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1.
Development ; 151(6)2024 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-38546043

RESUMEN

The timely degradation of proteins that regulate the cell cycle is essential for oocyte maturation. Oocytes are equipped to degrade proteins via the ubiquitin-proteasome system. In meiosis, anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin-ligase, is responsible for the degradation of proteins. Ubiquitin-conjugating enzyme E2 S (UBE2S), an E2 ubiquitin-conjugating enzyme, delivers ubiquitin to APC/C. APC/C has been extensively studied, but the functions of UBE2S in oocyte maturation and mouse fertility are not clear. In this study, we used Ube2s knockout mice to explore the role of UBE2S in mouse oocytes. Ube2s-deleted oocytes were characterized by meiosis I arrest with normal spindle assembly and spindle assembly checkpoint dynamics. However, the absence of UBE2S affected the activity of APC/C. Cyclin B1 and securin are two substrates of APC/C, and their levels were consistently high, resulting in the failure of homologous chromosome separation. Unexpectedly, the oocytes arrested in meiosis I could be fertilized and the embryos could become implanted normally, but died before embryonic day 10.5. In conclusion, our findings reveal an indispensable regulatory role of UBE2S in mouse oocyte meiosis and female fertility.


Asunto(s)
Puntos de Control de la Fase M del Ciclo Celular , Meiosis , Animales , Femenino , Ratones , Ciclosoma-Complejo Promotor de la Anafase/genética , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Oocitos/metabolismo , Ubiquitinas/metabolismo
2.
Development ; 150(14)2023 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-37485540

RESUMEN

Accurate chromosome segregation, monitored by the spindle assembly checkpoint (SAC), is crucial for the production of euploid cells. Previous in vitro studies by us and others showed that Mad2, a core member of the SAC, performs a checkpoint function in oocyte meiosis. Here, through an oocyte-specific knockout approach in mouse, we reconfirmed that Mad2-deficient oocytes exhibit an accelerated metaphase-to-anaphase transition caused by premature degradation of securin and cyclin B1 and subsequent activation of separase in meiosis I. However, it was surprising that the knockout mice were completely fertile and the resulting oocytes were euploid. In the absence of Mad2, other SAC proteins, including BubR1, Bub3 and Mad1, were normally recruited to the kinetochores, which likely explains the balanced chromosome separation. Further studies showed that the chromosome separation in Mad2-null oocytes was particularly sensitive to environmental changes and, when matured in vitro, showed chromosome misalignment, lagging chromosomes, and aneuploidy with premature separation of sister chromatids, which was exacerbated at a lower temperature. We reveal for the first time that Mad2 is dispensable for proper chromosome segregation but acts to mitigate environmental stress in meiotic oocytes.


Asunto(s)
Proteínas de Ciclo Celular , Huso Acromático , Animales , Ratones , Proteínas de Ciclo Celular/metabolismo , Huso Acromático/metabolismo , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Segregación Cromosómica/genética , Oocitos/metabolismo , Cinetocoros/metabolismo , Meiosis/genética
3.
Development ; 149(10)2022 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-35546066

RESUMEN

Mammalian early embryo cells have complex DNA repair mechanisms to maintain genomic integrity, and homologous recombination (HR) plays the main role in response to double-strand DNA breaks (DSBs) in these cells. Polo-like kinase 1 (PLK1) participates in the HR process and its overexpression has been shown to occur in a variety of human cancers. Nevertheless, the regulatory mechanism of PLK1 remains poorly understood, especially during the S and G2 phase. Here, we show that protein phosphatase 4 catalytic subunit (PPP4C) deletion causes severe female subfertility due to accumulation of DNA damage in oocytes and early embryos. PPP4C dephosphorylated PLK1 at the S137 site, negatively regulating its activity in the DSB response in early embryonic cells. Depletion of PPP4C induced sustained activity of PLK1 when cells exhibited DNA lesions that inhibited CHK2 and upregulated the activation of CDK1, resulting in inefficient loading of the essential HR factor RAD51. On the other hand, when inhibiting PLK1 in the S phase, DNA end resection was restricted. These results demonstrate that PPP4C orchestrates the switch between high-PLK1 and low-PLK1 periods, which couple the checkpoint to HR.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Recombinación , Animales , Proteínas de Ciclo Celular , Línea Celular , ADN/genética , Reparación del ADN por Unión de Extremidades , Reparación del ADN/genética , Desarrollo Embrionario/genética , Femenino , Recombinación Homóloga , Mamíferos/genética , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas , Quinasa Tipo Polo 1
4.
Histochem Cell Biol ; 2024 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-39093409

RESUMEN

Oocyte meiotic maturation failure and chromosome abnormality is one of the main causes of infertility, abortion, and diseases. The mono-orientation of sister chromatids during the first meiosis is important for ensuring accurate chromosome segregation in oocytes. MEIKIN is a germ cell-specific protein that can regulate the mono-orientation of sister chromatids and the protection of the centromeric cohesin complex during meiosis I. Here we found that MEIKIN is a maternal protein that was highly expressed in mouse oocytes before the metaphase I (MI) stage, but became degraded by the MII stage and dramatically reduced after fertilization. Strikingly, MEIKIN underwent phosphorylation modification after germinal vesicle breakdown (GVBD), indicating its possible function in subsequent cellular event regulation. We further showed that MEIKIN phosphorylation was mediated by PLK1 at its carboxyl terminal region and its C-terminus was its key functional domain. To clarify the biological significance of meikin degradation during later stages of oocyte maturation, exogenous expression of MEIKIN was employed, which showed that suppression of MEIKIN degradation resulted in chromosome misalignment, cyclin B1 and Securin degradation failure, and MI arrest through a spindle assembly checkpoint (SAC)-independent mechanism. Exogenous expression of MEIKIN also inhibited metaphase II (MII) exit and early embryo development. These results indicate that proper MEIKIN expression level and its C-terminal phosphorylation by PLK1 are critical for regulating the metaphase-anaphase transition in meiotic oocyte. The findings of this study are important for understanding the regulation of chromosome segregation and the prevention meiotic abnormality.

5.
Biol Reprod ; 108(3): 437-446, 2023 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-36503987

RESUMEN

As the most abundant organelles in oocytes, mitochondria play an important role in maintaining oocyte quality. Here, we report that March5, encoding a mitochondrial ubiquitin ligase that promotes mitochondrial elongation, plays a critical role in mouse oocyte meiotic maturation via regulating mitochondrial function. The subcellular localization of MARCH5 was similar to the mitochondrial distribution during mouse oocyte meiotic progression. Knockdown of March5 caused decreased ratios of the first polar body extrusion. March5-siRNA injection resulted in oocyte mitochondrial dysfunctions, manifested by increased reactive oxygen species, decreased ATP content as well as decreased mitochondrial membrane potential, leading to reduced ability of spindle formation and an increased ratio of kinetochore-microtubule detachment. Further study showed that the continuous activation of the spindle assembly checkpoint and the failure of Cyclin B1 degradation caused MI arrest and first polar body (PB1) extrusion failure in March5 knockdown oocytes. Taken together, our results demonstrated that March5 plays an essential role in mouse oocyte meiotic maturation, possibly via regulation of mitochondrial function and/or ubiquitination of microtubule dynamics- or cell cycle-regulating proteins.


Asunto(s)
Oogénesis , Ubiquitina-Proteína Ligasas , Animales , Ratones , Mitocondrias/metabolismo , Oocitos/metabolismo , Proteínas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
6.
FASEB J ; 36(3): e22210, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35167144

RESUMEN

Precise regulation of chromosome separation through spindle assembly checkpoint (SAC) during oocyte meiosis is critical for mammalian reproduction. The kinetochore plays an important role in the regulation of SAC through sensing microtubule tension imbalance or missing microtubule connections. Here, we report that kinetochore scaffold 1 (KNL1, also known as CASC5), an outer kinetochore protein, plays a critical role in the SAC function of mouse oocytes. KNL1 localized at kinetochores from GVBD to the MII stage, and microinjection of KNL1-siRNA caused accelerated metaphase-anaphase transition and premature first meiosis completion, producing aneuploid eggs. The SAC was prematurely silenced in the presence of unstable kinetochore-microtubule attachments and misaligned chromosomes in KNL1-depleted oocytes. Additionally, KNL1 and MPS1 had a synergistic effect on the activation and maintenance of SAC. Taken together, our results suggest that KNL1, as a kinetochore platform protein, stabilizes SAC to ensure timely anaphase entry and accurate chromosome segregation during oocyte meiotic maturation.


Asunto(s)
Puntos de Control de la Fase M del Ciclo Celular , Meiosis , Proteínas Asociadas a Microtúbulos/metabolismo , Oocitos/metabolismo , Oogénesis , Animales , Células Cultivadas , Femenino , Ratones , Ratones Endogámicos ICR , Proteínas Asociadas a Microtúbulos/genética , Oocitos/citología
7.
Exp Cell Res ; 416(1): 113135, 2022 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-35398309

RESUMEN

Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, cell migration and morphogenesis. Microtubule-associated RP/EB family member 2 (MAPRE2/EB2) is a highly conserved core component of +TIPs networks, but whether this molecule is required for mammalian meiotic progression is unknown. In this study, we investigated the expression and function of MAPRE2 during oocyte maturation. Our results showed that MAPRE2 was consistently expressed from germinal vesicle (GV) to metaphase II (MII) stages and that MAPRE2 was distributed in the cytoplasm of oocytes at GV stage and along the spindle at metaphase I (MI) and MII stages. Small interfering RNA-mediated knockdown of Mapre2 severely impaired microtubule stability, kinetochore-microtubule attachment, and chromosome alignment and subsequently caused spindle assembly checkpoint (SAC) activation and cyclin B1 nondegradation, leading to failure of chromosome segregation and first polar body extrusion. This study demonstrates for the first time that MAPRE2 plays an important role during mouse oocyte meiosis.


Asunto(s)
Meiosis , Huso Acromático , Animales , Segregación Cromosómica , Mamíferos , Metafase , Ratones , Oocitos/metabolismo , Huso Acromático/metabolismo
8.
J Cell Physiol ; 237(1): 730-742, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34189751

RESUMEN

In mammals, oocytes are arrested at G2/prophase for a long time, which is called germinal vesicle (GV) arrest. After puberty, fully-grown oocytes are stimulated by a gonadotropin surge to resume meiosis as indicated by GV breakdown (GVBD). CCNB1 is accumulated to a threshold level to trigger the activation of maturation promoting factor (MPF), inducing the G2/M transition. It is generally recognized that the anaphase-promoting complex/cyclosome (APC/C) and its cofactor CDH1 (also known as FZR1) regulates the accumulation/degradation of CCNB1. Here, by using small interfering RNA (siRNA) and messenger RNA (mRNA) microinjection, immunofluorescence and confocal microscopy, immunoprecipitation, time-lapse live imaging, and immunoblotting analysis, we showed that Septin 4 regulates the G2/M transition by regulating the accumulation of CCNB1 via APC/CCDC20 . Depletion of Septin 4 caused GV arrest by reducing CCNB1 accumulation. Unexpectedly, the expression level of CDC20 was higher in Septin 4 siRNA-injected oocytes than in control oocytes, but there was no significant change in the expression level of CDH1. Importantly, the reduced GVBD after Septin 4 depletion could be rescued not only by over-expressing CCNB1 but also could be partially rescued by depleting CDC20. Taken together, our results demonstrate that Septin 4 may play a critical role in meiotic G2/M transition by indirect regulation of CCNB1 stabilization in mouse oocytes.


Asunto(s)
Septinas , Maduración Sexual , Ciclosoma-Complejo Promotor de la Anafase/genética , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Animales , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mamíferos/metabolismo , Meiosis , Ratones , Oocitos/metabolismo , ARN Interferente Pequeño/metabolismo , Septinas/genética
9.
J Cell Physiol ; 237(12): 4477-4486, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36183380

RESUMEN

Miro1, a mitochondrial Rho GTPase1, is a kind of mitochondrial outer membrane protein involved in the regulation of mitochondrial anterograde transport and its subcellular distribution. Mitochondria influence reproductive processes of mammals in some aspects. Mitochondria are important for oocyte maturation, fertilization and embryonic development. The purpose of this study was to evaluate whether Miro1 regulates mouse oocyte maturation by altering mitochondrial homeostasis. We showed that Miro1 was expressed in mouse oocyte at different maturation stages. Miro1 mainly distributed in the cytoplasm and around the spindle during oocyte maturation. Small interference RNA-mediated Miro1 depletion caused significantly abnormal distribution of mitochondria and endoplasmic reticulum as well as mitochondrial dysfunction, resulting in severely impaired germinal vesicle breakdown (GVBD) of mouse oocytes. For those oocytes which went through GVBD in the Miro1-depleted group, part of them were inhibited in meiotic prophase I stage with abnormal chromosome arrangement and scattered spindle length. Our results suggest that Miro1 is essential for maintaining the maturation potential of mouse oocyte.


Asunto(s)
Meiosis , Mitocondrias , Oocitos , Proteínas de Unión al GTP rho , Animales , Femenino , Ratones , Embarazo , Homeostasis , Mitocondrias/fisiología , Oocitos/fisiología , Oogénesis , Proteínas de Unión al GTP rho/fisiología
10.
J Cell Physiol ; 237(9): 3661-3670, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35853150

RESUMEN

AZD1208, a pan-inhibitor that can effectively inhibit PIM kinase, is used for the treatment of advanced solid tumors and malignant lymphomas. Numerous studies have proved its curative effects while its potential cellular toxicity on reproduction was still little known. In this study, we investigated the toxic effects of AZD1208 on mouse oocytes. The results showed that AZD1208 treatment did not affect meiotic resumption, but postponed oocyte maturation as indicated by delayed first polar body extrusion. Further mechanistic study showed that AZD1208 treatment delayed spindle assembly. In addition, we found that oocytes treated with AZD1208 showed mitochondrial dysfunction. Abnormal mitochondrial clusters with decreased mitochondrial membrane potential were observed in oocytes during incubation in vitro. Moreover, increased oxidative stress was observed by testing the level of reactive oxygen species. In summary, our results suggest that AZD1208 treatment influences oocyte meiotic progression by causing mitochondrial dysfunctions and subsequent delayed spindle assembly.


Asunto(s)
Compuestos de Bifenilo , Oocitos , Animales , Compuestos de Bifenilo/farmacología , Meiosis , Ratones , Mitocondrias , Oocitos/metabolismo , Tiazolidinas/metabolismo
11.
J Cell Sci ; 133(3)2020 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-31964702

RESUMEN

Oocyte meiotic maturation failure is one of the major causes for female infertility. Meiotic resumption (the G2/M transition) and progression through metaphase I (MI) are two critical stages of oocyte meiotic maturation. Here, we report that centromere protein T (CENP-T), an internal kinetochore protein, plays a critical role in meiotic resumption of mouse oocytes. Depletion of CENP-T by siRNA injection increased the CDH1 (also known as FZR1) level, resulting in increased activity of the anaphase-promoting complex (APC)-CDH1 complex, and further leading to decreased levels of the cyclin protein CCNB1, attenuated maturation-promoting factor (MPF) activity, and finally severely compromised meiotic resumption. The impaired meiotic resumption caused by CENP-T depletion could be rescued by overexpression of exogenous CCNB1 or knockdown of endogenous CDH1. Overexpression of exogenous CENP-T resulted in decreased CDH1 levels, which accelerated the progression of G2/M transition, and accelerated meiotic cell cycle progression after germinal vesicle breakdown (GVBD). Unexpectedly, spindle organization after GVBD was not affected by the overexpression, but the distribution of chromosomes was affected. Our findings reveal a novel role for CENP-T in regulating meiotic progression by acting through CDH1.


Asunto(s)
Anafase , Meiosis , Animales , Cadherinas , Ciclina B1/genética , Femenino , Mesotelina , Metafase , Ratones , Oocitos
12.
Development ; 146(23)2019 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-31704793

RESUMEN

Chromosome segregation is driven by separase, activity of which is inhibited by binding to securin and cyclin B1/CDK1. In meiosis, premature separase activity will induce aneuploidy or abolish chromosome segregation owing to the untimely destruction of cohesin. Recently, we have proved that cyclin B2 can compensate for cyclin B1 in CDK1 activation for the oocyte meiosis G2/M transition. In the present study, we identify an interaction between cyclin B2/CDK1 and separase in mouse oocytes. We find that cyclin B2 degradation is required for separase activation during the metaphase I-anaphase I transition because the presence of stable cyclin B2 leads to failure of homologous chromosome separation and to metaphase I arrest, especially in the simultaneous absence of securin and cyclin B1. Moreover, non-phosphorylatable separase rescues the separation of homologous chromosomes in stable cyclin B2-arrested cyclin B1-null oocytes. Our results indicate that cyclin B2/CDK1 is also responsible for separase inhibition via inhibitory phosphorylation to regulate chromosome separation in oocyte meiosis, which may not occur in other cell types.


Asunto(s)
Anafase , Proteína Quinasa CDC2/metabolismo , Segregación Cromosómica , Ciclina B2/metabolismo , Metafase , Oocitos/metabolismo , Separasa/metabolismo , Animales , Proteína Quinasa CDC2/genética , Ciclina B2/genética , Femenino , Ratones , Ratones Noqueados , Oocitos/citología , Separasa/genética
13.
J Cell Physiol ; 236(10): 7001-7013, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-33724469

RESUMEN

Parathyroid hormone-related protein (PTHrP), the main cause of humoral hypercalcemia in malignancies, promotes cell proliferation and delays terminal cell maturation during embryonic development. Our previous study reported that PTHrP plays important roles in blastocyst formation, pluripotency gene expression, and histone acetylation during mouse preimplantation embryonic development. In this study, we further investigated the mechanism of preimplantation embryonic development regulated by PTHrP. Our results showed that Pthrp depletion decreased both the developmental rate of embryos at the cleavage stage and the cell number of morula-stage embryos. Pthrp-depleted embryos had significantly decreased levels of cyclin D1, phospho (p)-AKT (Thr308) and E2F1. However, Pthrp depletion did not cause significant changes in CDK4, ß-catenin or RUNX2 expression. In addition, our results indicated that Pthrp depletion promoted HDAC4 translocation from the cytoplasm to the nucleus in cleavage-stage embryos by stimulating the activity of protein phosphatase 2A (PP2A), which resulted in dephosphorylation of HDAC4. Taken together, these results suggest that PTHrP regulates cleavage division progression and blastocyst formation through the AKT/cyclin D1 pathway and that PTHrP modulates histone acetylation patterns through nuclear translocation of HDAC4 via PP2A-dependent HDAC4 dephosphorylation during preimplantation embryonic development in mice.


Asunto(s)
Blastocisto/metabolismo , Ciclina D1/metabolismo , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Acetilación , Transporte Activo de Núcleo Celular , Animales , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Histona Desacetilasas/genética , Ratones , Proteína Relacionada con la Hormona Paratiroidea/genética , Fosforilación , Proteína Fosfatasa 2/metabolismo , Transducción de Señal
14.
Am J Hum Genet ; 103(2): 188-199, 2018 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-30032984

RESUMEN

Acephalic spermatozoa syndrome is a severe teratozoospermia that leads to male infertility. Our previous work showed that biallelic SUN5 mutations are responsible for acephalic spermatozoa syndrome in about half of affected individuals, while pathogenic mechanisms in the other individuals remain to be elucidated. Here, we identified a homozygous nonsense mutation in the testis-specific gene PMFBP1 using whole-exome sequencing in a consanguineous family with two infertile brothers with acephalic spermatozoa syndrome. Sanger sequencing of PMFBP1 in ten additional infertile men with acephalic spermatozoa syndrome and without SUN5 mutations revealed two homozygous variants and one compound heterozygous variant. The disruption of Pmfbp1 in male mice led to infertility due to the production of acephalic spermatozoa and the disruption of PMFBP1's cooperation with SUN5 and SPATA6, which plays a role in connecting sperm head to the tail. PMFBP1 mutation-associated male infertility could be successfully overcome by intracytoplasmic sperm injection (ICSI) in both mouse and human. Thus, mutations in PMFBP1 are an important cause of infertility in men with acephalic spermatozoa syndrome.


Asunto(s)
Infertilidad Masculina/genética , Mutación/genética , Proteínas/genética , Espermatozoides/patología , Animales , Proteínas del Citoesqueleto/genética , Exoma/genética , Femenino , Homocigoto , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Inyecciones de Esperma Intracitoplasmáticas/métodos , Síndrome , Secuenciación del Exoma/métodos
15.
FASEB J ; 34(7): 8990-9002, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32449168

RESUMEN

Precise regulation of chromosome segregation during oocyte meiosis is of vital importance to mammalian reproduction. Anaphase promoting complex/cyclosome (APC/C) is reported to play an important role in metaphase-to-anaphase transition. Here we report that cell division cycle 23 (Cdc23, also known as APC8) plays a critical role in regulating the oocyte chromosome separation. Cdc23 localized on the meiotic spindle, and microinjection of Cdc23 siRNA caused decreased ratios of metaphase-to-anaphase transition. Loss of Cdc23 resulted in abnormal spindles, misaligned chromosomes, errors of homologous chromosome segregation, and production of aneuploid oocytes. Further study showed that inactivation of spindle assembly checkpoint and degradation of Cyclin B1 and securin were disturbed after Cdc23 knockdown. Furthermore, we found that inhibiting spindle assembly checkpoint protein Msp1 partly rescued the decreased polar body extrusion and reduced the accumulation of securin in Cdc23 knockdown oocytes. Taken together, our data demonstrate that Cdc23 is required for the chromosome segregation through regulating the spindle assembly checkpoint activity, and cyclin B1 and securin degradation in meiotic mouse oocytes.


Asunto(s)
Subunidad Apc8 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Segregación Cromosómica , Meiosis , Oocitos/fisiología , Huso Acromático/fisiología , Animales , Subunidad Apc8 del Ciclosoma-Complejo Promotor de la Anafase/genética , Proteínas de Ciclo Celular , Femenino , Ratones , Ratones Endogámicos ICR , Oocitos/citología
16.
J Cell Physiol ; 235(10): 7136-7145, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32030765

RESUMEN

Mammalian cyclin A1 is prominently expressed in testis and essential for meiosis in the male mouse, however, it shows weak expression in ovary, especially during oocyte maturation. To understand why cyclin A1 behaves in this way in the oocyte, we investigated the effect of cyclin A1 overexpression on mouse oocyte meiotic maturation. Our results revealed that cyclin A1 overexpression triggered meiotic resumption even in the presence of germinal vesicle breakdown inhibitor, milrinone. Nevertheless, the cyclin A1-overexpressed oocytes failed to extrude the first polar body but were completely arrested at metaphase I. Consequently, cyclin A1 overexpression destroyed the spindle morphology and chromosome alignment by inducing premature separation of chromosomes and sister chromatids. Therefore, cyclin A1 overexpression will prevent oocyte maturation although it can promote meiotic resumption. All these results show that decreased expression of cyclin A1 in oocytes may have an evolutional significance to keep long-lasting prophase arrest and orderly chromosome separation during oocyte meiotic maturation.


Asunto(s)
Segregación Cromosómica/genética , Segregación Cromosómica/fisiología , Ciclina A1/genética , Ciclina A1/metabolismo , Meiosis/genética , Meiosis/fisiología , Oocitos/metabolismo , Animales , Segregación Cromosómica/efectos de los fármacos , Femenino , Meiosis/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Milrinona/farmacología , Oocitos/citología , Oocitos/efectos de los fármacos , Oogénesis/efectos de los fármacos , Oogénesis/genética , Oogénesis/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Separasa/metabolismo , Regulación hacia Arriba
17.
J Cell Physiol ; 235(7-8): 5541-5554, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-31984513

RESUMEN

Cell division cycle protein, CDC6, is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from pro-metaphase I (MI) to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (germinal vesicle breakdown [GVBD]) through regulation of Cdh1 and cyclin B1 expression and CDK1 (CDC2) phosphorylation in a GVBD-inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ-tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation, and spindle assembly checkpoint (SAC) activation, leading to significant pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.


Asunto(s)
Proteínas de Ciclo Celular/genética , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Meiosis/genética , Proteínas Nucleares/genética , Oocitos/crecimiento & desarrollo , Anafase/genética , Animales , Centrosoma , Femenino , Puntos de Control de la Fase M del Ciclo Celular/genética , Metafase/genética , Ratones , Oocitos/metabolismo , Huso Acromático/genética
18.
Biochem Biophys Res Commun ; 527(1): 8-14, 2020 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-32446395

RESUMEN

Oocyte meiotic maturation failure and unfaithful chromosome segregation are major causes for female infertility. Here, we showed that CENP-W, a relatively novel member of the kinetochore protein family, was expressed in mouse oocytes from the germinal vesicle (GV) to metaphase II (MII) stages. Confocal microscopy revealed that CENP-W was localized in the germinal vesicle in the GV stage, and then became concentrated on kinetochores during oocyte maturation. Knockdown of CENP-W by specific siRNA injection in vitro caused kinetochore-microtubule detachment, resulting in severely defective spindles and misaligned chromosomes, leading to metaphase I arrest and failure of first polar body (PB1) extrusion. Correspondingly, spindle assembly checkpoint (SAC) activation was observed in CENP-W knockdown oocytes even after 10h of culture. Our results suggest that CENP-W acts as a kinetochore protein, which takes part in kinetochore-microtubule attachment, thus mediating the progression of oocyte meiotic maturation.


Asunto(s)
Cinetocoros/metabolismo , Meiosis , Microtúbulos/metabolismo , Oocitos/citología , Animales , Femenino , Ratones , Ratones Endogámicos ICR , Oocitos/metabolismo
19.
FASEB J ; 33(1): 1179-1187, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30110177

RESUMEN

N6-methyladenosine (m6A) is the most prevalent and reversible internal modification of mammalian messenger and noncoding RNAs mediated by specific m6A writer, reader, and eraser proteins. As an m6A writer, the methyltransferase-like 3-methyltransferase-like 14 (METTL14)-Wilms tumor 1-associated protein complex dynamically regulates m6A modification and plays important roles in diverse biologic processes. However, our knowledge about the complete functions of this RNA methyltransferase complex, the contributions of each component to the methylation, and their effects on different biologic pathways are still limited. By using both in vivo and in vitro models, we here report that METTL14 is indispensable for postimplantation embryonic development by facilitating the conversion from naive to primed state of the epiblast. Depletion of Mettl14 leads to conspicuous embryonic growth retardation from embryonic d 6.5, mainly as a result of resistance to differentiation, which further leads to embryonic lethality early in gestation. Our data highlight the critical function of METTL14 as an m6A modification regulator in orchestrating early mouse embryogenesis.-Meng, T.-G., Lu, X., Guo, L., Hou, G.-M., Ma, X.-S., Li, Q.-N., Huang, L., Fan, L.-H., Zhao, Z.-H., Ou, X.-H., OuYang, Y.-C., Schatten, H., Li, L., Wang, Z.-B., Sun, Q.-Y. Mettl14 is required for mouse postimplantation development by facilitating epiblast maturation.


Asunto(s)
Desarrollo Embrionario/genética , Estratos Germinativos/citología , Metiltransferasas/fisiología , Adenosina/análogos & derivados , Adenosina/genética , Animales , Sistemas CRISPR-Cas , Femenino , Perfilación de la Expresión Génica , Genes Letales , Metiltransferasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Madre Embrionarias de Ratones/citología , ARN Mensajero/genética
20.
Zygote ; 28(2): 97-102, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-31787133

RESUMEN

Synaptotagmin 1 (Syt1) is an abundant and important presynaptic vesicle protein that binds Ca2+ for the regulation of synaptic vesicle exocytosis. Our previous study reported its localization and function on spindle assembly in mouse oocyte meiotic maturation. The present study was designed to investigate the function of Syt1 during mouse oocyte activation and subsequent cortical granule exocytosis (CGE) using confocal microscopy, morpholinol-based knockdown and time-lapse live cell imaging. By employing live cell imaging, we first studied the dynamic process of CGE and calculated the time interval between [Ca2+]i rise and CGE after oocyte activation. We further showed that Syt1 was co-localized to cortical granules (CGs) at the oocyte cortex. After oocyte activation with SrCl2, the Syt1 distribution pattern was altered significantly, similar to the changes seen for the CGs. Knockdown of Syt1 inhibited [Ca2+]i oscillations, disrupted the F-actin distribution pattern and delayed the time of cortical reaction. In summary, as a synaptic vesicle protein and calcium sensor for exocytosis, Syt1 acts as an essential regulator in mouse oocyte activation events including the generation of Ca2+ signals and CGE.


Asunto(s)
Exocitosis , Sinaptotagmina I , Animales , Calcio/metabolismo , Femenino , Ratones , Oocitos/metabolismo , Oogénesis , Sinaptotagmina I/genética
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