Labelling of the chromatid body by [3H]uridine in rat pachytene spermatocytes

In this study it is shown that a cytoplasmic cell organelle, the chromatoid body, becomes labelled with [³H]uridine in the pachytene spermatocytes. The chromatoid body becomes labelled when the cells are first labelled for 2 h in the presence of [³H]uridine and thereafter chased for 9 h in the presence of unlabelled uridine. This labelling is inhibited by the specific RNA polymerase II inhibitor α-amanitin. Based on this it is suggested that part of the RNA synthesized in the pachytene spermatocytes is stored in the chromatoid body and transported to the postmeiotic spermatids where it is used in the differentiation of the spermatids.     

Incorporation of (3H)uridine by the chromatoid body during rat spermatogenesis

The in vitro incorporation of tritiated uridine into RNA by the spermatogenic cells of the rat has been analyzed by high-resolution autoradiography. Special attention has been focused on the unique cytoplasmic organelle, the chromatoid body. After a short labeling time (2 h), this organelle remains unlabeled in the vast majority of the early spermatids although the nuclei are labeled. When the 2-h incubation with (3H)uridine is followed by a 14-h chase, the chromatoid body is seen distinctly labeled in all spermatids during early spermiogenesis from step 1 to step 8. Very few grains are seen elsewhere in the cytoplasm of these cells. When RNA synthesis in the spermatid ceases, the chromatoid body also remains unlabeled. It is likely that the chromatoid body contains RNA which is synthesized in the nuclei of the spermatids. The function of this RNA as a stable messenger RNA needed for the regulation of late spermiogenesis is discussed.     

Ribonucleic acid synthesis in vitro in primary spermatocytes isolated from rat testis

The incorporation of [(3)H]uridine into RNA was studied quantitatively (by incorporation of [(3)H]uridine into acid-precipitable material) and qualitatively (by phenol extraction and electrophoretic separation of RNA in polyacrylamide gels) in preparations enriched in primary spermatocytes, obtained from testes of rats 26 or 32 days old. The rate of incorporation of [(3)H]uridine into RNA of isolated spermatocytes was constant during the first 8h of incubation, after which it decreased, but the decreased rate of incorporation was not reflected in a marked change in electrophoretic profiles of labelled RNA. In isolated spermatocytes, [(3)H]uridine was incorporated mainly into heterogeneous RNA with a low electrophoretic mobility. Most of this RNA was labile, as shown when further RNA synthesis was inhibited with actinomycin D. Spermatocytes in vivo also synthesized heterogeneous RNA with a low electrophoretic mobility. A low rate of incorporation of [(3)H]uridine into rRNA of isolated spermatocytes was observed. The cleavage of 32S precursor rRNA to 28S rRNA was probably retarded in spermatocytes in vitro as well as in vivo. RNA synthesis by preparations enriched in early spermatids or Sertoli cells was qualitatatively different from RNA synthesis by the spermatocyte preparations. It is concluded that isolated primary spermatocytes maintain a specific pattern of RNA synthesis, which resembles RNA synthesis in spermatocytes in vivo. Therefore isolated spermatocytes of the rat can be used for studying the possible regulation of RNA synthesis during the meiotic prophase.     

Biochemical evidence of haploid gene activity in spermatogenesis of the mouse

Mice were exposed to two X-ray doses of 300 and 100 R with 4 days interval in order to deplete the testes of spermatogonia and early meiotic cells. After X-ray treatment, the seminiferous tubules were labelled in culture with radioactive RNA precursors, dispersed into single cells by trypsin treatment and these were fractionated into several cell classes by velocity sedimentation at unit gravity in a Ficoll gradient. With this method quasi-homogeneous populations of middle-late pachytene spermatocytes and round spermatids (steps 1–8 of spermiogenesis) were obtained. RNA was extracted from these two cell types and analysed by linear sucrose gradient fractionation and by affinity chromatography on a poly(U)-Sepharose column. The results showed that round spermatids, as well as pachytene spermatocytes, synthesize both ribosomal RNA (rRNA) and poly(A)⁺ RNA (presumptive messenger RNA) (mRNA). The post-meiotic synthesis of RNA ceases completely in mid-spermiogenesis after nuclear elongation in spermatids has set in.     

Reaction of the chromatoid body with a monoclonal antibody to a rat histocompatibility antigen

The monoclonal antibody OX3 against a polymorphic class II antigen encoded by the major histocompatibility locus of the rat has been shown to cross-react with the chromatoid body during spermatogenesis. Using an indirect immunofluorescence assay on frozen, fixed testis sections, the antibody revealed a pattern of fluorescent speckling that correlated with specific stages of spermatogenesis. The positive material first appeared in late pachytene spermatocytes as multiple small spots. Larger dots appeared in all regions containing round spermatids, but, as the spermatids matured, only fine dots were seen. Mature spermatids were negative, as were all early cells (spermatogonia to early pachytene spermatocytes). When suspension of fixed testicular cells were tested, the activity was clearly associated with the chromatoid body adjacent to the nucleus in round spermatids and with multiple smaller structures encircling the nucleus in primary spermatocytes. These associations were confirmed in observations on immature testes at various ages. No reactivity was seen in testes of animals whose testes had previously been irradiated to render them aspermatogenic, nor in grc/grc rats in which spermatogenesis is arrested at the primary spermatocyte stage. Because the expression of this reactivity was seen even in rats that do not express the OX3 antigen on their somatic cells, this antibody should prove useful in determining the structure of this body, its origin and fate, and any possible role it may have in spermiogenesis.     

RNA synthesis during spermiogenesis in the sea urchin: a high resolution autoradiographic study of [3H]uridine incorporation

Summary

RNA synthesis has been studied during spermiogenesis of Paracentrotus lividus by high resolution autoradiography using [³H]uridine as a labeled precursor. Under the experimental conditions used [³H]uridine incorporation is detectable only at the early spermatid stage. Labeling is distributed mainly over the nucleus and it appears completely absent from the mitochondria. After RNase digestion radioactivity falls to a background level, demonstrating that RNA synthesis actually occurs in early spermatids of P. lividus. It follows that in P. lividus during spermiogenesis cell differentiation is not entirely dependent on stored premeiotic RNAs.     

In vitro effects of growth factors on rat germ cell RNA synthesis and their modulation by Sertoli cell-secreted proteins

In vitro rat germ cell RNA synthesis is influenced by growth factors. Basic fibroblast growth factor (0.1 to 100 ng/ml) increases [3H]uridine incorporation in round spermatids (RS) but not in pachytene spermatocytes (PS); this effect is potentiated by insulin (10 micrograms/ml) and blocked in the presence of Sertoli cell-secreted proteins (SCSP). Somatomedin C (0.1 to 100 ng/ml) exhibits a similar effect when used alone without an influence by SCSP. Transforming growth factor beta (0.1 to 10 ng/ml) acts on both cell types, but SCSP amplify this effect only in PS. These data suggest that growth factors synthesized in situ may play a role in the germ cell development and that their effects are modulated by SCSP.     

Influence of mycoplasma infection on the incorporation of different precursors into RNA components of tissue culture cells

Seven different tissue culture cells have been cultured with and without mycoplasma (M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows:

1. 1. When cells are labelled with [8-³H]guanosine or [5-³H]uridine there is some incorporation into host cell 28S and 18S rRNA, but it is less than into mycoplasma 23S and 16S rRNA. [8-³H]guanosine or [5-³H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7S RNA, but the incorporation into host cell 5S rRNA and low molecular weight RNA components (LMW RNA) is reduced.

2. 2. [5-³H]uracil is not incorporated into host cell RNA but into mycoplasma tRNA, 4.7S RNA, a mycoplasma low molecular weight RNA component M₁ and 23S and 16S rRNA.

3. 3. [³H]methyl groups are incorporated into mycoplasma tRNA, 23S and 16S rRNA, but not into host cell 28S, 18S, 5S rRNA nor into mycoplasma 4.7S RNA.

4. 4. With [³²P]orthophosphate or [³H]adenosine as precursors, the labelling is primarily in the host RNA.

Mycoplasma infection influences the labelling of RNA primarily by an effect on the utilization of the exogenously added radioactive RNA precursors, since the generation time of mycoplasma infected cells is about the same as that of uninfected cells. Mycoplasma infection may completely prevent the identification of LMW RNA components.     

The pattern of methylation of RNA in peripheral nerve of the chick during development

The pattern of the methylation of RNA was investigated in organ cultures of the sciatic nerve of the chicken. Nerve tissue from 14-day embryos, 17-day embryos and 3-day- old chicks was incubated with [methyl-³H]methionine or with [2-¹⁴C]uridine and [methyl-³H]methionine simultaneously for various periods of time. Subsequently, RNA was extracted from the tissues and the purified preparations were fractionated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the rapidly labelled RNA changed during the three developmental stages. The incorporation of both uridine and the methyl groups from methionine was highest in the‘heavy’RNA species of the 14-day embryonic nerve during the 0.5 and 1.0 h incubation periods. In contrast, in the nerves of 3-day-old chicks during a 0.5 h pulse with both precursors, methylation was almost entirely limited to the transfer RNA species. Furthermore, the incorporation of uridine in the nerves from 3-day-old animals revealed the presence of a heterogeneous population of rapidlylabelled, unmethylated species of RNA, most of which migrated between the smaller ribosomal RNA and transfer RNA components of the bulk RNA. The pattern of uridine incorporation and the methylation of the rapidly-labelled RNA of the 17-day embryonic nerve represented a transitional state between that of the 14-day embryos and that of the 3-day-old chicks. The 17-day embryonic stage of development corresponded to the phase of the onset of rapid deposition of myelin lipids in the sciatic nerve. Pulse-chase experiments on the embryonic nerves indicated that a number of methylated precursors of ribosomal RNA and labile, heterogeneous, probably DNA-like RNA were synthesized.     

Activité aromatase dans les cellules germinales mâles: quelle signification fonctionnelle?

The ability of the male gonad to convert androgens into estrogens is well known. According to age, aromatase activity has been already measured in immature and mature rat Leydig cells as well as in Sertoli cells. Recently, in different studies, a cytochrome P450_arom has even been immunolocalized not only in Leydig cells but also in germ cells of mouse, brown bear and rooster whereas in pig, ram and human the aromatase is mainly present in Leydig cells. Our purpose was to investigate the testicular cell distribution of cytochrome P450_arom mRNA in adult rat using RT-PCR. With 2 highly specific primers located on exons 8 and 9, we have been able to amplify a 289 bp aromatase fragment not only in Leydig cells and Sertoli cells but more importantly in highlyenriched preparations of pachytene spermatocytes, round spermatids and testicular spermatozoa. These amplified products showed 100% homology with the corresponding fragment of the rat ovary cDNA. In parallel, using an anti-human cytochrome P450_arom antibody we have demonstrated the presence of a 55 kDa protein in seminiferous tubules and crude germ cell (pachytene spermatocytes and round spermatids) preparation of the mature rat. After incubation with tritiated androstenedione, the aromatase activities in the microsomal fractions were 3.12±0.19 pmoles/mg/h in the testis, 1.25±0.13 in the seminiferous tubules and 1.53±0.15 in the crude germ cells. In purified testicular spermatozoa the aromatase activity was 2.96±0.69 pmoles/mg/h and found to be 5-fold higher when compared to that of either purified pachytene spermatocytes or round spermatids. Using a quantitative RT-PCR method with a standard cDNA 29 bp shorter, we have compared the amount of cytochrome P450_arom mRNA in mature rat Leydig cells and Sertoli cells. In purified Leydig cells from 90 day-old rats the P450_arom mRNA level was: 36.2±3.4×10^?3 amoles/μg RNA whereas in Sertoli cells the mRNA level was 10 fold lower. In pachytene spermatocytes, round spermatids and testicular spermatozoa the P450_arom mRNA levels were re pectively 367.2±76.6, 117.6±22.0 and <1×10^?3 amole/μg RNA. In conclusion we have demonstrated that the P450 aromatase is present not only in Sertoli cells and Leydig cells from mature rat testis but a biologically active aromatase exists also in germ cells (pachytene spermatocytes, round spermatids and spermatozoa). The existence of an additional source of estrogens within the genital tract of the male is now well documented and that suggests a putative role for these hormones during the male germ cell development.     

THE EFFECT OF CORDYCEPIN ON THE APPEARANCE OF [3H]RNA IN THE GOLDFISH OPTIC TECTUM FOLLOWING INTRAOCULAR INJECTION OF [3H]URIDINE

Abstract— Although biochemical and electron microscopic evidence has shown that RNA molecules may be found within axons, the origin of this RNA is not known. In order to determine if the RNA found in axons is synthesized in the nerve cell body and axonally transported, we have studied the effect of the RNA inhibitor cordycepin (3′-deoxyadenosine) on the retinal synthesis and axonal migration of radioactive RNA. Ten μg of cordycepin was injected into the right eye of 11 fish and 3 h later [³H]uridine was injected into the same eye. Twelve control fish were injected with [³H]uridine only and all fish were sacrificed 6 days later. Results of RNA extraction of retina and tecta showed that cordycepin decreased retinal RNA synthesis by approx 24%, while inhibiting the amount of [³H]RNA appearing in the contralateral tectum by 74%. Since the transport of RNA precursors was depressed by only 50%, (significantly different from the effect on RNA, P < 0.01) it seems unlikely that the action of cordycepin in decreasing tectal [³H]RNA levels was due solely to a decrease in the availability of labeled precursors for tectal RNA synthesis. For the purpose of blocking tectal RNA synthesis, 200 μg of cordycepin was injected intracranially several days after the intraocular injection of [³H]uridine. This route of cordycepin administration failed to significantly block the appearance of [³H]RNA in the tectum, suggesting that at least some of the [³H]RNA in the tectum was synthesized before arrival in the tectum itself. To be sure that cordycepin itself was not being transported, we injected cordycepin into the right eye of fish and 5 days later, injected fish intracranially with [³H]uridine. Autoradiograms were prepared and grains were counted over the fiber layers of left (experimental) and right (control) tecta. No significant difference was observed in the number of grains of left vs right tecta indicating that cordycepin itself is not axonally transported. These experiments support earlier findings from our laboratory which suggest that RNA may be axonally transported in goldfish optic fibers.     

STUDIES ON RAPIDLY LABELLED NUCLEAR RNA OF RAT BRAIN

—Methyl albumin kieselguhr chromatography (MAK) has been employed to separate rat brain nuclear RNA, labelled in vivo with [³H]uridine, into three major fractions. The first fraction (QI RNA) is ribosomal in nature for it has a high G + C/U ratio and is methylated by [methyl-³H] methionine. The other two fractions (Q2 RNA and TD RNA) are DNA-like for they exhibit a low G + C/U ratio and are labelled minimally by methionine. Pure ribosomal RNA chromatographs almost entirely in the Q1 RNA fraction. Labelling studies indicate that ribosomal RNA and DNA-like RNA behave differently. Initially, the label in the DNA-like RNA fractions increases rapidly and in a linear fashion for the first 30 min, but thereafter decreases rapidly and reaches a steady state level by 1 h and remains so up to at least the 2 h period. In contrast, the labelling of ribosomal RNA is much slower than that of DNA-like RNA during the first 30 min; however, unlike DNA-RNA, the labelling of ribosomal RNA still continues to increase linearly thereafter. Thus, during longer labelling periods, ribosomal RNA is labelled more rapidly than DNA-like RNA. It appears that the labelling of ribosomal RNA relative to DNA-like RNA is more rapid in liver than in brain.     

Expression of MAEL in nuage and non-nuage compartments of rat spermatogenic cells and colocalization with DDX4, DDX25 and MIWI

The functions of MAELSTROM protein (MAEL) in spermatogenesis are gradually being identified but the precise distribution of MAEL in spermatogenic cells during spermatogenesis has not yet been mapped. We studied the expression of MAEL in rat testis by immunofluorescence and immunoelectron microscopy (IEM). Immunofluorescence staining showed that MAEL was localized in intermitochondrial cement, irregularly-shaped perinuclear granules and satellite bodies of pachytene spermatocytes, and in chromatoid bodies of spermatids. The SBs appeared exclusively in pachytene spermatocytes at stages IX–X and were stained strongly for MAEL. In step 12–19 spermatids, many granules stained for MAEL but not DDX4. These granules were confirmed to be non-nuage structures, including mitochondria-associated granules, reticulated body, granulated body by IEM. In the neck region of late spermatids and sperm, MAEL-positive small granules were found. MAEL is colocalized with MIWI in nuage and non-nuage. The results suggest that MAEL seems to function in nuage and non-nuage structures and interacts with MIWI.     

Microcyst germination in the cellular slime mold, Polysphondylium pallidum: Effects of actinomycin D and cycloheximide on macromolecular synthesis and enzyme accumulation

Germination of microcysts of Polysphondylium pallidum is characterized by an immediate rapid increase in incorporation of [³H]leucine into protein which is cycloheximide-sensitive but unaffected by actinomycin D. Significant RNA synthesis, as measured by [³H]uridine incorporation, does not begin until approx. 2 h after the onset of germination. The increase in [³H]uridine incorporation is prevented by actinomycin D. Germination and the increase in alkaline phosphatase and β-glucosidase enzyme activities are prevented by cycloheximide but unaffected by actinomycin D. The data strongly imply the presence of stable RNA in dormant microcysts and indicate a requirement for a discrete period of protein synthesis for germination of microcysts of P. pallidum.     

Fucosylation of glycoproteins in rat spermatocytes and spermatids

Glycoprotein synthesis in pachytene spermatocytes and round spermatids, isolated from rat testes, was studied by analysis of the incorporation of (³H)-fucose. The isolated germ cells were capable of incorporating (³H)-fucose into cell-bound, acid-precipitable components for an incubation period of at least 23 hours (at 32°C). In young spermatids, engaged in the formation of the acrosome, (³H)-fucose was incorporated into more than 16 different glycoproteins within the molecular weight range of 20.000–100,000. A qualitatively similar set of glycoproteins was found to be labeled in spermatocytes. Radioautography showed that after 4 hr most of the incorporated radioactivity was present at one pole in the perinuclear zone of spermatocytes and spermatids, which could reflect incorporation of fucose in the Golgi apparatus. The newly fucosylated glycoproteins were associated with a particulate subcellular fraction (membrane fraction). Trypsin treatment of whole cells after 25 hours of incubation with (³H)-fucose, however, did not cause significant lysis of tritiated glycoproteins. From the results it was concluded that the majority of the newly fucosylated glycoproteins in spermatocytes and spermatids remained associated with an intracellular membrane system, presumably the Golgi apparatus and the vesicles that arise from this structure, to be deposited subsequently in proacrosomic granules and the acrosome. The results also suggest that initiation of the synthesis of spermatidal glycoproteins occurs during the prophase of meiosis in spermatocytes.     

Ligand-dependent modulation of membrane phospholipid metabolism in ConA-stimulated lymphocytes

Human peripheral lymphocytes were activated by ConA in serum-free culture medium, supplemented by BSA. Incorporation of [³H]thymidine into DNA, of [³H]uridine into RNA and of oleate or acetate into membrane phospholipids was investigated. DNA synthesis could be completely inhibited by αMM or by anti-ConA-IgG. Fab and F(ab)₂ fragments of the anti-ConA were equally active. When αMM or anti-ConA was added to cultures at different times after stimulation with ConA, incorporation of [³H]thymidine into DNA (measured after 72 h) could be prevented up to 6–8 h completely and up to 20–30 h partially. Incorporation of [³H]uridine into RNA could be arrested at any time of the culture up to 40 h at the level it had reached but did not reverse to the level of unstimulated cells for a long time. In contrast, incorporation of oleate into lecithin returned to the level of unstimulated cells within 2–3 h after removal of ConA. This suggests that the activation of the phospholipid turnover in stimulated cells is a direct consequence of the presence of the mitogen at the membrane and thus may be a critical initial event in lymphocyte activation.     

Late spermatocytes from immature rat testis isolation,electron microscopy,lectin agglutinability and capacity for glycoprotein and sulfogalactoglycerolipid biosynthesis

A method is described for preparing late spermatocytes form immature rat testes which yields about 2 · 10⁵ cells per testis a purity of 70–80% and a viability of over 90%. The spermatocytes are highly agglutinable by both concanavalin A and wheat germ agglutinin but no major difference in lectin-mediated agglutinability was observed between late spermatocytes, early spermatids and spermatozoa. Isolated spermatocytes were capable of incorporating [¹⁴C]glucosmine into glycoprotein at a linear rate for about 50 min at 30°C and contained a glycoprotein N-acetylglucosaminyltransferase (8.6 nmol/mg protein per h) and a glycoprotein fucosyltransferase (4.5 nmol/mg protein per h) previously described in partially purified adult mouse testicular germinal cells. A Golgi-rich fraction was prepared from isolated spermatocytes wchich was enriched 15-fold in the N-acetylglucosaminyltransferase, 19-fold in the fucosyltransferase and 3-fold in a galactosyltransferase. These studies showed that late spermatocytes were higly active in glycoprotein synthesis. Studies on the incorporation of [³⁵S]sulfate into sulfogalactoglycerolipid indicated that late spermatocytes were not the primary site of synthesis of this lipid although late spermatocytes were shown to be highly enriched in sulfogalactoglycerolipid (5 times the level in whole rat testis). Further, [³⁵S]sulfogalactoglycerolipid took 5 weeks to migrate from its site of synthesis to the epididymis. These studies suggest that sulfogalactoglycerolipid is sulfated at a spermatocyte cell stage prior to the late (pachytene and diplotene) seprmatocyte stage.