Gross anatomy of the gluteal and posterior thigh muscles in koalas based on their innervations.

Morphological and functional comparison of convergently-evolved traits in marsupials and eutherians is an important aspect of studying adaptive divergence in mammals. However, the anatomy of marsupials has been particularly difficult to evaluate for multiple reasons. First, previous studies on marsupial anatomy are often uniformly old and non-exhaustive. Second, muscle identification was historically based on muscle attachment sites, but attachment sites have since been declared insufficient for muscle identification due to extensive interspecific variation. For example, different names have been used for muscles that are now thought to be equivalent among several different species, which causes confusion. Therefore, descriptions of marsupial muscles have been inconsistent among previous studies, and their anatomical knowledge itself needs updating. In this study, the koala was selected as the representative marsupial, in part because koala locomotion may comprise primate (eutherian)-like and marsupial-like mechanics, making it an interesting phylogenetic group for studying adaptive divergence in mammals. Gross dissection of the lower limb muscles (the gluteal and the posterior thigh regions) was performed to permit precise muscle identification. We first resolved discrepancies among previous studies by identifying muscles according to their innervation; this recent, more reliable technique is based on the ontogenetic origin of the muscle, and it allows for comparison with other taxa (i.e., eutherians). We compared our findings with those of other marsupials and arboreal primates and identified traits common to both arboreal primates and marsupials as well as muscle morphological features unique to koalas.

Ovary of the southern hairy-nosed wombat (Lasiorhinus latifrons): its divergent structural organisation.

The organisation of the ovarian interstitial tissue in the southern hairy-nosed wombat Lasiorhinus latifrons was investigated. Unlike in most other marsupials, the outer cortical region of the ovary contains abundant luteinised interstitial tissue that largely occurs in discrete lobules, many of which contain a localised area of non-cellular, highly eosinophilic and periodic acid-Schiff-positive material. The findings suggest that the latter arises from the zona pellucida that surrounded the oocyte in growing follicles and that the luteinised interstitial tissue thus developed from transformed theca interna of degenerated atretic follicles. It is hypothesised that this tissue synthesises and secretes progestogens, which may result in the long, and variable, oestrous cycle length that has been found to occur in this species.

Interspecific diversity of testes mass and sperm morphology in the Philippine chrotomyine rodents: implications for differences in breeding systems across the species.

The high diversity of native Philippine murid rodents includes an old endemic group, the chrotomyines, which are the sister group of the Australasian hydromyines. Herein we detail their interspecific diversity of relative testes mass (RTM) and sperm morphology. We find that in chrotomyines, as in the Australasian hydromyines, testes mass relative to body mass differs by an order of magnitude across the species and ranges from a large RTM in Soricomys and Chrotomys species to a small RTM in Apomys. Sperm morphology is associated with these findings, with individuals in species of Soricomys and Chrotomys producing relatively larger spermatozoa with a prominent apical hook and long tail, whereas, by contrast, the Apomys species have a sperm head that either has a very short or no apical hook and a shorter tail. These findings indicate coevolution of RTM with sperm morphological traits across the species, with the marked interspecific differences in RTM suggesting differences in the intensity of intermale sperm competition and hence breeding system. Thus, we hypothesise that species of Soricomys and Chrotomys that produce more streamlined spermatozoa with longer tails have a polyandrous or promiscuous mating system, whereas the Apomys species, which produce smaller and less streamlined spermatozoa, may exhibit monogamy.

Mammalian development does not recapitulate suspected key transformations in the evolutionary detachment of the mammalian middle ear.

The ectotympanic, malleus and incus of the developing mammalian middle ear (ME) are initially attached to the dentary via Meckel's cartilage, betraying their origins from the primary jaw joint of land vertebrates. This recapitulation has prompted mostly unquantified suggestions that several suspected--but similarly unquantified--key evolutionary transformations leading to the mammalian ME are recapitulated in development, through negative allometry and posterior/medial displacement of ME bones relative to the jaw joint. Here we show, using µCT reconstructions, that neither allometric nor topological change is quantifiable in the pre-detachment ME development of six marsupials and two monotremes. Also, differential ME positioning in the two monotreme species is not recapitulated. This challenges the developmental prerequisites of widely cited evolutionary scenarios of definitive mammalian middle ear (DMME) evolution, highlighting the requirement for further fossil evidence to test these hypotheses. Possible association between rear molar eruption, full ME ossification and ME detachment in marsupials suggests functional divergence between dentary and ME as a trigger for developmental, and possibly also evolutionary, ME detachment. The stable positioning of the dentary and ME supports suggestions that a 'partial mammalian middle ear' as found in many mammaliaforms--probably with a cartilaginous Meckel's cartilage--represents the only developmentally plausible evolutionary DMME precursor.

Morphological diversity and evolution of the spermatozoon in the mouse-related clade of rodents.

Most species in the three highly speciose families of the mouse-related clade of rodents, the Muridae, Cricetidae, and Nesomyidae (superfamily Muroidea), have a highly complex sperm head in which there is an apical hook but there are few data available for the other related families of these rodents. In the current study, using light and electron microscopies, we investigated the structure of the spermatozoon in representative species of four other families within the mouse-related clade, the Dipodidae, Spalacidae, Pedetidae, and Heteromyidae, that diverged at or near the base of the muroid lineage. Our results indicate that a diverse array of sperm head shapes and tail lengths occurs but none of the species in the families Spalacidae, Dipodidae, or Pedetidae has a sperm head with an apical hook. By contrast, a rostrally extending apical hook is present in spermatozoa of members of the Family Heteromyidae which also invariably have comparatively long sperm tails. These findings suggest that the hook-shaped sperm head in the murid, cricetid, and nesomyid rodents evolved after divergence of this lineage from its common ancestor with the other families of the mouse-related clade, and that separate, and independent, convergent evolution of a similar sperm head form, and long sperm tail, occurred in the Heteromyidae.

Spermatozoa of the old endemic rodents of Australia - the possible functional significance of their ventral processes.

Spermatozoa of the plains mouse (Pseudomys australis), like those of most Australian old endemic rodents, contain, in addition to an apical hook, two further processes that extend from the upper concave surface of the head, the ventral processes. This study shows that these processes contain thiol-rich cytoskeletal proteins, which presumably help to maintain their rigidity during sperm transport, together with the overlying cell membrane having abundant intramembranous proteins. To determine the possible functional significance of these processes, an in vitro study of spermatozoon-zona binding was undertaken. The findings suggest that initial sperm binding occurs by way of the cell membrane over the acrosome of the apical hook and that, subsequently, the lateral surfaces of the ventral processes also become tightly bound to the zona matrix. These ventral processes may therefore have evolved to increase sperm adhesion to the outer zona surface and/or to enhance stabilisation of the spermatozoon at the time of zona binding and initial penetration of the egg coat.

Reproductive biology of an old endemic murid rodent of Australia, the Spinifex hopping mouse, Notomys alexis: adaptations for life in the arid zone.

The Spinifex hopping mouse (Notomys alexis Thomas, 1922) is an arid adapted Australo-Papuan old endemic rodent that undergoes boom and bust population cycles. In this communication, we review our findings on the timing of reproduction and the potential reproductive rate of this species. To investigate the first question, the reproductive condition of adult females, and occurrence of immatures, in a population on a cattle station in central Australia was determined and, for the second, data from a laboratory colony compared to those previously published on closely related species in other environments. The findings show that, at least in the population at the times that monitoring was performed, reproductive activity was only taking place in spring and early summer, whereas the laboratory study indicates that females have a similar gestation length and litter size to those of most close relatives occurring in other environments. Males have extremely small testes and store relatively few sperm. The findings suggest that Notomys alexis might show some seasonality of reproduction at least in this region of central Australia and that this species does not have a higher reproductive rate than that of related species in other, more predictable, environments.

Whole-body heat exposure induces membrane changes in spermatozoa from the cauda epididymidis of laboratory mice.

This study was carried out to determine if exposure to hot environmental temperatures had a direct, detrimental effect on sperm quality. For this the effect of whole-body heat exposure on epididymal spermatozoa of laboratory mice was investigated. C57BL/6 mice (n = 7) were housed in a microclimate chamber at 37 degrees C-38 degrees C for 8 h per day for three consecutive days, while control mice (n = 7) were kept at 23 degrees C-24 degrees C. Cauda epididymal spermatozoa were obtained 16 h after the last heat treatment. The results showed that sperm numbers were similar in the two groups (P = 0.23), but after heat treatment, a significant reduction in the percentage of motile sperm was present (P < 0.0001). Membrane changes of the spermatozoa were investigated by staining with phycoerythrin (PE)-conjugated Annexin V, which detects exteriorization of phosphotidylserine from the inner to the outer leaflet of the sperm plasma membrane, and 7-aminoactinomycin D (7-AAD), which binds to the sperm nucleus when the plasma membrane is damaged. The percentage of spermatozoa showing positive staining with Annexin V-PE or 7-AAD or both, was significantly higher (P < 0.05) in heat-exposed mice compared with controls. These results show that whole-body heat exposure to 37 degrees C-38 degrees C induces membrane changes in the epididymal spermatozoa of mice, which may lead to apoptosis.

Changes in the blood-CSF barrier in experimental traumatic brain injury.

PURPOSE: Elevation of intracranial pressure (ICP) is a major complication of traumatic brain injury (TBI), and cerebrospinal fluid (CSF) volume is a key factor in ICP regulation. Choroidal epithelial cells (CEC) form the blood-CSF barrier and their integrity is essential for controlling CSF production. In the current study, the morphology of the CEC was studied from 5 h to 28 day after TBI in the rat. METHODS: Male Sprague-Dawley rats were subjected to severe TBI using the impact-acceleration model, and the ultrastructure of the CEC was studied using transmission (TEM) and scanning (SEM) electron microscopy. RESULTS: Radical ultrastructural changes were seen by TEM in CEC in injured animals. At 5 h post-injury cell swelling and incipient cytoplasmic vacuoles were seen. At 24 h most severe changes were noted with extensive widening of intercellular clefts. At 7 day and 14 day post-injury, increased cytoplasmic electron density was evident. At 21 day, most microvilli had bulbous ends, and at 28 day cytoplasmic vacuoles were numerous with widened intercellular clefts. SEM revealed a continuum of changes in all injured animals and most conspicuous was the heterogeneity of surface features, with most cells showing bulbous and cup-shaped microvilli, burr-like processes and pits. Epiplexus cells were hypertrophic and more numerous. CONCLUSION: At 4 weeks after trauma, choroidal epithelial cells continued to show morphological alterations suggesting that brain homeostasis was still not restored.

Changes in distribution of basic nuclear proteins and chromatin organization during spermiogenesis in the greater bandicoot rat, Bandicota indica.

Male germ cells of the greater bandicoot rat, Bandicota indica, have recently been categorized into 12 spermiogenic steps based upon the morphological appearance of the acrosome and nucleus and the cell shape. In the present study, we have found that, in the Golgi and cap phases, round spermatid nuclei contain 10-nm to 30-nm chromatin fibers, and that the acrosomal granule forms a huge cap over the anterior pole of nucleus. In the acrosomal phase, many chromatin fibers are approximately 50 nm thick; these then thickened to 70-nm fibers and eventually became 90-nm chromatin cords that are tightly packed together into highly condensed chromatin, except where nuclear vacuoles occur. Immunocytochemistry and immunogold localization with anti-histones, anti-transition protein2, and anti-protamine antibodies suggest that histones remain throughout spermiogenesis, that transition proteins are present from step 7 spermatids and remain until the end of spermiogenesis, and that protamines appear at step 8. Spermatozoa from the cauda epididymidis have been analyzed by acid urea Triton X-100 polyacrylamide gel electrophoresis for basic nuclear proteins. The histones, H2A, H3, H2B, and H4, transitional protein2, and protamine are all present in sperm extracts. These findings suggest that, in these sperm of unusual morphology, both transition proteins and some histones are retained, a finding possibly related to the unusual nuclear form of sperm in this species.

Electron microscope study of blood-brain barrier opening induced by immunological targeting of the endothelial barrier antigen.

Barrier vessels in the central nervous system are lined with endothelial cells which constitute the blood-brain barrier (BBB) and show selective expression of certain biochemical markers. One of these, the endothelial barrier antigen (EBA), is specific to the rat. The exact role of EBA in the BBB is not known, although several studies have shown a correlation between the reduction in EBA expression in endothelial cells and the opening of the BBB. However, in these studies it was not possible to determine if EBA reduction was a primary event or was secondary to opening of the BBB. A recent light microscope study demonstrated that immunological targeting of EBA in vivo, by intravenous injection of a monoclonal antibody (anti-EBA), leads to acute and widespread opening of the BBB. In the current study we have employed this model together with tracer application and immunoperoxidase electron microscopy to determine the site of binding of the injected antibody and the route of opening of the BBB. The results showed that (a) the anti-EBA injected in vivo became bound to brain endothelial cells, principally to luminal membranes. (b) Endothelial cells showed widened intercellular junctions and increased cytoplasmic vesicles and vacuoles. (c) Many perivascular astrocytic processes were swollen. (d) The macromolecular tracer HRP was present in vesicles, vacuoles, widened paracellular clefts, the perivascular space and brain parenchyma. In conclusion, the in vivo targeting of EBA leads to opening of the BBB apparently via paracellular and transcellular routes. This model is useful for the study of vascular permeability in the CNS and experimental manipulation of the BBB. It may have a potential application in experimental studies on drug delivery throughout the CNS.