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R J Aitken

Publications and source records attributed to R J Aitken.

At least 37 records · Page 2Linked to original sources
PubMed2001

Analysis of reactive oxygen species generating systems in rat epididymal spermatozoa.

Epididymal sperm maturation culminates in the acquisition of functional competence by testicular spermatozoa. The expression of this functional state is dependent upon a redox-regulated, cAMP-mediated signal transduction cascade that controls the tyrosine phosphorylation status of the spermatozoa during capacitation. Analysis of superoxide anion (O2(-.)) generation by rat epididymal spermatozoa has revealed a two-component process involving electron leakage from the sperm mitochondria at complexes I and II and a plasma membrane NAD(P)H oxidoreductase. Following incubation in a glucose-, lactate-, and pyruvate-free medium (-GLP), O2(-.) generation was suppressed by 86% and 96% in caput and cauda spermatozoa, respectively. The addition of lactate, malate, or succinate to spermatozoa incubated in medium -GLP stimulated O2(-.) generation. This increase could be blocked by rotenone and oligomycin (R/O) in the presence of malate or lactate but not succinate. Stimulation with all three substrates, as well as spontaneous O2(-.) production in +GLP medium, was blocked by the flavoprotein inhibitor, diphenylene iodonium. Diphenylene iodonium, but not R/O, suppressed NAD(P)H-induced lucigenin-dependent chemiluminescence. This NAD(P)H-dependent enzyme resided in the sperm plasma membrane and its activity was regulated by zinc and uncharacterized cytosolic factors. Reverse transcription-polymerase chain reaction analysis indicated that the sperm NAD(P)H oxidoreductase complex is quite distinct from the equivalent leukocyte system.

Animals↗
PubMed2001

Detection of Ehrlichia platys in dogs in Australia.

OBJECTIVE: To describe the detection of Ehrlichia platys in free-roaming dogs in Central Australia. PROCEDURE: Blood samples were collected from four dogs and examined for bacterial 16S ribosomal DNA using Polymerase Chain Reaction (PCR)-based assays. The three positive samples obtained were then sequenced and identification of the PCR product carried out. As a result of all three samples being identical to or closely related to part of the 16S rRNA gene of E. platys, blood samples were subsequently obtained from a further 24 dogs. These samples were screened using a PCR-assay to determine the presence of Ehrlichia DNA using genus-specific primers. The positive samples obtained from the screening process were then subjected to a further PCR-assay using E. platys specific primers. RESULTS: Of 28 dogs sampled, Ehrlichia DNA was detected in the blood of 13 dogs. Sequencing of the amplicons obtained indicated a high homology with the 16S rRNA gene for E. platys. When the E. platys-specific PCR was performed for 10 of those dogs, the 678 bp product obtained from the PCR amplification confirmed the identification as part of the 16S rRNA gene of E. platys in all 10 dogs. CONCLUSION: This study reports for the first time Ehrlichia carriage by dogs in Australia. It also indicates the usefulness of the PCR technique in rapidly and accurately identifying diseases that are otherwise difficult to detect. By using universal primers directed against bacterial 16S ribosomal DNA and sequencing analysis, the detection of potentially pathogenic Ehrlichia organisms that had not previously been found in Australia has been made possible.

Animals↗
PubMed2001

Relative susceptibilities of mitochondrial and nuclear DNA to damage induced by hydrogen peroxide in two mouse germ cell lines.

The objective of this study was to determine the relative susceptibilities to the damaging effects of hydrogen peroxide of DNA in the mitochondrial and nuclear compartments of two murine germ cell lines. We used a quantitative polymerase chain reaction assay (QPCR) to measure gene- and mitochondrial-specific DNA damage and examined for the presence of alkali-labile sites using alkaline gel electrophoresis. No DNA damage was observed in a nuclear gene (beta-globin) in response to hydrogen peroxide treatment. In addition, no increase in alkali-labile sites was observed. However, mitochondrial DNA suffered extensive damage which increased in a dose-dependent manner. These results demonstrate that the nuclear DNA in these germ cell lines is relatively resistant to peroxide-mediated DNA damage, and that mitochondrial DNA is a sensitive biomarker for oxidative stress in these cells.

Animals↗
PubMed2001

Oxidative stress, DNA damage and the Y chromosome.

Recent advances in understanding of male infertility have implicated two major causative factors, oxidative stress and Y chromosome deletions. A major cause of oxidative stress appears to be the high rate of reactive oxygen species generation associated with the retention of excess residual cytoplasm in the sperm midpiece. Other possible causes include the redox cycling of xenobiotics, and antioxidant depletion or apoptosis. Oxidative stress induces peroxidative damage in the sperm plasma membrane and DNA damage in both the mitochondrial and nuclear genomes. Nuclear DNA damage in the germ line of the father may be associated with pathology in the offspring, including childhood cancer and infertility. Gene deletions on the non-recombining region of the Y chromosome account for the infertility observed in about 15% of patients with azoospermia and 5-10% of subjects with severe oligozoospermia. The Y chromosome is particularly susceptible to gene deletions because of the inability of the haploid genome to deploy recombination repair in retrieving lost genetic information. Aberrant recombination, defective chromatin packaging, abortive apoptosis and oxidative stress may all be involved in the aetiology of DNA damage in the germ line. The factors responsible for Y chromosome deletions in spermatozoa remain unresolved but may be one facet of a central reproductive problem: controlling the amount of oxidative stress experienced by germ cells during their differentiation and maturation in the male reproductive tract.

Animals↗
PubMed2000

Importance of sperm-to-epithelial cell contact for the capacitation of human spermatozoa in fallopian tube epithelial cell cocultures.

OBJECTIVE: To investigate the mechanisms involved in the stimulatory effect of fallopian tube epithelial cell coculture on sperm movement characteristics. DESIGN: Human spermatozoa were cultured with human fallopian tube epithelial cell monolayers. A microporous membrane was used to prevent sperm-to-epithelial cell contact. Sperm movement characteristics were measured at 4 and 24 hours. SETTING: University hospital and fertility center. PATIENT(S): Voluntary donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Movement characteristics of human spermatozoa. RESULT(S): Fallopian tube epithelial cell coculture increased sperm motility, curvilinear velocity, amplitude of lateral head displacement, and hyperactivated motility, mainly at 24 hours, compared with controls. These stimulatory effects were inhibited when a microporous membrane prevented cell-to-cell contact between sperm and fallopian tube epithelial cells. CONCLUSION(S): Physical contact between sperm and epithelial cells in coculture systems seems to be the main factor in stimulating sperm movement characteristics, and this could be the main mechanism of in vivo sperm capacitation.

Adult↗
PubMed2000

Impact of a deep saturation dive on semen quality.

The demonstration dive 'Aurora' has provided an opportunity to study the impact of extreme hyperbaric conditions on male fertility. This operation involved a 33-day diving programme during which divers were exposed to a maximum pressure of 4.6 Mega Pascals (Mpa) for 7 days. At days - 4, + 27, + 34, + 82 and + 263 relative to the initiation of the dive, semen samples were analysed to determine the quality of spermatogenesis and the functional competence of the spermatozoa. A dramatic fall in semen quality was observed in association with the dive and by day + 82 the potential fertility of the men was seriously compromised as evidenced by oligoasthenoteratozoospermic semen profiles and the poor fertilizing potential of the spermatozoa. These studies indicate, for the first time, that the severe hyperbaric conditions associated with deep saturation dives have a profound effect on male reproductive function.

Diving↗
PubMed2000

Selection of peptides targeting the human sperm surface using random peptide phage display identify ligands homologous to ZP3.

Analysis of the surface architecture of human spermatozoa is a necessary step in the development of new approaches to contraception and resolving the causes of human infertility. In this study we have utilized phage display technology to identify peptides that bind with high affinity to the surface of human spermatozoa. Fifteen- and twelve-mer random peptide phage display libraries were screened against paraformaldehyde-fixed spermatozoa and a number of sperm-binding peptides were identified. One peptide, M6, displayed a high level of affinity for the sperm surface and showed sequence homology with a dominant human ZP3 epitope (hZP 25-33). This peptide bound preferentially to the equatorial and post acrosomal domains of the sperm head and exhibited contraceptive activity by virtue of its capacity to impair the fusion of acrosome-reacted spermatozoa with the vitelline membrane of the oocyte. A similar form of contraceptive activity was also observed within an unrelated peptide, K6, derived from screening the 12-mer library. These results indicate that phage display technology is a powerful tool for developing reagents capable of targeting the human sperm surface, providing insights into the composition of this structure and the identity of targets susceptible to contraceptive attack and pathological disruption.

Adult↗
PubMed2000

Immunocontraception with zona pellucida proteins.

The prospect of an immunological approach to contraception that would disrupt the process of fertilisation itself has resulted in a considerable interest into research in this area. It has been known for some time that antibodies raised against the zona pellucida (ZP) can suppress fertility very effectively. However, the initial optimism of this approach has been marred by the appearance of an ovarian pathology characterised by disruption of folliculogenesis and depletion of the primordial follicle pool. Adverse auto-immune reactions have been observed in the ovaries of mice after the induction of immunity with mouse ZP3 epitopes. However, this was associated with lymphocytic infiltration of the ovarian stroma, which could be circumvented by careful selection of B-cell epitopes to induce reversible infertility. In order to identify similar epitopes on primate ZP3, epitope-mapping studies were performed and incorporated into chimeric vaccines that included a promiscuous T-helper cell epitope. Both single and triple peptide vaccines have been evaluated in vivo and no detrimental effects on ovarian function were observed. The resulting high titre antibodies bound exclusively to the ZP of marmoset and human ovarian sections and could suppress in vitro human sperm-egg binding by approximately 60%, but did not prevent pregnancy in actively immunised female marmosets. Thus, considerable research is still required to identify a combination of ZP3 epitopes that will induce infertility free of any unwanted side effects.

Animals↗
PubMed2000

In vitro culture of brushtail possum (Trichosurus vulpecula) epididymal epithelium and induction of epididymal sperm maturation in co-culture.

A medium modified from eutherian systems was used to culture epididymal epithelial cells of the brushtail possum (Trichosurus vulpecula) for more than 2 months. Epididymal tubule fragments from the caput, corpus and cauda epididymides were used to generate cell monolayers. All three epididymal cell culture systems supported maturational changes in marsupial spermatozoa and enabled immature possum spermatozoa to differentiate from a T-shape to a streamlined shape, accompanied by the development of progressive motility after co-culture with 7-day-old cultured epididymal cell monolayers. This epididymal cell and sperm co-culture system for marsupial species may facilitate the identification of specific epithelial factors that affect sperm maturation, particularly in a species in which morphological maturation is readily visible.

Animals↗
PubMed2000

Use of patches and whole body sampling for the assessment of dermal exposure.

There has been a growing awareness of the importance of dermal exposure in recent years. A wide range of techniques are employed to measure exposure, of which surrogate skin techniques such as patch sampling and whole body sampling are frequently used. One of the problems associated with dermal sampling is that different methods often produce different results due to differences in the principles involved in sample collection. As a consequence little progress towards establishing dermal exposure limits has been made. Both patches and clothing act as passive samplers and are intended to collect all of a substance deposited on them. This paper details the principles underlying patch and whole body sampling and outlines some of the advantages and disadvantages of each. A conceptual model has recently been proposed for dermal exposure and the role that surrogate techniques may play in the application of this model is discussed. Finally, suggestions are made as to how these techniques may be made more relevant and areas of future research highlighted.

Bandages↗
PubMed1999

Cloning, sequencing and site of origin of the rat sperm receptor protein, ZP3.

The ZP3 gene encodes for a zona glycoprotein that serves as both a cell-specific binding site for capacitated spermatozoa and an inducer of acrosomal exocytosis during fertilisation. In this study we have determined the nucleotide sequence of rat ZP3 (accession no. Y10823), predicted primary amino acid structure and determined the cellular origin of this molecule within the ovary. Rat ZP3 was found to have an open reading frame of 1272 nucleotides encoding a polypeptide chain of 424 amino acids that was expressed exclusively by the actively growing oocyte population. Rat ZP3 exhibited 91%, 78% and 66% identity with the mouse, hamster and human homologues, respectively. Key features of mouse ZP3, including the number and location of cysteine and proline residues and N-linked glycosylation sites, were also conserved in the rat homologue. The putative O-linked glycosylation sites, a series of serine residues at ZP3(329-334), were also conserved in rat and mouse ZP3, although immediately downstream of this site the amino acid sequences deviated over a short stretch of amino acids. The hydropathicity profile revealed two hydrophobic domains. The first was associated with a putative N-terminal signal sequence which is unusual in the rat in possessing a proline residue at the -1 position relative to the signal cleavage site, a feature it shares with human and marmoset ZP3 but not mouse. The second hydrophobic domain was observed at the C-terminus downstream of a TGF-beta type III receptor domain that appears to be common to all ZP3 sequences examined to date.

Amino Acid Sequence↗
PubMed1999

Outcome in patients with colorectal cancer managed by surgical trainees.

BACKGROUND: The surgeon is an important variable that influences outcome following colorectal cancer surgery. Operative training of suitable quality and quantity is essential if intersurgeon variation is to be reduced. The aim of this study was to examine the outcome of colorectal cancer surgery when a high proportion of the operations were performed by trainee surgeons. METHODS: A prospective 7-year (1989-1996) audit of 306 consecutive colorectal cancers referred to a single general surgeon with a colorectal interest was carried out. The outcome (anastomotic leakage, 30-day mortality rate, local recurrence and cancer-related survival) of operations performed by the consultant was compared with that of his trainees. RESULTS: Some 245 (92.5 per cent) of 265 patients undergoing laparotomy had a resection. Seventy (28.6 per cent) and 67 (27.3 per cent) of operations were performed by supervised and independent trainees respectively. There was no difference between the consultant, supervised and independent trainees for 30-day mortality rate (6.5, 6 and 4 per cent respectively), clinical anastomotic leakage rate (9, 2 and 5 per cent) and local recurrence rate (2, 3 and 7 per cent). There was no difference between the three groups for adjusted 5-year disease-related survival rates. CONCLUSION: Properly supervised trainees can resect a high proportion of colorectal cancers without compromising immediate outcome or long-term survival. Presented in part to the annual meeting of the Association of Surgeons of Great Britain and Ireland, Bournemouth, UK, April 1997, and published in abstract form as Br J Surg 1997; 84(Suppl): 56

Adult↗
PubMed1999

Design and evaluation of a ZP3 peptide vaccine in a homologous primate model.

The concept of a safe, immunocontraceptive vaccine using the zona pellucida glycoprotein 3 (ZP3) as an immunogen has been marred by the appearance of ovarian dysfunction in several species. However, careful selection of epitopes on mouse ZP3 have demonstrated that it is possible to segregate contraceptive bone marrow-derived (B)-cell epitopes from the cytotoxic thymus-derived (T)-cell epitopes thought to be responsible for inducing ovarian disease. B-cell epitopes on marmoset ZP3 (mstZP3) were identified by epitope mapping studies. Using a panel of polyclonal antibodies against recombinant mstZP3, an immunodominant epitope mstZP3(301-320) was identified. A chimeric peptide was co-linearly synthesized incorporating this sequence with a promiscuous tetanus toxoid T-helper cell epitope. Using the common marmoset (Callithrix jacchus) as an animal model, we have compared the consequences of active immunization with homologous recombinant mstZP3 and mstZP3(301-320) chimeric peptide vaccine. Long-term infertility was achieved using mstZP3 but at the expense of ovarian function. In contrast, no disruption to ovarian function was observed following mstZP3(301-320) immunization. Antibodies to this peptide immunolocalized to the zona pellucida of both marmoset and human ovarian sections and inhibited human sperm-zona binding by approximately 60% in vitro. However, in-vivo studies indicated that targeting a single ZP3 epitope was insufficient to reliably and consistently achieve a contraceptive effect.

Animals↗
PubMed1999

Phaeochromocytoma of the spermatic cord.

Phaeochromocytoma of the spermatic cord is very rare. It can arise anywhere in the distribution of cells of neural crest origin, but 80-90% arise in the adrenal medulla and only 3% are extra-abdominal. A small tumour may be asymptomatic because insufficient catecholamines are secreted to cause haemodynamic disturbance.

Genital Neoplasms, Male↗
PubMed1999

Measurement of human exposure to biologically relevant fractions of inhaled aerosols.

Aerosol sampling has evolved with changes in technology and our understanding of the importance of assessing the biologically relevant fractions of the total aerosol. During the past decade there has been international agreement on the definitions for the inhalable, thoracic, and respirable fractions and instruments have been developed to collect samples according to these conventions. These measurement techniques are now well established in the workplace and are increasingly being applied to assessments of non-occupational exposure, as the practical difficulties in obtaining samples over 24 hours have been solved. It is argued that multiple aerosol size fractions should be measured for hazardous substances--such as inorganic lead--in which inhaled material may either be absorbed in the alveoli (respirable) or cleared from the ciliated airways in the lung to the gut and then absorbed (thoracic and extrathoracic). Such measurements should improve the evaluation of the risk for inhaled lead, particularly for non-occupational exposure of children. Also, passive aerosol sampling techniques may enable measurements of non-occupational exposure to be made over several weeks and this would also help improve the reliability of the risk evaluation for aerosol toxicants with a long biological half life. However, further development of these techniques is required if they are to be practicable and biologically relevant.

Aerosols↗
PubMed1999

The Amoroso Lecture. The human spermatozoon--a cell in crisis?

A great deal of evidence has accumulated in recent years to suggest that there has been a gradual increase in male reproductive pathology over the past 30-40 years, as evidenced by increased rates of testicular cancer and declining semen quality. The hypothesis is advanced that this phenomenon is causally related to the ability of male germ cells to generate reactive oxygen metabolites. When produced in low levels, such metabolites are thought to enhance sperm function by stimulating DNA compaction and promoting a redox-regulated cAMP-mediated pathway that is central to the induction of sperm capacitation. When produced in excessive amounts, the same metabolites stimulate DNA fragmentation and a loss of sperm function associated with peroxidative damage to the sperm plasma membrane. Free radical-induced mutations in the male germ line may also be involved in the aetiology of childhood cancer and recent increases in the incidence of seminoma. In light of these considerations, establishing the mechanisms for free radical generation by the male germ line and determining the factors that influence this activity are important objectives for future research in this area.

Adult↗