Determination of phthalate esters in human semen.

Phthalate esters are a large group of chemical compounds used in the production of plastics, household articles, packages, cosmetics and plant pesticides. World production of phthalates is estimated to be several million tons a year. Recent observations indicate some mutagenic, cancerogenic and orchidotoxic effect of these compounds. Therefore, to assess the extent of risk it is imperative to have an adequate analytical method. The following is simple and relatively inexpensive. The study group consisted of 58 men.

Evaluation of HLA expression on gametogenic cells isolated from human testis.

Human germinal cells were isolated using a combined mechanical method and enzymatic digestion of testicular tissue. The attempt to fractionate the testicular cells by centrifugation in the continuous and discontinuous Percoll gradient was undertaken. The homogeneous (100%) fraction of late spermatids and enriched fractions of spermatocytes (up to 60%) and round spermatids (approx. 66%) were obtained. Cell-binding radioimmunoassay (CB-RIA) was used to evaluate the HLA expression both on heterogeneous suspensions and enriched fractions of testicular, germ cells using mouse monoclonal antibodies directed to Class I (HLA, -A, -B, -C) and Class II (HLA, -DR) determinants of MHC (main histocompatibility complex). Consistently negative results were obtained as well for heterogenous populations of germinal cells as their fractions in respect to both Class I and Class II antigenic determinants of HLA system.

HLA expression on human germinal cells.

Ejaculated human seminal plasma cells obtained from 46 healthy men and 48 infertile patients were tested for expression of HLA class I and II antigens by complement-mediated cytotoxicity, cell-binding radioimmunoassay (CB-RIA), and an ELISA involving monoclonal antibodies (mAbs). In a group of healthy men tested for HLA expression on human spermatozoa, 4 of 46 were positive for class I and II HLA antigens. However, the results were negative in a second examination of the same donors, possibly on account of alterations with time in the expression of various subpopulations of cells which are antigen positive or antigen negative. In a group of infertile patients, we found positive expression of HLA class I and II antigens in 7 of 48 men. The samples contained immature and mature sperm but no contaminating leucocytes or epithelial cells.

The non-mature sperm cells: evaluation of surface markers and interaction in in vitro cultures.

The ejaculated human seminal plasma cells may be classified into subpopulations on the basis of spermatogenesis or biological features. In this paper we divided these cells into two main groups: one of them is obviously predestinated to fertilization as mature sperm cells, viable and motile ejaculated cells. Second group of unknown biological role is very heterogeneous; simplifying its variety we call them non-mature sperm cells. Morphological studies by electron microscopy and evaluation of surface markers of these cells by monoclonal antibodies did not reveal any known biological features or markers for cells of leukocyte origin. They did not express any antigenic determinants of HLA system and they did not react with autologous or allogeneic lymphocytes.

Chlorinated pesticide residues in testicular tissue samples. Pesticides in human testicles.

Residues of chlorinated pesticides--HCB, alpha-, beta-, gamma-, delta-, epsilon-HCH isomers, heptachlor epoxide, DDE and DDT isomers in bilateral testicular biopsy material were determined in gas liquid chromatography with electron capture detector (GLC-ECD). The average levels of chlorinated pesticides residues occurring in 36 men are presented. Alpha-, delta-HCH and p,p'-DDE were observed in nearly all analyzed samples. Other analyzed compounds were observed in lower percentages, whereas o,p'-DDT and p,p'-DDT were not detected.

Blast transformation of lymphocytes induced by spermatozoa obtained from spermatocoele.

Human lymphocytes cultured for 6 days in the presence of autologous and allogeneic spermatozoa obtained from spermatocoele undergo blast transformation. The proliferation level in autologous combination is markedly higher than that in allogeneic one.

Simple, low-cost method for determination of selected chlorinated pesticides in fat samples.

A method is described for the quantitative and qualitative determination of selected chlorinated pesticides in fat samples. Pesticides residues are extracted with petroleum ether and separated from fat with concentrated H2SO4 instead of the commonly used adsorbents Florisil, alumina, or silica gel. Residues were analyzed by gas chromatography with electron capture detection. Recoveries of fortified samples were approximately 100%.

Stimulation of lymphocytes by spermatozoa-stimulated cells.

The mechanism of spermatozoa-lymphocyte interaction was studied using spermatozoa and lymphocytes obtained from 11 healthy individuals and from 2 patients with spermatocoele. The lymphocytes activated by spermatozoa in 3-day cultures were used as inducers of fresh, unstimulated autologous or allogeneic lymphocytes. In 9 cases of 13 tested sperm samples, a slight proliferative response of lymphocytes stimulated by spermatozoa-induced lymphocytes was observed. The final effect did not depend on whether an autologous or an allogeneic system was used in the first and the second incubation. A possible explanation of the observed reaction is discussed.

Content of chlorinated pesticides in human semen of a random population.

Residues of chlorinated pesticides--BHC isomers, DDT isomers, HCB and DMDT--in samples of human semen have been determined. Samples of semen were collected from a random population of patients sent to our department for the evaluation of their fertilizing ability. The contents of 50 samples of semen were analyzed individually for residues of chlorinated pesticides and their metabolites by gas-liquid chromatography. Of BHC isomers detected in the analyzed samples of semen, the highest absolute values were shown for epsilon-BHC (mean 0.005 microgram/g of sample). DMDT, op'DDT and pp'DDt were not detected in the studied samples. DDE was found in 20 samples on the average level of 0.003 microgram/g.

Comparison of the content of chlorinated pesticide residues in human semen, testicles and fat tissues.

Studies were undertaken to determine the residues of chlorinated pesticides in human semen, testicles and fat tissues. Samples of semen were taken from a random population and samples of tissues at surgery. A preliminary estimation was made to assess the level gradient of environmental pollutants in these types of specimens characterized by a different fat content. Pesticides were considered to show an affinity to fat. The results are an early stage in a programme for a detailed analysis of the effect of pesticides on human reproductive ability and on the value of genetic material.

T and B lymphocytes in the ontogenetic development of man.

Cord blood lymphocytes of premature and mature fetuses and peripheral blood of infants and adults were studied by rosette techniques and membrane immunofluorescence. Percentage of lymphocytes carrying surface immunoglobulins, receptors for complement and receptors for uncoated sheep erythrocytes or receptors for Fc IgG was determined. T lymphocyte receptors were found to appear later in ontogeny than the receptors typical of B lymphocytes. Receptors for Fc IgG were the earliest to appear: already in premature fetuses the percentage of lymphocytes resembled the one seen in healthy adults. However, the absolute number of T cells increased during development of fetuses and infants and slightly decreased in adults. On the other hand, the absolute number of B lymphocytes were highest in mature fetuses and decreased in subsequent age groups. In the course of human ontogeny a progressive increase was found in lymphocytes carrying full sets of T or B lymphocyte receptors, paralleled by a corresponding decrease in mean values of "null" lymphocytes free of detectable receptors.