Metabolism and defluorination of fluoroacetate in the brush-tailed possum (Trichosurus vulpecula).

The brush-tailed possum (T. vulpecula) from Western Australia was found to be nearly 150 times more resistant to fluoroacetate intoxication in vivo than the same species from South Australia. Acetone powder preparations from the liver of animals from both populations showed similar abilities to convert fluoroacetate into fluorocitrate. Aconitate hydratase activity in liver preparations from both Western Australian and South Australian animals was similarly and competitively inhibited by fluorocitrate. Both animals were capable of defluorinating fluoroacetate at similar rates by a glutathione-dependent enzymic mechanism resulting in the formation of free fluoride ion and S-carboxymethylcysteine. Glutathione was also capable of partial protection against the toxic effects of fluoroacetate in vitro by a further unelucidated mechanism.     

Putative osmolytes in the kidney of the Australian brush-tailed possum,Trichosurus vulpecula

The Australian brush-tailed possum, Trichosurus vulpecula, is capable of producing a moderately concentrated urine, at least up to 1300 mOsm l(-1). Kidneys of adult animals fed in captivity on a normal diet with ready access to water were analysed. The inner medullary regions were found to have moderately high concentrations of sodium (outer medulla, 367+/-37; inner medulla 975+/-93 mmol kg(-1) dry wt.), chloride (outer medulla 240+/-21; inner medulla 701+/-23 mmol kg(-1) dry wt.) and urea (outer medulla, 252+/-62; inner medulla, 714+/-69 mmol kg(-1) protein). When the animals were fed on a 'wet diet', amounts of these substances in the outer medulla and cortex were reduced, although with the exception of urea these changes were not significant. There were highly significant changes in amounts of Na(+), Cl(-) and urea in the inner medulla (Na(+), 566+/-7; Cl(-), 422+/-9 mmol kg(-1) dry wt.; urea 393+/-84 mmol kg(-1) protein). Likewise, the inner medulla of animals fed a 'dry diet' with limited access to water showed highly significant increases in the same substances (Na(+), 1213+/-167; Cl(-), 974+/-137 mmol kg(-1) dry wt.; urea, 1672+/-98 mmol kg(-1) protein). Inositol was found in the outer medulla (224+/-90 mmol kg(-1) protein) and inner medulla (282 mmol kg(-1) protein) as was sorbitol (outer medulla, 62+/-20; inner medulla, 274+/-72 mmol kg(-1) protein). Both these polyols were reduced in amount in renal tissue from 'wet diet' animals, and increased in 'dry diet' animals, but the changes were not statistically significant. The methylamines, betaine and glycerophosphorylcholine (GPC), showed a similar pattern, but both were significantly elevated in the inner medulla of 'dry diet' animals (betaine 154+/-57 to 315+/-29 mmol kg(-1) protein; GPC 35+/-7 to 47+/-10 mmol kg(-1) protein). It was concluded that in this marsupial the concentrating mechanism probably functions in a similar way to that in higher mammals, and that the mechanism of osmoprotection of the medulla of the kidney involves the same osmolytes. However, the high ratio of betaine to GPC in the inner medulla resembles the situation in the avian kidney.     

Spermiogenesis in the brush-tailed possum,Trichosurus vulpecula (Marsupialia)

Summary Acrosome development in the Australian Brush-tailed possum, Trichosurus vulpecula, displays a number of extraordinary features. This is particularly evident in the later stages of spermiogenesis, when the area of the nuclear surface bounded by the nuclear ring, and covered by the acrosome, is reduced considerable. As a result, the acrosomal material becomes located over its definitive position on the anterior third of the dorsal nuclear surface; in this process it is thrown into a series of folds, and a wide subacrosomal space is formed.Further changes around the time of spermiation result in the release of a spermatozoon in which a thin layer of acrosomal material is closely applied to the nucleus over the area of the definitive location of the acrosome, whilst its margins are greatly extended and project freely away from the nucleus. The latter feature does not appear to have been reported for the sperm of other mammals.The authors would like to thank Dr. D.J.H. Cockayne, Director of the Electron Microscope Unit, University of Sydney, for the generous provision of transmission electron microscope facilities, and Dr. M.R. Dickson, Electron Microscopist in charge, Biomedical Electron Microscope Unit, University of New South Wales, for the use of other facilities. Also, thanks are due to Miss Robin Arnold and Mrs. Eva Vassak of the Department of Histology and Embryology, University of Sydney, for their expert assistance. The assistance of the N.S.W. National Parks and Wildlife Service in the provision of permits to work on these native mammals is acknowledged     

Organic osmolytes in the developing kidney of the Australian brush-tailed possum, Trichosurus vulpecula

Developmental changes in the plasma and urine composition of 120 immature possums, Trichosurus vulpecula, were investigated and correlated with changes in the osmolyte composition of the kidneys. In early life, when the animal is confined in the pouch, the urine is more or less isosmotic with the plasma (300+/-70 mOsm kg(-1)), but rises steeply in animals more than 90 days old, up to 667+/-53 mOsm kg(-1) in the oldest age group. In parallel with this change there are corresponding increases in medullary osmolytes. Measured as mmol kg(-1) wet weight, sorbitol was found to increase from 2.1+/-0.8 to 9.7+/-2.2, myo-inositol from 10.9+/-6.2 to 33.6+/-11.7, while the methylamines glycerophosphorylcholine and betaine rose from 6.2+/-0.5 to 15.3+/-3.1 and 3.9+/-2.2 to 9.4+/-2.5 mmol kg(-1) wet weight respectively. Medullary taurine showed no significant changes with age in young possums, while urea increased from 11+/-4.6 to 49+/-7.1 mmol kg(-1) wet weight. These values are similar to, but less than, those found in adult kidneys, but probably further refinement of the concentrating ability of the kidney occurs after the young animal becomes independent.     

Structure of the oviducal epithelium of the brush-tailed possum (Trichosurus vulpecula)

Observations were made using scanning and transmission electron microscopy and were correlated with measurements of the mean surface area and the percentage area occupied by secretory cells to provide precise interpretation of the morphological data. In cyclic and anoestrous possums the oviducts were lined with elaborate folds of mucosa except in the portion of the isthmus adjacent to the uterus where the pattern was much simplified. The folds decreased in height and number after ovariectomy. Secretion was maximal between oestrus and Day 2 of the cycle when most ovulations take place. Extrusion of vesicles was observed in the ampulla and isthmus at this stage although the process was localized to some extent because groups of cells with actively extruding vesicles were interspersed with others which contained vesicles but otherwise appeared inactive. This is interpreted as a device whereby a constant supply of secretion can be maintained over several days. Secretory activity declined between Days 3 and 8 and the epithelium in mid-cycle (Days 8-18) appeared relatively quiescent. By Day 24 there was morphological and morphometric evidence of a return to oestrous conditions, although vesicles had not yet reappeared in secretory cells. Ciliogenesis was prevalent at this time and is probably related to the ovum transport that will occur several days later.     

Structure of the uterine luminal epithelium of the brush-tailed possum (Trichosurus vulpecula)

Morphology and morphometry of the luminal surface of the uterus of the brush-tailed possum were studied during the oestrous cycle, in anoestrous animals and after ovariectomy. At oestrus the secretory cells were small and the epithelium heavily ciliated. The relative surface area occupied by secretory cells reached a maximum on Day 13 when plasma progesterone concentrations are maximal. The mean apical surface area of the secretory cells also reached a maximum at this time. Both these measures decreased on Day 18 when involution of the epithelium was taking place. This process was essentially complete by Day 24 and was followed by extensive ciliogenesis. Secretory cells from anoestrous animals appeared to have an apical surface area similar to the minimum recorded during the oestrous cycle and extensive loss of cilia did not occur. Ovariectomy caused loss of ciliated cells and a reduction in the mean apical surface area to a dimension much smaller than that measured in intact animals.     

Expression of PGK-A in the Australian brush-tailed possum,Trichosurus vulpecula (Kerr), consistent with paternal X inactivation

An extensive survey of erythrocytes of marsupials other than kangaroos for electrophoretic variation in X-linked enzymes revealed two rare PGK-A phenotypes in the phalangerid Trichosurus vulpecula and one in Trichosurus caninus. Four putatively heterozygous females expressed only the variant allelic isozyme in some tissues but expressed a trace of the normal isozyme in others. A putatively hemizygous male expressed only the variant isozyme in all tissues. The phenotypic patterns were consistent with those observed in kangaroos known to exhibit partial or complete paternal X inactivation in cells of females. Two of the T. vulpecula were a mother and her female pouch young, further suggesting that paternal X inactivation occurs in T. vulpecula. This peculiar mechanism of dosage compensation may not be restricted to kangaroos.This work was supported by grants to D. W. C. from the Australian Research Grants Committee and Macquarie University, J. L. V. was the recipient of a Fulbright-Hays Award from the Australian-American Educational Foundation and a postgraduate fellowship from General Motors-Holden.     

The subcommissural organ of the adult and pouch-young brush-tailed possum (Trichosurus vulpecula)

Summary Scanning electron microscopy (SEM) was used to examine the surface features of the subcommissural organ (SCO) and the roof of the cerebral aqueduct of 35 adult and 33 pouch-young Trichosurus vulpecula. The animals were at various developmental stages and of both sexes. In the adult animals, the surfaces of the groove and the adjacent medial walls of the ridges of the SCO were characterized primarily by microvilli. Typically, Reissner's fibre was associated closely with the median groove of the SCO. The ridges and the paramedian grooves of the SCO were often heavily ciliated displaying many cerebrospinal fluid-contacting nerve processes. These processes were of varying lengths with terminal and preterminal varicosities. The observed morphology supports a hypothesis suggesting that under certain physiological and pathological conditions the flow of CSF may be directed away from the heavily ciliated ridges to the poorly ciliated groove containing the Reissner's fibre. In the youngest pouch-young animals there were no cilia, CSF-contacting nerve processes, nor supraependymal cells. Also the surface features of the SCO in the young assumed adult appearance before the adjacent roof of the cerebral aqueduct. These findings suggest the possibility that the SCO begins to function early in ontogenetic development. Acknowledgement. Technical assistance of Mrs. G. Hermanis is gratefully acknowledged.The author thanks the Director of Wild Life and National Parks, South Australia, for permission to use brush-tailed possums     

The effects of hypoosmotic infusion on the composition of renal tissue of the Australian brush-tailed possum Trichosurus vulpecula

Although the occurrence of organic osmolytes in the inner medulla of the marsupial kidney has been recently reported [Comp. Biochem. Physiol. (2002) 132B 635-644], changes in these substances, in response to water loading in vivo, has not been studied. Adult Trichosurus vulpecula, the Australian brush-tailed possum, were subjected to water deprivation for 48 h. Following anaesthesia and unilateral nephrectomy, the animals were perfused with hypo-osmotic saline (80 mmol l(-1); 1.5 ml min(-1)) for 60 min. This resulted in a rapid increase in urine volume and a corresponding fall in urine osmolality. At the end of the infusion the animals were killed and the second kidney removed. Analysis of the renal tissue revealed that water content of cortical, outer and inner medullary regions of the kidney increased slightly following infusion, while sodium, and chloride contents of all three regions fell. Potassium contents, on the other hand, were barely changed. Of the organic osmolytes determined, very significant decreases in the inner medulla, following infusion, were found for sorbitol (from 397+/-79 to 266+/-49 mmol kg(-1) protein), inositol (247+/-23 to 190+/-25 mmol kg(-1) protein), and betaine (464+/-70 to 356+/-21 mmol kg(-1) protein), while only inositol was significantly decreased in the outer medulla (197+/-22 to 150+/-16 mmol kg(-1) protein). Glycerophosphorylcholine levels were low throughout the kidney and were not significantly affected by the infusion. It was concluded that inositol and sorbitol play a significant role as compatible organic osmolytes in the possum kidney, while betaine functions as the principal counteracting osmolyte. Amino acid levels in the cortex and outer medulla showed no overall change in amount following infusion, although there were highly significant changes in individual amino acids. In the inner medulla there was a highly significant reduction in total amino acids with infusion, largely due to a fall in amounts of taurine (104+/-4 to 75+/-17 mmol kg(-1) protein), and glycine (97+/-15 to 71+/-18 mmol kg(-1) protein). A fall in free amino acid levels in the inner medulla appears to significantly contribute to the process of intracellular osmotic adjustment during an induced diuresis.     

A PMSG/GnRH method for the superovulation of the monovulatory brush-tailed possum (Trichosurus vulpecula)

Optimal ovarian stimulation with minimal degenerative changes was achieved in brush-tailed possums 3 days after a subcutaneous injection of 10 i.u. PMSG. This treatment alone did not result in ovulation and only rarely in oocyte maturation. Ovulation of 8-24 mature oocytes occurred when on the 3rd day after 10 i.u. PMSG the female received 3 intramuscular injections of 50 micrograms synthetic gonadotrophin-releasing hormone (GnRH) 90 min apart. Superovulation was achieved in immature, cycling, pregnant and lactating females, but not in animals in the preovulatory phase of a natural oestrous cycle.     

Ultrastructural changes in spermatozoa of the brush-tailed possum,Trichosurus vulpecula (Marsupialia), during epididymal transit

Summary The acrosome in spermatozoa from the caput epididymidis of the Australian Brush-tailed possum, Trichosurus vulpecula, typically forms a cup-like structure, sitting on the anterior third of the dorsal surface of the nucleus. The base of the acrosomal cup is narrowly separated from the nuclear surface, while the body of the cup projects voluminously away from the nucleus.During epididymal transit these pronounced marginal extensions of the acrosome are retracted towards the nucleus, and the electron dense acrosomal material undergoes a process of compaction within the plasma membrane of the head to produce the convex ovate form of the definitive acrosome. During this process a variety of bizarre forms of the acrosome are produced before its final configuration is attained.The authors would like to thank Dr. D.J.H. Cockayne, Director of the Electron Microscope Unit, University of Sydney, for the generous provision of transmission electron microscope facilities, and Dr. M.R. Dickson, Electron Microscopist in charge, Biomedical Electron Microscope Unit, University of New South Wales, for the use of other facilities. Thanks also are due to Miss Robin Arnold and Mrs. Eva Vassak of the Department of Histology and Embryology, University of Sydney, for their expert assistance. The assistance of the N.S.W. National Parks and Wildlife Service in the provision of permits to work on these native mammals is acknowledged     

Seasonal reproductive periodicity in the male Australian Brush-tailed possum (Trichosurus vulpecula)

The reproduction of the possum, Trichosurus vulpecula (Kerr), was investigated on Banks Peninsula, Canterbury, from 1963 to 1965. The majority of births occur in the autumn, but there is a minor breeding season in the spring. After undergoing an anoestrous period during the summer most females enter oestrus around April. A small proportion of females give birth to a second young during September or October. Reproductive periodicity was also found to occur in the male possum. Although spermatogenesis continues throughout the year in the sexually mature animal there is a marked change in the size of the prostatic urethra at particular periods of the year which correspond to those in which the majority of females are undergoing oestrus. The mean weight of the prostate shows a sixfold increase during the autumn. However only very small changes take place in the size of the testis and epididymis. Changes in size and activity of the prostate correspond with marked changes in the interstitial tissue of the testis. Leydig cells, responsible for the secretion of testosterone, show a great increase in number in animals in which the prostate is enlarged. Associated with the growth of the prostate are changes in the behaviour of the possum. The body weight and fat deposits of the male possum drop markedly at the time of the autumn breeding season.     

The origin of mitotic sex-chromosome association in the brush-tailed possum, Trichosurus vulpecula (marsupalia:phalangeridae)

Nonrandom associations between the sex chromosomes of the brush-tailed possum, Trichosurus vulpecula, were found to be due to association of nucleolar organizer regions (NOR's) on the X and Y chromosomes. NOR association was also observed between an autosome and the X chromosome. These findings, based on silver staining, are in contrast to the report of MURRAY (1977), who observed sex-chromosome association in this animal and indicated that these nonrandom associations may reflect an association between heterochromatic regions during interphase. We observed only two pairs of NOR's per cell in this animal, one autosomal and one on the sex chromosomes, rather than the several such regions observed by MURRAY, who used an N-banding technique. We discuss the problem of nonhomologous chromosome association in mammalian cells as influenced by heterochromatin and NOR's and find little support for nonhomologous chromosome associations at mitotic metaphase due to heterochromatin association.     

Projection of cervical dorsal root fibers to the medulla oblongata in the brush-tailed possum, Trichosurus vulpecula

This study describes the projection of cervical spinal afferent nerve fibers to the medulla in the brush-tailed possum, a marsupial mammal. After single dorsal roots (between C2 and T1) were cut in a series of animals, the Fink-Heimer method was used to demonstrate the projection fields of fibers entering the CNS via specific dorsal roots. In the high cervical spinal cord, afferent fibers from each dorsal root form a discrete layer in the dorsal funiculus. The flattened laminae from upper cervical levels are lateral and those from lower cervical levels are medial within the dorsal columns. All afferent fibers at this level are separated from gray matter by the corticospinal fibers in the dorsal funiculus. All cervical roots project throughout most of the length of the well-developed main cuneate nucleus in a loosely segmentotopic fashion. Fibers from rostral roots enter more lateral parts of the nucleus, and fibers from lower levels pass to more medial areas; but terminal projection fields are typically large and overlap extensively. At more rostral medullary levels, fibers from all cervical dorsal roots also reach the external cuneate nucleus. The spatial arrangement here is more complex and more extensively overlapped than in the cuneate nucleus. Rostral cervical root fibers reach ventral and ventrolateral areas of the external cuneate nucleus and continue to its rostral pole; more caudal root fibers project to more dorsal and medial regions within the nucleus. These results demonstrate that projection patterns of spinal afferents in this marsupial are similar to those seen in the few placental species for which detailed data concerning this system are available.     

Uterine oxytocin receptors in an Australian marsupial, the brushtail possum, Trichosurus vulpecula

1. Oxytocin receptors in the uterus of the brushtail possum (T. vulpecula) were characterized by radioreceptor assay and compared with those of the sheep and rat uterus. 2. A single oxytocin binding site was found with an affinity (Kd) and receptor concentration (Ro) of 3.0 +/- 0.8 nmol/l and 200 +/- 60 fmol/mg protein, respectively (SEM; n = 5). The receptor was stable at -20 degrees C; divalent ions were required for optimum binding. 3. Competitive displacement curves with related peptides showed the following order of specificity: vasotocin greater than oxytocin greater than mesotocin = arginine-vasopressin = [Thr4, Gly7]-oxytocin greater than lysine-vasopressin = isotocin much greater than [d(CH2)5, D-Phe2, Ile4, Ala9-NH2]-AVP. 4. It was concluded that oxytocin receptors in the possum have similar characteristics to those of placental mammals.     

Spontaneous or experimentally induced formation of a special zone in the adrenal cortex of the adult brush-tailed possum (Trichosurus vulpecula)

The cytology and ultrastructure of the hypertrophied special zone, which is formed spontaneously in the adrenal cortex of adult female brush-tailed possums (Trichosurus vulpecula), was compared to the adrenocortical tissue in adult males in which the special zone, normally absent, was induced following castration alone or by additional treatment with follicle-stimulating hormone (FSH). The special zone in females was situated between the zona fasciculata and the zona reticularis, the latter being a rudimentary zone in this species. Special zone tissue extended as a broad band parallel to and on one side of the adrenal medulla. In the luteal phase of the reproductive cycle, the special zone cells showed ultrastructural features commonly associated with steroidogenic tissues, with many mitochondria and compact masses of smooth endoplasmic reticulum. Cytoplasmic lipid inclusions were rarely observed. In lactating females, however, the special zone cells exhibited cytological and ultrastructural features suggestive of a transformation in their morphology broadly divided into two types of cells: (1) cells at the periphery of the special zone (closest to the zona fasciculata) showed variable quantities of lipid inclusions, mitochondria with dispersed cristae, and segregation of the smooth endoplasmic reticulum into compact masses; (2) cells within the more central regions showed an increasing abundance of lipid inclusions which in many cells became the dominant feature of the cytoplasm. These special zone cells contained very little smooth endoplasmic reticulum and their mitochondria contained few cristae together with amorphous granular material within the matrix. In castrated males, special zone tissue developed between the zona fasciculata and the zona reticularis, appearing initially as focal islands of cells (8 months postcastration) and later (11 months postcastration) expanding into a single zone, probably via the proliferation and differentiation of adjacent cells of the zona fasciculata and longitudinal growth of the special zone. Similar focal aggregations of special zone cells were induced after 14 days of FSH treatment given to 2-month castrated males. In all castrated and FSH-treated castrated males, the ultrastructure of special zone cells was similar to that of special zone cells in luteal-phase female possums. The findings suggest that the formation and cellular composition of the special zone is associated with changes in the pituitary-gonadal axis and that FSH plays a primary role in the differentiation of this tissue.