Transcription switch of two phosphoglycerate kinase genes during spermatogenesis as determined with mouse testis sections in situ.

In order to elucidate the mechanistic interpretations underlying differential expression of the two phosphoglycerate kinase (PGK) genes during mammalian spermatogenesis, localization of its mRNAs in mouse testis sections was determined by in situ hybridization. MRNA for nonsperm-type PGK-1 was identified in nongerminal Leydig and Sertoli cells, spermatogonia, and spermatocytes, but was not detected in spermatids. In contrast, mRNA for sperm-type PGK-2 was notable in leptotene spermatocytes, becoming most abundant in pachytene spermatocytes. It was amply present in spermatids only up to step 10, completely disappearing after step 12. It is possible to assume that a transcription switch of the two PGK genes ensued following the onset of meiosis. These findings taken together with previous observations indicate that differential expression of the two PGK genes during mammalian spermatogenesis is regulated at the transcriptional and post-transcriptional levels.

[Elimination of the genome of one of the parents prior to premeiotic DNA synthesis in a hybridogenic species of Rana esculenta]. / Eliminatsiia genoma odnogo iz roditelei do premeoiticheskogo sinteza DNK u gibridogennogo vida Rana esculenta.

A DNA-cytometric study was made of spermatogenesis of the hybridogenic European green frog R. esculenta, whose somatic cells have the ridibunda + lessonae genome. The DNA amount in the ridibunda genome is by 16% more than the lessonae one. The DNA content of esculenta somatic cells is exactly intermediate between those of both the parental species. On the contrary, the sperms (1c) and the primary spermatocytes (4c) of R. esculenta have the DNA content which corresponds to the size of the ridibunda genome. These findings are in a good agreement with the hypothesis of semiclonal inheritance. Furthermore, some hybridogenic males have also spermatogonia (2c) with only the ridibunda genome size, whereas the others have altogether diploid cells with the esculenta (i.e. ridibunda + lessonae) genome size. So, it can be suggested that the selective elimination of the lessonae genome and compensatory doubling of the ridibunda one may occur in spermatogonia of R. esculenta males before the premeiotic DNA synthesis. Meiosis, as it can be inferred from the DNA-cytometry data, proceeds in a usual way on the basis of the ridibunda genome.

The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies.

In order to assess the potential of protein gene product (PGP) 9.5 as a marker of the nervous and neuroendocrine systems, we examined its immunolocation in human, rat and guinea-pig tissues, using a rabbit polyclonal antiserum and two new mouse monoclonal antisera, I3C4 and 3IA3. Our results demonstrate immunoreactive PGP 9.5 in neurons and nerve fibres at all levels of the central and peripheral nervous system, in many neuroendocrine cells, in part of the renal tubule, in spermatogonia and Leydig cells of the testis, and in ova and in some cells of the pregnant and non-pregnant corpus luteum. In routinely processed tissues, standard immunohistochemical techniques using the polyclonal antibody demonstrated peripheral nerve fibres of all sizes with striking clarity.

Identification of the specific proteins associated with differentiation of spermatogonia in mice by two-dimensional gel electrophoresis.

Protein synthesis in testicular germ cells was studied during the differentiation of Type A spermatogonia in testes from adult cryptorchid mice to Intermediate and Type B spermatogonia in organ culture by two-dimensional gel electrophoresis. By comparing the patterns of Type A spermatogonia with those of differentiated (Intermediate and Type B) spermatogonia, we found two newly synthesized protein spots that correlated with differentiation. The isoelectric points (pIs) and molecular weights (MWs) of these proteins were estimated to be approximately pI 5.4, MW 67,000, and pI 5.8, MW 28,000, respectively.

[Dna organization in histone-free nuclei at various stages of rat spermatogenesis]. / Organizatsiia DNK v lishennykh gistonov iadrakh na nekotorykh stadiiakh spermatogeneza krysy.

Electron microscopy analysis of DNA organization in histone-depleted nuclei of rat spermatogonial cells was performed. It was shown that the rosette-like structures are one of the forms of the loop organization of the meiotic cell nuclear DNA. Ions of bivalent metal play an important role in stabilizing the rosette-like structures of meiotic cells. A scheme for the restructuring of the mitotic organization of nuclear DNA into the meiotic one is suggested.

Purification and characterization of a proline-rich secretory protein that is a precursor to a structural protein of an insect spermatophore.

The spermatophore or sperm sac of Tenebrio molitor (yellow mealworm beetle) is an acellular structure composed mostly of structural proteins, termed spermatophorins. The proteins are derived from the bean-shaped accessory reproductive glands of the male and are assembled into the multilayered structure within the ejaculatory duct. Homogenates of the secretory plug from this gland were used as immunogens for the production of monoclonal antibodies, including one identified as PL 21.1 which recognizes an antigen in the gland and the spermatophore. With the aid of gel filtration and immunoaffinity chromatography with a PL 21.1, we isolated a glandular secretory protein that is a precursor to a spermatophorin with similar electrophoretic mobility. On native polyacrylamide gels, the antigen from gland homogenates has an apparent molecular mass of 370 kDa. On sodium dodecyl sulfate gels, the antigen from the gland and that from the spermatophore have apparent molecular masses of 23 kDa. According to immunoblots of sodium dodecyl sulfate gels, the 23-kDa glandular antigen is organ-specific and adult-specific. By immunocytochemistry with PL 21.1, we found the antigens to be restricted to secretory vesicles of only one cell type in the gland and to a discrete layer in the outer wall of the spermatophore. The 23-kDa secretory antigen is distinguished by being high in glutamic acid/glutamine (15.4%) and in proline (25.2%).

Biochemical composition of seminal secretions with special reference to LDH activity in the reproductive tissues of the field crab, Paratelphusa hydrodromous (Herbst).

Biochemical composition of seminal secretions as well as the male and female reproductive tissues, with special reference to lactate dehydrogenase (LDH) activity, were determined in the field crab, Paratelphusa hydrodromous. The seminal secretions comprising spermatophores and spermatophore-carrying seminal plasma (hereafter referred to as seminal plasma) are rich in protein, free carbohydrates and lipids. The seminal plasma contains large quantities of free carbohydrates. LDH activity, as measured by UV spectrophotometric method, is very high within the spermatophores. Electrophoretic separation of LDH isozymes reveals the occurrence of 6 fractions in spermatophores, of which, the most conspicuous fraction resolves in between LDH3 and LDH4. The use of L-lactate as substrate and consideration of its relative mobility show that this fraction is homologous to the mammalian sperm-specific LDHx fraction. Among the 6 fractions, the majority of them, including LDHx, fall under M-type suggesting that they are mainly involved in the anaerobic metabolism of spermatozoa. When compared to anterior vas deferens and mid vas deferens, LDH activity is maximum in the posterior vas deferens which contains seminal secretions prior to their ejaculation. Though the LDH activity is found to be moderate in the spermathecal contents of freshly mated females, the carbohydrate reserves are very minimal. Interestingly, spermathecal contents are rich in lipid substances, thereby indicating that the spermatozoa, when stored in the spermatheca, may utilise fatty substances for oxidative metabolism.

Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures.

Sertoli-spermatogenic cell co-cultures prepared from sexually immature rats (20-22 days old) and maintained in serum-free, hormone/growth factor-supplemented medium were used to determine the cell-specific localization of the growth factor somatomedin-C (SM-C). SM-C localization studies were carried out by indirect immunofluorescence using a monoclonal antibody (sm-1.2) to SM-C. In cultured rat hepatocytes, Sertoli and testicular peritubular cells, SM-C immunoreactivity was observed as a diffuse distribution of discrete immunofluorescent granules. Radio-immunoassay experiments using a rabbit antibody against human SM-C showed that testicular peritubular cells and Sertoli cells in primary culture accumulated SM-C in the medium. In spermatogenic cells co-cultured with subjacent Sertoli cells, immunoreactive SM-C was associated with pachytene spermatocytes but not with spermatogonia or early meiotic prophase spermatocytes (leptotene or zygotene). Both Sertoli cells and pachytene spermatocytes displayed binding sites for exogenously added SM-C. SM-C6 binding to spermatocytes reaching an advanced stage of meiotic prophase suggests a possible role of this growth factor in the meiotic process.

Presence of ribonucleoproteins and basic proteins in the nuage and intermitochondrial bars of human spermatogonia.

Ultrastructural cytochemical study of the nuage in the human adult testis revealed that this structure was a cytoplasmic fine fibrillar electron-dense mass, similar to the chromatoid body of spermatids, in all spermatogonial types and spermatocytes. The nuage was often observed in relation with the nucleus or mitochondria. Cytochemical techniques showed staining affinity of the nuage for both ethanolic phosphotungstic acid and ethylene diamine tetra-acetic acid. The intermitochondrial bars were also stained with the two procedures. The results suggest that the nuage originates from the nucleus and migrates to the cytoplasm through nuclear pores, giving rise to the intermitochondrial bars.

Histone variants in rat spermatogonia and primary spermatocytes.

The levels and synthesis of histone variants have been directly measured in spermatogonia and in various stages of primary spermatocytes purified from the rat testis. These measurements were made possible by the development of a procedure, employing centrifugal elutriation and density gradient centrifugation, to separate highly enriched populations of such cells from immature rat testes at the early stages of spermatogenesis. The results show a difference in regulation of the synthesis and accumulation of testis-specific histones H1t, TH2A, TH2B, and TH3. TH3 is present and actively synthesized in A and B spermatogonia. The testis-enriched variants, H2A.X and H1a, are also present at their maximal levels in A spermatogonia. No detectable amounts of H1t, and at most, low levels of TH2A and TH2B could be found in spermatogonia. While TH2A and TH2B are already present and actively synthesized in early primary spermatocytes (around the preleptotene stage), H1t does not accumulate until the pachytene stage.

DNA analysis and sorting of rat testis cells using two-parameter flow cytometry.

By use of two-parameter flow cytometry of rat testis cell suspensions stained with mithramycin for DNA (the peak amplitude of the fluorescence signal versus total fluorescence intensity integrated over time), eight cell compartments could be distinguished without pre-enrichment of the samples. Cells in these compartments were identified by sorting and subsequent microscopic examination.

Immunocytochemical localization of an H2A variant in the spermatogenic cells of the mouse.

Immunocytochemical localization of protein "A," an H2A variant, has been carried out in the adult, neonatal, and embryonic spermatogenic cells of the mouse using the peroxidase-antiperoxidase technique. The results indicate an apparent enrichment of this protein in the meiotic cells of the adult testis. In addition, T-prospermatogonia present in the neonatal mouse and 16-day-old embryos were found to be immunoreactive. By contrast, Sertoli cells and other somatic elements of the neonatal and embryonic gonads were only weakly immunoreactive. These data suggest potential usefulness of protein "A" as a nuclear marker of the embryonic spermatogenic cells.

N-acetyl endorphin in rat spermatogonia and primary spermatocytes.

In previous reports modest levels of beta-endorphin have been found by radioimmunoassay in rat testis, and localized by immunofluorescence to the interstitial cells. We have confirmed these previous reports and extended them by showing that the majority of testicular endorphins are acetylated forms, N-acetyl gamma-endorphin, N-acetyl alpha-endorphin, and N-acetyl beta-endorphin1-27. In addition, N-acetylated endorphins are not found in interstitial cells, but are confined to spermatogonia and primary spermatocytes.

Nuclear testicular 1,25-dihydroxyvitamin D3 receptors in Sertoli cells and seminiferous tubules of adult rodents.

1,25(OH)2D3 receptors were studied in whole testes, Sertoli cells, seminiferous tubules, Leydig cells and spermatogonia of adult NMRI mice and SD rats. Specific reversible high affinity binding (KD 1.4 x 10(-10)M; Nmax 72 fmol/mg protein) by a 3.5 S macromolecule was demonstrated in whole testes, Sertoli cells and seminiferous tubules. With identical techniques, no receptors were found in Leydig cells despite previous reports of 1,25(OH)2D3 actions on Leydig cell function.

Male gonadotrophic factor in brain and blood of photoperiodically stimulated slugs.

A pulmonate male gonadotrophic factor (MGF) has been described that is released from cerebral ganglia of male-phase slugs (Limax maximus). This factor produces, directly or indirectly, an increase in spermatogonial proliferation as determined by in vivo incorporation of [3H]thymidine into gonadal DNA. In the present investigation MGF activity was demonstrated in saline homogenates of male-phase cerebral ganglia by injecting homogenates into immature slugs for 5 consecutive days and assaying gonadal [3H]thymidine incorporation on Day 7. Dose-response data indicate that daily administration of as little as 0.1 brain equivalent can produce a significant stimulation in incorporation. Comparison of brain homogenates from immature (short-day) and male-phase (long-day) animals has shown that male-phase cerebral ganglia contain substantially more MGF activity than immature ganglia. Similar injection experiments using slug blood plasma showed that activity is present in male-phase blood but not in the blood of short-day immatures. MGF activity in long-day brain homogenates and blood plasma was found to be associated with a molecular weight fraction of 50 to 100 kDa obtained by ultrafiltration. Activity could be reduced or destroyed by treatment with trypsin or by heating. The present findings suggest that MGF is a proteinaceous factor of substantial size. It appears that both the synthesis and the secretion of MFG are stimulated in slugs that are in their male developmental phase as a result of prior exposure to long-day photoperiods.

Equimolar proportions of somatic-type core histones in chromatin of spermatogonia.

[3H]Leucine incorporation into histones of seminiferous epithelial cells of hypophysectomized rats was used to calculate the molar proportions of the core histones of spermatogonia. The molar proportions H3:H2B:(H2A + protein A24):H4 are 1:1:1:1, viz. identical with those reported by others for somatic cells. Similar results were obtained when molar proportions of histones of seminiferous epithelial cells from immature rat testis (predominantly populated with spermatogonia) were determined by the dye-binding method. These data are relevant to mechanisms for the replacement of some of the core histones by variants during the primary spermatocyte stages.

Histochemical distribution of sudanophilic and unsaturated lipids in the testes of buffalo, goat, and ram.

The sudanophilic and unsaturated lipids have been histochemically localized and correlated with seminiferous epithelial cycle in the testes of buffalo (Bubalus bubalis), goat (Capra hircus) and ram (Ovis aries). All sudanophilic lipids are unsaturated. The masked lipids may be present either in small quantity or absent altogether. In the seminiferous tubules, 3 types of lipid bodies (L1, L2, L3) are present. L3 bodies in the buffalo are of relatively small size but are more in number. L2 bodies appear only in the elongated spermatids. All spermatogenic cells also have diffused sudanophilic lipids. Sertoli cells contain only L1 and L3. The lipids in the spermatids and Sertoli cells show cyclic variations reverse to each other. In the interstitial tissue, the amount of lipids is much less in goat and ram as compared to that in buffalo.

DNA content in the mouse spermatogonia B and spermatid nuclei during treatment with insecticides.

The DNA content in nuclei of germ cells: spermatogonia B and spermatides in the maturation phase of male mice receiving commercial insecticide preparations commonly used in this country was cytophotometrically measured. The following insecticides were tested: 1% Metox-30 solution, containing 30% of methoxychlorine (p,p-dimethoxydiphenyltrichlorethane), 0,3% solution of Sadofos-30 containing 30% of malathione (1,2-dicarboethoxyethyl-0,0-dimethyldithiophosphate) and 0,3% solution of Foschlor-50 containing about 50% trichlorfon (2,2,2-trichloro-1-hydroxyethyl-0,0-dimethyl-diphosphonate). It was found that a statistically significant number of spermatogonium B and spermatide nuclei in the phase of maturation of the animals treated with the insecticides exhibited an abnormal DNA content as compared with the nuclei of control animals. The chromatin of these nuclei is also more sensitive to acid hydrolysis in Feulgen reaction.

Phosphoglycopeptide, a major constituent of the spermatophoric plasma of the octopus (Octopus dofleini martini).

A phosphoglycopeptide, accounting for approximately 90% of the characteristically high content of acid-soluble organically-bound phosphorus in the octopus spermatophoric plasma (4 mg P/ml), was identified. Electrophoretic and chromatographic purification, followed by chemical and enzymic hydrolysis, yielded D-galactose phosphate as a degradation product. The galactose and peptide moieties of the compound were linked via a phosphoryl rather than a glycosidic linkage but the peptide was devoid of aromatic amino acids.