O16-3.SE
Mus musculus
Protocols: Ovaries were harvested from reproductively young and old mice in Leibovitz's L-15 medium (Life Technologies Corporation, Grand Island, NY) supplemented with 3 mg/ml polyvinylpyrrolidone (Sigma-Aldrich, St. Louis, MO) and 0.5% Pen Strep (Life Technologies) (L15/PVP). Ovaries were disrupted mechanically using insulin syringes and late primary to transitioning secondary stage follicles were identified based on size and morphology and collected. For short-term culture (maximum of 40 minutes), follicles were cultured in CZB media (EMD Millipore, Billerica, MA) overlaid with mineral oil in a humidified atmosphere of 5% CO2 in air at 37 oC. Follicles were imaged using the EVOS FL Auto Cell Imaging system, and the mean diameter was determined based on two perpendicular measurements from basement membrane-to-basement membrane. Two age cohorts of adult CB6F1 female mice were used: 6-12 week old mice (reproductively young; Envigo, Indianapolis, IN) and 14-17 month old mice (reproductively old; National Institute on Aging Aged Rodent Colony, Bethesda, MD). 14-17 month old mice were selected as the reproductively old cohort because it has been shown that a significant deterioration in egg quality and the ovarian microenvironment occurs at this age (Chiang et al. 2010; Briley et al. 2016). We acknowledge that we do not know whether the founder sub-strains used to produce the F1 animals in our study were identical between our young and old mouse cohorts. Although we can not rule out confounding effects due to potential sub-strain variability, we think this contribution is likely to be minimal given that most reproductive aging phenotypes are conserved across strains (i.e. follicle loss, increased egg aneuploidy, increased ovarian stromal fibrosis) (Chiang et al. 2010; Lister et al. 2010; Hirshfeld-Cytron et al. 2011; Merriman et al. 2012; Briley et al. 2016). Pre-pubertal (12-13 day old) CD-1 female mice (Envigo) were used to optimize nucleolar antibody staining conditions. All mice were housed in a controlled barrier facility at the University of Kansas Medical Center's (KUMC) Research Support Facility under constant temperature, humidity, and light (12h light/12h dark). Food and water were provided ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center and were in accordance with National Institutes of Health Guidelines. Follicles were isolated from reproductive young (n = 5) and old CB6F1 (n = 4) mice as mentioned above. Isolated follicles were placed individually in 5 μl droplets of L15/PVP under oil and imaged immediately. Individual follicles were snap frozen in 3.5 μl 2x reaction buffer supplied in the cDNA synthesis reagents. For each animal, the total time that elapsed between mouse euthanasia and snap frozen samples did not exceed 30 minutes. Construction of cDNA was done using the Smart-Seq v4 Ultra Low Input RNA kit (Clontech Laboratories Inc, Mountain View, CA) with 14 cycles of PCR according to manufacturer's instructions. Quality and yield of cDNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Adapter-ligated libraries were constructed from 1 ng cDNA using the Nextera XT kit (Illumina Inc, San Diego, CA) according to manufacturer's instructions. Resulting libraries were checked for quality and quantity using the Agilent 2100 Bioanalyzer and Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA).
ENA first public
2018-05-22
ENA last update
2017-08-25
External Id
SAMEA104226512
INSDC center alias
Stowers Institute for Medical Research
INSDC center name
Stowers Institute for Medical Research
INSDC first public
2018-05-22T17:03:04Z
INSDC last update
2017-08-25T10:56:47Z
INSDC status
public
Submitter Id
E-MTAB-5952:O16-3.SE
age
98 to 119
broker name
ArrayExpress
common name
house mouse
disease
normal
genotype
wild type genotype
individual
O16
organism part
mature ovarian follicle
phenotype
reproductively old
sample name
E-MTAB-5952:O16-3.SE
sampling site
follicle 3
sex
female
strain
CB6F1