Some effects of mouse urine on neonatal growth and reproduction

Anim. Behav.,1972,20, 499-506 SOME

EFFECTS

OF

MOUSE

URINE

ON

NEONATAL

GROWTH

AND

REPRODUCTION BY J. J. C O W L E Y & D. R. WISE

Department of Psychology, The Queen's University, Belfast Abstract. An experiment is described in which urine collected from female mice at different stages of the reproductive cycle and from adult male mice was applied to the nasal region of neonatal female mice. The topical application of pseudopregnant urine had an accelerating effect on growth while the application of urine from virgin female donors and mice in late pregnancy was associated with a slow rate of growth. Male urine accelerated the sexual maturation of the mice while the application of urine from virgin females delayed vaginal introitus and first oestrus. The application of both male and pseudopregnant urine was associated with a high incidence of pregnancy when adult. It is suggested that the urine of adult mice contains an active substance(s) which can affect the growth of young female mice and that the effect is dependent on the reproductive state of the donor. The block to pregnancy that occurs when a recently mated mouse is exposed to an unfamiliar adult male (Bruce 1959, 1960) can be effected by applying urine from an unfamiliar adult male direct to the external nares of the pregnant female, thus, demonstrating that the pheromone responsible for the block is present in bladderurine (Dominic 1964). Synchronization of oestrus occurs when mice that have been housed in allfemale communities are mated or exposed to the presence of a male. This can also be initiated by applying male urine directly to the nasal region (Marsden & Bronson 1964). The presence of the male and the topical application of the urine shorten the oestrous cycle (Whitten effect). Exposure of neonatal female mice to the presence of adult males accelerates sexual development (Castro 1967; Vandenbergh 1967) and the pheromone responsible has been shown to be airborne (Vandenbergh 1969). The presence of adult male and female mice was also observed to exercise a differentiating effect on the growth and behaviour in an open field test of young mice (Fullerton & Cowley 1971). The present study was aimed at ascertaining whether, as in the Bruce effect and Whitten effect, the active substance responsible for the growth changes was contained in the urine and whether the urine of mice at different stages of pregnancy and pseudopregnancy would affect the sexual and somatic development of young mice if topically applied to the external nares.

collected by holding the mouse over a large Petri dish and gently squeezing the flanks of the animal. The urine collected from a single mouse was diluted with 0.5 ml of distilled water. The mice from which urine was obtained are referred to as donors and those on which the urine was painted as recipients. The Donors

The donors were housed in groups of twelve in standard aluminium shoe box cages (41 • 16 x 10 cm). For each treatment the mice were housed together for 2 weeks prior to the commencement of the experiment. After collection of the urine, the mouse was marked with Gentian Violet and returned to the home cage. Donors were used on one, or at the most two, occasions and were housed in the same room as the recipients but some distance away. The donors, 150 days of age, made up the following samples: (i) proven males (N=24), (ii) virgin females (N=24), (iii) early pregnant females (N=36), (iv) late pregnant females (N=36), and (v) pseudopregnant females (N= 36). In order to have a quantity of urine available from mice at both the early and late stages of pregnancy, it was necessary to arrange the mating in such a way, that three sub-groups of donors were available in each of these and the pseudopregnant samples. Early pregnancy. A sample of donors was mated 2 days before the birth of the 'first' recipient babies. The urine from the mice was used for 7 days only, after which urine, was collected from mice which had in their turn

Methods

Urine collected from albino mice of the CS1 strain was applied to the external surface of the nose of mice of the same strain. The urine was 499

500

ANIMAL

BEHAVIOUR,

been mated 1 week after the first group. Mice mated 2 weeks after the first sample had been mated provided early pregnant urine for the last week of treatment. Late pregnancy. The collection of urine from late pregnant mice was arranged in much the same way. A sample was mated 1 week later than the mothers of the recipients. Thus, the donors were at day 14 of gestation when the recipients were born. Likewise, two other samples were mated 7 and 14 days after the first donors and these provided late pregnant urine for the 2nd and 3rd weeks of treatment. Pseudopregnaney. The procedure followed in mating the donors of this group was the same as that used for the early pregnant treatment. except that the mice were mated with vasectomized mice of the same strain. In all, urine was collected from seventy-two pregnant mice and thirty-six mice that had been mated with vasectomized males. The presence o f a vaginal plug was regarded as evidence of successful mating and the mice, that provided the early pregnant urine, were also regarded as pregnant on this basis. Donors providing urine in late pregnancy were identified as pregnant by both presence of vaginal plug and increase in body weight. The Recipients Thirty-five litters were allocated to seven treatments. The procedure followed was to allocate a mother on the day she gave birth (day 0) to a treatment. All the female mice born on one day were pooled and four selected, in such a way that the body weight of the litter was equivalent amongst the different treatments. Male and female babies not required for the experiment were discarded. Of the thirty-five litters, ten formed the two control groups. Half of these had distilled water applied to the nasal region while the others were stroked with a dry brush. The distilled water and the urine from the donors was painted on the dorsal surface of the nose of the recipients in the region of the nasal and frontal bones. Four or five gentle strokes with a soft water-colour paint brush were given while care was taken to avoid the mouth. The brushes were kept wet but not dripping. A similar brush was used and the same procedure followed in the 'dry brush' treatment. All treatments were given twice daily until the mice were 21 days of age. Fresh urine

20,

3

collected each morning from the donors, was applied immediately to the recipients and the remainder stored in a deep-freeze (--17~ until the afternoon when, after thawing, the second treatment of the day was given. Separate brushes were used for the different treatments and every care was exercised to prevent contamination of one with the urine of another. During treatments the mothers were removed to a separate container and returned to the young after all the babies had been treated. The babies were weighed weekly on a Mettler H balance until they were weaned at 21 days. Thereafter, they were weighed at 42 and 56 days of age. The mice were inspected daily and the age of vaginal introitus recorded and thereafter vaginal smears were taken each day for 20 days. Mice were housed in their original litters of four from the time they were weaned until mated at 70 days of age. They were mated with males of the same strain from the laboratory colony. The males were left with the females for 5 days and during this period an examination was made twice daily for vaginal plugs. Half of the animals, in each treatment and in which vaginal plugs were found, were killed some 14 days after the plugs were formed and the number of fetuses, and partially absorbed fetuses, counted. The remainder of the mice were housed singly and the number of offspring recorded. All mice were housed in an artificially-lit room (12-hr light) at a temperature of 21~ Throughout the experiment the mice were fed 41B cubed diet (Bruce 1950) and water was available at all times.

Results Weight Changes with Age Figure 1 shows the changes in body weight of the mice from birth to 56 days of age. The mean weight of the different treatments, at birth, are much the same but by 42 days the curves have diverged maximally. A Kruskal-Wallis one-way analysis of variance (Siegel 1956) shows an overall difference between the groups at both this age (P<0.001) and at 56 days of age (P<0-001). Table I sets out the mean weights and standard errors for the various treatments. At 42 days of age the control mice (dry brush) and the mice exposed to pseudopregnant urine were the heaviest but by 56 days of age the mice treated with male urine were heavier than the dry brush treatment mice, though not significantly so, and almost as heavy as those

COWLEY & WISE: MOUSE URINE AND NEONATAL GROWTH

I//v'j~:"_

// /ffr/u/

.... ,....,A

20 ~

/

,

,,,,-~

/// I."

10-

Dry brush o Dist w a t e r

9 'Male urine Virgin female urine v Early pregnant urine 9 Late pregnant urine 9 Pseudopregnant urine

Age (days)

Fig. 1. The effect of urine collectedfrom male and femalemiceat differentphases of their reproductivecycle on the bodyweightof recipients. exposed to pseudopregnant urine. The mice which grew at the slowest pace were those treated with urine from virgin females. These were lightest from 14 days and retained their position until 56 days of age. Topical application of urine from early pregnant mice was associated with more rapid growth, than that from late pregnancy. There was no statistically significant difference between the two control treatments but the mice treated with water were lighter at 42 days and they retained this position at 56 days of age.

Sexual Development Vaginal introitus. Table II shows that there is

501

an overall difference between treatments in the age of vaginal introitus. Topical application of male urine was associated with early vaginal opening and that from virgin females with a delayed opening of the vagina. Application of urine from pesudopregnant and early pregnant mice resulted in vaginal opening at an age which was comparable with that of the control samples but younger than that of the mice treated with virgin female urine. As with weight, the urine from the pseudopregnant and early pregnant mice appeared to have an accelerating effect on sexual development when compared with that of the mice treated with urine of virgin females and late pregnancy. Of these latter two samples, it is the former which is constantly less mature. Vaginal cornification. Table III shows the mean age (days) and standard error of the mice at first oestrus. The topical application of male urine accelerated the onset of first oestrus when compared with both control samples. The onset of first oestrus in mice receiving late pregnant urine was delayed, but not significantly so, compared with the control mice and the delay was not as great as that of the mice treated with virgin female urine (P<0.05). The mean age at first oestrus in the latter sample was 43.4 days compared with a mean age of 30-8 days in the male treated sample. In the other treatments, first oestrus occurred at varying ages between the two extremes. The topical application of early and late pregnant urine tended to accelerate the onset of first oestrus when compared with the onset in those mice treated with virgin female urine. In general mice treated with early pregnant and pseudopregnant urine had earlier vaginal cornification than mice treated with other female urines.

Reproduction. Only 45 per cent of the mice were found to have vaginal plugs. A chi-square test showed that there was an overall significant difference between treatments (P<0-05). Mice treated with pseudopregnant urine (73.7 per cent) and male urine (57.8 per cent) had a higher percentage of plugs than any of the other treatments. There were more vaginal plugs found in mice treated with distilled water (42.1 per cent) than those given dry-brush treatment (20 per cent). The difference between the two samples was not, however, significant. The presence of fetuses in the dissected mice was noted and, for the purpose of statistical comparison, the mice with fetuses present and

502

ANIMAL

BEHAVIOUR,

20,

3

Table I. Mean Body Weight at 42 and 56 Days of Age D a y s of age 42 days Treatment

D r y Brush

N Mean (g) (•

N

20

26.12 "58)

20

23.86

20

(• Water

19

M e a n (g) Age (4-SE) (days)

19

25.53

19

Virgin female urine

20

Early preg. urine

20

Late preg. urine

19

Pseudopreg. urine

19

19.37

20

24.81

20

23.95

19

25.86

20

Pseudopreg. urine

NS NS

<0"01 <0"025

NS <0"05

26.10

42 56

---

NS NS

<0-001 <0.001

NS NS

NS NS

<0.05 <0.025

28.40

42 56

--

--

<0.001 <0.001

NS NS

<0.01 NS

NS NS

20.63

42 56

---

-~

<0.001 <0.001

<0.01 <0.001

<0.001 <0.001

26.72

42 56

. .

<0.01 <0.025

NS <0.05

24-56

42 56

. .

. .

28.80

42 56

. .

. .

(•

(•

Late preg. urine

<0.001 <0"001

(•

(•

Early preg. urine

NS NS

(•

(•

Virgin female urine

NS NS

(•

(•

Male urine

42 56

(•

(•

Water

26.89 (•

(• Male urine

Treatment groups M a n n Whitney U tests

56 days

(•

---

. .

. .

. . . .

. . . .

. . . .

<0.001 <0'001 . .

. .

sn of means is in parentheses. Kruskal Wallis Analysis of Variance: P < 0.001. The P values in the table were obtained from M a n n Whitney U test comparisons. NS = n o t significant.

Table II. Age (Days) Differences in Vaginal Introitus Treatment M a n n Whitney U test Treatment

Dry brush

N

Mean sE

20

Kruskal Wallis P

Water

Male urine

Virgin female urine

Early preg. urine

Late preg. urine

Pseudopreg. urine

29"65

NS

<0.025

<0'025

NS

NS

NS

29"85

--

<0.025

<0.05

NS

NS

NS

<0.001

NS

<0.05

NS

<0.05

<0.01

NS

NS

(• Water

20 (•

Male urine

19

28.21 (=t=0.41)

Virgin female urine

20

Early preg. urine

20

Late preg. urine

19

--

--

33.70 (~1-15)

--

--

29"25 (=I=0"37)

.

.

30.05

.

.

Pseudopreg. urine

20

29.25 (4-0.35)

.

.

(+0.65)

<0.02

-.

0.01

. .

. .

. .

NS .

.

SE of means is in parentheses. P values based on Kruskal Wallis analysis of variance and M a n n Whitney U tests. N S = n o t significant.

COWLEY & WISE: MOUSE URINE AND NEONATAL GROWTH

503

Table HI. Mean Age (Days) at First Vaginal Cornitication

Treatment Mann Whitney U test Kruskal WallisP

Virgin female urine

Early preg. urine

Late preg. urine

<0.01

<0.01

NS

NS

NS

--

<0.01

<0.025

NS

NS

NS

--

--

<0.001

NS

<0.01

<0.05

43.40

m

_

--

<0-05

<0.01

32.40

.

.

<0.05

NS

36.47

.

.

32.45

.

.

N

Mean sE

Dry brush

20

33.70 (+0.82)

NS

Water

20

36-05 (~1.5D

Male urine

19

30.79 (+0.64)

Virgin female urine

20

Early preg. urine

20

Late preg. urine

19

Pseudopreg. urine

20

Treatment

(• (• (• (•

<0.001

Male Urine

Water

.

<0"01

. .

. .

. .

Pseudopreg. urine

<0.05 .

.

sE of means is in parentheses. P values based on Kruskal Wallis Analysis of Variance and Mann Whitney U tests. NS=not significant. mice which gave birth, were combined and compared with the number not pregnant. A chi-square analysis (Table IV) showed an overall difference in the proportion of mice pregnant (P<0.01). As in the previous analysis the mice treated with pseudopregnant urine (63.2 per cent) and male urine (57.9 per cent) showed a high percentage pregnant. Differences between the other treatments were not statistically significant. A number of mice which had vaginal plugs were not pregnant, though the differences between treatments were not at an acceptable level of statistical significance (Z2= 11.97, 0 . 1 0 > P>0.05). There was some indication that a higher proportion of the mice, which were administered early pregnant and virgin female urine in infancy, were less successful in implanting and giving birth than were mice in the male and pseudopregnant treatments. There was no evidence that the various treatments affected the survival of the offspring (Z2=8.71, 0 . 2 0 > P > 0 - 1 0 ) nor was there any evidence of a differential effect on the number of fetuses absorbed (Z2=5.37, 0 . 5 0 > P > 0 . 3 0 ) . Discussion

The results of this study suggest that an active

substance present in the urine of adult mice can modify the development of neonatal mice and that the urine of males and of females at different phases of the reproductive cycle act differentially in bringing about the maturational changes. Urine collected from adult virgin female mice and applied to the nasal region of neonatal female mice retarded the growth and sexual maturation of the recipients. A similar pattern of delayed growth had been observed when infant female mice were reared in proximity to adult females (Fullerton & Cowley 1971). The growth of infant female mice was not significantly accelerated during the first weeks of development by urine from male donors. When mature, mice in the groups given urine from pseudopregnant donors and from males were heavier than the mice in the other groups and their weight curves showed little evidence of having reached an asymptote. Mice treated with pseudopregnant urine were significantly heavier than the controls (dry brush) but the question as to whether male urine has a growth promoting action must remain open. In our earlier studies where the urine and stools of male donors passed directly into the cages of the recipients and an acceleration in the rate of growth of the female mice observed,

504

ANIMAL

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Table IV. Percentage of Mice with Vaginal Plugs, Plugs Present but not Pregnant, Pregnant and Successfully Rearing Litterss Treatment groups Dry brush Water Male urine Virgin femaleurine Early pregnancy urine Late pregnancy urine Pseudopregnancy urine

N

Vaginal plug present (~o)

20 19 19 20 20 19 19

4 8 11 9 9 8 14

Z2 df P

Vaginal Plug present--not Pregnant ( ~ )

(20) (42.1) (57.9) (45) (45) (42.1) (73-7)

0 4 3 6 7 4 4

12.61 6 < .05 Treatment groups

DB DB DB DB DB DB W W W W W MU MU MU MU VFU VFU VFU EPU EPU LPU

W MU VFU EPU EPU PPU MU VFU EPU LPU PPU VFU EPU LPU PPU EPU LPU PPU LPU PPU PPU

Pregnant* (~)

(0) (50) (27.3) (66-7) (77-8) (50) (28.6)

4 8 11 3 3 5 12

11.97 6 <0-10

(20) (42.1) (57.9) (15) (15) (26.3) (63"2) 21.29 6 <0.01

Zz

p

X2

p

X2

p

1.32 4.42 1.82 1.82 1.32 9.24 0.42 0.02 0.02 0 2.70 0.24 0.24 0-42 0.47 0 0"02 2.23 0"02 2.23 2.70

NS <0.025 <0.I0 <0.10 <0.10 <0.005 NS NS NS -<0.10 NS NS NS NS -NS <0"10 NS <0"10 <0"10

1.17 0.19 2.63 3.97 1-17 0.28 0.28 0.04 0.47 0 0.30 1.72 3.23 0.28 0.14 0 0"04 1.87 0"47 3"53 0.30

NS NS <0.10 <0.025 NS NS NS NS NS -NS <0.10 <0.05 NS NS -NS <0.10 NS <0"05 NS

1.32 4.42 0 0 0"09 5.82 0.42 2.32 2.32 0.47 0.95 6.04 6.04 2.70 0 0 0"23 7-62 0"23 7"62 5.22

NS <0.025 -NS <0.01 NS <0.10 <0.10 NS NS <0-01 <0.01 <0.10 --NS <0.005 NS <0.005 <0"025

NS =not significant. Differences tested by the Chi-square test for k independent samples. In the contingency tables (onetailed) DB=Dry Brush; W=Water; MU=Male Urine; VFU=Virgin Female Urine; EPU=Eady Pregnant Urine; LPU-----Late Pregnant Urine; PPU---Pseudopregnancy Urine. *Vaginal plug were not found in some of the pregnant mice. the stools m a y have provided a source o f nutrim e n t for the mice. L i e b e r m a n & Teich (1953) have shown that steroids are concentrated i n the u r i n e a n d stools of rodents a n d the latter are also k n o w n to be a source o f v i t a m i n H (Barnes, K w o n g & Fiala 1959). The results also indicate that the reproductive c o n d i t i o n o f the d o n o r is critical in effecting changes in growth a n d m a t u r a t i o n o f recipients. Whereas urine from virgin female mice retarded growth a n d m a t u r a t i o n , urine from pseudo-

p r e g n a n t d o n o r s accelerated growth. I n each ease the active substances reflect the h o r m o n a l c o n d i t i o n of the d o n o r a n d act differentially o n development. T h a t exogenous substances can act o n the nervous system at a n early age is suggested b y the work of M a i n a r d i (1963) a n d M a i n a r d i , M a r s a n & Pasquali (1965). Female mice reared with the father present showed a preference for males o f the same sub-species as the father when given a choice o f mates as adults. I f the

COWLEY & WISE: MOUSE URINE AND NEONATAL GROWTH odour of the adults had been blocked by strong perfuming, however, the discrimination did not occur. Further evidence of the imprinting effect on adult social behaviour is provided by the work of Marr & Gardner (1965) on the spraying of some young rats with methyl salicylate, and others with cologne. The results of our study suggest that the urine from donors had a similar imprinting effect in so far as the reproductive behaviour of the adult mice varied according to treatment received. All the mice were housed and reared in all female groups but those that had been treated with male or pseudopregnant urine had a higher proportion of vaginal plugs, and more pregnancies. Even when mating had occurred, as evidenced by the presence of plugs, the mice that had received urine from virgin, early pregnant and late pregnant donors were often not pregnant. The activity of adult rats may be affected by substances contained in the urine. Hemmingsen (1933) showed that the addition of late pregnancy urine to the drinking water of castrated male and spayed female rats increased their wheel running activity. Our own work has shown that urine from an adult female donor may modify the wheel activity of a recipient female and that the changes in activity varies with the reproductive state of the donor. The changes occur when the urine is fed to a region below the wheel and to which the recipient does not have access (Cowley & Wise 1970). G a r d et al. (1967) have shown that body movements of infant rats are increased by the presence of ammonia and our work (Cowley & Wise 1970) showed that proximity of soiled nesting material or urine collected from lactating mothers, may reduce the activity of infant rats. The response o f the animals to the soiled nesting and the urine varies according to chronological age and sex. The work of Harris (1964) on mice suggests that the sensitivity may be associated with the inductive action of endogenous sex hormones acting on the central nervous system at this early age. The nature of the substances in the urine and the pathways through which they act to change growth and behaviour is unknown. In the Bruce effect and in the Whitten effect it is the odour of the male mouse that is considered to be instrumental in blocking pregnancy and in synchronizing oestrus, and can be obtained by the direct application of urine from a male mouse. Vandenbergh (1969) has shown that the sexual maturation of immature female mice can be

505

accelerated by having mature males housed in proximity to them and that the transfer of soiled bedding from cages in which males had been living was effective in accelerating sexual development. The odour of the urine may have been responsible for the changes in growth and behaviour in this study, but it is possible that the active substance, or substances, whether nutritive or toxic, may have been absorbed or ingested. Acknowledgment We wish to thank Miss Hilda Bruce for her helpful criticism of this paper. REFERENCES Barnes, R. H., Kwong, E. & Fiala, G. (1959). Effects of the prevention of coprophagy in the rat. IV. Biotin. J. Nutr., 67, 599-610. Bruce, H. M. (1950). Feeding and rearing of laboratory animals. J. Hyg. Camb., 48, 171-183. Bruce, H. M. (1959). An extroceptive block to pregnancy in the mouse. Nature, Lond., 11t4, 105. Bruce, H. M. (1960). A block to pregnancy in the mouse caused by proximity of strange males. J. Reprod. Fert., 1, 96-103. Castro, B. M. (1967). Age of puberty in female mice: relationship to population density and the presence of adult males. Anais Acad. bras. Cient., 39, 289291. Cowley, J. J. & Wise, D. R. (1970). Pheromones, growth and behaviour. In: Chemical Influences on Behaviour: Ciba Foundation Study Group (Ed. by R. Porter & J. Birch). pp. 144-170. London: Churchill. Dominic, C. J. (1964). Source of the male odour causing pregnancy block in mice. J. Reprod. Fert., 8, 266-267. Fullerton, C. & Cowley, J. J. (1971). The differential effect of the presence of adult male and female mice on the growth and development of the young. J. genet. Psychol., 119, 89-98. Gard, C., Hard, E., Larsson, K. & Petersson, V. (1967). The relationship between sensory stimulation and gross motor behaviour during the post natal development in the rat. Anirn. Behav., 15, 563-567. Harris, G. W. (1964). Sex hormones, brain development and brain function. Endocrinology, 75, 627-648. Hemmingsen, A. M. (1933). Studies on the oestrusproducing hormone (oestrin). Skand. Arch. PhystoL, B 65, 97. Lieberman, S. & Teich, S. (1953). Recent trends in the biochemistry of the steroid hormones. Pharra. Rev., 5, 285-380. Mainardi, D. (1963). Speciazione nel topo fattori etologici determinanti barriere riproduttive tra Mus musculus domesticus em M. bactrianus. Rc. Ist. lomb. Sci. Lett., B 97, 135-142. Mainardi, D., Marsan, M. & Pasquali, A. (1965). Causation of sexual preferences of the house mouse. The behaviour of mice reared by parents whose odour was artificially altered. Atti. Soc. ltal. Sci. Nat., CIV, 325-338.

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Mart, J. N. & Gardner, L. E. (1965). Early olfactory experience and later social behaviour in the rat: preference, sexual responsiveness and care of young. J. genet. PsychoL, 107, 167-174. Marsden, H. M. & Bronson, F. A. (1964). Estrus synchrony in mice: alteration by exposure to male urine. Science, N.Y., 144, 1469. Siegel, S. (1956). Nonparametrie Statistics for the Behavioural Science. New York: McGraw-Hill.

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Vandenbergh, J. G. (1967). Effect of the presence of a male on the sexual maturation of female mice. Endocrinology, 81, 345-349. Vandenbergh, J. G. (1969). Male odour accelerates female maturation in mice. Endocrinology, 84, 658-660.

(Received 7 September 1971 ; revised 25 February 1972; MS. number: 1090)