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.     

Time of ovulation in the brushtail possum (Trichosurus vulpecula) following PMSG/LH induced ovulation

This study aimed to determine the timing of ovulation in response to a new induced ovulation regime for the monovulatory brushtail possum (Trichosurus vulpecula). Ovarian stimulation was achieved by a single subcutaneous injection of 15 i.u. pregnant mare serum gonadotrophin (PMSG). This treatment resulted in promotion of a large number (9-16) of Graafian follicles > 2 mm diameter on the ovaries. Seventy-two hours later a single intramuscular injection of 4 mg porcine luteinizing hormone (LH) was administered to induce ovulation. Groups of possums were killed 24 hr post-LH injection and subsequent groups were killed at 6-hr periods up to 48 hr later. Ovulation occurred from 30 hr to 42 hr post-LH. The ovulatory success increased from 3% at 30 hr post-LH to 83% at 48 hr post-LH. Oocytes were recovered primarily from the oviducts at 36 hr post-LH. Thereafter oocytes were recovered increasingly from the uteri and by 48 hr post-LH, were only found there. The implications of these observations for artificial breeding in possums are discussed.     

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.     

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.

     

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.     

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.     

Ultrastructural characteristics of the process of cornification in developing claws of the brushtail possum (Trichosurus vulpecula)

Cornification of developing claws in the brush possum has been analysed by electron microscopy and compared with the process in other tetrapods. Newborns from 3 to 60 days postparturition were studied. After formation of symmetric and round outgrowth in digits the epidermis becomes thicker in the dorsal with respect to the ventral digit tip. The claw elongates forming the unguis and a shorter subunguis. Spinosus keratinocytes in both unguis and subunguis accumulate tonofilaments that fill their cytoplasm. Keratohyaline‐like granules are formed in early stages of differentiation in both unguis and subunguis but they later disappear in highly cornified corneocytes. Tonofilaments become electron‐dense in keratinocytes of the precorneous layer in the large corneocytes of the unguis and in narrow corneocytes of the subunguis. Keratin bundles transform into an amorphous corneous material that embeds or masks the original keratin intermediate filaments. Nucleated corneocytes are accumulated in the unguis while thinner corneocytes are present in the subunguis. The latter contain a dense material, possibly containing high sulphur keratin associated proteins, as occurs during cornifcation of the cortex and cuticle hair cells and in the nail. The process of cornification of mammalian claws is compared with that of reptilian and avian claws.     

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.     

Physiological changes in the brushtail possum (Trichosurus vulpecula) following relocation from Armidale to Brisbane, Australia.

To determine the effect of relocation on the health of possums the body weights and hormone and immune responses of six male and nine female brushtail possums were monitored for 20 weeks following transfer from the environs of Armidale into enclosures in Brisbane. Over the first 6 weeks of captivity, male possums lost 11.0% of their original body weight and females lost 16.8%. The mean concentrations of plasma cortisol in the male and female possums were 14.5 and 29.4 ng/mL, respectively, and did not change over the 20-week period. Male and female possums displayed a similar pattern of thyroxine secretion over the 20 weeks, with low concentrations up to week seven (2.1 and 2.7 ng/mL, respectively) increasing to 6.9 and 5.8 ng/mL in weeks 7-12 (P < 0.005). This increase in the concentration of thyroxine corresponded with the increase in body weight. The number of white blood cells (WBCs) and the percentage of neutrophils increased from the capture to week 6-10. However, during the last 10 weeks of captivity the number of WBCs and the percentage of neutrophils decreased, indicating recovery of the immune system. This was in accord with the proliferative response of lymphocytes to the T cell mitogen PHA that increased from weeks 11-15 to weeks 16-20 in both male and female possums. The results above suggest that the Armidale possums, like the Brisbane possums, were stressed following their relocation; however, their immune systems were able to gradually recover as they adjusted to their new environment in Brisbane. The death rate of pouch young and of adult female possums after relocation was considerably higher in the Armidale possums compared to Brisbane possums. The mortality rate of Brisbane possums over the first 20 weeks of captivity was 8.3% and 19.6% for male and female possums, respectively, and for Armidale possums 16.6% and 47.1%, respectively. The possums transferred from the environs of Armidale into captivity in Brisbane were under greater stress than possums captured in Brisbane and placed in captivity in Brisbane.     

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.     

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.     

Some effects of breeding season and castration on the prostate and epididymis of the brushtail possum, Trichosurus vulpecula

In an attempt to understand the mechanism(s) responsible for the reported marked seasonal increase in prostatic, but not epididymal, weight in T. vulpecula, a number of parameters were measured in tissues from mature, entire males sampled within and outside of the breeding season and from castrates. Conditions for the measurement of cytosol androgen receptors were also established. The weight of both the prostate and the epididymis was significantly elevated in the breeding season but the relative increase in prostate weight was considerably greater. The increase in prostatic weight was associated with a decrease in DNA: g tissue and an increase in protein: DNA and RNA: DNA ratios, each indicative of cellular hypertrophy and/or accumulation of secretory product. In the epididymis there were no significant seasonal changes in RNA: DNA, protein: DNA or DNA: g tissue ratios. Low-capacity, high-affinity binding was demonstrated in the epididymal and prostatic cytosols and values for the equilibrium association constants and receptor concentrations were within the range reported for androgen receptors in eutherian species. The temperature sensitivity of the binding, steroid specificity and slow dissociation in the cold indicated that in both tissues cystosol receptor and not androgen-binding or serum-binding protein(s) were being measured. In prostatic, but not epididymal, cytosol a low level of progesterone binding was observed and was masked by triamcinolone acetonide. When expressed in terms of tissue DNA, cytosol androgen receptor level in the prostate only was elevated in the breeding season. Prostatic tissue showed a low level of 5 alpha-reductase in vitro which was not influenced by season. However, both tissues showed a high concentration of 5 alpha-dihydrotestosterone and in the prostate, where seasonal effects were measured, the concentration was higher in the breeding season. This indicates that although 5 alpha-dihydrotestosterone is the likely active androgen in the prostate it may be formed elsewhere. Part of the explanation for the increased growth of the prostate in the breeding season appears to be a change in receptor concentration coupled with elevated tissue androgen level.     

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.     

Adaptive biases in offspring sex ratios established before birth in a marsupial, the common brushtail possum Trichosurus vulpecula

Offspring sex ratios in the common brushtail possum are malebiased in many populations, and there is evidence that inter-populationdifferences in sex ratios represent adaptive responses to localconditions. However, how these biases are produced is not known.Using comparisons between populations with and without biasedoffspring sex ratios, we show that biases in this species arenot produced by sex-differential mortality between birth andweaning or sex-selective termination of pregnancy. Rather,adjustment in the sex ratio of offspring are evidently dueto shifts in the probability of conceiving male and femaleoffspring.