Project description:Two replicates of GV, GVBD, and MII oocytes were subjected to the 6-plex TMT labeling, HP-RP fractionation, and LC-MS/MS analysis. Two labeling experiments were performed for the total four replicates of each stage of oocytes.
Project description:The proteome changes were quantified in RibosomeRPL39L-/- spermatocytes and elongated spermatids using TMT 6-plex, and in spermatogonia, spermatocytes, round spermatids and elongated spermatids using TMT 10-plex by LC-MS/MS.
Project description:Arachidonic acid (ARA) treated and control oocytes in 3 replicates were subjected to the 6-plex TMT labeling, HP-RP fractionation, and LC-MS/MS analysis.
Project description:The proteome and phosphoproteomics changes were quantified in seminal plasma extracellular vesicles among healthy individuals with normal sperm, nonobstructive azoospermia and obstructive azoospermia using TMT 10-plex by LC-MS/MS.
Project description:Five replicates of GV, GVBD, and MII oocytes were subjected to the 15-plex TMT labeling, HP-RP fractionation, and LC-MS/MS analysis. For each replicate, 2,000 oocytes were collected from each of the GV, GVBD, and MII stages. Ti4+-IMAC was used to enrich phosphopeptides.
Project description:RNA-seq of single Fully Grown Oocytes using SMART-seq2. The oocytes were collected from female FVB mice, approximately. 44-48 h post-PMSG injection. The oocytes were microinjected with 1.3-1.7 μg/μL Kdm5b wild-type (WT) or catalytic mutant (CM) mRNA. Noninjected oocytes are denoted as “Noninj”. The oocytes were cultured overnight for approximately 16 h in M2 containing 20 μM Milrinone and 5% FBS and collected for sequencing.
Project description:To elucidate some of the tools involved in early embryonic reprogramming, the levels of gene transcripts believed to be of importance to epigenetic modifications, and chromatin remodeling were detected by oligonucleotide microarrays in in vivo matured MII oocytes and compared with fully in vivo grown GV oocytes.
Project description:Two major factors contributing to reduced fertility is use of exogenous hormones and old age. We use mouse model to study transcriptional and cell-cell communication changes upon superovulation and ageing in female reproductive cells - oocytes (OC) - and somatic cells - granulosa (GC) - surrounding them. Here, we are validating the results obtained by a polyA-biased method. Oocytes from naturally and superovulated mice were collected as follows: mice were sacrificed by cervical dislocation and oviducts dissected. Ampullas were torn to release the COCs into a 96 µl M2 media drop under mineral oil at room temperature. Then, 4ul of pre-heated 500 µg/ml hyaluronidase diluted in M2 media (final concentration in the drop 20 µg/ml) was added to separate the COCs into single units. The COCs were incubated for 10-20 min at 37oC and then mechanically separated into individual M2 drops using 115-124 μm glass retransfer pipette. Individual COCs were then washed in M2 once and incubated for less than 5 min with enzymatic mix Accutase at 37oC to further separate granulosa cells from the oocytes. Oocytes were washed twice with M2 media and once with DPBS before collection. Cells were immediately flash frozen in liquid nitrogen in individual 0.2ml thin wall PCR tubes and stored in -80oC until library preparation. SMARTer Stranded Total RNA - Low Input Mammalian kit was used for lysis, cDNA amplification and library preparation with 10 cycles for PCR 1 and 16 cycles for PCR 2. Samples were sequenced as recommended by the manufacturer on Novaseq600 with paired-end sequencing.