Duration of meiotic prophase stages during oogenesis in the newt Triturus cristatus cristatus]

The duration of the early stages of meiotic prophase was determined in the oogenesis of T. cristatus cristatus by means of autoradiography. The oocytes were being investigated during 39 days from the moment of 3H-thymidine injection. It was shown that preleptotene lasts 1--2, leptotene ca. 4, zygotene 5 and pachytene 26 days. When studying the preparations obtained 1 day after the injection of 3H-thymidine, the silver grains were found to be localized over the nuclei at all stages of meiotic prophase; this suggests the amplification of rDNA which begins in leptotene-zygotene and ends in early diplotene.     

Autoradiographic and cytophotometric study of DNA synthesis in the oocytes of the domestic hen at the preleptotene prophase stage of meiosis

3H-thymidine incorporation into the fowl oocytes was established radioautographically at the middle preleptotene, when chromosomes are condensed and associated in the complex chromocenters. According to the cytophotometry of Feulgen stained oocyte nuclei, their DNA value increases during preleptotene from 2 to 4c. So, the DNA synthesis observed is characteristic of chromosome reduplication, rather than of nuclear organizer amplification. The preleptotene should be considered as the initial stage of meiotic prophase because it involves spiralization and individualization of chromosomal threads. Both the analysis of literary data and of our own results enable us to conclude that in different species the meiotic chromosome reduplication may proceed in different periods between telophase of last gonial mitosis and the beginning of homologous chromosome conjugation.     

Centromere and telomere redistribution precedes homologue pairing and terminal synapsis initiation during prophase I of cattle spermatogenesis

Alterations in nuclear topology associated with meiotic chromosome pairing were studied in premeiotic cells and spermatocytes I of adult bovine males. To this end, we performed FISH with chromosome, pericentromeric satellite-DNA and telomere-specific probes in combination with immunostaining of synaptonemal complex proteins (SCP3, SCP1) on testis tissue sections. Nuclei of premeiotic cells (spermatogonia) exhibited a scattered telomere distribution while pericentromeres formed a few intranuclear clusters. We observed that the chromosome pairing process in cattle prophase I is preceded by repositioning of centromeres and telomeres to the nuclear periphery during preleptotene. Clustering of chromosome ends (bouquet formation) was observed during the transition from leptonema to zygonema and coincided with pairing of a sub-centromeric marker of bovine chromosomes 7. Dissolution of bouquet topology during zygonema left perinuclear telomeres scattered over the nuclear periphery at pachynema. SCP3 staining in frozen tissue sections revealed the appearance of this axial element protein in intranuclear aggregates during preleptotene, followed by extensive axial element formation during leptotene. Synapsis as revealed by SCP1 staining initiated peripherally at earliest zygotene, at this stage nuclei still contained numerous SCP3 clusters. Our observations reveal prominent non-homologous satellite-DNA associations in spermatogonia and indicate the conservation of topological features of the meiotic chromosome pairing process among mammals. The comparison of telomere dynamics in mouse and cattle prophase I suggests that a larger number of chromosomes prolongs the duration of the bouquet stage.     

Formation of cytoplasmic synaptonemal-like polycomplexes at leptotene and normal synaptonemal complexes at zygotene in Ascaris suum male meiosis

A thread-like (more than 70 cm long) testis of Ascaris suum, when examined under the light and electron microscope, reveals the linear succession of meiotic stages. Beginning from, at least, late leptotene, the spermatocytes are synchronous in their development. Thus within each transverse section of the testis all the spermatocytes are in the same stage. The spermatocytes at each stage of prophase I occupies several (4 to 10) cm of the whole testis length. — At leptotene, synaptonemal-like polycomplexes of lateral and central stacked elements are formed in the cytoplasm of spermatocytes. At late leptotene, the polycomplexes are attached to the external nuclear membrane. The polycomplexes disappear at zygotene. Slightly discernable axial cores are observed in the late leptotene chromosomes. The synaptonemal complexes (SCs) are formed at the zygotene stage, their structure being characteristically tripartite. The SCs disappear from the nuclei at the diffuse stage of prophase I. In other organisms completely developed polycomplexes of stacked lateral and central elements were never found during the presynaptic period of meiosis, although single or two parallel layers of aggregated central regions of SC were found in Neottiella meiocytes at the stage prior to chromosome pairing (Westergaard and von Wettstein, 1970, 1972). — First appearance of the polycomplexes in the cytoplasm insetead of the nucleus is also a novel fact. It is concluded that the polycomplexes at leptotene are formed by a self-assembly of the SC molecular material precociously synthesized in the cytoplasm. Two hypotheses regarding possible function and the further fate for leptotene polycomplexes are discussed.     

Chromosome and DNA methylation dynamics during meiosis in the autotetraploid Arabidopsis arenosa

Variation in chromosome number due to polyploidy can seriously compromise meiotic stability. In autopolyploids, the presence of more than two homologous chromosomes may result in complex pairing patterns and subsequent anomalous chromosome segregation. In this context, chromocenter, centromeric, telomeric and ribosomal DNA locus topology and DNA methylation patterns were investigated in the natural autotetraploid, Arabidopsis arenosa. The data show that homologous chromosome recognition and association initiates at telomeric domains in premeiotic interphase, followed by quadrivalent pairing of ribosomal 45S RNA gene loci (known as NORs) at leptotene. On the other hand, centromeric regions at early leptotene show pairwise associations rather than associations in fours. These pairwise associations are maintained throughout prophase I, and therefore likely to be related to the diploid-like behavior of A. arenosa chromosomes at metaphase I, where only bivalents are observed. In anthers, both cells at somatic interphase as well as at premeiotic interphase show 5-methylcytosine (5-mC) dispersed throughout the nucleus, contrasting with a preferential co-localization with chromocenters observed in vegetative nuclei. These results show for the first time that nuclear distribution patterns of 5-mC are simultaneously reshuffled in meiocytes and anther somatic cells. During prophase I, 5-mC is detected in extended chromatin fibers and chromocenters but interestingly is excluded from the NORs what correlates with the pairing pattern.     

Synaptonemal complexes,telomeric nucleoli and the karyosphere in achiasmatic oocyte meiosis of the carob moth

Synaptonemal complexes and telomeric nucleoli are involved in the spatial organization and regular distribution of homologous chromosomes in meiosis of the achiasmatic female carob moth. The bivalents are held together from zygotene to metaphase by the Synaptonemal complexes. These are attached to telomeric nucleoli which appear during early meiotic prophase and are unique to the oocyte. The telomeric nucleoli fuse during prophase and the chromosomes concentrate into a small karyosphere before prometaphase. During the final stages of prophase elements of the Synaptonemal complex are found in the periphery of the fibrillar region of the telomeric nucleoli.     

Abnormal meiosis in bisporic strains of white button mushroom Agaricus bisporus (Lange) imbach

A formerly developed method of microspreading of mushroom basidial nuclei was applied to study meiotic prophase I in bisporic white button mushroom (Agaricus bisporus) strains. Meiotic recombination and assemblage of axial structures (axial elements and synaptonemal complexes) of chromosomes in meiotic prophase I are interrelated. It is known that the frequency of meiotic recombination is reduced in the bisporic A. bisporus variety. We showed that formation of axial structures of meiotic chromosomes in bisporic strains of this mushroom was disrupted. The anomalous phenotypes in spread prophase nuclei are diverse. In leptotene and early zygotene, many nuclei contain abnormal, often short, and, as a rule, few chromosomal axial elements. The abnormalities in the formation of synaptonemal complexes at the zygotene-diplotene stage are of the same kind and even more pronounced. We discovered an important feature of meiosis in A. bisporus associated with fruit-body morphogenesis. Meiosis starting in basidia (meiocytes) of young closed fruit bodies is accompanied by disruption of chromatin condensation in prophase I and, probably, is arrested. After partial veil breakage, the course of meiosis normalizes. Preparations with clearly observable chromosomal axial structures can be obtained only at this stage of fruit-body development.     

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.     

Multiplication of nucleolar fibrillar centres and absence of rDNA amplification in mouse ooctye during meiotic prophase I

During meiotic prophase I the nucleolus of the mouse oocyte assumes a reticulate structure of ‘nucleolonema’ type. This change coincides with the appearance of several secondary fibrillar centres. The number of these centres at diplotene (97–113), largely exceeds that of nucleolar organizers (4c DNA = 20 NORs). The quantitatative analysis of autoradiographs after hybridization in situ with -³H-uridine labelled rRNA, enabled us to demonstrate that the multiplication of the fibrillar centres in mouse oocyte nucleolus during meiotic prophase I is not the result of an amplification of the rDNA. The number of silver grains in pachytene and diplotene nuclei was twice that counted for somatic cell and oogonium nuclei (2c DNA).     

Abnormal meiosis in bisporic mutants of white button mushroom Agaricus bisporus (Lange) Imbach

A formerly developed method of obtaining spread preparations of mushroom basidial nuclei was applied to study of meiotic prophase I in bisporic white button mushroom (Agaricus bisporus) strains. Meiotic recombination and assemblage of axial structures (axial elements and synaptonemal complexes) of chromosomes in meiotic prophase I are interrelated. It is known that the frequency of meiotic recombination is reduced in the bisporic A. bisporus variety. We showed that formation of axial structures of meiotic chromosomes in bisporic strains of this mushroom was disrupted. The phenotypes of disruptions in spread prophase nuclei are diverse. In leptotene and early zygotene, many nuclei contain abnormal, often short, and, as a rule, few chromosomal axial elements. The abnormalities in the formation of synaptonemal complexes at the zygotene-diplotene stage are of the same kind and even more pronounced. We discovered an important feature of meiosis in A. bisporus associated with fruit-body morphogenesis. Meiosis starting in basidia (meiocytes) of young closed fruit bodies is accompanied by disruption of chromatin condensation in prophase I and, probably, is arrested. After indusium breakage, the course of meiosis normalizes. Preparations with clearly observable chromosomal axial structures can be obtained only at this stage of fruit-body development.     

Autoradiographic study of RNA synthesis during meiotic prophase in the human oocyte

The incorporation of 3H-uridine in oogonia and oocytes during meiotic prophase I was studied in three human fetuses 13, 18, and 19 weeks old. Following a 40- or 60-min pulse, intense nuclear and nucleolar labeling was observed in oogonia. During the preleptotene chromosome condensation stage, the heteropycnotic masses were unlabeled, while numerous silver grains were seen on the filaments persisting around these masses. During leptotene, chromosomal and nucleolar RNA synthesis was significant, but less than that in the oogonia. The rate of incorporation declined rapidly during zygotene and fell to a very low level at early pachytene. Throughout pachytene no nucleolar RNA synthesis was observed. Chromosomal RNA synthesis progressively recovered during middle pachytene, was of moderate intensity at late pachytene, and increased again at early diplotene. Nucleolar RNA synthesis was very intense at early diplotene, at the same time as nucleolar size and basophilia increased.     

Analysis of germ cell populations in ejaculate of men infected with herpes simplex virus

Cytological and molecular genetics methods were used to study sperm from patients with sperm infected with herpes simplex virus (HSV) as indicated by virological and immunocytochemical tests. The following methods were used: (1) sperm analysis to evaluate the morphology and functional properties of sperm; (2) fluorescence in situ hybridization (FISH) with DNA probes specific for chromosomes 1, X, and Y to evaluate nondisjunction frequencies of these chromosomes in sperm; and (3) quantitative analysis of immature germ cells in the ejaculate to identify spermatogenic abnormalities. The total sperm count and the count of sperm with normal motility proved similar to the norm. FISH analysis demonstrated no difference in the nondisjunction frequency of chromosomes 1, X, and Y between infertile patients with HSV-infected sperm and fertile donors. Comparative quantitative analysis of immature germ cells from the ejaculate has demonstrated a significant and considerable (threefold) increase in the number of spermatocytes I at the prepachytene stages of prophase I (preleptotene, leptotene, and zygotene) in HSV patients compared to normal donors. At the same time, HSV patients demonstrated a significant decrease in the number of spermatocytes I, a decrease in the proportion of spermatocytes II and spermatids, and a twofold increase in the number of unidentifiable immature germ cells. The data obtained indicate a partial spermatogenic arrest at the early stages of meiotic prophase I in HSV patients, which prompts further research into the cellular mechanisms of abnormal spermatogenesis after viral infection in humans.     

Dense bodies in silver-stained spermatocytes of the chinese hamster: behavior and cytochemical nature

From the silver staining behavior of various organelles in the nucleus we have divided meiotic prophase (leptotene to the diffuse stage) of the male Chinese hamster into five stages. Components within the nucleus, such as synaptonemal complex (SC), sex bivalent (SB), nucleolus organizer regions (NORs), chromatin and the dense bodies, showed a characteristic feature in each stage of meiotic prophase. The lampbrush chromosome stage was found to be followed by the diffuse stage. The chromatin around SC began to be organized at early pachytene and formed a brush-like structure at late pachytene. During early prophase stages a dramatic change in SB morphology occurred. Three types of morphology of SB were recognized: (1) the XY pair with long synapsis and fusiform or diffuse thickening of the unpaired portions (late zygotene and early pachytene), (2) desynapsed, thread-like axes seen at midpachytene, and (3) multistranded, branched, and anastomosed axes seen at late pachytene.Two types of the dense body were found during meiotic prophase; the double body in early stage (leptotene to early pachytene) and the single body in later stages (mid pachytene to diffuse stage). The small precursors of the double body existed at early leptotene but they increased in size and also changed the silver stainability during zygotene, becoming the characteristic double body consisted of one light body (L-body) and one dark body (D-body). These two bodies can also be recognized after Giemsa or acridine orange (AO) staining. The L-body fluoresced reddish orange after AO staining. The single body, which is probably formed by amalgamation of the D- and the L-bodies, showed a staining reaction similar to that of the D-body.Data from pancreatic lipase and protease treatments suggest that the D-body contained a lipoprotein.     

Stage-specific damage to synaptonemal complexes and metaphase chromosomes induced by X rays in male mouse germ cells

Synaptonemal complexes reveal mutagen-induced effects in germ cell meiotic chromosomes. This study was aimed at characterizing relationships between damage to synaptonemal complexes and metaphase I chromosomes following radiation exposure at various stages of spermatogenesis. Male mice were irradiated with doses of 0, 2, or 4 Gy, and spermatocytes were harvested at times consistent with earlier exposures as spermatogonial stem cells, preleptotene cells (premeiotic DNA synthesis), or meiotic prophase cells. After stem-cell exposure, twice as many rearrangements were observed in synaptonemal complexes as in metaphase I chromosomes. Irradiation during premeiotic DNA synthesis resulted in dose-related increases in synaptonemal complex breakage and rearrangements (including novel forms) and in metaphase chromosomal aberrations. Following prophase exposure, various types and levels of damage to synaptonemal complexes and metaphase chromosomes were observed. Irradiation of zygotene cells led to high frequencies of chromosome multivalents in metaphase I without a correspondingly high level of damage in preceding prophase synaptonemal complexes. Thus irradiation of premeiotic and meiotic cells results in variable relationships between damage to synaptonemal complexes and metaphase chromosomes. Interpretations of these relationships are based upon what is known about both radiation clastogenesis and the structural/temporal relationships between synaptonemal complexes at prophase and chromosomes at metaphase I of meiosis.     

Ultrastructural characterization of the sex chromosomes during spermatogenesis of spiders having holocentric chromosomes and a long diffuse stage

An ultrastructural study has been made of spermatogenesis in two species of primitive spiders having holocentric chromosomes (Dysdera crocata, XO and Segestria florentia X₁X₂O). Analysis of the meiotic prophase shows a scarcity or absence of typical leptotene to pachytene stages. Only in D. crocata have synaptonemal complex (SC) remnants been seen, and these occurred in nuclei with an extreme chromatin decondensation. In both species typical early prophase stages have been replaced by nuclei lacking SC and with their chromatin almost completely decondensed, constituting a long and well-defined diffuse stage. Only nucleoli and the condensed sex chromosomes can be identified. — In S. florentina paired non-homologous sex chromosomes lack a junction lamina and thus clearly differ from the sex chromosomes of more evolved spiders with an X₁X₂O male sex determination mechanism. In the same species, sex chromosomes can be recognized during metaphase I due to their special structural details, while in D. crocata the X chromosome is not distinguishable from the autosomes at this stage. — The diffuse stage and particularly the structural characteristics of the sex chromosomes during meiotic prophase are reviewed and discussed in relation to the meiotic process in other arachnid groups.