Centromeres are epigenetically specified chromosomal sites that support kinetochore assembly and often embedded within large satellite DNA arrays. Recent telomere to telomere genome assemblies have revealed extensive variation in centromeric satellite arrays between chromosomes and between individuals, but the functional significance of this variation remains unclear. To determine how satellite array size influences centromere function, we generated hybrid mouse models in which homologous chromosomes with different array sizes are paired in meiosis I, creating array size asymmetry across each meiotic bivalent. When an extremely small array is paired with a moderate size array, we find that array size asymmetry leads to functional asymmetry in both centromere chromatin and interactions with spindle microtubules, lagging chromosomes in anaphase I, and increased aneuploidy in MII eggs. In contrast, pairing an extremely large array with a moderate array does not lead to functional centromere asymmetry. Together, these results suggest a threshold model in which centromere array size is tolerated across a broad range, but minimal arrays become functionally limiting when paired with larger arrays in meiosis.

Persistence of unrepaired DNA damage in oocytes is detrimental and may cause genetic aberrations, miscarriage, and infertility. RPA, an ssDNA-binding complex, is essential for various DNA-related processes. Here we report that RPA plays a novel role in DNA damage repair during postnatal oocyte development after meiotic recombination. To investigate the role of RPA during oogenesis, we inactivated RPA1 (replication protein A1), the largest subunit of the heterotrimeric RPA complex, specifically in oocytes using two germline-specific Cre drivers (Ddx4-Cre and Zp3-Cre). We find that depletion of RPA1 leads to the disassembly of the RPA complex, as evidenced by the absence of RPA2 and RPA3 in RPA1-deficient oocytes. Strikingly, severe DNA damage occurs in RPA1-deficient GV-stage oocytes. Loss of RPA in oocytes triggered the canonical DNA damage response mechanisms and pathways, such as activation of ATM, ATR, DNA-PK, and p53. In addition, the RPA deficiency causes chromosome misalignment at metaphase I and metaphase II stages of oocytes, which is consistent with altered transcript levels of genes involved in cytoskeleton organization in RPA1-deficient oocytes. Absence of the RPA complex in oocytes severely impairs folliculogenesis and leads to a significant reduction in oocyte number and female infertility. Our results demonstrate that RPA plays an unexpected role in DNA damage repair during mammalian folliculogenesis.

Mammalian centromeres direct faithful genetic inheritance and are typically characterized by regions of highly repetitive and rapidly evolving DNA. We focused on a mouse species, Mus pahari, that we found has evolved to house centromere-specifying centromere protein-A (CENP-A) nucleosomes at the nexus of a satellite repeat that we identified and termed π-satellite (π-sat), a small number of recruitment sites for CENP-B, and short stretches of perfect telomere repeats. One M. pahari chromosome, however, houses a radically divergent centromere harboring ~6 mega–base pairs of a homogenized π-sat–related repeat, π-sat B , that contains >20,000 functional CENP-B boxes. There, CENP-B abundance promotes accumulation of microtubule-binding components of the kinetochore and a microtubule-destabilizing kinesin of the inner centromere. We propose that the balance of pro- and anti-microtubule binding by the new centromere is what permits it to segregate during cell division with high fidelity alongside the older ones whose sequence creates a markedly different molecular composition.

To generate haploid gametes, germ cells undergo two consecutive meiotic divisions requiring key changes to the cell division machinery. Here, we demonstrate that the protease separase rewires key cell division processes at the meiosis I/II transition by cleaving the meiosis-specific protein Meikin. Separase proteolysis does not inactivate Meikin but instead alters its function to create a distinct activity state. Full-length Meikin and the C-terminal Meikin separase cleavage product both localize to kinetochores, bind to Plk1 kinase, and promote Rec8 cleavage, but our results reveal distinct roles for these proteins in controlling meiosis. Mutations that prevent Meikin cleavage or that conditionally inactivate Meikin at anaphase I result in defective meiosis II chromosome alignment in mouse oocytes. Finally, as oocytes exit meiosis, C-Meikin is eliminated by APC/C-mediated degradation prior to the first mitotic division. Thus, multiple regulatory events irreversibly modulate Meikin activity during successive meiotic divisions to rewire the cell division machinery at two distinct transitions.

Selfish centromere DNA sequences bias their transmission to the egg in female meiosis. Evolutionary theory suggests that centromere proteins evolve to suppress costs of this "centromere drive." In hybrid mouse models with genetically different maternal and paternal centromeres, selfish centromere DNA exploits a kinetochore pathway to recruit microtubule-destabilizing proteins that act as drive effectors. We show that such functional differences are suppressed by a parallel pathway for effector recruitment by heterochromatin, which is similar between centromeres in this system. Disrupting the kinetochore pathway with a divergent allele of CENP-C reduces functional differences between centromeres, whereas disrupting heterochromatin by CENP-B deletion amplifies the differences. Molecular evolution analyses using Murinae genomes identify adaptive evolution in proteins in both pathways. We propose that centromere proteins have recurrently evolved to minimize the kinetochore pathway, which is exploited by selfish DNA, relative to the heterochromatin pathway that equalizes centromeres, while maintaining essential functions.

SKP1 coordinates chromosome synapsis and meiotic recombination with progression through the first meiotic cell division in mouse.

SummarySelfish centromere DNA sequences bias their transmission to the egg in female meiosis. Evolutionary theory suggests that centromere proteins evolve to suppress costs of this “centromere drive”. In hybrid mouse models with genetically different maternal and paternal centromeres, selfish centromere DNA exploits a kinetochore pathway to recruit microtubule-destabilizing proteins that act as drive effectors. We show that such functional differences are suppressed by a parallel pathway for effector recruitment by heterochromatin, which is similar between centromeres in this system. Disrupting heterochromatin by CENP-B deletion amplifies functional differences between centromeres, whereas disrupting the kinetochore pathway with a divergent allele of CENP-C reduces the differences. Molecular evolution analyses using newly sequenced Murinae genomes identify adaptive evolution in proteins in both pathways. We propose that centromere proteins have recurrently evolved to minimize the kinetochore pathway, which is exploited by selfish DNA, relative to the heterochromatin pathway that equalizes centromeres, while maintaining essential functions.

The N6-methyladenosine (m6A) modification is the most prevalent internal RNA modification in eukaryotes. The majority of m6A sites are found in the last exon and 3' UTRs. Here we show that the nuclear m6A reader YTHDC1 is essential for embryo viability and germline development in mouse. Specifically, YTHDC1 is required for spermatogonial development in males and for oocyte growth and maturation in females; Ythdc1-deficient oocytes are blocked at the primary follicle stage. Strikingly, loss of YTHDC1 leads to extensive alternative polyadenylation in oocytes, altering 3' UTR length. Furthermore, YTHDC1 deficiency causes massive alternative splicing defects in oocytes. The majority of splicing defects in mutant oocytes are rescued by introducing wild-type, but not m6A-binding-deficient, YTHDC1. YTHDC1 is associated with the pre-mRNA 3' end processing factors CPSF6, SRSF3, and SRSF7. Thus, YTHDC1 plays a critical role in processing of pre-mRNA transcripts in the oocyte nucleus and may have similar non-redundant roles throughout fetal development.

Mice lacking Cnot6l , a deadenylase component of the CCR4–NOT complex, are viable, but females have ∼40% smaller litters. Cnot6l is a maternal-effect gene acting in maternal mRNA degradation. Removal of poly(A) tail is an important mechanism controlling eukaryotic mRNA turnover. The major eukaryotic deadenylase complex CCR4-NOT contains two deadenylase components, CCR4 and CAF1, for which mammalian CCR4 is encoded by Cnot6 or Cnot6l paralogs. We show that Cnot6l apparently supplies the majority of CCR4 in the maternal CCR4-NOT in mouse, hamster, and bovine oocytes. Deletion of Cnot6l yielded viable mice, but Cnot6l −/− females exhibited ∼40% smaller litter size. The main onset of the phenotype was post-zygotic: fertilized Cnot6l −/− eggs developed slower and arrested more frequently than Cnot6l +/− eggs, suggesting that maternal CNOT6L is necessary for accurate oocyte-to-embryo transition. Transcriptome analysis revealed major transcriptome changes in Cnot6l −/− ovulated eggs and one-cell zygotes. In contrast, minimal transcriptome changes in preovulatory Cnot6l −/− oocytes were consistent with reported Cnot6l mRNA dormancy. A minimal overlap between transcripts sensitive to decapping inhibition and Cnot6l loss suggests that decapping and CNOT6L-mediated deadenylation selectively target distinct subsets of mRNAs during oocyte-to-embryo transition in mouse.

Affiliations: 1 Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic 2 Bioinformatics Group, Division of Molecular Biology, Department of Biology, Faculty of Science, University of Zagreb, Horvatovac 102a, Zagreb, Croatia 3 Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan 4 Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan 5 Department of Biology, University of Pennsylvania, Philadelphia, 19104 USA 6 Current address: Berlin Institute for Medical Systems Biology, Max-Delbrück Center for Molecular Medicine, Germany 7 Current address: Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA

Retrotransposons are “copy-and-paste” insertional mutagens that substantially contribute to mammalian genome content. Retrotransposons often carry long terminal repeats (LTRs) for retrovirus-like reverse transcription and integration into the genome. We report an extraordinary impact of a group of LTRs from the mammalian endogenous retrovirus-related ERVL retrotransposon class on gene expression in the germline and beyond. In mouse, we identified more than 800 LTRs from ORR1, MT, MT2, and MLT families, which resemble mobile gene-remodeling platforms that supply promoters and first exons. The LTR-mediated gene remodeling also extends to hamster, human, and bovine oocytes. The LTRs function in a stage-specific manner during the oocyte-to-embryo transition by activating transcription, altering protein-coding sequences, producing noncoding RNAs, and even supporting evolution of new protein-coding genes. These functions result, for example, in recycling processed pseudogenes into mRNAs or lncRNAs with regulatory roles. The functional potential of the studied LTRs is even higher, because we show that dormant LTR promoter activity can rescue loss of an essential upstream promoter. We also report a novel protein-coding gene evolution—D6Ertd527e—in which an MT LTR provided a promoter and the 5′ exon with a functional start codon while the bulk of the protein-coding sequence evolved through a CAG repeat expansion. Altogether, ERVL LTRs provide molecular mechanisms for stochastically scanning, rewiring, and recycling genetic information on an extraordinary scale. ERVL LTRs thus offer means for a comprehensive survey of the genome's expression potential, tightly intertwining with gene expression and evolution in the germline.

The germ cell lineage is specified early in embryogenesis and undergoes complex developmental programs to generate gametes. Here, we conducted genetic studies to investigate the role of Utf1 (Undifferentiated embryonic cell transcription factor 1) in mouse germ cell development. Utf1 is expressed in pluripotent embryonic stem (ES) cells and regulates ES cell differentiation. In a proteomics screen, we identified UTF1 among 38 proteins including DNMT3L and DND1 that associate with chromatin in embryonic testes. We find that UTF1 is expressed in embryonic and newborn gonocytes and in a subset of early spermatogonia. Ubiquitous inactivation of Utf1 causes embryonic lethality in mice with a hybrid genetic background. Male mice with a germline-specific deletion of Utf1 resulting from Prdm1-Cre mediated recombination are born with significantly fewer gonocytes and exhibit defective spermatogenesis and reduced sperm count as young adults. These defects are ameliorated in older animals. These results demonstrate that UTF1 is required for embryonic development and regulates male germ cell development.

The oocyte-to-embryo transition entails genome activation and a dramatic reprogramming of gene expression that is required for continued development. Superimposed on genome activation and reprogramming is development of a transcriptionally repressive state at the level of chromatin structure. Inducing global histone hyperacetylation relieves this repression and histone deacetylases 1 and 2 (HDAC1 and HDAC2) are involved in establishing the repressive state. Because SIN3A is an HDAC1/2-containing complex, we investigated whether it is involved in reprogramming gene expression during the course of genome activation. We find that Sin3a mRNA is recruited during maturation and that inhibiting its recruitment not only inhibits development beyond the 2-cell stage but also compromises the fidelity of reprogramming gene expression. The SIN3A that is synthesized during oocyte maturation reaches a maximum level in the mid-1-cell embryo and is essentially absent by the mid-2-cell stage. Overexpressing SIN3A in 1-cell embryos has no obvious effect on pre- and postimplantation development. These results provide a mechanism by which reprogramming can occur using a maternally inherited transcription machinery, namely, recruitment of mRNAs encoding transcription factors and chromatin remodelers, such as SIN3A.

Maternal mRNAs in oocytes are remarkably stable. In mouse, oocyte maturation triggers a transition from mRNA stability to instability. This transition is a critical event in the oocyte-to-embryo transition in which a differentiated oocyte loses its identity as it is transformed into totipotent blastomeres. We previously demonstrated that phosphorylation of MSY2, an RNA-binding protein, and mobilization of mRNAs encoding the DCP1A-DCP2 decapping complex contribute to maternal mRNA destruction during meiotic maturation. We report here that Cnot7, Cnot6l, and Pan2, key components of deadenylation machinery, are also dormant maternal mRNAs that are recruited during oocyte maturation. Inhibiting the maturation-associated increase in CNOT7 (or CNOT6L) using a small interference RNA approach inhibits mRNA deadenylation, whereas inhibiting the increase in PAN2 has little effect. Reciprocally, expressing CNOT7 (or CNOT6L) in oocytes prevented from resuming meiosis initiates deadenylation of mRNAs. These effects on deadenylation are also observed when the total amount of poly (A) is quantified. Last, inhibiting the increase in CNOT7 protein results in an ~70% decrease in transcription in 2-cell embryos.

Menopause results from loss of ovarian function and marks the end of a woman's reproductive life. Alleles of the human SYCP2L locus are associated with age at natural menopause (ANM). SYCP2L is a paralogue of the synaptonemal complex protein SYCP2 and is expressed exclusively in oocytes. Here we report that SYCP2L localizes to centromeres of dictyate stage oocytes, which represent the limited pool of primordial oocytes that are formed perinatally and remain arrested till ovulation. Centromere localization of SYCP2L requires its C-terminal portion, which is missing in truncated variants resulting from low-frequency nonsense mutations identified in humans. Female mice lacking SYCP2L undergo a significantly higher progressive loss of oocytes with age compared with wild-type females and are less fertile. Specifically, the pool of primordial oocytes becomes more rapidly depleted in SYCP2L-deficient than in wild-type females, such that with aging, fewer oocytes undergo maturation in developing follicles. We find that a human SYCP2L intronic single nucleotide polymorphism (SNP) rs2153157, which is associated with ANM, changes the splicing efficiency of U12-type minor introns and may therefore regulate the steady-state amount of SYCP2L transcript. Furthermore, the more efficiently spliced allele of this intronic SNP in SYCP2L is associated with increased ANM. Our results suggest that SYCP2L promotes the survival of primordial oocytes and thus provide functional evidence for its association with ANM in humans.

A full-length cDNA of cyclin B was isolated from ovary of the mud crab (Scylla paramamosain) in this study. This transcript encodes a polypeptide of 401 amino acids, which is highly homologous to cyclin B protein family. Reverse transcription-PCR (RT-PCR) showed that cyclin B mRNA was expressed at highest levels in ovary of the mud crab. During the ovarian maturation process, real-time RT-PCR revealed that the abundance of cyclin B mRNA increased from the second stage (early-developing stage) to the fourth stage (nearly-ripe stage) and reached the peak level at the fifth stage (ripe stage). This result indicates the identified cyclin B gene might be related to the cell proliferation in ovary, both mitotically and meiotically. Immunohistochemistry showed that cyclin B protein was localized in the cytoplasm of prophase oocytes at the second stage while enriched in the nuclei of pro-metaphase oocytes at the fourth stage. It suggests the tested cyclin B protein might play different roles in ovary at the two stages.

The oocyte-to-zygote transition entails transforming a highly differentiated oocyte into totipotent blastomeres and represents one of the earliest obstacles that must be successfully hurdled for continued development. Degradation of maternal mRNAs, which likely lies at the heart of this transition, is characterized by a transition from mRNA stability to instability during oocyte maturation. Although phosphorylation of the oocyte-specific RNA-binding protein MSY2 during maturation is implicated in making maternal mRNAs more susceptible to degradation, mechanisms underlying mRNA degradation during oocyte maturation remain poorly understood. We report that DCP1A and DCP2, proteins responsible for decapping mRNA, are encoded by maternal mRNAs recruited for translation during maturation via cytoplasmic polyadenylation elements located in their 3' untranslated regions. Both DCP1A and DCP2 are phosphorylated during maturation, with CDC2A being the kinase likely responsible for both, although MAPK may be involved in DCP1A phosphorylation. Inhibiting accumulation of DCP1A and DCP2 by RNA interference or morpholinos decreases not only degradation of mRNAs during meiotic maturation but also transcription of the zygotic genome. The results indicate that maternally recruited DCP1A and DCP2 are critical players in the transition from mRNA stability to instability during meiotic maturation and that proper maternal mRNA degradation must be successful to execute the oocyte-to-zygote transition.

MicroRNAs (miRNAs) are small endogenous RNAs that typically imperfectly base pair with 3' untranslated regions (3'UTRs) and mediate translational repression and mRNA degradation. Dicer, which generates small RNAs in the miRNA and RNA interference (RNAi) pathways, is essential for meiotic maturation of mouse oocytes. We found that 3'UTRs of transcripts upregulated in Dicer1(-/-) oocytes are not enriched in miRNA binding sites, implicating a weak impact of miRNAs on the maternal transcriptome. Therefore, we tested the ability of endogenous miRNAs to mediate RNA-like cleavage or translational repression of reporter mRNAs. In contrast to somatic cells, endogenous miRNAs in oocytes poorly repressed translation of mRNA reporters, whereas their RNAi-like activity was much less affected. Reporter mRNA carrying let-7-binding sites failed to localize to P body-like structures in oocytes. Our data suggest that miRNA function is downregulated during oocyte development, an idea supported by normal melotic maturation of oocytes lacking Dgcr8, which is required for the miRNA but not the RNAi pathway (Suh et al. [1], this issue of Current Biology). Suppressing miRNA function during oocyte growth is likely an early event in reprogramming gene expression during the transition of a differentiated oocyte into pluripotent blastomeres of the embryo.

Vertebrate-type steroids, such as progesterone, have been identified in crustaceans. The physiological activity of progesterone during vitellogenesis is still not well understood. In this study, progesterone levels in the female mud crab, Scylla paramamosain, were determined by enzyme-linked immunosorbent assay. Peak levels of progesterone were detected during the previtellogenic stage in the hemolymph, ovary, and hepatopancreas, whereas the progesterone level decreased significantly in vitellogenic stage I. During vitellogenic stage II, progesterone levels rose again in the hemolymph and ovary, but continued to decrease in the hepatopancreas. By using western blotting, progesterone receptor (PR), with an apparent molecular weight of 70 kDa, was identified in the ovary during both vitellogenic stages I and II. By means of immunohistochemistry, PR was detected mainly in the follicle cells during vitellogenic stage I and in the nuclei of oocytes in vitellogenic stage II. Our results strongly suggest that progesterone promotes vitellogenesis in the mud crab, S. paramamosain via a classical genomic mechanism.

Crustacean neurons, obtained from the cerebral ganglion of the mud crab Scylla paramamosain, were successfully cultured in vitro. They maintained typical morphological characteristics and showed better outgrowth in modified Medium 199 (M199) medium than that in Liebowitz's L-15 medium. Fetal bovine serum (FBS), muscle extracts, and hemolymph of the mud crab S. paramamosain were added as supplements. Only 20% FBS could promote neuron outgrowth, while muscle extracts and hemolymph of S. paramamosain did not improve neuron outgrowth. For cell dissociation, both collagenase type I and trypsin worked well as determined by initial cell viability and following cell outgrowth potential. More than six kinds of cells with different morphological characteristics were identified in the neuron outgrowth. They were "small cells", "veilers", "branchers", "multipolar cells", "super-large cell", and "bipolar cells". Among all of the cells, bipolar cells were identified for the first time in crustacean neurons culture and they could live longer than other cells. The neurons could grow for more than a week before retraction and eventual degradation.