Captive breeding does not alter brain volume in a marsupial over a few generations.

Captive breeding followed by reintroduction to the wild is a common component of conservation management plans for various taxa. Although it is commonly used, captive breeding can result in morphological changes, including brain size decrease. Brain size reduction has been associated with behavioral changes in domestic animals, and such changes may negatively influence reintroduction success of captive-bred animals. Many marsupials are currently bred in captivity for reintroduction, yet the impacts of captive breeding on brain size have never been studied in this taxa. We investigated the impacts of a few generations (2-7) of captive breeding on brain volume in the stripe-faced dunnart (Sminthopsis macroura), and found that captive breeding in a relatively enriched environment did not cause any changes in brain volume. Nonetheless, we advocate that great care be taken to provide suitable husbandry conditions and to minimize the number of captive generations if marsupial reintroduction programs are to be successful.

A marsupial, Trichosurus vulpecula, DDX4/VASAGene (TvDDX4) of the DEAD box protein family: molecular conservation and germline expression.

The DDX4/VASA gene plays an important role in germ cell development in animals. We cloned and characterized a marsupial DDX4/VASA homolog (TvDDX4, 2,769 bps) from the possum and examined its expression in adult tissues at mRNA and protein levels. The isolated cDNA had a deduced 704 amino acid residues with significant homology to DDX4 from other animals, including mouse (86%) and human (87%). The DDX4 transcript was detected in the ovary and testis, and was undetectable in somatic tissues. The recombinant possum DDX4 protein (TvDDX4) was successfully produced in a bacterial expression system and used in polyclonal antibody generation. The recombinant TvDDX4 was detected by antibody against human DDX4 and mouse antibody against TvDDX4, but native possum TvDDX4 was only recognized by the possum antibody in the ovary and testis. Our results suggest a structural and functional conservation of DDX4 in marsupials and in mammals in the therian branches.

The production of mature oocytes from adult ovaries following primary follicle culture in a marsupial.

A model marsupial culture system has been developed whereby individual primary follicles, obtained from adult ovaries, can be grown in vitro to the antral stage and oocytes retrieved from these follicles can achieve nuclear maturation (metaphase II) in the presence of LH. Primary follicles isolated from adult Sminthopsis macroura ovaries were cultured individually in one of four systems: microdrops under oil, upright, inverted, or roller culture. After 6 days of culture, cumulus-oocyte complexes (COCs) were excised from early antral follicles and incubated for an additional 24 h to assess meiotic competence and the effects of LH and lithium on oocyte maturation. Histology and transmission electron microscopy established normal in vivo standards and verified oocyte and follicular integrity following culture. On day 6 of culture, follicle viability was significantly greater in the inverted system (73%) than in the other three systems (10-46%). The inverted system was the most effective in supporting development with follicles demonstrating progressive growth during culture and showing antral signs by day 4. Meiotic resumption during COC culture was facilitated by LH, but hindered by lithium. The ability to resume meiosis and progress to metaphase II was equivalent in oocytes retrieved following follicle culture and those matured in vivo. This study highlights the importance of oxygen and nutrient availability during marsupial follicle culture, and demonstrates for the first time that primary follicles isolated from adult mammalian ovaries can undergo normal growth and development in vitro, to produce mature, meiotically competent oocytes.

Progesterone concentration in the marsupial Sminthopsis macroura: relationship with the conceptus, uterine glandular regeneration and body weight.

Close examination of hormonal profiles and uterine morphology in the marsupial reproductive cycle highlights significant differences between pregnant and non-pregnant cycles. In the polyovular dasyurid marsupial Sminthopsis macroura, we identified changes associated with gestation by comparing ovarian and plasma progesterone concentrations, uterine weights, uterine epithelial mitoses, body weights and gestation lengths between pregnant and non-pregnant luteal phases. The plasma progesterone profile of S. macroura was biphasic, peaking during unilaminar blastocyst expansion and on the day of implantation. Periods of rapid embryonic development were associated with increasing plasma progesterone concentrations and animal body weight. For the first time in a polyovular marsupial, we identified 1) a correlation between ovarian progesterone concentration and conceptus number during the luteal phase just prior to implantation (total ovarian progesterone), indicating a conceptus influence on progesterone concentration; 2) a pulse of uterine epithelial mitotic activity at the time of implantation and 3) increased mitotic activity in pregnant animals during unilaminar blastocyst formation compared with non-pregnant animals. Gestation length was reduced by up to 15%, due to the loss of, or reduction in, the four-cell arrest and more rapid definitive blastocyst expansion. This is the first time a conceptus influence on gestation length has been identified in a dasyurid. This study provides further evidence for the modification of the luteal phase by pregnancy in S. macroura.

Uterine expression of cp4 gene homolog in the Stripe-faced Dunnart, Sminthopsis macroura: relationship with conceptus development and progesterone profile.

The marsupial conceptus is surrounded by a uterine-secreted shell coat for 60-80% of gestation. Coat protein 4 (CP4) is the only marsupial shell coat protein characterized and it has only been identified in one species, the Common Brushtail Possum. In this possum, uterine transcription and secretion of cp4 during the oestrous cycle is biphasic and associated with the stage of conceptus development. Here we cloned cp4 (sm-cp4) from a distantly related species, the Stripe-faced Dunnart (Sminthopsis macroura). Transcription of sm-cp4 and secretion of smCP4 were identified by semi-quantitative RT-PCR and immunohistochemistry, respectively. The effect of reproductive hormones on sm-cp4 transcription was investigated in vitro by treatment of uterine explant cultures with oestrogen and/or progesterone. In vivo, uterine expression of smCP4 was biphasic and associated with conceptus development. Uterine smCP4 expression (transcription and secretion) began during the pre-ovulatory period and continued post-ovulation during cleavage stages. Transcription of sm-cp4 continued during the unilaminar blastocyst stage, but smCP4 secretion was reduced during this stage. During the bilaminar blastocyst stage, both transcription of sm-cp4 and secretion of smCP4 were low before they both resumed during the trilaminar blastocyst stage, and continued during the embryo and fetal stages. In vitro uterine transcription of sm-cp4 increased after incubation with progesterone.

The Development of Integration in Marsupial and Placental Limbs.

The morphological interdependence of traits, or their integration, is commonly thought to influence their evolution. As such, study of morphological integration and the factors responsible for its generation form an important branch of the field of morphological evolution. However, most research to date on post-cranial morphological integration has focused on adult patterns of integration. This study investigates patterns of correlation (i.e., morphological integration) among skeletal elements of the fore- and hind limbs of developing marsupial and placental mammals. The goals of this study are to establish how patterns of limb integration vary over development in marsupials and placentals, and identify factors that are likely responsible for their generation. Our results indicate that although the overall pattern of correlation among limb elements is consistent with adult integration throughout mammalian development, correlations vary at the level of the individual element and stage. As a result, the relative integration among fore- and hind limb elements varies dynamically between stages during development in both marsupial and placental mammals. Therefore, adult integration studies of the limbs may not be indicative of developmental integration. Results are also consistent with integration during early limb development being more heavily influenced by genetic and developmental factors, and later by function. Additionally, results are generally consistent with a constraint on marsupial forelimb evolution caused by the functional requirements of the crawl to the teat that operates by limiting morphological variation before and at the time of birth, and not after.

Vesicle-associated protein 1: a novel ovarian immunocontraceptive target in the common brushtail possum, Trichosurus vulpecula.

Ovarian-based immunological research is currently restricted to proteins of the zona pellucida. This study examined the immunocontraceptive potential of a novel vesicle-associated protein, VAP1, previously isolated from the vesicle-rich hemisphere of the brushtail possum oocyte. Seven female possums were immunized against recombinant glutathione S-transferase-VAP1 fusion protein. Control animals (n=3) received antigen-free vaccinations. Following immunization, regular blood sampling determined the level and duration of immune response. Animals were monitored daily, pre- and post-immunization, to determine estrous cycling activity and the percentage of reproductive cycles yielding viable young. The reproductive tracts and somatic organs of VAP1-immunized (n=7), control-immunized (n=3) and non-immunized (n=5) animals were collected and examined by histology and transmission electron microscopy. VAP1 immunization caused a strong and sustained immune response. Elevated levels of VAP1 antibody binding were detected in sera following initial injections, and immune titers rose as boosters were administered. Immunization had no adverse effect upon animal behavior or body condition. Immunized females demonstrated no major change in annual estrous cycling activity; however, the percentage of reproductive cycles resulting in pouch young decreased significantly (P<0.05) by 40%. Histological and ultrastructural analyses revealed an abundance of lipid-like degradation bodies within the ooplasm of developing oocytes and the cytoplasm of failing uterine zygotes. Active macrophage invasion of enlarged endometrial glands was observed in the uteri of two females. Reproductive tract changes are discussed in relation to observed fertility decline. The results of this study indicate that VAP1 has exciting potential as an immunocontraceptive target for possum control in New Zealand.

Novel immunocontraceptive targets in mammals: uterine secretions and the conceptus; a marsupial approach.

We report the first immunocontraceptive trial in mammals using a uterine-secreted protein, the marsupial shell coat protein 4 (CP4). The marsupial shell coat, which surrounds the conceptus for 60-80% of gestation, is secreted by the uterine epithelium. Following immunization against glutathione S-transferase (GST)-CP4, the fertility of female common brushtail possums (n=6) was significantly reduced (P=0.000), and this reduction in fertility was positively correlated with the maximum GST-CP4 humoral immune response (P=0.025). Ultrastructural examination of the reproductive tract indicated that the cell-mediated immune response against GST-CP4 targeted the shell coat, the shell-free conceptus and the uterine glandular epithelium, thus preventing normal conceptus development and uterine secretion of shell coat proteins and nutrients. These results show that uterine-secreted proteins are promising immunocontraceptive targets, especially in pest mammal species, e.g. possum, rabbit and horse, that have uterine-secreted additions to embryonic coats, or that have late implantation requiring uterine nutrient provisioning from secretions.

Development in vitro of Marsupials: a comparative review of species and a timetable of cleavage and early blastocyst stages of development in Monodelphis domestica.

The development of marsupial oocytes and embryos in vitro is reviewed. Most stages of development have been cultured successfully, usually in a complex medium with added fetal calf serum. Simpler media without added serum have been developed for fertilization and cleavage in vitro. Culture systems have been established for oocyte maturation and fertilization in the grey short-tailed opossum and for cleavage from the zygote to the early expanding unilaminar blastocyst in a number of other marsupials. Survival in vitro of the unilaminar and early bilaminar blastocyst stages is limited in all species examined. In contrast, late bilaminar, trilaminar, embryonic and fetal stages develop at rates approximating those in vivo. More stages have been cultured successfully in Sminthopsis macroura than in any other species. It has been cultured from the late bilaminar blastocyst to within 18 h of birth. Stages of cleavage and unilaminar blastocyst formation of Monodelphis domestica timed by videotaping mating animals, proceeded at similar rates in vivo and in vitro. As in other marsupials, cleavage in this opossum is characterized by a polarized conceptus. This polarity is expressed in the distribution of organelles in the zygote and the localization of secretion of the extracellular matrix material into the cleavage cavity and of the initial cell-zona attachment. Because cell-cell adhesion follows cell-zona adhesion, a unilaminar blastocyst forms without the development of an intervening morula stage.

cDNA cloning, characterization, expression and recombinant protein production of leukemia inhibitory factor (LIF) from the marsupial, the brushtail possum (Trichosurus vulpecula).

A reverse transcription technique using RNA templates combined with polymerase chain reaction (RT-PCR) was used to clone the cDNA fragment encoding the amino acid sequence of mature LIF protein of the marsupial, the brushtail possum, Trichosurus vulpecula. A PCR product with expected size, of 546bp, and termed tvLIF, was obtained using cDNA reverse-transcribed from total RNA isolated from possum uterus. A genomic DNA fragment (about 650bp) between the specified primers was also amplified, indicating the similarity in structure and organization of this gene and LIF genes from studied eutherian species, although the full-length of its cDNA and genomic DNA needs to be further clarified. The deduced amino acid sequence of tvLIF shows a high level of sequence identity and similar molecular characteristics to eutherian LIF, which suggests similar biological actions of this molecule in this marsupial. Because the expression of LIF gene in other mammalian species has been found to be at very low levels and its transcripts cannot be detected by Northern hybridization analysis, the expression pattern of tvLIF in adult tissues and reproductive tracts during early development was investigated using the RT-PCR technique. Resultant products of the RT-PCR were further analyzed by Southern hybridization using tvLIF as a probe. tvLIF transcripts were detected in most of the adult tissues and in the reproductive tracts of pregnant females. These results lend support to the idea that LIF contributes to the maintenance of pregnancy in this marsupial.

A developmental investigation of the liver, bone marrow and spleen of the stripe-faced dunnart (Sminthopsis macroura).

The development of the liver, bone marrow and spleen have been investigated in the stripe-faced dunnart. At birth, the liver was undergoing haematopoiesis but the level declined rapidly and by day 50 after birth the liver was histologically mature. Both the bone marrow and spleen were non-haematopoietic at birth but initiated haematopoiesis shortly thereafter. Bone marrow was initially detected at day 11 postpartum. By 57 days after birth, adipocytes had infiltrated the marrow and were abundant by day 60 after birth. Mitotic cells were observed in remaining areas of marrow until at least 170 days postpartum. The spleen at birth was undifferentiated, with trabeculae appearing by day 42. Red and white pulp areas became apparent by day 43 and were well defined by day 57 after birth. In summary, the pattern of the development of the liver, bone marrow and spleen in the stripe-faced dunnart were similar to that observed in eutherians and other metatherians studied to date.

Cleavage in vivo and in culture in the Dasyurid marsupial Antechinus stuartii (macleay).

Cleavage in the brown marsupial mouse, Antechinus stuartii, from the zygote to the unilaminar blastocyst, was observed in vivo and in culture and in sections of embryos. The first three divisions were meridional and passed from the yolk pole to the opposite pole. Deutoplasmolysis, resulting in a distinct yolk mass, occurred during the first two divisions. Prior to the third and fourth divisions, the blastomeres elongated and flattened against the zona pellucida. The fourth division was latitudinal and resulted in two histologically distinct rings of eight blastomeres which were at first rounded and then became flattened against the zona. Further divisions and flattening of the blastomeres resulted in a complete unilaminar blastocyst by the time the blastocyst numbered 22 to 30 cells. Some expansion, causing compression of the zona and mucoid layers, occurred before completion of the blastocyst, but most expansion occurred once the blastocyst was complete. No histological differences could be detected between the blastomeres at this stage. Embryos were successfully cultured from the zygote to the rounded four-cell stage and from the flattened four-cell stage to the completed unilaminar blastocyst of around 32 cells. Total estimated cleavage times were slower in culture than in vivo, but the relative lengths of time for each division were approximately the same.

There is no highly conserved embryonic stage in the vertebrates: implications for current theories of evolution and development.

Embryos of different species of vertebrate share a common organisation and often look similar. Adult differences among species become more apparent through divergence at later stages. Some authors have suggested that members of most or all vertebrate clades pass through a virtually identical, conserved stage. This idea was promoted by Haeckel, and has recently been revived in the context of claims regarding the universality of developmental mechanisms. Thus embryonic resemblance at the tailbud stage has been linked with a conserved pattern of developmental gene expression - the zootype. Haeckel's drawings of the external morphology of various vertebrates remain the most comprehensive comparative data purporting to show a conserved stage. However, their accuracy has been questioned and only a narrow range of species was illustrated. In view of the current widespread interest in evolutionary developmental biology, and especially in the conservation of developmental mechanisms, re-examination of the extent of variation in vertebrate embryos is long overdue. We present here the first review of the external morphology of tailbud embryos, illustrated with original specimens from a wide range of vertebrate groups. We find that embryos at the tailbud stage - thought to correspond to a conserved stage - show variations in form due to allometry, heterochrony, and differences in body plan and somite number. These variations foreshadow important differences in adult body form. Contrary to recent claims that all vertebrate embryos pass through a stage when they are the same size, we find a greater than 10-fold variation in greatest length at the tailbud stage. Our survey seriously undermines the credibility of Haeckel's drawings, which depict not a conserved stage for vertebrates, but a stylised amniote embryo. In fact, the taxonomic level of greatest resemblance among vertebrate embryos is below the subphylum. The wide variation in morphology among vertebrate embryos is difficult to reconcile with the idea of a phyogenetically-conserved tailbud stage, and suggests that at least some developmental mechanisms are not highly constrained by the zootype. Our study also highlights the dangers of drawing general conclusions about vertebrate development from studies of gene expression in a small number of laboratory species.

The blastocyst epithelium is not a protoderm in dasyurid marsupials: a review of the evidence.

Evidence from studies of cleavage and blastocysts in dasyurids in reviewed to show that the unilaminar blastocyst is not a protoderm but consists of two cell types. Cleavage and blastocyst formation in marsupials has been most comprehensively studied in dasyurids, in which the secondary oocyte and zygote are polarized with respect to the position of the nucleus, cytoskeletal elements and cytoplasmic vesicles. Polarity is reinforced by fertilization. Early cleavage divisions are associated with the polarized elimination of a yolk mass and many vesicular structures into the perivitelline space. Because secretion of the vesicular structures, of which several types are found, facilitate blastomere-zona then blastomere-blastomere associations during cleavage, a unilaminar blastocyst forms without an intervening morula stage. Polarization of the cleavage cavity is related to the appearance of two cell lineages, pluriblast and trophoblast, at the 16-cell stage. In species in which the yolk mass persists, the tier of eight cells lying nearest the yolk mass forms the pluriblast (future embryonic and extra-embryonic lineages), and the other tier forms the trophoblast (future extra-embryonic ectoderm of the yolk sac). Thus, the unilaminar blastocyst epithelium is not a protoderm. Blastocyst expansion is associated with increased cell numbers mainly in the trophoblast. Pluriblast cells begin to increase just before the appearance of the hypoblast. The two cell populations can be distinguished by ultrastructural and histological features at the end of cleavage. During blastocyst expansion they differ in appearance and behaviour in vitro and in cell-doubling time. Two populations of cells have also been found in cleavage and unilaminar blastocyst stages in other marsupials, such as opossums, brushtail possums and bandicoots.

Development of early cell lineages in marsupial embryos: an overview.

All major embryonic and extra-embryonic cell lineages are established before implantation in marsupials. In reptiles, birds, monotremes and most marsupials, the zygote is polarized, sometimes markedly so, and the cleavage pattern in relation to the polarized state provides the mechanism for the generation of positional signals. These ensure that the embryonic cell lineages develop in the centre of the developing blastoderm or blastocyst epithelium and the extra-embryonic lineages at the periphery. The evolution of the vertebrate yolky egg was accompanied by a decreasing dependence on maternal determinants and increasing dependence on positional signals to determine cell fate. It is proposed that when a less yolky egg evolved, the mechanisms for determination of cell fate in a developing epithelium were retained. It is proposed that in marsupials, positional signals are involved in the determination of cell fate of embryonic and trophectoderm cells but do so in a two-dimensional epithelium not a three-dimensional morula. The next lineage formed is the primary endoderm which separates off from the primitive ectoderm in the embryoblast and eventually lines the blastocyst cavity. Positional signals are responsible for the determination of primary endoderm in eutherian mammals, birds and probably also marsupials. Order of cell division during cleavage provides a mechanism whereby some cells in the embryoblast of marsupials have earlier and greater contact with their neighbouring cells. The mechanism for determination of primary endoderm cells in the blastocyst epithelium is examined in the Virginia opossum and the stripe-faced dunnart.

The type and differentiation of cells in vitro from unilaminar and bilaminar blastocysts of two marsupials, Antechinus stuartii and Sminthopsis macroura.

The type and ability to differentiate in vitro of cells found in blastocysts of two marsupials were examined. Thirteen unilaminar blastocysts on Day 7 to Day 12 of gestation and 24 bilaminar blastocysts on Day 16 and Day 18 of gestation were collected from 11 brown antechinus, Antechinus stuartii. A total of of 77 unilaminar blastocysts on Day 5 and Day 6 of gestation and a total of 61 bilaminar blastocysts on Day 6 and Day 7 of gestation were collected from 40 stripe-faced dunnarts, Sminthopsis macroura. Pluriblast and trophoblast cells, confined to separate hemispheres, were found in unilaminar and bilaminar blastocysts, establishing that the blastocyst epithelium was not a protoderm. Hypoblast cells were found only in bilaminar blastocysts. Pluriblast and hypoblast cells did not differentiate or proliferate in up to eight weeks in culture. A small number of trophoblast cells transformed to a multinucleate state but the remainder did not proliferate or differentiate further. The presence of murine leukaemia inhibitory factor or medium conditioned by exposure to marsupial fibroblast feeder layer was not required for the maintenance of an undifferentiated state. Differentiation, proliferation and attachment of cells were not influenced by the presence of the yolk mass or the egg coats in culture. The time taken for attachment of dissociated cells varied significantly between cultures with no substrate and with collagen, fibronectin or laminin (P = 0.001, ANOVA) but did not vary significantly between substrates. The substrates did not influence the state of differentiation of the cells.

Embryonic development in culture of the marsupials Antechinus stuartii (Macleay) and Sminthopsis macroura (Spencer) during preimplantation stages.

Forty-nine blastocysts from 11 brown antechinus, Antechinus stuartii, and 96 blastocysts from 17 stripe-faced dunnarts, Sminthopsis macroura, were used to develop a culture system for embryos during preimplantation stages. Blastocysts of brown antechinus were collected on Days 6-9 for unilaminar stages, Days 16-21 for bilaminar stages and Days 20 and 21 for trilaminar stages. Blastocysts of stripe-faced dunnarts were collected on Day 6 for unilaminar stages, Days 6-8 for bilaminar stages and Day 8 for trilaminar stages. Culture media were Dulbecco's modified Eagle's medium (DMEM) with 4.5% glucose and Whittingham's T6 medium both of which were supplemented with 5, 10, 12.5 and 20% fetal calf serum (FCS). Antechinus serum (5%) and bovine serum albumin (0.1%, 0.2%) were also added to some media. Human amniotic fluid (HAF) and Monomed media were also tested. Blastocysts were cultured at 35 degrees C in 5% CO2 in air. DMEM + 10% FCS and HAF supported normal development for the longest periods and over the greatest range of stages. Developmental failure of blastocysts in vitro during expansion of the unilaminar blastocyst and formation of the bilaminar blastocyst suggests that these stages may be dependent on uterine signals. When cultured in DMEM + 10% FCS, the rate of development of bilaminar and trilaminar blastocysts into organogenesis was 4 h slower than in vivo in the stripe-faced dunnart and about 6 h slower than in vivo in the brown antechinus. Embryos of stripe-faced dunnarts were cultured to within 18 h of birth.

Plasma progesterone concentrations during pregnancy in the dasyurid marsupial, Antechinus stuartii: relationship with differentiation of the embryo.

Antechinus stuartii females were sacrificed at various stages of pregnancy timed from the first day that epithelial cell numbers declined in the urine (day 0). Embryos were recovered and their developmental stage assessed, corpora lutea (CL) were fixed for histological sections and the plasma was taken for progesterone determination. The rate of development of the embryo appeared to parallel the formation of the CL and the plasma progesterone concentrations: cleavage (less than day 6) and the expansion of the unilaminar blastocyst (less than day 14), were periods of slow development, during which the formation of the CL was incomplete and plasma progesterone was low [less than 6 ng mL-1 (19.1 nmol L-1)] though steadily increasing. After day 14 the CL reached its maximum size and plasma progesterone was maximally elevated [greater than or equal to 15 ng mL-1 (47.7 nmol L-1)] until parturition on day 27. Rapid differentiation and growth of the embryo occurred between day 22 and day 27.

A light microscopic study of oogenesis in the brushtail possum Trichosurus vulpecula.

Ovaries from young of the brushtail possum, Trichosurus vulpecula, were examined histologically and histochemically to determine stages of oogenesis. Groups of dividing oogonia were first present in a 13-day-old pouch young, with extensive oogonial proliferation after about 23 days of age. Meiosis was initiated in some oogonia by 48 days of age, and by 88 days numerous early primordial follicles were present. The first primary follicles had formed by 103 days of age. In oocytes of quiescent primordial follicles, dark granular material, which stained positively for protein, mainly occupied the perinuclear cytoplasm. Associated with the transition to primary follicles and continuing throughout oocyte growth was the production within the central cytoplasm of large clear vesicles and, to a lesser extent, lipid-like yolk bodies. The former were analogous to similar vesicles found in other species of marsupial, and have a putative role in development of the blastocyst. By contrast, yolk-like bodies are not found in all marsupial oocytes and their importance in development is unknown.