Beneficial Effects of Neurotrophin-4 Supplementation During in vitro Maturation of Porcine Cumulus-Oocyte Complexes and Subsequent Embryonic Development After Parthenogenetic Activation

Abstract

Neurotrophin-4 (NT-4) is a neurotrophic factor that plays an important role in follicular development and oocyte maturation. However, it is not yet known whether NT-4 is related to oocyte maturation and follicular development in pigs. This study aims to investigate the effects of NT-4 supplementation during in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after parthenogenetic activation (PA). First, NT-4 and its receptors (TrkB and p75NTR) were identified through fluorescent immunohistochemistry in porcine ovaries. NT-4 was mainly expressed in theca and granulosa cells; phospho-TrkB and total TrkB were expressed in theca cells, granulosa cells, and oocytes; p75NTR was expressed in all follicular cells. During IVM, the defined maturation medium was supplemented with various concentrations of NT-4 (0, 1, 10, and 100 ng/mL). After IVM, the nuclear maturation rate was significantly higher in the 10 and 100 ng/mL NT-4 treated groups than in the control. There was no significant difference in the intracellular reactive oxygen species levels in any group after IVM, but the 1 and 10 ng/mL NT-4 treatment groups showed a significant increase in the intracellular glutathione levels compared to the control. In matured cumulus cells, the 10 ng/mL NT-4 treatment group showed significantly increased cumulus expansion-related genes and epidermal growth factor (EGF) signaling pathway-related genes. In matured oocytes, the 10 ng/mL treatment group showed significantly increased expression of cell proliferation-related genes, antioxidant-related genes, and EGF signaling pathway-related genes. We also investigated the subsequent embryonic developmental competence of PA embryos. After PA, the cleavage rates significantly increased in the 10 and 100 ng/mL NT-4 treatment groups. Although there was no significant difference in the total cell number of blastocysts, only the 10 ng/mL NT-4 treatment group showed a higher blastocyst formation rate than the control group. Our findings suggest that supplementation with the 10 ng/mL NT-4 can enhance porcine oocyte maturation by interacting with the EGF receptor signaling pathway. In addition, we demonstrated for the first time that NT-4 is not only required for porcine follicular development, but also has beneficial effects on oocyte maturation and developmental competence of PA embryos.

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

“…In mice, the NGFR mRNA is expressed in all follicular cells (mural granulosa cells and CCs), except oocytes ( Kawamura et al, 2005 ); in cattle, the p75 NTR mRNA ( Da Silva et al, 2005 ) and protein ( Levanti et al, 2005 ) are expressed in both oocytes and CCs. In porcine studies, both p75 NTR mRNA ( Lee et al, 2007 ; Kim et al, 2021 ) and protein ( Levanti et al, 2005 ; Kim et al, 2021 ) have been detected in all follicular cells (granulosa cells, CCs, and oocytes). In our previous study, NT-4-related signaling pathways were identified in response to the MAPK/ERK pathway in matured porcine COCs by qPCR analysis ( Kim et al, 2021 ).…”

“…In porcine studies, both p75 NTR mRNA ( Lee et al, 2007 ; Kim et al, 2021 ) and protein ( Levanti et al, 2005 ; Kim et al, 2021 ) have been detected in all follicular cells (granulosa cells, CCs, and oocytes). In our previous study, NT-4-related signaling pathways were identified in response to the MAPK/ERK pathway in matured porcine COCs by qPCR analysis ( Kim et al, 2021 ). In the current study, the relative mRNA expression levels of NGFR and its downstream gene, NFKB1 , were analyzed in matured porcine COCs upon NT-4 treatment during IVM to confirm the involvement of the NT-4/p75 NTR -related signaling pathway.…”

“…Previous studies have demonstrated that the NT-4 mRNA and protein are expressed in granulosa cells, CCs, and oocytes ( Dissen et al, 1995 ; Dissen et al, 2002 ; Seifer et al, 2006 ; Kim et al, 2021 ). In particular, many studies have indicated that NT-4 improves follicular assembly in vitro ( Dissen et al, 1995 ; Ojeda et al, 2000 ; Anderson et al, 2002 ; Dissen et al, 2002 ; Harel et al, 2006 ; Farhi et al, 2011 ).…”

“…Porcine ovaries were collected at a local slaughterhouse and transferred to the laboratory within 3 h in 0.9% (w/v) NaCl at 37°C–39°C. Oocyte collection and IVM were performed as previously described ( Kim et al, 2021 ). Briefly, COCs were aspirated from 3–6-mm ovarian follicles using a 10-ml disposable syringe with an 18-G needle attached.…”

“…Our previous study determined the optimal NT-4 concentration for porcine IVM and measured the relative expression levels of TrkB-related genes ( Kim et al, 2021 ). In the present study, we investigated the physiological and functional roles of NT-4 in the maturation of porcine cumulus–oocyte complexes (COCs).…”