Effects of aqueous crude extract of Echeveria gibbiflora on mouse sperm function

ABSTRACT

The present study evaluates the possible antifertility effect of aqueous crude extract (OBACE) of Echeveria gibbiflora, a plant that belongs to the crassulaceae family, used in traditional Mexican medicine as a vaginal post coital rinse to prevent pregnancy and shown to have an immobilization/agglutination effect on sperm of different mammal species. We evaluated the effect of OBACE on functional parameters of mouse sperm, such as viability, capacitation, and acrosome reaction. In addition, due to the high concentrations of calcium bis-(hydrogen-1-malate) hexahydrate [Ca (C₄H₅O₅)₂•6H₂O] present in this plant extract, we evaluated its effect on Ca²⁺ influx in mouse sperm under capacitating conditions. Moreover, we determined the acute toxicity of OBACE and its in vivo effect in mouse sperm motility administering a single daily dose of 50 and 100 mg/kg during seven days, intraperitoneally. The sperm viability was not affected by the presence of different concentrations of OBACE, however, the capacitation and acrosome reaction suffered a significant decrease in a concentration-dependent manner, coinciding with the reduction of Ca²⁺ influx. Furthermore, OBACE displayed an LD₅₀ of 3,784.42 mg/kg and can be classified as a low toxic substance. Also, in vivo OBACE showed an inhibition of total and progressive motility on mouse sperm alongside a significant decrease of motility kinematic parameters and IVF rates. The results confirm the antifertility effect of this plant used in Mexican folk medicine. Further study on OBACE as a possible contraceptive treatment is warranted because of its activity and low in vivo toxicity.

Abbreviations: ALH: lateral amplitude; AP: acid phosphatase; BCF: beat frequency; BSA: bovine serum albumine; CTC: chlortetracycline; FDA: fluorescein diacetate; Fura-2 AM: fura-2-acetoxymethyl ester; HIV: human immunodeficiency virus; IVF: in vitro fertilization; OBACE: aqueous crude extract of Echeveria gibbiflora; PI: propidum iodide; SN: supernatant; VAP: average path velocity; VCL: track speed; VSL: straight line velocity

KEYWORDS:

• Calcium
• contraceptive
• Echeveria gibbiflora
• motility
• spermatozoa

Introduction

Presently, there is a lack of an ideal non-hormonal male contraceptive. Other options, like vasectomy, are drastic and irreversible. On one hand, the use of a condom seems to be more effective for prevention of HIV infection; nevertheless, its use has little acceptance between established couples [Lampiao 2011; Nieschlag 2009]. On the other hand, the use of hormonal male contraceptives has also been proposed, but requires more study before widespread adoption [Chao et al. 2014; Murdoch and Goldberg 2014]. These facts are meaningful to find new strategies for birth control, such as herbal contraceptives. A wide variety of herbal male contraceptives are known. Some of them are well described: the extract of leaves and stem of Carica papaya caused a drop in both sperm motility and pH of human semen [Nassar 1979]; the steroidal alkaloid of Tripterygium wilfordii caused disruptive changes in the acrosome membrane of human sperm and also arrested motility [Kanwar et al. 1988]; and the root bark extract of Ecballium ellaterum reduced the concentration and motility of human sperm [Qian 1987]. Despite these findings, a herbal male contraceptive that has the desired features of high activity, low toxicity, high specificity, ease of use, and does not impair libido is yet to discovered.

Echeveria gibbiflora is a plant that belongs to the Crassulaceae family. It is a perennial succulent rosette of 15 leaves around a prostrated thick stem, with paniculate inflorescences and rises up to 1 m during the dry season [Parra-Tabla et al. 1998]. This plant is also known under the common name of Donkey Ear (Oreja de Burro), and is present in wild stands and in rural gardens of Sierra Norte de Puebla and also in degraded forest areas in southern states of Mexico [Leszczyñska-Borys et al. 2005]. The main use of Echeveria gibbiflora is ornamental, however, it is popularly used in traditional Mexican medicine as a vaginal post coital rinse without side effects [Delgado et al. 1999; Reyes et al. 2002; Taboada et al. 1992]. The aqueous crude extract (OBACE) of this plant induced instantaneous immobilization/agglutination effects on human and bull spermatozoa, as well as a hypotonic-like effect, this effect is characterized by membrane distension, formation of a ’bubble-head’ followed by an increase in cell volume more than three times. [Hernández et al. 2001; Taboada et al. 1992]. In previous studies, bufadienolides, flavonoidic-glycosides, alkanes, alcohols, triterpenes, steroids, and phenolic compounds have been isolated from the plant crude extract [Waizel-Bucay and Waizel-Haiat 2009]. Furthermore, high concentrations of calcium bis-(hydrogen-1-malate) hexahydrate [Ca (C₄H₅O₅)₂•6H₂O] in OBACE have also been described and linked to the immobilization/agglutination and the hypotonic-like effects [Reyes et al. 2005]. Nevertheless, convincing evidence of a possible correlation was not found. For this reason, more studies are important to elucidate why OBACE produces immobilization/agglutination and hypotonic-like effects on mouse sperm. In this research, we assayed the in vitro effect of OBACE on different functional parameters of mouse sperm (viability, motility, capacitation, and acrosome reaction) with the purpose to show its possible antifertility effect on mouse sperm. In order to better understand the activity of OBACE on mouse sperm Ca²⁺ influx, toxicity and motility assays were conducted.

Results

Effect of OBACE on mouse sperm viability and acute toxicity

To determine the possible toxic effect of OBACE on mouse sperm, we evaluated the sperm viability by flow cytometry using a double staining FDA-PI technique (Figure 1). After a 30 minute incubation period, in the presence and the absence of different concentrations of OBACE, a very low toxic effect, even with the highest OBACE concentration, was observed. Treated mouse sperm showed a 2.5% and 7% decrease in the viability at concentrations of 0.25 and 0.5 mg/mL, respectively. Even at the highest concentration (1.0 mg/mL) only 13% of the cells had died.

Figure 1. Effect of different concentrations of aqueous crude extract of Echeveria gibbiflora (OBACE) against the viability of mouse spermatozoa. The immobilization/agglutination effect produced by OBACE suggests cellular death, nevertheless about 2.5% and 7% of the cells showed red fluorescence (non- viable) on the lowest concentrations (0.25 and 0.5 mg/mL), respectively, and even in the highest concentrations (1 mg/mL) only 13% of the cells showed red fluorescence. There was no meaningful difference compared with the control sample. p = 0.4767. Values are expressed as mean ± SEM (n = 3). Statistical significance of the results was determined using one-way ANOVA followed by Dunnett test. Differences were considered significant at p < 0.05.

The evaluation of the toxicity of OBACE was carried out using the same mouse strain. The OBACE LD₅₀ was 3784.42 mg/kg which can be classified as having low toxicity according to the classification of Loomis and Hayes [1996].

Effect of OBACE on mouse sperm capacitation, the acrosome reaction, and calcium influx

To determine if the immobilization/agglutination effect produced by OBACE has an impact on the fertilizing capacity of mouse sperm, we evaluated the capacitation with CTC staining (Figure 2). The samples were incubated in the presence and the absence of different concentrations of OBACE, the same used on viability experiments (0.25, 0.5, and 1.0 mg/mL), to allow the sperm capacitation. We found that there was an increase of the non-capacitated cells in the presence of OBACE. This phenomenon was concentration-dependent, and showed an increase, compared to the control, of about 20% at the lowest concentration (0.25 mg/mL), 25% at 0.5 mg/mL, and more than 50% at the highest concentration (1.0 mg/mL) (Figure 2). These results were significantly different than the controls.

Figure 2. Effect of aqueous crude extract of Echeveria gibbiflora (OBACE) on mouse sperm capacitation. Despite the low toxicity of OBACE, the sperm sample showed a significant number of cells which did not achieve capacitation, in the same way, OBACE showed an effect in a concentration dependent manner. Values are expressed as mean ± SEM (n = 3), p < 0.0001. Data analysis was performed using Dunnett’s multiple comparison test, p < 0.05. *means significant difference.

Once we demonstrated that the extract prevented sperm capacitation, we evaluated the effect of OBACE on the acrosome reaction using the acid phosphatase method [Pietrobon et al. 2001] (Figure 3). After 120 minutes incubation, under capacitating conditions in modified Tyrode medium, the acrosome reaction was induced by the addition of calcium ionophore A23187. OBACE was also added at different concentrations (0.25, 0.5, and 1.0 mg/mL). We found that there was a decrease on acquisition of acrosome reaction in a concentration-dependent manner. Compared to the control, the reduction was about 12% at the lowest concentration (0.25 mg/mL), 20% at 0.5 mg/mL, and more than 33% at the highest concentration (1 mg/mL). These results were significantly different than the controls.

Figure 3. Effect of aqueous crude extract of Echeveria gibbiflora (OBACE) on mouse sperm acrosome reaction. OBACE produced a significant number of cells which did not achieve acrosome reaction, compared with the control. The reduction was about 12% in the lowest concentration (0.25 mg/mL), 20% (0.5 mg/mL), and more than 33% at the highest concentration (1.0 mg/mL), in the same way, OBACE showed an effect in a concentration dependent manner. Values are expressed as mean ± SEM (n=3). p < 0.0001. Data analysis was performed using Dunnett’s multiple comparison test, p < 0.05. *means significant difference.

In order to acquire more evidence about how OBACE induces the agglutination/immobilization and hypotonic-like effects, we determined the sperm intracellular Ca²⁺ using a fluorescence indicator Fura-2 AM. The samples were incubated during 60 minutes under capacitating conditions in the presence and the absence of different concentrations of OBACE (0.25, 0.5, and 1.0 mg/mL) to induce and allow the calcium influx. The treatment groups showed a reduction of intracellular Ca²⁺ due to the presence of OBACE. Initially, the decrease of intracellular Ca²⁺ at the lowest OBACE concentration (0.25 mg/mL) (Figure 4A) was not significant compared with the control. However, after 10 minutes the difference was already significant. The intracellular Ca²⁺ decreased even further with increasing concentrations of OBACE (0.5 and 1.0 mg/mL) (Figure 4A). At the end of the recording (60 minutes), we could observe a significant Ca²⁺ decrease of 35% at 0.25 mg/mL, 57% at 0.5 mg/mL, and 62% at 1.0 mg/mL (Figure 4B). To confirm the effect of OBACE on Ca²⁺ influx we used NiCl₂ as a calcium channel blocker.

Figure 4. Effect of aqueous crude extract of Echeveria gibbiflora (OBACE) on Ca²⁺ influx of mouse sperm. Graph shows the effect of different concentrations of OBACE (0.25, 0.5, and 1 mg/mL) on mice sperm calcium efflux after 60 minutes of incubation under capacitating conditions. Changes in the calcium efflux was monitored during 60 minutes, displaying a concentration dependent manner effect (A), were a decrease of intracellular calcium was observed and it was more evident as the concentration of OBACE was rising. After 60 minutes of recording we can notice a clear significant difference between the control sample and the different concentrations (B). NiCl₂ was used as the negative control. Values are expressed as mean ± SEM (n = 3). p < 0.0002. Data analysis was performed using Dunnett’s multiple comparison test, p < 0.05. *means significant difference.

In vivo effect of OBACE and in vitro fertilization (IVF)

A single daily dose of 100 (group 1) and 200 (group 2) mg/kg of OBACE was administered intraperitoneally to the experimental groups, six male mice each, while the vehicle was administered to a control group of the same size. Taking into account that the epididymal transit time for mouse spermatozoa is five days [Dadoune and Alfonsi 1984], we decided to treat all groups for seven days. The effect of the extract on spermatozoa motility is shown in Figure 5. Both total and progressive motility decreased in mice treated with OBACE. On one hand, there was an important decrease of 36% and 40% in total motility for group 1 and 2, respectively (Figure 5A). On the other hand, a 12 % decrease (group 1) and almost a total loss of progressive motility (group 2) was observed for treated mouse sperm as compared to the control (Figure 5B). All sperm kinematic parameters: average path velocity (VAP), straight line velocity (VSL), track speed (VCL), lateral amplitude (ALH), and beat frequency (BCF), were decreased in the treated mouse (Figure 5C to G).

Figure 5. In vivo effect of aqueous crude extract of Echeveria gibbiflora (OBACE) on motility and kinematic parameters of mouse sperm from vas deferens. ■ control,

100 mg/kg and,

200 mg/kg. (A) Total motility, (B) progressive motility, (C) average path velocity (VAP), (D) straight line velocity (VSL), (E) track speed (VCL), (F) lateral amplitude (ALH), and (G) beat frequency (BCF). Values are the mean ± SEM, n = 6. p < 0.0001. Analysis was performed using Dunnett´s multiple comparison test, p < 0.05. *means significant difference.

After having shown the in vivo effect of OBACE on the kinematic parameters of sperm motility, we performed an IVF assay to determine if the sperm of mice treated with OBACE were able to fertilize an oocyte. Intraperitoneal administration of OBACE (see the in vivo effect of OBACE section) to the experimental groups was performed for IVF testing. Sperm from OBACE treated mice had IVF rates of 13.3% compared to that of 52.3% for the control (Table 1).

Table 1. Effect of aqueous crude extract of Echeveria gibbiflora (OBACE) on in vitro fertilization (IVF).

Group	IVF+ (%)	IVF- (%)
Control	52.38	47.62
Group A^1	13.33	86.67
Group B^2	13.33	86.67

Intraperitonially administration of ¹100 mg/kg; ²200 mg/kg.

Discussion

The agglutination/immobilization effect of Echeveria gibbiflora extract, as well as the hypotonic-like effect on different sperm from mammals (bull, human, and guinea pig) has been well-documented [Delgado et al. 1999; Hernández et al. 2001; Taboada et al. 1992], however, we do not know for certain if the immobilized-agglutinated spermatozoa with hypotonic-like effect were dead or alive. Experiments to determine the viability of the spermatozoa treated with OBACE were already performed [Reyes et al. 2002], but they were not conclusive due to the deposit of a sticky dense material on the plasma membrane, making impossible the measurement of cell viability by fluorescence microscopy. Despite this fact, we evaluated the viability of the mouse sperm cells with the same fluorescence double staining (FDA-PI) but using flow cytometry. The results showed that the mouse sperm cells were alive during the incubation time (30 minutes) with no significant difference compared with the control sample (Figure 1). This is a major finding, insomuch as the effect of OBACE only implies the immobilization/agglutination of the cell and does not compromise the plasma membrane integrity. Other plant extracts have shown similar effects as OBACE. Sapindus saponaria in human spermatozoa [Damke et al. 2013] produce sperm immobilization and cell death using concentrations from 1.25 to 10 mg/mL, indicating the toxicity of this extract. Also, an aqueous extract of Ricinus communis L [Nath et al. 2013] was tested on human sperm motility, showing 100% of sperm immobilization after 30 minutes of incubation, using a concentration of 300 mg/mL. The effect of different plant extracts mentioned above, reveals some differences compared with of OBACE performance: (1) OBACE has shown instantaneous agglutination/immobilization effect on sperm of different species [Delgado et al. 1999], and (2) OBACE does not affect significantly the sperm viability (Figure 1).

Because mouse sperm are alive during OBACE treatment, we assayed the acquisition of sperm capacitation and acrosome reaction, induced in vitro, in presence of OBACE. Capacitation represents a series of changes in sperm physiology to acquire fertilizing capacity [Aitken and Nixon 2013; Ferramosca and Zara 2014]. Such changes lead the spermatozoa to undergo a special motility pattern, known as hyperactivation, prior to fertilization [Boryshpolets et al. 2015; Santi et al. 2013]. Despite the fact that OBACE does not affect sperm viability (Figure 1), the capacitation was consistently inhibited, achieving significant differences in all concentrations compared to the control (Figure 2). The inhibition of sperm capacitation was expected, due to the immobilization/agglutination effect of OBACE, immobilized sperm will not undergo hypermotility, therefore, will not achieve capacitation [Darszon et al. 2005]. However, even today it is not clear if hypermotility is a consequence of capacitation or is a requirement for the spermatozoa to achieve capacitation [Alasmari et al. 2013; Eddy 2006].

As well as capacitation, the acrosome reaction is a physiological event necessary for normal fertilization. The acrosome reaction can be characterized by a morphological change caused by membrane fusion and the production of hybrid vesicles during which various enzymes and proteins are released from the acrosome [Okabe 2013, 2014]. The effect of OBACE against the acrosome reaction showed significant difference compared to the control sample. The induction of acrosome reaction with calcium ionophore A23187 was inhibited in a concentration-dependent manner (Figure 3). This result matches with the inhibition of capacitation produced by OBACE because both capacitation and acrosome reaction are Ca²⁺-dependent in mammalian sperm. There is supporting evidence that the acrosome reaction depends on the increase in intracellular Ca²⁺ concentration [Darszon and Hernández-Cruz 2014; Harayama 2013; Santi et al. 2013]. Therefore, we suggest that the presence of high concentrations of calcium bis-(hydrogen-1-malate) hexahydrate in OBACE [Reyes et al. 2005] is not directly related with the lack of acquisition of sperm capacitation and the further inhibition of the, Ca²⁺-dependent, acrosome reaction [Hernández et al. 2001]. This hypothesis is supported by the results of the measurement of the Ca²⁺ influx, where the presence of OBACE clearly diminished the calcium influx in a concentration-dependent manner (Figure 4A and B). Calcium has an important role in sperm motility. Knockout mice have demonstrated that at least four Ca²⁺ channels (CatSper1, CatSper2, Cav2.3, and PMCA4) may participate in the regulation of intracellular Ca²⁺ concentration and, therefore, are involved in the initiation of sperm motility. CatSper1 is a testis-specific voltage-gated Ca²⁺ entry channel localized at the sperm principal piece [Darszon et al. 2011; Eddy 2006]. In human sperm, failure to increase intracellular Ca²⁺ concentrations is directly related with failure to acquire hypermotility in sperm stimulated with Catsper agonist [Alasmari et al. 2013; Correia et al. 2015]. In that order of ideas, sperm unable to undergo hypermotility will not achieve the acrosome reaction due to lack of Ca²⁺ in the signaling pathway [Costello et al. 2009; Navarrete et al. 2015]. OBACE should block some calcium channels present in mouse sperm, thus should inhibit the acrosome reaction [Darszon et al. 2011].

The acute in vivo toxicity of OBACE and the LD₅₀ was found to be 3,784.42 mg/kg, showing that OBACE is a low toxic extract. This result is in accord with the low effect against the sperm viability described above. In addition, OBACE clearly inhibits the total and progressive motility of mouse sperm treated for seven days with a single daily dose of 100 or 200 mg/kg (Figure 5A and B). Furthermore, all kinematic parameters of motility (VAP, VSL, VCL, ALH, and BCF) were inhibited in sperm collected from the vas deferens (Figure 5C to G).

Other plants have been documented to exert an antifertility effect, Acacia nilotica shows in vivo activity against the sperm motility similar to those showed by OBACE, and the effect was achieved until 16 weeks of daily administration [Lampiao 2013]. This evidence is useful to compare our in vivo results obtained by OBACE administration, being that the effect on the sperm motility was achieved at relatively short-term administration (seven days).

In addition to motility tests, the fertilizing capacity of OBACE-treated sperm was examined, revealing that OBACE-treatment significantly reduced IVF rates. These results and those from the other in vitro tests display characteristics of a potential contraceptive.

In human and mouse sperm, high concentrations of extracellular Ca²⁺ inhibit sperm motility, nevertheless, the concentrations of Ca²⁺ needed to support sperm motility are not well elucidated and the information provided in many reports is contradictory [Bhoumik et al. 2014]. Additionally, in maturing goat spermatozoa it has been shown that sperm auto-agglutination is dependent on Ca²⁺ [Banerjee et al. 2006]. It is therefore possible to say that the presence of high concentrations of calcium bis-(hydrogen-1-malate) hexahydrate in OBACE have an impact on the immobilization/agglutination effects.

With respect to the hypotonic-like effect, we can hypothesize that: (1) the effect is due to the possible influx of taurine present in OBACE. In mouse and human sperm, taurine regulates many physiological functions as motility and osmolarity. In somatic cells taurine is related to the modulation of [Ca²⁺]ion movement through an osmotic-linked mechanism resulting in alteration of the composition and properties of the phospholipid bilayer [Schaffer et al. 2000; Yeung et al. 2003]. In addition, taurine has been found in the secretions of the mammalian reproductive tract, preserving the motility of the spermatozoa [Bidri and Choay 2003]. (2) OBACE may induce the inhibition of enzymes of the taurine synthesis (such as hypotaurine dehydrogenase EC 1.8.1.3.) [Sumizu 1962], and the absence of this amino acid and the accumulation of precursors may produce all the effects mentioned above.

In summary, data obtained from in vitro studies showed that OBACE exerts inhibition of mouse sperm capacitation, acrosome reaction, and Ca²⁺ influx in a concentration-dependent manner. Nevertheless, OBACE did not affect sperm viability. In vivo, OBACE showed low toxicity and inhibited all sperm kinematic motility parameters. These results contribute to the scientific validation of the antifertility effect of this plant in Mexican folk medicine and we consider it important to continue with detailed screening of the components of OBACE to elucidate the mechanism of induction of the hypotonic-like and the agglutination/immobilization effects on mammalian sperm.

Material and methods

Materials and reagents

All materials and reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA) or Mallinckrodt Baker (Phillipsburg, NJ, USA). Other chemicals used were of the highest purity available.

Aqueous extract preparation

Plant specimens were collected in different batches at several locations in the México valley according the national biodiversity rights. Voucher specimens documenting these collections have been deposited in the Instituto Mexicano del Seguro Social (IMSS) Herbarium under the code number 12213. Echeveria gibbiflora aqueous crude extract was prepared by grinding 300 g of fresh plant leaves and diluted with enough water to give 250 mL of aqueous crude extract. This extract was filtered through a gauze and centrifuged at 300 xg for 10 min. In order to remove ions that are extremely deleterious to sperm cells, the supernatant was dialyzed against two changes of deionized water (20 L each). The active fraction remained inside the dialysis bag and was lyophilized [Huacuja et al. 1985] for subsequent studies.

Animals procedures

All the experiments were carried out in male adult CD1 albino mice of 20-25 g weight. We obtained these rodents from the ENCB-IPN vivarium. The animals were housed in polypropylene cages at room temperature and under normal light/dark cycle. All animals were fed with a standard pellet diet and water ad libitum. The investigation and animal protocols were approved by The Institutional Ethics Committee of Escuela Nacional de Ciencias Biológicas of the IPN in 2014, in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (Bethesda, MD, USA).

Sperm preparation

Mouse sperm was collected from vas deferens according to a published procedure [Mújica et al. 1991]. Sperms were allowed to disperse for 10 min at 37°C in Tyrode medium of pH 7.6 (120 mM NaCl, 2.8 mM KCl, 11.9 mM NaHCO₃, 0.36 mM NaH₂PO₄, 0.49 mM MgCl₂, 0.25 mM sodium pyruvate, 20 mM sodium lactate). The spermatozoa concentration was 8-10×10⁶ spermatozoa/mL for each experiment. Parallel incubations with and without OBACE were started by the addition of the pre-warmed (37°C) medium.

Sperm viability evaluation

The sperm suspensions were incubated in Tyrode medium pH 7.6 at 37°C during 30 min in the presence and absence of different concentrations of OBACE (0.25, 0.5, and 1.0 mg/mL). Immediately after incubation, a double staining of FDA and PI [Jones and Senft 1985] was added to the sperm suspensions to evaluate the viability and the plasma membrane integrity. The effect of the different concentrations of OBACE on the sperm suspensions was measured by flow cytometry using a FACSAria cell sorter (Becton Dickinson Towson, MD, USA), equipped with a laser beam of 488 nm. The results were analyzed with the FACSDiva software (Becton Dickinson) [Díaz et al. 2004].

Sperm capacitation in vitro

The sperm cells were maintained at 37°C in a modified Tyrode’s medium (120 mM NaCl, 2.8 mM KCl, 11.9 mM NaHCO₃, 0.36 mM NaH₂PO₄, 0.49 mM MgCl₂, 0.25 mM sodium pyruvate, 20 mM sodium lactate, 3 mg/mL BSA, 1 mg/mL glucose, 1.7 mM CaCl₂). To achieve the capacitation, the mouse sperm were incubated 2 h with and without different concentrations of OBACE (0.25, 0.5, and 1 mg/mL). At the end of this time, the cells were stained with chlortetracycline (CTC) [Pietrobon et al. 2001]. The assay was performed by counting 200 cells per sample in high-magnification at 400 x in a Olympus BX50 photomicroscope equipped with phase contrast and epifluorescence. The samples were counted immediately, and the sperm status, characterized by the fluorescence pattern [Ward and Storey 1984], were also observed.

Sperm acrosome reaction in vitro

The sperm cells were maintained at 37°C in a modified Tyrode medium (120 mM NaCl, 2.8 mM KCl, 11.9 mM NaHCO₃, 0.36 mM NaH₂PO₄, 0.49 mM MgCl₂, 0.25 mM sodium pyruvate, 20 mM sodium lactate, 3 mg/mL BSA, 1 mg/mL glucose, 1.7 mM CaCl₂). Sperm were incubated for 2 h. Afterwards, 10 µM calcium ionophore A23187 was added to the sperm suspension. The same samples were also incubated with and without different concentrations of OBACE (0.25, 0.5, and 1 mg/mL), during 30 min. At the end of this time, the suspensions of acrosome reacted sperm were centrifuged (200 xg for 10 min) and the supernatants were used to measure the activity of acid phosphatase by a fluorometric method using 4-methylumbelliferyl phosphate as substrate at a concentration of 0.03 mg/mL in 0.05 M citrate buffer, pH 4.5 in a Synergy 2 Multi-Function Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA). After incubation for 30 min at 37°C, the reaction was stopped by adding 1 mL of 0.4 M glycine buffer, pH 10.4. One unit of enzymatic activity represents the amount of enzyme that catalyzes the release of 1 pmol of 4-methylumbelliferone/mL per h. The Tyrode medium was used as the blank [Pietrobon et al. 2001].

Ca²⁺ influx determination

The sperm cells were maintained at 37°C in a modified Tyrode medium (120 mM NaCl, 2.8 mM KCl, 11.9 mM NaHCO₃, 0.36 mM NaH₂PO₄, 0.49 mM MgCl₂, 0.25 mM sodium pyruvate, 20 mM sodium lactate, 3 mg/mL BSA, 1 mg/mL glucose, 1.7 mM CaCl₂). The sperm concentration was adjusted to 8 x 10⁶/mL followed by incubation for 60 min at 37ºC, 5% CO₂ in the same medium. Also, the same samples were incubated with and without different concentrations of OBACE (0.25, 0.5, and 1 mg/mL). During the last 15 min of capacitation, cells were loaded with 1 μM of Fura-2 AM (Molecular Probes) and 0.05% pluronic acid F-127 at 37ºC under 5% of CO₂, followed by a wash by centrifugation in the same medium at 500 xg for 5 min to eliminate the fluorescence background. The fluorescence measurement was carried out in a Synergy 2 Multi-Function Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA), at an excitation wavelength of 340-380 nm and recording emissions at 510 nm [Alasmari et al. 2013; Li et al. 2014; Tamburrino et al. 2014]. Changes were recorded during 60 min and the readings were interpolated in a Ca²⁺ calibration curve. A sample control with 5 μM of NiCl₂ to inhibit Ca²⁺ channels was present [Darszon et al. 2011; Lee et al. 1999].

Acute toxicity evaluation

The median lethal dose (LD₅₀) was determined in male CD1 mice weighing 22 ± 3 g. Groupings of three individuals were made and OBACE was administered intraperitoneally using isotonic saline solution as a vehicle (1/100 of the animal weight). The number of deaths was registered at 24 h, according with the method described by [Lorke 1983]. LD₅₀ was determined according to the protocol described by [Klassen 2001].

In vivo mouse sperm motility evaluation

To perform in vivo evaluations of OBACE, three groups were made, one control and two treatment groups (100 and 200 mg/kg), with six male CD1 mice per group weighing 22 ± 3 g. A daily OBACE dose was administered intraperitoneally for 7 d. After the administration period, the animals were sacrificed and the sperm cells were obtained from the vas deferens and gently mixed to evaluate the kinematic parameters of sperm motility using the TOX IVOS sperm analyzer system (Hamilton Thorne Biosciences, Beverly, MA, USA), software version 12.21[Cordero-Martínez et al. 2014]. Experiments were performed in triplicate.

In vitro fertilization assay

To perform IVF evaluations of OBACE, three groups were made, one control and two treatment groups (100 and 200 mg/kg), with six male CD1 mice per group weighing 22 ± 3 g. A daily OBACE dose was administered intraperitoneally for 7 days. After the administration period, the animals were sacrificed and the sperm cells were obtained from the vas deferens in KSOM medium and gently mixed to evaluate the morphology, motility, and viability. The sperm were capacitated during 1 h, to avoid variability between individuals, a pool sample was performed and 1x10⁵ cells was taken from each group to perform the IVF. Six female CD1 mice weighing 22 ± 3 g were superovulated with 5.0 IU of pregnant mare serum gonadotropin (PMSG) and after 48 h, they were also administrated with 5.0 UI of human chorionic gonandotropin (hCG). After 17 h the animals were sacrificed and the oviducts were removed and dissected free from surrounding tissues; a total of 51 oocytes were transferred into fertilization wells with KSOM medium, divided in three groups and incubated during 2 h at 37ºC [Liu et al. 2009].

IVF was performed by the addition of 1x10⁵ sperm cells into the fertilization wells and incubated during 17 h at 37ºC. Oocytes were stained with Hoesch 33342 (10 µg/ml) and placed in coverslips for examination using a microscope Zeiss AxioObserver equipped with Apotome 2 system (Zeiss, Mexico). A positive fertilization was determined by the presence of pronuclei, 2-cells stages or the presence of sperm in the oocyte cytoplasm [Neuber and Powers 2000].

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software). All data are shown as the mean±SEM. Statistical significance of the results was determined using one-way ANOVA followed by Dunnett test. Differences were considered significant at p<0.05.

Declaration of interest

CAA and JCF-A are fellows of the SNI-CONACyT. LR-P is a fellow of the COFAA-IPN and of the SNI-CONACyT. Authors declare that they have not any conflict of interest.

Acknowledgments

The authors express their gratitude to MVZ Ricardo Gaxiola Centeno for the handling of the experimental animals to carry out this study.

Additional information

Notes on contributors

Joaquín Cordero-Martínez

Drafted the paper and coached JGG-S and CES-A: JC-M; Performed the determination of in vitro motility: CA-A; Perform the determination of viability, capacitation, and acrosome reaction: JGG-S; Performed the determination of Ca²⁺ influx and acute toxicity: CES-A; Elaborated the plant extract and IVF assay: JCF-A; Performed the experimental design and thoroughly revised the manuscript: LRP.