Sodium butyrate promotes synthesis of testosterone and meiosis of hyperuricemic male mice

Abstract

Hyperuricemia (HUA) impaires spermatogenesis. This study was carried out, aiming to determine whether butyric acid (NaB) avoids the HUA-induced decline of sperm quality HUA mice were developed through intra-peritoneal injection of the potassium oxalate combined with intragastric uric acid (UA) and by tube feeding 300 mg·kg-1·d-1NaB. The effect of NaB on the reproduction of HUA male mice was determined by measuring sperm count, sperm motility and testosterone content. In addition, TM3 and GC-2 cells were treated with a solution containing 30 mg/dl UA and 1mM NaB. The effects of NaB on the sperm quality were evaluated with the expression level of the genes involving in LH/cAMP/PKA signaling pathway and meiosis, and that encoding OPRL1 receptor protein. Results showed that NaB improved sperm count, sperm motility, testosterone synthesis, and impaired spermatocyte meiosis via HUA. In addition, in vitro analysis showed that NaB activated the LH/cAMP/PKA signaling pathway of TM3 cells, promoted the synthesis of testosterone, up-regulated the content of pain-sensitive peptide receptor (OPRL1) on the surface of GC-2 cells, and promoted meiosis. NaB also promoted the utilization of ATP by GC-2 cells. We illustrated a close relationship between HUA and spermatogenesis defects. NaB-promoted the expression of the genes functioning in testis meiosis, and the testosterone content may aid to improving spermatogenesis quality.

Keywords: Hyperuricemia; Meiosis; Sodium butyrate; Sperm quality; Testosterone.

Fig. 1

The investigation of the reproductive toxicity and protective effect of NaB against HUA background in male mice. (a), weight change of the mice; (b), serum uric acid concentration (n = 6); (c), testicular coefficient (testicular weight/ weight ×100%, n = 6); (d), sperm count (n = 6); (e), sperm motility rate (n = 6); (f), testicular structure examined after hematoxylin and eosin staining (arrows pointing to the abnormal structurs, the decrease of spermatogenic cells). Data are presented as mean ± standard deviation. *, p < 0.05; **, p < 0.01. Scale bar = 50 μm.

Fig. 2

The effects of HUA and NaB on serum testosterone, GnRH, LH, and FSH levels. The concentrations of GnRH (a), LH (b), FSH (c) and testosterone (d) in serum were quantified using ELISA, and expressed as mean ± SD. *, p < 0.05, **, p < 0.01.

Fig. 3

NaB up-regulates the expression of LHR, CYP11A1s, and STAR proteins and genes in testis. (a), the expression levels of LHR, CYP11A1 and STAR proteins in testicular tissue; (b), (c) and (d), the transcript abundances of these genes in testicular tissue; (e), (f), (g) and (h), the protein contents of LHR, STAR and CYP11A1 in TM3 cells (n = 3); (i), (j) and (k), the transcript abundances of LHR, STAR and CYP11A1 genes in TM3 cells; (l), (m) and (n), the concentrations of intracellular cAMP, PKA and testosterone in the cell supernatant. Data are presented as mean ± SD. *, p < 0.05, **, p < 0.01. Scale bar = 50 μm. The arrows denote testicular interstitial cells.

Fig. 4

NaB enhances the meiosis of HUA mice. (a) and (c), the expression levels of PCNA and c-Kit protein in testicular tissue. The testicular tissue was stained with PCNA and c-Kit (red) and DAPI (blue). c-Kit play an important role in the development and differentiation of spermatogenic cells. Undifferentiated spermatogonia do not express c-Kit, but are expressed in differentiated spermatogonia, so it can be used as an indicator of whether spermatocytes are differentiated. Spermatogenic cells (spermatogonia and spermatocytes) in HUA group exhibits a diminished capacity of proliferation (scale = 50 μm) (arrows indicated). (b), (d), the expression levels of PCNA and c-Kit protein as was quantified using ImageJ (n = 3). Data are expressed as mean ± SD. *, p < 0.05; **, P < 0.01.

Fig. 5

NaB promotes the secretion of N/OFQ and increases the expression of genes associated with meiosis in spermatocytes. (a), the immunohistochemical detection of OPRL1 (N/OFQ receptor) and the immunofluorescence detection of WT-1 in testicular tissue. (b),and (c), the expression level of OPRL1 and WT-1 genes (n = 6). (d), (e) and (f), the serum level of N/OFQ. (g), the transcript abundances of meiosis-related genes, Smc1b, Smc4, Smc5, Sycp1, Sycp2, Dmc1, Stra8, Hspa1b, in spermatocytes. Data are expressed as mean ± SD. *, P < 0.05; **, P < 0.01. The scale bar = 50 μm.

Fig. 6

NaB improves ATP utilization and diminishes the level of oxidative stress. (a), (b), the mitochondrial membrane potential, ROS and Ga²⁺ levels. (c), (d) and (e), the expression levels of the mitochondrial membrane potential, ROS and Ga²⁺ as was quantified using ImageJ (n = 3). (f), the ATPase activity in GC-2 cells. (g) and (h), the level of cAMP and PKA in GC-2 cells. The data are presented as mean ± SD. *, P < 0.05; **, P < 0.01. The scale bar = 100 μm.