Journal of Chemical Ecology, Vol. 13, No. 9, 1987
VARIATIONS IN MOUSE (Mus musculus) URINARY VOLATILES DURING DIFFERENT PERIODS OF PREGNANCY A N D LACTATION
BOZENA JEMIOLO, FRANCA ANDREOLINI, DONALD WIESLER, and MILOS NOVOTNY Department of Chemistry lndiana University Bloomington, Indiana 47405 (Received August 15, 1986; accepted November 17, 1986)
Abstract--Mouse urine samples from different pregnancy and lactation periods were examined by capillary gas chromatography to assess variations in the volatile signals that may affect the endocrine function of other females. Statistically significant changes in the excretion of certain urinary volatiles were observed; from 26 readily quantifiable constituents, 14 appear to be under the endocrine control. These selected components, positively identified through mass spectrometry and retention data, and the synthetic standards are ketones, unsaturated alcohols, esters, and cyclic vinyl ethers. Key Words--Female mouse urine, pregnancy, lactation, urinary volatiles, mouse, Musmusculus.
INTRODUCTION
In the house mouse (Mus musculus), there are at least three female urinary chemosignals that can affect the reproduction status of other females: (1) the delay of puberty occasioned by treatment with urine from grouped females (Drickamer, 1974, 1977, 1983a); (2) the acceleration of puberty by the urine from estrus females (Drickamer 1982a); and, (3) the acceleration of puberty (Drickamer and Hoover, 1979) and prolongation of the estrous period (Hoover and Drickamer, 1979) through exposure to the urine from pregnant and lactating females. It has been suggested (Drickamer, 1982b) that some chemical substances in the urine of females may act as the general signals to other conspecifics regarding the adequacy of social and environmental conditions which are important for successful reproduction. 1941 0098-0331/87/0900-1941505.00/0 9 1987PlenumPublishingCorporation
1942
JEMIOLO ET AL.
Urine samples collected at different time periods during pregnancy and lactation are differentially effective in terms of the acceleration of the first estrus relative to control females treated with water (Drickamer, 1983b, 1984). The chemosignals from pregnant/lactating females seem to be present in the urine only during the last two thirds of pregnancy or lactation (Drickamer, 1983b). In addition, Cowley and Wise (1972) reported that the urine from pseudopregnant females applied to the nasal region of neonatal females has an accelerating effect of their growth, while the application of urine from females in late pregnancy is associated with a slow rate of growth. It is not yet known what set of hormonal and related physiological conditions within the female mice may reflect these changes in the chemical signals released by donor females. Since no chemical investigations on these signals have been conducted to date, their nature remains a subject of speculation. Earlier study by Drickamer and Hoover (1979) has shown that the urine from pregnant or lactating females, either applied daily on the extemal nares of the young animals or provided to them in a plastic (inaccessible) capsule, can accelerate their sexual maturation. In addition, the urine from pregnant or lactating females that is exposed to the ambient air remains effective for only one to three days (Drickamer, 1986). These findings suggest that the pregnancy/lactation chemosignal(s) is either an air-sensitive or a volatile substance. The chemosignals from pregnancy urine and that of lactating animals may or may not contain similar substances. Both chemical characterization and biological experiments are needed to elucidate this problem. Our previous successful uses of a headspace sampling technique (Novotny et al., 1974) and capillary gas chromatography-mass spectrometry to identify the male chemosignals responsible for aggression (Novotny et al., 1985), estrus synchronization (Jemiolo et al., 1986a), and the female signals causing the delay of puberty (Novotny et al., 1986) have prompted us to apply similar techniques to the identification of additional female chemosignals in this study. As the initial approach to this complex problem, the urinary profiles of volatile substances were quantitatively followed throughout the pregnancy and lactation periods. Statistically significant alterations in quantities of certain volatile components are reported here and correlated with the animals' endocrine status. The structures of these components were verified through their mass spectra and chromatographic behavior of authentic substances.
METHODS AND MATERIALS
All mice used as urine donors were from a randomly bred, closed colony of ICR/Alb (Mus musculus) purchased from Ward's Natural Science Establishment, Inc., Rochester, New York. Females were housed in plastic cages (12 • 28 x 27 cm), maintained at 21~ + 0.2~ 50-70% humidity, and a 12-
MOUSE URINARY VOLATILES
1943
hr light-12-hr dark daily regime. Unlimited amounts of Purina Mouse Chow (Ralston Purina Corp., St. Louis, Missouri) and water were supplied throughout the whole experimental period. Bedding was changed weekly. For use as urine donors, 120 virgin and 120 multiparous females, in total, were bred with fertile males. The occurrence of pregnancy was established by a daily examination for vaginal plugs (day 0). After insemination, each female was caged alone over the whole period of pregnancy. The urine from pregnant females was collected from days 2 to 7 (period 1), 8 to 12 (period 2), 13 to 15 (period 3), and from day 16 postplug to the last day before parturition (period 4). An additional 120 multiparous pregnant female mice were isolated into individual cages and checked daily for newborns. On the day of delivery, the litters were counted and reduced to 10 pups. Collection of urine from lactating females was done at the same periods as during pregnancy: days 2-7 (period 1), 8-12 (period 2), 13-15 (period 3), and from day 16 postpartum until the young were 21 days old. All females used as urine donors were 80-130 days of age. Fresh urine was collected by holding a mouse over a glass vial and gently squeezing the abdomen and flanks. Immediately after collection, samples were stored at - 2 0 ~ and analyzed within two to four days. All types of urine were analyzed three to six times by using each time a 1-ml sample obtained from different collections within the same experimental period. For each sample, the urine was collected and pooled from 8 to 10 pregnant or lactating females. As a standard sample, we used urine collected from virgin, nonreproducing females at 90-120 days of age, that were housed singly. Collection of the urine was carried out throughout all stages of the estrous cycle. The urinary volatiles were analyzed using the headspace technique, employing Tenax GC (a porous polymer) as the adsorption medium (Novotny et al., 1974). The volatiles were sparged from 1-ml urinary samples at room temperature with purified helium gas at a flow rate of 100 ml/min and adsorbed onto a precolumn packed with 4 mg of Tenax GC. The sample was subsequently desorbed in the heated injection port (220-240~ of a gas chromatograph (Perkin-Elmer, 3920 instrument, Norwalk, Connecticut) equipped with a flame ionization detector, and retrapped into a cooled section of a glass capillary column. The analytical column was a glass capillary (60 m x 0.25 mm, ID) of a sodalime type, coated statically with UCON 50-HB-2000 (Schwende et al., 1984a,b). While quantitative comparisons were obtained through peak integration routines (Sigma 10, Perkin-Elmer), identification of the individual profile constituents was established through a combined GC-MS (Hewlett-Packard, 5981 dodecapole mass spectrometer, Palo Alto, California) using electron impact ionization at 70 eV. Wherever feasible, authentic samples were either purchased or synthesized in the laboratory to verify agreement of spectral information and gas-chromatographic retention time.
1944
JEMIOLO ET AL.
Statistical Calculations. Statistical comparisons of the levels of excreted volatiles were made using one-way analysis of the variance (ANOVA) with Duncan's new multiple-range test (Zar, 1974). The probability level for statistical significance was set at P < 0.05.
RESULTS AND DISCUSSIONS The complexity of mouse urinary volatile profiles has been demonstrated in our previous studies (Novotny et al., 1980, 1984, 1986; Schwende et al., 1984a,b, 1986; Jemiolo et al., 1986a,b); genetic background, sex, endocrine status, etc., all seem to influence the quantitative proportions of various urinary constituents. The capillary chromatogram shown in Figure 1 is representative of a profile obtained from pregnant female ICR/Alb mice. From a large number of components, 26 substances are numbered; most have been positively identified by combined capillary gas chromatography-mass spectrometry and the retention measurements of authentic compounds (Table 1). Through careful visual inspection of the profiles obtained from different stages of pregnancy and lactation, as well as from nonreproducing females, we first established that 14 constituents showed obvious variations in their chro-
28
[
18 17
23242526
19
3O
b-
5O
70
I
I
90 TEMP (~
9,
110
130
1~
1~
I
I
I
I
20
30TIME (rain) 40
50
60
~therrr~l
, 70
FI6. t. Gas chromatogram of urine volatiles from ICR/Alb primiparous females at the 13th to 15th day of pregnancy (period 3).
1945
MOUSE URINARY VOLATILES
T A B L E 1. V O L A T I L E C O M P O U N D S IN U R I N E OF P R E G N A N T P R I M I P A R O U S ( A ) ; P R E G N A N T M U L T I P A R O t J S ( B ) ; AND L A C T A T I N G ( C ) F E M A L E M I C E
Changes in concentration of compounds during pregnancy and lactation Peak number
Structure
A
B
C
1
tool w t 126"
_
+b
_c
2
4-ethylcyclohexene
-
-
-
3
3 - m e t h y l - 1- b u t e n - 3 - o l
-
-
-
4
2 ethyl-5-methyl-fumn
-
-
-
5
3-hexanone
-
-
-
6
2-hexanone
-
+
-
7
m . w 126 a
-
+
-
8
4-heptanone
+
+
+
9
m . w 126 a
-
-
-
10
2 heptanone
+
-
+
11
n - p e n t y I acetate
+
+
+
12
methyl hexanoate
-
-
-
13
6-hepten-2-one
-
-
-
14
c i s - 2 - p e n t e n - 1-yl acetate
+
-
+
15
trans-5-hepten-2-one
+
-
+
16
trans-4-hepten-2-one
-
-
+
17
4-penten-l-ol
+
+
+
18
u n i d e n t i f i e d e s t e r o f t o o l w t 142
+
-
+
19
2,5-dimethylpyrazine
-
-
-
211
6 - m e t h y l - 6 - h e p t e n - 3 -one
+
+
-
21
6-methyl-5-hepten-3-one
+
+
-
22
dehydrobrevicomin
-
-
-
23
2-nonanone
-
-
-
24
benzaldehyde
-
-
-
25
unidentified
-
-
-
26
1-octen-3-ol
+
+
+
a P r e s u m e d i s o m e r i c c y c l i c v i n y l ethers u n i q u e to t h e m o u s e ( S c h w e n d e e t a l . , 1986); d e h y d r a t i o n products o f known 5,5 dimethyl-2-ethyltetrahydrofuran-2-ol. b C o n c e n t r a t i o n o f the c o m p o u n d d e p e n d e n t o n a p e r i o d o f p r e g n a n c y o r l a c t a t i o n . C C o n c e n t r a t i o n o f the c o m p o u n d d o e s not s e e m d e p e n d e n t o n a p e r i o d o f p r e g n a n c y o r l a c t a t i o n .
matographic peak areas (Table 1, see peaks numbered in bold type). The remaining substances listed in this table demonstrated relatively constant concentrations throughout all periods of pregnancy and lactation. The additional peaks in the chromatogram (Figure 1) have not been quantified for two reasons: (1) their small peak areas would not permit reliable measurements; and (2) for the early eluting constituents (substances with high volatility), peak areas are greatly affected by the sampling procedure.
A B C
A B C
A B C
A B C
A B C
A B C
10
15
16
20
21
A B C
Type of sample
8
Ketones 6
Peak designation
b10.7 • 1.9 ~5.4 • 0.9 4.5 • 0.6
b16.6 • 2.5 b'~9.9 • 1.3 5.2 • 0.6
25.9 • 2.4 17.6 • 2.2 a14.3 + 3.6
a9.8 • 1.5 6.8 ___ 1.5 ~3.6 • 1.0
~150.3 • 20.4 98.7 • 26.1 ~91.1 • 18.4
a3.3 _+ 0.6 a2.6 :k 0.3 1.0 • 0.0
3,9 • 1.2 42.4 • 0.5 1.4 _+ 0.2
1
"4.6 • 0.9 a3.4 • 0.2 3.2 • 0.4
a7.9 __. 1.3 b6.5 • 0.6 4,1 _+0,6
18.8 • 1.6 16.6 + 1.0 3.0 • 1.1
b4.5 • 0.9 4.2 • 1.0 0.5 • 0.2
b77.0 • 16.1 58.9 • 3.7 27.8 • 2.8
"2.3 • 0.2 b'al.5 • 0.4 "4.2 +_ 1.8
5,6 • 0.3 ~4.9 • 0.6 0.4 •
2
%.3 • 2.2 b8.4 • 2.1 4.6 • 0.6
a9.5 • 2.4 b'al0.6 +' 1.8 5.7 i 0.6
24.5 • 5.5 19.2 + 2.4 3.4 • 1.1
~'~6.7 • 0.8 4.8 • 0.7 0.6 • 0.2
~'"140.9 • 25.1 56.9 • 1.8 32.5 • 10.7
1.0 • 0.0 "2.7 • 1.1 1.6 • 0.6
4,6 • 0.6 ~'b3.2 + 0.5 0.5 •
3
Periods of pregnancy and lactation
b'a8.7 • 0.2 O9.5 • 0.6 5.0 __+0.3
b'al0.9 • 1.3 a13.0 • 1.0 5.9 • 0.7
21.9 • 6.5 12.4 • 1.1 2.4 • 0.4
b'~7.2 • 1.0 4.5 • 1.0 0.4 • 0.1
c211.1 _+ 30.7 109.1 • 20.4 13.9 • 1,3
1.0 + 0.1 bl.0 • 0.1 0.4 • 0.1
3.2 • 0.8 "2.4 + 0.6 0.4 • 0.1
4
TABLE 2. MEAN VALUE (+__SEM) OF PEAK AREAS (ARBITRARY UNITS) FOR VOLATILE COMPOUNDS FOUND IN URINE OF PREGNANT PRIMIPAROUS (A), MULTIPAROUS (B); AND LACTATING (C) FEMALES DURING DIFFERENT PERIODS OF PREGNANCY AND LACTATIONa
t" 9 r~ ~, V
~, ~:
b'a2.9 • 0.9 a l . l • 0.0 "3.1 • 0.3
35.5 • 7.4 "18.8 • 3.1 20.1 • 5.1
10.3 • 2.4 b'a6.1 • 1.4 6.8 • 1.5
A B C
A B C
C
B
A
6.8 • 1.6 "3.9 • 1.5 4.4 • 0.4
7.7 • 3.4 b'"7.7 • 1.7 3.4 • 0.3
29.3 • 7.9 b'a28.8 • 2.2 11.4 _--t-2.2
c'al.8 • 0.5 b2.4 • 0.4 bl.4 • 0.2
• 0.0 ~ a1.2 • 0.4 a2.9 • 0.7
23.9 • 5.0 a17.9 • 1.5 13.9 • 1.5
2.9 • 1.0 b5.7 • 1.0 0.4 •
1.2 • 0.5 a1.4 • 0.3 0.4 • 0.1
9.8 _+ 0.7 u9.8 _+ 0.9 2.4 + 0.9
43.4 + 7.3 b34.8 _+ 1.1 7.8 • 4.0
b4.7 • 0.0 b2.3 • 0.0 O0.5 • 0.0
"10.6 • 3.1 ~ • 1.7 0.4 • 0.1
a7.2 • 1.2 3.3 • 0.6 0.5 • 0.0
b'a3.4 + 0.7 3.5 • 0.8 0.8 • 0.01
02.1 • 0.2 a2.4 • 0.2 0.9 • 0.2
a8.3 • 1.9 4.3 • 0.8 0.2 •
b'~6.4 • 1.1 4.4 • 0.2 0.5 •
b4.9 • 1.2 2.7 • 0.2 0.3 • 0.1
c30.7 • 6.8 a10.3 • 1.4 0.2 •
b'a7.5 • 3.0 3.4 • 0.5 0.6 • 0.1
bl.9 • 0.3 1.5 • 0.1 0.7 • 0.1
aN = 3-6 runs for each average peak area value; 1 ml of urine collected from 8-10 females was used in one ran. Those means not connected by the same vertical lines are significantly different at the 0.05 level. Those means in rows not marked by the same superscript letter (a, b, c) are significantly different at the 0.05 level. If there are no superscript letters in a row, there are no significant differences among the means.
1
Dihydrofurans
26
2.6 + 0.6 al.5 • 0.4 a3.3 • 0.9
A B C
"5.3 • 0.8 4.3 • 1.0 ~3.4 • 0.2
A B C
18
Alcohols 17
"9.6 • 2.0 2.7 • 0.2 "3.7 • 1.2
a9.9 • 3.2 2.1 • 0.3 "7.3 • 2.8
A B C
A B C
14
Esters 11
4~ --O
< o >
Z
O
1948
JEMIOLO ET AL.
The 14 components exhibiting some dependence on the animals' endocrine status can be readily classified into one of four structurally distinct categories: ketones, esters, alcohols, and dihydrofurans (cyclic vinyl ethers). The components selected for a careful quantitative evaluation in the urine of pregnant primiparous (A), pregnantmultiparous (B), and lactating (C) females are listed in Table 2. A majority of the investigated urinary components tended to be more concentrated in pregnant primiparous than in either pregnant multiparous or lactating females. This trend was observed for all distinct periods; however, only in a few cases (Table 2) are the differences between the pregnant primiparous and pregnant multiparous animals statistically significant. On the other hand, Statistical Significance (P < 0.05) is obvious when comparing the levels of ketones, esters, alcohols, and dihydrofurans of lactating females with the pregnant animals. Substantial decreases were generally observed. The levels of the 14 investigated compounds changed clearly during the different periods of pregnancy and lactation. Most of these appeared at a higher average level during the first period of pregnancy or lactation than in the second. Some of the ketones and esters that decreased in concentration at the second period of pregnancy have shown a tendency toward elevation in their levels during the consecutive periods (Table 2). In the urine of lactating females, most ketones and all esters also decreased initially during the second period, but maintained the same levels throughout the rest of the experiment. The concentrations of alcohols in female urine have a tendency to increase during the last periods of pregnancy. Yet, the same substances drop after the first period of lactation and remain at low levels until parturition. In order to provide "reference levels" of the investigated urinary volatile constituents, samples from singly caged, nonreproducing females were analyzed under identical experimental conditions (Table 3). The average levels of the 14 volatile compounds found in the urine of pregnant and lactating females were then tabulated and compared to the same 14 peaks from urine of the nonreproducing females (control data). The percentage of the difference in the peak area of ketones, esters, alcohols, and dihydrofurans is shown in Figures 2-7. On the basis of our earlier work (Novotny et al., 1986), we have learned that the urine of group-caged females contains a similar set of volatile compounds, but the concentrations of such compounds were clearly different as compared to singly caged, nonreproducing animals. The data reflecting these percentage differences have been placed at the fight side of each figure (Figures 2-7) to provide an additional point of comparison. Seven ketones (peaks 6, 8, 10, 15, 16, 20, and 21 of Figure 1) varied in concentration in the urine of pregnant primiparous females (Figure 2). 2-Hexanone (6), 4-heptanone (8), and 2-heptanone (10) exhibited concentrations that were significantly (P < 0.05) higher, as compared to those in nonreproducing animals, throughout all distinct periods.
1949
MOUSE URINARY VOLATILES
TABLE 3. MEAN VALUE (-}-SEM) OF PEAK AREAS (ARBITRARY UNITS) FOR SELECTED VOLATILE COMPOUNDS FOUND IN URINE OF SINGLY CAGED, NONREPRODUCING FEMALESa
Peak designation 1 6 7 8 10 11 14 15 16 17 18 20 21 26
Structure mol wt 126 2-hexanone mol wt 126 4-heptanone 2-heptanone n-pentyl acetate
cis-2-penten-l-ylacetate trans-5-hepten-2-one trans-4-hepten-2-one 4-penten-l-ol mol wt 142 ester 6-methyl-6-hepten-3-one 6-methyl-5-hepten-3-one 1-octen-3-ol
Average levels 43.2 2.5 12.7 1.3 52.1 4.1 4.9 4.8 18.3 1,5 3.0 12.3 7.5 1.4
+ 2.7 + 0.4 _ 0.8 + 0.1 _+ 4.3 _+ 0.6 _+ 0,5 +_ 0.3 + 1.2 + 0.3 _+ 0.2 _+ 0.7 _+ 0.5 + 0.7
~N = 13-17 runs for each peak area value; 1 ml of urine collected from 10-20 females was used in one run.
The remaining ketones, trans-5-hepten-2-one (15), trans-4-hepten-2-one (16), 6-methyl-6-hepten-3-one (20), and 6-methyl-5-hepten-3-one (21) exhibited either an increase or decrease in concentration compared to nonreproducing females, but the range o f changes was not significant. In the urine o f group-caged females, the concentrations of 2-hexanone (6) and 4-heptanone (8) were similar to those appearing in nonreproducing animals. However, this type o f urine contained additional ketones, such as 2-heptanone (10), trans-5-hepten-2-one (15), and trans-4-hepten-2-one (16) in higher concentration than did the urine from nonreproducing females (Figure 2). The increases observed for ketones in the urine o f pregnant multiparous animals were less than in pregnant primiparous females. However, a trend toward more elevated levels o f 2-hexanone (6), 4-heptanone (8), and 2-heptanone (10) as compared to the control group of females was still noticeable (Figure 3). Two compounds, 6-methyl-6-hepten-3-one (20) and 6-methyl-5-hepten-3one (21), have become significantly suppressed in the pregnant multiparous animals as compared to nonreproducing females (Figure 3). F o r all the ketones, the most dramatic changes were seen in the urine o f the lactating females (Figure 4). There was a clear suppression o f these compounds, 9from 4 5 - 9 5 %, compared to nonreproducing females. That suppression
1950
JEMIOLO ET AL. PERIODS OF PREGNANCY PERCENT I
{
-
1 -
1
2 E - - 1
I
-
3 -
4 I
170 ~-
150 ~
130 110 '100
_z
z o
, F
GROUPED FEMALES - -
t
o,
21
V]
_z CONTROL 0
~
zo
zsi
2o
~'ERCENT
F~G. 2, Percentage difference in the peak areas of urinary ketones between pregnant primiparous and nonreproducing (control) females of ICR/Alb strain. Diagonally striped bars represent significant increases of ketones in pregnant and grouped females as compared to controls. was observed throughout all lactation periods. Two exceptions were noted: for 2-heptanone during the first period, and for 4-heptanone during the second period. The latter substance attained a level never noted before with any urine sample. The levels of esters are dealt with in Figure 5; n-pentyl acetate (11), 2penten-1-yl acetate (14), and an unidentified ester (18) showed both increases and decreases in urines of pregnant, lactating, and group-caged females as compared with the control. Here, the concentration of esters is variable and dependent on a type of urine and period of collection. Grouped and pregnant females produced these compounds in very high concentrations only during the first and last periods of pregnancy. In the mid-pregnancy intervals, these esters were found at a low level (similar to the control group). The urine collected at the first period of lactation (up to seven days) contained 80 % more 2-pentyl acetate (11) than did the urine from nonreproducing females. During the next three periods, the concentration of esters in the urine of females dropped significantly (Figure 5). The same type of comparison used for ketones and esters was also era-
MOUSE
URINARY
1 pERCENT
1951
YOLATILES
I
m]
PERIODS OF PREGNANCY 2 3 ~ f - - ]
4 [
m
GROUPED FEMALES F - - 7
i
170 16{) z
8
~
L ~6
Fro. 3. Percentage difference in the peak areas of urinary ketones between pregnant multiparous and nonreproducing (control) females of ICR/Alb strain. Diagonally striped bars represent significant increases and decreases of ketones in pregnant and grouped females as compared to controls. ployed for alcohols and dihydrofurans (see Figures 6 and 7). Extremely high levels of alcohols were found in the urine of pregnant females, both primiparous and multiparous (Figure 6). In both types of urine, an elevation of 4-penten-1ol (17) and 1-octen-3-ol (26) appeared during the last period of pregnancy rather than at the beginning. The urine of grouped females also contained alcohols in very high concentration. Only the urine from lactating females showed no changes in the concentration of alcohols, as compared to controls, during the last periods. However, in the first period of lactation, the levels of alcohols were significantly different from those of control females (Figure 6). The last group of investigated compounds, the dihydrofurans of molecular weight 126, were structurally postulated in a previous publication (Schwende et al., 1986). For these "mouse-specific" compounds, significant variations were found as well (Figure 7). Concentration of the dihydrofurans was decreased 20-45 % in the urine of pregnant primiparous females, while the samples from pregnant multiparous and lactating animals exhibited levels 35-80 % lower. Although a wealth of statistically significant data has been acquired here
1952
JEM~O~OET PERIODS OF L A C T A T I O N pERCENT r
-
I F~
-
2
I
3
i - -
1
4 l - - I
l
AL.
GROUPED FEMALES - - 1
VARIATIONS IN MOUSE (Mus musculus) URINARY VOLATILES DURING DIFFERENT PERIODS OF PREGNANCY A N D LACTATION
BOZENA JEMIOLO, FRANCA ANDREOLINI, DONALD WIESLER, and MILOS NOVOTNY Department of Chemistry lndiana University Bloomington, Indiana 47405 (Received August 15, 1986; accepted November 17, 1986)
Abstract--Mouse urine samples from different pregnancy and lactation periods were examined by capillary gas chromatography to assess variations in the volatile signals that may affect the endocrine function of other females. Statistically significant changes in the excretion of certain urinary volatiles were observed; from 26 readily quantifiable constituents, 14 appear to be under the endocrine control. These selected components, positively identified through mass spectrometry and retention data, and the synthetic standards are ketones, unsaturated alcohols, esters, and cyclic vinyl ethers. Key Words--Female mouse urine, pregnancy, lactation, urinary volatiles, mouse, Musmusculus.
INTRODUCTION
In the house mouse (Mus musculus), there are at least three female urinary chemosignals that can affect the reproduction status of other females: (1) the delay of puberty occasioned by treatment with urine from grouped females (Drickamer, 1974, 1977, 1983a); (2) the acceleration of puberty by the urine from estrus females (Drickamer 1982a); and, (3) the acceleration of puberty (Drickamer and Hoover, 1979) and prolongation of the estrous period (Hoover and Drickamer, 1979) through exposure to the urine from pregnant and lactating females. It has been suggested (Drickamer, 1982b) that some chemical substances in the urine of females may act as the general signals to other conspecifics regarding the adequacy of social and environmental conditions which are important for successful reproduction. 1941 0098-0331/87/0900-1941505.00/0 9 1987PlenumPublishingCorporation
1942
JEMIOLO ET AL.
Urine samples collected at different time periods during pregnancy and lactation are differentially effective in terms of the acceleration of the first estrus relative to control females treated with water (Drickamer, 1983b, 1984). The chemosignals from pregnant/lactating females seem to be present in the urine only during the last two thirds of pregnancy or lactation (Drickamer, 1983b). In addition, Cowley and Wise (1972) reported that the urine from pseudopregnant females applied to the nasal region of neonatal females has an accelerating effect of their growth, while the application of urine from females in late pregnancy is associated with a slow rate of growth. It is not yet known what set of hormonal and related physiological conditions within the female mice may reflect these changes in the chemical signals released by donor females. Since no chemical investigations on these signals have been conducted to date, their nature remains a subject of speculation. Earlier study by Drickamer and Hoover (1979) has shown that the urine from pregnant or lactating females, either applied daily on the extemal nares of the young animals or provided to them in a plastic (inaccessible) capsule, can accelerate their sexual maturation. In addition, the urine from pregnant or lactating females that is exposed to the ambient air remains effective for only one to three days (Drickamer, 1986). These findings suggest that the pregnancy/lactation chemosignal(s) is either an air-sensitive or a volatile substance. The chemosignals from pregnancy urine and that of lactating animals may or may not contain similar substances. Both chemical characterization and biological experiments are needed to elucidate this problem. Our previous successful uses of a headspace sampling technique (Novotny et al., 1974) and capillary gas chromatography-mass spectrometry to identify the male chemosignals responsible for aggression (Novotny et al., 1985), estrus synchronization (Jemiolo et al., 1986a), and the female signals causing the delay of puberty (Novotny et al., 1986) have prompted us to apply similar techniques to the identification of additional female chemosignals in this study. As the initial approach to this complex problem, the urinary profiles of volatile substances were quantitatively followed throughout the pregnancy and lactation periods. Statistically significant alterations in quantities of certain volatile components are reported here and correlated with the animals' endocrine status. The structures of these components were verified through their mass spectra and chromatographic behavior of authentic substances.
METHODS AND MATERIALS
All mice used as urine donors were from a randomly bred, closed colony of ICR/Alb (Mus musculus) purchased from Ward's Natural Science Establishment, Inc., Rochester, New York. Females were housed in plastic cages (12 • 28 x 27 cm), maintained at 21~ + 0.2~ 50-70% humidity, and a 12-
MOUSE URINARY VOLATILES
1943
hr light-12-hr dark daily regime. Unlimited amounts of Purina Mouse Chow (Ralston Purina Corp., St. Louis, Missouri) and water were supplied throughout the whole experimental period. Bedding was changed weekly. For use as urine donors, 120 virgin and 120 multiparous females, in total, were bred with fertile males. The occurrence of pregnancy was established by a daily examination for vaginal plugs (day 0). After insemination, each female was caged alone over the whole period of pregnancy. The urine from pregnant females was collected from days 2 to 7 (period 1), 8 to 12 (period 2), 13 to 15 (period 3), and from day 16 postplug to the last day before parturition (period 4). An additional 120 multiparous pregnant female mice were isolated into individual cages and checked daily for newborns. On the day of delivery, the litters were counted and reduced to 10 pups. Collection of urine from lactating females was done at the same periods as during pregnancy: days 2-7 (period 1), 8-12 (period 2), 13-15 (period 3), and from day 16 postpartum until the young were 21 days old. All females used as urine donors were 80-130 days of age. Fresh urine was collected by holding a mouse over a glass vial and gently squeezing the abdomen and flanks. Immediately after collection, samples were stored at - 2 0 ~ and analyzed within two to four days. All types of urine were analyzed three to six times by using each time a 1-ml sample obtained from different collections within the same experimental period. For each sample, the urine was collected and pooled from 8 to 10 pregnant or lactating females. As a standard sample, we used urine collected from virgin, nonreproducing females at 90-120 days of age, that were housed singly. Collection of the urine was carried out throughout all stages of the estrous cycle. The urinary volatiles were analyzed using the headspace technique, employing Tenax GC (a porous polymer) as the adsorption medium (Novotny et al., 1974). The volatiles were sparged from 1-ml urinary samples at room temperature with purified helium gas at a flow rate of 100 ml/min and adsorbed onto a precolumn packed with 4 mg of Tenax GC. The sample was subsequently desorbed in the heated injection port (220-240~ of a gas chromatograph (Perkin-Elmer, 3920 instrument, Norwalk, Connecticut) equipped with a flame ionization detector, and retrapped into a cooled section of a glass capillary column. The analytical column was a glass capillary (60 m x 0.25 mm, ID) of a sodalime type, coated statically with UCON 50-HB-2000 (Schwende et al., 1984a,b). While quantitative comparisons were obtained through peak integration routines (Sigma 10, Perkin-Elmer), identification of the individual profile constituents was established through a combined GC-MS (Hewlett-Packard, 5981 dodecapole mass spectrometer, Palo Alto, California) using electron impact ionization at 70 eV. Wherever feasible, authentic samples were either purchased or synthesized in the laboratory to verify agreement of spectral information and gas-chromatographic retention time.
1944
JEMIOLO ET AL.
Statistical Calculations. Statistical comparisons of the levels of excreted volatiles were made using one-way analysis of the variance (ANOVA) with Duncan's new multiple-range test (Zar, 1974). The probability level for statistical significance was set at P < 0.05.
RESULTS AND DISCUSSIONS The complexity of mouse urinary volatile profiles has been demonstrated in our previous studies (Novotny et al., 1980, 1984, 1986; Schwende et al., 1984a,b, 1986; Jemiolo et al., 1986a,b); genetic background, sex, endocrine status, etc., all seem to influence the quantitative proportions of various urinary constituents. The capillary chromatogram shown in Figure 1 is representative of a profile obtained from pregnant female ICR/Alb mice. From a large number of components, 26 substances are numbered; most have been positively identified by combined capillary gas chromatography-mass spectrometry and the retention measurements of authentic compounds (Table 1). Through careful visual inspection of the profiles obtained from different stages of pregnancy and lactation, as well as from nonreproducing females, we first established that 14 constituents showed obvious variations in their chro-
28
[
18 17
23242526
19
3O
b-
5O
70
I
I
90 TEMP (~
9,
110
130
1~
1~
I
I
I
I
20
30TIME (rain) 40
50
60
~therrr~l
, 70
FI6. t. Gas chromatogram of urine volatiles from ICR/Alb primiparous females at the 13th to 15th day of pregnancy (period 3).
1945
MOUSE URINARY VOLATILES
T A B L E 1. V O L A T I L E C O M P O U N D S IN U R I N E OF P R E G N A N T P R I M I P A R O U S ( A ) ; P R E G N A N T M U L T I P A R O t J S ( B ) ; AND L A C T A T I N G ( C ) F E M A L E M I C E
Changes in concentration of compounds during pregnancy and lactation Peak number
Structure
A
B
C
1
tool w t 126"
_
+b
_c
2
4-ethylcyclohexene
-
-
-
3
3 - m e t h y l - 1- b u t e n - 3 - o l
-
-
-
4
2 ethyl-5-methyl-fumn
-
-
-
5
3-hexanone
-
-
-
6
2-hexanone
-
+
-
7
m . w 126 a
-
+
-
8
4-heptanone
+
+
+
9
m . w 126 a
-
-
-
10
2 heptanone
+
-
+
11
n - p e n t y I acetate
+
+
+
12
methyl hexanoate
-
-
-
13
6-hepten-2-one
-
-
-
14
c i s - 2 - p e n t e n - 1-yl acetate
+
-
+
15
trans-5-hepten-2-one
+
-
+
16
trans-4-hepten-2-one
-
-
+
17
4-penten-l-ol
+
+
+
18
u n i d e n t i f i e d e s t e r o f t o o l w t 142
+
-
+
19
2,5-dimethylpyrazine
-
-
-
211
6 - m e t h y l - 6 - h e p t e n - 3 -one
+
+
-
21
6-methyl-5-hepten-3-one
+
+
-
22
dehydrobrevicomin
-
-
-
23
2-nonanone
-
-
-
24
benzaldehyde
-
-
-
25
unidentified
-
-
-
26
1-octen-3-ol
+
+
+
a P r e s u m e d i s o m e r i c c y c l i c v i n y l ethers u n i q u e to t h e m o u s e ( S c h w e n d e e t a l . , 1986); d e h y d r a t i o n products o f known 5,5 dimethyl-2-ethyltetrahydrofuran-2-ol. b C o n c e n t r a t i o n o f the c o m p o u n d d e p e n d e n t o n a p e r i o d o f p r e g n a n c y o r l a c t a t i o n . C C o n c e n t r a t i o n o f the c o m p o u n d d o e s not s e e m d e p e n d e n t o n a p e r i o d o f p r e g n a n c y o r l a c t a t i o n .
matographic peak areas (Table 1, see peaks numbered in bold type). The remaining substances listed in this table demonstrated relatively constant concentrations throughout all periods of pregnancy and lactation. The additional peaks in the chromatogram (Figure 1) have not been quantified for two reasons: (1) their small peak areas would not permit reliable measurements; and (2) for the early eluting constituents (substances with high volatility), peak areas are greatly affected by the sampling procedure.
A B C
A B C
A B C
A B C
A B C
A B C
10
15
16
20
21
A B C
Type of sample
8
Ketones 6
Peak designation
b10.7 • 1.9 ~5.4 • 0.9 4.5 • 0.6
b16.6 • 2.5 b'~9.9 • 1.3 5.2 • 0.6
25.9 • 2.4 17.6 • 2.2 a14.3 + 3.6
a9.8 • 1.5 6.8 ___ 1.5 ~3.6 • 1.0
~150.3 • 20.4 98.7 • 26.1 ~91.1 • 18.4
a3.3 _+ 0.6 a2.6 :k 0.3 1.0 • 0.0
3,9 • 1.2 42.4 • 0.5 1.4 _+ 0.2
1
"4.6 • 0.9 a3.4 • 0.2 3.2 • 0.4
a7.9 __. 1.3 b6.5 • 0.6 4,1 _+0,6
18.8 • 1.6 16.6 + 1.0 3.0 • 1.1
b4.5 • 0.9 4.2 • 1.0 0.5 • 0.2
b77.0 • 16.1 58.9 • 3.7 27.8 • 2.8
"2.3 • 0.2 b'al.5 • 0.4 "4.2 +_ 1.8
5,6 • 0.3 ~4.9 • 0.6 0.4 •
2
%.3 • 2.2 b8.4 • 2.1 4.6 • 0.6
a9.5 • 2.4 b'al0.6 +' 1.8 5.7 i 0.6
24.5 • 5.5 19.2 + 2.4 3.4 • 1.1
~'~6.7 • 0.8 4.8 • 0.7 0.6 • 0.2
~'"140.9 • 25.1 56.9 • 1.8 32.5 • 10.7
1.0 • 0.0 "2.7 • 1.1 1.6 • 0.6
4,6 • 0.6 ~'b3.2 + 0.5 0.5 •
3
Periods of pregnancy and lactation
b'a8.7 • 0.2 O9.5 • 0.6 5.0 __+0.3
b'al0.9 • 1.3 a13.0 • 1.0 5.9 • 0.7
21.9 • 6.5 12.4 • 1.1 2.4 • 0.4
b'~7.2 • 1.0 4.5 • 1.0 0.4 • 0.1
c211.1 _+ 30.7 109.1 • 20.4 13.9 • 1,3
1.0 + 0.1 bl.0 • 0.1 0.4 • 0.1
3.2 • 0.8 "2.4 + 0.6 0.4 • 0.1
4
TABLE 2. MEAN VALUE (+__SEM) OF PEAK AREAS (ARBITRARY UNITS) FOR VOLATILE COMPOUNDS FOUND IN URINE OF PREGNANT PRIMIPAROUS (A), MULTIPAROUS (B); AND LACTATING (C) FEMALES DURING DIFFERENT PERIODS OF PREGNANCY AND LACTATIONa
t" 9 r~ ~, V
~, ~:
b'a2.9 • 0.9 a l . l • 0.0 "3.1 • 0.3
35.5 • 7.4 "18.8 • 3.1 20.1 • 5.1
10.3 • 2.4 b'a6.1 • 1.4 6.8 • 1.5
A B C
A B C
C
B
A
6.8 • 1.6 "3.9 • 1.5 4.4 • 0.4
7.7 • 3.4 b'"7.7 • 1.7 3.4 • 0.3
29.3 • 7.9 b'a28.8 • 2.2 11.4 _--t-2.2
c'al.8 • 0.5 b2.4 • 0.4 bl.4 • 0.2
• 0.0 ~ a1.2 • 0.4 a2.9 • 0.7
23.9 • 5.0 a17.9 • 1.5 13.9 • 1.5
2.9 • 1.0 b5.7 • 1.0 0.4 •
1.2 • 0.5 a1.4 • 0.3 0.4 • 0.1
9.8 _+ 0.7 u9.8 _+ 0.9 2.4 + 0.9
43.4 + 7.3 b34.8 _+ 1.1 7.8 • 4.0
b4.7 • 0.0 b2.3 • 0.0 O0.5 • 0.0
"10.6 • 3.1 ~ • 1.7 0.4 • 0.1
a7.2 • 1.2 3.3 • 0.6 0.5 • 0.0
b'a3.4 + 0.7 3.5 • 0.8 0.8 • 0.01
02.1 • 0.2 a2.4 • 0.2 0.9 • 0.2
a8.3 • 1.9 4.3 • 0.8 0.2 •
b'~6.4 • 1.1 4.4 • 0.2 0.5 •
b4.9 • 1.2 2.7 • 0.2 0.3 • 0.1
c30.7 • 6.8 a10.3 • 1.4 0.2 •
b'a7.5 • 3.0 3.4 • 0.5 0.6 • 0.1
bl.9 • 0.3 1.5 • 0.1 0.7 • 0.1
aN = 3-6 runs for each average peak area value; 1 ml of urine collected from 8-10 females was used in one ran. Those means not connected by the same vertical lines are significantly different at the 0.05 level. Those means in rows not marked by the same superscript letter (a, b, c) are significantly different at the 0.05 level. If there are no superscript letters in a row, there are no significant differences among the means.
1
Dihydrofurans
26
2.6 + 0.6 al.5 • 0.4 a3.3 • 0.9
A B C
"5.3 • 0.8 4.3 • 1.0 ~3.4 • 0.2
A B C
18
Alcohols 17
"9.6 • 2.0 2.7 • 0.2 "3.7 • 1.2
a9.9 • 3.2 2.1 • 0.3 "7.3 • 2.8
A B C
A B C
14
Esters 11
4~ --O
< o >
Z
O
1948
JEMIOLO ET AL.
The 14 components exhibiting some dependence on the animals' endocrine status can be readily classified into one of four structurally distinct categories: ketones, esters, alcohols, and dihydrofurans (cyclic vinyl ethers). The components selected for a careful quantitative evaluation in the urine of pregnant primiparous (A), pregnantmultiparous (B), and lactating (C) females are listed in Table 2. A majority of the investigated urinary components tended to be more concentrated in pregnant primiparous than in either pregnant multiparous or lactating females. This trend was observed for all distinct periods; however, only in a few cases (Table 2) are the differences between the pregnant primiparous and pregnant multiparous animals statistically significant. On the other hand, Statistical Significance (P < 0.05) is obvious when comparing the levels of ketones, esters, alcohols, and dihydrofurans of lactating females with the pregnant animals. Substantial decreases were generally observed. The levels of the 14 investigated compounds changed clearly during the different periods of pregnancy and lactation. Most of these appeared at a higher average level during the first period of pregnancy or lactation than in the second. Some of the ketones and esters that decreased in concentration at the second period of pregnancy have shown a tendency toward elevation in their levels during the consecutive periods (Table 2). In the urine of lactating females, most ketones and all esters also decreased initially during the second period, but maintained the same levels throughout the rest of the experiment. The concentrations of alcohols in female urine have a tendency to increase during the last periods of pregnancy. Yet, the same substances drop after the first period of lactation and remain at low levels until parturition. In order to provide "reference levels" of the investigated urinary volatile constituents, samples from singly caged, nonreproducing females were analyzed under identical experimental conditions (Table 3). The average levels of the 14 volatile compounds found in the urine of pregnant and lactating females were then tabulated and compared to the same 14 peaks from urine of the nonreproducing females (control data). The percentage of the difference in the peak area of ketones, esters, alcohols, and dihydrofurans is shown in Figures 2-7. On the basis of our earlier work (Novotny et al., 1986), we have learned that the urine of group-caged females contains a similar set of volatile compounds, but the concentrations of such compounds were clearly different as compared to singly caged, nonreproducing animals. The data reflecting these percentage differences have been placed at the fight side of each figure (Figures 2-7) to provide an additional point of comparison. Seven ketones (peaks 6, 8, 10, 15, 16, 20, and 21 of Figure 1) varied in concentration in the urine of pregnant primiparous females (Figure 2). 2-Hexanone (6), 4-heptanone (8), and 2-heptanone (10) exhibited concentrations that were significantly (P < 0.05) higher, as compared to those in nonreproducing animals, throughout all distinct periods.
1949
MOUSE URINARY VOLATILES
TABLE 3. MEAN VALUE (-}-SEM) OF PEAK AREAS (ARBITRARY UNITS) FOR SELECTED VOLATILE COMPOUNDS FOUND IN URINE OF SINGLY CAGED, NONREPRODUCING FEMALESa
Peak designation 1 6 7 8 10 11 14 15 16 17 18 20 21 26
Structure mol wt 126 2-hexanone mol wt 126 4-heptanone 2-heptanone n-pentyl acetate
cis-2-penten-l-ylacetate trans-5-hepten-2-one trans-4-hepten-2-one 4-penten-l-ol mol wt 142 ester 6-methyl-6-hepten-3-one 6-methyl-5-hepten-3-one 1-octen-3-ol
Average levels 43.2 2.5 12.7 1.3 52.1 4.1 4.9 4.8 18.3 1,5 3.0 12.3 7.5 1.4
+ 2.7 + 0.4 _ 0.8 + 0.1 _+ 4.3 _+ 0.6 _+ 0,5 +_ 0.3 + 1.2 + 0.3 _+ 0.2 _+ 0.7 _+ 0.5 + 0.7
~N = 13-17 runs for each peak area value; 1 ml of urine collected from 10-20 females was used in one run.
The remaining ketones, trans-5-hepten-2-one (15), trans-4-hepten-2-one (16), 6-methyl-6-hepten-3-one (20), and 6-methyl-5-hepten-3-one (21) exhibited either an increase or decrease in concentration compared to nonreproducing females, but the range o f changes was not significant. In the urine o f group-caged females, the concentrations of 2-hexanone (6) and 4-heptanone (8) were similar to those appearing in nonreproducing animals. However, this type o f urine contained additional ketones, such as 2-heptanone (10), trans-5-hepten-2-one (15), and trans-4-hepten-2-one (16) in higher concentration than did the urine from nonreproducing females (Figure 2). The increases observed for ketones in the urine o f pregnant multiparous animals were less than in pregnant primiparous females. However, a trend toward more elevated levels o f 2-hexanone (6), 4-heptanone (8), and 2-heptanone (10) as compared to the control group of females was still noticeable (Figure 3). Two compounds, 6-methyl-6-hepten-3-one (20) and 6-methyl-5-hepten-3one (21), have become significantly suppressed in the pregnant multiparous animals as compared to nonreproducing females (Figure 3). F o r all the ketones, the most dramatic changes were seen in the urine o f the lactating females (Figure 4). There was a clear suppression o f these compounds, 9from 4 5 - 9 5 %, compared to nonreproducing females. That suppression
1950
JEMIOLO ET AL. PERIODS OF PREGNANCY PERCENT I
{
-
1 -
1
2 E - - 1
I
-
3 -
4 I
170 ~-
150 ~
130 110 '100
_z
z o
, F
GROUPED FEMALES - -
t
o,
21
V]
_z CONTROL 0
~
zo
zsi
2o
~'ERCENT
F~G. 2, Percentage difference in the peak areas of urinary ketones between pregnant primiparous and nonreproducing (control) females of ICR/Alb strain. Diagonally striped bars represent significant increases of ketones in pregnant and grouped females as compared to controls. was observed throughout all lactation periods. Two exceptions were noted: for 2-heptanone during the first period, and for 4-heptanone during the second period. The latter substance attained a level never noted before with any urine sample. The levels of esters are dealt with in Figure 5; n-pentyl acetate (11), 2penten-1-yl acetate (14), and an unidentified ester (18) showed both increases and decreases in urines of pregnant, lactating, and group-caged females as compared with the control. Here, the concentration of esters is variable and dependent on a type of urine and period of collection. Grouped and pregnant females produced these compounds in very high concentrations only during the first and last periods of pregnancy. In the mid-pregnancy intervals, these esters were found at a low level (similar to the control group). The urine collected at the first period of lactation (up to seven days) contained 80 % more 2-pentyl acetate (11) than did the urine from nonreproducing females. During the next three periods, the concentration of esters in the urine of females dropped significantly (Figure 5). The same type of comparison used for ketones and esters was also era-
MOUSE
URINARY
1 pERCENT
1951
YOLATILES
I
m]
PERIODS OF PREGNANCY 2 3 ~ f - - ]
4 [
m
GROUPED FEMALES F - - 7
i
170 16{) z
8
~
L ~6
Fro. 3. Percentage difference in the peak areas of urinary ketones between pregnant multiparous and nonreproducing (control) females of ICR/Alb strain. Diagonally striped bars represent significant increases and decreases of ketones in pregnant and grouped females as compared to controls. ployed for alcohols and dihydrofurans (see Figures 6 and 7). Extremely high levels of alcohols were found in the urine of pregnant females, both primiparous and multiparous (Figure 6). In both types of urine, an elevation of 4-penten-1ol (17) and 1-octen-3-ol (26) appeared during the last period of pregnancy rather than at the beginning. The urine of grouped females also contained alcohols in very high concentration. Only the urine from lactating females showed no changes in the concentration of alcohols, as compared to controls, during the last periods. However, in the first period of lactation, the levels of alcohols were significantly different from those of control females (Figure 6). The last group of investigated compounds, the dihydrofurans of molecular weight 126, were structurally postulated in a previous publication (Schwende et al., 1986). For these "mouse-specific" compounds, significant variations were found as well (Figure 7). Concentration of the dihydrofurans was decreased 20-45 % in the urine of pregnant primiparous females, while the samples from pregnant multiparous and lactating animals exhibited levels 35-80 % lower. Although a wealth of statistically significant data has been acquired here
1952
JEM~O~OET PERIODS OF L A C T A T I O N pERCENT r
-
I F~
-
2
I
3
i - -
1
4 l - - I
l
AL.
GROUPED FEMALES - - 1
