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Amongst all events which lead into a successful pregnancy in the mammalian female reproductive tract, preservation of sperm motility and viability as well as regulating its maturation are of high significance, especially in the species in which there are gaps of hors to days between mating and ovulation. Formation of sperm reservoir inside the female oviduct is a clever solution to accomplishment of this regulatory task. Reduction of the oviductal tube size located after the utero-tubal junction, decrease in sperm motility and binding of spermatozoa to mucosal epithelium are a combination of mechanisms observed to form and and maintain the reservoir (Suarez et al., 1991).
Oviductal reservoir provides a secure and effective setting for sperm to become fertilising competent via two different local systems. One is producing secretory fluid which contains various types of molecules, into the oviduct lumen and the other is the mucosal epithelium. Upon arrival to the oviductal region, spermatozoa closely associate with both the systems by bathing in the secreted fluid or establishing tight bonds with the apical epithelial membrane. Each of the systems contains a number of proteic factors reported to positively influence sperm properties beneficiary to fertilisation. HSP60 in the secretions maintains sperm viability and acrosome integrity via in vitro co-incubation (Boilard et al., 2004). HSPA8, a 70 kDa heat shock protein purified from the apical segment of oviductal epithelial cells maintains in vitro sperm viability (Elliott RM, 2009).
Existence of HSPA8 in extracellular space and observations regarding its positive effects on spermatozoa rendered further investigations on the possible effects of exogenous HSPA8 on important characteristics of spermatozoa. For this, a system was developed for in vitro incubation of boar spermatozoa with varying concentrations of bovine recombinant HSPA8 at different time intervals. HSPA8 concentrations were inspired by the previous study carried out by Elliott RM and colleagues (Elliott RM, 2009). The long-term (48 h) effects of HSPA8 on boar sperm viability as well as its short-term (15 min) exposure effect on sperm viability, mitochondrial activity and capacity to bind immortalised pig OEC monolayers (hTERT-OPEC) were studied. The effect of sperm pre-incubation under capacitating conditions on the ability of HSPA8 to increase sperm viability after brief exposure time (15 min) was also evaluated. Meanwhile, the specificity of the effect of HSPA8 on sperm viability was determined by evaluating the effect of Î±-tubulin and HSP70 as control proteins. Sperm viability was evaluated using two fluorescent viability assays; Calcein-AM & Ethidium-homodimer and SYBR-14 & Propidium iodide. Mitochondrial activity was evaluated by Jc-1 and chlortetracycline staining method was used to assess the capacitation status of boar spermatozoa.
As preliminary approach to the sperm viability enhancing mechanism/s of HSPA8, Fluorescence recovery after photobleaching (FRAP) technique was applied, aiming to discover the possible alterations in sperm membrane fluidity under the influence of HSPA8. Boar semen was chosen for establishing the system, first because there were other relevant studies with useful guiding background information and second, due to easy accessibility to plenty amount of freshly extended boar semen.
2.2. Methods and Materials:
Media, Reagents and cell line:
All chemicals used in the preparation of the media were purchased from Sigma-Aldrich Co. (UK) unless otherwise stated.
Tyrode's medium consisted of 3.1 mM KCL, 0.4 mMMgCl2, 6H2O, 2 mM CaCl2, 25mM NaHCO3, 10 mM HEPES, 0.3 mM NaH2PO42H2O. TALP consisted of Tyrode's medium supplemented with 12 mg/ml Bovine Serum Albumin, 21.6 mM. sodium lactate and 1 mM sodium pyrovate. DMEM/F12 HAM medium (500 ml) was supplemented with 10% fetal calf serum, 1% L-Glutamine, 1% Penicillin/Streptomycin, 160 ng/ml human recombinant insulin (Invitrogen, catalogue # 12585-014) and 7Âµl (1nM) 17-beta-estradiol (E2). Recombinant human stress-induced HSP70 (StressMarq, Catalog # SPR-115A). ODAF or 5-(N-octa-decanoyl) aminofluorescin was purchased from Molecular probes (Catalogue # D-109). Immortalized porcine oviductal epithelial cell line (TERT-OPEC) with stable epithelial phenotype described previously (Hombach-Klonisch et al., 2006).
2.2. Semen Preparation
Boar semen diluted and stored for 24 hours in BTS was obtained from a commercial artificial insemination station JSR Healthbred Limited, Thorpe Whillougby, Yorkshire,UK. On the day of experiment, diluted boar semen was washed using Percoll gradient technique (Holt and Harrison, 2002). Aliquots of 2-4 ml of diluted semen were layered over a two-step iso-metric Percoll gradient. The iso-metric Percoll gradient consisted of 2 ml 70% (v/v) Percoll overlaid with 2 ml 35% Percoll. The gradient was centrifuged at 200g for 15 min followed by 15 min centrifugation at 1000g. The supernatant was removed, and the pellet was resuspended in Tyrode's medium. Thereafter, semen samples were centrifuged for last time at 900g for 15 min and resuspended in Tyrode's medium. Sperm concentration was measured using a haemocytometer and adjusted to 5 x 106 spermatozoa with TALP, unless otherwise stated.
2.3.Evaluation of Sperm Parameters
2.3.1.Assessment of sperm viability
Sperm viability was evaluated using two fluorescent viability assays, Ethidium-Homodimer & Calcein-AM assay (Molecular Probes Kit, UK) and Propidium iodide & SYBR-14 assay (Molecular Probes Kit, UK). Live cells contain cytoplasmic esterase activity which can be used for enzymatic conversion of the nonfluorescent cell-permeant calcein-AM to the intensely fluorescent calcein. The polyanionic dye calcein can easily pass through intact live cells' membranes and by the esterase activity of the enzyme produces a high intensity uniform green fluorescence (ex/em ~495 nm/~515 nm). EthD is excluded by the intact plasma membrane of live cells and is merely able to enter cells with damaged membranes. Inside the cell it binds to nucleic acid and goes under a 40-fold enhancement of red fluorescence. Hence membrane damaged or dead cells exhibit a bright red fluorescence (ex/em ~495 nm/~635 nm) under the epifluorescence microscope.
The fluorescence-based viability assay, Propidium iodide and SYBR-14, is produced specifically for analyzing the viability of sperm. This combination of dyes label intracellular DNA. Live sperm cells with intact cell membranes fluoresce bright green (SYBR-14) and cells with damaged cell membranes fluoresce red (Propodium-Iodide). The fact that both dyes stain cellular nucleic acid prevents the vagueness of labelling different cellular components.
Dyes were added to 100 Âµl of semen aliquots with 5 x 106 spermatozoa to a final concentrations of 0.4ÂµM and 0.08 ÂµM, for Ethidium-Homodimer & Calcein-AM, and 12ÂµM and 4nM for Propodium-Iodide & SYBR-14, respectively. Samples were mixed and incubated for 30 min at 39Â°C in 5% CO2 for Ethidium-Homodimer & Calcein-AM assay and 15 min for Propodium-Iodide & SYBR-14 assay. A 10 Âµl aliquot of each preparation assay was placed on a slide and evaluated by fluorescence microscopy. Two hundred spermatozoa were counted per slide (3 replicates for each sample) and classified as live spermatozoa (green) and dead spermatozoa (red). Spermatozoa with intact membrane (live spermatozoa) showed green fluorescence due to Calcein-AM or SYBR-14 staining, depending on the assay. Spermatozoa with disrupted membranes (dead spermatozoa) showed red fluorescence due to Ethidium-Homodimer or Propodium-Iodide.
2.3.2. Assessment of sperm mitochondrial activity:
Mitochondrial activity in live cells based on electrochemical gradient across the mitochondrial inner membrane was evaluated by using a cationic, lipophilic dye, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide; Sigma-Aldrich Kit, UK).. JC-1 is a fluorescent dye which emits red (~ 590 nm) or green (~ 525 nm) depending on normal or collapsed mitochondrial potential respectively. Therefore, it is useful for determination of high or low mitochondrial activity in cells.
Due to the intact electrochemical potential gradient in normal cells, the dye forms red fluorescent aggregates (J-aggregates) in the mitochondrial matrix. Any insult that disrupts the mitochondrial membrane potential inhibits the JC-1 aggregation in the mitochondria and hence, the dye is distributed throughout the cell cytoplasm resulting in a shift from red (J-aggregates) to green fluorescence (JC-1 monomers). In sperm, the red JC-1 aggregates are mostly concentrated in midpiece due to the high density of mitochondria in there and to a less extent in the head.
JC-1 Kit was used according to manufacturer's instructions. Briefly, stock solutions of 200 x JC-1 (1mg/ml) were prepared in DMSO and stored in 25Âµl aliquots at -20Â°C. On the day of experiment, 25 Âµl aliquot of the 200x JC-1 stock solution was thawed and mixed by vortex in 4 ml of ultrapure water. The suspension was incubated for 1-2 minutes at room temperature to ensure that JC-1 is completely dissolved. Then, 1 ml of the JC-1 Staining Buffer 5x (supplied in the kit) was added to the suspension and mixed by inversion (staining solution). Two hundred Âµl of semen were added to 200 Âµl of staining solution, mixed and incubated for 20 minutes at 37 Â°C in a humidified atmosphere containing 5% CO2. Then, 10 ml of the 1x JC-1 Staining Buffer was prepared by diluting 1 ml of the JC-1 Staining Buffer 5x with 4 ml of water and kept on ice. The semen-staining suspension was centrifuged at 600 x g for 3-4 minutes at 2-8 Â°C. The supernatant was removed and the pellet was placed on ice. Then, the pellet was washed with 200Âµl of the ice-cold 1x JC-1 Staining Buffer and resuspended in 400 Âµl of the same solution. The sample was kept on ice and analyzed within 1 hour after staining.
To analyse mitochondrial activity in the live sperm population, the mitochondrial staining assay was combined with Propodium-Iodide staining (Garner et al., 1997). Spermatozoa that showed red fluorescence due to P.I were excluded from counting. Two hundred live spermatozoa per slide (3 replicates for each sample) were evaluated using an epifluorescence microscope.
2.3.3. Evaluation of sperm capacitation
The Chlortetracycline Hydrochloride (CTC, Sigma-Aldrich, UK) staining method was used to assess the capacitation status of boar sperm as described by Fazeli et al. (Fazeli et al., 1999). Briefly to perform CTC assay, a buffer containing 130mM NaCl and 20mM Tris (BDH) was prepared. The CTC staining solution was prepared by adding 750ÂµM CTC and 5mM D,L-cystein to the buffer solution. Then, the solution was filtered using a 0.22ÂµM filter and the pH was adjusted to 7.8. CTC assay was performed by mixing equal volumes of semen diluted in TALP and CTC staining solution. After 30 seconds, an equal volume of 2% Paraformaldehyde in PBS was added to the sample. Finally, 2 drops of an antifade material Citifluor (Citifluor Ltd., 18 Enfield Cloisters, Fanshaw Street, London) was added to each sample in order to preserve the fluorescence. Two hundred spermatozoa per slide (3 replicates for each sample) were evaluated immediately using an epifluorescence microscope. CTC assay showed 3 patterns of spermatozoa capacitated, uncapacitated and acrosome reacted spermatozoa as described by Wang et al. (Wang et al., 1995).
2.4.Sperm Binding Assay
2.4.1. Oviductal epithelial cells preparation
Boar oviductal epithelial cells monolayer (hTERT-EEC) were cultured in a 75-cm2 flask containing DMEM/F12 medium supplemented with 10% fetal calf serum, 1% L-Glutamine, 1% Penicillin/Streptomycin/Amphotericin, 160 ng/ml human recombinant insulin (Invitrogen, catalogue # 12585-014) and 7Âµl estradiol.
The flask was incubated at 39Â°C with 100% humidity and 5% CO2. Oviduct epithelial cells were cultured to confluency. The culture medium was refreshed every 48 hours. On the day of experiment, confluent cells were rinsed three times with PBS and detached from the flask wall by incubation with 3 ml Trypsin-EDTA solution for 10 min at 39Â°C. Detached cells were rinsed with 2 ml PBS for a couple of times to dissociate the cells. Then, 2 ml of DMEM/F12 medium containing fetal calf serum was immediately added to inhibit any further damaging action of Trypsin to the cells. Oviduct epithelial cells were centrifuged at 300g for 4 min. The supernatant was discarded and the cells were resuspended in 1ml of DMEM/F12 medium. Cell concentration was determined by a haemocytometer and adjusted to 2 x 106 cell/ml in DMEM/F12 medium supplemented with 10% fetal calf serum, 1% L-Glutamine, 1% PSN (antibiotics), 20 Âµl insulin and 7Âµl estradiol.
2.4.2. Sperm-Oviductal epithelial cells co-incubation
Percoll washed boar sperm samples were diluted and adjusted to a final concentration of 100 x 106 spermatozoa/ml in TALP. Following the methods used by Green et al. (Green et al., 2001). Briefly, two hundred microlitres of boar sperm samples and 200ml of oviduct epithelial cells were transferred to the same tube. The tubes were incubated at 39Â°C, 5% CO2 for 30 min on a rotator. To remove unbound or loosely attached spermatozoa, 400 ml of sperm-oviductal epithelial cell suspension was layered over a two-step Percoll gradient containing 1ml of 35% Percoll overlaid on the top of 1ml of 70% Percoll in a 15 ml polypropylene tube.
After centrifugation at 200 g for 3 min 3 layers of cell appeared. Non-motile, unbound spermatozoa and unattached oviductal epithelial cells located at the interface between the media and Percoll 30% (layer 1). Sperm-oviductal epithelial cell complexes located at the interface of the two Percoll layers (layer 2), and unattached highly motile spermatozoa sedimented at the bottom of the tube (layer 3). Cells from layers 1 and 2 were removed carefully using a pipette. Then, cells from the 2 layers were combined and diluted in 15 ml of PBS. Unattached spermatozoa were removed by centrifugation at 200 g for 5 min. The supernatant was discarded, the sperm-oviductal epithelial cells were resuspended in 500Âµl of PBS and fixed with 1% formaldehyde in PBS. In order to count the number of spermatozoa bound to epithelial cells, 10 Âµl of the fixed cell suspension was placed on slide under a cover slip.
2.5. Fluorescence recovery after photobleaching; a method to measure sperm membrane fluidity
Fluorescence recovery after photobleaching (FRAP) is an optical technique based on fluorescent contrast principals which is able to quantify the fluidity of molecular clusters gathered as a thin film. In other words, FRAP is a method for measuring the two dimensional lateral diffusion of fluorescently labelled molecules. It is a useful method in cell membrane studies investigating live cell membrane lipid and protein diffusion and fluidity.
The basics contributing to FRAP development lie in simple physics and biologic known facts. The biologic aspect dates back in 1972 when the original fluid mosaic model of membrane structure was proposed (Ladha et al., 1997). This described the membrane lipids and proteins as freely diffusing components within the membrane bilayer arrangement and Brownian motion was proposed as a contributing factor to membrane fluidity. The Brownian diffusion leads to the constant movement of molecules fro highly concentrated regions to those with lower density levels and this way reduces heterogeneity (Marguet et al., 2006).
From the physics point of view, fluorescent molecules loose their ability to emit photons after being exposed to frequent excitation and emission cycles. In other words, intense excitation light photobleaches fluorophores in an irreversible manner.
To briefly describe the FRAP technique: The target molecule in membrane whether lipid or protein are loaded with a non disturbing amount of fluorescent probe to produce a quantifiable signal after excitation with the appropriate wavelength (Wolfe et al., 1998).
Laser beam is focused on the membrane and the initial fluorescent intensity (prebleach intensity) is recorded.
The laser intensity is increased to 1000 folds for an extremely short period (mseconds) to permanently bleach the the fluorescent molecules inside the laser beam area.
Based on Brownian rules, the surrounding unbleached molecules travel to the bleached area and mix with photobleached molecules.
The recorded kinetics report:
Rate of diffusion (D) of the fluorescently labelled molecules within the membrane bilayer.
Rate of recovery (R) which represents the proportion of the freely diffusing fluorescent molecules.
In the current study, ODAF excited at 488nm, was used as a fluorescent lipid probe reporter to investigate the effect of HSPA8 on boar sperm membrane lipid mobility. Live and dead staining pattern is markedly different in samples stained with ODAF; live spermatozoa are always stained weakly whereas the dead and membrane disrupted ones absorb more of ODAF and appear in stronger fluorescence, particularly over the acrosome and mid piece domains. ODAF uptake was checked by a Zeiss epifluorescence microscopy fitted with a 100 x objective lens. Laser beam was produced by a water-cooled argon ion laser and bleaching time was 5 milliseconds at 0.2 mW. The laser beam was ~ 5 ÂµM. FRAP analysis was performed by COOLSNAPHQ / ICX285 camera. Excitation and emission filters at 406 and 530 nm were applied.
2.6.1. Effect of different concentrations and co-incubation times of HSPA8 on boar sperm viability
In attempt to evaluate the effect of HspA8 on boar sperm viability, different concentrations and different incubations times were tested. Aliquots of semen from 10 different boars diluted in TALP (5 x 106 spermatozoa/ml) were incubated with 0 (Control), 0.1, 0.5 and 1Âµg/ml of HspA8 at 39Â°C, 5% CO2 during 24 (T24) and 48 hours (T48) and at room temperature for 15 min (T0). Samples were stained with Ethidium-Homodimer and Calcein-AM as described above at T0, T24 and T48.
2.6.2. Effect of short exposure time of HSPA8 on boar sperm viability
To investigate if a short incubation time (15 min) is sufficient to extend sperm viability by HSPA8.
Diluted semen samples (5 x 106 spermatozoa/ml) of 7 boars were incubated with and without (Control) 0.5 Âµg/ml of HspA8 at room temperature for 15 min. and then, were stained with Ethidium-Homodimer/ Calcein-AM and Propodium-Iodide/SYBR-14 dyes separately, to determinate the viability of boar spermatozoa.
2.6.3. Effect of short exposure time of HSPA8 on boar sperm mitochondrial activity
In order to assess sperm mitochondrial activity under the immediate effect of HspA8, JC-1 staining protocol was performed on the samples incubated at room temperature with 0 and 0.5Âµg/ml HSPA8. Using epi-fluorescent microscope, two hundred live spermatozoa were counted in 3 replicates of each sample and the proportion spermatozoa with high and low mitochondrial activity was determined.
2.6.4. Effect of short exposure time of HSPA8 on pre-incubated boar sperm viability
In this experiment, we aimed to characterise the effect of sperm pre-incubation on HSPA8 ability to expand sperm viability during a 15min exposure period. A total of 6 boar semen samples was pre-incubated at different intervals: 0, 1, 2, 4 and 6 h. At each time-interval, pre-incubated sperm samples were mixed with and without (Control) 0.5Âµg/ml of HSPA8 for 15 min. Then, viability and capacitation status of each sample were determined separately.
2.6.5. Effect of HSPA8 on sperm-oviductal epithelia cell binding
To investigate the effect of HspA8 on boar sperm capacity to bind ovidutal epithelial cells, boar sperm samples adjusted to 100 x 106 sperm/ml concentration in TALP were incubated at room temperature with and without (Control) 0.5Âµg/ml of HspA8. Then, 200 ml of boar sperm samples with or without (Control) HSPA8 and 200ml of oviduct epithelial cells were transferred to the same tube. The tubes were incubated at 39Â°C, 5% CO2 for 30 min on a rotator. The experiment was performed as described in the materials and methods section. In order to count the number of spermatozoa bound to epithelial cells, 10 Âµl of the fixed sperm-oviductal epithelial cell suspension was placed on slide under a cover slip. The number of spermatozoa attached to 100 oviductal epithelial cells was counted by light microscopy in three replicates.
2.6.6. Effect of varying concentrations of stress-induced HSP70 and HSP70-HSPA8 combination on boar sperm viability
Percoll washed spermatozoa (n=9) were incubated in TALP containing varying concentrations of HSP70 (0.1, 0.5 and 1Âµg/ml) and combinations of 0.1-0.1, 0.5-0.1, 0.5-0.5 and 1-0.5Âµg/ml HSP-HSPA8 (3:2), respectively. A control sample with no protein as well as a sample with HSPA8 0.5Âµg/ ml were included for final comparison.
2.6.7. Lateral mobility of plasma membrane lipids in boar spermatozoa in response to the presence to HSPA8.
To observe the short exposure (15 min) effect of HSPA8 on sperm membrane fluidity , after determination of sperm viability % by PI and SYBR-14, equal volumes of washed boar sperm adjusted to 10 x 106 /ml in Tyrode's and 12.5 ÂµM ODAF in ethanol 2% were incubated for 15 min at room temperature. Then washed twice by centrifugation at 400 g for 10 min. Labelled spermatozoa were resuspended in Tyrode's 200Âµl containing 0 and 0.5Âµg/ml HSPA8 (Wolfe et al., 1998). After 15 min of incubation in room temperature FRAP analysis was done on spermatozoa samples.
The data were expressed in mean Â± SEM. ANOVA, Fisher's exact test was used to examine the effect of treatments within experimental designs. The level of significance was considered p â‰¤ 0.05.
3.1. Influence of HSPA8 on boar sperm viability, capacitation, mitochondrial activity and capacity to bind OECs
To test the effect of the extracellular bovine recombinant HSPA8 on boar sperm survival rate, washed spermatozoa from 10 different boars were co-incubated at 39Â° C in TALP medium (capacitating conditions) containing 0, 0.1, 0.5 and 1Âµg/ml HSPA8 for 24 (T24), 48 (T48) hours and at room temperature for 15 min (T0) and then, sperm viability was evaluated by Ethidium-homodimer & Calcein-AM at designated time intervals. Both long-term (24 and 48h) and short-term (15 min) sperm-HSPA8 co-incubation showed significant overall viability enhancement (pâ‰¤0.0001) regardless of the HSPA8 concentration. However, this positive survival enhancing effect appeared to be optimal at 0.5Âµg/ml at T0 and T24 and 1 Âµg/ml at T48. (Figure 8)
The outcome from brief sperm-HSPA8 exposure (15 min, T0) was pursued further by repeating the bioassays with the optimal concentration of HSPA8, 0.5Âµg/ml as discovered in the previous experiment and then sperm viability was evaluated using SYBR-14 & Propidium iodide along with the previous viability assay, Calcein-AM & Ethidium-homodimer for comparison and validation between sperm viability evaluated by two viability assays. As additional comparison, the spermatozoa were also incubated in the presence of Î±-tubulin 0.5Âµg/ml to evaluate the effectiveness of HSpA8 against other proteins.
The beneficial effects of HSPA8 on sperm viability over a 15 min exposure to spermatozoa were again evident assessed by SYBR-14 & Propidium iodide, as in the earlier experiment (p= 0.002). (Figure 9) Tubulin as a control protein treatment had no beneficial effect on maintaining sperm survival and it was concluded that the effect of HSPA8 on enhancing sperm survival must be specific.
To test the effect of sperm pre-incubation (capacitation) on the HSPA8 ability to extend sperm survival, pre-incubated spermatozoa (n=6) in capacitating conditions (TALP medium, 39Â°C) at 0, 1, 2, 4, 6 h were mixed with 0.5Âµg/ml HSPA8 for 15 min at room temperature. Then sperm viability along with capacitation status of live sperm population were evaluated by SYBR-14 & Propidium iodide and Chlortetracycline staining, respectively.
As depicted in Fig. 10A & B , 15 min exposure of HSPA8 to pre-incubated spermatozoa does not affect sperm capacitation process, whereas, the beneficial effect of HSPA8 on maintaining sperm survival decreased along with the increase in pre-incubation time and rise in the number of capacitated spermatozoa. The half life of the ability of HSPA8 to extend sperm viability over 15 min exposure time lasted no more than 4hours of sperm pre-incubation. Therefore, it was concluded that capacitated sperm is not responsive to the positive HSPA8 effects in respect to survival enhancement.
As HSPA8 had a positive effect on boar sperm viability over a brief period of 15 min, we hypothesised that its role might apply to other sperm characteristics. To test this hypothesis, a sperm mitochondrial activity bioassay was set up.
A total of 9 boar semen samples were briefly (15 min) exposed to 0.5Âµg/ml bovine recombinant HSPA8 before being evaluated by Jc-1 staining for mitochondrial activity assessment. A control treatment with no protein exposure was also set up. Exposure of spermatozoa to HSPA8 did alter sperm mitochondrial trans membrane potential and reduced mitochondrial activity (TALP control versus HSPA8 0.5Âµg/ml, p=0.002). (Figure 11)
The influence of HSPA8 on capacity of boar sperm to bind oviductal epithelial cells was assessed based on the assumption that the effect is brought to sperm membrane to enable higher bonding capacity. To test this hypothesis, washed spermatozoa (n=7) were briefly (15 min) incubated at room temperature in TALP with 0 and 0.5Âµg/ml HSPA8 and sperm-OEC binding assay was done as previously described in methods. Inclusion of HSPA8 in the incubating medium increased the number of spermatozoa attached to single epithelial cells (TALP control versus 0.5Âµg/ml HSPA8; p=0.003). (Figure 12)
3.2. Determination of HSPA8 effect specificity
Adding varying concentrations of HSP70 did not alter the sperm survival after 15 min exposure time in contrast to HSPA8 which had significant increasing effect on sperm viability, (Figure 13) neither, combination of HSP70 and HSPA8 at varying concentrations did seem to have any kind of positive or negative effects on boar sperm survival contrary to the positive effect of HSPA8. This suggested that HSP70 prevents HSPA8 from affecting sperm viability. (Figure 14)
3.3. FRAP data analysis:
Live pattern spermatozoa uniformly stained over the surface were analysed on both acrosome and postacrosome domains. Preliminary measurements of initial fluorescent intensity, diffusion coefficients (D) and percentage recovery (R%) for ODAF in acrosome and postacrosome domains are shown in table 2.
As shown in table 2, fifteen min exposure of spermatozoa to HSPA8, significantly increased D values obtained on acrosome and postacrosome of live pattern spermatozoa relative to non-exposed spermatozoa (p <0.001, 0.005 respectively). This concludes that the rate of diffusion at which labelled membrane molecules travel is higher when sperm is in contact with HSPA8 in the medium.
Live pattern spermatozoa showed recovery (R) % higher than 60% on both acrosome and postacrosome domains of control and test samples, with R% of test samples being significantly higher than that of control groups (p= 0.007, 0.009 for acrosome and postacrosome, respectively). Calculated 5 min. R% for acrosome and postacrosome domains revealed significantly higher values for test groups at 1, 2, 3 and 1,2 second time points, respectively relative to the control samples. Given R% values for 4 and 5 second time points for acrosome and 3,4 and 5 time points for postacrosome did not show any significant difference between samples with and without HSPA8. This indicated that in spermatozoa exposed to HSPA8 the moving proportion of fluorescently labelled molecules over acrosome and postacrosome regions were higher initially and gradually decreased with time and the largest R% was of the first seconds' on both domains figure ().
Surprisingly, the average recorded initial intensity of fluorescence on acrosome and postacrosome domains revealed approximately 2 fold higher values on spermatozoa in test samples relative to control ones which means staining was enhanced as the result of HSPA8 introduction. Considering equal conditions for all samples except for addition of HSPA8, this indicates the different reactions membrane molecules express in response to varying treatments.
This was performed based on the fact that these two proteins function together in vivo in a number of cell types. However, none of the studies demonstrated similar results compared to the positive immediate effect of HSPA8. Furthermore, it was concluded that HSP70 inhibits the immediate HSPA8 effect on sperm viability.
Formation of sperm reservoir in the mammalian oviductal tube necessitates a fine regulatory system for production and preservation of fertilising competent spermatozoa. Intimate association between sperm head and apical OEC membrane plays an important role in fulfilment of the regulatory mission. The responsible factors and involved mechanisms are not clear. However, a number of extracellular HSPs identified in oviductal secretions or apical membrane of oviducatal epithelium have been shown to induce regulatory effects on sperm motility, viability and acrosomal reaction (Elliott RM, 2009, Boilard et al., 2004). Elliott and colleagues reported that HSPA8 is present within a soluble protein extract from porcine apical oviductal epithelium (sAPM) and demonstrated that HSPA8 significantly enhances boar sperm viability (Elliott RM, 2009). They also showed that this effect is reproducible in vitro by a recombinant bovine HSPA8 on boar as well as bull spermatozoa. This observation suggested HSPA8 is biologically effective constituent of sAPM in the process of sperm storage.
HSPA8 is a member of the 70 kDa heat shock proteins which are the highest conserved protein molecules among species `(Garrido et al., 2001). Therefore, similar effects and functions of HSPA8 from different species origins are predictable.
In the current study, we clearly and consistently demonstrated that only 15 min exposure of boar spermatozoa to exogenous bovine recombinant HSPA8 is influential on boar spermatozoa as shown by increase in viability, whereas, outcomes with human recombinant HSP70, the inducible isoform and Î±-Tubulin did not show any significant increase in sperm viability. For any observed biologic effect in response to particular components of any composition, it is important to define the specificity of the response so that the outcome can be attributed to the particular factor studied. Therefore, protein control treatments were included in experimental design to assure the specificity of HSPA8 viability enhancing effect.
In previous studies, extracellular combinations of constitutive (HSPA8) and inducible (HSP70) forms of 70 kDa HSPs exhibited significant accelerating effects on early embryonic development in sea urchin (Browne et al., 2007). Accordingly, a range of 2:3 mixtures of HSPA8-HSP70 were used because it is similar to physiological conditions in many cell types (Browne et al., 2007). None of the protein concentration ranges had any viability enhancing effect in contrast to HSPA8, indicating that HSP70 negates the positive effect which is observed with individual HSPA8.
In addition, long-term influence of the protein on maintaining sperm survival was observed similar to previous reports. Although further investigation is required to decipher the mechanism of action, we are, by far, quite certain that the protein is exerting its viability enhancing effects on sperm membrane as validated by two viability assays based on their permeability to intact cell membrane.
Hereby, it is noteworthy to explain that the viability assays applied in this study, primarily determine cell membrane status rather than the whole intracellular survival pathways. Therefore, one can speculate that cell membrane disruption does not necessarily represent cell death, particularly that membrane is the first mechanical defensive barrier against environmental stress. Various factors from oxidation to mechanical stress e.g. centrifugation can impose reversible or irreversible physical/chemical damage to the membrane structure. Physical disruptions can include membrane pore formation, swelling and disturbed net water and ion transportation across the membrane as a result of conformational changes in lipid and protein components. Anyhow, since their major role is to modify harmful and preserve favourable protein conformational changes (Lanneau et al., 2008), it is rational to consider HSPs to be capable of repairing the reversible membrane damages.
Our observations from various experiments revealed quite consistent increase of around 10% in boar sperm viability which might highlight the ability of HSPA8 in reversing the nonfatal membrane alterations caused by insults imposed through the process of sperm transportation and preparation. In vivo, a very small number of spermatozoa inside the oviduct are tightly attached to OECs and this population holds the highest proportion of live spermatozoa compared to loosely attached or unattached sperm (Smith and Yanagimachi, 1990). Also, in vitro examination of sPAM proteins-sperm surface assoionciat revealed that small numbers of live spermatozoa became bound to the extracts (Elliott RM, 2009). Hence, it is likely that HSPA8 in the sPAM plays a selective role in associating with and affecting on sperm cells with highest membrane quality or at least those which are physiologically destined to stay alive. This indicates that apical epithelial membrane as the site of attachment, provides a hospitable environment with considerable energy resources for the bound sperm cells due to its effective components like HSPA8. However, the whole discussion here is based on the type of the viability assay used in our experiments. Application of viability assays with different mechanisms of action might reveal different useful results and is therefore strongly suggested.
On the other hand, Previous investigations on the effect of apical OEC extracts on sperm revealed its ability in maintaining low intracellular Ca2+ concentration (Boilard et al., 2002). Also, HSPA8 is shown to have the ability to interact with neural membrane calcium channels and induce rapid transmembrane calcium ion efflux over a brief period of 30 minutes (Smith, 1995) and cause low intracellular calcium concentrations. Therefore, there is a possibility that HSPA8 as the constitutive member of 70 kDa HSPs and an effective component of apical membranous OEC acts through similar pathways to retain and preserve sperm transmembrane potential at normal level and so the shift in cell membrane permeability and viability status follows.
In further investigations blockade of the viability enhancing effect of HSPA8 on sperm by sperm capacitation process was revealed. The entire biochemical and/or biophysical alterations in all aspects of sperm cell structure and function is collectively called capacitation (Hunter and Rodriguez-Martinez, 2004). Capacitation is the final maturation process that takes place gradually in vivo inside the oviduct and can be mimicked in vitro by using appropriate temperature and media. During capacitation, sperm membrane undergoes major structural and chemical alterations which are integral to fulfilment of the process. Hence, based on the hypothesis that HSPA8 targets sperm membrane to exert its positive effects, one can speculate that the induced membrane capacitation changes occur in a direction which is not consistent with optimal settings required for HSPA8 to induce any effects in that respect.
Moreover, sperm capacitation requires high intracellular calcium ion concentrations .This may as well counteract with low intracellular calcium induction by HSPA8. In addition, undergoing capacitation means fast approach to sperm death stage, therefore it is sensible to conclude that actions aiming to expand cell survival must skip or interfere before this steep downhill to death.
Speaking of cell membrane and viability, the fact that plasma membranes are fluid structures led into the following move. The basic structural unit of membrane lipid bilayer consists of various types of molecules (lipids, proteins and carbohydrates) with lipids having the highest proportion and forming the basic frame. These molecular assembly results from low energy interactions between individual units and this, in turn provides membrane organization based on free diffusion process. However, evidence indicates that membranous molecular diffusion is not free after all; various mechanisms, direct or indirect, constrain and regulate the movements (Marguet et al., 2006).
Membrane fluidity simply refers to molecular viscosity and lateral diffusion on membrane domains. Maintenance of fluidity is vital to cellular viability, function and reproduction (Wolfe et al., 1998, Hollan, 1996). In intact viable cells, lipids and proteins freely move in membrane plane with high diffusion coefficient, but any entity that causes constraints to the molecular movement leads into decreased membrane fluidity and functional disruptions in membrane. For instance, reduced temperature or rise in free oxygen radicals decrease membrane fluidity measures. Also, dead cells show lower diffusion coefficients compared to live ones (Wolfe et al., 1998).
We hypothesised that HSPA8 gets involved in a cascade through which alters membrane fluidity and membrane molecular movement patterns in favour of the cell's viability and functional restoration. FRAP analysis performed by ODAF which is a lipid reporter, revealed this speculation to be a useful step towards discovering the exact mechanism. Fluorescent recovery after photobleaching created invaluable preliminary information which demonstrated that movement of membrane molecules within the lipid bilayer is accelerated on the acrosome and postacrosome domains when exposed to HSPA8. In addition, the proportion of molecules which float in sperm head membrane rises under the effect of HSPA8. Consistent increase in boar sperm lipid membrane fluidity measures are integral characters of cell viability.
Wolfe, C. A. And colleagues studied fluidity measures of fixed sperm cells in bull spermatozoa. For this, they prelabelled the cells with the ODAF probe and then permeabilized them by formaldehyde fixation. FRAP analysis of all sperm membrane domains revealed diffusion coefficients significantly lower than that of nonfixed controls. Though the majority of the analyzed spermatozoa retained the live pattern of labelling (Wolfe et al., 1998). This can be an indicative to our hypothesis that not all cells with increased membrane permeability are totally non-functional ones and so better not to be referred to as dead cells. However, similar studies to ours using HSPA8 should be carried out on sperm tail and midpiece for further comparison.
So far, one can conclude that during sperm-HSPA8 exposure, this protein takes part in protection of the sperm membrane integrity by implementing its various chaperone functions like protein transfer, folding or even denatured protein refolding. In addition to the mechanisms described here, possibilities such as involvement of apoptotic/anti-apoptotic pathways, influence of HSPA8 on particular fluorescent stain leakage in or out of the cell might be considered. Having said all the above, considering the original role the heat shock proteins play as cell protectors such as limiting the cytotoxic damage and DNA break caused by cytokines and reactive oxygen species and preventing apoptosis and mitochondrial structure and function, the viability enhancing effect of the protein on sperm sounds justifiable.
Yet we do not know if exogenous HSPA8 gets into the cell and functions internally or induces cellular alterations from outside via mediators. There are a number of studies regarding exogenous HSP-cell association all of which have tracked protein location after they obtained positive effects from addition of the protein to their particular cell type of interest. Association of HSP70 with arterial smooth muscle cell happens through protein-membrane binding rather than protein internalisation (Johnson and Tytell, 1993). Similarly, incubation of spermatozoa with either HSP60 or HSPA8 leads into significant cell membrane-protein association. Considering the results obtained and speculations made so far, interaction of HSPA8 with sperm membrane seems very likely. Intimate association of protein with its particular target of action accelerates the outcome like increase in sperm viability in less than 30 minutes exposure time via interaction with its membrane.
However, other possibilities including HSPA8 internalisation or external action through mediator molecules should be considered too. Hence, the next step is to pinpoint the location at which exogenous HSPA8 associates with boar sperm.
The concept of intracellular calcium modulation through alterations in membrane arrangements can also be applied to the results obtained from the effect of HSPA8 on sperm mitochondrial activity. The security and maintenance of mitochondrial function relies on intact membrane integrity and electrical properties. Mitochondrial transmembrane potential indicates the level of mitochondrial activity and energy production. In this study, we detected lowered mitochondrial activity under the effect of HSPA8 by Jc-1 mitochondrial staining which reacts distinctively to high or low mitochondrial electrical activities. Although a qualitative assay, it partially shed light on the possible in vivo physiological effectors/mechanisms which lead in reduced sperm motility in the oviductal reservoir. However, further quantitative measurements can create useful information in this respect.
Last but not the least, is the ability of HSPA8 to augment sperm capacity to bind OECs. Establishment of direct sperm-OECs attachment seems to be a significant contributing factor in accomplishment of all the events favourable to effective maternal gamete interaction and eventually a fruitful fertilisation. This close contact facilitates signal exchange and effective interaction to improve and preserve sperm quality which is the principal goal in the formation of the oviductal sperm reservoir. Considering this significant role, there must exist physiologic effectors to boost this sort of cell-cell association to betterment of the reproductive outcome.
The results presented in this study suggest that brief exposure of boar spermatozoa to extracellular bovine recombinant HSPA8 results in significant increase in sperm viability, capacity to bind OECs and decrease in mitochondrial activity. Although this was an in vitro model, all of the effects created by HSPA8 on sperm seem to be similar to events happening in the oviductal sperm reservoir. Primary approaches have shed some light on likely mechanisms and relevant hypotheses which necessitate prospective thorough investigations.