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Background Oocyte fertilization and successful embryo implantation are key events marking the onset of pregnancy. In sexually reproducing organisms, embryogenesis begins with the fusion of two haploid gametes, each of which has undergone progressive stages of maturation. In the final stages of oocyte maturation, minimal transcriptional activity is present and regulation of gene expression occurs primarily at the post-transcriptional level. MicroRNAs (miRNA) are potent effectors of post-transcriptional gene silencing and recent evidence demonstrates that the miR-34 family of miRNA are involved in both spermatogenesis and early events of embryogenesis. Methods The profile of miR-34 miRNAs has not been characterized in gametes or embryos of Bos taurus . We therefore used quantitative reverse transcription PCR (qRT-PCR) to examine this family of miRNAs: miR-34a, -34b and -34c as well as their precursors in bovine gametes and in vitro produced embryos. Oocytes were aspirated from antral follicles of bovine ovaries, and sperm cells were isolated from semen samples of 10 bulls with unknown fertility status. Immature and in vitro matured oocytes, as well as cleaved embryos, were collected in pools. Gametes, embryos and ovarian and testis tissues were purified for RNA. Results All members of the miR-34 family are present in bovine spermatozoa, while only miR-34a and -34c are present in oocytes and cleaved (2-cell) embryos. Mir-34c demonstrates variation among different bulls and is consistently expressed throughout oocyte maturation and in the embryo. The primary transcript of the miR-34b/c bicistron is abundant in the testes and present in ovarian tissue but undetectable in oocytes and in mature spermatozoa. Conclusions The combination of these findings suggest that miR-34 miRNAs may be required in developing bovine gametes of both sexes, as well as in embryos, and that primary miR-34b/c processing takes place before the completion of gametogenesis. Individual variation in sperm miR-34 family abundance may offer potential as a biomarker of male bovine fertility.

Phase separation of substrates and effectors is proposed to enhance biological reaction rates and efficiency. Targeting protein for Xklp2 (TPX2) is an effector of branching microtubule nucleation in spindles and functions with the substrate tubulin by an unknown mechanism. Here we show that TPX2 phase separates into a co-condensate with tubulin, which mediates microtubule nucleation in vitro and in isolated cytosol. TPX2-tubulin co-condensation preferentially occurs on pre-existing microtubules, the site of branching microtubule nucleation, at the endogenous and physiologically relevant concentration of TPX2. Truncation and chimera versions of TPX2 suggest that TPX2-tubulin co-condensation enhances the efficiency of TPX2-mediated branching microtubule nucleation. Finally, the known inhibitor of TPX2, the importin-α/β heterodimer, regulates TPX2 condensation in vitro and, consequently, branching microtubule nucleation activity in isolated cytosol. Our study demonstrates how regulated phase separation can simultaneously enhance reaction efficiency and spatially coordinate microtubule nucleation, which may facilitate rapid and accurate spindle formation. The microtubule binding protein TPX2 enhances branching microtubule nucleation though the current mechanisms are unclear. Here, the authors show that TPX2 undergoes liquid-liquid phase separation and co-condensates with tubulin to enhance TPX2-mediated microtubule nucleation.

Fertilization occurs when sperm and egg recognize each other and fuse to form a new, genetically distinct organism. The molecular basis of sperm–egg recognition is unknown, but is likely to require interactions between receptor proteins displayed on their surface. Izumo1 is an essential sperm cell-surface protein, but its receptor on the egg has not been described. Here we identify folate receptor 4 (Folr4) as the receptor for Izumo1 on the mouse egg, and propose to rename it Juno. We show that the Izumo1–Juno interaction is conserved within several mammalian species, including humans. Female mice lacking Juno are infertile and Juno -deficient eggs do not fuse with normal sperm. Rapid shedding of Juno from the oolemma after fertilization suggests a mechanism for the membrane block to polyspermy, ensuring eggs normally fuse with just a single sperm. Our discovery of an essential receptor pair at the nexus of conception provides opportunities for the rational development of new fertility treatments and contraceptives. The egg receptor for Izumo, a sperm cell-surface protein required for male fertility, is identified here and renamed Juno; these findings show that the Izumo–Juno interaction is conserved within mammals, and open new opportunities for the development of fertility treatments and contraceptives. 'Conception protein' Juno identified in sperm Little is known about how sperm and egg specifically recognize each other. A sperm cell surface protein that is required for male fertility (called Izumo) was identified in 2005, but the identity of its partner on the egg has remained elusive. Gavin Wright and colleagues have now identified the egg receptor for Izumo, which they name Juno. Female mice lacking Juno are infertile and Juno-deficient eggs do not fuse with normal sperm. The authors show that the interaction is conserved within mammals, so these findings may open possible opportunities for the development of new fertility treatments and contraceptives.

Spindle microtubules are well known to orchestrate the segregation of chromosomes during egg development. But the other major cytoskeletal component, actin, has not been thought to play a role in this process. Mogessie and Schuh examined how chromosomes are segregated in mammalian cells (see the Perspective by Maiato and Ferrás). Instead of using an entirely microtubule-dependent mechanism, mammalian oocytes use a second spindle that is made of F-actin to segregate their chromosomes correctly. Actin associated with the spindle bundles microtubules into functional kinetochore fibers, the key structures that drive chromosome segregation. Increasing or decreasing the number of actin filaments in the spindle causes an imbalance in kinetochore fiber bundling, which results in chromosome segregation errors and aneuploidy, a frequent cause of miscarriage and Down syndrome in humans. Science , this issue p. eaal1647 ; see also p. 756 Actin prevents chromosome segregation errors by promoting the formation of functional kinetochore fibers. Chromosome segregation is driven by a spindle that is made of microtubules but is generally thought to be independent of actin. Here, we report an unexpected actin-dependent mechanism that drives the accurate alignment and segregation of chromosomes in mammalian eggs. Prominent actin filaments permeated the microtubule spindle in eggs of several mammalian species, including humans. Disrupting actin in mouse eggs led to significantly increased numbers of misaligned chromosomes as well as lagging chromosomes during meiosis I and II. We found that actin drives accurate chromosome segregation by promoting the formation of functional kinetochore fibers, the microtubule bundles that align and segregate the chromosomes. Thus, actin is essential to prevent chromosome segregation errors in eggs, which are a leading cause of miscarriages, infertility, and Down syndrome.

Genome-wide erasure of DNA cytosine-5 methylation has been reported to occur along the paternal pronucleus in fertilized oocytes in an apparently replication-independent manner, but the mechanism of this reprogramming process has remained enigmatic. Recently, considerable amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5-methylcytosine (5mC) by TET proteins, have been detected in certain mammalian tissues. 5hmC has been proposed as a potential intermediate in active DNA demethylation. Here, we show that in advanced pronuclear-stage zygotes the paternal pronucleus contains substantial amounts of 5hmC but lacks 5mC. The converse is true for the maternal pronucleus, which retains 5mC but shows little or no 5hmC signal. Importantly, 5hmC persists into mitotic one-cell, two-cell, and later cleavage-stage embryos, suggesting that 5mC oxidation is not followed immediately by genome-wide removal of 5hmC through excision repair pathways or other mechanisms. This conclusion is supported by bisulfite sequencing data, which shows only limited conversion of modified cytosines to cytosines at several gene loci. It is likely that 5mC oxidation is carried out by the Tet3 oxidase. Tet3, but not Tet1 or Tet2, was expressed at high levels in oocytes and zygotes, with rapidly declining levels at the two-cell stage. Our results show that 5mC oxidation is part of the early life cycle of mammals.

In an in vitro Xenopus extract system that can undergo cell cycles, mitosis spreads through the cytoplasm in ‘trigger waves’ of Cdk1 activation, which may reflect a more general biological mechanism related to action potentials and calcium waves. Mitosis control by Cdk1 activation trigger waves The Xenopus laevis egg is large in size yet mitosis is rapid and spatially coordinated, a feat that cannot be accounted for by spatial diffusion of the activating kinase Cdk1. Here, using an in vitro Xenopus extract system that can undergo cell cycles, Jeremy Chang and James Ferrell demonstrate that mitosis spreads through the cytoplasm through the propagation of 'trigger waves' of Cdk1 activation. These mitotic waves may reflect a more general biological concept also related to action potentials and calcium waves. Despite the large size of the Xenopus laevis egg (approximately 1.2 mm diameter), a fertilized egg rapidly proceeds through mitosis in a spatially coordinated fashion. Mitosis is initiated by a bistable system of regulatory proteins centred on Cdk1 (refs 1 , 2 ), raising the possibility that this spatial coordination could be achieved through trigger waves of Cdk1 activity 3 . Using an extract system that performs cell cycles in vitro , here we show that mitosis does spread through Xenopus cytoplasm via trigger waves, propagating at a linear speed of approximately 60 µm min −1 . Perturbing the feedback loops that give rise to the bistability of Cdk1 changes the speed and dynamics of the waves. Time-lapse imaging of intact eggs argues that trigger waves of Cdk1 activation are responsible for surface contraction waves, ripples in the cell cortex that precede cytokinesis 4 , 5 . These findings indicate that Cdk1 trigger waves help ensure the spatiotemporal coordination of mitosis in large eggs. Trigger waves may be an important general mechanism for coordinating biochemical events over large distances.

Environmental compounds including fungicides, plastics, pesticides, dioxin and hydrocarbons can promote the epigenetic transgenerational inheritance of adult-onset disease in future generation progeny following ancestral exposure during the critical period of fetal gonadal sex determination. This study examined the actions of the pesticide methoxychlor to promote the epigenetic transgenerational inheritance of adult-onset disease and associated differential DNA methylation regions (i.e. epimutations) in sperm. Gestating F0 generation female rats were transiently exposed to methoxychlor during fetal gonadal development (gestation days 8 to 14) and then adult-onset disease was evaluated in adult F1 and F3 (great-grand offspring) generation progeny for control (vehicle exposed) and methoxychlor lineage offspring. There were increases in the incidence of kidney disease, ovary disease, and obesity in the methoxychlor lineage animals. In females and males the incidence of disease increased in both the F1 and the F3 generations and the incidence of multiple disease increased in the F3 generation. There was increased disease incidence in F4 generation reverse outcross (female) offspring indicating disease transmission was primarily transmitted through the female germline. Analysis of the F3 generation sperm epigenome of the methoxychlor lineage males identified differentially DNA methylated regions (DMR) termed epimutations in a genome-wide gene promoters analysis. These epimutations were found to be methoxychlor exposure specific in comparison with other exposure specific sperm epimutation signatures. Observations indicate that the pesticide methoxychlor has the potential to promote the epigenetic transgenerational inheritance of disease and the sperm epimutations appear to provide exposure specific epigenetic biomarkers for transgenerational disease and ancestral environmental exposures.

Sperm acrosomal membrane proteins, such as Izumo sperm–egg fusion 1 (IZUMO1) and sperm acrosome-associated 6 (SPACA6), play essential roles in mammalian gamete binding or fusion. How their biosynthesis is regulated during spermiogenesis has largely remained elusive. Here, we show that 1700029I15Rik knockout male mice are severely subfertile and their spermatozoa do not fuse with eggs. 1700029I15Rik is a type-II transmembrane protein expressed in early round spermatids but not in mature spermatozoa. It interacts with proteins involved in N-linked glycosylation, disulfide isomerization, and endoplasmic reticulum (ER)–Golgi trafficking, suggesting a potential role in nascent protein processing. The ablation of 1700029I15Rik destabilizes non-catalytic subunits of the oligosaccharyltransferase (OST) complex that are pivotal for N-glycosylation. The knockout testes exhibit normal expression of sperm plasma membrane proteins, but decreased abundance of multiple acrosomal membrane proteins involved in fertilization. The knockout sperm show upregulated chaperones related to ER-associated degradation (ERAD) and elevated protein ubiquitination; strikingly, SPACA6 becomes undetectable. Our results support for a specific, 1700029I15Rik-mediated pathway underpinning the biosynthesis of acrosomal membrane proteins during spermiogenesis.

Diapause represents a major developmental switch in insects and is a seasonal adaptation that evolved as a specific subtype of dormancy in most insect species to ensure survival under unfavorable environmental conditions and synchronize populations. However, the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration in morphological, physiological, behavioral, and reproductive responses remains unclear. In the bivoltine strain of the silkworm Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother. Here, we show that the hierarchical pathway consists of a γ-aminobutyric acid (GABA)ergic and corazonin signaling system modulating progeny diapause induction via diapause hormone release, which may be finely tuned by the temperature-dependent expression of plasma membrane GABA transporter. Furthermore, this signaling pathway possesses similar features to the gonadotropin-releasing hormone (GnRH) signaling system for seasonal reproductive plasticity in vertebrates.

Mutations in CDCA7, the SNF2 family protein HELLS (LSH), or the DNA methyltransferase DNMT3b cause immunodeficiency–centromeric instability–facial anomalies (ICF) syndrome. While it has been speculated that DNA methylation defects cause this disease, little is known about the molecular function of CDCA7 and its functional relationship to HELLS and DNMT3b. Systematic analysis of how the cell cycle, H3K9 methylation, and the mitotic kinase Aurora B affect proteomic profiles of chromatin in Xenopus egg extracts revealed that HELLS and CDCA7 form a stoichiometric complex on chromatin, in a manner sensitive to Aurora B. Although HELLS alone fails to remodel nucleosomes, we demonstrate that the HELLS–CDCA7 complex possesses nucleosome remodeling activity. Furthermore, CDCA7 is essential for loading HELLS onto chromatin, and CDCA7 harboring patient ICF mutations fails to recruit the complex to chromatin. Together, our study identifies a unique bipartite nucleosome remodeling complex where the functional remodeling activity is split between two proteins and thus delineates the defective pathway in ICF syndrome.