Presence of an extra chromosome alters meiotic double-stranded break repair dynamics and MLH1 foci distribution in human oocytes.

Studies performed on human trisomic 21 oocytes have revealed that during meiosis, the three homologues 21 synapse and, in some cases, achieve what looks like a trivalent. This implies that meiotic recombination takes place among the three homologous chromosomes 21, and to some extent, crossovers form between them. To see how meiotic recombination is in the presence of an extra chromosome 21, we analyzed the distribution of three recombination markers (γH2AX, RPA, and MLH1) on trisomic 21 oocytes at pachynema and, in particular, on chromosomes 21. Results clearly show how the presence of an extra chromosome 21 alters meiotic recombination progression, leading to the presence of a higher number of early recombination markers at pachynema. Moreover, the distribution on these chromosomes 21 of some of these markers is different in aneuploid oocytes. Finally, there is a substantial increase in the number of MLH1 foci, a marker of most crossovers in mammals, which is related to the number of synapsed chromosomes in pachynema. Thus, bivalents 21 had fewer MLH1 foci than partial or total trivalents, suggesting a close relationship between synapsis and crossover designation. All of the data presented suggest that the presence of an extra chromosome alters meiotic recombination globally in aneuploid human oocytes.

Gene expression is altered after bisphenol A exposure in human fetal oocytes in vitro.

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 spontaneous abortion, abnormal karyotypes, the diminishing of oocyte survival, delay in meiotic progression and an elevated rate of MLH1 foci in vitro. The aim of this study is to characterize the gene expression of human fetal oocytes in culture as well as to evaluate the effect of BPA in cultured human oocytes. To accomplish our objective, 12 ovaries from 6 euploid fetuses were used. The ovarian fetal tissue was cultivated in two groups: control group and BPA group (BPA30 µM). The cultures were analyzed at T0 and after 7 (T7), 14 (T14) and 21 (T21) days of culture. Evaluation of gene expression was performed by real-time PCR (RT-PCR), with the evaluated genes being: Smc1ß, Sycp1 (pairing-synapsis), Spo11, Rpa, H2ax, Mlh1 and Blm [double-strand break (DSBs) generation, signaling and repair], Erα, Erß and Errγ (estrogen receptors), Stra8 and Nalp5 (markers of meiotic progression). Oocytes from ovaries cultured and treated with BPA show changes in the expression of Spo11, H2ax and Blm genes, with a significant increase from 3- to 5-fold (P≤ 0.05). Finally, Rpa, showed a 100-fold increment (P≤ 0.01). Erα, Erß and Errγ genes showed a BPA up-regulation of 2-4-fold in all of the culture times (P≤ 0.05). Oocytes exposed to BPA showed an up-regulation of genes involved in DSB generation, signaling and repair except by Mlh1. Thus, BPA can modify the gene expression pattern, which may explain the effects of BPA on female germ cells.

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.

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.