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Male-induced synchrony of estrus in deermice
GENERAL
AND
COMPARATWE
ENDOCRINOLOGY
Male-induced
$
634-637
(1964)
Synchrony
F. H. BRONSON The
Jackson
of AND’
Laboratory,
Received
in
Deermice’
H. M. MARSDEN Bar
July
Estrus
Harbor,
Maine
20, 1964
Relative numbers of inseminations occurring during the first 4 days after pairing were compared in deermice which had been housed 1, 4, or 10 females per cage for the 2 weeks preceding pairing. Females crowded 10 per cage before pairing showed a characteristic peak in estrous activity 3 days after pairing; those isolated before pairing showed no such peak; and females housed 4 per cage before pairing were intermediate. Topical (nose) exposure of females to male urine for 2 days before pairing accelerated attainment of estrus when compared with controls exposed to female urine. Male-induced synchrony of estrus in deermice appears to operate in a manner similar to that previously described for house mice.
Synchrony of estrus within groups of house mice, a phenomenon which is maleinduced and olfactory-mediated, has been described by Whitten (1956). Specifically, he found: (1) a peak number of inseminations on the t,hird night after pairing with fewer than expected on the first, second, and fourth nights; (2) a peak frequency of inseminations on the first night after pairing providing the females had been exposed to the “presence” of a male for 2 days prior to pairing; (3) that pre-pairing exposure to cages soiled by males somewhat altered the characteristic third day peak, indicating involvement of exteroceptive stimuli ; and (4) that such effects were somewhat dependent upon grouping of females prior to pairing (Whitten, 1959). Marsden and Bronson (1964)) also using house mice, altered the expected third day peak in inseminations by exposing females to male urine for 2 days before pairing, thus indicating a component of urine as the pheromone responsible for the effects found by Whitten. The purpose of the present experiments was to test for the presence of such male‘Supported by Public Health M-4481, CRT-5013, and HD-00767 tional Institutes of Health, Public
Service grants from the NaHealth Service. 634
induced synchrony in a nonmurine species of rodent, prairie deer mice (Peromyscus maniculatus bairdii). Specifically, females housed in different sized groups before pairing were compared with respect to t,he characteristics of their estrous cycles and the relative numbers of inseminations which occurred on each of the first 4 days after pairing. In addiCon, the ability of male urine to accelerate attainment of estrus was evaluated in this species. MATERIALS
AND
METHODS
Experiment I. All deermice were from a stock maintained in this laboratory for over 10 years. Females were grouped 4 per cage at weaning (2128 days) and paired at 75-35 days of age. Two weeks prior to pairing, 142 females were isolated and 137 were grouped 9 or 10 per clean cage. A total of 284 animals remained in groups of 4 in their original cages. Females were paired at 4 P.M. in clean cages with a male from a battery of 6-12month-old studs of proven fertility. Vaginal smears were examined at 9 .4.M. and 4 P.M. on the following 4 days. The criterion of insemination consisted of an average of 20 sperm per microscopic field ( ~264). Vaginal smears were taken daily for 8 days from an additional 50 animals of the same age (6 isolates, 24 grouped 4 per cage and 20 grouped 10 per cage) and were evaluated with respect to cycle characteristics.
OF
SYNCHRONY Ezperiment II. A total of 233 females were reared as in Experiment I. Two weeks before pairing, they were transferred to clean cages in groups of 9 or 10 per cage. All females in 12 cages (119 females) were individually exposed to male urine daily for the 2 days immediately preceding pairing. Such exposure consisted of placing one drop (about 0.05 ml) of urine, collected from mature males, on the general area of the external nares of each female 4 times a day at 2-hour intervals beginning at 9 A.M. Tmelve cages (114 females) were similarly treated with urine collected from mature females. Urine was collected indiscriminately from females in all stages of the reproductive cycle. Pairing and smearing procedures were idcnt,ical to Experiment I. Treatment, lvith urine in Expniment II and pairing and postpairing housing in both experiments were done in a 10 x la-foot room lighted from 5 A.M. to 7 P.M. and having a complete fresh air exchange 10 times per hour. 811 females used in the two experiments were moved into this room 2 reeks before pairing. Males were left in the room only as long as necessary: and, at all times during either experiment, a maximum limit of 200 animals was maintained in this room. All cages were 6 x 12 x 6-inch stainless steel. Statistical procedures consisted of testing, by F, frequency distributions of the numbers inseminated during the first 4 days after pairing for each type of treatment either against an expected (random) 25% per day or with each other. RESULTS
Ex-periwmf 1. About 749~ of the females housed 1. 4. or 10 per cage were inseminated within t,he first 4 clays after pairing with no significant differences in insemination rates
ESTRUR
633
between the three groups (Table I). The frequency dist,ributions of numbers insemtnated on each of the 4 days for each of the three groups differed significantly from an expected (random for a 4-day cycle) 2.5~~ per day at the 0.001 level. All groups were characterized by a low number of inseminations on the fourth clay. The dist’ributions across the first 3 days differed depending upon the degree of prior crowding. Animals housed one per cage before pairing showed little difference bet,ween each of the first 3 days ; those housed 10 per cage showed a strong tendency toward a third-day peak in inseminations; and t,hose housed 4 pei cage before pairing were intermediate with respect to a third-day peak. The dist,rihution of inseminations for females housed 40 per cage before pairing differed significantly from that of the 4 per cage group (10 < 0.01) and the isolate group (p < 0.~~~~. The frequency distributions of the grollps held I or 4 per cage were not significantly different. Vaginal cycles for 24 animals held 4 per cage showed a median cycle length of 3 days for 27 “definite” cycles in the 8-day period (19% were 3 days, 59% were 4 days: and 22% were 5 days in length). Wher, the groups held 1, 4, or 10 per cage were compared, there was a distinct impression oi longer, or more irregular, cycles in tile IO per cage group. Quantification of such ar? impression was difficult since smearing wab only conducted across an &day period:
TABLE 1 OF INSEMINSTIOXS DURING THE FIRST 4 D.~Ys AFTER P.URING 1, 1, OR 10 PER CAGE BOX 2 WEEKS BEFORE PAIRING (EXPERIMEX Ij OR 2 WEEKS AND EXPOSED T O MOLE OR FEK4LE URINE FOR 2 DY~YS BEFORE PaTRING (EXPERIMENT II) --
INSEMINATION RATES ‘L~VD DISTRIBUTION IN F-EXILES M-UNTUNED HELD 10 PER CAGE FOR
No.
Pre-pairino treatment Experiment, Housed Housed Housed Experiment Exposed Exposed
females
I l/cage 4/cage IO/cage II t,o 0 urine to ~3 urine
a %, by day,
of the number
which
No. inseminated
% Inseminstod
% hseminsted on day” 1 2 3 4 __-
142 285 13’7
111 202 9s
TS 71 7’
33 25 11
23 25 16
36 40 5!)
Y 10 13
114 119
86 92
ii 77
14 23
"7 43
50 "7
9 7
were
insemina,ted.
636
BROMSON
AND
however, one statistic which illustrates the difference was a comparison of the proportions of animals which showed no peak in epithelial cell activity for 5 consecutive days. A total of 10 of 20 animals in the 10 per cage group fell in such an arbitrary category, while only 2 of 24 of the 4 per cage group showed such prolonged periods of diestrum. One of 6 isolates showed 5 consecutive days with no period of estrus. Experiment II. Distributions of the relative numbers of inseminations across the first 4 days after pairing for both the male and female urine-treat,ed groups differ significantly (p < 0.001) from a random 25% per day. As in Experiment I, both distributions are characterized by low numbers inseminated during the fourth day. The two distributions differ significantly (p < 0.01) from each other; the group treated with female urine showing a third-day peak similar to the untreated 10 per cage group in Experiment I while the group exposed individually to male urine showed greater numbers on days 1 and 2. Estrus therefore occurred earlier after pairing if the females had been previously exposed to male urine. Two other aspects of insemination in colony-raised bairdii should be reported since they differ from most other laboratory rodents and, in addition, are relevant to the choice of methods in this study. First, insemination may occur during either the day or night. Of the 591 inseminations, 226 occurred between 4 P.M. and 9 A.M. (17 hours) and 365 between 9 A.M. and 4 P.M. (7 hours). These figures are not directly comparable because of the probable interact,ion between length of time between smearings and attainment of the arbitrary 20 per field criterion; however, they do indicate a lack of nocturnality in mating habits. Second, vaginal plugs are seldom found in association with inseminations and, in fact, may occasionally be found when t,here is no male present. A total of 6 solidified plugs, usually extruding 24 mm and always associated with estrous smears, were found in the absence of a male while examining 50 females for cycle characteristics.
MARSDEN
DISCUSSION
An arbitrary criterion of insemination (20 sperm per high-power field) was necessary in view of the lack of plug formation in bairdii. Past work (Bronson and Eleftheriou, 1963) with this criterion has yielded respectable pregnancy rates of 6090%. These figures, coupled with the fact, that an average of about 75% of the animals in the present study reached the criterion within 4 days, would tend to indicate that the method, even though arbitrary, is sound. Vaginal smearing is known to affect cycle characteristics; however, it can be pointed out that all groups were smeared in an identical manner. Insemination rates did not vary between groups. The fact that the social environment, male or female, has a profound effect on the estrous cycles of house mice and that many of the effects are olfactory-mediated is well documented (reviewed by Parkes and Bruce, 1961). Of specific interest here, in addition to the findings described in the introduction, are the suppression of estrus associated with crowding in all female groups (Whitten, 1957; Lamond, 1959) ; and the fact that the presence of a male tends to short,en the cycle (Whitten, 1958). While all of these studies have been done with laboratory strains of house mice, the present study evaluates the presence or absence of these phenomena in a member of a different family of rodents. Male-induced synchrony of estrus appears to operate similarly in deermice and house mice. Pairing of females which have been previously grouped is followed by a peak in estrous activity on t,he third day postpairing. In addition, the third-day peak may be moved forward, closer to the time of pairing, by pre-pairing exposure to male urine. The critical experiment of transection of the olfactory lobes to determine if these phenomena are olfactory-mediated has not been done in deermice; however, it is difficult to explain the action of male urine by any other mechanism. Two other aspects of the present data, even though not as well
SYNCHRONY
documented, appear to be similar in the two species. The consistently low number of inseminat.ions on the fourth day after pairing might indicate some shortening of the estrous cycle due t’o the male’s presence, but these data could also be influenced by the proportion of animals showing 3-day cycles. Second, some degree of estrus suppression due to crowding is indicated by the differences in cycle characteristics between females housed 4 and 10 per cage, The appearance of a third-day peak in estrous activity after pairing seems to be somewhat more dependent upon prior crowding in bnircl’ii than in house mice. The only comparable study (Whitten, 1959) found isolation for 2 weeks before pairing to incompletely suppress the characteristic third-day peak. The present study found no real difference bet,ween insemination rates on any of the first. 3 days after pairing if the females had experienced isolation. In summary, then. it would seem t,hat’ one function of males of both species, or, more specifically. of t,hr active principles in their urine, is to stimulate estrus and that this is brought about most effectively when estrous cycles are suppressed by crowding in all female groups.
OF
ESTRUB
632
REFERENCES H., AND ELEFTFTWERIOU, B. E. (!963j. Influence of strange males on implantation in the deermouse. Gen. Camp. Endocrinol. 3, 5W 518. LAMOND, D. R. (1959). Effect of stimulation derived from other animals of the same species 011 oestrous cycles in mice. J. Endocrinol. 18, 343349. MARSDEN, H. M., .~ND BROKSON, F. H, (1%X). Estrous synchrony in mice: Alteration by exposure to male urine. Science 144, 1469. PARICES, A. S., AND BRUCE, 33. M. (1961). Olfactory stimuli in mammalian reproduetioa. Xcience 136 1049-1054. WHITTEN, W. K. (1956). Modification of the oestrous cycle of the mouse by external stimuli associated with the male. .I, Endocrinol. I& 399-404. WHITTEN, TV. K. (1957). Effect of ext,eroceptive factors on the oestrous cycle of mice. biatw~ 180, 1436. WHITTEN: W. K. (1958). Modification of the oestrous cycle of the mouse by external stimuli associated with t,he male: Changes in the oestrous cycle determined by vaginal smears J. Endocrinol. 17, 307-313. WHITTEN, W. K. (1959). Occurrence of anoestrus in mice caged in groups. S. EndocrinoE. I 10% 107. BRONSON,
F.
AND
COMPARATWE
ENDOCRINOLOGY
Male-induced
$
634-637
(1964)
Synchrony
F. H. BRONSON The
Jackson
of AND’
Laboratory,
Received
in
Deermice’
H. M. MARSDEN Bar
July
Estrus
Harbor,
Maine
20, 1964
Relative numbers of inseminations occurring during the first 4 days after pairing were compared in deermice which had been housed 1, 4, or 10 females per cage for the 2 weeks preceding pairing. Females crowded 10 per cage before pairing showed a characteristic peak in estrous activity 3 days after pairing; those isolated before pairing showed no such peak; and females housed 4 per cage before pairing were intermediate. Topical (nose) exposure of females to male urine for 2 days before pairing accelerated attainment of estrus when compared with controls exposed to female urine. Male-induced synchrony of estrus in deermice appears to operate in a manner similar to that previously described for house mice.
Synchrony of estrus within groups of house mice, a phenomenon which is maleinduced and olfactory-mediated, has been described by Whitten (1956). Specifically, he found: (1) a peak number of inseminations on the t,hird night after pairing with fewer than expected on the first, second, and fourth nights; (2) a peak frequency of inseminations on the first night after pairing providing the females had been exposed to the “presence” of a male for 2 days prior to pairing; (3) that pre-pairing exposure to cages soiled by males somewhat altered the characteristic third day peak, indicating involvement of exteroceptive stimuli ; and (4) that such effects were somewhat dependent upon grouping of females prior to pairing (Whitten, 1959). Marsden and Bronson (1964)) also using house mice, altered the expected third day peak in inseminations by exposing females to male urine for 2 days before pairing, thus indicating a component of urine as the pheromone responsible for the effects found by Whitten. The purpose of the present experiments was to test for the presence of such male‘Supported by Public Health M-4481, CRT-5013, and HD-00767 tional Institutes of Health, Public
Service grants from the NaHealth Service. 634
induced synchrony in a nonmurine species of rodent, prairie deer mice (Peromyscus maniculatus bairdii). Specifically, females housed in different sized groups before pairing were compared with respect to t,he characteristics of their estrous cycles and the relative numbers of inseminations which occurred on each of the first 4 days after pairing. In addiCon, the ability of male urine to accelerate attainment of estrus was evaluated in this species. MATERIALS
AND
METHODS
Experiment I. All deermice were from a stock maintained in this laboratory for over 10 years. Females were grouped 4 per cage at weaning (2128 days) and paired at 75-35 days of age. Two weeks prior to pairing, 142 females were isolated and 137 were grouped 9 or 10 per clean cage. A total of 284 animals remained in groups of 4 in their original cages. Females were paired at 4 P.M. in clean cages with a male from a battery of 6-12month-old studs of proven fertility. Vaginal smears were examined at 9 .4.M. and 4 P.M. on the following 4 days. The criterion of insemination consisted of an average of 20 sperm per microscopic field ( ~264). Vaginal smears were taken daily for 8 days from an additional 50 animals of the same age (6 isolates, 24 grouped 4 per cage and 20 grouped 10 per cage) and were evaluated with respect to cycle characteristics.
OF
SYNCHRONY Ezperiment II. A total of 233 females were reared as in Experiment I. Two weeks before pairing, they were transferred to clean cages in groups of 9 or 10 per cage. All females in 12 cages (119 females) were individually exposed to male urine daily for the 2 days immediately preceding pairing. Such exposure consisted of placing one drop (about 0.05 ml) of urine, collected from mature males, on the general area of the external nares of each female 4 times a day at 2-hour intervals beginning at 9 A.M. Tmelve cages (114 females) were similarly treated with urine collected from mature females. Urine was collected indiscriminately from females in all stages of the reproductive cycle. Pairing and smearing procedures were idcnt,ical to Experiment I. Treatment, lvith urine in Expniment II and pairing and postpairing housing in both experiments were done in a 10 x la-foot room lighted from 5 A.M. to 7 P.M. and having a complete fresh air exchange 10 times per hour. 811 females used in the two experiments were moved into this room 2 reeks before pairing. Males were left in the room only as long as necessary: and, at all times during either experiment, a maximum limit of 200 animals was maintained in this room. All cages were 6 x 12 x 6-inch stainless steel. Statistical procedures consisted of testing, by F, frequency distributions of the numbers inseminated during the first 4 days after pairing for each type of treatment either against an expected (random) 25% per day or with each other. RESULTS
Ex-periwmf 1. About 749~ of the females housed 1. 4. or 10 per cage were inseminated within t,he first 4 clays after pairing with no significant differences in insemination rates
ESTRUR
633
between the three groups (Table I). The frequency dist,ributions of numbers insemtnated on each of the 4 days for each of the three groups differed significantly from an expected (random for a 4-day cycle) 2.5~~ per day at the 0.001 level. All groups were characterized by a low number of inseminations on the fourth clay. The dist’ributions across the first 3 days differed depending upon the degree of prior crowding. Animals housed one per cage before pairing showed little difference bet,ween each of the first 3 days ; those housed 10 per cage showed a strong tendency toward a third-day peak in inseminations; and t,hose housed 4 pei cage before pairing were intermediate with respect to a third-day peak. The dist,rihution of inseminations for females housed 40 per cage before pairing differed significantly from that of the 4 per cage group (10 < 0.01) and the isolate group (p < 0.~~~~. The frequency distributions of the grollps held I or 4 per cage were not significantly different. Vaginal cycles for 24 animals held 4 per cage showed a median cycle length of 3 days for 27 “definite” cycles in the 8-day period (19% were 3 days, 59% were 4 days: and 22% were 5 days in length). Wher, the groups held 1, 4, or 10 per cage were compared, there was a distinct impression oi longer, or more irregular, cycles in tile IO per cage group. Quantification of such ar? impression was difficult since smearing wab only conducted across an &day period:
TABLE 1 OF INSEMINSTIOXS DURING THE FIRST 4 D.~Ys AFTER P.URING 1, 1, OR 10 PER CAGE BOX 2 WEEKS BEFORE PAIRING (EXPERIMEX Ij OR 2 WEEKS AND EXPOSED T O MOLE OR FEK4LE URINE FOR 2 DY~YS BEFORE PaTRING (EXPERIMENT II) --
INSEMINATION RATES ‘L~VD DISTRIBUTION IN F-EXILES M-UNTUNED HELD 10 PER CAGE FOR
No.
Pre-pairino treatment Experiment, Housed Housed Housed Experiment Exposed Exposed
females
I l/cage 4/cage IO/cage II t,o 0 urine to ~3 urine
a %, by day,
of the number
which
No. inseminated
% Inseminstod
% hseminsted on day” 1 2 3 4 __-
142 285 13’7
111 202 9s
TS 71 7’
33 25 11
23 25 16
36 40 5!)
Y 10 13
114 119
86 92
ii 77
14 23
"7 43
50 "7
9 7
were
insemina,ted.
636
BROMSON
AND
however, one statistic which illustrates the difference was a comparison of the proportions of animals which showed no peak in epithelial cell activity for 5 consecutive days. A total of 10 of 20 animals in the 10 per cage group fell in such an arbitrary category, while only 2 of 24 of the 4 per cage group showed such prolonged periods of diestrum. One of 6 isolates showed 5 consecutive days with no period of estrus. Experiment II. Distributions of the relative numbers of inseminations across the first 4 days after pairing for both the male and female urine-treat,ed groups differ significantly (p < 0.001) from a random 25% per day. As in Experiment I, both distributions are characterized by low numbers inseminated during the fourth day. The two distributions differ significantly (p < 0.01) from each other; the group treated with female urine showing a third-day peak similar to the untreated 10 per cage group in Experiment I while the group exposed individually to male urine showed greater numbers on days 1 and 2. Estrus therefore occurred earlier after pairing if the females had been previously exposed to male urine. Two other aspects of insemination in colony-raised bairdii should be reported since they differ from most other laboratory rodents and, in addition, are relevant to the choice of methods in this study. First, insemination may occur during either the day or night. Of the 591 inseminations, 226 occurred between 4 P.M. and 9 A.M. (17 hours) and 365 between 9 A.M. and 4 P.M. (7 hours). These figures are not directly comparable because of the probable interact,ion between length of time between smearings and attainment of the arbitrary 20 per field criterion; however, they do indicate a lack of nocturnality in mating habits. Second, vaginal plugs are seldom found in association with inseminations and, in fact, may occasionally be found when t,here is no male present. A total of 6 solidified plugs, usually extruding 24 mm and always associated with estrous smears, were found in the absence of a male while examining 50 females for cycle characteristics.
MARSDEN
DISCUSSION
An arbitrary criterion of insemination (20 sperm per high-power field) was necessary in view of the lack of plug formation in bairdii. Past work (Bronson and Eleftheriou, 1963) with this criterion has yielded respectable pregnancy rates of 6090%. These figures, coupled with the fact, that an average of about 75% of the animals in the present study reached the criterion within 4 days, would tend to indicate that the method, even though arbitrary, is sound. Vaginal smearing is known to affect cycle characteristics; however, it can be pointed out that all groups were smeared in an identical manner. Insemination rates did not vary between groups. The fact that the social environment, male or female, has a profound effect on the estrous cycles of house mice and that many of the effects are olfactory-mediated is well documented (reviewed by Parkes and Bruce, 1961). Of specific interest here, in addition to the findings described in the introduction, are the suppression of estrus associated with crowding in all female groups (Whitten, 1957; Lamond, 1959) ; and the fact that the presence of a male tends to short,en the cycle (Whitten, 1958). While all of these studies have been done with laboratory strains of house mice, the present study evaluates the presence or absence of these phenomena in a member of a different family of rodents. Male-induced synchrony of estrus appears to operate similarly in deermice and house mice. Pairing of females which have been previously grouped is followed by a peak in estrous activity on t,he third day postpairing. In addition, the third-day peak may be moved forward, closer to the time of pairing, by pre-pairing exposure to male urine. The critical experiment of transection of the olfactory lobes to determine if these phenomena are olfactory-mediated has not been done in deermice; however, it is difficult to explain the action of male urine by any other mechanism. Two other aspects of the present data, even though not as well
SYNCHRONY
documented, appear to be similar in the two species. The consistently low number of inseminat.ions on the fourth day after pairing might indicate some shortening of the estrous cycle due t’o the male’s presence, but these data could also be influenced by the proportion of animals showing 3-day cycles. Second, some degree of estrus suppression due to crowding is indicated by the differences in cycle characteristics between females housed 4 and 10 per cage, The appearance of a third-day peak in estrous activity after pairing seems to be somewhat more dependent upon prior crowding in bnircl’ii than in house mice. The only comparable study (Whitten, 1959) found isolation for 2 weeks before pairing to incompletely suppress the characteristic third-day peak. The present study found no real difference bet,ween insemination rates on any of the first. 3 days after pairing if the females had experienced isolation. In summary, then. it would seem t,hat’ one function of males of both species, or, more specifically. of t,hr active principles in their urine, is to stimulate estrus and that this is brought about most effectively when estrous cycles are suppressed by crowding in all female groups.
OF
ESTRUB
632
REFERENCES H., AND ELEFTFTWERIOU, B. E. (!963j. Influence of strange males on implantation in the deermouse. Gen. Camp. Endocrinol. 3, 5W 518. LAMOND, D. R. (1959). Effect of stimulation derived from other animals of the same species 011 oestrous cycles in mice. J. Endocrinol. 18, 343349. MARSDEN, H. M., .~ND BROKSON, F. H, (1%X). Estrous synchrony in mice: Alteration by exposure to male urine. Science 144, 1469. PARICES, A. S., AND BRUCE, 33. M. (1961). Olfactory stimuli in mammalian reproduetioa. Xcience 136 1049-1054. WHITTEN, W. K. (1956). Modification of the oestrous cycle of the mouse by external stimuli associated with the male. .I, Endocrinol. I& 399-404. WHITTEN, TV. K. (1957). Effect of ext,eroceptive factors on the oestrous cycle of mice. biatw~ 180, 1436. WHITTEN: W. K. (1958). Modification of the oestrous cycle of the mouse by external stimuli associated with t,he male: Changes in the oestrous cycle determined by vaginal smears J. Endocrinol. 17, 307-313. WHITTEN, W. K. (1959). Occurrence of anoestrus in mice caged in groups. S. EndocrinoE. I 10% 107. BRONSON,
F.