Biological Management of Possums - In Vitro and In Vivo Models of Key Reproductive Events as Tools for Possum Biological Control

Introduction
Egg maturation
Sperm maturation (capacitation)
Fertilization
Embryo development
Applications
Table 1: Current Status of Models of Key Reproductive Events for the Possum
Conclusions
Acknowledgements
References

Session 1 Manipulation of Breeding

In Vitro and In Vivo Models of Key Reproductive Events as Tools for Possum Biological Control

Molinia FC¹, Glazier AM¹, Myers JV¹, Sidhu KS², Mate KE², Berg DK³, Smith JF³, Cowan PE⁴ and Rodger JC²

Cooperative Research Centre for the Conservation and Management of Marsupials¹Landcare Research, PO Box 69, Lincoln, 8152, New Zealand

²Department of Biological Sciences, Macquarie University, NSW 2109, Australia

³AgResearch Limited, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand

⁴Landcare Research, Private Bag 11052, Palmerston North, New Zealand

Introduction

Key reproductive events like egg and sperm maturation, fertilization and embryo development, are potential targets for disruption for effecting possum biocontrol. In recent years, the Marsupial CRC has focussed its extensive suite of assisted reproductive technologies on development of in vitro and in vivo models of these events. These models are the "tools" for: a) rapidly progressing identification of suitable reproduction-based targets; and b) cost-effective and efficient screening of emerging reproductive-function based vaccines (Molinia et al., 1999). An update on the development of these models is reported here; both in the context of new targets that warrant further investigation and their suitability for screening emerging vaccines.

Egg maturation

Possum eggs, like those of eutherians, remain arrested at the germinal vesicle stage until the immediately preovulatory period. During this time they increase dramatically in size, commence maturation of cytoplasmic components, complete the first meiotic division, and extrude the first polar body (Mate, 1996). Tammar wallaby eggs undergo spontaneous nuclear maturation when removed from the ovarian follicle and culture in vitro under suitable conditions (Mate and Rodger, 1993; Mate and Buist, 1999). A similar protocol has been established for the possum, which results in more than 50 percent of eggs collected from large (> 2 mm) ovarian follicles of PMSG-primed females reaching the metaphase II + 1 polar body stage, after in vitro maturation (IVM) for up to 48 hrs (Glazier et al., unpublished observations). Recent experiments have shown that these eggs are capable of being fertilized in vitro (detailed below).

Superovulation protocols have been developed for the possum, to increase the productivity of captive females, and as a model of in vivo egg maturation. During the breeding season, treatment with PMSG/pLH results in ovulation of 9-10 eggs per female (Glazier and Molinia, 1998), from 30 hr after pLH treatment (Glazier, 1999), and up to half of these are fertilized following artificial insemination (AI) (Molinia et al., 1998, Mate et al., 1998). For stimulation during seasonal anoestrus, a pFSH/pLH protocol has been developed, but nuclear maturation rates of eggs recovered, and fertilization rates following AI were low (Molinia et al., 2000). Work is in progress using a PMSG + pFSH/pLH protocol to optimise numbers of in vivo matured eggs from possums treated during seasonal anoestrus.

Sperm maturation (capacitation)

The process by which sperm acquires the capacity to fertilize an egg is termed capacitation. Co-incubation of sperm in vitro with oviduct epithelial cell monolayers (OECM) (Sidhu et al., 1999a), or conditioned media (CM) from oviduct explants (Sidhu et al., 1999b), generates large populations of highly motile, T-shaped sperm, a putative indicator of capacitation (Molinia et al., 1998; Jungnickel et al., 2000). Recently, computer-assisted semen analysis was used to characterise motility patterns of these "capacitated" sperm, no changes were observed which were consistent with those observed with eutherian sperm capacitation (Smith et al., 2001), despite these in vitro matured sperm having the capacity to fertilize eggs (Mate et al., 2000; Sidhu et al., 2001).

Sperm recovered from the oviducts 5-6 hr after insemination of superovulated possums with 50 million motile sperm/uterus, represent a population of in vivo matured or capacitated sperm. This is because these sperm are capable of fertilising eggs in vivo (Molinia et al., 1998; Jungnickel et al., 1999) and in vitro (Mate et al., 2000).

Fertilization

In research towards development of IVF for any Australian marsupial, our group was first to demonstrate sperm-zona pellucida (ZP) binding and penetration in vitro (Mate et al., 2000). This was achieved using in vivo matured eggs collected from superovulated females and in vivo capacitated sperm collected from the oviducts of inseminated possums (positive control), and in vitro capacitated sperm. More recently, sperm-egg binding has been achieved (Sidhu et al., 2001), even with in vitro matured eggs, evidenced by the presence of decondensing sperm in the egg cytoplasm, one of the very early stages of true fertilization. Conventional EMEM media and a newly formulated possum synthetic oviduct fluid (pSOF), based on measurements of the ionic and energy substrate composition of possum oviduct fluid collected in vivo (Berg et al., unpublished), was used in these experiments. However, this recent success is mostly attributed to the levels of media additives (like foetal calf serum of polyvinyl alcohol) used in the in vitro system. Work is now focussed on moving towards a totally in vitro system by optimising the media and culture conditions required to mature eggs, capacitate sperm and achieve monospermic fertilizations.

Methods to achieve fertilization in vivo are now well established. This involves timed insemination of possums after superovulation treatment with sperm either into the uterus (Molinia et al., 1998) or vagina (Mate et al., 1998). Fertile conceptions have also been achieved with frozen-thawed sperm (Molinia and Myers, 1999).

Embryo development

In vitro culture (IVC) systems for embryos are currently being developed. Recently fertilized eggs sourced from females following superovulation/AI, continue development to the 4-cell stage when cultured on OECMs in vitro. Early cleavage-staged embryos have also been cultured in vitro in conventional media and foetal calf serum, and continue development (Lynne Selwood, personal communication).

Multiple embryos up to the 4-cell stage can be reliably recovered using superovulation/AI (Molinia et al., 1998). If these are allowed to continue development in vivo, they progress to the unilaminar blastocyst stage and then degenerate. However, since possums are monovular, the hormonal milieu of these superovulated animals may be inadequate to sustain further embryo development. Natural mating of captive possums still remains the most reliable method for development of embryos to term in vivo. Experiments are underway to determine whether timed AI of possums after removal of pouch young (the current best way to synchronise oestrus) yields live young, and whether more females give birth if they are treated with exogenous hormones before AI to better synchronise oestrus. Ultimately, this will lead to development of protocols for preparing surrogate mothers for embryo transfer (ET), to assess whether the multiple embryos generated after superovulation/AI are indeed capable of development to term.

Applications

Table 1 summarises the current status of the development and application of models of key reproductive events for the possum. Egg and sperm maturation models are being used by our group to confirm localisation of potential contraceptive antigens, their accessibility to antibody attack and to identify their mode of action of the block to fertilization. Superovulation/AI technology is now routinely used as a primary screen to examine in vivo fertility of possums immunised with already identified egg (Duckworth et al., 2001), sperm (Harris et al., 2001), and new reproductive tract-based vaccines before progressing to full-scale natural breeding trials (Rodger, 2001). IVF technology, once optimised, promises to be the most efficient and cost-effective means to screen emerging vaccines "in a petri-dish" for their ability to disrupt fertilization (Molinia et al., 1999). Similarly, the establishment of improved embryo development models will rapidly facilitate testing of emerging vaccines that interfere with the embryo and its interaction with the reproductive tract (Selwood et al., 2001; Rodger, 2001).

Apart from their role as a screening tool, development of these models has vastly increased our knowledge of key reproductive processes, providing opportunities to identify unique targets. For example, possum reproductive tract secretions (specifically from the oviduct) have a critical role in conferring sperm capacitation (Sidhu et al., 1999b; Mate et al., 2000). More recently, possum oviduct glycoprotein (OGP) has been identified, and since OGP plays an important role in pre- and post-fertilization events in mammals (Verhage et al., 1998), it warrants further development as a promising oviduct target. From studies of fertilization in vitro, the critical role of calcium in sperm capacitation, and the mechanism of sperm-egg fusion are being investigated, since these are uniquely different to conventional eutherian systems and thus represent emerging new target areas for biocontrol (Rodger et al., 2001).

Table 1: Current Status of Models of Key Reproductive Events for the Possum

Key event	In vitro model	Status	In vivo model	Status	Application
Egg maturation	Egg IVM	Established	Superovulation Breeding season Seasonal anoestrus	Established In progress	Screen vaccines that interfere with the development of matured eggs
Sperm maturation (capacitation)	Sperm + CM or OECM culture	Established	Sperm recovered from oviducts after superovulation/AI	Established	Screen vaccines that interfere with the ability of sperm to fertilize eggs Identify key reproductive tract targets
Fertilization	IVF	Sperm-ZP binding, penetration and sperm-egg fusion	Superovulation/AI	Established	Screen vaccines that interfere with fertilization Identify key sperm-egg interaction sites as targets
Embryo development	Embryo IVC	In progress	Up to blastocyst stage Cleavage to birth stages	Established In progress	Screen vaccines that interfere with the developing embryo and its

Conclusions

Models of key reproductive events for the possum are established or under development. Already, some are being used for vaccine screening, while others have revealed unique reproduction-based mechanisms as promising targets. These "tools" are the key to rapidly progressing biocontrol strategies for the possum.

Acknowledgements

This work is funded by the Australian Government's Cooperative Research Centres Program, the New Zealand Ministry of Agriculture and Forestry, Policy Division and the New Zealand Foundation for Research, Science and Technology (Contract C09X0009).

References

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