RESUMEN
The repetitive sequences of DNA centromeric regions form the structural basis for kinetochore assembly. Recently they were found to be transcriptionally active in mitosis, with their RNAs providing noncoding functions. Here we explore the role, in mouse oocytes, of transcripts generated from within the minor satellite repeats. Depletion of minor satellite transcripts delayed progression through meiosis I by activation of the spindle assembly checkpoint. Arrested oocytes had poorly congressed chromosomes, and centromeres were frequently split by microtubules. Thus, we have demonstrated that the centromeric RNA plays a specific role in female meiosis I compared with mitosis and is required for maintaining the structural integrity of centromeres. This may contribute to the high aneuploidy rates observed in female meiosis.
Asunto(s)
ARN/metabolismo , Huso Acromático/metabolismo , Animales , Centrómero/genética , Centrómero/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Mitosis/genética , ARN/genética , Huso Acromático/genéticaRESUMEN
Mouse female meiotic spindles assemble from acentriolar microtubule-organizing centers (aMTOCs) that fragment into discrete foci. These are further sorted and clustered to form spindle poles, thus providing balanced forces for faithful chromosome segregation. To assess the impact of aMTOC biogenesis on spindle assembly, we genetically induced their precocious fragmentation in mouse oocytes using conditional overexpression of Plk4, a master microtubule-organizing center regulator. Excessive microtubule nucleation from these fragmented aMTOCs accelerated spindle assembly dynamics. Prematurely formed spindles promoted the breakage of three different fragilized bivalents, generated by the presence of recombined Lox P sites. Reducing the density of microtubules significantly diminished the extent of chromosome breakage. Thus, improper spindle forces can lead to widely described yet unexplained chromosomal structural anomalies with disruptive consequences on the ability of the gamete to transmit an uncorrupted genome.
Asunto(s)
Cromosomas de los Mamíferos/metabolismo , Edición Génica , Meiosis , Centro Organizador de los Microtúbulos/metabolismo , Oocitos/metabolismo , Huso Acromático/metabolismo , Animales , Cromosomas de los Mamíferos/genética , Femenino , Ratones , Ratones Transgénicos , Oocitos/citología , Huso Acromático/genéticaRESUMEN
In the first meiotic division (MI) of oocytes, the cortically positioned spindle causes bivalent segregation in which only the centre-facing homologue pairs are retained. 'Selfish' chromosomes are known to exist, which bias their spindle orientation and hence retention in the egg, a process known as 'meiotic drive'. Here we report on this phenomenon in oocytes from F1 hybrid mice, where parental strain differences in centromere size allows distinction of the two homologue pairs of a bivalent. Bivalents with centromere and kinetochore asymmetry show meiotic drive by rotating during prometaphase, in a process dependent on aurora kinase activity. Cortically positioned homologue pairs appear to be under greater stretch than their centre-facing partners. Additionally the cortex spindle-half contain a greater density of tubulin and microtubule organising centres. A model is presented in which meiotic drive is explained by the impact of microtubule force asymmetry on chromosomes with different sized centromeres and kinetochores.
Asunto(s)
Meiosis/fisiología , Centro Organizador de los Microtúbulos/fisiología , Oocitos/fisiología , Huso Acromático/fisiología , Tubulina (Proteína)/fisiología , Animales , Aurora Quinasas/metabolismo , Centrómero , Segregación Cromosómica , Cromosomas/metabolismo , Citocalasina B/antagonistas & inhibidores , Femenino , Cinetocoros/fisiología , Ratones , Ratones Endogámicos C57BL , Microinyecciones , Microtúbulos/fisiología , Proteínas Nucleares/metabolismo , Oocitos/citología , Oocitos/efectos de los fármacosRESUMEN
Initiated by luteinizing hormone and finalized by the fertilizing sperm, the mammalian oocyte completes its two meiotic divisions. The first division occurs in the mature Graafian follicle during the hours preceding ovulation and culminates in an extreme asymmetric cell division and the segregation of the two pairs of homologous chromosomes. The newly created mature egg rearrests at metaphase of the second meiotic division prior to ovulation and only completes meiosis following a Ca2+ signal initiated by the sperm at gamete fusion. Here, we review the cellular events that govern the passage of the oocyte through meiosis I with a focus on the role of the spindle assembly checkpoint in regulating its timing. In meiosis II, we examine how the egg achieves its arrest and how the fertilization Ca2+ signal allows the initiation of embryo development.
Asunto(s)
Mamíferos/fisiología , Meiosis/fisiología , Oocitos/citología , Animales , Señalización del Calcio , División Celular/fisiología , Segregación Cromosómica , Desarrollo Embrionario , Femenino , Fertilización/fisiología , Masculino , Ratones , Ovulación , Interacciones Espermatozoide-Óvulo , Huso Acromático/fisiologíaRESUMEN
The spindle assembly checkpoint (SAC) prevents chromosome missegregation by coupling anaphase onset with correct chromosome attachment and tension to microtubules. It does this by generating a diffusible signal from free kinetochores into the cytoplasm, inhibiting the anaphase-promoting complex (APC). The volume in which this signal remains effective is unknown. This raises the possibility that cell volume may be the reason the SAC is weak, and chromosome segregation error-prone, in mammalian oocytes. Here, by a process of serial bisection, we analyzed the influence of oocyte volume on the ability of the SAC to inhibit bivalent segregation in meiosis I. We were able to generate oocytes with cytoplasmic volumes reduced by 86% and observed changes in APC activity consistent with increased SAC control. However, bivalent biorientation remained uncoupled from APC activity, leading to error-prone chromosome segregation. We conclude that volume is one factor contributing to SAC weakness in oocytes. However, additional factors likely uncouple chromosome biorientation with APC activity.
Asunto(s)
Tamaño de la Célula , Segregación Cromosómica , Microtúbulos/metabolismo , Oocitos/metabolismo , Huso Acromático/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/genética , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Animales , Femenino , Regulación de la Expresión Génica , Gonadotropinas Equinas/farmacología , Cinetocoros/efectos de los fármacos , Cinetocoros/metabolismo , Cinetocoros/ultraestructura , Meiosis/genética , Ratones , Ratones Endogámicos C57BL , Microinyecciones , Microtúbulos/efectos de los fármacos , Microtúbulos/ultraestructura , Milrinona/farmacología , Nocodazol/farmacología , Oocitos/efectos de los fármacos , Oocitos/ultraestructura , ARN Complementario/genética , ARN Complementario/metabolismo , Huso Acromático/efectos de los fármacos , Huso Acromático/ultraestructuraRESUMEN
Mouse oocytes carrying DNA damage arrest in meiosis I, thereby preventing creation of embryos with deleterious mutations. The arrest is dependent on activation of the spindle assembly checkpoint, which results in anaphase-promoting complex (APC) inhibition. However, little is understood about how this checkpoint is engaged following DNA damage. Here, we find that within minutes of DNA damage checkpoint proteins are assembled at the kinetochore, not at damage sites along chromosome arms, such that the APC is fully inhibited within 30â min. Despite this robust response, there is no measurable loss in k-fibres, or tension across the bivalent. Through pharmacological inhibition we observed that the response is dependent on Mps1 kinase, aurora kinase and Haspin. Using oocyte-specific knockouts we find the response does not require the DNA damage response kinases ATM or ATR. Furthermore, checkpoint activation does not occur in response to DNA damage in fully mature eggs during meiosis II, despite the divisions being separated by just a few hours. Therefore, mouse oocytes have a unique ability to sense DNA damage rapidly by activating the checkpoint at their kinetochores.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN , Cinetocoros/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Meiosis , Oocitos/citología , Oocitos/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Animales , Aurora Quinasas/metabolismo , Centrómero/efectos de los fármacos , Centrómero/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinetocoros/efectos de los fármacos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Meiosis/efectos de los fármacos , Ratones , Modelos Biológicos , Oocitos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Accurate chromosome segregation is necessary so that genetic material is equally shared among daughter cells. However, maturing mammalian oocytes are particularly prone to chromosome segregation errors, making them a valuable tool for identifying the causes of mis-segregation. Factors such as aging, cohesion loss, DNA damage, and the roles of a plethora of kinetochore and cell cycle-related proteins are involved. To study chromosome segregation in oocytes in a live setting is an imaging challenge that requires advanced techniques. Here we describe a method for examining chromosomes in live oocytes in detail as they undergo maturation. Our method is based on tracking the "center of brightness" of fluorescently labeled chromosomes. Here we describe how to set up our software and run experiments on a Leica TCS SP8 confocal microscope, but the method would be transferable to other microscopes with computer-aided microscopy.
Asunto(s)
Cromosomas de los Mamíferos/metabolismo , Microscopía Confocal/métodos , Oocitos/fisiología , Imagen de Lapso de Tiempo/métodos , Animales , Cromosomas de los Mamíferos/genética , Femenino , Fluorescencia , Ratones , Microscopía Confocal/instrumentación , Programas Informáticos , Imagen de Lapso de Tiempo/instrumentaciónRESUMEN
Mouse oocytes respond to DNA damage by arresting in meiosis I through activity of the Spindle Assembly Checkpoint (SAC) and DNA Damage Response (DDR) pathways. It is currently not known if DNA damage is the primary trigger for arrest, or if the pathway is sensitive to levels of DNA damage experienced physiologically. Here, using follicular fluid from patients with the disease endometriosis, which affects 10% of women and is associated with reduced fertility, we find raised levels of Reactive Oxygen Species (ROS), which generate DNA damage and turn on the DDR-SAC pathway. Only follicular fluid from patients with endometriosis, and not controls, produced ROS and damaged DNA in the oocyte. This activated ATM kinase, leading to SAC mediated metaphase I arrest. Completion of meiosis I could be restored by ROS scavengers, showing this is the primary trigger for arrest and offering a novel clinical therapeutic treatment. This study establishes a clinical relevance to the DDR induced SAC in oocytes. It helps explain how oocytes respond to a highly prevalent human disease and the reduced fertility associated with endometriosis.
Asunto(s)
Daño del ADN , Endometriosis/patología , Líquido Folicular/química , Oocitos/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Endometriosis/metabolismo , Femenino , Líquido Folicular/metabolismo , Depuradores de Radicales Libres/farmacología , Humanos , Peróxido de Hidrógeno/toxicidad , Proteínas Mad2/antagonistas & inhibidores , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Morfolinos/metabolismo , Oocitos/citología , Oocitos/crecimiento & desarrollo , Especies Reactivas de Oxígeno/metabolismo , Huso Acromático/metabolismoRESUMEN
Whether the adult mammalian ovary contains oogonial stem cells (OSCs) is controversial. They have been isolated by a live-cell sorting method using the germ cell marker DDX4, which has previously been assumed to be cytoplasmic, not surface-bound. Furthermore their stem cell and germ cell characteristics remain disputed. Here we show that although OSC-like cells can be isolated from the ovary using an antibody to DDX4, there is no good in silico modelling to support the existence of a surface-bound DDX4. Furthermore these cells when isolated were not expressing DDX4, and did not initially possess germline identity. Despite these unremarkable beginnings, they acquired some pre-meiotic markers in culture, including DDX4, but critically never expressed oocyte-specific markers, and furthermore were not immortal but died after a few months. Our results suggest that freshly isolated OSCs are not germ stem cells, and are not being isolated by their DDX4 expression. However it may be that culture induces some pre-meiotic markers. In summary the present study offers weight to the dogma that the adult ovary is populated by a fixed number of oocytes and that adult de novo production is a rare or insignificant event.
Asunto(s)
ARN Helicasas DEAD-box/análisis , Células Germinativas/química , Células Germinativas/fisiología , Células Madre Oogoniales/química , Células Madre Oogoniales/fisiología , Ovario/citología , Animales , Femenino , Citometría de Flujo , Ratones Endogámicos C57BLRESUMEN
DNA damage acquired during meiosis can lead to infertility and miscarriage. Hence, it should be important for an oocyte to be able to detect and respond to such events in order to make a healthy egg. Here, the strategies taken by oocytes during their stages of growth to respond to DNA damaging events are reviewed. In particular, recent evidence of a novel pathway in fully grown oocytes helps prevent the formation of mature eggs with DNA damage. It has been found that fully grown germinal vesicle stage oocytes that have been DNA damaged do not arrest at this point in meiosis, but instead undergo meiotic resumption and stall during the first meiotic division. The Spindle Assembly Checkpoint, which is a well-known mitotic pathway employed by somatic cells to monitor chromosome attachment to spindle microtubules, appears to be utilised by oocytes also to respond to DNA damage. As such maturing oocytes are arrested at metaphase I due to an active Spindle Assembly Checkpoint. This is surprising given this checkpoint has been previously studied in oocytes and considered to be weak and ineffectual because of its poor ability to be activated in response to microtubule attachment errors. Therefore, the involvement of the Spindle Assembly Checkpoint in DNA damage responses of mature oocytes during meiosis I uncovers a novel second function for this ubiquitous cellular checkpoint.
Asunto(s)
Daño del ADN , Oocitos/fisiología , Huso Acromático/fisiología , Animales , Puntos de Control del Ciclo Celular , Femenino , Humanos , Oocitos/citologíaRESUMEN
Extensive damage to maternal DNA during meiosis causes infertility, birth defects and abortions. However, it is unknown if fully grown oocytes have a mechanism to prevent the creation of DNA-damaged embryos. Here we show that DNA damage activates a pathway involving the spindle assembly checkpoint (SAC) in response to chemically induced double strand breaks, UVB and ionizing radiation. DNA damage can occur either before or after nuclear envelope breakdown, and provides an effective block to anaphase-promoting complex activity, and consequently the formation of mature eggs. This contrasts with somatic cells, where DNA damage fails to affect mitotic progression. However, it uncovers a second function for the meiotic SAC, which in the context of detecting microtubule-kinetochore errors has hitherto been labelled as weak or ineffectual in mammalian oocytes. We propose that its essential role in the detection of DNA damage sheds new light on its biological purpose in mammalian female meiosis.
Asunto(s)
Puntos de Control del Ciclo Celular , Daño del ADN , Meiosis , Oocitos/citología , Huso Acromático/metabolismo , Animales , Daño del ADN/efectos de la radiación , Femenino , Meiosis/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Oocitos/metabolismo , Oocitos/efectos de la radiación , Huso Acromático/efectos de la radiación , Rayos UltravioletaRESUMEN
Currently, maternal aging in women, based on mouse models, is thought to raise oocyte aneuploidy rates, because chromosome cohesion deteriorates during prophase arrest, and Sgo2, a protector of centromeric cohesion, is lost. Here we show that the most common mouse strain, C57Bl6/J, is resistant to maternal aging, showing little increase in aneuploidy or Sgo2 loss. Instead it demonstrates significant kinetochore-associated loss in the spindle assembly checkpoint protein Mad2 and phosphorylated Aurora C, which is involved in microtubule-kinetochore error correction. Their loss affects the fidelity of bivalent segregation but only when spindle organization is impaired during oocyte maturation. These findings have an impact clinically regarding the handling of human oocytes ex vivo during assisted reproductive techniques and suggest there is a genetic basis to aneuploidy susceptibility.
Asunto(s)
Envejecimiento/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Oocitos/metabolismo , Huso Acromático/metabolismo , Aneuploidia , Animales , Aurora Quinasa C/metabolismo , Segregación Cromosómica , Femenino , Proteínas Mad2/metabolismo , Ratones , Ratones Endogámicos C57BL , Nocodazol/farmacología , Oocitos/efectos de los fármacos , Especificidad de la EspecieRESUMEN
The spindle assembly checkpoint (SAC) prevents aneuploidy by coupling anaphase onset, through anaphase-promoting complex (APC) activation, with chromosome attachment to spindle microtubules. Here, we examine APC activity in oocytes, noted for their susceptibility to chromosome mis-segregation during the first meiotic division (MI). We find that MI oocytes only contain sub-maximal APC activity, measured through cyclin B1-GFP degradation, because inhibition of SAC proteins when the APC is normally fully active increases cyclin B1 degradation twofold and reduces the length of this division by 2 h. In addition, inhibiting the SAC component Mps1 only when the APC is already active increases aneuploidy rates in the resulting egg by up to 30%. We therefore establish that the activities of SAC proteins and the APC co-exist in oocytes, and such concurrence has a vital role in reducing aneuploidy rates by extending MI, probably by allowing time for numerous erroneous microtubule attachments to be corrected.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Oocitos/metabolismo , Huso Acromático/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Segregación Cromosómica , Ciclina B1/metabolismo , Femenino , Puntos de Control de la Fase M del Ciclo Celular/genética , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Masculino , Ratones , Oocitos/citología , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Fizzy-related 1 (FZR1) is an activator of the Anaphase promoting complex/cyclosome (APC/C) and an important regulator of the mitotic cell division cycle. Using a germ-cell-specific conditional knockout model we examined its role in entry into meiosis and early meiotic events in both sexes. Loss of APC/C(FZR1) activity in the male germline led to both a mitotic and a meiotic testicular defect resulting in infertility due to the absence of mature spermatozoa. Spermatogonia in the prepubertal testes of such mice had abnormal proliferation and delayed entry into meiosis. Although early recombination events were initiated, male germ cells failed to progress beyond zygotene and underwent apoptosis. Loss of APC/C(FZR1) activity was associated with raised cyclin B1 levels, suggesting that CDK1 may trigger apoptosis. By contrast, female FZR1Δ mice were subfertile, with premature onset of ovarian failure by 5 months of age. Germ cell loss occurred embryonically in the ovary, around the time of the zygotene-pachytene transition, similar to that observed in males. In addition, the transition of primordial follicles into the growing follicle pool in the neonatal ovary was abnormal, such that the primordial follicles were prematurely depleted. We conclude that APC/C(FZR1) is an essential regulator of spermatogonial proliferation and early meiotic prophase I in both male and female germ cells and is therefore important in establishing the reproductive health of adult male and female mammals.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Cdh1/metabolismo , Animales , Proteínas Cdh1/deficiencia , Proteínas Cdh1/genética , Ciclina B1/metabolismo , Roturas del ADN de Doble Cadena , Femenino , Regulación del Desarrollo de la Expresión Génica , Infertilidad Femenina/genética , Infertilidad Femenina/metabolismo , Infertilidad Femenina/patología , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Masculino , Profase Meiótica I/genética , Profase Meiótica I/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Oogénesis/genética , Oogénesis/fisiología , Ovario/metabolismo , Ovario/patología , Embarazo , Caracteres Sexuales , Espermatogénesis/genética , Espermatogénesis/fisiología , Espermatogonias/citología , Espermatogonias/metabolismo , Testículo/metabolismo , Testículo/patologíaRESUMEN
As women get older their oocytes become susceptible to chromosome mis-segregation. This generates aneuploid embryos, leading to increased infertility and birth defects. Here we examined the provenance of aneuploidy by tracking chromosomes and their kinetochores in oocytes from young and aged mice. Changes consistent with chromosome cohesion deterioration were found with age, including increased interkinetochore distance and loss of the centromeric protector of cohesion SGO2 in metaphase II arrested (metII) eggs, as well as a rise in the number of weakly attached bivalents in meiosis I (MI) and lagging chromosomes at anaphase I. However, there were no MI errors in congression or biorientation. Instead, premature separation of dyads in meiosis II was the major segregation defect in aged eggs and these were associated with very low levels of SGO2. These data show that although considerable cohesion loss occurs during MI, its consequences are observed during meiosis II, when centromeric cohesion is needed to maintain dyad integrity.
Asunto(s)
Aneuploidia , Segregación Cromosómica/fisiología , Edad Materna , Meiosis/genética , Oocitos/citología , Anafase/fisiología , Animales , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromátides/fisiología , Femenino , Cinetocoros/fisiología , Metafase/fisiología , Ratones , Microscopía ConfocalRESUMEN
Mammalian oocytes are particularly error prone in segregating their chromosomes during their two meiotic divisions. This results in the creation of an embryo that has inherited the wrong number of chromosomes: it is aneuploid. The incidence of aneuploidy rises significantly with maternal age and so there is much interest in understanding this association and the underlying causes of aneuploidy. The spindle assembly checkpoint, a surveillance mechanism that operates in all cells to prevent chromosome mis-segregation, and the cohesive ties that hold those chromosomes together, have thus both been the subject of intensive investigation in oocytes. It is possible that a lowered sensitivity of the spindle assembly checkpoint to certain types of chromosome attachment error may endow oocytes with an innate susceptibility to aneuploidy, which is made worse by an age-related loss in the factors that hold the chromosomes together.
Asunto(s)
Aneuploidia , Mamíferos/genética , Óvulo/metabolismo , Animales , Segregación Cromosómica , Humanos , Meiosis , Óvulo/citología , Factores de TiempoRESUMEN
The integrity of male germ cell genome is critical for the correct progression of spermatogenesis, successful fertilization, and proper development of the offspring. Several DNA repair pathways exist in male germ cells. However, unlike somatic cells, key components of such pathways remain largely unidentified. Gametogenetin (GGN) is a testis-enriched protein that has been shown to bind to the DNA repair protein FANCL via yeast-two-hybrid assays. This finding and its testis-enriched expression pattern raise the possibility that GGN plays a role in DNA repair during spermatogenesis. Herein we demonstrated that the largest isoform GGN1 interacted with components of DNA repair machinery in the mouse testis. In addition to FANCL, GGN1 interacted with the critical component of the Fanconi Anemia (FA) pathway FANCD2 and a downstream component of the BRCA pathway, BRCC36. To define the physiological function of GGN, we generated a Ggn null mouse line. A complete loss of GGN resulted in embryonic lethality at the very earliest period of pre-implantation development, with no viable blastocysts observed. This finding was consistent with the observation that Ggn mRNA was also expressed in lower levels in the oocyte and pre-implantation embryos. Moreover, pachytene spermatocytes of the Ggn heterozygous knockout mice showed an increased incidence of unrepaired DNA double strand breaks (DSBs). Together, our results suggest that GGN plays a role in male meiotic DSB repair and is absolutely required for the survival of pre-implantation embryos.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Hormonas Testiculares/metabolismo , Animales , Células Cultivadas , Reparación del ADN/genética , Desarrollo Embrionario/genética , Femenino , Inmunoprecipitación , Masculino , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Hormonas Testiculares/genéticaRESUMEN
The quality of metaphase II oocytes deteriorates rapidly following ovulation as the result of an aging process associated with impaired fertilizing potential, disrupted developmental competence, and increased likelihood of embryonic resorption. Because oxidative stress accelerates the onset of apoptosis in oocytes and influences their capacity for fertilization, this study aimed to characterize the significance of such stress in the postovulatory aging of mouse oocytes in vitro. We investigated the ability of the potent antioxidant melatonin to arrest the aging process when used to supplement oocyte culture medium. This study demonstrated that oxidative stress may occur in oocytes after as little as 8 h in culture and coincides with the appearance of early apoptotic markers such as phosphatidylserine externalization, followed 16 h later by caspase activation (P < 0.05) and morphological evidence of oocyte senescence. Importantly, supplementation of oocyte culture medium with 1 mM melatonin was able to significantly relieve the time-dependent appearance of oxidative stress in oocytes (P < 0.05) and, as a result, significantly delay the onset of apoptosis (P < 0.05). Furthermore, melatonin supplementation extended the optimal window for fertilization of oocytes aged for 8 and 16 h in vitro (P < 0.05) and significantly improved the quality of the resulting embryos (P < 0.01). We conclude that melatonin may be a useful tool in a clinical setting to prevent the time-dependent deterioration of oocyte quality following prolonged culture in vitro.
Asunto(s)
Antioxidantes/farmacología , Senescencia Celular/efectos de los fármacos , Melatonina/farmacología , Oocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Cafeína/farmacología , Evaluación Preclínica de Medicamentos , Femenino , Fertilización In Vitro , Ratones , Ratones Endogámicos C57BL , Inhibidores de FosfodiesterasaRESUMEN
Mammalian oocytes spend the majority of their lives in a dormant state, residing in primordial follicles. This arrest, most analogous to the G2 stage of the mitotic cell cycle division, is only broken in the hours preceding ovulation, when a hormonal rise induces meiotic resumption and entry into the first meiotic division. At a molecular level, this event is triggered by CDK1 activity, and here, we examine how CDK1 is suppressed during meiotic arrest and raised for oocyte maturation. We focus on signaling: intercellular signaling between the oocyte and the somatic cells of the follicle, and spatial signaling involving the anaphase-promoting complex (APC) within the oocyte. Meiotic arrest is achieved through APC(FZR1)-mediated cyclin B1 degradation. Once meiotic resumption resumes, CDK1 levels rise, but its activity eventually needs to be suppressed for completion of the first meiotic division. This is achieved by APC(CDC20), whose activity is critically regulated by the spindle assembly checkpoint, and which induces both a loss in CDK1 activity as well as the cohesive ties holding chromosomes together.