3.1 Physiology and Reproduction

Research is aimed at gaining a basic understanding of the development of possum sexual organs and investigating ways to disrupt that development. This includes isolating, characterising and investigating the expression of genes involved in male and female sexual organ development. Experience gained in genetic investigation of other animals such as mice can be used in studying possum genetics. Some research has focussed on the structure and function of sexual and mammary organs. Other research has focussed on possum growth and thermoregulation with the aim of finding ways to disrupt those processes.

3.1.1

Programme Title:Isolation and characterisation of genes involved in sex determination and sperm formation in the brushtailed possum Trichosurus vulpecula

Programme Leader: Professor J Graves

Institution:La Trobe University

Summary

This programme aims to isolate and characterise possum genes involved in possum sex determination and sperm formation. Once genes are isolated, the function of the genes, and their potential as a biological control of possums will be examined.

In order for researchers to investigate various genes believed to be involved in sperm formation and sex determination, construction of libraries of genetic information was necessary. From the libraries, various genes were investigated and some being partially or completely cloned for the following possum genes; SRY, DAX1 RBM1, DAZ, UEB1Y and TSPY genes.

Objective 1:
Construction of possum gene libraries
Research Leader: Professor J Graves

Objective 2:
Isolation of genes involved in possum sex determination
Research Leaders: Professor J Graves/Dr D Hill

Objective 3:
Isolation of possum genes involved in spermatogenesis by identifying and cloning the possum homologues of several Y-borne genes thought to be involved in mammalian spermatogenesis.
Research Leaders: Professor J Graves/Dr D Hill

Objective 4:
Characterization of possum genes involved in sex determination
Research Leader: Professor J Graves

Objective 5:
Characterization of possum genes involved in spermatogenesis for homology with known human and mouse genes.
Research Leader: Professor J Graves

Description - Objective 1: Construction of possum gene libraries

To construct lambda and cosmid genomic libraries, as well as testis cDNA libraries, using DNA from the possum.

Approach & Outcomes

In order to make copies of genes involved in sex determination and sperm formation, it was necessary for researchers to construct lambda and cosmid genomic libraries, as well as testis cDNA libraries, using DNA from the possum.

The cosmid library from male possum genomic DNA and the cDNA library from adult possum testis DNA have now been constructed and contain sufficient recombinants for them to be considered representative, i.e. it is likely that any possum genomic gene or testis cDNA could be isolated from these libraries. The libraries have been "amplified" and put into long term storage. The possum cosmid library and testis cDNA library form a valuable resource, and are available to other researchers wanting to clone genes from possums.

Description - Objectives 2 & 4: Possum genes involved in sex determination

To isolate and characterise possum genes involved in sex determination.

Approach & Outcomes

The principal targets of this objective were the Y-borne gene SRY, known to be testis determining in eutherian mammals (mammals which develop placentas, which include humans and mice but not possums), and DAX1, a sex-reversing gene on the human X. Other genes of interest are SOX3, which appears to be the gene from which SRY evolved, and may be involved in sex determination, and SOX9, which has been identified as an autosomal (occurs on a chromosome other than the X or Y) sex-reversing gene, and now seems likely to play a major part in normal sex determination in mouse and human.

Possum SRY was partially cloned and characterised. Five cDNA clones were isolated from the cDNA library (constructed under objective 1), using the wallaby SRY clone. Sequencing proved that none of these clones was SRY, and no further clones could be obtained from the library. An alternative polymerase chain reaction (PCR) strategy was therefore followed. A search of databases revealed high homology of the cloned material found to the Dama wallaby Macropus eugenii SRY, and confirmed that the clone was undoubtedly the possum SRY.

Possum DAX1 was partially cloned and characterised. Five positive clones were obtained from the possum testis cDNA library, screened with human DAX1. The clones’ homology with human DAX1 at the 3' (poly-A) end, confirmed that these clones represent possum DAX1.

New information, as yet unpublished, suggests that the SRY-related gene SOX9 does play a conserved role in sex determination in mouse and chicken. It is likely that it interacts directly with SRY, or indirectly (perhaps through the X-borne progenitor of SRY, SOX3). When Southern analysis of male and female-derived possum DNA was performed, several shared bands, as well as the male-specific SRY gene, were observed. Some ofthese were present in an equal dosage in males and females, and may represent autosomal SOX genes, including SOX9. One band showed dosage differences, and may represent the X-borne SOX3 gene.

Description - Objectives 3 & 5: Possum genes involved in spermatogenesis

To isolate and characterise possum genes involved in spermatogenesis.

Approach & Outcomes

The major part of this objective was to clone and characterises the possum homologues of the Y-borne human candidate spermatogenesis genes RBM1 (formerly YRRM), DAZ and TSPY. Deletions of regions of the Y containing these genes in humans and mice produce normal but sterile males, showing specific blocks to meiosis, sperm maturation or motility. Candidate genes cloned from mouse include ZFY, UBE1Y, and SMCY, and those cloned from the human Y are TSPY and YRRM1.

Researchers have screened male and female DNA from a number of marsupials for male-specific sequences homologous to RBM1, DAZ and TSPY. Human RBM1 detects one or more male-specific bands in all species, implying the presence of a Y-borne copy of this gene in marsupials. DAZ and TSPY probes detected only shared bands in all marsupial species, implying that the marsupial homologues are autosomal. Therefore, DAZ and TSPY are likely to play a general role in both sexes, and are consequently of less interest than is RBM1.

Southern analysis of the possum homologues of ZFY and UBE1Y on the human/mouse Y suggests they have a role in spermatogenesis. The former proved to be autosomal, as researchers had discovered for other marsupials, but mouse UBE1Y detected a strong male-specific band in possum DNA. Two clones were obtained from a lambda genomic library screened with wallaby UBE1Y.

Possum RBM1 has been cloned and partially characterized. A cDNA library was screened with the wallaby RBM1 clone, and obtained seven positives, two of which were identical. Two clones have been restriction mapped, and homology of different restriction fragments to the wallaby RBM1 established. When one of these clones was used to probe genetic material containing male and female-derived possum DNA, male-specific bands were detected, after digesting with a range of restriction enzymes. This result could not be due to a single polymorphism and must confirm its origin on the Y chromosome.

Southern analysis using the possum RBM1 clone produces an intense male specific signal, suggesting that RBM1 sequences may be amplified on the Y, as they are in human and mouse (but not in tammar wallaby or Sminthopsis common name also fat-tailed dunnart). Initial sequencing results show that two independent cDNAs have different sequences, suggesting that more than one RBMI gene may be transcribed.

3.1.2

Programme Title:Isolation of possum gonadotropin-releasing hormone cDNA clones

Research Leader: Dr K McNatty

Institution:AgResearch

Summary

This programme aims to isolate genetic material responsible for producing possum gonadotropin-releasing hormone (GnRH), a hormone important in reproduction. Isolation of GnRH producing genetic material will assist researchers working on reproduction.

The possum GnRH gene has been difficult to clone. As a result of the difficulties with cloning GnRH, the objective was changed to obtaining genetic information about possum follicle stimulating hormone (FSH), which was more successful.

Description: Possum gonadotropin-releasing hormone (GnRH) gene

To isolate cDNA and genomic clones of the possum gonadotropin-releasing hormone (GnRH) gene.

Approach & Outcomes

Gonadotropin-releasing hormone (GnRH) is important in reproduction. Last year (June 1994 - June 1995) a possum hypothalamic cDNA library was produced and screened with anti-mGnRH antibodies. Two putative positive clones identified proved, upon DNA sequencing, to be "false positives". However, the DNA libraries produced will be a useful resource for the general possum research effort.

Continued work on investigating the feasibility of screening a possum genomic library with oligomers designed from other mammalian GnRH DNA sequences was done.

Reverse transcriptase polymerase chain reaction (RT-PCR) offers the advantage of being able to amplify from templates of very low abundance. The GnRH gene found in mammals is not highly conserved outside of the small sequence which made finding suitable primers difficult. Using RT-PCR redundant nested primers paired with a universal primer a number (ca. 30) of putative "GnRH" amplification products were cloned and sequenced but none proved to be GnRH.

This objective was extended to include obtaining cDNA clones of genes encoding the _ and ß chains of possum follicle stimulating hormone (FSH). This work has proven to be successful.

The FSH _ gene proved fairly straight forward to both amplify and clone. A cDNA clone has been obtained encoding the complete coding region of the possum FSH _ gene. Comparison of the possum FSH _ gene sequences with that of eutherian mammals shows 72-78% homology at the nucleotide level.

Amplification and cloning of the possum FSH ß gene was more difficult than the FSH _ gene. By varying the PCR conditions, in combination with using a variety of degenerate primers, a partial FSH ß cDNA sequence has been cloned and sequenced and shows 76% homology with rat FSH ß.

3.1.3

Programme Title:Biological control of possum reproduction

Research Leader: Dr K McNatty (Dr Lynne Selwood, La Trobe University)

Institution:AgResearch/(La Trobe University - collaborator)

Summary

This programme aims to gain a basic understanding of the process of sex determination in possums and investigate ways to disrupt this process. Possums are born sexually undifferentiated. The migration of germ cells (reproductive cells) in the early stages of sexual organ development, is essential for sexual differentiation. If changes to the sex differentiation process could be made, it may prove possible to produce animals all of the same sex or lead to sterile animals.

Two genes important in sex differientiation were partially isolated and characterised and their expression examined. Difficulties were encountered in isolating the genes because the process relied on information about gene sequences in other mammals. These were found not to correspond well with the possum gene sequence.

This study is part of a larger project for the researchers in this area.

Description: Possum gonadal development

To gain a basic understanding of possum gonadal development, focusing on the migration of primordial germ cells (PGC) into gonad development by:

  • isolating PGC specific marker genes c-kit, oct 3/4 and stem cell factor.

Approach & Outcomes

Possums are born sexually undifferentiated. The migration of germ cells from the yolk sac to the gonadal primordium (early stages of sexual organs), during embryonic development, is essential for sexual differentiation. A potential strategy for the control of reproduction in possums is to disrupt normal gonadal development by targeting germ cell migration, oogenesis and/or folliculogenesis. This may result in possums all of one sex, or which are sterile.

Two genes important for primordial germ cell migration (PGC) in eutherian mammals are stem cell factor, and its receptor gene, c-kit. A further important gene is the possum germ cell-specific marker gene, oct 3/4.

The method being used to obtain possum c-kit cDNA clones is reverse transcriptase polymerase chain reaction (RT-PCR) using c-kit specific primers based on sequences obtained from eutherian mammals.

First strand cDNA was synthesized from mRNA derived from gonads of cycling and pregnant adult and pouch young possums. A variety of PCR primer combinations corresponding to different parts of the c-kit gene were used in PCR reactions under different conditions on this cDNA. Over the past year a large number of candidate c-kit cDNAs have been amplified, cloned and analysed by nucleotide sequencing, the majority of these isolates were subsequently shown to be genes unrelated to c-kit.

The difficulties encountered in isolating possum c-kit cDNAs may be explained by the poor sequence homology between the gene in possum and other eutherian mammals, making it difficult to design PCR primers with high enough specificity. Recently a 347 base pair product from adult possum ovaries has been amplified and cloned. Nucleotide sequencing has confirmed that the cDNA product encodes amino acids 337 to 453 of the extracellular domain of the c-kit protein. The possum c-kit nucleotide sequence shows approximately 75% homology to this c-kit sequence in eutherian mammals. Possum specific PCR primers have now been designed based on this sequence and are being used in the RACE (Rapid Amplification of cDNA Ends) technique in order to isolate the complete c-kit cDNA. The 347bp possum c-kit sequence will be used as a probe in gene expression studies to determine the size and number of c-kit transcripts and the cellular location of the c-kit mRNA in possum tissues.

A collaborative project was established with Dr Lynne Selwood (La Trobe University, Melbourne, Australia) to isolate the possum germ cell-specific marker gene, oct 3/4.

The purpose of this work was to isolate the germ cell specific gene oct 3/4 from possums. Three fragments from possum genomic DNA, which are homologous to human oct 3/4 and mouse oct 3/4, were cloned. These collectively span a region of approximately 240 base pairs. Sequences from these clones showed the presence of at least two genes with high homology to eutherian oct 3/4 in the possum genome, although it is not known whether one or both of these is functional.

By chance, during this work, two RT-PCR products were cloned which were homologous to other known genes from other species. One of them is important for ovarian-pituitary signalling in eutherian mammals and it is likely to be useful for future studies.

Publications

Greenwood, P.J.; Seamer and Tisdall, D.J. (1996): Cloning, sequencing and expression of stem cell factor (c-kit ligand) cDNA of brushtail possum, Trichosurus vulpecula. Reproduction, Fertility and Development (in press).

3.1.4

Programme Title:The importance of the egg investments for long-term survival of embryos of the Australian brush-tailed possum

Research Leader: Dr L Selwood

Institution:La Trobe University

Summary

This programme aims to examine substances, unique to marsupials, that forms part of the egg coat. In possums, the egg coat has been found to be essential for embryonic survival.

The possum reproductive tract secretes two outer coats, mucoid and shell, around the fertilised egg and cells in the early embryo secrete an inner coat, the extracellular matrix (ECM). Interference with the ECM interferes with the separation of these two cell types.

A method to culture possum embryos and standards for normal development was established and the time of production of the egg coats was identified. The removal of the outer coats was found to prevent embryonic development in early embryos, stop maintenance of normal structures in older embryos and affect cells responsible for nourishing the embryo.

Objective 1:Egg coats and embryonic survival

Objective 2: Blastomere-zona adhesion and embryo survival

Description - Objective 1: Egg coats and embryonic survival

To determine the effects of removal of the egg shell/mucoid coat on embryonic survival in vitro and use collected coats to prepare proteins to raise antibodies.

Approach & Outcomes

Egg coat removal either by microdissection or by a combination of techniques was successfully developed. This allowed experimental analysis of the survival of coat-free embryos in vitro. There was marked cross reactivity between a polyclonal antibody and the possum mucoid coat, and less cross reaction between it and the shell coat.

The following usable sample stages were obtained: stages showing development of oocytes to identify time of production of ECM molecules in oogenesis, embryos during cleavage, unilaminar, bilaminar and trilaminar blastocyte stages, unfertilized eggs or failed embryos and other embryonic stages.

Egg coat removal either by microdissection or by a combination of techniques has been successfully developed. This allowed experimental analysis of the survival of coat-free embryos in vitro. Chemical, biochemical and microdissection techniques on the embryos of Monodelphis domestica (South American Possum), which have a similar coat structure to the brush-tailed possum were trailed. Although the techniques worked, microdissection was found to be the quickest, least intrusive techinque and possum embryos can be removed unharmed from the coats.

It has been determined when and which tissues produce the egg coats using a combination of electron microscopy (EM) and histochemistry. The Zona pellucida deposition occurred in primary oocytes in primary follicles in pouch young aged between 88 and 133 days. The mucoid layer was deposited on the egg in the oviduct and histochemistry showed that it was composed of glycoproteins and sulphated and acidmucopolysaccharides. The shell cost was 8.5 um wide in early cleavage stages and thinned to about 3um wide by the bilaminar blastocyte stage.

Removal of the outer egg coats (shell and outer mucoid coat) from cleavage stage embryos resulted in abnormalities in formation of the blastocyst epithelium especially in the trophoblast region. Coat-free blastocyst stages failed to maintain normal epithelia structures especially in trophoblast and hupoblast tissues. Embryonic growth reduction and failure occurred presumably because of the failure of the trophic lineages (trophoblast and hypoblast) of the conceptus.

Marked cross reactivity between Roberts polyclonal antibody (Roberts et al 1994) and the possum mucoid coat and less cross reaction between it and the shell coat was found. Streptavidin/biotin immunoperoxidase cytochemistry to sections of possum eggs and reproductive tracts confirmed the timing and location of mucoid and shell coat secretion and showed that different marsupial families had some common components in mucoid and coat secretions and the mucoid coat had common epitopes with all five species tested. Species tested were the fat-tailed and stripe-faced dunnarts, brush-tailed possum, domestic rabbit and laboratory mouse.

Description - Objective 2: Blastomere-zona adhesion and embryo survival

To identify the relationship between secretion of extracellular matrix (ECM) by blastomeres and normal blastomere-blastomere-zona adhesion in blastocyst formation.

Approach & Outcomes

Tests to determine whether the outer egg coats are essential for blastocyst construction during cleavage and maintenance of blastocyst form and function during unilaminar, bilaminar and trilaminar stages in the possum were carried out. During cleavage the rate of cell division appeared to progress as normal but other aspects of cleavage related to blastocyst formation were abnormal. In the blastocyst stages normal morphology was disrupted very soon after coat removal. Electron microscopy showed an apparent breakdown of osmotic relationships and a failure to maintain epithelial structure. Cells were progressively lost from the blastocyst epithelium, chiefly into the cleavage cavity.

Development of possum embryos in vitro has been examined during cleavage, unilaminar, bilaminar and trilaminar blastocyst stages and found to progress at similar rates to in vivo.

Publications

Frankenberg, S.; Newell, G. and Selwood, L. (1996): A timetable of oogenesis in the brushtail possum, Trichosurus vulpecula. Reproduction, Fertility and Development 8 (in press).

Roberts, C.T.; Selwood, L.; Leigh, C.M. and Breed, W.G. (submitted): Antiserum to the egg coats of the fat-tailed dunnart cross reacts with egg coats of other mammalian species.

Frankenberg, S. and Selwood, L. (in preparation): The role of the extra-cellular matrix in early embryonic development in possums: ultrastructure.

Conferences

Frankenberg, S.; Newell, G. and Selwood, L. (1995): A timetable of oogenesis in the brushtail possum. Boden Conference Proceedings.

Selwood, L. and Frankenberg, S. (1996): The significance of polarised conceptuses during cleavage in marsupials from three families, Phalangeridae, Dasyuridae and Didelphidae. Australian Mammal Society Proceedings.

Frankenberg, S. and Selwood, L. (1996): Early embryonic development in the brushtail possum. Australian Mammal Society Proceedings.

3.1.5

Programme Title:Possum gamete biology - sperm and female tract interactions

Programme Leader: Professor J C Rodger

Institution:University of Newcastle

Summary

This programme is part of a collaboration between the University of Newcastle and Manaaki Whenua Landcare Research, Lincoln.

This programme aims to continue to develop better artifical breeding techniques, examine the processes of in vivo sperm capacitation (the process in which sperm gain the ability to fertilise during passage through the female reproductive tract) and sperm transport in the possum.

Using new artificial breeding techniques developed, the timing and localisation of motile thumbtack (capacitated) spermatozoa in the female reproductive tract and the first measurements of sperm transport in vitro were obtained.

Using intrauterine laparoscopic artificial insemination (LAI) of PMSG/LH primed possums, highest numbers of motile thumbtack (capacitated) spermatozoa were recovered from the ampulla, mid-oviduct and isthmus around the time of ovulation. From the isthmus to the ampulla, an increasing proportion of spermatozoa were capacitated. This suggests that spermatozoa become capacitated in the isthmus then a wave of capacitated sperm progress further up to the site of fertilization (ampulla).

Objective 1:
Artificial breeding techniques for the possum
Research Leaders: Mr A Grazier & Dr F Molinia

Objective 2:
In vivo sperm capacitation period in the possum
Research Leaders: Dr F Molinia with Mr R Gibson, Hons Student

Objective 3:
Measure of possum sperm transport in vivo
Research Leader: Dr F Molinia

Objective 4:
Identifying whether regions of the possum female tract other than the oviduct have a critical role in capacitation
Research Leaders: Dr K Mate with Mr D Setiade MSc student

Description - Objective 1: Artificial breeding techniques for the possum

To focus on the continued improvement of methods to hormonally manipulate female possums and to achieve fertile conceptions after artificial insemination.

Approach & Outcomes

Work continued to focus on the improvement of methods to hormonally manipulate female possums and to achieve fertile conceptions following LAI. In particular to increase the yield of fertilized eggs or embryos produced per female.

The optimal dose of LH required to induce ovulation of multiple ovarian follicles in possums primed with Pregnant Mare Serum (PMSG) was established for New Zealand conditions.

LAI into the uterus of superovulated possums with spermatoza, yielded cleaved eggs. Electron microscopic examination of these eggs revealed evidence of sperm remnants in the cytoplasm, thereby indicating that the eggs were fertilized and not pathenogenetically activated. It was possible to inseminate one side of the tract with spermatozoa and the other with sperm free diluent and recover fertilized eggs from only the inseminated side.

Difficulties with embryo recovery rates were solved by the LAI procedure being performed at least 12 h prior to the onset of ovulation, thereby minimising stresses (e.g. anaesthetic or surgical) which could impede ovulation rate and the fact that spermatozoa were deposited directly into the vaginal cul-de-sac at a time which mimics natural mating.

Two superovulation protocols have been developed in the possum. Females may be primed with a single im injection of 15 iu PMSG on day 1 followed by 4 x 50ug im injections of GnRH, 1.5h apart or with a single im injection of 4mg LH on day 4. Ovulation of about 9 oocytes per female treated occurs between 24-36h or 30-42h after administration of the ovulatory stimulus (GnRH or LH) respectively. Oocytes recovered appear to be of correct nuclear maturation.

Intrauterine LAI of PMSG/GnRH or LH primed possums has been optimised following insemination with 2 million motile epididymal or electro-ejaculated spermatozoa per uterus. About 5-6 ovulations, and the recovery of 2-4 oocytes including at least 1 embryo (1-4 cell) per female were observed from these groups. The low egg and embryo retrieval rates may be attributed to trauma inherent in the superovulation/LAI procedure.

Intravaginal LAI of PMSG/LH primed possums with 100 million motile epididymal spermatozoa resulted in the recovery of 5-6 or 2-3 embryos when inseminations were performed at 6 or 12 and 18 h after LH treatment respectively.

Description - Objective 2, 3 & 4: In vivo sperm capacitation in the possum

To characterise the process of in vivo sperm capacitation and transport in the possum and to see if sequential exposure to oviduct and uterus of thumb tack sperm can be modelled in vitro.

Approach & Outcomes

This objective focused on determining the precise timing and localisation of capacitated possum spermatozoa following superovulation and intrauterine LAI. It has been assumed for some time that marsupial sperm undergo a functional maturation in the female reproductive tract equivalent to the process of capacitation in eutherian mammals, which is a prerequisite to fertilization.

Highly motile spermatozoa with thumbtack morphology were recovered from the oviducts and uteri of PMSG/LH treated females following intrauterine LAI with either electro-ejaculated or epididymal spermatozoa.With the concomitant recovery of fertilized eggs using this protocol, the attainment of sperm fertilizing capacity appears to be independent of the presence of seminal plasma.

This year a clamping method was devised to separate the oviduct (the site of fertilization and probably capacitation) into three equal segments. At 3h after LAI, motile thumbtack (capacitated) spermatozoa were recovered from all segments of the oviduct but in moving from the isthmus to the ampulla, an increasing proportion of capacitated spermatozoa were recovered. This was the same for 6 or 9h after LAI, except the concentration of spermatozoa in each segment was comparatively lower with increasing time. This suggests that spermatozoa become capacitated in the region immediately anterior to the uterus (isthmus), and then a wave of capacitated spermatozoa progressed further up the oviduct, to the site of fertilization as is the case with many eutherian spermatozoa.

Intrauterine LAI (4 million motile spermatozoa inseminated per uterus) was performed on PMSG/LH primed possums at 27h after LH. Haemocytometer measurements revealed that highest numbers of spermatozoa were recovered from the ampulla (5-20 thousand), mid-oviduct (5-15 thousand) and isthmus (20-60 thousand) at 3 and 6h after LAI which corresponds to the timing of the onset of ovulation (Objective 1). At 1.5 to 6h after LAI in excess of 250 thousand spermatozoa were recovered from the uteri but decreased to 50-100 thousand by 9 and 12h. Interestingly at 12h, about 100 thousand spermatozoa were recovered from the isthmus, perhaps a temporary storage site and presumably these spermatozoa were mobilised to fertilise eggs that ovulated at later times (up to 42h after LH-Objective l).

The in vivo environment of the female reproductive tract at 30-33h after LH treatment following PMSG/LH priming and LAI will generate capacitated spermatozoa. These reproductive tracts may be useful for developing oviduct/uterus organ culture to generate motile thumbtack (capacitated) spermatozoa in vitro.

Publications

Rodger, J.C. (1997) Towards fertility management of marsupials, a case study. Reproduction, Fertility and Development (submitted).

Molinia, F.C.; Gibson, R.J.; Brown, A.M. and Rodger, J.C. (1996): Successful fertilisation after laparoscopic intrauterine insemination of superovulated brushtail possums, Trichosurus vulpecula and tammar wallabies, Macropus eugenii (submitted).

Glazier, A.M. and Markham, L.J. (1996): A PMSG/LH method of superovulation in the marsupial brushtail possum, Trichosurus vulpecula (in draft).

Glazier, A.M. and Markham, L.J. (1996): Time of ovulation in superovulated brushtail possums, Trichosurus vulpecula (in draft).

Conferences

Molinia, F.C.; Gibson, R.J. and Rodger, J.C. (1995): Post-ovulatory intrauterine insemination of superovulated brushtail possums, Trichosurus vulpecula with epididymal spermatozoa. Proceedings of the Australian Society of Reproductive Biology, Melbourne, September 27, p147.

Glazier, A.M. and Markham, L.J. (1996): A PMSG/LH superovulation protocol for the brushtail possum, Trichosurus vulpecula. International Congress on Animal Reproduction, Sydney, June/July, Poster 4-3.

Molinia, F. C. and Rodger, J. C. (1996): Artificial insemination of superovulated brushtail possums and tammar wallabies. International Congress on Animal Reproduction, June/July, Poster 4-27.

Glazier, A.M. and Markham, L.J. (1996): Time of ovulation in the brushtail possum, Trichosurus vulpecula in response to a PMSG/LH hormone regime. 4th International Conference of Fertility Control for Wildlife Management, Great Keppel Island, July, Miniposter 28.

Rodger, J.C. (1996): Towards fertility management of marsupials, a case study. 4th International Conference of Fertility Control for Wildlife Management, Great Keppel Island, July, plenary paper.

3.1.6

Programme Title:The use of oxytocin and vasopressin antagonists for the disruption of reproduction in the brushtail possum

Research Leader: A/Professor C Sernia

Institution:The University of Queensland

Summary

This programme aims to examine two hormones, mesotocin and vasopressin which are involved in marsupial reproduction.

In the current year progress has been made in the localization of receptors in the ovary, kidney and adrenal. Mesotocin profiles for the oestrous cycle and pregnancy have been compiled.

The major conclusion which can be drawn from the results so far is that the research direction set up in the initial objectives is correct. That is, all the data supports the hypothesis that mesotocin and arginine vasopressin (AVP) are important in the reproductive events of producing steroid hormones, giving birth and lactation.

Objective 1:
Disruption of ovarian function in possums

Objective 2:
Prevention of parturition and lactation in possums

Description - Objective 1: Disruption of ovarian function in possums

To study the localisation of receptors in the ovary to determine which cells will be the targets of the antagonists.

Approach & Outcomes

In eutherian mammals the peptides oxytocin and AVP are involved in the regulation of ovarian function. Oxytocin and vasopressin receptors are present in the possum ovary and both vasopressin and mesotocin are produced by the corpus luteum. Therefore, it seems probable that these two peptides are important in possum ovarian function.

Progress has been made in the localization of receptors in the ovary, kidney and adrenal. Previously used radioreceptor assays showed that the ovary contained high concentrations of vasopressin receptors and few oxytocin receptors. The receptor density in the ovary was much higher at oestrous than during lactation, and higher in the adrenal glands of females than males.

By using receptor autoradiography, vasopressin receptors have been recognised in the corpus luteum, at a very high density, and in the larger follicles. Receptors were not found in the stroma. The corpus luteum and follicles are sites of high steroidogenic activity which should be affected by antagonists. Without steroidal support, ovulation and implantation should fail. The use of antagonists of oxytocin and vasopressin, as an effective means of controlling reproduction, seems to be supported. A similar investigation of the adrenals showed that both vasopressin and oxytocin receptors are present in the progesterone-synthesizing fasciculataand reticularis zones. Immunocytochemistry of the adrenal showed that oxytocin and vasopressin are produced locally at sites adjacent to receptor sites.

Description - Objective 2: Prevention of parturition and lactation in possums

To find out how oxytocin receptors change during gestation/parturition and the oestrus cycle and to research the normal plasma profiles of mesotocin and vasopressin during the oestrous cycle/gestation/parturition.

Approach & Outcomes

Plasma mesotocin profiles have been characterized and the effect of an oxytocin antagonist on ovulation, parturition and lactation.

A profile for mesotocin during the oestrous cycle and pregnancy has been obtained. Progesterone concentrations were measured and indicated the time of the luteal phase and confirm that an oestrous cycle/pregnancy was present. Mesotocin remains low until the end of the luteal phase. Then a sharp and steep peak in concentration occurs, coinciding with luteolysis, and where pregnancy was present, parturition. These results indicate that parturition involves mesotocin in some way not yet understood.

High concentrations of oxytocin receptors in the uterus and medial vagina of the possum were found. The marsupial uterus is also responsive to vasopressin. Although it is known that receptors are present in the possum uterus/vaginae, it is not known how they change during gestation/parturition and the oestrous cycle.

Characterisation of the plasma profile mesotocin and AVP during the oestrus cycle and pregnancy was a difficult task, but results were obtained which indicate that parturition involves mesotocin in some way not yet understood. Experiments with lactating possums infused with an oxytocin antagonist were inconclusive.

The major conclusion which can be drawn from the results so far is that the research direction set up in the initial objectives is correct. That is, all the data support the hypothesis that mesotocin and arginine vasopressin are important in the reproductive events of producing steroid hormones, giving birth and lactation.

Publications

Sernia, C.; Bathgate, R.A.D. and Gemmell, R.T. (1994): Mesotocin and arginine-vasopressin in the corpus luteum of an Australian marsupial, the brushtail possum, Trichosurus vulpecula. Journal of Comparative Endocrinology 93, pp.197-204.

Bathgate, R.A.D.; Sernia, C. and Gemmell, R.T. (1995): Regulation of neurohypophysial hormone secretion in an Australian marsupial. American Journal of Physiology 268, pp.R1319-R1326.

Bathgate, R.A.D. and Sernia, C. (1995): Characterization of vasopressin and oxytocin receptors in an Australian marsupial. Journal of Endocrinology 144, pp19-29.

Gemmell, R.T. and Sernia, C. (1995): Effect of changing from a short-day to long-day photoperiod on the breeding season of the brushtail possum, Trichosurus vulpecula. Journal of Experimental Zoology 273, pp.242-246.

Bathgate, R.; Parry, L.; Shaw, G.; Fletcher, T.; Renfree, M.; Gemmel, R.T. and Sernia, C. (1996): Comparative aspects of oxytocin-like hormones in marsupials. Advanced Experimental Medical Biology 395, pp.639-654.

Sernia, C.; Bathgate, R.A.D. and Mann, R. (1996): Receptors and immunohistochemical sites for mesotocin and vasopressin in the adrenal gland of the marsupial brushtailed possum. Journal of Comparative Endocrinology (in prep for submission).

Sernia, C.; Zeng, T. and Gemmell, R.T. (1996): Ontogeny of thyroxine receptors in the brushtailed possum (in prep).

Sernia, C.; Bathgate, R.A.D.; Zeng, T. and Gemmell, R.T. (1996): Plasma profiles of mesotocin during the oestrous cycle and pregnancy in the brushtail possum (in prep).

Sernia, C (1996): Neurohypophysial hormones in peripheral tissues of marsupials. Proceedings of Endocrine Society of Australia, Sydney, to be presented at a symposium on comparative endocrinology.

3.1.7

Programme Title:Structure and function of the reproductive tract in the female possum

PhD fellowship:Janet Crawford

Institution:University of Otago/AgResearch

Summary

The aim of this PhD fellowship is to examine the structure and function of the female reproductive tract associated with oestrus, mating and pregnancy in the possum.

Description: Structure and function of the female possum reproductive tract

To focus on the structure and function of the female reproductive tract associated with oestrus, mating and pregnancy in the possum by:

  • determining using light and electron microscopy, the cellular location of mucous secretory activity of the vaginal cul-de-sac, uterus and oviduct in the female possum;
  • using stereology to quantitatively define changes in the size of secretory epithelial cells (volume) and organelles associated with secretion (volume and membrane surface area) during oestrus, mating and pregnancy, and correlate these with hormonal changes over the reproductive cycle; and
  • collecting and characterising mucoid secretions.

Approach & Outcomes

The PhD student is part of the AgResearch programme called "The female possum reproductive tract and its secretions". The experimental work has largely been completed for the PhD this year, other than comparing the effect of oestradiol on an animal in the non-breeding season.

A preliminary study undertaken to assess changes in secretory cells, involved collection of tissue from oestradiol-treated and control animals during seasonal anoestrus. Electron microscopy was used for an overall description of cul-de-sac tissue at the epithelial layers. A variety of post-fixation solutions were tested to determine which best defined the various parts of the cells.

Oestradiol treatment induced a ten-fold increase in tissue size, demonstrating the high sensitivity of epithelial cells to stimulation by ovarian steroids.

A new biochemical method for accurately determining glycosaminoglycans (GAGs) content in the cul-de-sac tissue, and in mucus, has been developed. It has been shown that GAGs, known to be essential in uterine remodelling in eutherian mammals, increases over the follicular phase of the oestrous cycle.

3.1.8

Programme Title:The female possum reproductive tract and its secretions

Research Leader: Dr B McLeod

Institution:AgResearch

Summary

This programme aims to examine the changes in the female possum that are associated with oestrus, ovulation and mating.

Investigation of the changes that occur in reproductive tract tissue over the oestrus cycle was severely hampered by the inability to synchronise oestrus in groups of females, and to identify oestrus and/or ovulation in individual females. Recent development of laparoscopic monitoring of ovarian function will help overcome many of these problems.

Methods for examining cells lining the reproductive tract, and secretions were developed and tested for use with possum tissues.

Description: The biochemical and functional changes in the female reproductive tract

To characterise the biochemical and functional changes in the female reproductive tract associated with oestrus, mating and pregnancy in the possum and the feral rabbit by:

  • investigating, using light and electron microscopy, changes in mucous secretory cells of the vaginal cul-de-sac, uterus and the oviduct in the female possum and in the uterus and oviduct in the rabbit;
  • using stereology to characterise quantitative changes in the size, volume and ultrastructure of the epithelial cells associated with mucus secretion during oestrus, mating and pregnancy in the major reproductive tissues;
  • establishing culture media requirements for in vitro cell cultures of reproductive tract epithelium cells from possums and rabbits, and characterising them using cell surface glycoconjugates;
  • analysing cell cycle times of epithelial cells and investigating their response to steroid hormones in vitro and in vivo; and
  • collecting mucoid secretions and quantifying their protein, glycoprotein and proteoglycan content and identifying secretion products of epithelial cell cultures.

Approach & Outcomes

A major constraint on progress in the area of female possum reproduction is the inability to synchronise oestrus in groups of females, or to identify the day of oestrus in individual female possums. It has now been established that the traditional method of synchronising oestrus, by removal of pouch young and monitoring oestrus, by vaginal mucous changes are unreliable and can be misleading. Using laparoscopic observation of the ovaries, the time of ovulation can now be accurately identified.

These studies have established that removal of pouch young alone does not synchronise oestrus in the possum. More importantly, it shows that current methods of "identifying" the time of oestrus in this species (from vaginal mucous characteristics or from the presence of an enlarged reproductive tract) can be very misleading. The use of laparoscopy to monitor ovarian status overcomes many of these problems.

Initial attempts to culture epithelial cells in vitro, using culture media defined for short term incubation of marsupial tissue proved unsuitable for cell culture. A series of experiments investigating energy metabolism of the possum, to help define the culture media requirements was carried out. Using reformulated media developed on the basis of the findings from the energy metabolism studies, cell growth and proliferation in vitro has recently been established for both uterine and cul-de-sac tissue. Once established, cells have been maintained for up to 7 days.

Using a RT-PCR product, a 1kb segment of the possum oestrogen receptor gene, and an additional overlapping sequence of 1kb was cloned.

It has also been shown that follicle development can be stimulated by treatment with exogenous hormones. In a series of experiments, the time of ovulation has ranged from 8 days to 17 days after pouch young removal and the percentage of animals in each group that failed to ovulate has ranged from 0 to 77%. Neither the interval from pouch young removal to ovulation, nor the percentage of animals that fail to ovulate, could be related to the month of treatment.

Significant hypertrophy in both cul-de-sac and uterine tissue has frequently been recorded in animals that failed to ovulate. Enlargement is considered to be indicative of impending ovulation.

Researchers monitored changes in the numbers of cornified epithelial cells, and ferning patterns, in vaginal mucous. Preliminary analysis shows that patterns of mucous ferning usually associated with oestrus, were recorded in several animals that failed to ovulate.

Progesterone treatment is used to successfully synchronise oestrus in eutherian species. Treatment of possums had no effect on the time of ovulation. The administration of FSH resulted in a significant increase in ovarian weight, and in the weight of the uterus and cul-de-sac, all indicative of increased oestrogen secretion.

Using, laparoscopic observation of the ovaries, researchers are able to monitor the progressive development of preovulatory follicles and to predict the time of ovulation with some confidence.

Experiments were undertaken to assess the importance of the presence of a male on the incidence of ovulation. Researchers have demonstrated that presence of the male can be important for ovulation (increasing by 30 to 70%). Presence of males was found to have no effect on the synchrony of ovulation.

The mortality rate of possums within the colony is exceptionally low (less than 0.5%). The researchers have had requests for details of methods used and researchers have written a paper outlining their approach to maintaining possums in captivity.

Publications

Legge, M.; Hill, B.L.; Shackell, G.H. and McLeod, B.J. (1996): Uterine and vaginal cul-de-sac tissue glycosaminoglycans in the brushtail possum, Trichosurus vulpecula. Reproduction, Fertility and Development 8, (in press).

Shackell, G.H.; Norman, N.G.; McLeod, B J. and Hurst, P.R. (1996): A morphometric study of early ovarian development in pouch young of the brushtail possum, Trichosurus vulpecula. The Anatomical Record 244, (in press).

McLeod, B J.; Thompson, E.G.; Crawford, J.L. and Shackell, G.H. (1996): Successful group-housing of wild-caught brushtail possums, Trichosurus vulpecula. Animal Welfare 5, (in press).

Crawford, J.L.; Shackell, G.H.; Thompson, E.G.; McLeod, B.J. and Hurst, P.R. (1996): Monitoring pre-ovulatory follicle emergence, development and ovulation in brushtail possums, Trichosurus vulpecula by repeated laparoscopy. Journal of Reproduction and Fertility, (submitted).

Conferences

Shackell, G.H.; Norman, N.G.; McLeod, B.J. and Hurst, P. R. (1995): Folliculogenesis in the brushtail possum, Trichosurus vulpecula. The 12th Annual Meeting Australian and New Zealand Society for Comparative Physiology and Biochemistry, Christchurch.

Legge, M.; McLeod, B.J.; Mason, P.; Crawford, J.L. and Shackell, G.H. (1995): Energy substrate utilisation by the brushtail possum, Trichosurus vulpecula. The 12th Annual Meeting Australian and New Zealand Society for Comparative Physiology and Biochemistry, Christchurch.

McLeod, BJ. (1996): Wobbly Possum Syndrome. The 10th Biennial Conference New Zealand Association of Science Educators, Dunedin.

Reports

McLeod, B.J. (1995): Reproductive physiology of the brushtail possum. Research in Infectious Mycobacterium 3.

McLeod, B.J. (1995): Wobbly Possum Syndrome. Research in Infectious Mycobacterium 3.

3.1.9

Programme Title: Possum and other wild animals as reservoirs of bovine tuberculosis: improved strategies to control the spread of disease to domestic livestock

Programme Leader: Dr P E Cowan

Institution:Landcare

Summary

This programme aims to examine the effectiveness of fertility control for controlling possum populations in the wild, and look at chemicals known to change milk production in other animals and determining whether they have the same effect in possums.

Field trials were set up to assess the effectiveness of fertility control for controlling possum populations in the wild. Sterilisation treatments were successful at reducing breeding rates. Preliminary analysis indicates that the application of sterilisation treatments to individuals (either tubal ligation or sham operation) did not significantly depress capture rates compared with non-treated animals. In addition, sampling for the initial prevalence of Leptospira balcanica was done and investigations of suitable sites for radiotelemetry were completed.

Experiments aimed to assess the effects of chemical lactation inhibitors on milk production in possums showed the long-acting formulation of bromocriptine, at 25 mg/ml, was ineffective as an inhibitor of milk production in the possum.

Objective 1:
Field trial of reproductive inhibition of possums
Research Leader: Mr D Ramsey

Objective 2:
Milk characterisation and lactation inhibition in possums
Research Leader: Dr T Fletcher

Description - Objective 1: Field trial of reproductive inhibition of possums

To assess whether reproductive inhibition can effectively reduce possum population abundance in the wild by continuing a replicated field trial of reproductive inhibition by:

  • completing the establishment of seven study sites;
  • sterilising the first cohorts of females;
  • monitoring population dynamics by mark-release live-trapping; and
  • initiating radio-tracking studies of the effects of reproductive inhibition on possum behaviour.

Approach & Outcomes

Seven study populations were selected at sites so that they would contain approximately 80 female possums. Three levels of treatment were applied to study populations - 0%, 50% or 80% of female possums sterilised. Estimates of adult female possum numbers in the study sites were made. Population parameters such as body condition, sex ratio and capture probabilities were varied between populations at two of the sites.

Initial estimates of reproductive success of study populations indicate that treatments have been successful at reducing breeding rates. However, an overall depressed rate of breeding was recorded on non-treatment areas. The cause of this low breeding success is currently unknown but appears to be unrelated to the experimental treatments.

Preliminary analysis showed no difference in capture rates between sham-operated, tubally-ligated, or untreated animals. The surgical procedures did not, therefore, affect short term survival.

Samples of the initial prevalence of L. balcanica at non-treatment and treatment sites was undertaken.

Preliminary investigations of suitable sites for radiotelemetry were completed, including identification of an additional non-treatment site at Turitea.

Description - Objective 2: Milk characterisation and lactation inhibition in possums

To characterise the constituents of possum milk and the effects of lactational disruption on pouch young growth and survival at different stages of lactation to evaluate potential methods of biological control by:

  • completing cage and pen studies of the effects on possum pouch young growth and survival following interruption of lactation by cabergoline;
  • completing cage and pen studies of the effects on establishment of lactation in possums after bromocriptine treatment in late pregnancy; and
  • measuring the effects of a lactation modifier on possum milk composition and pouch young growth and survival.

Approach & Results

Treatment of female possums, with suckling pouch young of 16 -18 weeks of age, with 25 mg/ml of long-acting bromocriptine did not suppress milk production. This finding contrasts with previous studies (Jolly and Fletcher 1995a,b) using standard bromocriptine. This suggests that the long-acting preparation does not stimulate all the available dopamine receptors in the possum. A rebound increase in milk production 7 days after bromocriptine treatment was found which then declined to pre-treatment levels by day 15 of the experiment.

The cabergoline trial could not be completed because none of the pouch young survived to the required age.

Searches of agricultural and biomedical literature databases failed to identify any potentially useful and effective lactation modifiers, so the proposed trial did not proceed. It may not be possible to selectively alter milk composition of lactating possums by chemicals.

Publications

Jolly, S.E.; Morriss, G.A.; Scobie, S. and Cowan, P.E. (1996): Compsition of milk of the common brushtail possum, Trichosurus vulpecula (Marsupialia:Phalangeridae) concentrations of elements. Australian Journal of Zoology, 44 (in press).

Reports

Jolly, S.E.; Fletcher, T.P. (1995a): Possum biocontrol: Assessing the effects of lactation inhibitors on milk production in brushtail possum, Trichosurus bulpecula. Landcare Research Contract Report, LC9596/08 (unpublished) 12p.

Jolly, S.E.; Fletcher, T.P. (1995b): Possum biocontrol: Assessing the effects of lactation inhibitors on growth and survival of brushtail possum pouch young. Landcare Research Contract Report, LC9596/09 (unpublished) 13p.

Jolly, S.E.; Scobie, S.; Cowan, P.E.; (1995): Possum biocontrol: Assessing the effects of cholecalciferol on milk composition of brushtail possum pouch young. Landcare Research Contract Report, LC9596/10 (unpublished) 11p.

3.1.10

Programme Title:Use of immune system of mother against pouch young for biocontrol of possums

Research Leader: Professor D Cooper

Institution:Macquarie University

Summary

This programme aims to determine whether antibodies (part of the body’s defence system) to pouch bacteria occur in the serum, milk and other saliva of the mother possum and to clone the possum androgen receptor.

Several species of bacteria occurring naturally in the possum (from New Zealand and Australia) pouches were characterised. Investigations established that the pouch young is exposed to potentially pathogenic bacteria from as young as 1 day old.

About 95% of the androgen receptor gene, which is important in the masculinigation of tissues, was cloned and possible antigenic sites in the androgen receptor protein were identified.

Objective 1:Passive immunity to pouch bacteria in possums

Objective 2:Maternal immune attack upon the neonate in possums

Description - Objective 1: Passive immunity to pouch bacteria in possums

To determine whether antibodies to pouch bacteria occur in the serum, milk and other saliva of the mother possum. In addition researchers aimed to clone the possum androgen receptor by:

  • characterising the species of bacteria which occur in possums, devoting particular attention to those which could prove pathogenic to pouch young;
  • examining whether antibodies to pouch bacteria occur in the serum, milk and other secretions of the female; and
  • conducting investigations to establish whether there is transfer of the immunity to the pouch young.

Approach & Outcomes

The neonatal possum has no active immune system at birth. It develops gradually over a period of three months. Numerous species of bacteria can be found in the pouch of wallabies. Pouch bacteria could be used to spread control agents in genetically modified bacteria.

Possums were trapped in Australia and New Zealand and pouch swabs taken and analysed. Three groups were examined, possums with the presence or absence of possum young or juveniles

Identification of bacteria in the pouch was initially performed by using an API kit (Biomerieux Vitek) after some preliminary tests. Occasionally, the API system fails to give an acceptable identification and sequencing of the 16s rRNA gene is performed. The sequence information along with the information obtained from the API kit allows for a more accurate identification.

The results for Australia and New Zealand show the same trend. Skin organisms are predominant in all three categories of possums, enteric bacteria are more prevalent in pouches with a pouch young and environmental organisms (bacteria that are normally isolated from soil, water, plants etc.) are more prevalent in pouches without a pouch young. Despite the presence of pouch young, all categories of bacteria can occur in the pouch. Staphylococcus and Micrococcus species are a prominent part of the pouch microbiota. Enteric bacteria are more abundant in pouches carrying pouch young. Juvenile pouches contained no enteric bacteria. These findings established that the pouch young is exposed to potentially pathogenic bacteria from as young as 1 day old.

Description - Objective 2: Maternal immune attack upon the neonate in possums

To investigate possible candidate antigens concerned with sexual differentiation, e.g. androgen receptor, Mullerian inhibition factor, ZP3, c-kit, c-kit ligand, or those concerned with the immunological system, e.g. thymus lymphocyte antigens, erythrocyte antigens.

Approach & Outcomes

The androgen receptor is a candidate antigen to target the fertility of male pouch young because it is necessary for the masculinization of target tissues.

A 510bp possum RT-PCR product was cloned and sequenced. This clone was used to screen a possum pituitary cDNA library. No androgen receptor clones were detected using this probe. A human androgen receptor probe to the steroid binding domain was then used to rescreen a cDNA library. This probe detected a 1.5kb androgen receptor clone. The remaining portion of the androgen receptor was obtained by using RT-PCR to produce fragments of 630bp and 920bp. There is approximately 83% homology between human and possum AR at the nucleotide level and 93% homology at the amino acid level.

A programme called PEPTIDESTRUCTURE has been used to identify regions in the deduced amino acid sequence.

About 95% of the androgen receptor gene was cloned and possible antigenic sites in the androgen receptor protein were identified.

Conferences

Deakin, J.E. and Cooper, D.W. (1996): Cloning the androgen receptor gene in the common brushtail possum, Trichosurus vulpecula. Genetics Society of Australia 43rd Annual Conference, July 7-11.

Deakin, J.E.; Harrison, G.A. and Cooper, D.W. (1996): Cloning the androgen receptor gene in the common brushtail possum, Trichosurus vulpecula. The 1st Higher Degree Student Conference for the Department of Biological Sciences, University of Newcastle, July 19.

Deakin, J.E. (1995): Immunology of mother-pouch young relationships in brushtail possums. Macquarie University Postgraduate Conference, November.

3.1.11

Programme Title: Casein genes of the possum

PhD fellowship:Melanie Ginger

Institution:University of Otago

Summary

The aim of this PhD fellowship is to examine the casein (protein of milk) genes of the possum to investigate how lactation is controlled.

The caseins of possum milk were characterised and changes in their concentration throughout lactation were determined. Analysis of the genetic makeup of two major casein components was done. Screening of a possum genomic cosmid library was unsuccessful in isolating the genes of interest. However, construction and screening of two lambda genomic libraries have yielded clones for six milk protein genes including _- and ß-casein.

Description: The control of lactation in the possum

To investigate the control of lactation in the possum by understanding the control of lactation in the possum by:

  • characterising the caseins of possum milk;
  • determining any changes in the expression of caseins in possum milk during lactation;
  • obtaining molecular probes for the caseins of possum milk; and
  • determining critical regulatory features of the genes of the possum caseins.

Approach & Outcomes

The caseins of possum milk were characterised in detail and changes in their concentration throughout lactation were determined. Full length cDNA clones of two major casein components have been isolated and the complete DNA sequences of these clones have been determined. Screening of a possum genomic cosmid library was unsuccessful in isolating the genes of interest. However, construction and screening of two lambda genomic libraries have yielded clones for six milk protein genes including _- and ß-casein.

A possum mammary gland cDNA library was screened using cDNA clones of milk proteins of the tammar wallaby. Several full-length clones of possum _- and ß-casein were successfully isolated. The full-length sequence of these clones has been obtained by automated and manual DNA sequencing. The expression of _- and ß-casein appears to remain constant throughout lactation.

The regulatory regions of these two casein genes were examined. This required screening of a possum cosmid library to obtain genomic DNA clones of _- and ß-casein. Preliminary Southern blot analysis suggests that thesepossum casein genes span an area of approximately 15 kb. A polymerase chain reaction (PCR) -based screening protocol has been established as a preliminary screen of thirteen cosmid pools.

The two milk proteins, _- and ß-casein, were screened using a possum genomic cosmid library constructed by Dr Jerome Demmer. A PCR-based screening protocol was developed as a preliminary screen of thirteen cosmid pools. This work was unsuccessful and generated a large number of false positives, possibly due to cross-hybridisation between the cDNA probes and the cosmid vector.

Because of these difficulties, the cosmid library was abandoned and two lambda genomic libraries were constructed. High molecular weight DNA was prepared from kidney and spleen tissue and subjected to partial digestion with either Sau 3A or BamHI. Partially digested DNA was then size selected and ligated into a lambda Gem-12 vector. These ligation products were packaged and the primary libraries amplified to produce a final titre of 3.1 x 1010 and 1.3 x 1011 pfu per ml respectively.

A total of 2 x 106 pfu from both libraries was screened and has yielded several putative casein clones. In addition, four other milk protein genes (trichosurin, early lactation protein, late lactation protein and lysozyme) have been isolated from the Sau 3A library.

3.1.12

Programme Title:The role of the thyroid gland in growth and thermoregulation in the developing brushtail possum Trichosurus vulpecula

Research Leader: Associate Professor R Gemmell

Institution:University of Queensland

Summary

This programme aims to examine the role of the thyroid gland, and its hormones, on growth and thermoregulation in the developing possum.

Preliminary results of the research suggest that thyroxine from the mother, before the young can produce thyroxine, is vital for the development of the young. In the young possum, thermoregulation was altered by treatment with methimazole, an inhibitor of thyroid action. This resulted in a large fluctuation in body temperature at day 150, which suggests that the thyroid is essential for the young possum to display the temperature rhythm.

The surge in growth rate in the possum at day 96 post partum is likely to be initiated by thyroxine produced by the thyroid gland of the young. The results also suggest that possum young deprived of maternal thyroid hormones early in pouch life do not survive possibly due to impaired organ development.

Objective 1:
Control of growth in possums

Objective 2:
Control of thermoregulation in possums

Objective 3:
Disruption of growth and thermoregulation in possums

Description - Objective 1: Control of growth in possums

To continue work on developing an understanding of the role of the maternal and fetal thyroid glands on growth and development of the brushtail possum.

Approach & Outcomes

To determine the possible role of thyroxine on the organ development of the possum, tissues including lung, kidney and liver have been collected from possums at day 25, 50, 100, 150 post partum and the number of receptors for the thyroid hormones determined. The quantity of receptors in the three organs was similar at the four stages of development. The structure of these organs was examined with the light and electron microscopes to obtain developmental patterns and to make correlations with the plasma concentration of thyroxine and the number of receptors for the thyroid hormones. This morphological data is incorporated into the PhD thesis of W. Buaboocha.

It has been shown that the growth rate of the possum is approximately lg/day up to 100 days post partum and 20g/day for the remainder of the lactation period. This suggests that the increase in the growth rate about day 100 post partum is due to thyroxine secretion from the fetal thyroid gland.

There is already evidence that thyroxine passes across in the milk. To determine the effect of methimazole on the possum prior to day 100, minipumps containing methimazole were placed in mothers at days 10, 20 and 30 post partum. The young died between 30 and 90 days after insertion of the minipump. These preliminary results suggest that thyroxine from the mother, before the young can produce thyroxine, is vital for the development of the young.

Methimazole was administered via Alzet minipumps to adult and young possums at day 140 post partum for 28 days and young at day 100 post partum for 42 days and the effects of this treatment on plasma concentrations of thyroxine and on growth were monitored.

Methimazole was found to inhibit thyroxine production in 4 of the 6 adult (140 day) possums although it had no effect on body weight prior to day 156 post partum. The four young showed some depression in growth rates. However, the depression was not as marked as that at 100 day treated possums.

Methimazole treatment administered to 9 young (100 days) possums inhibited growth. The three surviving young (100 days) weighed less than the control possums after day 163 post partum. Therefore, it is likely that the surge in growth rates in the possum at day 96 post partum is initiated by thyroxine produced by the thyroid gland of the young.

Description - Objective 2: Control of thermoregulation in possums

To understand the role of the maternal and fetal thyroid glands in the development of thermoregulation in the brushtail possum by determining if the ability to thermoregulate correlates to the increase in thyroxine concentrations in the developing possum.

Approach and Outcomes

Gemmell and Cepon (1993) reported that the adult possum showed a circadian rhythm of body temperature with a peak in temperature around midnight and a nadir at noon. The young possum within the pouch displayed a circadian rhythm with the highest temperatures during the day and the lowest in the early evening. The normal developing possum can maintain a steady body temperature around 37°C between 140 and 167 days and displays a circadian rhythm between 157 and 190 days.

Methimazole (1-methyl-2-mercaptoimidazole) is a potent inhibitor of thyroid iodide peroxidase, which catalyses the initial step of thyroxine biosynthesis. Methimazole inhibits the growth of the possum and its ability to thermoregulate. Methimazole was given to 4 young possums at day 100 post partum. Two of the young died at days 131 and 139. Thus, body temperatures were obtained for two young possums. The methimazole treated possums had great difficulty in maintaining body temperature as was demonstrated by the large variation intemperature over 24 hours at about day 150 post partum, having a daily range of 10°C. Whereas the untreated possum obtained a steady body temperature between days 140 and 167 post partum the methimazole treated possums did not obtain a steady temperature.

The methimazole possums also changed the profile of temperature in which the lowest temperature was at midnight and the highest at midday at day 150 post partum compared with the normal adult rhythm of high temperatures at midnight and the nadir at midday by days 170 to 220. An adult circadian rhythm is observed in normal possums between days 157 and 195. Thus, treatment with methimazole does not delay the young possum obtaining the adult circadian rhythm for body temperature. However, the large fluctuation in body temperature of methimazole treated possums at day 150 would suggest that the thyroid is essential for the young possum to display the temperature rhythm.

Publications

Gemmell, R.T. (1995): Breeding biology of brushtail possums, Trichosurus vulpecula, (Marsupialia: Phalangeridae) in captivity. Australian Mammalogy 18, pp.1-7.

Buaboocha, W. and Gemmell, R.T. (1995): Thyroid gland development in the brushtail possum, Trichosurus vulpecula. Anatomical Record, 243, pp.254-260.

Gemmell, R.T. and Sernia, C. (1995): The effect of changing from a short-day to long-day photoperiod on the breeding season of the brushtail possum, Trichosurus vulpecula. Journal of Experimental Zoology 273, pp.242-246.

Baker, M.L.; Canfield, P.J.; Gemmell, R.T.; Spencer, P.B.S. and Agar, N.S. (1995): Erythrocyte metabolism in the koala, the common brushtail possum and the whiptail wallaby. Comparative Heamatology International 5, pp.163-169.

Buaboocha, W. and Gemmell, R.T. (1996): The effect of methimazole on the growth of the developing brushtail possum, Trichosurus vulpecula. Growth, Development and Ageing, (in press).

Conferences

Gemmell, R.T. (1995): The role of the thyroid gland in growth and thermoregulation in the developing brushtail possum, Trichosurus vulpecula. NSSC Possum Biocontrol Workshop, AgResearch, Wallaceville, New Zealand, p8.

Gemmell, R.T. (1995): The role of the thyroid gland in marsupial development. The 4th International Conference on Veterinary Perinatology, Cambridge, UK, A39.

Gemmell, R.T. (1995): The effect of relocation of brushtail possums, Trichosurus vulpecula from Adelaide to Brisbane on the initiation of the breeding season. Proceedings of the Australian Society for Reproductive Biology, Melbourne.

Gemmell, R.T. (1995): The role of the thyroid gland in the development of the marsupial bandicoot. Proceedings of the Australian Endocrine Society, Comparative Endocrinology Symposium, Melbourne.

Saunders, M.C.; Gale, J.; Gemmell, R.T. and Curlewis, J.D. (1995): Preliminary investigations of growth hormone in the brushtail possum. Proceedings of the Australian Endocrine Society, Melbourne.

Gemmell, R.T.;Turner, S.J. and Krause, W.J. (1995): The circadian rhythm of body temperature of three marsupial species. The 12th Meeting of the Australian and New Zealand Society for Comparative Physiology and Biochemistry, Christchurch, New Zealand.

Watson, C.M.; Johnston, P.G.; Hughes, R.L.; Gemmell, R.; Smith, M. and Cooper, D.W. (1995): X-chromosome involvement in marsupial sex differentiation. Proceedings of the Australian Mammal Society, Townsville.

Gemmell, R.T. and Buaboocha, W. (1996): Thyroid hormones and growth in the marsupial possum, Trichosurus vulpecula. The 3rd Congress of the Asia & Oceania Society for Comparative Endocrinology, Macquarie University, Sydney.

Gemmell, R.T. and Buaboocha, W. (1996): Thyroid hormones and growth in the marsupial possum, Trichosurus vulpecula. The 3rd Congress of the Asia & Oceania Society for Comparative Endocrinology, Macquarie University, Sydney.

W. Buaboocha is a PhD student. The title of her thesis will be "Development of the thyroid gland in the marsupial".

3.1.13

Programme Title: Purification and radioimmunoassay of prolactin and growth hormone (GH) from the brushtail possum

Research Leader: Dr J Curlewis

Institution:University of Queensland

Summary

This programme aims to purify and develop radioimmunoassays of prolactin and growth hormone.

The pituitary hormones prolactin and GH are essential for normal physiological processes such as lactation, growth and immune responses. Studies on the role of these hormones are difficult until hormone assays become available.

Macquarie University unexpectedly isolated a partial growth hormone clone from the possum pituitary cDNA library. From the partial clone, a PCR-BASED approach was used and two partial GH cDNA clones, encompassing 550bp of a total expected 725 bp, were isolated.

Difficulties were encountered with purification of prolactin from pituitary glands, so a molecular cloning strategy was adopted.

Several attempts were made to raise antibodies against possum GH. Rabbits and guinea pigs were immunised with possum GH. Sera was tested for possum GH and two of the guinea pigs gave high titres.

Problems have been encountered with iodinating possum GH and this has prevented the further development of the possum GH radioimmunoassay.

Objective 1: Purification of possum prolactin and growth hormone

Objective 2: Develop possum prolactin and growth hormone radioimmunoassay

Description - Objective 1: Purification of possum prolactin and growth hormone

To purify and partially characterise possum prolactin and growth hormone (GH), then use the purified hormones to develop prolactin and GH assays.

Approach and Outcomes

The pituitary hormones, prolactin and GH, are essential for normal physiological processes such as lactation, growth and immune responses. Studies on the role of these hormones are difficult until hormone assays become available.

Pituitary hormones were extracted and purified. Milligram quantities were recovered but very little prolactin obtained. After looking at several possible causes for low levels of prolactin, it was concluded that the reason is due to the very low prolactin content in possum pituitaries.

Because of the difficulties encountered with purification of prolactin from pituitary glands, it was decided to adopt a molecular cloning strategy with the eventual aim of producing a recombinant hormone. In initial experiments, 6 different primers, based on conserved regions of the mammalian prolactin sequence, were used in PCR with the possum pituitary cDNA library as the template. A number of primer combinations and annealing temperatures for PCR were tested in order to optimise reaction conditions. These initial attempts did not yield the appropriate sequence because the primers were not specific for the possum prolactin gene. A new pair of primers was used. The primers were based on the two most conserved sequences of pig prolactin, human prolactin and rat prolactin. Partial sequence of possum prolactin cDNA clones shows 70% homology at the DNA level with pig, human, chicken and rat prolactin genes.

Macquarie University unexpectedly isolated a partial growth hormone clone from the possum pituitary cDNA library. From the partial clone, a PCR based approach was used and two partial GH cDNA clones, encompassing 550bp of a total expected 725 bp, were isolated.

Description - Objective 2: Develop possum prolactin and growth hormones radioimmunoassays

To use the purified hormones produced under Objective 1 and once high titre antibodies are obtained, develop and test hormone assays to prove they can measure plasma hormone concentrations.

Approach and Outcomes

Researchers have made several attempts to raise antibodies against possum GH. Rabbits and guinea pigs were immunised with possum GH. Sera was tested for possum GH and two of the guinea pigs gave high titres. The remaining animals did not give high binding titres.

The antisera from the two guinea pigs show binding properties equivalent to anti-wallaby GH serum which forms the basis for wallaby GH assay. Problems have been encountered with iodinating possum GH and this has prevented researchers from further development of the possum GH radioimmunoassay. This is a problem in pituitary derived GH in a number of species.

Conferences

Saunders, M.C.; Gale, J.; Gemmell, R.T. and Curlewis, J.D. (1995): Preliminary investigations of growth hormone in the brushtail possum. Proceedings of the Endocrine Society of Australia, Melbourne, Abstract 90.

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