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L Selwood

Developing patterns in early embryogenesis are analysed in conceptuses from several families, including Dasyuridae, Phalangeridae, Macropodidae and Didelphidae, in which cleavage has been examined in some detail. Features common to cleavage and blastocyst formation, and in some cases to hypoblast formation, are used to develop an outline of possible mechanisms leading to axis formation and lineage allocation. Relevant features that have been described only in some species are also included. It is suggested that certain features of marsupial cleavage establish patterns in the developing blastocyst epithelia, pluriblast, trophoblast and hypoblast that contribute to axis formation and lineage allocation. All marsupials examined had a polarized oocyte or conceptus, the polarity of which was related to the conceptus embryonic-abembryonic axis and, eventually, the conceptus dorsal-ventral axis and the formation of the pluriblast (future embryo) and trophoblast. The embryonic dorsal-ventral and anterior-posterior axes were established after the allocation of hypoblast and epiblast. Mechanisms that appear to result in patterning of the developing epithelia leading to axis formation and lineage allocation are discussed, and include sperm entry point, gravity, conceptus polarity, differentials in cell-zona, cell-cell and cell-type (boundary effects) contacts, cell division order during cleavage and signals external to the conceptus. A model of the patterning effects is included. The applicability of these mechanisms to other amniotes, including eutherian mammals, is also examined.

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A Nation and L Selwood

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.

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L. Selwood and F. McCallum

Summary. Twenty-six female brown marsupial mice in a laboratory colony were mated at intervals ranging from 1 to 20 days between coitus and ovulation. The numbers of corpora lutea and normal embryos were counted.

A multiple regression model examined the parabolic relationship between the proportion of normal embryos and the time from coitus to ovulation. The proportion of normal embryos increased until a mean of 9·5 days and decreased thereafter. This relationship was independent of the year of breeding and the number of corpora lutea. After survival of spermatozoa for up to 13 days in the female reproductive tract, the fertility levels of females was 88–92%. Low fertility levels after 13 days appeared to be due to a decrease in the number of spermatozoa.

Reproductive tracts from 7 females killed after insemination and examined histologically showed many spermatozoa in the isthmus of the oviduct and the uterus at 5 days post coitum; spermatozoa confined to the isthmus between 6 and 13 days; and few spermatozoa in the isthmus at 14 days after copulation.

A comparison between the fertility levels in the females which had been inseminated once and a further 17 females which had been inseminated 2 or 3 times suggested that spermatozoa from 2nd and 3rd inseminations can contribute spermatozoa for fertilization. In these females fertility levels did not decline with time after the first mating.

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D Hickford and L Selwood

This study describes for the first time the spatial and temporal distribution of a growth factor and its receptors in uteri and conceptuses of a marsupial species during the peri-gastrulation period. Uteri (gravid and non-gravid) and blastocysts from 40 female stripe-faced dunnarts (Sminthopsis macroura) were collected over the peri-gastrula period (days 6.0-8.5) and stained immunohistochemically for transforming growth factor beta2 (TGFbeta2) and its receptors, TbetaRI and TbetaRII, to determine possible roles for TGFbeta2 in marsupial embryonic development. The events that occurred during the period examined included the appearance and proliferation of hypoblast and mesoderm, primitive streak and node formation, and early neurulation. Differences in TGFbeta2 quantities between gravid and non-gravid uteri reflect differences in uterine morphology, indicating a role for TGFbeta2 in endometrial remodelling. In blastocysts, large quantities of all three proteins in the trophectoderm during the node stage coincide with both blastocyst expansion before implantation and the appearance of multinucleated cells, indicating that TGFbeta2 plays a role in conceptus elongation and trophectoderm differentiation. In contrast, lack of TbetaRII in blastocysts during hypoblast formation and proliferation negates any role for TGFbeta2 in these processes, as both receptors are required for a response to TGFbeta2. High concentrations of TGFbeta2 but low concentrations of TbetaRII in blastocysts during early primitive streak formation indicate that paracrinal embryo-maternal signalling may be occurring, as blastocysts cannot respond to TGFbeta2 at this stage, but uteri could. A similar situation, but reversed, also occurs during primitive node formation.

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Y. P. Cruz and L. Selwood

Uterine samples from Antechinus stuartii on days 1, 4, 6, 8, 11, 13, 15, 18, 21 and 23 after ovulation were examined histologically. Animals were pregnant, nonpregnant and unmated, or nonpregnant and mated but found to have only unfertilized eggs on autopsy. The histological parameters used were thickness of the myometrium, endometrial stroma, and endometrial epithelium, and density of uterine stromal glands and of lymphocytes at the endometrial basal lamina. Overall, the fluctuation patterns of these parameters were superficially similar between pregnant and nonpregnant animals (mated or unmated). However, statistically significant differences were detected between pregnant and unmated nonpregnant animals in every parameter examined at nearly every time point except day 13. Comparison of these results with known data on embryonic stages, corpus luteum development and plasma progesterone concentrations revealed that the gravid uterus underwent histological changes co-incident with changes in both progesterone concentration and developmental delay or embryonic arrest. It was concluded that the uterus mediates progesterone-driven changes in embryonic developmental rate. Although determination of number of lymphocytes provided inconclusive evidence of cellular immunity against embryos, the possibility that embryonic signalling to the uterus occurs is discussed.

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Y. P. Cruz and L. Selwood

Uterine samples from pregnant Sminthopsis macroura representing the first 10 days of its 11 day gestation period and samples from non-pregnant animals were compared histologically and examined for differences in the following characteristics: thickness of the endometrial stroma, luminal epithelium, myometrium and glandular epithelium, and the density of stromal glands and number of lymphocytes at the endometrial basal lamina. A highly significant difference between gravid and non-gravid uteri with respect to thickness of the endometrial epithelium was found on day 3, when lineage divergence occurs between the pluriblast and trophoblast. The endometrial stroma was significantly thicker in pregnant animals on day 8, when the epiblast differentiates into ectoderm, endoderm and mesoderm. Other differences between gravid and non-gravid uteri were detected in myometrial thickness on days 1 and 5. Taken together, these results indicate that despite similar endocrinological profiles of pregnant and non-pregnant marsupials, there are subtle, but significant, differences in uterine histology. The observed concordance between histological differences and differentiative events in embryogenesis is considered as indicative of embryo–maternal signalling.

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YP Cruz, D Hickford and L Selwood

The inaccessibility of mammalian organogenesis stage embryos has precluded their widespread use in embryological and teratological studies. As organogenesis occurs during the last 1.5 days of the 10. 7 days of gestation in the stripe-faced dunnart (Sminthopsis macroura), the aim of the present study was to investigate whether day 9 and day 10 embryos and fetuses could be grown to term in vitro. High glucose Dulbecco's modified Eagle's medium with 10% fetal calf serum (FCS) supported embryonic growth for various periods of time, some to within 5 h of the predicted time of parturition. A roller culture system maintained at 35 degrees C was used to incubate organogenesis stage embryos (n = 43). Nine unincubated (control) embryos were either fixed for microscopic analysis or frozen for microprotein determination. The results of the present study indicate that with some optimization of the culture conditions (increasing oxygen in the gas phase in the culture tubes, replacing FCS with rat serum), it might be possible for organogenesis stage S. macroura embryos to be grown to term. A scoring scheme for assessing morphological development was devised for use as a standard in staging organogenesis stage embryos. This scheme reflects the highly compressed schedule of developmental events that occurs mainly during day 9 of gestation in S. macroura embryos. In comparison, during embryogenesis in Didelphis virginiana these developmental events occur from day 8 to day 10.5 of gestation, and birth occurs on day 13.

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E Menkhorst, N Ezard and L Selwood

Induced ovulation allows reproduction by otherwise infertile females, and is ideal for the captive breeding of endangered species where the population is aged or breeding is unsuccessful. A predictable time of ovulation after induction has not yet been achieved in polyovular marsupials. Ovulation was induced in Sminthopsis macroura using an initial injection of 20 IU equine serum gonadotrophin (eSG; Day 0), followed on Day 4 by either 20 IU eSG (n = 25) or 0.5 mg porcine luteinizing hormone (n = 26). I.p. hormone injection was given in the morning or early evening, and reproductive status was established prior to induction. Five non-cyclic animals began to cycle naturally following induction and one gave birth to a litter. The time of ovulation after the 1st injection (7.8 ± 0.9 days) was significantly shorter (P = 0.000) and less variable than the previous study, mimicked the timing of natural cycling, and both natural and induced animals ovulated in the early morning. In vitro oocyte movement through the oviduct, observed for the first time in a marsupial, occurred in pulses. We estimated one group of oocytes could travel the length of the oviduct in 40 min, but it was probably around 4 h. The entire ovulation time (including multiple ovulations) was estimated at 7.5 h. This study has achieved a predictable timing of ovulation after stimulation, and induced noncyclic animals to cycle naturally and give birth, providing a modified methodology for use in captive breeding programs of endangered dasyurid marsupial species with low fecundity.

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A Nation, S Cui and L Selwood

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.

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E M Menkhorst, S Cui and L Selwood

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.