Artemisia vulgaris extract causes precocious acrosome reaction and viability loss but low rate of membrane damage in mouse spermatozoa

Abstract

Several herbs including Artemisia are known to possess conceptive property. In the present study, mouse spermatozoa were incubated with ethanol extract of Artemisia vulgaris leaves. The effect of extract on acrosome exocytosis was studied by labeling spermatozoa with fluorescein isothiocyanate (FITC) peanut agglutinin and by staining with Coomassie blue. Viability and membrane integrity were studied by Trypan-blue staining and hypo-osmotic swelling test. Artemisia extract at very low concentration caused precocious acrosome reaction and loss of sperm viability. Acrosome reaction increased remarkably from 22.63% to 88.42% with increasing extract concentration from 0 to 2,000 µg/mL. However, the viability loss of spermatozoa was increased from 11.71% in control to 63.73% in samples treated, evaluated by Trypan-blue staining method. Membrane damage caused by the extract, evaluated by hypo-osmotic swelling test was even low, ranging from 2.27% to only 24.23%. These results indicate that Artemisia extract might block fertilization by causing precocious acrosome exocytosis in spermatozoa. A direct contraceptive effect was tested by injecting the plant extract into the vagina of female mice and then allowing them to mate with normal males. The treated female mice delivered significantly fewer litters in comparison to the control.

Keywords: Acrosome; Artemisia; Membrane damage; Spermatozoa; Viability.

Fig. 1.. Effect of Artemisia leaf extract on mouse sperm acrosome.

(A) Untreated mouse spermatozoa labeled with fluorescein isothiocyanate (FITC) peanut agglutinin (FPNA) and ethidium bromide (EtBr). Most of spermatozoa displayed crescent shaped acrosomes, labeled with FPNA (green, arrows) on the dorsal surface of nuclei. Nuclei were stained red by EtBr but not clearly visible in the microphograph, because they were overshadowed by bright green fluorescence of FPNA. (B) Mouse spermatozoa treated with 1,000 µg/mL extract. Few spermatozoa possessed acrosome (green, arrow). Sperm heads are labeled red with EtBr (arrowheads). (C) Mouse spermatozoa stained with coomassie blue. The acrosome appeared as thick blue line on the dorsal surface of sperm heads (arrow). Thick blue line was absent on acrosome reacted sperm heads (arrowhead). Magnified view of sperm heads with intact and lost acrosome are also shown in the insets. (D) Acrosome reaction of spermatozoa treated with different concentrations of A. vulgaris extract. Each plotted point represents a mean value from three experiments. The vertical lines indicate standard deviations. Open columns represent the vehicle controls. Bar in (A–C), 10 µm.

Fig. 2.. Effect of Artemisia extract on sperm viability and membrane damage.

(A) Trypan blue staining of mouse spermatozoa treated with 1,000 µg/mL extract. Dead spermatozoa showed nuclei stained with Trypan blue (arrow) due to leaky membrane. Live spermatozoa excluded dye pentration, showing unstained or light blue nuclei (arrowhead). (B) Mouse spermatozoa treated with 1,000 µg/mL extract and subjected to hypoosmotic swelling test. Membrane damaged spermatozoa (arrows) were unaffected by hypoosmotic medium and remained straight. (C) Untreated spermatozoa subjected to hypoosmotic swelling test. Live spermatozoa developed various deformations or bendings in the neck and tail regions (arrows) due to the presence of undamaged plasma membrane and endosmotic swelling (see text). (D) Viability of mouse spermatozoa treated with various concentrations of Artemisia leaf extract, evaluated by Trypan blue staining. Open columns represent the vehicle controls. (E) Membrane damage of spermatozoa treated with various concentrations of extract, evaluated by hypoosmotic swelling test. Bar in (A–C), 10 µm.