A candidate gene analysis and GWAS for genes associated with maternal nondisjunction of chromosome 21

Human nondisjunction errors in oocytes are the leading cause of pregnancy loss, and for pregnancies that continue to term, the leading cause of intellectual disabilities and birth defects. For the first time, we have conducted a candidate gene and genome-wide association study to identify genes associated with maternal nondisjunction of chromosome 21 as a first step to understand predisposing factors. A total of 2,186 study participants were genotyped on the HumanOmniExpressExome-8v1-2 array. These participants included 749 live birth offspring with standard trisomy 21 and 1,437 parents. Genotypes from the parents and child were then used to identify mothers with nondisjunction errors derived in the oocyte and to establish the type of error (meiosis I or meiosis II). We performed a unique set of subgroup comparisons designed to leverage our previous work suggesting that the etiologies of meiosis I and meiosis II nondisjunction differ for trisomy 21. For the candidate gene analysis, we selected genes associated with chromosome dynamics early in meiosis and genes associated with human global recombination counts. Several candidate genes showed strong associations with maternal nondisjunction of chromosome 21, demonstrating that genetic variants associated with normal variation in meiotic processes can be risk factors for nondisjunction. The genome-wide analysis also suggested several new potentially associated loci, although follow-up studies using independent samples are required

Error-Prone ZW Pairing and No Evidence for Meiotic Sex Chromosome Inactivation in the Chicken Germ Line

In the male mouse the X and Y chromosomes pair and recombine within the small pseudoautosomal region. Genes located on the unsynapsed segments of the X and Y are transcriptionally silenced at pachytene by Meiotic Sex Chromosome Inactivation (MSCI). The degree to which MSCI is conserved in other vertebrates is currently unclear. In the female chicken the ZW bivalent is thought to undergo a transient phase of full synapsis at pachytene, starting from the homologous ends and spreading through the heterologous regions. It has been proposed that the repair of the ZW DNA double-strand breaks (DSBs) is postponed until diplotene and that the ZW bivalent is subject to MSCI, which is independent of its synaptic status. Here we present a distinct model of meiotic pairing and silencing of the ZW pair during chicken oogenesis. We show that, in most oocytes, DNA DSB foci on the ZW are resolved by the end of pachytene and that the ZW desynapses in broad synchrony with the autosomes. We unexpectedly find that ZW pairing is highly error prone, with many oocytes failing to engage in ZW synapsis and crossover formation. Oocytes with unsynapsed Z and W chromosomes nevertheless progress to the diplotene stage, suggesting that a checkpoint does not operate during pachytene in the chicken germ line. Using a combination of epigenetic profiling and RNA–FISH analysis, we find no evidence for MSCI, associated with neither the asynaptic ZW, as described in mammals, nor the synaptic ZW. The lack of conservation of MSCI in the chicken reopens the debate about the evolution of MSCI and its driving forces

Fertilization in vitro increases non-disjunction during early cleavage divisions in a mouse model system

We have been studying an unusual mouse-the BALB/cWt (Wt) male-in which the Y chromosome is susceptible to high rates of mitotic non-disjunction, particularly at the first two cleavage divisions. As these are the same divisions that human embryos generated through assisted reproductive technology must complete in an artificial setting, analysis of the Wt Y chromosome allows us to examine the effect of fertilization and culture in vitro on mammalian chromosome segregation. We performed standard mouse IVF, cultured embryos in 5% CO2 in air or in a lowered oxygen atmosphere, and used fluorescence in-situ hybridization to examine the sex chromosome constitutions of 2-, 4-, 8- and 16-cell stage Wt Y-bearing embryos. We observed a significant increase in mosaic sex chromosome aneuploidy at each embryonic stage in embryos cultured in 5% CO2 in air, but under lowered oxygen conditions mosaicism returned to control (in-vivo) levels. Our results demonstrate that slight alterations in in-vitro conditions may have a considerable impact on the genetic quality of assisted reproductive technology-derived embryos and suggest that the genetic quality of embryos should be a fundamental concern in the development of new culture systems for clinical use

The bisphenol A experience: a primer for the analysis of environmental effects on mammalian reproduction

It is increasingly evident that environmental factors are a veritable Pandora's box from which new concerns and complications continue to emerge. Although previously considered the domain of toxicologists, it is now clear that an understanding of the effects of the environment on reproduction requires a far broader range of expertise and that, at least for endocrine-disrupting chemicals, many of the tenets of classical toxicology need to be revisited. Indeed, because of the wide range of reproductive effects induced by these chemicals, interest among reproductive biologists has grown rapidly: in 2000, the program for the annual Society for the Study of Reproduction meeting included a single minisymposium on the fetal origins of adult disease, one platform session on endocrine disruption, and 23 toxicology poster presentations. In contrast, environmental factors featured prominently at the 2009 meeting, with strong representation in the plenary, minisymposia, platform, and poster sessions. Clearly, a lot has happened in a decade, and environmental issues have become an increasingly important research focus for reproductive biologists. In this review, we summarize some of the inherent difficulties in assessing environmental effects on reproductive performance, focusing on the endocrine disruptor bisphenol A (BPA) to illustrate important emerging concerns. In addition, because the BPA experience serves as a prototype for scientific activism, public education, and advocacy, these issues are also discussed

Bisphenol A Exposure In Utero Disrupts Early Oogenesis in the Mouse

Estrogen plays an essential role in the growth and maturation of the mammalian oocyte, and recent studies suggest that it also influences follicle formation in the neonatal ovary. In the course of studies designed to assess the effect of the estrogenic chemical bisphenol A (BPA) on mammalian oogenesis, we uncovered an estrogenic effect at an even earlier stage of oocyte development—at the onset of meiosis in the fetal ovary. Pregnant mice were treated with low, environmentally relevant doses of BPA during mid-gestation to assess the effect of BPA on the developing ovary. Oocytes from exposed female fetuses displayed gross aberrations in meiotic prophase, including synaptic defects and increased levels of recombination. In the mature female, these aberrations were translated into an increase in aneuploid eggs and embryos. Surprisingly, we observed the same constellation of meiotic defects in fetal ovaries of mice homozygous for a targeted disruption of ERb, one of the two known estrogen receptors. This, coupled with the finding that BPA exposure elicited no additional effects in ERb null females, suggests that BPA exerts its effect on the early oocyte by interfering with the actions of ERb. Together, our results show that BPA can influence early meiotic events and, importantly, indicate that the oocyte itself may be directly responsive to estrogen during early oogenesis. This raises concern that brief exposures during fetal development to substances that mimic or antagonize the effects of estrogen may adversely influence oocyte development in the exposed female fetus

Evidence for paternal age-related alterations in meiotic chromosome dynamics in the mouse

Increasing age in a woman is a well-documented risk factor for meiotic errors, but the effect of paternal age is less clear. Although it is generally agreed that spermatogenesis declines with age, the mechanisms that account for this remain unclear. Because meiosis involves a complex and tightly regulated series of processes that include DNA replication, DNA repair, and cell cycle regulation, we postulated that the effects of age might be evident as an increase in the frequency of meiotic errors. Accordingly, we analyzed spermatogenesis in male mice of different ages, examining meiotic chromosome dynamics in spermatocytes at prophase, at metaphase I, and at metaphase II. Our analyses demonstrate that recombination levels are reduced in the first wave of spermatogenesis in juvenile mice but increase in older males. We also observed age-dependent increases in XY chromosome pairing failure at pachytene and in the frequency of prematurely separated autosomal homologs at metaphase I. However, we found no evidence of an age-related increase in aneuploidy at metaphase II, indicating that cells harboring meiotic errors are eliminated by cycle checkpoint mechanisms, regardless of paternal age. Taken together, our data suggest that advancing paternal age affects pairing, synapsis, and recombination between homologous chromosomes--and likely results in reduced sperm counts due to germ cell loss--but is not an important contributor to aneuploidy

Rapid generation of nested chromosomal deletions on mouse chromosome 2

Nested chromosomal deletions are powerful genetic tools. They are particularly suited for identifying essential genes in development either directly or by screening induced mutations against a deletion. To apply this approach to the functional analysis of mouse chromosome 2, a strategy for the rapid generation of nested deletions with Cre recombinase was developed and tested. A loxP site was targeted to the Notch1 gene on chromosome 2. A targeted line was cotransfected with a second loxP site and a plasmid for transient expression of Cre. Independent random integrations of the second loxP site onto the targeted chromosome in direct repeat orientation created multiple nested deletions. By virtue of targeting in an F 1 hybrid embryonic stem cell line, F 1 (129S1×Cast/Ei), the deletions could be verified and rapidly mapped. Ten deletions fell into seven size classes, with the largest extending six or seven centiMorgans. The cytology of the deletion chromosomes were determined by fluorescent in situ hybridization. Eight deletions were cytologically normal, but the two largest deletions had additional rearrangements. Three deletions, including the largest unrearranged deletion, have been transmitted through the germ line. Several endpoints also have been cloned by plasmid rescue. These experiments illustrate the means to rapidly create and map deletions anywhere in the mouse genome. They also demonstrate an improved method for generating nested deletions in embryonic stem cells