Karen E. Mate

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.

Manipulation of the fertility of marsupials for conservation of endangered species and control of over-abundant populations

Marsupials present a dichotomy in population management; the numbers of many Australian marsupial species have declined due to loss of habitat, competition from introduced herbivores and predation by introduced carnivores, but other species have become locally overabundant in Australia or are introduced pests in New Zealand. The manipulation of reproduction offers the means to increase or decrease productivity; however, considerable fundamental research is required before reproductive technologies can be applied to marsupials. Marsupials differ from eutherian mammals in several aspects of their reproduction including sex differentiation, gamete function and endocrinology, as well as in the relative lengths of gestation and lactation. Although these differences present unique problems in the application of reproductive technologies to marsupials, they also present unique opportunities for marsupial-specific fertility control. This paper summarises the assisted breeding technologies currently being applied to marsupials including superovulation, artificial insemination, in vitro fertilization and gene banking; unique marsupial targets for contraceptive intervention including gamete production, sperm capacitation, gamete surface antigens and embryonic development; and some options for the delivery of contraceptive vaccines to marsupial populations.

Immunocontraception of Eastern Grey kangaroos (Macropus giganteus) with recombinant brushtail possum (Trichosurus vulpecula) ZP3 protein

This study examined the potential of a recombinant marsupial zona pellucida 3 protein as a contraceptive vaccine for the Eastern Grey kangaroo, a marsupial that is locally overabundant in several regions of eastern Australia. First, a pilot study using porcine zona pellucidae (PZP) demonstrated that ZP proteins, primarily the ZP3 component of PZP, are highly immunogenic in the grey kangaroo and produce a long-lasting humoral response to a single immunisation, as found in other marsupials. Immunisation with 300 microg of a non-glycosylated recombinant brushtail possum ZP3 (recBP-ZP3) protein in complete Freunds adjuvant produced a similar, significant and sustained antibody response, and none of the immunised kangaroos (n=7) produced offspring during the following breeding season compared with four out of the six control animals. An epitope analysis of the B-cell response to recBP-ZP3 using a brushtail possum ZP3 identified numerous B-cell epitope regions clustered around the N- and C-terminal regions of the protein. Two regions of interest for further fertility vaccine development based on their immunogenicity and fertility trials and functional studies in other species were found to be immunogenic. These results suggest that immunocontraception based on targeting the ZP3 protein within the zona pellucida may be an effective strategy for fertility reduction in Eastern Grey kangaroos.

Sperm binding and penetration of the zona pellucida in vitro but not sperm–egg fusion in an Australian marsupial, the brushtail possum ( Trichosurus vulpecula )

Sperm capacitation and in vitro fertilisation (IVF) have been achieved in most eutherian mammals and American marsupials under relatively simple culture conditions. In contrast sperm capacitation in Australian marsupials has not been achieved in vitro and attempts at IVF have previously been characterised by a complete lack of sperm-zona pellucida (ZP) binding. Recently, co-culture of sperm with oviduct epithelial cell monolayers or with oviductal explant conditioned media has been shown to prolong the viability and motility of brushtail possum spermatozoa, as well as to induce capacitation-associated changes such as transformation of sperm to the T-shape orientation. In this study we report that these in vitro produced T-shaped sperm, and in vivo derived T-shaped sperm flushed from the oviduct of artificially inseminated possums as a control, are able to bind to and penetrate the ZP of approximately 25% of eggs recovered from PMSG/LH-superovulated possums in vitro. Development of ZP receptivity and penetrability towards sperm was also identified as a major factor affecting the outcome of IVF. Neither in vivo nor in vitro derived T-shaped sperm were able to bind to or penetrate the ZP if eggs were obtained from animals that were treated with pLH less than 76 h after PMSG. Thus this study provides preliminary evidence for the necessity of sperm-oviduct epithelial cell interactions for capacitation in Australian species and lends further support to the suggestion that the T-shape head orientation is indicative of sperm capacitation. Despite the occurrence of sperm-ZP binding and penetration, sperm-egg membrane fusion and egg activation were not observed. Although the factor(s) responsible for the lack of sperm-egg membrane fusion in the possum have not been identified it is possible that egg capacity for membrane fusion develops independently of zona receptivity and is defective in these eggs, or alternatively that membrane fusion requires strictly defined ionic conditions which are not provided by the IVF media used in this study.

Sequence and analysis of zona pellucida 2 cDNA (ZP2) from a marsupial, the brushtail possum, Trichosurus vulpecula

All mammalian eggs are surrounded by the zona pellucida, an extracellular coat involved in vital functions during fertilization and early development. The zona pellucida glycoproteins are promising antigenic targets for development of contraceptive vaccines to control pest populations of marsupials in Australia and New Zealand. Our current understanding of the function of the zona pellucida glycoproteins is based almost entirely on the mouse and may not be representative of gamete interactions in all eutherian or marsupial mammals. This study reports the isolation and characterization of the ZP2 gene from the brushtail possum (Trichosurus vulpecula). The brushtail possum ZP2 mRNA is 2,182 nucleotides long with an open reading frame coding for a polypeptide chain of 712 amino acids with a molecular mass of 79,542 d. The deduced amino acid sequence of possum ZP2 is 48 to 55% identical to that of eutherian mammals. It shares several structural characteristics including N‐linked glycosylation sites, location and number of cysteine residues, and hydropathy profile. The brushtail possum ZP2gene is expressed exclusively in the ovary. Further studies are planned to elucidate the specific site of ZP2 expression within the ovary and its function during fertilization in marsupials. Mol. Reprod. Dev. 51:322–329, 1998.

The immune response and fertility of koalas (Phascolarctos cinereus) immunised with porcine zonae pellucidae or recombinant brushtail possum ZP3 protein

To evaluate the potential contraceptive effect of immunisation with zona pellucida antigens, 50 free-ranging koalas were immunised with either porcine zonae pellucidae (PZP), recombinant brushtail possum ZP3 (recBP-ZP3) or buffer, in complete Freunds adjuvant. A single booster immunisation in incomplete Freunds adjuvant was administered 3-5 months later. Where possible animals were recaptured, reproductive status was assessed and blood was collected at 1-3-month intervals for the next 33 months. Forty-three koalas were recaptured at least three times allowing reliable assessments of their fertility. Fourteen animals were observed never to have a pouch young. Of the remaining 29 animals the reproductive productivity of PZP treated females was reduced compared with control and recBP-ZP3 treated females, in terms of both total number of young produced, and failure to produce further young in females of proven fertility. One month after the initial immunisation, serum antigen-specific antibody titres were higher in animals immunised with PZP or recBP-ZP3 compared to controls, and reached a plateau by 4 months. Antibody against the relevant immunising antigen was also detected in ovarian follicular fluid, uterine fluid and vaginal secretions. Epitope analysis suggested that immune responses other than antibodies directed against the ZP3 amino acid sequence were responsible for mediating infertility. The results demonstrate that the fertility of female koalas can be compromised by immunisation against zona pellucida antigens. However, unlike in the eastern grey kangaroo and the brushtail possum, immunisation with bacterial recombinant brushtail possum ZP3 did not compromise fertility in the koala.

Timing of zona pellucida formation in the tammar wallaby (Macropus eugenii) and brushtail possum (Trichosurus vulpecula)

The zona pellucida (ZP) is an extracellular coat that surrounds the mammalian egg, and serves as the primary recognition site for fertilizing spermatozoa. The timetable of ZP formation was examined in two marsupials, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula) using conventional histological methods, immunofluorescence and electron microscopy. Ovaries from tammar wallaby pouch young less than 80 days of age contained only primordial follicles with a single layer of flattened granulosa cells. There was no evidence of ZP formation until 98 days, when a small number of eggs surrounded by a single layer of cuboidal granulosa cells had a ZP detectable by periodic-acid-schiff staining and rabbit anti-pig ZP polyclonal antibody labelling. Possum ovaries at 108 and 114 days also contained a small number of eggs with a ZP and a single layer of cuboidal granulosa cells. The antibody also labelled the peripheral cytoplasm of oocytes at this stage and, occasionally, the granulosa cells. Antral follicles were first detected at 144 days in the wallaby and 125 days in the possum, and always contained an egg surrounded by a ZP. Ovaries from 147, 158, 165, 181, 184 and 210-day-old tammar wallabies contained a range of follicle types from primordial through early antrum formation. Electron microscopy confirmed observations made at the light microscope level. The ZP was first detectable in small primary follicles with a single layer of cuboidal granulosa cells in areas where microvilli had begun to form on the egg plasma membrane. Immunogold labelling indicated the egg cytoplasm as the origin of the ZP proteins. The ZP completely filled the space between the egg and the adjacent granulosa cells in preantral follicles, so that there was no perivitelline space.

Diagnosis and disclosure of a memory problem is associated with quality of life in community based older Australians with dementia.

BACKGROUND Identification of factors associated with quality of life (QoL) in people having dementia will help develop strategies for maintenance and improvement of patient QoL. This study examined the predictors of QoL in a community-dwelling population aged 75 years and over, with or without dementia. METHODS This was a cross-sectional study involving 169 GPs and 2,028 patients. Patients were interviewed to collect information on personal circumstances. Several instruments were administered including the WHOQOL-BREF (quality of life outcome measure), Geriatric Depression Scale, GPAQ (satisfaction with GP care), and the CAMCOG-R (cognitive function). Patients with a CAMCOG-R score < 80 were allocated to the dementia group. GPs provided an independent clinical judgment of cognitive function for each of their participating patients. RESULTS The dementia group had significantly lower QoL scores in all four domains of the WHOQOL-BREF (all p ≤ 0.002). The GDS score was negatively correlated with all four domains in the non-dementia group and with physical, psychological, and environmental QoL in the dementia group (all p < 0.001). Satisfaction with GP communication was positively associated with psychological QoL in the dementia group and all domains in the non-dementia group. Participants in the dementia group who had been given a diagnosis of a memory problem had significantly higher physical (2.05, 95% CI 0.36 to 3.74) and environmental (2.18, 95% CI 0.72 to 3.64) QoL. CONCLUSIONS Satisfaction with GP communication is associated with a higher QoL in their older patients. Diagnosis and disclosure of memory problems is associated with better QoL in people with dementia. Clinicians should not be deterred from discussing a memory diagnosis and plans for the future with patients.

Fertilization following intracytoplasmic sperm injection of in vivo and in vitro matured oocytes from an Australian marsupial, the tammar wallaby (Macropus eugenii).

Conventional in vitro fertilization (IVF) techniques have been unable to produce normal embryos in any Australian marsupial, largely owing to problems with the early stages of sperm-oocyte binding. This study has used intracytoplasmic sperm injection (ICSI) of in vivo and in vitro matured tammar wallaby oocytes to bypass these processes and achieve fertilization in vitro. The fertilization rate (i.e. development to the two-pronuclei stage) of in vivo and in vitro matured oocytes following ICSI and sham injection was assessed at 17-19 h after injection. Fertilization occurred in 48% (45/93) of in vivo matured oocytes that were injected with spermatozoa. Significantly fewer sham-injected oocytes (6/82, P < 0.005) and uninjected control oocytes (5/84, P < 0.005) formed two pronuclei. In a direct comparison, the numbers of in vivo and in vitro matured oocytes that formed two pronuclei after ICSI were 22/28 (78.6%) and 23/40 (57.6%), respectively, which are not significantly different. There was also no significant difference in the nuclear response of in vivo and in vitro matured oocytes to sham injection. The numbers of oocytes forming a single pronucleus after sham injection were 10/24 (41.7%) and 24/37 (64.9) for in vivo and in vitro matured oocytes, respectively. Immature germinal-vesicle-stage oocytes were unable to decondense sperm injected during ICSI or to form pronuclei. These results demonstrate that both in vitro and in vivo matured tammar wallaby oocytes can be fertilized by ICSI. The success of ICSI not only offers the opportunity for fundamental analysis of marsupial fertilization but could, in conjunction with development of appropriate culture conditions and embryo transfer technologies, contribute to increased production of offspring from rare or valuable marsupials.

Screening for Dementia in Primary Care: A Comparison of the GPCOG and the MMSE

Background/Aims: The General Practitioner Assessment of Cognition (GPCOG) is a brief cognitive test. This study compared the GPCOG to the Mini-Mental State Examination (MMSE), the most widely used test, in terms of their ability to detect likely dementia in primary care. Methods: General practitioners across three states in Australia recruited 2,028 elderly patients from the community. A research nurse administered the GPCOG and the MMSE, as well as the Cambridge Examination for Mental Disorders of the Elderly Cognitive Scale-Revised that we used to define likely dementia. Results: Overall, the GPCOG and the MMSE were similarly effective at detecting likely dementia. The GPCOG, however, had a higher sensitivity than the MMSE when using published cutpoints. Conclusion: The GPCOG is an effective screening tool for dementia in primary care. It appears to be a viable alternative to the MMSE, whilst also requiring less time to administer.