Transcriptomic profiling of neonatal mouse granulosa cells reveals new insights into primordial follicle activation

Abstract

The dormant population of ovarian primordial follicles is determined at birth and serves as the reservoir for future female fertility. Yet our understanding of the molecular, biochemical, and cellular processes underpinning primordial follicle activation remains limited. The survival of primordial follicles relies on the correct complement and morphology of granulosa cells, which provide signalling factors essential for oocyte and follicular survival. To investigate the contribution of granulosa cells in the primordial-to-primary follicle transition, gene expression profiles of granulosa cells undergoing early differentiation were assessed in a murine model. Ovaries from C57Bl/6 mice were enzymatically dissociated at time-points spanning the initial wave of primordial follicle activation. Post-natal day (PND) 1 ovaries yielded primordial granulosa cells, and PND4 ovaries yielded a mixed population of primordial and primary granulosa cells. The comparative transcriptome of granulosa cells at these time-points was generated via Illumina NextSeq 500 system which identified 131 significantly differentially expressed transcripts. The differential expression of eight of the transcripts was confirmed by RT-qPCR Following biological network mapping via Ingenuity Pathway Analysis, the functional expression of the protein products of three of the differentially expressed genes, namely FRZB, POD1 and ZFX, was investigated with in-situ immunolocalisation in PND4 mouse ovaries was investigated. Finally, evidence was provided that Wnt pathway antagonist, secreted frizzled-related protein 3 (FRZB), interacts with a suppressor of primordial follicle activation WNT3A and may be involved in promoting primordial follicle activation. This study highlights the dynamic changes in gene expression of granulosa cells during primordial follicle activation and provides evidence for a renewed focus into the Wnt signalling pathway’s role in primordial follicle activation.

Cocktails of NSAIDs and 17α Ethinylestradiol at Environmentally Relevant Doses in Drinking Water Alter Puberty Onset in Mice Intergenerationally

“…Analysis of PadI6 , Zp1 , Zp2 and Nlrp14 expression via a RT-qPCR in F1–D1 and F1–D2 oocytes confirmed these results ( Figure 10 F). The expression of several genes involved in primordial follicle activation [ 80 ] was modified in F1–D1 ovaries, such as Nlrp4f and Nlrp14 , that are downstream of Figla and Sohlh2 , respectively, in the promotion of secondary follicle oocyte growth [ 81 , 82 ]. Tcf21 ( Pod1 ) was upregulated in D2 ovaries.…”

Single-cell transcriptomics of staged oocytes and somatic cells reveal novel regulators of follicle activation

“…Historically, samples isolated using animal-age- or oocyte size-specific methods have been applied to the studies of ovarian follicle and oocyte development, for example, using different perinatal timepoints for collecting primordial or primary stage cells ( Pan et al 2005 , Ford et al 2021 ) or collecting size-specific oocytes to represent other follicular stages ( Veselovska et al 2015 , Lv et al 2021 ). These approaches allow for efficient sample collection and analysis in bulk quantity and have therefore established fundamental cellular events as well as identified critical players that drive follicle development.…”

“…Several of these targets have been characterized in follicle activation. For example, studies have identified the importance of WNT signaling in pre-granulosa to granulosa cell differentiation ( Ford et al 2021 , Habara et al 2021 ). Further, TGFB1 signaling mediates cell cycle arrest in pre-granulosa cells ( Hardy et al 2018 , Granados-Aparici et al 2019 ), and inhibiting TGFB1 signaling promotes follicle activation ( Wang et al 2014 ).…”

“…Studies on primordial follicles traditionally involve their manipulation at the tissue or organ level or via single cells collected using animal-age- or oocyte size-specific selection criteria ( Pan et al 2005 , Veselovska et al 2015 , Ford et al 2021 , Lv et al 2021 ). These approaches preclude the ability to study cells in precise follicle stage-specific populations because both animal-age and size-specific cells are heterogeneous with respect to follicle stages.…”

Making a good egg: human oocyte health, aging, and in vitro development

“…This effect is likely through its interaction with the mTORC1-KITL signaling pathway in pregranulosa cells and the KIT-PI3K signaling in oocytes ( 290 , 292 ). Recent studies suggest an involvement of WNT signaling during granulosa cell differentiation from squamous to cuboidal and PFA ( 293 , 294 ). Mouse studies have also suggested that the TNFα signaling through the receptor TNFR2 and downstream nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway would be key positive regulators of PFA.…”