Influence of Wilms' tumor suppressor gene WT1 on bovine Sertoli cells polarity and tight junctions via non-canonical WNT signaling pathway.

Sertoli cells (SCs) are polarized epithelial cells and provide a microenvironment for the development of germ cells (GCs). The Wilms' tumor suppressor gene WT1 which support spermatogenesis is expressed explicitly in SCs. This study investigated the effect of WT1 on the polarity and blood-testis barrier (BTB) formation of bovine SCs in order to provide theoretical and practical bases for the spermatogenic process in mammals. In this study, newborn calf SCs were used as research material, and the RNAi technique was used to knockdown the endogenous WT1. The results show that WT1 knockdown did not affect the proliferation ability of SCs, but down-regulated the expression of polarity-associated proteins (Par3, Par6b, and E-cadherin), junction-associated protein (occludin) and inhibits transcription of downstream genes (WNT4, JNK, αPKC, and CDC42) in non-canonical WNT signaling pathway. WT1 also altered ZO-1 and occludin protein distribution. Overexpression of WNT1 did not affect the expression of Par6b, E-cadherin, and occludin, whereas the non-canonical WNT signaling pathway inhibitors wnt-c59, CCG-1423, and GO-6983 down-regulated the expression of Par6b, E-cadherin, and occludin respectively. This study indicates that WT1 mediates the regulation of several proteins involved in bovine SCs polarity maintenance and intercellular tight junctions (TJ) by non-canonical WNT signaling pathway.

Thyroid hormone (T3) is involved in inhibiting the proliferation of newborn calf Sertoli cells via the PI3K/Akt signaling pathway in vitro.

The experiment was designed to study the effects of Thyroid hormone (T3) on the proliferation and differentiation of newborn calf Sertoli cells (SCs) to provide a theoretical and practical basis for increased testicular semen production. In this experiment, the cck8 method was used to detect the effects of different concentrations of T3 on the proliferation rate of newborn calf SCs. qPCR and Western Blot methods were used to explore the effects of T3 on the proliferation and differentiation of calves SCs and whether T3 through Wnt/ß-catenin and PI3K/Akt pathways can regulate the proliferation and differentiation of SCs. We found that dosage (T3) and time correlated with proliferation inhibition of SC. T3 inhibited the proliferation of SC by down-regulating cyclinD1, upregulating p21Cip, p27Kip1, and other cell-cycle factors. By up-regulating AR and down-regulating KRT-18, T3 promoted the maturated differentiation of SC. T3 could not affect the expression of ß-catenin in SC of newborn calf, indicating that T3 may not regulate SCs proliferation through the Wnt pathway. T3 also negatively regulated the gene expression and protein levels of some genes in the PI3K/Akt signaling pathway. We concluded that T3 inhibited newborn calf SCs proliferation through the PI3K/Akt signaling pathway and possibly promoted their differentiation.

Microcystin-leucine arginine (MC-LR) induced inflammatory response in bovine sertoli cell via TLR4/NF-kB signaling pathway.

Sertoli cells were treated with 0, 20, 40, 60 and 80 µg/L of MC-LR to investigate its toxic effects, mechanism of action and immune response of the cells. Our results revealed that treatment containing 20 µg/L of MC-LR was non-toxic to the cells. Treatments containing 40, 60 and 80 µg/L of MC-LR reduced the cell viability, induced nuclear morphological changes and downregulated the blood-testis barrier constituent proteins within 48 h after treatment. The toll-like receptor 4 (TLR4) and nuclear factor-kappaB (NF-kB) were activated and significantly (P < 0.05) upregulated in cells treated with 40, 60 and 80 µg/L of MC-LR compared to the control. The pro-inflammatory cytokines were upregulated within 48 h after treatment. However commencing from 72 h, upregulation of anti-inflammatory cytokines and expression of blood-testis barrier constituent proteins was observed. This study indicates that MC-LR induced inflammatory response in bovine Sertoli cell via activation of TLR4/NF-kB signaling pathway.

Sodium Selenite inhibits mitophagy, downregulation and mislocalization of blood-testis barrier proteins of bovine Sertoli cell exposed to microcystin-leucine arginine (MC-LR) via TLR4/NF-kB and mitochondrial signaling pathways blockage.

This study was conducted to investigate the ameliorative effect of selenium on microcystin-LR induced toxicity in bovine Sertoli cells. Bovine Sertoli cells were pretreated with selenium (Na2SeO3) for 24 h after which selenium pretreated and non-pretreated Sertoli cells were cultured in medium containing 10% heat activated fetal bovine serum FBS+ 80 µg/L MC-LR to assess its ameliorative effect on MC-LR toxicity. The results show that selenium pretreatment inhibited the MC-LR induced mitophagy, downregulation and mislocalization of blood-testis barrier constituent proteins in bovine Sertoli cells via NF-kB and cytochrome c release blockage. The observed downregulation of electron transport chain (ETC) related genes (mt-ND2, COX-1, COX-2) and upregulation of inflammatory cytokines (IL-6, TNF-α, IL-1ß, IFN-γ, IL-4, IL-10, 1 L-13, TGFß1) in non-pretreated cells exposed to MC-LR were ameliorated in selenium pretreated cells. There was no significant difference (P > 0.05) in the protein levels of blood-testis barrier constituent proteins (ZO-1, occludin, connexin-43, CTNNB1, N-cadherin) and mitochondria related genes (mt-ND2, COX-1, COX-2, ACAT1, mtTFA) of selenium pretreated Sertoli cell compared to the control. Taken together, we conclude that selenium inhibits MC-LR caused Mitophagy, downregulation and mislocalization of blood-testis barrier proteins of bovine Sertoli cell via mitochondrial and TLR4/NF-kB signaling pathways blockage.

Selenium (Na2SeO3) Upregulates Expression of Immune Genes and Blood-Testis Barrier Constituent Proteins of Bovine Sertoli Cell In Vitro.

Sertoli cells were isolated from newborn calves and cultured in a medium supplemented with 0, 0.25, 0.50, 0.75, and 1.00 mg/L of sodium selenite to study their immune stimulatory effect, influence on cell's viability, and expression of blood-testis barrier proteins (occludin, connexin-43, zonula occluden, E-cadherin) using quantitative PCR and western blot analyses. Results showed that medium supplemented with 0.50 mg/L of selenium significantly (P < 0.05) promoted cell viability, upregulated toll-like receptor gene (TLR4), anti-inflammatory cytokines (IL-4, IL-10, TGFß1), and expressions of blood-testis barrier proteins, and modulated expressions of pro-inflammatory cytokines (TNF-α, IL-1ß, IFN-γ). Sertoli cells grown in culture medium supplemented with 0.25 mg/L of selenium significantly upregulated TLR4, IL-4, IL-10, TGFß1, and blood-testis barrier proteins compared to the control group. Sodium selenite supplementation at 0.75 and 1.00 mg/L levels was cytotoxic and temporarily downregulated the expression of blood-testis barrier protein within 24 h after culture; however, commencing from 72 h post culture, increased cell viability and upregulation of expression of blood-testis barrier proteins were observed. In conclusion, the results of this study showed that selenium supplementation in the culture medium up to 0.50 mg/L concentration upregulates immune genes and blood-testis barrier constituent proteins of bovine Sertoli cells.

Effects of glial cell line-derived neurotrophic factor and leukemia-inhibitory factor on the behavior of two calf testis germline stem cell colony types.

Germline stem cells are the only such capable of transmitting genetic information in vivo. The isolation and culture of these cells in vitro provide a unique model to understand sperm differentiation and hence, spermatogenesis and male fertility. In this study, we isolated, purified, and cultured germline stem cells from the testes of newborn calves. Moreover, we investigated the effects of glial cell line-derived neurotrophic factor (GDNF) and leukemia-inhibitory factor (LIF) on their proliferation. Male calf germline stem cells were found to be pluripotent, and able to form grape-like and embryonic stem cell (ES)-like colonies when cultured. GDNF promoted proliferation of the former, whereas LIF induced growth of the latter. The grape-like colonies retained their germline stem cell characteristics, whereas the ES-like colonies demonstrated more primitive attributes. This investigation established a male calf germline stem cell culture model that may serve as a foundation for further studies aiming to understand the properties of such cells.