- Sperm in the female tract - transport, storage and capacitation
- Blocking embryonic development in the brushtail possum by targeting the mucoid and shell coats, the extracellular matrix and LIF protein
- Transfer of antibody from mother to pouch young after immunisation against androgen receptor
- Transmucosal delivery of peptides to possums
- Controlled breeding technologies
- Control of reproduction by targeting the possum pituitary gland through the use of GnRH-toxin conjugates
3.1 Physiology & Reproduction
| Programme Title: | Sperm in the female tract - transport, storage and capacitation |
| Programme Leader: | Professor John Rodger |
| Institution: | Marsupial CRC |
| 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:
Complete ultrastructural examination of sperm-oviduct cell and sperm-egg interactions in vivo. Continue IVF experiments using in vivo capacitated sperm, and EM of sperm-bound zona pellucida penetrated eggs from these experiments. Complete biochemical evaluation of (whole) oviduct fluid collected from possums during the pre- and post-ovulatory period, and compare with separate ampullary and isthmic fluid collected from possums during the same period.
Objective 2
| Objective Title: | In vitro studies |
| Research Leader: | Dr Kuldip Sidhu |
Description:
Complete ultrastructural examination of sperm-oviduct cell interactions in vitro using OECM sperm co-culture system. Continue IVF experiments using in vitro capacitated sperm from OECM and CM sperm co-culture systems, and EM of sperm-bound zona pellucida penetrated eggs from these experiments. Characterise motility patterns (Hobson Tracker), surface studies (lectins and monoclonal antibodies) and ultrastructure (EM) of definitive populations of in vitro capacitated sperm from CM sperm co-culture system. Identify and characterise oviduct secretory proteins and in the longer term, the genes that encode them. Raise antibodies against most promising proteins and examine effect of blocking proteins with anitbody on sperm function.
| 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.
| Programme Title: | Transfer of antibody from mother to pouch young after immunisation against androgen receptor |
| Programme Leader: | Professor Des Cooper |
| Institution: | Macquarie University |
| Programme Goal: | To study the of transfer of antibody from mother to offspring, using as antigen peptide from the androgen receptor molecule whose corresponding antibody could affect postnatal male sex differentiation. |
Objective 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.
| (i) | Androgen receptor (AR) peptide coupled to keyhold limpet haemocyanin (KLH) in Freund’s adjuvant. |
| (ii) | AR peptide coupled to Bovine Serum Albumin (BSA) in Freund’s adjuvant. |
| (iii) | 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 D.W. 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. We 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 used possums in the previous MAF funded project on mother-PY immunological relationships.)
| Programme Title: | Transmucosal delivery of peptides to possums |
| Programme Leader: | Dr Bernie McLeod |
| Institution: | AgResearch, Wallaceville |
| Programme Goal: | Assess methods of maximising absorption of peptides and proteins across the mucosa of the gastrointestinal tract of the possum using metabolic inhibitors and permeation enhancers to promote uptake. |
Description:
This objective will:
- Determine the rates of metabolism of a peptide (GnRH) and of a protein (BSA) in luminal fluid collected from different sites of the possum gastrointestinal tract and assess the effectiveness of bile salts and of carbopol to inhibit proteolysis.
- Establish the optimum conditions for maintaining preparations of possum gut mucosa in Ussing chambers and methods for monitoring rates of transfer of peptides and proteins across the tissue, and then assess the effectiveness of a permeation enhancers to increase mucosal permeability.
- With whole tissue preparations, determine the rates of in vitro absorption of a peptide (GnRH) and of a non-metabolisable marker (mannitol) across two regions of the gut, and assess the ability of at least two permeation enhancers to increase them.
One paper will be presented at a scientific conference and one paper will be submitted to a refereed international journal.
| Programme Title: | |
| Programme Leader: | Dr Jerome Demmer |
| Institution: | AgResearch, Wallaceville |
| Programme Goal: | To determine the classes and quantities of immunoglobulins milk, and the characterisation of the mechanism required to transport them into the milk for development of methods for the biological control of possums. |
Objective 1
| Objective Title: | Possum Immunoglobulins |
| Research Leader: | Dr Jerome Demmer |
Description:
This objective will:
- Develop an antibody raised against possum IgA produced by recombinant DNA methods.
- Develop methodology for quantifying the immunoglobulins present in possum milk.
- Determine the levels of immunoglobulins present in possum milk throughout lactation.
Objective 2
| Objective Title: | Mammary immunoglobulin receptors |
| Research Leader: | Dr Jerome Demmer |
This objective will:
- Determine the RNA expression patterns for the polymeric immunoglobulin receptor in the lactating mammary gland.
- Screen a possum mammary gland cDNA library with a molecular probe for possum immunoglobulin G receptors.
- Make a DNA construct for the possum polymeric immunoglobulin receptor that can be expressed in eukaryotic cells.
| Programme Title: | Controlled breeding technologies |
| Programme Leader: | Dr Bernie McLeod |
| Institution: | AgResearch, Wallaceville |
| Programme Goal: | To develop methods of synchronising oestrus, ovulation and mating in possums during the breeding season, and of inducing fertile ovulation during seasonal anoestrus. |
Objective 1
Description:
The aim of this objective is to;
- Investigate the pharmacokinetics of progesterone metabolism in ovariectomised possums and use this information to develop silastic progesterone implants for controlling oestrus.
- Evaluate the efficacy of exogenous FSH and LH/GnRH to induce follicle development and ovulation.
- Compare the effectiveness for induced follicle development of crude pituitary extracts (as a source of possum gonadotrophins) with purified hormones from other species.
One paper will be submitted to a refereed international journal
| Programme Title: | Control of reproduction by targeting the possum pituitary gland through the use of GnRH-toxin conjugates |
| Programme Leader: | Dr Andrew Fidler |
| Institution: | AgResearch, Wallaceville |
| Programme Goal: | To control reproduction by targeting the possum pituitary gland through the use of GnRH-toxin conjugates. |
Objective 1
Description:
To express the possum GnRH-receptor gene in Saccharomyces cerevisiae thereby developing a simple in vitro bioassay to monitor the biopotency of GnRH-conjugated toxins by:
- Cloning of possum GnRH-receptor coding sequence into yeast expression vectors.
- Expression of possum GnRH-receptor gene in yeast.
- Assaying the binding affinity of the recombinant GnRH-receptor for mammalian GnRH, chicken type II GnRH and a range of GnRH analogues.
- Screening actions of GnRH-toxins on yeast strains expressing the recombinant possum GnRH-receptor to select conjugates with high affinity and toxic activity.
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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