Commitment of Mitotic Cells to Meiosis during the G2 Phase of Premeiosis

In the developing anther, archesporial cells that proliferateby mitotic division are converted into meiotic cells duringthe premeiotic interphase. Experiments with explanted microsporocytesof Lilium and Trillium were made to obtain evidence for theconversion of mitotic to meiotic cells during the premeioticperiod. Explanted premeiotic cells were cultured through thedivision cycle at relatively high division frequencies and showeda variety of division types with respect to chromosomal events.The type of division depended on the premeiotic stage at whichthe cells were explanted. Cells in the G₁, S and early G² phasesunderwent mitotic division and formed a diad or binucleate monad.Cells explanted at the late G₂ phase were cultured throughoutthe normal meiotic cycle, which resulted in typical tetrad configuration. In microsporocytes explanted during the main part of the G₂interval, centromere behavior was meiotic, but chromosome pairingand chiasma formation were disturbed. Thus, she G₂ intervalwas shown to be critical for the commitment of mitotic cellsto meiotic division. Detailed analysis showed that the intracellularchanges that commit the cells to meiosis begin shortly aftercompletion of premeiotic DNA synthesis and that these changesare progressive and cumulative. (Received February 2, 1982; Accepted May 24, 1982)     

Studies on microspore development in liliaceous plants II. The behavior of explanted microspores of the lily, Lilium longiflorum

Uninucleate microspores of Lilium longiflorum from differentlengths of buds were explanted under various culture conditions,and their behavior was studied during subsequent cultures. Thenutritional conditions permitting survival of microspores wererelatively simple, but most of the living cells showed cytologicalabnormalities. A typical type of cell division could only beinduced in explanted microspores at the late G₁ phase of thecell cycle. For the normal development of microspores in culture,the microspore environment with respect to moisture was an importantfactor. The rate of the mitotic cycle of the cultured microsporeswas essentially the same as that for microspores grown undergreenhouse conditions. ¹Department of Medicine, Kochi Medical School, Nangoku, Kochi781-51, Japan. (Received March 25, 1980; )     

Monitoring meiosis in gametogenesis

Meiotic recombination is essential to hold homologous chromosomes together so that they can separate accurately in the formation of gametes, thus preventing fetal loss due to aneuploidy. How do germ cells know when they have finished genetic recombination and that it is time to enter the meiotic division phase, and what are the elements that signal the onset of the division phase? During spermatogenesis there is no arrest at the end of meiotic prophase (as there is in oogenesis) and signals for progress into the meiotic division phase may be closely related to events of chromosome pairing and recombination. Methods for culture of male germ cells have been used to show that spermatocytes become competent for some aspects of the division phase by the early pachytene stage, long before they would normally enter division. Evidence suggests that establishment of homologous chromosome pairing is one aspect of acquiring competence. Activation of the cell cycle regulator MPF also appears to be important, and there is a requirement for activity of topoisomerase II in order for spermatocytes to exit prophase and enter the meiotic division phase. Understanding how these molecular entities tie into monitoring the completion of recombination and meiotic progress will be instructive about important gametic safeguards preventing aberrant chromosome segregation and resultant aneuploidy.     

Studies on the behavior of meiotic protoplasts IV. Protoplasts isolated from microsporocytes of liliaceous plants

Enzymatic isolation of protoplasts from microsporocytes of various species of liliaceous plants is described along with some of the features of the isolated meiotic protoplasts. Protoplasts are produced with a high viability from coherent filaments of cells preculture for 24 hr during premeiosis and early meiotic prophase, but with low survival rates from free cells at late prophase. The suspensions of protoplasts contain multinucleate cells produced by spontaneous fusion at various frequencies up to 40%. In the enzyme solution meiotic protoplasts adhere to one another. When isolated at meiotic prophase, protoplasts may be cultured through the meiotic cycle.     

MEIOSIS,BLADE DEVELOPMENT,AND SEX DETERMINATION IN PORPHYRA PURPUREA (RHODOPHYTA)1

The discovery in the early 1980s that meiosis occurs during germination of conchospores of Porphyra yezoensis Ueda suggested that the sexually divided fronds of Porphyra purpurea (Roth) C. Agardh might similarly originate from meiotic segregation of a pair of sex-determining alleles during early sporeling development. After establishing conditions suitable for propagating P. purpurea in culture, observations on developing sporelings demonstrated that meiosis takes place during the first two divisions of the germinating conchospores. In the first division, the spore is split into an upper and lower cell. In the second, an anticlinal division in the upper cell yields two daughter cells situated one beside the other, and a periclinal division in the bottom cell gives two cells arranged one above the other. Thus, during normal development, the first four cells of the sporeling constitute a meiotic tetrad whose cells are arranged in a characteristic fashion. Stable color mutants of P. purpurea were isolated, genetically characterized, and used as genetic markers to follow the fate of individual cells of the tetrad during subsequent frond development. Nearly the entire blade of the mature thallus is derived from the two upper cells of the tetrad, with the two lower cells mostly giving rise to the rhizoidal holdfast region. Cell lineage boundaries laid down by the segregation of color alleles at meiosis corresponded perfectly with those later defined by sexual differentiation on the same fronds, strongly supporting the hypothesis that sex determination in P. purpurea is controlled by alleles at a segregating chromosomal locus.     

Radiation-induced mitotic and meiotic aneuploidy in the yeast Saccharomyces cerevisiae.

A number of genetic systems are described which in yeast may be used to monitor the induction of chromosome aneuploidy during both mitotic and meiotic cell division. Using these systems we have been able to demonstrate the induction of both monosomic and trisomic cells in mitotically dividing cells and disomic spores in meiotically dividing cells after both UV light and X-ray exposure. The frequency of UV-light-induced monosomic colonies were reduced by post-treatment with photoreactivity light and both UV-light- and X-ray-induced monosomic colonies were reduced by liquid holding post-treatment under non-nutrient conditions. Both responses indicate an involvement of DNA-repair mechanisms in the removal of lesions which may lead to monosomy in yeast. This was further confirmed by the response of an excision-defective yeast strain which showed considerably increased sensitivity to the induction of monosomic colonies by UV-light treatment at low doses. Yeast cultures irradiated at different stages of growth showed variation in their responses to both UV-light and X-rays, cells at the exponential phase of growth show maximum sensitivity to the induction of monosomic colonies at low doses whereas stationary phase cultures showed maximum induction of monosomic colonies at high does. The frequencies of X-ray-induced chromosome aneuploidy during meiosis leading to the production of disomic spores was shown to be dependent upon the stage of meiosis at which the yeast cells were exposed to radiation. Cells which had proceeded beyond the DNA synthetic stage of meiosis were shown to produce disomic spores at considerably lower radiation doses than those cells which had only recently been inoculated into sporulation medium. The results obtained suggest that the yeast sustem may be suitable for the study of sensitivities of the various stages of meiotic cell division to the induction of chromosome aneuploidy after radiation exposure.     

Meiotic division and fusion of nuclei in multinucleate cells from the induced fusion of meiotic protoplasts of liliaceous plants

Multinucleate protoplasts were produced from meiotic cells at the zygotene and pachytene stages in a lily andTrillium, and their meiotic divisions were followed during subsequent culture. In each multinucleate, a complete synchrony of nuclear division was maintained throughout the meiotic process, and chromosome behavior appeared normal up to the metaphase stage. In most dinucleates, chromosome segregation movement was organized in a common spindle, and the daughter nuclei at the telophase appeared to envelope each other in the newly formed nuclear membrane. The cell was divided into two daughter cells by a common cell plate. Trinucleates were similarly converted to two cells with a hexaploid number of chromosomes. Some of the di- and trinucleates subsequently completed the second meiotic division with the formation of typical tetrad configurations. In giant cells with more than several nuclei, chromosomes separated at random but reaggregated into one giant resting nucleus, with no later cytokinesis. The rate of meiotic development in multinucleates was relatively slower in cells which contained greater numbers of nuclei.     

Culture of intact Sertoli/germ cell units and isolated Sertoli cells from Squalus testis: I. Evidence of stage-related functions in vitro

As part of an ongoing program of research using the testis of the dogfish shark (Squalus acanthias) to characterize morphologic and functional changes during spermatogenesis, we have developed procedures for culturing intact spermatocysts (germ cell/Sertoli cell clones) and isolated Sertoli cells from premeiotic, meiotic, and postmeiotic stages of development. Phase contrast and light microscopy confirmed the stage and cellular composition of spermatocysts and showed that they retained their closed, spherical configuration for at least 15 d in culture. Stage-related variations in [3H]thymidine incorporation (premeiotic much greater than meiotic = postmeiotic) were observed, a pattern that was the same quantitatively and qualitatively after one or seven days of culture. [3H]Leucine-labeled protein synthesis was twofold greater in cultures with premeiotic spermatocysts than in cultures with more mature stages, whether medium or cysts were analyzed. Sertoli cells isolated from spermatocysts of different stages differed in size, shape, cytological appearance, ability to form flattened monolayers, and rate of DNA synthesis. One day after seeding, [3H]thymidine labeling of Sertoli cells corresponded to the pattern obtained with intact spermatocysts (premeiotic much greater than meiotic = postmeiotic); however, 7 days in culture effected a 40- to 200-fold increase in this parameter and altered the stage-dependent pattern (premeiotic = meiotic greater than postmeiotic). Also, when [3H]leucine-labeled macromolecules secreted by Sertoli cells from premeiotic versus meiotic stages were analyzed by polyacrylamide gel electrophoresis (PAGE), banding patterns differed. Initial results demonstrate the feasibility and potential of this in vitro system for studying qualitative and quantitative changes during spermatogenesis.     

The Microsporogenesis and Megasporogenesis of Soybean

This article deals with the morphological aspects of the process of megasporogenesis and microsporogenesis in soybean. The tempos of microsporogenesis in different anthers of the same flowers were compared, and it has been found that at the leptone- ma, zygonema, pachynema and diplonema of the first meiotic division there appeared a certain degree of synchrony, while at the uninuclear stage of the microspore a perfect synchrony was observed. The development of different pollen mother cells within the same anther was in most cases highly synchronized. The anther in which the PMCs were found to be in the'near stages of the meiotic divisions accounts for 7% only. The megasporocyte develops later than the mierosporoeyte. It enters into the leptotene stage or diplotene stage of the first meiotic division while the mierosporocyte has already finished the process of the meiotic division. Explantation of plates 1. A section of a partieal mierosporangium, mierospore mother cells and the cells of the anther wall. × 600 2. Mierospore mother cells in zygotene stage (bouquet stage). × 600 3. Pa- chytene stage of first meiotic division. ×530 4. Diakinesis of meiosis Ⅰ, the tapetal cells begin- ning to degenerate. ×900 5. Metaphase Ⅰ. ×630 6. Anaphase Ⅰ. ×630 7. Anaphase Ⅰ. ×370 8 Interphase, two-nucleated dyad condition with no intervening cell walls formed. ×630 9. Me- taphase Ⅱ. ×630 10. Beginning of telophase Ⅱ×370 11, Telophase Ⅱ, four microspore nuclei contained within the original microspore mother cell wall. ×630 12. Uninuclear microspore, ×630     

The presence of X- and Y-chromosomes in oocytes leads to impairment in the progression of the second meiotic division

The oocytes of B6.Y(TIR) sex-reversed female mice can be fertilized but the resultant embryos die at early cleavage stages. In the present study, we examined chromosome segregation at meiotic divisions in the oocytes of XY female mice, compared to those of XX littermates. The timing and frequency of oocyte maturation in culture were comparable between the oocytes from both types of females. At the first meiotic division, the X- and Y-chromosomes segregated independently and were retained in oocytes at equal frequencies. However, more oocytes retained the correct number of chromosomes than anticipated from random segregation. The oocytes that had reached MII-stage were activated by fertilization or incubation with SrCl(2). As expected, the majority of oocytes from XX females completed the second meiotic division and reached the 2-cell stage in 24 h. By contrast, more than half of oocytes from XY females initially remained at the MII-stage while the rest precociously entered interphase after SrCl(2) activation; very few oocytes were seen at the second anaphase or telophase and they often showed impairment of sister-chromatid separation. Eventually the majority of oocytes entered interphase and formed pronuclei, but very few reached the 2-cell stage. Similar results were obtained after fertilization. We conclude that the XY chromosomal composition in oocyte leads to impairment in the progression of the second meiotic division.     

Ontogeny of intruding non-periplasmodial tapetum in the wild chicory,Cichorium intybus (Compositae)

The tapetal development ofCichorium intybus L. is investigated using LM and TEM and discussed in relation to the development in other species. During the second meiotic division the tapetal cells become binucleate and lose their cell walls. They intrude the loculus at the time of microspore release from the meiotic callose walls, which means that a locular cavity is never present in this species. During pollen development they tightly junct the exine, especially near the tips of the spines. During the two-celled pollen grain stage they degenerate and most of their content turns into pollenkitt. Until anther dehiscence they keep their individuality, which means that these intruding tapetal cells never fuse to form a periplasmodium. The ultrastructural cytoplasmatic changes during this development are discussed in relation to possible functions.     

Behavior of germinal micronuclei under control of the somatic macronucleus during conjugation in Paramecium caudatum.

In conjugating pairs of Paramecium caudatum, the micronuclear events occur synchronously in both members of the pair. To find out whether micronuclear behavior is controlled by the somatic macronucleus or by the germinal micronucleus, and whether or not synchronization of micronuclear behavior is due to intercellular communication between conjugating cells, the behavior of the micronucleus was examined after removal of the macronuclei from either or both cells of a mating pair at various stages of conjugation. When macronuclei were removed from both cells of a pair, micronuclear development was arrested 1 to 1.5 hr after macronuclear removal. When the macronucleus of a micronucleate cell mating with an amicronucleate cell was removed later than 3 to 3.5 hr of conjugation, that is, an early stage of meiotic prophase of the micronucleus, micronuclear events occurred normally in the operated cell. These results suggest that most micronuclear events are under the control of the macronucleus and that the gene products provided by the macronucleus are transferable between mating cells. One such product is required for induction of micronuclear division and is provided just before metaphase of the first meiotic division of the micronucleus. This factor is effective at a lower concentration in the cytoplasm and/or is more transferable between mating cells than the factors required for other stages. This factor, which seems to be present at least until the stage of micronuclear disintegration, is able to induce repeated micronuclear division as long as it remains active. The factor can act on a micronucleus which has not passed through a meiotic prophase. Moreover, the results suggest the existence of a second factor which is provided by the macronucleus after the first meiotic division that inhibits further micronuclear division.     

Analysis of corneal development with monoclonal antibodies. I. Differentiation in isolated corneas

Monoclonal antibodies highly selective for developmentally regulated antigens present in the cornea (Zak and Linsenmayer, Dev. Biol. 99, 373-381, 1983) have been used to immunohistochemically evaluate differentiation in intact chick corneas cultured on the chorioallantoic membrane (CAM) of host embryos. One antibody is directed against the epithelial cell layer and the other is against the corneal stromal matrix. It has been established that both antigens recognized by the antibodies are expressed de novo in young explanted corneas and that the stromal matrix antigen is a product of the corneal fibroblasts. Thus expression of the antigens can be used as criteria for overt differentiation of the respective cell types. The antibodies have been employed to assess when the corneal epithelial and stromal cells become capable of autonomous differentiation within isolated corneas. To accomplish this, corneas of various ages were explanted with and without adjacent pericorneal tissues. The results indicate that, under the culture conditions employed, corneal stromal differentiation is dependent on the presence of the lens until stage 28 (51/2-6 days of development), which is the time when invasion of the stroma by pericorneal mesenchymal cells is initiated. After stage 28, the stromal matrix antigen was expressed by isolated corneas irrespective of the presence of the lens. Possibly the lens acts by maintaining the integrity of the corneal endothelial monolayer and thus promoting normal migration of pericorneal mesenchymal cells into the primary corneal stroma, where they undergo differentiation. Conversely, differentiation of the corneal epithelium was independent of any pericorneal structure from the earliest stage examined (41/2-5 days of development). It was even independent of overt stromal differentiation, thus suggesting an early and strong determination for this tissue.     

Morphogenesis of the synapton during yeast meiosis

The formation of the synapton (synaptonemal complex) was followed by an electron microscopic examination of large samples of Saccharomyces cerevisiae cells at various stages of meiosis. Three temperature-sensitive mutants were used, cdc4, cdc5 and cdc7, which undergo a slow but normal meiosis at 25° C. At the restrictive temperature of 34° C, cdc4 and cdc5 arrest at an advanced enough stage of meiosis to allow the study of synapton morphogenesis. Based on the frequencies of nuclear structures, we describe the formation of the central region and central elements of the synapton in the dense body, which may be part of the nucleolus. This process occurs during early meiotic stages, concomittantly with recombination commitment and premeiotic DNA replication. Mature synaptons usually appear after premeiotic S, at the pachytene stage, and later disappear. A possible intermediate stage in this disappearance is found in arrested cdc5 cells, which contain paired lateral elements without central elements. — Following the frequencies of spindle plaque configurations, we conclude that the plaques in meiosis duplicate once at the beginning of the main DNA replication, as is also observed prior to mitosis. In contrast to mitotic cells, however, meiotic plaques remain duplicated for a long period, until the synaptons disappear, and only then separate from each other to form a spindle. During late stages of the first meiotic division, the outer plates of the spindle plaques thicken, to duplicate later and give the second division spindles. The characteristically thick outer plate may have a role in the formations of the ascospore wall.     

Meiosis in NEUROSPORA CRASSA. II. Genetic and Cytological Characterization of Three Meiotic Mutants

Three recessive meiotic mutants, asc(DL95), asc(DL243) and asc(DL879), were detected by the abortion of many of their ascospores and were analyzed using both cytological and genetic methods. Even though asc(DL95), asc (DL243) and the previously studied meiotic mutant, mei-1 (Smith 1975; Lu and Galeazzi (1978), complement one another in crosses, they apparently do not recombine (DeLange and Griffiths (1980). Thus, they may represent alleles of the same gene or comprise a gene cluster. Ascospore abortion in these mutants is caused by abnormal disjunction of meiotic chromosomes. In crosses homozygous for asc(DL95), asc(DL879) or mei-1, both pairing of homologs and meiotic recombination frequencies are reduced. In each case, this primary defect is followed by the formation of univalents at metaphase I and their irregular segregation. The mutant asc(DL243) has a defect in ascus formation, and later in disjunction during the second meiotic and post-meiotic divisions. The first-acting defect before or during karyogamy results in the abortion of most cells. Some cells manage to proceed past this block. During the second meiotic division, most chromosomes of the few resulting asci are attached to only one of the two spindle-pole bodies. Disjunction at the post-meiotic division is also highly irregular. This mutant appears to be defective in the attachment of one spindle-pole body to a set of chromosomes. The defect may involve either a centromere-associated product or a spindle-pole body.     

THE EFFECT OF X-RAYS ON CHROMOSOMES IN DIFFERENT STAGES OF MEIOSIS

1. Pollen mother cells exposed to low dosages of x-rays at various stages show different frequencies of chromosome abnormalities in the first meiotic anaphase. 2. Maximum frequencies of abnormalities were obtained in buds irradiated in the pachytene stage of the meiotic prophase and in the preceding mitosis. 3. These results are taken to indicate that the x-ray-sensitive portions of the chromonemata are closely approximated in pairs in pachytene and in the early mitotic prophase. The significance of this in relation to non-homologous pairing is indicated. 4. From the nature of the chromosome configurations observed it is concluded that chromonemata are two-parted when they synapse and that a chromonematic division occurs between pachytene and anaphase and during the mitotic prophase. 5. The frequencies of abnormalities show a linear relationship to dosage. 6. The diameter of the sensitive volume of the chromonema is calculated and found to approximate the diameter of some known protein molecules. 7. The linkage mechanism is found to make up about 90 per cent of the total sensitive volume which corresponds with the approximate reduction in length of the chromonema from pachytene to anaphase. 8. The relation of these sensitive volumes to the gene is discussed.     

Culture of one-cell bovine embryos in explanted mouse oviduct and bovine oviductal epithelial cells

One-cell bovine embryos fertilized in vivo were cultured in TCM-199 and bovine oviductal epithelial cells, in TCM-199, or in explanted immature mouse oviducts supported by TCM-199 to compare development to the blastocyst stage. The morphological stage of development and cell number were determined following 144 hours of culture. Of the embryos that cleaved at least once, 52.6, 30.4 and 0.0% developed to the morula/blastocyst stage after culture in oviductal epithelial cells, in TCM-199 alone, or in explanted mouse oviducts, respectively. The mean total cell number for embryos cultured in oviductal epithelial cells (24.5) was higher than for embryos cultured in TCM-199 (12.8) or in explanted mouse oviducts (5.9; P<0.05). The mean cell number of embryos cultured in TCM-199 or in explanted mouse oviducts did not differ. The explanted immature mouse oviduct supported by TCM-199 did not provide an environment adequate for development of one-cell bovine embryos to the blastocyst stage. Development of one-cell bovine embryos was best supported by co-culture with oviductal epithelial cells in TCM-199 medium.     

Chromosome malsegregation and embryonic lethality induced by treatment of normally ovulated mouse oocytes with nocodazole

The mouse egg is ovulated with its nucleus arrested at the metaphase-II stage of meiosis. Sperm entry triggers the completion of the second meiotic division. It has been speculated that damage to the meiotic spindle of normally ovulated eggs at around the time of sperm entry could result in chromosome malsegregation and the death of conceptuses with numerical chromosome anomalies. This hypothesis was tested using nocodazole, a microtubule inhibitor. Nocodazole was administered either to maturing preovulatory oocytes or to normally ovulated eggs at one of the following stages: (1) the time of sperm entry, (2) early pronuclear stage, (3) pronuclear DNA synthesis, (4) prior to first cleavage division, (5) early 2-cell stage, or (6) prior to the second cleavage division. Little or no effect was observed for treatment times other than the time of sperm entry, when the egg is being activated to complete the second meiotic division. Remarkably high frequencies of embryonic lethality, expressed at around the time of implantation, were induced at this stage. Cytogenetic analysis of first cleavage metaphases of zygotes treated at the time of sperm entry revealed a high incidence of varied numerical chromosome anomalies, with changes in ploidy being predominant.     

A study of mitotic division fidelity and numerical chromosome changes in ageing Syrian hamster dermal cells

Events associated with culture ageing in Syrian hamster dermal cells have been studied from the time of culture isolation during continuous passage until they senesced and died. Microscopic examination of mitotic cells using differential staining of chromosome and spindle apparatus assessed the faithfulness of cell division. Other indicators of the quality of cell division were obtained from chromosome counts, micronucleus frequencies and incidences of binucleate cells. A loss of spindle fidelity and an increase in aneuploidy corresponded to the period of culture senescence. The data presented indicate that the loss of division fidelity and chromosome number instability is an important indicator of the progression of a mammalian culture to senescence under in vitro conditions. Such information may provide the basis of a model for the study of factors which modify mitotic fidelity and senescence and provide a methodology for monitoring the suitability of mammalian cultures for commercial usage.     

Growth of mouse oocytes to maturity from premeiotic germ cells in vitro

In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12-14 day- old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6-7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16-18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.