Sex steroids effects on the molting process of the helminth human parasite Trichinella spiralis

Abstract

We evaluated the in vitro effects of estradiol, progesterone, and testosterone on the molting process, which is the initial and crucial step in the development of the muscular larvae (ML or L1) to adult worm. Testosterone had no significative effect on the molting rate of the parasite, however, progesterone decreased the molting rate about a 50% in a concentration- and time-independent pattern, while estradiol had a slight effect (10%). The gene expression of caveolin-1, a specific gene used as a marker of parasite development, showed that progesterone and estradiol downregulated its expression, while protein expression was unaffected. By using flow citometry, a possible protein that is recognized by a commercial antiprogesterone receptor antibody was detected. These findings may have strong implications in the host-parasite coevolution, in the sex-associated susceptibility to this infection and could point out to possibilities to use antihormones to inhibit parasite development.

Figure 1

Dose-dependent curves of sex steroid hormones on in vitro molting of muscle larve of T. spiralis. One hundred muscle larve (ML) of T. spiralis were incubated for 36 hours with different concentrations of progesterone (P4), estradiol (E2), or testosterone (T4). Progesterone inhibited the molting rate of the muscle larva of T. spiralis in a concentration-independent pattern (a). Estradiol inhibit only at 50 nM concentration (b), while testosterone has no any effect on molting (c). Each point represents the mean (SD) of quintuplicate determinations of the number of larvae in molting process.

Figure 2

Time curves of T. spiralis molting process after exposure to progesterone (P4), estradiol (E2), and/or testosterone (T4). The parasites were cultured for 44 hours. Progesterone and estradiol had 35–50% of inhibitory effect on molting rate compared with control and testosterone. The maximum inhibitory effect was at 36 hr of culture. Each point represents the mean (SD) of 5 assays counting the number of molting larvae and the mean was obtained. **P < 0.05. The hormone concentration was as follows: P4, 100 mM; E2, 50 nM, and T4, 50 nM.

Figure 3

Micrographies of ML parasites cultures in presence of sexual steroids. Parasites cultured at 36 hr with progesterone (P4, 100 mM), estradiol (E2, 50 nM), or testosterone (T4, 50 nM) or in absence of sex steroid (control) were observed in Axiovert microscopy using 25x objective. In all cases, the old cuticle starts to detach (clear zone) at the apical or basal of the parasite (arrows).

Figure 4

Ts-Cav-1 gene expression in T. spiralis ML cultured in presence of sex steroids. A single band of 306 bp, corresponding to the caveolin-1 of T. spiralis, was detected in all parasites cultured at 36 hr (a). Progesterone and estradiol reduced the expression level of this gene at least 40%, while testosterone has no effect on the expression of this gene (b). Densitometric analysis is shown (b). 18 S was used as constitutive expression gene. Data are represented as mean ± SD. **P < 0.05.

Figure 5

Indirect immunofluorescence of Ts-Cav-1 protein expression in muscle larvae (ML) cultured. Four-micrometre sections from parasites cultured without or with sex steroids (P4, E2 or T4) were treated with anti-Ts-Cav-1 antibody and fluorescein isothiocyanate-conjugated goat anti-mouse antibody and observed under confocal laser microscopy. In all cases of parasite treated there was no changes on Ts-Cav-1 protein expression. Bars = 25 μm.

Figure 6

Progesterone receptor (PR) and host markers expression on T. spiralis cells. (a) T. spiralis cells were stained with anti-mCD3, anti-mCD19, and anti-mMac-1 (upper row); cultured in presence of media (green line), E2 (orange line), P4 (blue line), and T4 (pink line) and stained with anti-Cav-1 of T. spiralis and anti-PR specific antibodies (middle row). (b) Ovary-derived cells were stained with anti-PR antibody as a positive control for PR.