Non-invasive method to assess genotoxicity of nocodazole interfering with spindle formation in mammalian oocytes

Trisomies due to nondisjunction in oogenesis are still a major cause of genetic diseases in humans. In this study, we analysed spindle morphology of in vitro matured nocodazole-exposed mouse oocytes by novel non-invasive Polscope-microscopy, and compared images to those obtained by anti-tubulin immunofluorescence of fixed oocytes. Polscope revealed a reduction in the numbers of oocytes expressing a birefringent spindle, and alterations in spindle morphology at concentrations of nocodazole below those inducing detectable aberrations in immunofluorescence. Hyperploidy increased significantly at a concentration of 40 nM nocodazole in mouse metaphase II oocytes, similar to thresholds inducing nondisjunction in cultured human lymphocytes. In conclusion, Polscope represents a novel highly sensitive, non-invasive method to identify chemicals inducing severe spindle aberrations that predispose mammalian oocytes to nondisjunction. Polscope may provide information on the functionality of the spindle in experimental studies but is also compatible with clinical trials in human assisted reproduction due to its non-invasive nature.     

Mitochondrial activity is required for the expression of IME1, a regulator of meiosis in yeast

Sporulation in the yeast Saccharomyces cerevisiae occurs in diploid cells following starvation for glucose and nitrogen sources. A key gene in the regulation of the meiotic process is IME1. A well-documented fact is that respiration is necessary for sporulation. We now show that respiration is necessary for the expression of IME1. We suggest that glucose repression of meiosis is transduced through its effect on respiration, in a pathway separate from that of adenylyl cyclase.     

The Iml3 protein of the budding yeast is required for the prevention of precocious sister chromatid separation in meiosis I and for sister chromatid disjunction in meiosis II

The mitotic kinetochore of the budding yeast contains a number of proteins which are required for chromosome transmission but are non-essential for vegetative growth. We show that one such protein, Iml3, is essential for meiosis, in that the absence of this protein results in reduced spore viability, precocious sister chromatid segregation of artificial and natural chromosomes in meiosis I and chromosome non-disjunction in meiosis II.     

Ipl1/Aurora B kinase coordinates synaptonemal complex disassembly with cell cycle progression and crossover formation in budding yeast meiosis

Several protein kinases collaborate to orchestrate and integrate cellular and chromosomal events at the G2/M transition in both mitotic and meiotic cells. During the G2/M transition in meiosis, this includes the completion of crossover recombination, spindle formation, and synaptonemal complex (SC) breakdown. We identified Ipl1/Aurora B kinase as the main regulator of SC disassembly. Mutants lacking Ipl1 or its kinase activity assemble SCs with normal timing, but fail to dissociate the central element component Zip1, as well as its binding partner, Smt3/SUMO, from chromosomes in a timely fashion. Moreover, lack of Ipl1 activity causes delayed SC disassembly in a cdc5 as well as a CDC5-inducible ndt80 mutant. Crossover levels in the ipl1 mutant are similar to those observed in wild type, indicating that full SC disassembly is not a prerequisite for joint molecule resolution and subsequent crossover formation. Moreover, expression of meiosis I and meiosis II-specific B-type cyclins occur normally in ipl1 mutants, despite delayed formation of anaphase I spindles. These observations suggest that Ipl1 coordinates changes to meiotic chromosome structure with resolution of crossovers and cell cycle progression at the end of meiotic prophase.     

Elucidation of the developmental mechanism of ovarian mature cystic teratomas using B allele‐frequency plots of single nucleotide polymorphism array data

Ovarian mature cystic teratomas (MCTs) originate from post‐meiotic germ cells. Conventional methods such as karyotyping or short tandem repeat‐polymorphism analysis may be used to better classify MCTs, although such data would be insufficient. The aim of this study was to elucidate the origin of ovarian MCTs using B allele‐frequency (BAF) plots of single nucleotide polymorphism array data. MCTs can be classified in terms of the zygosity of the centromeres and distal chromosome regions. We evaluated the zygosity of all chromosomes from 38 MCT specimens using BAF plot data. BAF plots were used to determine the homozygous and heterozygous regions over the whole genome. Theoretically, MCTs originated from the fusion of two ova (previously referred to as type V MCTs) should have a mixed pattern of centromeric zygosity, that is, a combination of heterozygous and homozygous regions in the centromeric regions. However, no MCTs in this study met this criterion. We identified 13 type I MCTs, 14 type II MCTs, and 11 type III MCTs. In addition, BAF plots facilitated the construction of recombination maps at the whole‐genome level for type I and II MCTs. No crossover, especially in the short arms, contributed to the failure of meiosis I, resulting in type I MCTs. Crossover in all arms might assure the normal progress of meiosis in human oocytes. In conclusion, our findings indicate that BAF plots can elucidate the developmental mechanism of MCTs, and further serve as useful analytical tools for analyzing human oocyte meiosis, and related aberrations.     

46,XY,t（4;11）平衡易位携带者的减数分裂分析

利用不育症精液中的脱落生殖细胞对具46，XY，t（4；11）平衡易位的男性不育症进行了减数分裂分析，结果表明患者精液中具有大量的脱落生殖细胞（4000万／ml），其中精原细胞占3％，精母细胞占50％，精细胞占47％，呈减数分裂早期部分阻断，无精子产生。易位染色体在减数分裂中的配对形式以链状三价体（71．09％）为主，还有链状四价体（13．28％），完全配对的环状四价体占15．63％。配对位点不饱和以及染色体大片段易位所导致的位置效应可能是该患者无精子产生的原因。     

Age-associated alterations in the micromechanical properties of chromosomes in the mammalian egg

Purpose

The incidence of aneuploidy in eggs from women of advanced reproductive age can exceed 60 %, making the mammalian egg a unique model system to study the mechanisms of chromosome segregation errors.

Methods

Here we applied a novel biophysical chromosome stretching approach to quantify mechanical stiffness of meiotic chromosomes in the mammalian egg and then documented how these properties changed in a mouse model of physiologic reproductive aging.

Results

We found significant differences in chromosome micromechanics, and thus in higher order chromosome structure, coincident with advanced reproductive age, a time that is also unequivocally associated with an increase in egg aneuploidy.

Conclusions

These findings have important implications for both reproductive and cancer biology where aneuploidy plays a central role in aging related disease states.     

Ex-vivo assessment of chronic toxicity of low levels of cadmium on testicular meiotic cells

Using a validated model of culture of rat seminiferous tubules, we assessed the effects of 0.1, 1 and 10 μg/L cadmium (Cd) on spermatogenic cells over a 2‐week culture period. With concentrations of 1 and 10 μg/L in the culture medium, the Cd concentration in the cells, determined by ICP-MS, increased with concentration in the medium and the day of culture. Flow cytometric analysis enabled us to evaluate changes in the number of Sertoli cells and germ cells during the culture period. The number of Sertoli cells did not appear to be affected by Cd. By contrast, spermatogonia and meiotic cells were decreased by 1 and 10 μg/L Cd in a time and dose dependent manner. Stage distribution of the meiotic prophase I and qualitative study of the synaptonemal complexes (SC) at the pachytene stage were performed by immunocytochemistry with an anti SCP3 antibody. Cd caused a time-and-dose-dependent increase of total abnormalities, of fragmented SC and of asynapsis from concentration of 0.1 μg/L. Additionally, we observed a new SC abnormality, the “motheaten” SC. This abnormality is frequently associated with asynapsis and SC widening which increased with both the Cd concentration and the duration of exposure. This abnormality suggests that Cd disrupts the structure and function of proteins involved in pairing and/or meiotic recombination. These results show that Cd induces dose-and-time-dependent alterations of the meiotic process of spermatogenesis ex-vivo, and that the lowest metal concentration, which induces an adverse effect, may vary with the cell parameter studied.     

Premature ovarian insufficiency

The natural lifespan of the ovary is occasionally interrupted by pathological processes; some are known, but many are unknown. Premature ovarian insufficiency (POI) can be a devastating diagnosis for an adolescent or for someone who has yet to start a family. Common causes of POI include genetic and chromosomal defects, autoimmune damage, and cancer treatments. Knowledge of the pathogenesis of this condition and an awareness of contemporary hormone replacement and fertility options are required to design a multidisciplinary therapeutic approach comprising reproductive medicine, endocrinology, clinical psychology, and assisted fertility expertise.     

Polar body morphology is not predictive of its cell division origin

Purpose  

This study sought to determine whether morphology of simultaneously biopsied polar bodies is predictive of their cell division origin.