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SummaryThe decision by germ cells to differentiate and undergo either oogenesis or spermatogenesis takes place during embryonic development and Nanos plays an important role in this process. The present study was designed to investigate the expression patterns in rat of Nanos2-homologue protein in primordial germ cells (PGCs) over different embryonic developmental days as well as in spermatogonial stem cells (SSCs). Embryos from three different embryonic days (E8.5, E10.5, E11.5) and SSCs were isolated and used to detect Nanos2-homologue protein using immunocytochemistry, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Interestingly, Nanos2 expression was detected in PGCs at day E11.5 onwards and up to colonization of PGCs in the genital ridge of fetal gonads. No Nanos2 expression was found in PGCs during early embryonic days (E8.5 and 10.5). Furthermore, immunohistochemical and immunofluorescence data revealed that Nanos2 expression was restricted within a subpopulation of undifferentiated spermatogonia (As, single type A SSCs and Apr, paired type A SSCs). The same results were confirmed by our western blot and RT-PCR data, as Nanos2 protein and transcripts were detected only in PGCs from day E11.5 and in undifferentiated spermatogonia (As and Apr). Furthermore, Nanos2-positive cells were also immunodetected and sorted using flow cytometry from the THY1-positive SSCs population, and this strengthened the idea that these cells are stem cells. Our findings suggested that stage-specific expression of Nanos2 occurred on different embryonic developmental days, while during the postnatal period Nanos2 expression is restricted to As and Apr SSCs.
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Proteínas de Unión al ARN/genética , Espermatogonias/fisiología , Células Madre/fisiología , Animales , Embrión de Mamíferos/citología , Embrión de Mamíferos/fisiología , Femenino , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/fisiología , Masculino , Embarazo , Proteínas de Unión al ARN/metabolismo , Ratas Endogámicas , Espermatogonias/citología , Células Madre/citologíaRESUMEN
The present study investigated if the presence of encircling granulosa cells protected against di(2-ethylhexyl)phthalate (DEHP)-induced oxidative stress in rat oocytes cultured in vitro. Denuded oocytes and cumulus-oocyte complexes (COCs) were treated with or without various doses of DEHP (0.0, 25.0, 50.0, 100, 200, 400 and 800 µM) in vitro. Morphological apoptotic changes, levels of oxidative stress and reactive oxygen species (ROS), mitochondrial membrane potential, and expression levels of apoptotic markers (Bcl2, Bax, cytochrome c) were analyzed. Our results showed that DEHP induced morphological apoptotic changes in a dose-dependent manner in denuded oocytes cultured in vitro. The effective dose of DEHP (400 µg) significantly (P>0.05) increased oxidative stress by elevating ROS levels and the mitochondrial membrane potential with higher mRNA expression and protein levels of apoptotic markers (Bax, cytochrome c). Encircling granulosa cells protected oocytes from DEHP-induced morphological changes, increased oxidative stress and ROS levels, as well as increased expression of apoptotic markers. Taken together our data suggested that encircling granulosa cells protected oocytes against DEHP-induced apoptosis and that the presence of granulosa cells could act positively towards the survival of oocytes under in vitro culture conditions and may be helpful during assisted reproductive technique programmes.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Células de la Granulosa/fisiología , Oocitos/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Células Cultivadas , Dietilhexil Ftalato/toxicidad , Femenino , Expresión Génica/efectos de los fármacos , Células de la Granulosa/citología , Potencial de la Membrana Mitocondrial/fisiología , Oocitos/citología , Oocitos/metabolismo , Estrés Oxidativo/fisiología , Plastificantes/toxicidad , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Increased oxidative stress (OS) due to in vitro culture conditions can affect the quality of denuded eggs during various assisted reproductive technologies (ARTs). Presence of intact granulosa cells may protect eggs from OS damage under in vitro culture conditions. The present study was aimed to investigate whether encircling granulosa cells could protect against hydrogen peroxide (H2O2)-induced egg apoptosis in ovulated cumulus oocyte complexes (COCs) cultured in vitro. The OS was induced by exposing COCs as well as denuded eggs with various concentrations of H2O2 for 3 h in vitro. The morphological changes, total reactive oxygen species (ROS) as well as catalase expression, Bax/Bcl-2, cytochrome c levels and DNA fragmentation were analysed in COCs as well as denuded eggs. Our results suggest that H2O2 treatment induced morphological apoptotic features in a concentration-dependent manner in denuded eggs cultured in vitro. The 20 µM of H2O2 treatment induced OS by elevating total ROS level, reduced catalase and Bcl-2 expression levels with overexpression of Bax and cytochrome c and induced DNA fragmentation in denuded eggs cultured in vitro. The presence of encircling granulosa cells protected H2O2-induced morphological apoptotic features by preventing the increase of Bax, cytochrome c expression levels and DNA fragmentation in associated egg. However, 20 µM of H2O2 was sufficient to induce peripheral granulosa cell apoptosis in COCs and degeneration in few denuded eggs cultured in vitro. Taken together our data suggest that the presence of encircling granulosa cells could be beneficial to protect ovulated eggs from OS damage under in vitro culture conditions during various ART programs.
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Apoptosis/genética , Células de la Granulosa/metabolismo , Oocitos/metabolismo , Estrés Oxidativo/genética , Animales , Apoptosis/efectos de los fármacos , Catalasa/genética , Células Cultivadas , Fragmentación del ADN/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/citología , Células de la Granulosa/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Oocitos/citología , Oocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Ratas , Especies Reactivas de Oxígeno/metabolismo , Técnicas Reproductivas AsistidasRESUMEN
Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals.
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Apoptosis , Mamíferos/metabolismo , Oocitos/metabolismo , Animales , Humanos , Oocitos/citología , OogénesisRESUMEN
The present study was aimed to find out changes in signal molecules and maturation promoting factor (MPF) levels during meiotic cell cycle progression from diplotene and metaphase-II (M-II) arrest, a period during which oocyte achieves meiotic competency. Data suggest that high levels of adenosine 3'-5'-cyclic monophosphate (cAMP), guanosine 3'-5'-cyclic monophosphate (cGMP), and nitric oxide (NO) are associated with diplotene arrest, while reduction in their levels correlates with reduced MPF level and meiotic resumption from diplotene arrest. On the other hand, increased intracellular NO, calcium (Ca(2+) ) as well as hydrogen peroxide (H2 O2 ) levels correlate with decreased cAMP, Thr-161 phosphorylated cyclin-dependent kinase-1 (Cdk1) as well as cyclin B1 levels. The decreased Thr-161 phosphorylated Cdk1 and cyclin B1 level reduce MPF level leading to exit from M-II arrest in oocytes cultured in vitro. These data suggest that the decrease of cAMP level and increase of cytosolic free Ca(2+) as well as H2 O2 levels associate with the reduced MPF level and meiotic resumption from diplotene arrest. On the other hand, increase of NO, cGMP, Ca(2+) as well as H2 O2 levels are associated with reduced MPF and spontaneous exit from M-II arrest in rat oocytes cultured in vitro.
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Factor Promotor de Maduración/metabolismo , Meiosis , Oocitos/citología , Oocitos/metabolismo , Transducción de Señal , Animales , Proteína Quinasa CDC2/metabolismo , Calcio/metabolismo , Forma de la Célula , Cromosomas de los Mamíferos/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Ciclina B1/metabolismo , Citosol/metabolismo , Femenino , Fluorescencia , Peróxido de Hidrógeno/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Fosforilación , Fosfotreonina/metabolismo , RatasRESUMEN
Endometriosis is a common condition that affects 5% to 10% of women during their reproductive years, although the aetiology and pathophysiology are still unknown. This study aimed to create an endometriosis model in rats to investigate the efficacy of natural and synthetic medications in treating endometriosis. An in vivo endometriotic model was established using a surgical induction method and the endocrine-disrupting drug diethylstilbestrol (DES). In brief, the experiment is categorised into three different groups. Each group contains five rats. The first group had no surgery, while in the in the second group of rats (n = 5), two small tissue grafts were fixed at the right and left walls of the abdomen. But in the in the third group of rats (n = 5), two small pieces of tissue have been grafted on the right and left abdomen walls by surgically along with DES treatments. Noninvasive photoacoustic imaging (PAI) was employed in the study to measure factors such as haemoglobin levels, oxygen saturation, and the size of endometriotic lesions. Histopathological analysis was carried out utilising staining techniques such as Hematoxylin and Eosin, Masson's Trichrome, and Periodic Acid Schiff, as well as immunohistochemistry with marker antibodies. Molecular markers in uterine tissue were examined using Western blots and real-time PCR. The developed endometriosis rat model showed a significant increase in the expression of anti-apoptotic Bcl-2, angiogenic marker VEGF and pro-inflammatory (COX-2 and IL-6) protein markers. In contrast to the control group, the treatment group had considerably lower Caspase-3 expression levels. Photoacoustic imaging (PAI) data demonstrated a constant increase in lesion size, as well as a decrease in oxygen saturation levels. The findings suggest that the in vivo endometriosis rat model may accurately assess the efficacy of natural or synthetic endometriosis treatments. This model may help in the improvement of disease understanding and the development of targeted therapeutic drugs.
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Modelos Animales de Enfermedad , Endometriosis , Animales , Endometriosis/patología , Endometriosis/metabolismo , Femenino , Ratas , Factor A de Crecimiento Endotelial Vascular/metabolismo , Interleucina-6/metabolismo , Dietilestilbestrol/farmacología , Ratas Sprague-Dawley , Endometrio/patología , Endometrio/metabolismo , Endometrio/efectos de los fármacos , Caspasa 3/metabolismoRESUMEN
BACKGROUND: To achieve oocyte competence for successful fertilization, bidirectional communication between oocyte and granulosa cells is crucial. The acquisition of meiotic competency in oocyte is facilitated by various regulatory genes however, expression pattern of these genes is not well documented during meiotic transition from Metaphase-I to Metaphase-II stage. Therefore, the present research analyzed the expression pattern of regulatory genes that are involved in the transition from M-I to M-II stages in rat oocyte. METHODS: The analysis of the data was conducted by applying an array of bioinformatic tools. The investigation of gene group interactions was carried out by employing the STRING database, which relies on co-expression information. The gene ontology (GO) analysis was performed utilizing the comparative GO database. Functional annotation for GO and pathway enrichment analysis were performed for genes involved in networking. The GO obtained through computational simulations was subsequently validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. RESULTS: The findings of our study suggest that there is a distinct gene expression pattern in both the oocyte and granulosa cells. This pattern indicates that oocyte-secreted factors, such as BMP15 and GDF9, play a crucial role in regulating the progression of the meiotic cell cycle from the M-I to M-II stages. We have also examined the level of mRNA expression of genes including CYP11A1, CYP19A1, and STAR, which are crucial for the steroidogenesis. CONCLUSIONS: It is fascinating to observe that the oscillatory pattern of specific key genes may hold significance in the process of in vitro oocyte maturation, specifically during the transition from the M-I to M-II stage. It might be useful for determining biomarker genes and potential pathways that play a role in attaining oocyte competency, thereby aiding in the assessment of oocyte quality for the purpose of achieving successful fertilization.
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Oocitos , Ovario , Femenino , Animales , Ratas , Oocitos/fisiología , Células de la Granulosa/metabolismoRESUMEN
The current study sought to investigate the associations of common genetic risk variants with gestational diabetes mellitus (GDM) risk in the north Indian population and to evaluate their utility in identifying GDM cases. A case-control study, including 300 pregnant women, was included, and clinical and pathological information was collected. The amplification-refractory mutation system (ARMS) was used for genotyping four single nucleotide polymorphisms (SNPs), namely FTO (rs9939609), PPARG2 (rs1801282), SLC30A8 (rs13266634), and TCF7L2 (rs12255372). The odds ratio and confidence interval were determined for each SNP in different genetic models. Further, attributable risk, population penetrance, and relative risk were also calculated. The risk allele A of FTO (rs9939609) poses a two times higher risk of GDM (p = 0.02, OR = 2.5). The CG and GG genotypes of PPARG2 (rs1801282) have half a lower risk of GDM. In SLC30A8 (rs13266634), the recessive model analysis showed a two times higher risk of having GDM, while the recessive model (TT vs. GG + GT) analysis in TCF7L2 (rs12255372) indicates a lower risk of GDM. Finally, the relative risk, population penetrance, and attributable risk for risk allele in all four variants was higher in GDM mothers. All four polymorphisms were found to be significantly associated with BMI, HbA1c, and insulin. Our study first time confirmed a significant association with GDM for four variants, FTO, PPARG2, SLC30A8, and TCF7L2, in the North Indian population.
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Diabetes Gestacional , Predisposición Genética a la Enfermedad , Insulina , Polimorfismo de Nucleótido Simple , Proteína 2 Similar al Factor de Transcripción 7 , Transportador 8 de Zinc , Humanos , Femenino , Diabetes Gestacional/genética , Embarazo , Adulto , Estudios de Casos y Controles , Proteína 2 Similar al Factor de Transcripción 7/genética , Insulina/metabolismo , Transportador 8 de Zinc/genética , Transportador 8 de Zinc/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , PPAR gamma/genética , IndiaRESUMEN
AIMS: To study the impact of Mediator complex subunit 12 (MED12) gene variants on the encoded protein's function and pathogenic relevance for genesis of uterine leiomyoma's (ULs). METHODS: Mutational analysis in exon-2 of MED12 gene was performed by PCR amplification and DNA sequencing in 89 clinically diagnosed ULs tissues. Pathogenicity prediction of variation was performed by computational analysis. The functional effects of missense variation were done by quantity RT-PCR and western blot analysis. RESULT(S): Out of 89 samples, 40 (44.94%) had missense variation in 14 different CDS position of exon-2 of MED12 gene. Out of 40 missense variation, codon 44 had 25 (62.5%) looking as a hotspot region for mutation for ULs, because CDS position c130 and c131present at codon 44 that have necleotide change G>A, T, C at c130 and c131 have necleotide change G>A and C. We also find somenovel somatic mutations oncodon 36 (T > C), 38 (G>T) of exon-2 and 88 (G>C) of intron-2. No mutations were detected in uterine myometrium samples. Our computational analysis suggests that change in Med12c .131 G>A leads to single substitution of amino acid [Glycine (G) to Aspartate (D)] which has a pathogenic and lethal impact and may cause instability of MED12 protein. Further, analysis of extracellular matrix (ECM) component (MMP-2 & 9, COL4A2 and α-SMA) mRNA and protein expression levels in the set of ULs having MED12 mutation showed significantly higher expression of MMP-9 and α-SMA. CONCLUSION(S): The findings of present study suggest that missense variation in codon 44 of MED12 gene lead to the genesis of leiomyoma's through over-expression of MMP-9 of ECM pathway which could be therapeutically targeted for non-surgical management of ULs.
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The present study was aimed to find out whether an increase of cytosolic free calcium level induces egg apoptosis through mitochondria-caspase mediated pathway. To increase cytosolic free calcium level and morphological apoptotic changes, ovulated eggs were cultured in Ca(2+)/Mg(2+) free media-199 with or without various concentrations of calcium ionophore (0.5, 1, 2, 3, 4 µM) for 3 h in vitro. The morphological apoptotic changes, cytosolic free calcium level, hydrogen peroxide (H(2)O(2)) concentration, catalase activity, cytochrome c concentration, caspase-9 and caspase-3 activities and DNA fragmentation were analyzed. Calcium ionophore induced morphological apoptotic features in a concentration-dependent manner followed by degeneration at higher concentrations (3 and 4 µM). Calcium ionophore increased cytosolic free calcium level, induced generation of hydrogen peroxide (H(2)O(2)) and inhibited catalase activity in treated eggs. The increased H(2)O(2) concentration was associated with increased cytochrome c concentration, caspase-9 and caspase-3 activities that resulted in the induction of morphological features characteristic of egg apoptosis. The increased caspase-3 activity finally induced DNA fragmentation as evidenced by TUNEL positive staining in calcium ionophore-treated eggs. These findings suggest that high cytosolic free calcium level induces generation of H(2)O(2) that leads to egg apoptosis through mitochondria-caspase mediated pathway.
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Apoptosis , Calcio/metabolismo , Citosol/metabolismo , Mitocondrias/metabolismo , Oocitos/enzimología , Animales , Ionóforos de Calcio/farmacología , Señalización del Calcio , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Catalasa/metabolismo , Forma de la Célula/efectos de los fármacos , Tamaño de la Célula , Células Cultivadas , Citocromos c/metabolismo , Citosol/efectos de los fármacos , Fragmentación del ADN , Femenino , Peróxido de Hidrógeno/metabolismo , Oocitos/efectos de los fármacos , Oocitos/fisiología , RatasRESUMEN
OBJECTIVE: Present study was aimed to determine whether aqueous neem leaf extract (NLE) induces generation of reactive oxygen species (ROS) and apoptosis through mitochondria-mediated pathway in rat oocytes. DESIGN: A controlled prospective study. SETTING: Laboratory research setting at Department of Zoology of Banaras Hindu University. ANIMAL(S): Forty eight sexually immature female rats that were 20-30 days of age. INTERVENTION(S): Sexually immature female rats were fed palatable dose of NLE (10 mg/g dry feed palate) for 10 days and then subjected to superovulation induction protocol. Thereafter, rats were euthanized, ovulated cumulus oocyte complexes were collected from oviduct and oocytes were denuded. MAIN OUTCOME MEASURE(S): Rate of morphological apoptotic changes, measurement of hydrogen peroxide, nitric oxide and cytochrome c concentrations, caspase-9, caspases-3 activities and DNA fragmentation in oocytes. RESULTS: In vivo NLE treatment induced morphological apoptotic changes were associated with increased hydrogen peroxide, nitric oxide and cytochrome c concentrations, caspase-9, caspase-3 activities and DNA fragmentation in oocyte. CONCLUSION: NLE induces generation of ROS that leads to oocytes apoptosis through mitochondria-mediated pathway.
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Oocitos/citología , Oocitos/metabolismo , Extractos Vegetales/farmacología , Hojas de la Planta/química , Especies Reactivas de Oxígeno/metabolismo , Animales , Apoptosis/efectos de los fármacos , Azadirachta/química , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Citocromos c/metabolismo , Femenino , Peróxido de Hidrógeno/metabolismo , Óxido Nítrico/metabolismo , Oocitos/efectos de los fármacos , Extractos Vegetales/química , Ratas , SuperovulaciónRESUMEN
Meiotic cell cycle in mammalian oocytes is a dynamic process that involves several stop/go channels. The cell cycle arrest in oocyte occurs at various stages such as diplotene, metaphase-I (M-I), metaphase-II (M-II), and so called metaphase-like arrest (M-III). Leutinizing hormone surge induces meiotic resumption from diplotene arrest in follicular microenvironment by overriding several factors responsible for the maintenance of meiotic arrest. The inhibitory factors are synthesized in oocyte or in the associated follicular somatic cells and transferred to the oocyte. The major factors include hypoxanthine, cyclic adenosine 3', 5'-monophosphate, cyclic guanosine 3', 5'-monophosphate, reactive oxygen species, protein kinase A, and protein kinase C. In the presence of active protein kinases, epidermal-like growth factors are produced that activate mitogen-activated protein kinase in cumulus granulosa cells. The maturation promoting factor, cytostatic factors, and spindle assembly checkpoint proteins are also involved in that maintenance of arrest at various stages of meiotic cell cycle in mammalian oocytes. In this review, we briefly summarize the role of these factors in the maintenance of meiotic cell cycle arrest in mammalian oocytes.
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Mamíferos/metabolismo , Meiosis , Oocitos/citología , Animales , Humanos , Profase Meiótica I , MetafaseRESUMEN
Mammalian ovary is metabolically active organ and generates by-products such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) on an extraordinary scale. Both follicular somatic cells as well as oocyte generate ROS and RNS synchronously and their effects are neutralized by intricate array of antioxidants. ROS such as hydrogen peroxide (H(2)O(2)) and RNS such as nitric oxide (NO) act as signaling molecules and modulate various aspects of oocyte physiology including meiotic cell cycle arrest and resumption. Generation of intraoocyte H(2)O(2) can induce meiotic resumption from diplotene arrest probably by the activation of adenosine monophosphate (AMP)-activated protein kinase A (PRKA)-or Ca(2+)-mediated pathway. However, reduced intraoocyte NO level may inactivate guanylyl cyclase-mediated pathway that results in the reduced production of cyclic 3',5'-guanosine monophosphate (cGMP). The reduced level of cGMP results in the activation of cyclic 3',5'-adenosine monophosphate (cAMP)-phosphodiesterase 3A (PDE3A), which hydrolyses cAMP. The reduced intraoocyte cAMP results in the activation of maturation promoting factor (MPF) that finally induces meiotic resumption. Thus, a transient increase of intraoocyte H(2)O(2) level and decrease of NO level may signal meiotic resumption from diplotene arrest in mammalian oocytes.
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Meiosis , Profase Meiótica I , Oocitos/metabolismo , Animales , Humanos , Óxido Nítrico , Nucleótidos Cíclicos/metabolismo , Oocitos/citología , Especies Reactivas de OxígenoRESUMEN
Deoxyelephantopin (DOE), a phytochemical, extracted and purified from Elephantopus scaber, has been shown to exhibit antitumor activities. Objective of the present study was to investigate anti-tumor and apoptosis-inducing properties of DOE against uterine leiomyoma (UL) and to explore their molecular mechanisms. Primary cell cultures from fresh UL tissue were established and maintained up to 12 passages. The cells exhibited continuous proliferation with 24 -h doubling time until 12 passages and was then subjected to molecular characterization. The growth inhibitory effect of DOE on UL cells was confirmed by colony formation, cellular senescence, AO/PI and DAPI staining. Fluorescent-activated cell sorting (FACS) assay for apoptosis and cell cycle arrest analysis revealed that DOE significantly (p < 0.05) inhibited the UL cell proliferation via cell cycle arrest at G2/M phase and induced apoptosis via ROS production by lowering mitochondrial membrane potential. Subsequently, the DOE induced ROS was markedly attenuated by co-treatment of N-Acetyl-Cysteine (NAC). Our quantitative RT-PCR and western blot results showed up-regulation of Bax, Caspase-3 and down-regulation of Bcl2, P53, αSMA, COL4A2, VEGF, PCNA, Cyclin B1 and oncogenic lncRNAs (H19, HOTAIR, BANCR and ROR) in DOE treated UL cells which further strengthen our findings. In conclusion, DOE inhibits growth of UL cells via cell cycle arrest at G2/M phase, induces ROS-dependent caspase-3-mediated mitochondrial intrinsic apoptotic pathway and down-regulation of oncogenic lncRNA in UL cells. Our findings suggest that DOE deserves for further systematic investigation in the uterine leiomyoma animal model as a novel apoptosis inducer for potential applications in the prevention or treatment of uterine leiomyoma.
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Apoptosis/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Lactonas/farmacología , Leiomioma/tratamiento farmacológico , ARN Largo no Codificante/análisis , Especies Reactivas de Oxígeno/metabolismo , Sesquiterpenos/farmacología , Neoplasias Uterinas/tratamiento farmacológico , Adulto , Caspasa 3/fisiología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Matriz Extracelular/metabolismo , Femenino , Humanos , Lactonas/uso terapéutico , Leiomioma/metabolismo , Leiomioma/patología , ARN Largo no Codificante/fisiología , Sesquiterpenos/uso terapéutico , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologíaRESUMEN
Non-specific L-type calcium channel blockers, such as verapamil (> or =50 microM), induce metaphase-II (M-II) arrest and apoptosis in aged rat eggs cultured in Ca(2+)-deficient medium. However, the effects of extracellular Ca(2+) on verapamil-induced M-II arrest and apoptosis have not yet been reported. We have demonstrated that postovulatory aging induced exit from M-II arrest by extruding a second polar body, a morphological sign of spontaneous egg activation (SEA). Verapamil inhibited SEA and induced egg apoptosis in a dose-dependent manner in Ca(2+)-deficient medium. The initiation of apoptotic features was observed at 50 microM of verapamil. Extracellular Ca(2+) (1.80 mM) reduced intracellular H2O2 level, bax protein expression, caspase-3 activity, DNA fragmentation and protected against 50 microM, but not higher concentrations of > or =100 microM in verapamil-induced egg apoptosis. These results suggest that extracellular Ca(2+) ions have a role during SEA and protect against verapamil induced apoptosis in aged rat eggs.
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Apoptosis , Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Óvulo/efectos de los fármacos , Verapamilo/farmacología , Animales , Canales de Calcio Tipo L/metabolismo , Caspasa 3/metabolismo , Senescencia Celular , Peróxido de Hidrógeno/metabolismo , Metafase/efectos de los fármacos , Óvulo/fisiología , Ratas , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Increased oxidative stress (OS) due to ubiquitous exposure to di-(2-ethylhexyl) phthalate (DEHP) can affect the quality of oocytes by inducing apoptosis and hampering granulosa cell mediated steroidogenesis. This study was carried out to investigate whether DEHP induced OS affects steroidogenesis and induces apoptosis in rat ovarian granulosa cells. OS was induced by exposing granulosa cells to various concentrations of DEHP (0.0, 100, 200, 400 and 800 µM) for 72 h in vitro. Intracellular reactive oxygen species (ROS), oxidative stress (OS), mitochondrial membrane potential, cellular senescence, apoptosis, steroid hormones (estradiol & progesterone) and gene expression were analyzed. The results showed that an effective dose of DEHP (400 µg) significantly increased OS by elevating the ROS level, mitochondrial membrane potential, and ß-galactosidase activity with higher mRNA expression levels of apoptotic genes (Bax, cytochrome-c and caspase3) and a lower level of an anti-apoptotic gene (Bcl2) as compared to the control. Further, DEHP significantly (P > 0.05) decreased the level of steroid hormones (estradiol and progesterone) in a conditioned medium and this effect was reciprocated with a lower expression (P > 0.05) of steroidogenic responsive genes (Cyp11a1, Cyp19A1, Star, ERß1) in treated granulosa cells. Furthermore, co-treatment with N-Acetyl-Cysteine (NAC) rescues the effects of DEHP on OS, ROS, ß-galactosidase levels and gene expression activities. Altogether, these results suggest that DEHP induces oxidative stress via ROS generation and inhibits steroid synthesis via modulating steroidogenic responsive genes, which leads to the induction of apoptosis through the activation of Bax/Bcl-2-cytochrome-c and the caspase-3-mediated mitochondrial apoptotic pathway in rat granulosa cells.
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Nitric oxide (NO) acts as a major signal molecules and modulate physiology of mammalian oocytes. Ovarian follicles generate large amount of NO through nitric oxide synthase (NOS) pathway to maintain diplotene arrest in preovulatory oocytes. Removal of oocytes from follicular microenvironment or follicular rupture during ovulation disrupt the flow of NO from granulosa cells to the oocyte that results a transient decrease of oocyte cytoplasmic NO level. Decreased NO level reduces cyclic nucleotides level by inactivating guanylyl cyclases directly or indirectly. The reduced cyclic nucleotides level modulate specific phosphorylation status of cyclin-dependent kinase 1 (Cdk1) and triggers cyclin B1 degradation. These changes result in maturation promoting factor (MPF) destabilization that finally triggers meiotic resumption from diplotene as well as metaphase-II (M-II) arrest in most of the mammalian species.
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Meiosis/fisiología , Óxido Nítrico/metabolismo , Oocitos/citología , Oocitos/metabolismo , Animales , Femenino , Humanos , Profase Meiótica I/fisiología , Transducción de SeñalRESUMEN
The present study was aimed to find out whether roscovitine reduces phosphorylated Thr-161 of cyclin-dependent kinase 1 (Cdk1) level and induces egg apoptosis through Fas ligand (FasL)-mediated pathway. For this purpose, ovulated eggs were cultured in media 199 with or without various concentrations of roscovitine (0, 25, 50, 100, 200 µM) for 3 h in vitro. The morphological apoptotic changes, phosphorylation status of Cdk1, FasL concentration, caspase-8 and caspase-3 activities, and DNA fragmentation were analyzed. Data of the present study suggest that roscovitine significantly reduced Thr-161 phosphorylated Cdk1 level without altering the total level of Cdk1 and induced cytoplasmic fragmentation, a morphological apoptotic feature in a concentration-dependent manner. The roscovitine-induced cytoplasmic fragmentation was associated with increased FasL concentration. The increased FasL concentration induced caspase-8 followed by caspase-3 activities. The increased caspases activity finally induced DNA fragmentation in eggs that showed cytoplasmic fragmentation. Taken together, these results suggest that roscovitine reduced phosphorylated Thr-161 of Cdk1 level and induces apoptosis through FasL-mediated pathway in rat eggs cultured in vitro.
Asunto(s)
Apoptosis/efectos de los fármacos , Quinasas Ciclina-Dependientes/metabolismo , Proteína Ligando Fas/metabolismo , Óvulo/efectos de los fármacos , Purinas/farmacología , Animales , Proteína Quinasa CDC2 , Caspasa 3/metabolismo , Células Cultivadas , Citoplasma/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Óvulo/citología , Óvulo/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Ratas Endogámicas , Roscovitina , Treonina/metabolismoRESUMEN
BACKGROUND: Inhibition of cyclin-dependent kinases (Cdks) may result in meiotic cell cycle arrest and apoptosis in rat eggs in vitro. We aimed to find out whether roscovitine, a Cdk inhibitor, inhibits extrusion of second polar body (II PB) and induced egg apoptosis in vitro. METHODS: The metaphase-II (M-II) arrested eggs were collected from oviduct and exposed to various concentrations of roscovitine for 3h in vitro. The morphological changes, phosphorylation status of Cdk1, cyclin B1 level, hydrogen peroxide (H2O2), p53, Bax, Bcl2 and cytochrome c expressions, caspase-3 activity and DNA fragmentation were analyzed. RESULTS: We showed that the lower concentrations of roscovitine significantly reduced Thr-161 phosphorylated Cdk1 level and inhibited extrusion of II PB. The higher concentrations of roscovitine significantly reduced Thr-161 phosphorylated Cdk1 level but total Cdk as well as cyclin B1 levels remained high. Higher concentrations of roscovitine increased H2O2 level and expressions of p53, Bax and cytochrome c in treated eggs. The increased proapoptotic factors induced capsase-3 activity and thereby DNA fragmentation that finally resulted in cytoplasmic fragmentation, a morphological apoptotic feature. CONCLUSION: Our data suggest that roscovitine inhibited II PB extrusion possibly by reducing Thr-161 phosphorylated Cdk1 level and induced apoptosis through mitochondria-caspase-mediated apoptotic pathway in rat eggs cultured in vitro.
Asunto(s)
Apoptosis/efectos de los fármacos , Óvulo/efectos de los fármacos , Cuerpos Polares/efectos de los fármacos , Purinas/farmacología , Animales , Caspasa 3/metabolismo , Ciclina B1/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Citocromos c/metabolismo , Femenino , Metafase/efectos de los fármacos , Fosforilación , Ratas , Roscovitina , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Neem (Azadirachta indica L.) leaf has been widely used in ayurvedic system of medicine for fertility regulation for a long time. The molecular mechanism by which neem leaf regulates female fertility remains poorly understood. Animal studies suggest that aqueous neem leaf extract (NLE) induces reactive oxygen species (ROS) - mediated granulosa cell apoptosis. Granulosa cell apoptosis deprives oocytes from nutrients, survival factors and cell cycle proteins required for the achievement of meiotic competency of follicular oocytes prior to ovulation. Under this situation, follicular oocyte becomes more susceptible towards apoptosis after ovulation. The increased level of hydrogen peroxide (H2O2) inside the follicular fluid results in the transfer of H2O2 from follicular fluid to the oocyte. The increased level of H2O2 induces p53 activation and over expression of Bax protein that modulates mitochondrial membrane potential and trigger cytochrome c release. The increased cytosolic cytochrome c level induces caspase-9 and caspase-3 activities that trigger destruction of structural and specific proteins leading to DNA fragmentation and thereby oocyte apoptosis. Based on these animal studies, we propose that NLE induces generation of ROS and mitochondria-mediated apoptosis both in granulosa cells as well as in follicular oocyte. The induction of apoptosis deteriorates oocyte quality and thereby limits reproductive outcome in mammals.