfactory stimuli, such as the aggressionpromoting signal (Mugford and
Nowell, 1971) or a sex attraction pheromone (Bronson and Caroom, 1971). Based on these results, and the fact that individual molecules can function in both
releaser and primer activities (for a review see Novotny et al., 1990a), the preputial gland is a reasonable candidate for a source of estrus induction signal.
Since the earlier behavioral studies implicating preputial gland secretions to aggression (Mugford and Nowell, 1971) and sexual attraction (Bronson
and Caroom,
1971), some progress has been made in chemical analysis of these secretions.
E,E--farnesene and E-ß-farnesene (Figure 1) are two major
constituents (accounting for nearly 80% of its volatiles) of the male mouse preputial gland
secretions (Novotny et al., 1990b) excreted into urine. The
farnesenes are
odoriferous, terpene-like substances which occur in other natural sources: trail markers of red fire
ants (Van der Meer et al., 1981), alarm pheromones of aphids (Bowers et al., 1972) and even a defense substance of wild potato
plants against the aphids (Gibson and Pickett, 1983). The farnesenes were
also found in Mediterranean fruit flies (Baker et al., 1985) and
the
dorsal gland of the springbok (Berger et al., 1981).
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The concentration of farnesenes in male mouse preputial glands depends on the endocrine and dominance status of animals (Novotny et al., 1990b
). While
castrates
have lower levels of farnesenes than intact males, dominant males have higher concentrations
than
subordinate animals (Harvey et al., 1989). Both dominant and
subordinate males avoid marking areas soiled with stimulus samples containing either natural or
synthetic farnesenes (Jemiolo et al., 1992). The farnesenes also
are
attractive to female mice (Jemiolo et al., 1991).
Following a partial elucidation of the farnesenes' role as behaviorally active
chemosignals
(releasing pheromones; Harvey et al., 1989; Novotnyet al., 1990b; Jemiolo et al., 1991, 1992), we are now investigating their potential primer functions. The purpose of this
work
was to examine if farnesenes are the active components of the preputial gland in estrus induction
in
grouped females. The effect of synthetic farnesenes was evaluated along with preputial gland
homogenates from either intact or castrated male mice.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Sexually experienced female mice (M. domesticus) of the ICR/Alb strain (Harlan Sprague Dawley Inc., Indianapolis, IN), which were ~3.5 months old and exhibited normal 4-5 day estrous cycles, were used for the experiments. The animals were maintained under standard laboratory conditions (22 °C; relative humidity, 60-70%). A 12 h light: 12 h dark daily regime was followed, with lights on at 06.00 h. Formulated food (Purina Mills, Richmond, IN) and water were supplied ad libitum. The wood shavings used for bedding were changed weekly.
Two sets of experiments were conducted using densities of four (set I) and eight animals per
cage
(set II). The dimensions of cages were 12 x 27 x 17 cm. Prior to the exposure to
olfactory
stimuli, the animals were grouped together for 3 weeks to cause a degree of estrus suppression
(Whitten, 1959). Four treatment groups were employed for each set of
experiments: (i) distilled water
(serving as a control); (ii) an aqueous extract of preputial glands from intact males; (iii) an
aqueous
extract of preputial glands from castrated males; and (iv) an equal-part mixture of and
ß-farnesene dissolved in distilled water at 250 p.p.m. (total concentration). Preputial glands
collected from 40 intact and 39 castrated males of 4 months of age were homogenized with 40
and 39
ml saline solution respectively. (Castration was performed 1 month before the mice were
sacrificed.)
The homogenates were centrifuged (8500 g for 10 min) and their respective supernatants were
used
for treatments. The mixture of and ß-farnesene was synthesized in our laboratory as
follows:
commercially available trans-nerolidol (Aldrich, Milwaukee, WI) was heated in
hexamethylphosphoramide at 80 °C for 12 h to yield a mixture containing equal amounts of
E,E--farnesene and E-ß-farnesene with a purity of >95%, as judged by its analyses
by the
combined gas chromatography/mass spectrometry and proton nuclear magnetic resonance
spectrometry. One drop (~50 µl) of each stimulus sample was delivered
directly to
the nasal groove and external nares of the tested animals (held by hand) via a small plastic tube
connected to a microsyringe. Each animal was treated twice daily for 10 consecutive days, first in
the
morning (08.00-10.00 h), and then in the afternoon (16.00-17.00 h). After each treatment,
animals
were immediately returned to the original cage. The estrous status of each female mouse was
determined each morning for 10 days using the standard vaginal smear method (Rugh,
1990). A smear
with cornified cells suggested estrus, while the appearance of persistent leukocytes and/or mucus
in the
smear indicated diestrus. All animals were individually labeled for identification. Animals
exhibiting
estrus for two consecutive days were not recounted. Each animal treatment group was housed in
a
separate testing room free of males. In each treatment group, 4-5 cages of animals (16-20 animals
for
set I, 40 for set II) were used. All animals were tested just once.
The number of animals exhibiting estrus in the first 5 days of treatments and the mean
number of
estrous cycles during the whole 10 day experimental period were recorded and considered as
indicators for estrus synchronization and induction through the tested samples. The data were
analyzed
using 
2 analysis and two-way analysis of variance (ANOVA) (Zar,
1984). The probability level for a significant difference was set at 0.05.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Table 1 contains the data for estrus disposition during the first 5 days of experimental treatments, showing the influence of various stimuli on estrus induction and synchronization. It is clearly demonstrated here that a significantly higher percentage of females (P< 0.01) treated with the aqueous extract of intact preputial glands or the solution containing synthetic farnesenes attained their estrus as compared with the animals treated with the extract from castrates or the water control. However, no significant difference was detected between the control and the extract of castrate preputial glands (P> 0.05), verifying that the biological activity of the preputial gland is under gonadal control. Since the levels of farnesenes are androgendependent (Harvey et al., 1989
),
it is easy to explain why the castrate preputial gland is biologically ineffective. There was a
characteristic
days 3 and 4 peak of estrus (Marsden and Bronson, 1964) in the case of
positive stimuli (P<
0.01). For example, in set II, the total occurrence of estrus on days 3 and 4 for the intact preputial
group and the farnesene group was 47.5% (19/40) and 45% (18/40) respectively, while no such
`peak' was evident for the remaining two groups (15 and 27.5% for the water
control
and the castrate preputial gland respectively).
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Comparing the results from sets I and II, it can be seen that the percentage of females stimulated into estrus appears to be higher for the less crowded environment (P = 0.12), though the difference did not reach significance at P = 0.05 under the current experimental design. It has been known that the degree of estrus suppression in grouped females is density-dependent (Champlin, 1971
). Consequently, the effect of estrus induction by the odor stimuli is likely to be
related
to density.
Thus, in a high-density situation, a reversal of estrus suppression by a positive male stimulus
could be
partially counterbalanced by a signal of female origin (Whitten, 1959; Ma et al.,
1998) or
some other stress-related factors caused by overcrowding. Table 2 summarizes the data for frequency of estrous cycles during the whole 10-day experimental period. The data support long-term inductive effects of the stimuli on estrous cycles. In both density conditions, the intact preputial extract and synthetic farnesenes caused significantly more estrous cycles (P< 0.01), than the control (water) stimulus and the extract of castrate preputial glands. Between the two density sets, there was some tendency of the animals from the four-per-cage condition to exhibit more frequent cycles, although the differences were not significant (P = 0.07). Once again, female-originated inhibitory chemosignals produced by crowding may exert some counterbalance to the stimulatory effect of tested samples.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The results shown here verify the earlier reports (Gaunt, 1968; Chipman and Albrecht, 1974;
Marchlewska-Koj et al., 1990) on the effectiveness of preputial
gland
secretions in estrus
induction. The estrus induction phenomenon is clearly androgen-dependent, since this capacity
disappears after castration. The major androgen-dependent volatile constituents of the preputial
gland
secretion, the sequiterpenes and ß-farnesene, seem to account for most observed
biological
activity associated with this gland.
We have previously demonstrated that farnesenes have some behavioral roles. They are the
chemosignals causing territorial avoidance among male mice (Novotny et al.,
1990b; Jemiolo et al., 1992) and attracting females (Jemiolo et al., 1991). Here, we find their
function as primer pheromones. It is likely that these two compounds act as multipurpose
pheromones:
signaling pheromones for an immediate behavioral control and primer pheromones for a
physiological
regulation. This does not seem unreasonable, as we have seen previously a somewhat parallel
situation
with dehydro-exo-brevicomin and 2-(sec-butyl)-4,5-dihydrothiazole, two
compounds that are male-originated and androgen-dependent chemosignals. When added to
castrate
urine, these two compounds can act synergistically to potentiate inter-male aggression (Novotny et
al., 1985a), be attractive to females (Jemiolo et al., 1985) and induce estrus
synchronization (Jemiolo et al., 1986). We have previously
observed
(Jemiolo et al.,
1991) that the investigatory preference for synthetic farnesenes was stronger for
sexually
experienced
females than naive females. Sexual experience by females may thus also be important in the
process of
estrus induction. Nevertheless, estrous cycles of grouped virgin females were shown to be
successfully
induced by the stimuli from male mice (Marchlewska-Koj et al., 1990; Marsden and Bronson,
1964).
Both bladder urine (Bronson and Whitten, 1968) and preputial glands
[as
shown here and
elsewhere (Gaunt, 1968; Chipman and Albrecht, 1974; Marchlewska-Koj et al., 1990)]
contain chemosignals that are active in estrus induction. This suggests that estrus induction
signals can
come from multiple sources. Chipman and Albrecht (1974) pointed out
that in
addition to the urinary
factor, a preputial substance may become incorporated into the male void urine and thus enhance
the
stimulation of estrus. Our previous study demonstrated that two bladder urine trace components,
dehydro-exo-brevicomin and 2-(sec-butyl)-4,5dihydrothiazole, potentiate the
Whitten effect (Jemiolo et al., 1986). The results of this work
reveal
that the two urinary
farnesenes (produced in the preputial gland and excreted into the urine) are also active in estrus
induction. At the molecular level, this supports the notion of the additive nature of
chemosignaling from
two metabolically distinct sources.
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Acknowledgments |
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This work was supported by grant no. DC 02418 from the National Institute of Deafness and Communicative Disorders, US Department of Health and Human Services.
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Accepted January 7, 1999
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