C. M. Leigh

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.

Effect of cooling and cryopreservation on sperm motility and morphology of several species of marsupial

The effects of long-term cooling and freezing on sperm motility are described for six marsupial species: the fat-tailed dunnart, koala, brushtail possum, long-footed potoroo, northern brown bandicoot and ring-tailed possum. The effects of up to eight days of cooling at 4 degrees C on the motility of dunnart spermatozoa and the effect of cryopreservation on spermatozoa of the other species were determined. The cryoprotectant used was a Tris-citrate-fructose-egg yolk-glycerol diluent. The percentage and rating of sperm motility, and sperm structure, as determined by light microscopy, were investigated. Sperm motility in the fat-tailed dunnart was retained for up to six days when cooled to 4 degrees C, suggesting that sperm from this species have some degree of tolerance to cold shock. After this time, however, the percentage of motile spermatozoa and their motility rating declined. In all species except the fat-tailed dunnart, reinitiation of motility following cryopreservation occurred across a range of glycerol concentrations (4-17%). Cryoprotectant containing 6% and/or 8% glycerol resulted in little change of motility rating or of the percentage of live sperm after thawing, although there was some decline in the percentage of motile sperm. The unusual structural and motility characteristics of dunnart spermatozoa may account for the lack of success of sperm cryopreservation in this species.

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.

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 Meckels 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 Meckels cartilage—represents the only developmentally plausible evolutionary DMME precursor.

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.

Ultrastructure and motility of spermatozoa in the male reproductive tract of perameloid marsupials

The number, distribution, maturation, motility and ultrastructure of spermatozoa from both northern (Isoodon macrourus) and southern (Isoodon obesulus) brown bandicoots were examined. One epididymidis per animal was fixed for light microscopy and transmission electron microscopy, and the contralateral side was used for the determination of sperm number, distribution and motility. Sperm form was similar between the two species. Approximately 56 x 10(6) testicular sperm and 100 x 10(6) epididymal sperm per side were present in I. macrourus, about 60% of which were in the caudal region. Initiation of sperm nuclear rotation and loss of the cytoplasmic droplet was first observed in distal caput or proximal corpus segments along with slow progressive motility. In these sperm, dislocation and anterior movement of the sperm neck from the implantation fossa and the modification of the distal margins of the sperm acrosome were evident. Motility of cauda epididymidal spermatozoa was rapid and coordinated, movement was restricted to one plane, and lateral head displacement was marked. As media viscosity increased, sperm velocity decreased, as did the amplitude of the tail beat, its frequency, and lateral head displacement but, in viscous mineral oil and mixtures of media and prostatic exudate, extremely rapid sinusoidal motility occurred. This study has detailed unusual morphological changes in bandicoot sperm during epididymal maturation and has shown that, although bandicoot sperm differ morphologically from those of the dasyurids, particularly in relation to head-tail orientation and tail ultrastructure, they exhibit similar motility.

Ultrastructure and motility of spermatozoa in macropodid and potoroidid marsupials.

In order to gain some understanding of the significance of the morphological features of spermatozoa within the Macropodoidea, the motility of spermatozoa from two macropodids (Petrogale xanthopus and Dendrolagus matschiei) and the motility, number and distribution of spermatozoa from three potoroidids (Aepyprymnus rufescens, Bettongia penicillata and Potorous tridactylus) were examined. Sperm were collected by electro-ejaculation or from the cauda epididymides. Epididymides from the potoroidids were divided into 12 regions. One epididymidis per animal was fixed for light and transmission electron microscopy and, on the contralateral side, the number of sperm, their distribution and motility were determined. In general, spermatozoa of all five species differed markedly from one another in head and flagella dimensions. Spermatozoa from B. penicillata and P. tridactylus were significantly longer and broader and had a smaller acrosome relative to head length, and there was a radial displacement of dense fibres. They also progressed more rapidly in standard culture media. Spermatozoa from at least three species were able to alter their motility pattern in vitro as media viscosity increased. Sperm movement in all species appeared to be restricted to one plane and showed no evidence of rotation, whereas lateral head displacement was often pronounced; there was no evidence of a sinusoidal mode of progressive motility. Testicular and epididymal sperm numbers in A. rufescens and P. tridactylus were relatively high (approximately 17.5-50 x 10(6)). In A. rufescens, approximately 69% of all epididymal sperm were located in the cauda epididymidis compared with approximately 40% in P. tridactylus. This study demonstrated that marked radial displacement of the dense fibres is probably closely associated with the ability to develop a sinusoidal mode of progressive movement, and that this feature of the sperm tail structure is not just linked with sperm size. Sperm size, however, is associated with sperm velocity.

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.

Antiserum to the egg coats of the fat‐tailed dunnart (marsupialia, dasyuridae) cross‐reacts with egg coats of other marsupial and eutherian species

We are examining the extracellular coats of the brush-tailed possum as a possible target for an immunocontraceptive vaccine for biocontrol of this pest species in New Zealand. In this study we have compared the composition of the extracellular coats of the fat-tailed and stripe-faced dunnarts, brush-tailed possum, domestic rabbit, and laboratory mouse using histochemistry, immunocytochemistry, and immunofluorescence. The histochemistry of the luminal epithelium of the oviduct and mucoid coats of the marsupials and rabbit indicated that they contain acidic glycoproteins. Immunofluorescence showed that polyclonal antiserum raised against the extracellular coats of the oocyte and early embryo of the fat-tailed dunnart, cross-reacted with the extracellular coats of the oocytes of all five species. These results suggest that there are common epitopes on the extracellular coats of oocytes and early embryos of distinctly related therian species. Further work to characterise these proteins is required to determine whether there is close homology between the oviductal glycoproteins of these species.

Leaf oxalate content of Eucalyptus spp. and its implications for koalas (Phascolarctos cinereus) with oxalate nephrosis

Abstract. Oxalate nephrosis is a leading disease of the Mount Lofty Ranges koala population in South Australia, but the cause is unclear. In other herbivorous species, a common cause is high dietary oxalate; therefore this study aimed to determine the oxalate content of eucalypt leaves. Juvenile, semimature and mature leaves were collected during spring from eucalypt species eaten by koalas in the Mount Lofty Ranges and compared with those from Moggill, Queensland, where oxalate nephrosis has lower prevalence. Total oxalate was measured as oxalic acid by high-performance liquid chromatography. The oxalate content of eucalypts was low (<1% dry weight), but occasional Mount Lofty leaf samples had oxalate levels of 4.68–7.51% dry weight. Mount Lofty eucalypts were found to be higher in oxalate than those from Queensland (P < 0.001). In conclusion, dietary oxalate in eucalypt leaves is unlikely to be the primary cause of oxalate nephrosis in the Mount Lofty koala population. However, occasional higher oxalate levels could cause oxalate nephrosis in individual koalas or worsen disease in those already affected. Further studies on the seasonal variation of eucalypt leaf oxalate are needed to determine its role in the pathogenesis of oxalate nephrosis in koalas.