Introduction
Sesquiterpene lactones (SLs) constitute one of the largest biogenetically homogeneous groups of known secondary metabolites (Heinrich et al., Reference Heinrich, Robles, West, Ortiz de Montellano and Rodriguez1998). These natural compounds have a broad spectrum of biological activities, which are related to the great structural diversity. Cytotoxic effects on animal cells have been attributed to these compounds (Lee et al., Reference Lee, Huang, Piantadosi, Pagano and Geissman1971) as well as antimicrobial (Vega et al., Reference Vega, Wendel, Maria and Pelzer2009; Mohamed et al., Reference Mohamed, Hegazy, Abd El Aty, Ghabbour, Alsaid, Shahat and Paré2017), antiamoebic (Rodríguez-Expósito et al., Reference Rodríguez-Expósito, Nocchi, Reyes-Batlle, Sifaoui, Suárez-Gómez, Díaz-Marrero, Souto, Piñero, Fernández and Lorenzo-Morales2021), antiviral (Li et al., Reference Li, Zhou, Zhou, Chen, Yang, Bai, Wu, Peng and Zhao2005; Ozçelik et al., Reference Ozçelik, Gürbüz, Karaoglu and Yeşilada2009) and antiallergic effect (Lee et al., Reference Lee, Park, Nam, Kang, Hwang, Lee and Im2018). Hehner et al. (Reference Hehner, Heinrich, Bork, Vogt, Ratter, Lehmann, Schulze-Osthoff, Dröge and Schmitz1998) even demonstrated an inhibitory effect on immune and inflammatory responses.
Dehydroleucodine (DhL) is a sesquiterpene lactone of the guaianolide type, featuring an alpha-methylene butyrogamma ring connected to a seven-atom ring, which is fused to another exocyclic alpha beta-unsaturated cyclopentenone ring (Priestap et al., Reference Priestap, Abboud, Velandia, Lopez and Barbieri2011) as shown in Figure 1. DhL was isolated from the aerial parts of Artemisia douglasiana Besser, a plant used in traditional medicine. Studies with DhL have demonstrated gastric cytoprotective and antiulcer effects (Giordano et al., Reference Giordano, Guerreiro, Pestchanker, Guzman, Pastor and Guardia1990; Guardia et al., Reference Guardia, Guzman, Pestchanker, Guerreiro and Giordano1994; Penissi et al., Reference Penissi, Fogal, Guzman and Piezzi1998), justifying its use in popular medicine as an infusion.
Structure of Dehydroleucodine (DhL). It belongs to the group of guaianolides and has an alphamethylenebutyro-gamma-lactone ring connected to a seven-membered ring fused to an exocyclic alpha, beta-unsaturated cyclopentenone ring.
Assays on the cell cycle have shown that DhL causes an arrest in the G2 phase in meristematic cells of Allium cepa (Lopez et al., Reference Lopez, Giordano and Lopez2002), in myocytes from the blood vessels of cultured rats (Cruzado et al., Reference Cruzado, Castro, Fernandez, Gomez, Roque and Giordano2005; Polo et al., Reference Polo, Castro, Cruzado, Collino, Cuello-Carrión, Ciocca, Giordano, Ferrari and López2007), in cancer cells (Siriwan et al., Reference Siriwan, Miyawaki, Miyamoto, Naruse, Okazaki and Tamura2011), and affects mitotic clonal expansion to inhibit preadipocyte differentiation at the G0/G1 phase (Abood et al., Reference Abood, Veisaga, López and Barbieri2018). Similarly, Sánchez Toranzo et al. (Reference Sánchez Toranzo, Giordano, López and Bühler2007, Reference Sanchez Toranzo, Lopez, Zapata Martinez, Gramajo Buhler and Buhler2009) recorded its inhibitory effect on the meiotic cell cycle in Rhinella arenarum oocytes.
Interestingly, it was demonstrated that the use of DhL in mature bovine oocytes induces their activation, while exocytosis of cortical granules was observed in amphibians (Medina et al., Reference Medina, Bühler and Sánchez-Toranzo2014). Studies carried out in embryos obtained by intracytoplasmic sperm injection and somatic cell nuclear transfer, showed encouraging results when DhL was used in the activation of embryo development (Vichera et al., Reference Vichera, Alfonso, Duque, Silvestre, Pereyra Bonnet, Fernández-Martín and Salamone2010).
Assays by Suhaiman et al. (Reference Suhaiman, Carlos de-Rosas, Sartor, Palmada, Giordano and Lopez2011) administering DhL in the drinking water of male mice, did not show changes in the levels of testosterone or o-estradiol in plasma. However, capacitated epididymal sperm showed a higher percentage of binding to zone-free oocytes compared to controls.
Chinchillas are an endangered species, in their natural habitat and, in captivity, they present a low reproductive rate. Knowledge of their reproductive biology is essential to develop effective conservation strategies and improve reproductive protocols. While DhL has shown promising effects on several animal models by influencing exocytosis processes and oocyte activation, its impact on chinchilla reproduction could offer new possibilities for improving reproductive efficiency, which is essential for the preservation of this species in both natural habitats and captivity.
The present study constitutes the first on the effect of a sesquiterpene lactone on the physiology of the male gamete, showing an induction of the acrosome reaction (AR) in Chinchilla lanigera epididymal sperm. The results presented contribute to a better understanding of the molecular mechanisms involved in this process.
Materials and methods
Animals
The animals used in this study were provided by a commercial hatchery authorized for skin production. We selected 6 sexually mature male specimens, 10 to 12 months of age, with a body mass of 550–650 g. All animals were housed under a 12-h dark-light cycle at a constant temperature of 22 ± 1 °C and received water and food ad libitum. The slaughtered animals were sacrificed by cervical dislocation and then immediately transported to the laboratory in thermal containers to maintain a constant temperature.
Reagents and incubation media
PBS 1X (Dulbecco’s phosphate-buffered saline solution) Irvine Scientific_ (Santa Ana, CA, USA). Dimethyl sulfoxide (DMSO), N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89 dihydrochloride hydrate), quinacrine, 1-[6-[((17β)-3-Methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U73122 hydrate), dehydroleucodine (DhL) from Sigma Aldrich. Co. (St. Louis, MO, USA). DhL was used from a stock solution of 10 mM in DMSO. Coomassie Brilliant Blue G250 from Biopack® (CABA, Argentina).
Modified HTF medium (human tubal fluid)
To 750 ml of purified water, 5.931 g of NaCl, 0.35 g of potassium chloride (KCl), 0.05 g of magnesium sulphate heptahydrate (MgSO4.7H2O), 0.05 g of potassium dihydrogen phosphate (KH2PO4), 0.366 g of sodium bicarbonate (NaHCO3), 0.5 g of d-glucose, 0.036 g of sodium pyruvate, 0.3 g of calcium chloridedihydrate (CaCl2.2H2O) and 4.0 g of sodium dl-lactate (60% (v/v) syrup) were added. Ten µg of phenol red, 100 UI penicillin and 50 µg streptomycin sulphate were added to 1 ml of the above medium. pH was then adjusted to 7.4 with 1 mol/l hydrochloric acid (HCl).
Collection and preparation of spermatozoa
Immediately after slaughtering, spermatozoa were collected by puncture from the cauda epididymis. The samples were washed twice in PBS, centrifuged at 1000 × g for 10 minutes, and the pellet was resuspended with HTFm. All materials used for the sample collection and subsequent trials were under strict temperature control (37 ± 0.5 °C). We only worked with samples with a vitality percentage equal to or >80%, using the eosin test 0.5% (w/v) with an inverted microscopy at 400x. Counts were performed on 200 cells per replicate. Eosin enters only dead cells, staining them a pink colour, while in living cells, the plasma membrane prevents its penetration.
To assess motility patterns, we placed 10 µl of sperm suspensions on a clean and dry slide, covered with a 22 mm × 22 mm coverslip to ensure a 20.7 µm chamber depth, allowing free movement of the sperm. Observations were made on 200 cells in duplicate, with microscope of phase contrast at 400x.
Capacitation of spermatozoa: assay of DhL inducer effect
The washed spermatozoa were separated into aliquots (final concentration 3–5 × 106 cells/ml) and incubated with HTFm supplemented with 2.5 g/l albumin fraction V for 2.5 h at 37 °C in an atmosphere containing 95% relative humidity and 5% CO2. Previously capacitated sperm suspensions were treated with different concentrations (0–2 mM) of DhL for different incubation times (0–40 minutes). Reactions were stopped with buffered formalin 4% final concentration, and samples were processed for cytological staining and scanning electron microscopy.
Determination of signalling pathways involved in DhL-induced acrosome reaction
In order to determine the participation of cAMP-dependent PKA, Phospholipase A2 (PLA2) and Phospholipase C (PLC) in the AR induced by DhL, we used capacitated spermatozoa pretreated with various pharmacological inhibitors of these key enzymes: H89 (30 µM) – a PKA inhibitor, for 30 min; quinacrine (5 µM) – a PLA2 specific inhibitors, for 15 min; and U73122 (20 µM) – a PLC inhibitors, for 30 min. After incubation with the inhibitors, the AR was induced with 0.5 mM DhL.
When the protocol included the addition of two or more drugs, the sperm was washed in HTFm and centrifuged before addition of the next drug. All control experiments were performed under identical conditions. Negative controls were treated with the corresponding solvent. The doses and incubation times used in this study were selected as described in our previous study (Gramajo-Bühler et al., Reference Gramajo-Bühler, Zelarayán and Sánchez-Toranzo2016) and the same final concentrations were consistently used.
Evaluation of acrosomal status by Coomassie Blue staining
In all incubations, reactions were stopped by adding buffered formalin (final concentration 4%). Samples were fixed at 4°C for 10 minutes, then washed twice with a 0.1 M ammonium acetate solution at pH 9, followed by centrifuging for 5 minutes at 1200 × g. The smears were air-dried, hydrated in distilled water for 5 minutes, stained with Coomassie Blue G250 solution, and finally washed with distilled water for 5 minutes.
A total of 200 cells were analyzed by light microscopy at 1000x. A positive response was defined as the absence of acrosome, while a negative response was indicated by the presence of the acrosomal vesicle, visible as a brilliant blue band in the anterior region of the head.
Scanning electron microscopy (SEM)
Sperm cells were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 for 2 h at 4 °C. Subsequently, samples were post-fixed in 1% osmium tetroxide in phosphate buffer overnight, dehydrated in a graded series of alcohol solutions and 100% acetone, and finally desiccated using the critical point drying technique with CO2. After dehydration, samples were mounted on specimen holders for SEM (2 cm diameter metal cylinders) and coated with gold. Observations were made with a JEOL 35CF SEM.
Statistical analysis
All experiments were performed independently in duplicate. Means and standard errors were calculated for all data sets. Differences between groups were evaluated using two-way analysis of variance followed by Fisher’s exact test. A p value of <0.05 was considered statistically significant.
Results
Assay of the effect of dehydroleucodine on sperm
Given that previous studies have shown that this lactone decreases viability in tumour cells, we analyzed its effect on sperm viability and motility. Gametes were exposed for different time periods (0–40 minutes) to increasing concentrations of DhL (0–2 mM). The sperm suspensions incubated for 10 minutes with concentrations of up to 1 mM presented similar vitality percentages to the control. In contrast, gametes exposed to concentrations of 0.5 and 2 mM for longer times (20–40 minutes) showed a significant decrease in viable cell percentages (Figure 2A). Vitality percentages higher than 65% were recorded up to 20 minutes of incubation at all concentrations tested.
(A) Effect of Dehydroleucodine on the viability of sperm incubated for different time periods at 37 °C. The results are expressed as the mean ± SEM of five experiments performed in duplicate with different animals. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs control group (0 mM DhL). (B) Effect of Dehydroleucodine on the progressive motility of sperm. Samples were incubated with increasing concentrations of DhL at different times. The results are expressed as the mean ± SEM of four experiments performed in duplicate with different animals. Significant differences at the same concentrations for different incubation periods (a, b, c).
Sperm motility showed a significant decrease compared to the control at all concentrations tested. However, the suspensions incubated with 0.1 mM DhL presented similar values at all tested times. At higher DhL concentrations, lower percentages of cells with progressive motility were observed as a function of incubation time (Figure 2B).
Inducer effect of the acrosomal reaction
Taking into account the results of sperm viability, the samples were exposed to different concentrations of DhL (0–2 mM) for 10 and 20 minutes of incubation. The negative control (DMSO) triggered a spontaneous acrosomal reaction of 9%, and this percentage did not show significant variation in the trials.
When analyzing the percentage of acrosomal reaction (AR) as a function of incubation time, significant increases are observed at 10 minutes in all tested concentrations compared to the control. These values remained constant for up to 20 minutes of treatment (Figure 3).
Effect of different concentrations of dehydroleucodine (DHL) in capacitated epididymal sperm, incubated at different times at 37 °C. The control presents the same volume of DMSO. The values recorded at time 0 correspond to the initial SAR. The results are expressed as the average ± SEM of five experiments carried out in duplicate with different animals.
Suspensions treated with a concentration of 2 mM DhL recorded the highest AR percentages at both time periods (56.8% and 60.1%, respectively). However, there are no significant differences with the samples treated with 0.5 and 1 mM.
It is interesting to note that at all tested concentrations, AR percentages significantly higher than the negative control were recorded at all exposure times to DhL. Aliquots of the different treatments were processed for observation under electron microscopy to rule out false positives due to acrosome rupture or damage (Figure 4).
Electronic microphotography of epididymal sperm exposed to DhL. The acrosome is presented as a very noticeable structure (asterisk). Sperm with arrows have undergone complete exocytosis of their acrosomes.
Determination of signalling pathways activated with DhL
To determine the involvement of cAMP-dependent PKA and phospholipases (PLs) in the acrosome reaction (AR) induced by dehydroleucodine (DhL), we designed various protocols (see Materials and Methods) using DhL as an inducer. Capacitated spermatozoa were exposed to specific enzyme inhibitors before DhL treatment to identify the signalling pathways involved.
In the PKA inhibition assays with H89, AR percentages decreased to 49.9% compared to the DhL control (61.4%). Gametes treated with quinacrine showed AR percentages of 53.6%. However, significant differences were observed with the PI-PLC inhibitor, U73122, where AR percentages were 26.5% compared to the positive controls. Sperm suspensions sequentially exposed to all inhibitors before the addition of DhL exhibited acrosomal exocytosis percentages of 27.4%, with no significant differences compared to assays with U73122 alone (Figure 5).
Assessment of PKA/cAMP, PLA2 and PLC participation in the acrosomal reaction induced by Dehydroleucodine. Sperm suspensions were treated with inhibitors of signalling pathways (H89, Quinacrine and U73122) alone or in combination, prior to the addition of DhL. Negative control: DMSO. Positive control: DhL. The values recorded with the negative control represent the spontaneous acrosome reaction (SAR). The results are expressed as the mean ± SEM of five experiments performed in duplicate with different animals.
Discussion
Our results indicated that dehydroleucodine (DhL) is capable of inducing the acrosome reaction, altering the sperm motility pattern, and exerting cytotoxic effects in a dose- and time-dependent manner. The cytotoxicity of different sesquiterpene lactones (SLs) is known to depend on the presence of a lactone ring, with the unsaturated alpha-exo-methylene-gamma-lactone ring playing a crucial role, as observed in vitro.
Sperm suspensions incubated for 10 minutes with concentrations up to 1 mM showed viability percentages similar to those of the controls. However, extending the exposure time to 20–40 minutes and increasing the concentration to 2 mM resulted in a significant decrease in viable cells. Similar effects were observed in Rhinella arenarum embryos, where DhL concentrations of 0.5–2 mM led to 93% mortality (Moreno et al., Reference Moreno, Juárez and Pelzer2012).
The assessment of motility patterns revealed a detrimental dose- and time-dependent effect, with significant decreases at all concentrations tested. Notably, samples incubated with 0.5, 1, and 2 mM DhL showed significant differences within groups exposed to the same concentration at different times. These findings suggest that DhL has a direct negative impact on cell motility, which does not equally affect sperm viability.
Introducing a sesquiterpene lactone like DhL into the incubation medium of male gametes offers a valuable opportunity to study its effects under in vitro conditions. The ability to induce acrosomal exocytosis and identify the involved signal transduction pathways is crucial for evaluating sperm quality. Experimental designs limited to 20 minutes of exposure aimed to mitigate cytotoxic effects on cell viability while demonstrating that DhL induces the acrosome reaction at all tested concentrations.
When analyzing the results of DhL as an inducer, we observed significant decreases in AR percentages in samples treated with each inhibitor alone compared to the positive control. This indicates a minimal involvement of these signalling pathways. Evaluating the significance among different inhibitors, the inhibition percentages recorded with H89 and quinacrine did not show significant differences, whereas the inhibition of PLC with U73122 led to a marked decrease in AR percentage. Although similar studies in male gametes are lacking, analogous results were obtained in Rhinella arenarum oocytes (Zapata-Martínez et al., Reference Zapata-Martínez, Medina, Gramajo-Bühler and Sánchez-Toranzo2016). The authors suggested that DhL may activate IP-PLC, resulting in the production of IP3 and DAG, which mobilize calcium ions from cytoplasmic reserves. However, based on our studies, we cannot definitively confirm the same mechanism in sperm.
In summary, DhL demonstrates a significant potential to induce the acrosome reaction in epididymal sperm of Chinchilla lanigera and affect sperm motility in a dose- and time-dependent manner. While it directly impacts motility, its effect on viability is less pronounced. Further research is required to fully elucidate the mechanisms involved and their implications for male fertility. DhL’s ability to influence acrosomal exocytosis and signalling pathways makes it a valuable tool for in vitro studies of male gametes.
Acknowledgements
We would like to thank Dr. Luis Alberto López for his support throughout this study.
Competing interests
The authors declare none.
Funding
This work was supported by a grant from the Secretary of Science, Art and Technological Innovation of the National University of Tucumán-SCAIT (grant numbers 26D-617) and the National Agency for the Promotion of Science and Technology-FONCYT (grant numbers PICT-0272). We would like to thank Dr. Luis Alberto López for his support throughout this study.
Ethical standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.
