Prototherian Mammals Research Articles

Evolutionary Analysis of the Mammalian IL-17 Cytokine Family Suggests Conserved Roles in Female Fertility.

The interleukin-17 (IL-17) family includes pro-inflammatory cytokines IL-17A-F with important roles in mucosal defence, barrier integrity and tissue regeneration. IL-17A can be dysregulated in fertility complications, including pre-eclampsia, endometriosis and miscarriage. Because mammalian subclasses (eutherian, metatherian, and prototherian) have different related reproductive strategies, IL-17 genes and proteins were investigated in the three mammalian classes to explore their involvement in female fertility. Gene and protein sequences for IL-17s are found in eutherian, metatherian and prototherian mammals. Through synteny and multiple sequence protein alignment, the relationships among mammalian IL-17s were inferred. Publicly available datasets of early pregnancy stages and female fertility in therian mammals were collected and analysed to retrieve information on IL-17 expression. Synteny mapping and phylogenetic analyses allowed the classification of mammalian IL-17 family orthologs of human IL-17. Despite differences in their primary amino acid sequence, metatherian and prototherian IL-17s share the same tertiary structure as human IL-17s, suggesting similar functions. The analysis of available datasets for female fertility in therian mammals shows up-regulation of IL-17A and IL-17D during placentation. IL-17B and IL-17D are also found to be over-expressed in human fertility complication datasets, such as endometriosis or recurrent implantation failure. The conservation of the IL-17 gene and protein across mammals suggests similar functions in all the analysed species. Despite significant differences, the upregulation of IL-17 expression is associated with the establishment of pregnancy in eutherian and metatherian mammals. The dysregulation of IL-17s in human reproductive disorders suggests them as a potential therapeutic target.

Monotreme chromosomes: an introductory review

The three extant genera of the prototherian mammals, Ornithorhynchus (platypus), Tachyglossus (Australian echidna) and Zaglossus (New Guinea echidna), all have a mechanism of sex determination at odds with that seen in eutherian and metatherian mammals. Indeed, they stand apart from all vertebrates. Instead of the XX/XY, X1X2Y or ZZ/ZW systems seen in the majority of vertebrates the monotremes have a chain of nine (or ten) chromosomes present during meiosis in the male. This is believed to be the consequence of a presumed series of reciprocal translocations involving four autosomal pairs and the original X and Y chromosomes. The presence of this chain in all three genera indicates that a similar chain occurred in their common ancestor. This paper provides an overview of the search to unravel the mystery of this chain and to determine the identity of the sex chromosomes and members of the chain. The development of new techniques has hugely facilitated clarification of the findings of the earlier researchers. As a result, the chromosomes of the platypus and the echidna have now been individually described, the chain elements and/or sex chromosomes have been identified unambiguously and their order in the chain has been determined. The research reviewed here has also provided insights into the evolution of mammalian sex chromosomes and given new directions for unravelling dosage compensation and sex-determination mechanisms in mammals.

The Evolution of the DLK1-DIO3 Imprinted Domain in Mammals

A comprehensive, domain-wide comparative analysis of genomic imprinting between mammals that imprint and those that do not can provide valuable information about how and why imprinting evolved. The imprinting status, DNA methylation, and genomic landscape of the Dlk1-Dio3 cluster were determined in eutherian, metatherian, and prototherian mammals including tammar wallaby and platypus. Imprinting across the whole domain evolved after the divergence of eutherian from marsupial mammals and in eutherians is under strong purifying selection. The marsupial locus at 1.6 megabases, is double that of eutherians due to the accumulation of LINE repeats. Comparative sequence analysis of the domain in seven vertebrates determined evolutionary conserved regions common to particular sub-groups and to all vertebrates. The emergence of Dlk1-Dio3 imprinting in eutherians has occurred on the maternally inherited chromosome and is associated with region-specific resistance to expansion by repetitive elements and the local introduction of noncoding transcripts including microRNAs and C/D small nucleolar RNAs. A recent mammal-specific retrotransposition event led to the formation of a completely new gene only in the eutherian domain, which may have driven imprinting at the cluster.

Retrotransposon Silencing by DNA Methylation Can Drive Mammalian Genomic Imprinting

Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5′ region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.

Evolution of the “OR37” Subfamily of Olfactory Receptors: A Cross-Species Comparison

Genes encoding the olfactory receptors of the "OR37" subfamily of the mouse are characterized by special features including a clustered expression pattern, assembly in two distinct gene clusters, and highly conserved putative promoter motifs. Mining the rat and dog databases revealed that these two species possess highly conserved clusters of OR37 genes at two syntenic genomic loci. In a prototherian mammal, the platypus (Ornithorhynchus anatinus), none of the characteristic OR37 genes were found. Examination of a metatherian mammal, the gray short-tailed opossum (Monodelphis domestica) revealed seven canonical OR37 genes, all phylogenetically related to cluster II genes and also organized similar to cluster II of eutherian species. In addition, their 5' upstream regions comprised sequence motifs related to the putative regulatory sequences of cluster II genes. Typical cluster I OR37 genes were identified only in the eutherian mammals examined, including the evolutionary ancient anteater, wherein OR37 genes related to both clusters were present. Together, these results reveal novel information concerning the phylogenetic origin and important evolutionary steps of the mammalian-specific OR37 olfactory receptor family.

The origin and loss of the ubiquitin activating enzyme gene on the mammalian Y chromosome.

Mammalian sex chromosomes are thought to be descended from a homologous pair of autosomes: a testis-determining allele which defined the Y chromosome arose, recombination between the nascent X and Y chromosomes became restricted and the Y chromosome gradually lost its non-essential genetic functions. This model was originally inferred from the occurrence of few Y-linked genetic traits, pairing of the X and Y chromosomes during male meiosis and, more recently, the existence of X-Y homologous genes. The comparative analysis of such genes is a means by which the validity of this model can be evaluated. One well-studied example of an X-Y homologous gene is the ubiquitin activating enzyme gene ( UBE1 ), which is X-linked with a distinct Y-linked gene in many eutherian ('placental') and metatherian (marsupial) mammals. Nonetheless, no UBE1 homologue has yet been detected on the human Y chromosome. Here we describe a more extensive study of UBE1 homologues in primates and a prototherian mammal, the platypus. Our findings indicate that UBE1 lies within the X-Y pairing segment of the platypus but is absent from the human Y chromosome, having been lost from the Y chromosome during evolution of the primate lineage. Thus UBE1 illustrates the key steps of 'autosomal to X-specific' evolution of genes on the sex chromosomes.

Antigenic cross-reactions among immunoglobulin of diverse vertebrates (elasmobranchs to man) detected using xenoantisera

1. 1. Antisera raised in rabbits and goats against intact immunoglobulins or their constituent light and heavy chains from man, mouse and the galapagos shark ( Carcharhinus galapagenesis) were tested for their reactivity with immunoglobulins of elasmobranchs, other lower vertebrates and eutherian and prototherian mammals. 2. 2. Xenoantisera directed against human heavy chain isotypes allowed the serological identification of IgM and IgG immunoglobulins in the echidna ( Tachyglossus aculeatus), a monotreme which is one of the most primitive species of extant mammmals. 3. 3. The antisera to heavy chains reacted to varying degrees with purified immunoglobulins of non-mammalian species, including the chicken, teleost fish and elasmobranchs in a fashion that was specific for immunoglobulins, but was not related to defined human isotyplc markers. 4. 4. The reactions of some antisera seem to skip species known to possess homologous immunoglobulins. 5. 5. Antisera directed against isotyplc markers of human K and λ light chains reacted with shark light chains in a manner that was specific for light chain determinants but was not isotype-related. 6. 6. Antisera directed against heavy chains of either sharks or mammals reacted with heavy chains, but not with light chains of diverse species. 7. 7. A rabbit antiserum specific for shark light chain reacted with human and murine monoclonal λ chains and with two synthetic peptides corresponding to human VA Fr3 and Fr4 sequences. 8. 8. These results establish that a variety of antigenic markers including conformational and linear determinants can be shared among immunoglobulins of vertebrates species that had an ancestral divergence more than 400 million years ago.

The retinae of Prototherian mammals possess neuronal types that are characteristic of non-mammalian retinae

This study has shown that the retinae of Prototherian (egg-laying) mammals possess two neuronal types that are present in non-mammalian retinae, but absent or morphologically different in the retinae of Eutherian (placental) mammals. First, endogenous serotonin-like immunoreactivity has been localized in a population of presumptive amacrine cells in the platypus retina, the first such report in a mammalian retina. Second, the protein kinase C-immunoreactive (PKC-IR) bipolar cells in the echidna retina appear similar to the PKC-IR bipolars in the chicken retina, in that their dendrites give rise to a Landolt's club and their axons are multistratified. By contrast, the PKC-IR rod bipolar cells in the rabbit and in the brushtail possum, a Metatherian (marsupial) mammal, have no Landolt's clubs and their axons form terminal lobes in the innermost stratum of the inner plexiform layer.

Distribution of substance P in the enteric plexuses of the small intestine of the platypus, Ornithorhynchm anatinus

A study was made of substance P-like-immunoreactive nerves within the small intestine of platypus, an Australian prototherian mammal. Both immunoreactive nerve cell bodies and fibers were present. No immunoreactive fibers were found in mesenteric nerves or on intramural blood vessels, suggesting that extrinsic sensory neurons containing substance P do not innervate platypus ileum. This was further supported by the result of in vitro experiments. Although applied substance P (10(-9) M-3 x 10(-8) M) caused contraction of the longitudinal muscle, neither mesenteric nerve stimulation nor application of capsaicin caused contractions.

Sex chromosome homology and incomplete, tissue-specific X-inactivation suggest that monotremes represent an intermediate stage of mammalian sex chromosome evolution.

Female mammals have two X chromosomes and males have a single X and a smaller, male-determining Y chromosome. The dosage of X-linked gene products is equalized between the sexes by the genetic inactivation of one X chromosome in females. The characteristics of the mechanism of X-chromosome inactivation differ in eutherian and metatherian mammals, and it has been suggested that the metatherian system represents a more primitive stage. The present study of monotreme sex chromosomes and X-chromosome inactivation suggests that the prototherian mammals may represent an even more primitive stage. There is extensive G-band homology between the monotreme X and Y chromosomes, and differences in the patterns of replication of the two X chromosomes in females suggest that X inactivation is tissue specific and confined to the unpaired segment of the X. On the basis of these results, we propose a model for the differentiation of mammalian sex chromosomes and the evolution of the mechanism of X-chromosome inactivation. This model involves a gradual reduction of the Y chromosome and an accompanying gradual recruitment of (newly unpaired) X-linked loci under the control of a single inactivation center.

Determination of lysozyme activity at low levels with emphasis on the milk enzyme

A method is described for the determination of lysozyme (muramidase) activity, whereby sensitivity is maximized by incubation of the reaction mixture (sample, buffer, and substrate ( Micrococcus luteus)) over an extended period. This approach is made feasible by exploiting our observation that the lytic reaction follows simple kinetic order during this time (e.g., 700 min for bovine lysozyme and 960 min for the eggwhite enzyme at low concentrations). After this period, the reaction rates diminish, indicating biphasic behavior, and eventually become negligible. The kinetic order may vary with both the type of lysozyme and the buffer system used. The limit of detection for bovine milk lysozyme is 100 pg/ml reaction mixture, equivalent to 6 ng/ml milk, for a 50-μl sample (with reference to hen eggwhite lysozyme). With these limits, the method has proven valuable in our comparative studies, particularly for low levels of activity in bovine milk, but also in secretions and tissue extracts from various other eutherian, metatherian, and prototherian mammals. The method may also be applied to investigation of structure and function in modified forms of the enzyme.

Electrophoretic and binding behavior of steroid-binding proteins in the plasma of a prototherian mammal, Tachyglossus aculeatus.

Electrophoretic and binding behavior of steroid-binding proteins in the plasma of a prototherian mammal, Tachyglossus aculeatus.

Role of the adrenal cortex in the adaptation of the monotreme Tachyglossus aculeatus to low environmental temperature.

SUMMARY When exposed to a low ambient temperature of 5 °C, adrenalectomized echidnas were able to increase their metabolic rate and to maintain their body temperature within the normal range for no more than 48 h — less than 12 h in five out of six animals. Thereafter, activity, metabolic rate, cardiac rate and body temperature declined and the animals became torpid. When maintained with daily i.m. injections of 1–2 mg cortisol acetate/kg, adrenalectomized echidnas maintained activity and normal body temperature in the cold environment indefinitely. When cortisol injections were withheld and exposure to cold continued, normal body temperature was maintained for a further 10 days, after which it declined rapidly. The onset of torpor was always preceded by a marked fall in plasma glucose concentration, as occurred in normal, but fasted, echidnas after prolonged exposure to cold. Both cortisol and corticosterone have glucocorticoid activity in echidnas, and torpor was prevented in adrenalectomized echidnas by preventing the fall in plasma glucose with either intermittent injections or constant rate infusions of glucose solutions. The adrenal glands of normal echidnas exposed repeatedly to low environmental temperatures showed marked hypertrophy and increase in lipid content. It is concluded that adrenocortical secretions are necessary for the metabolic response to cold stress in these prototherian mammals, and a major role of the corticosteroids is in maintenance of normal blood glucose concentrations, presumably by enhancing hepatic gluconeogenesis.

The Classification of Nontherian Mammals

On the basis of a unique specialization of the braincase the four nontherian orders of mammals—the living Monotremata and the extinct Tricono-donta, Docodonta, and Multituberculata—are united into a single subclass, Prototheria. The Triconodonta and Docodonta are closely related and are grouped together in the infraclass Eotheria. The Monotremata and Multituberculata are only remotely allied to all other mammals and are each placed in a separate infraclass—Ornithodelphia for the monotremes and Allotheria for the Multituberculates. A classification of prototherian mammals to family is presented.