3, 3', 5-Triiodo-L-thyronine affects polarity proteins of bovine Sertoli cells via WT1/non-canonical Wnt signaling pathway.

To determine the role of 3, 3', 5-triiodo-L thyroxine (T3) in the differentiation of Sertoli cells (SCs) and the factors influencing maturity via the Wilms' tumor 1 (WT1)/non-canonical Wnt signaling pathway, high purity SCs were isolated from newborn calves' testes and cultured in vitro. The SCs were stimulated with T3, and co-treated with short interference (si) RNA to knockdown endogenous WT1 and non-canonical Wnt signalling inhibitor Wnt-c59. Our results suggested that the addition of different concentrations (0, 25, 50, and 100 nM) of T3 in the culture medium changed the expression of KRT-18 (SCs immature marker) and accelerated the differentiation of SCs. T3 (100 nM) treatment induced up-regulated expression of WT1 over time (p < 0.05), while the expression of polarity proteins (Par3, Par6b, and E-cadherin) and Wnt4 were affected to varying degrees (p < 0.05). SCs were treated simultaneously with T3 + Wnt-c59 and T3 + WT1 siRNA, and the results showed that T3 could affect the expression of polarity proteins via WT1/non-canonical Wnt signaling pathway. These data put together indicate that T3 plays a dependent role in the induction of bovine SCs differentiation via WT1/non-canonical Wnt signaling pathway in vitro. This study proposes for the first time that WT1 is a major target for T3.

Wilms' tumour 1 (WT1) negatively regulates the expression of connexin 43 via a non-canonical Wnt signalling pathway in cultured bovine Sertoli cells.

The gap junction protein connexin (Cx) 43 between adjacent Sertoli cells (SCs) is the main testicular factor regulating the growth and development of SCs, and plays a vital role in controlling cell differentiation and maturation. However, the endogenous testicular factors that regulate Cx43 and the downstream signalling pathways that mediate Cx43-dependent SC differentiation are unclear. In this study, high-purity SCs were isolated from newborn calves' testes by differential adherence. The SCs were then cultured invitro and treated with short interference RNA to knockdown endogenous Wilms' tumour 1 (WT1). In WT1-knockdown SCs, Cx43 expression was upregulated. To elucidate the intracellular signalling mechanism of Cx43 in the differentiation and maturation of immature SCs, SCs were treated simultaneously with non-canonical Wnt signalling inhibitors CCG-1423 and GO-6983; in these SCs, Cx43 expression was upregulated. Together, these data indicate that WT1 negatively regulates the expression of Cx43 produced from SCs via a non-canonical Wnt signalling pathway in cultured bovine SCs.

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.

Soluble immune complexes and immunoglobulin (IgG, IgA and IgM) levels in Nigerians with primary liver cell carcinoma.

Circulating soluble immune complexes and serum immunoglobulins (G,A and M) levels were determined in patients with primary liver cell carcinoma (PLCC) and healthy subjects by the polyethylene glycol precipitation and single radial immunodiffusion methods respectively. A considerably higher proportion of the patients than the controls had elevated levels of soluble immune complexes, IgG and IgM were significantly higher in the patients than the controls, that of IgA was lower. Correlation studies showed association between serum concentration of IgG, IgA and IgM and the levels of circulating soluble immume complexes. Several factors may influence our findings of elevated concentrations of soluble immune complexes and serum immunoglobulins G and M as well as the positive correlations between these indices. It could be as a result of increased rate of production and release of antigen from the tumour; enhanced interaction of antibody with membrane antigens at the tumour cell surface which promoted release of immune complexes or/and decreased rate of elimination of the complexes from the body of phagocytosis. That antibodies are required for the formation of immune complexes may explain our observation of increased levels of IgG and IgM.

Complement proteins and soluble immune complex levels in Nigerians having primary liver cell carcinoma.

Serum Complement (C3), proteins and circulating immune complex levels were estimated in Nigerians having primary liver cell carcinoma and control subjects by immunodiffusion in agar, biuret and polyethylene glycol precipitation methods respectively. The patients were observed to have diminished mean C3 and albumin concentrations whereas the mean total proteins, globulins and soluble immune complex levels were elevated. About 80 percent of the patients who had depressed serum C3 concentrations also had elevated levels of circulating immune complexes. There is however no correlation between the complement (C3) concentrations and the serum levels of immune complexes.

Complement proteins and soluble immune complex levels in Nigerians having primary liver cell carcinoma

Serum complement(C3); proteins and circulating immune complex levels were estimated in Nigerians having primary liver cell carcinoma and control subjects by immunodiffusion in agar; biuret and polyethylene glycol precipitation methods respectively. There is however no correlation between the complement (C3) concentrations and the serum levels of immune complexes

Serum immunoglobulin levels in normal, premature and postmature newborns and their mothers.

Immunoglobulin (Ig) levels (IgG, IgA, IgM and IgD) were measured in 92 paired maternal and cord sera by the modified single radial immunodiffusion method. There was a progressive increase in mean IgG levels as the gestational age increased which reached a maximum of 1771 mg/100 ml in the postmature babies. The mean IgG level of infants of 32--36 weeks' gestation was lowest (1395 mg/100 ml). The maternal IgG levels were generally higher than those of the cord sera. The mean maternal IgG level (1773 mg/100 ml) was considerably higher than the corresponding level in the cord sera of the lowest gestational age group. Such marked difference was not present in newborns who were of normal gestational ages. The mean IgA and IgM levels of the cord sera were low. There were no significant differences between the mean IgA and IgM levels of newborns in different gestational age groups, even when their mothers showed variations in mean IgA and IgM levels. There was no detectable level of IgD in the cord sera.