Red cell metabolism in a small dasyurid marsupial, the brown antechinus (Antechinus stuartii)

Red cell metabolism was examined in a small dasyurid marsupial, the brown antechinus Antechinus stuartii. There were significant differences in haematocrit with regard to sex and the season. Compared to other marsupials, the haemoglobin of A. stuartii was more susceptible to oxidation by acetylphenylhydrazine. However, the rate of methaemoglobin reduction was faster in A. stuartii. Our results appear to support a previous suggestion that methaemoglobin reducing ability is greater in those species whose haemoglobin is more susceptible to oxidative stress.     

The formation of the allantoic placenta in the golden hamster (Mesocricetus auratus)

Summary The processes of attachment, implantation and placentation in the golden hamster (Mesocricetus auratus) have been re-examined. Blastocyst attachment occurs at 4.0 days P.C. and complete interstitial implantation is attained by 5.5 days P.C. At 6.15 days P.C. the trophoblast of the träger breaks into the uterine lumen which becomes filled with blood clot. The advancing träger uses this blood clot as a bridge to reach the decidua basalis, lying on the opposite side of the uterine cavity, at 8.5 days P.C. Thus the uterine lumen becomes obliterated opposite each conceptus by the formation of the allantoic placentas which are vascularised by 8.5 days P.C. Non-glandular extensions of the glandular interlocular lumina penetrate between the decidua capsularis and the muscularis of the antimesometrial wall of the uterus from the 8th day P.C. onwards. On the 14th day P.C. the parietal wall of the yolk sac and its thin covering layer of decidua capsularis disappear to form a completely inverted yolk sac placenta. This places the yolk sac cavity in continuity with the non-glandular extensions of the uterine lumen and re-establishes complete continuity of the uterine lumen.     

Late postnatal development and differentiation of the ductus epididymidis in a dasyurid marsupial (Antechinus stuartii)

Summary The general histology and ultrastructural features of the developing ductus epididymidis were examined in the brown marsupial mouse, Antechinus stuartii, from April, when males were sexually immature, until August, when the adult males were involved in mating activities, just prior to the annual male die-off. Samples were also examined 3 and 6 months after the August die-off period in males kept in isolation from conspecifics during the prebreeding and breeding periods. In April, tubule diameter and epithelial height were largest in the caput and least in caudal segments but the reverse was observed thereafter. Epithelial height increased in caput segments in August and remained high in the post die-off samples. However, caput epithelial height and tubule diameters were low compared with the remainder of the duct from July until February. Luminal shape in caudal segments (10, 11 and 12) changed in June from circular to a narrow slit, and the epithelium became variable in height. The epididymal epithelium was undifferentiated with few cytoplasmic organelles in April. Differentiation occurred mostly from May to June in associaion with an increased abundance of cytoplasmic organelles, increasing prostatic weight and rising plasma androgen levels. Differentiated principal and basal cells were found in caput and corpus regions in May and in caudal segments in June in association with the de novo development of a brush border of microvilli. Few clear cells were seen in caput and corpus regions of the duct in May but they, and mitochondria-rich cells, were common throughout the duct from June. Development of the unusual structural features of the cauda epididymidis preceded the arrival of spermatozoa in June. The presence of degenerating spermatozoa and cytoplasmic droplets in the cauda at this time suggested that it was not yet capable of supporting sperm viability. There was no evidence to suggest that the presence of spermatozoa has a stimulatory effect on the epididymis. Intact sperm were observed throughout the duct from July. Free cytoplasmic droplets, which showed some evidence of degeneration, collected in large masses in the distal corpus/ proximal cauda epididymidis of adult males between aggregates of spermatozoa. Epididymal differentiation appeared complete by mid-July; few ultrastructural changes occurred after this time. Recruitment of spermatozoa into the epididymis ceased by August and was associated with a rapid decline in sperm content in the proximal caput segments. In the November and February samples, spermatozoa were present only in distal corpus and proximal cauda segments. As in some eutherian mammals, differentiation of the epididymis in A. stuartii occurs in a descending fashion from caput to cauda. Development is linked to the onset of fluid and androgen production from the testis, which is essential for developing and maintaining a suitable caudal environment for storage and survival of spermatozoa.     

Freeze-fracture observations on the visceral yolk sac placenta of rats, mice and hamsters

Summary Freeze-fracture replicas of visceral yolk sac from rats, mice and hamsters in late stages of gestation were studied by electron microscopy. Special attention was directed toward determining the types of junctional specializations that exist between the columnar endoderm cells of this placental membrane. In all three species, well-developed, zonular tight (occluding) junctions were found on the contiguous lateral surfaces of the endoderm cells. The tight junctional network, located in an immediate subluminal position, was from 0.2–0.5 m in depth and consisted at any point of 2–5, interconnecting, 9 m wide, strands (P-face) or shallow furrows (E-face). Patch-like aggregations of irregular intramembrane particles, characteristic of desmosomes (maculae adherentes), also were observed at scattered sites below the tight junctions. However, no evidence of gap (communicating) junctions was encountered. The endoderm cells of the rodent visceral yolk sac have been shown to play a central role in the selective transport of macromolecular substances from the maternal to the fetal system. Tight junctions may be vital to this endodermal cell function by preventing random paracellular fluxes of macromolecules.This investigation was supported by U.S. National Institutes of Health Grant HD-09585     

Red cell metabolism in a small dasyurid marsupial,the brown antechinus (Antechinus stuartii)

Red cell metabolism was examined in a small dasyurid marsupial, the brown antechinus Antechinus stuartii. There were significant differences in haematocrit with regard to sex and the season. Compared to other marsupials, the haemoglobin of A. stuartii was more susceptible to oxidation by acetylphenylhydrazine. However, the rate of methaemoglobin reduction was faster in A. stuartii. Our results appear to support a previous suggestion that methaemoglobin reducing ability is greater in those species whose haemoglobin is more susceptible to oxidative stress.     

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.     

Development of visual projections in the marsupial, Setonix brachyurus

Summary Retinal projections to the primary visual centres were studied following injection of tritiated proline into one eye in the Marsupial, Setonix brachyurus between 10 and 100 days postnatal and in adults. Initially, projections from the two eyes overlapped extensively, particularly between 20 and 50 days. There was a gradual refinement thereafter, including a segregation of inputs from the two eyes within both the lateral geniculate nucleus (LGN) and superior colliculus (SC) by 70 days. Such refinement in visual centres is discussed in relation to the concurrent emergence of retinal ganglion cell density gradients, a decrease in ganglion cell numbers, cell death in the ganglion cell layer and loss of optic axonal profiles.     

Spermiogenesis and spermiation in a marsupial, the tammar wallaby (Macropus eugenii)

Fourteen steps of spermatid development in the tammar wallaby (Macropus eugenii), from the newly formed spermatid to the release of the spermatozoon into the lumen of the seminiferous tubules, were recognised at the ultrastructural level using transmission and scanning electron microscopy. This study confirmed that although the main events are generally similar, the process of the differentiation of the spermatid in marsupials is notably different and relatively more complex than that in most studied eutherian mammals and birds. For example, the sperm head rotated twice in the late stage of spermiogenesis: the shape of the spermatid changed from a T-shape at step 10 into a streamlined shape in step 14, and then back to T-shape in the testicular spermatozoa. Some unique figures occurring during the spermiogenesis in other marsupial species, such as the presence of Sertoli cell spurs, the nuclear ring and the subacrosomal space, were also found in the tammar wallaby. However, an important new finding of this study was the development of the postacrosome complex (PAC), a special structure that was first evident as a line of electron dense material on the nuclear membrane of the step 7 spermatid. Subsequently it became a discontinuous line of electron particles, and migrated from the ventral side of the nucleus to the area just behind the posterior end of the acrosome, which was closely located to the sperm–egg fusion site proposed for Monodelphis domestica (Taggart et al. 1993). The PAC and its possible role in both American and Australian marsupials requires detailed examination. Distinct immature features were discovered in the wallaby testicular spermatozoa. A scoop shape of the acrosome was found on the testicular spermatozoa of the tammar wallaby, which was completely different to the compact button shape of acrosome in ejaculated spermatozoa. The fibre network found beneath the cytoplasm membrane of the midpiece of the ejaculated sperm also did not occur in the testicular spermatozoa, although the structure of the principal piece was fully formed and had no obvious morphological difference from that of the epididymal and ejaculated spermatozoa. The time frame of the formation of morphologically mature spermatozoa in the epididymis of the tammar wallaby needs to be determined by further studies.     

Testicular and epididymal development in the brown marsupial mouse, Antechinus stuartii (Dasyuridae, Marsupialia)

Reproductive tissues were collected monthly from male Antechinus stuartii during the first 5 months of post-partum development, a period corresponding to the time between birth and the initial increase in plasma androgen above non-detectable levels. The gonad appeared undifferentiated at day 3 after birth, but the basic structure of the testis (tunica albuginea, sex cords, stroma) was well established at 1 month of age. At this stage the developing sex cords contained a single layer of pre-Sertoli cells which surrounded a central core of gonocytes. Mitotic division of cells within the cords was common. Intertubular fetal Leydig cells, often observed in clumps, and perivascular and peritubular fetal Leydig cells were common and readily identified. By 2 months of age there was an obvious increase in cord diameter and the abundance of pre-Sertoli cells, while a marked reduction in the density of connective tissue cells and fetal Leydig cells was observed in the interstitium. Fetal Leydig cells appeared to persist only in close association with the developing seminiferous cords. Testicular size and the diameter and convolutions of the seminiferous cords increased substantially (two fold increase in cord diameter) by 3 months of age. Gonocytes had begun to migrate toward the basal lamina of the cords, and connective tissue cells and Leydig cells appeared in large numbers throughout the interstitium. By 4 and 5 months of age, gonocytes were commonly seen in contact with the basement membrane, and the cords remained non-patent. Leydig cell number and density increased greatly during these months. The epididymal epthelium remained undifferentiated throughout the first 5 months of development. Epithelial cells characteristically contained a large nucleus which occupied most of the cell, very little cytoplasm and few organelles. The diameter of the epididymal duct was similar throughout for the first 3 months of the study. In months 4 and 5 the diameter of the duct in caput and corpus regions increased, ahead of that of the cauda, possibly in relation to variations in androgen exposure at different regions along the developing duct. Further histological and quantitative studies on the growth and development of Leydig cells within the Dasyuridae are needed for comparision with eutherian mammals, which together with knowledge of the changing levels of fetal androgens may provide a greater understanding of the role of the different populations of Leydig cells in the differentiation of the testis and male reproductive tract. Marsupials from excellent animal models for such studies, since much of the early differentiation of the gonads and reproductive tract occurs in the pouch, rather than in utero, allowing easy access to young at this time.     

Immune interactions at the maternal-fetal interface: a focus on antigen presentation

Problem: Viruses and fetuses face similar immunologic challenges. Each must evade immune detection and destruction. The virus must avoid host recognition of intracellular infection; the fetus allogenic recognition. Each has manipulated the process of antigen presentation to allow survival in an immunologic environment otherwise predictably hostile. How have these approaches co‐evolved? What can they teach us about viral pathogenesis and immunologic interactions at the maternal‐fetal interface? Method of study: Review of relevant literature. Results: Special classical and non‐classical MHC class I products are spared from downregulation in the placenta and from viral immunoevasive strategies. Conclusions: Viruses rely upon some of the same strategies to avoid immune detection as do trophoblast cells. In the future, viral infections may prove a useful tool for studies of immunology at the maternal‐fetal interface.     

Redistribution of aquaporins in uterine epithelial cells at the time of implantation in the rat

The aim of this study was to investigate the presence of aquaporins 1, 4 and 5 in uterine epithelial cells during the early stages of pregnancy in the rat. Immunofluorescent and immunogold techniques showed that there was a shift of aquaporin 5 to the apical surface of uterine epithelial cells in the mesometrial pole of the uterus at the time of implantation. This study also revealed the absence of aquaporin 4 in the rat uterus during early pregnancy. Aquaporin 1 was observed in stromal blood vessels but there was no change in uterine epithelial cells during early pregnancy. These results suggest that aquaporin 5 plays an important role in the removal of uterine luminal fluid at the time of implantation in the rat and may contribute to the antimesometrial positioning of the implanting blastocyst.     

白血病抑制因子在小鼠子宫内膜不同部位的表达研究

目的探讨白血病抑制因子(LIF)在着床窗口期小鼠子宫内膜不同部位的表达。方法运用半定量逆转录聚合酶链反应(RT-PCR)和免疫组化技术,分别从mRNA水平和蛋白质水平检测LIF在孕4d小鼠子宫内膜着床位点及着床旁组织表达量及位置的分布。结果LIFmRNA及蛋白在胚泡着床位点较着床旁组织表达明显增高(P<0.05)。免疫组化检测结果显示LIF蛋白表达于子宫内膜间质细胞及腺上皮细胞胞桨。结论在着床窗口期,LIF作用的发挥主要是通过在着床位点高表达而促进胚泡着床、胚胎发育。     

In vivo dendritic cell depletion reduces breeding efficiency, affecting implantation and early placental development in mice

Implantation of mammalian embryos into their mother's uterus ensures optimal nourishment and protection throughout development. Complex molecular interactions characterize the implantation process, and an optimal synchronization of the components of this embryo-maternal dialogue is crucial for a successful reproductive outcome. In the present study, we investigated the role of dendritic cells (DC) during implantation process using a transgenic mouse system (DTRtg) that allows transient depletion of CD11c(+) cells in vivo through administration of diphtheria toxin. We observed that DC depletion impairs the implantation process, resulting in a reduced breeding efficiency. Furthermore, the maturity of uterine natural killer cells at dendritic cell knockout (DCKO) implantation sites was affected as well; as demonstrated by decreased perforin expression and reduced numbers of periodic-acid-Schiff (PAS)-positive cells. This was accompanied by disarrangements in decidual vascular development. In the present study, we were also able to identify a novel DC-dependent protein, phosphatidylinositol transfer protein beta (PITPbeta), involved in implantation and trophoblast development using a proteomic approach. Indeed, DCKO mice exhibited substantial anomalies in placental development, including hypocellularity of the spongiotrophoblast and labyrinthine layers and reduced numbers of trophoblast giant cells. Giant cells also down-regulated their expression of two characteristic markers of trophoblast differentiation, placental lactogen 1 and proliferin. In view of these findings, dendritic cells emerge as possible modulators in the orchestration of events leading to the establishment and maintenance of pregnancy.     

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.     

A light and electron microscopic study of the mouse visceral yolk sac endodermal cells in the middle and late embryonic periods, showing the possibility of definitive erythropoiesis

Hematological studies have revealed the importance of the visceral yolk sac (VYS) in the primitive erythropoiesis of mouse embryos at an early stage before day 12. We examined the possibility of the occurrence of extra-embryonic erythropoiesis at a stage later than embryonic day 12 by light and electron microscopic analyses. Surprisingly, a novel structure in the form of erythrocyte-like globules was observed in the VYS endodermal cells. They were consistently present in the VYS endodermal cells from embryonic day 12 until day 18 (birth is day 19), by immunocytochemical and enzyme histochemical analyses. They were immuno-positive for mouse erythrocyte antibody and also positive for the benzidine reaction showing the presence of hemoglobin. The erythrocyte-like globules were shown to be the erythrocytes present in the cytoplasm. These results indicated that erythropoiesis in the VYS endodermal cells continues from the early embryonic stage, as primitive erythropoiesis, until the late stage.     

Immune system cell markers in the northern brown bandicoot, Isoodon macrourus

This is the first study to report the presence of T and B lymphocyte markers and antigen presenting-like molecules in a marsupial bandicoot. Intra-cytoplasmic markers for CD3 and CD5, as well as surface Thy-1.1 and CD1a molecules were located in lymphocytes of T dependent regions of immuno-lymphoid tissue in the northern brown bandicoot using immunohistochemical techniques. Similarly, intra-cytoplasmic domains of CD79a, CD79b molecules and surface IgG molecules enabled characterisation of B lymphocytes and plasma cells. The phenotypic expression of these molecules parallels findings in eutherians, suggesting firstly the conservation of lineage epitopes for T and B subsets and secondly, the potential for similar functional properties of immune system cells between marsupials and eutherians. In addition, the presence of MHC class II and CD1a molecules on dendritic-like cells may indicate similar mechanisms for antigen processing and presentation as reported in eutherians. The use of such immune system cell markers will enable functional studies to characterise the marsupial immune system as well as ontogeny studies of immune competence.     

The effects of a transient increase in temperature on cell generation and cell death in the hippocampus and amygdala of the wallaby, Setonix brachyurus (quokka)

We examined the effects of a single exposure to a temperature elevation of 2° C for a 2 h period on the developmental processes of cell division and cell death in the hippocampus and the amygdala of the quokka. Animals aged postnatal day (P) 40–45 were injected with tritiated (³H-) thymidine and then exposed to either 37° C (normal) or 39° C (±0.2° C) in an incubator for a duration of 2 h. The young were then returned to the nipple and, after a period of 24 h, were sacrificed. Brains were sectioned and selected sections processed for autoradiography, and some were counterstained. Cell division taking place at the time of heating was estimated by counting ³H-thymidine-labelled cells and at the time of sacrifice by counting mitotic figures. Dying cells were visualised as pyknotic profiles in cresyl-violet-stained sections. In both the amygdala and the hippocampus, the number of ³H-thymidine-labelled cells (cells dividing in situ during the heating period) was significantly lower in the experimental than the control group. Such cells were glia in the amygdala and granule cells and glia in the hippocampus. However, the number of dying cells or mitotic figures (cells dividing at the time of sacrifice) did not differ significantly between the two groups. By comparison, the number of ³H-thymidine-labelled cells, dying cells or mitotic figures did not significantly differ in the diencephalon. Therefore, a brief exposure to a slight elevation in temperature results in an immediate alteration in cell division in the hippocampus and amygdala. These findings have implications for the role played by raised temperature, such as during virus infection, in producing developmental anomalies of the brain. Received: 17 November 1997 / Accepted: 5 May 1998     

Immune system cell markers in the northern brown bandicoot, Isoodon macrourus

This is the first study to report the presence of T and B lymphocyte markers and antigen presenting-like molecules in a marsupial bandicoot. Intra-cytoplasmic markers for CD3 and CD5, as well as surface Thy-1.1 and CD1a molecules were located in lymphocytes of T dependent regions of immuno-lymphoid tissue in the northern brown bandicoot using immunohistochemical techniques. Similarly, intra-cytoplasmic domains of CD79a, CD79b molecules and surface IgG molecules enabled characterisation of B lymphocytes and plasma cells. The phenotypic expression of these molecules parallels findings in eutherians, suggesting firstly the conservation of lineage epitopes for T and B subsets and secondly, the potential for similar functional properties of immune system cells between marsupials and eutherians. In addition, the presence of MHC class II and CD1a molecules on dendritic-like cells may indicate similar mechanisms for antigen processing and presentation as reported in eutherians. The use of such immune system cell markers will enable functional studies to characterise the marsupial immune system as well as ontogeny studies of immune competence.     

Developmental changes at the materno-embryonic interface in early pregnancy of the alpaca, Lamos pacos

This study analyses the manner in which trophoblast cells adhere to uterine epithelium and the subsequent interactions that contribute to the establishment of epitheliochorial placentation in the alpaca Lama pacos. Specimens at the luteal and follicular phases and at 22, 26, 30 and 45 days of pregnancy (op) were processed for morphological studies. On day 15 op, the blastocysts are completely free within the uterine lumen, with implantation starting around day 20. On days 22 and 26 of gestation, the trophoblast is apposed to the epithelial surface of the uterus, with areas of contact and adhesion by means of complex interdigitation. Implantation sites occur prevalently in the left uterine horn, but an expanded trophoblast also occupies large extensions of the right horn, where the maternofetal interaction shows peculiar areas of apposition. As development continues, attachment areas become more extensive. On days 30 and 45, many secretory granules can be seen in the uterine epithelium, while giant multinucleate cells appear interposed between the remaining trophoblast cells, showing intense alkaline phosphatase activity, deposits containing iron and PAS-positive granules. Placental lactogen hormone is not present within the cytoplasm of the binucleate or multinucleate trophoblast cells. By day 30 of gestation, the trophoblast layer is lined by an extraembryonic connective tissue that by day 45 is well vascularized, thus indicating the starting point of placental formation. Fetal and maternal capillaries indent the epithelium and the trophoblast, narrowing the specialized areas of exchange, which occur along the entire maternofetal interface, characterizing the diffuse nature of this placenta.