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.

Necropsy findings of koalas from the Mount Lofty Ranges population in South Australia.

OBJECTIVE: This study reports necropsy findings of koalas from the Mount Lofty Ranges region in order to identify health threats to this mainland South Australian population. METHODS: Koalas from the Mount Lofty Ranges region (n = 85) that had died or been euthanased on welfare grounds were examined at necropsy during 2012-13 at the School of Animal and Veterinary Sciences, University of Adelaide. Disease findings, approximate age, sex and body condition of koalas were recorded. Histopathological examination was undertaken on gross lesions and in suspect cases, skin scrapings taken for microscopy and PCR performed for Chlamydia pecorum detection. RESULTS: Traumatic injury was the most common necropsy finding (48/85; 57%), caused by motor vehicle accidents (35/48; 73%), canine attacks (11/48; 23%) or bushfire burns (2/48; 4%). Oxalate nephrosis (27/85; 32%) was also more common than other conditions. Infectious diseases included chlamydiosis (10/85; 12%) and sarcoptic mange (7/85; 8%). Marked testis asymmetry was evident in 11% (6/56) of males, with histopathology suggestive of atrophic change in four animals. Other pathological conditions included gastrointestinal disease (7/85; 8%) and respiratory disease (3/85; 4%). Almost half of the koalas (38/85; 45%) were found to have two or more abnormalities at necropsy. CONCLUSION: This study found trauma, mainly from motor vehicle accidents, and oxalate nephrosis to be the predominant causes of death and/or disease in koalas from the Mount Lofty Ranges region. Recent emergence of both clinical chlamydiosis and sarcoptic mange has also occurred, providing insight into the health status and causes of disease or injury in this South Australian mainland koala population.

Effects of whole-body heat on male germ cell development and sperm motility in the laboratory mouse.

This study investigated the effects of high temperatures on male germ cell development and epididymal sperm motility of laboratory mice. In Experiment 1, adult males (n=16) were exposed to whole-body heat of 37-38°C for 8h day(-1) for 3 consecutive days, whereas controls (n=4) were left at 23-24°C. In Experiment 2, adult mice (n=6) were exposed to 37-38°C for a single 8-h period with controls (n=6) left at 23-24°C. Experiment 2 was conducted as a continuation of previous study that showed changes in spermatozoa 16h after exposure to heat of 37-38°C for 8h day(-1) for 3 consecutive days. In the present study, in Experiment 1, high temperature reduced testes weights 16h and 14 days after exposure, whereas by Day 21 testes weights were similar to those in the control group (P=0.18). At 16h, 7 and 14 days after exposure, an increase in germ cell apoptosis was noticeable in early and late stages (I-VI and XI-XII) of the cycle of the seminiferous epithelium. However, apoptosis in intermediate stages (VII-X) was evident 16h after heat exposure (P<0.05), without any change at other time periods. By 21 days, there were no significant differences between heat-treated groups and controls. Considerably more caspase-3-positive germ cells occurred in heat-treated mice 16h after heat exposure compared with the control group (P<0.0001), whereas 8h after heat in Experiment 2, sperm motility was reduced with a higher percentage of spermatozoa showing membrane damage. In conclusion, the present study shows that whole-body heat of 37-38°C induces stage-specific germ cell apoptosis and membrane changes in spermatozoa; this may result in reduced fertility at particular times of exposure after heating.

Pathological features of oxalate nephrosis in a population of koalas (Phascolarctos cinereus) in South Australia.

The wild and captive koala population of the Mt Lofty Ranges in South Australia has a high level of renal dysfunction in which crystals consistent with calcium oxalate have been observed in the kidneys. This study aimed to describe the pathological features of the renal disease in this population, confirm the composition of renal crystals as calcium oxalate, and determine whether any age or sex predispositions exist for this disease. A total of 51 koalas (28 wild rescues, 23 captive) were examined at necropsy, of which 28 (55%) were found to have gross and/or histological evidence of oxalate nephrosis. Histopathological features included intratubular and interstitial inflammation, tubule dilation, glomerular atrophy, tubule loss, and cortical fibrosis. Calcium oxalate crystals were demonstrated using a combination of polarization microscopy, alizarin red S staining, infrared spectroscopy, and energy-dispersive X-ray analysis with scanning electron microscopy. Uric acid and phosphate deposits were also shown to be present but were associated with minimal histopathological changes. No significant differences were found between the numbers of affected captive and wild rescued koalas; also, there were no sex or age predispositions identified, but it was found that oxalate nephrosis may affect koalas <2 years of age. The findings of this study suggest that oxalate nephrosis is a leading disease in this koala population. Possible causes of this disease are currently under investigation.

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.

The mechanistic basis for sexual dysfunction in male transforming growth factor beta1 null mutant mice.

The cytokine transforming growth factor beta1 (TGFB1) is implicated in male sexual function. Previous behavioral studies show that Tgfb1 null mutant mice mount and display limited intromission behavior with receptive females but are unable to complete successful copulation. The studies presented here explore the physiologic basis for sexual dysfunction in Tgfb1 null mutant males. Scanning electron microscopy revealed that the surface of the penis in Tgfb1 null mutant males was abnormally coated in superficial keratinized epithelial cells. There was a significant reduction in protrusion of penile spines through the superficial tissue in Tgfb1 null mutant mice; in some mice, the spines were almost completely embedded. Histologic analysis revealed reduced skin thickness in the penis of Tgfb1 null mutant males. Nerve fibers, endothelial cells, smooth muscle actin, macrophages, and neuronal and inducible nitric oxide synthase were present in similar abundance and location in Tgfb1 null mutant mice compared with wild-type controls; however, an increase in collagen I deposition was detected. Behavioral studies revealed that Tgfb1 null mutant males undergo spontaneous noncontact erections, albeit at a reduced rate compared with control mice, and engage in less frequent genital grooming activity. These studies suggest that Tgfb1 null mutation may adversely influence copulatory behavior through effects on both altered structural integrity of the penile skin and impaired tissue compliance leading to erectile dysfunction.

Spawning dynamics and biomass estimates of an anchovy Engraulis australis population in contrasting gulf and shelf environments.

The spawning biomass of Australian anchovy Engraulis australis in gulf and shelf waters of South Australia was compared using the daily egg production method (DEPM). The total survey area was 128 700 km2 with recorded spawning areas in gulf and shelf waters of 4898 and 44 618 km2, respectively. High egg densities in the warm, shallow gulf waters were produced by small, young (<1 year old) E. australis that spawned relatively small batches of eggs (c. 855) approximately every 3 days. In cooler, deeper shelf waters, where larger, older E. australis are found, lower egg densities occurred despite individuals producing much larger batches of eggs (c. 15,572) approximately every 7 days. In shelf waters, the highest densities were recorded at inshore sampling stations. Spawning appeared to peak between 0000 and 0100 hours. Females were more abundant than males in samples from both gulf and shelf waters with sex ratios of 0.61 and 0.56, respectively. The spawning biomass of E. australis in shelf waters was 101 522 t, whereas the estimate for gulf waters was 25 374 t. Due to the differences in mean size of the spawning females, however, c. 6x10(9)E. australis were present in each region. The results support the hypothesis that variability in habitat conditions may directly influence E. australis reproduction. A large reserve of young fish in the relatively stable gulf environment may increase the resilience of the E. australis population in South Australia to unfavourable interannual changes in offshore environmental conditions.

Ovarian follicular superstimulation and oocyte maturation in the anoestrous southern hairy-nosed wombat, Lasiorhinus latifrons.

This study investigates the effect of three exogenous gonadotrophin regimens on ovarian follicular development in southern hairy-nosed wombats during the non-breeding season. Females were given either porcine follicle stimulating hormone (pFSH; total of 200 mg at 12 h intervals over 7 (Group 1), or 4 days (Group 2)), or pregnant mares' serum gonadotrophin (PMSG; single dose of 150 I.U. (Group 3)). In all treatment groups 25 mg of porcine luteinising hormone (pLH) was used to trigger maturation; Groups 1 and 2 received pLH 12 h after the final pFSH injection and Group 3 received pLH 72 h after PMSG. The results showed Group 1 produced significantly more follicles per ovary (5.91+/-1.28) than Group 2 (1.67+/-0.62), or Group 3 (2.17+/-1.16) at p<0.05. Control females received saline injections concurrently with the three treatment groups (n=6; 2 control animals for each treatment group). No follicular development occurred in any control female. Analysis of oocyte nuclear status revealed that while oocytes from all three treatment groups had resumed meiosis, only those in Group 1 (7-day pFSH/pLH treatment) progressed to metaphase II. These results have implications for the development of assisted breeding strategies in this species.

Variation of sperm head shape and tail length in a species of Australian hydromyine rodent: the spinifex hopping mouse, Notomys alexis.

In Australia, there are around 60 species of murid rodents that occur in the subfamily Hydromyinae, most of which produce highly complex, monomorphic, spermatozoa in which the head has an apical hook together with two ventral processes containing filamentous actin and a long tail of species-specific length. One of the few exceptions to this is the spinifex hopping mouse, Notomys alexis, whose spermatozoa have previously been shown to have pleiomorphic heads. In this study, the structural organisation of the sperm head has been investigated in more detail and the variability in length of the midpiece and total length of the sperm tail has been determined for this species. The findings confirm that pleiomorphic sperm heads are invariably present in these animals and that this variability is associated with that of the nucleus, although nuclear vacuoles were not evident. The total length of the sperm tail, as well as that of the midpiece, was also highly variable both within, as well as between, individual animals. The reason(s) for this high degree of variability in sperm morphology is not known but it may relate to a relaxation of the genetic control of sperm form owing to depressed levels of inter-male sperm competition.

Effect of exogenous gonadotrophins on ovarian morphology and oocyte maturation in the southern hairy nosed wombat Lasiohinus latifrons during the breeding season.

The effect of the exogenous administration of porcine follicle-stimulating hormone (pFSH) and pregnant mare serum gonadotrophin (PMSG) on ovarian follicular development and oocyte maturation in the southern hairy nosed wombat Lasiorhinus latifrons was investigated. Three experimental groups were administered pFSH at various doses and for different treatment lengths, followed by 25 mg porcine luteinising hormone (pLH) 12 h after the last dose of pFSH. Another group was given PMSG followed 72 h later by 25 mg pLH. Animals were killed 24 h after pLH. The left ovary was fixed for histology and the morphology of the antral follicles was determined, whereas follicular oocytes in the right ovary were aspirated, fixed, stained with 42,62-diamidino-2-phenylindole, and viewed for nuclear maturation. There was no significant difference in the mean number of ovarian follicles >1 mm, or in the size class of follicles assessed between control and experimental groups. However, a trend was observed suggesting a possible increase in follicles >3.0 mm in experimental groups compared with control animals. In all females administered exogenous porcine gonadotrophins, but not controls, some of the mural granulosa cells of large tertiary antral follicles had markedly enlarged nuclei (approximately 14 microm in diameter). All oocytes from the control group remained at the germinal vesicle stage, whereas approximately 40% of oocytes retrieved from the pFSH groups and 82.4% retrieved from the PMSG-primed animals had undergone germinal vesicle break down, with a small number reaching meiosis II. The present study shows that exogenous administration of either pFSH or PMSG to hairy nosed wombats can induce follicular growth and oocyte maturation. Such findings could be useful in the development of reproductive technology in this species.

Comparative study of sperm chromatin condensation in the excurrent ducts of the laboratory mouse Mus musculus and spinifex hopping mouse Notomys alexis.

In most mammals, post-testicular sperm maturation is completed in the caput and corpus epididymides, with storage occurring in the cauda epididymides. However, in the spinifex hopping mouse, Notomys alexis, epididymal sperm transit is rapid and some sperm storage occurs in the distal region of the vas deferens. The aim of the present study was to determine whether the rapid progression of sperm into the vas deferens in the hopping mouse results in late sperm maturation. To determine this, sperm nuclei from the epididymides and vasa deferentia of laboratory and hopping mice were compared for: (1) thiol content after staining with monobromobimane (mBBr); (2) chromatin resistance to acid denaturation following incubation with acetic alcohol and staining with acridine orange; and (3) chromatin resistance to in vitro decondensation after incubation with 1% sodium dodecyl sulfate (SDS). It was found that, whereas laboratory mouse sperm completed chromatin condensation by the time they reached the cauda epididymidis, hopping mouse sperm nuclei from the vas deferens showed significantly less mBBr fluorescence and a greater proportion of sperm were resistant to decondensation with SDS than those in the cauda epididymidis. Therefore, the results of the present study indicate that, unlike in the laboratory mouse, hopping mouse chromatin condensation of spermatozoa continues in the vas deferens and this may be due, at least in part, to rapid epididymal transit.

Morphogenesis of the fibrous sheath in the marsupial spermatozoon.

The spermatozoon fibrous sheath contains longitudinal columns and circumferential ribs. It surrounds the axoneme of the principal piece of the mammalian sperm tail, and may be important in sperm stability and motility. Here we describe its assembly during spermiogenesis in a marsupial, the brush-tail possum, and compare its structural organization with that of eutherian mammals, birds and reptiles. Transmission electron microscopy showed that possum fibrous sheath assembly is a multistep process extending in a distal-to-proximal direction along the axoneme from steps 4 to 14 of spermiogenesis. For the most part, assembly of the longitudinal columns occurs before that of the circumferential ribs. Immunohistochemical and immunogold labelling showed that fibrous sheath proteins are first present in the spermatid cytoplasm; at least some of the proteins of the sheath precursors differ from those in the mature fibrous sheath. That immunoreactivity develops after initiation of chromatin condensation suggests that fibrous sheath proteins, or their mRNAs, are stored within the spermatid cytoplasmic lobule prior to their assembly along the axoneme. These findings are similar to those in laboratory rats, and thus suggests that the mode of fibrous sheath assembly evolved in a common ancestor over 125 million years ago, prior to the divergence of marsupial and eutherian lineages.

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.

A comparative study of sperm production in two species of Australian arid zone rodents (Pseudomys australis, Notomys alexis) with marked differences in testis size.

The plains rat, Pseudomys australis, and the spinifex hopping mouse, Notomys alexis, show marked differences in the size of their testes and in the number of spermatozoa within the epididymides. In the present study, the dynamics of sperm production and the duration of sperm transit along the male excurrent ducts were compared between these two species. The durations of the cycle of the seminiferous epithelium, spermatogenesis and sperm transit were determined by tracking cells using autoradiography after [(3)H]thymidine incorporation. Daily sperm production was determined from counts of testicular spermatids after homogenization and further estimates of sperm transit were obtained by dividing sperm reserves within the various regions of the extratesticular ducts by the daily sperm production of the attached testis. In the plains rat, the mean duration of the cycle of the seminiferous epithelium was 11.2 days, the duration of spermatogenesis was 45 days, daily sperm production was 2.6 x 10(7) spermatozoa per gram of testis and epididymal transit of spermatozoa took approximately 9 days (caput 0.8 days; corpus 1.5 days; cauda 6.5 days). In contrast, in the hopping mouse, the mean duration of the cycle of the seminiferous epithelium was 14 days, the duration of spermatogenesis was 56 days and daily sperm production per gram of testis was < 1.0 x 10(7). Epididymal transit of spermatozoa was completed in about 4 days (caput + corpus < 1 day; cauda 3 days); however, spermatozoa may be stored for an additional 1.5-2.0 days in the vas deferens. These results indicate that, in addition to small testes, the hopping mouse shows a low efficiency of sperm production, a relatively long duration of spermatogenesis and rapid passage of spermatozoa through the epididymis, all of which contribute to low epididymal sperm counts. These data are considered in relation to interspecific differences in sperm competition.

Isolation and partial characterization of the outer dense fibres and fibrous sheath from the sperm tail of a marsupial: the brushtail possum (Trichosurus vulpecula).

The flagellum of a mammalian spermatozoon consists of a central axoneme surrounded by two cytoskeletal structures, the outer dense fibres and the fibrous sheath, which may aid in sperm motility or stability. In this study the outer dense fibres and fibrous sheath were isolated and partially characterized in a marsupial species, the brushtail possum (Trichosurus vulpecula). Spermatozoa from the cauda epididymidis were decapitated by sonication, and the head and tail fractions were separated by centrifugation over a 20, 40 and 60% (w/v) sucrose density gradient. After confirming sperm tail purity by Nomarski microscopy, the tails were incubated in either SDS-dithiothreitol to isolate the outer dense fibres or urea-dithiothreitol to isolate the fibrous sheaths. Purified outer dense fibres and fibrous sheaths were solubilized in SDS and beta-mercaptoethanol and proteins were separated by one-dimensional PAGE. Coomassie blue staining showed that the outer dense fibres were composed of seven major proteins (molecular masses: 73, 58, 55, 54, 52, 41 and 16 kDa), and the fibrous sheath was composed of 12 major proteins (molecular masses: 106, 76, 66, 62, 55, 53, 52, 46, 40, 30, 28 and 16 kDa). A polyclonal antibody to the fibrous sheath proteins showed strong crossreactivity with those of fibrous sheath from spermatozoa of several other marsupial species, as well as those from laboratory rats. Subsequent western blotting identified the immunoreactive 76 and 62 kDa proteins from all species, thus indicating their high conservation between species. No crossreactivity of the fibrous sheath antibody to any other cytoskeletal structures, including the outer dense fibres, mid-piece fibre network or connecting laminae, or to the acrosome or underlying subacrosomal material, was evident, indicating that the fibrous sheath proteins are localized to this structure alone. Further work is in progress to determine the extent of homology of these proteins to those in eutherian mammals.

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.

Protein composition of the ventral processes on the sperm head of Australian hydromyine rodents.

The sperm head of the plains rat, an Australian hydromyine rodent, is highly complex in structure and contains, in addition to an apical hook, two large ventral processes (VPs) that extend from its upper concave surface and that are largely composed of a huge extension of the sperm head cytoskeleton surrounded by postacrosomal dense lamina. In this study we have attempted to determine their protein composition. For this, the VPs were isolated, the proteins within them separated by SDS-PAGE, and the resultant polypeptide bands Western blotted and probed with antibodies against laboratory rat perforatorial and bull perinuclear theca sperm proteins. Antibodies were also used to determine the perforatorial and perinuclear theca proteins by immunogold labeling of transmission electron microscopic sections. The results indicate that the material within the VPs is largely composed of perforatorial cross-reacting proteins together with F-actin with the dominant protein being PERF 15. The perinuclear theca proteins are, by contrast, restricted to a narrow region adjacent to the acrosomal and nuclear membranes. In conclusion, this study has shown that the VPs of the spermatozoa of Australian rodents are perforatorial-like appendages that contain similar proteins to the perforatorium of the apical hook together with F-actin; their functional significance remains unknown.

Interspecific variation of zona pellucida glycoconjugates in several species of marsupial.

The zona pellucida glycoconjugate content of several marsupial species was investigated using differential lectin histochemistry. Ovaries from fat-tailed dunnarts, a southern brown bandicoot, grey short-tailed opossums, brushtail possums, ringtail possums, koalas and eastern grey kangaroos were fixed, embedded in paraffin wax, sectioned and stained with ten fluorescein isothiocyanate-conjugated lectins. Sections were also incubated with either neuraminidase or saponified, respectively, before incubation with the lectins to identify saccharide residues masked by sialic acids or O-acetyl groups on sialic acids. The zonae pellucidae surrounding the oocytes of the marsupials demonstrated interspecific variation in glycoconjugate content, with mannose-containing glycoconjugates exhibiting the greatest variation. Some of the zona pellucida glycoconjugates of all species, except those of the opossums, were masked by sialic acid with an increase in fluorescence with lectins from Arachis hypogea (PNA), and Glycine max (SBA), after desialylation. The disaccharide beta-galactose(1-4)N-acetyl-D-glucosamine appeared to be conformationally masked by O-acetyl groups of sialic acids in the zonae pellucidae of all species, with an increase in fluorescence with the lectin from Erythrina cristagalli (ECA), after saponification. Similar intensity and localization of beta-(1-4)-N-acetyl-D-glucosamine, as shown by staining of the lectin from Triticum vulgaris (WGA), to the inner and outer regions of the zona pellucida, were found to those reported in eutherian species. WGA fluorescence became uniform throughout the zonae pellucidae after saponification, indicating differential O-acetylation of sialic acids on the internal compartment of the zonae pellucidae.