Publications by authors named "Marilyn B Renfree"

174 Publications

Plasma progesterone secretion during gestation of the captive short-beaked echidna.

Reproduction 2021 Sep 6;162(4):267-275. Epub 2021 Sep 6.

School of Agriculture and Food Sciences, The University of Queensland, Gatton, Australia.

This study describes the progesterone profile during pregnancy in sexually mature female captive short-beaked echidnas (Tachyglossus aculeatus aculeatus). Echidnas were monitored daily by video surveillance to confirm key reproductive behaviour. Plasma samples were collected and pouch morphology was assessed three times a week. The pouch of the female echidna only develops during gestation and it was possible to create a four-stage grading system using the most distinguishable characteristics of pouch development. Maximum pouch development was associated with declining progesterone concentrations, with the pouch closing in a drawstring-like manner at oviposition. Control of pouch development in pregnant echidnas is not yet clear but later pouch development is associated with a decrease in progesterone and pouch closure and may be under mechanical influences of the egg or young in the pouch. The length of pregnancy was 16.7 ± 0.2 days with a 15.1 ± 1.0 days luteal phase followed by an incubation period in the pouch. Eggs could be detected in utero at least 4 days before oviposition. Plasma progesterone peaked at 10.5 ± 0.9 ng/mL within 12 days of mating but then declined to basal levels within 1 day of oviposition and remained basal throughout egg incubation, confirming that progesterone is elevated throughout pregnancy and that gestation does not extend beyond the luteal phase. After the loss of an egg or pouch young, most females entered a second oestrous cycle and ovulated, suggesting echidnas are seasonally polyoestrous. The duration of the luteal phase in the echidna corresponds with that observed in other mammals.
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http://dx.doi.org/10.1530/REP-21-0110DOI Listing
September 2021

Selection on phalanx development in the evolution of the bird wing.

Mol Biol Evol 2021 Jun 23. Epub 2021 Jun 23.

Animal Science & Health, Institute of Biology Leiden (IBL), Leiden University, 2333BE Leiden, the Netherlands Sylviusweg 72.

The frameshift hypothesis is a widely-accepted model of bird wing evolution. This hypothesis postulates a shift in positional values, or molecular-developmental identity, that caused a change in digit phenotype. The hypothesis synthesised developmental and palaeontological data on wing digit homology. The 'most anterior digit' (MAD) hypothesis presents an alternative view based on changes in transcriptional regulation in the limb. The molecular evidence for both hypotheses is that the most anterior digit expresses Hoxd13 but not Hoxd11 and Hoxd12. This digit I 'signature' is thought to characterise all amniotes. Here, we studied Hoxd expression patterns in a phylogenetic sample of 18 amniotes. Instead of a conserved molecular signature in digit I, we find wide variation of Hoxd11, Hoxd12 and Hoxd13 expression in digit I. Patterns of apoptosis, and Sox9 expression, a marker of the phalanx-forming region, suggest that phalanges were lost from wing digit IV because of early arrest of the phalanx-forming region followed by cell death. Finally, we show that multiple amniote lineages lost phalanges with no frameshift. Our findings suggest that the bird wing evolved by targeted loss of phalanges under selection. Consistent with our view, some recent phylogenies based on dinosaur fossils eliminate the need to postulate a frameshift in the first place. We suggest that the phenotype of the Archaeopteryx lithographica wing is also consistent with phalanx loss. More broadly, our results support a gradualist model of evolution based on tinkering with developmental gene expression.
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http://dx.doi.org/10.1093/molbev/msab150DOI Listing
June 2021

The Unique Penile Morphology of the Short-Beaked Echidna, Tachyglossus aculeatus.

Sex Dev 2021 29;15(4):262-271. Epub 2021 Apr 29.

School of BioSciences, University of Melbourne, Melbourne, Victoria, Australia.

Monotremes diverged from therian mammal ancestors approximately 184 million years ago and have a number of novel reproductive characteristics. One in particular is their penile morphology. There are differences between echidna and platypus phalluses, but both are somewhat similar in structure to the reptilian phallus. The echidna penis consists of 4 rosette glans, each of which contains a termination of the quadrifurcate urethra, but it appears that only 2 of the 4 glans become erect at any one time. Despite this, only a few historical references describe the structure of the echidna penis and none provides an explanation for the mechanisms of unilateral ejaculation. This study confirmed that the echidna penis contains many of the same overall structures and morphology as other mammalian penises and a number of features homologous with reptiles. The corpus cavernosum is well supplied with blood, extends up to the base of the glans penis and is primarily responsible for erection. However, the echidna possesses 2 distinct corpora spongiosa separated by a septum, each of which surround the urethra only distal to the initial urethral bifurcation in the glans penis. Together with the bifurcation of the main penile artery, this provides a mechanism by which blood flow could be directed to only one corpus spongiosum at a time to maintain an open urethra that supplies 2 of the 4 glans to facilitate unilateral ejaculation.
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http://dx.doi.org/10.1159/000515145DOI Listing
April 2021

Male germline development in the tammar wallaby, Macropus eugenii.

Reproduction 2021 03;161(3):333-341

School of BioSciences, The University of Melbourne, Melbourne, Victoria, Australia.

Male germ cells undergo two consecutive processes - pre-spermatogenesis and spermatogenesis - to generate mature sperm. In eutherian mammals, epigenetic information such as DNA methylation is dynamically reprogrammed during pre-spermatogenesis, before and during mitotic arrest. In mice, by the time germ cells resume mitosis, the majority of DNA methylation is reprogrammed. The tammar wallaby has a similar pattern of germ cell global DNA methylation reprogramming to that of the mouse during early pre-spermatogenesis. However, early male germline development in the tammar or in any marsupial has not been described previously, so it is unknown whether this is a general feature regulating male germline development or a more recent phenomenon in mammalian evolutionary history. To answer this, we examined germ cell nuclear morphology and mitotic arrest during male germline development in the tammar wallaby (Macropus eugenii), a marsupial that diverged from mice and humans around 160 million years ago. Tammar pro-spermatogonia proliferated after birth and entered mitotic arrest after day 30 postpartum (pp). At this time, they began moving towards the periphery of the testis cords and their nuclear size increased. Germ cells increased in number after day 100 pp which is the time that DNA methylation is known to be re-established in the tammar. This is similar to the pattern observed in the mouse, suggesting that resumption of germ cell mitosis and the timing of DNA methylation reprogramming are correlated and conserved across mammals and over long evolutionary timescales.
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http://dx.doi.org/10.1530/REP-20-0634DOI Listing
March 2021

Platypus and echidna genomes reveal mammalian biology and evolution.

Nature 2021 Apr 6;592(7856):756-762. Epub 2021 Jan 6.

Tree of Life Programme, Wellcome Sanger Institute, Cambridge, UK.

Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.
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http://dx.doi.org/10.1038/s41586-020-03039-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8081666PMC
April 2021

Transient role of the middle ear as a lower jaw support across mammals.

Elife 2020 06 30;9. Epub 2020 Jun 30.

Centre for Craniofacial and Regenerative Biology, King's College London, London, United Kingdom.

Mammals articulate their jaws using a novel joint between the dentary and squamosal bones. In eutherian mammals, this joint forms in the embryo, supporting feeding and vocalisation from birth. In contrast, marsupials and monotremes exhibit extreme altriciality and are born before the bones of the novel mammalian jaw joint form. These mammals need to rely on other mechanisms to allow them to feed. Here, we show that this vital function is carried out by the earlier developing, cartilaginous incus of the middle ear, abutting the cranial base to form a cranio-mandibular articulation. The nature of this articulation varies between monotremes and marsupials, with juvenile monotremes retaining a double articulation, similar to that of the fossil mammaliaform , while marsupials use a versican-rich matrix to stabilise the jaw against the cranial base. These findings provide novel insight into the evolution of mammals and the changing relationship between the jaw and ear.
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http://dx.doi.org/10.7554/eLife.57860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363448PMC
June 2020

Unique reproductive strategy in the swamp wallaby.

Proc Natl Acad Sci U S A 2020 03 2;117(11):5938-5942. Epub 2020 Mar 2.

School of BioSciences, The University of Melbourne, Parkville, VIC 3010, Australia.

Reproduction in mammals requires distinct cycles of ovulation, fertilization, pregnancy, and lactation often interspersed with periods of anoestrus when breeding does not occur. Macropodids, the largest extant species of marsupials, the kangaroos and wallabies, have a very different reproductive strategy to most eutherian mammals whereby young are born at a highly altricial stage of development with the majority of development occurring over a lengthy lactation period. Furthermore, the timings of ovulation and birth in some species occurs within a very short interval of each other (sometimes hours). Female swamp wallabies have an oestrous cycle shorter than their pregnancy length and were, therefore, speculated to mate and form a new embryo before birth thereby supporting two conceptuses at different stages of pregnancy. To confirm this, we used high-resolution ultrasound to monitor reproduction in swamp wallabies during pregnancy. Here, we show that females ovulate, mate, and form a new embryo prepartum while still carrying a full-term fetus in the contralateral uterus. This embryo enters embryonic diapause until the newborn leaves the pouch 9 mo later. Thus, combined with embryonic diapause, females are continuously pregnant and lactating at the same time throughout their reproductive life, a unique reproductive strategy that completely blurs the normal staged system of reproduction in mammals.
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http://dx.doi.org/10.1073/pnas.1922678117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084135PMC
March 2020

Discrete Hedgehog Factor Expression and Action in the Developing Phallus.

Int J Mol Sci 2020 Feb 12;21(4). Epub 2020 Feb 12.

School of BioSciences, The University of Melbourne, Parkville, VIC 3010, Australia.

Hypospadias is a failure of urethral closure within the penis occurring in 1 in 125 boys at birth and is increasing in frequency. While paracrine hedgehog signalling is implicated in the process of urethral closure, how these factors act on a tissue level to execute closure itself is unknown. This study aimed to understand the role of different hedgehog signalling members in urethral closure. The tammar wallaby () provides a unique system to understand urethral closure as it allows direct treatment of developing offspring because mothers give birth to young before urethral closure begins. Wallaby pouch young were treated with vehicle or oestradiol (known to induce hypospadias in males) and samples subjected to RNAseq for differential expression and gene ontology analyses. Localisation of Sonic Hedgehog (SHH) and Indian Hedgehog (IHH), as well as the transcription factor SOX9, were assessed in normal phallus tissue using immunofluorescence. Normal tissue culture explants were treated with SHH or IHH and analysed for , , PTCH1, GLI2, , and expression by qPCR. Gene ontology analysis showed enrichment for bone differentiation terms in male samples compared with either female samples or males treated with oestradiol. Expression of SHH and IHH localised to specific tissue areas during development, akin to their compartmentalised expression in developing bone. Treatment of phallus explants with SHH or IHH induced factor-specific expression of genes associated with bone differentiation. This reveals a potential developmental interaction involved in urethral closure that mimics bone differentiation and incorporates discrete hedgehog activity within the developing phallus and phallic urethra.
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http://dx.doi.org/10.3390/ijms21041237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072906PMC
February 2020

Transcriptomic Analysis of MAP3K1 and MAP3K4 in the Developing Marsupial Gonad.

Sex Dev 2019 1;13(4):195-204. Epub 2020 Feb 1.

MAPKs affect gonadal differentiation in mice and humans, but whether this applies to all mammals is as yet unknown. Thus, we investigated MAPK expression during gonadal differentiation and after treatment with oestrogen in a distantly related mammal, the marsupial tammar wallaby, using our model of oestrogen-induced gonadal sex reversal. High-throughput RNA-sequencing was carried out on gonads collected from developing tammar 2 days before birth to 8 days after birth to characterise MAPK and key sexual differentiation markers. Day 25 foetal testes were cultured for 120 h in control medium or medium supplemented with exogenous oestrogen and processed for RNA-seq to identify changes in gene expression in response to oestrogen. MAPK pathway genes in the tammar were highly conserved at the sequence and amino acid level with those of mice and humans. Marsupial MAP3K1 and MAP3K4 clustered together in a separate branch from eutherian mammals. There was a marked decrease in the expression of male-determining genes SOX9 and AMH and increase in the female marker FOXL2 in oestrogen-treated male gonads. Only MAP3K1 expression increased in male gonads in response to oestrogen while other MAPK genes remained unaffected. This study suggests that MAP3K1 can be influenced by exogenous oestrogens during gonadal differentiation in this marsupial.
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http://dx.doi.org/10.1159/000505799DOI Listing
August 2020

Hormonal and Molecular Regulation of Phallus Differentiation in a Marsupial Tammar Wallaby.

Genes (Basel) 2020 01 16;11(1). Epub 2020 Jan 16.

School of BioSciences, The University of Melbourne, Victoria 3010, Australia.

Congenital anomalies in phalluses caused by endocrine disruptors have gained a great deal of attention due to its annual increasing rate in males. However, the endocrine-driven molecular regulatory mechanism of abnormal phallus development is complex and remains largely unknown. Here, we review the direct effect of androgen and oestrogen on molecular regulation in phalluses using the marsupial tammar wallaby, whose phallus differentiation occurs after birth. We summarize and discuss the molecular mechanisms underlying phallus differentiation mediated by sonic hedgehog () at day 50 pp and phallus elongation mediated by insulin-like growth factor 1 ( and insulin-like growth factor binding protein 3 (, as well as multiple phallus-regulating genes expressed after day 50 pp. We also identify hormone-responsive long non-coding RNAs (lncRNAs) that are co-expressed with their neighboring coding genes. We show that the activation of and , mediated by balanced androgen receptor (AR) and estrogen receptor 1 (ESR1) signalling, initiates a complex regulatory network in males to constrain the timing of phallus differentiation and to activate the downstream genes that maintain urethral closure and phallus elongation at later stages.
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http://dx.doi.org/10.3390/genes11010106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017150PMC
January 2020

WOMEN IN REPRODUCTIVE SCIENCE: Reproduction down under.

Reproduction 2019 12;158(6):F127-F137

School of BioSciences, The University of Melbourne, Melbourne, Victoria, Australia.

Australia is home to a unique assembly of mammals - the marsupials and monotremes. Despite this uniqueness, they have been largely ignored by the biomedical scientific community, and yet study of marsupials has contributed to modern research on reproduction, development, evolution, conservation, molecular and comparative genomics. My lifetime passion for these long-neglected Australian fauna has led to unexpected discoveries and insights that challenged assumptions and opened up new areas of international research. I used a range of disciplinary expertise to pursue the study of these unique mammals. My main experimental species has been the tammar wallaby that I have used as a model species to investigate and understand not only biomedical problems but also to provide knowledge that is critical for the continued conservation and management of Australia's dwindling native mammals. This model provided more than a few surprises for me and my wonderful team of students, post-docs and collaborators about how hormones, genes and signalling molecules control reproductive biology and development in a wider context as well as how the interactions of the environment with mother and conceptus, with mother and fetus and mother and young ultimately control most aspects of successful reproduction in mammals.
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http://dx.doi.org/10.1530/REP-19-0230DOI Listing
December 2019

Women in reproductive science.

Reproduction 2019 12;158(6):E5-E7

School of BioSciences, The University of Melbourne, Melbourne, Victoria, Australia.

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http://dx.doi.org/10.1530/REP-19-0482DOI Listing
December 2019

EvoChromo: towards a synthesis of chromatin biology and evolution.

Development 2019 09 26;146(19). Epub 2019 Sep 26.

Conway Institute and School of Medicine, University College Dublin, Dublin 4, Ireland.

Over the past few years, interest in chromatin and its evolution has grown. To further advance these interests, we organized a workshop with the support of The Company of Biologists to debate the current state of knowledge regarding the origin and evolution of chromatin. This workshop led to prospective views on the development of a new field of research that we term 'EvoChromo'. In this short Spotlight article, we define the breadth and expected impact of this new area of scientific inquiry on our understanding of both chromatin and evolution.
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http://dx.doi.org/10.1242/dev.178962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376748PMC
September 2019

The tammar wallaby: a non-traditional animal model to study growth axis maturation.

Reprod Fertil Dev 2019 Jul;31(7):1276-1288

School of BioSciences, The University of Melbourne, Vic. 3010, Australia.

Maturation of the growth hormone (GH)/insulin-like growth factor 1 (IGF1) axis is a critical developmental event that becomes functional over the peripartum period in precocial eutherian mammals such as sheep. In mice and marsupials that give birth to altricial young, the GH/IGF1 axis matures well after birth, suggesting that functional maturation is associated with developmental stage, not parturition. Recent foster-forward studies in one marsupial, the tammar wallaby (Macropus eugenii), have corroborated this hypothesis. 'Fostering' tammar young not only markedly accelerates their development and growth rates, but also affects the timing of maturation of the growth axis compared with normal growing young, providing a novel non-traditional animal model for nutritional manipulation. This review discusses how nutrition affects the maturation of the growth axis in marsupials compared with traditional eutherian animal models.
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http://dx.doi.org/10.1071/RD18271DOI Listing
July 2019

Androgen and Oestrogen Affect the Expression of Long Non-Coding RNAs During Phallus Development in a Marsupial.

Noncoding RNA 2018 Dec 30;5(1). Epub 2018 Dec 30.

School of BioSciences, The University of Melbourne 3010, VIC, Australia.

There is increasing evidence that long non-coding RNAs (lncRNAs) are important for normal reproductive development, yet very few lncRNAs have been identified in phalluses so far. Unlike eutherians, phallus development in the marsupial tammar wallaby occurs post-natally, enabling manipulation not possible in eutherians in which differentiation occurs in utero. We treated with sex steroids to determine the effects of androgen and oestrogen on lncRNA expression during phallus development. Hormonal manipulations altered the coding and non-coding gene expression profile of phalluses. We identified several predicted co-regulatory lncRNAs that appear to be co-expressed with the hormone-responsive candidate genes regulating urethral closure and phallus growth, namely , and . Interestingly, more than 50% of -associated coding genes and lncRNAs were also associated with . In addition, we identified and validated three novel co-regulatory and hormone-responsive lncRNAs: and . was detected in the urethral epithelium of male phalluses and was downregulated by oestrogen in males. was downregulated by oestrogen treatment in male phalluses at day 50 post-partum (pp). was downregulated by adiol treatment in female phalluses but increased in male phalluses after castration. Thus, the expression pattern and hormone responsiveness of these lncRNAs suggests a physiological role in the development of the phallus.
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http://dx.doi.org/10.3390/ncrna5010003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468475PMC
December 2018

DNA methylation dynamics in the germline of the marsupial tammar wallaby, Macropus eugenii.

DNA Res 2019 Feb;26(1):85-94

School of BioSciences, The University of Melbourne, Melbourne, VIC, Australia.

Parent specific-DNA methylation is the genomic imprint that induces mono-allelic gene expression dependent on parental origin. Resetting of DNA methylation in the germ line is mediated by a genome-wide re-methylation following demethylation known as epigenetic reprogramming. Most of our understanding of epigenetic reprogramming in germ cells is based on studies in mice, but little is known about this in marsupials. We examined genome-wide changes in DNA methylation levels by measuring 5-methylcytosine expression, and mRNA expression and protein localization of the key enzyme DNA methyltransferase 3 L (DNMT3L) during germ cell development of the marsupial tammar wallaby, Macropus eugenii. Our data clearly showed that the relative timing of genome-wide changes in DNA methylation was conserved between the tammar and mouse, but in the tammar it all occurred post-natally. In the female tammar, genome-wide demethylation occurred in two phases, I and II, suggesting that there is an unidentified demethylation mechanism in this species. Although the localization pattern of DNMT3L in male germ cells differed, the expression patterns of DNMT3L were broadly conserved between tammar, mouse and human. Thus, the basic mechanisms of DNA methylation-reprogramming must have been established before the marsupial-eutherian mammal divergence over 160 Mya.
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http://dx.doi.org/10.1093/dnares/dsy040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379045PMC
February 2019

Effects of androgen and oestrogen on IGF pathways controlling phallus growth.

Reproduction 2019 01;157(1):1-12

School of BioSciences, The University of Melbourne, Victoria, Australia.

The development of the mammalian phallus involves hormone-dependent mesenchymal-epithelial signalling mechanisms that contribute to urethral closure and regulation of phallus elongation and growth. In marsupials, most differentiation and growth of the phallus occurs post-natally, making them amenable to direct hormone treatment. Expression of IGFs, FGFs, EFNB2, MAFB, DLX5 and AP-1 mRNAs in the phallus at day 50 post-partum (pp) were altered after treatment of tammar wallaby young from day 20 to 40 pp with androgen, oestrogen or after castration at day 25 pp. However, the most interesting changes occurred in the IGF pathway genes. Androgen treatment upregulated IGF1 in female phalluses and oestrogen treatment upregulated IGF1 in male phalluses, but it was downregulated by castration. IGFBP3 was higher in female phalluses and downregulated by androgen. IGF1 expression was higher in all untreated male than in female phalluses from day 50 to 150 pp, but IGFBP3 had the reverse pattern. At day 90 pp, when urethral closure in males is progressing and male phallus growth is accelerating. IGF1 and PCNA protein were only detected in the male urorectal septum, suggesting for the first time that closure and elongation may involve IGF1 activation of cell proliferation specifically in male phalluses. These effects of sex steroids on gene expression and on the IGF1 signalling pathway in particular, suggest that the developing phallus may be especially susceptible to perturbation by exogenous hormones.
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http://dx.doi.org/10.1530/REP-18-0416DOI Listing
January 2019

Identification of a novel antisense noncoding RNA, ALID, transcribed from the putative imprinting control region of marsupial IGF2R.

Epigenetics Chromatin 2018 09 29;11(1):55. Epub 2018 Sep 29.

School of BioSciences, The University of Melbourne, Victoria, 3010, Australia.

Background: Genomic imprinting leads to maternal expression of IGF2R in both mouse and opossum. In mouse, the antisense long noncoding (lnc) RNA Airn, which is paternally expressed from the differentially methylated region (DMR) in the second intron of Igf2r, is required to silence the paternal Igf2r. In opossum, however, intriguingly, the DMR was reported to be in a different downstream intron (intron 11) and there was no antisense lncRNA detected in previous analyses. Therefore, clarifying the imprinting mechanism of marsupial IGF2R is of great relevance for understanding the origin and evolution of genomic imprinting in the IGF2R locus. Thus, the antisense lncRNA associated with the marsupial DMR can be considered as the 'missing link'. In this study, we identified a novel antisense lncRNA, ALID, after detailed analysis of the IGF2R locus in an Australian marsupial, the tammar wallaby, Macropus eugenii, and compared it to that of the grey short-tailed opossum, Monodelphis domestica.

Results: Tammar IGF2R showed maternal expression and had a maternally methylated CpG island (CGI) in intron 12 as well as a promoter CGI without differential methylation, but none in the second intron. Re-analysis of the IGF2R of opossum detected the CGI in intron 12, not intron 11, as previously reported, confirming that the DMR in intron 12 is conserved between these marsupials and so is the putative imprinting control region of marsupial IGF2R. ALID is paternally expressed from the middle of the DMR and is approximately 650 bp long with a single exon structure that is extremely short compared to Airn. Hence, the lncRNA transcriptional overlap of the IGF2R promoter, which is essential for the Igf2r silencing in the mouse, is likely absent in tammar. This suggests that fundamental differences in the lncRNA-based silencing mechanisms evolved in eutherian and marsupial IGF2R and may reflect the lack of differential methylation in the promoter CGI of marsupial IGF2R.

Conclusions: Our study thus provides the best candidate factor for establishing paternal silencing of marsupial IGF2R without transcriptional overlap, which is distinct from the Igf2r silencing mechanism of Airn, but which may be analogous to the mode of action for the flanking Slc22a2 and Slc22a3 gene silencing in the mouse placenta.
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http://dx.doi.org/10.1186/s13072-018-0227-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162910PMC
September 2018

Embryos and embryonic stem cells from the white rhinoceros.

Nat Commun 2018 07 4;9(1):2589. Epub 2018 Jul 4.

Avantea, Laboratory of Reproductive Technologies, 26100, Cremona, Italy.

The northern white rhinoceros (NWR, Ceratotherium simum cottoni) is the most endangered mammal in the world with only two females surviving. Here we adapt existing assisted reproduction techniques (ART) to fertilize Southern White Rhinoceros (SWR) oocytes with NWR spermatozoa. We show that rhinoceros oocytes can be repeatedly recovered from live SWR females by transrectal ovum pick-up, matured, fertilized by intracytoplasmic sperm injection and developed to the blastocyst stage in vitro. Next, we generate hybrid rhinoceros embryos in vitro using gametes of NWR and SWR. We also establish embryonic stem cell lines from the SWR blastocysts. Blastocysts are cryopreserved for later embryo transfer. Our results indicate that ART could be a viable strategy to rescue genes from the iconic, almost extinct, northern white rhinoceros and may also have broader impact if applied with similar success to other endangered large mammalian species.
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http://dx.doi.org/10.1038/s41467-018-04959-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031672PMC
July 2018

Adaptation and conservation insights from the koala genome.

Nat Genet 2018 08 2;50(8):1102-1111. Epub 2018 Jul 2.

Sydney School of Veterinary Science, Faculty of Science, University of Sydney, Sydney, New South Wales, Australia.

The koala, the only extant species of the marsupial family Phascolarctidae, is classified as 'vulnerable' due to habitat loss and widespread disease. We sequenced the koala genome, producing a complete and contiguous marsupial reference genome, including centromeres. We reveal that the koala's ability to detoxify eucalypt foliage may be due to expansions within a cytochrome P450 gene family, and its ability to smell, taste and moderate ingestion of plant secondary metabolites may be due to expansions in the vomeronasal and taste receptors. We characterized novel lactation proteins that protect young in the pouch and annotated immune genes important for response to chlamydial disease. Historical demography showed a substantial population crash coincident with the decline of Australian megafauna, while contemporary populations had biogeographic boundaries and increased inbreeding in populations affected by historic translocations. We identified genetically diverse populations that require habitat corridors and instituting of translocation programs to aid the koala's survival in the wild.
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http://dx.doi.org/10.1038/s41588-018-0153-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197426PMC
August 2018

Conceptus Coats of Marsupials and Monotremes.

Curr Top Dev Biol 2018 23;130:357-377. Epub 2018 Apr 23.

School of BioSciences, University of Melbourne, Parkville, VIC, Australia. Electronic address:

Mammals evolved from oviparous reptiles that laid eggs in a dry, terrestrial environment, thus requiring large amounts of yolk to support development and tough, outer coats to protect them. Eutherian mammals such as humans and mice exhibit an "extreme" form of viviparity in which yolk and conceptus coats have become largely redundant. However, the "other" mammals-monotremes and marsupials-have retained and modified some features of reptilian development that provide valuable insights into the evolution of viviparity in mammals. Most striking of these are the conceptus coats, which include the zona pellucida, the mucoid coat, and the shell coat. We discuss current knowledge of these coats in monotremes and marsupials, their possible roles, and recently identified components such as the zona pellucida protein ZPAX, conceptus coat mucin (CCM), and nephronectin (NPNT).
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http://dx.doi.org/10.1016/bs.ctdb.2018.03.004DOI Listing
July 2019

Hormone-responsive genes in the SHH and WNT/β-catenin signaling pathways influence urethral closure and phallus growth.

Biol Reprod 2018 10;99(4):806-816

School of BioSciences, The University of Melbourne, Victoria, Australia.

Environmental endocrine disruptors (EEDs) that affect androgen or estrogen activity may disrupt gene regulation during phallus development to cause hypospadias or a masculinized clitoris. We treated developing male tammar wallabies with estrogen and females with androgen from day 20-40 postpartum (pp) during the androgen imprinting window of sensitivity. Estrogen inhibited phallus elongation but had no effect on urethral closure and did not significantly depress testicular androgen synthesis. Androgen treatment in females did not promote phallus elongation but initiated urethral closure. Phalluses were collected for transcriptome sequencing at day 50 pp when they first become sexually dimorphic to examine changes in two signaling pathways, sonic hedgehog (SHH) and wingless-type MMTV integration site family (WNT)/β-catenin. SHH mRNA and β-catenin were predominantly expressed in the urethral epithelium in the tammar phallus, as in eutherian mammals. Estrogen treatment and castration of males induced an upregulation of SHH, while androgen treatment downregulated SHH. These effects appear to be direct since we detected putative estrogen receptor α (ERα) and androgen receptor (AR) binding sites near SHH. WNT5A, like SHH, was downregulated by androgen, while WNT4 was upregulated in female phalluses after androgen treatment. After estrogen treatment, WIF1 and WNT7A were both downregulated in male phalluses. After castration, WNT9A was upregulated. These results suggest that SHH and WNT pathways are regulated by both estrogen and androgen to direct the proliferation and elongation of the phallus during differentiation. Their response to exogenous hormones makes these genes potential targets of EEDs in the etiology of abnormal phallus development including hypospadias.
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http://dx.doi.org/10.1093/biolre/ioy117DOI Listing
October 2018

The history of the discovery of embryonic diapause in mammals.

Biol Reprod 2018 07;99(1):242-251

School of BioSciences, The University of Melbourne, Parkville, Victoria Australia.

The first incidence of embryonic diapause in mammals was observed in the roe deer, Capreolus capreolus, in 1854 and confirmed in the early 1900s. Since then scientists have been fascinated by this phenomenon that allows a growing embryo to become arrested for up to 11 months and then reactivate and continue development with no ill effects. The study of diapause has required unraveling basic reproductive processes we now take for granted and has spanned some of the major checkpoints of reproductive biology from the identification of the sex hormones to the hypothalamic-pituitary axis to microRNA and exosomes. This review will describe the history of diapause from its origins to the current day, including its discovery and efforts to elucidate its mechanisms. It will also attempt to highlight the people involved who were instrumental in progressing this field over the last 160 years. The most recent confirmation of mammalian diapause was in the panda in 2009 and there are still multiple mammals where it has been predicted but not yet confirmed. Furthermore, there are many questions still unanswered which ensure that embryonic diapause will continue to be a topic of research for many years to come. Note that there have recently been several extensive reviews covering the recent advances in embryonic diapause, so they will be mentioned only briefly here. For further information refer to Renfree and Shaw 2014; Fenelon et al 2014; Renfree and Fenelon 2017, and references therein.
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http://dx.doi.org/10.1093/biolre/ioy112DOI Listing
July 2018

The Basal Radial Glia Occurs in Marsupials and Underlies the Evolution of an Expanded Neocortex in Therian Mammals.

Cereb Cortex 2018 01;28(1):145-157

Institute of Veterinary Anatomy, Histology and Embryology, University of Leipzig, 04103 Leipzig, Germany.

A hallmark of mammalian brain evolution is the emergence of the neocortex, which has expanded in all mammalian infraclasses (Eutheria, Marsupialia, Monotremata). In eutherians, neocortical neurons derive from distinct neural stem and progenitor cells (NPCs). However, precise data on the presence and abundance of the NPCs, especially of basal radial glia (bRG), in the neocortex of marsupials are lacking. This study characterized and quantified the NPCs in the developing neocortex of a marsupial, the tammar wallaby (Macropus eugenii). Our data demonstrate that its neocortex is characterized by high NPC diversity. Importantly, we show that bRG exist at high relative abundance in the tammar indicating that this cell type is not specific to the eutherian neocortex and that similar mechanisms may underlie the formation of an expanded neocortex in eutherian and marsupial mammals. We also show that bRG are likely to have been present in the therian ancestor, so did not emerge independently in the eutherian and marsupial lineages. Moreover, our data support the concept that changes in multiple parameters contribute to neocortex expansion and demonstrate the importance of bRG and other NPCs for the development and expansion of the mammalian neocortex.
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http://dx.doi.org/10.1093/cercor/bhw360DOI Listing
January 2018

Non-invasive placentation in the marsupials Macropus eugenii (Macropodidae) and Trichosurus vulpecula (Phalangeridae) involves redistribution of uterine Desmoglein-2.

Mol Reprod Dev 2018 01 15;85(1):72-82. Epub 2018 Jan 15.

School of Life and Environmental Sciences, University of Sydney, Sydney, New South Wales, Australia.

In mammalian pregnancy, the uterus is remodeled to become receptive to embryonic implantation. Since non-invasive placentation in marsupials is likely derived from invasive placentation, and is underpinned by intra-uterine conflict between mother and embryo, species with non-invasive placentation may employ a variety of molecular mechanisms to maintain an intact uterine epithelium and to prevent embryonic invasion. Identifying such modifications to the uterine epithelium of marsupial species with non-invasive placentation is key to understanding how conflict is mediated during pregnancy in different mammalian groups. Desmoglein-2, involved in maintaining lateral cell-cell adhesion of the uterine epithelium, is redistributed before implantation to facilitate embryo invasion in mammals with invasive placentation. We identified localization patterns of this cell adhesion molecule throughout pregnancy in two marsupial species with non-invasive placentation, the tammar wallaby (Macropus eugenii; Macropodidae), and the brushtail possum (Trichosurus vulpecula; Phalangeridae). Interestingly, Desmoglein-2 redistribution also occurs in both M. eugenii and T. vulpecula, suggesting that cell adhesion, and thus integrity of the uterine epithelium, is reduced during implantation regardless of placental type, and may be an important component of uterine remodeling. Desmoglein-2 also localizes to the mesenchymal stromal cells of M. eugenii and to epithelial cell nuclei in T. vulpecula, suggesting its involvement in cellular processes that are independent of adhesion and may compensate for reduced lateral adhesion in the uterine epithelium. We conclude that non-invasive placentation in marsupials involves diverse and complementary strategies to maintain an intact epithelial barrier.
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http://dx.doi.org/10.1002/mrd.22940DOI Listing
January 2018

Three-dimensional mammalian tooth development using diceCT.

Arch Oral Biol 2018 Jan 20;85:183-191. Epub 2017 Oct 20.

School of Biological Sciences, 18 Innovation Walk, Monash University, Victoria 3800, Australia; Geosciences, Museums Victoria, Melbourne, Victoria 3001, Australia.

Objective: This study aims to develop the Diffusible Iodine-based Contrast-Enhanced CT (diceCT) method for non-destructive imaging of both soft and mineralised tissues. We sought to document the 3D spatio-temporal pattern of mammalian tooth development including multiple tooth classes and generations, using the tammar wallaby (Macropus eugenii) as a model species.

Design: We took microCT scans of developing fetuses and pouch young stained using Lugol's Iodine (IKI) contrast agent. Stained versus unstained specimen comparisons were then made to investigate whether staining had improved visualisation of structures. Scan slices were compared to histological sections to confirm the identity of tissues and structures. Tissue layers were digitally segmented to create 3D models.

Results: DiceCT dramatically enhanced visual contrast of soft tissues, allowing differentiation between epithelial and mesenchymal layers. Subvolume scans at higher magnification achieved single-cell layer resolution within relatively large intact heads. We observed in-situ initiating teeth, which progressed through major stages of tooth development including morphogenesis and mineralisation. In addition, we traced the development of other mineralized and unmineralised tissues, such as the cranial bones and the brain, eye and olfactory system.

Conclusions: DiceCT was time- and cost-effective in producing complex 3D models of the entire dentition of the tammar wallaby at each developmental stage with tissue-level resolution. The 3D view of soft and mineralised tooth structures allowed us to define tooth class and generation from a developmental perspective. Additionally, the development of other organs can also be documented using the same scans, demonstrating the efficiency and versatility of this technique.
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http://dx.doi.org/10.1016/j.archoralbio.2017.10.018DOI Listing
January 2018

Expression of STRA8 is conserved in therian mammals but expression of CYP26B1 differs between marsupials and mice.

Biol Reprod 2017 Aug;97(2):217-229

School of BioSciences, The University of Melbourne, Victoria, Australia.

The first sign of mammalian germ cell sexual differentiation is the initiation of meiosis in females and of mitotic arrest in males. In the mouse, retinoic acid induces ovarian Stra8 expression and entry of germ cells into meiosis. In developing mouse testes, cytochrome P450 family 26, subfamily b, polypeptide 1 (CYP26B1) produced by the Sertoli cells degrades retinoic acid, preventing Stimulated by Retinoic Acid Gene 8 (Stra8), expression and inhibiting meiosis. However, in developing humans, there is no evidence that CYP26B1 acts a meiosis-inhibiting factor. We therefore examined aspects of the retinoic acid/STRA8/CYP26B1 pathway during gonadal development in the tammar wallaby, a marsupial, to understand whether retinoic acid stimulation of STRA8 and CYP26B1 degradation of retinoic acid was conserved between widely divergent mammals. In tammar ovaries, as in human ovaries and unlike the pattern in mice, CYP26B1 expression was not downregulated before the onset of meiosis. Exposure of pre-meiotic tammar ovaries to exogenous retinoic acid in vitro upregulated STRA8 expression compared to controls. We conclude that retinoic acid and STRA8 are conserved factors that control the initiation of meiosis amongst mammals but the role of CYP26B1 as a meiosis-inhibiting factor may be specific to rodents. The identity of the marsupial meiosis-inhibiting factor remains unknown.
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http://dx.doi.org/10.1093/biolre/iox083DOI Listing
August 2017

The enigma of embryonic diapause.

Development 2017 09;144(18):3199-3210

The Ottawa Hospital Research Institute, Ottawa, Ontario, Canada K1H8L6.

Embryonic diapause - a period of embryonic suspension at the blastocyst stage - is a fascinating phenomenon that occurs in over 130 species of mammals, ranging from bears and badgers to mice and marsupials. It might even occur in humans. During diapause, there is minimal cell division and greatly reduced metabolism, and development is put on hold. Yet there are no ill effects for the pregnancy when it eventually continues. Multiple factors can induce diapause, including seasonal supplies of food, temperature, photoperiod and lactation. The successful reactivation and continuation of pregnancy then requires a viable embryo, a receptive uterus and effective molecular communication between the two. But how do the blastocysts survive and remain viable during this period of time, which can be up to a year in some cases? And what are the signals that bring it out of suspended animation? Here, we provide an overview of the process of diapause and address these questions, focussing on recent molecular data.
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http://dx.doi.org/10.1242/dev.148213DOI Listing
September 2017

Molecular conservation of marsupial and eutherian placentation and lactation.

Elife 2017 09 12;6. Epub 2017 Sep 12.

Department of Genetics, Stanford University School of Medicine, Stanford, United States.

Eutherians are often mistakenly termed 'placental mammals', but marsupials also have a placenta to mediate early embryonic development. Lactation is necessary for both infant and fetal development in eutherians and marsupials, although marsupials have a far more complex milk repertoire that facilitates morphogenesis of developmentally immature young. In this study, we demonstrate that the anatomically simple tammar placenta expresses a dynamic molecular program that is reminiscent of eutherian placentation, including both fetal and maternal signals. Further, we provide evidence that genes facilitating fetal development and nutrient transport display convergent co-option by placental and mammary gland cell types to optimize offspring success.
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http://dx.doi.org/10.7554/eLife.27450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595433PMC
September 2017

Uterine molecular changes for non-invasive embryonic attachment in the marsupials Macropus eugenii (Macropodidae) and Trichosurus vulpecula (Phalangeridae).

Mol Reprod Dev 2017 Oct 7;84(10):1076-1085. Epub 2017 Aug 7.

School of Life and Environmental Sciences, University of Sydney, New South Wales, Australia.

Pregnancy in mammals requires remodeling of the uterus to become receptive to the implanting embryo. Remarkably similar morphological changes to the uterine epithelium occur in both eutherian and marsupial mammals, irrespective of placental type. Nevertheless, molecular differences in uterine remodeling indicate that the marsupial uterus employs maternal defences, including molecular reinforcement of the uterine epithelium, to regulate embryonic invasion. Non-invasive (epitheliochorial) embryonic attachment in marsupials likely evolved secondarily from invasive attachment, so uterine defences in these species may prevent embryonic invasion. We tested this hypothesis by identifying localization patterns of Talin, a key basal anchoring molecule, in the uterine epithelium during pregnancy in the tammar wallaby (Macropus eugenii; Macropodidae) and the brush tail possum (Trichosurus vulpecula; Phalangeridae). Embryonic attachment is non-invasive in both species, yet Talin undergoes a clear distributional change during pregnancy in M. eugenii, including recruitment to the base of the uterine epithelium just before attachment, that closely resembles that of invasive implantation in the marsupial species Sminthopsis crassicaudata. Basal localization occurs throughout pregnancy in T. vulpecula, although, as for M. eugenii, this pattern is most specific prior to attachment. Such molecular reinforcement of the uterine epithelium for non-invasive embryonic attachment in marsupials supports the hypothesis that less-invasive and non-invasive embryonic attachment in marsupials may have evolved via accrual of maternal defences. Recruitment of basal molecules, including Talin, to the uterine epithelium may have played a key role in this transition.
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http://dx.doi.org/10.1002/mrd.22861DOI Listing
October 2017
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