R. Golan

Immunochemical gel diffusion study of relationships between erythrocyte catalase of various species

Abstract 1. 1. The activity and starch gel electrophoretic mobility of catalase (EC 1.11.1.6) in red cells of several mammals and of two avian species has been examined. Among the mammals the highest catalase activity was observed in the red cells of Rhesus monkeys and the lowest in those of dogs. Very low activity was found in chicken red cells, while in quail erythrocytes no catalase activity was detected by the methods used. 2. 2. The method of Thorup for localization of catalase activity in starch gels has been adapted for agar gel double diffusion and immunoelectrophoresis. 3. 3. Hemolysates of the various species have been examined by immunodiffusion procedures with rabbit antiserum against non-hemoglobin protein fraction of human red cells. Catalases of all the animals examined (with the exception of rats and chicken) produced catalytically active enzyme-antibody complexes with this anti-serum, demonstrating the existance of common antigenic determinants. 4. 4. The mammals investigated could be divided into four groups of immunological relationships of red cell catalases: (1) man, Rhesus monkey, Vervet monkey; (2) horse, donkey, guinea pig; (3) goat, sheep, calf; (4) dog. Catalases from animals within each group produced patterns of complete identity on double diffusion. Intergroup differences of some antigenic determinants were demonstrated by production of spurring reactions between respective immuno-precipitates.

Spermatogenesis in the golden hamster during the first spermatogenic wave: A flow cytometric analysis

In the present study propidium iodide was used as a fluorescent dye to stain DNA of cells of hamster testicular origin and fluorescent intensities were analyzed by flow cytometry. We used hamster testicular cells from the first spermatogenic wave to observe the consecutive appearance of the different types of cells during puberty. At 12 days postpartum (dpp) diploid cells (including spermatogonia) predominated and some tetraploid cells were also present. Tetraploid spermatocytes increased dramatically by 21 dpp. The first haploid cells appeared at 21 dpp but substantial numbers were first present at 23 dpp. Immature haploid cells predominated at 32 dpp. Elongating condensing spermatids appeared at 34 dpp and spermatozoa began to leave the testis to enter the epididymidis at 36–38 dpp marking the end of the first round of spermatogenesis. Using acridine orange staining flow cytometry, chromatin condensation was followed by measuring fluorescence decrease from early round spermatids to spermatozoa obtained from the initial segment and from the cauda epididymides. The major portion of sperm chromatin condensation (88–90%) in the hamster occurred in the testis and only 10–12% occurred during epididymal sperm maturation. Spermatozoa in the initial segment of the epididymidis of the hamster contained a small amount of RNA that was no longer present in sperm of the cauda epididymidis, indicating that RNA was lost during epididymal sperm maturation in this species. Mol. Reprod. Dev. 55:205–211, 2000.

Chromatin condensation during spermiogenesis in the golden hamster (Mesocricetus aureus): a flow cytometric study.

DNA‐staining of hamster testis cell suspensions followed by flow cytometry demonstrated appearance of the first haploid cells at 23 days post partum (dpp) and of condensed chromatin (in elongated spermatids and spermatozoa) at 33–34 dpp. Mature spermatozoa were first observed in the caput epididymis at 36–37 dpp, thus completing the first spermatogenic wave. Testicular cell suspensions from animals from 23 to 38 dpp were stained with acridine orange, and flow cytometer gating was adjusted to include only the haploid cells. Acridine orange intercalated into double‐stranded DNA to produce green fluorescence. The decrease in green fluorescence intensity from 23 until 37 dpp was caused by changes in the binding of DNA to basic proteins in such a fashion as to impede the access of the dye to the DNA double helix. When the green fluorescence values (of the most advanced spermatids) were plotted against the age of the hamsters (in dpp) or the corresponding steps of spermiogenesis, the decrease in fluorescence could be seen to occur in three phases. The inflection point between the first and second phases was observed at about spermiogenesis step 7, consistent with the hypothesis that this represents removal of histone from the chromatin. The second phase presumably represents the period in which transition proteins are bound to the DNA. At approximately steps 15 or 16 a further inflection point was seen where protamines replaced the transition proteins. The red fluorescence produced when acridine orange bound to RNA in spermatids, increased early in spermiogenesis and decreased dramatically at 34 dpp, consistent with the fact that elongating spermatids discard the bulk of their cytoplasm during the maturation process. Mol. Reprod. Dev. 56:105–112, 2000.

Immunoelectrophoretic-chemical investigation of erythrocyte aspartate aminotransferase.

Abstract Aspartate aminotransferase of human red cells formed on immunoelectrophoresis a catalytically active enzyme-antibody complex, which was identified by a specific chromogenic reaction. Rabbit antiserum against non hemoglobin protein fraction of human red cells produced similar results and patterns of antigenic identity with erythrocyte aspartate aminotransferase of Rhesus and Vervet monkeys. Hemolysates of horse, calf, donkey, goat, sheep, dog, pig, guinea-pig, rat and rabbit produced negative results with this antiserum.

Use of confocal microscopy for the study of spermatogenesis

Spermatogenesis consists of spermatogonial proliferation, meiosis and spermatid differentiation. Laser scanning confocal microscopy (LSCM) may be used as an advanced analytical tool to follow spermatogenesis inside the seminiferous tubules without performing histological sections. For this purpose, separated seminiferous tubules are fixed in 0.5% paraformaldehyde, stained for DNA with propidium iodide and analyzed by LSCM. By producing longitudinal optical sections in the layer of spermatogonia, spermatocytes and spermatids, stage-specific changes in their structure may be followed within the tubules by LSCM. Longitudinal z-sections may be obtained to produce three-dimensional images of the seminiferous tubules. In addition, different proteins may be followed during spermatogenesis in a stage specific manner within the tubule by incubation of the fixed seminiferous tubules with appropriate antibodies. As an example of the spermatogenesis studies using described LSCM techniques, detailed examination of spermatogonia, spermatocytes and spermatids during golden hamster spermatogenesis is presented. LSCM analysis of c-kit and SC3 protein expression at different stages of hamster spermatogenesis is demonstrated.

Spermiogenesis defects in human: detection of transition proteins in semen from some infertile men

Semen samples from 60 infertile men were examined by flow cytometry following propidium iodide staining. Of these, 23 samples contained young haploid cells. Transition proteins (TP1 and/or TP2) were detected in 12 of these, using immunohistochemical staining. The presence of TPs in spermatids in semen indicates inhibition in the differentiation pathway from round spermatids to spermatozoa. Cells of this type were found in semen from patients with nonobstructive azoospermia, severe to extreme cases of oligozoospermia, asthenozoospermia and teratozoospermia.

Immunochemical study of relationships between erythrocyte 6-phosphogluconate dehydrogenase (6PGD, E.C. 1.1.1.44) of various mammalian species

Abstract 1. 1. 6-Phosphogluconate dehydrogenase (6PGD) activity and electrophoretic mobility were investigated in red cells of fourteen mammalian species. 2. 2. The comparative antigenic structure of 6PGD of various species was investigated by immunodiffusion with antisera prepared against non-hemoglobin protein fraction of human and bovine erythrocytes, coupled with a specific chromogenic reaction. 3. 3. Two common antigenic determinants were detected in 6PGD of representatives of primates, Bovidae and Equidae families. 4. 4. Only one of these determinants was found in the enzymes of members of Canidae and Caviidae families. 5. 5. The anti-human serum revealed also an antigenic determinant specific to 6PGD of man and Cercopithecidae, while the anti-bovine serum revealed an antigenic determinant specific to the Bovidae family. 6. 6. Two variants of human 6PGD (PDG A /PGD C and PGD C /PGD C ) produced on immunodiffusion reactions of complete identity with the usual phenotype (PGD A /PGD A ).