Polyploidy in the haplontic yeast Schizosaccharomyces pombe: construction and analysis of strains

The fission yeast Schizosaccharomyces pombe has a haplontic life cycle in which the diplophase is confined to the zygote. Through the use of one- and two-step protoplast fusions we show that the ploidy can be increased up to pentaploid. The polyploid fusion products are rather unstable and segregate cells of lower ploidies by gradual loss of chromosomes during mitotic divisions. The polyploid cells conjugate normally but are prone to arrest at various stages of meiosis (1-, 2- and 3-spored asci, binucleate spores) and/or produce inviable, most probably aneuploid, spores. Marker segregation in the complete tetrads indicates the multiple association of homologous chromosomes. In tetra- and penta-ploid meiosis, multispored (6- to 7-spored) asci are also produced, probably by postmeiotic division of the nuclei.     

Ultrastructure of the final nuclear maturation of bovine oocytes in vitro

Summary Cumulus-oocyte complexes were obtained from cow ovaries by aspiration from small (1–6 mm in diameter) antral follicles after slaughter. Complexes with a compact multilayered cumulus investment were cultured and subsequently processed for electron microscopy after various periods of culture. By morphological criteria the oocytes could be divided into the following sequence of meiotic stages. The oocyte nucleus I stage was characterized by a spherical nucleus located peripherally in the ooplasm while undulation of the nuclear envelope and initial chromatin condensation was seen at the oocyte nucleus II stage. The oocyte nucleus breakdown stage was characterized by formation of long slender projections from the nuclear envelope in which the envelope doubled back on itself, appearance of dense areas and haphazardly oriented microtubules in the nucleus, marked condensation of the chromatin, and dissolution of the nuclear envelope into irregular vesicles and tubules. The condensed chromatin I stage was characterized by the location of condensed chromatin configuration and uniformly oriented microtubules in a dense area peripherally in the ooplasm while the final condensed chromatin II stage was characterized by a gradual invasion of condensed chromatin configurations into a dense area combined with the presence of the first polar body in the perivitelline space.     

Paternal uniparental disomy of chromosome 14 and unique exchange of chromosome 7 in cases of spontaneous abortion

To investigate the involvement of uniparental disomies (UPDs) in spontaneous abortion, the polymorphic patterns of microsatellites on each chromosome were analyzed in 164 cases of abortion. Eighty-three of the 164 cases had chromosomal abnormalities. In 79 of the remaining 81 cases with normal karyotypes, the microsatellite analysis revealed that biparental patterns were present in the informative microsatellites in all chromosomes. In one of the remaining two cases, however, the polymorphic patterns of chromosome 14 appeared to be both of paternal origin. The patterns of the distal of the long arm were homozygous, and those of the remaining region were heterozygous. That is, this fetus had paternal UPD 14, originating from meiosis I nondisjunction. In the other case, the polymorphic patterns of the distal one third of the long arm of chromosome 7 were uniparental (maternal) in origin whereas those of the remaining region of this chromosome were biparental. These findings thus suggested that this chromosome might have originated from chromatid exchange between the long arms of paternal and maternal chromosome 7 at the first mitotic division. Microsatellite analysis, however, produced no evidence of duplication or deletion of any segments. The findings also suggest the possibility that some UPDs may cause spontaneous abortion.     

Natural translocation of a large segment of chromosome III to chromosome I in a laboratory strain of Saccharomyces cerevisiae

We have investigated chromosomal segregation during meiosis in a cross between two polymorphic haploid laboratory strains of Saccharomyces cerevisiae, FL100 and GRF18. These two strains have large chromosome-length polymorphisms for chromosomes I and III allowing for easy scoring of parental chromosomes after meiotic segregation. Chromosome III in the FL100 strain was 35 kb shorter than chromosome III in GRF18, while FL100 chromosome I was 40 kb larger than chromosome I in GRF18. Segregation analysis of chromosomes I and III in 50 tetrads showed an apparent association between chromosomes I and III, whereas only the original parental association of chromosomes I and III was found in the spores. By hybridization with chromosome-specific probes we have shown that the polymorphisms are due to a large translocation from chromosome III onto chromosome I in FL100. The translocated fragment is larger than 80 kb and was mapped between Ty and HML. In nine tetrads analyzed, chromosome-length polymorphisms which did not segregate according to Mendelian law were observed. Received: 31 January 1996 / 12 April 1996     

Initiation of the second meiotic division in Schizosaccharomyces pombe shares common functions with that of mitosis

Summary The cell-cycle start gene, cdc2 ⁺, and its activator, cdc25 ⁺, are required for the initiation of the second meiotic nuclear division. They exert their functions downstream of mes1, a gene presumed to act between meiosis I and meiosis II. weel ⁺, the inhibitor of cdc2 ⁺ in mitotic control, does not appear to play any role in this process.     

Distinct effects of the recurrent Mlh1G67R mutation on MMR functions, cancer, and meiosis

Mutations in the human DNA mismatch repair (MMR) gene MLH1 are associated with hereditary nonpolyposis colorectal cancer (Lynch syndrome, HNPCC) and a significant proportion of sporadic colorectal cancer. The inactivation of MLH1 results in the accumulation of somatic mutations in the genome of tumor cells and resistance to the genotoxic effects of a variety of DNA damaging agents. To study the effect of MLH1 missense mutations on cancer susceptibility, we generated a mouse line carrying the recurrent Mlh1(G67R) mutation that is located in one of the ATP-binding domains of Mlh1. Although the Mlh1(G67R) mutation resulted in DNA repair deficiency in homozygous mutant mice, it did not affect the MMR-mediated cellular response to DNA damage, including the apoptotic response of epithelial cells in the intestinal mucosa to cisplatin, which was defective in Mlh1(-/-) mice but remained normal in Mlh1(G67R/G67R) mice. Similar to Mlh1(-/-) mice, Mlh1(G67R/G67R) mutant mice displayed a strong cancer predisposition phenotype. However, in contrast to Mlh1(-/-) mice, Mlh1(G67R/G67R) mutant mice developed significantly fewer intestinal tumors, indicating that Mlh1 missense mutations can affect MMR tumor suppressor functions in a tissue-specific manner. In addition, Mlh1(G67R/G67R) mice were sterile because of the inability of the mutant Mlh1(G67R) protein to interact with meiotic chromosomes at pachynema, demonstrating that the ATPase activity of Mlh1 is essential for fertility in mammals.     

Role of gap junctions and protein kinase A during the development of oocyte maturational competence in Ayu (Plecoglossus altivelis)

Meiotic resumption in teleost oocytes is induced by a maturation-inducing hormone (MIH). The sensitivity of oocytes to MIH, also known as oocyte maturational competence (OMC), is induced by LH via mechanisms that are not fully understood. A previous study of Ayu (Plecoglossus altivelis) showed the presence of functional heterologous gap junctions (GJs) between oocytes and their surrounding granulosa cells. The objectives of this study were to determine the role of ovarian GJs and of protein kinase A (PKA) during the acquisition of OMC. We examined the effects of the specific GJ inhibitor carbenoxolone (CBX) and 18alpha-glycyrrhetinic acid (alpha-GA) on the LH-(hCG)-dependent acquisition of OMC and on MIH-(17,20beta-dihydroxy-4-pregnen-3-one)-dependent meiotic resumption; measured the cAMP content of ovarian follicles during the hCG-dependent acquisition of OMC; and determined the effects of PK activators and inhibitors on hCG-dependent OMC. Production of follicular cAMP increased during the hCG-dependent acquisition of OMC. Both GJ inhibitors and the PKA inhibitor H8-dihydrochloride, but not the PKC inhibitor GF109203X, suppressed the hCG-dependent acquisition of OMC in a dose-dependent manner. The PKA activator forskolin induced OMC with a similar potency to hCG. Unlike previous observations with teleosts where disruption of heterologous GJ either blocks or stimulates meiotic resumption, treatment with GJ inhibitors did not affect MIH-dependent meiotic resumption in maturationally competent follicles of Ayu. These observations suggest that ovarian GJs are essential for LH-dependent acquisition of OMC but not for MIH-dependent meiotic resumption, and that the stimulation of OMC by LH is mediated by cAMP-dependent PKA. They are also consistent with the view that a precise balance between GJ-mediated signals (positive or negative) and oocyte maturational readiness is required for hormonally regulated meiotic resumption.     

Evaluation of the phosphodiesterase 3 inhibitor ORG 9935 as a contraceptive in female macaques: initial trials

Background

The study was conducted to determine whether a phosphodiesterase (PDE) 3 inhibitor has potential as a novel contraceptive in primates.

Methods

Regularly cycling adult female cynomolgus macaques of proven fertility (n=16) were treated for 7 months with placebo (controls) or the PDE3 inhibitor ORG 9935 as a daily food treat (150 mg/kg) or as a weekly depot injection (150 mg/kg, sc). After 1 month, a male of proven fertility was introduced into each group. Females underwent weekly monitoring of progesterone (P) and ultrasound evaluation for pregnancy if P remained elevated (1.0 ng/mL) >3 weeks. ORG 9935 values were evaluated using high-performance liquid chromatography.

Results

Overall, the pregnancy rate in ORG 9935-treated monkeys (4/8, 50%) did not differ from controls (7/8, 88%; p=.5). However, no animal became pregnant in a cycle when the serum level of ORG 9935 exceeded 300 nmol/L. Moreover, two treated monkeys who mated throughout the treatment phase and did not conceive became pregnant within four cycles after stopping ORG 9935. The other two animals were discontinued prematurely from the protocol.

Conclusions

These results demonstrate that ORG 9935 may prevent pregnancy in primates at serum concentrations above 300 nmol/L and that the effect is reversible.