Evidence that gene expression of ovarian follicular tight junction proteins is regulated in vivo and in vitro in cattle

Abstract

Tight junctions (TJ) are common paracellular sealing structures that control the transport of water, ions, and macromolecules across cell layers. Because the role of TJ in bovine follicular development is unknown, we investigated the developmental and hormonal regulation of the transmembrane TJ protein, occludin (OCLN), and the cytoplasmic TJ proteins, TJ protein 1 (TJP1) and cingulin (CGN) in bovine granulosa cells (GC) and theca cells (TC). For this purpose, bovine GC and TC were isolated from large (>8 mm) and/or small (1 to 5 mm) follicles and either extracted for real-time PCR (qPCR) or cultured in vitro. The abundances of both and mRNA were greater ( < 0.05) in TC than GC, whereas the mRNA abundance was greater ( < 0.05) in GC than TC. The abundance of mRNA in both GC and TC was greater ( < 0.05) in small follicles compared with large follicles, whereas the GC of large follicles had less ( < 0.05) mRNA abundance than the GC of small follicles. The abundance of mRNA in GC or TC did not differ ( > 0.10) among follicle sizes. In vitro treatment with various growth factors known to affect ovarian folliculogenesis indicated that , , and were hormonally regulated. Fibroblast growth factor 9 (FGF9) decreased ( < 0.05) the and mRNA abundances. Tumor necrosis factor α (TNFα) and vascular endothelial growth factor A (VEGFA) increased ( < 0.05) the mRNA abundance but decreased ( < 0.05) the mRNA abundance. Dexamethasone (DEX) increased ( < 0.05) and mRNA abundances. Epidermal growth factor (EGF) decreased ( < 0.05) and dihydrotestosterone (DHT) increased ( < 0.05) the abundances of , , and mRNA. We propose that the downregulation of OCLN and other TJ proteins during follicular development could reduce barrier function, thereby participating in increasing follicle size by allowing for an increase in the volume of follicular fluid as well as by allowing additional serum factors into the follicular fluid that potentially may directly impact GC functions. The results of the current study indicate the following in cattle: 1) gene expression of TJ proteins (i.e., , , and ) differs between GC and TC and changes with follicle size, and 2) autocrine, paracrine, and endocrine regulators, such as FGF9, EGF, DHT, TNFα, and glucocorticoids, modulate , , and mRNA abundance in TC in vitro.

The role of tight junction proteins in ovarian follicular development and ovarian cancer

“…1D), and in vivo inhibition of VEGFA using soluble recombinant VEGF receptor proteins caused a decrease in theca CLDN5 protein and an increase in granulosa OCLN protein in and CGN mRNA in bovine theca cells (Zhang et al 2017). Previously, we reported that TC had 375 greater OCLN and TJP1 mRNA than GC whereas GC had greater CGN mRNA than TC of bovine follicles (Figure 2A; Zhang et al 2017). Therefore we conducted an additional study to determine if abundance of CGN mRNA in GC differed among estrogen-active and estrogen-378 inactive small, medium and/or large follicles in cattle, and found that there were no significant differences in GC CGN mRNA expression among estrogen-inactive follicles of different sizes ( Figure 2B).…”

“…CLDN5 protein localizes around granulosa cells in marmoset preantral follicles (Fig. 1D), and in vivo inhibition of VEGFA using soluble recombinant VEGF receptor proteins caused a decrease in theca CLDN5 protein and an increase in granulosa OCLN protein in and CGN mRNA in bovine theca cells (Zhang et al 2017). Previously, we reported that TC had 375 greater OCLN and TJP1 mRNA than GC whereas GC had greater CGN mRNA than TC of bovine follicles (Figure 2A; Zhang et al 2017).…”

“…In zebrafish mid-vitellogenic follicles, GDF9 357 decreased OCLN mRNA whereas activin A, BMP15 nor TGFβ had no effect (Clelland & Kelly 2011). Gene expression for the TJ proteins OCLN and TJP1 are more prevalent in bovine TC than GC, and both are decreased with follicular development, indicating a role of these TJ 360 proteins in bovine ovarian folliculogenesis (Zhang et al 2017) (Table 2). In contrast, in zebrafish ovaries, OCLN protein was found in both GC and TC and its content did not change during follicular development but was regulated by GDF9 in different follicular stages (Clelland & 363 Kelly 2010) ( Table 2).…”

“…VEGF and hCG decreased expression of TJ proteins (i.e., OCLN) during follicular development and CL formation in primates (Rodewald et al 2007, Herr 450 et al 2015, and it was suggested that VEGF altered luteal permeability via regulation of CLDN5 (Rodewald et al 2007). Recently, VEGFA was shown to increase OCLN mRNA and decrease CGN mRNA in theca cells of cattle (Zhang et al 2017). Therefore, hormones that 453 regulate vascular development may also be regulating TJ proteins.…”

“…In contrast, in zebrafish ovaries, OCLN protein was found in both GC and TC and its content did not change during follicular development but was regulated by GDF9 in different follicular stages (Clelland & 363 Kelly 2010) ( Table 2). A recent study in cattle revealed that TC gene expression of TJ proteins OCLN, TJP1 and CGN are regulated by autocrine, paracrine and endocrine regulators, such as fibroblast growth factor 9 (FGF9), epidermal growth factor (EGF), dihydrotestosterone (DHT), 366 tumor necrosis factor α (TNFα), VEGFA and glucocorticoids in vitro (Zhang et al 2017).…”