KLF1 regulates a diverse suite of genes to direct erythroid cell differentiation from bipotent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which KLF1 operates, we performed KLF1 ChIP-seq in the mouse. We found at least 945 sites in the genome of E14.5 fetal liver erythroid cells which are occupied by endogenous KLF1. Many of these recovered sites reside in erythroid gene promoters such as Hbb-b1, but the majority are distant to any known gene. Our data suggests KLF1 directly regulates most aspects of terminal erythroid differentiation including production of alpha-and beta-globin protein chains, heme biosynthesis, coordination of proliferation and anti-apoptotic pathways, and construction of the red cell membrane and cytoskeleton by functioning primarily as a transcriptional activator. Additionally, we suggest new mechanisms for KLF1 cooperation with other transcription factors, in particular the erythroid transcription factor GATA1, to maintain homeostasis in the erythroid compartment.

During apoptosis, Bak and Bax are activated by BH3-only proteins binding to the α2–α5 hydrophobic groove; Bax is also activated via a rear pocket. Here we report that antibodies can directly activate Bak and mitochondrial Bax by binding to the α1–α2 loop. A monoclonal antibody (clone 7D10) binds close to α1 in non-activated Bak to induce conformational change, oligomerization, and cytochrome c release. Anti-FLAG antibodies also activate Bak containing a FLAG epitope close to α1. An antibody (clone 3C10) to the Bax α1–α2 loop activates mitochondrial Bax, but blocks translocation of cytosolic Bax. Tethers within Bak show that 7D10 binding directly extricates α1; a structural model of the 7D10 Fab bound to Bak reveals the formation of a cavity under α1. Our identification of the α1–α2 loop as an activation site in Bak paves the way to develop intrabodies or small molecules that directly and selectively regulate these proteins.

There is some interest in how mammalian oocytes respond to different types of DNA damage because of the increasing expectation of fertility preservation in women undergoing chemotherapy. Double strand breaks (DSBs) induced by ionizing radiation and agents such as neocarzinostatin (NCS), and interstrand crosslinks (ICLs) induced by alkylating agents such as mitomycin C (MMC), are toxic DNA lesions that need to be repaired for cell survival. Here we examined the effects of NCS and MMC treatment on oocytes collected from antral follicles in mice, because potentially such oocytes are readily collected from ovaries and do not need to be in vitro grown to achieve meiotic competency. We found that oocytes were sensitive to NCS, such that this ionizing radiation mimetic blocked meiosis I and caused fragmented DNA. In contrast, MMC had no impact on the completion of either meiosis I or II, even at extremely high doses. However, oocytes treated with MMC did show γ-H2AX foci and following their in vitro maturation and parthenogenetic activation the development of the subsequent embryos was severely compromised. Addition of MMC to 1-cell embryos caused a similarly poor level of development, demonstrating oocytes have eventual sensitivity to this ICL-inducing agent but this does not occur during their meiotic division. In oocytes, the association of Fanconi Anemia protein, FANCD2, with sites of ICL lesions was not apparent until entry into the embryonic cell cycle. In conclusion, meiotic maturation of oocytes is sensitive to DSBs but not ICLs. The ability of oocytes to tolerate severe ICL damage and yet complete meiosis, means that this type of DNA lesion goes unrepaired in oocytes but impacts on subsequent embryo quality.

STUDY QUESTION Do mitochondria-targeted therapies reverse ageing- and oxidative stress-induced spindle defects in oocytes from mice and humans? SUMMARY ANSWER Exposure to MitoQ or BGP-15 during IVM protected against spindle and chromosomal defects in mouse oocytes exposed to oxidative stress or derived from reproductively aged mice whilst MitoQ promoted nuclear maturation and protected against chromosomal misalignments in human oocytes. WHAT IS KNOWN ALREADY Spindle and chromosomal abnormalities in oocytes are more prevalent with maternal aging, increasing the risk of aneuploidy, miscarriage and genetic disorders such as Down’s syndrome. The origin of compromised oocyte function may be founded in mitochondrial dysfunction and increased reactive oxygen species (ROS). STUDY DESIGN, SIZE, DURATION Oocytes from young and old mice were treated with MitoQ and/or BGP-15 during IVM. To directly induce mitochondrial dysfunction, oocytes were treated with H2O2, and then treated the MitoQ and/or BGP-15. Immature human oocytes were cultured with or without MitoQ. Each experiment was repeated at least three times, and data were analyzed by unpaired-sample t-test or chi-square test. PARTICIPANTS/MATERIALS, SETTING, METHODS Immature germinal vesicle (GV) stage oocytes from 1-, 12- and 18-month-old mice were obtained from preovulatory ovarian follicles. Oocytes were treated with MitoQ and/or BGP-15 during IVM. GV-stage human oocytes were cultured with or without MitoQ. Mitochondrial membrane potential and mitochondrial ROS were measured by live-cell imaging. Meiotic spindle and chromosome alignments were visualized by immunofluorescent labeling of fixed oocytes and the 3-dimensional images were analyzed by Imaris. MAIN RESULTS AND THE ROLE OF CHANCE MitoQ or BGP-15 during IVM protects against spindle and chromosomal defects in oocytes exposed to oxidative stress and in oocytes from aged mice (P < 0.001). In human oocytes, the presence of MitoQ during IVM promoted nuclear maturation and had a similar positive effect in protecting against chromosomal misalignments (P < 0.001). LIMITATIONS, REASONS FOR CAUTION Our study identifies two excellent candidates that may help to improve fertility in older women. However, these potential therapies must be tested for efficacy in clinical IVM systems, and undergo thorough examination of resultant offspring in preclinical models before utilization. WIDER IMPLICATIONS OF THE FINDINGS Our results using in-vitro systems for oocyte maturation in both mouse and human provide proof of principle that mitochondrially targeted molecules such as MitoQ and BGP-15 may represent a novel therapeutic approach against maternal aging-related spindle and chromosomal abnormalities. STUDY FUNDING/COMPETING INTEREST(S) The project was financially supported by the National Health and Medical Research Council and Australian Research Council, Australia. U.A.-Z. was supported by the Iraqi Higher Education and Scientific Research Ministry PhD scholarship and O.C. was supported by TUBITAK-1059B191601275. M.P.M. consults for MitoQ Inc. and holds patents in mitochondria-targeted therapies. R.L.R. is an inventor on patents relating to the use of BGP-15 to improve gamete quality. TRIAL REGISTRATION NUMBER N/A

The role of the Cdk regulatory protein cyclin A2 and its dynamics in female meiosis are unclear. Zhang et al. show that, unlike in mitosis, cyclin A2 persists during metaphase of meiosis II (MII). Cyclin A2 regulates microtubule stability, allows normal MII spindle formation, and prevents merotelic attachments and lagging chromosomes at MII exit.

Confocal Raman spectroscopy (CRS) can provide information about oocyte competency through measurement of changes in the macromolecular architecture during oocyte development and maturation. Hitherto most spectroscopic studies have been limited to fixed oocytes due to the inherent difficulties working with live cells. Here we report the first three-dimensional images of living murine oocytes using CRS. We show that fixation induces significant changes in the macromolecular chemistry compared to living oocytes. A band at 1602 cm−1, assigned to a marker for mitochondria function was found in living oocytes but absent from fixed oocytes providing an in vivo marker. Fixation resulted in significant changes in protein and nucleic acid bands and the spatial distribution of organelles. Raman imaging of Metaphase I and II (MI, MII) and germinal vesicle stage oocytes showed changes in nuclear organisation and cytoplasm macromolecular architecture during these development and maturation stages related to changes in chromosome condensation, mitochondria aggregation and lipid droplet numbers.

The integrity of male germ cell genome is critical for the correct progression of spermatogenesis, successful fertilization, and proper development of the offspring. Several DNA repair pathways exist in male germ cells. However, unlike somatic cells, key components of such pathways remain largely unidentified. Gametogenetin (GGN) is a testis-enriched protein that has been shown to bind to the DNA repair protein FANCL via yeast-two-hybrid assays. This finding and its testis-enriched expression pattern raise the possibility that GGN plays a role in DNA repair during spermatogenesis. Herein we demonstrated that the largest isoform GGN1 interacted with components of DNA repair machinery in the mouse testis. In addition to FANCL, GGN1 interacted with the critical component of the Fanconi Anemia (FA) pathway FANCD2 and a downstream component of the BRCA pathway, BRCC36. To define the physiological function of GGN, we generated a Ggn null mouse line. A complete loss of GGN resulted in embryonic lethality at the very earliest period of pre-implantation development, with no viable blastocysts observed. This finding was consistent with the observation that Ggn mRNA was also expressed in lower levels in the oocyte and pre-implantation embryos. Moreover, pachytene spermatocytes of the Ggn heterozygous knockout mice showed an increased incidence of unrepaired DNA double strand breaks (DSBs). Together, our results suggest that GGN plays a role in male meiotic DSB repair and is absolutely required for the survival of pre-implantation embryos.

Thermal scanning probe lithography (t-SPL) is a nanofabrication technique in which an immobilized thermolabile resist, such as polyphthalaldehyde (PPA), is locally vaporized by a heated atomic force microscope tip. Compared with other nanofabrication techniques, such as soft lithography and nanoimprinting lithography, t-SPL is more efficient and convenient as it does not involve time-consuming mask productions or complicated etching procedures, making it a promising candidate technique for the fast prototyping of nanoscale topographies for biological studies. Here, we established the direct use of PPA-coated surfaces as a cell culture substrate. We showed that PPA is biocompatible and that the deposition of allylamine by plasma polymerization on a silicon wafer before PPA coating can stabilize the immobilization of PPA in aqueous solutions. When seeded on PPA-coated surfaces, human mesenchymal stem cells (MSC) adhered, spread, and proliferated in a manner indistinguishable from cells cultured on glass surfaces. This allowed us to subsequently use t-SPL to generate nanotopographies for cell culture experiments. As a proof of concept, we analyzed the surface topography of bovine tendon sections, previously shown to induce morphogenesis and differentiation of MSC, by means of atomic force microscopy, and then "wrote" topographical data on PPA by means of t-SPL. The resulting substrate, matching the native tissue topography on the nanoscale, was directly used for MSC culture. The t-SPL substrate induced similar changes in cell morphology and focal adhesion formation in the MSC compared to native tendon sections, suggesting that t-SPL can rapidly generate cell culture substrates with complex and spatially accurate topographical signals. This technique may greatly accelerate the prototyping of models for the study of cell−matrix interactions.