Chromatin diminution and chromosome elimination in four Japanese hagfish species

Cytogenetic examination of four Japanese hagfish species belonging to the order Myxinida (Eptatretus okinoseanus, E. burgeri. Paramyxine atami, and Myxine garmani) revealed differences in chromosome number between germ cells (spermatocytes and spermatogonia) and somatic cells (liver, blood, gill, and kidney). The differences in chromosome number between spermatogonia (54, 52, 48, and 16) and somatic cells (34, 36, 34, and 14) were 20, 16, 14, and 2 in E. okinoseanus, E. burgeri, P. atami, and M. garmani, respectively. The amount of DNA in a somatic cell (2C) relative to that in a germ cell (2C) averaged 54.6% (E. okinoseanus type A), 44.9% (E. okinoseanus type B), 79.1% (E. burgeri), 60.0% (P. atami), and 70.2% (M. garmani). These results clearly indicate that chromosome elimination takes place during early cleavage in the four hagfish species of Myxinida living in Japanese waters, except in the ancestral germline cells. C-banding of metaphase chromosome preparations of germline and somatic cells from each hagfish species revealed that the C-band-positive chromatin in the ancestral somatic cells had been almost completely eliminated. Three patterns of elimination of this chromatin are discussed.     

Meiotic chromosome behaviour in male triploid transparent coloured crucian carp, Carassius auratus L.

Meiotic chromosome behaviour was investigated by surface-spreading, air-drying and thin sectioning testes for light and electron microscope examination in artificial triploid transparent coloured crucian carp ( Carassius auratus L.) produced by hydrostatic pressure shock. Unsynapsed univalents, synapsed bivalents and partially synapsed trivalents could be observed and distinguished from each other in the surface-spread spermatocytes. The pairing of the partially paired trivalents mainly occurred in the telomeric regions. Similarly, lateral elements of unsynapsed univalents, typical synapsed bivalents and triple pairing configurations having three lateral elements and two central elements in the trivalents were also observed in the thin sectioned pachytene spermatocytes. The metaphase I cells were mainly composed of univalents, bivalents and trivalents, but a few tetravalents, pentavalents and hexavalents were also found. The relationship between disturbed chromosome pairing and abnormal spermatogenesis is briefly discussed.     

Colchicine promotes a change in chromosome structure without loss of sister chromatid cohesion in prometaphase I-arrested bivalents

In somatic cells colchicine promotes the arrest of cell division at prometaphase, and chromosomes show a sequential loss of sister chromatid arm and centromere cohesion. In this study we used colchicine to analyse possible changes in chromosome structure and sister chromatid cohesion in prometaphase I-arrested bivalents of the katydid Pycnogaster cucullata. After silver staining we observed that in colchicine-arrested prometaphase I bivalents, and in contrast to what was found in control bivalents, sister kinetochores appeared individualised and sister chromatid axes were completely separated all along their length. However, this change in chromosome structure occurred without loss of sister chromatid arm cohesion. We also employed the MPM-2 monoclonal antibody against mitotic phosphoproteins on control and colchicine-treated spermatocytes. In control metaphase I bivalents this antibody labelled the tightly associated sister kinetochores and the interchromatid domain. By contrast, in colchicine-treated prometaphase I bivalents individualised sister kinetochores appeared labelled, but the interchromatid domain did not show labelling. These results support the notion that MPM-2 phosphoproteins, probably DNA topoisomerase IIalpha, located in the interchromatid domain act as "chromosomal staples" associating sister chromatid axes in metaphase I bivalents. The disappearance of these chromosomal staples would induce a change in chromosome structure, as reflected by the separation of sister kinetochores and sister axes, but without a concomitant loss of sister chromatid cohesion.     

Synaptonemal complex analysis in spermatocytes of diploid and triploid Chinese shrimp Fenneropenaeus chinensis

A modified surface spreading technique for synaptonemal complex (SC) analysis was tested to assess the process of chromosome synapsis in spermatocytes of diploid and induced triploid Fenneropenaeus chinensis. Spermatocytes of diploid shrimp showed typical morphological characteristics of eukaryote SC, with complete synapsis of bivalents. No recognizable bivalent associated with sex chromosomes was observed in spermatocytes of diploid shrimp. However, differences in morphology of SC, including unsynapsed univalents, bivalents, totally paired trivalents with non-homologous synapsis, partner switches and triple synapsis were identified at early pachytene stage of triploid spermatocytes. Triple synapsis was especially common at late pachytene stage in spermatocytes of triploid shrimp. The observed abnormal synapsis behavior of chromosomes in spermatocytes indicated that triploid male shrimp may find it difficult to develop normal haploid sperm.     

Sex-dependent frequency and type of autosomal univalency at the first meiotic metaphase in mouse germ cells

Univalents at the first meiotic metaphase in mouse spermatocytes occur mainly in the XY pair, making it difficult to compare the amounts of univalency in males and females. In this study, the amounts of autosomal univalency in male and female meiosis were compared using the model strain CBA-T6, in which univalency of the small marker autosome pair T6 has been shown to occur very frequently in spermatocytes. Mice from inbred CBA and DBA strains were also analysed. The total frequencies of univalency (sex chromosomes plus autosomes) in metaphase I spermatocytes were 45.6% in CBA, 36.9% in CBA-T6, and 37.3% in DBA males. The aneuploidy in metaphase II spermatocytes ranged from 1.4 to 3% in these strains, which was in agreement with previous findings that most primary spermatocytes with abnormal chromosome configurations are arrested in their development before metaphase II. In the CBA-T6 strain, autosomal univalency at metaphase I mostly involved chromosome pair T6; however, its frequency differed significantly between the sexes, amounting to 18.9% in spermatocytes and 4.3% in oocytes. In the CBA strain, autosomal univalents at metaphase I were seen in 7.7% of the spermatocytes and 1.4% of the oocytes and, in DBA mice, in 4.9% of the spermatocytes and 3.8% of the oocytes. However, in DBA oocytes, when univalency occurred it usually concerned a greater number of bivalents in one cell (range: 2-19 disjoined bivalents), a phenomenon very rare in males of this strain. This study shows that univalent formation differs between the male and female types of meiosis.     

Malorientation and abnormal segregation of chromosomes during recovery from colcemid and nocodazole

Reversal of meiotic arrest in crane-fly spermatocytes by U. V. irradiation of Colcemid-arrested cells or by rinsing Nocodazole-arrested cells in fresh buffer results in the induction of chromosome malorientation. Malorientations observed among Colcemid-recovering and Nocodazole-recovering spermatocytes at frequencies higher than normally observed in untreated cells included associations of sister kinetochores of half-bivalents with both spindle poles (amphitely), in contrast with associations of sisters with only one pole (syntely) as is usually found during the first meiotic division. In several cases, prior to anaphase onset, maloriented bivalents appeared unusually tilted with respect to the spindle axis, and during anaphase they gave rise to laggard half-bivalents that did not segregate during anaphase along with half-bivalents having proper syntelic orientation. The results parallel previous findings obtained during cold recovery, and the properties of the drugs used here suggest that their action on microtubules, although reversible, induces malorientation during recovery from meiotic arrest.     

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.     

Meiosis in a male with Down's syndrome

Summary The chromosomes in mitotic and meiotic phases were investigated in a male Down's syndrome case, aged 45. Information was obtained that based on blood and tunica vaginalis cultures, the somatic chromosome complement was found to possess 47 chromosomes with the standard 21-trisomy, and further that the majority of cells from biopsied testicular specimens examined showed the chromosome number 47 in spermatogonia, and 22 autosomal elements consisting of 21 bivalents and a trivalent, together with an X-Y bivalent in the first spermatocytes. The seminiferous tubules contained no mature spermatozoa.Contribution No. 688 from the Zoological Institute, Faculty of Science, Hokkaido University, Sapporo. This paper is dedicated to Professor Sajiro Makino, Zoological Institute, Hokkaido University, Sapporo, in honor of his sixtieth birthday, June 21, 1966.     

Temporal appearance of seasonal changes in numbers of Sertoli cells, Leydig cells, and germ cells in stallions

The temporal appearance of seasonal changes in numbers of Leydig, Sertoli, and germ cells was evaluated to determine if seasonally increased daily spermatozoan production might be preceded by changes in numbers of either of two somatic testicular cells. A significant increase in numbers of spermatogonia and Sertoli cells preceded the significant increase in number of Leydig cells in the approaching breeding season. Seasonal changes in parenchymal weight and in numbers of Sertoli cells, Leydig cells, and germ cells were maximal in May and June. Numbers of A or B spermatogonia in June were 2.4 to 2.5 times the number present in January. During the same time period, numbers of other germ cells, as well as Leydig cells and Sertoli cells, were increased by 1.5 to 1.9 times. The magnitude of change between January and March (first time period that the change was significant) was greater for A spermatogonia (1.7-fold) than for other cell types (1.3-fold to 1.5-fold). Hence, the need to accommodate more spermatogonial progeny might cause increased testicular size and number of somatic cells, including Sertoli cells. Season did not influence the rate of degeneration between A and B spermatogonia. However, in the breeding season, the conversion of B spermatogonia to primary spermatocytes was reduced. The lack of a seasonal difference in the ratio of primary spermatocytes per Sertoli cell was consistent with a limited capacity of individual Sertoli cells to accommodate primary spermatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

IGF-I, IGF-II and insulin promote differentiation of spermatogonia to primary spermatocytes in organ culture of newt testes.

Recombinant human insulin-like growth factors (rhIGF-I and rhIGF-II) and human insulin promoted the differentiation of spermatogonia into primary spermatocytes in newt testes fragments cultured in a chemically defined medium. The biological potency for promoting differentiation was dose-dependent for all the ligands with the highest potency displayed by IGF-I, followed by IGF-II, and the least by insulin. The difference in potency was larger between IGF-II and insulin than that between IGF-I and IGF-II. This order of biological potency was in good accordance with the order of affinity in binding specificity of [125I]IGF-I to the testicular membrane fractions: IGF-II and insulin competed the binding of [125I]IGF-I only at concentrations 20-fold and 100-fold higher, respectively, than IGF-I. Specific binding was observed in both somatic cells (mostly Sertoli cells) and germ cells (spermatogonia and primary spermatocytes), though the binding to somatic cells was about 2.7 times higher than that to germ cells. These results indicate that (1) specific binding sites for IGF-I are present in the newt testes, (2) IGF-II and insulin also bind to these receptors but to a lesser degree, and (3) IGF-II and insulin as well as IGF-I promote spermatogonial differentiation into primary spermatocytes by binding to the IGF-I receptor.     

Chromosome attachment to the spindle in crane-fly spermatocytes requires actin and is necessary to initiate the anaphase-onset checkpoint

Summary We investigated the possible involvement of actin in the attachment of chromosomes to spindles in crane-fly primary spermatocytes. In a previous study, cytochalasin D, an inhibitor of actin polymerisation, prevented bivalent attachment to microtubules when applied at prophase, but did not cause the detachment of already attached bivalents. We were able to detach the already attached bivalents by first treating prometaphase cells with an antitubulin drug, nocodazole, to disrupt spindle microtubules. 2 min after nocodazole addition, we added cytochalasin D, to disrupt actin filaments; then 2 min later nocodazole was removed, and the cells were kept in cytochalasin D until the time of normal anaphase. Double treatment with nocodazole and cytochalasin D blocked reattachment of bivalents to the spindle. Single treatment with nocodazole alone caused chromosome detachment but did not prevent reattachment when nocodazole was washed out. Extended treatment with cytochalasin D alone starting in prometaphase did not cause bivalents to detach from the spindle. These data suggest that actin is needed for attachment of bivalents to spindle microtubules. This protocol is relevant to the anaphase-onset checkpoint. From previous experiments it was argued that the anaphase-onset checkpoint recognises unattached chromosomes only after those chromosomes first interact with (become attached to) the spindle. Our experiments showed that anaphase disjunction occurred at normal times when bivalents were prevented from attaching to the spindle (by adding cytochalasin D in prophase), while anaphase disjunction was greatly delayed when previously attached bivalents were detached (with nocodazole) and then prevented from re-attaching (with cytochalasin D) in the double treated cells. Thus the anaphaseonset checkpoint recognises only those unattached bivalents that previously were attached to the spindle. Other results provided further indication that actin-microtubule interactions are important in spindle organisation. Nocodazole treatment for 4 min caused most microtubules to disappear: bivalents aggregated around remnant microtubules. When cytochalasin D treatment followed nocodazole treatment, remnant spindle microtubules were not seen, suggesting that actin interactions help stabilise those microtubules.Abbreviations CD cytochalasin D - NMBD nuclear-membrane breakdown - NOC nocodazole     

Pachytene chromosomes in male and female mice heterozygous for the Is(7;1)40H insertion

Pairing of pachytene chromosomes was studied in oocytes and spermatocytes of mice heterozygous for the male-sterile Is(7;1)40H insertion using light and electron microscopy for synaptonemal complex analysis in surface-spread, silver-stained preparations. The data comprised four males and four female embryos. The insertion/deletion configurations appeared as either two bivalents or one quadrivalent in both sexes, but the proportion of bivalents was higher in oocytes. Some insertion and deletion bivalents showed synaptic adjustment. The insertion/deletion configurations were associated with, or adjacent to, the XY bivalent in the majority of spermatocytes. End-to-end association of different bivalents was more frequent in oocytes than in spermatocytes. It is suggested that physiological differences between male and female gametocytes may lead to the difference in their reproductive potential.The authors warmly dedicate this paper to the Founder and Senior Editor of Chromosoma, Professor Hans Bauer, on the occasion of his 80th birthday.     

A pachytene map of the mouse spermatocyte

Pachytene chromomere maps of early and mid/late mouse spermatocytes have been prepared which permit exact identification of each bivalent. The average total number of chromomeres on the autosomal bivalents was 248 in the early cells and 184 in the mid/late. There was close correspondence between early and mid/late chromomeres in 122 locations. Comparisons of early pachytene chromomeres with published prometaphase dark G bands revealed 1.6 more chromomeres in the meiotic autosomal bivalents, with close correspondence of larger chromomeres and major mitotic bands. Fewer chromomeres were found in pachytene spermatocytes than had been seen in a previous study of pachytene oocytes. Comparisons of chromomeres of spermatocytes and oocytes revealed several differences.     

Meiotic chromosome structure. Kinetochores and chromatid cores in standard and B chromosomes of Arcyptera fusca (Orthoptera) revealed by silver staining.

The behaviour of two chromosome structures in silver-stained chromosomes was analyzed through the first meiotic division in spermatocytes of the acridoid species Arcyptera fusca. Results showed that at diakinesis kinetochores and chromatid cores are individualized while they associate in bivalents of metaphase I; only kinetochores and distal core spots associate in the sex chromosome. Metaphase I is characterized by morphological and localization changes of both kinetochores and cores which define the onset of anaphase I. These changes analyzed in both autosomes and in the sex chromosome allow us to distinguish among three different substages in metaphase I spermatocytes. B chromosomes may be present as univalents, bivalents, or trivalents. Metaphase I B univalents are characterized by separated cores except at their distal ends and individualized and flat sister kinetochores. At anaphase I sister kinetochores of lagging B chromatids remain connected through a silver-stained strand. The behaviour of cores and kinetochores of B bivalents is identical with that found in the autosomal bivalents. The differences in the morphology of kinetochores of every chromosome shown by B trivalents at metaphase I may be related to the balanced forces acting on the multivalent. The results show dramatic changes in chromosome organization of bivalents during metaphase I. These changes suggest that chromatid cores are not involved in the maintenance of bivalents. Moreover, the changes in morphology of kinetochores are independent of the stage of meiosis but correlate with the kind of division (amphitelic-syntelic) that chromosomes undergo.     

Complete chromomere map of mid/late pachytene human oocytes.

A complete chromomere map of the mid/late human pachytene oocyte has been developed from ovaries of 35 fetuses at 18-22 weeks gestation. Bivalents, which were all specifically identifiable, were more extended than comparable human spermatocyte bivalents. The total number of chromomeres found was 639, exceeding both the number of human pachytene spermatocytes and the number of mitotic bands seen in metaphase somatic chromosomes. Each oocyte bivalent contained more chromomeres than the number of corresponding prometaphase chromosome bands. Similarities and differences were noted in regional comparisons of chromomere distribution between oocytes and spermatocytes.     

Cytogenetic studies on natural populations of grasshoppers with special reference to B-chromosomes

In Atractomorpha bedeli the frequencies of males with B-chromosomes in the males from the Tofuro-ato population were 21, 30, 32, 40, and 26%, respectively, over five years. Numbers of B's (0 to 7) in the primary spermatocytes were usually constant within the follicles but were variable among the follicles within the individuals. In males from the Okazyo-ato population, 35% contained B's in their primary spermatocytes. The cells of the gastric caeca were observed in 8 males with B's in the primary spermatocytes but no cells of the gastric caeca contained B's. Cells of ovariole walls and those of gastric caeca were analysed in 80 females from the same population; none of them contained B's. These facts indicate that B's are present in the germ line but absent in the somatic line. The difference between the germ line and the somatic one was explained on the basis of elimination of B's from the somatic cells. The inter-follicular variation in number of B's was ascribed to mitotic non-disjunction in the germ line prior to differentiation of the follicles.Contribution No. 86 from the Institute of Biology, Oita University.     

FSH-initiated differentiation of newt spermatogonia to primary spermatocytes in germ-somatic cell reaggregates cultured within a collagen matrix

We previously cultured fragments of newt testes in chemically defined media and showed that mammalian follicle-stimulating hormone (FSH) stimulates proliferation of spermatogonia as well as their differentiation into primary spermatocytes (Ji et al., 1992; Abe and Ji, 1994). Next, we indicated in cultures composed of spermatogonia and somatic cells (mainly Sertoli cells) that FSH stimulates germ cell proliferation via Sertoli cells (Maekawa et al., 1995). However, the spermatogonia did not differentiate into primary spermatocytes, but instead died. In the present study, we embedded large reaggregates of spermatogonia and somatic cells (mainly Sertoli cells) within a collagen matrix and cultured the reaggregates on a filter that floated on chemically defined media containing FSH; in this revised culture system, spermatogonia proliferated and differentiated into primary spermatocytes. The viability and percentage of germ cells differentiating into primary spermatocytes were proportional to the percentage of somatic cells in the culture, indicating that differentiation of spermatogonia into primary spermatocytes is mediated by Sertoli cells.     

Damaging effect of antibiotics on the structure of synaptonemal complexes of meiotic chromosome of mice

The structure of synaptonemal complexes (SCs) of chromosomes of mouse primary spermatocytes were studied using electron microscopy on days 1, 10, and 36 after the completion of per os administration of drugs belonging to three groups of antibiotics: tetracyclins, macrolides, and fluoroquinolones. The antibiotics were administered to mice during ten days. At the substages of early and middle pachytene, heteromorphic SC bivalents and fragments of chromosome-core elements were detected in spermatocytes at all times studied after the administration of the antibiotics of three groups. As cells passed through the period from early to middle pachytene, the number of cells containing heteromorphic SC bivalents and the fragments of chromosome cores gradually decreased, which could be an indication of selection of cells with chromosomal aberrations. A high level of associations between the X chromosome and autosome bivalents (including heteromorphic ones) also favors this suggestion. A gradual decrease in the number of chromosomal aberrations was detected, as time elapsed from the completion of antibiotics administration. The study of sperm obtained from epididymises of males did not reveal significant differences in both morphology and motility of sperm between males of the control and experimental groups.