Cell Viability, Acrosomal Status and DNA Integrity in Porcine Sperm Permeabilized with Streptolysin O

The aim of this study is to determine the use of streptolysin O (SLO), to open pores on the plasmatic membrane of the swine sperm, to see the effect on the viability, functional status and integrity of nuclear chromatin. 30 ejaculates were used; immediately after collection, semen was diluted in a solution of BTS (2:1, v/v preheated to 35ºC). Two aliquots of semen samples were assigned to each group, of which: Group 1. 0.3 IU/ml SLO, Group 2. 0.6 IU/ml, Group 3 1.2 IU/ml, and Group 4 Control (PBS). All groups were incubated for 5 minutes at 37ºC, in SLO; after this period, the samples were incubated in 5% fetal bovine serum (FBS) for 15 minutes at 37ºC. Each treatment were evaluated before and after incubation with FBS (to close the pores), in duplicate. Sperm viability, acrosome status and integrity of the nuclear chromatin were evaluated with eosin nigrosine (NE), chlortetracycline (CTC) and acridine orange (AO) stains, respectively. The data obtained was analyzed by comparing proportions in each treatment using non-parametric module of STATISTICA V10.0 program. The Kruskal Wallis H test was used for the analysis. In assessing sperm viability each of the treatments were compared in between, and the resulting differences were also compared with the control group (iv). There was no difference in the proportion of viable sperm, using the concentration of 0.6 IU/ml, before and after sealing of pores (83.15±9.10 vs 91.52±4.83) (p>0.05). And mathematical trend indicates that, after sealing the pores, a higher sperm proportion were viable in both treatments. However, no statistical differences were found (P>0.05). Also no changes were found in patterns of acrosomal status and DNA integrity, suggesting that the SLO executes its permeabilizing effect at the level of sperm membrane.


INTRODUCTION
The Streptolysin O (SLO) is a toxin produced by Streptococcus pyogenes, it's a protein with a molecular weight of 60 kDa; belonging to group A of cytolytic toxins β-haemolytica, known as oxygen-labile and produced by a gram positive bacteria. Besides its pathogenic interest, SLO has been reported as an ideal tool for cell pores forming [1]. The SLO is toxic for eukaryotic cells, because of its affinity for cholesterol, but in controlled doses, has proven to be useful for live cell permeabilization [2,3]. In the case of sperm, the SLO has been used to study the mechanisms that regulate some physiological processes of the sperm as capacitation, the acrosome reaction and motility [4,5]. The SLO binds to cholesterol in the cytoplasmic membranes of animal cells [6], and subsequently participates in reversible pore formation, in the plasma membrane [6,7,8]. The poration with SLO has been used extensively to introduce external molecules, such as proteins, DNA, and RNA, into the intracellular compartment [9,10,11]. Fawcett et al. [3] conducted a research with SLO (0.2 IU / ml) to form pores in the cells membrane of mouse myocyte and introduce fluorescent macromolecules [3]. Other work with mouse sperm, shown that these cells remain viable in SLO medium with concentrations up to 0.6 IU / ml for short periods of time, showing greater viability than cells treated with lower concentrations and a longer exposure [4]. Other studies mention the use of SLO as a former of pores in the membrane, to introduce proteins in different cell types of the immune system; Moreover, after the sealing of pores, such cells showed similar viability rates to those of control cells that were not treated with SLO [9]. In a more recent work, Bo-Woong et al. [12] used SLO a dose of 5 IU/ ml as a medium for the introduction of exogenous DNA into mouse sperm and showed that there was an increase in the rate of transgenic embryo production and later litters than with intracytoplasmic sperm injection. The sperm cell is a complex and a good biological model to several studies; the opening of the pores can modify their structure and, therefore, serve as a vehicle for cryopreservation studies and biotechnology. The aim of this study is to determine the effect of streptolysin on cell viability, functional status and integrity of nuclear chromatin of swine sperm.

Reagents
Unless otherwise stated, all of the chemicals used in the experiments were analytical grade and were purchased from Sigma-Aldrich Co. (

Biological Material
The work took place from February to July 2014. Thirty ejaculates, which were analyzed before use, to check that met inclusion criteria. Procedures involving animals were approved by the Institutional Animal Care and Use Committee at the University of Veracruz, Mexico. Two-four years old clinically healthy boars without pathologies associated to the male reproductive tract and with a proven history of fertility after conventional AI with fresh extended semen were selected. In addition, boars followed a routine schedule of vaccination, de-worming and vitamins (mainly ADE) appropriate to the species. Boars were housed in individual pens, fed twice daily with 1.5 kg of concentrate that contained 15% of crude protein and 1.2 Mcal of metabolizable energy.

Collection
Semen was collected by the gloved hand technique. During the collection, semen was filtered through sterile gauze and only the spermrich fraction was used. Semen samples with 70% progressive motility and 80% normal sperm morphology and intact acrosome status (assessed by phase contrast microscopy, the sperm samples were fixed in 2% buffered glutaraldehyde).

Motility evaluation
To evaluate mass motility, a semen drop was placed in a glass slide at 37ºC, and was observed under microscope (Olympus CX41) 100 x magnification, for one minute. The estimate is based on intensity of waves, sorting have values 0-5; samples with less than 3 degrees, were not considered for this study. For individual motility analysis, a drop of diluted semen in saline, was placed on a glass slide at 37ºC, covered with a microscope was observed under microscope at 400 x magnification; was evaluated for progressively motile spermatozoa (scale 0-100%) [13].

Morphology
The morphology was evaluated with the technique of chinese ink, which is prepared by placing a volume 1: 8 v/v semen-ink and it was observed with a microscope at 400 x counting 200 cells per replicate [14].

Dilution
Immediately after the collection, the semen was diluted in a BTS solution (2: 1, v / v) preheated to 35ºC. Subsequently, the semen was transported in a container between 20-22ºC, to the Laboratory of Biology of Animal Reproduction FMVZ UV.

Sample preparation and groups description
Two aliquots with semen samples were assigned to each group with: Group 1. 0.3 IU/ml SLO, Group 2. 0.6 IU/ml, Group 3 1.2 IU/ml, and Group 4 Control (FBS). All samples were incubated for 5 minutes at 37ºC, in SLO; after this period, the samples were incubated in fetal bovine serum (FBS) at 5% for 15 minutes at 37ºC.

Cell permeabilization
Cells were permeabilized, using the protocol described by Fawcett et al. [3]. For the membrane permeabilization, three treatments were used with Streptolysin O (SLO) lyophilized Sigma Aldrich brand. The lyophilized streptolysin was dissolved in cold distilled water to 60 IU / ml and activated with 4 mM DTT at for 2 h at 37ºC and aliquots were stored at -20ºC until use. 100 ul of semen was added to each treatment and incubated for five minutes at 37ºC, washed twice by centrifugation at 1200 rpm for 3 minutes, the supernatant removed and resuspended in 1 ml PBS. Concentrations for the treatment with SLO were 0.3, 0.6 and 1.2 IU / ml.

Sealed method
For closing the pores, Fetal Bovine Serum (FBS) at 5% in PBS was used, to restore the integrity of cell membrane. The incubation was performed at 37ºC for 15 minutes [3].

Cell evaluation
Each experimental group was evaluated before and after incubation with FBS, this was done in duplicate. The sperm viability was evaluated by eosin nigrosine stain (NE), the acrosomal status of sperm by chlortetracycline technique (CTC), and the integrity of nuclear chromatin was evaluated by fluorescence technique with acridine orange (NA).

Sperm viability
The assessment of sperm viability was performed with eosin nigrosine staining (EN) (HYSEL ® ). Using a 1:8 dilution of the semen sample and dye, the sample mixture was incubated for 5 minutes at 37ºC and a smear was made on a slide. The slides were observed under phase contrast microscope at 400x, several fields were reviewed to count 200 cells, which showed the following pattern of coloration.  [14,15].

Images of Eosin Nigrosine staining (EN)
A

Acrosomal status
To determine the status of capacitation and acrosome reaction, It was used the technique described in 1993 by Das Gupta et al, which must presented the following fluorescence patterns: [16]. i) Uniform fluorescence over the whole head: Un-Capacitated and -acrosome-reacted. ii) Fluorescence-free band in the postacrosomal region: Capacitated. iii) Dull fluorescence over the entire head: capacitated and acrosome-reacted.

DNA integrity assessement
DNA integrity was assessed by fluorescence technique with acridine orange (OA). It was used as described by Tejada (1984). Covered the slide and immediately observed under the fluorescence microscope (Leica DM 020-518500 / LS) with filter blue excitation 405-455 nm, 400x, reviewed several fields to count 200 cells which must present the following fluorescence patterns:  Green sperm = intact DNA.  Orange sperm or red Sperm = denatured DNA [17].

Statistical analysis
The data obtained were analyzed using the nonparametric module V10.0 STATISTICA program; the parameters evaluated include: the percentage of the acrosomal status CTC technique, the percentage of plasma membrane integrity by eosin nigrosin technique and the percentage of the integrity of the nuclear chromatin and acridine orange technique. Fig. 1 shows the percentage of live sperm in each of the treatments and there was no statistically significant differences (P>0.05), among the groups. The percentage of live spermatozoa increased in all treatments after incubation with 5% FBS; however this increase was not significant (P>0.05). Moreover, as shown in Table 1, the value of the lowest sperm viability was 83.15±9.10 with the SLO 0.6 IU / ml treatment, before incubation with FBS, while the highest value (91.52±4.83) was found in the same concentration of SLO but after being incubated with SFB to seal the pores; the differences were not significant (P>0.05).

Acrosomal Status
A not significant (P>0.05) decrease in the percentage of un-capacitated and -acrosome reacted spermatozoa only in 1.2 IU / ml of SLO treatment group in comparison to control before and after incubation with FBS was observed. The differences among the groups are shown in Fig. 2. The highest value (83.68%) was for control treatment before incubation with FBS and the lowest (79.42%) was in the 1.2 IU / ml of SLO treatment group, after sealing of pores; However, these results are not significant (P<0.05) for any of the treatments due to the standard deviations are > 4.7% (Table 2).  Fig. 3 shows the effect of treatment on the integrity of the nuclear chromatin of spermatozoa. There was no statistically significant difference (P<0.05) between treatments before or after incubation with FBS. The percentages of cells with chromatin integrity remained > 98% in all treatments (Table 3) before and after sealing of pores. Although standard deviations were lower than in other techniques, but still no significant (P<0.05) difference was observed between control and treatment groups.

DISCUSION
In this study the effect of incubation with streptolysin O on sperm viability, indicated that there is no difference between treatments before and after sealing of the pores. The observed decrease in the percentage of viable sperm incubated with SLO for the permeabilization (Fig. 1), followed by an increase of the percentage of viable sperm after incubation with FBS for sealing pores, indicates SLO acts on the sperm plasma membrane, this allows the entry of eosin dye into the cell, similar to that described by Tartakoff [18], which states that certain dyes waterproof such as trypan blue and eosin, can be able to cross the plasma membrane of cells treated with toxins pores forming as the α-toxin, and the digitionina SLO and that prolonged exposure to such dyes causes staining in intact cells; however, in this study no statistical differences between treatments were found before and after the sealing of pores, contrary to what was reported by Johnson et al. [4], that using the same technique at a dose of 0.6 SLO IU / ml achieved 80% permeabilization in mouse sperm. Moreover, Michaut et al. [21] and Yunes et al. [19,20] conducted a study in human sperm and achieved to permeabilize 100% cells. These differences can be associated to the cholesterol found in the membrane, which differs significantly between species, being the lowest in pigs than in mouse or human and as mentioned above, the SLO is a toxin that acts by binding to cholesterol in plasma membranes, which have great concentration in these species. Fawcett et al. [3] found that the temperature, the dose and the period of exposure to the toxin, are factors that are associated with the effect of this on the membranes. Worked with mouse myocytes at a dose of 0.2 IU / ml at 37ºC for 5 min, and a higher percentage of viable cells when higher exposure periods and higher doses of the toxin used was found; In our study the incubation temperature was 37ºC and the time of 5 minutes, and it was found that the viability, status of acrosome integrity and integrity of nuclear chromatin, were maintained in high percentages in all treatments, similar to reported by these authors. To determine the effect of SLO on sperm physiology technique CTC, which indicates the state of capacitation and acrosome reaction was used to perform the study. No statistically significant differences between treatments before and after incubation with FBS for sealing; however, a decreasing trend is observed in the percentage of un-capacitated and acrosome reacted. This decrease can be attributed to the contact of the sperm and the seminal plasma and begins to develop such processes naturally [22].
In this study, porcine sperm permeabilization for 5 minutes with SLO 0.3, 0.6 and 1.2 IU / ml was found that the percentages of cells without capacitation and without acrosome reaction were of 80.34±7.8, 83.67±5.6 and 81.71±8.18 respectively, similar to those reported by Johnson et al. [4]; to permeabilize SLO mouse spermatozoa with 0.6 IU / ml for 5 minutes found that most of the cells remained with intact acrosome (75%) and that increasing the exposure time of 15 minutes the percentage decreased (44%). These results indicate that permeabilization with SLO performed in the plasma membrane and not in acrosomal membrane.
Until now there are no reports about the effect of the use upon the integrity of the nuclear chromatin of sperm cells SLO as permeabilizer; However, many authors report that individuals with spermatozoa exihibiting > 50% full chromatin are considered normally fertile. In our study, > 98% of spermatozoa were with intact chromatin in all treatments before and after incubation with FBS, which indicates that the SLO has no negative effect on the structural integrity of nuclear chromatin and permeabilized and sealed cells are able to fertilize.

CONCLUSION
In this work it was found that the SLO acts on the plasma membrane while the use of FBS as pore sealant does not affect sperm physiology. Finally no negative effect was observed in the use of the SLO in the structural integrity of DNA.