The ultrastructure of the pituitary and the adrenal gland of three newborn marsupials (Dasyurus hallucatus, Trichosurus vulpecula and Isoodon macrourus)

Summary The ultrastructure of the pituitary glands of the newborn northern native cat, brushtail possum and the northern brown bandicoot and the adrenal glands of the former two marsupials were examined to determine whether these endocrine glands were functional at birth.The anterior pituitary of all three species was well vascularised and many cells contained electron-dense, membrane bound granules. The adrenal glands were composed of two distinct cell populations. One group of cells possessed dark staining granules, normally observed in catecholamine-secreting cells, and the second group contained large amounts of smooth endoplasmic reticulum and mitochondria with tubulovesicular cristae, indicative of steroid hormone secreting cells.The ultrastructure of the cells of the fetal pituitary and adrenal of all three species would suggest that these glands are functional at birth and, as with many eutherians, the marsupial pituitary and adrenal gland may play an important role in determining the length of gestation.     

Ultrastructural identification of Merkel cells around the mouth of the newborn marsupial

Summary The ultrastructure of the epidermal cells surrounding the mouth of three newborn marsupial species, the Northern native cat Dasyurus hallucatus, the brush tail possum Trichosurus vulpecula and the Northern brown bandicoot Isoodon macrourus were examined. The presence of Merkel cells, highly sensitive touch receptors, would suggest that the sense of touch aids the relatively underdeveloped newborn marsupial to move from the urinogenital sinus to the pouch and to locate the teat.     

The embryological development and cytodifferentiation of the anterior pituitary in the marsupial, Isoodon macrourus

Summary Light and electron microscopy were used to study the development of the anterior pituitary gland from fetal stages to the end of pouch life in the marsupial I. macrourus.The early morphological development of the anterior pituitary in I. macrourus follows a similar pattern of events to that described for cutherians. Rathke's and Seesel's pouches were present in 101/2 day old embryos. At birth these pouches had formed a multi-chambered vesicle which was still connected to the stomodeum by a thin cord of tissue. A small number of granules (200–400 nm dia.) were found in cells at birth. These cells could not be classified on ultrastructural features but alcian blue-periodic acid Schiffs-orange G staining suggested one cell type was possibly a presumptive thyrotroph. There were no capillaries in the pars distalis at birth.The cords connecting Rathke's and Seessel's pouches to the stomodeum were located at the site of the periosteal bud of the developing basisphenoid which commenced to ossify at 7 days. At this stage presumptive thyrotrophs, gonadotrophs, and somatotrophs could be distinguished using alcian blue-periodic acid Schiffs-orange G staining. However, five cell types could be categorised at the same age using ultrastructural characteristics alone. Precise names for these cells are unavailable but two closely resemble presumptive mammotrophs and thyrotrophs described for another marsupial M. eugenii.By 13 days after birth the anterior pituitary of I. macrourus had become vascular and acidophils were concentrated in a posterior zone. There was little gross morphological change from 13 to 66 days after birth by which the time weaning has occurred.Cilia were seen in cells of the anterior pituitary and mitosis of granulated cells was observed from birth onwards.There is a considerable range of variation in pituitary cytogenesis amongst marsupials, and its functional significance awaits further investigation.     

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.     

Ultrastructure of the olfactory system of three newborn marsupial species

The ultrastructure of the olfactory apparatus of three newborn marsupial species, the northern native cat, the brushtail possum, and the northern brown bandicoot, were examined. The olfactory epithelium and olfactory bulbs were at a similar stage of development in all three species. Receptor cells with cilia were observed, and although the olfactory system undergoes further differentiation during pouch life and although the olfactory epithelium and bulb of the newborn differs from that of the adult, these facts do not preclude the ability of the newborn to detect smell. The presence of touch receptors around the mouth region and of sensory cells within the olfactory epithelium would suggest that touch and smell are two of the senses allowing the newborn marsupial to reach the pouch.     

Molecular identification of interleukin-2 in the lymphoid tissues of the common brushtail possum, Trichosurus vulpecula

The common brushtail possum (Trichosurus vulpecula) is an Australian marsupial. Here we describe the identification of possum interleukin-2 in mitogen-stimulated lymph node cells. We used a strategy of Rapid amplification of cDNA ends using probes designed from recently-sequenced marsupial genomes to identify the IL2 gene and then confirmed that IL-2 expression in possum immune tissue occurs in a similar manner to that in their eutherian counterparts. The predictive possum IL-2 peptide showed 28% and 35% amino acid sequence homology with the mouse and human IL-2 molecules, respectively, consistent with the divergence found within this cytokine family. Despite this low sequence identity, possum IL-2 still possessed the characteristic hallmarks of mammalian IL-2, such as a predicted signal peptide and conserved family motifs.     

Skin structure in newborn marsupials with focus on cutaneous gas exchange

A morphological and morphometric study of the skin of a variety of newborn marsupials (Dasyurus viverrinus, Monodelphis domestica, Trichosurus vulpecula, Isoodon obesulus, Perameles nasuta, Phascolarctos cinereus, Potorous tridactylus, Petrogale penicillata, Thylogale thetidi, Macropus dorsalis) and of a monotreme hatchling (Ornithorhynchus anatinus) was undertaken to assess the possibility of cutaneous gas exchange. Additionally, the lungs of some of these species were investigated to assess its structural degree at birth. The skin in the different newborn marsupials and the monotreme hatchling had a similar structure (no hair follicles and no sebaceous or perspiratory glands) and was in all cases less developed than the skin of altricial eutherians. The thickness of the entire skin (36–186 μm) and its different layers, epidermis (6–29 μm) and dermis (29–171 μm) varied among the marsupial species and reflected the differences in size and developmental degree of the neonates. In the skin of all marsupial neonates and the monotreme hatchling, numerous superficial cutaneous capillaries were encountered, some closely associated with the epidermis, indicating the possibility that the skin participated in gaseous exchange. The skin of the newborn D. viverrinus had the highest capillary volume density and shortest skin diffusion barrier of all marsupial neonates, suggesting that skin gas exchange in the dasyurid neonate might be the most pronounced. A graduation of the skin capillary density among the marsupial neonates inversely followed the respective lung structure and general developmental degree of the neonates.     

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.     

Passively Acquired Immunity in the Newborn of a Marsupial (Monodelphis domestica)

ABSTRACT: A colony of fully pedigreed Monodelphis domestica has been used to investigate the maternal-fetal relationship in this unique marsupial species. To determine how immunity is transferred from mothers to young in M. domestica, we hyperimmunized females with sheep red blood cells (SRBC) before and during gestation. Offspring from these females were collected at various times after birth, and saline extracts of the neonates were assayed for hemolysins against SRBC. Antibodies were present in extracts of newborn that had been allowed to suckle their mothers; none were detected in extracts of infants that were not allowed to suckle. Antibodies were present in the milk of immunized mothers, but were not detected in the milk of nonimmunized mothers. The titer of antibodies in the extracts of newborns generally increased proportionately to the time that the newborn had been allowed to suckle. We conclude that the transfer of passive immunity from mothers of M. domestica to their offspring occurs primarily via the milk.     

Uterine remodelling during pregnancy and pseudopregnancy in the brushtail possum (Trichosurus vulpecula; Phalangeridae)

The formation of a placenta is critical for successful mammalian pregnancy and requires remodelling of the uterine epithelium. In eutherian mammals, remodelling involves specific morphological changes that often correlate with the mode of embryonic attachment. Given the differences between marsupial and eutherian placentae, formation of a marsupial placenta may involve patterns of uterine remodelling that are different from those in eutherians. Here we present a detailed morphological study of the uterus of the brushtail possum (Trichosurus vulpecula; Phalangeridae) throughout pregnancy, using both scanning and transmission electron microscopy, to identify whether uterine changes in marsupials correlate with mode of embryonic attachment as they do in eutherian mammals. The uterine remodelling of T. vulpecula is similar to that of eutherian mammals with the same mode of embryonic attachment (non‐invasive, epitheliochorial placentation). The morphological similarities include development of large apical projections, and a decrease in the diffusion distance for haemotrophes around the period of embryonic attachment. Importantly, remodelling of the uterus in T. vulpecula during pregnancy differs from that of a marsupial species with non‐invasive attachment (Macropus eugenii; Macropodidae) but is similar to that of a marsupial with invasive attachment (Monodelphis domestica; Didelphidae). We conclude that modes of embryonic attachment may not be typified by a particular suite of uterine changes in marsupials, as is the case for eutherian mammals, and that uterine remodelling may instead reflect phylogenetic relationships between marsupial lineages.     

Why are human newborns so fat? Relationship between fatness and brain size at birth [retracted article]

The plumpness of the human newborn has long been recognized as a trait in need of explanation among researchers. Using a linear regression analysis, we find that head circumference is significantly and positively associated with BMI at birth, after gestational age and birthlength were controlled for, in a sample of 1,069 healthy liveborn routinely delivered at the University Hospital of Coimbra (partial correlation r = 0.409, P < 0.0001). This significant association is consistent with the idea that newborn fatness is related to the higher need of lipids in newborn humans as an energetic and plastic substrate during its accelerated brain growth period. As birthweight and birth head size are associated with head size and cognitive abilities in childhood and adult life, it could be postulated that these cognitive abilities could have acted as selective pressure responsible for the newborn fatness increase in our lineage. Am. J. Hum. Biol. 16:24–30, 2004. © 2003 Wiley‐Liss, Inc.     

Activity-dependent secretion of neuronal activity regulated pentraxin from vasopressin neurons into the systemic circulation

Neuronal activity regulated pentraxin (Narp) is a secreted, synaptic protein that has been implicated in modulating synaptic transmission. However, it is unclear how Narp secretion is regulated. Since we noted prominent Narp immunostaining in vasopressin neurons of the hypothalamus and in the posterior pituitary, we assessed whether it, like vasopressin, is released into the systemic circulation in an activity-dependent fashion. Consistent with this hypothesis, electron microscopic studies of the posterior pituitary demonstrated that Narp is located in secretory vesicles containing vasopressin. Using affinity chromatography, we detected Narp in plasma and found that these levels are markedly decreased by hypophysectomy. In addition, we confirmed that injection of a viral Narp construct into the hypothalamus restores plasma Narp levels in Narp knockout mice. In checking for activity-dependent secretion of Narp from the posterior pituitary, we found that several stimuli known to trigger vasopressin release, i.e. hypovolemia, dehydration and endotoxin, elevate plasma Narp levels. Taken together, these findings provide compelling evidence that Narp is secreted from vasopressin neurons in an activity-dependent fashion.     

Morphology and evolution of the oral shield in marsupial neonates including the newborn monito del monte (Dromiciops gliroides,Marsupialia Microbiotheria) pouch young

Newborn marsupials can be arranged into three grades of developmental complexity based on their external form, as well as based on their organ systems and their cytology. The dasyurids are considered the least developed marsupials at birth, while didelphids and peramelids are intermediate, and macropods are the most developed. Currently there is still little information on caenolestid and microbiotherid development at birth. Developmental stages can be graded as G1, G2 and G3, with G1 being the least developed at birth, and G3 the most developed. Marsupials are also characterized by having an extremely developed craniofacial region at birth compared with placentals. However, the facial region is also observed to vary in development between different marsupial groups at birth. The oral shield is a morphological structure observed in the oral region of the head during late embryological development, which will diminish shortly after birth. Morphological variation of the oral shield is observed and can be arranged by developmental complexity from greatly developed, reduced to vestigial. In its most developed state, the lips are fused, forming together with the rhinarium, a flattened ring around the buccal opening. In this study, we examine the external oral shield morphology in different species of newborn marsupials (dasyurids, peramelids, macropods and didelphids), including the newborn monito del monte young (Dromiciops gliroides – the sole survivor of the order Microbiotheria). The adaptive value of the oral shield structure is reviewed, and we discuss if this structure may be influenced by developmental stage of newborn, pouch cover, species relatedness, or other reproductive features. We observe that the oral shield structure is present in most species of Marsupialia and appears to be exclusively present in this infraclass. It has never been described in Monotremata or Eutherians. It is present in unrelated taxa (e.g. didelphids, dasyurids and microbiotherids). We observe that a well‐developed oral shield may be related to ultra altricial development at birth, large litter size (more than two), and is present in most species that lack a pouch in reproductive adult females or have a less prominent or less developed pouch with some exceptions. We try to explore the evolution of the oral shield structure using existing databases and our own observations to reconstruct likely ancestral character states that can then be used to estimate the evolutionary origin of this structure and if it was present in early mammals. We find that a simple to develop oral shield structure (type 2–3) may have been present in marsupial ancestors as well as in early therians, even though this structure is not present in the extant monotremes. This in turn may suggest that early marsupials may have had a very simple pouch or lacked a pouch as seen in some living marsupials, such as some dasyurids, didelphids and caenolestids. The study's results also suggest that different morphological stages of the oral shield and hindlimb development may be influenced by species size and reproductive strategy, and possibly by yet unknown species‐specific adaptations.     

Oxytocin/vasopressin-like immunoreactivity is present in the nervous system of hydra

Nerve cells have been found in hydra, which react with antisera to oxytocin, vasopressin and mesotocin. These nerve cells have a high density in the ectoderm of basal disk and tentacles and lower density in the ectoderm of peduncle, gastric region and hypostome. A very small number of nerve cells occur also in the endoderm of foot, gastric region and hypostome. By using a technique for simultaneous visualisation of nerve cells reacting with antisera to oxytocin and vasopressin, it can be shown that these nerve cells belong to a single population. In agreement with this, the staining of the nerve cells can be abolished by absorbing each antiserum with either oxytocin, vasopressin, [Lys⁸]vasopressin, vasotocin, mesotocin or isotocin, indicating that the antigenic determinant of hydra cross-reacts with those antibody subpopulations, which recognize common portions (sequence 1–2, 5–7, 9) of the oxytocin/vasopressin-like peptides. With radioimmunoassays that are specific for either oxytocin or vasopressin, only very low amounts of immunoreactivity were measured. In addition, the dilution curves in these assays were not parallel to the standards, indicating that the antigenic determinant of hydra is not oxytocin or vasopressin.The presence of oxytocin/vasopressin-like material in coelenterates, shows that this family of peptides is of great antiquity.     

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.     

Pathology of the neurohypophysis

Neurohypophysis is composed of nerve fibers and has an intimate morphologic and functional contact with the hypothalamus. Pathology of the posterior lobe of the pituitary gland may develop several clinical features such as diabetes insipidus, which is caused by infections, vascular events, surgical complications and benign and malignant tumors as well as metastasis. Inappropriate secretion of vasopressin (Schwartzbartter syndrome) is due to excessive vasopressin release from the posterior pituitary or the ectopic production by various tumors, mainly carcinoid of the lung.     

Postnatal lung and metabolic development in two marsupial and four eutherian species

Two marsupial species (Monodelphis domestica, Macropus eugenii) and four eutherian species (Mesocricetus auratus, Suncus murinus, Tupaia belangeri and Cavia aperea) were examined to compare and contrast the timing of lung and metabolic development during the postnatal maturation of the mammalian respiratory apparatus. Using light, scanning and transmission electron microscopy, the lung structural changes were correlated with indirect calorimetry to track the metabolic development. Marsupial and eutherian species followed the same pattern of mammalian lung development, but differed in the developmental pace. In the two newborn marsupial species, the lung parenchyma was at the early terminal sac stage, with large terminal air sacs, and the lung developed slowly. In contrast, the newborn eutherian species had more advanced lungs at the late terminal sac stage in altricial species (M. auratus, S. murinus) and at the alveolar stage in precocial species (T. belangeri, C. aperea). Postnatal lung development proceeded rapidly in eutherian species. The marsupial species had a low metabolic rate at birth and achieved adult metabolism late in postnatal development. In contrast, newborn eutherian species had high metabolic rates and reached adult metabolism during the first week of life. The time course of the metabolic development is thus tightly linked to the structural differentiation of the lungs and the timing of postnatal lung development. These differences in the neonatal lung structure and the timing of postnatal lung maturation between marsupial and eutherian species reflect their differing reproductive strategies.     

The Divergent Development of the Apical Ectodermal Ridge in the Marsupial Monodelphis domestica

Marsupials give birth after short gestation times to neonates that have an intriguing combination of precocial and altricial features, based on their functional necessity after birth. Perhaps most noticeably, marsupial newborns have highly developed forelimbs, which provide the propulsion necessary for the newborn's crawl to the teat. To achieve their advanced state at birth, the development of marsupial forelimbs is accelerated. The development of the newborn's hind limb, which plays no part in the crawl, is not accelerated, and is likely even delayed. Given the large differences in the rate of limb outgrowth among marsupials and placentals, we hypothesize that the pathways underlying the early development and outgrowth of marsupial limbs, especially that of their forelimbs, will also be divergent. As a first step toward testing this, we examine the development of one of the two major signaling centers of the developing limb, the apical ectodermal ridge (AER), in a marsupial, Monodelphis domestica. We found that, while both opossum limbs have reduced physical AER's, in the opossum forelimb this reduction has been taken to the extreme. Where the M. domestica forelimb should have an AER, it instead has only a few patches of disorganized cells. These results make the marsupial, M. domestica, the only known amniote (without reduced limbs) to exhibit no morphological AER. However, both M. domestica limbs normally express Fgf8, a molecular marker of the AER. Anat Rec 293:1325–1332, 2010. © 2010 Wiley‐Liss, Inc.     

The influence of age on the hypothalamo-neurohypophyseal system of the mouse: a quantitative ultrastructural analysis of the posterior pituitary

A quantitative ultrastructural study of the posterior pituitary was undertaken. The posterior pituitary is the storage site of the hormones vasopressin and oxytocin. Female C57BL/Icrfa^t mice which were under conditions of normal hydration were sampled at 8 and 32 months of age. There was a slight age-related decline in the volume fraction of cellular components with a corresponding increase in perivascular space. The proportion of neurosecretory endings and swellings showing autophagic activity (Dellman type II Herring bodies) increased significantly in the senescent mouse. The total pituicyte population showed a significant decrease in volume fraction, but with a significant increase in electron-dense types. Of the subcellular parameters examined, only the diameter of the neurosecretory granules showed a significant age-related decrease, the volume fraction remaining unaltered. The animals used in this study were from stocks found to be in a constant oestrus-metoestrus-like state unlikely to obscure changes in pituitary morphology. Over-all, the morphology of the posterior pituitary of the senescent female mouse under physiologically defined resting conditions indicates maintenance of the status quo.