Phases Of Meiosis Research Articles

DNA replication initiation by 6-DMAP treatment in maturing oocytes and dividing embryos from marine invertebrates.

6-dimethylaminopurine (6-DMAP), a potent protein kinase inhibitor, drives most cells into an interphasic stage. Experiments were undertaken with oocytes from three marine invertebrate species, i.e., Mytilus edulis, Spisula solidissima, and Strongylocentrotus droebachiensis, wherein oocytes were arrested at different phases of meiosis. 6-DMAP induced a continuous DNA synthesis in meiotic cells, whereas it allowed a single round of DNA replication in treated mitotic cells, regardless of species considered. The effects of 6-DMAP were accompanied in all cases by rephosphorylation on tyrosine of the p34cdc2 homolog, the M-phase promoting factor (MPF) catalytic subunit. The fact that 6-DMAP overcomes the inhibitory control of replication during meiosis suggests that this process depends upon protein phosphorylation, while DNA synthesis regulation in mitotic cells relies on 6-DMAP-insensitive events.

Numerical and structural chromosome aberrations induced by etoposide (VP16) during oocyte maturation of mice: transmission to one-cell zygotes and damage to dictyate oocytes.

The antineoplastic drug etoposide (ET) inhibits topoisomerase II (topo II) activity by forming a ternary complex (DNA-ET-topo II). This complex prevents the DNA-strand-rejoining activity of topo II and may result in structural chromosome aberrations. Inhibition of topo II activity may also predispose cells to aneuploidy because this enzyme is needed for removing regions of DNA catenation prior to chromosome segregation. Our objectives were to study the dose response for ET-induced numerical and structural chromosomal aberrations in mouse one-cell zygotes, to compare these data with those obtained from a contemporary metaphase II (MII) oocyte study and to evaluate the sensitivity of dictyate oocytes to ET-induced aneuploidy. ICR female mice were superovulated and injected i.p. with either 6% dimethylsulphoxide (controls) or 20, 40 or 60 mg/kg ET 2 h after human chorionic gonadotrophin (HCG). ICR males were paired (1:1) with females immediately after treatment. After 17 h the males were removed, and after 24 h the females with a vaginal plug were given colchicine. One-cell zygotes were harvested for cytogenetic analysis 17 h after colchicine. The percentages of hyperploid zygotes were 1.1, 5.7, 13.8 and 20.7 and of zygotes with structural aberrations were 2.5, 16.3, 37.7 and 64.7, for control, 20, 40 and 60 mg/kg ET respectively. The differences between each succeeding dose for both structural and numerical aberrations were statistically significant (P < 0.01). When the ET dose response aneuploidy data from zygotes were compared with similar data from a contemporary study involving metaphase II oocytes, the frequencies of hyperploidy were greater in zygotes than in oocytes. We conclude that when ET is administered during the preovulatory phase of meiosis, it is both an aneugen and a clastogen in mouse one-cell zygotes.

Stage-specific expression of B cell translocation gene 1 in rat testis

B Cell translocation gene 1 (BTG1) is a member of a new family of putative antiproliferative factors. They are characterized by their rapid, but transient, expression in response to factors that induce growth arrest and subsequent differentiation. In immature rat Sertoli cell cultures, BTG1 messenger RNA (mRNA) increases rapidly after FSH stimulation. We obtained the full-length coding sequence of rat BTG1 complementary DNA for Northern blot analysis and in situ hybridization to determine the temporal expression and spatial distribution of BTG1 mRNA in the rat testis. Northern analysis of isolated adult germ cells and in situ hybridization analysis of adult seminiferous epithelium demonstrated that BTG1 expression was first evident in late primary spermatocytes. The level of BTG1 mRNA was also elevated in secondary spermatocytes, but was maximal in postmeiotic round spermatids where levels were 5 times the background. BTG1 mRNA was not detectable in cells in the M phase of meiosis or spermatids undergoing nuclear elongation and condensation. The oscillation of BTG1 expression from the late prophase of the first meiotic division through spermatozoa release suggests BTG1 involvement in spermatogenesis. High levels of BTG1 mRNA at entry into terminal spermatid differentiation suggests a role consistent with that proposed for the BTG1 family of antiproliferative factors.

Stage-specific expression of B cell translocation gene 1 in rat testis.

B Cell translocation gene 1 (BTG1) is a member of a new family of putative antiproliferative factors. They are characterized by their rapid, but transient, expression in response to factors that induce growth arrest and subsequent differentiation. In immature rat Sertoli cell cultures, BTG1 messenger RNA (mRNA) increases rapidly after FSH stimulation. We obtained the full-length coding sequence of rat BTG1 complementary DNA for Northern blot analysis and in situ hybridization to determine the temporal expression and spatial distribution of BTG1 mRNA in the rat testis. Northern analysis of isolated adult germ cells and in situ hybridization analysis of adult seminiferous epithelium demonstrated that BTG1 expression was first evident in late primary spermatocytes. The level of BTG1 mRNA was also elevated in secondary spermatocytes, but was maximal in postmeiotic round spermatids where levels were 5 times the background. BTG1 mRNA was not detectable in cells in the M phase of meiosis or spermatids undergoing nuclear elongation and condensation. The oscillation of BTG1 expression from the late prophase of the first meiotic division through spermatozoa release suggests BTG1 involvement in spermatogenesis. High levels of BTG1 mRNA at entry into terminal spermatid differentiation suggests a role consistent with that proposed for the BTG1 family of antiproliferative factors.

Karyological investigations on the vampyrellid filose amoeba Lateromyxa gallica Hülsmann 1993

The nuclei of the vampyrellid filose amoeba Lateromyxa gallica were investigated in trophozoites, early digestive cysts, reproductive cysts, and in developing resting cysts. Trophozoites possess numerous, minute, spherical nuclei in interphase. In early digestive cysts the nuclei enlarge and the morphology of the nucleolus changes. The digestive cysts develop into reproductive cysts. Karyokinesis takes place synchronously. The spindle is intranuclear and acentrical. In metaphase, the chromosomes are arranged in a distinct equatorial plate. The nuclear envelope remains intact at least until telophase. Shortly after karyokinesis the trophozoites leave the cysts. Cytokinesis regularly takes place when the trophozoites invade cells of Oedogonium. Under unfavorable conditions the trophozoites as well as the digestive cysts are able to form resting cysts. Nuclei of digestive cysts which begin to develop into resting cysts were found in the pachytene phase of meiosis, proved by synaptonemal complexes. Karyokinesis, probably the second meiotic division, was detected when the resting cyst was almost fully developed. From the results of our investigations, the vampyrellid filose amoebae can be regarded as sexual.

Quantitation of sertoli cell-germ cell desmosome gap junctions in relation to meiotic divisions in the male rat

Desmosome-gap (D-G) junctions were quantified in relation to germ cell meiosis in the male, specifically to test the hypothesis that the loss of these junctions is related to successful passage of cells through diplotene phase of Meiosis I and the two cytokineses that follow. Such a hypothesis has been proposed as the cause for the resumption of meiosis that occurs prior to ovulation in the female. D-G junctions were quantified in pachytene spermatocytes (stage XII), diplotene spermatocytes (stage XII), secondary spermatocytes (stage XIV) and step 1 spermatids (stage I). These were referred to as the cells of interest as compared with spermatocytes (zygotene spermatocytes, zygotene spermatocytes, pachytene spermatocytes, pachytene spermatocytes) in the same stages, respectively, that served as controls termed control cells. Since gap junctions are not easily recognized in the average sectioned profile of a desmosome-gap junction, only the desmosomal component was quantified. The data were expressed as both numbers and length of junctions per tubule, per cell profile and per unit lineal membrane length to overcome errors inherent in the methodologies utilized. There was no indication that numbers of junctions changed specifically in the cells of interest after passage through diplotene suggesting that these junctions do not have a comparable role in meiotic continuance in the male as proposed for the female. Interestingly, the control cells always showed greater numbers and length of junctions than the cells of interest suggesting that junction may relate more to the period of initiation of meiosis than to its continuance.

Meiotic Studies in Pilocarpus pennatifolius Lem.(Rutaceae).

Pilocarpus pennatifolius shows a high frequency of fruits with abortive carpels. In order to verify the causes of the undevelopment of these carpels, seven wild specimens were analysed for their meiotic process. Meiotic abnormalities such as irregular chromosome segregation were observed in six specimens. Three specimens showed cytomixis in less than 1% of the analysed cells. This phenomenon was observed from the initial phases of meiosis to telophase I. These irregularities may have contributed, partly at least, to the reduction of pollen viability. However, they cannot justify for themselves the high rate of abortive carpels in this species. It is possible that other factors, not related to meiotic abnormalities, may be affecting the fertilization process.

Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800

Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.

Microsporogênese de Coffea canephora Pierre ex Froehner com número duplicado de cromossomos

Realizou-se o estudo do comportamento meiótico de C. canephora tetraplóide com 2n =44 cromossomos por tratar-se de uma espécie considerada possível ancestral de C. arabica (2n = 44): constatou-se, em 92,0% das células-mães de pólen, em todas as fases da microsporogênese estudada, 2n = 44 cromossomos. Em diacinese os cromossomos se apresentaram na forma de mono-, bi-, tri- e tetravalentes. Em metáfase 1, somente 13,16% das células apresentaram 22II sendo a seguinte a fórmula média do pareamento: 3,61I; 15,21II 0,71III e 1,93IV. As irregularidades anafásicas resumiram-se praticamente na disjunção desigual dos cromossomos para os pólos de 21-23, 20-24 e 19-25. Somente 37,65% das células apresentaram segregação normal de 22 cromossomos para cada pólo. Em anáfase II, observaram-se sete tipos diferentes de distribuição cromatídica e, também, somente em 26,0% das células foi encontrada distribuição normal dos cromossomos. Após a citocinese, foram observadas tríades (1,6%), tétrades (77,0%) e políades (21,4%). A inviabilidade dos grãos de pólen foi alta, 79,0%. Observações em cortes transversais medianos de frutos mostraram 43,4% do tipo normal, sendo 11,0% do tipo moca e 32,4% do chato. Em 56,6% dos frutos, não houve desenvolvimento de sementes, formando-se apenas perisperma.

Caffeine interactions with methyl methanesulphonate, hycanthone, benlate, and cadmium chloride in chromosomal meiotic segregation of Saccharomyces cerevisiae

Interactions of caffeine with chemicals known for their effects on chromosomal segregation during meiosis of Saccharomyces cerevisiae were studied. It appears that caffeine does interfere with the action of other compounds during the different phases of meiosis. Treatments with methyl methanesulphonate (MMS) and cadmium chloride (CdCl 2) resulted in a synergistic effect consisting of an increase in the frequency of recombination. The greatest effects were found on the induction of diploid spores: MMS, hycanthone, and distamycin demonstrated strong, benlate little synergistic action. CdCl 2 demonstrated antagonism to caffeine by counter-inhibiting its effect on the induction of diploids. Concerning disomic induction: caffeine reduced (or left unchanged) the effect on non-disjunction when MMS and hycanthone were used. Simple additive effects were caused in conjunction with distamycin, benlate, and (in small doses) CdCl 2. 2 mg of caffeine/ml in treatments with CdCl 2 resulted in a very high frequency of disomic clones.

Induction and survival of micronuclei in rat spermatids. Comparison of two meiotic micronucleus techniques using cyclophosphamide

The induction and survival of micronuclei (MN) in rat spermatids was studied by two different methods, the dissection method (DM) and the suspension method (SM). It was observed that MN are induced by cyclophosphamide in the S phase of meiosis, in preleptotene spermatocytes, and in an earlier cell stage, the type B spermatogonia. Both techniques showed that MN survive in spermatids at least 5 days. Advantages of the DM include the use of a DNA-specific fluorochrome for staining of MN, higher MN frequencies observed, and the possibility to gain detailed information of the kinetics of induction of MN. In the SM, slide preparation is simpler than in the DM, and several samples can be prepared simultaneously but the scoring of slides is time consuming. Improvements of the sampling system of the DM are suggested. For evaluation of clastogenic action of chemicals on male germ cells both techniques provide a simple and rapid approach.

Evidence for an absence of deleterious effects of ultrasound on human oocytes.

Animal and human data would suggest that ultrasound causes deleterious effects to oocytes during meiosis. We directly compared the fertilization rate and embryonic development following in vitro fertilization and embryo transfer of those oocytes exposed to ultrasound and those not exposed in the same patient. In 39 unscreened patients a combination of laparoscopy and ultrasound was used for oocyte recovery. Laparoscopy was performed first on the most accessible ovary (usually the right) and at least one oocyte was obtained. Ultrasound-guided oocyte recovery was successful in the other inaccessible ovary. To assess how oocytes obtained by ultrasound or laparoscopy related to the pregnancy rate, two groups of patients were evaluated in whom the embryos transferred either had been exposed to ultrasound or had not been. The fertilization and the embryo cleavage rates were not significantly different between the ultrasound-exposed and the unexposed groups. The pregnancy rate was also not significantly different [9 of 49 (18.4%) for ultrasound exposed versus 14 of 74 (18.9%) for unexposed]. There was one early spontaneous abortion in each group. Further analysis of a group of 40 patients, in whom the oocytes were exposed to ultrasound in situ, after the endogenous luteinizing hormone (LH) surge had begun 1-27 hr earlier, revealed that 6 became pregnant (15%). This preliminary study suggests that exposure of human oocytes to ultrasonic waves, either during the different phases of meiosis or after the completion of meiosis, did not significantly influence the developmental potential of the in vitro fertilized embryos.

Effect of a single exposure of organophosphorous insecticide, phosalone on sexual life cycle of alga Chlamydomonas reinhardtii

A single exposure of opposite mating types of the algae Chlamydomonas reinhardtii to an organophosphorous insecticide, phosalone, at 10 −3M (367 mg/L) concentration for 2 h delayed the meiotic division of the significant number of zygotes in the populations for the period of 5 days. All of these zygotes underwent division within a day following the delay period. The lag in meiosis of the zygotes appeared to be due to the delay in the commencement of the prophase, the first phase of meiosis. Treatment by phosalone did not influence the formation of gametes, young zygotes, and mature zygotes. Similarly, its exposure to female (mt ∗) or male (mt −) or to both the cell types (either before or after gametogenesis) did not result in differential or additive action on meiosis of the zygotes. Among the four stages of cell development, the stage during the fusion of gametes, appeared to be more sensitive to phosalone than the remaining three stages. However, these three stages (viz., before and after gematogenesis, and after fusion of gametes) did not exhibit differential sensitivity to phosalone.

ULTRASTRUCTURE OF MEIOSIS INDASYA BAILLOUVIANA(RHODOPHYTA). I. PROPHASE I1

ABSTRACTThis first of two papers on ultrastructural observations of meiosis in the red algaDasya baillouviana (Gmelin) Montague describes stages of prophase I of meiosis. Although the five stages of prophase were originally derived from light microscopic studies, the same stages were utilized for this study based on the developmental sequence of the synaptonemal complex, which has the same morphology and mode of development as those reported for other red algae. The cytoplasm in early prophase sporocytes was typically less electron dense than either vegetative cells or sporocytes in later stages of meiosis. The reduction in density suggests clearing of ribosomes and other cytoplasmic components prior to conversion from sporophyte to gametophyte control. Leptotene cells often had an amorphous, chromatin‐free area, function unknown, which was not obviously associated with any specific nuclear region. Diplotene cells were characterized by nuclei containing prominent ring‐shaped nucleoli composed of a dark staining ring of material surrounding an electron‐translucent “vacuole.” Packets of electron‐dense, fibrillar material were often noted in the cytoplasm of late prophase cells. These packets are thought to he “nuage,” a term applied to large cytoplasmic aggregations of RNA in germ cells of several other phyla. It is suggested that nuage may represent a new infusion of ribosomal and messenger RNA for post‐meiotic development. The division pales are established by late prophase and a single polar ring is found within each large “exclusion zone” in close association with a pore‐free area of nuclear envelope. Both annulate lamellae and small, numerous vesicles are located in the exclusion zones. The significance of the various aspects of prophase I is discussed with the overall observation that this phase of meiosis in red algae is very similar to the process in higher plant and animal cells.

The organization of DNA metabolism during the recombinational phase of meiosis with special reference to humans.

The organization of DNA metabolism during the recombinational phase of meiosis with special reference to humans.

Recent data on the examination of the mutagenic effect of a dinitro- o-cresol-containing pesticide by different test methods

The mutagenic effect of the 50% dinitro-o-cresol-containing pesticide Krezonit E was assessed by the following test methods: (1) human leukocyte cultures in vitro; (2) mouse bone-marrow chromosome preparations in vivo; and (3) examination of gametic mutations (a) in mouse test preparations, (b) with dominant lethality, and (c) by the examination of F1 embryonic chromosomes. According to the results the dinitro-o-cresol-containing pesticide is mutagenic. In all test systems aberrations both in the somatic and in the gametic cells could be detected. There was a significant increase in the rate of breaks and deletions and of the univalents in the diakinesis phase of meiosis found to be pathognomic by Schleiermacher [Humangenetik 3, 127–133 (1966)]. The chromosome aberrations of the F1 embryos were significantly higher in the first, second, and third week after the treatment of the male parent than in the control group. According to these findings the conclusion can be drawn that damage in the more mature forms of spermiogenesis can be better detected by examining the F1 embryonic chromosomes, while the aberrations of the early forms can be shown much better by dominant lethality.

Characterization of rat spermatocytes after plastic embedding.

Rat testicular tissue, perfused with glutaraldehyde, post-fixed with osmium and stained with toluidine blue, was studied to obtain information which could be used to characterize spermatocytes (also type B gonia and Step 1 spermatids) with the light microscope. Measurements of relative cell, nuclear sizes and absolute nuclear size are presented in graphic form, demonstrating the progressive growth found for spermatocytes. Early prophase spermatocytes (preleptotene, leptotene, zygotene) gradually increased in size. Pachytene cells showed no growth until Stage IV, at which point a dramatic size increase began and continued until the diplotene phase. Guidelines to identify a particular phase of meiosis were established for spermatocytes using primarily nuclear traits. Examination of longitudinal sections through Stages XIII, XIV and I were useful for comparing cells from Meiotic divisions (meta-, ana-, and telophases) I and II and also for differentiating secondary spermatocytes from Step 1 spermatids.

Membranous Structures Within Nuclei of Spermatocytes in the Adult Male Rat

In mid and late pachytene and diplotene phases of meiosis, rat spermatocytes demonstrated two types of unusual membrane modifications within the nucleus. These most commonly took the form of a membrane bounded saccule which faced the nuclear envelope. A 12–16 nm space containing dense material separated the saccule from the nuclear envelope. The sex vesicle or the components of the nucleolus were frequently seen lying adjacent to the saccule. The bleb, in contrast to the single walled saccule, was bounded by two membranes. Lying entirely within the nucleus, the bleb caused the nucleus to bulge outward. On the surface of the bleb which was exposed to the nuclear sap, numerous nuclear pores were observed. The contents of small blebs (0.5–1.5 µm in diameter) closely resembled the contents of the nucleus, whereas the contents of blebs larger than 1.5 µm in diameter showed certain similarities to cytoplasmic constituents. Most large blebs contained chromatoid-like material.

Meiotic Figures in the Testicular Biopsy of Subfertile Males

The phases and sequence of meiotic division are herein reviewed and illustrated as they occur in the human testis. Normally all phases of meiosis can be identified, and arrest at a phase is indicative of faulty spermatogenesis. The correlation of these arrest patterns with the clinical course and response to clinical therapy is now in progress. These meiotic studies can be undertaken in any laboratory with cytogenetic capabilities, and this study is intended to serve as a base line illustration of the normal.

Oxygen requirements for the maturation of hamster oocytes.

Summary. Hamster oocytes exhibited a sharply defined optimum of 5% O2 for maturation to metaphase II in vitro. Without the addition of O2, all of the oocytes were arrested during chromatin condensation. Under 10% O2, most of the oocytes were arrested at several phases of meiosis up to and including telophase I. Mammalian oocytes appear to require an oxygen concentration below 20% for optimal maturation in vitro. Thus, a greater proportion of mouse oocytes develop to metaphase II when the 02 concentration in the gas phase of the culture is reduced to 5 to 10% (Haidri, Miller & Gwatkin, 1971). Recently, Gwatkin & Haidri (1973) showed that hamster oocytes mature under 5% 02 but rapidly become necrotic under 20% 02. This prompted us to determine the effect of varying the 02 concentration on the proportion of hamster oocytes undergoing maturation to metaphase II. The results of these studies are reported in this communication. Employing methods already described (Gwatkin & Haidri, 1973), oocytes, free of cumulus cells, were collected under an air atmosphere from the enlarged follicles present in the ovaries of 9- to 10-week-old golden hamsters (Mesocricetus auratus) which had been injected intraperitoneally 3 days previously with 25 i.u. PMSG (Organon). These oocytes were cultured in medium GH-2 (Haidri & Gwatkin, 1973) in Microtest Plates (Cat. No. 3034, Falcon Plastics) without an oil cover. Medium GH-2 consists of a balanced salt solution, energy sources (pyruvate, lactate and glucose), twelve amino acids and crystalline bovine serum albumin. The amino acids and albumin were shown to be essential for maximum polar body formation by preovulatory oocytes (Haidri & Gwatkin, 1973). The three energy sources can be omitted without reducing the proportion of oocytes maturing (R. B. L. Gwatkin and A. A. Haidri, unpublished data). These needs are presumably met by gluconeogenesis from one or more of the amino acids. The cultures were placed in small desiccators, which were evacuated to 60 mm Hg and refilled three times with atmospheres containing various partial pressures of 02. The balance of each atmosphere was nitrogen, except for C02 which was kept constant at 5%. After 16 hr, the proportion of oocytes which had formed polar bodies was recorded. Text-figure 1 shows that under 5% 02, the optimum concentration, approximately 90% of the oocytes formed