Diplotene Stage Research Articles

Cytology of Puccinia Ruelliae

The teliospores of Puccinia ruelliae germinate readily at room temperature (25 2 C) without a rest period. The nuclear behavior in the basidium and basidiospore was studied under the light microscope. The diploid nucleus divides meiotically in the basidium showing leptotene, pachytene, diplotene and diakinesis stages. The haploid chromosome complement of the species is 5 (n = 5). A typical mitotic division in the haploid nucleus of basidiospores is also discernible.

Cytology of Puccinia ruelliae

SUMMARYThe teliospores of Puccinia ruelliae germinate readily at room temperature (25 ± 2 C) without a rest period. The nuclear behavior in the basidium and basidiospore was studied under the light microscope. The diploid nucleus divides meiotically in the basidium showing leptotene, pachytene, diplotene and diakinesis stages. The haploid chromosome complement of the species is 5 (n = 5). A typical mitotic division in the haploid nucleus of basidiospores is also discernible.

Development and Cytology of Anixiella endodonta

The homothallic fungus Anixiella endodonta exhibits typical sordariaceous centrum development. The ascocarp begins development as a coiled or looped ascogonium that becomes surrounded by hyphal branches. The surrounding hyphae produce the ascocarp wall and all the nonascogenous elements of the centrum. A cavity forms within the centrum. Paraphyses grow upward into the cavity prior to ascus formation and disappear as the asci enlarge. Crozier formation, ascus production, and cytology within the ascus follow the typical pattern. A diffuse diplotene stage is included. Unusually large spindle pole bodies are striking elements of the interphase II nucleus. The haploid chromosome number is six. The development and cytology of A. endodonia is compared with that of previously studied species of Anixiella and Gelasinospora. Similarities and differences indicate that the two genera are clearly related but are sufficiently distinct to warrant segregation.

Nucleolar organizers in human oocytes at meiotic prophase I, studied by the silver-NOR method and electron microscopy.

Use of the silver-NOR method to study the nucleolar organizers in human oocytes demonstrates that topographic and quantitative variations occur during meiotic prophase. In the oogonia nucleolus the nucleolar organizers are dispersed, whereas beginning at leptotene and throughout the remaining stages of meiotic prophase they occupy a marginal position in the nucleolus. At leptotene, a modal number of seven nucleolar organizers can be observed, whereas this number falls to 2.5 at pachytene and rises to ten at diplotene, thus showing that there is intense rRNA synthesis during the latter stage of meiosis. During pachytene, one end of the bivalents containing the ribosomal cistrons is always associated with the Ag-positive zone of the nucleolus. Observation of pachytene in the electron microscope shows that the secondary constriction region of D and G bivalents is constantly associated with the fibrillar center of the nucleolus. Comparison of these two methods of investigation reveals that the silver-stained regions of the nucleolus correspond to the fibrillar centers. The latter are surrounded by a layer of electron-dense fibrils corresponding to the zone of rDNA transcription. This electron-dense layer is absent during pachytene when the nucleolus displays spontaneous segregation of its components; this absence is related to temporary arrest of rDNA transcription. The affinity of the fibrillar centers for silver-NOR staining confirms that these structures contain ribosomal cistrons. During the diplotene stage, numerous micronucleoli are formed outside the nucleolar organizers of D and G chromosomes. Most of these micronucleoli present an Ag-positive granule on one of their margins, thus indicating that they contain an actively transcribed sequence of rDNA. This observation confirms the existence of amplification of ribosomal genes in the human oocyte.

Chromosome condensation and Hoechst 33258 fluorescence in meiotic chromosomes of the grasshopper Spathosternum prasiniferum (Walker)

Patterns of Hoechst 33258 fluorescence have been studied in grasshopper chromosomes. At metaphase of mitotic as well as meiotic divisions — when chromosomes were maximally compact — all the chromosomes fluoresced brightly but no differentially fluorescing regions were detected. However, when all the chromosomes, except the X, were highly extended at pachytene and diplotene stages a distinct differential fluorescence was observed: only the centromeres of the autosomal bivalents fluoresced brightly whereas the entire X univalent showed bright fluorescence. Restriction of differentially bright fluorescence to the more condensed regions of chromosomes suggests a modulatory role for chromosome condensation in the production of Hoechst fluorescence. This suggestion was further strengthened by the substantial quenching of fluorescence caused by removal of chromosomal proteins following treatment with H2SO4. Similarly, post-C-band-treatment staining with Hoechst also led to quenching, though now the centromeres of the chromosomes, including the X, retained their differential fluorescence. It is proposed, therefore, that in grasshopper chromosomes, H-fluorescence is modulated by chromosome condensation brought about by differential ratios of DNA/protein at different chromosome regions and at different division stages.

A sequential analysis of meiosis in the male mouse using a restricted spermatocyte population obtained by a hydroxyurea/triaziquone treatment

A method is described to restrict the spermatocyte population in mice and other rodents using hydroxyurea (HU) and triaziquone (T). HU affects cells in S-phase, whereas T is an agent especially active on spermatogonia and not on spermatocytes. An application of three i.p. HU injections with 12 h intervals, followed about nine days later by one i.p. T injection creates two large gaps in the spermatogenic line. The two gaps enclose a small, well-defined group of primary spermatocytes in meiotic interphase. - The development of the restricted spermatocyte population is followed day by day. The analysis of meiosis in male mice has revealed the correct sequence of meiotic, and especially prophase I stages. On account of clearly visible differences in chromosome morphology the diplotene stage could be divided into three periods. It is suggested to use the following nomenclature: pre-diffuse diplotene, diffuse diplotene and post-difuse diplotene. The experiment was also informative about the timing of the stages in spermatocyte development by correlating the days at which the successive stages were observed with the corresponding stage of the epithelial cycle. The calculation of the position and duration of the diffuse diplotene, enables us to put forward a proposal about the significance of the diffuse diplotene. - A combination of the HU/T method with cell separation techniques provides good perspectives for detailed biochemical studies on processes taking place during meiosis.

Intrafollicular control of oocyte maturation in the PIG

The mammalian oocyte becomes arrested at the diplotene stage of the first meiotic division during prenatal or early postnatal life. It remains arrested in meiosis until shortly before ovulation when the surge of gonadotropin induces resumption and completion of meiosis to the metaphase II stage. When oocytes are harvested from medium-sized or large follicles of pig and other species and cultured, they resume meiosis spontaneously indicating that the follicles exert an inhibitory influence on meiosis. To analyze the control of meiosis by follicular components, culture of isolated pig oocytes in the presence of follicular cells or follicular fluid (FF1) has been used as a model in this laboratory. An oocyte maturation inhibitor (OMI) has been isolated and partially purified by ultrafiltration and gel chromatography of FF1 and shown to be a polypetide with a molecular weight in the order of 2000 daltons. Physiological characterization has shown that the effect of OMI in vitro is reversible and that it can be overcome by luteinizing hormone (LH). The action of OMI requires the presence of cumulus cells surrounding the oocyte since it was found that denuded oocytes, stripped of cumulus cells, do not respond to OMI. Furthermore, when cumulus-enclosed oocytes were cultured, OMI inhibited the differentiation of the cumulus cells in terms of morphology and progesterone secretion in a dose-related manner. The inhibition of cumulus differentiation by OMI was reversible and could be overcome by LH. The results indicate that the effect of partially purified OMI upon meiosis may be mediated by the cumulus cells.

Method for the Preparation of Active Maturation Promoting Factor (MPF) from in vitro Matured Oocytes of Xenopus laevis

A method for the large scale extraction of Maturation Promoting Factor (MPF) from in vitro matured oocytes of Xenopus laevis is described. MPF has been previously described only as a component(s) of hormone-matured cytoplasm within amphibian oocytes (or eggs) which is able to induce the reinitiation of the meiotic process from late diplotene stage until second metaphase arrest, when microinjected into diplotene arrested (fully grown) recipient oocytes. Standard biochemical methods for the extraction and purification of this factor(s) haven been unsuccessful due to its extreme instability and sensitivity to dilution. The procedure is dependent upon the inclusion of sodium fluoride (NaF) in the extraction medium with its effect presumably due to its ability to inhibit phosphorprotein phosphatases. The successful preservation of MPF activity described in this report permits further attempts to be made to isolate and characterize this, to date, elusive cytoplasmic factor, which plays a key role in the complex cellular processes involved in the hormone-dependent differentiation of an oocyte into an egg.

Development and Cytology of Zopfia rhizophila

Zopfia rhizophila exhibits a modified Pleospora type of centrum development. The ascocarp begins development as a small stroma that differentiates into a dark cephalothecoid peridium and an internal mass of pseudoparenchyma. Within the internal pseudoparenchyma, some cells disintegrate. Others engender pseudoparaphyses, and still others give rise to ascogenous cells that produce ascogenous hyphae. Ascogonia are not formed. Croziers arise on the ascogenous hyphae. Ascus production and cytology follow the usual pattern and include a diffuse diplotene stage. The haploid chromosome number is 19±1. Ascospores are uninucleate when first formed and become multinucleate, centrally divided, and constricted. Later each cell cuts off a small uninucleate terminal cell. Spores are released by digestion of the ascus wall and are liberated when the peridium breaks along the sutures between cephalothecoid plates. Germination occurs from either end of the spore or through a break in the wall. The fungus is homothallic because it produces ascocarps from initially uninucleate single spores.

Localization of ribosomal cistrons in the quail oocyte during meiotic prophase I

The nucleolar organizers have been localized in the Japanese quail oocyte at pachytene and diplotene stages using hybridization in situ with rRNA. Autoradiography reveals that 8 microchromosomes, forming 4 bivalents at pachytene, contain ribosomal cistrons. The silver grains are located on the microchromosomes euchrpmatic segment, near the centromeric heterochromatin inserted in a chromocenter. This localization coincides with that of the nucleolar fibrillar center as revealed by electron microscopy. At advanced diplotene the silver grains, whose number is increased, are located over the central area of the nucleolus. This situation corresponds to that of the fibrillar centers which multiply at advanced diplotene and move to the central part of the nucleolus. Comparison of pachytene and diplotene grain counts suggests that moderate amplification of the ribosomal cistrons might take place during oogenesis in the quail.

Extensive pairing of the XY bivalent in mouse spermatocytes as visualized by whole-mount electron microscopy.

Autosomes and sex chromosomes of mouse spermatocytes were examined during zygotene, pachytene, and diplotene by a whole-mount electron-microscope technique after cell dispersion in a detergent solution (Nonidet-P40). Zygotene, pachytene, and diplotene stages can be adequately identified in the preparations. Thus, asynchronous side-by-side pairing of homologous autosomes, some of them displaying attached nucleoli, defines zygotene. Pachytene is identified by complete pairing of homologues. Diplotene is characterized by disjunction of bivalents (autosomes and sex chromosomes), lack of autosomal-attached nucleoli, divergent expansions observed at lateral element endings of disassembled synaptonemal complexes, end-to-end association of the XY pair and well defined outward deformations ('bulges') along sex chromosomal axial cores. X and Y chromosomes display at pachytene an extensive side-by-side pairing segment which decreases in length as meiotic prophase advances. Each sex chromosomal axial core appears double and is formed by close apposition of 2 nearly parallel elements displayed separately along the entire length of the chromosomal core. This double structural feature suggests that each sex chromosomal axial core is presumably composed of 2 chromatid axial cores, each of which, in turn, constitutes the respective lateral elements of short synaptonemal complexes observed at the unpaired segment.

Dissociation of Preovulatory Maturational Events in Rat Oocytes and Cumuli in the Presence of Dibutyryl Cyclic AMP

Mammalian oocytes enter the first meiotic division during pre‐natal life. Shortly thereafter the oocytes become arrested at the diplotene (dictyate) stage of the first meiotic division and they remain arrested until just prior to ovulation. The ovulatory LH‐surge leads to resumption of meiosis and the oocytes of preovulatory follicles undergo nuclear progression from dictyate to metaphase II (oocyte maturation). Although it is generally agreed that LH is responsible for the resumption of meiosis, the cellular mechanism for this hormonal effect remains to be elucidated. The immature oocyte in the follicle is surrounded by specialized granulosa cells comprising the cumulus oophorus. Morphological (Zamboni 1970) as well as biochemical (Biggers et al. 1967, Gwatkin and Andersen 1976) evidences suggest a role of the cumulus cells in the regulation of both oocyte growth and maturation. Following the ovulatory LH‐surge the cumulus surrounding the maturing oocyte is radically changed into a dispersed structure of cells embedded in a mucopolysaccharide matrix (e.g. Austin 1961). In a recent report isolated preovulatory follicles of PMSG‐injected immature rats were studied in an incubation system with chemically defined medium (Hillensjöet al. 1976). When LH was added to the medium the follicle‐enclosed oocyte resumed meiosis and the cumulus was transformed into a dispersed structure which was sensitive to hyaluronidase. The aim of this study was to see whether dibutyryl cyclic AMP (dbcAMP) would mimic the effects of LH on the follicle‐enclosed oocyte itself and on the cumulus oophorus.

Absence of rDNA amplification in the uninucleolate oocyte of the cockroach Blattella germanica (Oorthoptera: Blattidae).

Amplification of the genes coding for ribosomal RNA oocurs in the oocytes of a wide variety of organisms. In oocytes of various species of crickets (Orthoptera: Gryllidae) the amplified DNA is contained in a large extrachromosomal DNA body. Multiple nucleoli form about the periphery of the DNA body during the diplotene stage of meiosis I. In contrast to the general pattern of orthopteran oocytes, oocytes of the cockroach Blattella germanica demonstrate a single large nucleolus instead of many nucleoli. In order to determine whether the genes coding for rRNA are amplified in the oocytes of B. germanica, the relative amount of rDNA in oocytes was compared with the rDNA content of spermatocytes and somatic cells. An extrachromosomal DNA body similar to that present in crickets is not present in B. germanica. A satellite DNA band which contains nucleotide sequences complementary to rRNA accounts for approximately 3-5% of the total DNA in somatic and in male and female gametogenic tissues. Female cells contain approximately twice as much rDNA as do male cells. An XX-XO sex-determining mechanism is operative in B. germanica. In situ hybridization with rRNA indicates that the nucleolar organizer is located on one end of the X chromosome and that oocytes do not contain more than twice the amount of rDNA found in spermato cytes. The data indicate that rDNA is not amplified in the uninucleolate oocyte of B germanica.

Induction of meiosis in fetal mouse testis in vitro

Fetal mouse testes and ovaries with their urogenital connections were cultured singly or in pairs on Nuclepore filters. When a testis in which the sex was not yet morphologically detectable was cultured together with older ovaries containing germ cells which were progressing through the meiotic prophase, the male germ cells were triggered to enter meiosis. When older fetal testes in which the testicular cords have developed were cultured together with ovaries of the same age with germ cells in meiosis, the oocytes were prevented from reaching diplotene stage. It was concluded that the fetal male and female gonads secrete diffusable substances which influence germ cell differentiation. The male gonad secretes a “meiosis-preventing substance” (MPS) which can arrest the female germ cells within the meiotic prophase. The female gonad secretes a “meiosis-inducing substance” (MIS) which can trigger the nondifferentiated male germ cells to enter meiosis.

Cytochemical and Biochemical Analysis of Development ofUrechisOocytes

The echiuroid marine worm Urechis caupo is uniquely suited for the study of oogenesis. A relatively large quantity of oocytes at various developmental stages can be obtained and subjected to coordinated cytochemical and biochemical analysis Oocytes at the cluster, early diplotene, mid-diplotene, and diffuse diplotene or lampbrush stages are active in the synthesis and accumulation of ribosomal RNA, several proteins, carbohydrates, lipids, and also, perhaps, yolk constituents. Only cortical granule formation, which occurs during later stages of oogenesis, appears to be stage specific. Ribosomal RNA genes are also transcribed in the nucleolus of the mature oocytes or unfertilized eggs. However, the rate of production in these eggs appears to be regulated at the level of maturation of rRNA precursor molecules.

Gametangial meiosis in Phytophthora capsici

The report of Stephenson et al. that the nuclear divisions in the gametangia of Phytophthora capsici are mitotic, resembling the divisions in the vegetative hyphae, was not confirmed. Pachytene, diplotene, and diakinesis stages were observed in the oogonia. Metaphase and anaphase were followed by a second division without any increase in nuclear and chromosome size between the two divisions. The haploid chromosome number is nine and an association of four chromosomes is present in one or both parents.The association of four chromosomes observed at diakinesis and metaphase is evidence that pairing of homologous parts occurs during the first division and is thus proof that the gametangial divisions are meiotic.There was no evidence of a prolonged prophase in the first division of the zygote nucleus and an eight-nucleate oospore showed no evidence of reduction in nuclear size or nuclear elimination such as would be required to account for the homokaryotic condition of single-oospore cultures on the hypothesis of zygotic meiosis.The problems involved in obtaining reliable chromosome counts from somatic nuclei are discussed.

1 - Development of the Ovary and Oogenesis

This review of the literature deals with processes ranging from the migration of germ cells from the yolk sac to the germinal ridge, to the atresia of the Graafian follicle. The migration stage is followed by sex differentiation. Whereas oogonia disappear, oocytes develop as far as the diplotene (resting) stage of the meiotic prophase, and only during sexual maturity is meiosis resumed at the diakinesis stage. In the female, the second meiotic division is arrested in the metaphase until fertilization occurs. The factors controlling this are obscure, they may be connected with stimulating factors produced by sex differentiation, by the mesonephros or the rete ovarii; the resumption of meiosis after resting is controlled by gonadotrophins. The origin of the granulosa cells and of the zona pellucida is also discussed; the interdigitation of microvilli growing from the oocyte and from the granulosa cells into the zona pellucida may indicate the transfer of material from the mother to the embryo. For follicular growth, gonadotrophins are needed as a trigger, but then development proceeds wihout them during the preantral stage; in the later stages, however, they are needed permanently. The factors regulating th degeneration of germ cells (atresia) are poorly understood; they may be genetic (e.g., loss of one X chromosome), environmental (loss, or genetic incompatibility of granulosa cells, and gonadotrophins produce atresia, estrogens act in the opposite way), or metabolic. The purpose of atresia is to regulate litter size and to increase the supply of steroid hormones.

Incorporation of tritiated uridine during pachytene and diplotene stages in the oocytes of the Japanese quail (Coturnix coturnix japonica).

Incorporation of 3H-uridine was studied during pachytene and diplotene stages of quail oocytes. No labelling could be detected during early pachytene. During advanced and late pachytene, labelling simultaneously appeared on the macrochromosomes and on certain michromosomes in the zone where they emerge from the chromocentric surface periphery. The latter localization corresponds to the region of ribosomal RNA synthesis. At diplotene the same localizations were labelled with a considerably increased intensity.

Development and Cytology of Sordaria Humana

A coiled, septate, multinucleate ascogonium or its parent hypha produces branches that enfold the ascogonium and produce a small ball of cells with the ascogonium in the center. The outer layers of cells differentiate into the perithecial wall. As the radius of the perithecium doubles, the number of wall cells increases twoto threefold per section and each cell expands sufficiently in volume to maintain the spherical shape of the perithecium. Plectenchyma cells of the centrum produce paraphyses which grow upward into a subapical cavity that is present before the paraphyses originate. Above the cavity a dome-shaped region of meristematic cells produces the neck, ostiole, and periphyses. The ascogonium does not increase in size when the perithecium enlarges. Ascogenous hyphae develop from the ascogonium after the neck and ostiole and paraphyses have begun to form. Terminal branches of ascogenous hyphae produce croziers, which in turn produce asci. Cytology of the ascus is much like that in other pyrenomycetes and includes a diffuse diplotene stage. Spores are initially uninucleate, later binucleate. The fungus is homothallic. The haploid chromosome number is seven.

Heterochromatin and nucleolar organizers during first meiotic prophase in quail oocytes

The study of chromosomes in oocytes of the quail shows, at the pachytene stage, that microchromosomes are made of a euchromatic segment and a heterochromatic juxtacentromeric region. The heterochromatic regions of the microchromosomes amalgamate between themselves so as to constitute bulky chromocentres from which radiate the euchromatic segments which remain free. At late pachytene, nucleoli appear at the contact of these chromocentres. When the oocytes reach the diplotene stage, the nucleoli become quite large. They are stuck against chromocentres and establish a very close relationship with the euchromatic segments of the microchromosomes which surround or penetrate them. These observations lead one to think that the euchromatic segments of microchromosomes could be bearing nucleolar organizers. The close relations that the nucleolar organizers develop with the bulk of the nucleolus could explain its Feulgen-positive character in the quail.