RESUMEN
The in vitro effect of progesterone in T. canis larvae on their enlargement and motility were evaluated, together to the possible presence of progesterone receptors (PRs). T. canis larvae were cultured in RPMI-1640 with different concentrations of progesterone (0, 20, 40, 80, 400 and 800 ng/mL). Enlargement and increases in motility were dependent on the concentration only from 0 to 80 ng/mL (p < 0.05). The mean percentage of PR + cells in newly obtained larvae as measured by flow cytometry was 8.16 ± 0.4. The number of PR + cells increased depending on concentration from 0 to 80 ng/mL (p < 0.001). Cells obtained from larvae stimulated at any of the studied hormone concentrations showed greater mean fluorescence intensity when compared to non-stimulated cells. Additionally, the expression and location of PR + cells were determined in the larvae. The sequence of an amplicon (420-bp) obtained by PCR from T. canis larvae showed 100% homology with a gene fragment that codes for the PR of the dog. PR + cells were immunolocated using confocal microscopy in the intestinal region of the larvae that had been recently obtained. The results of this study show that T. canis larvae can recognize and respond to the presence of progesterone through a molecule possibly able to bind it. Since we previously observed a similar response to prolactin, we suggest that both hormones could participate sequentially in the reactivation of T. canis larvae in pregnant bitches.
Asunto(s)
Progesterona/farmacología , Progestinas/farmacología , Receptores de Progesterona/efectos de los fármacos , Toxocara canis/efectos de los fármacos , Animales , Secuencia de Bases , ADN de Helmintos/química , ADN de Helmintos/aislamiento & purificación , Perros , Femenino , Citometría de Flujo , Intestinos/parasitología , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Larva/fisiología , Ratones , Microscopía Confocal , Movimiento/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Receptores de Progesterona/análisis , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Toxocara canis/crecimiento & desarrollo , Toxocara canis/fisiologíaRESUMEN
Androgens have been shown to exert a cysticidal effect upon Taenia crassiceps, an experimental model of cysticercosis. To further inquire into this matter, the Taenia crassiceps model was used to evaluate the expression of several proteins after testosterone (T4) and dihydrotestosterone (DHT) in vitro treatment. Under 2-D proteomic maps, parasite extracts were resolved into approximately 130 proteins distributed in a molecular weight range of 10-250 kDa and isoelectrical point range of 3-10. The resultant proteomic pattern was analysed, and significant changes were observed in response to T4 and DHT. Based on our experience with electrophoretic patterns and proteomic maps of cytoskeletal proteins, alteration in the expression of isoforms of actin, tubulin and paramyosin and of other proteins was assessed. Considering that androgens may exert their biological activity in taeniids through the non-specific progesterone receptor membrane component (PGRMC), we harnessed bioinformatics to propose the identity of androgen-regulated proteins and establish their hypothetical physiological role in the parasites. These analyses yield a possible explanation of how androgens exert their cysticidal effects through changes in the expression of proteins involved in cytoskeletal rearrangement, dynamic vesicular traffic and transduction of intracellular signals.
Asunto(s)
Andrógenos/farmacología , Muerte Celular , Proteoma , Taenia/efectos de los fármacos , Taenia/fisiología , Actinas/genética , Animales , Biología Computacional , Cisticercosis/patología , Cysticercus/efectos de los fármacos , Cysticercus/fisiología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/genética , Dihidrotestosterona/farmacología , Femenino , Ratones , Ratones Endogámicos BALB C , Receptores de Progesterona/genética , Testosterona/farmacología , Tropomiosina/genética , Tubulina (Proteína)/genéticaRESUMEN
We evaluated the direct effects of progesterone on the morphology, maturation and behavior of Haemonchus contortus larvae in vitro. The presence and location of possible progesterone receptors in these larvae were also determined. The addition of 8ng/mL of progesterone to larval cultures over 10days reduced larval enlargement, while the addition of 160ng/mL of the hormone increased the enlargement. Up to 62% and 65% of the H. contortus larvae molted from third-stage larvae (L3) to fourth-stage larvae (L4) when cultured in RPMI-1640 media without hormone for 5 and 10days, respectively. The addition of different progesterone concentrations (1, 8, 16, 80 and 160ng/mL) to the larval cultures significantly inhibited the molting process within the same periods. The addition of 8ng/mL or higher progesterone concentrations to the cultures significantly increased larval motility (p<0.05) compared with unstimulated larvae. Flow cytometry showed the expression of progesterone receptors (P4-R) in 15% of the cells from newly isolated H. contortus larvae. When the larvae were cultured for 5days in the presence of the hormone, the percentage of P4-R+ cells remained the same. In contrast, unstimulated larvae showed a significant reduction in the number of P4-R+ cells. Using confocal microscopy, a greater concentration of P4-Rs was immunolocated in the anterior portion of the alimentary tract of the larvae, suggesting that the cells in this region are targeted by the hormone. The results of the present study show that H. contortus larvae have possible P4-Rs and respond to this hormone by inhibiting their molting process, thereby suggesting the participation of progesterone in the larval arrest phenomenon.
Asunto(s)
Haemonchus/efectos de los fármacos , Progesterona/administración & dosificación , Progestinas/administración & dosificación , Animales , Relación Dosis-Respuesta a Droga , Haemonchus/genética , Haemonchus/crecimiento & desarrollo , Haemonchus/metabolismo , Larva/efectos de los fármacos , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Muda/efectos de los fármacos , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismoRESUMEN
The in vitro effect of prolactin (PRL) on the growth and motility of Toxocara canis larvae was assessed. Additionally, the expression and location of prolactin receptors (PRL-Rs) were determined in the larvae. Larvae of T. canis were incubated with different concentrations of PRL for different periods of time. The stimulated larvae accelerated their enlargement and increased their motility. The mean percentage of PRL-R+ cells in non-stimulated larvae, measured by flow cytometry was 7.3±0.3%. Compared with non-stimulated larvae, the mean fluorescence intensity (p<0.05) increased in larvae incubated with 40ng/mL of PRL for 10 days. A 465-bp length fragment was amplified from larvae gDNA by PCR. The sequence of this fragment showed 99% similarity with the gene fragment that codes for the PRL-R of the domestic dog. A high concentration of PRL-Rs was immune-located in the posterior region of the larval intestine; therefore, the intestinal cells in this region were most likely the targets for this hormone. Based on these results, PRL-Rs were identified in T. canis larvae, and the in vitro stimulation with PRL increased the number of these receptors, accelerated the growth and modified the activity of larvae. All of the above suggest that T. canis larvae are evolutionarily adapted to recognize the PRL of their definitive host and furthermore might explain the reactivation of tissue-arrested larvae during the gestation of bitches, which does not occur in gestating females of other species.