Biomedical subjects

D Harkiss

Publications and source records attributed to D Harkiss.

11 recordsLinked to original sources

PubMed2000

Differential expression of estrogen receptor-alpha and -beta and androgen receptor in the ovaries of marmosets and humans.

Estrogens and androgens are essential for the maturation of the ovarian follicle and normal fertility in the female. We have used antibodies specific for both forms of estrogen receptor (alpha [ERalpha] and beta [ERbeta]) and androgen receptor (AR) to investigate the pattern of receptor expression in ovaries obtained from women and from a New World primate, the Common marmoset (Callthrix jacchus). On Western blots, three antibodies directed against different peptides within human ERbeta all recognized recombinant human (h) ERbeta but did not bind to recombinant hERalpha. The ERbeta protein was extracted from human ovary and prostate and marmoset ovary. In marmoset and human ovaries, ERbeta protein was detected in the nuclei of granulosa cells in all sizes of follicle (both before and after formation of the antrum), and it was also detected in thecal cells, corpora lutea, surface epithelium, and stroma. In contrast, ERalpha protein was not detected in the nuclei of granulosa cells in preantral follicles, was low/absent from stromal and thecal cells, but was expressed in granulosa cells of antral follicles and in the surface epithelium. The pattern of expression of AR protein more closely resembled that of ERbeta than ERalpha. In conclusion, three independent antibodies have demonstrated convincingly that ERbeta is expressed in a wide range of cells in the primate ovary. Granulosa cells in preantral follicles could contain ERbeta:beta dimers. In antral follicles, however, ERalpha is also expressed, and the formation of homo- or heterodimers containing ERalpha may influence the pattern of gene activation within these cells.

Amino Acid Sequence↗

PubMed1998

On the cellular mechanisms by which the bicarbonate ion mediates the extragenomic action of progesterone on human spermatozoa.

Progesterone exerts an extragenomic action on human spermatozoa, inducing a rapid calcium transient in the acrosomal domain of these cells and enhancing their potential for fertilization. This response is known to exhibit an absolute dependence on the presence of extracellular bicarbonate, although the mechanisms underlying this interaction are not understood. In this study, bicarbonate was found to exert a dose-dependent impact on the ability of progesterone to promote sperm-oocyte fusion in the absence of any collateral effect on sperm motility. The loss of sperm function in bicarbonate-free medium was associated with a failure to produce reactive oxygen species, an impaired capacity to exhibit redox-associated changes in tyrosine phosphorylation, and an apparent incapacity to generate normal calcium transients on exposure to progesterone. These defects were not related to a cAMP deficiency but were associated with a significant fall in intracellular pH. If cytosolic pH was chemically buffered into the normal range, then the spermatozoa regained every element of their response to progesterone. These results emphasize the importance of an alkaline intracellular milieu for the extragenomic action of progesterone on human spermatozoa and stress the fundamental difference between intracellular and extracellular sources of bicarbonate in maintaining the proton balance within such cells.

Acrosome↗

PubMed1998

Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa.

Reactive oxygen metabolites are known to disrupt sperm-oocyte fusion, sperm movement, and DNA integrity; however, the relative sensitivities of these elements to oxidative stress are unknown. In this study these factors were assessed in human spermatozoa exposed to increasing levels of oxidative stress achieved through the stimulation of endogenous oxidant generation with NADPH or direct exposure to hydrogen peroxide. At low levels of oxidative stress, DNA fragmentation was significantly reduced while the rates of sperm-oocyte fusion were significantly enhanced. As the level of oxidative stress increased, the spermatozoa exhibited significantly elevated levels of DNA damage (p < 0.001) and yet continued to express an enhanced capacity for sperm-oocyte fusion. At the highest levels of oxidative stress, extremely high rates of DNA fragmentation were observed but the spermatozoa exhibited a parallel loss in their capacities for movement and oocyte fusion. These studies emphasize how redox mechanisms can either enhance or disrupt the functional and genomic integrity of human spermatozoa depending on the intensity of the oxidative stimulus. Because these qualities are affected at different rates, spermatozoa exhibiting significant DNA damage are still capable of fertilizing the oocyte. These results may have long-term implications for the safety of assisted conception procedures in cases associated with oxidative stress.

DNA↗

PubMed1998

A novel signal transduction cascade in capacitating human spermatozoa characterised by a redox-regulated, cAMP-mediated induction of tyrosine phosphorylation.

Capacitation is a priming event that renders mammalian spermatozoa responsive to signals originating from the cumulus-oocyte complex. The attainment of a capacitated state is dependent upon an increase in tyrosine phosphorylation and results in the acquisition of responsiveness to physiological agonists such as progesterone and ZP3. In this study we have shown that this capacitation-dependent increase in tyrosine phosphorylation is controlled by a unique redox-regulated, cAMP-mediated, signal transduction cascade. Either stimulation of reactive oxygen species generation or elevation of intracellular cAMP induced increases in phosphotyrosine expression by human spermatozoa and enhanced their responsiveness to progesterone. Ultimate convergence of the redox- and cAMP-regulated pathways was indicated by the ability of the protein kinase A inhibitor, H89, to block both modes of signal transduction. Furthermore, the fact that the redox-regulated pathway could be silenced by catalase, while this enzyme had no effect on the cAMP-mediated response, indicated that oxidant generation must lie upstream from cAMP in the reaction sequence. In keeping with this conclusion, a functional association was demonstrated between the redox status of human spermatozoa and their cAMP content. The continuous production of reactive oxygen species was also shown to be necessary for the protein kinase A-tyrosine phosphorylation axis to remain functional. If the generation of oxidising conditions during capacitation was prevented with 2-mercaptoethanol, 2-deoxyglucose or the flavoprotein inhibitor, diphenylene iodonium, then cAMP could no longer trigger tyrosine phosphorylation. These data support a model for human sperm capacitation as a redox-regulated process, involving a unique sequence of interactive events including reactive oxygen species production, elevation of intracellular cAMP, stimulation of protein kinase A and the induction of tyrosine phosphorylation. This is the first report of such a signal transduction cascade and may have implications for the functional significance of reactive oxygen metabolites in other cell types.

Catalase↗

PubMed1996

The extragenomic action of progesterone on human spermatozoa is influenced by redox regulated changes in tyrosine phosphorylation during capacitation.

Capacitation had no effect on the ability of progesterone to elicit a rapid calcium transient in the acrosomal domain of human spermatozoa but had a marked influence of the ability of this steroid to induce a biological response. The development of this responsiveness to progesterone appeared to be redox regulated in that it was promoted by the stimulation of reactive oxygen species generation and inhibited by the presence of antioxidants, including catalase and membrane permeant thiols. The ability of redox conditions to influence the biological responsiveness of human spermatozoa did not involve changes in the dynamics of the calcium transients induced by progesterone but was causally linked with clear differences in tyrosine phosphorylation. We conclude that the ability of human spermatozoa to respond to the calcium transients induced by progesterone depends on a background of phosphotyrosine expression that can be profoundly influenced by the redox status of the spermatozoa during capacitation.

Humans↗

PubMed1994

Analysis of the extent to which sperm movement can predict the results of ionophore-enhanced functional assays of the acrosome reaction and sperm-oocyte fusion.

This study has examined the extent to which the information generated by ionophore-enhanced bioassays of the acrosome reaction and sperm-oocyte fusion might be predicted from the computer-aided analysis of sperm motility. Strong correlations (r approximately 0.7) were observed between specific components of sperm movement in semen and the potential for A23187-induced sperm-oocyte fusion, generating a stepwise regression coefficient of R = 0.663 on the bais of two criteria, percentage progressive motility and amplitude of sperm lateral head displacement (ALH). The movement characteristics of the spermatozoa recovered from the Percoll gradients gave an even higher R value of 0.838 on the basis of four variables (percentage rapid, average path velocity, straightness and ALH). In contrast, the ability of human spermatozoa to undergo acrosome reaction in response to A23187 exhibited a limited correlation with sperm movement, whether these measurements were made in the original semen sample or following Percoll purification (R approximately 0.4). These results have diagnostic implications, since sperm-oocyte fusion and the acrosome reaction clearly differ in their relative dependence on sperm motility. In practical terms, it should be noted that the computer-aided analysis of sperm movement was shown to provide up to 70% of the information generated by the more laboured assessment of sperm-oocyte fusion.

Acrosome↗

PubMed1993

Analysis of lipid peroxidation mechanisms in human spermatozoa.

The mechanisms by which ferrous ion promoters induce malondialdehyde generation by human spermatozoa have been investigated in order to provide a rational basis for the quantification and interpretation of lipid peroxidation assays. Incubation of human spermatozoa with a ferrous ion promoter in the presence of thiobarbituric acid (TBA) led to the generation of the bone fide malondialdehyde-TBA adduct. The importance of iron in the stimulation of lipid peroxidation was emphasized by the ability of Desferal and EDTA to suppress malondialdehyde generation. Paradoxically, when the concentration of EDTA relative to iron was equimolar or greater, the suppression of malondialdehyde formation was accompanied by the generation of hydroxyl radicals. These results suggested that the addition of promoter did not effect the first-chain initiation of lipid peroxidation but favored an alternative mechanism involving the catalytic decomposition of pre-existing lipid peroxides. This conclusion was reinforced by the inability of reagents that would limit the formation (superoxide dismutase and/or catalase) or availability (mannitol, formate) of hydroxyl radicals, to influence malondialdehyde generation. While hydroxyl radicals were not directly involved in Fe(2+)-promoted malondialdehyde generation, the existence of significant correlations between reactive oxygen species production and the outcome of the TBA assay, suggested that Fenton chemistry might be important in the initiation of peroxidative damage. It is proposed that the impeded propagation of peroxidation initiated by Fenton or Haber Weiss reactions would lead to the accumulation of lipid peroxides in the spermatozoa and it is these peroxides that are induced to decompose during the Fe(2+)-promoted TBA assay, stimulating a lipoperoxidative chain reaction and malondialdehyde formation.

Deferoxamine↗

PubMed1993

Use of a xanthine oxidase free radical generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa.

The reaction between xanthine and xanthine oxidase results in the univalent and divalent reduction of dioxygen to generate superoxide (O2-.) and hydrogen peroxide (H2O2), respectively. With the aid of this system, the direct effect of reactive oxygen species (ROS) on human sperm function has been investigated. A protocol involving the addition of xanthine oxidase to the reaction mixture at 0 and 15 min resulted in a loss of motility involving every component of sperm movement examined. Lower doses of xanthine oxidase, which did not influence sperm motility, were also found to suppress the competence of human spermatozoa to exhibit oocyte fusion in response to the ionophore, A23187. The reactive oxygen species responsible for the disruption of human sperm function was not influenced by the presence of superoxide dismutase (SOD) or scavengers of hypochlorous acid or hydroxyl radicals. However, the cytotoxic species was shown to be extremely stable and could be completely eliminated by catalase, which selectively eliminates H2O2. Confirmation that it is H2O2, and not O2-., which is cytotoxic to human spermatozoa was obtained in studies in which the direct addition of this oxidant was shown to influence both the movement of human spermatozoa and their competence for oocyte fusion. These results carry implications for the diagnosis of defective sperm function and the design of optimized culture media for the treatment of male factor infertility.

Calcimycin↗

PubMed1993

Relationship between iron-catalysed lipid peroxidation potential and human sperm function.

The relationship between lipid peroxidation and the functional competence of human spermatozoa has been investigated in a cohort of 31 infertility patients. Lipid peroxidation was assessed using a sensitive fluorometric assay for the generation of malondialdehyde in response to the presence of a ferrous ion promoter. Sperm function was evaluated by monitoring the movement characteristics of these cells and their capacity for sperm-oocyte fusion. Each sample was separated into high- and low-density sperm populations on discontinuous, two-step (40%:80%), Percoll gradients prior to analysis. The way in which individual ejaculates fractionated on these gradients was highly positively correlated (P < 0.001) with the lipoperoxidation status of the spermatozoa; the greater the potential for malondialdehyde generation, the higher the proportion of cells entering the low density region of the gradients. The lipoperoxidation potential of the freshly prepared spermatozoa was also highly predictive (P = 0.0001) of their capacity for movement at 3 and 24 h and their ability to exhibit sperm-oocyte fusion in response to the ionophore A23187. The potential for malondialdehyde generation in the 40% and 80% Percoll fractions was positively associated with midpiece abnormalities in the spermatozoa. These results emphasize the importance of lipid peroxidation in the pathophysiology of male infertility and suggest a mechanism by which such damage might arise.

Female↗

PubMed1992

Immunosuppression by human seminal plasma--extracellular organelles (prostasomes) modulate activity of phagocytic cells.

PROBLEM: Prostasomes are trilamellar to multilamellar vesicles produced by the acinar cells of the human prostate and are present in appreciable amounts in normal human semen. The aim of this work was to study the effect of prostasomes on human polymorphonuclear cell and monocyte function. METHODS: Functional activity of human neutrophils and monocytes was studied after incubation with prostasomes isolated from normal human seminal plasma. The following functional tests were employed: ability to ingest latex particles and opsonized bacteria (St. aureus) and ability to generate superoxide anion in response to PMA and FMLP. The latter was determined by measuring the superoxide-dismutase inhibitable reduction of ferricytochrome c to ferrocytochrome c at 550 nm. Expression of cell surface markers and interactions of prostasomes with cells were studied by cytofluorimetry. RESULTS: We show that prostasomes bind rapidly to the leukocyte cell membrane followed by internalization of adsorbed material. Interactions of prostasomes with neutrophils and monocytes inhibits their ability to phagocytose latex particles. The ability to ingest opsonized bacteria is, however, not impaired. Our results also show that incubation of leukocytes with prostasomes effectively inhibits superoxide anion generation in response to activation by PMA and FMLP. CONCLUSIONS: Prostasomes may play a complementary role to other immunosuppressive factors contained in the human semen. They may protect sperm cells from deleterious effects of phagocytosing cells, prolong their life, and consequently enhance the chance of conception. At the same time prostasomes may have a permissive effect on sexually transmitted diseases.

Cell Adhesion↗

PubMedDate not supplied

Sperm penetration into a hyaluronic acid polymer as a means of monitoring functional competence.

The diagnostic significance of sperm penetration assays based on a commercially available hyaluronate preparation (Sperm Select) has been investigated in the male partners of 77 couples characterized by a normal female partner. Sperm penetration into hyaluronate was highly correlated with the ability of the same sperm populations to penetrate bovine cervical mucus and, moreover, depended on the same attributes of semen quality, including the morphology of the spermatozoa, their number, and their motility as reflected by their mean path velocity. Stepwise multiple regression analyses employing these independent variables generated r values of 0.821 to 0.931, depending on the criterion of hyaluronate penetration used; path velocity was consistently the most informative variable according to the standardized regression coefficients. The relationship between hyaluronate penetration and sperm movement was so close that multiple regression equations could be generated that were capable of accounting for up to 76% of the variance in sperm velocity measurements obtained with a computerized image analysis system. Regression equations could also be generated using the hyaluronate penetration data that could account for 65% of the variance observed in an A23187-enhanced zona-free hamster oocyte penetration test, including the successful identification of the subpopulation of patients in whom 0% oocyte penetration had been recorded. Within the same data set, independent variables based on bovine cervical mucus penetration could only account for 43.5% of the variance in sperm-oocyte fusion. Hyaluronate penetration therefore appears to offer a simple, objective means of generating information on the functional competence of human spermatozoa that should find a role in routine diagnostic services where the more specialized tests are not available.

Animals↗