The role of photoperiod on the initiation of the breeding season of the brushtail possum (Trichosurus vulpecula).

The role of photoperiod on the initiation of the breeding season of brushtail possums was investigated in possums housed in three light regimens: a short-day, a natural and a long-day photoperiod. Seven possums were housed in a natural photoperiod. Four possums were transferred to a short-day photoperiod (10 h light, 14 h dark) and eight possums to a long-day photoperiod (14 h light, 10 h dark) on 22 November, when the daylength was 13.34 h. The first rises in plasma progesterone concentrations were observed on 9 January +/- 9 days (n = 4), 11 March +/- 6 days (n = 7) and 6 May +/- 6 days (n = 8), for possums held in short-day, natural or long-day photoperiods respectively. Similarly, births were observed on 12 January and 14 February in the short-day group, from 3 March to 8 May for the natural photoperiod group, and from 5 May to 8 August for the long-day group. These results suggest that photoperiod is important in the timing of the breeding season. However, annual breeding will commence in a nonstimulatory long-day photoperiod. Thus a long-day photoperiod does not prevent breeding activity.     

The development of thermoregulation in the marsupial brushtail possum Trichosurus vulpecula

• 1.1. The adult possums showed a circadian rhythm of body temperature with a peak in temperature around midnight and a nadir at noon.
• 2.2. The young possum within the pouch displayed a circadian rhythm with the highest temperatures during the day and the lowest in the early evening.
• 3.3. Although the body temperature of the young possum exceeded that of the mother occasionally, for the major part of the 24 hr the body temperature of the young was lower than that of the mother.
• 4.4. The young possum could maintain a steady body temperature between 140 and 167 days post partum. A circadian rhythm of temperature was observed between 157–190 days post partum.
• 5.5. All adipose tissue examined with the light and electron microscope had the morphology of white adipose tissue.

     

Influence of daylength on the initiation of the breeding season of the marsupial possum, Trichosurus vulpecula

In Queensland, possums in the wild and in captivity first give birth during March and continue to give birth throughout the year until November. In this study the effect of short daylengths on the initiation of breeding activity was examined. One male and 4 female possums were transferred from an outside enclosure into a light control room (10 h light, 14 h dark) on 22 November. A control group of possums was housed in outside enclosures. The possums held in the light room gave birth 81.2 +/- 14.7 days (s.e.m.) after being placed in the short-day photoperiod. The control group gave birth 133.8 +/- 9.8 days after 22 November. This result suggests that photoperiod plays a role in the initiation of the breeding season of the brushtail possum.     

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.     

Morphological trends in the Common brushtail possum, Trichosurus vulpecula, in New Zealand

Over 300 specimens of the Australian introduced Common brushtail possum, taken from throughout its range in New Zealand, were measured. A north-south trend in skull size was found, which conforms with Bergmann's rule. This trend probably evolved during less than 50 years (30-35 generations), and is not significantly different from the present Australian trend for skull size in this species. Males are slightly larger (1.8% in skull length) than females.     

Sexual dimorphism and synchrony of breeding: variation in polygyny potential among populations in the common brushtail possum, Trichosurus vulpecula

Male-biased sexual dimorphism in both size and body mass isa common characteristic of many polygynous mammals and is oftenattributed to sexual selection favoring large males. The degreeof dimorphism is thought to be related to the potential forsome males to monopolize access to estrous females, which isin turn related to the distribution of receptive females inspace and time. In the present study, we investigated the relationshipbetween the temporal distribution of breeding females and thedegree of mass dimorphism among 11 populations of the commonbrushtail possum from northern Australia. Breeding patternsvaried from complete aseasonality in some populations to a seasonalconcentration of births within 2 or 3 months in others. We predictedthat in populations in which mating opportunities were distributedthroughout the year, dominant males would be able to monopolizeaccess to larger numbers of estrous females than in populationsin which matings were more seasonal, and in such populations,large body size in males would be favored. We found that dimorphismwas related to seasonality of breeding, being greatest in populationswith a more aseasonal pattern. Mean body mass of male possumsalso decreased with increasing population density. Populationdensity may influence the degree of breeding synchrony withinpopulations, particularly in locations with a more seasonalclimate. The present study is the first to demonstrate plasticityin mass dimorphism in response to local variation in the synchronyof breeding in a mammal species.     

Changes in prolactin in peripheral plasma during lactation in the brushtail possum Trichosurus vulpecula

A heterologous double-antibody radioimmunoassay has been validated for prolactin in plasma and pituitary preparations of T. vulpecula. Serial dilutions of crude pituitary homogenates and plasmas from several marsupials and purified prolactin from the tammar, Macropus eugenii, showed parallel dose response curves. In both male and female possums plasma prolactin concentrations increased in response to a single intravenous injection of thyrotrophin releasing hormone. Plasma prolactin concentrations were measured in six lactating females (June-November) and in four non-lactating females (July-October). In the following year prolactin levels were also measured in 11 possums with young less than 50 days old and in 24 possums with young aged between 100 and 145 days. In early lactation prolactin concentrations were low (less than 8 ng/ml) but increased to high levels (greater than 30 ng/ml) by 120 days and remained high until about 160 days of lactation. Thereafter concentrations declined although the young continued to take milk from the mother for a further 30-50 days. The changes in plasma prolactin concentrations throughout lactation are very similar to those described for the tammar, and this unusual pattern appears to be common to marsupials. Non-lactating possums showed no consistent changes in plasma prolactin concentrations between July and October.     

Chemical characterization of milk oligosaccharides of the common brushtail possum (Trichosurus vulpecula)

Structural characterizations of marsupial milk oligosaccharides have been performed in only three species: the tammar wallaby, the red kangaroo and the koala. To clarify the homology and heterogeneity of milk oligosaccharides among marsupials, 21 oligosaccharides of the milk carbohydrate fraction of the common brushtail possum were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of mid-lactation milk and characterized by ¹H-nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The structures of the 7 neutral oligosaccharides were Gal(β1-3)Gal(β1-4)Glc (3’-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3”, 3’-digalactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I), Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl lacto-N-novopentaose I), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)Gal(β1-4)Glc (galactosyl lacto-N-novopentaose II). The structures of the 14 acidic oligosaccharides detected were Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Glc (sialyl 3’-galactosyllactose), Gal(β1-3)(O-3-sulfate)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate a) Gal(β1-3)[Gal(β1-4)(O-3-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)(?3-O-sulfate)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)[Gal(β1-4)(?3-O-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(?3-O-sulphate)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)(?3-O-sulphate)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)(?3-O-sulphate)GlcNAc(β1-6)]Gal(β1-4)Glc and Gal(β1-3)Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl sialyl lacto-N-novopentaose b). No fucosyl oligosaccharides were detected. Galactosyl lacto-N-novopentaose II, lacto-N-novopentaose I sulfate a, lacto-N-novopentaose I sulfate b and galactosyl sialyl lacto-N-novopentaose b are novel oligosaccharides. The results are compared with those of previous studies on marsupial milk oligosaccharides.     

Differences in steroid metabolism by testis, prostate and epididymis in the immature and adult possum (Trichosurus vulpecula)

Immature possums 126-195 days old and adults over 1 year old were used. Testicular homogenates from immature possums converted [3H]progesterone to nine different products, of which greater than 63% were 5 alpha-reduced androstane metabolites. The major product from adult testis was testosterone in yields greater than 60%. While metabolism of [3H]testosterone by the epididymis of immature possum was minimal, in adults 5 alpha-reduced products constituted greater than 80% of the yield. In contrast, prostatic tissue from adults converted less than 4% of [3H]testosterone to 5 alpha-reduced products, while the yields were greater than 80% from prostates of immature animals. The results showed that like in rodents, the testis of immature possum has a high 5 alpha-reductase and low 17 beta-hydroxysteroid dehydrogenase activity, which reverses in the adult state. The implications of the findings are discussed.     

Leptospirosis serology in the common brushtail possum (Trichosurus vulpecula) from urban Sydney, Australia

The common brushtail possum (Trichosurus vulpecula) is indeed a common marsupial in major cities of Australia. This species is known to be susceptible to leptospirosis and often lives in close contact with humans, raising concerns about the potential for transmission of this disease in urban areas. A total of 192 brushtail possum blood samples were collected from 136 individuals in suburban areas of metropolitan Sydney from November 2002 to November 2004. Sera were screened against a reference panel of 21 Leptospira spp. using the microscopic agglutination test. Leptospiral antibodies were detected in 9.6% (13/136) of tested brushtail possums and represented two serovars; antibodies to Leptospira interrogans serovar Hardjo were most frequently identified (11/136). A representative of the exotic sero-group Ballum, most likely serovar Arborea, was found in two of 136 brushtail possums. Exposure to leptospirosis seemed to be associated with age, as older animals had a higher incidence, but there was no distinction in relation to gender. Antibody prevalence varied between the different sampling sites and seropositive animals were clustered and restricted to a few sites. These data support the possible role of brushtail possums as a maintenance host for Leptospira spp. in urban environments and also identified them as a previously unknown and potential source of serovar Arborea.     

Endogenous type D retrovirus in a marsupial, the common brushtail possum (Trichosurus vulpecula)

We have sequenced and characterized an endogenous type D retrovirus, which we have named TvERV(D), from the genome of an Australian marsupial, the common brushtail possum (Trichosurus vulpecula). Intact TvERV(D) gag, pro, pol, and env open reading frames were detected in the possum genome. TvERV(D) was classified as a type D retrovirus, most closely related to those of Old World monkeys, New World monkeys, and mice, based on phylogenetic analyses and genetic organization. Approximately 30 TvERV(D) proviruses are present in the genomes of possums, as detected by Southern hybridization. However, variability in fragment patterns between possums was observed and suggests recent (or ongoing) retrotranspositional activity.     

Determination of steroidogenic potential of ovarian cells of the brushtail possum (Trichosurus vulpecula)

The ovary of the brushtail possum (Trichosurus vulpecula) secretes steroids; however, little is known about the identity of the steroidogenic cells in the ovary. The aim of the present study was to determine the identity of the ovarian cell types expressing mRNAs encoding proteins important for steroidogenesis and determine at what stage of follicular development they are expressed. The genes examined were those for steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein (StAR), cytochrome p450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase/Delta5,Delta4 isomerase (3betaHSD), cytochrome p45017alphahydroxylase (p45017alphaOH), and p450 aromatase (p450arom). None of the genes examined were expressed in oocytes at any stage of follicular development. SF-1 was expressed in granulosa cells from the type 2 or the primary stage of development and thereafter to the preovulatory stage. In addition, the theca interna of small and medium-size antral but not preovulatory follicles and the interstitial glands and corpora lutea expressed SF-1 mRNA. Granulosa cells of preantral and small to medium-size antral follicles were not capable of synthesizing steroids from cholesterol because they did not contain p450scc mRNA. However, granulosa cells of many of the small to medium-size antral follicles expressed p450arom and 3betaHSD mRNA. The interstitial glands, theca interna, and corpus luteum expressed StAR, p450scc, 3betaHSD, and p45017alphaOH mRNA, suggesting that these tissues are capable of synthesizing progestins and androgens. The corpus luteum expressed p450arom, indicating that this tissue also has the potential to secrete estrogens in this species.     

Protein and peptide degradation in the intestine of the common brushtail possum (Trichosurus vulpecula)

A protein (bovine serum albumin: BSA) and a peptide (luteinizing hormone releasing hormone: LHRH) were used to evaluate proteolytic activity in the intestine of common brushtail possums (Marsupiala, Trichosurus vulpecula). Luminal and mucosal extracts were isolated from the duodenum, jejunum, ileum, caecum, proximal colon and distal colon, their protein content assessed and specific activities in metabolising LHRH and BSA determined in vitro. The degradation of LHRH by luminal extracts was compared with that by the pancreatic enzymes, chymotrypsin, trypsin, and elastase. The protein concentration (microg x mg-1) of mucosal extract in the duodenum was higher ( P<0.05) than in the proximal colon, but that of luminal extracts did not differ significantly between regions. Proteolytic activity of luminal extracts was greater ( P<0.01) in the jejunum and ileum than in the hindgut. In the small intestine, proteolytic activity of luminal enzymes far exceeded that of mucosal enzymes ( P<0.05). All three pancreatic enzymes hydrolysed LHRH, but chymotrypsin had the greatest activity. This study has demonstrated that, in possums, proteolysis occurs primarily in the small intestine through luminal enzymes, with chymotrypsin playing a major role. The possum hindgut contributes little to the metabolism of peptides and proteins, identifying it as a potential site to target for their absorption following oral delivery.     

Cloning and characterisation of a zona pellucida 3 cDNA from a marsupial, the brushtail possum Trichosurus vulpecula

The mammalian zona pellucida (ZP) is an extracellular glycoprotein coat that plays vital roles throughout fertilisation and preimplantation development. Like that of eutherian mammals the brushtail possum ZP is composed of three glycosylated proteins of 137 kDa, 92 kDa and 62 kDa. The 62 kDa protein is a ZP3 orthologue based on its nucleotide and deduced amino acid sequence. The brushtail possum ZP3 cDNA isolated in this study is 1305 nucleotides with an open reading frame encoding a 422 amino acid peptide of 45.7 kDa. Possum ZP3 has a 46% amino acid identity with eutherian ZP3 and shares similar structural characteristics including 12 conserved cysteine residues, N-linked glycosylation sites and hydrophobic regions. Like human and rabbit ZP1 an altered furin cleavage site upstream of the C-terminal hydrophobic domain also occurs in possum ZP3 (S-R-K-R), suggestive of processing by a furin-related endoprotease. Expression of brushtail possum ZP3 is limited to the ovary. Characterisation of brushtail possum ZP3 will enable examination of its functional role in marsupial fertilisation and its effectiveness as an immunocontraceptive agent.     

Sperm transport in the female reproductive tract of the brushtail possum, Trichosurus vulpecula, following superovulation and artificial insemination

This study investigated sperm transport following superovulation and artificial insemination (AI) in the common brushtail possum, Trichosurus vulpecula. Females were superovulated by treatment with 15 IU pregnant mare serum gonadotrophin (PMSG) then 4 mg luteinizing hormone (LH) 78 h later. Inseminations were performed 27 h after LH (4 million motile spermatozoa/uterus). At 1.5, 3, 6, 9 and 12 h after AI (n=5 per group), females were euthanised and reproductive tracts removed for examination and flushed for sperm. No ovulations had occurred by 1.5 h, but 20% of animals had ovulated by 3 or 6 h, and 80% by 9 or 12 h. The mean numbers of spermatozoa recovered ranged from 249 to 275x10(3) in the uterus; 16-51x10(3) in the isthmus; 8-11x10(3) in the middle segment; and 6-16x10(3) in the ampulla at 1.5, 3 and 6 h after AI. Sperm numbers in all regions decreased at later times (P<0.05) except the isthmus, where 100x10(3) sperm were recovered by 12 h. Highly motile thumbtack sperm (a putative indicator of capacitation in marsupials), were recovered from the isthmus (20%), middle segment (50%) and ampulla (90%) at all sampling times, but not from the uterus. The epithelium of the oviduct segments contained mucus-secreting and ciliated cells and peak secretory activity was observed in the ampulla at 6 h. At 3, 6 and 12 h, many spermatozoa were found in epithelial folds within the isthmus. The present study has provided basic information on sperm transport and storage events within the female reproductive tract of T. vulpecula following superovulation and AI. It is concluded that this model may be useful to better understand pre-fertilization sperm maturation events in the possum, which could facilitate the development of IVF technology.