Meiosis-activating sterol protects oocytes from precocious chromosome segregation

BACKGROUND: Follicular fluid meiosis-activating sterol (FF-MAS) overcomes hypoxanthine (HX)-mediated meiotic arrest in mammalian oocytes. METHODS: In order to determine whether chromosome segregation was normal in oocytes matured in FF-MAS, the development, chromosomal constitution and chromosome alignment was analysed in spontaneously matured as well as HX-arrested mouse oocytes cultured in the absence or presence of FF-MAS. RESULTS: FF-MAS-induced meiotic maturation was significantly less effective compared with spontaneous maturation in supporting cytokinesis ( approximately 40 and approximately 90% polar body formation respectively). The majority of oocytes stimulated by FF-MAS to overcome the HX block developed to metaphase II (MII), but 23.4% of meiosis II oocytes were diploid. Chromosomes were well aligned on the spindle, and hyperploidy was low in spontaneously matured oocytes and HX-arrested oocytes cultured with or without FF-MAS. Unexpectedly, almost 40% of spontaneously matured MII oocytes contained chromatids/monads. Precocious loss of chromatid cohesion was significantly reduced in spontaneously matured as well as HX-arrested oocytes cultured in the presence of FF-MAS but not lanosterol. CONCLUSIONS: FF-MAS induces full nuclear maturation to MII, and chromosomes segregate with high fidelity. However, in delayed FF-MAS-stimulated meiotic maturation, anaphase I may occur in the absence of cytokinesis. FF-MAS appears to protect mammalian oocytes from precocious chromatid segregation.     

Control of homologous chromosome division in the mammalian oocyte

Homologous chromosomes are segregated during the first meioticdivision (meiosis I). Unfortunately, human oocytes are particularlysusceptible to mis-segregation errors, so generating aneuploid,often non-viable, embryos. Here we review the cell biology ofmeiosis I and how homolog disjunction is regulated for mammalianoocytes. We focus on the activity of the anaphase-promotingcomplex/cyclosome (APC/C), which is responsible for timely degradationof the cohesin component, REC8 and the cyclin B regulatory subunitof maturation-promoting factor, both essential steps for meiosisI completion. In particular, we examine the role played by thespindle assembly checkpoint in controlling the APC/C activity,and in so doing ensuring accurate disjunction of homologs.     

Meiotic aneuploidy in the XXY mouse: evidence that a compromised testicular environment increases the incidence of meiotic errors.

Male mammals with two X chromosomes are sterile due to the loss of virtually all germ cells in the differentiating testis. The survival of rare germ cells, however, can give rise to patches of normal-appearing spermatogenesis in the adult testis. Intracytoplasmic sperm injection (ICSI) makes possible the establishment of a pregnancy using spermatozoa from severely oligozoospermic men and, indeed, has been successful using spermatozoa from human 47,XXY (Klinefelter syndrome) males. The risk of an abnormal pregnancy, however, may be significantly increased since several studies have demonstrated elevated levels of aneuploidy in spermatozoa from Klinefelter syndrome men. This has been suggested to reflect the consequences of meiotic segregation in XXY germ cells; however, it is also possible that it is a consequence of abnormalities in meiotic regulation in the XXY testis. We have addressed this question experimentally in the XXY male mouse. Analysis of testicular spermatozoa from XXY and control males demonstrates a significant increase in meiotic aneuploidy in the XXY mouse. Since previous studies have demonstrated that germ cells in the adult XXY testis are exclusively XY, the meiotic abnormalities observed must be attributable to segregation errors in XY germ cells. These findings have potential significance for ICSI pregnancies using spermatozoa from other types of male factor infertility patients, since they raise the possibility that increased meiotic errors are a generalized feature of the severely oligozoospermic testis.     

Evolution of the meiotic prophase and of the chromosome pairing process during human fetal ovarian development

BACKGROUND: Studies on human oocytes in prophase I are limited due to the difficulty in obtaining the sample. However, a complete study of meiotic prophase evolution and the homologue pairing process is necessary to try to understand the implication of oogenesis in the origin of human aneuploidy. METHODS: A complete analysis of meiotic prophase progression comprising the long developmental time period during which meiotic prophase takes place, based on the analysis of a total of 8603 oocytes in prophase I from 15 different cases is presented. The pairing process of chromosomes 13 and 18 is also described. RESULTS: The findings significantly relate for the first time the evolution of meiotic prophase to fetal development. Although for both chromosomes 13 and 18 a high pairing efficiency is found, pairing failure at the pachytene stage has been observed in 0.1% of oocytes. However, errors at the diplotene stage are substantially increased, suggesting that complete, premature disjunction of the homologues commonly occurs. Moreover, pre-meiotic errors are also described. CONCLUSIONS: Our findings show that homologous chromosomes pair very efficiently, but the high frequency of complete, premature homologue separation found at diplotene suggests that mechanisms other than the pairing process could be more likely to lead to the high aneuploidy rate observed in human oocytes.     

Influence of follicular fluid meiosis-activating sterol on aneuploidy rate and precocious chromatid segregation in aged mouse oocytes

BACKGROUND: Follicular fluid meiosis-activating sterol (FF-MAS) protects young oocytes from precocious chromatid separation (predivision). Reduced expression of cohesion and checkpoint proteins and predivision has been hypothesized to occur in age-related aneuploidy in oocytes. METHODS: To know whether FF-MAS also protects aged oocytes from predivision and from age-related non-disjunction, we analysed chromosome constitution in mouse oocytes matured spontaneously with or without 10 microM FF-MAS and in hypoxanthine (HX)-arrested young and aged oocytes induced to resume maturation by FF-MAS. Messenger RNA for checkpoint protein MAD2 and cohesion protein SMC1beta was compared between oocytes matured with or without FF-MAS. RESULTS: Aged oocytes possessed many bivalents with single distal chiasma at meiosis I. Predivision was especially high in aged oocytes cultured sub-optimally to metaphase II in alpha-minimum essential medium (alpha-MEM). FF-MAS reduced predivision significantly (P < 0.001) but neither reduced non-disjunction nor induced aneuploidy in aged oocytes. Polyploidy was high in FF-MAS-stimulated maturation, in particular in the aged oocytes (P > 0.001). Relative levels of Smc1beta mRNA appeared increased by maturation in FF-MAS, and mitochondrial clustering was restored. CONCLUSIONS: Sister chromatids of aged oocytes appear to be highly susceptible to precocious chromatid separation, especially when maturation is under sub-optimal conditions, e.g. in the absence of cumulus and FF-MAS. This may relate to some loss of chromatid cohesion during ageing. FF-MAS protects aged oocytes from predivision during maturation, possibly by supporting Smc1beta expression, thus reducing risks of meiotic errors, but it cannot prevent age-related non-disjunction. Aged oocytes appear prone to loss of co-ordination between nuclear maturation and cytokinesis suggesting age-related relaxed cell cycle control.     

The influence of cooling on the oranization of meiotic spindle of the mouse oocyte

The effect of cooling on the organization of the microtubulesystem of the mouse oocyte has been investigated. Cooling to25, 18 or 4°C for varying periods of time resulted in aprogressive disassembly of the spindle and the dispersal ofthe chromosomes. The extent of the changes observed was greaterat lower temperatures and with longer periods of exposure. Transferof oocytes from either 37 or 4 to 24°C resulted in the rapidand transient appearance of polar asters and of multiple cytoplasmicasters associated with the pericentriolar material present atthe spindle poles and in the cytocortex of the oocyte. Thistransient aster formation appeared to be driven by the elevatedlevels of free tubulin released during spindle disassembly.An apparent reversal of many of the changes induced by low temperaturewas observed in many but not all oocytes on their restorationto 37°C for 1 h. These results have implications for ourunderstanding of microtubule organization in the oocyte, andfor the handling of oocytes during IVF and GIFT therapeuticprocedures and during the cryopreservation of oocytes.     

The high incidence of meiotic errors increases with decreased sperm count in severe male factor infertilities

BACKGROUND:The high frequency of aneuploidy sperm raises concerns that there may be an increased incidence of aneuploid offspring in ICSI programmes. In order to assess the role that chromosome complement plays in normal and abnormal fertility, detailed molecular cytogenetic studies must be done on sperm samples from men with normal and abnormal fertility. METHODS: To understand more clearly the cytogenetic make-up of sperm from oligoasthenoteratozoospermic (OAT) patients, multi-colour fluorescence in situ hybridization was used to determine numerical chromosome abnormalities. RESULTS: Increased aneuploidy frequencies for chromosomes 13, 18, 21, X and Y were detected in sperm from OAT patients. The frequencies of diploidy also increased. There were no differences in non-disjunction at meiosis I compared to meiosis II. Sperm count inversely correlated with the frequencies of diploidy, aneuploidies for chromosomes 13 and 21 in OAT patients. Twenty-two cycles of ICSI and 18 embryo transfers were performed in 20 couples. Only three cases achieved successful pregnancies. CONCLUSIONS: A higher incidence of meiotic errors and lower sperm counts was found in sperm from OAT patients.     

Interference with the C-terminal structure of MARF1 causes defective oocyte meiotic division and female infertility in mice

The Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510150, China; 3Collaborative Innovation Center of Genetics and Development, Fudan University, Shanghai 200433, China; 4Key Laboratory of Model Animal Research, Nanjing Medical University, Nanjing, Jiangsu 211166, China     

Mouse oocyte meiotic resumption and polar body extrusion in vitro are differentially influenced by FSH, epidermal growth factor and meiosis-activating sterol

BACKGROUND: In this study, we compared the relative ability of FSH (100 mIU/ml), epidermal growth factor (EGF) (10 ng/ml), and follicular-fluid meiosis-activating sterol (FF-MAS, 10 micromol/l) to induce meiotic resumption and polar body I (PBI) extrusion in mouse oocytes. METHODS: Cumulus-enclosed oocytes (CEO) were co-incubated with meiosis-arresting agents, including 4 mmol/l hypoxanthine (Hx), 0.3 mmol/l dibutyryl cAMP (dbcAMP), and 8.5 micromol/l cilostamide, a selective inhibitor of the oocyte-specific phosphodiesterase 3 (PDE 3). RESULTS: In Hx-treated oocytes, FSH, EGF and FF-MAS induced meiosis resumption at very high rates, but only FSH and EGF also promoted PBI extrusion with high frequency. In experiments conducted in the presence of dbcAMP, FF-MAS was unable to promote an increase in germinal vesicle breakdown (GVBD) rate, whereas FSH and EGF generated a response similar to the Hx groups. Neither FSH, EGF nor FF-MAS caused any change in the meiotic status of CEO when meiotic arrest at the germinal vesicle (GV) stage was maintained by cilostamide. In the presence of Hx, naked oocytes (NkO) co-cultured with their cumulus cells were able to respond to the GVBD-inducing effect of FSH and EGF by resuming meiosis at high rate. CONCLUSIONS: Collectively, these results indicate that: (i) a signal triggered in cumulus cells by either FSH or EGF, but not necessarily coincident with FF-MAS, may contribute to meiotic maturation, supporting GVBD and extrusion of PBI; (ii) the transmission of this signal can occur in a paracrine fashion, at least with reference to the breakdown of the GV. It also appears that concomitant regulation of intra-oocyte cAMP degradation is a prerequisite for meiosis resumption.     

Participation of EML6 in the regulation of oocyte meiotic progression in mice

The generation of a high-quality egg for reproduction requires faithful segregation of chromosome during oocyte meiosis. Here, we report that echinoderm microtubule-associated protein like 6 (EML6) is highly expressed in oocytes, and responsible for accurate segregation of homologous chromosomes in mice. Quantitative real-time RT-PCR and immunohistochemistry analyses revealed that EML6 was predominantly expressed by oocytes in the ovaries. Whole mount immunofluorescent staining showed that EML6 was colocalized with spindle microtubules in oocytes at various stages after meiotic resumption. This specialized localization was disrupted by nocodazole, the microtubule destabilizer, while enhanced by Taxol, a microtubule stabilizing reagent. In vivo knockdown of Eml6 expression by the specific siRNA resulted in chromosome misalignment and alteration in spindle dimension at both metaphase Ⅰ and Ⅱ stages, as well as the increased aneuploidy in the mature oocytes. Thus, these data suggest that EML family proteins participate in the control of oocyte meiotic division.     

Sperm aneuploidy and meiotic sex chromosome configurations in an infertile XYY male

BACKGROUND: There is little information regarding the behaviour of the extra Y chromosome during meiosis I in men with 47,XYY karyotypes and the segregation of the sex chromosomes in sperm. We applied immunofluorescent and FISH techniques to study the relationship between the sex chromosome configuration in meiotic germ cells and the segregation pattern in sperm, both isolated from semen samples of a 47,XYY infertile man. METHODS: The sex chromosome configuration of pachytene germ cells was determined by immunostaining pachytene nuclei for synaptonemal complex protein 3 (SCP3) and SCP1. FISH was subsequently performed to identify the sex chromosomes and chromosome 18 in pachytene cells. Dual- and triple-color FISH was performed on sperm to analyse aneuploidy for chromosomes 13, 18, 21, X, and Y. RESULTS: 46,XY/47,XYY mosaic pachytene cells were observed (22.2% vs. 77.8%, respectively). The XYY trivalent, and X+YY configurations were most common. While the majority of sperm were of normal chromosomal constitution, an increase in sex and autosome disomy was observed. CONCLUSIONS: The level of germ cell moscaicism and their meiotic sex chromosome configurations may determine sperm aneuploidy rate and fertility status in 47,XYY men. Our approach of immunostaining meiotic cells in the ejaculate is a novel method for investigating spermatogenesis in infertile men.     

小鼠卵母细胞减数分裂过程中活化Pak1的表达及其与染色体分离的相关性

目的研究活化的Pak1在小鼠卵母细胞减数分裂进程中的表达、亚细胞定位及其与染色体分离的相关性。方法用Western blot方法半定量分析活化的Pak1(~(Ser204)位点磷酸化,pPak1~(Ser204))在小鼠卵母细胞减数分裂各个时期的蛋白表达量;用免疫荧光染色法分析减数分裂进程中pPak1~(Ser204)的亚细胞定位模式及其与纺锤体和微管组织中心(MTOC)的时空相关性。结果 pPak1~(Ser204)在小鼠卵母细胞减数分裂重启时(GVBD)开始表达,其表达量随减数分裂进程逐渐升高,在第一次减数分裂中期(MⅠ)时达到峰值并持续保持至第二次减数分裂中期(MⅡ)。在第一次减数分裂前中期(pro-MⅠ)、MⅠ和MⅡ期,pPak1~(Ser204)与MTOC核心蛋白pericentrin和γ-tubulin共同定位于纺锤体两极,pPak1~(Ser204)同时存在于染色体上;在第一次减数分裂后期(AI)到末期(TelI)进程中pPak1~(Ser204)离开MTOC和染色体,聚集在收缩环部位。结论 pPak1~(Ser204)在卵母细胞减数分裂恢复时开始表达,是一种MTOC相关蛋白,可能通过参与纺锤体结构的形成和维持而调控染色体的分离。     

A constitutional complex chromosome rearrangement involving meiotic arrest in an azoospermic male: case report

Complex chromosome rearrangements are rare aberrations that frequently lead to reproductive failure and that may hinder assisted reproduction. A 25-year-old azoospermic male was studied cytogenetically with synaptonemal complex analysis of spermatocytes from a testicular biopsy and fluorescence in situ hybridization (FISH) of lymphocytes. The spermatocytes showed a pentavalent plus a univalent chromosome. Cell death occurred mainly at advanced pachytene stages. The sex chromosomes were involved in the multiple, as shown by their typical axial excrescences. Two autosomal pairs, including an acrocentric chromosome (15), were also involved in the multiple. FISH allowed the definite identification of all the involved chromosomes. An inverted chromosome 12 is translocated with most of one long arm of chromosome 15, while the centromeric piece of this chromosome 15 is translocated with Yqh, forming a small marker chromosome t(15;Y). The euchromatic part of the Y chromosome is joined to the remaining piece of chromosome 12, forming a neo-Y chromosome. The patient shows azoospermia and a normal phenotype. The disruption of spermatogenesis is hypothetically due to the extent of asynaptic segments and to sex-body association during pachytene. This CCR occurred 'de novo' during paternal spermatogenesis. Meiotic analysis and FISH are valuable diagnostic tools in these cases.     

Comparison of male and female meiotic segregation patterns in translocation heterozygotes: a case study in an animal model (Sus scrofa domestica L.)

BACKGROUND: The comparison of male and female meiotic segregation patterns for individuals carrying identical reciprocal translocations has been rarely reported in mammalian species. The main comparative study involving males and females with comparable genetic background has been performed in the mouse. Swine is another relevant animal model species for meiotic studies. Here we present the segregation patterns determined for sows carrying one of the two following reciprocal translocations: 38, XX, rcp(3;15)(q27;q13), and 38, XX, rcp(12;14)(q13;q21). These segregation data were compared to those previously obtained for closely related boars carrying the same balanced chromosomal rearrangements. METHODS: Dual colour in situ hybridization of whole chromosome painting probes was carried out on metaphases of in vitro-matured oocytes II. Segregation results were obtained for 118 and 206 metaphases II respectively for the two translocations. RESULTS: Significant differences between sexes were demonstrated for both rearrangements. For instance, for the 3/15 translocation, the chromosomally unbalanced gametes were of different origin: preponderance of the adjacent-I segregation in the male (31.4%), and of the adjacent-II (14.3%) and 3:1 (14.3%) segregations in females. For the 12/14 translocation, the proportion of balanced gametes was greater in males than in females (75.9 and 59.4% respectively). CONCLUSION: This study is a new scientific contribution to compare the segregation patterns of male and female carriers of identical chromosomal rearrangements. The results obtained are consistent with those previously reported in mice. Hypotheses to interpret the observed differences between the two translocations, as well as between the male and female segregation patterns, are formulated and discussed.     

Analysis using dual-colour fluorescencein situ hybridization of meiotic chromosome segregation in male mice heterozygous for a reciprocal translocation

Meiotic chromosome behaviour was investigated in male mice heterozygous for the translocation T(7;16)67H. At metaphase I, chain-of-four quadrivalents were present in approximately 80% of the spermatocytes; the bulk of remaining cells contained a ring quadrivalent, with only a few having either a trivalent plus univalent configuration or two bivalents. A low rate of non-disjunction, approximately 5%, was found through analysis of C-banded metaphase II spermatocytes. Using fluorescencein situ hybridization with differentially labelled whole chromosome paints, a wide array of segregation products were observed at metaphase II, depending on whether they arose from alternate, adjacent I, adjacent II orientation at metaphase I or were uninformative for alternative/adjacent I because of the presence of a chiasma in an interstitial pairing segment. Some 62% of the cells fell into this latter category, with only small proportions clearly arising through alternate (1.8%) or adjacent I (0.7%) orientations. Approximately 30% of the cells contained the products of adjacent II orientation. Consideration of the data suggested that most of these cells arose from metaphase I cells that contained a chain quadrivalent. Ring quadrivalents appeared predominantly to orientate in an alternate/adjacent I manner.for publication by J. S. (Pat) Heslop-Harrison     

Unique t(Y;1)(q12;q12) reciprocal translocation with loss of the heterochromatic region of chromosome 1 in a male with azoospermia due to meiotic arrest: a case report

A de novo reciprocal translocation 46,X,t(Y;1)(q12;q12) was found in an azoospermic male with meiotic arrest. Cytogenetics and fluorescent in situ hybridization (FISH) were used to define the karyotype, translocation breakpoints and homologue pairing. SRY (Yp), Yq11.2-AZF regions, DAZ gene copies and the distal Yq12 heterochromatin were studied by PCR and restriction analysis using sequence-tagged sites and single nucleotide variants. High resolution GTL, CBL and DA-DAPI staining revealed a (Y;1) translocation in all metaphases and a normal karyotype in the patient's father. FISH showed the presence of the distal Yq12 heterochromatic region in der(1) and loss of the heterochromatic region of chromosome 1. PCR demonstrated the intactness of the Y chromosome, including the SRY locus, AZF regions, DAZ genes and distal heterochromatin. A significant decrease (P = 0.005) of Xp/Yp pairing (18.6%), as compared with controls (65.7%), was found in arrested primary spermatocytes, and cell culture and mRNA expression studies confirmed an irreversible arrest at meiosis I, with induction of apoptosis and removal of germ cells by Sertoli cells. We characterized a de novo t(Y;1)(q12;q12) balanced reciprocal translocation with loss of the heterochromatic region of chromosome 1, that caused unpairing of sex chromosomes followed by meiosis I arrest, apoptotic degeneration of germ cells and azoospermia.