Partner preference of intact and ovariectomized female gray short-tailed opossums (Monodelphis domestica).

Intact, ovariectomized and ovariectomized estradiol (E)-treated female gray short-tailed opossums were placed in a test situation in which they could choose between an intact and a castrated male. Intact females chose to visit intact males first and visited them more frequently and spent more time with intact than with castrated males. Ovariectomized (OVX) females did not show this preference for visiting intact males over castrates. When compared to OVX females with blank implants, OVX females with E implants spent less time with castrated males. Like intact females, OVX and OVX-E-treated females preferred to stay in close proximity to but not actually in the cage of intact rather than castrated males. To our knowledge, this is the first experimental study of partner preference and its relationship to hormonal condition in a female marsupial.

Urinary progesterone in free-ranging red howler monkeys (Alouatta seniculus): preliminary observations of the estrous cycle and gestation.

The goals of this study were to develop and validate a radioimmunoassay (RIA) for measurement of unconjugated progesterone (P) concentrations in the urine of red howler monkeys (Alouatta seniculus) and to use urinary P profiles to characterize the reproductive cycle of this species. Analysis of P profiles from two females provided a preliminary estimate of the length of the estrous cycle (mean days +/- S.E.M. = 29.5 +/- 1.5; n = 2), and indicated that one female red howler copulated throughout two apparent estrous cycles. Urinary P concentrations during two confirmed pregnancies (211.8 +/- 29.7 ng P/ml) were higher (P < 0.05) than during the luteal phase (77.4 +/- 10.6 ng P/ml; n = 4) of the cycle.

Serodiagnostic potential of culture filtrate antigens of Mycobacterium tuberculosis.

Our studies of the humoral responses of tuberculosis (TB) patients have defined the repertoire of culture filtrate antigens of Mycobacterium tuberculosis that are recognized by antibodies from cavitary and noncavitary TB patients and demonstrated that the profile of antigens recognized changes with disease progression (K. Samanich et al., J. Infect. Dis. 178:1534-1538, 1998). We have identified several antigens with strong serodiagnostic potential. In the present study we have evaluated the reactivity of cohorts of human immunodeficiency virus (HIV)-negative, smear-positive; HIV-negative, smear-negative; and HIV-infected TB patients, with three of the candidate antigens, an 88-kDa protein, antigen (Ag) 85C, and MPT32, and compared the reactivity of the same patient cohort with the 38-kDa antigen and Ag 85A. We have also compared the reactivity of native Ag 85C and MPT32 with their recombinant counterparts. The evaluation of the reactivity was done by a modified enzyme-linked immunosorbent assay described earlier (S. Laal et al., Clin. Diag. Lab. Immunol. 4:49-56, 1997), in which all sera are preadsorbed against Escherichia coli lysates to reduce the levels of cross-reactive antibodies. Our results demonstrate that (i) antigens identified on the basis of their reactivity with TB patients' sera provide high sensitivities for serodiagnosis, (ii) recombinant Ag 85C and MPT32, expressed in E. coli, show reduced reactivity with human TB sera, and (iii) of the panel of antigens tested, the 88-kDa protein is the most promising candidate for serodiagnosis of TB in HIV-infected individuals. Moreover, these results reaffirm that both the extent of the disease and the bacterial load may play a role in determining the antigen profile recognized by antibodies.

Evidence for spontaneous postlactational estrus in gray short-tailed opossums (Monodelphis domestica).

Previous studies of the gray short-tailed opossum have shown that ovarian activity and estrus are induced by male pheromones, but we recently documented urogenital sinus (UGS) estrus in postlactational females despite their isolation from the male stimuli known to be associated with induced estrus. Body weights and UGS smears were collected after removal of pups in midlactation (19-37 days postpartum), after weaning (55-61 days postpartum), or after pheromone exposure. Estradiol was measured by RIA in plasma samples collected from dams during lactation, after separation from pups, and at estrus. Average days to UGS estrus from pup removal or initial pheromone exposure differed (P<0.05) only between the midlactation and pheromone exposure groups. Postlactational females showed a decrease in body weight from the time of pup removal or weaning to estrus, which contrasts with the increase seen in pheromonally stimulated females. Plasma estradiol was elevated at estrus in all groups, and females that were paired with males at postlactational estrus mated and produced litters. This study demonstrates that gray short-tailed opossums consistently experience estrus within 2 wk of weaning their young and that postlactational estrus appears to be hormonally and behaviorally equivalent to estrus induced by direct exposure to male pheromones.

Energetics of offspring production: a comparison of a marsupial (Monodelphis domestica) and a eutherian (Mesocricetus auratus).

This study compares the energetic cost of reproduction during gestation and lactation of a eutherian, the golden hamster (Mesocricetus auratus), and a similar-sized (60,120 g) marsupial, the gray short-tailed opossum (Monodelphis domestica). Food consumption was monitored in 20 reproductively active (RA) opossums and 16 RA hamsters from conception to weaning at equivalent intervals in 19 non-reproductive (NR) opossums and 21 NR hamsters, all maintained within their zone of thermoneutrality (30 degrees C). Total energy assimilated from conception to weaning [opossums: 1261.3 +/- 28.0 Kcal (1 Kcal = 4.1868 J) and hamsters: 1647.5 +/- 60.6 Kcal] was positively correlated with litter size and mass per young in both species. Maternal mass-specific assimilated energy was significantly greater in hamsters than in opossums during gestation (P < 0.001), but not during lactation or from conception to weaning (P > 0.05). Efficiency of offspring production (energy stored in young/incremental energy in RA females) was higher in hamsters than in opossums and, in both species, it was higher during lactation than in gestation. The energetic cost of reproduction (per young per day) was higher in hamsters than in opossums. The marsupial mode of reproduction, as seen in opossums, yields young at lower cost but requires a longer reproductive period than is the case for a similar-sized eutherian.

Vomeronasal organ removal blocks pheromonal induction of estrus in gray short-tailed opossums (Monodelphis domestica).

Estrus is induced in female gray short-tailed opossums (Monodelphis domestica) by exposure to male chemical signals (pheromones). Isolated females remain anestrous, but direct exposure to a male or his scent-marked cage induces estrus within 4-6 days. The objective of this study was to investigate the importance of the vomeronasal organ in detection of and response to estrus-inducing pheromones. The vomeronasal organ was surgically removed through the palate from 8 females (VNX); 5 females (SHAM) underwent sham surgeries in which the vomeronasal organ was exposed but not removed. After a 10-day recovery period, females were placed into male scent-marked cages. Body weight and urogenital sinus cytology were monitored throughout the experiment. All females were anesthetized and perfused with 4% paraformaldehyde 12-13 days after initial pheromone exposure. Vomeronasal organ ablation was evaluated histologically in decalcified snouts. In addition, deafferentation of the accessory olfactory bulb was confirmed by use of a lectin stain specific for the vomeronasal nerve and the glomerular layer of the bulb. All females classified as completely VNX (n = 5) remained anestrous throughout the pheromonal exposure. Incompletely VNX females (n = 2) and all SHAM animals exhibited estrus within 7 days of pheromone stimulation. At perfusion, the mean uterine weight (280.71 +/- 95.6 mg/85 g BW) of SHAM females was greater (p < 0.05) than that of unresponsive, VNX females (133.33 +/- 31.14 mg/85 g). This study demonstrates that the vomeronasal organ is an essential component for transduction of male pheromones required for induction of estrus in a marsupial species.

Gestation and placentation in two New World opossums: Didelphis virginiana and Monodelphis domestica.

Estrus, gestation, and placentation were studied in the Virginia opossum (Didelphis virginiana) and the gray short-tailed oppossum (Monodelphis domestica), representatives of the prototypic, New World marsupial family Didelphidae. Didelphis is a large (3,000 g), pouched opossum that exhibits a 28-day estrous cycle, spontaneous estrus and ovulation, and a high ovulation rate (60 ova per cycle). Monodelphis is a small (100 g), pouchless opossum with male-induced estrus and ovulation and a moderate ovulation rate (12 ova per cycle). Implantation occurs on day 10 of the 13-day gestation period in Didelphis and on day 12 of the 15-day gestation in Monodelphis. Gestation and placentation, including the ultrastructural features of trophoblastic apposition to the uterine mucosa, are very similar in Didelphis and Monodelphis. Differences, such as a larger increase in uterine volume and a more highly folded endometrium following implantation in Monodelphis are minor. They appear to be related to the smaller maternal body size and the relatively larger uterine load of embryos in Monodelphis. Because body size is one of the major variables among New World marsupials, the descriptions of gestation and placentation presented here for Didelphis and Monodelphis, provide a basis for comparison in future studies of didelphid marsupials.

Estrogen in peripheral plasma during postnatal development in gray short-tailed opossums.

Plasma samples obtained from gray short-tailed opossums (Monodelphis domestica) at selected ages through adulthood were assayed for estrogen (E). Levels of E in one mixed-sex plasma pool of animals aged postnatal day (pd) 4 and one of two mixed-sex plasma pools of animals aged pd 8 were over 300 pg/ml. On pd 16, E levels in males and females averaged 30 and 47 pg/ml, respectively. While no significant sex differences in E levels were seen on pd 30 or pd 60, mean E levels for animals on pd 30 were significantly higher (275 pg/ml in males and 181 pg/ml in females) than on pd 60 (78 pg/ml in males and 85 pg/ml in females) or pd 145 (adults). In adult animals, estrogen levels in females averaged 54 pg/ml; all adult male E levels were below the limit of sensitivity of the assay. Maternal E levels, which did not vary significantly by age of litter, averaged 10 pg/ml overall. These findings are discussed with respect to possible significance of high E levels in developing marsupials for sexual differentiation and general brain development.

Sexual maturation in female gray short-tailed opossums, Monodelphis domestica, is dependent upon male stimuli.

The development of physiological responsiveness to male stimuli in peripubertal female gray short-tailed opossums was examined in this study. Prepubertal females were exposed directly or indirectly to male chemicals (odors) or to no male stimuli. All females exposed directly to deposited male stimuli (22/22) showed estrus at a mean (+/- SEM) age of 127 +/- 2 days. Only 59% (13/22) of females exposed indirectly showed estrus, and at an older mean age of 162 +/- 5 days (p less than 0.01). Vaginal estrus was not observed in any of the females isolated from male stimuli (0/11). Direct exposure of prepubertal females to male odors was associated with higher uterine weights compared to those of indirectly exposed and isolated females. Reproductive success, measured as litter production, of peripubertal females greater than 140 days old was comparable to adult reproductive success. This study has shown that reproductive maturation in pubertal female opossums requires male stimuli, that this stimulus appears to be pheromonal, and that direct exposure to deposited male odors is the most effective stimulus for female reproductive maturation.

Evidence for high levels of androgen in peripheral plasma during postnatal development in a marsupial: the gray short-tailed opossum (Monodelphis domestica).

Plasma samples were assayed for androgen in gray short-tailed opossums (Monodelphis domestica) on the day of birth and at selected ages through adulthood. Levels of androgen in mixed-sex plasma pools of animals 4 and 8 days of age were higher than in either sex at all other ages examined. At postnatal Days 16, 30, and 60 (weaning), levels of androgen were equivalent in males and females and as high as in adult males. In both sexes, androgen levels were lower at postnatal Day 84 (juveniles) than at younger ages; after puberty, levels were significantly higher in males than in females. These findings are discussed with respect to similarities and differences between marsupials and eutherians in hormonal environment during the perinatal period and with respect to the possible role of androgens in sexual differentiation of the gray opossum brain.

Prostaglandin F in reproductive tissues collected during the luteal phase of the estrous cycle and at parturition in Virginia opossums (Didelphis virginiana ).

Prostaglandin F(2alpha) (PGF(2alpha)) plays a role in the regression of the corpus luteum (CL) in a number of placental mammals. However, the mechanism of luteal regression has not been extensively studied in marsupials. The objectives of this study were to characterize changes in concentrations of PGF(2alpha) within utero-ovarian (UO) tissue/venous plasma during the luteal phase of the estrous cycle in Virginia opossums, to correlate these changes with those of plasma progesterone (P(4)), and to characterize the peripheral pattern of 13,14-dihydro-15-keto-PGF(2alpha) (PGFM) in parturient opossums. Ovaries, uteri, UO venous plasma and peripheral plasma were collected on Days 5, 9 and 12 after induced ovulation (n = 3 to 4 opossums/group). In addition, concentrations of PGFM were measured in peripheral plasma collected from two opossums during late gestation (Days 7,9,11 and 12) and at parturition (Day 13). Concentrations of P(4), PGFM and PGF(2alpha) in tissue homogenates and plasma samples were estimated by radioimmunoassay. In nonpregnant opossums, peripheral P(4) levels were highest on Day 5 (38.8 +/- 11.1 ng/ml, x +/- SEM) declined on Day 9 (22.6 +/- 7.4 ng/ml), and were at basal levels by Day 12 (2.4 +/- 0.7 ng/ml). Endometrial concentrations of PGF(2alpha) increased (P = 0.056) from Day 5 (15.7 +/- 4.1 ng/g) to Day 9 (92.1 +/- 61.0 ng/g) and were maintained to Day 12 (97.2 +/- 25.7 ng/g). Prostaglandin F(2alpha) concentrations in UO plasma increased (P < 0.01) from Day 5 (143.1 +/- 32.7 pg/ml) to Day 12 (333.0 +/- 32.4 pg/ml). Prostaglandin F(2alpha) concentrations in ovarian tissue followed a similar pattern and were correlated with UO concentrations (r = 0.708, P < 0.05). In pregnant opossums, the highest levels of peripheral PGFM were recorded in the peripartum period, when luteal regression would also be expected to occur. The negative temporal relationship between peripheral concentrations of P(4) and concentrations of PGF(2alpha) in UO tissue/venous plasma observed in this preliminary study is consistent with the notion that PGF(2alpha) from the ovary and/or uterus may play a role in CL regression in the opossum.

Seasonal energetics of opossums (Didelphis virginiana) in Ohio.

1. Female opossums acclimatized to each of the four seasons were not significantly different (P greater than 0.05) in oxygen consumption at a Ta of 0 degrees C; the overall average was 0.66 +/- 0.1 ml O2/g-hr. 2. Body weight decreased by 27% from autumn to spring; pelage density increased in winter, but thermal conductance changed very little between seasons. 3. Respirometry revealed no evidence of thermoregulatory adjustments in opossums acclimatized to winter in Ohio.

Effects of removal in late pregnancy of the corpus luteum, graafian follicle or ovaries on plasma progesterone, oestradiol, LH, parturition and post-partum oestrus in the tammar wallaby, Macropus eugenii.

Concentrations of oestradiol-17 beta, progesterone, and luteinizing hormone (LH) were measured in plasma collected at 6- to 12-h intervals from tammars around the time of parturition and post-partum oestrus. Parturition occurred on Day 26 or 27 after reactivation of lactation-delayed pregnancy and coincided with a precipitous decline in progesterone levels. A sharp rise in oestradiol, from basal concentrations of less than 10 pg/ml to a peak of 13 to 32 pg/ml, as well as oestrus, followed the drop in progesterone by 8.3 and 9.8 h, respectively. The LH surge was dependent on the oestradiol rise and followed it by 7 h. Ovulation followed mating by about 30 h and the LH surge by 24 h. Removal of the ovary with the large Graafian follicle prevented the oestradiol rise, oestrus and the LH surge, but not parturition. Peripartum changes in peripheral oestradiol do not appear to be involved in initiation of parturition but the oestradiol rise and associated change in the oestradiol:progesterone ratio are important signals for post-partum oestrus and the LH surge.

Oestradiol in follicular fluid and in utero-ovarian venous and peripheral plasma during parturition and postpartum oestrus in the tammar, Macropus eugenii.

Oestradiol-17 beta was measured in plasma and follicular fluid by a radioimmunoassay validated for the tammar. Plasma was sampled from the peripheral circulation and both utero-ovarian veins to assess the contribution of oestradiol from the corpus luteum in one ovary and from the Graafian follicle in the other during late pregnancy, parturition, post-partum oestrus and ovulation. Concentrations of oestradiol in the peripheral plasma and in the vein draining the CL remained less than 5 pg/ml on all days except the day of parturition, when they were 12.7 pg/ml and 14.3 pg/ml respectively. Mean oestradiol concentration in the vein draining the Graafian follicle was significantly higher than the mean peripheral concentration on Days 24-27. The highest average concentration (36.5 pg/ml) occurred on Day 27 coincident with parturition and before oestrus. These latter concentrations were significantly correlated with the concentration and total oestradiol in the fluid of the preovulatory Graafian follicle (r = 0.73 and r = 0.82 respectively), thus providing strong evidence that the Graafian follicle is the main source of oestradiol in the peripheral circulation at this time.

Luteal and follicular populations in the ovary of the opossum (Didelphis virginiana) after ovulation.

The maximum diameters of all luteal and antral follicular structures were measured in opossum ovaries on Days 3, 7 and 11 after oestrus, and follicles were classed as developing or atretic. Ovarian weights and luteal diameters were equivalent in comparisons of pregnant and non-pregnant animals on each day. The number of CL (range 45-85) per animal per cycle indicated a very high ovulation rate for a mammal. Luteinized follicles (1-4 per ovary) were identified in all Day-3 ovaries. Ovarian weight, luteal diameter and follicular diameter were greater on Day 7 than on Days 3 or 11. More antral follicles occurred on Day 11 (120 +/- 10.7, s.e.m.) than Day 3 (77.8 +/- 8.8), although the percentage of atretic antral follicles also increased from 20% to 50 and 57% on Days 7 and 11. These increases were not accompanied by an increase in the number of developing antral follicles (58.1 +/- 4.0), thus indicating a mid-luteal increase in the rate of follicular recruitment, of growth and of atresia in the opossum.

Husbandry of a small breeding colony of opossums (Didelphis virginiana).

A small breeding colony of opossums was established in a semi-enclosed structure. Minor modifications of pre-existing pens permitted efficient and productive management of the opossums. Dry dog food was provided, and large amounts of shredded paper was used for nesting material. Vaginal smears were taken from opossums that were restrained only by their tail, a procedure that allowed one person to obtain and interpret vaginal smears from 20 females in less than 2 hours per day. Pregnancy resulted from 90% of the copulations when the females were mated at proestrus. This productive breeding colony was established quickly by obtaining lactating females from the wild in late winter and early spring.

Uterine histology and reproductive cycles in pregnant and non-pregnant opossums, Didelphis virginiana.

Uteri (N = 18) were collected on Days 3, 7, and 11 of the oestrous cycle or gestation. Reproductive cycles were monitored by vaginal cytology in captive opossums. There was no difference in length of cycles between reproductive states (29.2 +/- 1.0 (s.e.m.) days). All uteri on Day 3 or Day 7 were similar in total volume, endometrial width, and glandular abundance. However, on Day 11 endometrial widths were significantly less in pregnant animals and stroma appeared diffuse and flocculent in the non-pregnant uteri. The chorio-vitelline membrane induced no change in the size, conformation, or structure of the uterine epithelium. In some areas, trophoblast cells or their cellular fragments were attached to the epithelium, indicating that the embryonic-maternal relationship might entail adhesion or interdigitation as well as apposition. A mucopolysaccharide layer also characterized this interface of heterologous tissues.

Estradiol and progesterone profiles indicate a lack of endocrine recognition of pregnancy in the opossum.

Concentrations of estradiol and progesterone in blood collected during the 12.5-day gestation period of the Virginia opossum were not significantly different from those during equivalent days of the estrous cycle. Progesterone was correlated with an index of corpora luteral mass. Ratio of estradiol to progesterone were highest 3 to 4 days before estrus and on the day of parturition.