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Ribo-depleted total RNA-seq of oocytes in 3M mice after natural and superovulation

ABSTRACT: Ribo-depleted total RNA-seq of oocytes in 3M mice after natural and superovulation

PROVIDER: PRJEB67724 | ENA |

REPOSITORIES: ENA

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Ribo-depleted total RNA-seq of oocytes in 3M mice after natural and superovulation

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.

2024-09-22 | E-MTAB-13474 | biostudies-arrayexpress

RNA-seq of paired oocytes and granulosa cells from 3M and 12M old mice after natural and superovulation

Project description:Oocytes and granulosa cells were collected after natural and superovulation in young (3 month-old) and old (12 month-old) mice while conserving pairing information between oocyte and granulosa cells from each cumulus-oocyte-complex (COC). This experimental design allows for the analysis of the effects of ageing and / or superovulation on the transcriptome of COCs. The naming convention for samples is as follows: [ovulation, NO/SO][age, 3M/12M][cell, OC/GC][COC number], meaning that NO12MOC01 and NO12MGC01 are from the same COC. Oocyte and granulosa cell samples were processed for full-length cytoplasmic RNA amplification using slightly modified SMART-seq2 protocol. After amplification of cDNA and library preparation, samples were sequenced on NextSeq 550 using Single-Ended 75bp High-Output kit.

2024-09-22 | E-MTAB-13479 | biostudies-arrayexpress

RNA-seq of paired oocytes and granulosa cells from 3M and 12M old mice after natural and superovulation

Project description:RNA-seq of paired oocytes and granulosa cells from 3M and 12M old mice after natural and superovulation

| PRJEB67722 | ENA

Genome-wide DNA methylation assessment in mouse oocytes: effects of in vitro growth, superovulation and sexual immaturity

Project description:There is legitimate concern over epigenetic abnormalities that could be induced by procedures applied during assisted reproduction, necessitating epigenetic assessment of new methodologies. The effect of in vitro follicle culture (IFC) and superovulation on DNA methylation in the oocyte is not fully known. Here, we present the first genome-wide analysis of oocyte DNA methylation in IFC, superovulated mouse oocytes and, uniquely, we compare these data with naturally ovulated counterparts. Globally, the distinctive methylation landscape of oocytes, comprising alternating hyper- and hypomethylated domains, is preserved irrespective of the procedure. The conservation of methylation extends to the germline differential methylated regions (DMRs) of imprinted genes, necessary for their monoallelic expression in the embryo after fertilisation. However, we do detect specific, consistent and coherent differences in DNA methylation between IFC and oocytes from other groups, and between oocytes obtained after superovulation from pre-pubertal compared with sexually mature females. Several methylation differences span entire transcription units, consistent with effects on methylation downstream of transcriptional differences. Amongst these, we found alterations in Sox5, Zfp521 and Tcf4, genes for transcripton factors related to nervous system development. These observations also suggest transcriptional and epigenetic differences between oocytes from the initial and later phases of follicle recruitment. Our results provide an important reference for the use of in vitro growth and maturation, particularly from prepubertal females, in assisted reproductive technologies or fertility preservation.

2019-12-23 | GSE128656 | GEO

RNA-seq of paired granulosa cells and morulas or blastocysts from naturally and superovulated 3M old mice

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. In this experiment, we are testing how different GC transcriptional profiles correlate with embryo quality. 3M old C57BL6/J mice were naturally or superovulated and COCs singularized as described above. Granulosa cells were immediately flash frozen and matched oocytes were divided into individual drops of CARD media under mineral oil for fertilization. The oocytes were incubated in 37oC, 5% CO2 incubator for 30-40 min prior to fertilization. Frozen Mus musculus (C57BL6/J) male sperm straws were thawed and capacitated in Fertiup media for 30 min. Fertilization was achieved by combining 2.5 µl of activated sperm with 20 µl CARD media drop containing a single oocyte and incubated at 37oC, 5% CO2 for 3 hours. Then, oocytes were individually washed in Human Tubal Fluid (HTF) media drops and transferred to 20 µl HFT drops for overnight culture. After 24 hours, fertilization rate was evaluated by 2-cell stage and embryos transferred to G1+ media for further development. Early morula stage embryos were continuously collected based on observation 62-64 h post-fertilization. For late blastocysts, fertilization media was additionally changed to G2+ after 48h post- fertilization and blastocysts collected at 108h post-fertilization. For collection, the embryos were washed in DPBS and flash frozen. Granulosa cells and embryo samples were further processed by Smart-seq2, the cDNA amplification PCR cycle number for embryos was 16-17.

2024-09-22 | E-MTAB-13480 | biostudies-arrayexpress

PC-3M-1E8 transcriptome

Project description:PC-3M-1E8 transcriptome

| PRJNA600512 | ENA

Setaria viridis strain:NMU.00626. 3m | cultivar:NMU.00626. 3m

Project description:Setaria viridis strain:NMU.00626. 3m | cultivar:NMU.00626. 3m Genome sequencing

| PRJNA334511 | ENA

The trade-off hypothesis of oocyte quantity and quality in gonadotropin-stimulated ovarian regime: proteome profile of superovulated vs naturally ovulated mouse oocytes

Project description:In order to clarify if there is a tradeoff between quantity and quality of oocytes retrieved after ovarian stimulation, we compared the protein expression of mouse oocytes and embryos with vs without a background of ovarian stimulation using gonadotropins, administered according to the standard protocol in use since more than 50 years (PMID 13185277; PMID 13475597). The data we gathered contribute to a more complete understanding of embryonic gene expression, by taking into account the effect of the commonly used superovulation.

2021-12-15 | PXD021331 | Pride

The trade-off hypothesis of oocyte quantity and quality in gonadotropin-stimulated ovarian regime: proteome profile of superovulated vs naturally ovulated mouse oocytes

2021-12-14 | PXD026347 | Pride

Genome-wide DNA methylation assessment in mouse oocytes: effects of in vitro growth, superovulation and sexual immaturity

Project description:Genome-wide DNA methylation assessment in mouse oocytes: effects of in vitro growth, superovulation and sexual immaturity

| PRJNA528432 | ENA