Rate Of Meiosis Research Articles

Role of PTGS2-generated PGE2 during gonadotrophin-induced bovine oocyte maturation and cumulus cell expansion

Prostaglandin E2 (PGE2) is an autocrine/paracrine factor which mediates gonadotrophin (Gn) stimulation of cumulus expansion and oocyte maturation in rodents. Its role in bovine oocyte maturation is less characterized. This study detected PTGS2 (COX2) and PGE synthases (PTGES1, PTGES2 and PTGES3) in bovine cumulus–oocyte complexes (COC). Only PTGS2 and PTGES1 expression changed during maturation. In Gn-free media, no cumulus expansion and ∼45% nuclear maturation was achieved, while Gn-induced maturation showed full cumulus expansion (score 3) and ∼87% maturation. PGE2 supplementation without Gn induced mild cumulus expansion (score 0.5–1) but increased nuclear maturation to levels similar to those obtained with Gn alone. In the presence of Gn, exogenous PGE2 did not affect expansion or nuclear maturation and subsequent embryo development. Treatment with PTGS2 selective inhibitor (NS398), PTGS2-specific siRNA or PTGER2-receptor antagonist (AH6809) resulted in ∼20–25% reduction in nuclear maturation. NS398 and AH6809 did not affect cumulus expansion. Most oocytes not reaching metaphase of second meiosis (MII) following NS398, AH6809 and PTGS2-specific siRNA treatments were at MI. After longer maturation, NS398-treated oocytes had normal MII rate and uncompromised embryo development. PGE2 has a limited role in cumulus expansion in bovine COC but is important for the timing of Gn-induced nuclear maturation.We confirmed that genes involved in the synthesis of prostaglandin E2 (PGE2) are expressed by cumulus–oocyte complexes (or eggs) of cows and that PGE2 is synthesized during oocyte maturation in the presence of gonadotrophin hormones. When we inhibited synthesis of PGE2 or blocked its receptors, oocyte maturation, but not cumulus expansion, was compromised. Further investigation showed that oocyte maturation is delayed but not arrested when PGE2 synthesis is inhibited. On the other hand, addition of exogenous PGE2 induced a high maturation rate and mild cumulus expansion only in the absence of gonadotrophin stimulation, and had no effect in the presence of gonadotrophin.

270 HIGH GASEOUS PRESSURE PRETREATMENT IN IN VITRO MATURATION OF CANINE OOCYTES

Stress processes, such as hydrostatic pressure treatment of oocytes in different species, have been reported to increased embryo rate following in vitro maturation (IVM). However, studies on high gaseous pressure (HGP) pretreatment in IVM of oocytes from domestic animals are lacking in the literature. This experiment aimed to test HGP pretreatment of canine oocytes to increase meiosis achievement (metaphase II) after IVM. A total of 502 canine oocytes (6 replicates) were used in this study. Ovaries from 15 bitches were obtained from local shelters or rescue organizations after ovariohysterectomy. Sample collection was blind as to reproductive stage and dog age. The ovaries were transported to the laboratory in 0.9% NaCl and were processed within 3 h of collection. The ovarian cortex was sliced and washed in PBS with 1% FCS to release cumulus–oocyte complexes. Grade 1 and 2 cumulus–oocyte complexes were selected for IVM and randomly distributed into 3 treatment groups: HGP (oocytes placed in PBS and subjected to pressure chamber; 206 oocytes), ambient control (oocytes maintained in TCM-HEPES at room temperature for 60 min; 130 oocytes), and laboratory protocol (oocytes IVM after morphologic selection; 166 oocytes). The average pressure, initial and final temperature, and duration of oocytes in the HGP pretreatment were 76.19 atm (±0.92), 32.20°C (±5.17) and 27.71°C (±3.17), and 60 min, respectively. In vitro maturation was carried out for 72 h at 37°C in a high-glucose medium, consisting of TCM-199 with 2.2 mg mL–1 of sodium bicarbonate (11150, Gibco, Grand Island, NY, USA), and supplemented with 0.1% polyvinyl alcohol (P-8136, Sigma, St. Louis, MO, USA), 0.991 mg mL–1 of glucose (108337, Merck, Darmstadt, Germany), 50 µg mL–1 of gentamicin, 22 µg mL–1 of pyruvic acid, 20 µg mL–1 of oestradiol (E-8875, Sigma), 0.5 µg mL–1 of FSH (Folltropin-V, Vetepharm Inca), 0.03 IU mL–1 of hCG (Chorulon®, Intervet, Kenilworth, NJ, USA), under 20% oxygen tension. The number of oocytes at each stage (prophase to metaphase II) was recorded according to the morphology of nuclear content after staining with Hoechst 33342. For comparison purposes of nuclear maturation in oocytes, data were analysed by Fisher’s exact test. Differences at a P-value ≤0.05 were considered significant. Oocytes from the HGP, ambient control, and laboratory protocol groups had similar meiotic progression to the metaphase II stage (metaphase I–anaphase I–telophase I–metaphase II), and were 35.4% (73/206), 30.8% (40/130), and 34.9% (58/166), respectively (P ≥ 0.05). The proportion of oocytes without chromatin or having an irregular organisation was not different among groups. In conclusion, results indicate that HGP pretreatment as used in this experiment did not improve meiosis rates in IVM canine oocytes. Further investigations to understand the significance of HGP pretreatment in IVM and in vitro production of canine embryos are ongoing in our laboratory.

A transcriptional autoregulatory loop forKIN28-CCL1 andSRB10-SRB11, each encoding RNA polymerase II CTD kinase-cyclin pair, stimulates the meiotic development ofS. cerevisiae

Alkalization of the medium is associated with and required for the cellular development to meiosis and sporulation in the yeast Saccharomyces cerevisiae. To elucidate the molecular mechanisms for the significance of external alkalization, we isolated mutants defective in division arrest at G1 phase under an alkaline condition. The mutants obtained had recessive alleles of SRB10 encoding the cyclin (SRB11)-dependent protein kinase that phosphorylates the CTD domain of the largest subunit of RNA polymerase II and negatively regulates the transcriptional initiation of certain genes. A delta srb11 deletion mutant showed the same cell cycle defect. When shifted to alkali, wild-type cells decreased transcript levels of G1-cyclin genes (CLN1 to CLN3) and KIN28-CCL1 (encoding another CTD kinase-cyclin pair which, in contrast, stimulates the promoter clearance and transcriptional elongation in most genes), resulting in the accumulation of G1 cells and the hypophosphorylated form of RNA polymerase II and in an increase in cell size. However, under the same conditions, a delta srb10 mutant was defective in these events, except the downregulation of CLN1 and CLN2. The delta srb10 mutation also influenced on the transcript levels of meiosis-inducing genes called IME1 and IME2: the mutation elevated the transcript level of IME1 but reduced that of IME2, resulting in partial defects in premeiotic DNA synthesis and meiosis. Overexpression of KIN28 and CCL1 in wild-type cells impaired the alkali-induced G1 arrest and the rate of meiosis and elevated the transcript levels of SRB11 and IME1. These results indicate that a transcriptional autoregulatory loop for KIN28-CCL1 and SRB10-SRB11 is important for G1 arrest and meiosis. We also found that environmental conditions for meiosis finely regulate the transcript levels of KIN28 and CCL1, such that nitrogen starvation first elevates them but subsequent alkalization of medium decreases them.

Meiotic nondisjunction in oocytes from aged Djungarian hamsters correlates with an alteration in meiosis rate but not in univalent formation.

The effect of maternal ageing on the meiotic rate, on chiasma and univalent frequency as well as on heteroploidy in secondary oocytes from Djungarian hamsters was examined. The frequency of hyperhaploid oocytes increased from 0.6% in young (8-14 weeks) to 2.8% in middle-aged (26-46 weeks) and reached 3.6% in the oldest females (49-75 weeks). On the basis of malsegregated bivalents per oocyte, nondisjunction occurred most often in the middle-aged group (5.42 X 10(-2) bivalents per oocyte). Hereby, the large meta- and submetacentric A-D chromosomes were preferentially involved. Furthermore, the pattern of nondisjunction was not different from that expected on the basis of chromosome length or induced by colchicine. The large A-D chromosomes did not show any alteration in chiasma or univalent frequency. Terminalized chiasmata were only detected in the E group and univalents increased slightly, but not significantly in the small chromosomes (G group). At higher ages, both chromosome groups were not preferentially involved in nondisjunction. Presegregation slightly increased with age and affected more or less all bivalents, whereas the incidence of diploidy significantly decreased. With respect to the rate of meiosis in oocytes from aged females, the resumption was delayed at metaphase I. Our data suggest that failures in the control of oocyte proliferation are involved in nondisjunction rather than the "production-line." Furthermore, a model is proposed to explain nondisjunction of specific bivalents at certain maternal ages.

A model relating the incidence of meiotic trisomy to maternal age

A model is developed to explain the well-documented increase in the incidence of meiotic trisomies with increasing maternal age. This theoretical framework applies to all chromosomes, of which trisomy 21 (responsible for Down's syndrome in humans) is considered as a special case; the model can also be readily extended to trisomies of other mammals. The basic mechanism proposed links the hormonal environment of the oocyte to the durations of certain stages of meiosis. Changes in the hormonal environment, especially through aging, can slow the overall rate of meiosis, lengthening the interval from the resumption of meiosis in dictyotene until anaphase I. This extends the period in which homologous chromosomes are vulnerable to premature separation, increasing the probability of an unequal distribution of chromosomes in the first meiotic division. Testable predictions of the model are presented and discussed.

The relation of temperature to preleptotene chromosome contraction in Lilium

Preleptotene chromosome contraction was observed in individuals of Lilium longiflorum “Ace” grown at high temperatures in a glasshouse and at moderate or cool temperatures in a lathhouse. The lathhouse plants exhibited an extremely high degree of preleptotene contraction and the glasshouse plants an extremely low degree of preleptotene contraction. It is known that high temperature increases and low temperature decreases the rate of meiosis. Therefore it is proposed that the amounts of preleptotene contraction observed in lathhouse and glasshouse plants reflect the relative durations of the preleptotene contraction stages in these individuals. Differences in degree of chromosome contraction among microsporocytes in different regions of the anther may be attributed to the effects of axial and transverse gradients of metabolism on duration of the preleptotene contraction state. Any genotype that increases or decreases microsporocyte sensitivity to temperature, or otherwise alters the duration of the preleptotene spiralization period, should influence the degree of preleptotene contraction.

Populationsgenetische Untersuchungen anSaccharomyces Cerevisiae. II. Der Einfluß von Esterase-Loci auf Genfrequenzverändernde Faktoren

The influence of two esterase loci, each possessing two alleles (Est1s,Est1f andEst2s,Est2f), on the rate of reproduction in haploid yeasts (Saccharomyces cerevisiae) during the vegetative phase, as well as on the rate of meiosis and on the vitality of spores was investigated. Alleles of theEst1 locus had no apparent effect on the rate of vegetative reproduction. In contrast, genotypes with theEst2f allele possessed a definite selective advantage. Whereas theEst2 locus had no demonstrable influence on meiosis,Est1s/Est1s homozygotes, in contrast to the other two genotypes, exhibited an increased rate of meiosis. A connection between spore lethality and those esterases in question could not be demonstrated.

Rates of meiosis, spindle irregulartities and microsporocyte dividion in Bromus trinii x B. carinatus.

1. In the hybrid, Bromus trinii × Bromus carinatus, anthers were studied in which the proportion of microsporocytes in meiosis-I to those in meiosis-II ranged from 88∶1 to 1∶26. The different stages of meiosis occurred in various frequencies in individual anthers. 2. When anthers reach a certain stage of development the range of meiotic stages increases markedly. It is suggested that the rate of meiosis is altered, and variable among individual microsporocytes. 3. Multipolar spindles were observed at metaphase-I, metaphase-II, and multinucleate configurations at prophase-II and telophase-II. The separation of one nucleus into two was observed at late anaphase-I, prophase-II, late anaphase-II and telophase-II. 4. The multipolar spindles were generally tripolar. At prophase-II microsporocytes with 3 nuclei were most common, 4 nuclei were infrequent, 5 were rare. At telophase-II there were 2–7 nuclei per microsporocyte; 5 nuclei were most commonly found. Spindle poles were typically well separated; in multinucleate microsporocytes the nuclei were generally well separated. 5. The frequencies of multinucleate prophase-II microsporocytes varied from 0 to 36% in 41 anthers from 7 plants. The frequencies of telophase-II multinucleate microsporocytes ranged from 0 to 40% in the same anthers. There were no obvious differences between plants. 6. No correlation was found between frequencies of extra nuclei and lagging chromosomes. Therefore extra nuclei are believed to arise from subdivisions of existing nuclei; the formation of the latter is attributed to the previous establishment of spindle poles. 7. It is suggested that multipolar spindles and supernumerary nuclei arise from splitting or extra division of spindle organizers which may occur at various times throughout the meiotic cycles. It is further suggested that spindle organizers repel one another, most effectively during spindle formation, but to some extent also during other stages of meiosis. 8. Because multinucleate microsporocytes are generally found in older anthers, a relationship is suggested between the spindle irregularities that produce multipolar spindles and multinucleate microsporocytes and an altered, irregular rate of meiosis. Generally spindle aberrations are most often present at prophase-II when this stage is relatively infrequent, and at telophase-II when this stage is relatively abundant; however, the possible effect of differences in ages of anthers precludes the suggestion of a relationship between duration of meiotic stages and spindle irregularity. Frequencies of spindle aberrations are not necessarily constant in individual anthers, but may change during the course of the meiotic divisions. 9. Microsporocytes may divide normally into dyads at telophase-I and tetrads at telophase-II, or microsporocytes may remain single cells throughout meiosis. Extreme variations were found among individual anthers in the rate of microsporocyte division. Cell division tends to occur normally in young anthers and to be delayed in older anthers. Normal and delayed division are described. 10. The presence of lagging chromosomes is related to a somewhat delayed microsporocyte division at prophase-II and telophase-II. The presence of extra nuclei is related to a very striking delay in division at prophase-II and telophase-II. Possible reasons for this delay are discussed. 11. A description is given of various spindle types observed in undivided microsporocytes at metaphase-II, and of the telophase-II configurations expected to result from these. It is suggested that the various types of spindles may originate by means of splitting of compound spindle organizers.