3.1 Physiology & Reproduction
3.1.1 PBC 200
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| Programme Goal: | To develop GnRH toxin conjugates which will sterilise both male and female possums without causing any other side-effects leading to ill-health. |
| Objective 1 | |
| Objective Title: | Synthesis of GnRH analogue-polkweed antiviral protein (PAP) |
| Research Leader: | Dr T Nett - Colorado State University |
| Description: | Dr Nett will prepare 2ng GnRH-PAP conjugate for testing using methodology he has developed. |
| Objective 2 | |
| Objective Title: | Testing the toxicity effects on possums |
| Research Leader: | Dr D Eckery |
| Description: | PAP clone, GnRH-PAP, GnRH alone and saline will be tested on both male and female possums (n=3 males, 3 females per treatment group). |
| Objective 3 | |
| Objective Title: | Assessment of comparative bindability of 125I-GnRH and GnRH-PAP |
| Research Leader: | Dr D Eckery |
| Description: | Recover piturity glands, gonads, sliver, heart and brain from 100 possums and prepare 30,000 g homogenates to test bindability of 125I-GnRH. Establish characteristics of binding and determine ability of a number of known analogues and GnRH-PAP to competer for 125I-GnRH binding. |
| Objective 4 | |
| Objective Title: | Efficacy testing of GnRH-PAP conjugate |
| Research Leader: | Dr D Eckery |
| Description: | Test effect of different doses of GnRH-PAP conjugate in vivo to suppress plasma concentrations of FSH add LH and measure antibody titres against GnRH and/or toxin. |
Objective 5
| Objective Title: | Development of a bioassay system | |
| Research Leader: | Dr D Eckery | |
| Description: | Develop either an ovine or possum pituitary cell culture system as a bioassay for the GnRH-toxin system. |
3.1.2 PBC 201
|
| Programme Goal: | To better understand the cellular basis of sperm transport, storage and capacitation in the possum female tract and thereby (a) identify promising biocontrol targets in these processes and (b) generate in vivo and in vitro sources of capacitated sperm for IVF. |
Objective 1
| Objective Title: | In vivo studies |
| Research Leader: | Dr Frank Molinia |
Description:
Using the superovulation/AI system already established, and with on-going refinements of this protocol, this objective will establish the role of the female tract in preparing sperm for fertilization. A critical element is the definitive establishment of the fully capacitated sperm. Although motile "T-shape" sperm is one indicator of capacitation, studies in other marsupials suggest that sperm surface modifications are involved in achieving the capability to bind to, and then penetrate the egg coat. Researchers have established that sperm are stored in the isthmus of the oviduct and their subsequent release appears to be related to ovulation induced changes in the oviduct environment. Finally, the proteins of the oviduct secretions, likely agents for inducing capacitation, will be identified and characterised.
Objective 2
| Objective Title: | In vitro studies |
| Research Leader: | Dr Kuldip Sidhu |
Description:
Preliminary in vitro studies have been conducted using: whole organ culture, explants and oviduct epithelial cell monolayers and conditioned media from these culture. Under these in vitro culture conditions, changes to possum sperm associated with capacitation such as conversion of motile sperm to "T-shape" and alterations to acrosome stability were observed. This objective plans to establish the optimum systems for sperm-oviduct co-culture and better characterise the changes that sperm undergo during culture. This will establish if specific cell-cell interactions and/or paracrine regulation by products of the oviduct are involved in capacitation. Antibodies will be raised against the most promising products and the effects of blocking proteins with antibody on sperm function will be examined.
3.1.3 PBC 202
|
| Programme Goal: | To complete the project to identify features of early embryonic development in possums that are unique to marsupials and critical for embryonic survival and are related to molecules produced by the egg, embryo or reproductive tract. |
Objective 1:
| Objective Title: | Egg coats and embryonic survival |
| Research Leader: | Dr Lynne Selwood |
Description:
To determine the effect of removal of the shell/mucoid coat on embryonic survival in vitro; to use collected coats to prepare proteins to raise antibodies. Because the shell is a marsupial-specific protein deposited on the egg, with no homologue in eutherians and the mucoid coat has a homologue in the rabbit, these proteins are ideal for marsupial contraception and may provide a rabbit control mechanism as well. In other marsupials, egg coats are essential for long term embryonic survival in vitro. Development of coat-free embryos will be analysed by electron microscopy and will result in submission of paper and report MAF Policy in October 1997.
Objective 2:
| Objective Title: | Blastomere-zona adhesion and embryo survival |
| Research Leader: | Dr Lynne Selwood |
Description:
To identify the relationship between secretion of extracellular matrix (ECM) by blastomeres (identified in 1994) and normal blastomere-blastomere-zona adhesion in blastocyst formation. These processes are unique to marsupials and monotremes, essential to normal embryo formation and are implicated in the separation of embryonic and trophoblast lineages. ECM deficient embryos will be analysed by EM to determine cause of embryonic failure after development in vitro. Initial steps undertaken in preparation of the OCT4 probes for identification of embryonic versus trophoblast lineages. Submission of publication on the role of ECM in development of normal embryos and report to MAF Policy in October 1997.
3.1.4 PBC 203
|
| Programme Goal: | To identify and evaluate antigenic proteins involved in essential, early embryonic developmental processes in the possum: Oocyte ECM (extracellular matrix), egg coats, LIF (leukaemia inhibitory factor). |
Objective 1
| Objective Title: | ECM and normal blastocyst formation. |
| Research Leader: | Dr Lynne Selwood |
Description:
Collection of ECM and prepare an antibody against it. The researchers previous project determined that the ECM is formed in the oocyte during oogenesis and secreted by the conceptus during early cleavage and that polarised secretion of ECM is essential for normal blastocyst development. Principal research components will be:
- collection of ovaries for DNA, RNA and protein;
- segregation and collection of ECM vesicular material from oocytes;
- separation of ECM proteins on gels;
- raising of antibodies to ECM;
- immunohistochemistry of ovaries and embryos to confirm ECM identity; and
- confirmation of embryo normality using OCT 3/4 gene probe by in situ hybridisation.
The second year of the project will involve sequencing ECM proteins and cloning of ECM genes to produce proteins for immunisation. A report on cross reactivity between the antibody and the oocyte and embryonic ECM will be presented at the end of 8 months. Work for a paper on this will be completed at the end of the 1998 breeding season (October).
Objective 2
| Objective Title: | Coat antibodies, coat production and embryo development. |
| Research Leader: | Dr Lynne Selwood |
Description:
Determine the effect of antibodies raised previously on coat production in the tract and embryo development in vitro. If mucoid and shell coats are to be used as targets in contraception, the effects would be two-fold; to inhibit production of the coats in the tract and to interfere with normal embryonic function as previously shown. The research will consist of cross matching coat antibodies with coat proteins run on gels to identify coat layer; collection of tracts, embryonic material and coats and tissues for DNA and RNA; immunohistochemistry of the tract and embryos; sequencing of coat proteins; testing the effects of coat antibodies on coat production in oviduct and uterine monolayers and on embryos in vitro. The protein sequences will be used for gene cloning and protein production for immunisation in 1998/9. A paper on the effect of coat antibodies on coat production and embryo development in vitro and on the nature of coat proteins will come from this work.
Objective 3
| Objective Title: | Cloning the possum LIF gene |
| Research Leader: | Dr Lynne Selwood/Mr Shuliang Cui |
Description:
Clone the possum LIF gene. LIF protein is a polyfunctional cytokine which in the mouse suppresses differentiation of embryonic stem cells in vitro, stimulates proliferation of leukaemic cells and is essential for implantation. Hypoblast cells do not develop in its absence and it is secreted by extraembryonic cells as well as by the uterus. Its function in marsupials is unknown but Mr Cui et al (unpublished) have shown that it is a powerful suppressor of murine ES cell differentiation. It has been cloned recently in a marsupial S. crassicaudata and its in vivo expression and ability to maintain pluripotency assessed by Mr Cui (Ph.D candidate).
The research will involve:
- southern analysis of possum DNA;
- preparation and screening of a possum genomic DNA library to isolate the LIF gene; and
- assessment of RT-PCR technique to clone the possum LIF.
A report on Southern analysis of and screening of Possum DNA using S. crassicaudata or mouse probes will be finished by June 1998. Completion of cloning of possum LIF, expression and purification of possum LIF protein and some analysis of function will occur in 1998/9.
3.1.5 PBC 204
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| Programme Goal: | To study the of transfer of antibody from mother to offspring, using an antigen peptide from the androgen receptor molecule whose corresponding antibody could affect postnatal male sex differentiation. |
Objectives 1
| Objective Title: | Mother-to-offspring anti-AR antibody transfer |
| Research Leader: | Professor Des Cooper |
Description:
Females are immunised before the breeding season using a 13 amino acid AR peptide using one of three different immunisation regimes:
- Androgen receptor (AR) peptide coupled to Keyhold Limpet Cyanin (KLH) in Freund's adjuvant;
- AR peptide coupled to Bovine Serum Albumin (BSA) in Freund's adjuvant; or
- AR peptide coupled to KLH without adjuvant.
Antibodies to both the AR peptide and KLH are to be measured in maternal serum, milk and pouch young serum. Female offspring are to be removed as soon as they can be sexed which allows milk collection. Male offspring are retained to study their antibody levels and their later sexual development.
Objective 2
| Objective Title: | Anti-AR antibodies and male sex differentiation |
| Research Leader: | Professor Des Cooper |
Description:
The male pouch young which are exposed to anti-AR antibodies may have some of their specific male characters compromised, anatomically, physiologically, or behaviourally. The researchers will search for anatomical changes by histological examination of all male specific structures in 90 day old pouch young. Physiological examination will involve estimation of steroid and pituitary hormone levels in males after weaning.
Observations on sexual behaviour, especially mating behaviour, will take place from one year onwards, the earliest at which male possums can mature. (In the second half of 1997 the histological and physiological work will use possums in the previous MAF funded project on mother-PY immunological relationships.)
3.1.6 PBC 205
|
| Programme Goal: | To investigate the absorption of antigens and peptides across the mucosal tissue of the gastrointestinal tract in the possum, and to develop methods that inhibit their metabolism and enhance their stability, permeation and uptake. |
Objective 1
| Objective Title: | Transmucosal delivery of peptides to possums |
| Research Leader: | Dr Bernie McLeod |
Description:
Assess the methods of maximising the absorption of bioactive peptides and proteins across the mucosa of the gastrointestinal tract of the possum, using both in vitro and in vivo methods to determine sites of metabolism and to assess the efficacy of permeation enhancers and bioadhesives to promote uptake. This objective will be achieved by:
- Determining regional differences in pH and in peptide metabolism and absorption in the gastrointestinal tract.
- Evaluating the effectiveness of metabolic inhibitors on peptide stability within the gastrointestinal tract.
- Testing the effectiveness of permeation enhancers on antigen and peptide absorption.
- Determining the efficacy of bioadhesives to increase the rate and extent of peptide absorption.
Submit one paper to a refereed international journal and produce an annual report to MAF Policy.
3.1.7 PBC 206
|
| Programme Goal: | The aim of this research is to identify the classes of immunoglobulins present in possum milk and to determine the receptor pathways required for transporting these molecules across the mammary epithelium during lactation. |
Objective 1
| Objective Title: | Immunoglobulins in possum milk | |
| Research Leader: | Dr Jerome Demmer |
Description:
The possum pouch young receives immunoglobulins from the milk of its mother over the first 100-120 days of lactation. These immunoglobulins compensate for the immature immune system present in the pouch young at this time. In eutherian mammals the predominant class of immunoglobulins in colostrum varies between species. In cattle, where immunoglobulins are transferred to the calf after birth (i.e. via colostrum), IgG is the predominant class, whereas in the pig, where immunoglobulin transfer occurs predominantly during pregnancy (i.e. across the placenta), IgA is the major immunoglobulin in colostrum. It is not known which classes of immunoglobulins are in possum milk, but it seems likely that IgG will be present. In these experiments the levels and types of immunoglobulins in possum whey will be identified with anti-sera to IgG and IgA produced at AgResearch Wallaceville. The types of immunoglobulin identified in possum milk will determine the receptors chosen for further analysis in Objective 2.
Objective 2
| Objective Title: | Cloning of mammary immunoglobulin receptors | |
| Research Leader: | Dr Jerome Demmer |
Description:
Transmembrane receptors, specific to each class of immunoglobulins, are required to transport these molecules across the mammary epithelium and into milk. In eutherian mammals, IgA and IgM are transferred by the polymeric immunoglobulin receptor (pIgR), whilst both high and low affinity gamma immunoglobulin receptors (FcR-I, -II and -III) transport IgG. DNA sequences for these receptors have been determined for several species of eutherian mammals. The researchers will use this information in polymerase chain reaction (PCR) and cDNA cloning procedures to isolate DNA probes for possum immunoglobulin receptors present in mammary tissues. These immunoglobulin receptor probes will then be used to establish the pattern of RNA expression in possum mammary tissues during lactation. The cloned receptors can then be used to initiate in vitro functional studies examining how to best target these receptors for possum biocontrol.
3.1.8 PBC 207
|
| Programme Goal: | To establish reliable and predictable methods of synchronising oestrus and ovulation in possums that result in natural mating, normal pregnancy and birth, and to quantify parameters of reproductive tract remodelling that are essential for reproductive success. |
Objective 1
| Objective Title: | Controlled breeding technologies | |
| Research Leader: | Dr Bernie McLeod |
Description:
Evaluation of methods of synchronising oestrus, ovulation and mating in female possums during the breeding season, of inducing ovulation in the non-breeding season and of superovulation. This will be achieved by:
- Determining the magnitude and duration of the preovulatory LH and FSH surge in naturally cycling possums.
- Evaluating the steroidal control of oestrus in possums by administration of exogenous hormones during seasonal anoestrus.
- Determining the efficacy of prostaglandins to induce luteolysis at different times during the luteal phase.
- Assessing the effects of exogenous FSH and LH of various species, to induce follicle development and superovulation.
- Evaluating the functional competence of corpora lutea following induced ovulation.
- Accessing mating and pregnancy success at induced/synchronised ovulations.
Present one paper at a scientific conference and submit one paper to a refereed international journal and produce an annual report to MAF Policy.
3.1.9 PBC 209
|
| Programme Goal: | To sterilise male and female possums by inhibiting germ cell maturation in neonatal life through antibodies targeting the growth factor SCF and its receptor c-kit. |
Objective 1
| Objective Title: | C-kit: Cloning, expression and bioassay. |
| Research Leader: | Dr Andrew Fidler |
Description:
Possum c-kit has proven to be a difficult gene to clone. At present a partial cDNA clone, corresponding to about one third of the extracellular domain of this transmembrane receptor, has been cloned and sequenced but a clone of the entire extracellular domain is required for expression in E. coli and the subsequent raising of antibodies for passive immunisation experiments. The completion of the cloning of the possum c-kit coding region is the first aim of this objective.
An in vitro bioassay is required to test both the bioactivity of recombinant possum SCF and the inactivating action of antibodies raised against both recombinant SCF and its receptor c-kit. The establishment of a cell-line based bioassay is the second aim of this objective.
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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