Bisamide Derivative of Dicarboxylic Acid Contributes to Restoration of Testicular Tissue Function and Influences Spermatogonial Stem Cells in Metabolic Disorders

Abstract

Metabolic syndrome can lead to several challenging complications including degeneration of the pancreas and hypogonadism. Recently, we have shown that Bisamide Derivative of Dicarboxylic Acid (BDDA) can contribute to pancreatic restoration in mice with metabolic disorders via its positive effects on lipid and glucose metabolism, and by increasing the numbers of pancreatic stem cells. In the present study, we hypothesized that BDDA might also be effective in restoring hypogonadism caused by metabolic syndrome. Experiments were performed on male C57BL/6 mice with hypogonadism, where metabolic disorders have been introduced by a combination of streptozotocin treatment and high fat diet. Using a combination of histological and biochemical methods along with a flow cytometric analysis of stem and progenitor cell markers, we evaluated the biological effects of BDDA on testicular tissue, germ cells, spermatogonial stem cells in vitro and in vivo, as well as on fertility. We demonstrate that in mice with metabolic disorders, BDDA has positive effects on spermatogenesis and restores fertility. We also show that BDDA exerts its therapeutic effects by reducing inflammation and by modulating spermatogonial stem cells. Thus, our results suggest that BDDA could represent a promising lead compound for the development of novel therapeutics able to stimulate regeneration of the testicular tissue and to restore fertility in hypogonadism resulting from complications of metabolic syndrome.

Keywords: bisamide derivative of dicarboxylic acid; hypogonadism; infertility; metabolic disorders; regeneration; spermatogenesis; spermatogonial stem cells.

FIGURE 1

Graphical scheme of the protocol for inducing MD.

FIGURE 2

Structural formula of Bisamide Derivative of Dicarboxylic Acid (BDDA).

FIGURE 3

Photomicrographs of representative testis sections (A–C) obtained from male C57BL/6 mice on d70. The arrows indicate “seed balls.” Tissue was stained with hematoxylin-eosin. (A) Section from control group; (B) Section from mice with MD; (C) Section from mice with MD treated with BDDA between d49 and d70. At least 10 photomicrographs of the testicular tissue at × 400 magnification were taken for each experimental animal from all experimental groups (group 1, 2, and 3). Scale bar 50 μm. (D,E) Total number of spermatozoa and the percentage of mobile forms of spermatozoa in the epididymis of male C57BL/6 mice with MD on d70. (F) Free testosterone level in blood serum on d70. (G) Fertility index of mice with MD on d80. Groups: intact—control group from intact mice, MD—mice with MD, MD + BDDA—mice with MD treated with BDDA. Results are presented as the mean ± SEM. *Significance of difference compared with control (p < 0.05); •significance of difference compared with the MD group (p < 0.05).

FIGURE 4

Characterization of endothelial progenitors, epithelial precursors, and spermatogonial stem cells isolated from testis of male C57BL/6 mice on d70. Cells were analyzed by flow cytometry using antibodies against CD45, CD31, Sca1, CD49f, CD117, CD90, CD51, CD24, and CD52. Dot plots are representative for three independent experiments with the mean from three independent experiments. (A) Isotype control for IgG2a APC/IgG2b APC-Cy7. (B) Phenotype establishment and qualitative analysis of CD31/CD45 expression. (C) Phenotype establishment and qualitative analysis of Sca1/CD49f expression. (D) Isotype control for IgG2a Per-Cy5.5/IgG2b PE-Cy7. (E) Phenotype establishment and qualitative analysis of CD117/CD90 expression. (F) Histogram of isotype control for IgG2b PE. (G) Histogram of CD51 PE expression. (H) Isotype control for IgG2a APC/IgG2b FITC. (I) Phenotype establishment and qualitative analysis of CD24/CD52 expression.

FIGURE 5

Characterization of epithelial progenitors, endothelial progenitor cells, and spermatogonial stem cells isolated from testes of male C57BL/6 mice on d70 before and after culture (% of all stained mononuclear cells). Cells were analyzed by flow cytometry using antibodies against CD45, CD31, CD34, Sca1, CD49f, CD117, CD29, CD90, CD51, CD24, and CD52. Dot plots are representative for three independent experiments with the mean from three independent experiments. (A) The number of epithelial precursors (CD45^–CD31^–Sca1⁺CD49f⁺) before and after culture. (B) The number of endothelial progenitor cells (CD45^–CD31⁺ CD34⁺) before and after culture. (C) The number of spermatogonial stem cells 1 (CD117^–CD29⁺CD90⁺) before and after culture. (D) The number of spermatogonial stem cells 2 (CD117⁺CD29⁺CD90⁺) before and after culture. (E) The number of spermatogonial stem cells 3 (CD51^–CD24⁺CD52⁺) before and after culture. ^∗Significance of difference compared with each group before culture (p < 0.05). Groups: intact—cells from intact mice, MD—cells from mice with MD, MD + BDDA—cells from mice with MD treated by BDDA.