Metachromatic staining of human sperm nuclei after reduction of disulphide bonds.

Intra- and interprotamine cross-linking by disulphide bonds are the main factors responsible for the highly compact and stable structure of chromatin in mammalian spermatozoa. Unfixed or methanol fixed smears of human sperm and sperm suspensions from fertile donors and oligospermic patients were subjected to a reductive cleavage of disulphide bonds by using 2-mercaptoethanol (ME) or dithiothreitol (DTT). Untreated (control) and ME or DTT treated samples were stained with toluidine blue (TB) and examined in light microscopy; spectral characteristics of TB stained sperm nuclei were also analyzed. Untreated smears from fertile donors showed an orthochromatic (pale blue) staining of most sperm heads, while a variable proportion of metachromatic nuclei was found in spermatozoa from patients with oligospermia. After treatment with DTT followed by TB staining, fixed and unfixed smears showed metachromatic sperm heads. ME treatment only induced a scarce colour shift, whereas a striking metachromatic reaction and variable nuclear swelling were observed in DTT treated sperm suspensions. These results indicate that after cleavage of disulphide bonds, phosphate groups from chromatin DNA are unmasked and available for TB binding and metachromatic staining.

Hyperviscosity and hypofunction of the seminal vesicles.

The study was designed to determine whether hyperviscosity of the semen sample is related to dysfunction of the male accessory glands. It was carried out on men who consecutively attended an infertility clinic between June 1989 and June 1991, and the men were grouped according to viscosity of semen samples (normal viscosity or higher viscosity). Semen samples from 229 infertility patients were studied. From these, 155 had normal viscosity and 74 showed hyperviscosity. The effect of hyperviscosity of semen samples on seminal quality and the function of the prostate was evaluated by acid phosphatase measurement, and the seminal vesicles by measurement of corrected fructose. Sperm motility (grades II-III), sperm vitality, and corrected fructose were significantly reduced in samples with high viscosity (p < .05). A high prevalence of hyperviscosity in semen samples was associated with only hypofunction of the seminal vesicles. In fact, 36.5% of subjects with hyperviscosity showed reduced levels of corrected fructose. The same association with hyperviscosity was not observed when only hypofunction of the prostate was present, or when hypofunction of both prostate and seminal vesicles was present (P:NS). Further analysis showed that high viscosity is observed mainly when corrected seminal fructose levels were below 1.5 mg/mL x 10(6) spz/mL. It would appear that hyperviscosity affects sperm motility and is associated with hypofunction of the seminal vesicles.

Leucocyte populations in semen and male accessory gland function: relationship with antisperm antibodies and seminal quality.

Semen samples from 279 infertility patients attending an Immunological Centre were analysed to evaluate the relationship between the populations of leucocytes, seminal quality, antisperm antibodies, and seminal vesicle function. The most frequent finding between leucocytospermic samples was asthenozoospermia (57%), whereas in non-leucocytospermic samples normozoospermia was the most frequent finding (47%). In the samples with asthenozoospermia, granulocytes predominated, whereas in those with oligozoospermia and azoospermia a reduction in the number of macrophages and lymphocytes was observed, suggesting an obstructive process at the level of epididymis and/or vas deferens where these leucocytes are mostly produced. In the case of hypofunction of the seminal vesicles there was a predominance in granulocytes. The increased levels of each type of leucocytes affected seminal quality only when seminal vesicles were affected. Only the elevated granulocytes count was related to a decrease in sperm motility. In those samples with leucocytospermia, positive antisperm antibodies (ASA) were associated with low sperm motility, low sperm normal morphology, and low value of seminal corrected fructose, whereas, in the absence of leucocytospermia, ASA, were more related to low sperm counts. These data suggest that granulocytes were more related to seminal vesicles dysfunction and sperm motility changes, and that ASA may be observed in the presence or absence of leucocytospermia.

Effect of isotypes of antisperm antibodies on semen quality.

A direct immunobead test (IBT) was performed on 233 men who attended an immunological centre. Thirty-four (14.6%) of these men were found to be positive (greater than 20% binding) for antisperm antibodies (ASA). IgA, IgG and IgM were the most common sperm-associated immunoglobulins. In 50% of men with ASA asthenozoospermia, teratozoospermia, leukocytospermia or hypofunction of the seminal vesicles was observed. Semen parameters were altered most frequently when IgM was present in association with IgA and/or IgG. This suggests that there is an active inflammatory process in the reproductive tract, as evidenced by leukocytospermia, and this could be responsible for the abnormal semen parameters. ASA generation could be a consequence of this process rather than being the cause of the abnormal semen quality. If ASA do affect fertility, this could take place in the female reproductive tract.

Leukocytospermia and function of the seminal vesicles on seminal quality.

OBJECTIVE: To determine possible relationships between number of leukocytes, function of seminal vesicles, and seminal quality. DESIGN: The study was carried out on men who consecutively attended an infertility clinic between June 1989 to June 1991. SETTING: This study was conducted in a private immunological center for infertility, a tertiary care center, The Centro Immunológico-Sección Esterilidad y Reproducción. PATIENTS: Semen samples from 280 infertility patients attending an Immunological Center for Infertility were analyzed. MAIN OUTCOME MEASURE: We evaluated the effect of leukocytospermia in the presence of normal or abnormal function of seminal vesicles on seminal quality. RESULTS: Sperm count, percent of motile sperm, and percent of sperm vitality were significantly reduced when both leukocytospermia and hypofunction of seminal vesicles were present (P less than 0.01). Leukocytospermic subjects with normal function of seminal vesicles showed similar seminal parameters to those nonleukocytspermics. The incidence of subjects with antisperm antibodies measured by direct immunobeads was significantly higher in leukocytospermic men with hypofunction of seminal vesicles. No differences in the incidence of antisperm antibodies with nonleukocytospermic samples were observed in those with both leukocytospermia and normal function of seminal vesicles. CONCLUSIONS: These data provide evidence that white blood cells were deleterious for seminal quality when seminal vesicles were also affected.

[Role of the immune response in male infertility]. / Papel de la respuesta inmune en la esterilidad masculina.

In guinea pigs using testis homogenate with Complete Freund's Adjuvant as antigen, a close correlation between cellular immunity (CI) and orchitis injury was found. High titres of antisperm cytophilic antibodies (ACA) were found in serum simultaneously with gonadal lesions. Cytotoxic effects of ACA were investigated using a purified eluate of the antibody from macrophage surfaces and injecting it by subalbuginea route or incubating it with germinal cells or spermatozoa. The IgG2 nature of ACA was demonstrated by immunochemical techniques. Specific studies showed that the cytotoxic effect was not due to immune complexes. Ultrastructural studies of macrophage-ACA-spermatozoa complex by scanning and transmission electron microscopy showed the different stages of ACA-mediated sperm phagocytosis. "Thermal orchitis" was developed in guinea pigs as a model to induce orchitis without adjuvant. Isolation and identification of the sequential cell populations in the granuloma showed macrophages as the main cell type. T cells were seen to increase while B cells decreased and were substituted by plasma cells. Potentiality of unilateral sperm cord torsion for autosensitization against the other gonad was studied; orchiectomy of the injured gonad prevented the orchitis lesion and avoided the immune response. Infertile men were studied immunologically. Patients with genital infection or sperm duct obstruction exhibited positive antisperm autoimmunity tests. Cl alterations were detected early and persisted for many years, while humoral antisperm antibodies were not detected during more than two years. Azoospermic patients showed autosensitization in 50% of the cases. Finally, Percoll gradients were used to isolate and identify non spermatic round cells from semen samples of asthenozoospermic men.(ABSTRACT TRUNCATED AT 250 WORDS)

Papel de la respuesta inmune en la esterilidad masculina. / [Role of the immune response in male infertility]

In guinea pigs using testis homogenate with Complete Freunds Adjuvant as antigen, a close correlation between cellular immunity (CI) and orchitis injury was found. High titres of antisperm cytophilic antibodies (ACA) were found in serum simultaneously with gonadal lesions. Cytotoxic effects of ACA were investigated using a purified eluate of the antibody from macrophage surfaces and injecting it by subalbuginea route or incubating it with germinal cells or spermatozoa. The IgG2 nature of ACA was demonstrated by immunochemical techniques. Specific studies showed that the cytotoxic effect was not due to immune complexes. Ultrastructural studies of macrophage-ACA-spermatozoa complex by scanning and transmission electron microscopy showed the different stages of ACA-mediated sperm phagocytosis. [quot ]Thermal orchitis[quot ] was developed in guinea pigs as a model to induce orchitis without adjuvant. Isolation and identification of the sequential cell populations in the granuloma showed macrophages as the main cell type. T cells were seen to increase while B cells decreased and were substituted by plasma cells. Potentiality of unilateral sperm cord torsion for autosensitization against the other gonad was studied; orchiectomy of the injured gonad prevented the orchitis lesion and avoided the immune response. Infertile men were studied immunologically. Patients with genital infection or sperm duct obstruction exhibited positive antisperm autoimmunity tests. Cl alterations were detected early and persisted for many years, while humoral antisperm antibodies were not detected during more than two years. Azoospermic patients showed autosensitization in 50

of the cases. Finally, Percoll gradients were used to isolate and identify non spermatic round cells from semen samples of asthenozoospermic men.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophages and tumor growth. II. Cell kinetics at the site of allogeneic tumor growth.

The sponge matrix allograft model made possible the growth of an AKR lymphoma in a certain percentage of BALB/c mice making progressors (tumor-bearing) and regressors (tumor-rejecting) simultaneously available. Mice bearing either an AKR kidney allograft or a sponge alone were used as controls. The cell population infiltrating the sponge was evaluated 2, 5, 10, 15, and 21 days after subcutaneous implantation. It consisted of macrophages, lymphocytes, and neutrophils. There was no difference between groups on Day 2 and Day 5. From Day 10 onwards, tumor growth was evident with a clear cut separation between progressors and regressors. The latter behaved like the two control groups except for a significant increase in lymphocytes on Day 21. The progressors showed a striking increase in total cell count from Day 10 onwards. Macrophages were the major population with a maximum value of 201 X 10(6) as compared to 8 X 10(6) in the regressors; their phagocytic and lysosomal activity remained similar in all groups. The lymphocytes showed no variation in absolute numbers but, because of the high cell count in progressors, their ratio to macrophages reached 1:50 on Day 10, making up only 2% of the cell population. Neutrophils were significantly increased in progressors as compared with the other groups. It can be concluded that in the presence of a foreign body reaction a marked influx of macrophages accompanies allogeneic tumor growth.

Correlation of immune complexes with serum cytotoxic activity induced by antisperm cytophilic antibodies.

The object of the present work was to investigate: a) The cytotoxic activity of cytophilic antibody in sera from guinea pigs sensitized with the testicular antigen; b) The presence of immune complexes. In vitro cytotoxicity induced by hyperimmune sera or by the cytophilic antibodies eluted from the corresponding macrophages, was demonstrated after incubation with either germinal cells or spermatozoa. Marked cytotoxicity was also observed in vivo when sera or antibodies were injected into the tests, as shown by sequential histologic studies. Circulating immune complexes, as tested by the 125I-C1q binding assay, were not detected in these sera. On the contrary, high levels of circulating immune complexes were found when the polyethyleneglycol precipitation test was used. It can be concluded that the cytotoxic effect induced by cytophilic antibodies of IgG2 nature, may be associated with polyethyleneglycol precipitating immune complexes.

Correlation of immune complexes with serum cytotoxic activity induced by antisperm cytophilic antibodies.

The object of the present work was to investigate: a) The cytotoxic activity of cytophilic antibody in sera from guinea pigs sensitized with the testicular antigen; b) The presence of immune complexes. In vitro cytotoxicity induced by hyperimmune sera or by the cytophilic antibodies eluted from the corresponding macrophages, was demonstrated after incubation with either germinal cells or spermatozoa. Marked cytotoxicity was also observed in vivo when sera or antibodies were injected into the tests, as shown by sequential histologic studies. Circulating immune complexes, as tested by the 125I-C1q binding assay, were not detected in these sera. On the contrary, high levels of circulating immune complexes were found when the polyethyleneglycol precipitation test was used. It can be concluded that the cytotoxic effect induced by cytophilic antibodies of IgG2 nature, may be associated with polyethyleneglycol precipitating immune complexes.

Thymectomy does not affect tumour rejection and cytophilic activity in a murine allogeneic lymphoma model.

An experimental model was devised in which an AKR lymphoma is conditioned to grow in BALB mice leading to reproducible tumor incidence which makes tumor-bearing (progressor) and tumor-rejecting (regressor) animals simultaneously available. The object of this paper was to determine the effect of neonatal thymectomy (xT) on allogeneic tumor incidence and on anti-tumor cytophilic activity. The latter was determined by the adherence of lymphoma cells to guinea pig peritoneal macrophages previously incubated with preheated mouse serum. The results obtained, in 2-3 month old animals, show no difference in lethal tumor incidence between xT and intact mice, 36% (14/39) vs. 39% (14/36). Neither did xT alter the significant increase in cytophilic antibodies detected in regressor serum, 115 +/- 15 (S.E.) vs. 106+/- 22 0/00 macrophages bearing tumor cells as compared to control values in either xT or normal serum, 53 +/- 3 vs 52 +/- 3. This background cytophilic activity was not significantly altered in progressor serum of either xT or intact mice, 36 +/- 5 vs 65 +/- 6. The specificity of the antitumor cytophilic antibodies was determined by the negative results obtained when a different tumor was used as target cell. It can be concluded that ant-tumor cytophilic antibodies are detectable in regressor but not in progressor serum. Thymectomy in this model does not alter either in vivo tumor incidence or humoral cytophilic activity. Since no thymic remnants were encountered at autopsy, it is postulated that AKR lymphoma cells, which proved to be neoplastic T cells, are capable of rendering T-immunocompetent a thymectomized allogeneic BALB host.