Abstract
MTT is widely used in biology as a probe for cell viability by virtue of its ability to generate deposits of insoluble formazan at sites of intense oxidoreductase activity. This response is generally held to reflect mitochondrial redox activity; however, extra-mitochondrial MTT reduction has also been recorded in certain cell types. Given this background, we set out to determine the major sites of formazan deposition in mammalian spermatozoa. In the mouse, most MTT reduction took place within the extensive mitochondrial gyres, with a single minor site of formazan deposition on the sperm head. By contrast, human spermatozoa generally displayed small disorganized midpieces exhibiting moderate MTT reduction activity accompanied by a major extra-mitochondrial formazan deposit on various locations in the sperm head from the neck to the anterior acrosome. Equine spermatozoa presented a combination of these two patterns, with major formazan deposition in the mitochondria accompanied by an extra-mitochondrial formazan deposit in around 20% of cells. The functionality of human spermatozoa was positively associated with the presence of an extra-mitochondrial formazan granule. Subsequent studies indicated that this extra-mitochondrial activity was suppressed by the presence of diphenylene iodonium, zinc, 2-deoxyglucose, co-enzyme Q, an SOD mimetic and NADPH oxidase inhibitors. We conclude that the pattern of MTT reduction to formazan by spermatozoa is species specific and conveys significant information about the relative importance of mitochondrial vs extra-mitochondrial redox activity that, in turn, defines the functional qualities of these cells.
Introduction
MTT is a vital dye that has been used extensively in biological research to assess cell viability. The general principle of the assay is that this membrane permeant, yellow compound, passes across the cell plasma membrane, whereupon its positively charged quaternary tetrazole ring core facilitates uptake by the mitochondria. In this location, the MTT becomes reduced by oxidoreductases, predominantly succinate dehydrogenase, to generate dark purple formazan deposits which can be readily extracted and measured. Since only viable cells will have active mitochondria capable of effecting MTT reduction, the assay is thought to provide an accurate, sensitive measure of cell viability (Kumar et al. 2018).
Given this background, MTT reduction has recently been employed to measure the vitality and functionality of spermatozoa in a variety of species including sea urchin (Resgalla et al. 2018), chicken (Pranay Kumar et al. 2019), buffalo (Iqbal et al. 2010), horse (Aziz et al. 2005), bull (Aziz 2006) rat (Ohtani et al. 2004), rabbit (Halo et al. 2019) and man (Nasr-Esfahani et al. 2002). In the latter study, MTT formazan granules and spikes were observed in the midpiece of the spermatozoa consistent with a mitochondrial localization for the oxidoreductases that bring about MTT reduction. However, detailed examination of the pattern of formazan deposition in boar spermatozoa (van den Berg 2015) suggested that this explanation might not completely cover the various pathways that are actually employed by these cells to reduce MTT. This conclusion was reached because, in these preliminary studies, clear evidence of extra-mitochondrial formazan deposition in boar spermatozoa was obtained. There have previously been reports of extra-mitochondrial sites of MTT reduction in various cell types, but the view that MTT is reduced by mitochondrial dehydrogenases still dominates the literature (Berridge & Tan 1993, Liu et al. 1997, Liu 1999, Bernas & Dobrucki 2002, Berridge et al. 2005). Spermatozoa are unique in that their mitochondria are clearly compartmentalized and, as a result, these cells are excellent models for studying the sites and significance of MTT reduction. It is also apparent that the spermatozoa of different species, and even different members of the same genus, differ markedly in the extent to which they are dependent on mitochondrial oxidative phosphorylation or glycolysis to meet their energy needs (Tourmente et al. 2015). In this study, we have, therefore, examined the patterns of MTT reduction in the spermatozoa of three separate species (mouse, human and horse) and investigated some the functional correlates and causative mechanisms associated with this activity.
Materials and methods
Chemicals
MTT, Nitroblue Tetrazolium (NBT) and all other chemicals used in this study were obtained from Sigma-Aldrich, unless otherwise stated. The NADPH oxidase inhibitors ML171 (2-acetylphenothiazine; Cat # S5304), VAS2870 [7-(2-benzoxazolylthio)-3-(phenylmethyl)-3H-1,2,3-triazolo[4,5-d]pyrimidine; Cat # 19205] and GKT137831 [2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione; Cat #17764] were from Sapphire Bioscience (Redfern Australia). A mouse MAB to CD59 was purchased from Abcam (Cat # ab9183), while an HRP-conjugated secondary antibody was obtained from SantaCruz Biotechnology.
Preparation of spermatozoa
Mouse
All experimental procedures were conducted on mice with the approval of the University of Newcastle’s Animal Care and Ethics Committee. Mouse spermatozoa were back-flushed from the cauda epididymis as previously described (Smith et al. 2013).
Stallion
Stallion ponies were used with the authorization of the University of Newcastle Animal Care and Ethics Committee. Semen was collected using a pony-sized Missouri artificial vagina (Minitube Australia, Ballarat, Vic, Australia) and processed as previously described (Swegen et al. 2019).
Human
In the case of human spermatozoa, Institutional and State Government ethical approval was secured for the use of human semen samples for the purposes of this research. Semen samples from unselected donors were fractionated on discontinuous two-step Percoll gradients (80/40%), as previously described (Houston et al. 2015), and finally resuspended at 20 × 106/mL. Percoll gradient centrifugation separates the entire sperm population into two broad fractions: a high-density fraction of good quality spermatozoa characterized by excellent morphology, vigorous motility, a high capacity for fertilization and low levels of oxidative stress and a low-density fraction comprising poor quality spermatozoa, where all of these attributes are reversed (Aitken & Clarkson 1988, Yao et al. 1996).
Sperm incubation and capacitation
Capacitation was inhibited by incubating the cells in bicarbonate-free medium and stimulated by exposing the cells to a mixture of pentoxifylline (1 mM) and dbcAMP (1 mM) as described (Redgrove et al. 2013). For all species, spermatozoa were finally suspended in Biggers, Whitten and Whittingham medium (BWW; Biggers et al. 1971) supplemented with 1 mg/mL polyvinyl alcohol (PVA), 5 units/mL penicillin and 5 mg/mL streptomycin, 20 mM HEPES and an osmolarity of 290–310 mosmol/kg at pH 7.4. Sperm vitality was determined via the incorporation of Eosin Y into non-viable cells and assessment of this staining via light microscopy (pink = dead, no stain = viable).
MTT staining of spermatozoa
For every assay, a fresh stock solution of MTT was made up at 5 mg/mL in BWW and added to a 100 µL aliquot of the sperm suspension at a final concentration of 0.5 mg/mL. The spermatozoa were then incubated at room temperature (~20°C), in darkness, for 1 h. A small volume (8 µL) of this suspension was then placed on a slide and initially viewed using a 40× objective and transmitted light. MTT staining was then scored using an oil emersion 100× objective and the location of the MTT granules determined in 100 cells following fixation with 4% paraformaldehyde. The locations were identified as follows: acrosomal, equatorial, post-acrosomal, neck, midpiece (mitochondria), tail and none (zero staining). The data on extra-mitochondrial formazan granule formation were expressed as a percentage of cells in which MTT had been reduced to formazan in the midpiece and were therefore viable.
A number of reagents were assessed for their impact on MTT reduction by incubation with the spermatozoa for 20 min in darkness before the addition of MTT. The inhibitors comprised DPI (10 µM), zinc (0.5 mM), the antioxidant co-enzyme Q (0.1 and 1.0 mM), three NADPH oxidase inhibitors including ML171, VAS2870 and GKT137831 (all at 10 µM) and the SOD mimetic, MnTMPyP (50 and 100 µM). The impact of glycolytic substrates on MTT reduction was also examined by comparing the patterns of MTT reduction observed after 45 min incubation in normal medium BWW, BWW lacking glucose and BWW in which the glucose was replaced with an equimolar (1.7 mM) concentration of 2-deoxyglucose.
Sperm suspension in hypotonic medium
One of the procedures used to determine the location of MTT reductase activity involved permeabilizing Percoll purified spermatozoa using a hypotonic buffer (10 mM potassium phosphate, 1% PVA, pH 7.4) as described by Storey and Kayne (1975). Percoll-purified spermatozoa were incubated for 1.5 h at room temperature in the hypotonic medium in darkness. They were then washed by repeated (3×) cycles of centrifugation (500 g for 5 min) and finally resuspended at 5 × 106/mL in BWW. NADH or NADPH (2 mM) was then added followed 5 min later by MTT (0.5 mg/mL). After 1 h incubation in darkness at room temperature, the cells were scored for formazan deposition as described previously. Loss of the plasma membrane was confirmed by staining the spermatozoa with an antibody, diluted 1:100, targeting a plasma membrane complement regulatory factor (CD59) for 1 h at room temperature.
Transmission electron microscopy
Specimens were fixed in 2.5% (v/v) glutaraldehyde (ProSciTech, Queensland, Australia) pelleted (500 g), washed and post-fixed in 1% (v/v) osmium tetroxide (pH 7.4) for 2 h. They were then processed for transmission electron microscopy as described (Lu et al. 2017). Ultra-thin sections at a thickness of 60 nm were cut and stained with aqueous saturated uranyl acetate and Reynolds’ lead citrate (BDH Chemicals, Poole, England) and examined under a JEM-2100EX II transmission electron microscope (JEOL, Tokyo, Japan) at 80 kV.
Scanning electron microscopy
For SEM specimens, spermatozoa were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide, followed by dehydration through a graded series of ethanol solutions. Dried specimens were mounted on anodised aluminium stubs, coated with gold-palladium using a SPI Module™ Sputter Coater and then examined under a Philips XL30 scanning electron microscope (Philips).
Sperm–egg binding
Human zonae pellucidae were preserved in high-salt medium comprising 1.5 M MgCl2, 0.1% dextran, 0.01 mM HEPES buffer and 0.1% polyvinyl alcohol and maintained at 4°C until use (Yanagimachi et al. 1979). These zonae pellucidae were washed 3× in BWW prior to use in the sperm binding assay. Human spermatozoa were prepared at a concentration of 5 × 106/mL and 25 µL aliquots placed under liquid paraffin. A single zona pellucida was then added to each droplet and incubated for 1 h. Following co-incubation, the oocytes were washed 3× by serial aspiration through a fine bore glass micropipette to remove any unbound or loosely adhered sperm. MTT was then added to the washed zonae containing bound spermatozoa. The concentration of MTT in this instance was 0.2 mg/mL, which was established in dose-dependent studies as the lowest concentration of MTT compatible with detectable formazan deposition in spermatozoa. After 45 min incubation, the preparations were scored in order to assess the presence and position of the major extra-mitochondrial formazan granule.
Fractionation of MTT activity
A fresh solution of 1% n-dodecyl β-maltoside was made up in 50 mM Tris pH 7.8. Eight to 12 samples of human semen were separated on a Percoll gradient and cell pellets from the high-density fraction were pooled and cell concentration determined. The spermatozoa were then pelleted at 500 g for 5 min and resuspended in 100 to 150 µL of 1% n-dodecyl β-maltoside. The sample was placed at 4ºC on a rotating wheel to mix for 1 h, cleared by centrifugation at 10,000 g for 10 min and the supernatant removed and placed in a new tube. This protein preparation was then either stored at −20°C until use or a small aliquot was removed for MTT and NBT staining using identical procedures.
Native acrylamide gel separation was achieved using conventional PAGE procedures except that SDS was omitted from both the 4% stacking gel and the 7.5% resolving gel. Proteins were then resolved at 4°C for 3–4 h at 80 V. Following electrophoresis, the gels were allowed to equilibrate in 50 mM Tris buffer (pH 7.8) for 15 min on a shaker at 4°C. MTT (0.5 mg/mL final) or NBT (0.94 mg/mL final) was then added and incubated in darkness on a shaker at 4°C. The gels were ultimately exposed to NADH (1 mM final) and incubated in darkness, on a shaker, overnight at 4°C.
For FPLC analysis, spermatozoa were extracted with 1% n-dodecyl β-maltoside and the extracts run through a Superose-6 column at a flow rate of 0.25 mL/min in order to collect 50 × 0.5 mL fractions. NADH (1 mM) was subsequently added to each fraction along with MTT or NBT at a final concentration of 1.25 mM. The fractions were incubated for 1 h in darkness and absorbance measured at 550 nm.
Statistical analysis
The data were analysed statistically using JMP software (SAS institute, Cary, NC). Where appropriate, one- or two-way ANOVA and linear regression analyses were performed. In the case of one-way ANOVA, comparison of group mean values with the corresponding controls was achieved using Dunnett’s test. These parametric methods were only used if the data distribution was normal according to the Anderson–Darling Goodness-of-Fit test, while the assumed homogeneity of variances was checked using the Bartlett test. Where the data were not normally distributed, non-parametric methods were employed. The nature of the specific tests used for each component of this study are clearly indicated in the Figure legends. All graphs represent means ±s.e. and, unless otherwise indicated, all experiments were conducted on at least three independent samples.
Results
Patterns of MTT reduction in mouse, human and equine spermatozoa
Analysis of the cellular sites of MTT reduction revealed contrasting patterns in different species. In mouse spermatozoa, the mitochondrial gyres were clearly labelled, indicating the presence of intense MTT reductase activity in this site, in keeping with the highly organized nature of the long sperm midpiece in this species and its capacity for mitochondrial ATP generation (Carey et al. 1981; Fig. 1A). In addition, a majority of caudal epididymal spermatozoa displayed a single, small extra-mitochondrial formazan deposit in association with the sperm head (Fig. 1A and B, arrowed). This pattern was reversed in human spermatozoa. In normal cells recovered from the high-density region of Percoll gradients, there were minor formazan deposits within the few disorganized mitochondrial gyres visible in the short midpiece. However, in addition, a majority of these cells (87.0 ± 3.1%; n = 15) also displayed a large extra-mitochondrial formazan deposit associated with the neck and, particularly, the head region of the spermatozoa (Fig. 1C, D, E and F, arrowed). In a majority of spermatozoa, only a single extra-mitochondrial formazan deposit was observed but occasionally cells were identified with more than one granule, as illustrated in Fig. 1G.
Reduction of MTT to insoluble formazan deposits in mammalian spermatozoa. (A and B) Mouse spermatozoa: note the anticipated reduction of MTT in the mitochondrial gyres dominating the midpiece of the cell as well as a single small point of extra-mitochondrial reductase activity associated with the sperm head (arrowed). (C, D, E and F) Human spermatozoa: note the disorganized uneven reduction of MTT in the short sperm midpiece but the formation of a very large single extra-mitochondrial formazan granule on the sperm head (arrowed) which could be found in various anatomical locations from the neck to the acrosomal domain. (G) Occasional spermatozoa could be found with two large extra-mitochondrial granules; however, this was the exception rather than the rule.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Reduction of MTT to insoluble formazan deposits in mammalian spermatozoa. (A and B) Mouse spermatozoa: note the anticipated reduction of MTT in the mitochondrial gyres dominating the midpiece of the cell as well as a single small point of extra-mitochondrial reductase activity associated with the sperm head (arrowed). (C, D, E and F) Human spermatozoa: note the disorganized uneven reduction of MTT in the short sperm midpiece but the formation of a very large single extra-mitochondrial formazan granule on the sperm head (arrowed) which could be found in various anatomical locations from the neck to the acrosomal domain. (G) Occasional spermatozoa could be found with two large extra-mitochondrial granules; however, this was the exception rather than the rule.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Reduction of MTT to insoluble formazan deposits in mammalian spermatozoa. (A and B) Mouse spermatozoa: note the anticipated reduction of MTT in the mitochondrial gyres dominating the midpiece of the cell as well as a single small point of extra-mitochondrial reductase activity associated with the sperm head (arrowed). (C, D, E and F) Human spermatozoa: note the disorganized uneven reduction of MTT in the short sperm midpiece but the formation of a very large single extra-mitochondrial formazan granule on the sperm head (arrowed) which could be found in various anatomical locations from the neck to the acrosomal domain. (G) Occasional spermatozoa could be found with two large extra-mitochondrial granules; however, this was the exception rather than the rule.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Equine spermatozoa presented their own unique profile of staining following incubation with MTT. These cells are notorious for their dependence on oxidative phosphorylation (Gibb et al. 2014) and, as a reflection of this, exhibited long intensely stained midpieces with large numbers of active mitochondrial gyres (Fig. 2A, B, C and D). In this respect, they resembled mouse spermatozoa. However, around 20% of cells (17.0 ± 2.9; n = 8) also exhibited an extra-mitochondrial formazan deposit on the sperm head that was intermediate in size between that observed in human and mouse spermatozoa (Fig. 2A, B, C and D).
Equine spermatozoa. (A, B, C and D) Equine spermatozoa showed very strong labelling of the midpiece, as befits their dependence on oxidative phosphorylation coupled with a single extra-mitochondrial formazan deposit in various locations on the sperm head that was intermediate in size between the mouse and the human.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Equine spermatozoa. (A, B, C and D) Equine spermatozoa showed very strong labelling of the midpiece, as befits their dependence on oxidative phosphorylation coupled with a single extra-mitochondrial formazan deposit in various locations on the sperm head that was intermediate in size between the mouse and the human.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Equine spermatozoa. (A, B, C and D) Equine spermatozoa showed very strong labelling of the midpiece, as befits their dependence on oxidative phosphorylation coupled with a single extra-mitochondrial formazan deposit in various locations on the sperm head that was intermediate in size between the mouse and the human.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
MTT reduction in permeabilized cells
In order to provide more information on the precise site of MTT reduction, human spermatozoa were permeabilized in hypotonic media as described in Materials and Methods and incubated with MTT and either NADH or NADPH as a source of reducing equivalents. Under these hypotonic conditions, the plasma membrane ruptured but left the subcellular structure of the spermatozoa intact (Storey & Kayne 1975). Immunocytochemical localization of CD59, a surface-expressed complement regulatory protein, was used to confirm loss of the plasma membrane under these conditions. Following exposure to hypotonic medium, the normal pattern of surface CD59 labelling was lost and the only site of immunoreactivity remaining was the inner acrosomal membrane, which is known to express this complement regulatory factor (Supplementary Fig. 1, see section on supplementary materials given at the end of this article) (Cummerson et al. 2006). In the absence of NAD(P)H, no formazan deposition was observed following exposure to MTT, as these permeabilized cell were non-viable and therefore incapable of generating the co-enzymes (NADH or NADPH) needed to reduce the probe. However, in the presence of exogenous NADH, 90.6 ± 1.8% of permeabilized cells demonstrated the presence of extra-mitochondrial formazan granules, while in the presence of NADPH the percentage of positive cells was reduced to 64.3 ± 1.8% (P < 0.001). The formation of formazan granules in this permeabilized model was confirmed by scanning electron microscopy (Fig. 3A, B and C), clearly suggesting that the site of MTT reduction was sub-plasmalemmal. Transmission electron microscopy confirmed this assumption and revealed the presence of extra-mitochondrial formazan granules within the acrosomal vesicle (Fig. 3D, E, F and G).
Scanning and transmission electron microscopy of the extra-mitochondrial formazan deposit in human spermatozoa. (A, B and C) Scanning electron microscopy of formazan deposition in NADH-treated demembranated human spermatozoa demonstrating that the generation of this extra-mitochondrial MTT signal is not dependent on the presence of a plasma membrane. (D, E, F and G) Transmission electron microscopy of human spermatozoa illustrating the presence of the formazan granules (arrowed) within the acrosomal vesicle.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Scanning and transmission electron microscopy of the extra-mitochondrial formazan deposit in human spermatozoa. (A, B and C) Scanning electron microscopy of formazan deposition in NADH-treated demembranated human spermatozoa demonstrating that the generation of this extra-mitochondrial MTT signal is not dependent on the presence of a plasma membrane. (D, E, F and G) Transmission electron microscopy of human spermatozoa illustrating the presence of the formazan granules (arrowed) within the acrosomal vesicle.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Scanning and transmission electron microscopy of the extra-mitochondrial formazan deposit in human spermatozoa. (A, B and C) Scanning electron microscopy of formazan deposition in NADH-treated demembranated human spermatozoa demonstrating that the generation of this extra-mitochondrial MTT signal is not dependent on the presence of a plasma membrane. (D, E, F and G) Transmission electron microscopy of human spermatozoa illustrating the presence of the formazan granules (arrowed) within the acrosomal vesicle.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Extra-mitochondrial MTT reduction reflects semen quality
Extra-mitochondrial formazan deposits were generated rapidly in high-quality spermatozoa recovered from the high-density region of Percoll gradients with around 35% of cells possessing granules on the sperm head, from the neck to the acrosome, after 30 min (Fig. 4A). Two-way ANOVA revealed a highly significant difference between high- and low-density Percoll fractions (P > 0.001) in the absence of any significant impact of time between 30 and 120 min. The proportion of cells possessing extra-mitochondrial formazan deposits was also highly correlated with the motile sperm counts recorded in these high-density Percoll fractions (R2 = 0.76; n = 13; Fig. 4B) again emphasizing the relationship between extra-mitochondrial formazan deposition and sperm quality.
Extra-mitochondrial formazan deposition in relation to sperm quality. (A) When spermatozoa were separated into high- and low-quality sperm populations on discontinuous Percoll gradients, it was evident that extra-mitochondrial granule formation was an attribute of functional spermatozoa. Within 60 min, a majority (60–70%) of high quality spermatozoa had generated an extra-mitochondrial formazan granule on the sperm head, whereas in poor quality sperm populations this property was only observed in around 20% of cells; this difference was highly significantly different (P < 0.001) at all time points from 30 to 120 min; two-way ANOVA (B) within the high-quality Percoll populations there was a highly significant correlation (R2 = 0.76; n = 13) between the proportion of spermatozoa carrying extra-mitochondrial formazan deposits and motile sperm count. (C) The percentage of high-quality viable spermatozoa exhibiting extra-mitochondrial granule formation in the acrosomal domain increased significantly (P < 0.001) with time and was maximal after 90 min incubation; one-way ANOVA. (D) Incubation of human spermatozoa under conditions where capacitation was either encouraged with dbcAMP and pentoxifylline or suppressed by the omission of bicarbonate ion revealed a significant increase in the percentage of cells with an acrosomal location (P < 0.05 one-way ANOVA) under capacitating conditions and corresponding declines in the percentage exhibiting formazan deposition in the postacrosome/neck or the complete absence of extra-mitochondrial granules. All data are presented as means ± s.e. (*P < 0.05; ***P < 0.001). Non Cap, not capacitated; Cap, capacitated; Con, Control.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Extra-mitochondrial formazan deposition in relation to sperm quality. (A) When spermatozoa were separated into high- and low-quality sperm populations on discontinuous Percoll gradients, it was evident that extra-mitochondrial granule formation was an attribute of functional spermatozoa. Within 60 min, a majority (60–70%) of high quality spermatozoa had generated an extra-mitochondrial formazan granule on the sperm head, whereas in poor quality sperm populations this property was only observed in around 20% of cells; this difference was highly significantly different (P < 0.001) at all time points from 30 to 120 min; two-way ANOVA (B) within the high-quality Percoll populations there was a highly significant correlation (R2 = 0.76; n = 13) between the proportion of spermatozoa carrying extra-mitochondrial formazan deposits and motile sperm count. (C) The percentage of high-quality viable spermatozoa exhibiting extra-mitochondrial granule formation in the acrosomal domain increased significantly (P < 0.001) with time and was maximal after 90 min incubation; one-way ANOVA. (D) Incubation of human spermatozoa under conditions where capacitation was either encouraged with dbcAMP and pentoxifylline or suppressed by the omission of bicarbonate ion revealed a significant increase in the percentage of cells with an acrosomal location (P < 0.05 one-way ANOVA) under capacitating conditions and corresponding declines in the percentage exhibiting formazan deposition in the postacrosome/neck or the complete absence of extra-mitochondrial granules. All data are presented as means ± s.e. (*P < 0.05; ***P < 0.001). Non Cap, not capacitated; Cap, capacitated; Con, Control.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Extra-mitochondrial formazan deposition in relation to sperm quality. (A) When spermatozoa were separated into high- and low-quality sperm populations on discontinuous Percoll gradients, it was evident that extra-mitochondrial granule formation was an attribute of functional spermatozoa. Within 60 min, a majority (60–70%) of high quality spermatozoa had generated an extra-mitochondrial formazan granule on the sperm head, whereas in poor quality sperm populations this property was only observed in around 20% of cells; this difference was highly significantly different (P < 0.001) at all time points from 30 to 120 min; two-way ANOVA (B) within the high-quality Percoll populations there was a highly significant correlation (R2 = 0.76; n = 13) between the proportion of spermatozoa carrying extra-mitochondrial formazan deposits and motile sperm count. (C) The percentage of high-quality viable spermatozoa exhibiting extra-mitochondrial granule formation in the acrosomal domain increased significantly (P < 0.001) with time and was maximal after 90 min incubation; one-way ANOVA. (D) Incubation of human spermatozoa under conditions where capacitation was either encouraged with dbcAMP and pentoxifylline or suppressed by the omission of bicarbonate ion revealed a significant increase in the percentage of cells with an acrosomal location (P < 0.05 one-way ANOVA) under capacitating conditions and corresponding declines in the percentage exhibiting formazan deposition in the postacrosome/neck or the complete absence of extra-mitochondrial granules. All data are presented as means ± s.e. (*P < 0.05; ***P < 0.001). Non Cap, not capacitated; Cap, capacitated; Con, Control.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
The MTT formazan deposit shifts position during sperm capacitation
A particularly fascinating feature of extra-mitochondrial formazan deposition was that the position of the granule shifted in an anterior direction during sperm capacitation. Hence, if spermatozoa were incubated under capacitating conditions, there was a significant shift in the location of the formazan deposit to the anterior acrosome (P < 0.001; Fig. 4C and D) and corresponding reductions in the percentage of cells exhibiting extra-mitochondrial formazan deposits in the postacrosomal/neck region or lacking such deposits altogether (Fig. 4E and F). Video evidence for the anterior movement of the extra-mitochondrial granule was subsequently generated, as illustrated in Fig. 5, which includes a video file.
Video of stained human spermatozoa showing the movement of the large extra-mitochondrial formazan deposit (circled) in an anterior direction (A, B, C and D) – Video 1.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Video of stained human spermatozoa showing the movement of the large extra-mitochondrial formazan deposit (circled) in an anterior direction (A, B, C and D) – Video 1.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Video of stained human spermatozoa showing the movement of the large extra-mitochondrial formazan deposit (circled) in an anterior direction (A, B, C and D) – Video 1.
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
MTT reduction and sperm-egg binding
The ability of human spermatozoa to recognize and bind to the surface of the zona pellucida is capacitation dependent and involves the anterior migration of lipid rafts containing zona recognition complexes so that they ultimately come to occupy a forward position commensurate with the mediation sperm-zona recognition and binding (Reid et al. 2011). Since the extra-mitochondrial formazan deposit also appeared to migrate in an anterior direction during capacitation, it was of interest to determine whether such anterior migration is essential if spermatozoa are to bind to the zona surface. This analysis revealed that spermatozoa exhibiting a competence to bind to the zona pellucida invariably possessed an extra-mitochondrial formazan deposit on the sperm head; viable spermatozoa exhibiting mitochondrial MTT reduction but no extra-mitochondrial formazan deposit did not bind to the zona pellucida even though such cells made up 12.6 ± 0.6% of the unbound sperm population. However, the precise location of the extra-mitochondrial formazan deposit on the sperm head (neck, postacrosomal or acrosomal) did not appear to determine the zona binding capacity of the spermatozoa. Thus spermatozoa exhibiting extra-mitochondrial formazan deposits on the neck, postacrosomal domain or the acrosome, could all bind to the zona pellucida Overall, the zona binding capacity of spermatozoa that exhibited evidence of extra-mitochondrial granule deposition, regardless of location, was significantly greater than cells possessing no granule (P < 0.05) (Fig. 6).
Sperm function and extra-mitochondrial formazan deposition. An analysis of sperm binding to the human zona pellucida demonstrated a significant difference (P < 0.05) between samples that exhibited evidence of extra-mitochondrial granule formation and those that did not (pooled t-test); the latter failing to bind to the zona surface. Analysis of the spermatozoa that did exhibit zona binding capacity indicated that the precise site of formazan granule deposition did not determine the functionality of the spermatozoa in this context. Spermatozoa exhibiting formazan deposits in the acrosomal, equatorial or postacrosomal domains was all competent to bind to the zona surface (n = 9). All data are presented as means ± s.e. (*P < 0.05).
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Sperm function and extra-mitochondrial formazan deposition. An analysis of sperm binding to the human zona pellucida demonstrated a significant difference (P < 0.05) between samples that exhibited evidence of extra-mitochondrial granule formation and those that did not (pooled t-test); the latter failing to bind to the zona surface. Analysis of the spermatozoa that did exhibit zona binding capacity indicated that the precise site of formazan granule deposition did not determine the functionality of the spermatozoa in this context. Spermatozoa exhibiting formazan deposits in the acrosomal, equatorial or postacrosomal domains was all competent to bind to the zona surface (n = 9). All data are presented as means ± s.e. (*P < 0.05).
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Sperm function and extra-mitochondrial formazan deposition. An analysis of sperm binding to the human zona pellucida demonstrated a significant difference (P < 0.05) between samples that exhibited evidence of extra-mitochondrial granule formation and those that did not (pooled t-test); the latter failing to bind to the zona surface. Analysis of the spermatozoa that did exhibit zona binding capacity indicated that the precise site of formazan granule deposition did not determine the functionality of the spermatozoa in this context. Spermatozoa exhibiting formazan deposits in the acrosomal, equatorial or postacrosomal domains was all competent to bind to the zona surface (n = 9). All data are presented as means ± s.e. (*P < 0.05).
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Regulation of MTT reduction
There are two major ways in which MTT can be reduced; enzymatically through the action of oxidoreductases or via electron donation from free radicals such as superoxide anion. The importance of flavoproteins in achieving MTT reduction, both within mitochondria and in the extra-mitochondrial space, was indicated by the ability of DPI (10 µM), a flavoprotein inhibitor, to completely suppress formazan formation at both sites (Fig. 7A). This inhibition tells us about the importance of flavoproteins in MTT reduction, but since flavin groups are components of the mitochondrial electron transport chain, cytoplasmic oxidoreductases and free radical-generating NADPH oxidases, it does not help us resolve the source of this activity. The fact that extra-mitochondrial formazan deposition was significantly inhibited (P < 0.001) by Zn (0.5 mM) (Fig. 7B) is in keeping with the involvement of NADPH oxidase activity in this context, given recent studies suggesting that Zn is an inhibitor of NOX5, the major oxidase in human spermatozoa (Ghanbari et al. 2019). The mechanisms involved in extra-mitochondrial formazan deposition were also dependent on the presence of glucose in the incubation medium, since its replacement with 2-deoxyglucose impaired both the viability of the spermatozoa (P < 0.05) and the formation of extra-mitochondrial formazan deposits (P < 0.05) within viable cells (Fig. 7C and D) presumably via the impairment of NADH (glycolysis) or NADPH (hexose monophosphate shunt) availability.
Regulation of formazan deposition in human spermatozoa. (A) A 20 min exposure to DPI (10 µM), a flavoprotein inhibitor, prevented any subsequent reduction of MTT by human spermatozoa. Thus, following exposure to this reagent, neither mitochondrial nor extra-mitochondrial formazan deposition was observed on incubation with MTT (P < 0,05; Kolmogorov–Smirnov test). (B) Exposure to zinc (0.5 mM) for 20 min significantly (P < 0.001; n = 5; one-way ANOVA) reduced the subsequent formation of extra-mitochondrial formazan deposits in viable cells possessing labelled midpieces on addition of MTT. (C) Exposure to BWW medium in which the glucose component had been replaced with an equimolar amount of 2-deoxyglucose showed a significant loss of vitality and (D) a significant reduction in the percentage of viable cells exhibiting extra-mitochondrial formazan deposits (P < 0.05; n = 3; one-way ANOVA). All data are presented as means ± s.e. (*P < 0.05; ***P < 0.001).
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Regulation of formazan deposition in human spermatozoa. (A) A 20 min exposure to DPI (10 µM), a flavoprotein inhibitor, prevented any subsequent reduction of MTT by human spermatozoa. Thus, following exposure to this reagent, neither mitochondrial nor extra-mitochondrial formazan deposition was observed on incubation with MTT (P < 0,05; Kolmogorov–Smirnov test). (B) Exposure to zinc (0.5 mM) for 20 min significantly (P < 0.001; n = 5; one-way ANOVA) reduced the subsequent formation of extra-mitochondrial formazan deposits in viable cells possessing labelled midpieces on addition of MTT. (C) Exposure to BWW medium in which the glucose component had been replaced with an equimolar amount of 2-deoxyglucose showed a significant loss of vitality and (D) a significant reduction in the percentage of viable cells exhibiting extra-mitochondrial formazan deposits (P < 0.05; n = 3; one-way ANOVA). All data are presented as means ± s.e. (*P < 0.05; ***P < 0.001).
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Regulation of formazan deposition in human spermatozoa. (A) A 20 min exposure to DPI (10 µM), a flavoprotein inhibitor, prevented any subsequent reduction of MTT by human spermatozoa. Thus, following exposure to this reagent, neither mitochondrial nor extra-mitochondrial formazan deposition was observed on incubation with MTT (P < 0,05; Kolmogorov–Smirnov test). (B) Exposure to zinc (0.5 mM) for 20 min significantly (P < 0.001; n = 5; one-way ANOVA) reduced the subsequent formation of extra-mitochondrial formazan deposits in viable cells possessing labelled midpieces on addition of MTT. (C) Exposure to BWW medium in which the glucose component had been replaced with an equimolar amount of 2-deoxyglucose showed a significant loss of vitality and (D) a significant reduction in the percentage of viable cells exhibiting extra-mitochondrial formazan deposits (P < 0.05; n = 3; one-way ANOVA). All data are presented as means ± s.e. (*P < 0.05; ***P < 0.001).
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Stained human spermatazoa showing the movement of the large extra-mitochondrial formazan deposit in an anterior direction. This video (http://movie-usa.glencoesoftware.com/video/10.1530/REP-20-0205/video-1) is available from the online version of the article at https://doi.org/10.1530/REP-20-0205.
Download Video 1
The involvement of free radicals in the formation of extra-mitochondrial formazan deposits was also indicated by the ability of the antioxidant co-enzyme Q to induce a suppression of extra-mitochondrial formazan deposition following incubation with MTT (P < 0.01) with no loss of viability (Fig. 8A and B). Similarly, if the spermatozoa were permeabilized in hypotonic media and treated with MTT in the presence of NADH (1 mM), the same reduction in formazan deposition was observed with co-enzyme Q (P < 0.001; Fig. 8C). A specific role for superoxide in MTT reduction was also suggested by the ability of the SOD mimetic, MnTMPyP (100 µM), to achieve a 40% suppression in extra-mitochondrial formazan deposition (59.4 ± 7.0% of the activity observed in the DMSO control; P < 0.01; n = 5; Wilcoxon test).
Impact of the antioxidant, co-enzyme Q, on patterns of MTT reduction in human spermatozoa. Co-enzyme Q had no impact on cell viability (A); however, it did significantly (P < 0.01; n = 6; one-way ANOVA) reduce the percentage of viable cells exhibiting extra-mitochondrial MTT deposits (B), as well as the percentage of permeabilized cells exhibiting extra-mitochondrial formazan deposits following stimulation with 1 mM NADH for 1 h (P < 0.001; n = 6; one-way ANOVA) (C). (D) Fractionation of 1% n-dodecyl β-maltoside extracts of permeabilized human spermatozoa on a Superose-6 FPLC column. Each fraction was examined its capacity to reduce NBT or MTT in the presence of NADH (1 mM). (E) Electrophoresis of the same sperm extracts by native gel electrophoresis revealed protein bands of very similar mobility following incubation with NBT or MTT in the presence of NADH (1 mM). All data are presented as means ± s.e. (*P < 0.05; **P < 0.01; ***P < 0.001).
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Impact of the antioxidant, co-enzyme Q, on patterns of MTT reduction in human spermatozoa. Co-enzyme Q had no impact on cell viability (A); however, it did significantly (P < 0.01; n = 6; one-way ANOVA) reduce the percentage of viable cells exhibiting extra-mitochondrial MTT deposits (B), as well as the percentage of permeabilized cells exhibiting extra-mitochondrial formazan deposits following stimulation with 1 mM NADH for 1 h (P < 0.001; n = 6; one-way ANOVA) (C). (D) Fractionation of 1% n-dodecyl β-maltoside extracts of permeabilized human spermatozoa on a Superose-6 FPLC column. Each fraction was examined its capacity to reduce NBT or MTT in the presence of NADH (1 mM). (E) Electrophoresis of the same sperm extracts by native gel electrophoresis revealed protein bands of very similar mobility following incubation with NBT or MTT in the presence of NADH (1 mM). All data are presented as means ± s.e. (*P < 0.05; **P < 0.01; ***P < 0.001).
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
Impact of the antioxidant, co-enzyme Q, on patterns of MTT reduction in human spermatozoa. Co-enzyme Q had no impact on cell viability (A); however, it did significantly (P < 0.01; n = 6; one-way ANOVA) reduce the percentage of viable cells exhibiting extra-mitochondrial MTT deposits (B), as well as the percentage of permeabilized cells exhibiting extra-mitochondrial formazan deposits following stimulation with 1 mM NADH for 1 h (P < 0.001; n = 6; one-way ANOVA) (C). (D) Fractionation of 1% n-dodecyl β-maltoside extracts of permeabilized human spermatozoa on a Superose-6 FPLC column. Each fraction was examined its capacity to reduce NBT or MTT in the presence of NADH (1 mM). (E) Electrophoresis of the same sperm extracts by native gel electrophoresis revealed protein bands of very similar mobility following incubation with NBT or MTT in the presence of NADH (1 mM). All data are presented as means ± s.e. (*P < 0.05; **P < 0.01; ***P < 0.001).
Citation: Reproduction 160, 3; 10.1530/REP-20-0205
The possibility that NADPH oxidase (NOX) activity was involved in extra-mitochondrial formazan deposition was further explored using a set of three NOX inhibitors (VA2780, ML171 and GKT137831). These studies were carried on human spermatozoa and involved incubating the Percoll-purified cells in the presence of inhibitor (10 µM) for 20 min prior to the addition of MTT. After a 1-h incubation, the cells were fixed in paraformaldehyde (4%) and scored. This analysis revealed a 40% suppression of extra-mitochondrial granule deposition in viable cells with GKT137831 (59.8 ± 7.1% of the activity observed in the DMSO control; P < 0.01; n = 6; Wilcoxon test) but not with either of the other NOX inhibitors assessed.
Comparison of NBT and MTT reduction
Because the MTT-like probe, NBT, has also been used as a method for assessing the quality of human spermatozoa (Esfandiari et al. 2003), including the generation of reactive oxygen species (ROS) (Aitken et al. 2013), a comparison was conducted between this compound and MTT in terms of the entities responsible for the reductive activity. Human spermatozoa were demembranated under hypotonic conditions, extracted with 1% n-dodecyl β-maltoside and fractioned by FPLC on a Superose-6 column. Probing each fraction with MTT and NADH revealed a clear major peak of activity followed by a smaller peak (Fig. 8E). The elution profile of MTT reductase activity very closely resembled that observed with NBT as substrate (Fig. 8E), suggesting that similar mechanisms were involved. To confirm this, MTT/NBT reduction was also examined using native gels lacking SDS. Under these circumstances, a pattern of MTT reduction was again observed that closely matched the pattern observed with NBT in the presence of NADH (Fig. 8E), again suggesting that similar entities are involved.
Discussion
MTT has been widely adopted as a sensitive probe for assessing cell viability because of its responsiveness to oxidoreductase activity within the mitochondrial electron transport chain. However, most studies with this probe have used spectrophotometry to assess the outcome of the assay on a cell population basis and, as a result, have provided no information on the cellular site of MTT reduction and its underlying biochemistry (Stockert et al. 2018). While it is generally acknowledged that the major sites of MTT reduction are within the mitochondria, alternative foci of formazan deposition have also been reported as a consequence of extra-mitochondrial redox activity, involving enzymes such as glyceraldehyde-3-phosphate dehydrogenase (Takahashi et al. 2002), xanthine oxidase (Burdon et al. 1993) and NADPH/NADH oxidases (Berridge & Tan 1993). It has also been suggested that formazan generated as a consequence of extra-mitochondrial MTT reduction accumulates in lipid droplets (Diaz et al. 2007, Stockert et al. 2012). Spermatozoa are interesting cell types for determining sites of MTT reduction because they are so compartmentalized, with the mitochondria entirely confined to the midpiece of the cell and possess an ultrastructural architecture that does not include the overt presence of lipid droplets.
Microscopic examination of mouse spermatozoa revealed the anticipated pattern of formazan deposition following incubation with MTT, with a vast majority of the MTT-reductase activity confined to the mitochondrial gyres, symmetrically arranged in the long sperm midpiece and only a small trace of extra-mitochondrial activity associated with the sperm head (Fig. 1A and B). With human spermatozoa, the pattern of formazan deposition was quite different. In these cells, the short disorganized midpieces possessed a few formazan-positive mitochondria; however, a large single extra-mitochondrial formazan granule located in various positions along the sperm head dominated the histochemical landscape (Fig. 1C, D, E, F and G). Stallion spermatozoa presented an intermediate picture between these two extremes. Like mouse spermatozoa, they possessed long, highly organized midpieces with multiple mitochondrial gyres that actively reduced MTT to generate insoluble formazan deposits, entirely in keeping with the dependence of stallion spermatozoa on oxidative phosphorylation (Gibb et al. 2014). However, they were also like human spermatozoa in generating a single extra-mitochondrial formazan deposit that was located at various positions on the sperm head. This granule was not as large as that seen in human spermatozoa and was only present on around 20% of cells incubated with MTT for 1 h in medium BWW. The only other species to be examined with respect to patterns of MTT reduction is the boar (van den Berg 2015). In this species, the spermatozoa were shown to possess long well-organized midpieces which were heavily labelled with formazan, as befits a species whose spermatozoa are also heavily dependent on oxidative phosphorylation to meet their energy demands (Nesci et al. 2020). However, boar spermatozoa also possessed extra-mitochondrial formazan deposits which closely resemble those observed in equine spermatozoa. In the case of the boar, a vast majority of spermatozoa (80–95%) were found to possess extra-mitochondrial granules, which developed over a 1–2 h incubation period. These inter-species differences in the pattern extra-mitochondrial formazan deposition suggest the existence of corresponding differences in sperm metabolism which may be significant in understanding the mechanisms by which these cells express their functional competence.
Aside from highlighting differences in extra-mitochondrial MTT reductase activity, these results also emphasize the existence of inter-species variation in mitochondrial function, highlighting the paucity of human spermatozoa in this regard. Although both mouse and equine spermatozoa possessed long metabolically active mitochondria, we traditionally think of the latter as being the more dependent on oxidative phosphorylation (Gibb et al. 2014). However, certain species of mouse are also heavily dependent on oxidative phosphorylation to meet their functional needs (Tourmente et al. 2015). It transpires that the oxidative phosphorylation/glycolysis ratio is a highly dynamic property of spermatozoa that is subject to considerable evolutionary pressure associated with sperm competition. Within the genus Mus, M. musculus exhibits a particularly low respiratory rate, low ATP generation and low sperm velocity. This species clearly has the mitochondrial machinery for oxidative phosphorylation, but evolution has driven sperm metabolism in this particular species towards a higher dependence on glycolysis (Tourmente et al. 2015).
In terms of the mechanisms underpinning inter-species differences in extra-mitochondrial formazan deposition, one possible explanation is that, according to the NCBI database, human, boar and stallion spermatozoa all possess a calcium-dependent, free radical-generating NADPH oxidase (NOX5) while mouse spermatozoa do not. The ROS generated by this oxidase are thought to play a key role in mediating the redox regulation of sperm capacitation (Aitken et al. 1997, Musset et al. 2012). The specific involvement of NOX5 in extra-mitochondrial MTT reduction by human spermatozoa is suggested by the impact of NOX inhibitors: ML171 (particularly active against NOX1; Kumar et al. 2019) and VAS2870 (particularly active against NOX2) were both inactive in this regard , while GKT137831 (active against NOX1, NOX4 and NOX5; Altenhöfer et al. 2015) could effectively suppress extra-mitochondrial MTT reduction. In rat and mouse spermatozoa, capacitation is still redox regulated (Lewis & Aitken 2001, Vernet et al. 2001, Ecroyd et al. 2003); however, the source of the ROS is clearly not NOX5.
The oxidative drive for capacitation is a consistent feature of all mammalian spermatozoa that have been examined to date, including man, rat, mouse, buffalo, bull, pig and stallion (de Lamirande & Gagnon 1993, Aitken et al. 1996, 1998, Lewis & Aitken 2001, Ecroyd et al. 2003, Aitken & Curry 2011, Aitken & Nixon 2013, Aitken & Drevet 2020). Creation of an oxidative environment appears to be critical for such processes, namely the stimulation of adenylyl cyclase, the suppression of tyrosine phosphatase activity and the removal of cholesterol from the sperm plasma membrane. It is also possible that NOX5 generates the ROS required by cyclooxygenase (Marqués et al. 2020) to generate prostaglandins which have, in turn, been linked with capacitation, at least in human spermatozoa (Aitken et al. 1986). Overexpression of NOX5 has been linked with the oxidative stress and poor sperm motility associated with asthenozoospermia (Vatannejad et al. 2019) only, in this case, the excess ROS may be stimulating aberrant lipid peroxidation via the lipoxygenase pathway (Walters et al. 2018).
The tentative suggestion that extra-mitochondrial formazan deposition in human spermatozoa reflects NOX5-mediated ROS generating activity is suggested by the following observations: (1) extra-mitochondrial formazan deposition is suppressed by zinc and DPI, both of which suppress NADPH oxidase activity (Vernet et al. 2001, Donà et al. 2011); (2) the NADPH oxidase inhibitor GKT137831 (a recognized NOX5 inhibitor) also significantly suppresses extra-mitochondrial formazan deposition in human spermatozoa; (3) antioxidant strategies including exposure to an SOD mimetic and or co-enzyme Q also significantly suppress this activity and (4) extra-mitochondrial formazan deposition is reduced when glucose is replaced by 2-deoxyglucose, thereby reducing the ability of the spermatozoa to generate NADPH via the pentose phosphate pathway (PPP) which is directly involved in the regulation of sperm capacitation (Urner & Sakkas 2005). It is also worth noting that, while the formation of extra-mitochondrial formazan granules is clearly not a plasma membrane activity, the precise location of the granule is variable. In many cells its formation is associated with the post acrosomal membrane or the outer acrosomal membrane, while transmission electron microscopy clearly revealed evidence of granule formation within the acrosomal vesicle itself. Such a variable location is in keeping with the MTT reductant being a small molecular mass diffusible molecule such as superoxide anion rather than an oxidoreductase protein with a molecular mass of >30 kDa.
Despite such supportive data, it would be unwise to exclude other potential contributors to MTT reduction given the presence of powerful extra-mitochondrial reductases in mammalian spermatozoa (Baker et al. 2004, 2005). In this context, we have previously shown that cytochrome P450 reductase is responsible for reducing another tetrazolium salt, WST-1, [(2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium] in rat spermatozoa (Baker et al. 2004) and that this enzyme is known to reduce MTT directly (Yim et al. 2005). Indeed, we have previously made the argument that oxidoreductases are powerful mediators of NBT reduction (Aitken 2018) and in this study we show that similar protein entities are involved in reducing both NBT and MTT, even though their patterns of formazan deposition are very different; NBT generating a diffuse staining of the cytoplasm (Esfandiari et al. 2003) rather than the discrete granules seen with MTT. We also know from previous studies that NADPH P450 reductase resides primarily on the outer acrosomal membranes of round and elongating spermatids in species such as the mouse and, possibly, the bull and ram (Cotman et al. 2004). Such a localization would certainly be in keeping with a role for this enzyme in extra-mitochondrial MTT reduction in mammalian spermatozoa.
Although the inhibitory action of SOD mimetics might suggest the involvement free radical generation in formazan formation, it is also possible that superoxide generation is a consequence rather than a cause of this process. The reduction of tetrazolium salts involves free radical intermediates that are capable of donating electrons to ground state oxygen to generate superoxide as an artefactual consequence of probe activation. This kind of chemistry has been seen with other ‘superoxide’ detection probes such as lucigenin and WST-1 (Baker et al. 2004, 2005) and is potentially a feature of MTT reduction as well. So, NAD(P)H-dependent reductases and ROS-generating oxidases are both potential contributors to the extra-mitochondrial formazan deposition seen in the spermatozoa of certain species. However, the fact that mouse spermatozoa show little evidence of extra-mitochondrial formazan deposition despite the presence of NADPH cytochrome P450 reductase in their acrosomal membranes (Cotman et al. 2004) suggests that superoxide generation by oxidases such as NOX5 might be the more critical factor.
Regardless of whether reductases or oxygen free radicals are responsible for extra-mitochondrial MTT reduction in spermatozoa, the existence of such activity raises interesting questions about NAD(P)H availability within this highly specialized cell. The enzymes associated with glycolysis are bound to the fibrous sheath in the principal piece of the sperm tail where they release ATP directly to the axoneme in order to meet the energy demands associated with sperm movement (Krisfalusi et al. 2006). Under these circumstances, how would the NADH generated via glycolysis in the sperm tail provide reducing equivalents to the sperm head? Alternatively, the enzymes associated with the PPP, which are responsible for generating NADPH, are located in the post acrosomal region of the sperm head (Luna et al. 2016) and, moreover, this pathway is upregulated during capacitation (Urner & Sakkas 2005). This may explain why extra-mitochondrial MTT reduction appears to be initiated in the postacrosomal/neck region of the spermatozoa, leading to the formation of a large formazan granule that then moves anteriorly as the spermatozoa capacitate.
In conclusion, the formation of extra-mitochondrial formazan deposits following MTT reduction follows a species-specific pattern and in human spermatozoa this activity appears to reflect the overall quality of these cells. More studies are now needed to explore further the diagnostic significance of MTT reduction in the mitochondria and extra-mitochondrial space in different species, as well as the relative importance of oxygen radicals and oxidoreductase activity in the generation of this activity.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/REP-20-0205.
Declaration of interest
Prof R John Aitken is on the editorial board of Reproduction. Prof Aitken was not involved in the review or editorial processing of this paper on which he is listed as an author. The other authors have nothing to disclose.
Funding
This work was supported by the Priority Research Centre for Reproductive Science at the University of Newcastle, NSW.
Author contribution statement
R J A conceived the study, designed the experiments and generated the first draft of the manuscript. D G, L K, E T, M L, and A W conducted the experimental work and Z G organized the provision of equine spermatozoa and participated in the experimental design and manuscript preparation.
Acknowledgements
The authors gratefully acknowledge Ms Jodi Powell, for her work in organizing the human semen donor panel, and the staff of the Animal Services Unit, University of Newcastle, for their assistance with animal care and maintenance. The authors also thank the Analytical and Biomolecular Research Facility (ABRF) of the University of Newcastle for access to its flow cytometry core.
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