RNA-seq of paired oocytes and granulosa cells from 3M and 12M old mice after natural and superovulation
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PROVIDER: PRJEB67722 | ENA |
REPOSITORIES: ENA
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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
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
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
Project description:Ribo-depleted total RNA-seq of oocytes in 3M mice after natural and superovulation
| PRJEB67724 | ENA
Project description:Two major factors contributing to reduced fertility is use of exogenous hormones and old age. In previous experiments, we used 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 use granulosa cell samples from human patients undergoing in vitro fertilization to test how aged hormonally stimulated cell transcriptional profiles correspond to ones in mice. Total RNA was extracted from granulosa cell samples, collected during IVF procedures, using TRIzol. Further, RNA was cleaned from DNA with DNA-free™ DNA Removal Kit. ~1 µg of RNA was used to construct libraries with Truseq Stranded mRNA kit and barcoded with IDT for Illumina-TruSeq DNA and RNA UD Indexes. Samples were sequenced on Nextseq200 (Illumina) with 100bp paired-end sequencing.
2024-09-22 | E-MTAB-13496 | biostudies-arrayexpress
Project description:RNA-seq of paired granulosa cells and morulas or blastocysts from naturally and superovulated 3M old mice
| PRJEB77920 | ENA
Project description:Limited oocyte and ovarian reserve in vivo or chemo-therapy leads to reproductive aging or premature aging and associated diseases including infertility. Excitingly, oocytes have been successfully derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) by ectopic expression of transcription factors, showing great potential in fertility preservation or restoration. The accessible granulosa cells are a type of somatic cells that interact and evolve with oocyte development during folliculogenesis. Further, with stem cells-like property, granulosa cells are amenable to reprogramming to generate iPSCs and have been the first used for clone animals. These prompted us to explore the potential of granulosa cells in derivation of germ cells. Meanwhile, the strict genome fidelity required for germ cells inspired us to test reprograming by complete small chemicals, which avoids genetic manipulation, cell transfection and destruction of embryos. Here we show that somatic granulosa cells of adult mouse ovaries can be converted to germ cells and functional oocytes that reproduce fertile pups. We are able to consistently induce granulosa cells to pluripotent state (gPSCs) like ESCs in both developmental competence and molecular signatures. Notably, crotonic sodium-facilitated crotonylation is critical not only for pure small chemicals-based reprogramming of granulosa cells to gPSCs, but also confers the gPSCs with high germline capacity. Consequently, the gPSCs and the derived primordial germ-cell like cells hold longer telomeres and maintain high genomic stability which is critical for germ cells. Taken together, we efficiently generate high quality gPSCs and functional oocytes from adult granulosa cells by significantly improving chemical reprograming approach.
2019-03-06 | GSE127833 | GEO
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
Project description:Comparison of mitochondrial DNA (mtDNA) methylation patterns in oocytes, blastocysts and ovarian granulosa cells indicates hitherto unsuspected dynamics. Oocytes and blastocysts recovered from animals subjected to ovarian stimulation and from abattoir ovaries were analyzed using bisulphite transformation of DNA followed by whole genome sequencing. The number of mtDNA copies is quite high in oocytes (200–400,000) and early embryos, resulting in very high coverage (> 3000 X) and very low p values for each of 716 cytosine-based nucleosides. Methylation was lowest in oocytes, following by blastocysts then granulosa cells and was not restricted to CpG sites but found also at CHG and CHH sites not distributed randomly. Analysis of the corresponding genomic DNA also revealed numerous methylated CHG and CHH sites, particularly in oocytes. In association with ovarian stimulation, mtDNA methylation was lower in oocytes than in blastocysts, suggesting a developmental function. RNA analysis of mitochondria encoded genes showed a significant inverse correlation between methylation and expression level for all sequences. Methylation was more extensive in mature animals than in pre-pubertal animals and the profile in granulosa cells differed strikingly from those in oocytes and blastocysts. Although preliminary, the results suggest that mtDNA methylation might play a role during gametogenesis and might be subject to regulation according to animal environment and/or age.
2019-08-26 | GSE122599 | GEO
Project description:Derivation of functional oocytes from granulosa cells
| PRJNA525610 | ENA