Instability of (CTG)•(CAG) microsatellite trinucleotide repeat (TNR) sequences is responsible for more than one dozen neurological or neuromuscular diseases. TNR instability during DNA synthesis is thought to involve slipped strand or hairpin structures in template or nascent DNA strands, although direct evidence for hairpin formation in human cells is lacking. We have used targeted recombination to create a series of isogenic HeLa cell lines in which (CTG)•(CAG) repeats are replicated from an ectopic copy of the c-myc replication origin. In this system the tendency of chromosomal (CTG)•(CAG) tracts to expand or contract was affected by origin location and the leading or lagging strand replication orientation of the repeats, and instability was enhanced by prolonged cell culture, increasing TNR length, and replication inhibition. Hairpin cleavage by synthetic zinc finger nucleases in these cells has provided the first direct evidence for the formation of hairpin structures during replication in vivo.

The structure of replication origins in metazoans is only nominally similar to that in model organisms, such as Saccharomyces cerevisiae. By contrast to the compact origins of budding yeast, in metazoans multiple elements act as replication start sites or control replication efficiency. We first reported that replication forks diverge from an origin 5 to the human c-myc gene and that a 2.4-kb core fragment of the origin displays autonomous replicating sequence activity in plasmids and replicator activity at an ectopic chromosomal site. Here we have used clonal HeLa cell lines containing mutated c-myc origin constructs integrated at the same chromosomal location to identify elements important for DNA replication. Replication activity was measured before or after integration of the wild-type or mutated origins using PCR-based nascent DNA abundance assays. We find that deletions of several segments of the c-myc origin, including the DNA unwinding element and transcription factor binding sites, substantially reduced replicator activity, whereas deletion of the c-myc promoter P 1 had only a modest effect. Substitution mutagenesis indicated that the sequence of the DNA unwinding element, rather than the spacing of flanking sequences, is critical. These results identify multiple functional elements essential for c-myc replicator activity.The initiation of eukaryotic DNA synthesis is precisely regulated both at the G 1 /S phase checkpoint of the mitotic cycle and by mechanisms that control the order of replicon firing (reviewed in references 9, 18, and 21). For Escherichia coli, mammalian viruses, and the budding yeast Saccharomyces cerevisiae the initiation of DNA replication is controlled by transacting initiator proteins that interact with cis-acting DNA replicator sequences. For S. cerevisiae, replicators encompass 100 to 200 bp and include the major replication origin sites where DNA synthesis begins (8). These replicators contain an essential ϳ11-bp autonomous replicating sequence (ARS) consensus sequence (ACS) that binds the origin recognition complex (ORC) to nucleate formation of prereplication complexes. By comparison, the domains that control replication in the fission yeast Schizosaccharomyces pombe are 500 to 1,500 bp in size and comprise multiple regions that add synergistically to origin activity (11,22,28,48). The sequences that contribute to origin activity in S. pombe are heterogeneous, although AϩT-rich potential binding sites for spORC have been found in several origins (10,30,36). Hence, despite notable differences in the structures of their respective ORC orthologs, the dispersed replication origins of metazoan cells more closely resemble those of S. pombe than those of S. cerevisiae in that multiple elements distributed over large distances act as replication start sites or control replication (3,4,13,15,16,27,40,53,56,58,62).Replication begins in the 5Ј flanking region of the human c-myc gene, and a 2.4-kb core fragment of the c-myc origin displays plasmid ARS activity in transfected cells and in...

Women with polycystic ovary syndrome (PCOS) are at increased risk of miscarriage, which often accompanies the hyperandrogenism and insulin resistance seen in these patients. However, neither the combinatorial interaction between these two PCOS-related etiological factors nor the mechanisms of their actions in the uterus during pregnancy are well understood. We hypothesized that hyperandrogensim and insulin resistance exert a causative role in miscarriage by inducing defects in uterine function that are accompanied by mitochondrial-mediated oxidative stress, inflammation, and perturbed gene expression. Here, we tested this hypothesis by studying the metabolic, endocrine, and uterine abnormalities in pregnant rats after exposure to daily injection of 5α-dihydrotestosterone (DHT; 1.66 mg·kg body wt−1·day−1) and/or insulin (6.0 IU/day) from gestational day 7.5 to 13.5. We showed that whereas DHT-exposed and insulin-exposed pregnant rats presented impaired insulin sensitivity, DHT + insulin-exposed pregnant rats exhibited hyperandrogenism and peripheral insulin resistance, which mirrors pregnant PCOS patients. Compared with controls, hyperandrogenism and insulin resistance in the dam were associated with alterations in uterine morphology and aberrant expression of genes responsible for decidualization ( Prl8a2, Fxyd2, and Mt1g), placentation ( Fcgr3 and Tpbpa), angiogenesis ( Flt1, Angpt1, Angpt2, Ho1, Ccl2, Ccl5, Cxcl9, and Cxcl10) and insulin signaling (Akt, Gsk3, and Gluts). Moreover, we observed changes in uterine mitochondrial function and homeostasis (i.e., mitochondrial DNA copy number and the expression of genes responsible for mitochondrial fusion, fission, biogenesis, and mitophagy) and suppression of both oxidative and antioxidative defenses (i.e., reactive oxygen species, Nrf2 signaling, and interactive networks of antioxidative stress responses) in response to the hyperandrogenism and insulin resistance. These findings demonstrate that hyperandrogenism and insulin resistance induce mitochondria-mediated damage and a resulting imbalance between oxidative and antioxidative stress responses in the gravid uterus.

Key pointsr Women with polycystic ovary syndrome (PCOS) commonly suffer from miscarriage, but the underlying mechanisms remain unknown.r Herein, pregnant rats chronically treated with 5α-dihydrotestosterone (DHT) and insulin exhibited hyperandrogenism and insulin resistance, as well as increased fetal loss, and these features are strikingly similar to those observed in pregnant PCOS patients.r Fetal loss in our DHT+insulin-treated pregnant rats was associated with mitochondrial dysfunction, disturbed superoxide dismutase 1 and Keap1/Nrf2 antioxidant responses, over-production of reactive oxygen species (ROS) and impaired formation of the placenta.r Chronic treatment of pregnant rats with DHT or insulin alone indicated that DHT triggered many of the molecular pathways leading to placental abnormalities and fetal loss, whereas insulin often exerted distinct effects on placental gene expression compared to co-treatment with DHT and insulin.r Treatment of DHT+insulin-treated pregnant rats with the antioxidant N-acetylcysteine improved fetal survival but was deleterious in normal pregnant rats.r Our results provide insight into the fetal loss associated with hyperandrogenism and insulin resistance in women and suggest that physiological levels of ROS are required for normal placental formation and fetal survival during pregnancy.Yuehui Zhang is a professor and chief physician in the . She carried out postdoctoral research training in reproductive endocrinology at the University of Gothenburg, Sweden and obstetrics and gynaecology at the University of Pennsylvania, USA. Her research aims are to uncover the cellular and molecular mechanisms in aetiologies of polycystic ovary syndrome (PCOS) and other gynaecological diseases, as well as to develop an effective treatment for PCOS patients.Abstract Women with polycystic ovary syndrome (PCOS) commonly suffer from miscarriage, but the underlying mechanism of PCOS-induced fetal loss during pregnancy remains obscure and specific therapies are lacking. We used pregnant rats treated with 5α-dihydrotestosterone (DHT) and insulin to investigate the impact of hyperandrogenism and insulin resistance on fetal survival and to determine the molecular link between PCOS conditions and placental dysfunction during pregnancy. Our study shows that pregnant rats chronically treated with a combination of DHT and insulin exhibited endocrine aberrations such as hyperandrogenism and insulin resistance that are strikingly similar to those in pregnant PCOS patients. Of pathophysiological significance, DHT+insulin-treated pregnant rats had greater fetal loss and subsequently decreased litter sizes compared to normal pregnant rats. This negative effect was accompanied by impaired trophoblast differentiation, increased glycogen accumulation, and decreased angiogenesis in the placenta. Mechanistically, we report that over-production of reactive oxygen species (ROS) in the placenta, mitochondrial dysfunction, and disturbed superoxide dismutase 1 (SOD1) and Keap1/Nrf2 antioxidant responses constitute im...

Previous studies in rats showed that maternal exposure to 5α-dihydrotestosterone (DHT) and insulin (INS) from gestational day 7.5 to 13.5 induces hyperandrogenism and insulin resistance (HAIR) and subsequently leads to placental insufficiency and fetal loss. We therefore hypothesized that maternal HAIR triggers ferroptosis in the uterus and placenta in association with fetal loss in pregnant rats. Compared with controls, we found that co-exposure to DHT and INS led to decreased levels of Gpx4 and glutathione (GSH), increased GSH+glutathione disulfide (GSSG) and malondialdehyde (MDA), aberrant expression of ferroptosis-associated genes (Acsl4, Tfrc, Slc7a11, and Gclc), increased iron deposition, and activated ERK/p38/JNK phosphorylation in the gravid uterus. However, in the placenta, DHT and INS exposure only partially altered the expression of ferroptosis-related markers (e.g., Gpx4, GSH+GSSG, MDA, Gls2 and Slc7a11 mRNAs, and phosphorylated p38 levels). In the uteri co-exposed to DHT and INS, we also observed shrunken mitochondria with electron-dense cristae, and increased Dpp4 mRNA expression. In contrast, in placentas co-exposed to DHT and INS we found decreased Dpp4 mRNA expression and increased Cisd1 mRNA expression. Further, DHT+INS-exposed pregnant rats exhibited decreased apoptosis in the uterus and increased necroptosis in the placenta. Our findings suggest that maternal HAIR causes the activation of ferroptosis in the gravid uterus and placenta, although this is mediated via different mechanisms operating at the molecular and cellular levels. Furthermore, our data suggest other cell death pathways may play a role in coordinating or compensating for HAIR-induced ferroptosis when the gravid uterus and placenta are dysfunctional.