Maria Teresa Zenzes

Detection of benzo(a)pyrene diol epoxide–DNA adducts in sperm of men exposed to cigarette smoke

OBJECTIVE To determine whether the adducts formed when benzo(a)pyrene, a diol epoxide derivative, binds covalently to DNA (BPDE-DNA adducts) are detectable in the sperm of men who smoke cigarettes. DESIGN Prospective study. SETTING The Toronto Hospital IVF-ET program. PATIENT(S) Twenty-three patients with normal seminal parameters: 11 smokers (20.6 +/- 0.7 cigarettes per day) and 12 nonsmokers. INTERVENTION(S) Semen samples obtained by masturbation. MAIN OUTCOME MEASURE(S) Seminal plasma samples were assessed for cotinine by RIA. Sperm were treated with dithiothreitol to release disulfide bonds and allow for DNA binding, then exposed to an anti-BPDE monoclonal antibody, a biotinylated antibody, and streptavidin-conjugated peroxidase. Staining intensity scores, determined in 100 cells per individual, were correlated with seminal plasma cotinine levels, a marker of smoking. RESULT(S) Cotinine levels correlated highly with the number of cigarettes smoked per day. Mean cotinine levels and mean staining intensity scores were higher in smokers than in nonsmokers. Staining intensity correlated highly with cotinine levels. CONCLUSION(S) We demonstrated, for the first time, that BPDE-DNA adducts in sperm cells are increased by smoking; we also detected comparatively high levels in nonsmokers, which indicates that environmental exposure also is substantial. The formation of adducts in spermatozoa is a potential source of transmissible prezygotic DNA damage.

Pattern of activity of nucleolus organizers during spermatogenesis in mammals as analyzed by silver-staining.

Silver-staining in the nuclei and chromosomes of spermatogenesis of four species of mammals (Man, Mus musculus, Rattus norvegicus, and Cavia cobaya) was investigated qualitatively and quantitatively. These species show a very similar pattern of activity of the nucleolus organizer regions (NORs) during the various stages of spermatogenesis. Silver precipitates are detectable in growing spermatogonia and up until the pachytene stage of meiotic prophase. During the meiotic metaphases I and II and during interkinesis silver-stainability disappears completely. A resumption of silverstainability occurs in round spermatids indicating a postmeiotic reactivation of NORs. This process does not persist beyond the early elongation phase. The quantitative determination of the silver-covered areas in relation to the total nuclear areas reveals minor differences between the species investigated with regard to the times and extents of maximum activation. The known localizations of the NORs in the karyotypes of the species investigated was confirmed using metaphase-preparations derived from somatic tissues.

Studies on the function of H-Y antigen: dissociation and reorganization experiments on rat gonadal tissue

On circumstantial evidence, H-Y antigen is assumed to be responsible for the differentiation of the undetermined gonadal anlage into a testis. A direct approach to test the function of H-Y antigen is provided by Moscona-type experiments. Applying a modified technique of in vitro reassociation of cell suspensions, we obtained the following results: (1) dissociated newborn rat gonads, both testis and ovary, reorganize into histotypic structures; (2) under exposure to anti-H-Y antiserum, testicular cells reassociate into ovarian follicular-like organization; (3) anti-H-1 antiserum by itself does not prevent the testicular cells from forming tubular structures. It is concluded that H-Y antigen acts as a differentiation between preferably or exclusively on the cell elements participating in the formation of the seminiferous cords.

Activation of mouse ribosomal RNA genes at the 2-cell stage.

SummaryThe Ag-AS method, developed by Goodpasture and Bloom (1975) stains transcriptionally active nucleolus organizer regions (NORs) on the chromosomes and in the interphase nuclei. Metaphases and interphase nuclei of early mouse embryos (unfertilized eggs, pronucleus stages, 2-, 4-, 8-cell stages, and morulae) were subjected to silver-staining. First staining of a single chromosome bearing an NOR was observed at the 2-cell stage. At the 4-cell stage 4–6 chromosomes, and at the 8-cell stage invariably all the 6 chromosomes known to bear NORs, respond positively to silver-staining. These results indicate that during mouse embryogenesis ribosomal RNA genes start to function at the 2-cell stage. The polar body does not respond to silver-staining, which supports the view that the polar body genome remains inactive.

Organization in vitro of ovarian cells into testicular structures

SummaryWhile it has been shown previously (Zenzes et al., 1978; Ohno et al., 1978) that when dissociated testicular cells are exposed to anti-H-Y antiserum in vitro they are prevented from reorganizing into testicular structures, forming ovarian follicular structures instead, the most conclusive evidence for the action of H-Y antigen would be the conversion of ovarian cells into testicular organization. Testing for H-Y antigen of the medium collected from cultivated testicular cells revealed a positive reaction. Dissociated ovarian cells of newborn rats cultivated in this medium reorganize into testicular structures. It is concluded that H-Y antigen is responsible for this histomorphologic change.

Binding studies of H-Y antigen in rat tissues

SummaryThe binding capacity for H-Y antigen was studied in various rat tissues of both sexes. In nongonadal tissues (liver, kidney, brain, epidermis) binding could not be demonstrated. In contrast, the gonads are able to bind exogenously supplied H-Y antigen. In the ovary, the binding capacity remains unchanged in newborn and adult animals, while in the testis, this capacity decreases with age. A receptor like that of a proteohormone is assumed to exist in the gonads but not in other tissues. In nongonadal tissues, H-Y antigen apparently is present only if the cell itself synthesizes the antigen. The H-Y antigen receptor of the gonads is not sex-specific. Thus, the primary sex differentiation depends on whether H-Y antigen is synthesized in the organism.

Evidence for postmeiotic expression of ribosomal RNA genes during male gametogenesis

SummaryPre- and postmeiotic stages of male gametogenesis of 10 different vertebrate species belonging to mammals, birds, amphibians, and fishes were subjected to the Ag-AS staining technique (Goodpasture and Bloom, 1975). A uniform pattern of silver-staining is observable during spermatogenesis of the different vertebrate species. Silver-staining is present in spermatogonia and during the whole period of meiotic prophase, but totally absent during diakinesis and metaphase II of meiosis. In early spermatids silver-staining reappears and only disappears around the beginning of elongation of the spermatid nucleus. Since the Ag-AS technique is believed to stain only transcriptionally active nucleolus organizer regions, our findings indicate that ribosomal RNA genes become reactivated in the haploid spermatid.

Studies on H-Y antigen in different cell fractions of the testis during pubescence

SummaryVarious cell types of the rat testis during pubescence, including germ, Sertoli, and Leydig cells, were partially enriched. The fractions were tested for the presence, binding, and secretion of H-Y antigen. The main results are: Immature germ cells are H-Y antigen-negative until the late diploid stages, and late primary spermatocytes or spermatids become positive; the somatic cells of the gonad are positive at all ages examined (18 days old to adulthood). Secretion of H-Y antigen is restricted to the Sertoli cell fraction. Binding of externally supplied antigen takes place on Leydig cells; the Sertoli cell surface will be saturated because of active secretion; there is no binding to germ cells. Thus, immature germ cells seem to be the only H-Y antigen-negative cells of the male organism, and the Sertoli cells seem to be the only ones to secrete H-Y antigen.

Appearance of hCG-receptor after conversion of newborn ovarian cells into testicular structures by H-Y antigen in vitro

SummaryIn a previous report (Zenzes et al., 1978 b) it was shown that dissociated ovarian cells of newborn rats in vitro, if exposed to H-Y antigen, reorganize into testicular structures. The current study was designed to see whether this morphological conversion also results in a functional conversion. The LH/hCG receptor was used as a parameter characteristic for the newborn testis, but not for the newborn ovary. In the converted ovary, the LH/hCG receptor becomes detectable a few hours after onset of the culture and remains continuously present afterward. The appearance of this receptor may be due to a hormone-like action of H-Y antigen.

The testis as a secretory organ for H-Y antigen

SummaryAfter cultivation of dissociated rat testicular tissues, H-Y antigen is detectable in the medium; this is not the case if nongonadal male tissues are incubated. Release of H-Y antigen by testis cells is inhibited by the addition of cycloheximide. All tissues still type H-Y positive after culture. It is assumed that the testis actively secretes H-Y antigen. This assumption is supported by the finding that the amount of H-Y antigen in the epididymal fluid increases with the age of the animals.