Addition of granulosa cells collected from differential follicle stages supports development of oocytes derived from porcine early antral follicles

Self Cite

Xanthan gum (XG) and locust bean gum (LBG) are nontoxic polysaccharides that produce culture substrates. The present study examined the effect of XG-LBG gel on in vitro bovine oocyte growth and gene expression in granulosa cells. Oocytes and granulosa cell complexes (OGCs) were cultured in vitro on plastic culture plate (Plate) or XG-LBG gel for 16 days. OGCs formed a dome-like cavity surrounding the oocytes on plate but formed a spherical follicle structure on XG-LBG gel. The total granulosa cell numbers of the OGCs and their survival rate was greater for OGCs cultured on XG-LBG gel than for those cultured on plate. Oocytes grown on XG-LBG gels had higher lipid and mitochondrial content, as well as a larger diameter, than their plate counterparts. When oocytes grown in vitro were subjected to in vitro maturation and fertilization, the normal fertilization rate was significantly higher for oocytes developed on XG-LBG gel than that of oocytes cultured on the plate counterpart. RNAseq of the granulosa cells revealed that genes associated with focal adhesion, phosphatidylinositol 3'-kinase-Akt and Hippo signaling, and regulation of actin cytoskeleton were upregulated in granulosa cells of OGCs cultured on XG-LBG gel compared with those cultured on plate. K E Y W O R D S bovine, early antral follicle, locust bean gum, oocyte, xanthan gum 1 | INTRODUCTION Bovine embryos are produced from oocytes collected from antral follicles (AFs; 3-5 mm in diameter), but the number of large AFs is limited. Establishing methods for in vitro oocyte growth from small AFs has a great impact on expanding oocyte resources for domestic animals, humans, and endangered wild animals; however, the efficiency of in vitro oocyte development is still low, and oocytes grown in vitro generally have poor developmental competence compared with oocytes collected from large AFs. The major differences between plastic-based in vitro culture conditions versus intra-follicular environments include the presence of focal adhesions, interactions with the extracellular matrix (ECM), and stiffness of the substrate. Although the medium compositions and hormones in in vitro oocyte culture conditions have been studied, the stiffness of the culture substrate has not been extensively studied. Regarding simple modifications to the cell culture conditions, Munakata et al. (2017) grew porcine oocyte granulosa cell complexes

Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Effects of a gel culture system made of polysaccharides (xanthan gum and locust bean gum) on in vitro bovine oocyte development and gene expression of the granulosa cells

Sugimoto

Inoue

Tanaka

et al. 2021

Self Cite

“…For this purpose, mural granulosa cells were cultured with labeled CTB, and the effects of BMP15, GDF9, and FGF8 were determined. Given the well‐documented synergistic interaction between BMP15 and GDF9 (Sugiura et al, 2023), we also examined the effects of cotreatment with both BMP15 and GDF9. As shown in Figure 6, while the fluorescence intensities were not affected by the individual treatment with any of the ODPFs (Figure 6a,b), cotreatment with both BMP15 and GDF9 exhibited significant enhancement in CTB uptake (Figure 6c,d).…”

Section: Resultsmentioning

confidence: 99%

“…The precise coordination of these factors is crucial for the normal development and function of follicles and, therefore, for normal female fertility. It is widely accepted that oocytes play a central role in this coordination (Emori & Sugiura, 2014; Eppig et al, 2002; Russell et al, 2016; Sugiura et al, 2023). Oocyte‐derived paracrine factors (ODPFs) regulate the follicular response to gonadotropins by affecting the expression of gonadotropin receptors in granulosa cells of mice (Eppig et al, 1997; Otsuka et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Dynamics of extracellular vesicle uptake by mural granulosa cells in mice

Edure,

Matsuno,

Matsushita

et al. 2024

Extracellular vesicles (EVs) play an important role in the development and function of mammalian ovarian follicles. However, the mechanisms by which they are taken up by the follicular granulosa cells remain unclear. In addition, while oocytes play a pivotal role in follicular development, the possible interactions between oocyte‐derived paracrine factors (ODPFs) and EV signals are unknown. Therefore, this study aimed to elucidate the mechanism of EV uptake and the effects of ODPFs on EV uptake by follicular somatic mural granulosa cells in mice. Fluorescence‐labeled transferrin (TRF) and cholera toxin B (CTB), substrates for clathrin‐ and caveolae‐mediated endocytosis, respectively, were taken up by mural granulosa cells in vitro. Their uptake was inhibited by Pitstop 2 and genistein, inhibitors of clathrin and caveolae pathways, respectively. Mural granulosa cells took up EVs, and this uptake was suppressed by Pitstop 2 and genistein. Moreover, ODPFs promoted the uptake of EVs and CTB, but not TRF, by mural granulosa cells. These results suggest that mural granulosa cells take up EVs through both clathrin‐ and caveolae‐mediated endocytosis and that oocytes may promote caveolae‐mediated endocytosis to facilitate the uptake of EVs.

“…Concurrently, P 4 is secreted through GCs in response to LH levels (D. Zhang et al, 2019). Adding GCs from more mature follicles to the oocyte and cumulus complex increases oocyte metabolism and promotes their growth (Ishiguro et al, 2018). In contrast, GC apoptosis reduces the quality of oocytes and embryos (Almeida et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Effects of BAMBI on luteinized follicular granulosa cell proliferation and steroid hormone production in sheep

Zhang

Guo

et al. 2023

Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) regulates mammalian ovarian follicle growth and maturation; however, its effect on luteinized granulosa cells (LGCs) in sheep ovarian follicles remains unknown.Here we explored the regulatory role of LGC functions and steroid hormone synthesis by BAMBI. Multiple sequence alignment revealed that the sheep BAMBI gene sequence was relatively conserved. Sheep LGCs were strongly positive for BAMBI. LGC proliferation increased when BAMBI was silenced and decreased when BAMBI was overexpressed. After BAMBI overexpression, the expression of CASP3, CASP8, CASP9, and BAX significantly increased, whereas that of BCL2 and the ratio of BCL2/BAX expression decreased. The opposite was observed after BAMBI silencing. CDKN1A, CCND1, and CCND2 were downregulated with BAMBI overexpression and upregulated with BAMBI silencing. Expression of steroid hormonerelated genes (CYP11A1, STAR, and 3BHSD), except CYP19A1, significantly increased after BAMBI overexpression. Moreover, estrogen and progesterone secretion increased after BAMBI overexpression and decreased after BAMBI interference.The effect of the exogenous addition of bone morphogenetic protein 2 (BMP2) on GCs was similar to that of BAMBI overexpression. In conclusion, BAMBI can regulate the proliferation and steroid hormone synthesis of sheep LGCs, and BMP2 can affectLGCs as an activator of BAMBI. These findings provide a basis for further research on the physiological role of BAMBI.