Hydrogen Sulfide and/or Ammonia Reduces Spermatozoa Motility through AMPK/AKT Related Pathways

Abstract

A number of emerging studies suggest that air pollutants such as hydrogen sulfide (H₂S) and ammonia (NH₃) may cause a decline in spermatozoa motility. The impact and underlying mechanisms are currently unknown. Boar spermatozoa (in vitro) and peripubertal male mice (in vivo) were exposed to H₂S and/or NH₃ to evaluate the impact on spermatozoa motility. Na₂S and/or NH₄Cl reduced the motility of boar spermatozoa in vitro. Na₂S and/or NH₄Cl disrupted multiple signaling pathways including decreasing Na⁺/K⁺ ATPase activity and protein kinase B (AKT) levels, activating Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and phosphatase and tensin homolog deleted on chromosome ten (PTEN), and increasing reactive oxygen species (ROS) to diminish boar spermatozoa motility. The increase in ROS might have activated PTEN, which in turn diminished AKT activation. The ATP deficiency (indicated by reduction in Na⁺/K⁺ ATPase activity), transforming growth factor (TGF_β) activated kinase-1 (TAK1) activation, and AKT deactivation stimulated AMPK, which caused a decline in boar spermatozoa motility. Simultaneously, the deactivation of AKT might play some role in the reduction of boar spermatozoa motility. Furthermore, Na₂S and/or NH₄Cl declined the motility of mouse spermatozoa without affecting mouse body weight gain in vivo. Findings of the present study suggest that H₂S and/or NH₃ are adversely associated with spermatozoa motility.

Figure 1

(A) Spermatozoa motility determined by CASA. Y-axis = % of total cells, X axis = the treatment concentration (μM). n = 6–8. (B) Spermatozoa viability. Y-axis = % of total cells, X-axis = the treatment concentration (μM). n = 6–8. (C) Protein levels of apoptotic markers detected by Western blotting. n = 3. (D) The abnormality of boar spermatozoa detected by eosin Y staining. n = 5. (E) Spermatozoon plasma membrane phosphatidylserine (PS) externalization. n = 4. (F) The mitochondrial membrane potential (△Ψm) was measured by the specific probe JC-1 using flow cytometry. Y-axis = % of total cells with high membrane potential, X-axis = the treatment concentration (μM) n = 4. (G) Spermatozoa capacitation status detected by CTC staining. Y-axis = % of total cells, X axis = the treatment concentration (μM). n = 4.

Figure 2

(A) ROS levels in all the treatments by H₂DCFDA kit using flow cytometry. Y-axis = the fluorescent intensity (relative unit). X-axis = the treatment concentration (μM). ^a,b,c Means not sharing a common superscript are different (p < 0.05). n = 3. (B) Protein levels of SOD, GPX, and catalase detected by Western blotting. n = 3.

Figure 3

Protein levels of AMPK and p-AMPK (n = 3) (A), TAK1 (n = 3) (B) detected by Western blotting. (C) Total ATPase activity measured by spectrophotometry. Y-axis = the activity (U/mg protein), and X-axis = the treatment concentration (μM). ^a,b,c Means not sharing a common superscript are different (p < 0.05). n = 4. (D) Protein levels of ATPase 5β detected by Western blotting. n = 3. (E) ATP addition on spermatozoa motility. Y-axis = % of total cells, X-axis = the treatment concentration (μM). *Means significant at p < 0.05 compared to the same treatment without ATP addition (ATP-0). n = 3.

Figure 4. Protein levels of PI3K, AKT, p-AKT, ERK, p-ERK, PTEN, and p-PTEN detected by Western blotting.

n = 3.

Figure 5

Protein levels of PI3K (A), p-AKT (B) and ERK (C) detected by IHF. Scale bar = 50 μm. n = 3.

Figure 6. (A)

Mouse spermatozoa motility determined by CASA. Y-axis = % of total cells, X-axis = the treatment concentration mg/kg B.W. (p < 0.05). n = 8. (B) The abnormality of mouse spermatozoa detected by eosin Y staining. ^a,b,c Means not sharing a common superscript are different (p < 0.05). (C) Mouse body weight. Y-axis = body weight (g), X-axis = the treatment time (day). n ≥ 4.

Figure 7. H₂S and/or NH₃ regulation of spermatozoa motility through multiple signaling pathways.