Estrous synchrony: Modulation of ovarian cycle length by female pheromones

Physiology& Behavior, Vol. 32, pp. 701-705.Copyright©PergamonPress Ltd., 1984.Printed in the U.S.A.

0031-9384/84$3.00 + .00

Estrous Synchrony: Modulation of Ovarian Cycle Length by Female Pheromones M A R T H A K. M c C L I N T O C K

Department o f Behavioral Sciences, University o f Chicago 5730 South Woodlawn Ave., Chicago, IL 60637 q* Received 20 N o v e m b e r 1983 McCLINTOCK, M. K. Estrous synchrony: Modulation of ovarian cycle length by female pheromones. PHYSIOL BEHAV 32(5)701-705, 1984.--Airborne chemosignals from different phases of the rat's estrous cycle had opposing effects on the timing of the estrous cycle which were consistent with a coupled oscillator model of ovarian synchrony. Preovulatory odors shortened or phase advanced the ovarian cycle, whereas ovulatory odors lengthened or phase delayed the cycle. Female pheromones Synchrony Lordosis Estrous cycle

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IN rats, human and non-human primates, hamsters, and opossums, females that live together synchronize their ovarian cycles [6, 11, 13, 17, 18, 24, 26, 27, 29, 30]. This is a striking example of the social control of fertility and may well be a mechanism for ensuring that the costly investments of reproduction are coordinated with an appropriate social and physical environment [19,20]. The rhythmicity of the ovarian cycle can be conceptualized as a simple clock or oscillator [7,36]. The same sequence of ovarian events is repeated spontaneously: follicular development, ovulation and formation of corpora lutea. The timing of these repeated ovarian events is not determined solely by internal physiological mechanisms. In a socially breeding species, such as the rat, it is also affected by the reproductive condition and ovarian cycles of other females in the social group [12, 19, 22]. When the periodicity of an oscillator is influenced by another oscillator, termed coupled oscillators, then they may become relatively coordinated or synchronized [34]. Given an oscillator model of the ovarian cycle, ovarian synchrony can thus be thought of as the coupling or mutual entrainment of a population of oscillators. Since estrous synchrony occurs when female rats simply share a recirculated air supply [18], female rats must be producing airborne chemosignals that couple the ovarian cycles within a social group and alter the timing of the estrous cycle. Studies of other cycles that are mutually or environmentally entrained (e.g., circadian activity rhythms, cardiac pacemakers, fish-f'm movements, and cicada emergence cycles) have documented a variety of coupling mechanisms that can generate synchrony [4, 7, 14, 31]. A common element among these mechanisms is the presence of two opposing effects: one that phase advances or shortens cycle length and another that phase_ delays or increases cycle length. In the present study, a coupled oscillator model was used to examine the effects of different estrous cycle odors as part of the coupling mechanism that synchronizes the ovarian cycles of socially interacting females.

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Three hormonally distinct phases of the rat's estrous cycle were identified as points in the cycle which could produce different chemosignals: diestrus (preovulatory or follicular phase), proestrus (ovulatory phase), and metestrus (postovulatory or "luteal" phase) [23]. Each of these phases was used to generate a constant odor that was presented to a set of female rats. This technique is similar to that used to study circadian and neural entrainment [5,25]; the stimulus that normally generates entrainment is presented constantly, at different intensities, rather than cyclically. The focus is on the coupling mechanism itself, that is, the effect of the entraining stimulus on the periodicity of the cycle. METHOD

Animal Colony Female Sprague-Dawley rats (virgins, 125-140 days of age) were housed singly. The colony room was ventilated With 10 room changes per hour, maintained at 22__.2°C and lit with bright white light (1900-0900) and continuous dim red light. Food and water were available ad lib.

Estrous Cycle Measurement Estrous cycles were monitored via dally vaginal lavage taken at the midpoint of the dark phase (1300 hr). The proportion of nucleated (N), cornified (C), and leukocytic (L) cells was recorded for each vaginal lavage, generating 9 distinct types of vaginal smears. The analysis and recording of all vaginal smears was done by individuals who were unfamiliar with the working hypothesis and ignorant of the type of odors being presented. The phase, length, and regularity of each cycle was determined by the sequence of these smears [15, 16, 22, 32, 35]. Regular 4-day cycles began with two smears that contained primarily epithelial cells (/>80%) followed by two smears that contained leukocytes (~>20%) and were designated proestrus, estrus, metestrus, and diestrus I respectively. The

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Fifth day of a 5-day cycle was designated diestrus II. In cycles that were 6 days in length or longer, an extended luteal phase was indicated by an uninterrupted sequence of smears containing leukocytes. An extended follicular phase was indicated by an uninterrupted sequence of smears containing primarily epithelial cells. The lordosis reflex was measured by daily manual palpation. The strength (0-4) and habituation (1-2) of the reflex were recorded and combined into a 9-point scale which measured the intensity of the reflex.

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Estrous cycle odors were presented to females living in one of two wind tunnels. Each wind tunnel was made from three large boxes connected in series with tubing and a fan that pulled air through them [22]. The first box was designated the upwind box, the second, the source box, and the third, the downwind box. Smoke tests demonstrated that baffles, placed in front of the intake ports, deflected the airstream into each living compartment. Plexiglas covers ensured light-dark cycles identical to that of the colony room (14:10, lights on 1900). Pine chip bedding was changed every 4 days in the upwind and downwind boxes. The bedding in the source box was changed only weekly, ensuring a strong nonvariable odor source. In order to mimic the signal-to-noise ratio of a large breeding cage or rat burrow, colony room air was pulled through an intake valve in the upwind box (10 CFM) and mixed with odors of the upwind control group to form a background odor. The air then flowed through the middle source box where the experimental odors were added, through the downwind experimental box, and was then exhausted directly out of the room.

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Procedure The upwind and downwind boxes both housed six female rats living in individual compartments. Each female had regular 4- or 5-day estrous cycles (based on 1 month of observation in the colony) and was matched with another female for individual differences in the proportion of Cycle types; one member of each matched-pair lived upwind of the source box and the other lived downwind in the same position relative to the airstream. Constant odors from the three different phases of the estrous cycle were created within the source box by using vaginal smear records to select females from the colony that were expected to enter a particular phase of the estrous cycle on the following day. These females were put in the source box for 24 hours and then returned to the colony and replaced with another set of females that were just then entering the same phase of the estrous cycle. Animals in the source box were exchanged at lights-on. Therefore, the constant odors came from proestrus-early estrus, metestrus-early diestrus I, and diestrus I-diestrus II or -early .proestrus. The accuracy of each prediction was verified using the continuous vaginal smear records of the colony. Animals in the downwind box were exposed to two odors in succession. Another set of downwind animals in the second wind tunnel were exposed to the same two odors, but in the reverse order. This design established several types of controls: (1) The matched females living upwind established the baseline effect produced by the background odors and controlled for the effects of age, time living in the wind tunnel, and auditory communication. (2) A comparison of each

CYCLE LENGTH

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FIG. 1. The effect of estrous cycle odors on the distribution of estrous cycle lengths. Each graph combines data from 12 individuals during an 18-22 day observation period. These population differences were also significant for individual females (see text).

subject to herself during exposure to two different constant odors controlled for individual differences in responsiveness. (3) Matching the downwind females in the different wind tunnels and reversing the sequence in which odors were presented controlled for the order of presentation and for the time spent in the wind tunnel. RESULTS

Estrous Cycle Length Constant diestrous and constant proestrous odors had opposite effects on the timing of the estrous cycle. All o f the females exposed to constant diestrous odor had regular 4-day estrous cycles (4.0±0.0 days; see Fig. 1). The estrous cycles were shorter and more regular than they were when these same females were exposed to the background odors (p<~0.01, sign test). They were also shorter than the estrous cycles of the upwind females (4.4±0.1 days, p~<0.01, sign test) and those living alone in the colony (4.5±0. I days. p~<0.01, sign test); note that individuals housed in a single cage in a colony room are essentially isolated [1]. Thus, the typical variability in cycle length found in either grouped or

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FIG. 2. Effect of estrous cycle odors on group synchrony. One hundred percent is the highest synchrony score possible in a group of five (i.e., when each individual is in estrus on the same day with both cornified epithelial cells in the vaginal smear and a strong lordosis reflex). A score of fitly percent is the threshold for synchrony [19]. Scores above this level indicate that the majorityof females in the group are in estrus on the same day.

isolated females was reduced. The cycles were more likely to be phase advanced, but only to the 4-day limit which has been demonstrated to be the lower bound of the range in the length of the rat's ovarian cycle [10]. Constant proestrous odor lengthened the estrous cycles of downwind females. Their cycles were longer and more irregular than when they were exposed to constant metestrous odor (5.4_+0.2 days, p ~<0.01, sign test; see Fig. 1). They were also longer than those of females living upwind (4.4--+0.1 days, p~<0.01, sign test) or in the colony (4.5__-0.1 days, p~<0.01, sign test; see Fig. 1). Thus, proestrous odors increased the variability in cycle length by increasing the probability of long, phase delayed cycles. Constant metestrous odor was essentially *'neutral" when presented against the background odor. Adding metestrous odor to the airstream did not produce estrous cycles that were any different from those produced by background odors alone (4.4-+0.1 days, NS, sign test). In addition, the cycles of the upwind control groups in each wind tunnel were not significantly different from each other (NS, sign test; see Fig. 1).

Length of Estrous Cycle Phase Estrous cycles that were lengthened by exposure to proestrous odors had extended sequences of smears containing leukocytes rather than epithelial cells (p ~<0.05, sign test). The short 4-day estrous cycles that resulted from exposure to diestrous odors were more likely to have only one day with epithelial cells in the smear than were the 4-day cycles when those females were exposed to background odors (p<~0.035, sign test). Metestrous odors did not alter the length of estrous cycle phase (NS, sign test).

Estrous Synchrony In this study, estrous synchrony among the females living together in a wind tunnel box could be used as another indicator of the potency of estrous cycle odors. Because each odor was constant, odors that altered the timing of individual estrous cycles were expected to disrupt the process of synchronization within the group by masking or overriding the group's own olfactory signals and decoupling the cycles. Estrous synchrony is demonstrated graphically by plotting the number of females in a group that are in a particular phase of the estrous cycle as a function of time (see Fig. 2). The reference point for these synchrony plots was proestrus, the time of ovulation and mating. A group was considered synchronized when at least 50%of its members were at the same phase of the estrous cycle on the same day [19]. Usually, 50% to 80% of the group becomes synchronized, a value that fluctuates as individuals join and leave the group [11, 17, 18] (see Fig. 2: Windtunnel A, Background Odor). Both constant proestrous and constant diestrous odor disrupted the process of synchronization within the groups living in the downwind box, but did so for different reasons. Diestrous odor locked each animal into a regular 4-day cycle, both when an animal was phase delayed or phase advanced by one day (Fig. 2: Windtunnel A) and when the group was at an artificially high and predetermined level of synchrony (Windtunnel B). Thus, the group was locked at the level of synchrony it happened to be at when diestrous odor was first presented. Constant proestrous odor also disrupted the process of synchronization within the downwind group (Fig. 2: Windtunnels C and D). In this case, synchronization was disrupted because the variance in cycle length was substantially increased; when variance in periodicity is high, synchrony is precluded [36]. Single pulses of odor also affected the timing of the cycle and synchrony within the group. A l-day pulse of proestrous odor (Fig. 2: Day 16, Windtunnel D) phase delayed the cycles and disrupted synchrony. A 1-day pulse of diestrous odor in the midst of constant proestrous odor (Day 34, Windtunnel D) temporarily synchronized the group. In addition, it is striking that a disrupted group resynchronized over the time span of three potential cycles, even during exposure to constant metestrous odor (Fig. 2: Windtunnel C). This is the time that it usually takes for synchrony to develop among females that have been housed together recently [ 17,18]. This temporal constancy is particularly convincing evidence that metestrous odor does not alter the process of synchronization and is a "neutral" odor against a background of mixed colony odors.

Lordosis Reflex There was no statistically significant effect of any estrous cycle odor on the average daily strength of the lordosis reflex to manual palpation: diestrous odor (3.4), proestrous odor (6.4), and metestrous odor (5.7), with upwind control groups (3.5, 5.4, and 6.4 respectively); NS, matched-pairs signedranks test.

DISCUSSION Female rats produce at least two airborne chemosignals in the course of an estrous cycle which alter the estrous cycle length of other female rats. These two odors have opposing

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effects on estrous cycle length. Follicular or diestrous odor shortens the cycle, apparently by shortening the luteal phase of the cycle or facilitating ovulation. Ovulatory or proestrous odor lengthens the estrous cycle, apparently by lengthening the luteal phase or suppressing ovulation. The effects on the length of estrous cycle phase and hence the timing of ovulation are only inferred from the sequence of vaginal smears and need to be verified by direct measurement of hormonal response to each type of odor. Previous studies of female chemosignals have used an odor source that combined stimuli from all phases of the ovarian cycle [3, 8, 9, 33]. Most of the variable and subtle effects previously reported may have been confounded by the use of a stimulus which contained a mixture of signals with opposing effects. For example, female rat urine, pooled from all phases of the estrous cycle, can shorten the cycle, but only if the exposed rats have been isolated from rat odors since they were weaned. This is consistent with the present results, since the effect of diestrous odor is greater than that of proestrous odor. If diestrous urine were presented alone, it would be unopposed and therefore would have a more potent effect on the timing of the estrous cycle than a pooled mixture does. Furthermore, the dichotomization of species into those in which ovarian cyclicity is suppressed by female pheromones and those species in which it is enhanced may be an artificial

distinction [2, 21, 28]. These data indicate that both suppressing and enhancing signals may be found within a single species, although the effect of one may predominate in a particular circumstance. These data confirm the appropriateness of a coupled oscillator model of ovarian synchrony within a group of females. They invite further examination of the mechanisms that generate estrous synchrony in the rat, including a determination of the phase response curves for each odor, and a test of the model in the variety of species that also manifest ovarian synchrony [20]. Furthermore, they suggest that it will be possible to determine whether synchrony is the result of mutual entrainment or entrainment to the rhythm of a dominant individual or Zeitgeber within the group [21]. At the individual level, these data also indicate that fertility, that is, the probability of ovulation, can be both increased and decreased by pheromones derived from other females.

ACKNOWLEDGEMENTS I am indebted to T. Larson Butler, R. B. Church, S. Cogswell, C. Hedricks, J. Herman, J. LeFevre, M. Mostert and D. Wolf for data collection and to T. Larson Butler for graphics, This work was supported by NSF BNS 80-19496 and NIA PHS R23AGO2408.

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