Vitrification of mouse germinal vesicle oocytes: effect of treatment temperature and egg yolk on chromatin and spindle normality and cumulus integrity.
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The success rates for cryopreservation of immature oocytes from several species including human remain low, in contrast to major improvements with mature oocytes. In this study, a new approach has been developed using a short exposure ultra-rapid freezing protocol, examining the effect of temperature and egg yolk (two factors which may be expected to influence membrane flexibility) on the cryostability of immature mouse oocytes and cumulus complexes. These two factors were tested in various patterns for their cryoprotective effect using ethylene glycol as the principal cryoprotectant. The results showed that 37 degrees C pre- and post-freeze exposure significantly improved both survival and normal spindle configuration after in-vitro maturation. Egg yolk was found to produce further beneficial effects on both the oocyte and cumulus cell integrity, with the best effects being obtained at 37 degrees C with inclusion of egg yolk both before and after the freezing. This protocol produced > 80% normal survival post-thaw with intact and attached cumulus complex, 84% maturation rate and 45% normal metaphase configuration. In summary, a unique combination of high survival and meiotic normality together with good preservation of the attached cumulus cell mass has been achieved using a simple new vitrification procedure. (+info)
CYS3, a hotspot of meiotic recombination in Saccharomyces cerevisiae. Effects of heterozygosity and mismatch repair functions on gene conversion and recombination intermediates.
(26/6057)
We have examined meiotic recombination at the CYS3 locus. Genetic analysis indicates that CYS3 is a hotspot of meiotic gene conversion, with a putative 5'-3' polarity gradient of conversion frequencies. This gradient is relieved in the presence of msh2 and pms1 mutations, indicating an involvement of mismatch repair functions in meiotic recombination. To investigate the role of mismatch repair proteins in meiotic recombination, we performed a physical analysis of meiotic DNA in wild-type and msh2 pms1 strains in the presence or absence of allelic differences at CYS3. Neither the mutations in CYS3 nor the absence of mismatch repair functions affects the frequency and distribution of nearby recombination-initiating DNA double-strand breaks (DSBs). Processing of DSBs is also similar in msh2 pms1 and wild-type strains. We conclude that mismatch repair functions do not control the distribution of meiotic gene conversion events at the initiating steps. In the MSH2 PMS1 background, strains heteroallelic for frameshift mutations in CYS3 exhibit a frequency of gene conversion greater than that observed for either marker alone. Physical analysis revealed no modification in the formation of DSBs, suggesting that this marker effect results from subsequent processing events that are not yet understood. (+info)
Suppressor analysis of the Saccharomyces cerevisiae gene REC104 reveals a genetic interaction with REC102.
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REC104 is a gene required for the initiation of meiotic recombination in Saccharomyces cerevisiae. To better understand the role of REC104 in meiosis, we used an in vitro mutagenesis technique to create a set of temperature-conditional mutations in REC104 and used one ts allele (rec104-8) in a screen for high-copy suppressors. An increased dosage of the early exchange gene REC102 was found to suppress the conditional recombinational reduction in rec104-8 as well as in several other conditional rec104 alleles. However, no suppression was observed for a null allele of REC104, indicating that the suppression by REC102 is not "bypass" suppression. Overexpression of the early meiotic genes REC114, RAD50, HOP1, and RED1 fails to suppress any of the rec104 conditional alleles, indicating that the suppression might be specific to REC102. (+info)
The role of the mismatch repair machinery in regulating mitotic and meiotic recombination between diverged sequences in yeast.
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Nonidentical recombination substrates recombine less efficiently than do identical substrates in yeast, and much of this inhibition can be attributed to action of the mismatch repair (MMR) machinery. In this study an intron-based inverted repeat assay system has been used to directly compare the rates of mitotic and meiotic recombination between pairs of 350-bp substrates varying from 82% to 100% in sequence identity. The recombination rate data indicate that sequence divergence impacts mitotic and meiotic recombination similarly, although subtle differences are evident. In addition to assessing recombination rates as a function of sequence divergence, the endpoints of mitotic and meiotic recombination events involving 94%-identical substrates were determined by DNA sequencing. The endpoint analysis indicates that the extent of meiotic heteroduplex DNA formed in a MMR-defective strain is 65% longer than that formed in a wild-type strain. These data are consistent with a model in which the MMR machinery interferes with the formation and/or extension of heteroduplex intermediates during recombination. (+info)
Two structural variants of Nek2 kinase, termed Nek2A and Nek2B, are differentially expressed in Xenopus tissues and development.
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Nek2 kinase, a NIMA-related kinase, has been suggested to play both meiotic and mitotic roles in mammals, but its function(s) during development is poorly understood. We have isolated here cDNAs encoding a Xenopus homolog of mammalian Nek2 and have shown that Xenopus Nek2 has two structural variants, termed Nek2A and Nek2B. Nek2A, most likely a C-terminally spliced form, corresponds to the previously described human and mouse Nek2, while Nek2B is most probably a novel, C-terminally unspliced form of Nek2. As a consequence of this (probable) alternative splicing, Nek2B lacks the C-terminal 70-amino-acid sequence of Nek2A, which contains a PEST sequence (or a motif for rapid degradation). Western blot analysis reveals that Nek2A is expressed predominantly in the testis (presumably in spermatocytes) and very weakly in the stomach and, during development, only after the neurula stage. By contrast, Nek2B is expressed mainly in the ovary and in both primary and secondary oocytes and early embryos up to the neurula stage. These results suggest that Nek2A and Nek2B may play both meiotic and mitotic roles, but in a spatially and temporally complementary manner during Xenopus development, and that Nek2B, rather than Nek2A (or the conventional form of Nek2), may play an important role in early development. We discuss the possibility that a counterpart of Xenopus Nek2B might also exist and function in early mammalian development. (+info)
mRNAs encoding a von Ebner's-like protein and the Huntington disease protein are induced in rat male germ cells by Sertoli cells.
(30/6057)
The success of spermatogenesis is dependent upon closely coordinated interactions between Sertoli cells and germ cells. To identify specific molecules that mediate interactions between somatic cells and germ cells in the rat testis, Sertoli cell-germ cell co-cultures and mRNA differential display were used. Two cDNAs, clone 1 (660 nucleotides) and clone 2 (390 nucleotides) were up-regulated when Sertoli cells were co-cultured with pachytene spermatocytes or round spermatids. Northern blot analyses confirmed the differential display expression patterns. Sequence analyses indicated that clone 1 was similar to a von Ebner's gland protein (87% at the nucleotide level and 80% at the amino acid level) and clone 2 was identical to a region of the Huntington disease protein. The von Ebner's-like protein mRNA was induced after 4 h of co-culture, while the Huntington disease protein required 18 h of co-culture for expression. The von Ebner's-like protein was induced in germ cells by a secreted Sertoli cell factor(s) smaller than 10 kDa that is sensitive to freezing and thawing or boiling. The Huntington disease protein was induced in germ cells by a Sertoli cell secreted factor(s) larger than 10 kDa which survives freezing and thawing, but is inactivated by boiling. (+info)
Antioxidants reversibly inhibit the spontaneous resumption of meiosis.
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We previously showed that the cell-permeant antioxidant 2(3)-tert-butyl-4-hydroxyanisole (BHA) inhibited germinal vesicle breakdown (GVBD) in oocyte-cumulus complexes (OCC) of the rat. The objective of the present studies was to assess other antioxidants and whether such inhibition was reversible. Spontaneous GVBD in OCC incubated for 2 h was significantly inhibited (P < 0.005) by nordihydroguaiaretic acid (NDGA; GVBD = 19.4%), BHA (GVBD = 25.7%), octyl gallate (OG; GVBD = 52.2%), ethoxyquin (EQ; GVBD = 58.8%), 2, 6-di-tert-butyl-hydroxymethyl phenol (TBHMP; GVBD = 59%), butylated hydroxytoluene (BHT; GVBD = 59.5%), and tert-butyl hydroperoxide (TBHP; GVBD = 60.0%). Other antioxidants that produced lower but significant (P < 0.05) inhibition of oocyte maturation included propyl gallate (PG; GVBD = 70.3%), 2,4,5-trihydroxybutrophenone (THBP; GVBD = 71.4%), and lauryl gallate (LG; GVBD = 71.4%). Antioxidants that had no effect on oocyte maturation at the same concentration (100 microM) included ascorbic acid, vitamin E, and Trolox. Inhibition of GVBD was evident for up to 8 h of incubation of OCC and denuded oocytes (DO) with BHA or NDGA and was reversed by washing. NDGA was less potent than BHA for inhibition of GVBD in DO, unlike that seen with OCC. Oocyte maturation was induced by incubation of follicles for 3 h with human chorionic gonadotropin (hCG), and this response was inhibited by BHA or NDGA. These findings support the conclusion that cell-permeant antioxidants inhibit spontaneous resumption of meiosis, which may implicate a role of oxygen radicals in oocyte maturation. (+info)
Phosphorylation of histone H3 is required for proper chromosome condensation and segregation.
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Phosphorylation of histone H3 at serine 10 occurs during mitosis in diverse eukaryotes and correlates closely with mitotic and meiotic chromosome condensation. To better understand the function of H3 phosphorylation in vivo, we created strains of Tetrahymena in which a mutant H3 gene (S10A) was the only gene encoding the major H3 protein. Although both micronuclei and macronuclei contain H3 in typical nucleosomal structures, defects in nuclear divisions were restricted to mitotically dividing micronuclei; macronuclei, which are amitotic, showed no defects. Strains lacking phosphorylated H3 showed abnormal chromosome segregation, resulting in extensive chromosome loss during mitosis. During meiosis, micronuclei underwent abnormal chromosome condensation and failed to faithfully transmit chromosomes. These results demonstrate that H3 serine 10 phosphorylation is causally linked to chromosome condensation and segregation in vivo and is required for proper chromosome dynamics. (+info)