Physiological and Functional Roles of Neurotrophin-4 During In Vitro Maturation of Porcine Cumulus–Oocyte Complexes

Abstract

Neurotrophin-4 (NT-4), a granulosa cell-derived factor and a member of the neurotrophin family, is known to promote follicular development and oocyte maturation in mammals. However, the physiological and functional roles of NT-4 in porcine ovarian development are not yet known. The aim of this study was to investigate the physiological role of NT-4-related signaling in the in vitro maturation (IVM) of porcine cumulus–oocyte complexes (COCs). The NT-4 protein and its receptors were detected in matured porcine COCs via immunofluorescence analysis. NT-4 was shown to promote the maturation of COCs by upregulating NFKB1 transcription via the neurotrophin/p75NTR signaling pathway. Notably, the mRNA expression levels of the oocyte-secreted factors GDF9 and BMP15, sperm–oocyte interaction regulator CD9, and DNA methylase DNMT3A were significantly upregulated in NT-4-treated than in untreated porcine oocytes. Concurrently, there were no significant differences in the levels of total and phosphorylated epidermal growth factor receptor and p38 mitogen-activated protein kinase between NT-4-treated and untreated cumulus cells (CCs); however, the level of phosphorylated ERK1/2 was significantly higher in NT-4-treated CCs. Both total and phosphorylated ERK1/2 levels were significantly higher in NT-4-treated than in untreated oocytes. In addition, NT-4 improved subsequent embryonic development after in vitro fertilization and somatic cell nuclear transfer. Therefore, the physiological and functional roles of NT-4 in porcine ovarian development include the promotion of oocyte maturation, CC expansion, and ERK1/2 phosphorylation in porcine COCs during IVM.

Neurotrophin-4 promotes the specification of trophectoderm lineage after parthenogenetic activation and enhances porcine early embryonic development

“…Since the present study is the first to show the localization of neurotrophic factors and their receptors in porcine PA blastocysts, further studies are needed to confirm their localization patterns across different stages of porcine embryonic development. According to our previous study, NT-4, total-TrkB, phospho-TrkB, and p75 NTR were localized in both the nuclei and cytoplasm of porcine cumulus cells, whereas they were only localized to ooplasms of in vitro matured-porcine oocytes ( Kim et al, 2022 ). Therefore, our present study demonstrated that the localization patterns of NT-4 and its receptors are cell-type specific.…”

“…Recombinant human NT-4 (450-04) was purchased from PeproTech (Rocky Hill, NJ, United States). NT-4 protein was used after being dissolved in Dulbecco’s phosphate-buffered saline (DPBS; LB001-02; WELGENE, Gyeongsan, Gyeongsangbuk-do, Republic of Korea) with 0.1% (w/v) bovine serum albumin, as previously described ( Kim et al, 2022 ). Verteporfin (HY-B0146; MedChemExpress, Princeton, NJ, United States), a yes-associated protein (YAP) inhibitor, was then dissolved in dimethyl sulfoxide (DMSO; Sigma Aldrich Corporation, St. Louis, MO, United States).…”

“…NT-4 is a neurotrophic factor related to the transforming growth factor β superfamily ( Seifer et al, 2002 ). It is an intra-ovarian growth factor derived from granulosa cells in the mammalian ovary and promotes oocyte maturation in pigs ( Kim et al, 2021 ; Kim et al, 2022 ). Among the families of neurotrophins, both BDNF and NT-4 bind to tropomyosin receptor tyrosine kinase B (TrkB), a high-affinity receptor, and p75 neurotrophin receptor (p75 NTR ), a low-affinity receptor, and these are known to play physiologically similar roles in cortical development ( McAllister et al, 1995 ) and oocyte maturation ( Seifer et al, 2002 ).…”

“…Our previous studies have demonstrated the effects of NT-4 on IVM of oocytes in the porcine reproductive system ( Kim et al, 2021 ; Kim et al, 2022 ). As NT-4 plays an important role in porcine follicular development and oocyte maturation, we further evaluated and confirmed whether NT-4 improved IVP efficiency by affecting early embryonic development.…”