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Biomedical subjects

E S Robinson

Publications and source records attributed to E S Robinson.

At least 37 records · Page 2Linked to original sources
PubMed1995

Developmental progression of Gpd expression from the inactive X chromosome of the Virginia opossum.

Metatherian (marsupial) mammals possess a non-random form of X-chromosome inactivation in which the paternally-derived X is always the one inactivated. To examine the progression of X-linked gene expression during metatherian development, we compared relative levels of the maternally and paternally encoded Gpd gene products in heterozygous female Virginia opossums (Didelphis virginiana) across a major portion of the developmental period. Panels of tissues obtained from fetuses, newborns, and pouch young were examined via polyacrylamide gel electrophoresis of the G6PD protein. As in adults, G6PD phenotypes in these developmental stages were highly skewed in favor of the maternal allele product, but in some tissues there was a marked increase in paternal allele expression with advancing developmental age. However, even by 42 days of post-partum development, expression of the paternal Gpd allele had not attained the adult, tissue-specific activity pattern. Our findings indicate remarkable developmental changes in the activity of the paternal allele in several tissues/organs continuing well into mid pouch-life stages and beyond. Specifically we found that 1) a substantially repressed paternal Gpd gene is present in the cells of female stage 29 fetuses and later developmental stages, 2) the activity state of the paternal Gpd gene is not fixed during early embryonic development in this species, 3) major changes in paternal Gpd expression occur in advanced developmental stages and comprise a maturation of the gene expression pattern during ontogeny, and 4) alterations of paternal Gpd allele activity during development occur in a tissue-specific manner.

Alleles↗
PubMed1995

Ultraviolet radiation-induced skin lesions in laboratory opossums (Monodelphis domestica) exposed from the weanling stage.

After repeated exposure to ultraviolet radiation, adult laboratory opossums (Monodelphis domestica) may develop hyperplasia and neoplasia of the shaved skin. We exposed opossums from the weanling stage (8-10 weeks after birth) and compared the incidence of lesions at designated time points with those observed following introduction of adults (around 5 months old) to the same irradiation protocol (125 J/m2 of UVB three times each week for up to 40 weeks). The overall incidence of hyperplasia and neoplasia among opossums exposed from the weanling stage was markedly lower than among animals first exposed as adults, and hyperkeratosis and sarcoma were not observed. Although freckling was widespread, cutaneous melanocytic nevi were rare among animals first exposed as weanlings; however, one animal without freckling developed malignant melanoma with presumptive metastasis to the spleen. The basis of the lowered lesion incidence among weanling-introduced animals is not clear; it may be that cutaneous immunity to ultraviolet radiation damage is more efficient in juveniles than in adults.

Age Factors↗
PubMed1995

UV-induced melanoma. A karyotype with a single translocation is stable after allografting and metastasis.

Metastatic melanoma cell lines were derived from a lymph node of a laboratory opossum, Monodelphis domestica, which had been exposed to mid-wavelength ultraviolet radiation (UVB) initially as a suckling young, and subsequently as a shaved juvenile and adult. The melanoma cell lines were dendritic and pigmented in vitro and contained a t(6;8)(p13;q13) as the only cytogenetic abnormality. The translocation was detected in 15% of primary cultures (passage 2) from the affected lymph node and in 100% of two ring-clone-derived lines, L1 and L2. The breakpoint or resulting partial trisomy of chromosomes 8 may have played a functional role in the tumorigenesis or metastasis of the tumor. The t(6;8) served as a convenient cytogenetic marker for allogeneic grafting studies in Monodelphis. The L2 cells were allografted subcutaneously (s.c.) into genetically diverse suckling young at 3 weeks of age and resulted in the growth of invasive, pigmented, primary and metastatic lesions affecting lymph nodes, lung, and other tissues. Metastatic variant cell lines, M1 and M3, were derived from the affected lungs of two animals and both lines demonstrated the same t(6;8), without additional numerical or structural chromosomal abnormalities. The maintenance of karyotypic stability with a single translocation during in vivo tumor growth and dissemination in this new allografting model is quiet remarkable, as most human metastatic melanomas exhibit multiple structural and numerical cytogenetic abnormalities.

Animals↗
PubMed1995

A new allogeneic model for metastatic melanoma.

Metastatic melanoma cells, clonally derived from an affected lymph node of an ultraviolet-irradiated laboratory opossum, were allografted subcutaneously into suckling young, juveniles and adults to determine their tumorigenicity and metastatic potential. All injected 1- and 3-week-old suckling young survived well beyond weaning at 8 weeks. One died 12 weeks after injection from the effects of rampant metastatic involvement, while the rest were killed 13 to 26 weeks after injection. At necropsy, most animals showed extensive primary tumour growth, many showed metastasis to nodes and/or lungs, and in some there was dissemination to distant sites including liver and spleen. Animals injected as juveniles or adults rejected the allografts. Injection of allogeneic malignant melanoma cells during early postnatal development facilitates successful, long-term allografting and metastasis without concomitant immunosuppressive agents. Developmental lack of self-recognition (immunological immaturity) or induced tolerance may be responsible. This unique model system will be useful for further metastasis studies and may be valuable for investigations of novel antineoplastic therapies.

Animals↗
PubMed1994

Malignant melanoma in ultraviolet irradiated laboratory opossums: initiation in suckling young, metastasis in adults, and xenograft behavior in nude mice.

Litters of suckling young of the laboratory opossum (Monodelphis domestica) were irradiated with UV light from sunlamps with a spectral emission peak at 302 nm (UVB) to induce melanocytic nevi. Total doses of 0.87-5.0 kJ/m2 were divided equally among up to 14 exposures during the 19 days from birth. Of 358 sucklings exposed, 217 survived to weaning, and 22 (10%) possessed a nevus when shaved and examined at or after weaning. Affected animals were then exposed 3 times/week to 125 J/m2 of UVB for up to 45 weeks to promote progression to malignancy. Nevi of 8 of the 20 chronically-exposed animals progressed to malignant melanoma with metastases to lymph node(s). Cell cultures were prepared from affected nodes to confirm that pigmented nodal cells were metastatic melanomas. One established cell line (TD15L) contained highly pigmented, dendritic, malignant melanoma cells. These cells, injected s.c. as xenogeneic grafts into athymic nude mice, remained viable in the subcutis and were moderately tumorigenic in the dermis. UVR exposure of Monodelphis sucklings is a novel, effective, and proficient way of initiating melanocytic lesions for studies on susceptibility and progression to melanoma, and the cell lines derived from these melanomas will provide promising new reagents for chemotherapy and immunotherapy investigations.

Animals↗
PubMed1994

Susceptibility to ultraviolet-induced corneal sarcomas is highly heritable in a laboratory opossum model.

The laboratory opossum, Monodelphis domestica, develops hyperplasia and neoplasia of the corneal stroma after repeated exposure to low doses of ultraviolet radiation (UVR) (non-erythemic in skin). We exposed adult animals from genetically diverse families within our colony to determine whether there are any heritable components to the risk of this form of eye cancer. From about 5 months of age, animals were exposed 3 times a week to a dose of about 125 J/m2 of UVB (spectral peak = 302 nm). Thirty-three sibships (151 individuals) completed at least 30 weeks of the protocol and 137 individuals completed 45 weeks. For genetic analysis, each animal was classified at 30 and 45 weeks as affected with corneal sarcoma or not. Heritabilities were estimated for the dichotomous eye-tumor trait which was highly heritable at both time points. This eye-cancer model system is valuable as a source of material for in vitro studies of angiogenesis and neoplastic transformation, for in vivo studies of tumor therapy and prevention, and for further research on the genetic determinants of cancer.

Animals↗
PubMed1994

Timetable of in vivo embryonic development in the grey short-tailed opossum (Monodelphis domestica).

The timing of development was examined in 496 embryos from female Monodelphis domestica collected at known time intervals after video recorded mating. Ovulation occurred approximately 20 hr (day 1) after mating, and fertilization was observed by 24 hr. Transport through the oviducts was rapid, and pronuclear stage embryos were recovered from the uterus as early as 24 hr after mating. Second cleavage had occurred by 55 hr after mating. Three-celled embryos were among those collected on day 3 after mating, indicating that asynchronous cleavage of blastomeres can occur from the two-cell stage. The four-cell stage persisted for approximately 24 hr, and embryos that had undergone third cleavage were first recovered 74 hr after mating. Embryos that had undergone fourth to fifth cleavage were found 96-100 hr (4 days) after mating and complete unilaminar blastocysts by 5.5 days after mating. Primary endoderm formed from an already distinct embryonic area of the unilaminar blastocyst early on day 7 after mating. Formation of the bilaminar blastocyst was completed rapidly, on day 7 after mating. The primitive streak appeared on day 10 after mating, and organogenesis rapidly ensued on a timetable similar to that reported for Didelphis virginiana (McCrady, 1938). Close contact with the maternal circulation was established on day 11 and by day 12 maternal and embryonic tissues could not be separated without damage. The length of the gestation period from fertilization to birth was approximately 13.5 days. These observations provide the basis for further embryological cellular and molecular studies of this species as a laboratory model for marsupial development.

Animals↗
PubMed1994

Genetic analysis of ultraviolet radiation-induced skin hyperplasia and neoplasia in a laboratory marsupial model (Monodelphis domestica).

Monodelphis domestica, the laboratory opossum, develops hyperplasia and neoplasia of shaved skin after repeated exposure to ultraviolet radiation (UVR). We exposed Monodelphis from genetically diverse families within our colony to determine whether there are any heritable components to the risk of two distinct skin lesion phenotypes-melanocytic nevus (MN) and advanced hyperkeratosis (HK). From about 5 months of age, animals were shaved and exposed three times a week to a dose of about 125 J/m2 of UVR (spectral peak, 302 nm; range, 280-400 nm). Of 33 sibships (151 individuals) that completed at least 30 weeks of the protocol, 137 completed 45 weeks. For genetic analyses, each animal was classified at 30 and 45 weeks as affected with MN and HK or not affected. Heritabilities were estimated using a variance decomposition approach. Susceptibility to MN showed no significant evidence for a genetic component at 30 or 45 weeks. In sharp contrast, susceptibility to HK was under virtually complete genetic control (heritability, 0.999; P < 0.001) at 30 weeks, and had a moderately high heritability (0.702; P < 0.001) at 45 weeks. We conclude that this model has great potential for identifying genes that confer susceptibility to UVR-induced skin lesions and for investigating environmental factors that may contribute to the increasing incidence of skin cancer in human populations.

Animals↗
PubMed1994

HPRT activity in embryos of a South American opossum Monodelphis domestica.

Marsupial females show preferential paternal X-inactivation. However, the time at which X-inactivation occurs in early development has not yet been determined. A double microassay which measures the activities of X-linked hypoxanthine phosphoribosyl transferase (HPRT) and the autosomally-coded adenine phosphoribosyl transferase (APRT) from the same sample was performed on a collection of embryos from a South American opossum Monodelphis domestica. The embryos ranged in age from the 2-cell stage to the bilaminar blastocyst stage. The results indicate that their embryonic HPRT and APRT are not expressed until just before the unilaminar blastocyst stage in M. domestica. This is at a later stage of development than that in the mouse where embryonic HPRT and APRT expression first occurs at the 4-8-cell stage. It is concluded that HPRT is an uniformative enzyme for assessing X chromosome activity in cleaving embryos of M. domestica. The widespread distribution of HPRT:APRT ratios after the unilaminar blastocyst stage also makes it difficult to draw conclusions about the state of X chromosome activity in early marsupial development.

Adenine Phosphoribosyltransferase↗
PubMed1994

X-chromosome replication patterns in adult, newborn and prenatal opossums.

Somatic cells from the opossums Monodelphis domestica and Didelphis virginiana were labelled with 5-bromodeoxyuridine (BrdU), treated with colchicine, stained with acridine orange and examined using fluorescence microscopy. BrdU-incorporated metaphase spreads from females of M. domestica at developmental stages from late bilaminar blastocysts to adults showed replication asynchrony of the two (acrocentric) X chromosomes. The long arm of one X chromosome was the latest replicating region in the entire chromosome complement and is presumed to represent transcriptional inactivation and X dosage compensation. The minute short arm of the same X, which contains a nucleolar organizer region, was earlier replicating and synchronous with the short arm of its homologue and is thus assumed to escape inactivation. BrdU-incorporated spreads from cells of fetuses, neonates and adults of D. virginiana also showed a late replicating (submetacentric) X chromosome. The pattern was different from that of M. domestica because of the different morphology and the presence of large blocks of constitutive heterochromatin in both homologues. The timing and pattern of replication of the single X in males of both species resembled the earlier replicating X in females. The array of molecular techniques now available offers the best means for investigating X-chromosome replication and activity states of X-linked genes in the earliest stages of marsupial embryogenesis.

Animals↗
PubMed1994

Blood collection and surgical procedures for the laboratory opossum (Monodelphis domestica).

Procedures have been developed for collecting blood samples from the laboratory opossum (Monodelphis domestica) and for conducting survival surgery on adults. Blood samples up to 1 ml were collected routinely by cardiac puncture of animals anesthetized by inhalation of methoxyflurane or halothane with minimal risk; however, halothane required more careful monitoring. Surgical procedures such as partial hepatectomy unilateral hysterectomy, and tail wound repair were safely accomplished on animals anesthetized with methoxyflurane. Other surgical procedures including enucleation and intra-abdominal vasectomy were carried out successfully with an inhalation/injection (pentobarbital-atropine-ketamine) protocol that was successful also for recipient surrogate females in embryo transfer experiments.

Anesthesia↗
PubMed1993

Cell lines derived from ultraviolet radiation-induced benign melanocytic nevi in Monodelphis domestica exhibit cytogenetic aneuploidy.

The gray short-tailed opossum, Monodelphis domestica, develops dermal melanocytic nevi (MN) after long-term chronic exposure to UVB (midwavelength ultraviolet radiation) alone. We developed cell lines from six UVB-induced dermal benign melanocytic lesion biopsies. One of the MN was determined histologically to be a benign melanoma (BM), whereas the remainder were benign melanocytic hyperplasias (MH). The cell lines were not tumorigenic when injected subcutaneously into athymic nude mice. Protein extracts prepared from these cell lines were analyzed electrophoretically on polyacrylamide gels and protease zymograms in preliminary attempts to identify protein and protease markers for pathogenesis. Cytogenetic analyses showed that half (three of six) of the MN cell lines exhibited aneuploidy involving extra copies of chromosomes 3, 5, 7, and/or 8. This result suggests that nonrandom aneuploidy can be an early event in chronic UVB induction of benign dermal melanocytic lesions. Karyotyping also showed a centromeric variant of chromosome 7 in some animals, which was confirmed to be constitutional. These Monodelphis cell lines will be valuable reagents for future studies of UVB-induced damage to mammalian skin.

Aneuploidy↗
PubMed1993

The unique stability of the marsupial sperm acrosomal membranes examined by unprotected freeze-thawing and treatment with the detergent Triton X-100.

In this study of the unique stability of the marsupial acrosome, experiments were carried out on the acrosomes of spermatozoa of the tammar wallaby (Macropus eugenii), common brushtail possum (Trichosurus vulpecula) and grey short-tailed opossum (Monodelphis domestica). Light microscopy showed that 4% of opossum and 15% of possum and wallaby spermatozoa lost their acrosomes after freeze-thawing. Electron microscopy revealed that freeze-thawing also induced changes in the acrosomal matrix of some acrosome intact spermatozoa. In both possum and wallaby, freeze-thawing increased the number of spermatozoa with vesiculation of the acrosomal matrix. Freeze-thawing disrupted the plasma membrane of spermatozoa but the acrosomal membranes remained intact. Immediately on addition of high concentrations of TX-100 (0.02% and 0.04%) there was significant loss of acrosomes and motility in possum and wallaby spermatozoa. Lower concentrations of TX-100 (< or = 0.01%) did not affect motility for up to 30 min in all three species, and there was no significant loss of acrosomes. Although loss of acrosomes did not occur under mild detergent treatment, 56% of wallaby and 70% of possum spermatozoa had altered acrosomes after 30 min in 0.01% TX-100. Electron microscopy revealed that acrosomes were undergoing a vesiculation process similar to that seen after freeze-thawing. Often the plasma membrane of detergent-treated spermatozoa was disrupted and had formed plasma membrane vesicles. However, the acrosomal membranes remained intact despite major changes to the acrosomal matrix. The study confirmed the remarkable stability of the marsupial acrosome and suggested that this is probably based in the acrosomal membranes.

Acrosome↗
PubMed1991

Mammary glands in male marsupials. 2. Development of teat primordia in Didelphis virginiana and Monodelphis domestica.

Young and adults of both sexes of two didelphid marsupials, Didelphis virginiana and Monodelphis domestica, were examined externally for evidence of mammary gland development. Female young possessed teat numbers typical of adult females (13-15 in D. virginiana; 11-13 in M. domestica). Male young showed variable teat numbers which were always low compared with females, with the majority possessing 2-4 in anterior positions. Teats were also present in adult males of both species, in similar numbers and locations to those of young males. There are no previous reports of the presence of teats in any adult male marsupials. No mammary primordia in males have been recorded at any stage of development in the most thoroughly studied Australian marsupials. Our findings strengthen the view that there is a dichotomy between the two marsupial lineages in the regulation of male mammary gland expression.

Aging↗
PubMed1990

Mammary glands in male marsupials: I. Primordia in neonatal opossums Didelphis virginiana and Monodelphis domestica.

Neonates of the American didelphid marsupials Didelphis virginiana and Monodelphis domestica were sexed by karyotype and histologically examined on the day of birth. Mammary anlagen were found in both sexes of both species, but the neonatal males had less than one-third of the full female complement of mammary glands. Male neonates of both species also had paired scrotal bulges anterior to the genital tubercle but these were never present in females, once again raising the question of whether the pouch and scrotum are homologous structures. Mammary anlagen are not found in male neonates of the Australian marsupial species so far studied, which suggests a dichotomy in the control of some aspects of sexual differentiation in the two marsupial lineages.

Animals↗
PubMed1988

DNA methylation in the developing marsupial embryo.

Marsupial development differs from early development of placental mammals in that the blastocyst is unilaminar, so that both embryonic and extraembryonic cells are derived from a single layer of cells (protoderm) which faces the blastocyst cavity. Also, all cells in female marsupial conceptuses so far examined show preferential paternal X-inactivation. To test for a possible correlation between cell position, paternal X-inactivation and DNA hypomethylation, marsupial DNA preparations from three regions, embryo, vascular yolk sac and avascular yolk sac, were digested with methyl-specific restriction endonucleases, separated on agarose gels and end-labelled with 32P-dCTP. The size distribution of the fragments obtained indicated three levels of methylation: high methylation of embryonic DNA, intermediate levels of methylation of vascular yolk sac DNA and hypomethylation of avascular yolk sac DNA. The degree of methylation of repeat sequences, observed as discrete bands in end-labelled HpaII digests, was correlated with the overall methylation of tissue DNA. Thus, the difference in methylation in embryonic and extraembryonic DNA was similar to that described for the mouse conceptus, and the outside cell position of marsupial fetal precursor cells did not correlate with hypomethylation. HpaII tiny fragments, which indicate the presence of CpG-rich islands of DNA, were evident in the marsupial digests. In the mouse DNA, these islands are associated with gene transcription and provide one route to cloning of unique gene sequences.

Animals↗