The sequential appearance of components of the synaptonemal complex during meiosis of the female rat.

This paper describes the light microscopy (LM) and electron microscopy (EM) localization of synaptonemal complex (SC) antigens in oocytes of rats. For this purpose, we used monoclonal antibodies (Mabs) that recognize components of 30 + 33, 125, and 190 kDa antigens of SCs of rat spermatocytes. The LM localization was performed by immunofluorescence and the EM localization by immunogold staining. The reaction of the Mabs with oocytes was similar to the reaction with spermatocytes, but weaker. The 30 + 33 kDa as well as the 190 kDa antigens could always be demonstrated if axial elements of the SC were present, irrespective of whether these were paired or unpaired. Thus, these antigens could be detected from leptotene--early zygotene until diplotene. The 190-kDa antigen appeared in a diffuse manner just before the appearance of the 30 + 33 kDa antigens. The 30 + 33 kDa antigens were not only detected in the axial elements of SCs but also in characteristic aggregates, which appeared in zygotene and persisted until after the SCs had disappeared. Such aggregates had rarely been observed in spermatocytes. The 125 kDa antigen was only present in the tripartite segments of SCs, at the inner edge of the lateral elements. Thus, the reaction of the Mab against the 125 kDa antigen was detectable in zygotene, pachytene, and very early diplotene. It appeared later than 30 + 33 kDa and 190 kDa antigens and it disappeared earlier. We found that several steps of the immunostaining procedure could cause variation in the intensity of the Mab reaction.     

Two major components of synaptonemal complexes are specific for meiotic prophase nuclei

Monoclonal antibody II52F10 was elicited against purified synaptonemal complexes (SCs); it recognizes two major components of the lateral elements of SCs, namely an M_r=30 000 and an M_r=33000 protein. We studied the distribution of the antigens of II52F10 within tissues and cells of the male rat by immunoblot analysis and immuno-cytochemical techniques. Nuclear proteins from various cell types, including spermatogonia and spermatids, did not react with antibody II52F10 on immunoblots; the same holds for proteins from isolated mitotic chromosomes. As expected, an M_r=30 000 and an M_r=33 000 protein from spermatocyte nuclei did react with the antibody. In cryostat sections of liver, brain, muscle and gut we could not detect any reaction with II52F10. In the testis the reaction was confined to SCs or SC fragments. Partly on the basis of indirect evidence we identified the antigen-containing cells as zygotene up to and including post-diffuse diplotene spermatocytes. The persistence of some antigen-containing fragments in the earliest stages of spermatids could not be excluded. We conclude that the lateral elements (LEs) of SCs are not assembled by rearrangement of pre-existing components of the nucleus: at least two of their major components are newly synthesized, presumably during zygotene. Furthermore we conclude partly from indirect evidence that the major components of the LEs of SCs are not involved in the chromosome condensation processes that take place during the earliest stages of meiotic prophase.Abbreviations BSA bovine serum albumin - CE central element - FITC fluorescein isothiocyanate - LE lateral element - PBS phosphate-buffered saline (140 mM NaCl, 10 mM sodium phosphate, pH 7.3) - SC synaptonemal complex - TBST Tris-buffered saline with Tween (50 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.05% Tween-20)     

Identification and immunochemical characterization of spermatogenic cell surface antigens that appear during early meiotic prophase

Three spermatogenic cell populations isolated from prepuberal mice--type B spermatogonia, preleptotene spermatocytes, and leptotene/zygotene spermatocytes--were used to elicit distinct polyclonal antisera. Surface binding specificities were determined for purified IgGs by indirect immunofluorescence and rosette assays on live cells. Binding activities were assayed both before and after absorptions with a variety of somatic and spermatogenic cells. Each of these antisera binds to surface antigens that are present on germ cells throughout spermatogenesis and are not shared by splenocytes, thymocytes, and erythrocytes. Only the antiserum raised against leptotene and zygotene spermatocytes (ALZ) recognizes a stage-specific subset of surface determinants. After appropriate absorptions, ALZ binds to the surface of early pachytene spermatocytes and germ cells at subsequent stages of differentiation, including vas deferens spermatozoa. Antigens which react with this absorbed IgG are not detected on the surface of spermatogonia or meiotic cells prior to pachynema, including leptotene and zygotene spermatocytes. The observed binding specificities may result from the synthesis of one or more surface molecules during the early meiotic stages, followed by delayed insertion into the plasma membrane during the pachytene stage of meiotic prophase. Stage-specific antigens recognized by ALZ, including both protein and probably lipid, have been localized immunochemically on nitrocellulose blots from one-dimensional SDS gels. A dithiothreitol-sensitive constituent (Mr approximately 39,000) recognized by ALZ has been identified as the major protein determinant present in early meiotic cells but absent in 8-day-old seminiferous cell suspensions containing spermatogonia and Sertoli cells. This determinant is present in populations of preleptotene, leptotene/zygotene, and early pachytene spermatocytes isolated from 17-day-old animals, an observation consistent with the hypothesis of delayed insertion into the plasma membrane.     

Localisation of RAD50 and MRE11 in spermatocyte nuclei of mouse and rat

Synaptonemal complexes (SCs) are zipperlike structures that are assembled between homologous chromosomes during meiotic prophase. They consist of two axial elements (AEs) (one along each of the two homologous chromosomes), which, in mature SCs, are connected by numerous transverse filaments along their length. Several proteins involved in the later steps of meiotic recombination most probably function in close association with the AEs of SCs, because the proteins involved in these steps have all been localised along AEs or SCs by immunocytochemical methods. It is not known at which step in meiotic recombination this association with the AEs is established. In order to shed some light on this issue, we analysed the localisation of two proteins that are involved in early steps of meiotic recombination, RAD50 and MRE11, relative to AEs and SCs by immunofluorescence labelling of paraffin sections of the mouse testis, using affinity-purified polyclonal antibodies against RAD50 and MRE11, and monoclonal and polyclonal antibodies against SC components. The localisation patterns of MRE11 and RAD50 within spermatocytes were very similar. MRE11 and RAD50 appeared in high abundance in preleptotene spermatocytes, just before SC components could be detected. From preleptotene until early zygotene they were present throughout the nucleus. In mid and late zygotene, MRE11 and RAD50 concentrated in distinct areas; in early pachytene the two proteins had almost disappeared from the nucleus, except from the sex vesicle (the chromatin of the XY bivalent), where they persisted in high abundance until diplotene. We propose that MRE11 and RAD50, together with other proteins, prepare chromatin throughout the early meiotic prophase nucleus for the initiation of meiotic recombination. Possibly, only a small fraction of the RAD50- and MRE11-containing (pre)recombination complexes associates transiently with AEs, where further steps in meiotic recombination can take place. Received: 16 November 1999; in revised form: 29 December 1999 / Accepted: 3 January 2000     

普通小麦联会复合体发育过程的电镜观察

以改进的去污剂微铺展技术制备普通小麦减数分裂联会复合体标本,并对联会复合体发育的全过程作了详细的电镜观察和描述。结果表明,小麦SC以多点式起始方式于偶线期开始形成;随SC的发育,新的SC形成和已有SC片断的延伸并存;此外,在同一核内不同二价体之间,染色体配对和SC形成并不同步;SC成熟于粗线期,而以破碎方式解体,消失于双线期。在偶线期还观察到由同祖配对形成的多价体,但在随后阶段中这些多价体消失。对Ph基因的可能作用机制作了分析和讨论。     

Determination of daily sperm production in stallion testes by enumeration of germ cells in homogenates

Thirty adult stallion testes were selected with high (n = 15) and low (n = 15) Daily Sperm Production (DSP)/testis. Parenchymal samples were prepared for morphometric analysis, and the numbers of germ cells and Sertoli cells were determined. Testicular samples were homogenized, and germ cells and Sertoli cells were enumerated using phase contrast microscopy. Numbers of germ cells and Sertoli cells and potential DSP during spermatogenesis were determined. Significant correlations existed between morphometric and homogenate determinations of number per testis of preleptotene, leptotene plus zygotene primary spermatocytes (r = 0.58; P < 0.001), pachytene plus diplotene primary spermatocytes (r = 0.67; P < 0.0001), all primary spermatocytes (r = 0.67; P < 0.0001), round spermatids (r = 0.72; P < 0.0001), and Sertoli cells (r = 0.70; P < 0.0001). Significant correlations (P < 0.0001) existed between morphometric and homogenate determination of DSP/testis based on preleptotene, leptotene plus zygotene primary spermatocytes (r = 0.78), pachytene plus diplotene primary spermatocytes (r = 0.88), and round spermatids (r = 0.85). Using morphometric determination as the standard, the sensitivity (i.e., ability to detect low DSP/testis) and specificity (i.e., ability to detect high DSP/testis) by homogenate enumeration of germ cells was 81 and 93% for round spermatids, 100 and 24% for pachytene plus diplotene primary spermatocytes, and 67 and 87% for preleptotene, leptotene plus zygotene primary spermatocytes, respectively. Enumeration of primary spermatocytes in homogenates was less accurate than enumeration of round or elongated spermatids. Enumeration of round and elongated spermatids in homogenates was a rapid and useful method for determining DSP in horses, and it may prove to be a useful technique for quantitating potential DSP from testicular biopsies.     

DNA double-strand breaks and gamma-H2AX signaling in the testis

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated at serine 139 and forms gamma-H2AX foci at the sites of damage. These foci then play a role in recruiting DNA repair and damage-response factors and changing chromatin structure to accurately repair the damaged DNA. These gamma-H2AX foci appear in response to irradiation and genotoxic stress and during V(D)J recombination and meiotic recombination. Independent of irradiation, gamma-H2AX occurs in all intermediate and B spermatogonia and in preleptotene to zygotene spermatocytes. Type A spermatogonia and round spermatids do not exhibit gamma-H2AX foci but show homogeneous nuclear gamma-H2AX staining, whereas in pachytene spermatocytes gamma-H2AX is only present in the sex vesicle. In response to ionizing radiation, gamma-H2AX foci are generated in spermatogonia, spermatocytes, and round spermatids. In irradiated spermatogonia, gamma-H2AX interacts with p53, which induces spermatogonial apoptosis. These events are independent of the DNA-dependent protein kinase (DNA-PK). Irradiation-independent nuclear gamma-H2AX staining in leptotene spermatocytes demonstrates a function for gamma-H2AX during meiosis. gamma-H2AX staining in intermediate and B spermatogonia, preleptotene spermatocytes, and sex vesicles and round spermatids, however, indicates that the function of H2AX phosphorylation during spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA double-strand breaks.     

A change in the phosphorylation pattern of the 30000–33000 M r synaptonemal complex proteins of the rat between early and mid-pachytene

The lateral elements (LEs) of synaptonemal complexes (SCs) of the rat contain major components with relative electrophoretic mobilities (M _r , s) of 30000–33000, which are the products of a single gene. After one-dimensional separation of SC proteins on polyacrylamide-SDS gels, these components show up as two major bands, whereas upon two-dimensional electrophoresis they are resolved in at least 24 spots, which focus at pH 6.5 to 9.5. In this paper we show that these spots represent phosphorylation variants. For the analysis of the phosphorylation of the 30000-to 33000-M _r SC components during progression through meiotic prophase, we developed a procedure for isolation of fractions of testicular cells of the rat that are enriched in separate stages of meiotic prophase. Analysis of the 30000-to 33000-M _r SC components in these fractions by two-dimensional electrophoresis and immunoblotting showed that phosphorylated variants of the 30000-to 33000-M _r SC proteins occur throughout meiotic prophase. However, the extent of phosphorylation changes between early and mid-pachytene, when one phosphate group is probably added to each of the variants.     

Immunochemical localization of contractile proteins in mammalian meiotic chromosomes

Summary Smooth muscle heavy myosin and actin have been detected in mouse and rat meiotic chromosomes, by indirect immunofluorescence performed on testis cryostat sections and isolated germ cells. Both contractile proteins are detectable in the nuclei of meiotic cells during the first prophase. The appearance and disappearance time of myosin and actin, however, is not synchronous. While actin is visible in small spots from resting to late diplotene spermatocytes, myosin appears as filaments in the primary spermatocytes from the zygotene to the early stage of diplotene. The number of myosin filaments in the pachytene spermatocytes corresponds to the number of bivalent chromosomes, whereas actin spots constantly outnumber the pairing chromosomes by two units. These immunochemical observations suggest that the two contractile proteins are associated with the synaptonemal complex (SC). Myosin seems to be associated with the central region of the SC, while actin is present in its basal knob which is in connection with the nuclear membrane. The difference in number between myosin filaments and actin spots appears to be related to the peculiar behaviour of the pairing sex chromosomes. The presence of contractile proteins in the nuclei of primary spermatocytes seems to suggest that they might play a role in the process of pairing of homologous chromosomes.     

Identification of two major components of the lateral elements of synaptonemal complexes of the rat

This paper describes the identification of two major components of the lateral elements of synaptonemal complexes of the rat by immunocytochemical techniques. We prepared monoclonal antibodies against synaptonemal complexes (SCs) by immunization of mice with purified SCs. One of these antibodies, II52F10, reacts with a 30 and a 33 kDa polypeptide, which are major components of purified SCs. Using this antibody, we studied the localization of its antigens light microscopically, by means of the indirect immunoperoxidase technique, as well as ultrastructurally, by means of the immunogold labeling technique. The immunolocalization was carried out on whole-mount preparations of lysed spermatocytes. The antibody reacts with paired as well as unpaired segments of zygotene, pachytene and diplotene SCs. In light microscopic preparations, the attachment plaques, particularly those of late pachytene and diplotene SCs, also appear to react strongly. In electron micrographs the lateral elements in paired as well as unpaired segments could be seen to react. No reaction was observed in the attachment plaques; however, in late pachytene and diplotene SCs the swollen terminal segments of the lateral elements did react with the antibody. Thus, we conclude that a 30 and a 33 kDa polypeptide make part of the lateral elements of synaptonemal complexes of the rat.     

Synaptonemal complex antigen location and conservation

The axial cores of chromosomes in the meiotic prophase nuclei of most sexually reproducing organisms play a pivotal role in the arrangement of chromatin, in the synapsis of homologous chromosomes, in the process of genetic recombination, and in the disjunction of chromosomes. We report an immunogold analysis of the axial cores and the synaptonemal complexes (SC) using two mouse monoclonal antibodies raised against isolated rat SCs. In Western blots of purified SCs, antibody II52F10 recognizes a 30- and a 33-kD peptide (Heyting, C., P. B. Moens, W. van Raamsdonk, A. J. J. Dietrich, A. C. G. Vink, and E. J. W. Redeker, 1987, Eur. J. Cell Biol., 43: 148-154). In spreads of rat spermatocyte nuclei it produces gold grains over the cores of autosomal and sex chromosomes. The cores label lightly during the chromosome pairing stage (zygotene) of early meiotic prophase and they become more intensely labeled when they are parallel aligned as the lateral elements of the SC during pachytene (55 grains/micron SC). Statistical analysis of electronically recorded gold grain positions shows that the two means of the bimodal gold grain distribution coincide with the centers of the lateral elements. At diplotene, when the cores separate, the antigen is still detected along the length of the core and the enlarged ends are heavily labeled. Shadow-cast SC preparations show that recombination nodules are not labeled. The continued presence suggests that the antigens serve a continuing function in the cores, such as chromatin binding, and/or structural integrity. Antibody III15B8, which does not recognize the 30- and 33-kD peptides, produces gold grains predominantly between the lateral elements. The grain distribution is bimodal with the mean of each peak just inside the pairing face of the lateral element. The antigen is present where and while the cores of the homologous chromosomes are paired. From the location and the timing, it is assumed that the antigen recognized by III15B8 functions in chromosome pairing at meiotic prophase. The two anti-rat SC antibodies label rat and mouse SCs but not rabbit or dog SCs. A positive control using human CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) anti-centromere serum gives equivalent labeling of SC centromeres in the rat, mouse, rabbit, and dog. It is concluded that the SC antigens recognized by II52F10 and III15B8 are not widely conserved. The two antibodies do not bind to cellular or nuclear components of somatic cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of nuclear pore distribution in freeze-fracture replicas of seminiferous tubules isolated by transillumination.

Transilluminated seminiferous tubules were staged and utilized to determine the distribution of nuclear pore complexes in seminiferous tubules of the rat. Segments of seminiferous tubules of adult albino rats were separated and identified (in stages VII-VIII, IX-XI, XII-XIV, and V-VI), and then processed by freeze-fracture. Type A spermatogonia, the only spermatogonia located in seminiferous segments possessing stages IX-XI and XII-XIV, are oval cells in contact with the basal lamina. They either exhibit a random distribution of nuclear pores or a slight degree of clumping. Type B spermatogonia, found in segments possessing stages V-VI, exhibit, instead, a noticeable pore clustering. The identification of intermediate spermatogonia was not undertaken in this study. Preleptotene spermatocytes are easily identified in freeze-fracture by their location in segments with stages VII-VIII, by their arrangement in numerous groups between the basal lamina and the pachytene spermatocytes, and by their comparatively small size. They exhibit noticeable pore clustering. Leptotene (segments containing stages IX-XI) and zygotene (XII-XIV) spermatocytes show a more homogeneous distribution of nuclear pores. Pachytene spermatocytes are identified by their large size, by consistent detachment from the basal lamina and by being rather numerous and found in all the stages explored. Diplotene spermatocytes have the largest nuclei of all germ cells. They are always detached from the basal lamina and found only in seminiferous segments containing stage XIII. Pachytenes display a regular geometric array of pore aggregation with striking clustering, whereas diplotene nuclear pores takes on a random distribution. Secondary spermatocytes, only present in stage XIV intermingled with metaphase-anaphase profiles, are characterized in replicas by a paucity of evenly distributed nuclear pores.     

Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).     

Developmental control of sumoylation pathway proteins in mouse male germ cells

Protein sumoylation regulates a variety of nuclear functions and has been postulated to be involved in meiotic chromosome dynamics as well as other processes of spermatogenesis. Here, the expression and distribution of sumoylation pathway genes and proteins were determined in mouse male germ cells, with a particular emphasis on prophase I of meiosis. Immunofluorescence microscopy revealed that SUMO1, SUMO2/3 and UBE2I (also known as UBC9) were localized to the XY body in pachytene and diplotene spermatocytes, while only SUMO2/3 and UBE2I were detected near centromeres in metaphase I spermatocytes. Quantitative RT-PCR and Western blotting were used to examine the expression of sumoylation pathway genes and proteins in enriched preparations of leptotene/zygotene spermatocytes, prepubertal and adult pachytene spermatocytes, as well as round spermatids. Two general expression profiles emerged from these data. The first profile, where expression was more prominent during meiosis, identified sumoylation pathway participants that could be involved in meiotic chromosome dynamics. The second profile, elevated expression in post-meiotic spermatids, suggested proteins that could be involved in spermiogenesis-related sumoylation events. In addition to revealing differential expression of protein sumoylation mediators, which suggests differential functioning, these data demonstrate the dynamic nature of SUMO metabolism during spermatogenesis.     

Fine structure of the early stages of spermatogenesis in the Pacific oyster, Crassostrea gigas (Mollusca, Bivalvia)

The aim of this study is to describe the early stages of spermatogenesis of the Pacific oyster Crassostrea gigas using both light and electron microscopy. The gonad is formed by gonadal tubules invaginated in a connective tissue constituting a storage tissue. Myoepithelial cells surround each gonadal tubule and are associated with an acellular matrix delimiting the outer part of the tubule, the inner part is composed by intragonadal somatic cells associated with germinal lineage. Two types of spermatogonia are identified, where type I spermatogonia (Spg I) are large, scarce and pale cells leaned against the base of the tubule (nuclear diameter: 5.5+/-0.5 microm). Type II spermatogonia (Spg II) are clustered and dark cells which appear smaller than type I (nuclear diameter: 4.3+/-0.3 microm). The aspect of nuage-like material in cytoplasm is described from pale spermatogonia to primary spermatocytes (nuclear diameter: pachytene 3.6+/-0.3 microm, diplotene 3.4+/-0.3 microm), while no structure related to a chromatoid body was observed in oyster spermatocytes and spermatids.     

Synaptonemal complexes of Xenopus laevis.

Synaptonemal complexes (SCs) have been analyzed in spread Xenopus spermatocytes and oocytes. They showed all the usual features of animal SCs in addition to a high incidence of centromere mismatching. A centriole pair is visible throughout zygotene and pachytene. At zygotene the ends of SCs are markedly thickened and are clustered at the nuclear periphery.     

Synaptonemal complexes are integral components of the isolated mouse spermatocyte nuclear matrix

Synaptonemal complexes (SCs) have been isolated as integral components of the nuclear matrix from purified mouse pachytene spermatocytes. These nuclear synaptonemal complex-matrices are prepared by extracting Triton X-100-treated nuclei with low (0.2 M) and high (1.0 or 2.0 M) NaCl, DNase I, and RNase A to remove 85% of the nuclear proteins, 97% of the RNA, and 99% of the DNA. Studies with the light and electron microscopes indicate that these matrices, while lacking a distinct lamina, contain nuclear pores interconnected by a fiber network, residual nucleoli, and interchromatin fibers. In addition, the pachytene spermatocyte matrices contain residual XY heterochromatin and the principal components of the SCs, including two lateral elements, a central element, a presumptive centromere, and attachment plaques. These SCs are preserved within the matrix and retain their structural association with the pore-fiber complex, even when subjected to strong dissociating conditions. Nuclear matrices from pachytene spermatocytes and spermatids (steps 1-8), when analyzed by SDS PAGE, contain an array of polypeptides distinct from those of mouse liver nuclear matrices. Proteins of spermatogenic matrices range in Mr from 8,000 to approximately 150,000. The prominent lamina proteins (Mr approximately 60,000-70,000) of somatic nuclear matrices are either absent or represent only a minor part of the spermatogenic matrix. The polypeptide composition of the pachytene spermatocyte and spermatid matrices are similar, although minor quantitative and qualitative differences are evident. These observations suggest that the SC constituents may consist of a heterogeneous group of proteins present in low proportion relative to total matrix proteins, or they may be retained, but in a different form, within the spermatid matrix.     

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.     

Clastogenic effects of cis-diamminedichloroplatinum. II. Induction of chromosomal aberrations in primary spermatocytes and spermatogonial stem cells of mice

The clastogenic effect of the anticancer drug cis-diamminedichloroplatinum (II) (cisplatin) on meiotic prophase in primary spermatocytes and on spermatogonial stem cells of male (101/E1 x C3H/E1)F1 mice was studied. The intraperitoneal doses of cisplatin tested were 5.0, 7.5 and 10.0 mg/kg. Chromosomal aberrations were examined at diakinesis-metaphase 1 of meiosis 1-13 days after treatment, representing cells treated at diplotene, pachytene, zygotene, leptotene an preleptotene. Reciprocal translocations were evaluated 63-70 days after treatment, representing treated stem-cell spermatogonia. Cisplatin had a toxic effect in zygotene to preleptotene of meiosis, as indicated by the significant reduction in testicular weight. At diplotene, pachytene and zygotene no enhancement of aberrations was found. An increase in aberrant cells was observed during leptotene with preleptotene being the most sensitive stage. The dose-response relationship for aberrant cells was linear on day 13 after treatment. It is concluded that, like mitomycin C (Adler, 1976), cisplatin primarily caused aberrations during the premeiotic phase of DNA synthesis. No significant increase of translocation multivalents was found after treatment of stem-cell spermatogonia.     

Quantitation of Sertoli cell-germ cell desmosome gap junctions in relation to meiotic divisions in the male rat.

Desmosome-gap (D-G) junctions were quantified in relation to germ cell meiosis in the male, specifically to test the hypothesis that the loss of these junctions is related to successful passage of cells through diplotene phase of Meiosis I and the two cytokineses that follow. Such a hypothesis has been proposed as the cause for the resumption of meiosis that occurs prior to ovulation in the female. D-G junctions were quantified in pachytene spermatocytes (stage XII), diplotene spermatocytes (stage XII), secondary spermatocytes (stage XIV) and step 1 spermatids (stage I). These were referred to as the cells of interest as compared with spermatocytes (zygotene spermatocytes, zygotene spermatocytes, pachytene spermatocytes, pachytene spermatocytes) in the same stages, respectively, that served as controls termed control cells. Since gap junctions are not easily recognized in the average sectioned profile of a desmosome-gap junction, only the desmosomal component was quantified. The data were expressed as both numbers and length of junctions per tubule, per cell profile and per unit lineal membrane length to overcome errors inherent in the methodologies utilized. There was no indication that numbers of junctions changed specifically in the cells of interest after passage through diplotene suggesting that these junctions do not have a comparable role in meiotic continuance in the male as proposed for the female. Interestingly, the control cells always showed greater numbers and length of junctions than the cells of interest suggesting that junction may relate more to the period of initiation of meiosis than to its continuance.