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SRX11489325: GSM5460450: Lept18; Mus musculus; RNA-Seq
1 ILLUMINA (NextSeq 500) run: 10.4M spots, 790.6M bases, 262.4Mb downloads

Submitted by: NCBI (GEO)
Study: Leptin Signalling in the Ovary of Diet-Induced Obese Mice Regulates Activation of Nod-Like Receptor Protein 3 Inflammasome
show Abstracthide Abstract
Obesity leads to ovarian dysfunction and the establishment of local leptin resistance. The aim of our study was to characterise levels of Nod-Like Receptor Protein 3 (NLRP3) inflammasome activation during obesity progression in the mouse ovaries and liver and test the putative role of leptin on its regulation. C57BL/6J mice were treated with equine chorionic gonadotropin (eCG) or human chorionic gonadotropin (hCG) for oestrous cycle synchronisation and ovaries collection. In diet-induced obesity (DIO) model, mice were fed chow diet (CD) or high fat diet (HFD) for 4 or 16 weeks (wk), whereas in hyperleptinemic model (LEPT), mice were injected with leptin for 16 days (16L) or saline (16C) and in the genetic obese leptin-deficient ob/ob (+/? and -/-) animals were fed CD for 4wk. Either ovaries and liver were collected, as well as cumulus cells (CCs) after superovulation from DIO and LEPT. In DIO protocol, protein expression of NLRP3 inflammasome components was increased in 4wk HFD, but decreased in 16wk HFD. Moreover LEPT and ob/ob models revealed NLRP3 and IL-1? upregulation in 16L and downregulation in ob/ob. Transcriptome analysis of CC showed common genes between LEPT and 4wk HFD modulating NLRP3 inflammasome. Moreover analysis in the liver showed upregulation of NLRP3 protein only after 16wk HFD, but also the downregulation of NLRP3 protein in ob/ob-/-. We showed the link between leptin signalling and NLRP3 inflammasome activation in the ovary throughout obesity progression in mice, elucidating the molecular mechanisms underpinning ovarian failure in maternal obesity. Overall design: A total of 34 samples were analyzed including 3 from leptin treatment controls, 5 from leptin treated, 5 chow diet fed for 4 weeks, 5 high-fat diet fed for 4 weeks, 6 chow diet fed for 16 weeks, 7 high-fat diet fed for 16 weeks, and 3 low weight gainers after high-fat diet for 16 weeks.
Sample: Lept18
SAMN20293099 • SRS9529096 • All experiments • All runs
Organism: Mus musculus
Library:
Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Cumulus cells were collected into RLT buffer (1053393, Qiagen, Hilden, Germany) and kept in -80oC until library generation. mRNA was captured using Smart-seq2 oligo-dT pre-annealed to magnetic beads (MyOne C1, Invitrogen, Carlsbad, California, USA). The beads were resuspended in 10 μl of reverse transcriptase mix (100 U, SuperScript II, Invitrogen; 10 U, RNAsin, Promega, Madison, Wisconsin, USA), 1 × Superscript II First-Strand Buffer, 2.5 mM ditiotreitol (DTT, Invitrogen), 1 M betaine (Sigma Aldrich), 9 mM magnesium chloride (MgCl2, Invitrogen), 1 μM Template-Switching Oligo (Exiqon, Vedbaek, Denmark), 1 mM dNTP mix (Roche) and incubated for 60 min at 42 °C followed by 30 min at 50 °C and 10 min at 60 °C (Picelli et al., 2013, 2014). Amplification of the cDNA was then undertaken after adding 11 μl of 2 × KAPA HiFi HotStart ReadyMix and 1 μl of 2 μM ISPCR primer (Picelli et al., 2013, 2014), followed by the cycle: 98 °C for 3 min, then 9 cycles of 98 °C for 15 s, 67 °C for 20 s, 72 °C for 6 min and finally 72 °C for 5 min. Finally, the cDNA was purified using similar volume of AMPure beads (Beckman Coulter, Brea, California, USA) and eluted into 20 μl of water. All libraries were prepared from 100 to 200 pg of cDNA using the Nextera XT Kit (Illumina, San Diego, California, USA), regarding the manufacturer's instructions. The final cDNA library was purified using a 0.7:1 volumetric ratio of AMPure beads before pooling and sequencing.
Experiment attributes:
GEO Accession: GSM5460450
Links:
Runs: 1 run, 10.4M spots, 790.6M bases, 262.4Mb
Run# of Spots# of BasesSizePublished
SRR1518230310,402,195790.6M262.4Mb2021-11-02

ID:
15397790

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