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.     

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.     

Developmental staging in a marsupial Dasyurus hallucatus

Summary In a marsupial, Dasyurus hallucatus, pouchyoung of various ages from newborn to 55 days were embedded in wax and serially sectioned. On the basis of the relative development of external and internal characteristics, they were placed in the Carnegie staging system developed by Streeter and elaborated by O'Rahilly and associates. Birth occurred at stage 15, and the end of embryogenesis (stage 23) was reached about day 33. Whereas stage 23 is characterised in all eutherians by the closure of the secondary palate, this occurs before stage 15 in D. hallucatus. Since most other characters of the newborn are at a stage 15 level of development, there has been a relative acceleration of development of the secondary palate (and forelimb) in D. hallucatus that allows it to suckle and breathe at the same time. Between D. hallucatus and eutherians, there is general agreement in the sequence of development and in the relative degree of most structures at each stage. Further marsupials should be examined to see if the minor differences noted are peculiar to D. hallucatus or apply to marsupials generally.Abbreviations I II III IIII ventricles - AC anterior commissure - AD adenohypophysis - BS blood sinus - BT basitrabecular - CD cerebral peduncles - CE cervical swelling - CH cerebral hemisphere - CI central nucleus of inferior colliculus - CO cortical plate - CP cerebellar plate - CR cerebellar ridge - CX choroid plexus - DI diencephalon - DT dorsal thalamus - EC external capsule - EM eye muscle - EP epithalamus - ES epiphysis - ET epitrichium - EY eye - FL forelimb - GG gasserian ganglion - GU gut - HB habenular nucleus - HE heart - HP hippocampus - HS head shield - HY hypothalamus - IC internal capsule - IE inner ear - IL intermediate layer - IN inferior colliculus - IO inferior olivary nucleus - IX glossopharyngeal nerve - LF lateral forebrain bundle - LG lateral geniculate nucleus - LI liver - LO lamina orbitonasalis - LR lateral recess - LS lateral striatal ridge - MB mamillary bodies - ME mesencephalon - MI mitral cells - MS medial striatal ridge - NE neurohypophysis - NO neocortex - NP nasal passage - NS nasal septum - OB olfactory bulb - OC optic chiasm - OG otic ganglion - ON olfactory nerve - OP optic nerve - OS orbitosphenoid - OT optic stalk - PA paraseptal cartilage - PC posterior commissure - PL paleocortex - PR parachordal cartilage - RH rhombencephalon - RI rib - RL rhombic lip - SC superior colliculus - SH pectoral girdle - SI sulcus interhemisphericus - SM stria medullaris thalami - SP septum - ST striatum - TE telencephalon - TO tongue - TR trabecular cartilage - TU turbinal - VE velum transversum - VL ventricular layer - VN vomeronasal nerves - VT ventral thalamus     

The early development of the eye of the pouch-young of the marsupial Dasyurus hallucatus

Summary The development of the eye of the pouch-young of the marsuspial native cat Dasyurus hallucatus from birth to day 30 is described. Following the staging system used by other authors, the stage of development of the eye at birth is comparable to that of a six-week human embryo, a 33 day rhesus embryo or a 14 day rat embryo. In contrast to eutherian mammals where development progresses from one stage to another at approximately the same rate, in the native cat the rate tends to progressively decrease. All of the neural development of the native cat eye occurs while the young is in the pouch.Abbreviations used in Figures. The bar in each figure is 100 A annular vessel - AM acellular matrix of presumptive cornea - C conus vascularis - CC transient layer of Chievitz - CE corneal pithlium - CM ciliary muscle - CS closed space - D diencephaton - E eyelid - EM eye muscles - EN endothelium - H hyaloid vessels - ICB region of future iris (I) and ciliary body (CB) - L lens - LE lens epithelium - LB lens body - IF inner fold - INL inner neuroblastic layer - M mesoderm - MS marginal sinus - NF nerve fibres - OC optic cup - OF otter fold - ON optic nerve - ONL outer neuroblastic layer - OS optic stalk - P pigment - PLF primary lens fibres - PM pupillary membrane - SP substantia propia - VB vitreous body     

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.     

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.     

Cloning and structural analysis of IgM (μ chain) and the heavy chain V region repertoire in the marsupial Monodelphis domestica

To address the question of the Ig isotype repertoire of non placental mammals, we have examined the Ig expression in the marsupial Monodelphis domestica (grey short tailed opossum). Screening of an opossum spleen cDNA library has previously led to the isolation of full length clones for opossum IgG (γ chain), IgE ( chain) and IgA (α chain). We now present the isolation of several cDNA clones encoding the entire constant regions of the opossum IgM (μ chain). A comparative analysis of the amino acid sequences for IgM from various animal species showed that opossum IgM, within the various animals studied, is the most divergent member of its Ig class. However, it still conforms to the general structure of IgM in other vertebrates. Four Ig classes have now been identified in opossum and only one isotype is apparently present within each Ig class, IgM, IgG, IgA and IgE. Opossum has previously been shown to have a limited VH region diversity, with only two V gene families. Both of these belong to the group III of mammalian VH sequences. This limitation in variability is to some extent compensated for by a large variation in D, P and N regions, both in size and in sequence. However, evidence for the expression of only two functional J segments has so far been detected, which indicates a rather limited diversity also of the J segments in the opossum.     

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.     

Differentiation of the gonads and initiation of mammary gland and scrotum development in the brushtail possum Trichosurus vulpecula (Marsupialia)

The first appearance of the mammary and scrotal primordia and the sexual differentiation of the gonads of the brushtail possum, Trichosurus vulpecula, are described. Primordial germ cells were first observed, in fetuses of 7.5 mm crown-rump length, in the gonadal ridges and migrating up the dorsal mesentery. Mammary primordia were first observed in fetuses of 11 mm, and scrotal primordia in those of 12 mm crown-rump length. These structures were diagnostic of female and male brushtail possums respectively. Processus vaginales and gubernacula showed sexual dimorphism, being better developed and appearing earlier in males than in females. Sexual differentiation of the gonads occurred after the appearance of mammary and scrotal primordia, the testes being first recognisable in a 14.5-mm fetus and the ovaries postnatally. Birth occurred between the stages of 14 and 15 mm crown-rump length. These observations appear to indicate that the development of mammary and scrotal primordia are not under gonadal hormonal control, but under direct genetic control, as suggested for the tammar wallaby by previous authors.     

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.     

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.     

B淋巴细胞发育分化中的特征性表面分子

B淋巴细胞是体内唯一能够产生抗体的细胞,介导体液免疫.B淋巴细胞发育分化是一个十分有序的过程,大体分为五个阶段,祖B细胞、前B细胞、未成熟B细胞、过渡B细胞和成熟B细胞.每个阶段均有特征性的表面标志,其表面分子的表达呈规律性改变.各发育分化阶段特征性表面分子表达异常可引起某些疾病,虽然目前国外对B淋巴细胞发育分化情况有较多研究,但对于B淋巴细胞发育分化过程中的一些问题及对某些疾病的影响与相关机制仍不明确,需进一步研究.     

Production of interleukin-1 in a South American opossum (Monodelphis domestica)

A homologous thymocyte costimulatory assay using thymocytes from a South American opossum (Monodelphis domestica) detected and measured interleukin-1 (IL-1). Opossum IL-1 was obtained from lipopolysac-charide-stimulated macrophage and skin cultures and its molecular weight was determined to be 15,000 to 17,000. Opossum IL-1 did not stimulate proliferation of murine thymocytes; conversely, neither human nor murine IL-1 stimulated opossum thymocytes. Anti-human IL-1 antibodies were also not reactive with opossum IL-1. These observations indicate that there is no serological and functional crossreactivity between the opossum (marsupial mammals) and human and mouse (eutherian mammals) in an IL-1/thymocyte system. This is unusual because such crossreactivity occurs between rodents and distant, nonmammalian species. M. domestica has been used in our laboratory as a model for photobiological research; studies on IL-1 may provide insight into the relationship of immunosuppression and tumorigenesis induced by ultraviolet radiation.     

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.     

Morphometry and allometry of the postnatal marsupial lung development: an ultrastructural study

An utrastructural morphometric study of the postnatally remodelling lungs of the quokka wallaby (Setonix brachyurus) was undertaken. Allometric scaling of the volumes of the parenchymal components against body mass was performed. Most parameters showed a positive correlation with body mass in all the developmental stages, except the volume of type II pneumocytes during the alveolar stage. The interstitial tissue and type II cell volumes increased slightly faster than body mass in the saccular stage, their growth rates declining in the alveolar stage. Conversely, type I pneumocyte volumes increased markedly in both the saccular and alveolar stages. Both capillary and endothelial volumes as well as the capillary and airspace surface areas showed highest rates of increase during the alveolar stage, at which time the rate was notably higher than that of the body mass. The pulmonary diffusion capacity increased gradually, the rate being highest in the alveolar stage and the adult values attained were comparable to those of eutherians.     

The initial stages of development of the retinocollicular projection in the wallaby (Macropus eugenii): distribution of ganglion cells in the retina and their axons in the superior colliculus

The time course of ingrowth of retinal projections to the superior colliculus in the marsupial mammal, the wallaby (Macropus eugenii), was determined by anterograde labelling of axons from the eye with horseradish peroxidase, from birth to 46 days, when axons cover the colliculus contralaterally and ipsilaterally. The position of retinal ganglion cells giving rise to these projections over this period was determined in fixed tissue by retrograde labelling from the colliculus with a carbocyanine dye. Axons first reach the rostrolateral contralateral colliculus 4 days after birth and extend caudally and medially, reaching the caudal pole at 18 days and the far caudomedial pole at 46 days. The first contralaterally projecting cells are in the central dorsal and temporal retina, followed by cells in the nasal and finally the ventral retina. They are distributed closer to the periphery with increasing age. The first sign of a visual streak appears by 18 days. Axons reach the ipsilateral colliculus a day later than contralateral axons and come from a similar region of the retina. The sparser ipsilateral projection reaches the caudal and medial collicular margins by 46 days but by 16–18 days, ganglion cells giving rise to this transient projection are already concentrated in the temporoventral retina. The orderly recruitment of ganglion cells from retinotopically appropriate regions of the retina as axons advance across the contralateral colliculus suggests that the projection is topographically ordered from the beginning. The ipsilateral projection is less ordered as cells are located in the temporoventral crescent at a time when their axons are still transiently covering the colliculus prior to becoming restricted to the rostral colliculus. Features of mature retinal topography such as the visual streak and the location of ipsilaterally projecting cells begin to be established very early in development, before the period of ganglion cell loss and long before eye opening at 140 days.     

Retinotopic order in the optic nerve and superior colliculus during development of the retinocollicular projection in the wallaby ( Macropus eugenii )

 Retinotopic order of optic axons in the optic nerve and superior colliculus of the marsupial mammal, the wallaby (Macropus eugenii), has been examined and compared during development of the retinocollicular projection to investigate the role of order in the nerve in map formation. Small groups of axons from different retinal quadrants were labelled in vivo with a carbocyanine dye from just after axons first reached the colliculus to when the projection was mature. The distribution and branching patterns of axons and their arbors on the colliculus were assessed quantitatively during this period, as was the degree of order in the nerve. Initially, axons accumulated in coarse retinotopic order in the colliculus, with little branching and no sign of arborization to form terminal zones. Axons labelled from deposits covering a mean of 2.2% of the retina reached a mean collicular coverage of around 30% at 41–47 days, at which time they began arborizing in their retinotopically correct positions. By 55 days axons from all retinal quadrants had formed terminal zones in their retinotopically correct positions. Axons did not arborise in incorrect positions as has been reported in the rat. By 61–68 days coverage had decreased to around 10%. By 90–95 days only axons suppying terminal zones were present and terminal zones were smaller. In the nerve, axons showed a coarse and consistent order throughout development. This order was retinotopic only immediately behind the eye. Temporal and nasal axons occupied corresponding halves of the nerve along its course. Axons from dorsal and ventral retina shifted from dorsal and ventral positions in the nerve, respectively, to opposite sides of the nerve just before the chiasm. This would assist in positioning them in the appropriate lateral and medial optic tracts, respectively, in the positions they occupied as they approached the colliculus. However, the position in the nerve was not related to the ability to arborize in the correct collicular position. In particular, the increase in retinotopic order in the colliculus late in development was not accompanied by an increase in order in the nerve. Since the final organization in the colliculus shows greater order than is ever seen in the nerve, additional mechanisms must be involved in the maturation of the collicular map. Accepted: 17 May 1997     

Sexual differentiation of the urogenital system of the fetal and neonatal tammar wallaby, Macropus eugenii

In male tammar wallabies, the scrotum is the first organ to become sexually differentiated, 4–5 days before birth (day 22 of gestation). This is followed by enlargement of the gubernaculum and processus vaginalis one day before birth. However the indifferent gonad does not show any signs of testicular cord formation or androgen production until later, at around the time of birth; this is more pronounced at 2 days post-partum (p.p.), when the testis takes on a characteristic rounded appearance. Primordial germ cells proliferate throughout the testis at this time, although the testis does not become significantly heavier than the ovary until around 80 days p.p.. In females, the appearance of the mammary glands is the first sign of sexual differentiation 4–5 days before birth. The indifferent gonad first shows signs of developing an ovarian cortex and medulla 7 days after birth. The migrating germ cells are confined to the cortex, and first start to enter meiosis about 25 days after birth. The Wolffian (mesonephric) ducts are patent to the urogenital sinus in fetuses at day 21 of gestation. In the female they have started to regress by 10 days p.p. and only rudiments remain by day 25 p.p.. The Müllerian (paramesonephric) ducts develop adjacent to the cranial pole of the mesonephros at about day 25 of gestation and grow caudally to meet the urogenital sinus between days 2 and 7 p.p.. The Müllerian duct of the female develops a prominent ostium abdominale by day 9 p.p., but this structure has completely regressed in males by day 13 p.p.. The testis and ovary both migrate caudally, together with the adjacent mesonephros, at about day 10 p.p.. The ovaries remain around the level of lumbar vertebra 4 after about day 7 p.p., while the testes continue to descend. The testes enter the internal inguinal ring at about day 25 p.p., about the time that prostatic buds first appear in the urogenital sinus, and are in the inguinal canal from days 25 to 36 p.p.. They enter the scrotum at around day 36 p.p., and testicular descent is complete by days 65–72 p.p.. Melanin develops in the tunica vaginalis 72 days after birth. The overall development of the urogenital system in this marsupial is similar to that of eutherians but the sequence of events differs, with some aspects of genital differentiation preceding gonadal differentiation, apparently because they are directly controlled by X-linked genes, rather than indirectly controlled by gonadal steroids.