RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Zuogui Pill (ZGP) is a traditional herbal formula of Chinese Medicine with a long history of use in alleviating ovarian aging. AIM OF THE STUDY: To examine the impact of ZGP on oxidative stress and the stemness of oogonial stem cells (OSCs) in cyclophosphamide (CTX)-induced ovarian aging, as well as its molecular mechanisms involving the nuclear factor erythroid 2-related factor 2 (Nrf2, NFE2L2)/heme oxygenase-1 (HO-1, Hmox1) pathway. MATERIALS AND METHODS: Female Sprague-Dawley (SD) rats were randomly divided into seven groups: control, model (CTX), estradiol valerate (EV, 0.103 mg/kg), ZGP-L (low dose Zuogui Pill, 1.851 g/kg), ZGP-H (high dose Zuogui Pill, 3.702 g/kg), ML385 (30 mg/kg), and ML385+ZGP-L. After CTX modeling, the EV, ZGP-L, ZGP-H, and ML385+ZGP-L groups were treated by gavage for 8 weeks, while the ML385 and ML385+ZGP-L groups were administered the Nrf2 antagonist ML385 twice a week. OSCs were isolated after modeling and then treated with drug serum containing 10% ZGP or 10 µM ML385. The general conditions of the rats, including body weight, ovarian weight/body weight ratio, and estrous cycle, were observed. Ovarian ultrastructure, follicle and corpus luteum counts were assessed via hematoxylin and eosin (H&E) staining. Serum hormone levels were measured using enzyme-linked immunosorbent assay (ELISA). Nrf2/HO-1 pathway, stem cell, germ cell, and cell cycle biomarkers were analyzed by qPCR and Western blot. Cell viability was assessed by cell counting kit-8 (CCK-8) assay. Oxidative stress biomarkers were evaluated using flow cytometry and assay kits. Immunofluorescence was employed to detect and locate OSCs in the ovary, quantify the average fluorescence intensity, and identify OSCs. RESULTS: After ZGP treatment, rats with CTX-induced ovarian aging exhibited improved general condition, increased body weight, higher total ovarian weight to body weight ratio, and a restoration of the estrous cycle similar to the control group. Serum levels of estradiol (E2) and follicle stimulating hormone (FSH), two sex hormones, were also improved. Ovarian ultrastructure and follicle count at all stages showed improvement. Moreover, the viability and proliferation capacity of OSCs were enhanced following ZGP intervention. The Nrf2/HO-1 pathway was found to be down-regulated in CTX-induced aging ovarian OSCs. However, ZGP reversed this effect by activating the expression of Nrf2, HO-1, and NAD(P)H oxidoreductase 1 (NQO1), increasing the activity of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), and reducing the accumulation of malonaldehyde (MDA) and reactive oxygen species (ROS), thus restoring resistance to oxidative stress. Additionally, ZGP improved the cell cycle of OSCs, up-regulated the expression of Cyclin D1 and Cyclin E1, restored cell stemness, promoted proliferation, enhanced the expression of cell stemness markers octamer-binding transcription factor 4 (Oct4) and mouse VASA homolog (MVH), and down-regulated the expression of P21, thereby inhibiting apoptosis. The therapeutic effects of ZGP against oxidative stress and restoration of cell stemness were attenuated following inhibition of the Nrf2 signaling pathway using ML385. CONCLUSIONS: ZGP protected against CTX-induced ovarian aging by restoring normal ovarian function, alleviating oxidative stress in aging OSCs, promoting OSCs proliferation, and restoring their stemness in rats, possibly through regulating the Nrf2/HO-1 pathway.
Assuntos
Medicamentos de Ervas Chinesas , Células-Tronco de Oogônios , Ovário , Estresse Oxidativo , Transdução de Sinais , Animais , Feminino , Ratos , Envelhecimento/efeitos dos fármacos , Ciclofosfamida , Medicamentos de Ervas Chinesas/farmacologia , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Células-Tronco de Oogônios/efeitos dos fármacos , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ovário/patologia , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
The ovarian germline stem cells(OGSCs) cultured in the optimized culture system were used as the research object to observe the effect of Tripterygium glycosides(TG) on OGSCs and explore the mechanism of reproductive toxicity by the Notch signaling pathway. Cell counting kit-8(CCK-8) was used to observe the viability level of OGSCs in mice cultured in vitro by TG of 3.75, 7.5, and 15 µg·mL~(-1). Immunofluorescence technology and reverse transcription-polymerase chain reaction(RT-PCR) were used to detect the protein and gene expression level of OGSCs marker mouse vasa homologue(MVH) and octamer-binding transcription factor 4(Oct4) by TG of 3.75 µg·mL~(-1). RT-PCR detected the gene expression of neurogenic locus Notch homolog protein 1(Notch1), Hes family BHLH transcription factor 1(Hes1), and jagged canonical Notch ligand 1(Jagged1). The RNA was extracted for transcriptome analysis to analyze the mechanism of action of TG intervention on OGSCs. 3.75 µg·mL~(-1) of TG was combined with 40 ng·mL~(-1) Notch signaling pathway γ-secretagocin agonist jagged canonical notch ligand(Jagged) for administration. CCK-8 was used to detect the viability level of OGSCs. Double immunofluorescence technology was used to detect the protein co-expression of MVH with Hes1, Notch1, and Jagged1. The results showed that compared with the blank group, the TG administration group significantly inhibited the activity of OGSCs(P<0.01 or P<0.001). It could reduce the protein and gene expression of OGSC markers, namely MVH and Oct4(P<0.05, P<0.01, or P<0.001). It could significantly inhibit the gene expression of Notch1, Hes1, and Jagged1(P<0.001). Transcriptomic analysis showed that TG affected the growth and proliferation of OGSCs by intervening Jagged1, a ligand associated with the Notch signaling pathway. The experimental results showed that the combination of Notch signaling pathway γ-secretagorein agonist Jagged could significantly alleviate the decrease in OGSC viability induced by TG(P<0.001) and significantly increased the OGSC viability compared with the TG group(P<0.001). It also could significantly increase the co-expression of MVH/Jagged1, MVH/Hes1, and MVH/Notch1 proteins(P<0.01 or P<0.001). It suggested that TG play the role of γ-secretagorease inhibitors by downregulating the OGSC markers including MVH and Oct4 and Notch signaling pathway molecules such as Notch1, Hes1, and Jagged1, participate in the OGSC pathway, and mediate reproductive toxicity caused by the Notch signaling pathway.
Assuntos
Células-Tronco de Oogônios , Camundongos , Animais , Células-Tronco de Oogônios/metabolismo , Tripterygium , Ligantes , Transdução de SinaisAssuntos
Flavonoides , Células-Tronco de Oogônios , Flavonoides/farmacologia , Sêmen , Transdução de Sinais , FetoRESUMO
Highly potent animal stem cells either self renew or launch complex differentiation programs, using mechanisms that are only partly understood. Drosophila female germline stem cells (GSCs) perpetuate without change over evolutionary time and generate cystoblast daughters that develop into nurse cells and oocytes. Cystoblasts initiate differentiation by generating a transient syncytial state, the germline cyst, and by increasing pericentromeric H3K9me3 modification, actions likely to suppress transposable element activity. Relatively open GSC chromatin is further restricted by Polycomb repression of testis or somatic cell-expressed genes briefly active in early female germ cells. Subsequently, Neijre/CBP and Myc help upregulate growth and reprogram GSC metabolism by altering mitochondrial transmembrane transport, gluconeogenesis, and other processes. In all these respects GSC differentiation resembles development of the totipotent zygote. We propose that the totipotent stem cell state was shaped by the need to resist transposon activity over evolutionary timescales.
Most animals are made up of two cell types: germline stem cells, which give rise to reproductive cells (egg and sperm) and pass their DNA to the next generation, and somatic cells, which make up the rest of the body. Transposable elements fragments of DNA that can copy themselves and integrate into different parts of the genome can greatly disrupt the integrity of the germ cell genome. Systems involving small RNAs and DNA methylation, which respectively modify the sequence and structure of the genome, can protect germ cells from the activity of transposable elements. While these systems have been studied extensively in late germ cells, less is known about how they work in germ cells generated early on in development. To investigate, Pang et al. studied the germline stem cells that give rise to eggs in female fruit flies. Techniques that measure DNA modifications showed that these germline stem cells and the cells they give rise to early on are better protected against transposable elements. This is likely due to the unusual cell cycle of early germ cells, which display a very short initial growth phase and special DNA replication timing during the synthesis phase. Until now, the purpose of these long-known cell cycle differences between early and late germ cells was not understood. Experiments also showed known transposable element defences are upregulated before the cell division that produces reproductive cells. DNA becomes more densely packed and germ cells connect with one another, forming germline 'cysts' that allow them to share small RNAs that can suppress transposable elements. Pang et al. propose that these changes compensate for the loss of enhanced repression that occurs in the earlier stem cell stage. Very similar changes also take place in the cells generated from fertilized eggs and in mammalian reproductive cells. Further experiments investigated how these changes impact the transition from stem cell to egg cell, revealing that germline stem cells express a wide diversity of genes, including most genes whose transcripts will be stored in the mature egg later on. Another type of cell produced by germline stem cells known as nurse cells, which synthesize most of the contents of the egg, dramatically upregulate genes supporting growth. Meanwhile, 25% of genes initially expressed in germline stem cells are switched off during the transition, partly due to a mechanism called Polycomb-mediated repression. The findings advance fundamental knowledge of how germline stem cells become egg cells, and could lead to important findings in developmental biology. Furthermore, understanding that for practical applications germline stem cells do not need to retain transposable element controls designed for evolutionary time scales means that removing them may make it easier to obtain and manipulate new stem cell lines and to develop new medical therapies.
Assuntos
Proteínas de Drosophila , Células-Tronco de Oogônios , Animais , Masculino , Drosophila/genética , Cromatina/metabolismo , Células-Tronco de Oogônios/metabolismo , Proteínas de Drosophila/metabolismo , Células-Tronco/metabolismo , Diferenciação Celular/genética , Células Germinativas/metabolismo , Expressão Gênica , Biologia , Drosophila melanogaster/metabolismoRESUMO
Physiological status, particularly dietary input, has major impacts on the Drosophila melanogaster ovarian germline stem cell lineage. Moreover, several studies have shed light on the role that inter-organ communication plays in coordinating whole-organism responses to changes in physiology. For example, nutrient-sensing signaling pathways function within the fat body to regulate germline stem cells and their progeny in the ovary. Together with its incredible genetic and cell biological toolkits, Drosophila serves as an amenable model organism to use for uncovering molecular mechanisms that underlie physiological control of adult stem cells. In this methods chapter, we describe a general dietary manipulation paradigm, genetic manipulation of adult adipocytes, and whole-mount ovary immunofluorescence to investigate physiological control of germline stem cells.
Assuntos
Proteínas de Drosophila , Células-Tronco de Oogônios , Animais , Feminino , Drosophila/metabolismo , Drosophila melanogaster/genética , Células-Tronco de Oogônios/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ovário/metabolismo , Células Germinativas/metabolismoRESUMO
BACKGROUND: Metformin as a first-line clinical anti-diabetic agent prolongs the lifespan of model animals and promotes cell proliferation. However, the molecular mechanisms underlying the proliferative phenotype, especially in epigenetics, have rarely been reported. The aim of this study was to investigate the physiological effects of metformin on female germline stem cells (FGSCs) in vivo and in vitro, uncover ß-hydroxybutyrylation epigenetic modification roles of metformin and identify the mechanism of histone H2B Lys5 ß-hydroxybutyrylation (H2BK5bhb) in Gata-binding protein 2 (Gata2)-mediated proliferation promotion of FGSCs. METHODS: The physiological effects of metformin were evaluated by intraperitoneal injection and histomorphology. The phenotype and mechanism studies were explored by cell counting, cell viability, cell proliferation assay and protein modification omics, transcriptomics, chromatin immunoprecipitation sequencing in FGSCs in vitro. RESULTS: We found that metformin treatment increased the number of FGSCs, promoted follicular development in mouse ovaries and enhanced the proliferative activity of FGSCs in vitro. Quantitative omics analysis of protein modifications revealed that H2BK5bhb was increased after metformin treatment of FGSCs. In combination with H2BK5bhb chromatin immunoprecipitation and transcriptome sequencing, we found that Gata2 might be a target gene for metformin to regulate FGSC development. Subsequent experiments showed that Gata2 promoted FGSC proliferation. CONCLUSION: Our results provide novel mechanistic understanding of metformin in FGSCs by combining histone epigenetics and phenotypic analyses, which highlight the role of the metformin-H2BK5bhb-Gata2 pathway in cell fate determination and regulation.
Assuntos
Metformina , Células-Tronco de Oogônios , Animais , Camundongos , Histonas/metabolismo , Metformina/farmacologia , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Fator de Transcrição GATA2/farmacologia , Proliferação de CélulasRESUMO
Female germline stem cells (FGSCs) are a group of rare undifferentiated cells found in ovarian cortex, which have the unique ability to self-renew and differentiate. Stable maintenance and proliferation of FGSCs in culture are pivotal for clinic research. However, conventional 2-D (dimension) culture systems are limited in their ability to mimic the ovarian microenvironment during in vitro studies. To establish a suitable in vitro microenvironment for FGSCs, we conducted experiments using a Matrigel-based 3-D culture system. This involves testing different dilution ratios, medium compositions, and co-culture cells to find the optimal conditions for FGSCs maintenance and proliferation. Our results demonstrated the feasibility of using Matrigel as a FGSCs 3-D culture media. Moreover, co-culturing FGSCs with some types of cells in the Matrigel-based 3-D culture system had the potential to form ovarian organoids. Meanwhile, the safety of Matrigel was confirmed in vivo through transplantation experiment, which suggests the potential for clinic research.
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Células-Tronco de Oogônios , Feminino , Humanos , Ovário , Técnicas de CoculturaRESUMO
Germline stem cells (GSCs) are the only cell population capable of passing genetic information to offspring, making them attractive targets in reproductive biology and fertility research. However, it is generally more difficult to introduce exogenous biomolecules into GSCs than other cell types, impeding the exploration and manipulation of these cells for biomedical purposes. Herein, semiconductor polymer dots (Pdots)-based nanocomplex Pdot-siRNA is developed and achieves effective knockdown of target genes in female germline stem cells (FGSCs). Advantage of high fluorescence brightness of Pdots is taken for comprehensive investigation of their cellular uptake, intracellular trafficking, and exocytosis in FGSCs. Importantly, Pdots show excellent biocompatibility and minimally disturb the differentiation of FGSCs. Intracellular Pdots escape from the lysosomes and undergo active exocytosis, which makes them ideal nanocarriers for bioactive cargos. Moreover, Pdot-siRNA can penetrate into 3D ovarian organoids derived from FGSCs and down-regulate the expression levels of target genes. This study investigates the interface between a type of theranostic nanoparticles and FGSCs for the first time and sheds light on the manipulation and medical application of FGSCs.
Assuntos
Nanopartículas , Células-Tronco de Oogônios , Pontos Quânticos , Polímeros , Semicondutores , ExocitoseRESUMO
Many adult somatic stem cell lineages are comprised of subpopulations that differ in gene expression, mitotic activity, and differentiation status. In this study, we explored if cellular heterogeneity also exists within oogonial stem cells (OSCs), and how chronological aging impacts OSCs. In OSCs isolated from mouse ovaries by flow cytometry and established in culture, we identified subpopulations of OSCs that could be separated based on differential expression of stage-specific embryonic antigen 1 (SSEA1) and cluster of differentiation 61 (CD61). Levels of aldehyde dehydrogenase (ALDH) activity were inversely related to OSC differentiation, whereas commitment of OSCs to differentiation through transcriptional activation of stimulated by retinoic acid gene 8 was marked by a decline in ALDH activity and in SSEA1 expression. Analysis of OSCs freshly isolated from ovaries of mice between 3 and 20 months of age revealed that these subpopulations were present and persisted throughout adult life. However, expression of developmental pluripotency associated 3 (Dppa3), an epigenetic modifier that promotes OSC differentiation into oocytes, was lost as the mice transitioned from a time of reproductive compromise (10 months) to reproductive failure (15 months). Further analysis showed that OSCs from aged females could be established in culture, and that once established the cultured cells reactivated Dppa3 expression and the capacity for oogenesis. Analysis of single-nucleus RNA sequence data sets generated from ovaries of women in their 20s versus those in their late 40s to early 50s showed that the frequency of DPPA3-expressing cells decreased with advancing age, and this was paralleled by reduced expression of several key meiotic differentiation genes. These data support the existence of OSC subpopulations that differ in gene expression profiles and differentiation status. In addition, an age-related decrease in Dppa3/DPPA3 expression, which is conserved between mice and humans, may play a role in loss of the ability of OSCs to maintain oogenesis with age.
Assuntos
Células-Tronco de Oogônios , Ovário , Humanos , Adulto , Feminino , Camundongos , Animais , Idoso , Células-Tronco de Oogônios/metabolismo , Oócitos/fisiologia , Oogênese , Envelhecimento , Proteínas Cromossômicas não Histona/metabolismoRESUMO
Ovarian organoids, based on female germline stem cells (FGSCs), are nowadays widely applied for reproductive medicine screening and exploring the potential mechanisms during mammalian oogenesis. However, there are still key issues that urgently need to be resolved in ovarian organoid technology, one of which is to establish a culture system that effectively expands FGSCs in vitro, as well as maintaining the unipotentcy of FGSCs to differentiate into oocytes. Here, FGSCs were EED226 treated and processed for examination of proliferation and differentiation in vitro. According to the results, EED226 specifically increased FGSC survival by decreasing the enrichment of H3K27me3 on Oct4 promoter and exon, as well as enhancing OCT4 expression and inhibiting P53 and P63 expression. Notably, we also found that FGSCs with EED226 treatment differentiated into more oocytes during oogenesis in vitro, and the resultant oocytes maintained a low level of P63 versus control at early stage development. These results demonstrated that inhibition of EED activity appeared to promote the survival of FGSCs and markedly inhibited their apoptosis during in vitro differentiation. As a result of our study, we propose an effective culture strategy to culture FGSCs and obtain oocytes in vitro, which provides a new vision for oogenesis in vitro.
Assuntos
Células-Tronco de Oogônios , Animais , Células-Tronco de Oogônios/metabolismo , Sobrevivência Celular , Proliferação de Células , Oócitos , Oogênese , Diferenciação Celular , MamíferosRESUMO
It has been generally accepted that the number of oocyte pool in mammalian ovaries is limited and irreversibly consumed throughout the adulthood until menopause, which has been challenged by the existence of female germline stem cells (FGSCs) and their differentiation potentials into oocytes through mitosis. However, there have been a few reports about the existence of porcine FGSCs (pFGSCs) in the neonatal piglet ovarian tissues. In this study, the pFGSCs were isolated from the one day post partum (1 dpp) piglet ovaries by a differential anchoring velocity method combined with the magnetic cell sorting (MACS) using VASA antibody. The gene expression levels and in vitro differentiation potentials of pFGSCs were subsequently analyzed. The results showed that Oct4, C-kit, Vasa, Stella, Ifitm3 and Dazl were expressed in the pFGSCs. A small portion of pFGSCs (2.81 ± 0.76%) spontaneously differentiated into oocyte-like cells (OLCs) with a mean diameter of 50 µm and gene expressions of Vasa, Ifitm3, Blimp1, Gdf9, Zp3, Dazl and Stella. Compared with that of the spontaneous differentiation system, the differentiation rates of pFGSCs into OLCs were significantly increased after the co-supplementations of porcine follicular fluid (PFF) and retinoic acid (RA). Taken together, these above results revealed the direct evidences for the existence of pFGSCs in 1 dpp piglet ovaries and the in vitro differentiation potential of pFGSCs into OLCs, benefiting future research related to the in vitro establishment of livestock FGSCs and the in vitro differentiation of pFGSCs.
Assuntos
Células-Tronco de Oogônios , Feminino , Animais , Suínos , Oócitos/metabolismo , Ovário , Diferenciação Celular , Células Germinativas/metabolismo , MamíferosRESUMO
OBJECTIVES: Oogonial stem cells (OSCs) are germ cells that can sustain neo-oogenesis to replenish the pool of primary follicles in adult ovaries. In lower vertebrates, fresh oocytes are produced by numerous OSCs through mitosis and meiosis during each reproduction cycle, but the OSCs in adult mammals are rare. The birds have retained many conserved features and developed unique features of ovarian physiology during evolution, and the presence of OSCs within avian species remain unknown. MATERIALS AND METHODS: In this study, we investigated the existence and function of OSCs in adult chickens. The chicken OSCs were isolated and expanded in culture. We then used cell transplantation system to evaluate their potential for migration and differentiation in vivo. RESULTS: DDX4/SSEA1-positive OSCs were identified in both the cortex and medulla of the adult chicken ovary. These putative OSCs undergo meiosis in the reproductively active ovary. Furthermore, the isolated OSCs were expanded in vitro for months and found to express germline markers similar to those of primordial germ cells. When transplanted into the bloodstream of recipient embryos, these OSCs efficiently migrated into developing gonads, initiated meiosis, and then derived oocytes in postnatal ovaries. CONCLUSIONS: This study has confirmed the presence of functional OSCs in birds for the first time. The identification of chicken OSCs has great potential for improving egg laying and preserving endangered species.
Assuntos
Células-Tronco de Oogônios , Ovário , Feminino , Animais , Galinhas , Células-Tronco de Oogônios/fisiologia , Oócitos , Oogênese , MamíferosRESUMO
Recent studies have shown that mitochondrial morphology can modulate organelle function and greatly affect stem cell behavior, thus affecting tissue homeostasis. As such, we previously showed that the accumulation of fragmented mitochondria in aged Drosophila ovarian germline stem cells (GSCs) contributes to age-dependent GSC loss. However, standard immunofluorescence methods to examine mitochondrial morphology yield images with insufficient resolution for rigorous analysis, while 3-dimensional electron microscopy examination of mitochondrial morphology is labor intensive and allows only limited sampling of mitochondria. To overcome these issues, we utilized the expansion microscopy technique to expand GSC samples by 4-fold in combination with mitochondrial immunofluorescence labeling. Here, we present a simple, inexpensive method for nanoscale optical imaging of mitochondria in the germline. This protocol may be beneficial for studies that require visualization of mitochondria or other fine subcellular structures in the Drosophila ovary.
Assuntos
Proteínas de Drosophila , Células-Tronco de Oogônios , Animais , Feminino , Drosophila , Microscopia , MitocôndriasRESUMO
Sex Ratio chromosomes in Drosophila pseudoobscura are selfish X chromosome variants associated with 3 nonoverlapping inversions. In the male germline, Sex Ratio chromosomes distort the segregation of X and Y chromosomes (99:1), thereby skewing progeny sex ratio. In the female germline, segregation of Sex Ratio chromosomes is mendelian (50:50), but nonoverlapping inversions strongly suppress recombination establishing a 26-Mb haplotype (constituting â¼20% of the haploid genome). Rare crossover events located between nonoverlapping inversions can disrupt this haplotype, and recombinants have sometimes been found in natural populations. We recently reported on the first lab-generated Sex Ratio recombinants occurring at a rate of 0.0012 crossovers per female meiosis. An improved experimental design presented here reveals that these recombination events were at least 4 times more frequent than previously estimated. Furthermore, recombination events were strongly clustered, indicating that the majority arose from mitotic exchange in female germline stem cells and not from meiotic crossing-over in primary oocytes. Finally, asymmetric recovery of complementary recombinants was consistent with unequal exchange causing the recombination-induced viability defects. Incorporating these experimental results into population models for Sex Ratio chromosome evolution provided a substantially better fit to natural population frequencies and allowed maintenance of the highly differentiated 26-Mb Sex Ratio haplotype without invoking strong epistatic selection. This study provides the first estimate of spontaneous mitotic exchange for naturally occurring chromosomes in Drosophila female germline stem cells, reveals a much higher Sex Ratio chromosome recombination rate, and develops a mathematical model that accurately predicts the rarity of recombinant Sex Ratio chromosomes in natural populations.
Assuntos
Drosophila , Células-Tronco de Oogônios , Animais , Drosophila/genética , Razão de Masculinidade , Cromossomos Sexuais , Meiose/genética , Recombinação GenéticaRESUMO
Germline stem cells (GSCs) are critical for the reproduction of an organism. The self-renewal and differentiation of GSCs must be tightly controlled to avoid uncontrolled stem cell proliferation or premature stem cell differentiation. However, how the self-renewal and differentiation of GSCs are properly controlled is not fully understood. Here, we find that the novel intrinsic factor Yun is required for female GSC maintenance in Drosophila. GSCs undergo precocious differentiation due to de-repression of differentiation factor Bam by defective BMP/Dpp signaling in the absence of yun. Mechanistically, Yun associates with and stabilizes Thickveins (Tkv), the type I receptor of Dpp/BMP signaling. Finally, ectopic expression of a constitutively active Tkv (TkvQD) completely suppresses GSC loss caused by yun depletion. Collectively, these data demonstrate that Yun functions through Tkv to maintain GSC fate. Our results provide new insight into the regulatory mechanisms of how stem cell maintenance is properly controlled.
Assuntos
Proteínas de Drosophila , Células-Tronco de Oogônios , Animais , Diferenciação Celular/fisiologia , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Células Germinativas , Fator Intrínseco/metabolismo , Células-Tronco de Oogônios/metabolismo , Ovário/metabolismo , Proteínas Serina-Treonina Quinases , Receptores de Superfície Celular/metabolismoRESUMO
The delay of ovarian aging and the fertility preservation of cancer patients are the eternal themes in the field of reproductive medicine. Acting as the pacemaker of female physiological aging, ovary is also considered as the principle player of cancer, cardiovascular diseases, cerebrovascular diseases, neurodegenerative diseases and etc. However, its aging mechanism and preventive measures are still unclear. Some researchers attempt to activate endogenous ovarian female germline stem cells (FGSCs) to restore ovarian function, as the most promising approach. FGSCs are stem cells in the adult ovaries that can be infinitely self-renewing and have the potential of committed differention. This review aims to elucidate FGSCs aging mechanism from multiple perspectives such as niches, immune disorder, chronic inflammation and oxidative stress. Therefore, the rebuilding nichs of FGSCs, regulation of immune dysfunction, anti-inflammation and oxidative stress remission are expected to restore or replenish FGSCs, ultimately to delay ovarian aging.
Assuntos
Células-Tronco de Oogônios , Envelhecimento , Proliferação de Células , Feminino , Humanos , Ovário , Células-TroncoRESUMO
Female germline stem cells (FGSCs) have been successfully isolated and characterized from postnatal mammalian and human ovarian tissues. However, the effects and mechanisms of action of natural small-molecule compounds on FGSCs are largely unknown. Here, we found that daidzein promoted the viability and proliferation of FGSCs. To elucidate the mechanism underlying this, we performed RNA-Sequence in daidzein-treated FGSCs and controls. The results showed that there were 153 upregulated and 156 downregulated genes in daidzein treatment. We confirmed the expression of some genes related to cell proliferation in the sequencing results by RT-PCR, such as Type C lectin domain family 11 member a (Clec11a), Mucin1 (Muc1), Glutathione peroxidase 3 (Gpx3), and Tet methylcytosine dioxygenase 1 (Tet1). The high expression of Clec11a at the protein level after daidzein treatment was also confirmed by western blotting. Furthermore, recombinant mouse Clec11a (rmClec11a) protein was shown to promote the viability and proliferation of FGSCs. However, knockdown of Clec11a inhibited the viability and proliferation of FGSCs, which could not be rescued by the administration of daidzein. These results indicate that daidzein promoted the viability and proliferation of FGSCs through Clec11a. In addition, both daidzein and rmClec11a activated the Akt signaling pathway in FGSCs. However, Clec11a knockdown inhibited this pathway, which could not be rescued by daidzein administration. Taken together, our findings revealed that daidzein activates the Akt signaling pathway to promote cell viability and proliferation through upregulating Clec11a. This study should deepen our understanding of the developmental mechanism of FGSCs and female infertility.
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Isoflavonas , Células-Tronco de Oogônios , Animais , Feminino , Humanos , Camundongos , Proliferação de Células , Isoflavonas/farmacologia , Isoflavonas/metabolismo , Mamíferos/metabolismo , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/farmacologia , Células-Tronco de Oogônios/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Crescimento de Células Hematopoéticas/metabolismo , Lectinas Tipo C/metabolismo , Regulação para CimaRESUMO
Polycystic ovary syndrome (PCOS) is a common disease caused by complex endocrine and metabolic abnormalities in women of childbearing age. Metformin is the most widely used oral hypoglycemic drug in clinic. In recent years, metformin has been used in the treatment of PCOS, but its mechanism is not clear. In this study, we aimed to investigate the effect of metformin on PCOS and its mechanism through PCOS mouse model. Female C57BL/6J mice aged 4-5 weeks were intragastrically given letrozole (1 mg/kg daily) combined with a high-fat diet (HFD) for 21 days to establish the PCOS model. After modeling, metformin (200 mg/kg daily) was intragastrically administered. One month later, the body weight and oral glucose tolerance test (OGTT) were measured. Hematoxylin eosin (H&E) staining was used to detect the pathological changes of ovary. The serum levels of anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), E2 and testosterone (T) were measured by ELISA. The expression of DDX4/MVH was detected by immunohistochemistry. DDX4/MVH and PCNA were co-labeled by immunofluorescence. The protein levels of DDX4/MVH, PCNA, cyclin D2, AMPK and mTOR were detected by Western blot. The results showed that after metformin treatment, the body weights of PCOS mice were gradually returned to normal, glucose tolerance was significantly improved, serum E2 levels were increased, while AMH, LH, T levels and LH/FSH ratio were decreased. Ovarian polycystic lesions were reduced with reduced atresia follicles. Furthermore, the number of proliferative female germline stem cells (FGSCs) and levels of proliferation related proteins (PCNA, cyclin D2) were significantly increased, and the p-mTOR and p-AMPK levels were markedly up-regulated. These results suggest that metformin treatment not only improves hyperandrogenemia, glucose intolerance and polycystic ovarian lesions in PCOS, but also activates the function of FGSCs. The underlying mechanism may be related to the phosphorylation of AMPK and mTOR. These findings provide new evidence to use metformin in the treatment of PCOS and follicular development disorder.