HSP90 maintains boar spermatozoa motility and mitochondrial membrane potential during heat stress

A B S T R A C T
Heat Shock Proteins (HSP) is a family of proteins that protects cells from high temperatures. The present work aimed to elucidate the role that HSP90 exerts on boar sperm incubated under heat stress conditions on viability, total motility (TM), progressive motility (PM), acrosome status, mitochondrial membrane potential and plasma membrane lipid organization. Sperm were in- cubated in non-capacitating conditions (Tyrode’s basal medium or TBM) for 3, 8 and 24 h or incapacitating conditions (Tyrode’s complete medium or TCM) for 4 h at 38.5 °C or 40 °C (Heatstress) in the presence or absence of 5 or 20 μM of 17-AAG, a specific HSP90 inhibitor. Spermviability was not affected by the presence of 17-AAG in any condition tested compared with its own control (at the same temperature and incubation time). In non-capacitating conditions TM (22.7 ± 4.1 vs. 1.9 ± 1.1; % mean ± SEM), PM (3.1 ± 0.9 vs. 0) and high mitochondrial membrane potential (19.5 ± 2.2 vs. 11.8 ± 0.8) decreased significantly in sperm incubated at 40 °C for 24 h in the presence of 20 μM 17-AAG (control vs. 20 μM 17-AAG, respectively;p < 0.05). In sperm incubated at 38.5 °C only a mild decrease in TM was observed (48.7 ± 3.1vs. 32.1 ± 4.8; control vs. 20 μM 17-AAG, respectively; p < 0.05). However, under capaci- tating conditions none of the sperm parameters studied were affected by 17-AAG after 4 h ofincubation. These results demonstrate for the first time the role of HSP90 in the maintenance of boar sperm motility and mitochondrial membrane potential during prolonged heat stress in non- capacitating conditions.

1.Introduction
Sperm functions such as motility, capacitation or acrosome reaction are required to successfully achieve fertilization (Aitken and Nixon, 2013; Okabe, 2013). These events are regulated by multiple well-orchestrated pathways involving different types of proteins being the kinase family the most deeply studied (Visconti et al., 2011; Buffone et al., 2014; Hurtado de Llera et al., 2016; Jin and Yang, 2017). Recently, more attention is being put on the role that heat shock proteins (HSP) may play on mammalian sperm functions. HSP are highly conserved proteins among mammalian species and are classified into several families according to their molecular weight: HSP100 (HSPH), HSP90 (HSPC), HSP70 (HSPA), HSP60 (HSPD), HSP40 and HSP27 (HSPB) (Buchner, 1996;Bukau and Horwich, 1998). These proteins have been shown to be activated in response to heat stress (Ellis, 1987; Welch, 1993; Ellis, 1996) preventing protein denaturation and aggregation, thus helping to maintain proteins’ functions (Bukau and Horwich, 1998). However, several studies have demonstrated the implication of HSP in fertilization-related events. For instance, HSP90 isphosphorylated during capacitation in human and mice sperm (Ecroyd et al., 2003) and has also been implicated in progesterone- induced sperm hyperactivation and acrosome reaction in humans (Li et al., 2014; Sagare-Patil et al., 2017). Volpe et al. (2008) reported for the first time the presence of HSP90 in boar sperm, and it has been demonstrated to influence boar sperm motility (Huang et al., 2000) and capacitation (Hou et al., 2008). Nevertheless, at the present moment, there are no studies that demonstrate whether HSP90 constitutes a defensive mechanism against heat stress in boar sperm. Taking in account that heat stress is one of the major insults that impair spermatogenesis in boar, resulting in a diminished litter size and severe economic loss (Wettemann and Desjardins, 1979; Cameron and Blackshaw, 1980; Wettemann and Bazer, 1985), more effort needs to be put in understanding the defensive mechanisms that could be activated.Hence, the objective of the present study was to investigate the role of HSP90 in vitro during heat stress (40 °C) in capacitating and non-capacitating conditions in boar sperm. After this stress, different boar sperm parameters such as viability, motility, acrosome status, mitochondrial membrane potential and plasma membrane lipid organization were studied in presence and absence of 17-AAG, a HSP90 inhibitor.

2.Materials and methods
Propidium iodide (PI), SYBR-14, M540 and YoPro-1 probes were purchased from Molecular Probes (Leiden, The Netherlands); PNA-FITC was from Sigma-Aldrich® (St Louis, MI, USA) and JC-1 probe from Life Technologies Ltd. (Grand Island, NY, USA); Coulter Isoton II Diluent from Beckman coulter Inc. (Brea, CA, USA); 17-(Allylamino)-17-desmethoxygeldanamycin (17-AAG) was purchased from Enzo Life Sciences (East Farmingdale, NY, USA).Tyrode’s basal medium (TBM) was prepared as following: 96 mM NaCl, 4.7 mM KCl, 0.4 mM MgSO4, 0.3 mM NaH2PO4, 5.5 mM glucose, 1 mM sodium pyruvate, 21.6 mM sodium lactate, 20 mM HEPES and 5 mM EGTA. A variant of EGTA-free TBM medium was made by adding 1 mM CaCl2, 15 mM NaHCO3 and 3 mg/ml BSA termed Tyrode’s complete medium (TCM). All media were made on the day of use and adjusted to a pH of 7.45.Ejaculates were purchased from a commercial boar station (Tecnogenext, S.L, Mérida, Spain). Duroc boars were maintained according to institutional and European regulations. All boars were housed in individual pens in an environmentally controlled building (15–25 °C) and received the same diet. Fresh ejaculates were collected with the gloved hand technique and stored at 17 °C. To avoid individual variability between boars, for each experiment, semen from 3 different boars were randomly mixed (33 boars in different combinations were used in this study), centrifuged at 2400g for 4 min, washed with PBS and diluted in TBM or TCM toachieve a final concentration of 50 × 106/ml. After sperm dilution, one ml aliquots of the sample were placed in 24 well plates and the inhibitor (at 5 μM or 20 μM) was added using previously validated concentrations in human sperm (Li et al., 2014; Sagare-Patil et al., 2017).

Control samples were supplemented with 0.2% dimethylsulfoxide (DMSO) to mimic the conditions of the inhibitor- added samples; then, the samples were incubated at 38.5 °C or 40 °C for 3, 8 and 24 h in TBM and for 4 h in TCM.A total of 2 μl of sample were placed in a pre-warmed counting chamber (Leja; Luzernestraat, The Netherlands) and analyzed using a CASA system (ISAS®, Proiser R + D, Paterna, Valencia, Spain). Sperm motility was assessed with a microscope (Nikon Eclipse 50i) equipped with a 10 x negative-phase contrast objective and a heated stage at 38.5 °C. Analysis was based on the examination of25 consecutive digitalized images and at least 300 spermatozoa per sample were analyzed. After acquiring four representative fields in a random distribution, the following sperm motility descriptors were recorded: total motility (TM) and progressive motility (PM).Flow cytometry analysis was performed using an ACEA NovoCyte TM flow cytometer (ACEA Biosciences, Inc., San Diego, CA, USA) equipped with Blue/Red Laser (488/640 nm) and signal was collected in 3 channels with different band pass filter. Data were analysed using ACEA Novo Express TM software.Sperm viability was performed as described previously (Hurtado de Llera et al., 2012). SYBR-14 (5 μl) and PI (10 μl) were added to 500 μl of diluted semen (final concentration of SYBR-14 20 nM and PI 5 μM) and incubated for 15 min at room temperature in the dark. Fluorescence was detected using a 525/620 nm band pass filter, respectively. Viable spermatozoa were expressed as the averageof the percentage of SYBR14-positive and PI-negative spermatozoa.Sperm plasma membrane lipid organization was assessed by staining with M540 and YoPro-1 as described previously (Martin- Hidalgo et al., 2011); 1.87 μl of YoPro-1 were added to 500 μl of diluted semen and incubated at 38.5 °C for 15 min. Just before analysis, 3 μl of M540 were added to each sample (final concentration of M540 6 μM and YoPro-1 75 nM) and incubated for 2 min at38.5 °C.

Fluorescence was detected using a 585/525 nm band pass filter, respectively. Results were expressed as the average of thepercentage of viable cells with unstable membrane: M540-positive and YoPro-1-negative.The acrosomal status of spermatozoa was assessed after staining with PNA-FITC and PI (Martin-Hidalgo et al., 2011). PNA-FITC (10 μl) and PI (2.5 μl) were added to 100 μl of each semen sample (final concentration of PNA-FITC 2 μg/ml and PI 5 μM) and were incubated at room temperature in the dark for 5 min; 400 μl of isotonic buffered diluent was added before flow cytometry analysis.The fluorescence of PNA-FITC was collected using a 525/620 nm band pass filter, respectively. Viable spermatozoa with acrosome reacted or damaged are expressed as the average of the percentage of PNA-positive and PI-negative spermatozoa.The mitochondrial membrane potential was evaluated using the specific probe JC-1 as described previously (Martin-Hidalgo et al., 2011). In brief, 3 μl of JC-1 were added to 500 μl of diluted semen (final concentration of 0.9 μM) and incubated at 38.5 °C for 30 min. Fluorescence was collected using a 525/585 nm band pass filter. Results are expressed as the average of the percentage of spermatozoa exhibiting high mitochondrial membrane potential (orange-stained cells).Data distribution was first examined using the Shaphiro-Wilk test. A one-way ANOVA was used to compare values; for non- Gaussian samples the nonparametric test, Kruskal-Wallis ANOVA on ranks was used. Differences between samples (control and treatments) at the same temperature and incubation time were determined using Dunnett’s post-hoc method or Dunn’s post-hocmethod if the group sizes were not equal. The level of significance was set at p < 0.05. Analyses were performed using SigmaPlotsoftware (ver. 12.0) for windows (Systat Software, Chicago, IL).

3.Results
To investigate the possible role of HSP90 under heat stress conditions, spermatozoa were incubated at 40 °C during 3, 8 and 24 h in TBM in presence or absence of 17-AAG, a specific HSP90 inhibitor. Sperm viability remained unaffected despite 17-AAG addition, prolonged incubation or increased temperature (Table 1; p > 0.05). Additionally, inhibition of HSP90 did not affect total or pro- gressive motility of sperm after 3 and 8 h of incubation at 38.5 °C or 40 °C. After 24 h at 38.5 °C, TM decreased significantly (48.7 ± 3.1 vs. 32.1 ± 4.8; control vs. 20 μM 17-AAG, respectively; p < 0.05). However, the decrease on TM at 40 °C was morepronounced in sperm incubated for 24 h (22.7 ± 4.1 vs. 1.9 ± 1.1; % mean ± SEM) (control vs. 20 μM 17-AAG, respectively; p < 0.05, Table 1). PM decreased significantly after HSP90 inhibition for 24 h at 40 °C (3.1 ± 0.9 vs. 0; control vs. 20 μM 17-AAG, respectively at 24 h; p < 0.05) and a decrease in PM at 24 h of HSP90 inhibition was observed in sperm incubated at 38.5 °C but was not statistically significant. No differences were observed when sperm were incubated with 5 μM of 17-AAG compared with its own control (p > 0.05).Next, we investigated if HSP90 was implicated in the regulation of different sperm processes such as acrosome integrity main- tenance, mitochondrial membrane potential and plasma membrane lipid organization. As no differences were observed in motility and viability at 3 h (Table 1), this incubation time was not included.The acrosomal membrane status and plasma membrane lipid organization of spermatozoa did not change in presence of 17-AAG compared to sperm incubated in TBM (Table 2). However, the population of spermatozoa presenting high mitochondrial membrane potential decreased in presence of 17-AGG at 40 °C for 24 h (19.5 ± 2.2 vs. 11.8 ± 0.8; control vs. 20 μM 17-AAG, respectively;p < 0.05) (Table 3). No differences were observed when sperm were incubated with 5 μM of 17-AAG compared with its own control(p > 0.05).Finally, we investigated the role of HSP90 under standard capacitating conditions. For this purpose, spermatozoa were incubated in presence of calcium, bicarbonate and BSA (TCM) for 4 h at 38.5 °C and 40 °C. Total motility, progressive motility and viability were unaffected by 17-AGG at 5 or 20 μM in spermatozoa incubated in TCM (Table 4). Also, the parameters evaluated by flow cytometry remained unchanged by the presence of 17-AAG (Tables 5 and 6).Values are percentages of total spermatozoa (mean ± standard error of the mean, SEM).VUM: percentage of viable spermatozoa with an unstable plasma membrane; PNA+/PI−: percentage of viable spermatozoa with a damaged acrosome. No significant differences were found between treatments at the same temperature and incubation time (p > 0.05). n = 3.

4.Discussion
In this work we demonstrate the key role of HSP90 in the maintenance of boar spermatozoa motility and membrane mi- tochondrial potential under non-capacitating conditions during heat stress (40 °C).To the best of our knowledge, this is the first time that HSP90 is studied in boar spermatozoa under heat stress conditions; previously Huang et al.(2000) studied the role of HSP90 on boar spermatozoa motility at physiological temperature. These authors demonstrated that inhibition of HSP90 by geldanamycin (HSP90 inhibitor) triggered a pronounced decrease on sperm motility within 15 min after the onset of the incubation. Contrary to these results, we only observed a significant decrease in sperm motility after 24 h of incubation in non-capacitating conditions at physiological temperature. This discrepancy could be attributed to the use of different HSP90 inhibitors as 17-AAG is more specific and less toxic than geldanamycin (Powers and Workman, 2007). In this regard, it has to be noted that a negative effect of geldanamycin on boar sperm viability cannot be overruled, as sperm viability was not evaluated in the work performed by Huang et al. (2000). By contrary, in our conditions in boar spermatozoa, we clearly confirm that the HSP90 inhibitor 17-AAG does not possess any side effect that compromise sperm viability.Regarding the effect of HSP90 inhibitor on motility of boar spermatozoa incubated under capacitating conditions, we did not observe significant changes in sperm incubated at 38.5 °C or 40 °C in presence of 17-AAG at 5 or 20 μM compared with its own control. Interestingly, we observed a lack of effect of HSP90 inhibitor at 5 or 20 μM on boar sperm motility under capacitating conditions, which is in agreement with results from Li et al. (2014) and Sagare et al. (2017). These authors reported that inhibition of HSP90 does not induce significant changes on motility in human spermatozoa incubated up to 3 h under capacitating conditions. The role that HSP90 may play in capacitation is still controversial; Hou et al. (2008) reported that inhibition of HSP90 with geldanamycin consistently enhanced capacitation-related phenomena in boar spermatozoa, including nitric oxide (NO) production and acrosome reaction induction using A23187. Also, an increase on tyrosine phosphorylation of HSP90 has been described in rat and human spermatozoa under capacitating conditions (Ecroyd et al., 2003).

Conversely, in our study in boar spermatozoa under capacitating conditions, inhibition of HSP90 using 17-AAG at 5 or 20 μM didnot enhance spontaneous acrosome reaction and did not induce significant changes in viability, plasma membrane lipid organization and mitochondrial membrane potential at 38.5 °C or 40 °C. On the other hand, our findings are compatible with the report by Sagare et al. (2017) and Li et al. (2014), who did not find differences in capacitation and spontaneous acrosome reaction in human sper- matozoa incubated for 3 h in capacitating conditions in presence of HSP90 inhibitors. Our results suggest that the inhibition of HSP90 in boar spermatozoa does not affect either the classical processes related to sperm functions under standard capacitating conditions or when subjected to heat stress.However, under non-capacitating conditions, HSP90 inhibition with 17-AAG at 20 μM induces a significant decrease in high mitochondrial membrane potential in boar spermatozoa subjected to heat stress. This drop in the high mitochondrial membranepotential could also explain the decrease observed in motility in spermatozoa incubated for 24 h at 40 °C. This hypothesis is sup- ported by the work performed by Guo et al. (2017) who demonstrated that mitochondrial oxidative phosphorylation (OXPHOS) is necessary for the maintenance of boar sperm motility. It has to be noted that in somatic cells the HSP90 family ensures a correct mitochondrial function (Altieri, 2013) by its interaction with multiple proteins, including protein kinases and phosphatases (Taipale et al., 2010). In boar sperm, HSP90 has been localized in the midpiece and tail (Volpe et al., 2008), indicating that HSP90 is likely present in the mitochondria. In this sense, Hamilton et al. (2016) studied the mitochondria function of ram epididymis under heat stress and they found a decrease in mitochondrial membrane potential and an increase in reactive oxygen species production (ROS). Based in these works and our findings, we postulate that HSP90 is important to maintain sperm motility and mitochondrial function in boar spermatozoa subjected to prolonged heat stress.However, the decrease in motility caused by inhibition of HSP90 by 17-AAG at 20 μM after 24 h in sperm incubated at 38.5 °C innon-capacitating conditions was not associated with changes in the mitochondrial membrane potential, suggesting other non-mi- tochondrial pathways involved in the effect of 17-AAG on sperm motility.

In conclusion, we demonstrated a protective effect of HSP90 in boar spermatozoa against heat stress by maintaining mi- tochondrial membrane potential and motility in non-capacitating conditions. However, more studies are required to fully elucidate candidate proteins that interact with HSP90 as well as the pathways implicated in the effects mediated by 17-AAG HSP90 in boar spermatozoa.