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doi: 10.1038/srep28012.

IL-6 mediates differentiation disorder during spermatogenesis in obesity-associated inflammation by affecting the expression of Zfp637 through the SOCS3/STAT3 pathway

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IL-6 mediates differentiation disorder during spermatogenesis in obesity-associated inflammation by affecting the expression of Zfp637 through the SOCS3/STAT3 pathway

Guizhen Huang et al. Sci Rep. .

Abstract

Zfp637 is a recently identified zinc finger protein, and its functions remain largely unknown. Here, we innovatively demonstrate the effects of Zfp637 on the differentiation of mouse spermatogonia and on its downstream target gene SOX2 in vitro. Obesity has been recognized as a chronic inflammatory disease that leads to decreased sexual function and sexual development disorders. We observed higher levels of IL-6 in serum and testis homogenates from obese mice compared with control mice. We also demonstrated that high levels of IL-6 inhibited Zfp637 expression, and we elucidated the underlying mechanisms. SOCS3 overexpression and STAT3 phosphorylation inhibitor (AG490) were used to investigate the function of the SOCS3/STAT3 pathway during this process. Our results showed that exposure of mouse spermatogonial cells to high levels of IL-6 inhibited Zfp637 expression by increasing SOCS3 expression and inhibiting the phosphorylation of STAT3, further reducing cellular differentiation. Consistent with the in vitro results, we observed increasing expression levels of SOCS3 and SOX2, but a reduction of Zfp637 expression, in obese mouse testes. In conclusion, Zfp637 plays a crucial role in spermatogenesis by downregulating SOX2 expression, and IL-6 can decrease the expression of Zfp637 through the SOCS3/STAT3 signaling pathway.

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Figures

Figure 1
Figure 1. Spermatogenesis is dependent on regular expression of Zfp637.
(A) ICC showing that Zfp637 is highly expressed and localizes in the nucleus of GC-1 spg cells. (B) IHC, (C) qRT-PCR and (D) western blot analysis were performed to evaluate the expression of Zfp637 in mouse testis tissue. (E) Effects of OE-Zfp637 in GC-1 spg cells, Zfp637 expression knockdown by siRNA-Zfp63, and the expression of TH2B detected by qRT-PCR. (F) ICC was used to measure the expression of the specific haploid cell marker, protamine 1.
Figure 2
Figure 2. Zfp637 regulates the differentiation of spermatogonia by inhibiting the expression of SOX2.
(A) ICC showing that Zfp637 and SOX2 are both expressed in GC-1 spg cells and localize in the nucleus. (B) Immunoprecipitation showing that Zfp637 and SOX2 protein do not physically interact with each other. (C) There is one Zfp637 binding site in the SOX2 promoter. (D) EMSA showing the formation of Zfp637 complexes with a probe derived from the SOX2 promoter. (E) Western blot showing that the expression of SOX2 is inversely correlated with Zfp637 expression. (F) Expression of SOX2 in mouse testicles by IHC. (G) Prevention of spermatogonial cell differentiation by overexpression of SOX2.
Figure 3
Figure 3. IL-6 can directly disrupt spermatogenesis by inhibiting the expression of Zfp637 in obese individuals.
The expressions of protamine 1 (A), TH2B (B) and Zfp637 (C) were measured by ICC, qRT-PCR and western blot, respectively, after a 48-h treatment with Leptin. (D) ELISA demonstrating elevated IL-6 levels in obese mice. (E) The expression levels of protamine 1, (F) TH2B and (G) Zfp637 measured by ICC-IF, qRT-PCR and western blot, respectively, after a 24-h treatment with IL-6.
Figure 4
Figure 4. IL-6 can directly disrupt spermatogenesis by inhibiting the expression of Zfp637 in obese individuals.
(A,B) Cell morphology and expression of protamine 1 in GC-1 spg cells transfected with pcDNA-Zfp637 and pcDNA-Vector, respectively, after a 24-h treatment with IL-6. (C,D) Cell morphology and expression of protamine 1 in GC-1 spg cells transfected with siRNA-Zfp637 and si-NC, respectively, after a 24-h treatment with IL-6.
Figure 5
Figure 5. IL-6 reduces the expression of Zfp637 via the SOCS3/pSTAT3 signaling pathway.
(A) Activity of the SOCS3/STAT3 signaling pathway after exposure of GC-1 spg cells to IL-6 for 24 hours. * and #p < 0.01 compared with the IL-6 = 0 ng/ml group. ** and ##p < 0.0001 compared with the IL-6 = 0 ng/ml group. (B) Expression of STAT3, pSTAT3 and Zfp637 in GC-1 spg cells after OE-SOCS3. *p < 0.001. #p < 0.001. (C) IHC staining to measure the expression of SOCS3 in normal and obese mouse testes. (D) Cells treated with AG490 (10 nM, 2 hours) prior to exposure to IL-6. 1, 2, 3 and 4, p < 0.001 compared with the blank group. 1*, 2*, 3* and 4* p < 0.001 compared with the blank group. (E) Overexpression of Zfp637 did not influence the expression of STAT3, pSTAT3 or SOCS3.

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