RESUMEN
Serine protease 50 (PRSS50/TSP50) is highly expressed in spermatocytes. Our study investigated its role in testicular development and spermatogenesis. Initially, PRSS50 knockdown was observed to impair DNA synthesis in spermatocytes. To further explore this, we generated PRSS50 knockout ( Prss50 -/- ) mice ( Mus musculus), which exhibited abnormal spermatid nuclear compression and reduced male fertility. Furthermore, dysplastic seminiferous tubules and decreased sex hormones were observed in 4-week-old Prss50 -/- mice, accompanied by meiotic progression defects and increased apoptosis of spermatogenic cells. Mechanistic analysis indicated that PRSS50 deletion resulted in increased phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and elevated levels of MAP kinase phosphatase 3 (MKP3), a specific ERK antagonist, potentially accounting for testicular dysplasia in adolescent Prss50 -/- mice. Taken together, these findings suggest that PRSS50 plays an important role in testicular development and spermatogenesis, with the MKP3/ERK signaling pathway playing a significant role in this process.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Meiosis , Ratones Noqueados , Espermatozoides , Animales , Masculino , Ratones , Meiosis/fisiología , Espermatozoides/fisiología , Espermatogénesis/fisiología , Fosfatasa 6 de Especificidad Dual/genética , Fosfatasa 6 de Especificidad Dual/metabolismo , Testículo/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
Extracellular signal-regulated protein kinase 5 (Erk5), a member of the mitogen-activated protein kinase (MAPK) family, is ubiquitously expressed in all eukaryotic cells and is implicated in the various mitotic processes such as cell survival, proliferation, migration, and differentiation. However, the potential functional roles of Erk5 in oocyte meiosis have not been fully determined. In this study, we document that ERK5 participates in the meiotic maturation of mouse oocytes by regulating the spindle assembly to ensure the meiotic progression. We unexpectedly found that phosphorylated ERK5 was localized in the spindle pole region at metaphase I and II stages by immunostaining analysis. Inhibition of ERK5 activity using its specific inhibitor XMD8-92 dramatically reduced the incidence of first polar body extrusion. In addition, inhibition of ERK5 evoked the spindle assembly checkpoint to arrest oocytes at metaphase I stage by impairing the spindle assembly, chromosome alignment and kinetochore-microtubule attachment. Mechanically, over-strengthened microtubule stability was shown to disrupt the microtubule dynamics and thus compromise the spindle assembly in ERK5-inhibited oocytes. Conversely, overexpression of ERK5 caused decreased level of acetylated α-tubulin and spindle defects. Collectively, we conclude that ERK5 plays an important role in the oocyte meiotic maturation by regulating microtubule dynamics and spindle assembly.
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Meiosis , Proteína Quinasa 7 Activada por Mitógenos , Oocitos , Huso Acromático , Animales , Oocitos/fisiología , Meiosis/fisiología , Ratones , Huso Acromático/fisiología , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/genética , FemeninoRESUMEN
In many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in Caenorhabditis elegans oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF, but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Meiosis , Oocitos , Tubulina (Proteína) , Proteína de Unión al GTP ran , Animales , Anafase , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Cromatina/metabolismo , Segregación Cromosómica , Guanosina Trifosfato/metabolismo , Meiosis/fisiología , Oocitos/metabolismo , Prometafase , Proteína de Unión al GTP ran/metabolismo , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
BACKGROUND: Helicase for meiosis 1 (HFM1), a putative DNA helicase expressed in germ-line cells, has been reported to be closely associated with premature ovarian insufficiency (POI). However, the underlying molecular mechanism has not been clearly elucidated. The aim of this study was to investigate the function of HFM1 in the first meiotic prophase of mouse oocytes. RESULTS: The results suggested that the deficiency of HFM1 resulting in increased apoptosis and depletion of oocytes in mice, while the oocytes were arrested in the pachytene stage of the first meiotic prophase. In addition, impaired DNA double-strand break repair and disrupted synapsis were observed in the absence of HFM1. Further investigation revealed that knockout of HFM1 promoted ubiquitination and degradation of FUS protein mediated by FBXW11. Additionally, the depletion of HFM1 altered the intranuclear localization of FUS and regulated meiotic- and oocyte development-related genes in oocytes by modulating the expression of BRCA1. CONCLUSIONS: These findings elaborated that the critical role of HFM1 in orchestrating the regulation of DNA double-strand break repair and synapsis to ensure meiosis procession and primordial follicle formation. This study provided insights into the pathogenesis of POI and highlighted the importance of HFM1 in maintaining proper meiotic function in mouse oocytes.
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Profase Meiótica I , Oocitos , Ubiquitinación , Animales , Femenino , Ratones , Apoptosis/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Meiosis/fisiología , Profase Meiótica I/fisiología , Ratones Noqueados , Oocitos/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/genéticaRESUMEN
Estrogen is an important hormone that plays a role in regulating follicle development and oocyte maturation. Transzonal projections (TZPs) act as communication bridges between follicle somatic cells and oocytes, and their dynamic changes are critical for oocyte development and maturation. However, the roles and mechanisms of estrogen in regulating TZPs during follicular development are not yet understood. We found that the proportion of oocytes spontaneously resuming meiosis increases as the follicle grows, which is accompanied by rising estrogen levels in follicles and decreasing TZPs in cumulus-oocyte complex. To further explore the effect of elevated estrogen levels on TZP assembly, additional estrogen was added to the culture system. The increased estrogen level significantly decreased the mRNA and protein expression levels of TZP assembly-related genes. Subsequent research revealed that TZP regulation by estrogen was mediated by the membrane receptor GPER and downstream ERK1/2 signaling pathway. In summary, our study suggests that estrogen may regulate goat oocyte meiosis arrest by decreasing TZP numbers via estrogen-mediated GPER activation during follicle development.
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Células del Cúmulo , Estrógenos , Cabras , Oocitos , Folículo Ovárico , Receptores de Estrógenos , Receptores Acoplados a Proteínas G , Animales , Oocitos/metabolismo , Oocitos/citología , Femenino , Células del Cúmulo/metabolismo , Células del Cúmulo/citología , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Estrógenos/metabolismo , Estrógenos/metabolismo , Folículo Ovárico/metabolismo , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/citología , Meiosis/fisiología , Sistema de Señalización de MAP Quinasas/fisiologíaRESUMEN
Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this age-related decline remain unclear. To gain insights into this phenomenon, we applied plexDIA, a multiplexed data-independent acquisition, single-cell mass spectrometry method, to analyze the proteome of oocytes from both young women and women of advanced maternal age. Our findings primarily revealed distinct proteomic profiles between immature fully grown germinal vesicle and mature metaphase II oocytes. Importantly, we further show that a woman's age is associated with changes in her oocyte proteome. Specifically, when compared to oocytes obtained from young women, advanced maternal age oocytes exhibited lower levels of the proteasome and TRiC complex, as well as other key regulators of proteostasis and meiosis. This suggests that aging adversely affects the proteostasis and meiosis networks in human oocytes. The proteins identified in this study hold potential as targets for improving oocyte quality and may guide future studies into the molecular processes underlying oocyte aging.
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Edad Materna , Meiosis , Oocitos , Proteoma , Proteómica , Proteostasis , Análisis de la Célula Individual , Humanos , Oocitos/metabolismo , Oocitos/citología , Femenino , Meiosis/fisiología , Adulto , Proteómica/métodos , Análisis de la Célula Individual/métodos , Proteoma/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Persona de Mediana EdadRESUMEN
Oocyte maturation and early embryonic development are key steps in the reproductive physiology of female mammals, and any error in this process can adversely affect reproductive development. Recent studies have shown that epigenetic modifications of histones play important roles in the regulation of oocyte meiosis and quality assurance of early embryonic development. Histone deacetylase 11 (HDAC11) is the smallest known member of the histone deacetylases (HDACs) family, and inhibition of HDAC11 activity significantly suppresses the rate of oocyte maturation, as well as the development of 8-cell and blastocyst embryos at the embryonic stage. This paper focuses on recent progress on the important role of HDAC11 in the regulation of mammalian oocyte maturation and early embryonic development, hoping to gain insights into the key roles played by epitope-modifying proteins represented by HDAC11 in the regulation of mammalian reproduction and their molecular mechanisms.
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Desarrollo Embrionario , Histona Desacetilasas , Oocitos , Animales , Oocitos/fisiología , Desarrollo Embrionario/fisiología , Histona Desacetilasas/metabolismo , Histona Desacetilasas/fisiología , Histona Desacetilasas/genética , Femenino , Humanos , Oogénesis/fisiología , Mamíferos/embriología , Meiosis/fisiologíaRESUMEN
A dispersed cytoplasmic distribution of mitochondria is a hallmark of normal cellular organization. Here, we have utilized the expression of exogenous Trak2 in mouse oocytes and embryos to disrupt the dispersed distribution of mitochondria by driving them into a large cytoplasmic aggregate. Our findings reveal that aggregated mitochondria have minimal impact on asymmetric meiotic cell divisions of the oocyte. In contrast, aggregated mitochondria during the first mitotic division result in daughter cells with unequal sizes and increased micronuclei. Further, in two-cell embryos, microtubule-mediated centering properties of the mitochondrial aggregate prevent nuclear centration, distort nuclear shape, and inhibit DNA synthesis and the onset of embryonic transcription. These findings demonstrate the motor protein-mediated distribution of mitochondria throughout the cytoplasm is highly regulated and is an essential feature of cytoplasmic organization to ensure optimal cell function.
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Blastocisto , Núcleo Celular , Mitocondrias , Oocitos , Animales , Mitocondrias/metabolismo , Blastocisto/metabolismo , Blastocisto/citología , Ratones , Núcleo Celular/metabolismo , Oocitos/metabolismo , Oocitos/citología , Femenino , Desarrollo Embrionario/fisiología , Microtúbulos/metabolismo , Mitosis , Meiosis/fisiologíaRESUMEN
The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. We have previously implicated the outer kinetochore protein SPC105R/KNL1 in driving meiosis I chromosome segregation through lateral attachments to microtubules and coorientation of sister centromeres. To identify the domains of SPC105R that are critical for meiotic chromosome segregation, an RNAi-resistant gene expression system was developed. We found that the SPC105R C-terminal domain (aa 1284-1960) is necessary and sufficient for recruiting NDC80 to the kinetochore and building the outer kinetochore. Furthermore, the C-terminal domain recruits BUBR1, which in turn recruits the cohesion protection proteins MEI-S332 and PP2A. Of the remaining 1283 amino acids, we found the first 473 are most important for meiosis. The first 123 amino acids of the N-terminal half of SPC105R contain the conserved SLRK and RISF motifs that are targets of PP1 and Aurora B kinase and are most important for regulating the stability of microtubule attachments and maintaining metaphase I arrest. The region between amino acids 124 and 473 are required for lateral microtubule attachments and biorientation of homologues, which are critical for accurate chromosome segregation in meiosis I.
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Segregación Cromosómica , Proteínas de Drosophila , Cinetocoros , Meiosis , Microtúbulos , Oocitos , Cinetocoros/metabolismo , Animales , Meiosis/fisiología , Oocitos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Femenino , Centrómero/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Aurora Quinasa B/metabolismo , Aurora Quinasa B/genéticaRESUMEN
Kif16A, a member of the kinesin-3 family of motor proteins, has been shown to play crucial roles in inducing mitotic arrest, apoptosis, and mitotic cell death. However, its roles during oocyte meiotic maturation have not been fully defined. In this study, we report that Kif16A exhibits unique accumulation on the spindle apparatus and colocalizes with microtubule fibers during mouse oocyte meiotic maturation. Targeted depletion of Kif16A using gene-targeting siRNA disrupts the progression of the meiotic cell cycle. Furthermore, Kif16A depletion leads to aberrant spindle assembly and chromosome misalignment in oocytes. Our findings also indicate that Kif16A depletion reduces tubulin acetylation levels and compromises microtubule resistance to depolymerizing drugs, suggesting its crucial role in microtubule stability maintenance. Notably, we find that the depletion of Kif16A results in a notably elevated incidence of defective kinetochore-microtubule attachments and the absence of BubR1 localization at kinetochores, suggesting a critical role for Kif16A in the activation of the spindle assembly checkpoint (SAC) activity. Additionally, we observe that Kif16A is indispensable for proper actin filament distribution, thereby impacting spindle migration. In summary, our findings demonstrate that Kif16A plays a pivotal role in regulating microtubule and actin dynamics crucial for ensuring both spindle assembly and migration during mouse oocyte meiotic maturation.
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Cinesinas , Meiosis , Microtúbulos , Oocitos , Huso Acromático , Animales , Cinesinas/metabolismo , Cinesinas/genética , Meiosis/fisiología , Oocitos/metabolismo , Microtúbulos/metabolismo , Ratones , Huso Acromático/metabolismo , Femenino , Actinas/metabolismo , Cinetocoros/metabolismoRESUMEN
Arf6 is a member of ADP-ribosylation factor (Arf) family, which is widely implicated in the regulation of multiple physiological processes including endocytic recycling, cytoskeletal organization, and membrane trafficking during mitosis. In this study, we investigated the potential relationship between Arf6 and aging-related oocyte quality, and its roles on organelle rearrangement and cytoskeleton dynamics in porcine oocytes. Arf6 expressed in porcine oocytes throughout meiotic maturation, and it decreased in aged oocytes. Disruption of Arf6 led to the failure of cumulus expansion and polar body extrusion. Further analysis indicated that Arf6 modulated ac-tubulin for meiotic spindle organization and microtubule stability. Besides, Arf6 regulated cofilin phosphorylation and fascin for actin assembly, which further affected spindle migration, indicating the roles of Arf6 on cytoskeleton dynamics. Moreover, the lack of Arf6 activity caused the dysfunction of Golgi and ER for protein synthesis and signal transduction. Mitochondrial dysfunction was also observed in Arf6-deficient porcine oocytes, which was supported by the increased ROS level and abnormal membrane potential. In conclusion, our results reported that insufficient Arf6 was related to aging-induced oocyte quality decline through spindle organization, actin assembly, and organelle rearrangement in porcine oocytes.
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Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP , Oocitos , Animales , Oocitos/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Porcinos , Femenino , Meiosis/fisiología , Huso Acromático/metabolismo , Envejecimiento/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Anti-Müllerian hormone (AMH) is a key paracrine/autocrine factor regulating folliculogenesis in the postnatal ovary. As antral follicles mature to the preovulatory stage, AMH production tends to be limited to cumulus cells. Therefore, the present study investigated the role of cumulus cell-derived AMH in supporting maturation and competence of the enclosed oocyte. Cumulus-oocyte complexes (COCs) were isolated from antral follicles of rhesus macaque ovaries for in vitro maturation with or without AMH depletion. Oocyte meiotic status and embryo cleavage after in vitro fertilization were assessed. In vitro maturation with AMH depletion was also performed using COCs from antral follicles of human ovarian tissue. Oocyte maturation and morphology were evaluated. The direct AMH action on mural granulosa cells of the preovulatory follicle was further assessed using human granulosa cells cultured with or without AMH supplementation. More macaque COCs produced metaphase II oocytes with AMH depletion than those of the control culture. However, preimplantation embryonic development after in vitro fertilization was comparable between oocytes derived from COCs cultured with AMH depletion and controls. Oocytes resumed meiosis in human COCs cultured with AMH depletion and exhibited a typical spindle structure. The confluency and cell number decreased in granulosa cells cultured with AMH supplementation relative to the control culture. AMH treatment did not induce cell death in cultured human granulosa cells. Data suggest that reduced AMH action in COCs could be beneficial for oocyte maturation. Cumulus cell-derived AMH is not essential for supporting oocyte competence or mural granulosa cell viability.
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Hormona Antimülleriana , Células del Cúmulo , Técnicas de Maduración In Vitro de los Oocitos , Macaca mulatta , Oocitos , Hormona Antimülleriana/metabolismo , Oocitos/metabolismo , Oocitos/citología , Oocitos/efectos de los fármacos , Femenino , Células del Cúmulo/metabolismo , Células del Cúmulo/citología , Células del Cúmulo/efectos de los fármacos , Animales , Humanos , Técnicas de Maduración In Vitro de los Oocitos/métodos , Oogénesis/fisiología , Oogénesis/efectos de los fármacos , Células Cultivadas , Fertilización In Vitro/métodos , Meiosis/fisiología , Meiosis/efectos de los fármacos , Células de la Granulosa/metabolismo , Células de la Granulosa/citología , Folículo Ovárico/metabolismo , Folículo Ovárico/citología , Folículo Ovárico/fisiología , Desarrollo Embrionario/fisiologíaRESUMEN
Meiosis is a highly complex process significantly influenced by transcriptional regulation. However, studies on the mechanisms that govern transcriptomic changes during meiosis, especially in prophase I, are limited. Here, we performed single-cell ATAC-seq of human testis tissues and observed reprogramming during the transition from zygotene to pachytene spermatocytes. This event, conserved in mice, involved the deactivation of genes associated with meiosis after reprogramming and the activation of those related to spermatogenesis before their functional onset. Furthermore, we identified 282 transcriptional regulators (TRs) that underwent activation or deactivation subsequent to this process. Evidence suggested that physical contact signals from Sertoli cells may regulate these TRs in spermatocytes, while secreted ENHO signals may alter metabolic patterns in these cells. Our results further indicated that defective transcriptional reprogramming may be associated with non-obstructive azoospermia (NOA). This study revealed the importance of both physical contact and secreted signals between Sertoli cells and germ cells in meiotic progression.
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Comunicación Celular , Meiosis , Animales , Masculino , Ratones , Meiosis/fisiología , Humanos , Células de Sertoli/metabolismo , Células de Sertoli/fisiología , Testículo/metabolismo , Testículo/citología , Espermatogénesis/fisiología , Regulación de la Expresión Génica , Azoospermia/genética , Transcripción Genética , ARN Citoplasmático Pequeño/genética , ARN Citoplasmático Pequeño/metabolismo , Análisis de Expresión Génica de una Sola CélulaRESUMEN
RESEARCH QUESTION: Can immature oocytes vitrified and warmed using a short protocol survive and resume meiosis? DESIGN: This study examined modifications of oocyte vitrification and warming protocols that reduce the length of exposure to vitrification and warming solutions. In total, 561 germinal vesicles and 218 metaphase I oocytes that were immature at oocyte retrieval were vitrified at room temperature for 2 min. Warming was performed at 37°C for 2 min. Resumption of meiotic activity was evaluated after 24 and 48 h of culture. Two different commercially available vitrification and warming kits were used for comparison. RESULTS: Ninety-five percent of germinal vesicles survived, with no difference observed between the kits. The survival of metaphase I oocytes was, on average, 95.4% and did not differ significantly between the kits. Of the 533 germinal vesicles that survived, 491 converted to metaphase I oocytes (92.1%). After culture for 48 h, 54.4% converted to metaphase II oocytes. In addition, of the 208 metaphase I oocytes that survived warming, 84.1% converted to metaphase II oocytes after 24 h of culture. These maturation rates were similar to those of non-vitrified oocytes. CONCLUSIONS: Vitrification and warming of oocytes at different nuclear maturation stages can be performed with 2 min of exposure to hypertonic solution and 2 min of exposure to hypotonic solution, respectively. This approach reduces exposure of the oocytes to room temperature during dehydration and rehydration. Warming in 0.5M sucrose helps to maintain and support the potential of oocytes to resume nuclear meiotic activity, and conversion from germinal vesicles to metaphase I and metaphase II oocytes.
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Criopreservación , Meiosis , Oocitos , Vitrificación , Oocitos/citología , Oocitos/fisiología , Humanos , Meiosis/fisiología , Femenino , Criopreservación/métodos , Supervivencia Celular , Técnicas de Maduración In Vitro de los Oocitos/métodos , AdultoRESUMEN
Extensive research has been conducted on the role of CXCR3 in immune responses and inflammation. However, the role of CXCR3 in the reproductive system, particularly in oocyte development, remains unknown. In this study, we present findings on the involvement of CXCR3 in the meiotic division process of mouse oocytes. We found CXCR3 was expressed consistently throughout the entire maturation process of mouse oocyte. Inhibition of CXCR3 impaired the asymmetric division of oocyte, while the injection of Cxcr3 mRNA was capable of restoring these defects. Further study showed that inhibition of CXCR3 perturbed spindle migration by affecting LIMK/cofilin pathway-mediated actin remodeling. Knockout of CXCR3 led to an upregulation of actin-binding protein and an increased ATP level in GV-stage oocytes, while maintaining normal actin dynamics during the process of meiosis. Additionally, we noticed the expression level of DYNLT1 is markedly elevated in CXCR3-null oocytes. DYNLT1 bound with the Arp2/3 complex, and knockdown of DYNLT1 in CXCR3-null oocytes impaired the organization of cytoplasmic actin, suggesting the regulatory role of DYNLT1 in actin organization, and the compensatory expression of DYNLT1 may contribute to maintain normal actin dynamics in CXCR3-knockout oocytes. In summary, our findings provide insights into the intricate network of actin dynamics associated with CXCR3 during oocyte meiosis.
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Actinas , Oocitos , Receptores CXCR3 , Animales , Oocitos/metabolismo , Oocitos/fisiología , Ratones , Actinas/metabolismo , Actinas/genética , Receptores CXCR3/metabolismo , Receptores CXCR3/genética , Femenino , Meiosis/fisiología , Ratones NoqueadosRESUMEN
Coatomer Protein Complex-II (COPII) mediates anterograde vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Here, we report that the COPII coatomer complex is constructed dependent on a small GTPase, Sar1, in spermatocytes before and during Drosophila male meiosis. COPII-containing foci co-localized with transitional endoplasmic reticulum (tER)-Golgi units. They showed dynamic distribution along astral microtubules and accumulated around the spindle pole, but they were not localized on the cleavage furrow (CF) sites. The depletion of the four COPII coatomer subunits, Sec16, or Sar1 that regulate COPII assembly resulted in multinucleated cell production after meiosis, suggesting that cytokinesis failed in both or either of the meiotic divisions. Although contractile actomyosin and anilloseptin rings were formed once plasma membrane ingression was initiated, they were frequently removed from the plasma membrane during furrowing. We explored the factors conveyed toward the CF sites in the membrane via COPII-mediated vesicles. DE-cadherin-containing vesicles were formed depending on Sar1 and were accumulated in the cleavage sites. Furthermore, COPII depletion inhibited de novo plasma membrane insertion. These findings suggest that COPII vesicles supply the factors essential for the anchoring and/or constriction of the contractile rings at cleavage sites during male meiosis in Drosophila.
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Vesículas Cubiertas por Proteínas de Revestimiento , Citocinesis , Proteínas de Drosophila , Meiosis , Proteínas de Transporte Vesicular , Animales , Masculino , Cadherinas/metabolismo , Membrana Celular/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Citocinesis/fisiología , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Meiosis/fisiología , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Espermatocitos/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Heat stress induces testicular oxidative stress, impairs spermatogenesis, and increases the risk of male infertility. Recent studies have highlighted the antioxidative properties of the Sestrins family in reducing cellular oxidative damage. However, the role of Sestrins (Sestrin1, 2, and 3) in the testicular response to heat stress remains unclear. Here, we found that Sestrin2 and 3 were highly expressed in the testis relative to Sestrin1. Then, the Sestrin2-/- and Sestrin3-/- mice were generated by CRISPR/Cas9 to investigate the role of them on spermatogenesis after heat stress. Our data showed that Sestrin2-/- and Sestrin3-/- mice testes exhibited more severe damage manifested by exacerbated loss of germ cells and higher levels of oxidative stress as compared to wild-type counterparts after heat stress. Notably, Sestrin2-/- and Sestrin3-/- mice underwent a remarkable increase in heat-induced spermatocyte apoptosis than that of controls. Furthermore, the transcriptome landscape of spermatocytes and chromosome spreading showed that loss of Sestrin2 and Sestrin3 exacerbated meiotic failure by compromising DNA double-strand breaks repair after heat stress. Taken together, our work demonstrated a critical protective function of Sestrin2 and Sestrin3 in mitigating the impairments of spermatogenesis against heat stress.
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Respuesta al Choque Térmico , Meiosis , Ratones Noqueados , Espermatogénesis , Animales , Masculino , Espermatogénesis/fisiología , Espermatogénesis/genética , Ratones , Meiosis/fisiología , Respuesta al Choque Térmico/fisiología , Sestrinas/genética , Sestrinas/metabolismo , Estrés Oxidativo/fisiología , Testículo/metabolismo , Espermatocitos/metabolismo , Apoptosis/fisiologíaRESUMEN
PLD1 has been implicated in cytoskeletal reorganization and vesicle trafficking in somatic cells; however, its function remains unclear in oocyte meiosis. Herein, we found PLD1 stably expresses in mouse oocytes meiosis, with direct interaction with spindle, RAB11A+ vesicles and macroautophagic/autophagic vacuoles. The genetic or chemical inhibition of PLD1 disturbed MTOC clustering, spindle assembly and its cortical migration, also decreased PtdIns(4,5)P2, phosphorylated CFL1 (p-CFL1 [Ser3]) and ACTR2, and their local distribution on MTOC, spindle and vesicles. Furthermore in PLD1-suppressed oocytes, vesicle size was significantly reduced while F-actin density was dramatically increased in the cytoplasm, the asymmetric distribution of autophagic vacuoles was broken and the whole autophagic process was substantially enhanced, as illustrated with characteristic changes in autophagosomes, autolysosome formation and levels of ATG5, BECN1, LC3-II, SQSTM1 and UB. Exogenous administration of PtdIns(4,5)P2 or overexpression of CFL1 hyperphosphorylation mutant (CFL1S3E) could significantly improve polar MTOC focusing and spindle structure in PLD1-depleted oocytes, whereas overexpression of ACTR2 could rescue not only MTOC clustering, and spindle assembly but also its asymmetric positioning. Interestingly, autophagy activation induced similar defects in spindle structure and positioning; instead, its inhibition alleviated the alterations in PLD1-depleted oocytes, and this was highly attributed to the restored levels of PtdIns(4,5)P2, ACTR2 and p-CFL1 (Ser3). Together, PLD1 promotes spindle assembly and migration in oocyte meiosis, by maintaining rational levels of ACTR2, PtdIns(4,5)P2 and p-CFL1 (Ser3) in a manner of modulating autophagy flux. This study for the first time introduces a unique perspective on autophagic activity and function in oocyte meiotic development.Abbreviations: ACTR2/ARP2: actin related protein 2; ACTR3/ARP3: actin related protein 3; ATG5: autophagy related 5; Baf-A1: bafilomycin A1; BFA: brefeldin A; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GV: germinal vesicle; GVBD: germinal vesicle breakdown; IVM: in vitro maturation; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MI: metaphase of meiosis I; MII: metaphase of meiosis II; MO: morpholino; MTOC: microtubule-organizing center; MTOR: mechanistic target of rapamycin kinase; PB1: first polar body; PLA: proximity ligation assay; PLD1: phospholipase D1; PtdIns(4,5)P2/PIP2: phosphatidylinositol 4,5-bisphosphate; RAB11A: RAB11A, member RAS oncogene family; RPS6KB1/S6K1: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TUBA/α-tubulin: tubulin alpha; TUBG/γ-tubulin: tubulin gamma; UB: ubiquitin; WASL/N-WASP: WASP like actin nucleation promoting factor.
Asunto(s)
Autofagia , Meiosis , Oocitos , Fosfolipasa D , Huso Acromático , Animales , Autofagia/fisiología , Autofagia/genética , Oocitos/metabolismo , Meiosis/fisiología , Huso Acromático/metabolismo , Ratones , Femenino , Fosfolipasa D/metabolismo , Fosfolipasa D/genética , Movimiento Celular/fisiología , FosforilaciónRESUMEN
Polo-like kinase I (Plk1) is a highly conserved seronine/threonine kinase essential in meiosis and mitosis for spindle formation and cytokinesis. Here, through temporal application of Plk1 inhibitors, we identify a new role for Plk1 in the establishment of cortical polarity essential for highly asymmetric cell divisions of oocyte meiosis. Application of Plk1 inhibitors in late metaphase I abolishes pPlk1 from spindle poles and prevents the induction of actin polymerization at the cortex through inhibition of local recruitment of Cdc42 and Neuronal Wiskott-Aldrich Syndrome protein (N-WASP). By contrast, an already established polar actin cortex is insensitive to Plk1 inhibitors, but if the polar cortex is first depolymerized, Plk1 inhibitors completely prevent its restoration. Thus, Plk1 is essential for establishment but not maintenance of cortical actin polarity. These findings indicate that Plk1 regulates recruitment of Cdc42 and N-Wasp to coordinate cortical polarity and asymmetric cell division.
Asunto(s)
Actinas , Meiosis , Oocitos , Actinas/genética , Actinas/fisiología , Meiosis/genética , Meiosis/fisiología , Oocitos/fisiología , Polimerizacion , Proteínas Serina-Treonina Quinasas , Quinasa Tipo Polo 1RESUMEN
Deficiency in meiotic recombination leads to aberrant chromosome disjunction during meiosis, often resulting in the lethality of gametes or genetic disorders due to aneuploidy formation. Budding yeasts lacking Spo11, which is essential for initiation of meiotic recombination, produce many inviable spores in meiosis, while very rarely all sets of 16 chromosomes are coincidentally assorted into gametes to form viable spores. We induced meiosis in a spo11∆ diploid, in which homolog pairs can be distinguished by single nucleotide polymorphisms and determined whole-genome sequences of their exceptionally viable spores. We detected no homologous recombination in the viable spores of spo11∆ diploid. Point mutations were fewer in spo11∆ than in wild-type. We observed spo11∆ viable spores carrying a complete diploid set of homolog pairs or haploid spores with a complete haploid set of homologs but with aneuploidy in some chromosomes. In the latter, we found the chromosome-dependence in the aneuploid incidence, which was positively and negatively influenced by the chromosome length and the impact of dosage-sensitive genes, respectively. Selection of aneuploidy during meiosis II or mitosis after spore germination was also chromosome dependent. These results suggest a pathway by which specific chromosomes are more prone to cause aneuploidy, as observed in Down syndrome.