- 3.1.1 PBC 220: Possum population responses to fertility control
- 3.1.2 PBC 221: Blocking embryonic development in brushtail possums
- 3.1.3 PBC 223: Control of reproduction in possums by targeting the pituitary gland through the use of GnRH-toxin conjugates
- 3.1.4 PBC 224: Control of reproduction in possums by targeting the oocyte-specific growth factors, GDF-9 and GDF-9B
- 3.1.5 PBC 225: Controlled oestrus and synchronised breeding in possums to facilitate the testing of new biocontrol agents
3.1 Physiology & Reproduction
3.1.1 PBC 220: Possum population responses to fertility control
| Programme Title: | Possum population responses to fertility control |
| Programme Leader: | Phil Cowan |
| Institution: | Landcare Research New Zealand Ltd |
Programme Goal: The research aims to determine whether methods of biological management of possums based on inhibiting fertility will result in the levels of population suppression predicted by modelling and required for conservation and Tb management. This essential research continues and further develops existing research to assess the effects of fertility control on components of population dynamics (mortality, recruitment) in wild possum populations. Analysis of the first 5 years of population data from this research has indicated that 50 percent and 80 percent sterility of adult females can successfully suppress local recruitment (female young born that survive to breeding age) by an average of 57 percent and 77 percent, respectively. These levels of sterility were also sufficient, in the absence of immigration, to reduce possum density. This phase of the experiment has now been completed.
The declines in population densities have only been modest, as predicted by modelling, as they rely on natural mortality. For that reason, only those potential compensatory processes likely to operate in the absence of significant reductions in density have been characterised (e.g. increased survival of sterile females) and their impact on the efficacy of fertility control assessed. Recent modelling (e.g., Bayliss & Choquenot (1999) Royal Society of New Zealand Miscellaneous Series 56:19-23) suggests that the most effective application of bait-delivered fertility control will be in suppressing population recovery after conventional control. Therefore, it is essential to define fully the effects of possible density-dependent processes that could compensate for sterility treatments at low densities, such as would exist after conventional control with toxins. These compensatory processes (e.g., increase in juvenile survival rates) are most likely to be apparent at low population density, where competition for resources is minimal.
Current models of fertility control in possums (e.g., Barlow et al. (1997) Wildlife Research 24: 129-141) suggest that the expected level of population suppression is critically dependent on whether density-dependence is expressed through mortality or recruitment. From this field experiment, we will collect data on age-specific mortality and recruitment parameters that form the basis of these key parameter inputs for the models. The results from this field experiment will be used to build new, and refine existing, models on effects of fertility control on possum populations, particularly to improve predictions on the rate of recovery of possum populations and integration of conventional control technologies with fertility control.
This project will assist with meeting NSSC goals by:
- identifying target levels of fertility control needed to reduce possum populations to meet conservation and Tb goals;
- demonstrating in the field the potential efficacy of fertility control for possum management;
- clarifying the potential impact of compensation by wild possum populations on the efficacy of fertility control; and
- defining optimum ways to integrate fertility control and conventional control technologies for possum management.
This information will be available by 2004/5, and will be critical in field-testing of `prototype' fertility control products that may be available by that time.
Objective 1
Objective Title: The effects of fertility control on the recovery rates of possum populations.
Research Leader: Dr Dave Ramsey
Description:
Determine the relationship between level of sterility in possum populations and the rates at which populations recover after control, by:
- estimating, by capture-mark-release methods, population size, breeding, recruitment, survival and the rate of population recovery in study populations after experimental reductions in population density by 70 percent where 0 percent, 50 percent or 80 percent of female possums are sterilised;
- extending and improving a stochastic age-structured population model, using parameters estimated from the field experiment, to project the effects of fertility control on population dynamics beyond the timescale of the current field experiment.
Produce milestone and annual reports and give one conference presentation.
3.1.2 PBC 221: Blocking embryonic development in brushtail possums
| Programme Title: | Blocking embryonic development in brushtail possums |
| Programme Leader: | Lynne Selwood |
| Institution: | The University of Melbourne |
Programme Goal: Three highly possum-specific targets already cloned, will be tested to determine their immune response and their effects on embryonic development in the possum. Three further identified, possum specific targets will also be cloned.
Rationale: Unlike other aspects of reproduction, possum embryonic development has many unique developmental features that make suitable targets for controlling reproduction. Our previous projects have examined three of these:
- The proteins associated with polarised vesicles (VAPs) that form during oogenesis and are responsible for the first lineage allocation into pluriblast (embryonic) and trophoblast (placental) tissues (Frankenberg and Selwood, 1998, 2001). We have identified 10 vesicle-associated molecules, 9 VAPs and hyaluronate, and consider four of these are worth further testing because of possum specificity and/or embryo lethality effects (Vap5, Vap7, vasa protein, and alpha fetoprotein).
- The outer egg coats (CPs), that are essential for normal embryonic development (Selwood, 2000) and for maintenance of blastocyst epithelia in the possum (Selwood et al, 1998). We have isolated 7 shell coat proteins of which amino acid sequences have been obtained for 5 and three unique proteins (CP2, CP4 and CP5) are suitable for further studies.
- Leukaemia inhibitory factor (LIF) that is present in the possum at the time of implantation (Cui and Selwood, 2000) and is essential for implantation and proliferation of primordial germ cells (PGC). In each group, genes have been cloned and recombinant protein expressed and purified for testing in possums. We expect that these target molecules will be possum specific and not affect non-target species such a birds and other mammals.
Future intentions and progress expected: All target molecules should be tested for fertility control in colony animals by 2004, in animals in limited field trials (for selected targets) by 2006 and adapted to the best delivery system by 2010.
Objective 1
Objective Title: Assessment and Cloning of Vesicle-Associated Proteins (VAPs)
Research Leader: Prof. Lynne Selwood
Dr Shuliang Cui
Description
Aims: The gene vap5 has been already cloned (Selwood and Cui, in preparation) and a recombinant protein preparation protocol established for it. It is a protein with extensive possum unique sequences that was isolated from vesicle-rich oocyte fractions. We will determine whetherVAP5 recombinant protein initiates an immune response in the possum and begin assessment of its effect on embryonic development and oogenesis in vivo and in in vitro assays. VAP7 is another unique possum protein identified that we will clone and develop its recombinant protein technology for subsequent testing.
Outcomes: Because these proteins are unique to possums and have a significant role to play in lineage allocation in marsupial embryonic development, we expect response to them to include inhibition of normal lineage allocation in embryos. Because they are formed very early in oogenesis in primordial follicles, or earlier, it is possible that an immune response to them would also target oogenesis. A paper on vap5 would be submitted by June 2002 and one on vap 7 would be in progress.
Objective 2
Objective Title: Assessment and cloning of Coat Proteins
Research Leader: Prof. Lynne Selwood
Dr Shuliang Cui
Description
Aims: For the coat protein (CP4) that has already been cloned, characterised and expressed as a recombinant protein (Cui and Selwood, in prep.), we will test its ability to raise an immune response in possums, examine its effect on breeding in possums and examine its secretion pattern and the effect if its antibody on uterine tissues in vitro. This coat protein has major unique sequences and is expected to be a novel protein. Two other coat proteins, CP2 and CP5, have been identified and are also appear to have major unique sequences. We will clone, characterise and prepare the recombinant protein for one of these.
Outcomes: Because the shell coat is marsupial specific and we have demonstrated earlier that it is essential for normal blastocyst formation and for maintenance of blastocyst epithelia (Selwood, 2000), we expect an immune response to the shell coat proteins to terminate embryonic development well before implantation stages. In support of the essential role of the shell, earlier studies (Casey, 2000) have also shown that shell coat secretion continues in the possum until the late trilaminar blastocyst stage. We plan to have submitted a paper on cp4 and have begun preparation of a paper on cp2/5 by June 2002.
Objective 3
Objective Title: Assessment of Leukemia Inhibitory Factor (LIF)
Research Leader: Dr Shuliang Cui and Professor Lynne Selwood
Description
On the basis of what is known of LIF biology, interfering with normal LIF function should affect normal embryonic development at implantation which occurs at the flat embryo stage in the possum (Fletcher and Selwood, 2000) and also primordial germ cell (PGC) proliferation (Matsui et al., 1992) which starts in the possum at the primitive streak stages, prior to implantation around day 10-12.
Aims: Using the possum LIF gene and recombinant protein already produced we will determine whether LIF protein induces an immune response in possums and whether it affects embryonic development at implantation, and gametogenesis in vivo and in vitro.
Outcomes: As LIF is essential for implantation and PGC cell proliferation we expect the immune response to LIF to cause abortion of conceptuses at the flat embryo stage and germ cell proliferation to be reduced or lost. A paper on LIF function in vivo and in vitro will be submitted by June 2002.
3.1.3 PBC 223: Control of reproduction in possums by targeting the pituitary gland through the use of GnRH-toxin conjugates
| Programme Title: | Control of reproduction in possums by targeting the pituitary gland through the use of GnRH-toxin conjugates |
| Programme Leader: | Dr Doug Eckery |
| Institution: | AgResearch |
Programme Goal: To sterilise male and female possums by specifically ablating pituitary gonadotrophe cells through the use of GnRH-toxin conjugates.
Context: The work outlined in this proposal complements our FRST and MAF Policy funded research that focuses on understanding pituitary function in possums and targeting toxins to the pituitary gland. Our research to date has established that the use of GnRH-toxins to control fertility in possums is feasible and we have developed the assays and methodologies necessary to test the effects of GnRH-toxins in vivo. The work in this proposal is aimed at testing the effects of GnRH-toxins on reproductive function in possums, producing a recombinant GnRH-toxin and identifying alternative toxins for use in this technology.
Our research programme is closely linked to the possum research programme at AgResearch, Invermay where the formulation and delivery of baits containing putative sterilising reagents that we discover will be tested. In addition, the possum colony at Invermay is a valuable resource that we and other groups are becoming increasingly dependent upon.
Rationale: The majority of methods being investigated to control fertility in possums rely on the immune system to be effective. We are also looking at such methods, however, despite many years of research in a variety of species, the development of an effective fertility control vaccine has yet to be achieved. Therefore, we feel there is a need to explore other methods of fertility control that do not rely on the immune system. To this end, we have been investigating pituitary function and the use of gonadotrophin-releasing hormone (GnRH) to deliver toxins specifically to cells in the pituitary gland to cause permanent sterility in possums. Results from our previous MAF Policy contracts have been successful in providing a fundamental understanding of pituitary function and the interactions between GnRH and its receptor, including binding characteristics and rate of internalisation; and provided evidence that a toxin (pokeweed antiviral protein; PAP) conjugated to GnRH (GnRH-PAP) is capable of specifically killing possum pituitary gonadotrophe cells in culture. Thus, the feasibility for using GnRH-toxins to control fertility in possums has been established and now remains to be tested. Professor Terry Nett, Colorado State University (CSU), who is pioneering this technology for controlling fertility in animals has agreed to have one of our technical staff work in his laboratory for 4-6 weeks. This will enable us to obtain the large amounts of GnRH-PAP needed to do in vivo studies and to obtain new reagents and learn the techniques required to ensure the quality of GnRH-PAP and its effectiveness. Other work in this proposal will explore the possibility of producing a recombinant GnRH-PAP fusion protein. If successful, this would represent an essentially unlimited source of GnRH-PAP and possibly provide new insights into how such a reagent might be delivered to possums. We have formed a collaborative agreement with experts in toxicology at AgResearch, Ruakura who will assist us in exploring the use of alternative toxins in this technology. We are very encouraged by the enthusiasm they have expressed and their positive attitude towards the success of this programme. We also plan to hire a post-doctoral fellow to work in this programme whose primary area of focus will be on the discovery of new toxins to use in hormone-toxin conjugates.
Objective 1
Objective Title: Production and testing of GnRH-PAP.
Research Leader: Dr Doug Eckery.
Description:
The aims of this objective are to produce a large amount (~300mg) of GnRH-PAP for testing in possums and to learn new techniques for assessing the quality, stability and effectiveness of GnRH-toxin conjugates. It is proposed that Evelyn Bauer, who has been working for the last year and a half on the GnRH-toxin project, visit the laboratory of Prof. Terry Nett at Colorado State University to learn to make GnRH-PAP, perform the quality controls and learn other techniques relevant to the testing of GnRH-toxins in vitro. For example, she will learn how to perform an ELISA for measuring intact GnRH-PAP that will then be used to determine the stability of the conjugate in possum serum. She will also learn how to work with immortal cell lines that express the GnRH receptor (L_T2 and _T3 cells) for future screening and testing of new batches of GnRH-PAP and other GnRH-toxin conjugates. These techniques and reagents will be established in our laboratory at Wallaceville. In addition, we will determine the in vivo ED50 for GnRH in possums to help decide on appropriate doses of GnRH-PAP to use.
Objective 2
Objective Title: Recombinant GnRH-PAP.
Research Leader: Dr Jun Sheng Lin.
Description
In a recent paper, a group in France reported the production of GnRH-PAP as a recombinant fusion protein that was able to specifically kill cells expressing the GnRH receptor. These results present the possibility of having an unlimited source of GnRH-PAP and this use of recombinant protein technology may lead to new ways of delivering hormone-toxin conjugates. Thus, the aim of this objective is to produce biologically active recombinant GnRH-PAP.
Objective 3
Objective Title: Alternative toxins.
Research Leader: Dr Lloyd Moore.
Description
The toxin we are currently using, PAP, is highly efficient, but also a relatively large protein that may be difficult to deliver in a bait. Our aim in this objective is to find new toxins that are small molecular weight and suitable for conjugation to GnRH. our colleagues at AgResearch, Ruakura have recommended we start with quinone derivatives.
3.1.4 PBC 224: Control of reproduction in possums by targeting the oocyte-specific growth factors, GDF-9 and GDF-9B
| Programme Title: | Control of reproduction in possums by targeting the oocyte-specific growth factors, GDF-9 and GDF-9B |
| Programme Leader: | Dr Doug Eckery |
| Institution: | AgResearch |
Programme Goal: To sterilise female possums by immunising them against GDF-9 and /or GDF-9B.
Context: The work proposed in this bid complements our FRST and MAF Policy funded research that focuses on understanding reproductive biology in possums and controlling their fertility. Growth and differentiation factor-9B was recently found to be the gene responsible for mutant phenotypes observed in Inverdale sheep. This discovery has been of great interest to the international scientific community and has the potential to lead to the development of new treatments for enhancing and blocking fertility. In collaboration with colleagues at the University of Helsinki, our group has filed patents for the use of GDF-9 and GDF-9B as sterility agents.
Rationale: In past communications with MAF Policy, we have indicated that moving into research involving GDF-9 and -9B was being considered. To date, we have cloned and sequenced cDNAs for both of these growth factors in possums and have determined the ontogeny of their expression during development of the ovary in pouch young and at the various stages of follicular growth. We have found, as in other species, that both GDF-9 and -9B are oocyte-specific, but of particular interest was the observation that GDF-9B is expressed at an earlier stage of follicular development than in all other species reported to date. To us, this earlier expression represents the possibility of targeting the pool of non-growing follicles within the ovaries, or in other words, all the eggs within the ovaries. Recent work within our group has identified a region of the GDF-9B protein that is non-conserved between species, but appears to be very important for function. In sheep that were immunised against a peptide representing this region of GDF-9B, follicular growth was severely suppressed and the sheep became sterile. Follow-up studies showed that the induction of sterility was very rapid (e.g. less than 14 days). The fact that the sterile Inverdale sheep phenotype can be induced pharmacologically is a significant finding and these results form the most promising lead yet for a candidate that causes sterility using the immune system. These results are very exciting and present the possibility of developing a permanent form of fertility control that is species-specific.
Objective 1
Objective Title: Immunisation of possums against GDF-9 and GDF-9B
Research Leader: Dr Doug Eckery
Description:
Both GDF-9 and GDF-9B are oocyte-specific growth factors. Mutations in these growth factors or their absence can lead to infertility by blocking early follicular growth. The aim of this work is to determine the effects of immunising possums against these growth factors on ovarian function and reproductive activity.
3.1.5 PBC 225: Controlled oestrus and synchronised breeding in possums to facilitate the testing of new biocontrol agents
| Programme Title: | Controlled oestrus and synchronised breeding in possums to facilitate the testing of new biocontrol agents |
| Programme Leader: | Dr Bernie McLeod |
| Institution: | AgResearch |
Programme Goal: To synchronise oestrus and mating in possums to aid in the effective testing of biocontrol agents.
Rationale: Prerequisite to the field-testing of putative biocontrol agents in a situation where potentially critical elements (e.g. the stage of the oestrous cycle when the biocontrol agent is applied) cannot be controlled, will be the need to determine their effectiveness in large groups of possums under tightly controlled conditions. This will require the ability to synchronise oestrus and mating. Currently, we are unable to achieve this in possums. Our research to date has shown that traditional methods of oestrus synchronisation do not work in possums. It has highlighted significant differences in the hormonal control of follicle development and ovulation in possums, some of which have been confirmed at the cellular level by the allied FRST-funded programme (Genetic and Hormonal Control of Ovulation, AgResearch Wallaceville).
We have recently taken the view that, due to their long follicular phase compared with that in other animals in which hormonal control of ovulation is used with considerable success (e.g. sheep, cattle, goats, deer), there needs to be two parts to a strategy for synchronising ovulation in possums. Firstly, a treatment is required that will synchronise the emergence of a preovulatory follicle in a group of animals, and secondly, another treatment must then be given to synchronously induce the rupture (ovulation) of these follicles. The most successful hormonal treatment that we have developed to date, results in the presence of a presumptive preovulatory follicle (approx. 6mm diameter) on a single day (Day 7 after steroid implant removal), in 80 percent of animals treated. The objective of this programme is to (i) assess the viability of these induced preovulatory follicles and (ii) develop a protocol that will consistently induce they synchronous ovulation of these induced follicles.
This programme is aimed at providing a tool to evaluate the effectiveness of new biocontrol agents. The value of a biocontrol agent will be measured as the percentage of animals that it affects in a possum population. The ability to synchronise mating will provide the means of testing an agent on large numbers of possums at a known and predetermined age, or at a known and predetermined stage of their reproductive cycle.
Objective 1
Objective Title: Hormonal control of follicle development.
Research Leader: Dr B J McLeod.
Description:
We have developed a steroid-hormone regimen that will synchronise the emergence of a preovulatory follicle in a high percentage of possums. In this objective, the viability of the follicles (their ability to ovulate and be fertilised) will be assessed. The expression of mRNA for pituitary hormone receptors in both induced and spontaneously developing preovulatory follicles will be determined, to gain insight into ovulatory mechanisms in the possums.
Objective 2
Objective Title: Synchronised ovulation and mating.
Research Leader: Dr B J McLeod
Description
Hormonal treatments given to mimic the preovulatory LH surge fail to induce ovulation of large presumptive preovulatory follicles in possums. In this objective we will determine whether there are hormonal components other than the preovulatory LH surge, that are required for ovulation to occur. Further, being able to successfully synchronise the time of ovulation is not the endpoint to this objective. The synchronised oestrus must be accompanied by expression of oestrus and fertile mating.
Contact for Enquiries
Farm Monitoring Programme Manager
Monitoring and Evaluation
MAF Policy
PO Box 2526
Wellington
NEW ZEALAND
Phone: +64 4 894 0623
Fax: +64 4 894 0741
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