Lyn A. Hinds

Postnatal development of the telencephalon of the tammar wallaby (Macropus eugenii). An accessible model of neocortical differentiation.

SummaryThe sequence of development of cell layers in the neocortex of the tammar has been followed from 24 days gestation to 213 days postnatal. The tammar is born at 27 days gestation and the major period of its development occurs during the subsequent 250 days, most of this time being spent within the pouch. Although the pattern of differentiation of the cell layers appears to resemble that described for many Eutherian mammals, the neocortex is at an embryonic 2 layered stage at birth and a cortical plate is not present throughout the telencephalon until 10–15 days postnatal. A transient subplate zone, presenting a characteristic appearance with widely spaced rows of cells aligned parallel to the cortical surface, develops between 20 and 70 days postnatal, but no secondary proliferative region is seen in the subventricular zone of the dorso-lateral wall.Preliminary experiments with (3H)-thymidine injections indicate that the cortical plate follows the “inside-out” pattern of development described in many Eutherian mammals and that the oldest neurons are found in the parallel cell rows of the subplate zone. The importance of the late differentiation of the neocortex in relation to the time of birth and the resulting usefulness of the tammar as an experimental model of cortical development is discussed.

Chicken GnRH II occurs together with mammalian GnRH in a South American species of marsupial (Monodelphis domestica)

Two molecular forms of gonadotropin-releasing hormone (GnRH) were demonstrated in hypothalamic extracts of M. domestica using high performance liquid chromatography and radioimmunoassay with specific GnRH antisera. One form eluted in the same position as synthetic mammalian GnRH and was quantified equally by two mammalian GnRH antisera, while the second form coeluted with synthetic chicken GnRH II and was quantified equally with two chicken GnRH II antisera. The finding of chicken GnRH II in a South American species of marsupial, which has previously been reported in some Australian species of marsupial and in species of Aves, Reptilia, Amphibia, Osteichthyes and Chondrichthyes, supports our hypothesis that this widespread structural variant may represent an early evolved and conserved form of GnRH.

A longitudinal study of the protein components of marsupial milk from birth to weaning in the tammar wallaby (Macropus eugenii)

The major milk whey proteins of the tammar wallaby (Macropus eugenii) have been identified over the total period of lactation using proteomic analysis techniques comprising two-dimensional electrophoresis, comparative image analysis, matrix assisted laser desorption ionisation mass spectrometry (MALDI MS), de novo peptide sequencing and cross species protein matching. Samples were collected at the periods coinciding with major milestones of immunological development in the developing marsupial and in the four phases of milk production, specifically, Days 0, 5 (Phase 1); 27, 68 (Phase 2A); 137, 174 (Phase 2B) and 250 (Phase 3). Major changes in the protein content of marsupial milk whey correlated with the changing needs of the pouch young for stages in growth and development. We have shown that the levels of milk whey proteins vary with the developmental stage of the young animal, with a high number of proteins detected in early and late milk compared with the middle phases of lactation. Over 41 proteins were confidently identified, of which most had known roles in immunological protection. Proteins providing immunological protection across the lactation period included transferrin, beta2 microglobulin, haptoglobulin and a 78kDa glucose regulated protein. Immunoglobulin IgJ linker chain and a known antimicrobial cathelicidin, were only detected for the first 100-137 days, after which time Complement B factor was found to be present (Phase 2B). The changes which correlated with development and growth in the pouch young were reflected by the presence of proteins such as an alpha-fetoprotein like protein and clusterin found in early milk (Phase 1-2A) and two unknown proteins which were apparent in very early mammary gland secretions. This is the first comprehensive proteomic study of the major whey proteins of a marsupial across the entire period of lactation and provides fundamental data on proteins secreted by the mammary gland during key stages of immunological development of the young animal.

Anti‐fertility effect of levonorgestrel and quinestrol in Brandt's voles (Lasiopodomys brandtii)

The combination of levonorgestrel and quinestrol (EP-1) has been shown to have anti-fertility effects on several wild rodents, but the mechanism underlying these effects is poorly understood. We investigated the effects of EP-1 and each of its components, levonorgestrel (P) and quinestrol (E), on the fertility of Brandts voles (Lasiopodomys brandtii) by using a gastric gavage method. The doses for EP-1, E and P were 1, 0.34 and 0.66 mg/kg body weight, respectively. Male voles (n = 98) were treated daily for 5 or 14 days, then the testes and epididymides were collected, weighed and examined histologically at 30 (D30), 60 (D60) or 90 (D90) days after the end of treatment. Four males were allowed to mate with normal females at D90. Female voles (n = 75) were treated for 3 days and a further 3 days after a 7-day interval. The uteri and ovaries were weighed and examined histologically at 15 (D15), 30 (D30) or 75 (D75) days after the end of treatment. Each of three females were mated with fertile males at D30 and D75, respectively. Our results indicated that quinestrol (E) significantly decreased the sperm numbers in the testes as well as the weight of the testes and epididymides, with both of these tissues showing obvious structural abnormalities, and significantly reduced the litter size and the pup weight for females mated with males of the E treatment group. For female voles, treatment with E, P or EP-1 resulted in no marked influence on the fertility status. These data indicate that quinestrol (E) alone has a significant anti-fertility effect on male Brandts voles, but is ineffective in combination with levonorgestrel (P).

Differential temporal expression of milk miRNA during the lactation cycle of the marsupial tammar wallaby ( Macropus eugenii )

BackgroundLactation is a key aspect of mammalian evolution for adaptation of various reproductive strategies along different mammalian lineages. Marsupials, such as tammar wallaby, adopted a short gestation and a relatively long lactation cycle, the newborn is immature at birth and significant development occurs postnatally during lactation. Continuous changes of tammar milk composition may contribute to development and immune protection of pouch young. Here, in order to address the putative contribution of newly identified secretory milk miRNA in these processes, high throughput sequencing of miRNAs collected from tammar milk at different time points of lactation was conducted. A comparative analysis was performed to find distribution of miRNA in milk and blood serum of lactating wallaby.ResultsResults showed that high levels of miRNA secreted in milk and allowed the identification of differentially expressed milk miRNAs during the lactation cycle as putative markers of mammary gland activity and functional candidate signals to assist growth and timed development of the young. Comparative analysis of miRNA distribution in milk and blood serum suggests that milk miRNAs are primarily expressed from mammary gland rather than transferred from maternal circulating blood, likely through a new putative exosomal secretory pathway. In contrast, highly expressed milk miRNAs could be detected at significantly higher levels in neonate blood serum in comparison to adult blood, suggesting milk miRNAs may be absorbed through the gut of the young.ConclusionThe function of miRNA in mammary gland development and secretory activity has been proposed, but results from the current study also support a differential role of milk miRNA in regulation of development in the pouch young, revealing a new potential molecular communication between mother and young during mammalian lactation.

Prospects for the future: is there a role for virally vectored immunocontraception in vertebrate pest management?

Virally vectored immunocontraception (VVIC) has been studied and promoted as an alternative to lethal methods for vertebrate pest control in Australia and New Zealand. Virally vectored immunocontraception offers a potentially humane and species-specific control method with potential for a good benefit–cost outcome, but its applicability for broad-scale management remains unknown. We present case studies for the house mouse, European rabbit, red fox and common brushtail possum and describe the current status of research into the use of VVIC as a broad-scale pest-management tool. All case studies indicated that there are significant problems with delivery and efficacy. The current state of development suggests that VVIC is not presently a viable alternative for the management of these vertebrate pests, and it is highly unlikely that this will change in the foreseeable future. An absence of benefit–cost data also hinders decision-making, and until benefit–cost data become available it will not be clear if there are short- or long-term benefits resulting from the use of VVIC for broad-scale pest management.

Fertility control of rodent pests

Ricefield rats (Rattus argentiventer) in south-east Asian rice fields and house mice (Mus domesticus) in Australian grain fields are major pest species. They cause damage before and after harvest and carry zoonotic diseases. For both species, management techniques have been pursued using the approach of immunocontraceptive vaccination. We review results from a series of enclosure and field studies conducted with these species to assess the effects of fertility control in small rodents. In the experiments, fertility control was simulated by tubal ligation, ovariectomy or progesterone treatment. A once-off sterilisation of 50–75% of enclosed founder females considerably reduced reproductive output of ricefield rat populations until the end of the reproductive period. In house mice, similar success was achieved when a sterility level of 67% of female founders and offspring was maintained. Repeated antifertility treatments are required because of the much longer breeding period of house mice versus ricefield rats. Comparing the results of enclosure trials with the outcome of simulation models suggests that partial compensation of treatment effects can occur through enhanced reproduction of the remaining fertile females and improved survival of juveniles. However, such compensatory effects as well as behavioural consequences of sterility in field populations are not likely to prevent the management effect at the population level. The challenge for effective fertility control of small rodents in the field is the wide-scale delivery of an antifertility treatment to founders at the beginning of the breeding season and to fertile immigrants that are recruited into the population, which otherwise contribute to the reproductive output at the population level. Future research efforts should focus on species-specific techniques and on agents that can be effectively delivered via bait.

Prospects for virally vectored immunocontraception in the control of wild house mice (Mus domesticus)

The wild house mouse (Mus domesticus) is not native to Australia and was introduced from Europe with early settlement. It undergoes periodic population explosions or plagues, which place significant economic and social burdens on agricultural communities. Present control mechanisms rely on improvements to farm hygiene and the use of rodenticides. This review covers over a decade of work on the use of virally vectored immunocontraception (VVIC) as an adjunct method of controlling mouse populations. Two viral vectors, ectromelia virus (ECTV) and murine cytomegalovirus (MCMV) have been tested as potential VVIC vectors: MCMV has been the most widely studied vector because it is endemic to Australia; ECTV less so because its use would have required the introduction of a new pathogen into the Australian environment. Issues such as efficacy, antigen choice, resistance, transmission, species specificity and safety of VVIC are discussed. In broad terms, both vectors when expressing murine zona pellucida 3 (mZP3) induced long-term infertility in most directly inoculated female mice. Whereas innate and acquired resistance to MCMV may be a barrier to VVIC, the most significant barrier appears to be the attenuation seen in MCMV-based vectors. This attenuation is likely to prevent sufficient transmission for broad-scale use. Should this issue be overcome, VVIC has the potential to contribute to the control of house mouse populations in Australia.

A review of complementary mechanisms which protect the developing marsupial pouch young.

Marsupials are born without a functioning adaptive immune system, into a non-sterile environment where they continue to develop. This review examines the extent of exposure of pouch young to microorganisms and describes the protective mechanisms that are complementary to adaptive immunity in the developing young. Complementary protective mechanisms include the role of the innate immune system and maternal protection strategies, such as immune compounds in milk, prenatal transfer of immunoglobulins, antimicrobial compounds secreted in the pouch, and chemical or mechanical cleaning of the pouch and pouch young.

Proteomic analysis of early lactation milk of the tammar wallaby (Macropus eugenii).

We report the successful use of 2D electrophoresis, MALDI MS/MS and chemical derivatisation protocols of guanidination and sulfonation to identify over 100 protein spots present in early marsupial milk (tammar wallaby) at 40 days lactation, where a limited translated genomic database is publicly available for cross species matching and protein identification. Of the proteins identified, 25 matched to 6 existing marsupial milk protein sequences in the NCBI database; another 6 were identified with high confidence to other mammals and have not previously been identified in marsupial milk. By using chemical derivatisation, the reliable identification of a further 81 proteins was achieved. The identified proteins could be grouped into three main functional categories - transport, nutrition and immune protection. All these proteins play a potential role in determining growth and immunological protection of the highly altricial marsupial young at 40 days after birth.