Diplotene Stage Research Articles

Microtubule Organization, Acetylation, and Nucleation in Xenopus laevis Oocytes: II. A Developmental Transition in Microtubule Organization during Early Diplotene

Confocal immunofluorescence microscopy of ovaries from juvenile frogs revealed changes in the organization, acetylation, and nucleation, of microtubules (MTs), and redistribution of γ-tubulin (γ-TB), during early oogenesis in Xenopus laevis. Interphase oogonia contained sparse, radially organized, MT arrays and prominent centrosomes. Acetylated MTs were not commonly found in oogonia. In contrast, small (∼12-25 μm), postmitotic (stage 0) oocytes contained dense, highly polarized, MT networks that exhibited little or no evidence of radial organization. Examination of stage 0 oocytes stained with antibodies to γ-TB, in conjunction with assays of MT nucleation activity, revealed that stage 0 oocytes do contain active centrosomes. In addition, stage 0 oocytes contained numerous acetylated MTs, suggesting that arrest in meiotic prophase is accompanied by MT stabilization. Early stage I oocytes (diameters from ∼35-50 μm) exhibited a rounded morphology and contained a dispersed, apparently disordered, MT array with a substantial population of acetylated MTs. Examination of stage I oocytes stained with γ-TB antibodies revealed that this centrosomal protein was present in multiple cytoplasmic foci which did not function as MTOCs following cold-induced MT disassembly. The results presented indicate that the maternal centrosome is inactivated during early stage I, roughly coincident with the onset of the diplotene stage of meiotic prophase and prior to assembly of the mitochondrial mass. Our observations place constraints on the role of MTs and the maternal centrosome during specification of the animal-vegetal axis of Xenopus oocytes and raise questions regarding the mechanisms by which MT assembly and organization are regulated during oocyte differentiation.

Spermatogenesis in the urodele Amhystoma dumerilii.

The male reproductive cycle of this paedomorphic species that occurs only in Lake Pátzcuaro, Michoacán, México, was investigated by documenting changes in germinal cells during the spermatogenic cycle. Cysts of germ cells divide synchronously to complete spermatogenesis during September through December, with the proportion of evacuated cysts or cysts containing spermatozoa increasing during this period. The chromatin changes during prophase I of meiosis reveal the usual leptotene, zygotene, pachytene, and diplotene stages. A basal body at the caudal end of the spermatozoan head connects to the flagellum. After spermiation, empty cysts contain a granular substance. Spermatogenesis in this species follows an annual cycle like other north temperate salamanders, rather than the continuous spermatogenesis of some tropical salamanders. © 1994 Wiley-Liss, Inc.

Cell–cell interaction and oocyte growth

In most mammals, oocytes initiate meiosis in late fetal life; by the time of birthe they have already entered the diplotene stage of prophase I of meiosis and becaome arrested thereafter at the dictyate state(Baker, 1972). At this stage they became surrounded by a few nonproliferating flat follicle cells forming a unit called the resting or primordial follicle.

A balanced autosomal reciprocal translocation in an azoospermic bull

This appears to be the first reported case of a bull with a balanced autosomal reciprocal translocation associated with azoospermia. The analysis includes somatic chromosome banding, conventional meiotic analysis, and electron microscopy of synaptonemal complexes (SCs). The karyotype of the bull was found to be 60,XY,rcp(8;13)(q11;q24). Electron microscopy of SCs in microspread pachytene spermatocytes revealed a high incidence of terminal asynapsis of the smallest arm of the quadrivalent. Most quadrivalents with such asynapsis and few with nonhomologous synapsis showed associations with the XY sex bivalent, leading to complete meiotic arrest at late pachynema. Except for one diakinetic cell, no diplotene and subsequent stages were encountered in air-dried meiotic preparations. The presence of degenerating primary spermatocytes in SC preparations, as well as in testicular sections, and the absence of spermatozoa in ejaculates confirm the chromosomally derived male sterility of the bull. X-chromosome reactivation, evidenced by the cytomorphological reversal of associated sex bivalents, appeared to be the initial step in the degeneration of spermatocytes. Consequently, the formation of a separate, fully developed XY body, which was previously demonstrated on the periphery of spermatocyte nuclei in fertile bulls, could not be attained in this case. Extensive end-to-end association of autosomal bivalents in meiotically arrested, as well as degenerating, spermatocytes was a consistent and unique observation of this study. Such associations may lead to enhanced reactivation of the X chromosome.

Ultrastructural evidence for a triple structure of the lateral element of the synaptonemal complex

This study describes composition and localization of several substructures of the synaptonemal complex (SC) using different techniques. The techniques which were used were surface spreading, critical point drying of isolated SCs, and sectioning of Lowicryl embedded testis material. The lateral elements (LEs) of the SC appear to be composed of three lateral substructures: two morphologically identical major strands and a third strand which is considerably thinner. The thinner strand is localized on the inner side of the two major strands of the lateral element. In late pachytene/early diplotene stages when the SC starts to desintegrate more than three strands can be observed in the LEs. A model is presented and the function of the different substructures is speculated upon.

Sexually differentiated mechanisms of sterility in interspecific hybrids between Oryzias latipes and O. curvinotus.

Fertility of interspecific hybrids between Oryzias latipes and O. curvinotus was examined. F1 females were able to lay eggs but males were sterile. Histological examination of the ovaries of hybrids revealed that oogenesis does not proceed normally in spite of the apparent fertility. Most oocytes degenerated at the pachytene stage of the meiotic prophase, and only a few entered the diplotene stage to develop into ova. Hybrid males could induce females to spawn eggs, an indication that they had differentiated completely into true males. However, they did not produce fertile sperm. Most germ cells in testes of hybrids passes through almost the entire process of spermatogenesis, but deviations from the normal course of events were observed during spermiogenesis. The condensation of chromatin in spermatids occurred, but the diameters of sperm heads were about 1.5-fold larger than those of normal ones. Prominent abnormalities were apparent in the quantity and arrangement of microtubules in the cytoplasm. Abnormal spermatozoa were phagocytized by Sertoli cells. These observations indicate that the mechanisms of impaired gametogenesis in these interspecific hybrids are sexually differentiated.

REVIEW The genetic control and germ cell kinetics of the female and male germ line in mammals including man*

The female germ line (germ cell lineage, Keimbahn) is provided with only one proliferation wave, the oogenic, whereas male gametogenesis involves two successive waves: prespermatogenic, which corresponds to the female proliferation wave, and spermatogenesis, which is responsible for the immense number of male gametes produced in mature testes. Both male proliferation systems are linked by the transitional or T prospermatogonia. Using the reverse percentage of labelled metaphases method, it has been shown that the first differences between female and male germ cells can be identified by the end of the first wave, when oogonia and multiplying or M prospermatogonia are proliferating. This prenatal first wave of proliferation of male germ cells was also demonstrated in man and ceases around the 22nd week of pregnancy. Spermatogenesis involves a stock of stem cells (stem spermatogonia), a flexibly reacting pool of undifferentiated spermatogonia and several generations of differentiating spermatogonia, which proliferate almost exponentially. Furthermore, it consists of spermatocytes and haploid spermatids transforming into spermatozoa. The oocytes pass through the preleptotene stage, synthesizing DNA, and thereafter traverse the meiotic prophase up to the diplotene stage. In mammals they act as 'pre-embryos' in a similar but to a lesser degree than oocytes of amphibia and insects. The maternal chromosomes are largely responsible for the development of the embryo, the paternal genome for the development of the extra-embryonic tissue. The synthesis of transgenic animals is a powerful weapon in the armoury of geneticists, as has recently been demonstrated: a 14 kb genomic DNA fragment (Sry) is sufficient to induce testis differentiation and subsequent male development when introduced into chromosomally female mouse embryos.

The Expression of c-kit Protein during Oogenesis and Early Embryonic Development1

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor and was shown to be allelic with the white-spotting locus (W) of the mouse. Mutations at the W locus have pleiotropic effects on the development of hematopoietic stem cells, melanoblasts, and primordial germ cells. In order to elucidate the role of c-kit protein in gametogenesis and oocyte maturation, we have examined immunohistochemically the expression of c-kit in the ovaries of mice at late fetal and postnatal stages, and in early embryos. By the avidin-biotin-peroxidase (ABC) method using rat anti-mouse c-kit monoclonal antibody, the c-kit protein was detected in ovaries after the time of birth, but not before. The expression of c-kit was observed mainly on the surface of oocytes, but not in granulosa cells nor in interstitial regions. Oocytes of primordial to fully grown Graafian follicles showed the c-kit protein. When ovulation was induced by hCG, the expression of c-kit in ovulated unfertilized oocytes was weaker than in oocytes of Graafian follicles. In 1-cell embryos the c-kit protein was still observed, but with cell division its expression further decreased, and it was not detected in embryos of 4-cell, 8-cell, and morula stages. In summary, the highest expression of c-kit was observed on the surface of oocytes arrested in the diplotene stage of meiotic prophase. With ovulation and the resumption of meiotic maturation, its expression declined. These results suggest that the c-kit protein may play some role in meiotic arrest, oocyte growth, and oocyte maturation.

Unique State of Sexual Dimorphism of Crossing-Over in Diplotene Spermatocytes and Oocytes of Mesocricetus Brandti, a Species with Neonatal Oogenesis1

Sexual dimorphism of recombination has been held by classic genetic theory to disfavor the heterogametic sex. Assessment of chiasma frequencies at the diplotene stage of meiosis has been used as a valid measure of this concept and in many species has revealed, as expected, an increased frequency in female vs. male germ cells. Mesocricetus brandti, a species currently used in investigations of gonadal regression, photoperiods, and hibernation, was found by this measure to be an exception to this rule, with an average of 29.81 chiasmata in spermatocytes and 23.16 in oocytes. Sites of crossing-over unique to each sex were also detected. Oogenesis occurs in this species as a postnatal phenomenon and is suggested as a possible critical factor for the exceptional recombinant behavior.

Regulation of oocyte number during oocyte differentiation in the lizard Podarcis sicula

This paper concerns the differentiation process of germ cells from oogonia to primary follicles in the lizard Podarcis sicula. The study was carried out at the morphological level and using a cytophotometric analysis for determining the number of differentiating germ cells undergoing degeneration. The progressive disorganization of the germ cell clusters during the early diplotene stage and the role played by the prefollicular cells during this process are described. Oocyte degeneration has been observed between the mid-zygotene and the early diplotene stages. When the primary follicle (oocyte plus follicular cells) is being formed, the degeneration process stops and the oocyte undergoes regular growth and ovulation.

Synaptonemal complex formation in rye (Secale cereale) heterozygous for telomeric C-bands

Zygotene–pachytene nuclei from a rye line heterozygous for most terminal C-bands were found by electron microscopic spreading analysis to have unequal lateral elements at most synaptonemal complex telomeres. An unpaired lateral element protruded beyond the end of the synaptonemal complex at each such telomere. Another rye line with only one C-band heterozygous telomere had only one uneven synaptonemal complex telomere. The length differences between paired lateral elements in heterozygotes (both total complement and individual synaptonemal complexes) was considerably less than the difference in DNA content or somatic metaphase chromosome size between C-band positive and C-band negative lines. There was no evidence of a synaptic adjustment effect reducing the telomere length differences in later pachytene–diplotene stage nuclei. Heterozygosity for the 1RL terminal C-band resulted in a slight reduction in chiasma frequency in that arm but no shift in chiasma position.Key words: rye, C-bands, telomeres, chromosome pairing, synaptonemal complex.

Synaptonemal complex transformations in rye microsporocytes at the diplotene stage of meiosis

The process of synaptonemal complex degradation during diplotene was studied in spreads of rye microsporocytes stained with silver nitrate at pH 3.5–4.5 and 6.0–8.0. Two different patterns of the synaptonemal complex degradation process have been observed, depending on the two staining procedures used. Progressive synaptonemal complex fragmentation observable at the higher pH appeared to be absent in staining at pH 3.5–4.5: thin connecting threads have been found in the "gaps" between the synaptonemal complex segments. Complete tracing of the synaptonemal complex degradation process was attempted and revealed the following successive steps: (i) local repulsion of lateral elements; (ii) lateral element looping in the regions of repulsion; (iii) extension of the loops; (iv) transformation of the extended loops into coils of irregular shape with a diameter of about 2 μm and a pitch of about 1.2 μm; and (v) formation of paired beanlike thickenings on a gyral coil. In asynaptic mutant sy-9 unpaired lateral elements are transformed without looping into a similar coil but with single beanlike thickenings. We conclude that synaptonemal complex lateral element loops at diplotene are invisible after the routine silver staining of microspreads (at pH about 6 and higher) and look like gaps between discontinuous synaptonemal complex segments, thus simulating the process of synaptonemal complex fragmentation.Key words: synaptonemal complex, diplotene, rye.

Paragonimus ohirai: Identification of nucleolar organizer regions (NORs) and silver nitrate staining pattern in spermatogenesis

The identification of nucleolus organizer regions (NORs) and the qualitative description of silver nitrate staining patterns in spermatogenesis of Paragonimus ohirai are herein reported. The NORs of P. ohirai are located on the short arm of chromosome 4 and are associated with a large amount of constitutive heterochromatin. Silver (Ag) staining was detected from the interphase stage until the prometaphase stage of mitosis in spermatogonial cells but was absent at metaphase. On the other hand, during meiosis Ag-staining of NORs occurred from prophase to the diplotene stage, then was absent from meiotic metaphase I to anaphase II and was again demonstrable in early spermatid nuclei. The appearance and disappearance of NOR during Paragonimus spermatogenesis is similar to that described for vertebrate species except during mitotic metaphase.

Ultrastructural Observations on the Origin and Differentiation of Somatic Cells during Gonadal Development in the Frog Rana nigromaculata: (frog/gonadal development/sex differentiation).

The process of gonad development in the frog Rana nigromaculata was observed using the electron microscope. The gonadal medulla was formed by the proliferation and displacement of the epithelial cells within the primordial gonad, and a distinct continuity was observed between the cortical and medullary cells. Sex differentiation of the gonad occurred directly from the sexually indifferent primordial gonads. In the rudimentary testes, the continuity between the cortical and medullary regions increased closer, and the intermingling of cortical and medullary cells was evident. The inner region of the cortex developed into a cord-like structure and subsequently differentiated into rudimentary seminiferous tubules. The medulla differentiated into the testicular rete and efferent duct. In the rudimentary ovaries, the cortex and medulla were separated and the ovarian cavity was formed in the medullary region. In the cortex, the cortical cells surrounding oocytes which had reached the diplotene stage, differentiated into follicular cells. The intrusion of mesenchymal or blastemal cells derived from extragonadal regions into the cortex or medulla was never observed. These findings do not support Witschi's cortico-medullary antagonistic theory of sex differentiation.

Presence and distribution of specific prosome antigens change as a function of embryonic development and tissue-type differentiation in Pleurodeles waltl.

The prosomes, biochemically well characterized small RNA-protein complexes, found associated with mRNA in all eukaryotic cells tested, have been identified as maternal components in sea urchin and chick embryos. In this study, we investigated their presence and cytolocalization in the oocytes and embryos of Pleurodeles waltl by immunoblot analysis and immunofluorescence, using monoclonal antibodies prepared against duck prosome proteins. Of the four antibodies tested, three recognized the corresponding antigens in oocyte total protein extracts. Immunofluorescence analysis, using the three prosomal antibodies, demonstrated a drastic change in the localization of the prosome antigens, which changed from the cytoplasm to the nucleus during oogenesis. In the nucleus, in diplotene stages, prosomal antigens appeared to be associated with the lampbrush chromosomes and the nuclear matrix. During embryogenesis, the subcellular distribution of the prosome antigens was a function of development and differentiation: in the cleavage stages up to the mid-blastula they were localized in the cytoplasm and on the plasma membrane, while in the late blastula, gastrula and neurula they were in the nucleus. Interestingly, one of the prosome antigens, p31K, was found to be in a different location in certain cells in the animal pole of the mid-blastula and was absent in the neural tissue in the neurula. In still later stages, in the free-swimming larva, all three antigens were localized in the cytoplasm, specifically in certain cell types in the epidermal tissues. Furthermore, they were sectorially distributed in the cytoplasm. These data taken together indicate the possible presence of tissue-type-specific prosome antigens in Pleurodeles. Differentiation-dependent subcellular localization of the prosome antigens suggests a cell-compartment-related multiple function of prosomes.

An autoradiographic and cytophotometric study of oogenesis in a pulmonate snail, Planorbarius corneus

The spatial and temporal patterns of macromolecular syntheses in oocytes and somatic auxiliary cells of the snail Planorbarius corneus have been investigated by autoradiography and cytophotometry. Oogenesis has been divided into three stages, comprising early meiosis up to diplotene (stage I), previtellogenetic growth phase (stage II), and vitellogenesis (stage III). No DNA synthesis was found in any oocyte stage. In stage-I oocytes, only nucleoli were found labelled with 3H-uridine. Oocyte nuclei of stage II and III actively synthesize RNA in nucleoli and chromosomes. The most intense incorporation of uridine in chromatin probably occurs during the previtellogenesis — vitellogenesis transition period during which cytological findings suggest well developed lampbrush chromosomes. RNA synthesis in amphinucleoli of stage-III oocytes is restricted to basophilic nucleolar parts, whereas acidophilic parts (protein bodies) neither synthesize nor store RNA. During vitellogenesis oocytes incorporate amino acids into yolk platelet proteins. Radioactive proteins are found in yolk platelet precursors 5 h after injection of the tracer and in yolk platelets 3 h thereafter. The labelling pattern suggests that oocytes synthesize certain hitherto unidentified yolk components. No evidence for the participation of follicle cells in synthesis and transport of vitellogenic proteins has been obtained from autoradiography. Cytological findings suggest an important role for these cells in oogenesis. They are highly active in RNA and protein synthesis. Cellular differentiation is accompanied by polyploidization of the nuclei which attain a highest DNA content of 256 c. Polyploidization probably occurs in incremental steps as indicated by complete endomitotic chromosomal cycles. Autoradiographs show that, during vitellogenesis, oocytes do not incorporate significant amounts of glucose, and only certain follicle cells were labelled with glucose, probably indicating the synthesis of glycogen.

Evidence for the sexual bipotentiality of spermatogonia in the fish, Oryzias latipes.

The origin of the oocyte-like cells (testis-ova) induced by the administration of estradiol in the testis of the fish, Oryzias latipes, was examined by light and electron microscopy. A small number of testis-ova at the zygotene stage was seen on the sixth day after the beginning of treatment with estradiol, and on the 12th day testis-ova at the pachytene and diplotene stages were observed. Ultrastructural observations revealed that the testis-ova at the zygotene stage were organized as cysts and that all testis-ova within a given cyst developed synchronously. The number of these testis-ova in each cyst varied but cysts containing 16-20 or 26-30 testis-ova were most frequently observed. Spermatogonia of O. latipes are classified morphologically into two types: the type A and the type B spermatogonia (Grier, '76 Cell Tissue Res., 168:419-431). Type A spermatogonia, which were separated from each other by Sertoli cells, were of the stem type and type B spermatogonia appeared to divide clonally 9-10 times in each cyst before entering meiosis. The present observations on zygotene testis-ova indicate that the testis-ova originate from type B spermatogonia which have divided clonally four or five times, suggesting that male germ cells remain sexually bipotent through the early stages of development of type B spermatogonia.

Studies on manifold abnormalities at meiosis in Petunia (2n=14).

Anomalus meiotic behaviour was recorded in two plants of P. axillaris (Lam.) B.S.P. (2n=14). Meiotic irregularity was associated with diploid chromosome number in one plant, while in the other, chromosome instability and aneuploidy prevailed. Abnormalities such as persistent stickiness of chromosomes from late diplotene stage onwards, diffused arrangement of chromosomes at first metaphase, multipolar spindle abnormality, extreme variability in size of pollen mother cells and pollen grains, and syncyte formation were recorded. Chromosome stickiness observed in one category was attributed to a recessive gene, the expression of which was influenced by temperature, while variation in chromosome number and multiple meiotic anomalies recorded in the other may be due to spindle abnormalities at premeiotic mitotic cell divisions and at meiosis I.

Studies on radiation-induced chromosome aberrations in mouse spermatocytes I. Stage specificity and dose-response relationships of chromosome aberrations induced in mouse primary spermatocytes following X-irradiation

The cytological analysis of chromosome aberrations induced at diplotene, mid-pachytene, zygotene and leptotene stages following X-irradiation was performed at diakinesis-metaphase I in mouse spermatocytes. The dose-response relationships fitted well to linear equations for deletion-type aberrations at each stage, and to linear-quadratic equations for exchange-type aberrations at all stages except for leptotene. The radiosensitivity to chromosome aberration induction tended to increase gradually with progression through synaptic and post-synaptic stages, diplotene being the most sensitive. Chromatid exchanges were hardly observed at leptotene, the aberrations being mainly isochromatid fragments. On the contrary, chromatid exchanges and isochromatid deletions were mainly observed at later stages (zygotene-diplotene). The specificity of chromosome aberration induction in primary spermatocytes might be influenced by chromatin organization and chromosomal configuration peculiar to meiotic cells.

Biochemical Transmitters Regulating the Arrest and Resumption of Meiosis in Oocytes

Publisher Summary This chapter discusses biochemical transmitters regulating the arrest and resumption of meiosis in oocytes. Germ cells migrate to the genital ridge from the yolk sac region during early embryonic development. In the genital ridge, the female germ cells start to divide and differentiate into oogonia. They enter meiosis and become primary oocytes. Nuclear division progresses to the diplotene stage of the first meiotic prophase and is arrested. The chromosomes decondense and are distributed diffusely throughout the oocyte nucleus. Progression of meiosis to the diplotene stage occurs before or shortly after birth. The oocytes may remain arrested at the dictyate stage for a prolonged period. Subsequently, a follicle develops enclosing the oocyte that contains a large clear nucleus designated as the germinal vesicle. The resumption of meiosis follows a sequence of programmed events while the oocytes are situated within the preovulatory follicles. The process is designated as oocyte maturation and is characterized by a series of biochemical, morphological, and functional changes that take place within the nucleus, highlighted by various events: (1) dissolution of the nuclear membranes manifested as germinal vesicle breakdown (GVBD), (2) chromatin condensation and the formation of distinct chromosomes, (3) formation of the first meiotic spindle, (4) translocation of the spindle to the peripheral region, (5) formation and extrusion of the first polar body, (6) formation and positioning of the second meiotic division, (7) rearrest at the second metaphase.