Asynapsis and unreduced gamete formation in a Trifolium interspecific hybrid

Background Unreduced gametes, a driving force in the widespread polyploidization and speciation of flowering plants, occur relatively frequently in interspecific or intergeneric hybrids. Studies of the mechanisms leading to 2n gamete formation, mainly in the wheat tribe Triticeae have shown that unreductional meiosis is often associated with chromosome asynapsis during the first meiotic division. The present study explored the mechanisms of meiotic nonreduction leading to functional unreduced gametes in an interspecific Trifolium (clover) hybrid with three sub-genomes from T. ambiguum and one sub-genome from T. occidentale. Results Unreductional meiosis leading to 2n gametes occurred when there was a high frequency of asynapsis during the first meiotic division. In this hybrid, approximately 39% of chromosomes were unpaired at metaphase I. Within the same cell at anaphase I, sister chromatids of univalents underwent precocious separation and formed laggard chromatids whereas paired chromosomes segregated without separation of sister chromatids as in normal meiosis. This asynchrony was frequently accompanied by incomplete or no movement of chromosomes toward the poles and restitution leading to unreduced chromosome constitutions. Reductional meiosis was restored in progeny where asynapsis frequencies were low. Two progeny plants with approximately 5 and 7% of unpaired chromosomes at metaphase I showed full restoration of reductional meiosis. Conclusions The study revealed that formation of 2n gametes occurred when asynapsis (univalent) frequency at meiosis I was high, and that normal gamete production was restored in the next generation when asynapsis frequencies were low. Asynapsis-dependent 2n gamete formation, previously supported by evidence largely from wheat and its relatives and grasshopper, is also applicable to hybrids from the dicotyledonous plant genus Trifolium. The present results align well with those from these widely divergent organisms and strongly suggest common molecular mechanisms involved in unreduced gamete formation.

Effects of Epidermal Growth Factor and Myo-Inositol on Nuclear Configuration and Subsequent Embryonic Development of Sheep Oocytes

Growth factors and vitamin-like substances have great positive importance in most biological interactions in the cellular level. The addition of these elements in the culture media will increase the yield of the resulting embryos and improve quality. We examined the effects of epidermal growth factor (EGF) and myo-inositol (MI) on meiotic maturation and yields of blastocyst of Awassi sheep oocyte across two experiments. The oocytes obtained were subjected into three treatments: A (without EGF nor MI), B (10 ng/ml EGF + 20 mmol/l MI) and C (50 ng/ml EGF +40 mmol/l MI). Oocytes were then cultured in Ham's F-10 medium supplemented with 5% (v: v) fetal calf serum and 40 ng/ml follicle - stimulating hormone. In the first experiment, during the 27-h culture period, the oocytes were assessed for germinal vesicle break down, metaphase-I and metaphase-II stages across three-time intervals (9, 21 and 27-h). Results of the experiment showed that EGF and MI enhanced the rates of germinal vesicle break down phase (1.53%; 27-h interval; lowest value), metaphase-I (33.87%; 21-h interval) and metaphase-II (89.23%; 27-h interval). In the second experiment, the oocytes incubated in treatment B achieved the highest rates of cleavage (81.96%), 2-8 cell (62.35%) and blastocyst (45.09%). It is concluded from the present study that incubating sheep oocytes in culture media containing a cocktail of EGF (10 ng/ml) and MI (20 mmol/l) significantly improves the rates of metaphase-II, fertilization and blastocyst rates.

Meiotic Aberrations Leading To Aneuploidy In Cauliflower (Brassica Oleracea L. var. Botrytis)

Aneuploid cauliflower plants (Brassica oleracea L. var. botrytis) display abnormal curd phenotypes causing serious commercial problems in offspring populations. Despite extensive breeding efforts, selection of genotypes producing euploid gametes remains unsuccessful due to unknown genetic and environmental factors. To reveal the origin of aneuploid gametes, we analyzed chromosome pairing, chiasma formation and chromosome segregation in pollen mother cells of selected cauliflower genotypes. To this end we compared different genotypes exhibiting Low with < 5%, Moderate with 5-10% and High with > 10% aberrant offspring. Microscopic observations revealed regular chromosome pairing at pachytene. However, cells at diakinesis and metaphase I showed variable numbers of univalents, suggesting that chiasma formation during meiotic prophase is incomplete or disrupted and results in a partial desynaptic phenotype. Cells at anaphase I – telophase II exhibited various degrees of unbalanced chromosome numbers explaining the aneuploid offspring. Immunofluorescence probed with an MLH1 antibody demonstrated fluorescent foci in all genotypes, but their lower numbers do not correspond to the putative sites of chiasmata. Interchromosomal connections between chromosomes and bivalents are common at diakinesis and metaphase I, and they contain centromeric and 45S rDNA tandem repeats, but such threads seemed not to affect proper disjoin of the half bivalents at anaphase I. Moreover, male meiosis in the arabidopsis APETALA1/ CAULIFLOWER double mutant with the typical cauliflower phenotype did show interchromosomal connections, but there were no indications for partial desynapsis. We now hypothesize that the occurrence of desynapsis in cauliflower is a developmental out-of-phase phenomenon partially or completely controlled by genes involved in flower and curd development.

CENP-V is required for proper chromosome segregation through interaction with spindle microtubules in mouse oocytes

Proper chromosome segregation is essential to avoid aneuploidy, yet this process fails with increasing age in mammalian oocytes. Here we report a role for the scarcely described protein CENP-V in oocyte spindle formation and chromosome segregation. We show that depending on the oocyte maturation state, CENP-V localizes to centromeres, to microtubule organizing centers, and to spindle microtubules. We find that Cenp-V−/− oocytes feature severe deficiencies, including metaphase I arrest, strongly reduced polar body extrusion, increased numbers of mis-aligned chromosomes and aneuploidy, multipolar spindles, unfocused spindle poles and loss of kinetochore spindle fibres. We also show that CENP-V protein binds, diffuses along, and bundles microtubules in vitro. The spindle assembly checkpoint arrests about half of metaphase I Cenp-V−/− oocytes from young adults only. This finding suggests checkpoint weakening in ageing oocytes, which mature despite carrying mis-aligned chromosomes. Thus, CENP-V is a microtubule bundling protein crucial to faithful oocyte meiosis, and Cenp-V−/− oocytes reveal age-dependent weakening of the spindle assembly checkpoint.

The Culture and Correlative Electron Microscopy of Pollen Mother Cells in Meiosis: Development of Techniques and Some Observations on Selected Topics

<p>Reliable techniques for the living cell culture and correlative light and electron microscopy (EM) of meiotic pollen mother cells (PMCs) of Iris spuria, Allium triquetrum and Tradescantia flumenensis are described in detail. Living PMCs were successfully cultured in a slide chamber on agar/sucrose medium. Cells were covered with an inert oil to prevent their dehydration, and some cells were cultured from metaphase I to tetrad cell formation over a 20 hour period. Other PMCs were fixed with glutaraldehyde and flat embedded using a modification of the agar sandwich technique of Mole-Bajer and Bajer (1968). This technique was developed to permit the preselection of PMCs at known meiotic stages, for subsequent EM examination. Serial thin sections were cut at known planes of section; and 3-D reconstructions of MT distribution, and MT counts from transverse sections were completed. It was also possible to examine sections of an Iris anaphase I PMC which had been previously studied in life. Anaphase I and II chromosome velocities were analysed in the three species. Mean velocities were approximately 0.5 mu m/min with some variation from cell to cell and between sister half-spindles. In Allium anaphase I there was also variation in chromosome velocity within the half-spindle; and this variation was found not to be related to chromosome position on the metaphase I plate. Spindle elongation was zero in Allium anaphase I and in Iris anaphase II, but was detectable in Allium anaphase II (40%) and in "Iris anaphase I (l5%). The extent of spindle elongation in Tradescantia could not be determined. The kinetochore region in the first meiotic division consisted of two closely appressed, but structurally (and functionally) distinct, sister kinetochores. At meiosis II, the two sister kinetochores were separate from each other and faced opposite poles. The kinetochore arrangement probably changes from side-by-side (meiosis I) to back-to-back (meiosis II) during chromosome recondensation at prophase II in these cells. Bundles of non-kinetochore microtubules (nkMTs) span the interzone between sister chromosome units at metaphase I and II and anaphase II. Bundles of kinetochore MTs (kMTs) do not increase in divergence at any stage of meiosis studied; there was little interaction between nkMTs and kMTs, and MT-MT cross bridges were rare. These observations are not consistent with models of chromosome movement based on MT sliding or zipping. No relationship was found between nkMT distribution and spindle elongation, and the several different nkMT distributions which have been reported for other cell types may be variations on a structural theme. Spindle endoplasmic reticulum (ER) in meiosis II was found to be derived largely from invaginations and evaginations of the nuclear envelope. Growth of existing spindle ER was proposed to account for the doubling in the amount of ER observed between interphase and prometaphase II. Randomly oriented elements of ER, in early prometaphase II spindles may become passively aligned along the interpolar axis and then actively transported polewards at later stages of prometaphase II and metaphase II. Suggestions for future research are offered.</p>

The Culture and Correlative Electron Microscopy of Pollen Mother Cells in Meiosis: Development of Techniques and Some Observations on Selected Topics

<p>Reliable techniques for the living cell culture and correlative light and electron microscopy (EM) of meiotic pollen mother cells (PMCs) of Iris spuria, Allium triquetrum and Tradescantia flumenensis are described in detail. Living PMCs were successfully cultured in a slide chamber on agar/sucrose medium. Cells were covered with an inert oil to prevent their dehydration, and some cells were cultured from metaphase I to tetrad cell formation over a 20 hour period. Other PMCs were fixed with glutaraldehyde and flat embedded using a modification of the agar sandwich technique of Mole-Bajer and Bajer (1968). This technique was developed to permit the preselection of PMCs at known meiotic stages, for subsequent EM examination. Serial thin sections were cut at known planes of section; and 3-D reconstructions of MT distribution, and MT counts from transverse sections were completed. It was also possible to examine sections of an Iris anaphase I PMC which had been previously studied in life. Anaphase I and II chromosome velocities were analysed in the three species. Mean velocities were approximately 0.5 mu m/min with some variation from cell to cell and between sister half-spindles. In Allium anaphase I there was also variation in chromosome velocity within the half-spindle; and this variation was found not to be related to chromosome position on the metaphase I plate. Spindle elongation was zero in Allium anaphase I and in Iris anaphase II, but was detectable in Allium anaphase II (40%) and in "Iris anaphase I (l5%). The extent of spindle elongation in Tradescantia could not be determined. The kinetochore region in the first meiotic division consisted of two closely appressed, but structurally (and functionally) distinct, sister kinetochores. At meiosis II, the two sister kinetochores were separate from each other and faced opposite poles. The kinetochore arrangement probably changes from side-by-side (meiosis I) to back-to-back (meiosis II) during chromosome recondensation at prophase II in these cells. Bundles of non-kinetochore microtubules (nkMTs) span the interzone between sister chromosome units at metaphase I and II and anaphase II. Bundles of kinetochore MTs (kMTs) do not increase in divergence at any stage of meiosis studied; there was little interaction between nkMTs and kMTs, and MT-MT cross bridges were rare. These observations are not consistent with models of chromosome movement based on MT sliding or zipping. No relationship was found between nkMT distribution and spindle elongation, and the several different nkMT distributions which have been reported for other cell types may be variations on a structural theme. Spindle endoplasmic reticulum (ER) in meiosis II was found to be derived largely from invaginations and evaginations of the nuclear envelope. Growth of existing spindle ER was proposed to account for the doubling in the amount of ER observed between interphase and prometaphase II. Randomly oriented elements of ER, in early prometaphase II spindles may become passively aligned along the interpolar axis and then actively transported polewards at later stages of prometaphase II and metaphase II. Suggestions for future research are offered.</p>

Fertilization and early embryonic development of in vitro matured metaphase I oocytes in patients with unexpected low oocyte maturity rate

Summary To determine the fertilization and embryonic potential of immature metaphase I (MI) oocytes in patients with low oocyte maturity rate in whom the percentage of mature oocytes obtained was less than 75% of the total retrieved ones. In vivo matured metaphase II (MII) oocytes (MII-ICSI, n = 244), and in vitro matured MI oocytes (MI-MII-ICSI, n = 202) underwent an intracytoplasmic sperm injection (ICSI) procedure. Maturation rate, fertilization rate and early embryonic development were compared in both groups. In total, 683 oocytes were collected from 117 ICSI cycles of 117 patients. Among them, 244 (35.7%) were mature MII and 259 (37.9%) were MI after the denudation process. Of those 259 MI oocytes, 202 (77.9%) progressed to MII oocytes after an incubation period of 18–24 h. The maturation rate was 77.9%. Fertilization rate was found to be significantly higher in the rescued in vitro matured MI oocyte group when compared with the in vivo matured MII oocyte group (41.6% vs 25.8%; P = 0.0006). However, no significant difference was observed in terms of cleavage rates on days 2 and 3 between the groups (P = 0.9126 and P = 0.5031, respectively). There may be unidentified in vivo factors on the oocyte maturation causing low developmental capacity in spite of high fertilization rates in the group of patients with low oocyte maturity rate. Furthermore, studies are needed to determine the appropriate culture characteristics as well as culture period and ICSI timing of these oocytes.

M-Phase oscillations in PP1 activity are essential for accurate progression through mammalian oocyte meiosis

Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-Phase transitions (e.g., G2/M). Protein Phosphatase 1 (PP1) is one of these phosphatases, with oscillations in activity driving mitotic M-Phase entry, progression, and exit, with evidence from a variety of experimental systems pointing to roles in meiosis as well. Here we report that PP1 is important for M-Phase transitions through mouse oocyte meiosis. Employing a unique small-molecule approach to inhibit or activate PP1 at distinct phases of mouse oocyte meiosis, we found that aberrations in normal cyclical PP1 activity leads to meiotic abnormalities. We report here that temporal control of PP1 activity is essential for G2/M transition, metaphase I/anaphase I transition, and the formation of a normal metaphase II oocyte. Our data also reveal that inappropriate activation of PP1 is more deleterious at G2/M transition than at prometaphase I-to-metaphase I, and that an active pool of PP1 during prometaphase I is vital for metaphase I/anaphase I transition and metaphase II chromosome alignment. Taken together, these results establish that loss of oscillations in PP1 activity causes a range of severe meiotic defects, pointing to essential roles for PP1 in oocytes and female fertility, and more broadly, M-Phase regulation.