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1.
EBioMedicine ; 106: 105263, 2024 Aug.
Article in English | MEDLINE | ID: mdl-39067135

ABSTRACT

BACKGROUND: The KITL-KIT interaction is known as an important initiator in oocyte activation through the downstream pathway of PI3K-AKT-FOXO3 signalling. Previous studies utilising germ cell-specific Kit mutant knockin and kinase domain knockout models with Vasa-Cre suggested the crucial role of KIT in oocyte activation at the primordial follicle stage. METHODS: We utilised mice with complete postnatal deletion of KIT expression in oocytes via Gdf9-iCre and conducted analyses on ovarian follicle development, specific markers, hormone assays, and fertility outcomes. FINDINGS: Our findings reveal contrasting phenotypes compared to previous mouse models with prenatal deletion of Kit. Specifically, postnatal deletion of Kit exhibit no defects in germ cell nest breakdown, follicle activation, and folliculogenesis during development. Remarkably, upon reaching full maturity, mice with postnatal deletion of Kit experience a complete loss of ovarian reserve, growing follicles, and ovarian function. Furthermore, mice display smaller ovarian size and weight, delayed folliculogenesis, and phenotypes indicative of primary ovarian insufficiency (POI), including elevated serum levels of FSH, reduced AMH, and absence of ovarian follicles, ultimately resulting in infertility. Additionally, the ovaries exhibit randomly distributed expression of granulosa and theca cell markers such as Inhibin α, ACVR2B, and LHR. Notably, there is the uncontrolled expression of p-SMAD3 and Ki67 throughout the ovarian sections, along with the widespread presence of luteinised stroma cells and cleaved Caspase-3-positive dying cells. INTERPRETATION: These genetic studies underscore the indispensable role of KIT in oocytes for maintaining the survival of ovarian follicles and ensuring the reproductive lifespan. FUNDING: This work was supported by National Institutes of Health grant R01HD096042 and startup funds from UNMC (S.Y.K.).


Subject(s)
Oocytes , Ovarian Follicle , Proto-Oncogene Proteins c-kit , Animals , Female , Mice , Biomarkers , Cell Survival/genetics , Growth Differentiation Factor 9/metabolism , Growth Differentiation Factor 9/genetics , Mice, Knockout , Oocytes/metabolism , Ovarian Follicle/metabolism , Primary Ovarian Insufficiency/metabolism , Primary Ovarian Insufficiency/genetics , Proto-Oncogene Proteins c-kit/metabolism , Proto-Oncogene Proteins c-kit/genetics , Reproduction
2.
Biol Reprod ; 110(4): 772-781, 2024 Apr 11.
Article in English | MEDLINE | ID: mdl-38195246

ABSTRACT

Cisplatin, a platinum-containing alkylating agent, is used in the treatment of various tumors owing to its potent antitumor activity. However, it causes permanent and adverse effects, particularly hearing loss and depletion of ovarian reserve. Until recently, there were no clinically available protective agents to mitigate the adverse side effects of cisplatin-induced cytotoxicity. In 2022, sodium thiosulfate (STS) was approved by the Food and Drug Administration for mitigating hearing loss in children and adolescents undergoing cisplatin treatment. Consequently, our investigation aimed to determine if STS could protect ovarian reserve against cisplatin-induced gonadotoxicity. In an ex vivo culture, the cisplatin-only group exhibited a loss of primordial follicles, while post-STS administration after cisplatin exposure effectively protected primordial follicles. However, when post-STS was administrated either 6 or 4 h after cisplatin exposure, it did not confer protection against cisplatin-induced gonadotoxicity in postnatal day 7 or adolescent mouse models. Immunofluorescence assays using γH2AX and cPARP revealed that oocytes within primordial follicles exhibited DNA damage after cisplatin exposure, irrespective of post-STS administration. This underscores the rapid and heightened sensitivity of oocytes to gonadotoxicity. In addition, oocytes demonstrated an increased expression of pCHK2 rather than pERK, suggesting that the pathway leading to oocyte death differs from the pathway observed in the inner ear cell death following cisplatin exposure. These results imply that while the administration of STS after cisplatin is highly beneficial in preventing hearing loss, it does not confer a protective effect on the ovaries in mouse models.


Subject(s)
Antineoplastic Agents , Hearing Loss , Ovarian Reserve , Thiosulfates , Mice , Child , Female , Animals , Adolescent , Humans , Cisplatin/toxicity , Antineoplastic Agents/toxicity , Hearing Loss/chemically induced
3.
Int J Mol Sci ; 22(6)2021 Mar 13.
Article in English | MEDLINE | ID: mdl-33805821

ABSTRACT

Drought is the most serious abiotic stress, which significantly reduces crop productivity. The phytohormone ABA plays a pivotal role in regulating stomatal closing upon drought stress. Here, we characterized the physiological function of AtBBD1, which has bifunctional nuclease activity, on drought stress. We found that AtBBD1 localized to the nucleus and cytoplasm, and was expressed strongly in trichomes and stomatal guard cells of leaves, based on promoter:GUS constructs. Expression analyses revealed that AtBBD1 and AtBBD2 are induced early and strongly by ABA and drought, and that AtBBD1 is also strongly responsive to JA. We then compared phenotypes of two AtBBD1-overexpression lines (AtBBD1-OX), single knockout atbbd1, and double knockout atbbd1/atbbd2 plants under drought conditions. We did not observe any phenotypic difference among them under normal growth conditions, while OX lines had greatly enhanced drought tolerance, lower transpirational water loss, and higher proline content than the WT and KOs. Moreover, by measuring seed germination rate and the stomatal aperture after ABA treatment, we found that AtBBD1-OX and atbbd1 plants showed significantly higher and lower ABA-sensitivity, respectively, than the WT. RNA sequencing analysis of AtBBD1-OX and atbbd1 plants under PEG-induced drought stress showed that overexpression of AtBBD1 enhances the expression of key regulatory genes in the ABA-mediated drought signaling cascade, particularly by inducing genes related to ABA biosynthesis, downstream transcription factors, and other regulatory proteins, conferring AtBBD1-OXs with drought tolerance. Taken together, we suggest that AtBBD1 functions as a novel positive regulator of drought responses by enhancing the expression of ABA- and drought stress-responsive genes as well as by increasing proline content.


Subject(s)
Abscisic Acid/metabolism , Adaptation, Physiological/genetics , Arabidopsis Proteins/genetics , Arabidopsis/genetics , Endonucleases/genetics , Gene Expression Regulation, Plant , Abscisic Acid/pharmacology , Arabidopsis/drug effects , Arabidopsis/enzymology , Arabidopsis Proteins/agonists , Arabidopsis Proteins/antagonists & inhibitors , Arabidopsis Proteins/metabolism , Cell Nucleus/metabolism , Cyclopentanes/metabolism , Cyclopentanes/pharmacology , Cytoplasm/metabolism , Droughts , Endonucleases/antagonists & inhibitors , Endonucleases/metabolism , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/metabolism , Oxylipins/metabolism , Oxylipins/pharmacology , Plant Cells/drug effects , Plant Cells/enzymology , Plant Growth Regulators/metabolism , Plant Growth Regulators/pharmacology , Plant Leaves/drug effects , Plant Leaves/enzymology , Plant Leaves/genetics , Plant Stomata/drug effects , Plant Stomata/enzymology , Plant Stomata/genetics , Plants, Genetically Modified , Proline/metabolism , Stress, Physiological/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Water/metabolism
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