A sperm-epididymal cell co-culture was shown to be capable of inducing the in vitro maturation of... more A sperm-epididymal cell co-culture was shown to be capable of inducing the in vitro maturation of spermatozoa from a marsupial species, the tammar wallaby (Macropus eugenii ). This system was able to maintain wallaby epididymal epithelial cells in vitro for more than 2 months. The system also enabled immature wallaby spermatozoa to differentiate from a T-shaped to a streamlined form, accompanied by the development of progressive motility after co-culture with epididymal cell monolayers that had been cultured for 7 days. The addition of inhibitors of actin polymerization (latrunculin A or B) to the co-culture system showed that wallaby sperm maturation was impaired by the interruption of actin organization within the immature spermatozoa. These results indicate that actin filaments play a significant role in sperm transformation during post-testicular maturation in marsupials. These observations also indicate that the marsupial coculture system has the potential to greatly increase understanding of sperm-epididymal cell interactions and the mechanism of sperm maturation in these species.
In this observational case report, we share our experience of achieving >40% LDL cholesterol r... more In this observational case report, we share our experience of achieving >40% LDL cholesterol reduction in four Chinese homozygous familial hypercholesterolaemia children below 8 years of age with a triple combination of atorvastatin, probucol, and ezetimibe for >6 years. Within a follow-up duration of 6-13 years, this triple therapy achieved significant reduction of LDL cholesterol as well as an impressive regression of xanthomas in all paediatric cases. All the children remained free from treatment-related adverse responses and cardiovascular events throughout follow-up.
We investigated the cycle of the seminiferous epithelium in a marsupial, namely the brushtail pos... more We investigated the cycle of the seminiferous epithelium in a marsupial, namely the brushtail possum (Trichosurus vulpecula), using semithin sections of seminiferous tubules embedded in Spurr's resin. Using 14 steps of spermatid development as markers, we were able to class tubular cross-sections into 10 well-defined stages of the seminiferous epithelial cycle. The duration of one cycle was 13.5 days, as determined by injections of [(3)H]-thymidine and autoradiographic examination of the most advanced sperm cells at 2 h and 17 days after injection. The durations of stages I-X were 21.4, 66.4, 54.1, 47.0, 29.8, 28.5, 25.3, 25.0, 12.0 and 15.9 h, respectively, estimated by the relative percentage of occurrence of each stage. It was estimated that the life spans of the main germ cells were as follows: type B spermatogonia, 5.4 days; primary spermatocytes, 16.7 days; secondary spermatocytes, 0.7 days; and spermatids, 21.4 days. The results suggest that the kinetics of spermatogenesi...
Rapid input-restricted change in gene expression is an important aspect of synaptic plasticity re... more Rapid input-restricted change in gene expression is an important aspect of synaptic plasticity requiring complex mechanisms of post-transcriptional mRNA trafficking and regulation. Small non-coding miRNA are uniquely poised to support these functions by providing a nucleic-acid-based specificity component for universal-sequence-dependent RNA binding complexes. We investigated the subcellular distribution of these molecules in resting and potassium chloride depolarized human neuroblasts, and found both selective enrichment and depletion in neurites. Depolarization was associated with a neurite-restricted decrease in miRNA expression; a subset of these molecules was recovered from the depolarization medium in nuclease resistant extracellular exosomes. These vesicles were enriched with primate specific miRNA and the synaptic-plasticity-associated protein MAP1b. These findings further support a role for miRNA as neural plasticity regulators, as they are compartmentalized in neurons and ...
This study was undertaken to identify potential molecular markers of acrosomal biogenesis and pos... more This study was undertaken to identify potential molecular markers of acrosomal biogenesis and post-testicular maturation in marsupials, using the tammar wallaby as a model species. A two-step sperm extraction procedure yielded two protein extracts of apparent acrosomal origin and a tail extract. The extracts were analysed by SDS-PAGE under reducing conditions. Several prominent polypeptide bands (45, 38 and 33 kDa) appeared common to both acrosomal extracts. Antiserum raised against the 33 kDa polypeptide from the inner acrosomal membrane matrix (IAMM) extract showed immunoreactivity with 45, 38 and 33 kDa polypeptides in both acrosomal extracts, indicating that the 33 kDa polypeptide was related to the proteins in the 45 and 38 kDa bands. Therefore, the antiserum was used as a molecular probe. Indirect immunofluorescence indicated that the acrosome was the major location of the 33 kDa polypeptide. This contention was confirmed by ultrastructural study: immunogold labelling indicated that the 33 kDa polypeptide associated with acrosomal matrix components throughout acrosomal development in the testes and throughout posttesticular maturation in the epididymis. The label clearly delineated the changing morphology of the maturing marsupial acrosome. This is the first study to use immunocytochemical techniques to chart testicular and post-testicular development of any sperm organelle in a marsupial. As a result of this study, a 33 kDa molecular marker of marsupial acrosome differentiation and maturation has been identified. It may be possible to chart similar events in other marsupial species and identify opportunities for manipulating fertility.
Recent studies indicate that subacrosomal proteins are necessary for the attachment of the acroso... more Recent studies indicate that subacrosomal proteins are necessary for the attachment of the acrosome onto the nucleus during sperm formation, and for the stability of the nuclear membrane during fertilization. For the first time, subacrosomal proteins have been isolated from a marsupial species, the tammar wallaby (Macropus eugenii), using a method developed in our laboratory. Whole ejaculated spermatozoa were fractionated into head and tail sections by ultrasonication to extract subacrosomal proteins. The heads (> 95% purity) were then isolated from tail sections using centrifugation with a three-step discontinuous sucrose gradient (35, 68 and 75% (w/v). The heads were treated with 0.1% (v/v) Triton X-100 which stripped off the acrosome, but not the subacrosomal proteins, from the head. The proteins were finally extracted by 100 mmol NaOH l-1. Four prominent subacrosomal polypeptides, with molecular masses of 45, 38, 33 and 29 kDa, were recognized from the SDS-PAGE gel. The localization of these polypeptides (particularly the 45 kDa polypeptide) was confirmed by fluorescent and immunogold labelling with polyclonal antibodies raised in mice against the obtained polypeptides. In wallaby testes, the 45 kDa polypeptide was detected as early as at the step 3 spermatid and was mainly associated with the membrane of the newly formed acrosome vesicle. This polypeptide was also found on the acrosomal membrane of all older spermatids. The 45 kDa polypeptide was found on the acrosomal region of the spermatozoa collected from the caput, corpus and cauda of the epididymis. The similarity of the sperm anatomy of the tammar wallaby with that of other marsupials, such as the brushtail possum, implies that this procedure could be applied effectively to other marsupial species with minor modification.
Microsomal triglyceride transfer protein (MTP) is one of the promising targets for the therapy of... more Microsomal triglyceride transfer protein (MTP) is one of the promising targets for the therapy of dyslipidemia and MTP inhibition can lead to robust plasma low-density lipoprotein cholesterol (LDL-C) reduction. Lomitapide, a small-molecule MTP inhibitor, was recently approved by the US FDA as an additional treatment for homozygous familial hypercholesterolemia (hoFH). However, liver-related side effects, including hepatic fat accumulation and transaminase elevations, are the main safety concerns associated with MTP inhibitors. Here, we review recent knowledge on the mechanisms underlying liver toxicity of MTP inhibitors. The contribution of altered levels of intracellular triglycerides, cholesteryl esters, and free cholesterols toward cellular dysfunction is specifically addressed. On this basis, therapies targeted to attenuate cellular lipid accumulation, to reduce risk factors for non-alcoholic fatty liver disease (NAFLD) (i.e., insulin resistance and oxidative stress) and to specifically inhibit intestinal MTP may be useful for ameliorating liver damage induced by MTP inhibitors. In particular, weight loss through lifestyle interventions is expected to be the most effective and safest way to minimize the undesirable side effects. Specific dietary supplementation might also have protective effects against hepatosteatosis. Despite that, to date, few clinical data support these therapeutic options in MTP inhibition-related liver damage, such proposed approaches may be further explored in the future for their use in preventing unwanted effects of MTP inhibitors.
Current methods of morphological analysis do not permit detailed imaging of individual myelinated... more Current methods of morphological analysis do not permit detailed imaging of individual myelinated fibres over substantial lengths without disruption of neighbouring, potentially significant, cellular and extracellular relationships. We report a new method which overcomes this limitation by combining aldehyde-induced fluorescence with confocal microscopy. Myelin fluorescence was intense relative to that from other tissue components, enabling individual myelinated nerve fibres to be traced for distances of many millimeters in whole PNS nerve trunks. Images obtained with a Bio-Rad MRC-600 confocal laser scanning microscope clearly displayed features of PNS and CNS myelinated fibres including nodes of Ranvier; fibre diameter; sheath thickness and contour; branch points at nodes; as well as (in the PNS) Schmidt-Lanterman incisures and the position of Schwann cell nuclei. Direct comparisons using the same specimens (whole nerve trunks; also teased fibres) showed confocal imaging to be markedly superior to conventional fluorescence microscopy in terms of contrast, apparent resolution and resistance to photobleaching. Development of the fluorophore was examined systematically in sciatic nerves of young adult rats. In separate experiments, animals were perfused systemically using (1) 5% glutaraldehyde; (2) Karnovsky's solution; (3) 4% paraformaldehyde; buffered with either 0.1M sodium phosphate or sodium cacodylate (pH 7.4). The concentration of glutaraldehyde in the fixative solution was the principal determinant of fluorescence intensity. Confocal imaging was achieved immediately following perfusion with 5% glutaraldehyde or Karnovsky's. Fluorescence intensity increased markedly during overnight storage in these fixatives and continued to increase during subsequent storage in buffer alone. The fluorophore was stable and resistant to fading during storage (15 months at least), enabling data collection over extended periods. To demonstrate application of the method in neuropathology, individual fibres in transected sciatic nerve trunks were traced through multiple successive internodes: Classical features of Wallerian degeneration (axonal swelling and debris; ovoid formation and incisure changes; variation among fibres in the extent of degeneration) were displayed. The method is compatible with subsequent ultrastructural examination and will complement existing methods of investigation of myelinated fibre anatomy and pathology, particularly where preservation of 3-dimensional relationships or elucidation of spatial gradients are required.
A variety of media were compared for their ability to sustain the motility of tammar wallaby sper... more A variety of media were compared for their ability to sustain the motility of tammar wallaby spermatozoa over an 8-h period following swim-up from coagulated semen. The study demonstrated that a modified Tyrode's solution, Biggers, Whitter and Whittingham medium (BWW) was significantly better than any of the other assessed media in supporting wallaby sperm motility. After 8 h of incubation in BWW, motility was maintained at 79.3 ± 9.3%, with 77.0 ± 10.4% rapid and 65.7 ± 8.7% progressively motile spermatozoa. By contrast, motility was <10% at the same 8-h time point in all of the other media assessed. After 2 h of incubation in BWW, tammar spermatozoa consumed more oxygen than their counterparts in PBS (52.0 ± 2.7 vs 75.0 ± 6.6 μL per 108 spermatozoa per 2 h; P < 0.001). Motility was not enhanced in any of these media by the addition of 5 mM N-acetyl-D-glucosamine, the major energy substrate in wallaby semen. However, addition of dibutyryl cAMP and pentoxifylline in BWW resulted in the extremely rapid induction of hyperactivated motility in the entire sperm population. This burst of hyperactivated motility was entirely dependent on calcium in BWW and significantly inhibited by calmidazolium, a calmodulin inhibitor. A set of computer-assisted sperm analysis parameters were identified that permitted the accurate quantification of hyperactivation rates in this species. This is the first comparative analysis of media for harvesting and incubating marsupial spermatozoa and the first record of hyperactivated motility in any marsupial species.
The ultrastructure of 12 steps of spermatid development and the process of spermiation are descri... more The ultrastructure of 12 steps of spermatid development and the process of spermiation are described for the Japanese quail in order to clarify the classification proposed for determining the stages of the cycle of the seminiferous epithelium (Lin et al. 1990) and to assess disagreements in the literature about sperm development in birds. It was concluded that acrosomal development involves the formation of proacrosomal granules which do not contain dense granules like the mammalian acrosome. Material which forms the perforatorium initially accumulates as a nuclear granule before appearing in the subacrosomal space. A circular and longitudinal manchette develop sequentially during nuclear elongation. Microtubules of the circular manchette initially form around several parts of the spherical nucleus of step 4 spermatids and subsequently occur most frequently around the narrowest regions of the elongating nucleus. Fibrous sheath development starts in step 2 spermatids indicating that ...
It is concluded that in the birds which have been studied there is a well-defined, regular cycle ... more It is concluded that in the birds which have been studied there is a well-defined, regular cycle of the seminiferous epithelium, and that spermatogenesis involves synchronization of the activities of germ cells within and between successive generations to produce cellular associations of the seminiferous epithelium which are essentially similar to those described in mammals. However, the area of a cellular association is smaller in birds than in most (and probably all) mammals so that numerous cellular associations are present in a cross-section of a seminiferous tubule. As in primates, in which a cellular association also covers a small area of seminiferous tubule, the wave of spermatogenesis in the Japanese quail follows a spiral path along and around a seminiferous tubule with consecutive stages of the cycle always occurring as adjacent stages along the tubule. The literature indicates that there are fewer mitotic divisions during spermatogonial proliferation in birds than has be...
Journal of experimental zoology. Part A, Comparative experimental biology, 2004
Despite considerable advances in our understanding of the molecular mechanisms regulating eutheri... more Despite considerable advances in our understanding of the molecular mechanisms regulating eutherian sperm function, there is a paucity of such knowledge for the Metatheria. In eutherian spermatozoa, the attainment of functional competence is associated with a redox-regulated, cAMP-mediated tyrosine phosphorylation cascade, activated during capacitation. In this report we investigate whether tammar wallaby (Macropus eugenii) spermatozoa possess a similar signal transduction pathway. Western blot analysis of phosphotyrosine expression in caudal and ejaculated populations of tammar spermatozoa revealed that elevation of intracellular cAMP levels, but not exposure to oxidants or NADPH, induced a dramatic increase in the overall level of tyrosine phosphorylation. Washed, ejaculated spermatozoa exhibited more pronounced increases in tyrosine phosphorylation than unwashed sperm populations. Localisation of tyrosine phosphorylation by immunocytochemistry showed that phosphotyrosine residues...
The ultrastructure of 12 steps of spermatid development and the process of spermiation are descri... more The ultrastructure of 12 steps of spermatid development and the process of spermiation are described for the Japanese quail in order to clarify the classification proposed for determining the stages of the cycle of the seminiferous epithelium (Lin et al. 1990) and to assess disagreements in the literature about sperm development in birds. It was concluded that acrosomal development involves the formation of proacrosomal granules which do not contain dense granules like the mammalian acrosome. Material which forms the perforatorium initially accumulates as a nuclear granule before appearing in the subacrosomal space. A circular and longitudinal manchette develop sequentially during nuclear elongation. Microtubules of the circular manchette initially form around several parts of the spherical nucleus of step 4 spermatids and subsequently occur most frequently around the narrowest regions of the elongating nucleus. Fibrous sheath development starts in step 2 spermatids indicating that ...
The ultrastructure of cells of the seminiferous epithelium is described in both triploid ZZZ and ... more The ultrastructure of cells of the seminiferous epithelium is described in both triploid ZZZ and ZZW fowls, especially cytological aberrations influencing the development of morphologically abnormal spermatids and spermatozoa. Mitotic divisions in triploid fowls are similar to those in diploid fowls, but meiosis in both the ZZZ and ZZW triploid fowl is abnormal and many defective gametes are produced. The majority of spermatids have structural abnormalities and this is attributed to the aneuploidy expected from meiotic chromosome pairing in a triploid. Some spermatids were observed to possess an irregularly shaped head, 2 centriolar complexes, 2 acrosomes and 2 tails and this type differentiated into large-headed spermatoza. Such spermatids and spermatozoa are suggested to be near diploid due to their large nuclear size and 2 sets of organelles. Other spermatids, observed with only 1 centriolar complex, 1 acrosome and 1 tail, are suggested to be near haploid. Differentiation of thes...
On leaving the testis, spermatozoa can neither swim nor achieve fertilization of the oocyte. Thes... more On leaving the testis, spermatozoa can neither swim nor achieve fertilization of the oocyte. These functional properties are acquired as spermatozoa transit down the epididymis. The epididymal microenvironment in which spermatozoa undergo their maturation is constantly changing in a carefully orchestrated sequence. Determining the molecular basis of sperm maturation in the epididymis is central to our attempts to understand male infertility and is due largely to the failure of spermatozoa to acquire functionality during epididymal transit. We postulated that these sequential changes are important in sperm maturation and we aimed to characterize the molecular composition of the fluid within defined regions of the epididymis. We confirmed the presence of modulators of mitochondrial function in murine epididymal fluid consistent with a role in the acquisition of sperm function. Materials/Methods: Mouse sperm preparation and mitochondrial membrane potential detection: mouse spermatozoa were extracted from regions of epididymis representing different maturational stages. The fluorescent probe JC 1 was used to determine the mitochondrial membrane potential (MMP). Epididymal fluid preparation: mouse epididymides from the caput region were minced in 50 mM Tris buffer to release the spermatozoa and fluid. After centrifugation to pellet down particulate matter and cells, the fluid obtained was incubated at 100°C for 10 min prior to repeat centrifugation (16,000 g; 5 min). Fast protein liquid chromatography (FPLC): The resultant fluid preparation was fractionated by ion exchange FPLC on a Mono Q 5/50 GL column. Fractions were dialyzed and spin-dried at 60°C. Bioactivity of FPLC fractions: Fractions were resuspended in BWW medium, incubated with mature mouse sperm and mitochondrial status assayed. Results: Initial results revealed that the biochemical changes that accompany sperm maturation involve the activation of sperm mitochondria. Immature spermatozoa from the caput epididymis displayed a low MMP but mature spermatozoa from the cauda epididymis actively maintained a high MMP. However, by washing the cells free of epididymal fluid, immature spermatozoa acquired a high MMP 94.3 ± 2.2% versus the control value of 10.7 ± 4.7%; n = 3, with incubating caudal cells in caput epididymal fluid suppressed MMP (5.3 ± 5.5% versus 99.7 ± 0.6). These results strongly suggested that fluid from the caput epididymis contains a mitochondrial inhibitor and that activation of mitochondrial activity is due to the removal or inactivation of this inhibitor of mitochondrial function. Incubation of ejaculated human spermatozoa in murine epididymal fluid tested the species specificity of this mitochondrial inhibitor. Thus, when human spermatozoa with high MMP (99.7 ± 0.3%) were treated with caput epididymal fluid, their MMP was systematically suppressed (16.7 ± 3.5%; n = 3). Conclusion: In this study, we have demonstrated that epididymal sperm maturation in the mouse involves the inactivation or loss of an inhibitor of sperm MMP. Further
Gonadal development in fowls aged from 1 day to more than 4.5 years was studied in 63 ZZW and 16 ... more Gonadal development in fowls aged from 1 day to more than 4.5 years was studied in 63 ZZW and 16 ZZZ triploid crossbreds and compared with normal diploid males (ZZ) and females (ZW). In the ZZW fowl, the right gonad developed into a testis (although this occurred earlier in the ZZ genotype), and a structurally-abnormal excurrent duct system containing some malformed spermatids and spermatozoa was associated with the gonad of young adults. The left gonad was an ovotestes at hatching and no excurrent ducts were associated with it. The ovarian component was much less developed than that in the ZW genotype-it started to degenerate by 1 week of age, and most of the oocytes had disappeared by about 3 weeks of age. The seminiferous tubules developed in the medullary region, but only abnormal spermatozoa were produced. Leukocytes infiltrated both gonads at about 9 months of age and the seminiferous epithelium had degenerated in most fowls over 1 year old. In ZZZ fowl, gonadal and excurrent duct development was normal, but occurred earlier than in the ZZ genotype. However, meiosis and spermiogenesis were abnormal and malformed spermatozoa were produced. The heads of spermatozoa from the ductus deferens were about 1.4-times tonger in the ZZZ genotype than in the ZZ genotype, indicating that the former may be producing some diploid spermatozoa.
The metabolism, rate of intracellular accumulation of sugars, motility and ultrastructure of ejac... more The metabolism, rate of intracellular accumulation of sugars, motility and ultrastructure of ejaculated tammar sperm were impaired by swim-up into artificial media, particularly when the cells were subsequently exposed to N-acetyl-D-glucosamine (NAG). The inclusion of hyaluronate, serum albumin, catalase or Desferal in swim-up media helped prevent deterioration of sperm motility, but failed to prevent detrimental NAG-induced metabolic and ultrastructural changes. However, the sperm were unavoidably diluted during swim-up into artificial media and their behavioural properties were modified by dilution. Thus, sperm collected from the cauda epididymidis were immotile and their rate of oxygen uptake was low in undiluted caudal epididymal semen (CES). Nevertheless, these sperm were viable, and vigorous motility was induced by 5-to 50-fold dilution in Krebs-Ringer phosphate (KRP). Sperm respiration also dramatically increased with moderate dilution (5or 15-fold) in KRP, but decreased again at higher rates (50-fold). This suggested that motility and the metabolic properties of tammar sperm are modified both by dilution and on leaving the suppressing conditions of the epididymis. Diluted tammar epididymal sperm also displayed a Pasteur effect, but rapidly lost capacity for motility in an oxygen-depleted atmosphere. It was concluded that swim-up procedures compromise ejaculated tammar sperm by promoting dilution-induced changes. This may alter the permeability of the membrane with loss of the enzymes that process the ammoniagenerated during the metabolism of NAG in seminal plasma. Subsequent exposure to NAG further promotes ultrastructural damage culminating in loss of viability.
A sperm-epididymal cell co-culture was shown to be capable of inducing the in vitro maturation of... more A sperm-epididymal cell co-culture was shown to be capable of inducing the in vitro maturation of spermatozoa from a marsupial species, the tammar wallaby (Macropus eugenii ). This system was able to maintain wallaby epididymal epithelial cells in vitro for more than 2 months. The system also enabled immature wallaby spermatozoa to differentiate from a T-shaped to a streamlined form, accompanied by the development of progressive motility after co-culture with epididymal cell monolayers that had been cultured for 7 days. The addition of inhibitors of actin polymerization (latrunculin A or B) to the co-culture system showed that wallaby sperm maturation was impaired by the interruption of actin organization within the immature spermatozoa. These results indicate that actin filaments play a significant role in sperm transformation during post-testicular maturation in marsupials. These observations also indicate that the marsupial coculture system has the potential to greatly increase understanding of sperm-epididymal cell interactions and the mechanism of sperm maturation in these species.
In this observational case report, we share our experience of achieving >40% LDL cholesterol r... more In this observational case report, we share our experience of achieving >40% LDL cholesterol reduction in four Chinese homozygous familial hypercholesterolaemia children below 8 years of age with a triple combination of atorvastatin, probucol, and ezetimibe for >6 years. Within a follow-up duration of 6-13 years, this triple therapy achieved significant reduction of LDL cholesterol as well as an impressive regression of xanthomas in all paediatric cases. All the children remained free from treatment-related adverse responses and cardiovascular events throughout follow-up.
We investigated the cycle of the seminiferous epithelium in a marsupial, namely the brushtail pos... more We investigated the cycle of the seminiferous epithelium in a marsupial, namely the brushtail possum (Trichosurus vulpecula), using semithin sections of seminiferous tubules embedded in Spurr's resin. Using 14 steps of spermatid development as markers, we were able to class tubular cross-sections into 10 well-defined stages of the seminiferous epithelial cycle. The duration of one cycle was 13.5 days, as determined by injections of [(3)H]-thymidine and autoradiographic examination of the most advanced sperm cells at 2 h and 17 days after injection. The durations of stages I-X were 21.4, 66.4, 54.1, 47.0, 29.8, 28.5, 25.3, 25.0, 12.0 and 15.9 h, respectively, estimated by the relative percentage of occurrence of each stage. It was estimated that the life spans of the main germ cells were as follows: type B spermatogonia, 5.4 days; primary spermatocytes, 16.7 days; secondary spermatocytes, 0.7 days; and spermatids, 21.4 days. The results suggest that the kinetics of spermatogenesi...
Rapid input-restricted change in gene expression is an important aspect of synaptic plasticity re... more Rapid input-restricted change in gene expression is an important aspect of synaptic plasticity requiring complex mechanisms of post-transcriptional mRNA trafficking and regulation. Small non-coding miRNA are uniquely poised to support these functions by providing a nucleic-acid-based specificity component for universal-sequence-dependent RNA binding complexes. We investigated the subcellular distribution of these molecules in resting and potassium chloride depolarized human neuroblasts, and found both selective enrichment and depletion in neurites. Depolarization was associated with a neurite-restricted decrease in miRNA expression; a subset of these molecules was recovered from the depolarization medium in nuclease resistant extracellular exosomes. These vesicles were enriched with primate specific miRNA and the synaptic-plasticity-associated protein MAP1b. These findings further support a role for miRNA as neural plasticity regulators, as they are compartmentalized in neurons and ...
This study was undertaken to identify potential molecular markers of acrosomal biogenesis and pos... more This study was undertaken to identify potential molecular markers of acrosomal biogenesis and post-testicular maturation in marsupials, using the tammar wallaby as a model species. A two-step sperm extraction procedure yielded two protein extracts of apparent acrosomal origin and a tail extract. The extracts were analysed by SDS-PAGE under reducing conditions. Several prominent polypeptide bands (45, 38 and 33 kDa) appeared common to both acrosomal extracts. Antiserum raised against the 33 kDa polypeptide from the inner acrosomal membrane matrix (IAMM) extract showed immunoreactivity with 45, 38 and 33 kDa polypeptides in both acrosomal extracts, indicating that the 33 kDa polypeptide was related to the proteins in the 45 and 38 kDa bands. Therefore, the antiserum was used as a molecular probe. Indirect immunofluorescence indicated that the acrosome was the major location of the 33 kDa polypeptide. This contention was confirmed by ultrastructural study: immunogold labelling indicated that the 33 kDa polypeptide associated with acrosomal matrix components throughout acrosomal development in the testes and throughout posttesticular maturation in the epididymis. The label clearly delineated the changing morphology of the maturing marsupial acrosome. This is the first study to use immunocytochemical techniques to chart testicular and post-testicular development of any sperm organelle in a marsupial. As a result of this study, a 33 kDa molecular marker of marsupial acrosome differentiation and maturation has been identified. It may be possible to chart similar events in other marsupial species and identify opportunities for manipulating fertility.
Recent studies indicate that subacrosomal proteins are necessary for the attachment of the acroso... more Recent studies indicate that subacrosomal proteins are necessary for the attachment of the acrosome onto the nucleus during sperm formation, and for the stability of the nuclear membrane during fertilization. For the first time, subacrosomal proteins have been isolated from a marsupial species, the tammar wallaby (Macropus eugenii), using a method developed in our laboratory. Whole ejaculated spermatozoa were fractionated into head and tail sections by ultrasonication to extract subacrosomal proteins. The heads (> 95% purity) were then isolated from tail sections using centrifugation with a three-step discontinuous sucrose gradient (35, 68 and 75% (w/v). The heads were treated with 0.1% (v/v) Triton X-100 which stripped off the acrosome, but not the subacrosomal proteins, from the head. The proteins were finally extracted by 100 mmol NaOH l-1. Four prominent subacrosomal polypeptides, with molecular masses of 45, 38, 33 and 29 kDa, were recognized from the SDS-PAGE gel. The localization of these polypeptides (particularly the 45 kDa polypeptide) was confirmed by fluorescent and immunogold labelling with polyclonal antibodies raised in mice against the obtained polypeptides. In wallaby testes, the 45 kDa polypeptide was detected as early as at the step 3 spermatid and was mainly associated with the membrane of the newly formed acrosome vesicle. This polypeptide was also found on the acrosomal membrane of all older spermatids. The 45 kDa polypeptide was found on the acrosomal region of the spermatozoa collected from the caput, corpus and cauda of the epididymis. The similarity of the sperm anatomy of the tammar wallaby with that of other marsupials, such as the brushtail possum, implies that this procedure could be applied effectively to other marsupial species with minor modification.
Microsomal triglyceride transfer protein (MTP) is one of the promising targets for the therapy of... more Microsomal triglyceride transfer protein (MTP) is one of the promising targets for the therapy of dyslipidemia and MTP inhibition can lead to robust plasma low-density lipoprotein cholesterol (LDL-C) reduction. Lomitapide, a small-molecule MTP inhibitor, was recently approved by the US FDA as an additional treatment for homozygous familial hypercholesterolemia (hoFH). However, liver-related side effects, including hepatic fat accumulation and transaminase elevations, are the main safety concerns associated with MTP inhibitors. Here, we review recent knowledge on the mechanisms underlying liver toxicity of MTP inhibitors. The contribution of altered levels of intracellular triglycerides, cholesteryl esters, and free cholesterols toward cellular dysfunction is specifically addressed. On this basis, therapies targeted to attenuate cellular lipid accumulation, to reduce risk factors for non-alcoholic fatty liver disease (NAFLD) (i.e., insulin resistance and oxidative stress) and to specifically inhibit intestinal MTP may be useful for ameliorating liver damage induced by MTP inhibitors. In particular, weight loss through lifestyle interventions is expected to be the most effective and safest way to minimize the undesirable side effects. Specific dietary supplementation might also have protective effects against hepatosteatosis. Despite that, to date, few clinical data support these therapeutic options in MTP inhibition-related liver damage, such proposed approaches may be further explored in the future for their use in preventing unwanted effects of MTP inhibitors.
Current methods of morphological analysis do not permit detailed imaging of individual myelinated... more Current methods of morphological analysis do not permit detailed imaging of individual myelinated fibres over substantial lengths without disruption of neighbouring, potentially significant, cellular and extracellular relationships. We report a new method which overcomes this limitation by combining aldehyde-induced fluorescence with confocal microscopy. Myelin fluorescence was intense relative to that from other tissue components, enabling individual myelinated nerve fibres to be traced for distances of many millimeters in whole PNS nerve trunks. Images obtained with a Bio-Rad MRC-600 confocal laser scanning microscope clearly displayed features of PNS and CNS myelinated fibres including nodes of Ranvier; fibre diameter; sheath thickness and contour; branch points at nodes; as well as (in the PNS) Schmidt-Lanterman incisures and the position of Schwann cell nuclei. Direct comparisons using the same specimens (whole nerve trunks; also teased fibres) showed confocal imaging to be markedly superior to conventional fluorescence microscopy in terms of contrast, apparent resolution and resistance to photobleaching. Development of the fluorophore was examined systematically in sciatic nerves of young adult rats. In separate experiments, animals were perfused systemically using (1) 5% glutaraldehyde; (2) Karnovsky's solution; (3) 4% paraformaldehyde; buffered with either 0.1M sodium phosphate or sodium cacodylate (pH 7.4). The concentration of glutaraldehyde in the fixative solution was the principal determinant of fluorescence intensity. Confocal imaging was achieved immediately following perfusion with 5% glutaraldehyde or Karnovsky's. Fluorescence intensity increased markedly during overnight storage in these fixatives and continued to increase during subsequent storage in buffer alone. The fluorophore was stable and resistant to fading during storage (15 months at least), enabling data collection over extended periods. To demonstrate application of the method in neuropathology, individual fibres in transected sciatic nerve trunks were traced through multiple successive internodes: Classical features of Wallerian degeneration (axonal swelling and debris; ovoid formation and incisure changes; variation among fibres in the extent of degeneration) were displayed. The method is compatible with subsequent ultrastructural examination and will complement existing methods of investigation of myelinated fibre anatomy and pathology, particularly where preservation of 3-dimensional relationships or elucidation of spatial gradients are required.
A variety of media were compared for their ability to sustain the motility of tammar wallaby sper... more A variety of media were compared for their ability to sustain the motility of tammar wallaby spermatozoa over an 8-h period following swim-up from coagulated semen. The study demonstrated that a modified Tyrode's solution, Biggers, Whitter and Whittingham medium (BWW) was significantly better than any of the other assessed media in supporting wallaby sperm motility. After 8 h of incubation in BWW, motility was maintained at 79.3 ± 9.3%, with 77.0 ± 10.4% rapid and 65.7 ± 8.7% progressively motile spermatozoa. By contrast, motility was <10% at the same 8-h time point in all of the other media assessed. After 2 h of incubation in BWW, tammar spermatozoa consumed more oxygen than their counterparts in PBS (52.0 ± 2.7 vs 75.0 ± 6.6 μL per 108 spermatozoa per 2 h; P < 0.001). Motility was not enhanced in any of these media by the addition of 5 mM N-acetyl-D-glucosamine, the major energy substrate in wallaby semen. However, addition of dibutyryl cAMP and pentoxifylline in BWW resulted in the extremely rapid induction of hyperactivated motility in the entire sperm population. This burst of hyperactivated motility was entirely dependent on calcium in BWW and significantly inhibited by calmidazolium, a calmodulin inhibitor. A set of computer-assisted sperm analysis parameters were identified that permitted the accurate quantification of hyperactivation rates in this species. This is the first comparative analysis of media for harvesting and incubating marsupial spermatozoa and the first record of hyperactivated motility in any marsupial species.
The ultrastructure of 12 steps of spermatid development and the process of spermiation are descri... more The ultrastructure of 12 steps of spermatid development and the process of spermiation are described for the Japanese quail in order to clarify the classification proposed for determining the stages of the cycle of the seminiferous epithelium (Lin et al. 1990) and to assess disagreements in the literature about sperm development in birds. It was concluded that acrosomal development involves the formation of proacrosomal granules which do not contain dense granules like the mammalian acrosome. Material which forms the perforatorium initially accumulates as a nuclear granule before appearing in the subacrosomal space. A circular and longitudinal manchette develop sequentially during nuclear elongation. Microtubules of the circular manchette initially form around several parts of the spherical nucleus of step 4 spermatids and subsequently occur most frequently around the narrowest regions of the elongating nucleus. Fibrous sheath development starts in step 2 spermatids indicating that ...
It is concluded that in the birds which have been studied there is a well-defined, regular cycle ... more It is concluded that in the birds which have been studied there is a well-defined, regular cycle of the seminiferous epithelium, and that spermatogenesis involves synchronization of the activities of germ cells within and between successive generations to produce cellular associations of the seminiferous epithelium which are essentially similar to those described in mammals. However, the area of a cellular association is smaller in birds than in most (and probably all) mammals so that numerous cellular associations are present in a cross-section of a seminiferous tubule. As in primates, in which a cellular association also covers a small area of seminiferous tubule, the wave of spermatogenesis in the Japanese quail follows a spiral path along and around a seminiferous tubule with consecutive stages of the cycle always occurring as adjacent stages along the tubule. The literature indicates that there are fewer mitotic divisions during spermatogonial proliferation in birds than has be...
Journal of experimental zoology. Part A, Comparative experimental biology, 2004
Despite considerable advances in our understanding of the molecular mechanisms regulating eutheri... more Despite considerable advances in our understanding of the molecular mechanisms regulating eutherian sperm function, there is a paucity of such knowledge for the Metatheria. In eutherian spermatozoa, the attainment of functional competence is associated with a redox-regulated, cAMP-mediated tyrosine phosphorylation cascade, activated during capacitation. In this report we investigate whether tammar wallaby (Macropus eugenii) spermatozoa possess a similar signal transduction pathway. Western blot analysis of phosphotyrosine expression in caudal and ejaculated populations of tammar spermatozoa revealed that elevation of intracellular cAMP levels, but not exposure to oxidants or NADPH, induced a dramatic increase in the overall level of tyrosine phosphorylation. Washed, ejaculated spermatozoa exhibited more pronounced increases in tyrosine phosphorylation than unwashed sperm populations. Localisation of tyrosine phosphorylation by immunocytochemistry showed that phosphotyrosine residues...
The ultrastructure of 12 steps of spermatid development and the process of spermiation are descri... more The ultrastructure of 12 steps of spermatid development and the process of spermiation are described for the Japanese quail in order to clarify the classification proposed for determining the stages of the cycle of the seminiferous epithelium (Lin et al. 1990) and to assess disagreements in the literature about sperm development in birds. It was concluded that acrosomal development involves the formation of proacrosomal granules which do not contain dense granules like the mammalian acrosome. Material which forms the perforatorium initially accumulates as a nuclear granule before appearing in the subacrosomal space. A circular and longitudinal manchette develop sequentially during nuclear elongation. Microtubules of the circular manchette initially form around several parts of the spherical nucleus of step 4 spermatids and subsequently occur most frequently around the narrowest regions of the elongating nucleus. Fibrous sheath development starts in step 2 spermatids indicating that ...
The ultrastructure of cells of the seminiferous epithelium is described in both triploid ZZZ and ... more The ultrastructure of cells of the seminiferous epithelium is described in both triploid ZZZ and ZZW fowls, especially cytological aberrations influencing the development of morphologically abnormal spermatids and spermatozoa. Mitotic divisions in triploid fowls are similar to those in diploid fowls, but meiosis in both the ZZZ and ZZW triploid fowl is abnormal and many defective gametes are produced. The majority of spermatids have structural abnormalities and this is attributed to the aneuploidy expected from meiotic chromosome pairing in a triploid. Some spermatids were observed to possess an irregularly shaped head, 2 centriolar complexes, 2 acrosomes and 2 tails and this type differentiated into large-headed spermatoza. Such spermatids and spermatozoa are suggested to be near diploid due to their large nuclear size and 2 sets of organelles. Other spermatids, observed with only 1 centriolar complex, 1 acrosome and 1 tail, are suggested to be near haploid. Differentiation of thes...
On leaving the testis, spermatozoa can neither swim nor achieve fertilization of the oocyte. Thes... more On leaving the testis, spermatozoa can neither swim nor achieve fertilization of the oocyte. These functional properties are acquired as spermatozoa transit down the epididymis. The epididymal microenvironment in which spermatozoa undergo their maturation is constantly changing in a carefully orchestrated sequence. Determining the molecular basis of sperm maturation in the epididymis is central to our attempts to understand male infertility and is due largely to the failure of spermatozoa to acquire functionality during epididymal transit. We postulated that these sequential changes are important in sperm maturation and we aimed to characterize the molecular composition of the fluid within defined regions of the epididymis. We confirmed the presence of modulators of mitochondrial function in murine epididymal fluid consistent with a role in the acquisition of sperm function. Materials/Methods: Mouse sperm preparation and mitochondrial membrane potential detection: mouse spermatozoa were extracted from regions of epididymis representing different maturational stages. The fluorescent probe JC 1 was used to determine the mitochondrial membrane potential (MMP). Epididymal fluid preparation: mouse epididymides from the caput region were minced in 50 mM Tris buffer to release the spermatozoa and fluid. After centrifugation to pellet down particulate matter and cells, the fluid obtained was incubated at 100°C for 10 min prior to repeat centrifugation (16,000 g; 5 min). Fast protein liquid chromatography (FPLC): The resultant fluid preparation was fractionated by ion exchange FPLC on a Mono Q 5/50 GL column. Fractions were dialyzed and spin-dried at 60°C. Bioactivity of FPLC fractions: Fractions were resuspended in BWW medium, incubated with mature mouse sperm and mitochondrial status assayed. Results: Initial results revealed that the biochemical changes that accompany sperm maturation involve the activation of sperm mitochondria. Immature spermatozoa from the caput epididymis displayed a low MMP but mature spermatozoa from the cauda epididymis actively maintained a high MMP. However, by washing the cells free of epididymal fluid, immature spermatozoa acquired a high MMP 94.3 ± 2.2% versus the control value of 10.7 ± 4.7%; n = 3, with incubating caudal cells in caput epididymal fluid suppressed MMP (5.3 ± 5.5% versus 99.7 ± 0.6). These results strongly suggested that fluid from the caput epididymis contains a mitochondrial inhibitor and that activation of mitochondrial activity is due to the removal or inactivation of this inhibitor of mitochondrial function. Incubation of ejaculated human spermatozoa in murine epididymal fluid tested the species specificity of this mitochondrial inhibitor. Thus, when human spermatozoa with high MMP (99.7 ± 0.3%) were treated with caput epididymal fluid, their MMP was systematically suppressed (16.7 ± 3.5%; n = 3). Conclusion: In this study, we have demonstrated that epididymal sperm maturation in the mouse involves the inactivation or loss of an inhibitor of sperm MMP. Further
Gonadal development in fowls aged from 1 day to more than 4.5 years was studied in 63 ZZW and 16 ... more Gonadal development in fowls aged from 1 day to more than 4.5 years was studied in 63 ZZW and 16 ZZZ triploid crossbreds and compared with normal diploid males (ZZ) and females (ZW). In the ZZW fowl, the right gonad developed into a testis (although this occurred earlier in the ZZ genotype), and a structurally-abnormal excurrent duct system containing some malformed spermatids and spermatozoa was associated with the gonad of young adults. The left gonad was an ovotestes at hatching and no excurrent ducts were associated with it. The ovarian component was much less developed than that in the ZW genotype-it started to degenerate by 1 week of age, and most of the oocytes had disappeared by about 3 weeks of age. The seminiferous tubules developed in the medullary region, but only abnormal spermatozoa were produced. Leukocytes infiltrated both gonads at about 9 months of age and the seminiferous epithelium had degenerated in most fowls over 1 year old. In ZZZ fowl, gonadal and excurrent duct development was normal, but occurred earlier than in the ZZ genotype. However, meiosis and spermiogenesis were abnormal and malformed spermatozoa were produced. The heads of spermatozoa from the ductus deferens were about 1.4-times tonger in the ZZZ genotype than in the ZZ genotype, indicating that the former may be producing some diploid spermatozoa.
The metabolism, rate of intracellular accumulation of sugars, motility and ultrastructure of ejac... more The metabolism, rate of intracellular accumulation of sugars, motility and ultrastructure of ejaculated tammar sperm were impaired by swim-up into artificial media, particularly when the cells were subsequently exposed to N-acetyl-D-glucosamine (NAG). The inclusion of hyaluronate, serum albumin, catalase or Desferal in swim-up media helped prevent deterioration of sperm motility, but failed to prevent detrimental NAG-induced metabolic and ultrastructural changes. However, the sperm were unavoidably diluted during swim-up into artificial media and their behavioural properties were modified by dilution. Thus, sperm collected from the cauda epididymidis were immotile and their rate of oxygen uptake was low in undiluted caudal epididymal semen (CES). Nevertheless, these sperm were viable, and vigorous motility was induced by 5-to 50-fold dilution in Krebs-Ringer phosphate (KRP). Sperm respiration also dramatically increased with moderate dilution (5or 15-fold) in KRP, but decreased again at higher rates (50-fold). This suggested that motility and the metabolic properties of tammar sperm are modified both by dilution and on leaving the suppressing conditions of the epididymis. Diluted tammar epididymal sperm also displayed a Pasteur effect, but rapidly lost capacity for motility in an oxygen-depleted atmosphere. It was concluded that swim-up procedures compromise ejaculated tammar sperm by promoting dilution-induced changes. This may alter the permeability of the membrane with loss of the enzymes that process the ammoniagenerated during the metabolism of NAG in seminal plasma. Subsequent exposure to NAG further promotes ultrastructural damage culminating in loss of viability.
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