Pseudogenization of NK3 homeobox 2 (Nkx3.2) in monotremes provides insight into unique gastric anatomy and physiology.

The enzymatic breakdown and regulation of food passage through the vertebrate antral stomach and pyloric sphincter (antropyloric region) is a trait conserved over 450 million years. Development of the structures involved is underpinned by a highly conserved signalling pathway involving the hedgehog, bone morphogenetic protein and Wingless/Int-1 (Wnt) protein families. Monotremes are one of the few vertebrate lineages where acid-based digestion has been lost, and this is consistent with the lack of genes for hydrochloric acid secretion and gastric enzymes in the genomes of the platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus) . Furthermore, these species feature unique gastric phenotypes, both with truncated and aglandular antral stomachs and the platypus with no pylorus. Here, we explore the genetic underpinning of monotreme gastric phenotypes, investigating genes important in antropyloric development using the newest monotreme genomes (mOrnAna1.pri.v4 and mTacAcu1) together with RNA-seq data. We found that the pathway constituents are generally conserved, but surprisingly, NK3 homeobox 2 (Nkx3.2) was pseudogenized in both platypus and echidna. We speculate that the unique sequence evolution of Grem1 and Bmp4 sequences in the echidna lineage may correlate with their pyloric-like restriction and that the convergent loss of gastric acid and stomach size genotypes and phenotypes in teleost and monotreme lineages may be a result of eco-evolutionary dynamics. These findings reflect the effects of gene loss on phenotypic evolution and further elucidate the genetic control of monotreme stomach anatomy and physiology.

The shingled girl: Catherine Janet Hill and her contributions to embryology.

Catherine J. Hill is best remembered for her dedication to cataloguing the comprehensive embryological collection of her father J. P. Hill. Yet, her own research, during the interwar years, is little known. She made a significant contribution to interpreting the autonomic innervation of the gut, work that was presented to The Royal Society and earned her a PhD. Working in her father's laboratory, she then set about solving the sequence of secretions from the tubal epithelium and uterine glands that contributed the two layers of egg albumen and three shell layers of the monotreme egg. She was also the first to understand twinning in the marmoset and how two embryos came to share a single extraembryonic coelom, work that often is credited to J. P. Hill. Here. I explain how that happened and explore the context in which she and other female scientists worked at the time.

Comparative genomics of the T cell receptor µ locus in marsupials and monotremes.

T cells are a primary component of the vertebrate adaptive immune system. There are three mammalian T cell lineages based on their T cell receptors (TCR). The αß T cells and γδ T cells are ancient and found broadly in vertebrates. The more recently discovered γµ T cells are uniquely mammalian and only found in marsupials and monotremes. In this study, we compare the TCRµ locus (TRM) across the genomes of two marsupials, the gray short-tailed opossum and Tasmanian devil, and one monotreme, the platypus. These analyses revealed lineage-specific duplications, common to all non-eutherian mammals described. There is conserved synteny in the TRM loci of both marsupials but not in the monotreme. Our results are consistent with an ancestral cluster organization which was present in the last common mammalian ancestor which underwent lineage-specific duplications and divergence among the non-eutherian mammals.

Chiropterans Are a Hotspot for Horizontal Transfer of DNA Transposons in Mammalia.

Horizontal transfer of transposable elements (TEs) is an important mechanism contributing to genetic diversity and innovation. Bats (order Chiroptera) have repeatedly been shown to experience horizontal transfer of TEs at what appears to be a high rate compared with other mammals. We investigated the occurrence of horizontally transferred (HT) DNA transposons involving bats. We found over 200 putative HT elements within bats; 16 transposons were shared across distantly related mammalian clades, and 2 other elements were shared with a fish and two lizard species. Our results indicate that bats are a hotspot for horizontal transfer of DNA transposons. These events broadly coincide with the diversification of several bat clades, supporting the hypothesis that DNA transposon invasions have contributed to genetic diversification of bats.

Convergent evolutionary shifts in rhodopsin retinal release explain shared opsin repertoires in monotremes and crocodilians.

The visual ecology of early mammals remains poorly resolved. Studies of ancestral photopigments suggest an ancient transition from nocturnal to more crepuscular conditions. By contrast, the phenotypic shifts following the split of monotremes and therians-which lost their SWS1 and SWS2 opsins, respectively-are less clear. To address this, we obtained new phenotypic data on the photopigments of extant and ancestral monotremes. We then generated functional data for another vertebrate group that shares the same photopigment repertoire as monotremes: the crocodilians. By characterizing resurrected ancient pigments, we show that the ancestral monotreme underwent a dramatic acceleration in its rhodopsin retinal release rate. Moreover, this change was likely mediated by three residue replacements, two of which also arose on the ancestral branch of crocodilians, which exhibit similarly accelerated retinal release. Despite this parallelism in retinal release, we detected minimal to moderate changes in the spectral tuning of cone visual pigments in these groups. Our results imply that ancestral forms of monotremes and crocodilians independently underwent niche expansion to encompass quickly changing light conditions. This scenario-which accords with reported crepuscular activity in extant monotremes-may help account for their loss of the ultraviolet-sensitive SWS1 pigment but retention of the blue-sensitive SWS2.

Evolution of the Short Form of DNMT3A, DNMT3A2, Occurred in the Common Ancestor of Mammals.

Genomic imprinting is found in marsupial and eutherian mammals, but not in monotremes. While the primary regulator of genomic imprinting in eutherians is differential DNA methylation between parental alleles, conserved imprinted genes in marsupials tend to lack DNA methylation at their promoters. DNA methylation at eutherian imprinted genes is mainly catalyzed by a DNA methyltransferase (DNMT) enzyme, DNMT3A. There are two isoforms of eutherian DNMT3A: DNMT3A and DNMT3A2. DNMT3A2 is the primary isoform for establishing DNA methylation at eutherian imprinted genes and is essential for eutherian genomic imprinting. In this study, we investigated whether DNMT3A2 is also present in the two other mammalian lineages, marsupials and monotremes. We identified DNMT3A2 in both marsupials and monotremes, although imprinting has not been identified in monotremes. By analyzing genomic sequences and transcriptome data across vertebrates, we concluded that the evolution of DNMT3A2 occurred in the common ancestor of mammals. In addition, DNMT3A/3A2 gene and protein expression during gametogenesis showed distinct sexual dimorphisms in a marsupial, the tammar wallaby, and this pattern coincided with the sex-specific DNA methylation reprogramming in this species as it does in mice. Our results show that DNMT3A2 is present in all mammalian groups and suggests that the basic DNMT3A/3A2-based DNA methylation mechanism is conserved at least in therian mammals.

Evolution of milk oligosaccharides: Origin and selectivity of the ratio of milk oligosaccharides to lactose among mammals.

BACKGROUND: The carbohydrate fraction of mammalian milk is constituted of lactose and oligosaccharides, most of which contain a lactose unit at their reducing ends. Although lactose is the predominant saccharide in the milk of most eutherians, oligosaccharides significantly predominate over lactose in the milk of monotremes and marsupials. SCOPE OF REVIEW: This review describes the most likely process by which lactose and milk oligosaccharides were acquired during the evolution of mammals and the mechanisms by which these saccharides are digested and absorbed by the suckling neonates. MAJOR CONCLUSIONS: During the evolution of mammals, c-type lysozyme evolved to α-lactalbumin. This permitted the biosynthesis of lactose by modulating the substrate specificity of ß4galactosyltransferase 1, thus enabling the concomitant biosynthesis of milk oligosaccharides through the activities of several glycosyltransferases using lactose as an acceptor. In most eutherian mammals the digestion of lactose to glucose and galactose is achieved through the action of intestinal lactase (ß-galactosidase), which is located within the small intestinal brush border. This enzyme, however, is absent in neonatal monotremes and macropod marsupials. It has therefore been proposed that in these species the absorption of milk oligosaccharides is achieved by pinocytosis or endocytosis, after which digestion occurs through the actions of several lysosomal acid glycosidases. This process would enable the milk oligosaccharides of monotremes and marsupials to be utilized as a significant energy source for the suckling neonates. GENERAL SIGNIFICANCE: The evolution and significance of milk oligosaccharides is discussed in relation to the evolution of mammals.

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

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 Morganucodon, 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.

The defining feature of all mammals is how the jaw works. Fish, reptiles and other animals with backbones have a lower jaw made of many bones fused together, one of which connects to the upper jaw. The lower jaw in mammals, however, is made of a single bone that connects with the upper jaw using a completely unique jaw joint. This new joint emerged as the ancestors of all mammals split from the reptiles around 200 million years ago. The bones that formed the original jaw joint ended up in the middle ear in mammals and switched to a role in hearing. Nowadays, there are three types of mammals: the placentals, marsupials and monotremes (the egg laying mammals). In mice, humans and other placental mammals, the skeleton of the adult jaw joint forms in the embryo before birth. However, marsupials (such as kangaroos and opossums) and monotremes (platypuses and echidnas) are born at a much earlier embryonic stage, before the adult jaw joint has formed. It is therefore unclear how newborn marsupials and monotremes are able to move their jaws to feed on milk from their mother. Anthwal et al. compared how the jaw develops in mice, opossums, platypuses and echidnas before and after the adult jaw joint becomes functional. The experiments showed that young echidnas, platypuses and opossums use their middle ear bones to articulate the lower jaw with the head before the adult jaw joint forms. In young opossums, the ear bones form a cushion to support the jaw. In juvenile platypuses a double joint is evident, with the ear bones forming a joint at the same time as the newly formed adult jaw joint, similar to the situation observed in fossils of mammal ancestors. The experiments also indicated that mice and other placental mammals may potentially use their ear bones to support the jaw before birth. These findings shed light on why the ear and jaw have such a close connection in mammals. In humans, the ear and jaw bones are still connected by ligaments, explaining why trauma to the jaw joint can cause dislocation of the ear bones. Similarly, defects in the development of the jaw can impact the ear, such as in Treacher Collins Syndrome, where in some cases the jaw joint fails to form and the ear bones appear to try and take this role. Understanding how the ear and jaw evolved will help us understand why they look like they do and why a defect in one can have knock-on consequences for the other.

Extended cleavage specificities of two mast cell chymase-related proteases and one granzyme B-like protease from the platypus, a monotreme.

Mast cells (MCs) are inflammatory cells primarily found in tissues in close contact with the external environment, such as the skin and the intestinal mucosa. They store large amounts of active components in cytoplasmic granules, ready for rapid release. The major protein content of these granules is proteases, which can account for up to 35 % of the total cellular protein. Depending on their primary cleavage specificity, they can generally be subdivided into chymases and tryptases. Here we present the extended cleavage specificities of two such proteases from the platypus. Both of them show an extended chymotrypsin-like specificity almost identical to other mammalian MC chymases. This suggests that MC chymotryptic enzymes have been conserved, both in structure and extended cleavage specificity, for more than 200 million years, indicating major functions in MC-dependent physiological processes. We have also studied a third closely related protease, originating from the same chymase locus whose cleavage specificity is closely related to the apoptosis-inducing protease from cytotoxic T cells, granzyme B. The presence of both a chymase and granzyme B in all studied mammals indicates that these two proteases bordering the locus are the founding members of this locus.

Structural and mechanistic insights into EchAMP: A antimicrobial protein from the Echidna milk.

BACKGROUND: Antibiotic resistance is a problem that necessitates the identification of new antimicrobial molecules. Milk is known to have molecules with antimicrobial properties (AMPs). Echidna Antimicrobial Protein (EchAMP) is one such lactation specific AMP exclusively found in the milk of Echidna, an egg-laying mammal geographically restricted to Australia and New Guinea. Previous studies established that EchAMP exhibits substantial bacteriostatic activity against multiple bacterial genera. However, the subsequent structural and functional studies were hindered due to the unavailability of pure protein. RESULTS: In this study, we expressed EchAMP protein using a heterologous expression system and successfully purified it to >95% homogeneity. The purified recombinant protein exhibits bacteriolytic activity against both Gram-positive and Gram-negative bacteria as confirmed by live-dead staining and scanning electron microscopy. Structurally, this AMP belongs to the family of intrinsically disordered proteins (IDPs) as deciphered by the circular-dichroism, tryptophan fluorescence, and NMR spectroscopy. Nonetheless, EchAMP has the propensity to acquire structure with amphipathic molecules, or membrane mimics like SDS, lipopolysaccharides, and liposomes as again observed through multiple spectroscopic techniques. CONCLUSIONS: Recombinant EchAMP exhibits broad-spectrum bacteriolytic activity by compromising the bacterial cell membrane integrity. Hence, we propose that this intrinsically disordered antimicrobial protein interact with the bacterial cell membrane and undergoes conformational changes to form channels in the membrane resulting in cell lysis. GENERAL SIGNIFICANCE: EchAMP, the evolutionarily conserved, lactation specific AMP from an oviparous mammal may find application as a broad-spectrum antimicrobial against pathogens that affect mammary gland or otherwise cause routine infections in humans and livestock.

Soft tissue sarcoma in a short-beaked echidna (Tachyglossus aculeatus).

CASE REPORT: An adult male short-beaked echidna in poor body condition was found with a 25 × 12 mm round, ulcerated and bleeding mass on the left side of the face at the base of the beak. The animal responded well to initial supportive care and was referred to a specialist wildlife centre for further assessment and treatment. Clinical pathology showed moderate neutrophilia, mild anaemia, mild elevation in liver enzymes (ALT, AST and ALP) and mild azotaemia. Initial clinical differential diagnoses for the facial mass favoured an inflammatory rather than a neoplastic lesion, based on previous reports. Examination of an incisional biopsy identified a malignant spindle cell proliferation (sarcoma) not amenable to complete surgical excision. The animal was euthanased on humane grounds. Immunohistochemical assessment of the mass showed it to be negative for cytokeratin, desmin, smooth muscle actin, periaxin and MAC387 antibody labelling. Definitive histogenesis was undetermined and a final diagnosis of poorly differentiated sarcoma, unlikely to be of muscle, Schwann cell or histiocytic origin, was made. CONCLUSION: Reports of neoplasia in prototherian mammals (monotremes) are rare. To the authors' knowledge this is the first report of such a tumour in a monotreme species and the first immunohistochemical characterisation of a stromal tumour in these animals. The malignant nature of this tumour contrasts with a previous report of benign neoplasia (fibroma) associated with the beak. Although rare, malignant neoplasia should be included in the differential diagnoses of mass lesions in monotremes, despite inflammatory or traumatic mass lesions being more commonly reported.

Conceptus Coats of Marsupials and Monotremes.

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).

Structural characterization of a novel monotreme-specific protein with antimicrobial activity from the milk of the platypus.

Monotreme lactation protein (MLP) is a recently identified protein with antimicrobial activity. It is present in the milk of monotremes and is unique to this lineage. To characterize MLP and to gain insight into the potential role of this protein in the evolution of lactation, the crystal structure of duck-billed platypus (Ornithorhynchus anatinus) MLP was determined at 1.82 Šresolution. This is the first structure to be reported for this novel, mammalian antibacterial protein. MLP was expressed as a FLAG epitope-tagged protein in mammalian cells and crystallized readily, with at least three space groups being observed (P1, C2 and P21). A 1.82 Šresolution native data set was collected from a crystal in space group P1, with unit-cell parameters a = 51.2, b = 59.7, c = 63.1 Å, α = 80.15, ß = 82.98, γ = 89.27°. The structure was solved by SAD phasing using a protein crystal derivatized with mercury in space group C2, with unit-cell parameters a = 92.7, b = 73.2, c = 56.5 Å, ß = 90.28°. MLP comprises a monomer of 12 helices and two short ß-strands, with much of the N-terminus composed of loop regions. The crystal structure of MLP reveals no three-dimensional similarity to any known structures and reveals a heretofore unseen fold, supporting the idea that monotremes may be a rich source for the identification of novel proteins. It is hypothesized that MLP in monotreme milk has evolved to specifically support the unusual lactation strategy of this lineage and may have played a central role in the evolution of these mammals.

Male Mutation Bias Is the Main Force Shaping Chromosomal Substitution Rates in Monotreme Mammals.

In many species, spermatogenesis involves more cell divisions than oogenesis, and the male germline, therefore, accumulates more DNA replication errors, a phenomenon known as male mutation bias. The extent of male mutation bias (α) is estimated by comparing substitution rates of the X, Y, and autosomal chromosomes, as these chromosomes spend different proportions of their time in the germlines of the two sexes. Male mutation bias has been characterized in placental and marsupial mammals as well as birds, but analyses in monotremes failed to detect any such bias. Monotremes are an ancient lineage of egg-laying mammals with distinct biological properties, which include unique germline features. Here, we sought to assess the presence and potential characteristics of male mutation bias in platypus and the short-beaked echidna based on substitution rate analyses of X, Y, and autosomes. We established the presence of moderate male mutation bias in monotremes, corresponding to an α value of 2.12-3.69. Given that it has been unclear what proportion of the variation in substitution rates on the different chromosomal classes is really due to differential number of replications, we analyzed the influence of other confounding forces (selection, replication-timing, etc.) and found that male mutation bias is the main force explaining the between-chromosome classes differences in substitution rates. Finally, we estimated the proportion of variation at the gene level in substitution rates that is owing to replication effects and found that this phenomenon can explain >68% of these variations in monotremes, and in control species, rodents, and primates.

Comparative cranial morphology in living and extinct platypuses: Feeding behavior, electroreception, and loss of teeth.

The modern platypus, Ornithorhynchus anatinus, has an eye structure similar to aquatic mammals; however, platypuses also have a "sixth sense" associated with the bill electro- and mechanoreception that they use without opening their eyes underwater. We hypothesize that Ornithorhynchus and the Miocene taxon Obdurodon have different sensory capacities, which may have resulted from differences in foraging behavior. To estimate differences in foraging, sensory systems, and anatomical divergence between these monotremes, we compared their skull morphologies. Results indicate that the bill of Obdurodon is more dorsally deflected than that of Ornithorhynchus, suggesting a pelagic foraging behavior in Obdurodon compared to the bottom-feeding behavior in Ornithorhynchus. The infraorbital foramen of Obdurodon, through which the maxillary nerve passes sensory data from the bill to the brain, is relatively less developed than that of Ornithorhynchus. Whereas bill-focused sensory perception was likely shared among Mesozoic monotremes, the highly developed electrosensory system of Ornithorhynchus may represent an adaptation to foraging in cloudy water. Computed tomography imagery indicates that the enlarged infraorbital canal of Ornithorhynchus restricts the space available for maxillary tooth roots. Hence, loss of functional teeth in Ornithorhynchus may possibly have resulted from a shift in foraging behavior and coordinate elaboration of the electroreceptive sensory system. Well-developed electroreceptivity in monotremes is known at least as far back as the early Cretaceous; however, there are differences in the extent of elaboration of the feature among members of the ornithorhynchid lineage.

Comparative Myology and Evolution of Marsupials and Other Vertebrates, With Notes on Complexity, Bauplan, and "Scala Naturae".

Opossums are frequent subjects of developmental studies because marsupials share developmental features not seen in placentals and because Didelphimorpha is the sister-group of other extant Marsupialia. But is the adult marsupial muscular system markedly different from that of placentals or is it, like the skeletal system, very similar? We provide, for the first time, a brief description of all head and limb muscles of Didelphis virginiana based on our dissections and using a unifying nomenclature by integrating the data gathered in our long-term project on the development, homologies, and evolution of the muscles of all major vertebrate taxa. Our data indicate that there were many more muscle synapomorphic changes from the last common ancestor (LCA) of amniotes to the mammalian LCA (63) and from this LCA to the LCA of extant therians (48) than from this latter LCA to the LCA of extant placentals (10 or 11). Importantly, Didelphis is anatomically more plesiomorphic (only 14 changes from LCA of extant therians) than are rats (37 changes) and humans (63 changes), but its musculature is more complex (193 muscles) than that of humans (only 180 muscles). Of the 194 muscles of Didelphis, 172 (89%) are present in rats, meaning that their adult muscle anatomy is indeed very similar. This similarity supports the existence of a common, easy recognizable therian Bauplan, but one that is caused by developmental constraints and by evolutionary change driven by the needs of the embryos/neonates, rather than by a "goal" toward a specific adult plan/"archetype," as the name Bauplan suggests. Anat Rec, 299:1224-1255, 2016. © 2016 Wiley Periodicals, Inc.

Anterior commissure versus corpus callosum: A quantitative comparison across mammals.

Mammals rely on two major pathways to transfer information between the two hemispheres of the brain: the anterior commissure and the corpus callosum. Metatheria and monotremes rely exclusively on the anterior commissure for interhemispheric transfer between the isocortices and olfactory allocortices of each side, whereas Eutheria use a combination of the anterior commissure and an additional pathway exclusive to Eutheria, the corpus callosum. Midline cross-sectional area of the anterior commissure and corpus callosum were measured in a range of mammals from all three infraclasses and plotted against brain volume to determine how midline anterior commissure area and its size relative to the corpus callosum vary with brain size and taxon. In Metatheria, the square root of anterior commissure area rises in almost direct proportion with the cube root of brain volume (i.e. the ratio of the two is relatively constant), whereas among Eutheria the ratio of the square root of anterior commissure area to the cube root of brain volume declines slightly with increasing brain size. The total of isocortical and olfactory allocortical commissure area rises more rapidly with increasing brain volume among Eutheria than among Metatheria. This means that the midline isocortical and olfactory allocortical commissural area of metatherians with large brains (about 70 ml) is only about 50% of that among eutherians with similarly sized brains. On the other hand, isocortical and olfactory allocortical commissural area is similar in Metatheria and Eutheria at brain volumes around 1 ml. Among the Eutheria, some groups make less use of the anterior commissure pathway than do others: soricomorphs, rodents and cetaceans have smaller anterior commissures for their brain size than do afrosoricids, erinaceomorphs and proboscideans. The findings suggest that use of the anterior commissural route for isocortical commissural connections may have placed limitations on interhemispheric transfer of information among the metatherians, but only when brain size reaches 50 ml or more.

Morbidity and mortality of monotremes admitted to the Australian Wildlife Health Centre, Healesville Sanctuary, Australia, 2000-2014.

OBJECTIVE: The medical records of individual monotremes admitted to the Australian Wildlife Health Centre from 2000 to 2014 were reviewed to determine the causes of morbidity and mortality. RESULTS: During this period, a total of 38 platypus (Ornithorhyncus anatanus) and 273 short-beaked echidnas (Tachyglossus aculeatus) were examined. Trauma was the most significant reason for monotreme admissions, accounting for 73.7% of platypus cases and 90.1% of short-beaked echidna cases. Within the category of trauma, entanglement (28.6%) and unknown trauma (28.6%) were most significant for platypus, while impact with motor vehicle (73.2%) and domestic dog attack (14.2%) were the most significant subcategories for short-beaked echidnas. CONCLUSION: Indirect anthropogenic factors are a significant cause of morbidity and mortality of monotremes in Victoria, Australia.

Identification and characterisation of synaptonemal complex genes in monotremes.

The platypus and echidna are the only extant species belonging to the clade of monotremata, the most basal mammalian lineage. The platypus is particularly well known for its mix of mammalian and reptilian characteristics and work in recent years has revealed this also extends to the genetic level. Amongst the monotreme specific features is the unique multiple sex chromosome system (5X4Y in the echidna and 5X5Y in the platypus), which forms a chain in meiosis. This raises questions about sex chromosome organisation at meiosis, including whether there has been changes in genes coding for synaptonemal complex proteins which are involved in homologous synapsis. Here we investigate the key structural components of the synaptonemal complex in platypus and echidna, synaptonemal complex proteins 1, 2 and 3 (SYCP1, SYCP2 and SYCP3). SYCP1 and SYCP2 orthologues are present, conserved and expressed in platypus testis. SYCP3 in contrast is highly diverged, but key residues required for self-association are conserved, while those required for tetramer stabilisation and DNA binding are missing. We also discovered a second SYCP3-like gene (SYCP3-like) in the same region. Comparison with the recently published Y-borne SYCP3 amino acid sequences revealed that SYCP3Y is more similar to SYCP3 in other mammals than the monotreme autosomal SYCP3. It is currently unclear if these changes in the SYCP3 gene repertoire are related to meiotic organisation of the extraordinary monotreme sex chromosome system.

Immunohistochemical analysis of pancreatic islets of platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus ssp.).

Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. ß-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide (PP)-cells were mainly found in the islet periphery. Distinct PP-rich (PP-lobe) and PP-poor areas (non-PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and ß-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall, monotreme pancreata share the feature of consisting of distinct PP-poor and PP-rich islets with other mammals. A higher number of islets and α- or ß-cell only islets are shared between monotremes and birds. The islets of monotremes were larger than those of birds but smaller compared with therian mammals. This may indicate a trend of having fewer larger islets comprising several endocrine cell types during mammalian evolution.