Biomedical subjects

P F Watson

Publications and source records attributed to P F Watson.

At least 73 records · Page 4Linked to original sources

PubMed1997

The pattern of cytokine mRNA expression in ovarian endometriomata.

Intraovarian cytokines play a pivotal role in the normal growth and development of the ovarian follicle. The purpose of this study was to investigate the pattern of cytokine mRNA expression in ovarian endometriomata. A total of 10 patients with histologically confirmed endometriomata undergoing surgery formed the study group while nine patients undergoing sterilization with no evidence of a cyst in the ovary formed the control group. Biopsies of the ovary were obtained at surgery and stored in liquid nitrogen until processed by reverse transcription-polymerase chain reaction amplification to identify the presence of mRNA for interleukin (IL)-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, tumour necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma). IL-6 and IL-10 mRNA were expressed by nine and seven patients respectively in the endometriosis group compared with three and one patients in the control group; this difference was significant (P < 0.05). IL-1 alpha mRNA was expressed by seven of 10 patients with endometriosis but by only one of the control group; this was again significantly different (P < 0.04). Ovarian IL-2 and IL-4 mRNA were not expressed in either group. There was no significant difference in the expression of IL-8, IL-13, IFN-gamma and TNF-alpha mRNA in the two groups. These findings suggest that abnormal local expression of certain cytokines may contribute to the development of endometriomata.

Adult↗

PubMed1997

Detection of tyrosinase autoantibodies in patients with vitiligo using 35S-labeled recombinant human tyrosinase in a radioimmunoassay.

Tyrosinase antibodies recently have been reported to occur frequently in patients with vitiligo. We describe the detection of tyrosinase antibodies in vitiligo patients using in vitro 35S-labeled human tyrosinase in a radioimmunoassay. Of 46 vitiligo sera examined in the assay, five (10.9%) were found to be positive for tyrosinase antibodies. In contrast, 20 control sera and sera from 10 patients with Hashimoto's thyroiditis were negative. Four of the sera positive in the radioimmunoassay were also positive in an ELISA using mushroom tyrosinase as antigen. Absorption studies indicated that pre-incubation with mushroom tyrosinase absorbed out the immunoreactivity of the positive sera in the radioimmunoassay, suggesting cross-reactivity, but this absorption was never complete, indicating that there are tyrosinase antibodies in human sera that do not react with the mushroom protein. There was no obvious association between the presence of tyrosinase antibodies and the age of the patients (range: 22-62 y), their duration of disease (range: 5-20 y), or the type of vitiligo (one segmental, one symmetrical/periorificial, three symmetrical), although the three patients with the highest antibody levels also had an associated autoimmune disorder (one with Graves' disease; two with autoimmune hypothyroidism). The results confirm that tyrosinase autoantibodies are present in the sera of vitiligo patients but at a low frequency. The technique described is sensitive and quantitative and allows the detection of conformational epitopes. It will be useful in longitudinal studies to determine the relation between the clinical features of vitiligo and tyrosinase antibody levels.

Adult↗

PubMed1997

Analysis of immunoglobulin G kappa antithyroid peroxidase antibodies from different tissues in Hashimoto's thyroiditis.

Antibodies (Ab) to thyroid peroxidase (TPO) are common in patients with autoimmune thyroid disease and may play a role in disease pathogenesis. We have prepared immunoglobulin G kappa (IgG kappa) and IgG lambda phage display combinatorial libraries from the cervical (thyroid-draining) lymph nodes of 2 Hashimoto's thyroiditis patients and from the thyroid of 1 patient. After selection with purified recombinant human TPO, up to 10 high affinity IgG kappa clones from each tissue source were analyzed further. No IgG lambda Fab were detected in the patient with the highest TPO Ab titer. Sequence analysis of the clones showed restricted heavy and light chain usage, similar to that in previously published TPO-reactive Fabs. This was despite the substantially larger sizes of the initial libraries, the use of lymph node tissue to generate libraries, and the analysis of the repertoire in patients with Hashimoto's thyroiditis rather than Graves' disease. There was overall similarity in sequences obtained from lymph node and thyroid libraries, with higher levels of somatic hypermutation in the former. The Fab inhibited binding of serum TPO Ab from five patients by 55-95%. These data together with those from previous reports indicate that although there is no unique Ab gene usage, there is the recurrent presence of certain variable regions in the high affinity TPO Ab response.

Amino Acid Sequence↗

PubMed1997

Detection of IL-12, IL-13, and IL-15 messenger ribonucleic acid in the thyroid of patients with autoimmune thyroid disease.

IL-12, IL-13, and IL-15 are important cytokines that have not been studied previously in human autoimmune thyroid diseases. By applying RT-PCR on RNA extracted from tissue samples, we have investigated in vivo gene expression of these cytokines in multinodular goiter (MNG), Graves' disease (GD), and Hashimoto's thyroiditis (HT). In addition, in vitro studies were carried out using the transformed human thyroid cell line, HT-ori3, and primary thyroid cell cultures derived from patients with GD. These cells were used either unstimulated or stimulated for 12 h with TSH, IL-1, or interferon-gamma (IFN-gamma). IL-12 p40 gene expression was identified in 2 of 10 MNG samples, 6 of 12 GD, and 3 of 4 HT. HT-ori3 and primary thyroid cell cultures were positive for IL-12 p40 messenger RNA (mRNA) expression in both unstimulated and stimulated cell cultures. IL-13 mRNA was expressed in 2 MNG, 9 GD, and 1 HT sample. Both HT-ori3 and primary thyroid cultures expressed IL-13 after TSH, IL-1, or IFN-gamma stimulation; unstimulated primary cultures of thyroid cells, however, did not express IL-13. IL-15 gene expression was detected in 8 MNG, 8 GD, and 4 HT samples. HT-ori3 and primary thyroid cell cultures, stimulated with TSH, IL-1, or IFN-gamma, showed expression of this cytokine. Unstimulated cells showed only a weak expression. Our results indicate that the cytokine patterns in the various diseases studied are heterogeneous; that thyroid cells can express IL-12, IL-13, and IL-15 mRNA in culture, particularly after TSH, IL-1, or IFN-gamma stimulation; and that IL-15 is expressed in most of the tissue samples studied.

Autoimmune Diseases↗

PubMed1997

Analysis of the T cell receptor V alpha repertoire in Hashimoto's thyroiditis: evidence for the restricted accumulation of CD8+ T cells in the absence of CD4+ T cell restriction.

There has been considerable interest in the possible restriction of the TCR repertoire in autoimmune disorders, because it would have important therapeutic implications. Using ribonucleic acid derived from matched peripheral blood lymphocytes (PBL), intrathyroidal lymphocytes (ITL), and CD4- and CD8-selected ITL from three patients with Hashimoto's thyroiditis (HT), we carried out reverse transcription-PCR analysis of TCR V alpha family usage. No evidence was found for V alpha family restriction in the PBL, ITL, CD4-selected ITL, or CD8-selected ITL. However, restriction was frequent in the CD8-selected ITL after denaturation/reannealing of the PCR products followed by nondenaturing PAGE; similar restriction was uncommon in PBL, CD8-selected PBL, ITL, or CD4-selected ITL. V alpha 3 and V alpha 6 TCR chains from CD8-selected ITL bands from one patient were cloned and sequenced. There was marked sequence restriction, particularly within the ITL V alpha 6 TCR chains, in which 14 of 15 homoduplex band sequences used the J4 segment and had an identical V/N/J junction amino acid (but not nucleotide) sequence. Sequence restriction was not detected in matched CD8-selected PBL material. These data show that there is a marked restriction of V alpha chain usage in the CD8+ (but not CD4+) T cells in the Hashimoto's thyroid, with clonal expansion of some sequences.

Adult↗

PubMed1997

Immunoglobulin G kappa antithyroid peroxidase antibodies in Hashimoto's thyroiditis: epitope-mapping analysis.

Patients with autoimmune thyroid disease frequently have high affinity antibodies to thyroid peroxidase (TPO), although the role they play in disease pathogenesis is not known. We have previously prepared 37 monoclonal anti-TPO IgG kappa Fab fragments from two patients with Hashimoto's thyroiditis and demonstrated the similarity of these Fab sequences to those published previously, mainly derived from patients with Graves' disease. In this paper, we described epitope mapping of these Fabs using a previously characterized panel of murine monoclonal antibody (mAb) and show that the Fabs bind to two neighboring epitopes on native TPO. Although the epitope-mapping method differs from that used to characterize previously published TPO-reactive Fab sequences, it indicates a similarly restricted response to neighboring epitopes in both Graves' disease and Hashimoto's thyroiditis. The epitope mapping included mAb 47, which binds to a linear TPO peptide of known sequence in addition to native TPO. Although TPO-reactive Fab did not inhibit the binding of mAb 47, mAb 47 did inhibit the binding of Fab, indicating the likely site of the immunodominant region on native TPO. These results confirm the restricted nature of TPO antibody and further delineate the immunodominant region of native TPO as defined by the mAb.

Animals↗

PubMed1996

Cloning and analysis of IgG kappa and IgG lambda anti-thyroglobulin autoantibodies from a patient with Hashimoto's thyroiditis: evidence for in vivo antigen-driven repertoire selection.

Antibodies to thyroglobulin (Tg) are commonly found in patients with the autoimmune thyroid diseases Graves' disease and Hashimoto's thyroiditis as well as in individuals with apparently normal thyroid function. Although it is not clear how Tg Abs are involved in the pathology of the diseases, the study and analysis of these Abs may nevertheless be instructive in allowing the development of an Ab response to an autoimmune disease-associated self Ag to be followed. We have prepared IgG kappa and lambda phage display combinatorial libraries from the cervical lymph node of a single Hashimoto's thyroiditis patient with a high anti-Tg titer. These were selected with purified human Tg, and 10 IgG kappa and 9 IgG lambda clones were analyzed further. Sequence analysis of the clones showed a very highly restricted heavy chain usage and a less restricted light chain usage. There was a variable degree of divergence from germ-line sequence in the light chain sequences, with a clear relationship between relatively higher affinity of the Fab for human Tg and an increased degree of somatic hypermutation. The Tg-selected Fab did not bind to Tg from other species, to reduced denatured Tg, or to thyroid peroxidase. The Fab inhibited patient serum binding to human Tg by between 39 and 79%. In summary, we have isolated 19 high affinity, human Tg-specific Fab and shown that the relative affinity of the Fab is directly related to the pattern of somatic hypermutation.

Amino Acid Sequence↗

PubMed1996

A possible mechanism of action for fertilization promoting peptide, a TRH-related tripeptide that promotes capacitation and fertilizing ability in mammalian spermatozoa.

Fertilization promoting peptide (FPP), a tripeptide structurally related to thyrotrophin releasing hormone (TRH), has been shown to stimulate capacitation and fertilizing ability in both mouse and human spermatozoa, but the mechanisms of action involved in these responses are currently unknown. In the present study utilizing epididymal mouse spermatozoa, we have compared the ability of FPP, TRH, and pyroglutamylphenylalanineprolineamide (an uncharged structurally related tripeptide found in seminal plasma) to stimulate capacitation. At 50 nM, the mean concentration of FPP found in human seminal plasma, only FPP produced a significant response. This suggests that if a receptor is involved, it is one distinct from the TRH receptor. A significant response to FPP required the presence of extracellular Ca2+, with 90 microns Ca2+ being sufficient to support a stimulation of capacitation. The addition of FPP to suspensions at later stages of capacitation indicated that the nature of the response changed, such that addition of FPP to capacitated suspensions inhibited spontaneous acrosome reactions; however, FPP-treated cells were still able to undergo acrosomal exocytosis in response to progesterone, a physiological agonist of acrosomal exocytosis. Because earlier studies had identified a similar capacitation-related change in response to adenosine, being stimulatory early in capacitation and inhibitory later in capacitation, we investigated the possibility that FPP and adenosine might be acting via the same pathway. The combination of FPP plus adenosine, whether used at low, non-stimulatory concentrations or high, maximally-stimulatory concentrations, was more effective in promoting capacitation than either compound used individually. As observed with FPP, addition of adenosine to capacitated cells inhibited spontaneous acrosome loss but did not inhibit exocytosis in response to progesterone. This suggests that the two molecules are affecting a common pathway. Since adenosine, acting via specific cell surface receptors, can stimulate fertilizing ability and adenylate cyclase activity in uncapacitated cells and then inhibit enzyme activity in capacitated cells, we propose that FPP may act by modulating the adenylate cyclase/cyclic AMP signal transduction pathway. In vivo, FPP, which would contact spermatozoa at ejaculation and probably remain bound to cells for some time, could stimulate capacitation as the spermatozoa ascend the female tract; adenosine, present in seminal plasma and the female tract, could either augment FPP's action or replace it if FPP is lost from the cell surface. We therefore suggest that FPP and adenosine, by modulating adenylate cyclase activity to promote capacitation but inhibit spontaneous acrosomal exocytosis, may provide an endogenous mechanism that helps to optimize the fertilizing potential of the few sperm cells that reach the site of fertilization in vivo.

Animals↗

PubMed1996

Cytokines and thyroid function.

Cytokines play a crucial role in autoimmune thyroid disease (ATD) through various mechanisms. They are produced in the thyroid by intrathyroidal inflammatory cells, in particular lymphocytes, as well as by the thyroid follicular cells (TFC) themselves and may thus act in a cascade to enhance the autoimmune process (Fig. 1). Cytokines upregulate the inflammatory reaction through stimulation of both T and B cells, resulting in antibody production and tissue injury. In addition, intrathyroidal cytokines induce immunological changes in TFC including enhancement of both major histocompatibility complex (MHC) class I and class II molecule expression, and upregulation of adhesion and complement regulatory molecule expression. Cytokines can also modulate both growth and function of TFC and have a role in extrathyroidal complications of ATD, most importantly thyroid-associated ophthalmopathy (TAO), where they induce fibroblast proliferation and enhance the production of glycosaminoglycans (GAG), resulting in proptosis and the other clinical features of the disease. In addition to these effects, exogenous administration of cytokines has been associated with impairment of thyroid function ranging from the appearance of autoantibodies alone to the development of frank thyroid dysfunction. Cytokines have also been implicated in subacute thyroiditis (SAT) and amiodarone-induced thyroid dysfunction, as well as in thyroid function abnormalities occurring in patients with non-thyroidal illnesses (NTI). Genetic variations in cytokine genes represent potential risk factors for ATD, and disease associations have been described for polymorphisms in IL-1ra and TNF beta genes. Recent experimental evidence suggests the possibility of novel cytokine-based therapeutic approaches for ATD and its complications, in particular TAO.

Animals↗

PubMed1996

Intrathyroidal cytokine gene expression in Hashimoto's thyroiditis.

Hashimoto's thyroiditis (HT) is an organ-specific autoimmune disease in which cytokines are likely to have a role in the initiation and perpetuation of the disease. Using the reverse transcription-polymerase chain reaction (RT-PCR) we analysed the cytokine profile in four HT tissue samples. Furthermore, cell fractionation was carried out on two tissue samples and cytokine profile was studied in CD4+ and CD8+ T cells, in addition to the residual cellular infiltrate composed of CD4- and CD8- cells. Our results showed IL-1 beta, IL-4 and IL-6 mRNA expression in three out of four tissue samples, whereas IL-1 alpha, IL-2, IL-8, IL-10, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) were expressed in all tissue samples studied. Expression of IL-1 alpha and IL-1 beta was absent in both CD4+ and CD8+ subsets. However, IL-2, IL-4, IL-6, IL-8, IFN-gamma and TNF-alpha mRNA were detected in both CD4+ and CD8+ subsets. IL-10 was expressed in the CD4+ subset in one sample, whereas it was negative in both CD8+ fractions. All the cytokines studied were expressed in the residual infiltrate. These results suggest a mixed Th1 and Th2 response in HT, both of which may have an important role in the pathophysiology of the thyroid destructive process through cell-mediated cytotoxicity, and/or humoral autoimmunity manifested by autoantibody production.

Adult↗

PubMed1996

Fertilization promoting peptide, a tripeptide similar to thyrotrophin-releasing hormone, stimulates the capacitation and fertilizing ability of human spermatozoa in vitro.

Recent studies have demonstrated that a prostatic tripeptide similar in structure to thyrotrophin-releasing hormone (TRH) can stimulate the in-vitro capacitation and fertilizing ability of epididymal mouse spermatozoa. Therefore we have proposed that this tripeptide be referred to as fertilization promoting peptide (FPP). Using chlortetracycline fluorescence analysis and the hamster oocyte penetration test (HOPT), we have obtained evidence that FPP can also promote the capacitation and fertilizing ability of ejaculated human spermatozoa in vitro. FPP (25-200 nM) caused a significant increase in the proportion of B-pattern uncapacitated cells, with no significant stimulation of acrosomal exocytosis. Comparison of FPP with two structurally similar tripeptides, TRH and pyroglutamyl phenylalanylprolineamide, at 50 nM revealed that only FPP could significantly promote capacitation. Finally, after a brief exposure to progesterone to induce acrosomal exocytosis in capacitated cells, FPP-treated suspensions penetrated a significantly higher proportion of oocytes than the untreated controls when assessed in the HOPT. The presence of FPP in human seminal plasma at concentrations similar to those used here suggests that, in vivo, FPP may play a positive role in promoting human sperm function.

Animals↗

PubMed1996

Surface area and volume measurements for ram and human spermatozoa.

Surface area and volume measurements were made for ram and human spermatozoa. Measurements were made using different techniques in an attempt to confirm estimates arrived at by independent methodologies. Sperm head projected surface areas were calculated from published formulae using linear micrometry measurements or were obtained directly by image analysis. Total surface areas were calculated as twice the projected head area plus the flagella area, estimated from published dimensions. Spermatozoon surface area was also measured from electron micrographs using a stereological method. Micrometric methods gave values of 135 microns2 for ram spermatozoa and 106 microns2 for human spermatozoa. Stereology methods gave values of 142 microns2 for ram spermatozoa and 106.5 microns2 for human spermatozoa, offering good agreement between the two methods. Sperm volumes were estimated by stereology and by radiolabel volume exclusion methods. From stereology, ram sperm volume was 13.3 microns3 and human sperm volume was 22.2 microns3. From the volume exclusion method, ram sperm volume was estimated as 25 microns3. Attempts to measure total volume and water volume simultaneously using and adaptation of this method were unsuccessful. Separate estimation of water volume for ram spermatozoa gave a value or 12.8 microns3, suggesting a 50% water space. Results from this study are compared with existing published values.

Animals↗

PubMed1995

Flow cytometric detection of bicarbonate-induced changes in lectin binding in boar and ram sperm populations.

Boar and ram spermatozoa were incubated in Tyrode's medium in the presence or absence of bicarbonate/CO2, a component believed essential for capacitation. At intervals, samples were stained with a range of FITC-lectins to detect changes in surface glycoconjugates, using a rapid staining technique to avoid problems of lectin toxicity. The samples were then analysed directly by flow cytometry, using propidium iodide to distinguish dead cells. In the presence of bicarbonate, a live subpopulation of spermatozoa developed, which in both animal species showed higher binding affinities towards Phaseolus Vulgaris Agglutinin (PHA-E), Sophora Japonica Agglutinin (SJA), and Soybean Agglutinin (SBA), and lower binding affinity towards Erythrina Cristagalli Lectin (ECL). In boar samples, the modified subpopulation reached a maximum after 3 hr incubation, whereas in ram samples it maximized after 1.5 hr. No changes were seen when spermatozoa were incubated in bicarbonate-free medium. The bicarbonate-induced changes in lectin binding were not due to the onset of acrosome reactions, because spermatozoa induced to undergo acrosome reactions with the ionophore A23187 displayed very different lectin-binding patterns. Tested on boar spermatozoa, seminal plasma not only inhibited but reversed the bicarbonate-induced development of the modified subpopulation. EGTA also inhibited development of boar sperm subpopulations; excess Ca2+ was unable to overcome this inhibition, suggesting that multivalent metal ions might be involved in bicarbonate's action. We conclude that bicarbonate causes a loss of surface coating material with affinity for ECL and an unmasking of binding sites for SBA, SJA and PHA-E. A modified subpopulation of live spermatozoa is thereby established, which appears to maximize at a rate in accord with reported capacitation times.

Animals↗

PubMed1995

Localization of intracellular calcium during the acrosome reaction in ram spermatozoa.

Calcium was identified by a pyroantimonate-osmium fixation technique in ram spermatozoa undergoing a spontaneous acrosome reaction induced by incubation of diluted semen at 39 degrees C. Intracellular calcium was only detected in diluted spermatozoa and increased in amount and distribution over 4 hr At 4 hr, the majority of the spermatozoa displayed ultrastructural evidence of an acrosome reaction. Calcium was initially evident on the outer acrosomal membrane in multiparticulate clusters, which were seen to be located on scalloped crests of acrosomal membrane as fusion developed; it was also located in the region of the acrosomal ridge beneath the outer acrosomal membrane. Vesiculation commenced just anterior to the equatorial segment and proceeded anteriorly. As vesiculation advanced, calcium particles became associated with the periphery of the vesicles attached in the region of the fusion between the two membranes, but were never seen inside the vesicles. The equatorial segment was not labelled until much later in the reaction, at which time calcium particles were also evident on the nuclear membrane; vesiculation of the equatorial segment was also noted at this time. Dense labelling of the postacrosomal dense lamina was seen in all incubated spermatozoa. At the anterior margin of this structure the labelling was seen to be in a "sawtooth" arrangement. The disposition of the calcium both temporally and spatially is discussed in relation to its possible mechanisms in bringing about membrane fusion.

Acrosome↗

PubMed1995

Determination of water permeability coefficient and its activation energy for rabbit spermatozoa.

Critical tonicity (tonicity at which 50% of cells swell beyond their maximum volume-to-surface area ratio and lyse) of rabbit spermatozoa was measured as 45.6 mOsm at 22 degrees C. To determine the temperature effect on critical tonicity, cells were equilibrated to 15, 25, and 35 degrees C and critical tonicity was measured as 52.9, 42.2, and 32.4 mOsm, respectively. The time taken for rabbit sperm to achieve lysis at these temperatures was measured by exposing cells for increasing times to distilled water. From these results values for the permeability coefficient of rabbit spermatozoa to water (Lp) and its activation energy (Ea) were calculated. At 25 degrees C, Lp was 0.63 micron/min/atm and was clearly temperature dependent; Ea was 17.8 kcal/mol. Rabbit spermatozoa appear to have a low Lp and high Ea, the opposite of the situation seen with spermatozoa from all other species examined to date which have high Lp and low Ea. Nevertheless, the values obtained permit modeling of cooling rates for cell survival during cryopreservation in keeping with cooling rates commonly employed.

Animals↗

PubMed1995

Fracture phenomena in an isotonic salt solution during freezing and their elimination using glycerol.

Thermal stress and consequent fracture in frozen organs or cell suspensions have been proposed to be two causes of cell cryoinjury. A specific device was developed to study the thermal stress and the fracture phenomena during a slow cooling process of isotonic NaCl solutions with different concentrations of glycerol (cryoprotectant) in a cylindrical tube. It was shown from the experimental results that glycerol significantly influenced the solidification process of the ternary solutions and reduced the thermal stress. The higher the initial glycerol concentration, the lower the thermal stress in the frozen solutions. Glycerol concentrations over 0.3 M were sufficient to eliminate the fracture of the frozen solutions under the present experimental conditions. To explain the action of glycerol in reducing the thermal stress and preventing the ice fracture, a further study on ice crystal formation and growth of ice in these solutions was undertaken using cryomicroscopy. It is known from previous studies that an increase of initial glycerol concentration reduced frozen fraction of water in the solution at any given low temperature due to colligative properties of solution, which reduced the total ice volume expansion during water solidification. The present cryomicroscopic investigation showed that under a fixed cooling condition the different initial glycerol concentrations induced the different microstructures of the frozen solutions at not only a given low temperature but also a given frozen fraction of water. It has been known that ice volume expansion during solidification is a major factor causing the thermal stress and the interior microstructure is critical for the mechanical strength of a solid. Therefore, functions of glycerol in reducing the total ice volume expansion during water solidification and in influencing interior microstructure of the ice may contribute to reduce the thermal stress and prevent the fracture in the frozen solutions.

Animals↗

PubMed1995

Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function.

New research on the cooling and cryopreservation of mammalian spermatozoa is reviewed in the context of the older literature. Cryoinjury to a variety of cell organelles is regarded as being due to the two major stresses of cryopreservation, i.e., the change in temperature, and the formation and dissolution of ice and its consequences. Since the cryopreservation process involves departure of the cells from and return to body temperature, both cold shock and warm shock are included as potential stresses to be considered, as well as the stages involving cooling below the freezing point of the medium. The causes of cryoinjury are reconsidered and new concepts concerning the influence of osmotic stress are presented. Heterogeneity of the sperm population is discussed in the context of the success with which spermatozoa can be cryopreserved between and within ejaculates and individuals. The functional state of frozen and thawed spermatozoa is examined on the basis of published results of structural and functional tests of sperm competence. The hypothesis is advanced that cryopreserved mammalian spermatozoa are in a state resembling capacitation, which accounts for their relatively reduced longevity and their readiness to undergo egg penetration without incubation. The importance of this to the utilization of cryopreserved spermatozoa is examined, and proposals are made for new avenues of research to overcome these problems.

Animals↗

PubMed1995

Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol.

Use of a cryoprotective agent is indispensable to prevent injury to human spermatozoa during the cryopreservation process. However, addition of cryoprotective agents to spermatozoa before cooling and their removal after warming may create severe osmotic stress for the cells, resulting in injury. The objective of this study was to test the hypothesis that the degree (or magnitude) of human sperm volume excursion can be used as an independent indicator to evaluate and predict possible osmotic injury to spermatozoa during the addition and removal of cryoprotective agents. Glycerol was used as a model cryoprotective agent in the present study. To test this hypothesis, first the tolerance limits of spermatozoa to swelling in hypo-osmotic solutions (iso-osmotic medium diluted with water) and to shrinkage in hyperosmotic solutions (iso-osmotic medium with sucrose) were determined. Sperm plasma membrane integrity was measured by fluorescent staining, and sperm motility was assessed by computer-assisted semen analysis before, during and after the anisosomotic exposure. The result indicate firstly that motility was much more sensitive to anisosmotic conditions than membrane integrity, and secondly that motility was substantially more sensitive to hypotonic than to hypertonic conditions. Based on the experimental data, osmotic injury as a function of sperm volume excursion (swelling or shrinking) was determined. The second step, using these sperm volume excursion limits and previously measured glycerol and water permeability coefficients of human spermatozoa, was to predict, by computer simulation, the cell osmotic injury caused by different procedures for the addition and removal of glycerol. The predicted sperm injury was confirmed by experiment. Based on this study, an analytical methodology has been developed for predicting optimal protocols to reduce osmotic injury associated with the addition and removal of hypertonic concentrations of glycerol in human spermatozoa.

Buffers↗