Effects of chronic blood loss in a marsupial (Monodelphis domestica).

Monodelphis domestica, the gray short-tailed opossum, is used increasingly as an animal model in studies that require repeated blood sampling. Consequently, it is important to establish safe bleeding regimens. We investigated the effects of repeated blood loss on various hematologic values and on different organs in this species. Approximately 2 ml of blood were taken weekly from 20 animals for 13 weeks. The animals were then necropsied; members of an age- and sex-matched control group were bled (2 ml) once and necropsied immediately to obtain baseline data. Ultimately, each animal in the experimental group lost approximately three times its total blood volume. After the first bleeding in the chronically bled group, the red blood cell counts, hemoglobin, and hematocrit values decreased significantly but remained constant thereafter. In another experimental group bled only once, the hematologic values rose to higher than baseline levels after a rest of 2 weeks. Thereafter the values slowly returned to baseline levels. A notable increase in Howell-Jolly bodies occurred in the chronically bled group. Histologically, there was marked erythroid hyperplasia in the bone marrow and extramedullary hematopoiesis in the spleen, but none in the liver. Because there were no obvious detrimental physiologic effects, we conclude that M. domestica is markedly tolerant of chronic blood loss.     

Ontogeny of the thymus gland of a marsupial (Monodelphis domestica).

The gray short-tailed opossum (Monodelphis domestica) has recently been established as a laboratory animal; consequently, its utility as an animal model is enhanced as more of its basic biologic characteristics become defined. To this end, we studied the ontogeny of the thymus gland in M. domestica ranging in age from the day of birth to over three years. In contrast to most Australian marsupials, M. domestica possesses only a thoracic thymus. The thymus is large in neonates and remains large until young adulthood (six months of age). It has a well-defined cortex, medulla and Hassall's corpuscles. At about six months of age, the thymuses of most animals begin to atrophy, the cortex and medulla being replaced by fat. By 28 months of age, about 50% of animals have thymuses that are completely atrophied. Thus, the ontogeny of the thymus of M. domestica appears to be similar to that of eutherian mammals.     

Severely reduced recombination in females of the South American marsupial Monodelphis domestica.

The gray short-tailed opossum, Monodelphis domestica, is a model species for studying the underlying mechanisms that govern recombination. We have identified a new marker, PI (protease inhibitor), in M. domestica and demonstrate its linkage to AK1 (adenylate kinase 1). The rate of recombination in females of this species is approximately one fifth the rate in males, as might be predicted from the difference in chiasma frequency between the two sexes.     

Meiotic sex chromosome inactivation in the marsupial Monodelphis domestica

In eutherian mammals, the X and Y chromosomes undergo meiotic sex chromosome inactivation (MSCI) during spermatogenesis in males. However, following fertilization, both the paternally (Xp) and maternally (Xm) inherited X chromosomes are active in the inner cell mass of the female blastocyst, and then random inactivation of one X chromosome occurs in each cell, leading to a mosaic pattern of X-chromosome activity in adult female tissues. In contrast, marsupial females show a nonrandom pattern of X chromosome activity, with repression of the Xp in all somatic tissues. Here, we show that MSCI also occurs during spermatogenesis in marsupials in a manner similar to, but more stable than that in eutherians. These findings support the suggestion that MSCI may have provided the basis for an early dosage compensation mechanism in mammals based solely on gametogenic events, and that random X-chromosome inactivation during embryogenesis may have evolved subsequently in eutherian mammals.     

Trunk movements during locomotion in the marsupial Monodelphis domestica (didelphidae)

The small didelphid cmarsupial, Monodelphis domestica, uses a lateral sequence walk during slow treadmill locomotion and gradually shifts to a trot as speed increases. At higher speeds it changes abruptly to a half-bound. Cinematographic records suggest significant lateral bending but no sagittal bending of the trunk during the slow walk and a reduced amount of lateral bending during the fast walk. There is slight lteral, but no sagittal, bending during the trot. Sagittal bending is obvious during the half-bound, but no lateral bending is evident. Cineradiography confirms that the vertebral column of the trunk bends laterally during the slow walk. Bending occurs throughout the trunk region, but seems to be most pronounced in the anterior lumbar region. Associated with this bending of the trunk is substantial rotation of the pelvic girdle in the plane of yaw. Pelvic rotation is synchronized with the locomotor cycle of hindlimbs. Each side of the pelvis rotates forward during the recovery phase of the ipsilateral hindlimb and backward during the contact phase of this limb. Information on locomotor trunk movements in other limbed tetrapods is limited. The pattern of trunk bending found in Monodelphis, however, is consistent with that reported in the placental mammal Felis catus and in some lepidosaurian reptiles. This suggests that sagittal bending did not replace lateral bending during the evolution of mammals, as is sometimes suggested. Rather, bending in the vertical plane seems to have been added to lateral bleeding when the ancestors of extant mammals acquired galloping and bounding capabilities.     

Gonadal sex differentiation in the neonatal marsupial, Monodelphis domestica

A quantitative and histological study of the gonads of newborn grey short-tailed opossums, Monodelphis domestica, is described. The pups were karyotyped, and comparisons were made within litters segregating for XX and XY sex chromosomes. A total of four litters including 25 pups were available. On the day of birth, developing testes were significantly larger than the ovaries of litter mates, and testes could be histologically distinguished by the formation of sex cords and a tunica albuginea. The data suggest that in this marsupial species gonadal differentiation may be initiated in utero.     

Heterochrony in somitogenesis rate in a model marsupial,Monodelphis domestica

Marsupial newborns are highly altricial and also show a wide array of shifts in the rate or timing of developmental events so that certain neonatal structures are quite mature. One particularly notable feature is the steep gradient in development along the anterior–posterior axis such that anterior structures are generally well developed relative to posterior ones. Here, we study somitogenesis in the marsupial, Monodelphis domestica, and document two heterochronies that may be important in generating the unusual body plan of the newborn marsupial. First, we demonstrate a 4‐fold change in somitogenesis rate along the anterior–posterior axis, which appears to be due to somitogenesis slowing posteriorly. Second, we show that somitogenesis, particularly in the cervical region, initiates earlier in Monodelphis relative to other developmental events in the embryo. The early initiation of somitogenesis may contribute to the early development of the cervical region and forelimbs. Other elements of somitogenesis appear to be conserved. When compared to mouse, we see similar expression of genes involved in the clock and wavefront, and genes of the Wnt, Notch, and fibroblast growth factor (FGF) pathways also cycle in Monodelphis. Further, we could not discern differences in somite maturation rate along the anterior–posterior axis in Monodelphis, and thus rate of maturation of the somites does not appear to contribute to the steep anterior–posterior gradient.     

piRNA-like RNAs in the marsupial Monodelphis domestica identify transcription clusters and likely marsupial transposon targets

PIWI-interacting RNAs (piRNAs) are a recently discovered class of small noncoding RNAs that have been detected in human, mouse, rat, zebrafish, and Drosophila genomes. We have utilized a size-directed small-RNA cloning procedure to clone and map more than 300 candidate piRNA-like small RNAs in the genome of the marsupial species Monodelphis domestica. Our results are consistent with those from other species in that the piRNA-like candidate sequences range in size from 28 to 31 nucleotides, show a pronounced preference for uridine at the 5′ end, are transcribed from a few large clusters, appear to target transposons, and display virtually no sequence conservation.     

Chicken GnRH II occurs together with mammalian GnRH in a South American species of marsupial (Monodelphis domestica)

Two molecular forms of gonadotropin-releasing hormone (GnRH) were demonstrated in hypothalamic extracts of M. domestica using high performance liquid chromatography and radioimmunoassay with specific GnRH antisera. One form eluted in the same position as synthetic mammalian GnRH and was quantified equally by two mammalian GnRH antisera, while the second form coeluted with synthetic chicken GnRH II and was quantified equally with two chicken GnRH II antisera. The finding of chicken GnRH II in a South American species of marsupial, which has previously been reported in some Australian species of marsupial and in species of Aves, Reptilia, Amphibia, Osteichthyes and Chondrichthyes, supports our hypothesis that this widespread structural variant may represent an early evolved and conserved form of GnRH.     

Absence of a significant mixed lymphocyte reaction in a marsupial (Monodelphis domestica)

Previously we reported preliminary results suggesting that the marsupial Monodelphis domestica fails to exhibit a mixed lymphocyte reaction with allogeneic lymphocytes. To test whether this observation is simply a matter of a response too weak to detect, but capable of being augmented by immunization, we performed mixed lymphocyte culture tests on 23 of these animals that had been immunized with lymphocytes. Despite the fact that all recipients were sensitized to the lymphocytes of the donors, none of the animals had a substantial mixed lymphocyte response. Significant stimulation was noted with the mitogen concanavalin A; thus, the T cells were immunologically competent. It seems likely that the failure of this species to exhibit a significant mixed lymphocyte response is due to T cells whose ontogeny differs from that of the T cells of eutherian mammals.     

Chronic cold exposure increases liver oxidative capacity in the marsupial Monodelphis domestica

Marsupials lack brown adipose tissue, and therefore rely exclusively on other tissues for thermogenesis. To determine the magnitude of phenotypic plasticity of the liver in response to changing metabolic demand, gray short-tailed opossums (M. domestica) were exposed to thermoneutral (28 degrees C) or cold (9-12 degrees C) conditions continuously for 6 weeks. Half of each group was also endurance trained with a treadmill program during their respective temperature exposure. Mass specific summit metabolism (VO(2)summit) increased 11% following cold acclimation, though there was no significant main effect by training on VO(2)summit. To estimate the contribution of the liver to whole animal oxidative activity, we determined liver mass, mitochondrial volume density, and total mitochondrial volume. Relative liver mass was 48% greater in cold-acclimated animals, whereas training had no effect on liver mass. The stereological analysis of hepatocyte ultrastructure suggests the percentage of intracellular volumes remained unchanged in response to either aerobic challenge. Thus, following cold-acclimation, there is a 20% increase in the total mitochondrial volume of the liver. This increase could account for nearly half (44%) of the observed increase in whole animal VO(2)summit following cold exposure.     

The developmental reduction of the marsupial coracoid: A case study in Monodelphis domestica

During their embryogenesis, marsupials develop a unique structure, the shoulder arch, which provides the structural and muscle‐attachment support necessary for the newborn's crawl to the teat. One of the most pronounced and important aspects of the shoulder arch is an enlarged coracoid. After marsupial newborns reach the teat, the shoulder arch is remodeled and the coracoid is reduced to a small process on the scapula. Although an understanding of marsupial coracoid reduction has the potential to provide insights into both, marsupial evolution and the origin of mammals, little is known about the morphological and cellular processes controlling this process. To remedy this situation, this study examined the morphological and cellular mechanisms behind coracoid reduction in the gray short‐tailed opossum, Monodelphis domestica. A quantitative, morphometric study of shoulder girdle development revealed that the coracoid is reduced in size relative to other aspects of the shoulder girdle by growing at a slower rate. Using a series of molecular assays for cell death, no evidence was found for programmed cell death playing a role in the reduction of coracoid size in marsupials (in contrast to hypotheses of previous researchers). Although it is likely the case that coracoid growth is reduced through a relatively lower rate of cellular proliferation, differences in proliferative rates in the coracoid and scapula were not great enough to be quantified using standard molecular assays. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

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.     

A VpreB3 homologue in a marsupial, the gray short-tailed opossum, Monodelphis domestica

A VpreB surrogate light (SL) chain was identified for the first time in a marsupial, the opossum Monodelphis domestica. Comparing the opossum VpreB to homologues from eutherian (placental mammals) and avian species supported the marsupial gene being VpreB3. VpreB3 is a protein that is not known to traffic to the cell surface as part of the pre-B cell receptor. Rather, VpreB3 associates with nascent immunoglobulin chains in the endoplasmic reticulum. Homologues of other known SL chains VpreB1, VpreB2, and λ5, which are found in eutherian mammals, were not found in the opossum genome, nor have they been identified in the genomes of nonmammals. VpreB3 likely evolved from earlier gene duplication, independent of that which generated VpreB1 and VpreB2 in eutherians. The apparent absence of VpreB1, VpreB2, and λ5 in marsupials suggests that an extracellular pre-B cell receptor containing SL chains, as it has been defined in humans and mice, may be unique to eutherian mammals. In contrast, the conservation of VpreB3 in marsupials and its presence in nonmammals is consistent with previous hypotheses that it is playing a more primordial role in B cell development.