Late Diakinesis Research Articles

Ultrastructure and Dynamics of Synaptonemal Complex Components During Meiotic Pairing and Synapsis of Standard (A) and Accessory (B) Rye Chromosomes.

During prophase I a meiosis-specific proteinaceous tripartite structure, the synaptonemal complex (SC), forms a scaffold to connect homologous chromosomes along their lengths. This process, called synapsis, is required in most organisms to promote recombination between homologs facilitating genetic variability and correct chromosome segregations during anaphase I. Recent studies in various organisms ranging from yeast to mammals identified several proteins involved in SC formation. However, the process of SC disassembly remains largely enigmatic. In this study we determined the structural changes during SC formation and disassembly in rye meiocytes containing accessory (B) chromosomes. The use of electron and super-resolution microscopy (3D-SIM) combined with immunohistochemistry and FISH allowed us to monitor the structural changes during prophase I. Visualization of the proteins ASY1, ZYP1, NSE4A, and HEI10 revealed an extensive SC remodeling during prophase I. The ultrastructural investigations of the dynamics of these four proteins showed that the SC disassembly is accompanied by the retraction of the lateral and axial elements from the central region of the SC. In addition, SC fragmentation and the formation of ball-like SC structures occur at late diakinesis. Moreover, we show that the SC composition of rye B chromosomes does not differ from that of the standard (A) chromosome complement. Our ultrastructural investigations indicate that the dynamic behavior of the studied proteins is involved in SC formation and synapsis. In addition, they fulfill also functions during desynapsis and chromosome condensation to realize proper recombination and homolog separation. We propose a model for the homologous chromosome behavior during prophase I based on the observed dynamics of ASY1, ZYP1, NSE4A, and HEI10.

Chromosome painting in meiosis reveals pairing of specific chromosomes in polyploid Solanum species.

Analysis of chromosome pairing has been an important tool to assess the genetic similarity of homologous and homoeologous chromosomes in polyploids. However, it is technically challenging to monitor the pairing of specific chromosomes in polyploid species, especially for plant species with a large number of small chromosomes. We developed oligonucleotide-based painting probes for four different potato chromosomes. We demonstrate that these probes are robust enough to monitor a single chromosome throughout the prophase I of meiosis in polyploid Solanum species. Cultivated potato (Solanum tuberosum, 2n = 4x = 48) is an autotetraploid. We demonstrate that the four copies of each potato chromosome pair as a quadrivalent in 66-78% of the meiotic cells at the pachytene stage. Solanum demissum (2n = 6x = 72) is a hexaploid and has been controversial regarding its nature as an autopolyploid or allopolyploid. Interestingly, no hexavalent pairing was observed in meiosis. Instead, we observed three independent bivalents in 83-98% of the meiotic cells at late diakinesis and early metaphase I for the four chromosomes. These results suggest that S. demissum has evolved into a cytologically stable state with predominantly bivalent pairing in meiosis.

Cohesion and centromere activity are required for phosphorylation of histone H3 in maize.

Haspin-mediated phosphorylation of histone H3 at threonine 3 (H3T3ph) promotes proper deposition of Aurora B at the inner centromere to ensure faithful chromosome segregation in metazoans. However, the function of H3T3ph remains relatively unexplored in plants. Here, we show that in maize (Zea mays L.) mitotic cells, H3T3ph is concentrated at pericentromeric and centromeric regions. Additional weak H3T3ph signals occur between cohered sister chromatids at prometaphase. Immunostaining on dicentric chromosomes reveals that an inactive centromere cannot maintain H3T3ph at metaphase, indicating that a functional centromere is required for H3T3 phosphorylation. H3T3ph locates at a newly formed centromeric region that lacks detectable CentC sequences and strongly reduced CRM and ZmBs repeat sequences at metaphase II. These results suggest that centromeric localization of H3T3ph is not dependent on centromeric sequences. In maize meiocytes, H3T3 phosphorylation occurs at the late diakinesisand extends to the entire chromosome at metaphase I, but is exclusively limited to the centromere at metaphase II. The H3T3ph signals are absent in the afd1 (absence of first division) and sgo1 (shugoshin) mutants during meiosis II when the sister chromatids exhibit random distribution. Further, we show that H3T3ph is mainly located at the pericentromere during meiotic prophase II but is restricted to the inner centromere at metaphase II. We propose that this relocation of H3T3ph depends on tension at the centromere and is required to promote bi-orientation of sister chromatids.

First report on karyotypic and morphometric analysis of a defoliator beetle, Zygogramma bicolorata (Coleoptera: Chrysomelidae) from Jammu region (J&K), India

In the present study, the chromosomes of a defoliator beetle, Zygogramma bicolorata were studied from Jammu region (J&K). These beetles are bio-control agents for controlling the populations of poisonous weed Parthenium hysterophorus. Chromosome morphology, karyotype and morphometric data of chromosomes was done and the diploid number of the species was found to be 24. Meiotic observations included the pachytene, diplotene, late diakinesis and metaphase I bivalent stage. The sex chromosomes (XYp) included the submetacentric X chromosome and a small acrocentric Y chromosome. The study is useful in chromosomal evolution in the family Chrysomelidae and evolution of sex chromosomes in Coleoptera.

Abnormal meiotic chromosome behavior and gametic variation induced by intersectional hybridization in Populus L.

Abnormal meiotic chromosome behavior and pollen variation in two intersectional hybrids between Populus simonii Carr. (section Tacamahaca Spach) and Populus euphratica Oliv. (section Turanga Bge.) were investigated by the squashed technique and indirect immunofluorescence. Both hybrids produced abnormal flower buds characterized by a primary bud with an accessary bud at frequencies of 39.0 and 17.8 %, respectively. Chromosome pairing analysis at late diakinesis showed average configurations 20.31 I + 7.04 II + 1.42 III + 0.04 IV in hybrid “Xhu-DK” and 19.18 I + 7.16 II + 1.45 III + 0.04 IV in hybrid “Xhu-TL”, indicating high divergence between their parent species. Meiotic abnormalities, including precocious chromosome migration, lagging chromosomes, chromosome bridges, and micronuclei, were observed at high frequency, resulting in imbalanced chromosome segregation and chromosome elimination and caused high gamete sterility. Some micronuclei triggered formation of minispindles in Metaphase II, which were responsible for the formation of microcytes. Fused and tripolar spindles in the second meiotic division developed into dyads and triads, respectively, leading to viable first meiotic division restitution (FDR) type unreduced pollen formation. However, Xhu-TL was completely sterile because the high frequency of precocious cytokinesis during the second division prevented unreduced pollen formation though spindle fusion. Xhu-DK produced 29.6 % viable pollen grains. The diameter of viable pollen ranged from 19.6 to 64.9 μm, suggesting that Xhu-DK might be capable of producing unreduced pollen or pollen with a higher ploidy level. The strategy of using the distant hybrids for chromosome introgression and polyploidy breeding in Populus L. was considered.

Colchicine induced asynaptic chromosomal behavior at meiosis in Allium cepa L.

Two asynaptic plants were identified during meiosis from 0.2 % colchicine treated inflorescences for 12 h in Allium cepa. The asynaptic behavior could be noticed in early as well as at late diakinesis / metaphase-I. The chiasma frequency was very low as compared to control. At telophase-I/II, univalents created high abnormality. Unequal distributions, laggards, tripolar and micronuclei were found in asynaptic plants. Pollen fertility was found to be very low (55.1 and 52.6 %). Observations suggest that colchicine treatment impacts the meiosis of Allium cepa, suggesting that chromosomal associations in asynaptic plants are independent of meiotic behavior in the control.

Secretion of paracrine factors enabling expansion of cumulus cells is developmentally regulated in pig oocytes.

To demonstrate secretion of cumulus expansion-enabling factor (CEEF) by porcine oocytes, we used an interspecies testing system. Porcine oocytes were used to condition culture medium, and the presence of CEEF was tested using mouse oocytectomized complexes (OOX), which require CEEF for expansion. Follicle-stimulating hormone-stimulated expansion and synthesis of hyaluronic acid (HA) by mouse OOX were assessed after 18 h of culture in media conditioned by porcine oocytes: 1) at different stages of maturation and 2) in which maturation was inhibited with a specific inhibitor of cdk-kinases, butyrolactone I. Fully grown (GV-germinal vesicle), late-diakinesis (LD), metaphase I (MI), and metaphase II (MII) oocytes were prepared by culture of oocyte-cumulus complexes (OCC) for 0, 22, 27, and 42 h, respectively. To block GV breakdown, porcine oocytes were cultured for 27 h in medium supplemented with butyrolactone I (50 microM). Medium conditioned by oocytes in GV, LD, and after butyrolactone I block allowed full expansion of >90% of mouse OOX, whereas oocytes in MI and MII caused disintegration of mouse OOX without cumulus mucification. To measure synthesis of HA by cumulus cells, 25 mouse OOX were cultured in the conditioned media in the presence of 2.5 microCi of D-[6-(3)H]glucosamine hydrochloride. After 18 h, incorporation of the [(3)H]glucosamine into HA was determined either in complexes (retained HA) or in medium plus complexes (total HA). Total HA accumulation by mouse OOX was not different from that of intact OCC. However, oocytes in GV, LD, and after butyrolactone I treatment enabled mouse OOX to retain significantly more HA within the complex than oocytes in MI and MII. The results indicate that secretion of factors that promote the retention of HA within the complex is developmentally regulated during oocyte maturation.

Selective requirement for Cdc25C protein synthesis during meiotic progression in porcine oocytes.

Fundamental differences between meiosis and mitosis suggest that the shared central cell cycle machinery may be regulated differently during the two division cycles. This paper focuses on unique features of Cdc25C protein function during meiotic progression. We report on the existence of oocyte-specific CDC25C transcripts that differ from their somatic counterparts in the 3' untranslated region. While CDC25C mRNA levels remain constant in fully-grown oocytes, corresponding protein levels increase progressively during maturation to a maximum at metaphase II. Elevation of Cdc25C protein levels in G2-oocytes by mRNA injection failed to increase MPF-kinase levels or to induce premature entry into M-phase. Likewise, antisense-induced arrest of translation (translational arrest) had no effect on chromosome condensation, nucleolar disassembly, or nuclear membrane contraction. By contrast, translational arrest inhibited subsequent events including membrane disassembly and spindle formation. Neither up- nor down-regulation of Cdc25C synthesis after metaphase I plate formation influenced progression to metaphase II. However, translational arrest during metaphase resulted in incomplete chromosome decondensation and abnormal pronuclear membrane assembly after activation. We conclude that Cdc25 protein, translated from unique transcripts, is preferentially located in the oocyte nucleus and is essential for progress through late diakinesis. Subsequently, new synthesis of Cdc25C protein is required for the orderly transition from meiotic to mitotic cell division.

Chromosome movement during meiotic prophase in crane-fly spermatocytes: IV. Actin and the effects of cytochalasin D

Cytochalasin D (CD) was applied to crane-fly spermatocytes at late diakinesis with the aim of perturbing actin structure and actin function, thereby testing the hypothesis that intranuclear chromosome movement during late diakinesis is actin-based. Isolated tests were incubated in a range of CD concentrations (2-100 microM) for 1 or 2 h. None of those treatments resulted in cessation of prophase movements in living cells. An immediate effect of 10-100 microM CD at late diakinesis was the formation of highly refractile, actin-containing cables within the nonchromosomal nucleoplasm. No such cables were observed in vehicle-treated control cells. CD treatments caused autosomal bivalents in unusually large numbers of spermatocytes to become aggregated into densely-packed clusters; for example, with 40 microM CD about 80% of late diakinesis spermatocytes had clustered autosomes, vs. about 25% clustering in untreated cells. We conclude from these data that the mechanism of chromosome positioning at the nuclear envelope is CD-sensitive. Rhodamine-conjugates of phalloidin and DNase I were used to assess the status of actin in untreated cells as well as the effect of CD on actin distribution. Differences in nucleoplasmic staining with phalloidin and DNase I conjugates suggest that nucleoplasm at late diakinesis contains actin in a nonfilamentous form.

Stage-specific expression of the immunophilin FKBP59 messenger ribonucleic acid and protein during differentiation of male germ cells in rabbits and rats.

FKBP59 is an immunophilin that binds the immunosuppressant drugs FK506 and rapamycin. It is a 90-kDa heat shock protein (hsp90)-binding protein that was originally discovered as a member of steroid receptor complexes. FKBP59 is ubiquitous and well conserved, and it appears to be a multifunctional protein. It has peptidylprolyl cis-trans-isomerase activity and therefore may be involved in protein folding as a molecular chaperon. FKBP59 also includes in its structure a tetratricopeptide repeat (TPR) motif and could have a function in the cell division process. In situ hybridization experiments revealed an overexpression of FKBP59 mRNA in rabbit and rat testes in comparison with other organs. This high level of expression was restricted to germ cells of the seminiferous epithelium. Increasing levels of FKBP59 mRNA became obvious from the midpachytene stage, and the strongest signal was observed in the late pachytene, diplotene, and diakinesis primary spermatocytes. The expression then declined progressively in postmeiotic early spermatids. High expression of FKBP59 mRNA did not occur in earlier and later germ cell stages. During prepubertal development, Northern blot and in situ hybridization of rat testes examined at various postnatal ages revealed that FKBP59 mRNA was not expressed at over a basal level until the pachytene stage. High expression of the FKBP59 protein was demonstrated in the rabbit testis by Western blot and was localized by immunohistochemistry from late pachytene spermatocytes to round spermatids. The cell type-specific and developmental stage-specific expression of FKBP59 at a restricted period of male germ cell differentiation suggests that FKBP59 is involved in a specific function during the cell division process.

Fertilisation competence of bovine normally matured or aged oocytes derived from different antral follicles: morphology, protein synthesis, H1 and MBP kinase activity.

We have investigated the fertilisation competence, protein synthesis, histone H1 kinase and myelin basic protein (MBP) kinase activities in three categories of bovine oocytes (derived from three size categories of follicles: M--medium, 2.5-5.0 mm; S--small, 1.5-2.5 mm; T--tiny, 1.0-1.5 mm). In contrast to more or less normal meiotic maturation (85.6%) and fertilisation (70.8%) of M oocytes cultured for 24 h, the fertilisation of M oocytes cultured for 40 h was associated with increased rates of retarded male pronuclear development and retention of the second polar body. The S and T oocytes cultured for 24 h or 40 h were mostly arrested at defective late diakinesis-metaphase I (77.5-100%) stage. After fertilisation of S and T oocytes cultured for 24 h no polar body was extruded and formation of one, three or four female pronuclei, together with mostly normal male pronuclei, was observed. The fertilisation of S and T oocytes after 40 h culture resulted in a higher number of female and a decreased number of male pronuclei. A major change in the pattern of protein synthesis was associated with the resumption of meiosis. There were no significant differences in the profile of protein synthesis between oocyte categories in all groups either matured or fertilised. The H1 kinase activity reached comparable increased levels in oocytes of all categories matured for 24 h and decreased during the 40 h culture, most significantly in M oocytes. The MBP kinase activity was at approximately the same high level in all categories of oocytes after 24 h of culture and remained stable until 40 h. The fertilisation after 24 h of culture resulted, in M oocytes, in low levels of both H1 and MBP kinase activities; in S oocytes, only H1 kinase was completely inactivated while MBP kinase activity decreased to some extent; in T oocytes, both H1 and MBP kinase activity decreased. Fertilisation of all oocyte categories after 40 h culture resulted in complete inactivation of both these kinases to their basal levels.

The absence of chiasma terminalization and inverted meiosis in males and females of Myrmus miriformis Fn. (Corizidae, Heteroptera)

Terminalization of chiasmata and orientation of bivalents were analysed in males and females of Myrmus miriformis Fn. (Corizidae, Heteroptera). In spermatogenesis, the frequencies of the largest autosome bivalent with medial, subterminal and terminal chiasmata were compared at diplotene, early and late diakinesis and metaphase I. The bivalent displayed a single subterminal or medial chiasma in nearly 90 per cent of cells at diplotene. The frequency of such bivalents was still over 80 per cent at metaphase I, indicating the absence of complete chiasma terminalization. It is possible that chiasmata do not terminalize at all. In oogenesis, all bivalents at metaphase I displayed subterminal or medial chiasmata, indicating that terminalization is also absent in females. In both males and females all bivalents co-orientated at metaphase I, the telomeres of homologous chromosomes being orientated towards opposite spindle poles. Hence, they underwent prereduction in meiosis; no evidence for an inverted meiosis could be found.

The absence of chiasma terminalization and inverted meiosis in males and females of Myrmus miriformis Fn. (Corizidae, Heteroptera)

Terminalization of chiasmata and orientation of bivalents were analysed in males and females of Myrmus miriformis Fn. (Corizidae, Heteroptera). In spermatogenesis, the frequencies of the largest autosome bivalent with medial, subterminal and terminal chiasmata were compared at diplotene, early and late diakinesis and metaphase I. The bivalent displayed a single subterminal or medial chiasma in nearly 90 per cent of cells at diplotene. The frequency of such bivalents was still over 80 per cent at metaphase I, indicating the absence of complete chiasma terminalization. It is possible that chiasmata do not terminalize at all. In oogenesis, all bivalents at metaphase I displayed subterminal or medial chiasmata, indicating that terminalization is also absent in females. In both males and females all bivalents co-orientated at metaphase I, the telomeres of homologous chromosomes being orientated towards opposite spindle poles. Hence, they underwent prereduction in meiosis; no evidence for an inverted meiosis could be found.

Co-culture with pig membrana granulosa cells modulates the activity of cdc2 and MAP kinase in maturing cattle oocytes.

Bovine cumulus-enclosed oocytes, initially cultured up to diakinesis (8 h of initial culture) or metaphase I (12 h of initial culture), were subsequently co-cultured for 6 h in contact with pig membrana granulosa (PMG) cells and then assayed for histone H1 and MAP kinase activities. In addition, the phosphorylation state of ERK 1,2 proteins was determined by Western blotting. The alterations in nuclear envelope breakdown, meiotic spindle formation and the patterns of chromosome condensation were analysed by immunofluorescence and transmission electron microscopy. The diakinesis-stage oocytes (initially cultured for 8 h) already possessed high histone H1 kinase and MAP kinase activities that were correlated with condensed and partially individualised chromosomes. The ERK 1 and most ERK 2 proteins were partly phosphorylated. Following the 6 h co-culture of these oocytes with PMG a rapid decrease in MAP kinase activity and a slower decrease in histone H1 kinase occurred, as well as ERK 1 and ERK 2 dephosphorylation. Both kinase activities and ERK 1,2 phosphorylation were fully restored following the release of the oocytes from co-culture and a subsequent culture in the absence of PMG. Moreover, the clumped bivalents were reindividualised and 56% of these oocytes reached metaphase II after 20 h of culture without PMG. The metaphase I oocytes, initially cultured for 12 h, displayed a fusiform meiotic spindle and a metaphase array of chromosomal bivalents, accompanied by high levels of both histone H1 and MAP kinase activity. Co-culture of MI oocytes with PMG abolished the activity of both kinases and caused the dephosphorylation of ERK 1 and ERK 2. Furthermore, the spindle microtubules were depolymerised and the chromosomal bivalents clumped into a single mass. Neither of the protein kinase activities nor the meiotic spindle were restored following subsequent culture in the absence of PMG for up to 20 h. These observations indicate that under in vitro conditions membrana granulosa cells can cause a prompt decrease in histone H1 and MAP kinase activities, and metaphase I oocytes. While these events are fully reversible in late diakinesis oocytes, metaphase I oocytes did not complete maturation after release from co-culture.

Correspondence between C-banding and Ag-NOR in the Sex Chromosomes of Belostoma oxyurum (Belostomatidae, Heteroptera).

The mitotic and meiotic sex chromosomes of Belostoma oxyurum have been analyzed by C-banding and Ag-NOR staining. In mitotic prometaphases the Y chromosome and both telomeric regions of the X are C positive. However, at meiosis one telomeric region of the X chromosome presents always a C positive band, while the other telomeric region of this chromosome and the whole of the Y chromosome are light C banded during diplotene, the diffuse stage and early diakinesis. All these bands appear C positive by late diakinesis. In mitotic prometaphases, a positive Ag-NOR is revealed at one telomeric region of the X chromosome, but not in the Y chromosome. Besides, some intra and/or interindividual variation in NOR expression in the X chromosome is encountered. No positive Ag-NOR band is observed at meiosis.A comparison of the results obtained in mitotic and meiotic cells after C- and Ag-NOR banding lead to the conclusion that in Belostoma oxyurum the intermediate staining of the C-bands of the sex chromosomes at meiosis is related to both the DNA composition (repetitive DNA, rRNA genes) and its packaging (cycle of condensation).

Chromosome pairing and potential for intergeneric recombination in some hypotetraploid somatic hybrids of Lycopersicon esculentum (+) Solanum tuberosum

Three somatic hybrids resulting from protoplast fusions of a diploid kanamycin-resistant line of tomato (Lycopersicon esculentum) and a dihaploid hygromycin-resistant transformant of a monohaploid potato (Solanum tuberosum) line were used for a cytogenetic study on chromosome pairing and meiotic recombination. Chromosome counts in root-tip meristem cells revealed two hypotetraploids with chromosome complements of 2n = 46 and one with 2n = 47. Electron microscope analyses of synaptonemal complex spreads of hypotonically burst protoplasts at mid prophase I showed abundant exchanges of pairing partners in multivalents involving as many as eight chromosomes. In the cells at late pachytene recombination nodules were found in multivalents on both sides of pairing partner exchanges, indicating recombination at both homologous and homoeologous sites. Light microscope observations of pollen mother cells at late diakinesis and metaphase I also revealed multivalents, though their occurrence in low frequencies betrays the reduction of multivalent number and complexity. Precocious separation of half bivalents at metaphase I and lagging of univalents at anaphase I were observed frequently. Bridges, which may result from an apparent inversion loop found in the synaptonemal complexes of a mid prophase I nucleus, were also quite common at anaphase I, though the expected accompanying fragments could be detected in only a few cells. Most striking were the high frequencies of first division restitution in preparations at metaphase II/anaphase II, giving rise to unreduced gametes. In spite of the expected high numbers of balanced haploid and diploid gametes, male fertility, as revealed by pollen staining, was found to be negligible.

Correction of chlorophyll deficiency in alloplasmic male sterileBrassica junceathrough recombination between chloroplast genomes

SummaryBrassica junceacv. Pusa Bold carryingB. oxyrrhinacytoplasm (oxycytoplasm) was male sterile and chlorotic under field conditions at low temperature (Prakash & Chopra, 1990). Leaf protoplasts of the chlorotic male sterile alloplasmic line (2n = 36) were fused with hypocotyl protoplasts of green male fertile,B. junceacv. RLM-198 (2n = 36) using polyethylene glycol. Of the 1043 plants regenerated from 10 fusion experiments, 123 had ‘gigas’ features and were identified as presumptive fusion products. Among field-grown population, one plant was dark green even at low temperatures and male sterile. It possessed 72 chromosomes which formed 36 bivalents at late diakinesis of meiosis-I. This plant was back-crossed toB. junceacv. Pusa Bold (the maintainer line) for three successive generations. One male sterile, normal green BC3 progeny plant with 2n = 36 was analyzed for organelle constitution. Probing its total DNA with the mitochondrial gene for cytochrome oxidase subunit I revealed that it possessed mitochondria ofB. oxyrrhina. Southern hybridization pattern with the gene for ribulose bisphosphate carboxylase oxygenase-large subunit (rbcL) revealed that the chloroplast genome of the chlorophyll deficiencycorrected plant had characteristics of bothB. junceaandB. oxyrrhina. The deficiency correction has been attributed to recombination between chloroplast genomes of the two species.

Semi-Rigidity of Chain Trivalents as a Factor Contributing to their Adjacent Orientation at Metaphase I in Triploid Petunia.

In pollen mother cells of triploid Petunia axillaris (Lam.) B. S. P. at the stage of late diakinesis some of the chain trivalents revealed an unusual form of rigidity. These are here described as “hook-shaped”, although earlier documented as ‘L’ shaped chain trivalents (Reddi and Padmaja 1979). The other point considered was whether a similar form of rigidity is also true for some rod bivalents, where the single chiasma was terminalized. Such“ paranormal bivalents” were in fact observed. The corresponding observation of hook-shaped trivalents at late diakinesis is interpreted to be a factor that could limit the frequency of alternate orientation and incidentally increase the frequency of adjacent orientation of such chains at metaphase I.

Effects of cyclic AMP on the spontaneous meiotic maturation of cumulus-free bovine oocytes cultured in chemically defined medium.

The rate of spontaneous meiotic maturation and the period of commitment to this process were determined in bovine oocytes devoid of surrounding cumulus cells, cultured in chemically defined medium with bovine serum albumin in the absence of serum. The effects of compounds that are known to elevate levels of intracellular cyclic adenosine monophosphate (cAMP) on the resumption and progression of meiosis were investigated. Bovine oocytes were mass-harvested, denuded of cumulus cells, and cultured in 2A-BMOC medium supplemented with 0.5% bovine serum albumin. Intracellular cAMP levels were indirectly modified using 8-bromo-cAMP, dibutyryl cAMP (dbcAMP), forskolin, or 3-isobutyl-1-methyl xanthine (IBMX). Meiotic maturation was scored cytogenetically. Ninety percent of denuded bovine oocytes mature after 24 h, with 65% progressing beyond anaphase I. These oocytes remain at the germinal vesicle (GV) stage for up to 8 h in culture. GV breakdown (GVBD) occurs in 40.5% of oocytes at 9 h. The peak times for the different meiotic stages were 12 h for diakinesis, 15 h for late diakinesis to metaphase I, 20 h for metaphase I, and 24 h for telophase I. By 48 h, most had reached metaphase II. There is a 2-h lag period between the time at which they become irreversibly committed to mature (at 7 h) and when they demonstrate GVBD (at 9 h). Incubation for 12 h with high concentrations of 8-bromo-cAMP and forskolin significantly inhibited GVBD, while the effect of dbcAMP was similar but less pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)

Does autocatalytic amplification of maturation-promoting factor (MPF) exist in mammalian oocytes?

The method of polyethylene-glycol-induced fusion of mammalian oocytes was applied to study maturation-promoting factor (MPF) activity. After homologous fusions of one maturing--late diakinesis (LD), metaphase I (MI)--pig or mouse oocyte to one, two, or three immature-germinal vesicle (GV)--oocytes, giant cells were cultured in control or cycloheximide supplemented medium for 3 hours. The occurrence of germinal vesicle breakdown (GVBD) and premature chromosome condensation (PCC) served as a control of MPF activity. In giant cells composed of one maturing and one, two or three immature oocytes, GVBD and PCC were observed in all cases after cultivation in the control medium. In the presence of cycloheximide, the completion of GVBD and PCC remained high when one maturing and one immature oocyte were fused (83.7% and 95.7% of GVBD in pig and mouse, respectively). However, in giant cells composed of one maturing and up to three immature oocytes, all GVs were broken down only occasionally (4.8% and 11.7% in pig and mouse, respectively). These results suggest that in pig and mouse oocytes MPF does not amplify autocatalytically, but requires active protein synthesis for its production.