Maria M. Viveiros

Zygote arrest 1 ( Zar1 ) is a novel maternal-effect gene critical for the oocyte-to-embryo transition

The female gamete (the oocyte) serves the distinct purpose of transmitting the maternal genome and other maternal factors that are critical for post-ovulation events. Through the identification and characterization of oocyte-specific factors, we are beginning to appreciate the diverse functions of oocytes in ovarian folliculogenesis, fertilization and embryogenesis. To understand these processes further, we identified genes called zygote arrest 1 (Zar1 and ZAR1 in mouse and human, respectively) as novel oocyte-specific genes. These encode proteins of 361 amino acids and 424 amino acids, respectively, which share 59% amino-acid identity and an atypical plant homeo-domain (PHD) motif. Although Zar1-null (Zar1−/−) mice are viable and grossly normal, Zar1−/− females are infertile. Ovarian development and oogenesis through the early stages of fertilization are evidently unimpaired, but most embryos from Zar1−/− females arrest at the one-cell stage. Distinct pronuclei form and DNA replication initiates, but the maternal and paternal genomes remain separate in arrested zygotes. Fewer than 20% of the embryos derived from Zar1−/− females progress to the two-cell stage and show marked reduction in the synthesis of the transcription-requiring complex, and no embryos develop to the four-cell stage. Thus, Zar1 is the first identified oocyte-specific maternal-effect gene that functions at the oocyte-to-embryo transition and, as such, offers new insights into the initiation of embryonic development and fertility control in mammals.

Regulation of Mammalian Oocyte Maturation

Oocyte maturation stands uniquely at the foundation and culmination of developmental and reproductive biology. The processes comprising maturation are essential for the transition from a gamete to an embryo which, upon fertilization is competent to give rise to a healthy new individual. This chapter describes three oocyte maturational processes: (1) nuclear maturation, (2) epigenetic maturation, and (3) cytoplasmic maturation. Nuclear maturation refers to the resumption of meiosis and its progression to metaphase II. Epigenetic maturation occurs during oocyte growth and refers to genomic modifications that regulate gene expression during oocyte development and postfertilization. Cytoplasmic maturation includes processes, exclusive of meiosis, that prepare the oocyte for fertilization and embryogenesis and occur throughout oocyte growth and concurrently with nuclear maturation.

Loss of Maternal ATRX Results in Centromere Instability and Aneuploidy in the Mammalian Oocyte and Pre-Implantation Embryo

The α-thalassemia/mental retardation X-linked protein (ATRX) is a chromatin-remodeling factor known to regulate DNA methylation at repetitive sequences of the human genome. We have previously demonstrated that ATRX binds to pericentric heterochromatin domains in mouse oocytes at the metaphase II stage where it is involved in mediating chromosome alignment at the meiotic spindle. However, the role of ATRX in the functional differentiation of chromatin structure during meiosis is not known. To test ATRX function in the germ line, we developed an oocyte-specific transgenic RNAi knockdown mouse model. Our results demonstrate that ATRX is required for heterochromatin formation and maintenance of chromosome stability during meiosis. During prophase I arrest, ATRX is necessary to recruit the transcriptional regulator DAXX (death domain associated protein) to pericentric heterochromatin. At the metaphase II stage, transgenic ATRX-RNAi oocytes exhibit abnormal chromosome morphology associated with reduced phosphorylation of histone 3 at serine 10 as well as chromosome segregation defects leading to aneuploidy and severely reduced fertility. Notably, a large proportion of ATRX-depleted oocytes and 1-cell stage embryos exhibit chromosome fragments and centromeric DNA–containing micronuclei. Our results provide novel evidence indicating that ATRX is required for centromere stability and the epigenetic control of heterochromatin function during meiosis and the transition to the first mitosis.

H1FOO Is Coupled to the Initiation of Oocytic Growth

Abstract We previously reported the discovery of a novel mammalian H1 linker histone termed H1FOO (formerly H1OO), a replacement H1, the expression of which is restricted to the growing/ maturing oocyte and to the zygote. The significance of this pre-embryonic H1 draws on its substantial orthologous conservation, singular structural attributes, selectivity for the germ cell lineage, prolonged nucleosomal residence, and apparent predominance among germ cell H1s. Herein, we report that the intronic, single-copy, five-exon (≥5301 base pair) H1foo gene maps to chromosome 6 and that the corresponding primary H1foo transcript gives rise to two distinct, alternatively spliced mRNA species (H1fooα and H1fooβ). The expression of the oocytic H1FOO transcript and protein proved temporally coupled to the recruitment of resting primordial follicles into a developing primary follicular cohort and thus to the critical transition marking the onset of oocytic growth. The corresponding potential protein isoforms (H1FOOα and H1FOOβ), both nuclear localization sequence-endowed but export consensus sequence-free and possessing a significant net positive charge, localized primarily to perinucleolar heterochromatin in the oocytic germinal vesicle. Further investigation will be required to define the functional role of the H1FOO protein in the ordering of the chromatin of early mammalian development as well as its potential role in defining the primordial-to-primary follicle transition.

NEDD1 is crucial for meiotic spindle stability and accurate chromosome segregation in mammalian oocytes.

Defects in meiotic spindle structure contribute to chromosome segregation errors leading to genomic instability in oocytes and embryos upon fertilization. In this study, we analyzed the mechanisms that control spindle microtubule nucleation and stability in mammalian oocytes, and identified NEDD1/GCP-WD as a key regulator. NEDD1 specifically co-localizes with gamma-tubulin and pericentrin at microtubule-organizing centers (MTOCs) in mouse oocytes arrested at prophase-I. During metaphase-I and metaphase-II, the protein remains associated with MTOCs, in a pericentrin dependent manner. Notably, knockdown of Nedd1 transcripts using specific siRNAs resulted in a high incidence (65-70%) of metaphase-I arrest. The arrested oocytes were characterized by disrupted meiotic spindle structure, reduced microtubule density and significant chromosome misalignment. Detection of MAD2 at kinetochores indicated an absence of stable chromosome-microtubule attachment as well as activation of the spindle assembly checkpoint (SAC). Importantly, the disruption of meiotic spindle stability was associated with decreased gamma-tubulin at MTOCs in NEDD1-depleted oocytes, as well as a high frequency of chromosome non-disjunction errors leading to aneuploidy (50%) in the oocytes that did progress to metaphase-II. This study demonstrates that NEDD1 is an essential component of acentriolar oocyte MTOCs, which functions in the regulation of meiotic spindle stability. Moreover, it underscores that disruption of spindle stability in oocytes can lead to chromosomes segregation errors that are not fully resolved by SAC.

Characterization of Protein Kinase C-δ in Mouse Oocytes Throughout Meiotic Maturation and Following Egg Activation

Abstract Changes in protein kinase C (PKC) activity influence the progression of meiosis; however, the specific function of the various PKC isoforms in female gametes is not known. In the current study, the protein expression and subcellular distribution profile of PKC-delta (PKC-δ), a novel isoform of the PKC family, was determined in mouse oocytes undergoing meiotic maturation and following egg activation. The full-length protein was observed as a doublet (76 and 78 kDa) on Western blot analysis. A smaller (47 kDa) carboxyl-terminal fragment, presumably the truncated catalytic domain of PKC-δ, was also strongly expressed. Both the full-length protein and the catalytic fragment became phosphorylated coincident with the resumption of meiosis and remained phosphorylated throughout metaphase II (MII) arrest. Immunofluorescence staining showed PKC-δ distributed diffusely throughout the cytoplasm of oocytes during maturation and associated with the spindle apparatus during the first meiotic division. Discrete foci of the protein also localized with the chromosomes in some mature eggs. Following the completion of meiosis, PKC-δ became dephosphorylated within 2 h of in vitro fertilization or parthenogenetic activation. The protein also accumulated in the nuclei of early embryos and was phosphorylated during M-phase of the initial mitotic cleavage division. By the two-cell stage, expression of the truncated catalytic fragment was minimal. These data demonstrate that the subcellular distribution and posttranslational modification of PKC-δ is cell cycle dependent, suggesting that its activity and/or function likely vary with the progression of meiosis and egg activation.

Role of ATRX in chromatin structure and function: implications for chromosome instability and human disease

Functional differentiation of chromatin structure is essential for the control of gene expression, nuclear architecture, and chromosome stability. Compelling evidence indicates that alterations in chromatin remodeling proteins play an important role in the pathogenesis of human disease. Among these, α-thalassemia mental retardation X-linked protein (ATRX) has recently emerged as a critical factor involved in heterochromatin formation at mammalian centromeres and telomeres as well as facultative heterochromatin on the murine inactive X chromosome. Mutations in human ATRX result in an X-linked neurodevelopmental condition with various degrees of gonadal dysgenesis (ATRX syndrome). Patients with ATRX syndrome may exhibit skewed X chromosome inactivation (XCI) patterns, and ATRX-deficient mice exhibit abnormal imprinted XCI in the trophoblast cell line. Non-random or skewed XCI can potentially affect both the onset and severity of X-linked disease. Notably, failure to establish epigenetic modifications associated with the inactive X chromosome (Xi) results in several conditions that exhibit genomic and chromosome instability such as fragile X syndrome as well as cancer development. Insight into the molecular mechanisms of ATRX function and its interacting partners in different tissues will no doubt contribute to our understanding of the pathogenesis of ATRX syndrome as well as the epigenetic origins of aneuploidy. In turn, this knowledge will be essential for the identification of novel drug targets and diagnostic tools for cancer progression as well as the therapeutic management of global epigenetic changes commonly associated with malignant neoplastic transformation.

Protein kinase C delta (PKCδ) interacts with microtubule organizing center (MTOC)-associated proteins and participates in meiotic spindle organization

Defects in meiotic spindle structure can lead to chromosome segregation errors and genomic instability. In this study the potential role of protein kinase C delta (PKCdelta) on meiotic spindle organization was evaluated in mouse oocytes. PKCdelta was previously shown to be phosphorylated during meiotic maturation and concentrate on the meiotic spindle during metaphases I and II. Currently we show that when phosphorylated on Threonine 505 (pPKCdelta(Thr505)), within the activation loop of its C4 domain, PKCdelta expression was restricted to the meiotic spindle poles and a few specific cytoplasmic foci. In addition, pPKCdelta(Thr505) co-localized with two key microtubule organizing center (MTOC)-associated proteins, pericentrin and gamma-tubulin. An interaction between pPKCdelta(Thr505) and pericentrin as well as gamma-tubulin was confirmed by co-immunoprecipitation analysis using both fetal fibroblast cells and oocytes. Notably, targeted knockdown of PKCdelta expression in oocytes using short interfering RNAs effectively reduced pPKCdelta(Thr505) protein expression at MTOCs and leads to a significant (P < 0.05) disruption of meiotic spindle organization and chromosome alignment during MI and MII. Moreover, both gamma-tubulin and pericentrin expression at MTOCs were decreased in pPKCdelta(Thr505)-depleted oocytes. In sum, these results indicate that pPKCdelta(Thr505) interacts with MTOC-associated proteins and plays a role in meiotic spindle organization in mammalian oocytes.

ATRX contributes to epigenetic asymmetry and silencing of major satellite transcripts in the maternal genome of the mouse embryo

A striking proportion of human cleavage-stage embryos exhibit chromosome instability (CIN). Notably, until now, no experimental model has been described to determine the origin and mechanisms of complex chromosomal rearrangements. Here, we examined mouse embryos deficient for the chromatin remodeling protein ATRX to determine the cellular mechanisms activated in response to CIN. We demonstrate that ATRX is required for silencing of major satellite transcripts in the maternal genome, where it confers epigenetic asymmetry to pericentric heterochromatin during the transition to the first mitosis. This stage is also characterized by a striking kinetochore size asymmetry established by differences in CENP-C protein between the parental genomes. Loss of ATRX results in increased centromeric mitotic recombination, a high frequency of sister chromatid exchanges and double strand DNA breaks, indicating the formation of mitotic recombination break points. ATRX-deficient embryos exhibit a twofold increase in transcripts for aurora kinase B, the centromeric cohesin ESCO2, DNMT1, the ubiquitin-ligase (DZIP3) and the histone methyl transferase (EHMT1). Thus, loss of ATRX activates a pathway that integrates epigenetic modifications and DNA repair in response to chromosome breaks. These results reveal the cellular response of the cleavage-stage embryo to CIN and uncover a mechanism by which centromeric fission induces the formation of large-scale chromosomal rearrangements. Our results have important implications to determine the epigenetic origins of CIN that lead to congenital birth defects and early pregnancy loss, as well as the mechanisms involved in the oocyte to embryo transition. HIGHLIGHTED ARTICLE: The chromatin remodelling protein ATRX is transmitted to the early zygote through the maternal germ line and is required to silence major satellite transcripts and control chromosome stability.

Histone hyperacetylation during meiosis interferes with large-scale chromatin remodeling, axial chromatid condensation and sister chromatid separation in the mammalian oocyte.

Histone acetylation regulates higher-order chromatin structure and function and is critical for the control of gene expression. Histone deacetylase inhibitors (HDACi) are currently under investigation as novel cancer therapeutic drugs. Here, we show that female germ cells are extremely susceptible to chromatin changes induced by HDACi. Our results indicate that exposure to trichostatin A (TSA) at nanomolar levels interferes with major chromatin remodeling events in the mammalian oocyte leading to chromosome instability. High resolution analysis of chromatin structure and live-cell imaging revealed a striking euchromatin decondensation associated with histone H4 hyperacetylation following exposure to 15 nM TSA in >90% of pre-ovulatory oocytes. Dynamic changes in large-scale chromatin structure were detected after 2 h of exposure and result in the formation of misaligned chromosomes in >75% (P<0.05) of in vitro matured oocytes showing chromosome lagging as well as abnormal sister chromatid separation at anaphase I. Abnormal axial chromatid condensation during meiosis results in the formation of elongated chromosomes exhibiting hyperacetylation of histone H4 at lysine 5 and lysine 16 at interstitial chromosome segments, but not pericentric heterochromatin, while highly decondensed bivalents exhibit prominent histone H3 phosphorylation at centromeric domains. Notably, no changes were observed in the chromosomal localization of the condensin protein SMC4. These results indicate that HDAC activity is required for proper chromosome condensation in the mammalian oocyte and that HDACi may induce abnormal chromosome segregation by interfering with both chromosome-microtubule interactions, as well as sister chromatid separation. Thus, HDACi, proposed for cancer therapy, may disrupt the epigenetic status of female germ cells, predisposing oocytes to aneuploidy at previously unrecognized low doses.