Toxic metabolites, Sertoli cells and Y chromosome related genes are potentially linked to the reproductive toxicity induced by mequindox

Abstract

Mequindox (MEQ) is a relatively new synthetic antibacterial agent widely applied in China since the 1980s. However, its reproductive toxicity has not been adequately performed. In the present study, four groups of male Kunming mice (10 mice/group) were fed diets containing MEQ (0, 25, 55 and 110 mg/kg in the diet) for up to 18 months. The results show that M4 could pass through the blood-testis barrier (BTB), and demonstrate that Sertoli cells (SCs) are the main toxic target for MEQ to induce spermatogenesis deficiency. Furthermore, adrenal toxicity, adverse effects on the hypothalamic-pituitary-testicular axis (HPTA) and Leydig cells, as well as the expression of genes related to steroid biosynthesis and cholesterol transport, were responsible for the alterations in sex hormones in the serum of male mice after exposure to MEQ. Additionally, the changed levels of Y chromosome microdeletion related genes, such as DDX3Y, HSF2, Sly and Ssty2 in the testis might be a mechanism for the inhibition of spermatogenesis induced by MEQ. The present study illustrates for the first time the toxic metabolites of MEQ in testis of mice, and suggests that SCs, sex hormones and Y chromosome microdeletion genes are involved in reproductive toxicity mediated by MEQ in vivo.

Keywords: Sertoli cells; Y chromosome microdeletion; blood-testis barrier; mequindox; reproductive toxicity.

Figure 1. Chemical structure of mequindox (MEQ)

Figure 2

Body weight (A) and coefficient of testis (B) in male mice after the administration of MEQ for 18 months. ^* p< 0.05, and ^**p< 0.01 in comparison with control. Values represent mean ± SD (n = 10).

Figure 3. Selected microphotographs of testes in male mice after the administration of MEQ for 18 months (400×)

(A) Testes from the control group. (B) Testes from the 55 mg/kg MEQ group. Red arrow shows broadened interstitial testicular tissue and blue arrow shows decreased number of spermatogenic cells in the lumen. (C) Testes from the 110 mg/kg MEQ group. Red arrow shows broadened interstitial testicular tissue and blue arrow shows necrosis of spermatogonia and spermatocytes in the lumen.

Figure 4. Ultrastructure of sperm in male mice after the administration of MEQ for 18 months (scale bar = 1 μm)

(A) Sperm from the control group. (B) Sperm from the 55 mg/kg MEQ group. Arrow shows abnormal morphology, fracture and dissolution. (C) Sperm from the 110 mg/kg MEQ group. Arrow shows abnormal morphology and dissolution of the membrane.

Figure 5. Ultrastructure of SCs in male mice after the administration of MEQ for 18 months (scale bar = 2 μm)

(A) SCs from the control group. (B) SCs from the 110 mg/kg MEQ group. Blue arrow shows a few vacuoles in cytoplasm and red arrows show the dissolution and fragmentation of nuclear.

Figure 6. Accurate EICs of the prototype and metabolites of MEQ in the serum and testes of male mice by LC/MS-IT-TOF analysis

(A) Serum from the control group. (B) Serum from the 110 mg/kg MEQ group. (C) Testis from the control group. (D) Testis from the 110mg/kg MEQ group. (E) The accurate MS2 spectra of M4 and M8, respectively. (F) The chemical structure of M4 is 2-isoethanol 1-desoxymequindox, and M8 is 2-isoethanol 4-desoxymequindox.

Figure 7

Alterations to hormonal levels of (A) FSH (mIU/mL), (B) LH (mIU/mL), (C) GnRH (ng/mL), and (D) T (nmol/L) in the serum of male mice after the administration of MEQ for 18 months. ^* p< 0.05, and ^** p< 0.01 in comparison with control. Values represent mean ± SD (n = 10).

Figure 8. Alterations in 3β-HSD, 17β-HSD, CYP17, FSH-R, INH-α, INH-β B, LH-R, P450Scc, StAR, and CYP19 expression in mouse testes after the administration of MEQ for 18 months

^* p< 0.05, and ^** p< 0.01 in comparison with control. Values represent mean ± SD (n = 10).

Figure 9. lterations in DDX3Y, HSF2, Ssty2, and Sly expression in mouse testis after the administration of MEQ for 18 months

^* p< 0.05, and ^** p< 0.01 in comparison with control. Values represent mean ± SD (n = 10).

Figure 10. Hypothalamic-pituitary-testicular axis (HPTA), testosterone (T) biosynthetic pathway and potential metabolism of MEQ in the male mice

The schematic diagram indicates the structure and functions of HPTA (spermatogenesis and T biosynthesis), and shows the reactions leading from cholesterol to T including the originating cell (Leydig cells), corresponding enzymes, and organelles in which the reactions are carried out. 17β-HSD, 17-beta-hydroxysteroid dehydrogenase; 3β-HSD, 3-beta-hydroxysteroid dehydrogenase; CYP17, cytochrome P450c17 subfamily a; CYP19, cytochrome P450c19 subfamily a; FSH, follicle-stimulating hormone; LH, luteinizing hormone; FSH-R, follicle-stimulating hormone receptor; GnRH, gonadotropin-releasing hormone (GnRH); INH-α, inhibin subfamily α; INH-β B, inhibin subfamily β B; LH-R, luteinizing hormone receptor; P450scc, cholesterol side-chain cleavage enzyme; StAR, steroidogenic acute regulatory protein.

Figure 11. The proposed mechanisms of reproductive toxicity in mouse caused by MEQ

SCs are the main toxic target for MEQ to induce TJ disruption and morphologically abnormal sperm. The disrupted TJs further result in the transport of M4 into the testis where it exerts toxic effects on spermatogenesis. Additionally, Y chromosome microdeletion related genes, such as DDX3Y, HSF2, Sly, and Ssty2 in the testis play important roles in the MEQ-induced inhibition of spermatogenesis. Moreover, the MEQ-mediated alteration of sex hormones may result from adrenal toxicity, adverse effects on the HPTA, Leydig cells and SCs, as well as the down-regulated expression of genes responsible for steroid biosynthesis and cholesterol transport. Briefly, SCs, Y chromosome microdeletion related genes and changes to sex hormones levels are involved in the reproductive toxicity mediated by MEQ in vivo.