Cocultured porcine granulosa cells respond to excess non-esterified fatty acids during in vitro maturation

Abstract

Background Non-esterified fatty acids (NEFAs) are one of the main lipid components of follicular fluid at concentrations that depend on circulating levels. Elevated levels of NEFAs impair oocyte quality, development potential, and may subsequently influence the metabolism and reproductive fitness of offspring. Granulosa cells (GCs) are the follicular cells that are closely communicating with the oocyte. However, the responses of GCs exposed to high levels of NEFAs when cocultured with cumulus-oocyte complexes (COCs), and how they attenuate the negative effects of NEFAs on oocytes, are unclear. Results To better understand this protective effect, monolayers of porcine GCs were cocultured with COCs during in vitro maturation (IVM) in the presence of elevated levels of NEFAs. Genomic expression analysis was conducted to explore the responses of the GCs to the elevated levels of NEFAs. After limma algorithm analysis, 1,013 genes were differentially expressed between GCs cultured with and without elevated NEFAs. Among them, 438 genes were upregulated and 575 were downregulated. The differentially expressed genes were enriched in pathways related to metabolism, inflammation, and epithelial-mesenchymal transition. Conclusions The pathways and upstream regulators suggested that the cocultured GCs responded to the elevated NEFAs with (1) inhibition of the transition from granulosa to luteal cell, (2) interactions of metabolism change, anti-inflammation, mitochondrial function, and cell transition, (3) intercommunication with cocultured COCs of anti-inflammatory factors.

Metabolism of fatty acids in follicular cells, oocytes, and blastocysts

“…Supplementation of NEFA also had negative effects on porcine granulosa cell proliferation and viability and increased the lipid content in granulosa and cumulus cells ( Ogawa et al 2018 , Pawlak et al 2020 , Shibahara et al 2020 ). Besides, the high level of NEFA altered transcriptomic pattern which is enriched in pathways related to metabolism, inflammation, and impaired mitochondrial function in granulosa cells ( Shi & Sirard 2021 a ). After ovulation, the epithelial–mesenchymal transition (EMT) of granulosa cells to luteal cells occurs, which is associated with changes in estradiol and progesterone production.…”

“…After ovulation, the epithelial–mesenchymal transition (EMT) of granulosa cells to luteal cells occurs, which is associated with changes in estradiol and progesterone production. Inhibition of EMT and altered steroidogenesis in response to high levels of NEFA partly explain the anovulation and impaired follicle development observed in pathological nutritional status ( Foong et al 2006 , Chaput & Sirard 2020 , Shi & Sirard 2021 a ). Moreover, supplementation with NEFA during IVM also decreased the blastocyst formation ( Shibahara et al 2020 , Shi & Sirard 2021 c ).…”

“…In pigs, transcriptome and DNA methylation patterns of blastocysts from oocytes exposed to NEFA were also altered, and affected pathways were mainly related to metabolism and inflammation ( Shi & Sirard 2021 c ). Besides, transcriptomic analysis of GCs cocultured with COCs showed that metabolism, inflammatory responses, mitochondrial dysfunctions, and the EMT were all affected by the elevated level of NEFA ( Shi & Sirard 2021 a ). The differences and similarities in the effects of high levels of NEFA in different species are listed in Table 2 .…”

“…In recent years, more studies focused on the effects of elevated levels of NEFA on granulosa cells, cumulus cells, oocytes, and early embryo development in various species ( Mu et al 2001 , Leroy et al 2005 , Aardema et al 2011 , Van Hoeck et al 2011 , Aardema et al 2013 , Desmet et al 2016 , Sutton-McDowall et al 2016 , Aardema et al 2017 , Itami et al 2018 , Ogawa et al 2018 , Shi & Sirard 2021 a ). Additionally, ovarian dysfunctions induced by high fat diets were also investigated in humans ( Chavarro et al 2007 ), mice ( Yi et al 2012 ) and cattle ( Zachut et al 2010 ).…”

Integrative analysis of transcriptome complexity in pig granulosa cells by long-read isoform sequencing

“…Numerous articles are conducted to explore GCs-related gene functions as well as the precise mechanisms. Previous studies have suggested that miRNAs ( Tian et al, 2020 ; Toms et al, 2021 ), lncRNAs ( Hu et al, 2021 ; Meng et al, 2021 ; Ruszkowska et al, 2018 ; Zhang et al, 2019 ) and mRNAs ( Kulus et al, 2021 ; LaVoie, 2017 ; Shi & Sirard, 2021 ) have critical effects on modulating GCs function along with follicular development in pigs. In addition, for precursor-messenger RNAs (pre-mRNAs), their post-transcriptional modifications like alternative polyadenylation (APA) and alternative splicing (AS) enrich the proteome diversity and have critical effect in different tissues and different developmental stages ( Baralle & Giudice, 2017 ; Di Giammartino, Nishida & Manley, 2011 ; Gruber & Zavolan, 2019 ).…”