The highly divergent spermatozoon of the Palawan spiny rat, Maxomys panglima has an extremely long tail.

CONTEXT: Sperm morphology varies greatly across mammalian species and this variability is especially evident in murid rodents with both sperm head shape and tail length being sexually selected traits. The Palawan spiny rat, Maxomys panglima has a longer sperm tail than that currently recorded for any other mammalian species. AIMS: The aim of the current study was to determine the sperm morphology of an individual Palawan spiny rat, M. panglima . METHODS: Light and transmission electron microscopy were carried out. KEY RESULTS: We found that the sperm tail of M. panglima has an average length of 380µm with the midpiece being approximately 185µm in length with comparatively small mitochondria but very large coarse fibres. Furthermore, the sperm head has a less acutely flexed apical hook than that of most other murid rodents including those of several other Maxomys species. CONCLUSIONS: The Palawan spiny rat has a highly divergent sperm morphology with an extremely long tail. It may turn out to be an important species for testing various hypotheses of sperm form and function in mammals. IMPLICATIONS: These findings suggest markedly different selective pressures may have resulted in this unique sperm morphology, the functional significance of which remains to be determined.

Sperm morphology in the Malagasy rodents (Muroidea: Nesomyinae).

The morphology of the spermatozoon of representative species of the subfamily Nesomyinae (Muroidea: Nesomyidae), a monophyletic group of rodents endemic to Madagascar, was examined by light and electron microscopy to determine the sperm head shape and tail length across the species. Marked interspecific differences were found to occur in both the form of the sperm head and length of the tail. The species that possess a sperm head with an apical hook, which largely contains acrosomal material, generally displayed longer sperm tails, and a species with a spatulate sperm head had the shortest tail. The association between sperm head shape and tail length mirrors that previously found in Eurasian and Australasian murine rodents. Thus, the repeated association between sperm head shape and tail length across these groups of muroid rodents clearly indicates a functional relationship between these two features. A comparison of sperm morphology of the nesomyines to that of related muroid rodents on the mainland of Africa suggests that the possession of an apical hook is the ancestral condition.

Unusual germ cell organization in the seminiferous epithelium of a murid rodent from southern Asia, the greater bandicoot rat, Bandicota Indica.

In the greater bandicoot rat, Bandicota indica, of south-east Asia, nine cell associations were documented in the testicular seminiferous epithelium. In about 10% of the tubule cross sections two or more cell associations occurred and, furthermore, some of the generations of germ cells within the cell associations were sometimes either out of phase, or missing, in the tubule cross sections. These features, together with the fact that this species has a highly pleiomorphic sperm head shape, are somewhat reminiscent of those of the seminiferous epithelium in humans and some other primates but not of common laboratory rodents. This species could thus be a good model for investigating irregular patterns of spermatogenesis in naturally occurring wild species of rodent.

Studies on sperm storage in the vas deferens of the spinifex hopping mouse (Notomys alexis).

The cauda epididymidis, with its relatively cool temperature (32-35 degrees C), is considered to be the main site of sperm storage in male mammals. However, in the adult male spinifex hopping mouse, Notomys alexis, similar numbers of spermatozoa are found in the vas deferens to those in the cauda epididymidis. The present study shows that, unlike in the laboratory mouse in which spermatozoa of the vas deferens are found mainly in the epididymal region of the duct, spermatozoa in the hopping mouse are localized mainly to the middle and urethral regions of the vas deferens which lies in the inguinal and lower abdominal region of the body cavity. After ligation of the vas deferens close to its connection with the epididymis, many spermatozoa in the vas deferens retain the potential for motility for up to 2 weeks, indicating that the viability of spermatozoa is not compromised by being restricted to core body temperature. This urethral region of the vas deferens, in which spermatozoa reside, has a highly divergent structural organization compared with that of common laboratory rodents in which there is an expanded lumen with a network of epithelial folds. Ultrastructural observations of the cells lining the duct indicate that there are not any marked differences in morphology compared with the cells lining the duct in common laboratory murids, but the infoldings of the vas deferens of the hopping mouse are highly vascular which might facilitate supply of oxygen and nutrients to the spermatozoa residing in the lumen.

Glycoconjugates on the surface of epididymal spermatozoa in a marsupial, the brushtail possum, Trichosurus vulpecula.

Variation in localization and distribution of saccharides on the sperm surface of a marsupial, the brushtail possum, Trichosurus vulpecula, was compared between spermatozoa from the caput and cauda epididymides. Spermatozoa were subjected to the following treatments: (i) unfixed and fixed spermatozoa were stained with fluorescein-labelled lectins; (ii) unfixed spermatozoa were incubated with lectins for determination of agglutination; and (iii) spermatozoa were incubated with detergent to remove the plasmalemma, the glycoproteins were separated on SDS-PAGE and western blots were stained with biotinylated lectins. Many of the fluorescein isothiocyanate (FITC)-labelled lectins bound selectively to the sperm surface, and marked differences were found in lectin staining affinity between caput and cauda epididymal spermatozoa. Incubation of spermatozoa from the cauda epididymidis with neuraminidase reversed many of the differences in staining of the cauda epididymal spermatozoa, indicating masking of some terminal saccharides by sialic acid. Agglutination of spermatozoa from the caput epididymidis occurred after incubation with Concanavalin A (ConA) and soybean agglutinin (SBA), but agglutination was less extensive for spermatozoa from the cauda epididymidis. Western blot analysis indicated several ConA-positive bands in caput sperm extracts, but fewer positive bands in the cauda sperm extracts, whereas SBA stained four bands from caput but none from the cauda epididymal spermatozoa. These results demonstrate extensive glycosylation of the surface proteins of spermatozoa from the caput epididymidis and significant differences in spermatozoa from the cauda epididymidis. In general, the findings indicate similar glycosylation of the surface of marsupial spermatozoa to those from eutherian mammals despite marked differences in their morphology and early divergence of marsupials from eutherian mammals. It would appear that this situation differs markedly from that in sub-mammalian vertebrates.

The structural organisation of sperm head components of the wombat and koala (suborder: Vombatiformes): an enigma amongst marsupials.

The sperm head structural organisation of the koala and hairy-nosed wombat, and the effects of varying concentrations of the ionic detergent, Triton X-100, on its component parts, were determined by electron microscopy. Although alike in form between the 2 species, the sperm nucleus of the former, but not the latter, had nuclear vacuoles and appeared to be more easily dispersed by the Triton X-100. The structure of the acrosome of the spermatozoon was similar between the 2 species and, in both, previously undescribed thin posterior and lateral segments were found to be present. It is suggested that this thin segment may facilitate sperm-zona and/or sperm-oolemma binding and fusion.

Immunocytochemical identification of islet cells containing calcitonin gene-related peptide-like immunoreactivity in the plains rat pancreas (Pseudomys australis).

BACKGROUND/AIM: Calcitonin gene-related peptide (CGRP) is a product of alternative RNA processing of the calcitonin gene. Its dual occurrence in nerves and endocrine cells makes it likely to be both a neuropeptide and an endocrine (paracrine) messenger. The purpose of this study was to determine whether CGRP-containing cells are present in the pancreas of the plains rat (Pseudomys australis). METHODS: Paraffin embedded tissue from four adult plains rats was sectioned and stained using an immunocytochemical avidin-biotin-peroxidase (ABC) method. The sections were incubated with rabbit polyclonal rat CGRP (Peninsula Labs, Belmont, CA) diluted 1:1000, in a solution consisting of 1% bovine serum albumin and 0.05% sodium azide in 0.1 mol/L of PBST overnight in a humid chamber at room temperature. Immunolocalization was shown in the islets of Langerhans tissue. A negative control using normal serum was used to test the specificity of the individual immunoreaction and on all occasions there was no immunostaining. RESULTS: For the purpose of colocalization of CGRP immunoreactive cells, mirror images of stained sections were stained for CGRP and insulin, somatostatin, glucagon and pancreatic polypeptide. The immunostained cells of the same islet were assessed for colocalization and found to be identical with insulin cells. CONCLUSION: The dual localisation of CGRP immunoreactivity in neuronal and endocrine elements provide further evidence for both a neurocrine and hormonal role for CGRP in endocrine and exocrine functions. In the plains rats, we have shown CGRP to be an endocrine peptide.

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.

Immunocytochemical identification of cells containing insulin, glucagon, somatostatin, and pancreatic polypeptide in islets of Langerhans of the wombat pancreas with electron microscopy.

The endocrine pancreas of the southern hairy-nosed wombat (Lasiorhinus latifrons) was investigated by means of electron-microscope immunocytochemistry using the protein A--gold technique on London resin (LR) white-embedded tissue. The primary antibodies used were raised against insulin, glucagon, somatostatin and pancreatic polypeptide. The morphology of the secretory granules differed in the four cell types. The insulin cells are pleiomorphic, and the secretory granules composed of an electron-dense core surrounded by an electron-lucent halo. The glucagon cells possess granules of varying density. Somatostatin cells have large, less dense secretory granules. The pancreatic polypeptide cells show small dense secretory granules. It is essential that it be corroborated by immunocytochemical data at the light or preferably electron-microscopical level for the definite identification of endocrine cell types. Recent developments in immuno-electron-microscopic techniques have contributed to a better knowledge of cells responsible for the secretion of a wide variety of hormones, as in this study.

Successful embryo transfer in a small dasyurid marsupial, Sminthopsis crassicaudata.

A technique for the transfer of early embryos from the uterus of donors to recipient females of a small dasyurid marsupial, Sminthopsis crassicaudata , is described. When the recipients were killed 3 to 5 d later, a few expanded blastocysts were found to be present in the uteri of most of the females provided that corpora lutea were present in the ovaries. This study demonstrates successful embryo transfer in a small marsupial species and supports the view that secretion of the endometrium due to luteal activity may be necessary for development of the expanded unilaminar blastocyst stage.

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.

Sphero-echinocytosis of human red blood cells caused by snake, red-back spider, bee and blue-ringed octopus venoms and its inhibition by snake sera.

It was found that bee (Apis mellifera) venom, red-back spider (Latrodectus mactans) venom, blue-ringed octopus (Hapalochlaena maculosa) venom, ten different snake venoms, phospholipase A2 and four snake toxins caused sphero-echinocytosis of human red blood cells at 200 ng/ml. Most venoms and toxins lost the ability to deform human red blood cells when their components of less than mol. wt 10,000 were applied. In a number of cases the sphero-echinocytotic effect was also inhibited by blood sera of Notechis scutatus and Pseudonaja textilis.

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.

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.

Effect of cryopreservation on development and ultrastructure of preimplantation embryos from the dasyurid marsupial Sminthopsis crassicaudata.

The effect of different cryopreservation methods on the development and ultrastructure of preimplantation embryos of Sminthopsis crassicaudata, a small carnivorous marsupial and member of the family Dasyuridae, was investigated. Females were primed with 1 iu pregnant mares' serum gonadotrophin to induce oestrus and ovulation. Mating generally ensued and, approximately 6 days after priming, embryos were collected and cultured in 5% CO2 in air at 35 degrees C for 18-22 h in either Dulbecco's modified Eagles medium (DMEM) with high glucose or human tubal fluid medium (HTF), both supplemented with 10% fetal calf serum. Cleavage rates were higher in DMEM than in HTF. One slow and two ultrarapid freezing methods were used. Two out of 12 (17%) embryos cleaved in culture after freezing and thawing using the slow regimen, compared with six of 16 (38%) non-frozen controls. In addition, two of 11 (18%) embryos cleaved in culture following ultrarapid freezing and thawing by one of the two methods, compared to 31 of 41 (76%) non-frozen controls. Most of the embryos appeared morphologically normal under the light microscope after freezing and thawing by the slow regimen, but considerable variation in the degree of ultrastructural damage to the cellular organelles was evident with the transmission electron microscope. The rather low rate of cleavage after freezing and thawing was probably due, at least in part, to ultrastructural damage of the cells.

Unusual nuclear structure of the spermatozoon in a marsupial, Sminthopsis crassicaudata.

The organization of sperm chromatin in the dasyurid marsupial, Sminthopsis crassicaudata, was investigated using various morphological techniques. Transmission electron microscopy indicates two quite distinct chromatin regions became evident late in spermiogenesis with an outer globular region containing blocks of very electron-dense chromatin. Fluorescent light microscopical studies after staining with DNA dyes and 7-amino actinomycin D of testicular, caput, and cauda epididymal spermatozoa showed that this region fluoresced less brightly than the rest of the nucleus, indicating the presence of fewer DNA binding sites. Freeze fracture showed that the chromatin in most of the nucleus had randomly arranged particles of various sizes, but that of the outer region was composed entirely of small particles. This outer region was more resistant to low concentrations of the ionic detergent, SDS, whereas both guanidine hydrochloride and urea together with sodium chloride generally dispersed all the chromatin except that in the outer globular region and in a localized area of the nucleus beneath the acrosome. This study has thus revealed that the outer globular chromatin of these spermatozoa responds differently to ionic detergents and protein denaturing agents and has a different chromatin organization than most of the rest of the nucleus. The significance of these differences remains, however, to be determined.

The endocrine pancreas in the Australian wedge-tailed eagle (Aquila audax)--an immunocytochemical study.

The endocrine pancreas of the Australian wedge-tailed eagle, Aquila audax, was investigated by means of immunocytochemistry using the PAP method at the light microscopic level. Serial paraffin sections were stained with haematoxylin and eosin, aldehyde fuchsin, and Grimelius' silver impregnation method in each of the lobes-dorsal, ventral, third and splenic lobes to show the morphology of the islet, B cells, and A cells respectively. The islets were of the 'mixed type' with no segregation into 'light' and 'dark' islets as in other birds studies (Falkmer and Ostberg, 1977). Semithin serial plastic sections were stained individually with primary antibodies for insulin, glucagon, somatostatin and pancreatic polypeptide (PP) in each of the four lobes. Positive regional differences noted were an increased concentration of insulin, glucagon, somatostatin and pancreatic polypeptide cells in the splenic lobe. Pancreatic polypeptide cells were present in all the lobes. There was no reciprocal relationship between PP cells and glucagon cells in the splenic lobe. These results in the feral bird (Aquila audax) differed from those of previous studies in domestic fowl pancreas (Phasianidae) (Tomita et al., 1985; Falkmer, 1985).

Marsupial fertilization: some further ultrastructural observations on the dasyurid Sminthopsis crassicaudata.

Various morphological aspects of in vivo egg maturation and sperm-egg interaction were investigated in the Australian marsupial Sminthopsis crassicaudata with the transmission and scanning electron microscopes. Cortical granules invariably occurred in primary oocytes, with the number increasing after resumption of the first meiotic division. They generally occurred close to the oolemma, including the region near the oocyte nucleus. After mating, spermatozoa with intact acrosomes, which had a homogeneous electron-dense matrix, were found on the outer zona surface, but loss of acrosomal contents had occurred by the time of zona penetration. Sperm incorporation into the egg took place at the metaphase II stage of meiosis, and, at this time, cortical granules disappeared from the egg cortex. Sperm heads with condensed chromatin in the egg cytoplasm had an electron-dense layer of subacrosomal material over part of the dorsal nuclear surface, but no membranes were present around these incorporated spermatozoa. Sperm chromatin decondensation resulted in an elevation of egg cytoplasm, and the cell membrane over this area lacked microvilli. The pronuclear envelope was not laid down until after chromatin decondensation had occurred. By this time the fertilized egg had reached the uterus, and a smooth, electron-dense, shell membrane had been deposited. These observations, together with our previous findings, indicate that some of the processes of sperm-egg interaction are similar to those in eutherian mammals, whereas others appear highly divergent.

A light-microscopic immunocytochemical study of the endocrine pancreas in the Australian fat-tailed dunnart (Sminthopsis crassicaudata).

The endocrine pancreas of the Australian fat-tailed dunnart (Sminthopsis crassicaudata) was investigated by means of immunocytochemistry using the avidin-biotin-peroxidase technique. This was a light microscopic study using this established technique and has not been previously investigated. Serial paraffin sections were stained individually with primary antibodies for anti-porcine glucagon, anti-beef pork insulin, anti-human somatostatin, and anti-avian pancreatic polypeptide (APP), anti-bovine pancreatic polypeptide (BPP), anti-serotonin, anti-porcine motilin, showing the same islet. Cells immunoreactive to porcine glucagon, porcine insulin, human somatostatin, APP, BPP were found in endocrine islets, but BPP and APP also appear to be scattered amidst the exocrine portion. Immunoreactive cells were not observed with serotonin and anti-porcine motilin. All controls were negative. These results in the dunnart pancreas has shown four types of pancreatic endocrine cells. It has also shown that the structure of PP may more closely resemble BPP than APP. This study can be related to studies in echidnas (Tachyglossus aculeatus) and Australian possum (Trichosurus vulpecula). This is a part of an immunocytochemical study investigating the endocrine pancreas in Australian mammals.

A light-microscopic immunocytochemical study of the endocrine pancreas in the Australian brush-tailed possum (Trichosurus vulpecula).

The endocrine pancreas of the Australian brush-tailed possum (Trichosurus vulpecula) was investigated by means of immunocytochemistry using the avidin-biotin-peroxidase technique. This was a light microscopic study using this established technique. Serial paraffin sections were stained individually with primary antibodies for glucagon, insulin, somatostatin, and pancreatic polypeptide (PP), showing the same islet. Cells immunoreactive to glucagon, insulin, somatostatin and PP were found in endocrine islets. PP cells appear to be scattered amidst the exocrine portion also. Insulin immunoreactive cells were located in the central region of islet, glucagon in the periphery, somatostatin in periphery and had elongated processes. PP cells were more sparse and located both in the periphery of islet and amidst the exocrine tissue. These results can then be related to a similar study in the same marsupial, but using the immunofluorescence technique and to studies in other marsupials such as grey kangaroo (Macropus fuliginosus) fat-tailed dunnart (Sminthopsis crasicaudata) and the American opossum (Didelphis virginiana). These investigations are part of a study in Australian mammals.