R-Spondin 2 and WNT/CTNNB1 Signaling Pathways Are Required for Porcine Follicle Development and In Vitro Maturation.

Seon-Ung Hwang,Junchul David Yoon,Hyerin Choi,Sang-Hwan Hyun,Dongjin Oh,Eunhye Kim,Mirae Kim,Lian Cai

doi:10.3390/ani11030709

Seon-Ung Hwang, Junchul David Yoon + Show 6 more

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https://doi.org/10.3390/ani11030709

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Abstract

Simple SummaryA recent mouse study reported that R-spondin2 promotes follicular development by activating WNT/Catenin beta-1 (CTNNB1) signaling in granulated cells. Our results demonstrate that RSPO2, CTNNB1, G-protein coupled receptor 4 (LGR4), and G-protein coupled receptor 5 (LGR5) factors play a major role in porcine follicular development, and epidermal growth factor receptor (EGFR) signaling is essential for in vitro maturation. The R-spondin2 and WNT/CTNNB1 signaling pathways are involved in porcine follicle development, and it is expected that the EGFR-ERK signaling pathway is also involved.The secretion of oocyte-derived paracrine factors, such as R-spondin2, is an essential mechanism for follicle growth by promoting the proliferation and differentiation of cumulus cells around oocytes. In the present study, we aimed to identify the effect of R-spondin2 during follicular development. First, R-spondin2-related factors (R-spondin2, CTNNB1, LGR4, and LGR5) were identified through immunofluorescence in porcine ovarian tissue. CTNNB1 was expressed in ooplasm, and CTNNB1 and LGR4 were expressed in granulosa cells. In addition, R-spondin2, LGR4, and LGR5 were expressed in the theca interna. These results imply that these proteins play a major role in porcine follicular development. In addition, the effects of R-spondin2 on the in vitro maturation process of porcine cumulus oocyte complexes and subsequent embryonic development were confirmed. A treatment of 100 ng/mL R-spondin2 in the in vitro maturation (IVM) process increased nuclear maturation and increased the expression of EGFR mRNA in cumulus cells. The EGFR-ERK signal is essential for oocyte maturation, ovulation, and luteinization. R-spondin2 treatment also increased the expression of CTNNB1 and EGFR in primary cultured cumulus cells. In conclusion, RSPO2 and WNT/CTNNB1 signaling pathways are required for porcine follicle development and are predicted to be involved in the EGFR-ERK signaling pathway.

Highlights

In vitro maturation (IVM) is a widely used model that effectively mimics the growth of follicles in vivo
roof plate-specific spondin2 (RSPO2) was mainly identified in theca interna (T.I.), and Catenin Beta 1 (CTNNB1) was expressed in Granulosa cells and ooplasm
This study is the first to confirm the localization of RSPO2, CTNNB1, LGR4, and LGR5 in porcine ovary and to confirm the effect of RSPO2 in the porcine in vitro maturation (IVM) process

Summary

Introduction

In vitro maturation (IVM) is a widely used model that effectively mimics the growth of follicles in vivo. At least three oocyte-derived factors have been identified to date to promote the growth of granulosa cells: roof plate-specific spondin (RSPO2), growth differentiation factor-9, and bone morphogenic protein-15 [1]. Both Growth differentiation factor-9 (GDF9) and RSPO2 have been found to be essential for granulosa cell proliferation: RSPO2 activates WNT/Catenin Beta 1 (CTNNB1) signaling, and GDF9 signaling has been reported to act through the Bone morphogenetic protein receptor type II (BMPR2)/Activin receptor type-1B (ALK4) heterodimer receptor and the Mothers against decapentaplegic homolog 2/3 (SMAD2/3) pathway in granulosa cells [2,3]. RSPO2 is one of the four distinct members of the RSPO protein family (RSPO1–4), which show amino acid identities greater than 40% to 60% and are expected to exhibit structural homology [5]

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