School of BioSciences - Research Publications

Permanent URI for this collection

Search Results

Now showing 1 - 10 of 67
  • Item
    No Preview Available
    Marsupials have monoallelic MEST expression with a conserved antisense lncRNA but MEST is not imprinted
    Ishihara, T ; Suzuki, S ; Newman, TA ; Fenelon, JC ; Griffith, OW ; Shaw, G ; Renfree, MB (SPRINGERNATURE, 2024-01)
    The imprinted isoform of the Mest gene in mice is involved in key mammalian traits such as placental and fetal growth, maternal care and mammary gland maturation. The imprinted isoform has a distinct differentially methylated region (DMR) at its promoter in eutherian mammals but in marsupials, there are no differentially methylated CpG islands between the parental alleles. Here, we examined similarities and differences in the MEST gene locus across mammals using a marsupial, the tammar wallaby, a monotreme, the platypus, and a eutherian, the mouse, to investigate how imprinting of this gene evolved in mammals. By confirming the presence of the short isoform in all mammalian groups (which is imprinted in eutherians), this study suggests that an alternative promoter for the short isoform evolved at the MEST gene locus in the common ancestor of mammals. In the tammar, the short isoform of MEST shared the putative promoter CpG island with an antisense lncRNA previously identified in humans and an isoform of a neighbouring gene CEP41. The antisense lncRNA was expressed in tammar sperm, as seen in humans. This suggested that the conserved lncRNA might be important in the establishment of MEST imprinting in therian mammals, but it was not imprinted in the tammar. In contrast to previous studies, this study shows that MEST is not imprinted in marsupials. MEST imprinting in eutherians, therefore must have occurred after the marsupial-eutherian split with the acquisition of a key epigenetic imprinting control region, the differentially methylated CpG islands between the parental alleles.
  • Item
    No Preview Available
    An evaluation of DNA sample source and molecular markers to determine gender in the short-beaked echidna (Tachyglossus aculeatus)
    Hulse, L ; Tomsett, C ; Roser, A ; Clark, L ; Meer, H ; Pyne, M ; Fenelon, JC ; Renfree, MB ; Johnston, S (WILEY, 2024-01)
    The short-beaked echidna is sexually monomorphic such that gender identification without veterinary intervention is challenging. The aim of this study was to evaluate and compare the most optimal noninvasive genetic source by extracting echidna genomic DNA (gDNA) from fecal scats, plucked hair, and quills to perform genetic sex testing using a range of molecular markers. Sex determination of 14 captive short-beaked echidnas was determined by amplifying isolated DNA from noninvasive samples, targeting two Y chromosome (male-specific) genes (mediator complex subunit 26 Y-gametologue [CRSPY] and anti-Müllerian hormone Y-gametologue [AMHY]), in addition to four confirmed sex-specific RADseq markers. Results of noninvasive samples were compared with blood samples and clinical records. Receiver operating characteristic curves were used to assess accuracy of sex determination of markers for each sample type. The gender of the echidnas was successfully identified on 75% of occasions using fecal samples, 90.6% occasions using hair, and 84.6% occasions with quills. Overall, the male-specific RADseq markers accurately identified the sex of echidnas with all sample types for 90% of animals; compared with 81.5% using CRSPY, and 82.0% using AMHY to identify sex. Collection of hair, quills, and feces provides a useful alternative to invasively collected samples, however, the accuracy of results depends on sample type and genetic marker selected. We found gender determination in the short-beaked echidna was most accurate using four male-specific RADseq markers on gDNA isolated from blood and hair. The noninvasive genetic sexing techniques documented here will inform and facilitate husbandry and genetic management of captive echidna populations.
  • Item
    Thumbnail Image
    Getting out of a mammalian egg: the egg tooth and caruncle of the echidna
    Fenelon, JC ; Bennetts, A ; Anthwal, N ; Pyne, M ; Johnston, SD ; Evans, AR ; Tucker, AS ; Renfree, MB (ACADEMIC PRESS INC ELSEVIER SCIENCE, 2023-03)
    In the echidna, after development in utero, the egg is laid in the pouch and incubated for 10 days. During this time, the fetuses develop an egg tooth and caruncle to help them hatch. Using rare and unprecedented access to limited echidna pre- and post-hatching tissues, development of the egg tooth and caruncle were assessed by micro-CT, histology and immunofluorescence. Unlike therian tooth germs that develop by placode invagination, the echidna egg tooth developed by evagination, similar to the first teeth in some reptiles and fish. The egg tooth ankylosed to the premaxilla, rather than forming a tooth root with ligamentous attachment found in other mammals, with loss of the egg tooth associated with high levels of activity odontoclasts and apoptosis. The caruncle formed as a separate mineralisation from the adjacent nasal capsule, and as observed in birds and turtles, the nasal region epithelium on top of the nose expressed markers of cornification. Together, this highlights that the monotreme egg tooth shares many similarities with typical reptilian teeth, suggesting that this tooth has been conserved from a common ancestor of mammals and reptiles.
  • Item
    Thumbnail Image
    Genetic sex test for the short-beaked echidna (Tachyglossus aculeatus)
    Keating, SE ; Fenelon, JC ; Pyne, M ; Pinto, BJ ; Guzman-Mendez, IA ; Johnston, SD ; Renfree, MB ; Gamble, T (SPRINGER, 2022-04-30)
    Monotremes (echidnas and platypus) possess five X and four or five Y sex chromosomes, respectively, that evolved independently from the sex chromosomes found in therian mammals. While the platypus has obvious venomous spurs in the male, the short-beaked echidna (Tachyglossus aculeatus) lacks easily identifiable sexually dimorphic characteristics, making it difficult to sex adults out of the breeding season and almost impossible to sex juveniles or embryonic material. Here, we used restriction-site associated DNA sequencing (RADseq) to identify novel sex-specific markers in the short-beaked echidna. We identified and validated a subset of male-specific markers that can be used as a non-invasive genetic sex test for the short-beaked echidna. We also assessed how laboratory conditions, including DNA extraction protocol and number of PCR cycles, can influence the outcome of genetic sex tests. The combined use of these markers will provide a valuable toolkit for researchers, conservationists, and zoo-keepers to reliably and non-invasively determine sex in the short-beaked echidna.
  • Item
    Thumbnail Image
    The Unique Penile Morphology of the Short-Beaked Echidna, Tachyglossus aculeatus
    Fenelon, JC ; McElrea, C ; Shaw, G ; Evans, AR ; Pyne, M ; Johnston, SD ; Renfree, MB (KARGER, 2021-09)
    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.
  • Item
    Thumbnail Image
    Investigating the utility of using fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas (Tachyglossus aculeatus).
    Dutton-Regester, KJ ; Roser, A ; Meer, H ; Russell, FA ; Pyne, M ; Renfree, MB ; Johnston, SD ; Keeley, T (Elsevier, 2023-01-01)
    This study demonstrates the utility of the analysis of fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas. Over three breeding seasons daily fecal samples were collected from female echidnas (n = 8) that were monitored continuously by video surveillance to confirm key reproductive events. Fecal progesterone metabolite concentrations were elevated above baseline values (448.0 ± 156.3 ng/g) during pregnancy and the luteal phase. However, compared to plasma progesterone the rise in fecal progesterone metabolite concentrations after copulation was delayed (3.3 ± 0.4 versus 8.3 ± 0.6 days, respectively), such that pregnancy was more reliably detected in its latter half when using fecal samples. Mating and oviposition were observed for 14 of the 19 pregnancies resulting in an estimated gestation of 16.7 ± 0.2 days (range 16.0-18.1 d). The estrogen enzyme-immunoassays tested (n = 3) in this study were not suitable for the fecal samples of the echidna. Fecal progesterone metabolites are an effective tool for confirming the timing and occurrence of estrous cycles in captive echidna colonies and can assist zookeepers in identifying possible causes of sub-optimal reproductive success without the unnecessary stress of repeated capture and anaesthesia for blood collection.
  • Item
    Thumbnail Image
    Plasma progesterone secretion during gestation of the captive short-beaked echidna
    Dutton-Regester, K ; Keeley, T ; Fenelon, JC ; Roser, A ; Meer, H ; Hill, A ; Pyne, M ; Renfree, MB ; Johnston, S (BIOSCIENTIFICA LTD, 2021-10)
    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.
  • Item
    No Preview Available
    Comparing the potential for maternal-fetal signalling in oviparous and viviparous lizards
    Wen, J ; Ishihara, T ; Renfree, MB ; Griffith, OW (ROYAL SOC, 2022-12-05)
    The evolution of a placenta requires several steps including changing the timing of reproductive events, facilitating nutrient exchange, and the capacity for maternal-fetal communication. To understand the evolution of maternal-fetal communication, we used ligand-receptor gene expression as a proxy for the potential for cross-talk in a live-bearing lizard (Pseudemoia entrecasteauxii) and homologous tissues in a related egg-laying lizard (Lampropholis guichenoti). Approximately 70% of expressed ligand/receptor genes were shared by both species. Gene ontology (GO) analysis showed that there was no GO-enrichment in the fetal membranes of the egg-laying species, but live-bearing fetal tissues were significantly enriched for 50 GO-terms. Differences in enrichment suggest that the evolution of viviparity involved reinforcing specific signalling pathways, perhaps to support fetal control of placentation. One identified change was in transforming growth factor beta signalling. Using immunohistochemistry, we show the production of the signalling molecule inhibin beta B (INHBB) occurs in viviparous fetal membranes but was absent in closely related egg-laying tissues, suggesting that the evolution of viviparity may have involved changes to signalling via this pathway. We argue that maternal-fetal signalling evolved through co-opting expressed signalling molecules and recruiting new signalling molecules to support the complex developmental changes required to support a fetus in utero. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.
  • Item
    Thumbnail Image
    Validation of a non-invasive assessment technique for quantifying faecal glucocorticoid metabolite concentrations in the short-beaked echidna (Tachyglossus aculeatus)
    Russell, FA ; Johnston, SD ; Hill, A ; Roser, A ; Meer, H ; Fenelon, JC ; Renfree, MB ; Keeley, T (ACADEMIC PRESS INC ELSEVIER SCIENCE, 2022-10-01)
    The monotreme adrenocortical response to stress may not rely as heavily on the hypothalamic-pituitaryadrenal (HPA) axis compared to other mammals. This study aimed to validate a technique in which glucocorticoid metabolites could be quantified non-invasively in short-beaked echidna faeces by examining the secretion of glucocorticoids (GC) using an adrenocorticotrophic hormone (ACTH) challenge on sexually mature captive echidnas. Echidnas were housed individually for 15 days, with the ACTH challenge occurring on day five. Blood samples were collected on day five during the challenge and faecal samples were collected each morning for the 15 days. Both sample types were analysed for glucocorticoids (GC) or its metabolites. Plasma corticosterone concentrations increased significantly after 30 min and 60 min relative to time 0, whilst plasma cortisol concentrations increased significantly after 60 min. The ACTH challenge also resulted in an increase in glucocorticoid metabolite concentration in faecal samples from four of the six echidnas detected one to two days post ACTH injection, thereby validating a non-invasive method to assess adrenal response in the echidna. These results confirm that echidnas respond to a synthetic ACTH challenge in a similar manner to that of eutherian species indicating that echidnas appear to use the HPA axis in their stress response.
  • Item
    Thumbnail Image
    Proteomics and Deep Sequencing Comparison of Seasonally Active Venom Glands in the Platypus Reveals Novel Venom Peptides and Distinct Expression Profiles
    Wong, ESW ; Morgenstern, D ; Mofiz, E ; Gombert, S ; Morris, KM ; Temple-Smith, P ; Renfree, MB ; Whittington, CM ; King, GF ; Warren, WC ; Papenfuss, AT ; Belov, K (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012-11)
    The platypus is a venomous monotreme. Male platypuses possess a spur on their hind legs that is connected to glands in the pelvic region. They produce venom only during the breeding season, presumably to fight off conspecifics. We have taken advantage of this unique seasonal production of venom to compare the transcriptomes of in- and out-of-season venom glands, in conjunction with proteomic analysis, to identify previously undiscovered venom genes. Comparison of the venom glands revealed distinct gene expression profiles that are consistent with changes in venom gland morphology and venom volumes in and out of the breeding season. Venom proteins were identified through shot-gun sequenced venom proteomes of three animals using RNA-seq-derived transcripts for peptide-spectral matching. 5,157 genes were expressed in the venom glands, 1,821 genes were up-regulated in the in-season gland, and 10 proteins were identified in the venom. New classes of platypus-venom proteins identified included antimicrobials, amide oxidase, serpin protease inhibitor, proteins associated with the mammalian stress response pathway, cytokines, and other immune molecules. Five putative toxins have only been identified in platypus venom: growth differentiation factor 15, nucleobindin-2, CD55, a CXC-chemokine, and corticotropin-releasing factor-binding protein. These novel venom proteins have potential biomedical and therapeutic applications and provide insights into venom evolution.