Rad9a is involved in chromatin decondensation and post-zygotic embryo development in mice.

Zygotic chromatin undergoes extensive reprogramming immediately after fertilization. It is generally accepted that maternal factors control this process. However, little is known about the underlying mechanisms. Here we report that maternal RAD9A, a key protein in DNA damage response pathway, is involved in post-zygotic embryo development, via a mouse model with conditional depletion of Rad9a alleles in oocytes of primordial follicles. Post-zygotic losses originate from delayed zygotic chromatin decondensation after depletion of maternal RAD9A. Pronucleus formation and DNA replication of most mutant zygotes are therefore deferred, which subsequently trigger the G2/M checkpoint and arrest development of most mutant zygotes. Delayed zygotic chromatin decondensation could also lead to increased reabsorption of post-implantation mutant embryos. In addition, our data indicate that delayed zygotic chromatin decondensation may be attributed to deferred epigenetic modification of histone in paternal chromatin after fertilization, as fertilization and resumption of secondary meiosis in mutant oocytes were both normal. More interestingly, most mutant oocytes could not support development beyond one-cell stage after parthenogenetic activation. Therefore, RAD9A may also play an important role in maternal chromatin reprogramming. In summary, our data reveal an important role of RAD9A in zygotic chromatin reprogramming and female fertility.

CENP-A regulates chromosome segregation during the first meiosis of mouse oocytes.

Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A (CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.

Protective effect of antioxidants on the pre-maturation aging of mouse oocytes.

Pre-maturation aging of immature oocytes may adversely affect the fate of an oocyte. Oxidative stress is one of the most detrimental factors affecting oocyte developmental competence and maturation during aging. In this study, experiments were designed to examine whether supplementation of antioxidants in a culture medium could protect immature mouse oocytes from damages caused by oxidative stress. Mouse oocytes at germinal vesicle stage were prevented from meiosis resumption and cultured in a medium with or without antioxidants for 12-36 h to allow oocytes to undergo aging. After aging, oocytes were cultured for maturation. Nuclear maturation, mitochondria activity, spindle morphology and DNA integrity were examined after maturation. It was found that antioxidants had protective effects on the oocytes in terms of nuclear maturation, functional mitochondria, spindle morphology and DNA integrity. As aging time was prolonged from 12 to 36 h, the protective effect of antioxidants became more obvious. However, as compared with oocytes without aging, it was found that aging significantly inhibited nuclear maturation, impaired mitochondria function, and damaged the spindle and DNA. These results indicate that pre-maturation aging is detrimental to oocytes' competence to undergo maturation and other cellular activities, and antioxidants can protect oocytes from damages caused by aging.

CENP-A Regulates Chromosome Segregation during the First Meiosis of Mouse Oocytes / 华中科技大学学报（医学）（英德文版）

Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules.Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy,thus kinetochores are critical for faithful segregation of chromosomes.Centromere protein A (CENP-A) is an important component of the inner kinetochore·plate.Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores,leading to abnormal chromosome segregation,aneuploidy and apoptosis in cells.Here we report the expression and function of CENP-A during mouse oocyte meiosis.Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs.Thus,our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.

Rad9a is required for spermatogonia differentiation in mice.

Spermatogenesis in testes requires precise spermatogonia differentiation. Spermatocytes lacking the Rad9a gene are arrested in pachytene prophase, implying a possible role for RAD9A in spermatogonia differentiation. However, numerous RAD9A-positive pachytene spermatocytes are still observed in mouse testes following Rad9a excision using the Stra8-Cre system, and it is unclear whether Rad9a deletion in spermatogonia interrupts differentiation. Here, we generated a mouse model in which Rad9a was specifically deleted in spermatogonial stem cells (SSCs) using Cre recombinase expression driven by the germ cell-specific Vasa promoter. Adult Rad9a-null male mice were infertile as a result of completely blocked spermatogonia differentiation. No early spermatocytes were detected in mutant testicular cords of 9-day-old mice. Mutant spermatogonia were prone to apoptosis, although proliferation rates were unaffected. Rad9a deletion also resulted in malformation of seminiferous tubules, in which cells assembled irregularly into clusters, and malformation led to testicular cord disruption. Our findings suggest that Rad9a is indispensable for spermatogonia differentiation and testicular development in mice.

N6-Methyladenosine Sequencing Highlights the Involvement of mRNA Methylation in Oocyte Meiotic Maturation and Embryo Development by Regulating Translation in Xenopus laevis.

During the oogenesis of Xenopus laevis, oocytes accumulate maternal materials for early embryo development. As the transcription activity of the oocyte is silenced at the fully grown stage and the global genome is reactivated only by the mid-blastula embryo stage, the translation of maternal mRNAs accumulated during oocyte growth should be accurately regulated. Previous evidence has illustrated that the poly(A) tail length and RNA binding elements mediate RNA translation regulation in the oocyte. Recently, RNA methylation has been found to exist in various systems. In this study, we sequenced the N6-methyladenosine (m6A) modified mRNAs in fully grown germinal vesicle-stage and metaphase II-stage oocytes. As a result, we identified 4207 mRNAs with m6A peaks in germinal vesicle-stage or metaphase II-stage oocytes. When we integrated the mRNA methylation data with transcriptome and proteome data, we found that the highly methylated mRNAs showed significantly lower protein levels than those of the hypomethylated mRNAs, although the RNA levels showed no significant difference. We also found that the hypomethylated mRNAs were mainly enriched in the cell cycle and translation pathways, whereas the highly methylated mRNAs were mainly associated with protein phosphorylation. Our results suggest that oocyte mRNA methylation can regulate cellular translation and cell division during oocyte meiotic maturation and early embryo development.

Nek11 regulates asymmetric cell division during mouse oocyte meiotic maturation.

Nek11, a member of the never in mitosis gene A (NIMA) family, is activated in somatic cells associated with G1/S or G2/M arrest. However, its function in meiosis is unknown. In this research, the expression, localization and functions of NEK11 in the mouse oocyte meiotic maturation were examined. Western blotting indicated that NEK11S was the major NEK11 protein in mouse oocyte. MYC-tagged Nek11 mRNA microinjection and immunofluorescent staining showed that NEK11 was localized to the meiotic spindles at MI and MII stage. Knockdown of Nek11 by microinjection of siRNA did not affect germinal vesicle breakdown (GVBD) and the first polar body extrusion, but caused formation of 2-cell-like eggs. These results demonstrate that Nek11 regulates asymmetric cell division during oocyte meiotic maturation.

Cep55 regulates spindle organization and cell cycle progression in meiotic oocyte.

Cep55 is a relatively novel member of the centrosomal protein family. Here, we show that Cep55 is expressed in mouse oocytes from the germinal vesicle (GV) to metaphase II (MII) stages. Immuostaining and confocal microscopy as well as time lapse live imaging after injection of mRNA encoding fusion protein of Cep55 and GFP identified that Cep55 was localized to the meiotic spindle, especially to the spindle poles at metaphase, while it was concentrated at the midbody in telophase in meiotic oocytes. Knockdown of Cep55 by specific siRNA injection caused the dissociation of γ-tubulin from the spindle poles, resulting in severely defective spindles and misaligned chromosomes, leading to metaphase I arrest and failure of first polar body (PB1) extrusion. Correspondingly, cyclin B accumulation and spindle assembly checkpoint (SAC) activation were observed in Cep55 knockdown oocytes. Our results suggest that Cep55 may act as an MTOC-associated protein regulating spindle organization, and thus cell cycle progression during mouse oocyte meiotic maturation.

Cyclin B3 controls anaphase onset independent of spindle assembly checkpoint in meiotic oocytes.

Cyclin B3 is a relatively new member of the cyclin family whose functions are little known. We found that depletion of cyclin B3 inhibited metaphase-anaphase transition as indicated by a well-sustained MI spindle and cyclin B1 expression in meiotic oocytes after extended culture. This effect was independent of spindle assembly checkpoint activity, since both Bub3 and BubR1 signals were not observed at kinetochores in MI-arrested cells. The metaphase I arrest was not rescued by either Mad2 knockdown or cdc20 overexpression, but it was rescued by securin RNAi. We conclude that cyclin B3 controls the metaphase-anaphase transition by activating APC/C(cdc20) in meiotic oocytes, a process that does not rely on SAC activity.

Nuf2 is required for chromosome segregation during mouse oocyte meiotic maturation.

Nuf2 plays an important role in kinetochore-microtubule attachment and thus is involved in regulation of the spindle assembly checkpoint in mitosis. In this study, we examined the localization and function of Nuf2 during mouse oocyte meiotic maturation. Myc6-Nuf2 mRNA injection and immunofluorescent staining showed that Nuf2 localized to kinetochores from germinal vesicle breakdown to metaphase I stages, while it disappeared from the kinetochores at the anaphase I stage, but relocated to kinetochores at the MII stage. Overexpression of Nuf2 caused defective spindles, misaligned chromosomes, and activated spindle assembly checkpoint, and thus inhibited chromosome segregation and metaphase-anaphase transition in oocyte meiosis. Conversely, precocious polar body extrusion was observed in the presence of misaligned chromosomes and abnormal spindle formation in Nuf2 knock-down oocytes, causing aneuploidy. Our data suggest that Nuf2 is a critical regulator of meiotic cell cycle progression in mammalian oocytes.

Casein kinase 1 (α, δ and ε) localize at the spindle poles, but may not be essential for mammalian oocyte meiotic progression.

CK1 (casein kinase 1) is a family of serine/threonine protein kinase that is ubiquitously expressed in eukaryotic organism. CK1 members are involved in the regulation of many cellular processes. Particularly, CK1 was reported to phosphorylate Rec8 subunits of cohesin complex and regulate chromosome segregation in meiosis in budding yeast and fission yeast. (1-3) Here we investigated the expression, subcellular localization and potential functions of CK1α, CK1δ and CK1ε during mouse oocyte meiotic maturation. We found that CK1α, CK1δ and CK1ε all concentrated at the spindle poles and co-localized with γ-tubulin in oocytes at both metaphase I (MI) and metaphase II (MII) stages. However, depletion of CK1 by RNAi or overexpression of wild type or kinase-dead CK1 showed no effects on either spindle organization or chromosome segregation during oocyte meiotic maturation. Thus, CK1 is not the kinase that phosphorylates Rec8 cohesin in mammalian oocytes, and CK1 may not be essential for spindle organization and meiotic progression although they localize at spindle poles.

Deletion of Mylk1 in oocytes causes delayed morula-to-blastocyst transition and reduced fertility without affecting folliculogenesis and oocyte maturation in mice.

The mammalian oocyte undergoes two rounds of asymmetric cell divisions during meiotic maturation and fertilization. Acentric spindle positioning and cortical polarity are two major factors involved in asymmetric cell division, both of which are thought to depend on the dynamic interaction between myosin II and actin filaments. Myosin light chain kinase (MLCK), encoded by the Mylk1 gene, could directly phosphorylate and activate myosin II. To determine whether MLCK was required for oocyte asymmetric division, we specifically disrupted the Mylk1 gene in oocytes by Cre-loxP conditional knockout system. We found that Mylk1 mutant female mice showed severe subfertility. Unexpectedly, contrary to previously reported in vitro findings, our data showed that oocyte meiotic maturation including spindle organization, polarity establishment, homologous chromosomes separation, and polar body extrusion were not affected in Mylk1(fl/fl);GCre(+) females. Follicular development, ovulation, and early embryonic development up to compact morula occurred normally in Mylk1(fl/fl);GCre(+) females, but deletion of MLCK caused delayed morula-to-blastocyst transition. More than a third of embryos were at morula stage at 3.5 Days Postcoitum in vivo. The delayed embryos could develop further to early blastocyst stage in vitro on Day 4 when most control embryos reached expanded blastocysts. Our findings provide evidence that MLCK is linked to timely blastocyst formation, though it is dispensable for oocyte meiotic maturation.

Scaffold subunit Aalpha of PP2A is essential for female meiosis and fertility in mice.

Ppp2r1a encodes the scaffold subunit Aalpha of protein phosphatase 2A (PP2A), which is an important and ubiquitously expressed serine threonine phosphatase family and plays a critical role in many fundamental cellular processes. To identify the physiological role of PP2A in female germ cell meiosis, we selectively disrupted Ppp2r1a expression in oocytes by using the Cre-Loxp conditional knockout system. Here we report for the first time that oocyte-specific deletion of Ppp2r1a led to severe female subfertility without affecting follicle survival, growth, and ovulation. PP2A-Aalpha was essential for regulating oocyte meiotic maturation because depletion of PP2A-Aalpha facilitated germinal vesicle breakdown, causing elongation of the MII spindle and precocious separation of sister chromatids. The resulting eggs had high risk of aneuploidy, though they could be fertilized, leading to defective embryonic development and thus subfertility. Our findings provide strong evidence that PP2A-Aalpha within the oocyte plays an indispensable role in oocyte meiotic maturation, though it is dispensable for folliculogenesis in the mouse ovary.

Arrested human embryos are more likely to have abnormal chromosomes than developing embryos from women of advanced maternal age.

BACKGROUND: Aneuploidy is one of the major factors that result in low efficiency in human infertility treatment by in vitro fertilization (IVF). The development of DNA microarray technology allows for aneuploidy screening by analyzing all 23 pairs of chromosomes in human embryos. All chromosome screening for aneuploidy is more accurate than partial chromosome screening, as errors can occur in any chromosome. Currently, chromosome screening for aneuploidy is performed in developing embryos, mainly blastocysts. It has not been performed in arrested embryos and/or compared between developing embryos and arrested embryos from the same IVF cycle. METHODS: The present study was designed to examine all chromosomes in blastocysts and arrested embryos from the same cycle in patients of advanced maternal ages. Embryos were produced by routine IVF procedures. A total of 90 embryos (45 blastocysts and 45 arrested embryos) from 17 patients were biopsied and analyzed by the Agilent DNA array platform. RESULTS: It was found that 50% of the embryos developed to blastocyst stage; however, only 15.6% of the embryos (both blastocyst and arrested) were euploid, and most (84.4%) of the embryos had chromosomal abnormalities. Further analysis indicated that 28.9% of blastocysts were euploid and 71.1% were aneuploid. By contrast, only one (2.2%) arrested embryo was euploid while others (97.8%) were aneuploid. The prevalence of multiple chromosomal abnormalities in the aneuploid embryos was also higher in the arrested embryos than in the blastocysts. CONCLUSIONS: These results indicate that high proportions of human embryos from patients of advanced maternal age are aneuploid, and the arrested embryos are more likely to have abnormal chromosomes than developing embryos.

Maternal diabetes causes abnormal dynamic changes of endoplasmic reticulum during mouse oocyte maturation and early embryo development.

BACKGROUND: The adverse effects of maternal diabetes on oocyte maturation and embryo development have been reported. METHODS: In this study, we used time-lapse live cell imaging confocal microscopy to investigate the dynamic changes of ER and the effects of diabetes on the ER's structural dynamics during oocyte maturation, fertilization and early embryo development. RESULTS: We report that the ER first became remodeled into a dense ring around the developing MI spindle, and then surrounded the spindle during migration to the cortex. ER reorganization during mouse early embryo development was characterized by striking localization around the pronuclei in the equatorial section, in addition to larger areas of fluorescence deeper within the cytoplasm. In contrast, in diabetic mice, the ER displayed a significantly higher percentage of homogeneous distribution patterns throughout the entire ooplasm during oocyte maturation and early embryo development. In addition, a higher frequency of large ER aggregations was detected in GV oocytes and two cell embryos from diabetic mice. CONCLUSIONS: These results suggest that the diabetic condition adversely affects the ER distribution pattern during mouse oocyte maturation and early embryo development.

Overexpression of SETß, a protein localizing to centromeres, causes precocious separation of chromatids during the first meiosis of mouse oocytes.

Chromosome segregation in mammalian oocyte meiosis is an error-prone process, and any mistake in this process may result in aneuploidy, which is the main cause of infertility, abortion and many genetic diseases. It is now well known that shugoshin and protein phosphatase 2A (PP2A) play important roles in the protection of centromeric cohesion during the first meiosis. PP2A can antagonize the phosphorylation of rec8, a member of the cohesin complex, at the centromeres and thus prevent cleavage of rec8 and so maintain the cohesion of chromatids. SETß is a protein that physically interacts with shugoshin and inhibits PP2A activity. We thus hypothesized that SETß might regulate cohesion protection and chromosome segregation during oocyte meiotic maturation. Here we report for the first time the expression, subcellular localization and functions of SETß during mouse oocyte meiosis. Immunoblotting analysis showed that the expression level of SETß was stable from the germinal vesicle stage to the MII stage of oocyte meiosis. Immunofluorescence analysis showed SETß accumulation in the nucleus at the germinal vesicle stage, whereas it was targeted mainly to the inner centromere area and faintly localized to the interchromatid axes from germinal vesicle breakdown to MI stages. At the MII stage, SETß still localized to the inner centromere area, but could relocalize to kinetochores in a process perhaps dependent on the tension on the centromeres. SETß partly colocalized with PP2A at the inner centromere area. Overexpression of SETß in mouse oocytes caused precocious separation of sister chromatids, but depletion of SETß by RNAi showed little effects on the meiotic maturation process. Taken together, our results suggest that SETß, even though it localizes to centromeres, might not be essential for chromosome separation during mouse oocyte meiotic maturation, although its forced overexpression causes premature chromatid separation.

The distribution and possible role of ERK8 in mouse oocyte meiotic maturation and early embryo cleavage.

It is well known that extracellular signal-regulated kinase 8 (ERK8) plays pivotal roles in various mitotic events. But its physiological roles in oocyte meiotic maturation remain unclear. In this study, we found that although no specific ERK8 signal was detected in oocyte at the germinal vesicle stage, ERK8 began to migrate to the periphery of chromosomes shortly after germinal vesicle breakdown. At prometaphase I, metaphase I (MI), anaphase I, telophase I, and metaphase II (MII) stages, ERK8 was stably detected at the spindles. By taxol treatment, we clarified that the ERK8 signal was stained on the spindle fibers as well as microtubule asters in MI and MII oocytes. In fertilized eggs, the ERK8 signal was not observed in the two pronuclei stages. At prometaphase, metaphase, and anaphase of the first mitosis, ERK8 was detected on the mitotic spindle. ERK8 knock down by antibody microinjection and specific siRNA caused abnormal spindles, failed chromosome congression, and decreased first polar body extrusion. Taken together, our results suggest that ERK8 plays an important role in spindle organization during mouse oocyte meiotic maturation and early embryo cleavage.

Knockdown of UCHL5IP causes abnormalities in γ-tubulin localisation, spindle organisation and chromosome alignment in mouse oocyte meiotic maturation.

UCHL5IP is one of the subunits of the haus complex, which is important for microtubule generation, spindle bipolarity and accurate chromosome segregation in Drosophila and human mitotic cells. In this study, the expression and localisation of UCHL5IP were explored, as well as its functions in mouse oocyte meiotic maturation. The results showed that the UCHL5IP protein level was consistent during oocyte maturation and it was localised to the meiotic spindle in MI and MII stages. Knockdown of UCHL5IP led to spindle defects, chromosome misalignment and disruption of γ-tubulin localisation in the spindle poles. These results suggest that UCHL5IP plays critical roles in spindle formation during mouse oocyte meiotic maturation.

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis.

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.

The roles of parathyroid hormone-like hormone during mouse preimplantation embryonic development.

Parathyroid hormone-like hormone (PTHLH) was first identified as a parathyroid hormone (PTH)-like factor responsible for humoral hypercalcemia in malignancies in the 1980s. Previous studies demonstrated that PTHLH is expressed in multiple tissues and is an important regulator of cellular and organ growth, development, migration, differentiation, and survival. However, there is a lack of data on the expression and function of PTHLH during preimplantation embryonic development. In this study, we investigated the expression characteristics and functions of PTHLH during mouse preimplantation embryonic development. The results show that Pthlh is expressed in mouse oocytes and preimplantation embryos at all developmental stages, with the highest expression at the MII stage of the oocytes and the lowest expression at the blastocyst stage of the preimplantation embryos. The siRNA-mediated depletion of Pthlh at the MII stage oocytes or the 1-cell stage embryos significantly decreased the blastocyst formation rate, while this effect could be corrected by culturing the Pthlh depleted embryos in the medium containing PTHLH protein. Moreover, expression of the pluripotency-related genes Nanog and Pou5f1 was significantly reduced in Pthlh-depleted embryos at the morula stage. Additionally, histone acetylation patterns were altered by Pthlh depletion. These results suggest that PTHLH plays important roles during mouse preimplantation embryonic development.