Uterine epithelial remodelling during pregnancy in the marsupial Monodelphis domestica (Didelphidae): Implications for mammalian placental evolution

Mammalian pregnancy involves remodelling of the uterine epithelium to enable placentation. In marsupials, such remodelling has probably played a key role in the transition from ancestral invasive placentation to non-invasive placentation. Identifying uterine alterations that are unique to marsupials with non-invasive placentation can thus elucidate mechanisms of marsupial placental evolution. We identified apical alterations to uterine epithelial cells prior to implantation in Monodelphis domestica, a member of the least derived living marsupial clade (Didelphidae) with invasive (endotheliochorial) placentation. We then compared these traits with those of Macropus eugenii (Macropodidae) and Trichosurus vulpecula (Phalangeridae), both with non-invasive placentation, to identify which alterations to the uterine epithelium are ancestral and which facilitate secondarily evolved non-invasive placentation. In M. domestica, remodelling of the uterine epithelium involves reduced cellular heterogeneity and development of uterodome-like cells, suggesting that similar alterations may also have occurred in the marsupial common ancestor. These alterations also overlap with those of both T. vulpecula and Ma. eugenii, suggesting that the placental shift from invasive to non-invasive placentation in marsupials involves essential, conserved characteristics, irrespective of placental mode. However, unique apical alterations of both T. vulpecula and Ma. eugenii, relative to M. domestica, imply that lineage-specific alterations underpin the evolutionary shift to non-invasive placentation in marsupials.     

Epithelial cadherin disassociates from the lateral plasma membrane of uterine epithelial cells throughout pregnancy in a marsupial

The uterine luminal epithelium is the first site of contact between fetal and maternal tissues during therian pregnancy and must undergo specialised changes for implantation of the blastocyst to be successful. These changes, collectively termed the plasma membrane transformation (PMT), allow the blastocyst to attach to the uterine epithelium preceding the formation of a placenta. There are similarities in the morphological and molecular changes occurring in live‐bearing eutherian species during the PMT studied so far. Similar cellular remodelling occurs in a marsupial species, the fat‐tailed dunnart (Sminthopsis crassicaudata), despite the divergence of marsupials from eutherian mammals over 130 mya, which resulted in the evolution of distinct reproductive strategies. Adhesion molecules along the lateral plasma membrane of uterine epithelium provide a barrier to invasion by the embryo. We thus characterised the presence and change in distribution of epithelial cadherin (E‐cadherin) in uterine epithelium from non‐pregnant fat‐tailed dunnarts and compared it to dunnarts in early‐, mid‐ and late‐stage pregnancy. E‐cadherin staining is localised to the lateral plasma membrane in uterine epithelium from non‐pregnant and early‐stage pregnant dunnarts. The E‐cadherin staining is cytoplasmic in epithelium from uteri of mid‐ and late‐stage pregnant dunnarts. This loss of localised staining suggests that the adherens junction dissociates from the lateral plasma membrane, allowing for invasion between the epithelial cells by the blastocyst. As the changes during pregnancy to cadherin were similar in the laboratory rat with highly invasive (haemochorial) placentation, a live‐bearing lizard species with non‐invasive (epitheliochorial) placentation and a marsupial, the fat‐tailed dunnart, which has invasive (endotheliochorial) placentation, we suggest that the molecular mechanisms allowing for successful pregnancy are conserved among mammals during the early stages of pregnancy regardless of placental invasiveness.     

Uterine morphology during diapause and early pregnancy in the tammar wallaby (Macropus eugenii)

In mammals, embryonic diapause, or suspension of embryonic development, occurs when embryos at the blastocyst stage are arrested in growth and metabolism. In the tammar wallaby (Macropus eugenii), there are two separate uteri, only one of which becomes gravid with the single conceptus at a post‐partum oestrus, so changes during pregnancy can be compared between the gravid and non‐gravid uterus within the same individual. Maintenance of the viable blastocyst and inhibition of further conceptus growth during diapause in the tammar is completely dependent on the uterine environment. Although the specific endocrine and seasonal signals are well established, much less is known about the cellular changes required to create this environment. Here we present the first detailed study of uterine morphology during diapause and early pregnancy of the tammar wallaby. We combined transmission electron microscopy and light microscopy to describe the histological and ultrastructural changes to luminal and glandular epithelial cells. At entry into diapause after the post‐partum oestrus and formation of the new conceptus, there was an increase in abundance of organelles associated with respiration in the endometrial cells of the newly gravid uterus, particularly in the endoplasmic reticulum and mitochondria, as well as an increase in secretory activity. Organelle changes and active secretion then ceased in these cells as they became quiescent and remained so for the duration of diapause. In contrast, cells of the non‐gravid, post‐partum, contralateral uterus underwent sloughing and remodelling during this time and some organelle changes in glandular epithelial cells continued throughout diapause, suggesting these cells are not completely quiescent during diapause, although no active secretion occurred. These findings demonstrate that diapause, like pregnancy, is under unilateral endocrine control in the tammar, and that preparation for and maintenance of diapause requires substantial changes to uterine endometrial cell ultrastructure and activity.     

Uterine focal adhesion dynamics during pregnancy in a marsupial (Sminthopsis crassicaudata; Dasyuridae)

Alterations to the basal attachment points between the epithelium of the uterus and the underlying tissue in early pregnancy affect how easily the epithelium can be invaded by the implanting embryo. Attachment points‐ focal adhesions‐ disassemble to facilitate highly invasive implantation in rats, but species with less invasive implantation, including marsupials, may require different basal alterations for successful pregnancy. Here we used immunofluorescence microscopy and Western blotting to conduct the first study of basal plasma membrane dynamics in the uterus during marsupial pregnancy. We describe localisation patterns of two key anchoring molecules, talin and paxillin, throughout pregnancy in the fat‐tailed dunnart (Sminthopsis crassicaudata; Dasyuridae). Basal staining of both molecules occurs in early pregnancy, as it does in the rat. However, unlike rats, there is strong basal localisation of talin and paxillin just before implantation in S. crassicaudata, indicating that focal adhesions do not disassemble during pregnancy in this species, and that molecular reinforcement of the epithelium may be a maternal strategy to regulate invasion. Additionally, talin and paxillin do not co‐localise at all stages of pregnancy as they do in the rat. Different localisation patterns among mammalian species demonstrate that not all early pregnancy changes are ubiquitous in mammalian pregnancy, as changes to the basal plasma membrane of the epithelium, in particular, may instead be dependent on mode of implantation. Anat Rec, 300:1150–1159, 2017. © 2016 Wiley Periodicals, Inc.     

Posterior pituitary of the newborn marsupial possum,Trichosurus vulpecula

The fetal anterior pituitary-adrenal axis is thought to be involved in the initiation of birth in both eutherian and marsupial mammals. Little is known about the structure and function of the posterior pituitary at birth in the marsupial. Immunocytochemistry, high pressure liquid chromatography, and radioimmunoassay were used to identify vasopressin and mesotocin in the posterior pituitary of a newborn marsupial, the brushtail possum, Trichosurus vulpecula. The concentrations of vasopressin and mesotocin in the head of the newborn possum were 0.34 and 0.28 ng, respectively. The concentration of vasopressin was always greater than that of mesotocin, and the amounts of neuropeptides present in the head increased as the possum developed. © 1993 Wiley-Liss Inc.     

Lung Development of Monotremes: Evidence for the Mammalian Morphotype

The reproductive strategies and the extent of development of neonates differ markedly between the three extant mammalian groups: the Monotremata, Marsupialia, and Eutheria. Monotremes and marsupials produce highly altricial offspring whereas the neonates of eutherian mammals range from altricial to precocial. The ability of the newborn mammal to leave the environment in which it developed depends highly on the degree of maturation of the cardio‐respiratory system at the time of birth. The lung structure is thus a reflection of the metabolic capacity of neonates. The lung development in monotremes (Ornithorhynchus anatinus, Tachyglossus aculeatus), in one marsupial (Monodelphis domestica), and one altricial eutherian (Suncus murinus) species was examined. The results and additional data from the literature were integrated into a morphotype reconstruction of the lung structure of the mammalian neonate. The lung parenchyma of monotremes and marsupials was at the early terminal air sac stage at birth, with large terminal air sacs. The lung developed slowly. In contrast, altricial eutherian neonates had more advanced lungs at the late terminal air sac stage and postnatally, lung maturation proceeded rapidly. The mammalian lung is highly conserved in many respects between monotreme, marsupial, and eutherian species and the structural differences in the neonatal lungs can be explained mainly by different developmental rates. The lung structure of newborn marsupials and monotremes thus resembles the ancestral condition of the mammalian lung at birth, whereas the eutherian newborns have a more mature lung structure. Anat Rec, 2009. © 2008 Wiley‐Liss, Inc.     

Embryonic-maternal cell interactions at implantation in the fat-tailed dunnart,a dasyurid marsupial

Background: In marsupials implantation occurs about two-thirds the way through the short gestation before which time the embryo is surrounded by the permeable shell membrane which prevents physical contact between the trophoblast and uterine epithelium. Although the trophoblast has been shown to be invasive to varying degrees in several species of marsupials, the ultrastructure of the embryonic-uterine cell interactions at the time of implantation has not been described in this group. Methods: Thick plastic sections and transmission electron microscopy were employed to investigate the cellular interactions at implantation in the fat-tailed dunnart (Sminthopsis crassicaudata), a dasyurid Australian marsupial. Results: Our results show that epithelial penetration begins when the embryo is at the late presomite/early somite stage. In the trilaminar region of the yolk sac (TYS), trophoblast cells adjacent to the embryo form desmosomes with uterine epithelial cells and also appear to fuse with them to form hybrid cells, the cytoplasm of which resembles that of trophoblast. Later in the TYS, as the placenta develops, trophoblast microvilli and larger cell processes invaginate, and interdigitate with, the highly folded maternal epithelium but do not invade it. At this time in the bilaminar, or avascular, yolk sac (BYS), multinucleate trophoblast giant cells (TGCs) from an annular region adjacent to the sinus terminalis intrude between, and possibly fuse with, the maternal epithelium. The invading TGCs spread laterally above the residual basal lamina before migrating into the stroma. Conclusions: In this species of marsupial at least, the cell interactions at the time of implantation are similar to those seen in some eutherian species despite the fact that the fetal chorion is of yolk sac rather than allantoic origin. © 1994 Wiley-Liss, Inc.     

Invasive Cells in the Placentome of Andean Populations of Mabuya: An Endotheliochorial Contribution to the Placenta?

New world lizards of the genus Mabuya have the most specialized level of placentotrophy among reptiles known to date, and related to that, they have the most complex allantoplacenta characterized by a series of morphological specializations that converge with those known for eutherian mammals. One of these specializations is the placentome that is found in the embryonic pole of the incubation chamber. In the mature allantoplacenta, this structure is morphologically the most complex, which could support an important amount of nutrient exchange between mother and fetus. According to the relationship between the chorioallantois and the syncytial uterine epithelia, the placenta of Mabuya populations shows some interesting similarities to the synepitheliochorial type. Recently, cells of chorionic origin have been found invading the syncytial uterine epithelium, and in very close proximity with uterine blood vessels. In this study, we describe the relationship between these invasive chorionic cells, the uterine syncytium, and the subjacent blood vessels of several populations of this genus, by means of high resolution optical microscopy and transmission electron microscopy. Cell groups originating from the chorion, of variable size and shape, penetrate the uterine syncytial epithelium extending complex cytoplasmic projections that come in contact with uterine capillaries and form an extensive and complex double‐membrane system that surrounds the capillary. The close relationship between the chorion and the maternal circulation suggests that the Mabuya placentome shows some characteristics of an endotheliochorial placenta. This finding constitutes so far the only documented example of an endotheliochorial placentation in Reptilia. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

The search for a marsupial XIC reveals a break with vertebrate synteny

X-chromosome inactivation (XCI) evolved in mammals to deal with X-chromosome dosage imbalance between the XX female and the XY male. In eutherian mammals, random XCI of the soma requires a master regulatory locus known as the ‘X-inactivation center’ (XIC/Xic), wherein lies the noncoding XIST/Xist silencer RNA and its regulatory antisense Tsix gene. By contrast, marsupial XCI is imprinted to occur on the paternal X chromosome. To determine whether marsupials and eutherians share the XIC-driven mechanism, we search for the sequence equivalents in the genome of the South American opossum, Monodelphis domestica. Positional cloning and bioinformatic analysis reveal several interesting findings. First, protein-coding genes that flank the eutherian XIC are well-conserved in M. domestica, as well as in chicken, frog, and pufferfish. However, in M. domestica we fail to identify any recognizable XIST or TSIX equivalents. Moreover, cytogenetic mapping shows a surprising break in synteny with eutherian mammals and other vertebrates. Therefore, during the evolution of the marsupial X chromosome, one or more rearrangements broke up an otherwise evolutionarily conserved block of vertebrate genes. The failure to find XIST/TSIX in M. domestica may suggest that the ancestral XIC is too divergent to allow for detection by current methods. Alternatively, the XIC may have arisen relatively late in mammalian evolution, possibly in eutherians with the emergence of random XCI. The latter argues that marsupial XCI does not require XIST and opens the search for alternative mechanisms of dosage compensation.     

The Unique Paired Retinal Vessels of the Gray Short‐Tailed Opossum (Monodelphis domestica) and Their Relationship to Astrocytes and Microglial Cells

In marsupials that possess a retinal vasculature, the arterial and venous segments, down to the smallest calibre capillaries, have been shown to occur in pairs. This pattern is seen in the marsupial central nervous system (CNS) but not in other tissues in this group or in any tissues in eutherian mammals. The present study aimed to determine if the gray short‐tailed opossum (Monodelphis domestica), a south American marsupial, possesses double retinal vessels. Secondly, we investigated the relationship between vessels and astrocytes and microglia, which are known to play pivotal roles in the blood retinal barrier and immune surveillance respectively. Eyes from M. domestica between 2 months and 33 months of age were examined by bright field and fluorescein angiography, resin histology, and wholemount immunostaining. Retinal vessels in this marsupial always occur in closely related pairs with the arteriolar limb usually on the vitread aspect. Branches penetrate the retina to form layers of paired capillaries as far as the outer nuclear layer. Dense networks of GFAP⁺ astrocytes enveloped the vitread aspect of vessels. No particularly strong association with blood vessels and ramified Iba1⁺ and Ib4⁺ microglia was noted. M. domestica possessed the unusual paired vasculature and capillary loops arrangement previously described in the marsupial CNS. These observations in a small laboratory‐friendly marsupial open up new frontiers to investigate the factors that regulate paired blood vessel development and the functional significance of this arrangement when compared to the anastomotic pattern observed in the retina of eutherian mammals. Anat Rec, 300:1391–1400, 2017. © 2017 Wiley Periodicals, Inc.     

Lack of Evidence for Complement-Dependent Cytotoxic Antibodies to Fetal Paternally Derived Antigens in the Marsupial Macropus eugenii (Tammar Wallaby)

ABSTRACT: A total of 241 serum samples from 145 parous tammar wallabies (Macropus eugenii) were screened for presence of antibodies to paternally derived antigens of the fetus. These samples were taken at different stages in late pregnancy after placental contact was intimate and after birth. Complement-dependent cytotoxicity tests were unable to detect any specific antibodies. It is concluded that the yolk sac placenta of M. eugenii does not allow intimate enough contact between fetal tissues and the maternal circulation to induce formation of cytotoxic antibodies by its mother. This is in contrast to eutherian mammals, in which such production of cytotoxic antibodies occurs frequently as a result of pregnancy. Together with other data it is suggested that the short implantation period in M. eugenii, which is common to all marsupials, has probably not evolved to prevent maternal immune attack upon the conceptus.     

Isolation and comparison of the IgM heavy chain constant regions from Australian (Trichosurus vulpecula) and American (Monodelphis domestica) marsupials

cDNAs encoding IgM heavy chain constant region (Cμ) were isolated from two metatherians (marsupials) — the Australian common brushtail possum (Trichosurus vulpecula) and the South American grey short-tailed opossum (Monodelphis domestica). Analysis of the sequences suggested that they correspond to the secreted form of Cμ in both species. The domain size and structure of the marsupial Cμ sequences were compared with other Cμ sequences and a high degree of conservation throughout vertebrate evolution was observed. Amino acid sequence comparisons revealed a marked level of sequence similarity between the two marsupial sequences (79%), relatively high similarity between the marsupials and eutherians (63%), and lower similarities between marsupials and birds (45%), marsupials and amphibians (47%), marsupials and reptiles (45%) and marsupials and fish (37%). These data allow the incorporation of metatherians into the study of mammalian IgM evolution.     

Ultrastructure of the placenta of the tammar wallaby,Macropus eugenii: comparison with the grey short-tailed opossum,Monodelphis domestica

The ultrastructure of the tammar placenta was studied throughout pregnancy. The uterine epithelium grows from a columnar to an enlarged, undulating epithelium between early gestation and mid-gestation when the shell coat that surrounds the marsupial conceptus ruptures. Trophectoderm and uterine epithelium do not form syncytia, nor does invasion of the endometrium occur at any stage of pregnancy. Uterine secretion is provided to both the bilaminar and the trilaminar side of the yolk sac placenta up to birth. Fenestrations, abundant vesicles and lumenal processes of maternal capillaries, as well as deep basal folds of the uterine epithelium, suggest that there is transfer of hemotrophes adjacent to both parts of the yolk sac. In contrast, in the grey short-tailed opossum, these structures are lacking. The yolk sacs of adjacent embryos fuse to form a common yolk sac cavity, thus losing most of the bilaminar yolk sac. The bilaminar and trilaminar components of the yolk sac placenta of the tammar are less different in structure and function than those of the grey short-tailed opossum, but both types are fully functional placentas. The extended secretory phase of the tammar uterus and the maternal recognition of early pregnancy appear to be derived characters of macropodid marsupials.     

The testis and its excurrent ducts in American caenolestid and didelphid marsupials

The present study examines and compares the structure of the testis and its excurrent ducts in a caenolestid and four didelphid marsupials. Of particular interest was the site of sperm pairing in the epididymis and whether this feature, shared by both American marsupial families but not by any Australian marsupial, was associated with changes in the morphology of the duct. In contrast to the testes of most Australian marsupials, except the peramelids (bandicoots), the intertubular space in the American marsupials was filled by Leydig cells (around 20% of testis volume). The opossum testes were unusual compared with those of eutherian mammals in that histological sections of individual seminiferous tubules contained only a single cellular association irrespective of the length of the tubule sectioned. The rete testis, as in the Australian dasyurids (devil, quoll, etc.), was a simple branching duct system that arose deep within the testis and emerged as a single duct at the testicular hilus. This arrangement is completely different from that in kangaroos and Australian possums, indicating a diversity of rete form in the marsupials similar to that seen in eutherian mammals. The rete emptied into a single, essentially straight, efferent duct that became convoluted towards the epididymis, where it formed a distinct structure adjacent to the caput epididymidis. The efferent ducts were highly variable in diameter and epithelial height, suggesting that the duct was not of uniform character along its length, or that the initial single duct had divided to form ducts of different characters. Sperm pairs were first seen in the proximal cauda epididymidis, and their appearance was correlated with changes in the character of the duct and its epithelium. The distal ductus deferens of Caenolestes, in contrast to those of the didelphids and indeed all other marsupials, was a convoluted ampulla-like structure adjacent to the prostate gland. In the other marsupials the only accessory sex glands are a segmented prostate and bulbourethral glands.     

The role of oxytocin and regulation of uterine oxytocin receptors in pregnant marsupials

The oxytocin-like peptide of most Australian marsupials is mesotocin, which differs from oxytocin by a single amino acid. This substitution has no functional significance as both peptides have equivalent affinity for and biological activity on the marsupial oxytocin-like receptor. A role for mesotocin in marsupial parturition has been demonstrated in the tammar wallaby where plasma mesotocin concentrations increase less than one minute before birth. Infusion of an oxytocin receptor antagonist at the end of gestation disrupts normal parturition, probably by preventing mesotocin from stimulating uterine contractions. In the absence of mesotocin receptor activation, a peripartum surge in prostaglandins is delayed which suggests a functional relationship between mesotocin, prostaglandin release and luteolysis. Female marsupials have anatomically separate uteri and in monovular species, such as the tammar wallaby, only one uterus is gravid with a single fetus whereas the contralateral uterus remains non-gravid. We have used this unique animal model to differentiate systemic and fetal-specific factors in the regulation of uterine function during pregnancy. The gravid uterus in the tammar wallaby becomes increasingly sensitive to mesotocin as gestation proceeds, with the maximum contractile response observed at term. This is reflected in a large increase in mesotocin receptor concentrations in the gravid uterus, and a downregulation in the non-gravid uterus in late pregnancy. The upregulation in myometrial mesotocin receptors is pregnancy-specific and independent of systemic steroids. One factor that may influence mesotocin receptor upregulation in the gravid uterus in late pregnancy is mechanical stretch of the uterus caused by the growing fetus. Our data highlight that a local fetal influence is more important than systemic factors in the regulation of mesotocin receptors in the tammar wallaby.     

Evidence for an early appearance of modern post-switch isotypes in mammalian evolution; cloning of IgE,IgG and IgA from the marsupial Monodelphis domestica

In birds, reptiles and amphibians the IgY isotype exhibits the functional characteristics of both of IgG and IgE. Hence, the gene for IgY most likely duplicated some time during early mammalian evolution and formed the ancestor of present day IgG and IgE. To address the question of when IgY duplicated and formed two functionally distinct isotypes, and to study when IgG and IgA lost their second constant domains, we have examined the Ig expression in a non-placental mammal, the marsupial Monodelphis domestica (grey short-tailed opossum). Screening of an opossum spleen cDNA library revealed the presence of all three isotypes in marsupials. cDNA clones encoding the entire constant regions of opossum IgE (ϵ chain), IgG (γ chain) and IgA (α chain) were isolated, and their nucleotide sequences were determined. A comparative analysis of the amino acid sequences for IgY, IgA, IgE and IgG from various animal species showed that opossum IgE, IgG and IgA on the phylogenetic tree form branches clearly separated from their eutherian counterparts. However, they still conform to the general structure found in eutherian IgE, IgG and IgA. Our findings indicate that all the major evolutionary changes in the Ig isotype repertoire, and in basic Ig structure that have occurred since the evolutionary separation of mammals from the early reptile lineages, occurred prior to the evolutionary separation of marsupials and placental mammals.     

Physical mapping of the elephant X chromosome: conservation of gene order over 105 million years

All therian mammals (eutherians and marsupials) have an XX female/XY male sex chromosome system or some variant of it. The X and Y evolved from a homologous pair of autosomes over the 166 million years since therian mammals diverged from monotremes. Comparing the sex chromosomes of eutherians and marsupials defined an ancient X conserved region that is shared between species of these mammalian clades. However, the eutherian X (and the Y) was augmented by a recent addition (XAR) that is autosomal in marsupials. XAR is part of the X in primates, rodents, and artiodactyls (which belong to the eutherian clade Boreoeutheria), but it is uncertain whether XAR is part of the X chromosome in more distantly related eutherian mammals. Here we report on the gene content and order on the X of the elephant (Loxodonta africana)—a representative of Afrotheria, a basal endemic clade of African mammals—and compare these findings to those of other documented eutherian species. A total of 17 genes were mapped to the elephant X chromosome. Our results support the hypothesis that the eutherian X and Y chromosomes were augmented by the addition of autosomal material prior to eutherian radiation. Not only does the elephant X bear the same suite of genes as other eutherian X chromosomes, but gene order appears to have been maintained across 105 million years of evolution, perhaps reflecting strong constraints posed by the eutherian X inactivation system.