Human meiotic progression and recombination are affected by Bisphenol A exposure during in vitro human oocyte development.

BACKGROUND: Bisphenol A (BPA) is a 'weak' endocrine disruptor. The effect of BPA on human reproduction is controversial but has been related to meiotic anomalies, recurrent miscarriages and abnormal karyotypes. METHODS: To evaluate the effects of BPA on survival, pairing-synapsis and meiotic recombination of human fetal oocytes, 21 510 oocytes from 12 cultured fetal ovaries were analyzed. Ovaries were cultured for 7, 14 or 21 days in control medium, dimethylsulfoxide-medium, BPA-medium and estradiol (E(2))-medium. Meiotic pairing-synapsis and recombination were studied by immunofluorescence against lateral element protein, central element protein of the synaptonemal complex and chromosome axis cohesin REC8. Mismatch repair protein, MLH1, was used as a crossover (CO) marker. Meiotic progression was analyzed following the number of surviving oocytes at different meiotic stages found in each culture time and condition, and the total number of MLH1 foci found in oocytes from cultured ovaries. RESULTS: Oocyte survival in vitro decreased with the addition of BPA to the medium (1 µM or greater). Oocyte degeneration was up to five times higher when BPA was added to culture medium. Moreover, oocytes exposed to BPA concentrations of 10 µM or higher presented approximately two times more MLH1 foci than unexposed cultured oocytes (P = 0.01). This was also observed in chromosome 21 from BPA-exposed oocytes, which had double the average number of MLH1 foci found in control oocytes (P = 0.001). E(2) was used as a positive control of estrogen receptors activity, and E(2) addition to the medium had similar effects on meiotic progression of oocytes from cultured ovaries. CONCLUSIONS: Our findings show that BPA concentrations of 1 µM or higher decrease the survival of human fetal oocytes in vitro, and concentrations of 10 µM or higher increase MLH1 foci number. MLH1 is considered a CO marker, and thus an increase in MLH1 foci could indicate an increase in COs in BPA-exposed oocytes. These data suggest that BPA can act as a toxic substance, which has particular implications for human females and the critical events of meiotic prophase, such as pairing-synapsis and recombination processes, as well as oocyte survival.

Dynamics of cohesin proteins REC8, STAG3, SMC1 beta and SMC3 are consistent with a role in sister chromatid cohesion during meiosis in human oocytes.

BACKGROUND: Sister chromatid cohesion is essential for ordered chromosome segregation at mitosis and meiosis. This is carried out by cohesin complexes, comprising four proteins, which seem to form a ring-like complex. Data from animal models suggest that loss of sister chromatid cohesion may be involved in age-related non-disjunction in human oocytes. Here, we describe the distribution of cohesins throughout meiosis in human oocytes. METHODS: We used immunofluorescence in human oocytes at different meiotic stages to detect cohesin subunits REC8, STAG3, SMC1 beta and SMC3, [also synaptonemal complex (SC) protein 3 and shugoshin 1]. Samples from euploid fetuses and adult women were collected, and 51 metaphase I (MI) and 113 metaphase II (MII) oocytes analyzed. SMC1 beta transcript levels were quantified in 85 maturing germinal vesicle (GV) oocytes from 34 women aged 19-43 years by real-time PCR. RESULTS: At prophase I, cohesin subunits REC8, STAG3, SMC1 beta and SMC3 overlapped with the lateral element of the SC. Short cohesin fibers are observed in the oocyte nucleus during dictyate arrest. All four subunits are observed at centromeres and along chromosomal arms, except at chiasmata, at MI and are present at centromeric domains from anaphase I to MII. SMC1 beta transcripts were detected (with high inter-sample variability) in GV oocytes but no correlation between SMC1 beta mRNA levels and age was found. CONCLUSIONS: The dynamics of cohesins REC8, STAG3, SMC1 beta and SMC3 suggest their participation in sister chromatid cohesion throughout the whole meiotic process in human oocytes. Our data do not support the view that decreased levels of SMC1 beta gene expression in older women are involved in age-related non-disjunction.

Cytogenetic analyses of human oocytes provide new data on non-disjunction mechanisms and the origin of trisomy 16.

BACKGROUND: Nowadays, oocyte donation is an extended practise in IVF programmes. However, to date, little information on aneuploidy frequency in oocytes from donors is available. Aneuploidy is one of the major causes of embryo and fetal wastage as well as of congenital mental and developmental disabilities. It is known that most aneuploidies are due to non-disjunction events occurring in the maternal germ line. Linkage studies have associated abnormal patterns of meiotic recombination to the origin of the non-disjunction event in many aneuploid conditions. METHODS AND RESULTS: In the present study, we analyse the frequency of chromosome imbalances in a series of metaphase I (MI; n = 44) and metaphase II (MII; n = 103) oocytes from 140 young donors (aged from 18 to 35 years, mean age 26.6) after hormone-induced superovulation. The aneuploidy frequency found in MII oocytes was 12.6%, and both whole-chromosome non-disjunction (1.94%) and premature separation of sister chromatids (PSSC) (12.6%) have been found. The chromosomes involved have been identified by multiplex fluorescent in situ hybridization (FISH). Achiasmate chromosomes have been identified in MI oocytes (9.1%), with most of them corresponding to chromosome 16 (6.8%). For this reason, the meiotic recombination pattern of chromosome 16 has been analysed in prophase I oocytes (n = 81) by immunofluorescence staining against MLH1 protein and subsequent FISH with specific probes. Our results show a percentage of oocytes with non-crossover bivalent 16 (2.5%) and a high percentage of bivalents 16 with a single exchange (19.8%). CONCLUSIONS: In the present study, we report the finding of a considerable frequency of aneuploidy in oocytes from young donors, with the frequency of PSSC being higher than the frequency of whole-chromosome non-disjunction. In addition, we report vulnerable patterns of meiotic recombination in chromosome 16 that may be at risk of leading to a non-disjunction event. This gives new data on the susceptibility of the control population to conceive a trisomic 16 embryo.

A new culture technique that allows in vitro meiotic prophase development of fetal human oocytes.

BACKGROUND: Little is known about the mechanisms that regulate meiosis in the human female fetus as a result of the technical difficulties in obtaining samples. Currently, there is no technique for human fetal oocyte culture that permits the maintenance of fetal ovarian tissue in vitro which allows the progression of meiosis in a reproducible and standardized way. METHODS: Meiotic progression was analyzed following pairing-synapsis and recombination progress. A total of 7119 oocytes were studied and analyzed. The proteins used to evaluate meiotic progression were: REC8, SYCP1, SYCP3 and MLH1, studied by immunofluorescence. Four different sample disaggregating methods were used, two enzymatic (trypsin and collagenase + hyaluronidase) and two mechanical (puncture and ovarian fragments). Two different culture media were used, control media and stem cell factor (SCF)-supplemented media. The oocytes were studied at initial time T0, and then at T7, T14 and T21 days after culture. RESULTS: The mechanical methods increased the total number of oocytes found at the different times of culture and decreased the number of degenerated oocytes. Independently of the disaggregation method used, oocytes cultured with SCF-supplemented media showed a higher proportion of viable oocytes and fewer degenerated cells at all culture timepoints. No evidence of abnormal homologous chromosome synapsis was observed. Meiotic recombination was only observed in oocytes mechanically disaggregated and cultured with supplemented media. CONCLUSIONS: The oocytes obtained by mechanical disaggregating methods and cultured with SCF-supplemented media are able to follow pairing-synapsis and recombination, comparable to oocytes in vivo. The culture conditions described herein confirm the methodology as a standardized and reproducible method.

ATR, BRCA1 and gammaH2AX localize to unsynapsed chromosomes at the pachytene stage in human oocytes.

Asynapsis of homologous chromosomes at the pachytene stage has been associated with gametogenic failure and infertility, but the cellular mechanisms involved are currently unknown in human meiocytes. In mice, the protein encoded by the breast-cancer susceptibility gene Brca1 has been described to direct kinase ATR (ataxia telangiectasia and Rad3 related) to any unpaired DNA at the pachytene stage, where ATR triggers H2AX phosphorylation, resulting in the silencing of those chromosomes. In this study, the distribution of ATR, BRCA1 and the phosphorylated histone gammaH2AX is assessed by immunofluorescence in human oocytes and it is found that they localize at unpaired chromosomes at the pachytene stage. Evidence is shown to propose that BRCA1, ATR and gammaH2AX in the human may be part of a system such as the one previously described in mouse, which signals unsynapsed chromosomes at pachytene and may lead to their silencing.

Maternal origin of the human aneuploidies. Are homolog synapsis and recombination to blame? Notes (learned) from the underbelly.

Aneuploidy is the leading cause of mental deficiency in human newborns. Indirect studies suggest that, in most of the cases, the extra chromosome comes from an inaccurate meiotic division. But, particularly, all results seem to indicate that oogenesis is more prone to err than is spermatogenesis. Unfortunately, due to the time-frame in which meiosis takes place in the mammalian males and females, most of the studies performed so far have focused on analyzing male meiosis. Recently, some studies focusing on human meiosis have been published. Some of them revealed important sex-specific differences that may be involved in the predominant involvement of the human female in the genesis of aneuploidy. In this article, the current knowledge we have about human female meiotic synapsis and recombination is summarized and we try to relate it to the human aneuploidy origin.