Flow cytometric determination of binding of monoclonal antibodies to human spermatozoa

Summary. Although several monoclonal antibodies to sperm antigens are available, only few systematical reports on the appearance of distinct antibody binding structures in spermatozoa of infertile patients are available. We studied the binding frequency of six monoclonal antibodies (TÜS-1, -2, -10, -17, -19, -20) by means of a flow cytometer in different semen samples and their possible relationship to other sperm parameters, in particular to the motility parameters as determined by computer-assisted semen analysis. The percentage of vital spermatozoa binding the different antibodies was 21.5% (TÜS-2) to 52.1% (TÜS-20). The percentage binding was higher in samples with normozoospermia than in those with oligo-asthenoteratozoospermia. A significant correlation occurred between sperm concentration and the percentage of spermatozoa stained by TÜS-2 and TÜS-17; between motile spermatozoa and those binding TÜS-17 and TÜS-20; the velocity average path and those binding TÜS-17; an inverse correlation occurred between the morphologically-abnormal spermatozoa and those binding TÜS-20; an inverse correlation between TÜS-17 and acrosin. We conclude from our results that although the antigens of the antibodies concerned are not yet characterized, the determination of antibody binding will give additional information on the functional status during capacitation and acrosome reaction of spermatozoa.     

Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies

Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry (FCM) and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies (MoAbs), which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase (cAMP-dependent protein kinase II, PRKAR2A), evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins.     

Flow cytometric evaluation of Thomsen-Friedenreich antigen on transitional cell cancer using monoclonal antibody

Summary In 31 transitional cell cancer (TCC) tissues and 5 normal bladder muscosae (NBM), we compared the results of flow cytometry (FCM) and immunohistochemical examination in evaluating the expression of Thomsen-Friedenreich antigen (T-Ag) using a monoclonal antibody. On immunohistochemical examination, 14 (45%) cancer tissues showed T-Ag, while 7 (23%) cancer tissues and all NBM showed only cryptic T-Ag, which was detected only after neurminidase treatment. Ten (32%) high grade cancer tissues showed neither T-Ag nor cryptic T-Ag. On FCM the T-Ag positive cells (TPC) and the T-Ag positive cells after neuraminidase treatment (nTPC) were counted in fresh cell suspensions. FCM was more sensitive than immunohistochemical study in detecting T-Ag. Additionally, FCM revealed that some tumors had both T-Ag and cryptic T-Ag at the same time. The ratio of nTPC to TPC was well correlated with the stage or grade of the tumor and may be a more reliable marker of TCC than the expression of T-Ag assessed by immunohistochemical techniques.     

Localization of binding sites of naturally occurring antisperm antibodies on human spermatozoa by immunofluorescence

Antisperm antibodies (ASA) may affect sperm motility, acrosome reaction, sperm penetration of cervical mucus, binding to the zona pellucida, and sperm-egg fusion. We investigated the localization of ASA of infertile men or men after vasectomy bound on the sperm surface using an immunofluorescence method. Binding occurred in the acrosomal region, midpiece, and tail. Most of the ASA in both groups of patients bound to the midpiece alone or in combination with other regions of spermatozoa. Only few ASA samples showed binding to all the three sperm regions. A combination of binding to the acrosomal region and to the midpiece was never observed. In infertile patients with ASA, the binding site was compared with sperm parameters. ASA binding to the sperm head influenced the acrosome reaction. Binding of ASA on tail and/or midpiece was not associated with a significant alteration of viability and motility. Immunofluorescence appears to be a valuable tool in the diagnosis of immune infertility, in particular when impairment of the acrosome activity is suggested.     

Flow cytometric analysis of the nuclear DNA content of hepatocellular carcinoma

The nuclear DNA content of 77 resected specimens from 65 cases of hepatocellular carcinoma (HCC) was measured by means of flow cytometry. The DNA index (DI) was calculated and the correlation between the DNA ploidy pattern and clinicopathological findings was studied. In the cases of HCC with a diameter of less than 5 cm, the 3-year survival rate of the aneuploid cases was 44.5 per cent, which was significantly lower the 91.4 per cent of the diploid cases (p<0.001). Serum AFP levels were over 1000 ng/ml in 46.4 per cent of the aneuploid tumors and 18.5 per cent of the diploid tumors (p<0.05). The DI’s were investigated in several sites of the same tumor and no difference was seen among the different sites in 16 out of 17 tumors. From 8 recurrent cases out of 12 who underwent a second resection, seven did not show any significant differences in DI from their primary tumor. On the other hand, four cases of second primary tumors showed different DI’s to those of their first primary tumor. Intra-hepatic metastatic tumors exhibited the same DI’s as their primary tumors. Thus, the nuclear DNA ploidy pattern may serve as a stable and valuable marker in predicting the malignant potential and prognosis of HCC.     

The effect of anaesthesia and surgery for benign disease of the upper urinary tract on circulating leucocyte subpopulations identified with monoclonal antibodies

Summary Circulating lymphocyte subpopulations were monitored, using monoclonal antibodies and flow cytometry, in six patients undergoing surgery for benign disease of the upper urinary tract. A significant decrease in the total number of circulating lymphocytes was observed. This could be attributed to a significant decrease of T cells of both major subsets — the so-called T helper (Th) and T suppressor/cytotoxic (Ts) subpopulations. When the results of the T cell subsets were expressed as a ratio (leu-3a⁺/leu2a⁺, T helper/suppressor) no significant change was noted. In contrast neither B cells nor natural killer (NK) and antibody dependent killer (K) cells were significantly affected. This selective loss of T cells from the circulation may be relevant to post operative infection and should be considered in the course of immunological monitoring.Supported by the Cancer Research Campaign     

Identification of rectal ganglion cells using monoclonal antibodies

A monoclonal antibody has been developed that reacts specifically with ganglion cells of the myenteric and submucous plexuses of the gastrointestinal tract. This antibody also recognizes an axonal antigen that is distributed throughout the circular muscle of normal colon and rectum. Aganglionic segments of colon and rectum from patients with Hirschsprung's disease showed no specific antibody binding using a fluorescent labelled assay. The use of monoclonal antibodies should provide further insights into the pathophysiology of Hirschsprung's disease and allied disorders.     

Production and characterization of new monoclonal antibodies to human osteoclasts

Summary Two monoclonal antibodies raised against human osteoclastoma were found to show antiosteoclastic activity on frozen sections of tumor. Immunoreactivity was localized on the membrane surface. These antibodies exhibited no activity against tissue macrophages and human visceral tissue except kidney, where they stained tubules but not glomeruli. In addition, no activity was observed against rabbit or rat osteoclasts, suggesting that they might react with unique epitopes on human osteoclasts.     

Identification of osteoblast-specific monoclonal antibodies

Summary A series of four antibodies against rat osteoblasts have been produced using the hybridoma technique. After bone cells isolated from newborn rat calvariae by a sequential digestion procedure were cultured for 3 days, the cells were trypsinized and further maintained in rotation cultures overnight. Out of the cultured bone cells alkaline phosphatase-positive cells were sorted by flow cytometry and used as immunogens. The clones secreting the antibodies were selected on the basis of the abilities of these antibodies to bind to the bone cells but not to fibroblasts from neonatal rat head skins, in an enzyme-linked immunosorbent assay. Clones of two hybridomas, designated AOB-1 and AOB-2, were used to characterize the antigenic determinant(s) in osteogenic cells. The antibody showed the reactivity with isolated alkaline phosphatase-positive cells, osteogenic tissue cells in newborn rat calvaria, and mandibula, but not with the cells in head skin, lung, kidney, liver, or stomach as determined by immunofluorescence study. Western blot analysis has identified the antigenic determinants possessing apparent molecular weights of 210,000, 110,000, 65,000, 58,000, 40,000, 36,000, 32,000, 28,000, 25,000, 17,000, and 15,000 of osteoblast-rich monolayer cultured cells. According to the cell surface detection with biotin-avidin protein blotting technique, these fractions appear to be present as components of the cell surface of the osteoblast.     

Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of 'bench-top' flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa.     

Gallic and carnosic acids improve quality of frozen‐thawed ram spermatozoa

The objective was to determine effects of gallic acid (GA) and carnosic acid (CA), present in carob pods and rosemary extract respectively, on frozen‐thawed ram spermatozoa. Thirty ejaculates were collected from five Merino rams, pooled, diluted in Tris‐based extender and divided into five equal portions containing: 0.05 or 2 mM of GA; 0.05 or 0.2 mM of CA; or no additive (control). Extended semen was equilibrated at +4°C, loaded into straws, held 5 cm above liquid nitrogen for 12 min then plunged. Computer‐aided sperm analysis was used to assess motility, whereas flow cytometry was used to assess high mitochondrial membrane potential (HMMP) and percentages of spermatozoa with plasma membrane and acrosome integrity (PMAI). Spermatozoa supplemented with 2 mM GA had greater total motility than control spermatozoa (39.9 ± 3.01 vs. 29.2 ± 1.31%, mean ± SEM, p < .05). The PMAI was greatest in 0.2 mM CA (13.3 ± 0.68%), whereas HMMP was highest in 0.05 mM CA but lowest in control (22.9 ± 4.95 and 11.4 ± 3.64% respectively; p < .05). In conclusion, for cryopreservation of ram semen in Tris‐based extender, supplementation with 2 mM GA increased post‐thaw motility, whereas supplementation with 0.05 mM CA enhanced mitochondrial function.     

Flow cytometric evaluation of the acrosome reaction of human spermatozoa: a new method using a photoactivated supravitaI stain

A flow cytometric assay using a double-stain method for the measurement of the acrosome reaction of human spermatozoa is described. The use of a stable photoactivated stain, ethidium monoazide, allowed evaluation of the viability of spermatozoa. This stain was more stable in fixed samples than propidium iodide, which is not bound covalently to DNA and is therefore removed readily during the washing procedure. The permeabilized acrosome was labelled with Pisum sativum agglutinin conjugated with fluoroisothiocyanate. Since this lectin binds to the acrosome and acrosomal contents, a decrease in the fluorescence intensity allows the cytometric evaluation of the acrosome reaction.
Microscopic analysis and flow cytometric analysis were well correlated and cell sorting was performed to ensure the homogeneity of each different subpopulation encountered.     

Sperm antigens and reactivity of antisperm monoclonal antibodies in elisa

Summary. Several types of sperm antigenic suspensions as well as the whole sperm, either methanol-fixed or air-dried, were checked for intensity of binding to monoclonal antisperm antibodies with known characteristics of reactivity to sperm. The activity of sperm antigen—antibody binding was measured by elisa (enzyme-linked immunosorbent assay) and compared in several variations (parallely run) of the assay where different types of sperm antigen preparations were applied. The obtained results were then evaluated for statistical significance in Wilcox test. It was shown that antibody reactivity was markedly higher in experiments where the whole sperm was coated in a solid-phase in comparison to results obtained with adhered different sperm antigenic suspensions. However, one exception was noted, where the results from elisa, run with sperm organic extract, were (statistically) insignificantly lower than those obtained with the whole sperm. Therefore, organic sperm extracts (containing mostly glycolipids) can be a valuable alternative to screening for antisperm antibody activity and/or infertility background.     

Influence of mitochondrial membrane potential of spermatozoa on in vitro fertilisation outcome

To determine whether the outcome of in vitro fertilisation (IVF) is influenced by the percentage of spermatozoa with functional mitochondria, a total of 91 random couples undergoing IVF were included. Mitochondrial function was determined by flow cytometry and expressed as percentage of spermatozoa. Conventional sperm parameters were studied by light microscopy. Reproductive outcome parameters were fertilisation rate, embryo quality and clinical pregnancy. It was found that the fertilisation rate was correlated with the percentage of spermatozoa (r = 0.24, P = 0.01) as well as with the percentage of highly motile spermatozoa. However, we did not find any relationship between the percentage of spermatozoa and embryo quality. Nevertheless, no patient who exhibited less than 64% of spermatozoa achieved pregnancy. It is concluded that determination of Δψ(m) provides accurate information to guide physicians to identify male patients for whom IVF will be unlikely to result in pregnancy. Therefore, we suggest that the percentage of spermatozoa may contribute to identify the most appropriate treatment for an individual patient.     

Flow cytometric analysis of the DNA content of thymomas

In this study, the prognostic value of determining the nuclear DNA content of thymomas by flow cytometry was evaluated. Of a total 31 resected thymomas, 10 (32%) showed DNA aneuploidy, the presence of which was significantly correlated with an advanced clinical stage of disease. The patients with an aneuploid tumor had a poorer prognosis than those with a diploid tumor, demonstrating a survival rate of 50% at 7 postoperative years, which was considerably less favorable than that of the patients with a diploid tumor, being 100% in the same period (p<0.05). Moreover, patients with a high DNA index (DI), i.e., a DI1.5, tended to have a poorer prognosis than those with a low DI. These findings indicated that the DNA content can be an important prognostic index in patients with thymomas.     

Flow cytometric analysis of localized adenocarcinoma of the prostate: the use of archival DNA analysis in conjunction with pathological grading to predict clinical outcome following radical retropubic prostatectomy

Fifty-four specimens from patients undergoing radical prostatectomy for clinically confined prostate cancer between 1983 and 1987 were reviewed to determine the potential for flow cytometric (FCM) analysis of DNA ploidy and replication rate to predict disease recurrence. Each specimen was deparaffinized for FCM analysis and the pathology slides were reviewed by a single pathologist. FCM characteristics were correlated with pathological grade and stage, and both were correlated with disease status. In this series of patients, routine FCM analysis of DNA ploidy and replication rate failed to significantly enhance the ability of standard histopathological grading to predict disease recurrence in patients having clinically localized prostate cancer. Aneuploid tumors pathologically confined to the prostate did not appear to negatively affect prognosis.     

Immunomodulation of dog islets using a cocktail of monoclonal antibodies

Islet allografts are particularly vulnerable to rejection, and current immunosuppressive agents are deleterious to their function. They are, however, highly suitable for ‘immunomodulation’, i.e., the removal or inactivation of passenger leukocytes to reduce their immunogenicity. For this purpose we have used 3 rat anti-dog monoclonal antibodies (Mabs) which are synergistic for leukocytolysis in the presence of autologous dog serum. Spleen cells or purified islets treated with these Mabs together with autologous serum were tested in mixed leukocyte and islet co-culture assays. The stimulatory properties of the Mab-pretreated splenocytes or islets were markedly reduced; moreover, the Mab cytolytic activity was shown to be confined to the leukocyte target cells and did not affect islet secretory function upon glucose stimulation. We conclude that this method of modifying the immunogenicity of dog islets could lead to successful islet grafting in vivo, allowing the reduction of conventional immunosuppression. Successful in vivo studies in this model, which are currently in progress, could have implications for clinical islet transplantation.     

Flow cytometric DNA analysis of thyroid carcinoma

Abnormal DNA content has been considered as an additional criterion for determining the biological behavior of a tumor. Flowcytometric DNA analysis was done on 121 patients with thyroid carcinoma encountered during the period between 1975 and 1987. Tumor tissues were sampled from paraffin-embedded blocks and the histology of thyroid carcinoma found to consist of 91 papillary, 23 follicular, 2 medullary, 1 squamous cell and 4 anaplastic carcinomas. The incidence of aneuploidy in thyroid carcinoma was 7.4 per cent (9 patients) while that of diploidy was 92.6 per cent (112 patients). The aneuploid specimens consisted of 6 papillary, 1 follicular, 1 medullary and 1 anaplastic carcinomas and, of 4 anaplastic carcinoma patients with subsequent death within 6 months, only 1 was aneuploid. As an indicator of proliferative potential, S-phase fraction (SPF) was also determined by flow cytometry, but this could not be used as an independent prognostic factor. The aneuploid patients showed a significantly decreased survival rate (p<0.01). Thus, although DNA measurement proved useful for predicting the survival of aneuploid patients, there is some discrepancy between DNA content and the biological behavior of the tumor.     

Analysis of experimental immune synovitis cartilage using monoclonal antibodies reactive to rabbit proteoglycan

This study details the macromolecular changes in cartilage involving proteoglycan molecules in an animal model of rheumatoid arthritis. In experimental chronic immune synovitis, fluorescein-conjugated mouse IgG and three monoclonal antibodies (MAbs 2G2, 2E9, and 6C9) portraying differing fine antigenic specificity for rabbit cartilage proteoglycan monomer were utilized to detail alterations in cartilage proteoglycan. In normal and IgG-immune animals, fluorescein isothiocynate (FITC)-conjugated MAbs 2G2 and 2E9 stained cellular/pericellular (C/PC) region intensely. FITC-MAb 2G2 stained cartilage interterritorial matrix as well. FITC-MAb 6C9 stained only C/PC area lightly but did not stain matrix. A marked decrease in staining intensity with FITC-MAb 2G2 was noted in cartilage sections derived from animals with immune synovitis. A corresponding increase in staining of cartilage was noted with FITC-MAb 6C9. The augmented staining of articular cartilage with FITC-MAb 6C9 was most prominent in femoral condyle tissue sections, which corresponded to the cartilaginous area, with the greatest severity in gross pathology. There was a slight augmentation of staining with FITC-MAb 2E9, especially in the C/PC area of medial/femoral cartilage. In addition, the animals with immune synovitis showed abortive cartilage repair exemplified by the presence of chondrocyte cloning (up to 20 cells) which correlated with increased FITC-MAb 2G2 staining. The differential MAb staining patterns of cartilaginous tissues obtained utilizing FITC-conjugated monoclonal antibodies with known fine antigenic specificity indicates a modulation of proteoglycans involving predominantly core protein epitopes in the articular cartilage of animals with chronic immune synovitis.