Characterization of RNA synthesis in mid-pachytene spermatocytes of the rat

In this paper the RNA synthesis in mid-pachytene spermatocytes of the rat has been studied. The results show that RNA synthesized by these cells is mostly high molecular weight RNA comparable to HnRNA of somatic cells. In comparison with the rapid metabolism of HnRNA in somatic cells, HnRNA in pachytene spermatocytes is stable and remains in the nucleus for a considerable time. About 30% of this HnRNA contains a poly(A) sequence, although no difference in the rate of metabolism between poly(A)+ and poly(A)− RNA was observed. Based on these results it is suggested that at least a part of the RNA which is synthesized by pachytene spermatocytes is stored in the cells and utilized later during spermatogenesis when the RNA synthesis of the spermatids ceases, but protein synthesis is still active for about 2 weeks.     

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.     

Chronic cyclophosphamide treatment alters the expression of stress response genes in rat male germ cells

Increases in the survival rate of men treated with chemotherapeutic drugs and their desire to have children precipitate concerns about the effects of these drugs on germ cells. Azoospermia, oligospermia, and infertility are common outcomes resulting from treatment with cyclophosphamide, an alkylating agent. Exposure of male rats to cyclophosphamide results in dose-dependent and time-specific adverse effects on progeny outcome. Elucidation of the effects of chronic low-dose cyclophosphamide treatment on the expression of stress response genes in male germ cells may provide insight into the mechanisms underlying such adverse effects. Male rats were gavaged with saline or cyclophosphamide (6 mg/kg) for 4-5 wk; pachytene spermatocytes, round spermatids, and elongating spermatids were isolated; RNA was extracted and probed on cDNA arrays containing 216 cDNAs. After saline treatment, 125 stress response genes were expressed in pachytene spermatocytes (57% of genes studied), 122 in round spermatids (56%), and 83 in elongating spermatids (38%). Cyclophosphamide treatment reduced the number of genes detected in all germ cell types. The predominant effect of chronic cyclophosphamide exposure was to decrease the expression level of genes in pachytene spermatocytes (34% of genes studied), round spermatids (29%), and elongating spermatids (4%). In elongating spermatids only, drug treatment increased the expression of 8% of the genes studied. The expression profiles of genes involved in DNA repair, posttranslational modification, and antioxidant defense in male germ cells were altered by chronic cyclophosphamide treatment. We hypothesize that the effects of cyclophosphamide exposure on germ cell gene expression during spermatogenesis may have adverse consequences on male fertility and progeny outcome.     

Labelling of the chromatoid body by [H]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.     

Gene expression during mammalian spermatogenesis. II. Evidence for stage-specific differences in mRNA populations

Gene expression during murine spermatogenesis has been studied using highly enriched populations of cells obtained by velocity sedimentation at unit gravity and further purified by density gradient centrifugation through Percoll. Polypeptides whose synthesis was directed by total cytoplasmic RNA from round spermatids, pachytene spermatocytes, primitive type A spermatogonia, and Sertoli cells in cell-free translation systems have been compared by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. At the level of detection provided by the electrophoretic methods used, each population of cells contained mRNAs encoding over 200 polypeptides, many of which were present in high abundance in all four cell types. However, for each cell type examined, a minimum of 5-10% of these polypeptides appear to be either specific to or greatly enriched within a particular cell type. Analysis of the polysomal and nonpolysomal cell fractions from pachytene spermatocytes and round spermatids revealed that the two compartments share many identical mRNAs but specific mRNAs are selectively compartmentalized between the cell fractions and between the two cell types. Movement between compartments was seen; e.g., some polypeptides encoded by mRNA found primarily in the nonpolysomal fraction of pachytene cells were later seen in the polysomal fraction from round spermatids. Virtually every other combination was also observed. These results suggest that the control of gene expression at the level of selective production of mRNA and selective utilization of mRNA are among the mechanisms involved in regulation of spermatogenic cell differentiation.     

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.     

DNA polymerase-beta and poly(ADP)ribose polymerase mRNAs are differentially expressed during the development of male germinal cells.

We have examined the steady-state mRNA levels in spermatogenic cells of two nuclear enzymes that appear to be involved in DNA repair, DNA polymerase-beta (pol-beta) and poly(ADP)ribose polymerase (PADPRP). Two pol-beta mRNAs of 1.3 kb and 1.4 kb were detected in extracts from mouse testes. In leptotene/zygotene spermatocytes a low level of the 1.4-kb mRNA was observed. Both pol-beta mRNAs were found in meiotic pachytene spermatocytes, with the 1.3-kb form being more abundant. In contrast, the 1.4-kb form was more abundant in haploid round spermatids. Polysome gradient analyses indicated that the two pol-beta mRNAs were predominantly present in the nonpolysomal fractions of spermatocytes. In round spermatids, a larger fraction of the 1.4-kb pol-beta mRNA was associated with polysomes, correlating well with the higher levels of pol-beta enzyme detected during spermiogenesis. The pattern of PADPRP mRNA expression differed from the expression of pol-beta mRNA. The two PADPRP mRNAs of 3.7 and 3.8 kb were present in type A and type B spermatogonia, reached their highest levels in pachytene spermatocytes, and were greatly reduced in haploid round and elongating spermatids. Most of the pachytene spermatocyte PADPRP and mRNAs were present in polysomes, whereas a greater percentage of PADPRP mRNAs in round spermatids were detected in the nonpolysomal fractions. This finding correlates with the immunocytochemical nuclear localization of this enzyme in pachytene spermatocytes. These data demonstrate that different developmental patterns of mRNA expression and translational regulation exist for the pol-beta and PADPRP mRNAs during differentiation of male germinal cells.     

Separation of mouse testis cells by equilibrium density centrifugation in Renografin gradients

Mouse testis cells have been separated by equilibrium density centrifugation in gradients of Renografin. Intact testis cells were not damaged by the separation procedure provided that, following separation, the osmolarity was reduced gradually. The various cell types were identified microscopically and by ³H-thymidine labelling with similar results. The present technique has demonstrated that significant variations in cell density occur during spermatogenesis. Approximately ten-fold enrichments of nearly all testis cell types were achieved by equilibrium density separation of testis cell suspensions. More homogeneous cell populations were prepared by density gradient centrifugation of cell fractions obtained from velocity sedimentation separations. Overall enrichments of spermatogonia, by 29-fold; pachytene spermatocytes, 45-fold; dividing meiotic cells, 170-fold; round spermatids, 30-fold; step 11–13 elongating spermatids, 12-fold; Leydig cells, 70-fold; and cytoplasmic fragments, 55-fold, were obtained. In this study, a method for preparation of cell suspensions was also developed to produce higher yields of spermatogonia and young primary spermatocytes; however, the density distribution of these cells was altered.     

Pachytene spermatocytes can achieve meiotic process in vitro

Highly enriched pachytene spermatocytes prepared from adult rats by centrifugal elutriation were tested for their capacity to enter the meiotic process when cocultured with 20-day old rat Sertoli cells. This was traced by phase-contrast microscopy and by DNA flow cytometry. We also performed a Northern blot analysis using the mouse protamine I cDNA as a probe, the expression of which being restricted to spermatids. Our results demonstrate that pachytene spermatocytes cocultured with Sertoli cells developed into spermatids. The number of pachytene spermatocytes entering meiosis was affected neither by growth factors nor by hormones. However these later were required in long term cocultures for the maintenance of cell integrity and viability.     

Immunochemical identification of multiple cell surface antigens appearing during specific stages of mouse spermatogenesis

Cell surface antigens that appear in a defined temporal sequence during mouse spermatogenesis were previously detected serologically, but not identified biochemically, with four heterologous antibodies prepared against purified populations of pachytene spermatocytes (AP), round spermatids (ARS), vas deferens spermatozoa (AVDS), and mixed seminiferous cells (ASC) [Millette and Bellvé, J Cell Biol 74:86–97, 1977]. These antigens have now been identified immunochemically on nitrocellulose blots from SDS polyacrylamide gels. Three antisera (AP, ARS, and ASC) recognize a similar subset of determinants on one-dimensional immunoblots of germ cells and plasma membranes prepared from a mixed population of late spermatogenic cells. Comparisons of minor bands to reveal differences among these antisera. AVDS exhibits the least complex binding pattern. The results indicate that at least ten surface constituents appear during the pachytene stage of meiosis, coincident with a period of maximal RNA and protein synthesis [Monesi, Exp Cell Res 39:197–224, 1965]. Furthermore, two-dimensional immunoblot comparisons of plasma membranes isolated from pachytene spermatocytes and round spermatids reveal differences between surface determinants detectable at these two spermatogenic stages. For example, ASC recognizes two newly described proteins that are restricted to pachytene spermatocytes (? M_r 57,000, pI 6.45) and to round spermatids (? M_r 39,500, pI 4.85), respectively.     

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.     

In vitro synthesis of RNA by Xenopus spermatogenic cells I. Evidence for polyadenylated and non-polyadenylated RNA synthesis in different cell populations.

Premeiotic and postmeiotic (haploid) gene expression during spermatogenesis in the anuran, Xenopus laevis, was studied by analyzing the accumulation of radioactively labelled cytoplasmic polyadenylated [poly (A +)] and non-polyadenylated [poly (A -)] RNAs. Dissociated spermatogenic cells were labelled and maintained in an in vitro system capable of supporting cell differentiation. Labelled cells were separated by density gradient centrifugation into subpopulations enriched for individual spermatogenic stages. RNA was extracted and purified from each cell fraction, and separated into poly (A +) and poly (A -) species. Comparison of poly (A +) to non-poly (A) radioactivity in cells labelled with tritiated uridine or adenosine demonstrated that (1) all cell fractions produced significant quantities of polyadenylated RNA relative to total RNA synthesis; and (2) that a cell fraction enriched for pachytene spermatocyte RNA contained up to 15% of total cytoplasmic and 35% of total polysomal RNA labelled as poly (A +) containing species. RNA was also characterized by sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. All cell types showed typical poly (A -) peaks of 4S, 18S and 28S, corresponding to tRNA (4S) and rRNAs (18, 28S) respectively. Spermatids and spermatozoa had additional absorbance peaks at 13 and 21S which cosedimented with Xenopus oocyte mitochondrial rRNA. Patterns of incorporation of uridine and adenosine into poly (A +) RNA in all germ cell fractions tested were complex. In all cases, major areas of radioactivity were found in a broad band sedimenting between 6-17S. Spermatid fractions showed a prominent peak of incorporation at 6-8S, while pachytene cells also showed heavier poly (A +) peaks in the 17-25S region. A non-polyadenylated RNA species sedimenting at 6-8S with a relatively rapid rate of turnover was also observed in spermatids. From these results it is concluded that synthesis of transfer, ribosomal, and putative messenger RNA species continues in spermatogenic cells throughout all but the very last stages of spermatogenesis in Xenopus.     

RIBONUCLEIC ACID SYNTHESIS DURING MITOSIS AND MEIOSIS IN THE MOUSE TESTIS

The pattern of ribonucleic acid synthesis during germ cell development, from the stem cell to the mature spermatid, was studied in the mouse testis, by using uridine-H³ or cytidine-H³ labeling and autoradiography. Incorporation of tritiated precursors into the RNA occurs in spermatogonia, resting primary spermatocytes (RPS), throughout the second half of pachytene stage up to early diplotene, and in the Sertoli cells. Cells in leptotene, zygotene, and in the first half of pachytene stage do not synthesize RNA. No RNA synthesis was detected in meiotic stages later than diplotene, with the exception of a very low rate of incorporation in a fraction of secondary spermatocytes and very early spermatids. At long intervals after administration of the tracer, as labeled cells develop to more mature stages, late stages of spermatogenesis also become labeled. The last structures to become labeled are the residual bodies of Regaud. Thus, the RNA synthesized during the active meiotic stages is partially retained within the cell during further development. The rate of RNA synthesis declines gradually with the maturation from type A to intermediate to type B spermatogonia and to resting primary spermatocytes. "Dormant" type A spermatogonia synthesize little or no RNA. The incorporation of RNA precursors occurs exclusively within the nucleus: at later postinjection intervals the cytoplasm also becomes labeled. In spermatogonia all mitotic stages, except metaphase and anaphase, were shown to incorporate uridine-H³. RNA synthesis is then a continuous process throughout the cell division cycle in spermatogonia (generation time about 30 hours), and stops only for a very short interval (1 hour) during metaphase and anaphase.     

The immunolocalization of small nuclear ribonucleoprotein particles in testicular cells during the cycle of the seminiferous epithelium of the adult rat

The objective of this study was to determine the cellular and subcellular distribution of small nuclear ribonucleoprotein particles (snRNPs) in the adult rat testis in relation to the different cell types at the various stages of the cycle of the seminiferous epithelium. The distribution of snRNPs in the nucleus and cytoplasm of germ cells was quantitated in an attempt to correlate RNA processing with morphological and functional changes occurring during the development of these cells. Light-microscopic immunoperoxidase staining of rat testes with polyclonal anti-Sm and monoclonal anti-Y12 antibodies localized spliceosome snRNPs in the nuclei and cytoplasm of germ cells up to step 10 spermatids. Nuclear staining was intense in Sertoli cells, spermatogonia, spermatocytes, and in the early steps of round spermatid development. Although comparatively weaker, cytoplasmic staining for snRNPs was strongest in mid and late pachytene spermatocytes and early round spermatids. Quantitative electron-microscopic immunogold labeling of Lowicryl embedded testicular sections confirmed the light-microscopic observations but additionally showed that the snRNP content peaked in the cytoplasm of midpachytene spermatocytes and in the nuclei of late pachytene spermatocytes. The immunogold label tended to aggregate into distinct loci over the nuclear chromatin. The chromatoid body of spermatids and spermatocytes and the finely granular material in the interstices of mitochondrial aggregates of spermatocytes were found to be additional sites of snRNP localization and were intensely labeled. This colocalization suggests that these dense cytoplasmic structures may be functionally related. Anti-U1 snRNP antibodies applied to frozen sections showed the same LM localization pattern as spliceosome snRNPs. Anti-U3 snRNP antibodies applied to frozen sections stained nucleoli of germ cells where pre-rRNA is spliced.     

A strategy for an improved separation of mammalian spermatids

A collection procedure has been developed to improve the homogeneity of mammalian spermatid populations separated by elutriation. Trypsinizied ram testis cells were elutriated at 18C. Every cell population was eluted by progressive changes in the flow rate and/or rotor speed, instead of by abrupt changes, to reduce the contamination by cells from the next population. Pure populations were collected alternating with mixed populations corresponding to the overlap between two adjacent pure populations. Furthermore, each pure population was collected into two subfractions, the second of which, contamined by cells from the following population, was pooled with the following fraction. In less than 2 hr after castration, three populations of at least 1 × 10⁸ viable round or elongated or elongating spermatids were obtained with respective purities of 95%, 82%, and 99% of the nucleated cells. In addition, two mixed populations containing only two adjacent spermatid types (round plus elongating spermatids: 98%; elongated plus elongating spermatids: 98%) were obtained, as well as a population containing around 60% pachytene spermatocytes.