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a Department of Evolution, Ecology, and Organismal Biology, The Ohio State University, Columbus, Ohio 43210
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Estrus can be induced by allowing females to nuzzle [3, 4] male scentmarks, particularly those obtained from a male's suprasternal gland [5]. Nuzzling delivers nonvolatile chemical signals to the vomeronasal organ [3], which contains the peripheral sensory receptors of the accessory olfactory system. Surgical removal of the vomeronasal organ prevents induction of estrus by male scentmarks alone [6]. However, cautery ablation of the vomeronasal organ fails to prevent ovarian activation and ovulation in females that are caged with males [7].
Urogenital sinus (UGS) estrus is evident after 5–10 days of pheromone exposure, and estrous females typically copulate on the second night of pairing [8]. Females will not ovulate unless paired with a male [2], but copulation is not required to induce ovulation in paired females [9]. Lactation lasts for 8 wk, and during the first 2 wk the pups are firmly attached to a teat.
In order to obtain anestrous, reproductively experienced females for ongoing studies of pheromonal induction of estrus and ovulation, we removed litters from primiparous females midway (3–5 wk) through lactation. These postlactational females remained individually caged in a mixed-sex colony for 2–3 wk before the next planned exposure to male pheromone. Unexpectedly, UGS cytology from the first series of females treated in this manner (but isolated from males and male scentmarks) indicated that they were in estrus 7–10 days after removal of nursing young. UGS estrus persisted for 2–5 days, after which females remained anestrous. To further examine postlactational estrus in opossums, we continued to collect UGS cytology and body weight data from dams following early removal or natural weaning of pups. We also collected blood samples to monitor estradiol levels and tested whether postlactational estrus was associated with behavioral receptivity and production of young.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Opossums were housed in individual polycarbonate cages (30 x 30 x 15 cm) in a mixed-sex colony in an American Association for Accreditation of Laboratory Animal Care-approved facility in the Botany and Zoology Building at The Ohio State University. The animal room was maintained on a 14L:10D cycle, and room temperature was 25–28°C; food (Fox Reproduction Food; Milk Specialties Co., New Holstein, WI) and acidified water (pH = 4.2) were provided ad libitum. Males (9–27 mo old) and females (5–23 mo old) were caged individually and held on different shelving racks, separated by at least 2 m. Incidental transfer of pheromones in male scentmarks to females was avoided by handling females and their cages prior to handling males or male cages. Long-term records show that under these conditions of isolation from males, adult females remain anestrous ([1], unpublished results). All experimental procedures were conducted according to an Institutional Laboratory Animal Care and Use Committee-approved protocol.
Pheromonal Exposure, Detection of UGS Estrus, and Mating
Females were exposed to estrus-inducing pheromones by a standardized procedure routinely used in all of our pheromone experiments since 1996 [5]. Scentmarks are first collected by rubbing four sides of a 7-ml glass vial on the suprasternal gland of a breeding male. The vial is then inverted and screwed onto a stand (i.e., a vial cap glued to a plastic Petri dish) and placed daily in each female's cage. Females actively nuzzle the scentmarked vials, particularly during the first 5 min of a 30-min exposure [5]. The onset of estrus is monitored by UGS smears collected every 1–3 days; body weights are recorded when smears are collected. Estrus is indicated by a thick smear of keratinized cells [9, 10]. Females are monitored for 2 wk after initial pheromone exposure or until expression of UGS estrus. This procedure is designed to provide an effective, essential, and discrete estrus-inducing stimulus to females that are also exposed to the ambient sounds and volatile odors of a mixed-sex colony.
At 1300 h on the first day of UGS estrus, pheromone-stimulated or postlactational females were paired with males, and time-lapse video recording monitored sexual behavior and copulation. Males were returned to their own cages after copulation. Female body weight was recorded every 1–3 days during the 15-day gestation, and females were checked 1–2 times per day for newborn litters beginning 12 days following copulation. Females often cannibalize small litters (n < 4), leaving no evidence of parturition, and thus our records provide conservative estimates of reproductive success.
Experimental Groups
After our initial observations of postlactational estrus, females were randomly assigned to either the midlactation group, in which pups were removed from dams midway through lactation (3–5 wk postpartum, n = 23 litters), or to the weaned group, in which young were weaned at the natural time, 55–61 days postpartum (n = 23 litters). Females in both groups were monitored by UGS cytology following separation from their young. All midlactation pups and 8 weaned litters were killed; the remaining weaned pups were incorporated into the colony. The midlactation and weaned groups consisted of 36 dams nursing either their first (midlactation = 19, weaned = 11) or second (midlactation = 4, weaned = 12) litter. Ten females were used twice, once in each group. Analysis of changes in body mass and time to estrus within each postlactation group revealed no differences (P > 0.05, t-test) between females used once and females used twice, or between first and second litters, so these data were combined for analysis. We continued to collect smears from all females twice a week for at least 3 wk after postlactational metestrus to confirm that females remained anestrous.
Twenty-two primiparous, postlactational, anestrous females were then assigned to the pheromone exposure group, given the standard pheromone exposure, and mated at UGS estrus. Six females from the weaned group exhibiting postlactational, UGS estrus were also paired with males. Reproductive behavior, copulation, and birth of young in these 6 females were documented as for all females in the pheromone exposure group.
Blood Sampling and RIA
Blood samples (200–600 µl) were collected from a lateral tail vein of dams 1–24 h before removal of pups at midlactation (n = 9) or weaning (n = 10), at least once 3–10 days after separation from pups, and during UGS estrus. Blood was also collected from females in the pheromone exposure group prior to initial exposure and on the first day of UGS estrus (n = 19). Estradiol (E) was extracted with diethyl ether from 25–100 µl of plasma. Concentrations of E were measured using an RIA previously validated for opossums [11]. Assay sensitivity is 2 pg/200 µl of plasma. Intra- and interassay coefficients of variation averaged 6.9% and 13.7%, respectively. Concentrations of E measured in plasma samples were corrected for plasma blank, but not for procedural losses (12.6%). All samples were assayed in duplicate except for 7 single samples wherein plasma volume and expected E concentrations were limiting.
Mean values are expressed ± SEM. Differences in body mass change and mean E levels within a group were analyzed with a paired t-test and a repeated-measures ANOVA, respectively. Differences among groups were tested with a Kruskal-Wallis ANOVA or a Tukey's multiple comparisons test [12].
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RESULTS |
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Reproductive activation, as evidenced in elevated plasma E, was observed in all three groups (Table 1) with no significant differences in E concentration at UGS estrus. In the midlactation group, E increased from a low of 7.1 ± 1.7 pg/ml at pup removal to 24.5 ± 2.6 pg/ml at estrus (Fig. 1). The increase in E between weaning and UGS estrus was not significant (Fig. 1), and mean E levels at weaning and during anestrus did not differ from a mean of 15.3 ± 3.13 pg/ml measured in samples collected from anestrous females just prior to pheromone exposure. Plasma E concentrations were correlated (P < 0.05) with days from estrus in the midlactation group (Fig. 2A) but not in the weaned group (Fig. 2B). Body mass of the pheromone exposure females increased 6.0% from the day of first exposure to UGS estrus (P < 0.05). Females in the two postlactational groups did not exhibit this increase in mass at estrus, and in fact both groups showed a decrease (P < 0.05) relative to pup removal or weaning.
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The mating pattern and fertility of 6 females in postlactational estrus were similar to those of primiparous females following postlactational anestrus and subsequent pheromonal induction of estrus. Five of six females in postlactational estrus copulated within 2 nights of pairing compared to 16 of 19 in the pheromone exposure group. Three of five postlactational females produced and nursed litters (7.0 ± 1.9 young per litter) compared to 12 of 13 females (8.7 ± 1.1 young per litter) that mated after pheromonal induction of estrus (Table 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Heretofore, direct exposure to males or male scentmarks were the only reliable stimuli known for induction of estrus in adult females. It now appears that opossums are similar to other mammals such as the pig [13] and the domestic cat [14, 15], which exhibit postlactational estrus following gestational and lactational anestrus. Prairie voles (Microtus ochrogaster) resemble opossums in that females lack an estrous cycle and are reliant on male stimuli for ovarian activation, except after birth of a litter [16]. However, voles differ from opossums in that spontaneous estrus in voles is expressed immediately postpartum, but with a shorter period of receptivity relative to that seen during male-induced estrus [17].
Although the brief gestation of marsupials does not interrupt the estrous cycle as in eutherian mammals [18, 19], there is no evidence of postpartum estrus and conception in didelphid marsupials, including the Virginia opossum (Didelphis virginiana). Virginia opossums show postlactational estrus and mate within 2–15 days of removal of young [20–22] or at 97 days postpartum, just prior to natural weaning (96–104 days postpartum) [23].
The differences noted between midlactation and weaned groups might be related to the fact that young opossums (M. domestica) feed on solid food from 6 wk of age [24, 25] and dams probably experience a gradual reduction in suckling stimulus during the final 2 wk of lactation. By contrast, young were removed from dams in the midlactation group at 3–5 wk of age when demands on maternal energy are increasing [25] and young are nursing continuously, except when intermittently left in the nest. This apparent difference in suckling stimulus between the 2 groups might explain the longer interval from pup removal to estrus in the midlactation group. Also, the positive correlation between E levels and days to estrus in the midlactational group probably reflects greater similarity in the physiological status of females associated with the abrupt cessation of suckling at pup removal compared to the seemingly variable and gradual reduction in this stimulus in the weaned group. Plasma E levels were greater than 10 pg/ml in 7 of 10 blood samples collected from females prior to weaning but in only 3 of 9 samples collected prior to pup removal at midlactation, a difference reflected in the higher mean E levels of females at weaning (Fig. 1).
The mean E levels at UGS estrus determined in this study are lower than the mean (± SEM) of 47.9 (± 10.6) pg/ml reported for female opossums after 5 days of exposure to male-soiled cages [26], and they might reflect proestrous rather than maximal, preovulatory levels of E. Nonetheless, E levels in midlactation and pheromone exposure females increased significantly between pup removal or pheromone exposure and UGS estrus, and females in both the weaned and pheromone exposure groups mated and ovulated (as confirmed by birth of a litter). Thus, the levels reported here are indicative of ovarian activation culminating in ovulation.
The reduction in body mass in females at postlactational estrus was unexpected, because opossums consistently show a 4–6% increase in body mass with pheromonal induction of estrus, an apparent estrogenic effect of ovarian activation [6, 25, 27]. The most plausible explanation for the observed weight loss between weaning (or pup removal) and postlactational estrus is involution of mammary tissue and a loss of body water. Dams at weaning have higher body water content and consequently weigh 23% more than nonreproductive control females [28]. Thus, even if dams lose only half of this lactational body water between weaning and postlactational estrus, the reduction in mass would exceed or mask the expected increase due to estrogenic water retention at estrus.
Pheromones could influence the onset of estrus in postlactational females, and we cannot rule out this possibility since females in this study were exposed to volatile odors from both males and females. A study by Pelengaris et al. [7] indicates that volatile odor cues detected by the main olfactory system affect reproductive function in female opossums. These researchers demonstrated that ablation of the main olfactory epithelium by irrigation with zinc sulfate severely impaired sexual behavior and ovulation in females tested with unfamiliar males. While airborne odors are not sufficient to induce estrus in non-postlactational females, the reproductive axis of postlactational females might be more sensitive to volatile pheromones, perhaps due to recent removal of an inhibitory suckling stimulus.
Relatively little is known of the reproductive ecology of the gray short-tailed opossum in its native habitat [29, 30], and nothing is known of the role of olfactory communication therein. It now appears that after an initial male-induced estrus at puberty and subsequent conception, pregnancy, and lactation, females would experience spontaneous estrus and produce a second litter shortly after weaning their first litter. Thus, adult females living under favorable environmental conditions in the wild are probably pregnant, lactating, or experiencing postlactational induction of estrus. Limited evidence from the field supports this notion, as pregnant opossums (M. domestica) were collected in Brazil during all months of the year, although at a lower frequency during the driest months of July through November [30]. If females wean or lose their litters during times of poor environmental conditions and suffer a decline in physical condition, they might experience postlactational anestrus. Females might then disperse to better habitat or await the return of precipitation and improved food resources sufficient to restore breeding condition. Under such conditions, anestrous females would probably require access to male scentmarks for nuzzling and induction of estrus.
Perhaps the most intriguing aspect of our recent findings is that the opossum hypothalamo-hypophyseal-gonadal axis is regulated by at least two exogenous stimuli. Male pheromones activate female reproduction, as does the removal of suckling young. The peripheral and central sensory pathways associated with chemical versus mechanical signals must be quite distinct, yet each ultimately affects gonadotrophic hormone secretion and produces comparable ovarian and behavioral responses.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NSF grant IBN9616588 and The Ohio State University. 
2 Correspondence: John D. Harder, Department of Evolution, Ecology, and Organismal Biology, The Ohio State University, 1735 Neil Ave., Columbus, OH 43210. FAX: 614 292 2030; harder.2{at}osu.edu
Accepted: January 19, 2000.
Received: December 16, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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