Dataset Information

Functional and transcriptomic dose-response analysis of follicle-stimulating hormone in ex vivo mouse folliculogenesis and oogenesis

ABSTRACT: The gonadotropin-dependent phase of ovarian folliculogenesis primarily requires follicle-stimulating hormone (FSH) to support one or multiple early antral follicles, dependent on the species, to mature fully and support steroidogenesis, oogenesis, and ovulation, which critically sustain female reproductive cycles and fertility. At molecular levels, FSH binds to its membrane receptor, FSHR, in granulosa cells to activate a suite of genes and signal transduction pathways. Both insufficient FSH caused by genetic or non-genetic reasons and excessive FSH used for ovarian stimulation in assisted reproductive technology (ART) can cause poor female reproductive outcomes, but the underlying mechanisms remain elusive. Here, we used an ex vivo folliculogenesis and oogenesis system along with single-follicle and -oocyte RNA sequencing analysis and other approaches to investigate the effects of different concentrations of FSH on key follicular events. The results revealed that a minimum FSH threshold is required for follicle maturation, and such threshold is moderately variable among individual follicles. FSH at the subthreshold, threshold, and suprathreshold levels induced distinct expression patterns of follicle maturation-related genes and the entire follicular transcriptomics. The RNA-seq analysis identified several new genes and signaling pathways that may critically regulate follicle maturation. The treatment of excessive FSH resulted in multiple ovarian disorders including premature luteinization, high production of androgen and proinflammatory factors, and reduced expression of energy metabolism-related genes in oocytes. Together, our study enables a better understanding of the gonadotropin-dependent folliculogenesis, and the novel findings shed crucial insights into how ovarian stimulation with high doses of FSH used in ART impact follicular health, oocyte quality, pregnancy success, and systemic health.

ORGANISM(S): Mus musculus

PROVIDER: GSE249196 | GEO | 2024/06/05

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Vitrification is a method for long-term biological sample cryopreservation without causing intra- and extra-cellular ice formation. We recently established a novel closed vitrification system to cryopreserve mouse ovarian follicles. Using the 3D alginate hydrogel encapsulated in vitro follicle growth (eIVFG) method, we have demonstrated that compared to freshly-harvested follicles, vitrified follicles have normal follicle and oocyte reproductive outcomes. Our recent study further demonstrated the faithful preservation of molecular signatures of follicle-stimulating hormone (FSH)-stimulated follicle maturation in vitrified follicles. However, it is unknown whether ovulation, another crucial gonadotropin-dependent follicular event, and involved ovulatory gene regulatory pathways are well conserved in vitrified follicles. Fresh and vitrified follicles grown for 8 days by eIVFG were collected at 0, 1, 4 and 8-hour post human chorionic gonadotropin (hCG) treatment for the single-follicle RNA sequencing. Principal component analysis (PCA) and Pearson’s correlation analysis revealed that vitrified follicles have similar transcriptomic profiles to fresh follicles. Furthermore, the expression of several genes essential for ovulation were comparable between vitrified and fresh follicles. In summary, these results demonstrate that our closed vitrification system preserves follicular transcriptomic dynamics during ex vivo ovulation. Together with our previous findings that vitrification preserves FSH-stimulated follicle maturation, the integration of vitrification of immature follicles and eIVFG has a great potential to serve as an additional fertilization preservation approach for young cancer patients and endangerers species. Moreover, follicle vitrification enables a high-content ovarian follicle biobank, which can greatly improve the throughput of eIVFG for studying ovulation biology, anovulatory disease, toxicology, and novel contraceptive drug development targeting ovulation.

Project description:Vitrification is a method for long-term biological sample cryopreservation that transforms cells into a glass-like state by cooling without causing intra- and extra-cellular ice formation, which is a major driver of cell cryoinjury. Using an alginate hydrogel encapsulated in vitro follicle growth (eIVFG) system, we have recently demonstrated that compared to freshly-harvested follicles, vitrified follicles have normal follicle and oocyte reproductive outcomes as well as comparable expression levels of several genes that are essential for gonadotropin-dependent folliculogenesis and oogenesis. However, it is unknown whether vitrification preserves the molecular signatures of folliculogenesis at the whole transcriptomic level, which is the primary research focus in this study. Multilayered secondary follicles with a diameter of 130 -160 µm were mechanically isolated from 16-day-old CD-1 female mice and vitrified using the closed vitrification method. After a 2-week storage in liquid nitrogen, vitrified follicles were warmed, and together with freshly-harvested follicles, cultured for 8 days using the eIVFG method. Next, mature antral follicles from day 8 were collected for single-follicle RNA sequencing using the SMART-seq RNA-seq method. Principal component analysis (PCA) and Pearson’s correlation analysis revealed that vitrified follicles have similar transcriptomic profiles to fresh follicles. Using the criterion of fold change >2 or <0.5 and a false discovery rate (FDR) adjusted p-value < 0.05, there were 35 differentially expressed genes out of a total of 11,806 detected genes between vitrified and fresh follicles, among which 17 were up-regulated and 18 were down-regulated in vitrified follicles. To the best of our knowledge, none of those differentially expressed genes identified between vitrified and fresh follicles have been shown to be critical to folliculogenesis and oogenesis. Meanwhile, gene ontology (GO) and KEGG pathway analysis over these 35 genes using the functional annotation tool DAVID revealed that no GO terms or signaling pathways were significantly enriched. Furthermore, the expression of several genes essential for the gonadotropin-dependent folliculogenesis and oogenesis were comparable between vitrified and fresh follicles, including granulosa cell-specific genes (Cyp19a1, Amh, Inha, Inhba, Inhbb, and Fshr), theca cell-specific genes (Cyp17a1, Col1a2, and Col3a1), and oocyte-specific genes (Gdf9, Bmp15, Zp1, Zp2, and Zp3). Equally important, Lhcgr, the gene that is primarily expressed in theca cells of preantral follicles and then greatly induced in mural granulosa cells in antral follicles to prepare for the luteinizing hormone (LH) surge and ovulation, also had comparable mRNA expression levels between vitrified and fresh follicles. Taken together, these results demonstrate that vitrification preserves the follicular cell transcriptome and molecular signatures of gonadotropin-dependent folliculogenesis in the eIVFG system. In conclusion, our study re-evaluates individual follicle cryopreservation by using the closed vitrification method in a more in-depth perspective and proves that vitrification preserves the follicular cell transcriptome in the eIVFG system, thus providing a robust model for fertility preservation, conservation of endangered species, and also establishing a high-content ovarian follicle biobank for studying ovarian biology and female reproductive toxicology.

Project description:Background: The initial formation of follicular antrum (iFFA) is a milestone in folliculogenesis, which serves as a boundary between gonadotropin (GTH) -independent folliculogenesis and GTH-dependent folliculogenesis and endows the follicle's ability to acquire response to GTHs and continue to survive. However, iFFA is a barren field in folliculogenesis research, which do not match its physiological significance. Methods:In this study, transcriptome sequencing was used to comprehensively analyze the changes in the life activities of cells in the ovary during iFFA, and then the regulatory mechanism of iFFA was deduced based on the results of omics analysis, and it was systematically verified by experiments. Result: Herein, we conducted in vivo and in vitro experiments to studied iFFA in a mouse model, and revealed that: (1) iFFA is a physiological process of active cell proliferation, increased energy expenditure, enhanced secretory activity and increased moisture absorption, accompanied by complex changes in epigenetic modification, bioclock regulation, and cell junctions; (2) iFFA and blastocoel formation may be conservatively regulated in fluid accumulation, and tight junctions, ion pumps and aquaporins underlie the structural, kinetic and transport foundations for fluid accumulation during iFAA, respectively; (3) mTOR-CNP is the main signal cascade regulating iFFA, it initiated iFFA by targeting tight junction/ion pumps/aquaporins and follicular cell proliferation; (4) FSH controls iFFA by targeting the intra-ovarian mTOR-CNP cascade; (5) mTOR activator could be used as an improver to increase the efficiency of in vitro follicular culture technology. Conclusion: Overall, this study elucidated the changes in follicular cells' life activities in preparation for iFFA, proposed the model of "Dam-Pump-Pipe" to explain the accumulation of follicular fluid, and confirmed that FSH-mTOR-CNP-Tight junction/Ion pumps/Aquaporins is the core cascade controlling iFFA. These findings would help to fill in the gaps in iFFA research, and enrich the theory of folliculogenesis regulation.

Project description:Dibutyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), and benzyl butyl phthalate (BBP) are three phthalates commonly found in consumer products, including the plastic coating of pharmaceuticals and personal care products. Folliculogenesis, a tightly regulated process occurring in the ovary, is the maturation of an immature primordial follicle to a mature antral follicle. Follicles house the oocyte and antral follicles specifically play a crucial role in ovarian steroidogenesis and ovulation. DBP, BBP, and DEHP have been associated with inhibited antral follicle growth in vitro, decreased ovulation rates in vitro, and decreased antral follicle counts in women. However, little is known about the effects of a three-phthalate mixture on antral follicles in vivo. The objective of this study was to evaluate the effects of a human relevant mixture of DBP, BBP, and DEHP on ovarian follicles through proteome profiling analysis. CD-1 female mice (60 days old) were pipet fed tocopherol stripped corn oil (vehicle control) only or a phthalate mixture (52% DBP, 36% DEHP, and 12% BBP dissolved in vehicle) which modeled human follicular fluid concentrations. The mice were treated with 32µg/kg/day (PHT Mix 32; cumulative estimate in general population) and 500µg/kg/day (PHT Mix 500; cumulative estimate in occupationally exposed individuals) for 10 consecutive days. Proteome profiling of antral follicles (>250µm) was performed via label-free tandem mass spectrometry. A total of 5,417 antral follicle proteins were identified in the three groups, of which 194 were differentially abundant between the vehicle and PHT Mix 32 group, and 136 between the vehicle and PHT Mix 500 group. Gene ontology analysis revealed that the two treatments of the phthalate mixture upregulate and downregulate distinctive processes, supporting non-monotonic effects of phthalates on the antral follicle proteome. Taken together, these results reveal that a human relevant mixture of DBP, BBP, and DEHP alters the antral follicle proteome and merits further evaluation to elucidate the molecular mechanisms by which phthalates cause negative reproductive outcomes.

Dataset Information

Functional and transcriptomic dose-response analysis of follicle-stimulating hormone in ex vivo mouse folliculogenesis and oogenesis

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets