Gray Short-tailed Opossum Monodelphis Domestica Research Articles

Growth pattern of the middle ear in the gray short-tailed opossum, Monodelphis domestica

We studied the mass growth trajectories of middle ear ossicles and tympanic membrane and oval window area in 19 specimens of postnatal ages 30–180 days of the gray short-tailed opossum Monodelphis domestica. We weighed the skull mass and the mass of the three middle ear ossicles with appropriate balances. Using a binocular microscope provided with a grid, we measured the length of malleus and incus, as well as the longest axis and the one perpendicular to it on both the tympanic membrane and the stapes footplate. The size variation was studied with least squares regression analyses between various measurements. The incus and stapes change little in mass after 40 days of postnatal life, while the malleus does, reaching maximum mass at around 100 PND (postnatal days). This modularity in growth trajectory is in contrast with the shared evolutionary origin of malleus and incus from branchial arch 1. The maturation of the middle – and as indicated by previous work, that of the inner ear – is coupled with the improvement of hearing sensitivity at low and high frequencies after the initial onset of hearing at 29 PND.

γδ T cells are the predominant T cell type in opossum mammaries during lactation

Milk provides mammalian neonates with nutritional support and passive immunity. This is particularly true in marsupials where young are born highly altricial and lacking many components of a fully functional adaptive immune system. Here we investigated the T cell populations in the mammaries of a lactating marsupial, the gray short-tailed opossum Monodelphis domestica. Immunohistochemistry confirmed the presence of T cells within the opossum mammaries throughout lactation. Results of quantifying transcript abundance for lymphocyte markers are consistent with γδ T cells being the most common T cell type within lactating mammaries. Numbers of γδ T cells appear to peak early during the first postnatal week, and then decline throughout lactation until weaning. In contrast, numbers of αβ T cells and γμ T cells appear to be low to non-existent in the lactating mammaries. The results support an ancient and conserved role of immune cells in the evolution and function of mammalian mammary tissue.

Transcriptomic Changes Associated with Pregnancy in a Marsupial, the Gray Short-Tailed Opossum Monodelphis domestica.

Live birth has emerged as a reproductive strategy many times across vertebrate evolution; however, mammals account for the majority of viviparous vertebrates. Marsupials are a mammalian lineage that last shared a common ancestor with eutherians (placental mammals) over 148 million years ago. Marsupials are noted for giving birth to highly altricial young after a short gestation, and represent humans’ most distant viviparous mammalian relatives. Here we ask what insight can be gained into the evolution of viviparity in mammals specifically and vertebrates in general by analyzing the global uterine transcriptome in a marsupial. Transcriptome analyses were performed using NextGen sequencing of uterine RNA samples from the gray short-tailed opossum, Monodelphis domestica. Samples were collected from late stage pregnant, virgin, and non-pregnant experienced breeders. Three different algorithms were used to determine differential expression, and results were confirmed by quantitative PCR. Over 900 opossum gene transcripts were found to be significantly more abundant in the pregnant uterus than non-pregnant, and over 1400 less so. Most with increased abundance were genes related to metabolism, immune systems processes, and transport. This is the first study to characterize the transcriptomic differences between pregnant, non-pregnant breeders, and virgin marsupial uteruses and helps to establish a set of pregnancy-associated genes in the opossum. These observations allowed for comparative analyses of the differentially transcribed genes with other mammalian and non-mammalian viviparous species, revealing similarities in pregnancy related gene expression over 300 million years of amniote evolution.

The opossum MHC genomic region revisited.

The gray short-tailed opossum Monodelphis domestica is one of the few marsupial species for which a high quality whole genome sequence is available and the major histocompatibility complex (MHC) region has been annotated. Previous analyses revealed only a single locus within the opossum MHC region, designated Modo-UA1, with the features expected for encoding a functionally classical class I α-chain. Nine other class I genes found within the MHC are highly divergent and have features usually associated with non-classical roles. The original annotation, however, was based on an early version of the opossum genome assembly. More recent analyses of allelic variation in individual opossums revealed too many Modo-UA1 sequences per individual to be accounted for by a single MHC class I locus found in the genome assembly. A reanalysis of a later generation assembly, MonDom5, revealed the presence of two additional loci, now designated Modo-UA3 and UA4, in a region that was expanded and more complete than in the earlier assembly. Modo-UA1, UA3, and UA4 are all transcribed, although Modo-UA4 transcripts are rarer. Modo-UA4 is also relatively non-polymorphic. Evidence presented support the accuracy of the later assembly and the existence of three related class I genes in the opossum, making opossums more typical of mammals and most tetrapods by having multiple apparent classical MHC class I loci.

Formation of 5α-reduced androgens in the testes and urogenital tract of the grey short-tailed opossum, Monodelphis domestica

Testicular 5alpha-reduced androgens, largely 5alpha-androstane-3alpha,17beta-diol (androstanediol), are responsible for virilisation of pouch young in one marsupial (the tammar wallaby), but are not formed until later in development in another marsupial (the brushtail possum) and in rodents. Because the mechanism of virilisation of the urogenital tract in the grey short-tailed opossum Monodelphis domestica has never been defined, androgen formation and metabolism were investigated in this species. Testis fragments from grey short-tailed opossums of a wide range of ages were incubated with [3H]-progesterone and the metabolites were separated by high-performance liquid chromatography (HPLC). The only 19-carbon metabolites identified in the youngest ages (5-26 days) and the major metabolites in adult testes were testosterone and androstenedione. At 30, 42 and 49 days of age, dihydrotestosterone and small amounts of androstanediol were present. Time-sequence studies indicated that dihydrotestosterone and androstanediol were formed from the 5alpha-reduction (and 3-keto reduction) of testosterone. In a second series of experiments, tissue fragments of a variety of urogenital tract tissues were incubated with [3H]-testosterone and the metabolites separated by HPLC. During the interval in which male urogenital tract differentiation takes place in this species (between Days 15 and 28), the major metabolite identified was dihydrotestosterone. We conclude that the timing of 5alpha-reductase expression in the testes of the grey short-tailed possum resembles that of rodents and the brushtail possum rather than that of the tammar wallaby and that dihydrotestosterone is probably the intracellular androgen responsible for virilisation of the urogenital tract in this species.

Evolutionary dynamics of transposable elements in the short-tailed opossum Monodelphis domestica

The genome of the gray short-tailed opossum Monodelphis domestica is notable for its large size ( approximately 3.6 Gb). We characterized nearly 500 families of interspersed repeats from the Monodelphis. They cover approximately 52% of the genome, higher than in any other amniotic lineage studied to date, and may account for the unusually large genome size. In comparison to other mammals, Monodelphis is significantly rich in non-LTR retrotransposons from the LINE-1, CR1, and RTE families, with >29% of the genome sequence comprised of copies of these elements. Monodelphis has at least four families of RTE, and we report support for horizontal transfer of this non-LTR retrotransposon. In addition to short interspersed elements (SINEs) mobilized by L1, we found several families of SINEs that appear to use RTE elements for mobilization. In contrast to L1-mobilized SINEs, the RTE-mobilized SINEs in Monodelphis appear to shift from G+C-rich to G+C-low regions with time. Endogenous retroviruses have colonized approximately 10% of the opossum genome. We found that their density is enhanced in centromeric and/or telomeric regions of most Monodelphis chromosomes. We identified 83 new families of ancient repeats that are highly conserved across amniotic lineages, including 14 LINE-derived repeats; and a novel SINE element, MER131, that may have been exapted as a highly conserved functional noncoding RNA, and whose emergence dates back to approximately 300 million years ago. Many of these conserved repeats are also present in human, and are highly over-represented in predicted cis-regulatory modules. Seventy-six of the 83 families are present in chicken in addition to mammals.

The cremasteric neuromuscular complex in male and female grey short-tailed opossums (Monodelphis domestica)

In marsupials, both sexes possess a cremaster muscle, that of the female being attached to the mammary gland and surrounding subcutaneous tissues. In the grey short-tailed opossum, the muscle in both sexes has a distinct proximal origin from the anterior superior iliac spine. The number of muscle fibres is higher in male than in female opossums on average, but the difference is not statistically significant. Retrograde tracing, achieved by injecting B-cholera toxin into the muscle, showed that labelled neurons occupied several spinal cord segments, the bulk being in L2–L4. There was no overall sex difference in the number of labelled neurons. It is concluded that cremaster muscles are present in both sexes of the grey short-tailed opossum Monodelphis domestica , contain approximately equal numbers of fibres, and differ only in their insertion target. Since the motor innervation also shows no dimorphisms in this species, it is concluded that the whole neuromuscular complex is homologous in the two sexes. This is in marked contrast to the situation in adult eutherian mammals where the muscle is possessed by males only, and where the cremasteric nucleus is highly dimorphic in terms of motor neuron numbers.

Organization of the Olfactory and Respiratory Skeleton in the Nose of the Gray Short-Tailed Opossum Monodelphis domestica

The internal nasal skeleton in Monodelphis domestica, the gray short-tailed opossum, primarily supports olfactory and respiratory epithelia, the vomeronasal organ, and the nasal gland. This scaffold is built by the median mesethmoid, and the paired vomer and ethmoid bones. The mesethmoid ossifies within the nasal septum cartilage. The bilateral ethmoid segregates respiratory and olfactory regions, and its geometry offers insight into the functional, developmental, and genomic organization of the nose. It forms through partial coalescence of separate elements known as turbinals, which in Monodelphis comprise the maxilloturbinal, nasoturbinal, five endoturbinals, and two ectoturbinals. Geometry of the ethmoid increases respiratory mucosal surface area by a factor of six and olfactory mucosal surface by nearly an order of magnitude. Respiratory epithelium warms and humidifies inspired air, recovers moisture as air is exhaled, and may help mediate brain temperature. In contrast, the olfactory skeleton functions as a series of small funnels that support growth of new olfactory neurons throughout life. Olfactory mucosa lines the mouth of each funnel, forming blind olfactory recesses known as the ethmoid cells, and neuronal axons are funneled from the epithelium through tiny olfactory foramina in the cribriform plate, into close proximity with target glomeruli in the olfactory bulb of the brain where each axon makes its first synapse. The skeleton may thus mediate topological correspondence between odorant receptor areas in the nose with particular glomeruli in the olfactory bulb, enabling growth throughout life of new olfactory neurons and proper targeting by their axons. The geometric arrangement of odorant receptors suggests that a measure of volatility may be a component in the peripheral olfactory code, and that corresponding glomeruli may function in temporal signal processing. Supporting visualizations for this study are available online at www.DigiMorph.org.

Footfall patterns and interlimb co‐ordination in opossums (Family Didelphidae): evidence for the evolution of diagonal‐sequence walking gaits in primates

Most primates typically use a diagonal‐sequence footfall pattern during walking. This footfall pattern, which is unusual for mammals, is believed to have originated in ancestral primates in association with the use of grasping extremities for movement and foraging on thin, flexible branches. This theory was tested by comparing gait parameters between the grey short‐tailed opossum Monodelphis domestica and the woolly opossum Caluromys philander, two didelphid marsupials that are strongly differentiated in grasping morphology of the extremities and in their reliance on foraging strategies involving thin branches. One hundred and thirty gait cycles were analysed quantitatively from videotapes of subjects moving quadrupedally on a runway and on poles of different diameters (7 and 28 mm). Duty factor (i.e. duration of the stance phase as a percentage of the stride period) for the forelimb and hindlimb, as well as diagonality (i.e. phase relationship between the forelimb and hindlimb cycles), were calculated for each of these symmetrical gait cycles. We found that the highly terrestrial Monodelphis, like most other non‐primate mammals, relies primarily on lateral‐sequence walking gaits on both runway and poles, and has relatively higher forelimb duty factors. Like primates, the highly arboreal Caluromys uses primarily diagonal‐sequence walking gaits on the runway and pole, with relatively higher hindlimb duty factors and diagonality. The fact that the woolly opossum, a marsupial with primate‐like feet that moves and forages mainly on thin branches, uses primarily diagonal‐sequence gaits when walking supports the view that primate gaits evolved to meet the demands of locomotion on narrow supports. This also demonstrates the functional role of a grasping foot, in association with relatively higher hindlimb duty factors, protraction, and substrate reaction forces, in the production of such walking gaits.

The vestibular primary afferents and the vestibulospinal projections in the developing and adult opossum, Monodelphis domestica.

The gray short-tailed opossum Monodelphis domestica is born in a very immature state (eyes and ears closed, budlike hindlimbs, etc.) 14 days post-coitum, but it can locomote with its forelimbs from the mother's genital aperture to a nipple to which it attaches. The forelimb movements allowing this behavior may be the expression of central pattern generators in the spinal cord, but sensory clues must guide it. One such system may be the vestibular system, which senses linear and angular acceleration and has a strong influence on posture and balance at rest and during locomotion in adult animals. Using the neuronal tracer DiI, we have looked at the vestibular primary afferents and the vestibular nuclear projections to the cervical spinal cord in newborn and postnatal opossums, as well as in the adult animal. The projections in the adult opossum conform to those described for other mammals. Fibers of the vestibular portion of the eighth nerve distribute to all four vestibular nuclei and toward the cerebellar primordium on the day of birth. In addition, some of the fibers project to the contralateral vestibular ganglion, a projection that is not found in the adult opossum. Projections from the lateral, medial and inferior vestibular nuclei to the cervical cord are also present in the newborn. Although we cannot exclude the possibility that chemical and/or tactile guidance is used for directing the movements of the newborn opossum, our results support the hypothesis that the vestibular system may be directly involved in the control of these movements. However, not all components of the system are equally developed at birth, and the circuit from the utricle to the lateral vestibular nucleus and from the latter to the cervical cord may be better formed than that from the semicircular canals to the medial and inferior vestibular nuclei to the cervical cord.

Perivitelline space of marsupial oocytes: Extracellular matrix of the unfertilized oocyte and formation of a cortical granule envelope following the cortical reaction

The purpose of this study was to characterize the structure of the vestments surrounding unfertilized and cortical granule-reacted oocytes from a marsupial, the grey short-tailed opossum Monodelphis domestica and to determine if a cortical granule envelope (CGE) forms in the perivitelline space (PVS) following the cortical reaction. Unfertilized oocytes collected from mature ovarian follicles and oviducal oocytes that had undergone a cortical reaction were fixed for electron microscopy in the presence of ruthenium red which stabilizes extracellular matrices (ECM) and facilitates demonstration of a CGE. Unfertilized oocytes were surrounded by a zona pellucida and had a PVS which contained a thick ECM comprised of granules and filaments. This matrix appeared to attach to the oolemma and was structurally similar to matrices reported previously in the PVS of unfertilized oocytes from eutherian mammals and two other marsupials, the Virginia opossum and the fat-tailed dunnart. The cortex of unfertilized oocytes contained cortical granules which were absent in oocytes recovered from the oviducts of mated females. Oviducal oocytes which lacked cortical granules exhibited a new coat within the PVS between the zona pellucida and the tips of the oocyte microvilli. This coat, the CGE, appeared structurally similar to CGEs described previously around fertilized eutherian oocytes. The CGE of the grey short-tailed opossum is approximately 1 microns thick and is made up of numerous small dense granules. The coats of the opossum oocyte are compared to those present around other marsupial and eutherian oocytes.

The oocyte of a new world marsupial, Monodelphis domestica: structure, formation, and function of the enveloping mucoid layer.

Ovulated oocytes of the gray short-tailed opossum Monodelphis domestica are surrounded by a thin zona pellucida and are devoid of a cumulus oophorus. In the ampulla of the oviduct, oocytes acquire a thick mucoid layer composed of concentrically arranged fibrillar material. Exocytosis by the secretory cells of the oviductal epithelium occurs in the region of the oviduct adjacent to the egg. This suggests that the oocyte-zona-mucus layer complex may influence the oviductal epithelium to secrete. During secretion, fibrillar contents of the secretion granules appear to be transformed into membranous material which presumably becomes fibrillar again as it is incorporated into the forming mucoid layer. Spermatozoa (which are known to pair in the cauda epididymis) are found in pairs and with intact acrosomes in the mucoid layer of fertilized eggs. This suggests that spermatozoa of Mondelphis remain paired until they reach the zona pellucida and that the acrosome functions in zona binding and/or penetration.