Biological Management of Possums - Developmental Targets for Fertility Control in the Brushtail Possum

Abstract
Introduction
Vesicle Associated Molecules (VAMs)
Coat Proteins (CPs)
Leukemia inhibitory factor (LIF)
Other Molecules
Conclusions and Future Directions
Acknowledgements
References
Table 1 Targeted Developmental Molecules in the Brushtail Possum
Lynne Selwood - Figure 1

Developmental Targets for Fertility Control in the Brushtail Possum

Shuliang Cui, Nicole Griffith, Kamani Nanayakkara, Y.Cruz* & Lynne Selwood

Department of Zoology, University of Melbourne, Vic. 3010 Australia

*Department of Biology, Oberlin College, Oberlin, OH 44074-1082, USA

Abstract

In order to achieve fertility control in the brushtail possum, molecules active during embryonic development have been chosen as targets for immunocontraception and fertility control. In most cases, the molecules chosen were selected because they were:- essential for normal development, had marsupial-specific components, acted at different stages of pre-implantation development, acted before development of a functional nervous system.

The target molecules can be roughly divided based on the expected time of action during pre-implantation development, into vesicle associated molecules (VAM), that act during oogenesis and cleavage, coat proteins (CP) that are important during late cleavage and blastocyst stages and leukemia inhibitory factor (LIF) that acts during implantation.

Introduction

Despite the fact that the brushtail possum has one young at a time , it is an efficient reproducer. In the first few cycles in the breeding season between March and June about 70 percent of females will have PY and a further ~15 percent will conceive during the rest of the season which ends about October. Survival during pregnancy, which lasts for 17 day, and lactation, which lasts for ~ 200 days, is high (Fletcher and Selwood, 2000).

essential for normal development;
had marsupial-specific components;
acted at different stages of pre-implantation development;
acted before development of a functional nervous system.

Selection of marsupial-specific molecules makes the chance of other species being affected less likely. Molecules were also selected because they were important for different stages of development, so that a variety of developmental processes are affected. For humane reasons, molecules that play a role before the nervous system is fully developed and functional were also selected. In earlier projects the molecules and some of their roles in embryonic development were identified. In the current project the genes for target proteins have been cloned and their expression patterns determined to establish time of action.

Figure 1 shows the times when these molecules can be expected to act in embryonic development at known times after ovulation (Duckworth et al,1998). The vesicle associated molecules (VAMs) are important during oogenesis and during cleavage and blastocyst formation (Frankenberg and Selwood, 1998, 2001). The coat proteins (CPs) are secreted by the oviduct and uterus and are particularly important during the blastocyst stages of pregnancy (Selwood, 2000). The leukemia inhibitory factor protein (LIF) is known to be essential over the implantation period (Cui and Selwood, 2000).

Vesicle Associated Molecules (VAMs)

These are molecules that are present early in development, are often formed or accumulated in the oocyte during oogenesis and have a role in early possum embryos. Several have already been identified (Table 1) by enzyme digestion, immuno-staining and by differential sequence analysis of proteins bands obtained by polyacrylamide gel electrophoresis (PAGE) from fractions of oocytes. The polarised secretion of oocyte vesicles into the cleavage cavity is uniquely characteristic of marsupials and is responsible for separating the cleaving conceptus into future embryonic (pluriblast) and trophoblast cells. The pluriblast later forms the epiblast which expresses OCT3/4, a marker for normal possum embryonic development (Frankenberg et al., 2001). Our experiments have identified some of these molecules and cloned the genes for those proteins that appeared to be unique or have unique sequences.

VAMs, located in clear vesicles, are first found in the oocyte in the ovary during early oogenesis when the possum is between 88 and 100 days old (Frankenberg et al., 1996). Vesicles progressively accumulate in the oocyte and become located at one pole of the oocyte after fertilization (Frankenberg and Selwood, 1998, 2001). During early cleavage most of them are shed into the cleavage cavity. Their presence in the cavity is detectable as a fibrous and granular material embedded in a transparent extracellular matrix. At least one of the ECM molecules is hyaluronate, some are plasma proteins and others are vesicle associated proteins (VAPs). Short-term (7day) culture of primary and secondary follicles have been established, so that we can determine whether VAPs are endogenous to the oocyte or formed exogenously. Short-term follicle and cumulus oocyte complex culture protocols have been established for both the possum and the stripe-faced dunnart following the techniques of Butcher and Ullman (1996) and Selwood and VandeBerg (1992) respectively so that we can run year-round assays if necessary. Immunostaining of some plasma proteins suggests that they are formed exogenously.

We have isolated some VAPs by running comparative PAGE of vesicle-rich and vesicle-poor fractions and characterised them using a variety of techniques. Using this system we have identified 9 bands that are unique or more intense in the vesicle-rich section. We were unable to sequence bands from the vesicle-poor fraction because insufficient protein was present. Sequence analysis was used to identify the known proteins, or deduce the oligonucleotide sequence. This sequence was then used for molecular cloning. Two cDNAs encoding vap5 and vap7, have been cloned from ovarian tissue. Analysis of these two genes revealed that VAP5 and VAP7 are novel proteins and they have unique features in molecular constitution. Recombinant protein expression is in progress for vap5 and the sequence identity of vap5 and vap7 is still being assessed

Coat Proteins (CPs)

We have shown in previous projects that the two outer coats of the possum conceptus, mucoid and shell coat are secreted by the oviduct and uterus respectively. Because uterine glands extend into the utero-tubal junction, shell coat secretion occurs here as well. The time of secretion is associated with particular stages of the reproductive cycle and is possibly hormonally dependent. We have established that shell coat secretion continues until the time of implantation and is not restricted to the peri-ovular period (Casey and Selwood, in preparation) as suggested in earlier studies (see review, Selwood, 2000). The coats appear to play a minor role during early cleavage, but in late cleavage are essential for normal blastocyst development and epithelial maintenance (Casey and Selwood, in preparation). As the shell coat, in particular is unique to marsupials, it is a very suitable target for immunocontraception.

PAGE analysis followed by amino acid sequencing of CPs identified 5 proteins of which the fourth, (cp4) has been cloned. Sequence analysis is in progress and ANGIS reveals extensive regions without homology to other proteins. The recombinant protein has been expressed and used to generate a polyclonal antibody, which will be used for further characterisation of the protein. Expression studies shows that possum cp4 expression builds up during pre-implantation stages, supporting the evidence from ultrastructural and volumetric analysis on shell coat protein secretion that coat secretion continues until near to implantation.

An in vitro system of uterine monolayers established over 7-14 days following the techniques of Sherman (1976) has been established for the possum, to test the effect of the antibody on CP4 secretion and to determine whether coat secretion is hormonally dependent.

Leukemia inhibitory factor (LIF)

We have previously reported tvLIF cloning and characterisation (Cui and Selwood, 2000). Conditions for expression of tvLIF in bacteria were further optimised and purified tvLIF was used to immunise mice for generation of monoclonal antibodies. All 37 positive cell lines established by ELISA narrowed down to 4 strongly positive cell lines by ELISA and Western blot screening. These cell lines have been maintained for production of anti-tvLIF antibodies. A preliminary study of the binding capacity of the monoclonal antibody detected a 65 kDa native LIF protein in uterine tissue.

The tvLIF gene is expressed in the uterine tissue constantly at a basal level. Semi-quantitative studies show that tvLIF is expressed in the uterus between the early unilaminar blastocyst and the early fetal stage during embryonic development. It is interesting that LIF expression is strongest at implantation even though implantation in the possum is extremely superficial. These findings on LIF confirm our previous observations that implantation does not occurs later than the trilaminar blastocysts stage as reported in other studies.

An assay system to test the effect of LIF protein and antibody in vitro has been developed using a possum pouch young (PY) fibroblast cell line. Two substances known to up-regulate LIF expression and protein secretion, tumour necrosis factor (TNF_) and lipopolysaccharide (LPS) have been used. PAGE and Western blotting analysis has shown that LPS upregulates LIF but TNF has no significant effect.

Other Molecules

We have also established that early pregnancy factor(EPF), an immuno-suppressant molecule that is present from the zygote stage to immediately prior to birth in other mammals and the stripe-faced dunnart (Cruz et a, 2001) is present in the pregnant possum at the flat embryo stage and is absent in non pregnant possums undergoing the luteal phase of the oestrous cycle.

Conclusions and Future Directions

A total of 22 candidate molecules were identified by polyacrylamide gel electrophoresis (PAGE), amino acid sequencing, immunostaining, enzyme digestion, cloning and gene expression studies. Of these, 13 have not been further investigated because they were in too small a quantity to sequence or the sequence was ambiguous or too closely aligned to similar molecules in other species. Nine have been targeted because they have sufficient possum specificity and developmental significance to make them suitable to test whether they have an effect on possum fertility. These molecules areVAP5, VAP7, _ fetoprotein, hyaluronate-associated protein, CP2, CP4 and CP5 and LIF and LIF receptor protein. Expression and other studies have indicated that their time of action will effectively cover the pre-implantation period.

For future projects we would target the proteins that we know most about at present, VAP5, CP4 and LIF. For these we would examine whether possums mount and immune response to them, and further examine their expression patterns and effect in vitro. Proteins with a suitable profile would then have their effect on fertility tested in vivo in the possum.

Acknowledgements

This work was supported by the Ministry of Agriculture and Forestry (MAFPOL) New Zealand.

References

Butcher L and Ullmann SL (1996). Culture of pre-antral ovarian follicles in the grey short-tailed opossum Monodelphis domestica. Reprod. Fertil. Devel. 8, 491-508.

Cui, S. and Selwood, L. (2000). cDNA cloning, characterisation, expression and recombinant protein production of leukemia inhibitory factor (LIF) from the marsupial, the brushtail possum, Trichosurus vulpecula. Gene, 243, 167-178

Duckworth, J.A., Scobie, S., Jones, D.E. and Selwood, L. (1998). Determination of oestrus and mating in captive female Brushtail Possums, Trichosurus vulpecula (Marsupialia: Phalangeridae) from urine samples. Aust. J. Zool. 46, 547-555.

Fletcher, T.P. and Selwood, L. (2000). Possum Reproduction and Development. In "The Brushtail Possum in New Zealand". (Ed T. Montague), pp. 62-80. Landcare Press, New Zealand.

Frankenberg, S. and Selwood, L. (1998). An ultrastructural study of the role of an extracellular matrix during cleavage in a marsupial, the brushtail possum. Mol. Reprod. Dev. 50, 420-433.

Frankenberg, S. and Selwood, L. (2001). Ultrastructure of oogenesis in the brushtail possum. Molecular Reproduction and Development. 58, 297-306.

Frankenberg, S., Newell, G. and Selwood, G. (1996). A light microscopic study of oogenesis in the brushtail possum, Trichosurus vulpecula.. Reprod. Fertil. Devel. 8, 491-508.

Frankenberg, S., Tisdall, D. and Selwood, L. (2001). Identification of a homolog6ue of POU5F1 (OCT3/4) in a marsupial the brushtail possum. Molecular Reproduction and Development.58, 255-261.

Selwood, L. (2000). Marsupial egg and embryo coats. Cells, Tissues, Organs. 166, 208-219

Selwood L. and VandeBerg, JW (1992). The influence of incubation temperature on oocyte maturation, parthenogenetic and embryonic development in vitro of the marsupial Monodelphis domestica. Animal Reprod. Sci. 29, 99-116.

Sherman, M (1978), Implantation of mouse blastocysts in vitro. in Methods in Mammalian Reproduction, Ed J. Daniel, Academic Press NY. pp.247-258.

Table 1 Targeted Developmental Molecules in the Brushtail Possum

AASA, amino acid sequence analysis; ECM, extracellular matrix; GE, gene expression, I-S, immuno-staining

Stage of Action	Type	Examples	Method of Identification
Oogenesis, cleavage, early blastocyst	ECM Plasma proteins VAP	Hyaluronate Serum albumin Immunoglobulin Inter _ trypsin inhibitor _ fetoprotein VAP5 VAP7	Enzyme action AASA, I-S AASA, I-S I-S I-S AASA, cloning AASA, cloning
Late cleavage, blastocyst stages	Coat proteins	CP4 CP5	AASA, cloning, GE AASA
Later blastocyst stages, flat embryo	Implantation proteins	LIF	Cloning, I-S, GE
All	Pregnancy maintenance	EPF	I-S, serum and ovary

Figure 1. A diagrammatic representation of target molecules identified in possum development and their estimated time of action during pre-implantation stages of pregnancy, oogenesis and oestrus. Tlbl, trilaminar blastocyst. Unbroken line, known time of action; broken line, estimated time of possible action.

Lynne Selwood - Figure 1

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