BIOLOGY OF Male

REPRODUCTION and37, Female

1066-1074 Genotype
(1987) Mediate Pheromonal Regulation

of the Mouse Estrous Cycle’

DAISY DELGADO DE LEON and MARYLYNN S. BARKLEY2

Department of Animal Physiology

University of California, Davis
Davis, California 95616

ABSTRACT
Estrous cyclicity was studied to examine the possibility that strain differences in the regularity of the mouse
estrous cycle are the result of different olfactory signals produced by the male. Females with regular estrous
cycles (lines E and Si) were housed in the olfactory presence of males from a line with irregular cycles (line
CN-) or in the presence of males of their own line (used as a control). Females with irregular cycles (line CN-)
were housed in the presence of males from a line with regular cycles (line E) or were exposed to males of their
own line. The regularity of the estrous cycle decreased in line E females (regular cycles) when exposed to line
CN- males (irregular cycles). The decreased regularity of line E cyclicity resulted from an increased period of
diestrus, i.e., lengthening of the cycle. In contrast, line Si females (regular cycles) did not show any change in
estrous cyclicity when exposed to line CN- males. The period of diestrus increased in line CN-females when
they were exposed to line E males. These results provide evidence that 1) the genotype of the male can
influence the regularity of the estrous cycle, and 2) the genotype of the female regulates her responsiveness to
environmental factors (e.g., male odor).

INTRODUCTION
Hereditary factors also affect the regularity of the
Pheromones play an active role in the reproductive mouse estrous cycle (Nobunaga, 1973). In a previous
physiology of the laboratory mouse. While the rat study (Barkley and Bradford, 1981), estrous cyclicity
typically exhibits 4- or 5-day estrous cycles, the was examined in lines of mice successfully selected
mouse cycle ranges from 3 to 7 days, and pseudo- for small litters, large litters, high embryo survival,
pregnancies are frequently observed as a function of and rapid post-weaning weight gain. Improved repro-
the olfactory environment in which the females are ductive performance (high embryo survival, large
housed (Dewar, 1959; Whitten, 1959; Marsden and litters) was associated with increased regularity of the
Bronson, 1965; Champlin, 1971; Ryan and Schwartz, cycle, whereas poor teproductive performance (small
1977). Two different phenomena interact to produce litters, embryo loss with selection for gain) was cor-
such variation: 1) the acceleration or induction of related with prolonged cycles and anestrus. This
cyclicity by factor(s) in male urine; and 2) suppres- strain variation in estrous cyclicity presumably arises
sion of estrous cyclicity by chemosignal(s) produced from differences in endocrine regulation and/or
among groups of females. These effects on the mouse variation in responsiveness to environmental factors.
estrous cycle are presumed to be olfactory-mediated The purpose of the present study was to investigate
since exposure to urine from males or grouped whether strain differences in the regularity of the
females can elicit the same responses as when the cycle are the result of different pheromonal signals
animals are actually present (Marsden and Bronson, produced by the male.
1964; Bronson and Whitten, 1968; Champlin, 1971).
MATERIALS AND METHODS

Animals
Accepted May 19, 1987. The mice used in this study consisted of three
Received February 28, 1986.
genetically selected strains (E, Si, and CN-) derived
‘This work was supported by PHS GM08666.
2 Reprint requests.
from the same base population produced by crossing

1066
 GENOTYPE, MALE PHEROMONES AND ESTROUS CYCLICITY 1067

four inbred lines (C57BL/6, AKR, C3H, and DBA/2). of vaginal fluid were again performed for another 4
Line E was selected for high embryo survival (Brad- wk. At the end of this time, new males of the original
ford, 1969); lines Si and CN- were selected for large strain (E) were housed with the females, which
or small litters, respectively (Bradford, 1968, 1971). subsequently were monitored for estrous cyclicity for
As a correlated response to selection, lines E and Si a third period of 4 wk.
have regular estrous cycles, whereas line CN- has B. Twenty-nine females of line E initially were
irregular cycles (Barkley and Bradford, 1981). Mice housed 3/cage for 4 wk with a line CN- male, and
of all three lines were housed in the same animal daily observations of vaginal fluid were taken. After
room maintained at 21-23#{176}C and 14L:1OD (lights on this period, the male was exchanged for one of strain
from 0500-1900 h). Food (Purina Rodent Chow, E, and a second 4-wk observation period ensued. Sub-
Agronomics, Woodland, CA) and water were supplied sequently, line E females were exposed to males of
ad libitum. the original strain (CN-), and estrous cyclicity was
Animals were weaned at 21-2 5 days of age accord- monitored during this last 4-wk period. This is the
ing to size and were caged by sex (4-6/cage). At 6 same design as described above, but the order of ex-
wk of age, females were permanently identified by posure of females to the two strains of males is the
notching one ear and were then housed in cages reverse of Group IA.
divided by a wire mesh. Three females were placed on Group II: A and B. Twenty-one line 51 (regular
one side of a divided cage and one male was housed cycles) females per group (A or B) were subjected to
on the other side to expose the females to maximum the same treatment as described for Groups IA and
male pheromonal stimulation. IB, i.e., Group IIA was first exposed to line Si males,
then to CN- males, and finally to new Si males; Group
Estrous Cyclicity IIB was first exposed to CN- males, then to Si

After females were placed in divided cages, a 2-wk males, and finally to new CN- males.
period elapsed before vaginal smears were taken daily Group III: A and B. Twenty-six line CN- (irregu-
by saline lavage between 0900 and 1100 h. The vagi- lar cycles) females per group (A or B) were subjected
nal fluid obtained was examined in stained (new to the same treatment as described for Groups IA
methylene blue), wet preparations. The following and IB, i.e., Group lilA was first exposed to line CN-
criteria were used for identification of cycle stages: males, then to line E males, and finally to new line CN-
diestrus (DE)-leukocytes or leukocytes and some males; Group IIIB was first exposed to line E males,
nucleated cells; proestrus (PE)-nucleated cells or then to line CN- males, and finally to new line E males.
nucleated and cornified cells; estrus (E)-cornified Table 1 summarizes the design of Experiment I.
cells; metestrus (ME )-cornified cells and leuko cytes. Throughout the remaining text, treatment groups will
Females from all three strains were observed for a be identified by condition number (1-6), e.g., in
12-wk period. Two parameters were studied: 1) the figures illustrating experimental results.
percentage of time spent in each phase of the cycle, Experiment I was designed to determine 1) the
and 2) the total number of cycles completed during estrous cycle characteristics of females housed with
the 4-wk period of observation. males of their own strain, 2) any modification
in estrous cyclicity resulting from changing the
Experimental Design: Experiment 1 strain of male to which the female was exposed, and
3) whether any change observed during the second ex-
In a split-block design, females of each line initially
posure could be reversed by returning the female to the
were exposed to males of their own (Group A) or a
strain of male with which she was originally housed.
different (Group B) strain. Groups of mice repre-
sentative of each strain were assigned randomly to
one of the following treatments:
Experiment II
Group I: A. Twenty-nine females of line E (regu-
lar cycles) were housed as previously described with a To determine the effect of aging on estrous cycle
male of their own line (E); daily observations of dynamics, a study of line E females (regular cycles)
vaginal fluid were performed for 4 wk. At the end of and line CN- females (irregular cycles) was per-
this period, the strain of the male was replaced by formed. Females representative of each line were ex-
one of line CN- (irregular cycles). Daily observations posed in alternating fashion only to males of their
1068 DE LEON AND BARKLEY

TABLE 1. Design of Experiment I.

Period
of exposure
Condition Exposure Male genotype (days)

Group A 1 1 Female strain X male strain Same strain 1-28

2 2 Female strain X male strain Different strain 29-5 7
3 3 Female strain X male strain Same strain 58-84

Group B 4 1 Female strain X male strain Different strain 1-28

5 2 Female strain X male strain Same strain 29-5 7
6 3 Female strain X male strain Different strain 58-84

own strain. Estrous cyclicity was monitored for considered, i.e., the nested model was not collapsed to
12 wk, i.e., 3 consecutive periods of 28 days increase the power of the test for lines. The Bonferroni
equivalent to exposures 1, 2, and 3 in Experiment multiple comparisons procedure was used to assess dif-
I. Females were exposed first to one intrastrain ferences between group means (Neter and Wasserman,
male, then to another intrastrain male, and, finally, 1974).
to a third intrastrain male. RESU LTS

Shown in Table 2 is the percentage of time line E

Statistical A nalysis or CN- females spent in each stage of the cycle, and
Data were subjected to repeated measures analyses the total number of cycles exhibited by these females
of variance by the hierarchical (or nested) model, when exposed only to males of their own strain.
where Increasing age did not affect the amount of time
‘jk bt+lI+CJ+eIJk, females of either line spent in metestrus during the
84 days studied. However, the percentage of time
= the observation of the kth female within the spent in other stages of the estrous cycle changed as
jth cage within the ith line, the females aged. Females of both lines spent less
the overall
= mean, time in PE and E with increasing age, and the propor-
1: = effect of the ith line, tion of time spent in DE increased. The number of
C1 = effect of the jth cage within the ith line, cycles completed was similar during each of the three
e#{149}1k
= error (remainder). phases of this study. Of interest is the similarity of
estrous cycle regularity in both lines E and CN-. This
Although hierarchical analyses of variance indi- reflects an increase in estrous cyclicity in line CN-
cated a significant effect of cage within line, this was that occurred when females were housed in the same
restricted to the proportion of days spent in DE in cage with a CN- male as opposed to being housed in
lines E and Si. A closer inspection of line E data re- a cage separate from but next to a cage containing
vealed that one Group IA female in condition 2 in CN- males (Barkley and Bradford, 1981).
each of two cages spent 71 and 57% of the observa- The effect of male strain on the composition of
tion period in DE, respectively, as compared to the the estrous cycle in line E females (regular cycles)
line average of 26% for this condition. A similar in- is shown in Figures 1 and 2. When line E females were
spection of line Si data showed that three females first exposed to line E males (Group IA, condition 1),
in Group hA, condition 2, and three females in highly regular estrous cycles occurred (Fig. 1, panel
Group IIB, condition 4, spent 52 and 57% of the ob- A). In contrast, irregular cycles and periods of
servation period in DE, respectively, compared to the anestrus were seen when line E females were first
line average of 64 and 68% for Group hA, condition exposed to line CN- males (Group IB, condition 4;
2, and Group IIB, condition 4, respectively. Thus, the Fig. 1, panel B). Interestingly, females first exposed
significant cage-within-line effect was attributed to to line CN- males showed an improvement in estrous
only 8 of 152 females in Experiment II. Nevertheless, cyclicity when they were subsequently exposed to
the effect of the jth cage within the ith line (Cr1) was line E males (Group IB, condition 5; Fig. 1, panel C).
 GENOTYPE, MALE PHEROMONES AND ESTROUS CYCLICITY 1069

TABLE 2. Effect of aging on estrous cycle composition and frequency in lines E and CN._.a

. Mean number
Mean proportion ± SEM
of cycles
Exposure N Diestrus Proestrus Estrus Metestrus ± SEMb

Line E 1 (Days 1-28) 11 0.42 ± 0.02 0.18 ± 0.01 0.18 ± 0.01 0.22 ± 0.03 4.00 ± 0.30
2 (Days 29-56) 11 0.48 ± 0.05 0.14 ± 0.01 0.15 ± 0.02 0.22 ± 0.04 3.55 ± 0.43
3 (Days 57-84) 11 0.56 ± 0.05 0.14 ± 0.01 0.13 ± 0.02 0.17 ± 0.02 3.18 ± 0.48

Line CN- 1 (Days 1-28) 12 0.45 ± 0.04 0.18 ± 0.02 0.21 ± 0.02 0.16 ± 0.02 3.91 ± 0.43
2 (Days 29-56) 12 0.50 ± 0.06 0.16 ± 0.02 0.18 ± 0.02 0.18 ± 0.02 3.42 ± 0.53
3 (Days 57-84) 12 0.60 ± 0.03 0.14 ± 0.02 0.10 ± 0.01 0.16 ± 0.01 3.17 ± 0.44

aBased on percentage of days/exposure spent in each phase of the cycle.

bBased on 28 days/exposure.

As shown in Figure 2, the amount of time spent in of DE in the subsequent exposure to a line CN-
DE increased significantly (p’0.01) when line E male. Group IA females first exposed to line E males
females were first paired with a line CN- male had shorter periods of DE than Group lB females
(Group IB, condition 4). The time spent in PE and E exposed to line E males after housing with line CN-
decreased during this treatment (p0.001 and males (condition3 vs. condition 5; p0.O5), i.e., the
p0.0i, respectively). The duration of ME was simi- initial exposure to a line CN- male lengthened the
lar regardless of which strain of male the female was duration of DE.
exposed to for the first time. A comparison of the When line E females were exposed only to line E
duration of DE in Group lB females initially exposed males during a 12-wk period, DE increased with age
to line CN- males (condition 4) with that in Group (Table 2). An opposite phenomenon was observed in
IA females exposed to line CN- males after housing line E females exposed to line CN- males after pre-
with line E males (condition 2) shows that DE was vious housing with a line E male: in both Groups IA
significantly shortened in condition 2 (p0.O5), i.e., and IB, the duration of DE decreased with increasing
prior exposure to a line E male decreased the length age. Furthermore, line E females spent a longer time

A. E2X Ed C. EIxEd

DE

Condition 1 (days 1-28) Condition 4 (days 1-28) Condition 5 (days 29-56)

Group 1A Group lB Group lB

FIG. 1. Pattern of estrous cyclicity in line E females initially exposed to line E (Panel A) or line CN- males (Panel B). Panel C represents the
pattern of estrous cyclicity in line E females housed with line E males after an initial exposure to line CN- males. The data in each panel represent
the complete record of estrous cyclicity for individual females housed 3/cage (N=3 cages). The data are expressed as complete cycles beginning with
diestrus (DE) followed by proestrus (PE), estrus (E), and metestrus (ME). Dashed lines represent an incomplete cycle or a stage of the cycle that was
not observed (i.e., PE followed the next day by ME).
1070 DE LEON AND BARKLEY

LINE E by exposure to a CN- male. Estrous cycle frequency

Exposure 1 Exposure 2 Exposure 3 was also the same in all conditions, except condition
70 Group IA #{149}
Diestrus 2, where the total number of cycles increased (Table
U Proestrus 3, p0.Oi).
60 Estrus The effect of male genotype on estrous cycle com-
0 Metestrus position in line CN- females is presented in Figure 4.
50
In contrast to line E females, line CN- females
1 showed no significant differences in any of the cycle
40
stages when first exposed to a male from a different
line. Only a few differences in estrous cyclicity were
30-
seen in line CN- females when they were exposed to
line E males. However, when exposed to a line CN-

11
01 20
01
01

U to
U
LINE Si
C)
0) 2 3 Exposure 1 Exposure 2 Exposure 3
01 Group hA
Group lB 70
70
01
01 Diestrus
C 60
01 60 U Proestrus
0
I- Estrus
01
50
50-
c:i
L 40
40

30
30

01 20
20 -
01
01

0 I0
10 -
0
>.
C.)
0
01 2
>.
4 5 6 01
0 Group IIB
Condition
01
FIG. 2. Effect of male genotype on estrous cycle composition in
C
line E mice. Conditions 1, 2, and 3 represent females (N=29) first 01
a
exposed to a male of their own strain (line E) in which regular cycles
occur, then to a male from a strain with irregular cycles (Line CN-), a-
and finally housed with a new male from the original strain (E). Condi-
tions 4, 5, and 6 represent another group of females (N=29) for which
the order of exposure to males of the two strains (E, CN-) was re-
versed. The bars indicate the mean ± SEM of the percentage days/cycle
phase.
30

20
in PE and ME when exposed to line CN- males after
a prior exposure to line E males (Fig. 2, condition 2 10
vs. condition 4, pO.Oi and p0.O0i, respectively).
They also spent a longer period in ME when the strain 0
4 5
of the male to which they were first exposed
Condition
was their own (line E; condition 3 vs. condition 5,
FIG. 3. Effect of male genotype on estrous cycle composition in
pO .05). line SI. Conditions 1, 2, and 3 represent females (N21) first exposed
Estrous cyclicity in line Si females (regular cycles) to Si males, then to CN- males, and finally to Si males. Conditions
4, 5, and 6 represent females for which the order of exposure to males
was stable in all of the conditions studied (Fig. 3), of the two strains (Si, CN-) was reversed. The bars indicate the mean
i.e., the composition of the cycle was not influenced ± SEM of the percentage days/cycle phase.
 GENOTYPE, MALE PHEROMONES AND ESTROUS CYCLICITY 1071

TABLE 3. Effect of male genotype on the number of complete estrous

cycles than females in condition 4 (p’0.05). Con-
cycles during 28 days.
sistent with all the other parameters examined, male
Mean num- genotype did not change the total number of cycles
Genotype ber cycles in line Si except when Si females were initially ex-
Group Condition combination N ± SEM
posed to Si males and subsequently exposed to line
IA 1 (Days 1-28) E X E 29 3.24 ± 0.21 CN- males (condition i vs. 2, p0.05).
2 (Days 29-57) E X CN- 29 1.77 ± 0.20
3 (Days 58-84) E X E 29 2.31 ± 0.20
DISCUSSION
HA 1 (Days 1-28) Si X Si 21 2.41 ± 0.33
2 (Days 29-57) Si X CN- 21 3.60 ± 0.37 It is well established that olfactory stimuli modu-
3 (Days 58-84) 51 X Si 21 2.55 ± 0.44 late estrous rhythmicity in the mouse. When females
lilA 1 (Days 1-28) CN- X CN- 26 4.23 ± 0.31 are isolated, a great percentage of them exhibit irregu-
2 (Days 29-57) CN- X E 26 4.29 ± 0.29
3 (Days 58-84) CN- X CN- 26 2.43 ± 0.29

lB 4 (Days 1-28) E X CN- 29 1.45 ± 0.30 LINE CN-

5 (Days 29-57) E X E 29 3.46 ± 0.20
Exposure 1 Exposure 2 Exposure 3
6 (Days 58-84) E X CN- 29 2.90 ± 0.19
7#{176} Group lIlA
lIB 4 (Days
5 (Days
1-28)
29-57)
Si
Si
X CN-
Si
21
21
2.55 ± 0.36 #{149}
X 3.25 ± 0.35
60 Proestrus
6 (Days 58-84) Si X CN- 21 2.33 ± 0.36
#{149}
IIIB 4 (Days 1-28) CN- X E 26 3.40 ± 0.27 50 1-
0 Meteetrue
5 (Days 29-57) CN- X CN- 26 3.33 ± 0.34
6 (Days 58-84) CN- X E 26 3.07 ± 0.30
40

30

01 20
0)
01
male, the time spent in DE increased if the line CN-
female was first exposed to a line E male (condition 2 U l0
U
vs. 4, p’0.05). This effect was also seen even when
C)
the CN- female was initially exposed to a male of her S.. 0
0)

own strain (condition 4 vs. 6, p0.05; condition 5 01

0 Group IIIB
70
vs. 6, p0.0i). The duration of E decreased in condi-
01
tion 2 vs. 4 and in condition 3 vs. 5 (p0.O5). C
01 60
The total number of cycles completed in each U
I-
01
condition is summarized in Table 3. Line E (Group I) a.
50
females showed a significant decrease in the number
of cycles they completed when exposed to line CN- 40
males (condition i vs. 4, p0.01; condition 1 vs. 2,
p’O.01). An increased number of cycles occurred 30

when line E females were exposed to males of their

own line immediately after exposure to CN- males 20

(condition 2 vs 3, p0.05; condition 4 vs. 5, p0.01).

tO
Line CN- (Group III) females also showed an in-
crease in the number of cycles they completed when
0
initially exposed to a line CN- male (condition 1) vs. 5
a line E male (condition 4) (p0.05). Other changes Condition
in the number of cycles observed in line CN- females
FIG. 4. Effect of male genotype on estrous cycle composition in
were seen when the male was changed: females in line CN-. Conditions 1, 2, and 3 represent Group lIlA females (N26)
condition 1 had more cycles than females in condi- first exposed to CN- males, then to E males, and finally to CN- males.
Conditions 4, 5, and 6 represent Group IIIB females for which the
tion 6, as did females in condition 2 vs. conditions 4
order of exposure to males of the two strains (CN-, E) was reversed.
and 6 (p0.05). Females in condition 3 had fewer The bars indicate the mean ± SEM of the percentage days/cycle phase.
1072 DE LEON AND BARKLEY

lar or prolonged cycles (Whitten, 1958). However, by the observation that the prolongation of ME in
when females are placed in close proximity to a male, line E females in conditions 2 and 6 is abolished when
a significant proportion of them exhibit short (4-6 line E females are first housed with line E males and
days) and regular cycles. The estrous cycle in the then exposed to castrated line CN- males (DeLeon
mouse, and other species, reflects a series of sponta- and Barkley, 1986). The change in female responsive-
neous ovarian events: follicular growth, ovulation, ness to presumably androgen-mediated male olfactory
and formation of the corpora lutea (CL) (McClintock, stimuli seems to result from the previous exposure
1983). These changes can be detected by studying to a male of her own strain. This suggests that
vaginal cytology. An alteration in the timing of any changes in follicular growth rate may occur as a func-
of these ovarian events can suppress, enhance, or tion of the male to which the female is first exposed.
synchronize estrous cyclicity. Pheromones seem to If exposure to line E males stimulates follicular
modulate the length of the ovarian cycle by 1) in- growth rate to a greater extent than does exposure to
creasing or decreasing follicular growth, 2) delaying line CN- males, then previous exposure to line E
or advancing ovulation, or 3) varying the lifespan of males followed by subsequent exposure to CN-
the CL. Within a cycle, variation in the proportion of males may reduce ovarian E2 production, thereby
types of vaginal smears (PE, E, ME, DE) can indicate inducing a delay in ovulation. Such a change in
those ovarian processes altered by exposure to male ovarian function could result in vaginal smears charac-
pheromones. teristic of ME (McClintock, 1983).
The effect of male genotype on the composition of The lengthening of ME in line E females when ex-
the estrous cycle in mice selected for high embryo posure to a line E male precedes exposure to a line
survival is dramatic. When line E females are first CN- male is even more fascinating in view of the
exposed to line CN- males, a marked increase in DE observation that ME does not change with age in the
occurs, and prolonged cycles and periods of anestrus intrastrain exposure condition (Table 2). Both
are consistently observed. Even longer periods of DE strains of mice (E and CN-) responded similarly
and fewer cycles occur after exposure of line E when housing with males of their own strain was
females to castrated line CN- males (DeLeon and alternated during each of the three phases of study,
Barkley, 1986). This suggests that testicular factors i.e., the periods of ME in both line E and CN- were
are involved in the CN- male’s mediation of the line consistent when these females were exposed to males
E female’s estrous cyclicity. Lengthening of the of their own strain during each of the three phases
estrous cycle indicates either a prolonged function of of study. With increasing age, the period of DE
luteal tissue or a delay in the preovulatory estradiol lengthened and PE and E decreased concomitantly.
(E2) surge. In the first case, progesterone (P) secreted These changes in the proportion of time spent in each
from luteal tissue can retard follicular development phase of the cycle had no significant effect on cycle
and delay the gonadotropin surge. Alternatively, a frequency in either mouse strain. In a longitudinal
delay in the preovulatory E2 iurge lengthens the study of estrous cyclicity, Nelson et al. (1982) found
follicular phase and increases the period during which that aging female mice (13-16 mo of age) show a
cornified cells are present in vaginal smears. It should decrease in cycle frequency along with persistent
be noted, however, that a delay in the estrogen rise vaginal cell cornification. The time at which females
would also extend the leukocytic phase, thereby from different mouse strains show these aging charac-
making it difficult to use vaginal smears to distinguish teristics varies considerably (6-16 mo-Jones and
between prolonged function of luteal tissue and re- Krohn, 1961; Parkening et al,, 1980; Felicio et al.,
tarded E2 secretion. For example, aging C57BL/6J 1984). The females used in the present study ex-
females show an increase in the length of DE, which hibited a cycle frequency (4.0 and 3.9 for E and
is correlated with a delay in the elevation of E2 prior CN-, respectively) that compares well with the cycle
to the LH surge (Nelson et al., 1981). frequency of C57BL/6J mice at 6 mo of age (4.2); at
The reduced frequency of long cycles and the ab- this age, C57BL/6J mice are in their period of maxi-
sence of anestrus when line E females are exposed to mum cycle frequency and do not require the presence
line CN- males after a previous exposure to line E of male olfactory cues to cycle regularly (Nelson et
males could be indicative of a change in female sensi- al., 1982), In fact, beyond 5 mo of age, but not
tivity to pheromonal cues. This idea is strengthened before, C57BL/6J females cycle regularly when
 GENOTYPE, MALE PHEROMONES AND ESTROUS CYCLICITY 1073

housed alone (Nelson et al., 1982; Felicio et al., line E males has no effect on cyclicity. In contrast,
i984). Although inconclusive, analyses of reproduc- initial exposure of the CN- females to line E males
tive capacity in lines E and CN- suggest that, unlike reduces cyclicity, and this effect is not overcome by
C57BL/6J females, cycle regularity declines signifi- subsequent exposure to males of their own strain
cantly before 6 mo of age and may remain dependent (Table 3). In other words, the irregular cyclicity
on male olfactory signals. characteristic of line CN- is enhanced after exposure
In contrast to line E females, in which exposure to interstrain males. Although male genotype
to an interstrain male produced dramatic changes plays a major role in the regulation of estrous
in estrous cyclicity, this parameter and the total num- cyclicity, the proximity of the female to male phero-
ber of cycles completed were stable in line Si females monal information is also important. For example,
in all the conditions studied. This relative difference shorter cycles are produced when line CN- females
in responsiveness to male pheromonal cues is sugges- cohabit with line E males than when CN- females
tive of interstrain genetic variation. Marsden and are housed next to but not in the same cage with line
Bronson (1965) studied the role of the male in CN- males (Barkley and Bradford, 1981). Further
estrous cycle synchronization and demonstrated that study is needed to determine whether this is an age-
C57BL/6J females were unresponsive to males from specific phenomenon.
a different strain (CBA/J). In a series of studies The present study indicates that the relationship
designed to investigate the influence of pheromonal between genotype and estrous cyclicity is complex.
facilitation of ovulation in different strains of mice, In general, the results support the concept that
Eleftheriou et al. (1973) showed that the presence of estrous cyclicity is regulated by a mixture of chemical
a male increased ovulation in females from strains signals rather than a single, predominant compound.
C3 HeB/FeJ and BALB/cJ, but not in females from Evidence for this theory is provided by the observa-
strains DBA/2J and C57BL/6J. Zarrow et al. (1973) tion that line CN- males elaborate stimuli sufficient
also observed that males from different strains were to maintain cyclicity in both line CN- and Si
able to facilitate ovulation in females from strains females, yet this information does not support
SWR/J, BALB/cJ, and C57BL/6J, but males of the cyclicity in line E females. Thus it can be argued that
C57BL/6J strain failed to elicit this response. In line CN- and Si females are more responsive to
general, these studies demonstrated that genetic dif- pheromonal stimulation by CN- males than are line
ferences could affect the response of the female to a E females. Another possibility is that line Si females
male from a different strain as well as the capacity of do not require male pheromonal stimuli to cycle
a male to produce a pheromone that could induce a regularly, whereas line CN- and F females must be
response by a female. In a previous study (1972), provided male olfactory cues to maintain estrous
Eleftheriou and coinvestigators concluded that the cyclicity. In other words, at an earlier age, line Si
presence of male pheromones in the SWR/J strain was females may resemble the C57BL/6J female who be-
determined by a single dominant gene and proposed comes able to cycle regularly when housed alone.
the presence of alleles that determine levels of phero- Alternatively, the attenuated cyclicity in line E
monal activity in males. females after an initial exposure to line CN- males
An intriguing possibility is that the impaired may arise from an enhanced ability to respond to the
ability of line CN- males to induce estrous cyclicity full complement of male chemical communication,
in line E females (Fig. i, panel B) is a heritable trait including inhibitory signals. At another level of com-
similar to that demonstrated by Zarrow et al. (1973) plexity, initial exposure to an intrastrain male may
in line C57BL/6J. Such a trait could have segregated produce a priming effect that influences cyclicity
in line CN-, which is derived from a 4-way cross of during the period of exposure to an interstrain
lines C57BL/6J, DBA/2, AKR, and C3H. However, at male. This idea is supported by the observa-
least some component of pheromonal stimulation of tion that after exposure to intrastrain males,
estrous cyclicity has been retained in line CN- males, DE is shortened when line E or Si females are housed
because in a first encounter they are more effective with line CN- males.
in improving cycle regularity in line CN- females In summary, the results of this study provide
than are line E males. If line CN- females are initially indirect evidence that pheromonal signals originating
exposed to line CN- males, subsequent exposure to from the male are complex in nature and may have
1074 DE LEON AND BARKLEY

separate genetic controls. While the genotype of the different strains of mice. J Endocrinol 57:363-70
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