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From the * Department of Biology, University of
York, York, United Kingdom; and the Genus
Freezing Unit, Llanrhydd, Ruthin, United Kingdom.
| Correspondence to: Dr Henry J. Leese, Department of Biology, University of York, York YO10 5YW, United Kingdom (e-mail: hjl1{at}york.ac.uk). |
| Received for publication June 12, 2007; accepted for publication November 21, 2007. |
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Abstract |
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Key words: Fertility, semen analysis, sperm, oxygen consumption
Sperm motility is supported by adenosine triphosphate (ATP), which is supplied to dynein/ATPase in the flagellum. The majority of ATP produced by spermatozoa is used to support motility (Kamp et al, 1996; Minelli et al, 1999). Bohnensack and Halangk (1986) determined that 75% of the ATP produced by bovine spermatozoa was used in this way. ATP can be formed by 2 processes: oxidative respiration and glycolysis, both of which occur in bovine spermatozoa (Krzyzosiak et al, 1999). Oxidative respiration occurs within the mitochondria, located in the midpiece of the spermatozoon, and results, in the case of glucose, in 36 ATPs being formed per molecule. Since the distance between the mitochondria and the distal tip of the flagellum is approximately 40 to 50 µm, it is unlikely that ATP generated in the midpiece could diffuse the length of the flagellum (while being consumed en route) and supply enough energy to support motility (Turner, 2003). It is more likely that ATP used by the distal end of the flagellum is produced locally by glycolysis. Hexokinase (the first enzyme of glycolysis) has been localized to the membranes of the spermatozoon head, the flagellum, and the mitochondria (ie, midpiece; Travis et al, 1998), suggesting that glycolysis could occur in these regions. The discovery that glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Westhoff and Kamp, 1997) and the other glycolytic enzymes downstream from GAPDH (Storey and Kayne, 1975) are bound to the fibrous sheath of the flagellum provides further evidence that glycolysis occurs in this region. For each molecule of glucose metabolized by glycolysis there is a net yield of 2 ATP. However, to the best of our knowledge, there are no reports in which both oxidative respiration and glycolysis have been measured in the same sample of spermatozoa to determine total ATP formation.
In the mitochondria, electrons are pumped across the inner mitochondrial membrane and then return via ATP synthase, forming ATP from adenosine diphosphate (ADP) and inorganic phosphate. A significant proportion of the total oxygen consumption is used to maintain the mitochondrial proton gradient. However, this system is never fully coupled, as the mitochondrial membrane is permeable to protons, a phenomenon termed the proton leak. In thymocytes, for example, 39% of the total respiration rate can be attributed to the proton leak (Buttgereit and Brand, 1995).
The Clark electrode is a well-established method for the measurement of oxygen consumption, and it has been used successfully to measure oxygen consumption of many tissues and cells, including bovine spermatozoa (Schoff and First, 1995). Cells or tissue are added to a sealed incubation chamber and stirred at high speed to ensure the even distribution of oxygen. Oxygen diffuses through a Teflon membrane separating the medium from the base of the incubation chamber. The oxygen is reduced by a cathode and causes a current to flow to an anode, which is proportional to the partial pressure of oxygen in the medium. However, owing to the high speed at which the medium is stirred, the spermatozoa are likely to be subjected to unphysiologic forces, which may prevent their normal motility.
The oxygen biosensor system (OBS) is a 96-well plate in which a fluorescent, oxygen-sensitive compound (tris 1,7-diphenyl-1,10 phenanthroline ruthenium (II) chloride) is embedded in a gas-permeable and hydrophobic matrix permanently attached to the bottom of each well. Incubation medium is added to the wells, and as the oxygen in the well is depleted, the fluorescence intensity increases in a concentration-dependent manner. This is recorded in a temperature-controlled, time-lapse fluorescence plate reader. The OBS was designed to be used for screening assays for drug discovery, and it has not been used to measure oxygen consumption by spermatozoa. It offers several advantages over the Clark electrode: spermatozoa are allowed to "swim" normally, sample throughput is greatly increased, and simultaneous reading of many samples is possible.
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Materials and Methods |
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Preparation of Fresh Spermatozoa
Semen from proven fertility bulls was diluted 1:5 with Eqcellsire
immediately after collection and transported overnight to the laboratory at
ambient temperature. To separate spermatozoa with intact membranes, semen was
centrifuged on a density gradient. Semen was mixed 1:1 (v/v) with Optiprep
(Sigma-Aldrich, Gillingham, United Kingdom) to produce a medium with a density
of 1.16 g/mL. A medium with a density of 1.15 g/mL was prepared by mixing
5.775 g Optiprep with 5.726 g Eqcellsire. A density gradient was formed by
layering 1 mL of the 1.15 g/mL medium over 8 mL of the 1.16 g/mL medium, which
was centrifuged at 1000 x g for 15 minutes. Membrane-permeable
spermatozoa form a pellet, whereas those with intact membranes form a layer at
the top of the density gradient (Revell, personal communication). The
membrane-intact spermatozoa were removed from the density gradient and kept at
37°C in a water bath. The concentration of spermatozoa was determined
using a Neubauer chamber.
Clark Electrode
The Clark electrode (Rank Brothers, Cambridge, United Kingdom) was
calibrated using distilled water. Prewarmed Eqcellsire (400 µl) was added
to the incubation chamber of the electrode and incubated at 39°C. The
plunger was lowered into the incubation chamber to form an airtight seal. Once
a steady linear reading was obtained, 100 µl resuspended membrane-intact
spermatozoa was added through the plunger. The final concentration of
spermatozoa in the incubation chamber was 2 x 107/ml. Oxygen
consumption was calculated from the initial gradient following spermatozoa
addition and expressed as ZO2 (µl/108
spermatozoa/h).
Oxygen Biosensor Plate
A 96-well oxygen biosensor plate (BD Biosciences) was allowed to
equilibrate overnight at 39°C and then placed in a fluorescence plate
reader (BMG Labtech, Offenburg, Germany) equilibrated for 30 minutes at
39°C. Excitation was at 485 nm from the base of the plate, and emitted
light was detected at 612 nm also from the base of the plate to reduce
possible scatter caused by the medium or the spermatozoa. A blank reading of
the plate was taken to allow each well to be referenced against its own
initial signal. To serve as 20% oxygen controls, 310 µL prewarmed distilled
water, Eqcellsire, and Eqcellsire plus 2 µg/mL oligomycin was added to
separate wells in triplicate. As a 0% oxygen control, 310 µL prewarmed,
fresh 100 mM Na2SO3 was added to 3 wells. To all
"sample" wells, 210 µL Eqcellsire or Eqcellsire supplemented
with 2 µg/ml oligomycin was added. Serial readings were taken every 2
minutes for 18 minutes. Finally, 100 µL resuspended membrane-intact
spermatozoa was then added to the sample wells to give a concentration of
spermatozoa in each well of 5 x 107/mL. The plate was sealed
using polymerase chain reaction foil seals, and serial readings were taken
every 2 minutes for 30 minutes. After the last reading, the OBS plate was
immediately put onto ice, 300 µL sample was removed from each well and
centrifuged for 5 minutes at 15 000 x g. The supernatant was
stored at –80°C for analysis of lactate production.
Proton Leak
The oxygen consumption due to ATP turnover can be measured using
oligomycin. This blocks proton flow through the F0 particle, which
is needed for ATP synthesis by the F1 particle in the mitochondria.
Several ejaculates from the same bull were used to determine the saturating
concentration of oligomycin. Spermatozoa were incubated in the Clark electrode
with a range of oligomycin concentrations: 0.002, 0.02, 0.2, 2, and 20
µg/mL. The oligomycin was dissolved in DMSO. A DMSO control experiment was
included.
Nonmitochondrial Oxygen Consumption
The oxygen consumption due to nonmitochondrial processes (such as NADPH
oxidase, cytochrome P450, and oxalate oxidase) can be measured using
myxothiazol, which inhibits the transfer of electrons from complex III to
cytochrome c in the electron transport chain. Spermatozoa were
incubated in the Clark electrode with a range of myxothiazol concentrations:
1.6, 16, and 160 nM. The myxothiazol was dissolved in DMSO. A DMSO control
experiment was included.
Lactate Production
Samples were thawed and analyzed using an automated analyzer (Cobas Mira;
Roche Diagnostics, Sussex, United Kingdom). The reaction was carried out as
shown in Equation 1. The assay is based on the conversion of NAD+
to NADH and H+ by lactate dehydrogenase.
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| (1) |
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Results |
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Clark Electrode
ZO2 (µl oxygen consumed/108 spermatozoa/h) was
calculated for each experiment, and the mean value was determined. The mean
ZO2 values for the following bulls (±SEM) were: Belgian blue
32.3 ± 4.7 (n = 3), Simmental 29 ± 3.21 (n = 11), and Charolais
39.7 ± 5.69 (n = 3; Figure
1).
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Frozen/Thawed vs Fresh Spermatozoa
The oxygen consumption of frozen/thawed and fresh spermatozoa from 3 bulls
was measured using the OBS. The mean ZO2 values of the
frozen/thawed spermatozoa were: bull 1, 39.9 ± 2.5 (n = 9); bull 2,
26.6 ± 0.67 (n = 3), and bull 3, 28.3 ± 0.79 (n = 3). The mean
ZO2 values of the fresh spermatozoa were: bull 1, 35.8 ±
6.51 (n = 5), bull 2, 17.8 ± 3.51 (n = 5), and bull 3, 13.0 ±
0.81 (n = 5). The data are shown in Figure
2. There was no significant difference between oxygen consumption
of frozen/thawed and fresh spermatozoa for bull 1 and bull 2 (Mann-Whitney
U). However, oxygen consumption of frozen/thawed spermatozoa was
significantly higher (P < .05) than fresh spermatozoa for bull 3
(Mann-Whitney U).
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Proton Leak
Increasing concentrations of oligomycin were added to spermatozoa in the
Clark electrode to determine the concentration needed to block the electron
transport chain. The average decrease in ZO2 after the addition of
oligomycin was calculated as the percentage of original respiration
(Figure 3). The maximum effect
of oligomycin on oxygen consumption was seen by 0.2 µg/mL. The mean
percentage of the original respiration that was sensitive to oligomycin was
23.9% ± 2.5% (calculated from data using oligomycin at 0.2, 2, and 20
µg/mL) for the bull tested in this set of experiments. Using the OBS, the
oxygen consumption of fresh spermatozoa collected from 4 bulls was calculated
with and without 2 µg/mL oligomycin. The difference between the 2 values
corresponds to oxygen consumption due to ATP turnover. The percentage of total
respiration due to proton leak was: bull 1, 42.6 ± 6.57; bull 2, 36.1
± 5.75; bull 3, 16.0 ± 1.3; and bull 4, 24.8 ± 5.82 (n =
5 for each bull; Figure 4). As
seen in Figure 4, the greater
the oxygen consumption, the smaller the proportion due to
non–ATP-forming processes. This appears to be because the absolute value
of proton leak does not vary much between individuals
(Figure 5).
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Nonmitochondrial Oxygen Consumption
At all concentrations tested, myxothiazol reduced oxygen consumption to 0,
indicating there was no detectable nonmitochondrial oxygen consumption in
bovine spermatozoa.
ATP Production
The mean amount of ATP formed from glycolysis was calculated on the basis
that 1 mol lactate forms 1 mol ATP, shown in Equation 2.
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| (2) |
ATP formation due to oxygen consumption was calculated on the basis that 1 mol oxygen forms 4.8 mol ATP (P/Omax = 2.4; Brand, 2005).
The mean amounts of ATP formed by oxygen consumption were: bull 1, 1.99 ± 0.26; bull 2, 2.41 ± 0.27; bull 3, 8.09 ± 1.51; and bull 4, 3.65 ± 0.83 µmol ATP/108 spermatozoa/h (n = 5 for each bull; Figure 5).
Total ATP production (ATP from oxygen consumption + ATP from glycolysis) was: bull 1, 2.6 ± 0.41; bull 2, 3.63 ± 0.34; bull 3, 9.71 ± 1.65; and bull 4, 5.93 ± 1.61 µmol ATP/108 spermatozoa/h (n = 5 for each bull; Figure 5). A comparison of ATP turnover measured in different cell types is shown in Table 1.
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NRR Compared With Oxygen Consumption
The oxygen consumption of 20 bulls from the breeds Limousin, Belgian Blue,
Charolais (all beef breeds), and Holstein (dairy breed) were measured and
compared with their NRRs. Figure
6 shows NRR against ZO2 for all the bulls tested. The
association between NRR and ATP formation (measured in the previously
mentioned experiments) was also compared. To test the correlation between
ZO2/ATP formation and NRR, the data were normalized (by taking the
square root) and then applied in the Pearson product-moment correlation test.
The results are summarized in Tables
2 and
3.
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Discussion |
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As spermatozoa have limited energy stores, they rely upon substrates taken
up from their environment for ATP generation. The yield of ATP from oxidative
respiration is approximately 18 times greater than that by glycolysis, and it
is therefore likely that under aerobic conditions, spermatozoa produce ATP
mostly by aerobic means. The presence of a phosphagen shuttle—an
energetic link between ATP formed in the mitochondria and ATP used by
dynein-ATPases, while maintaining an appropriate 
GATP for
ATPase function (phosphocreatine shuttle)—has been demonstrated in human
(Yeung et al, 1996), sea
urchin (Tombes and Shapiro,
1987), and rooster (Wallimann
et al, 1986) spermatozoa. However, this system is not present in
bull spermatozoa (Kamp et al,
1996). Hence, it is likely that bovine spermatozoa have a greater
reliance on glycolysis to supply ATP to the distal end of the flagellum, since
diffusion of ATP formed by the mitochondria may not be sufficient. The present
experiments showed that glycolysis accounts for 20% to 44% of ATP formed by
bovine spermatozoa (Figure 5),
suggesting that the lack of a phosphagen shuttle is compensated for by high
glycolytic ATP production in the flagellum
(Figure 5). The value of 20% to
44% of ATP production accounted for by glycolysis is comparable with those
obtained by Guppy et al (2002)
for proliferating MCF-7 breast cancer cells (20% glycolytic and 80%
oxidative). Total ATP production by bovine spermatozoa varied from 2.21 (bull
1) to 8.09 (bull 3) µmol ATP/108 spermatozoa/h.
Table 1 shows a comparison of
ATP production between different cell types. The values of ATP production by
bovine spermatozoa measured in the present study are similar to those measured
by Inskeep and Hammerstedt
(1985) using a
luciferin-luciferase assay (Table
1). In comparison to classically "hardworking" cells,
such as heart cells (0.05 µmol ATP/108 cell/h/cell volume), and
highly proliferative cells, such as breast cancer cells (0.067 µmol
ATP/108 cell/h/cell volume), spermatozoa produce 3 million times
more ATP per cell volume, an exceptionally high rate of ATP production.
Motility consumes 75% of the ATP produced by spermatozoa
(Bohnensack and Halangk, 1986);
this high rate of ATP production enables spermatozoa to sustain a high level
of motility for a prolonged period of time (up to 3 days in the bovine). Once
ejaculated, contractions of the female tract and flagella beating of the
spermatozoa enable them to pass through the cervical mucus and uterotubal
junction. The spermatozoa then free themselves from the oviductal reservoir
and, ultimately, hyperactivation causes a whiplashlike flagella beating, which
helps penetration of the egg vestments.
ATP is known to act as a signaling molecule on ciliated airway and oviduct epithelium, increasing cilary beat frequency via a calcium-dependent pathway (Barrera et al, 2004; Stutts et al, 1992), and ATP added apically or basally to oviduct epithelial cells can modulate the formation of oviductal fluid (Downing et al, 1997). As well as providing energy for the spermatozoa to swim to the site of fertilization in the oviduct, ATP may, therefore, be acting as a signaling molecule, increasing ciliary beat frequency and helping move the spermatozoa through the oviduct or release them from the oviductal reservoir. This mechanism could also help move the ovulated oocyte, which relies on ciliary motion and contractions of the oviduct muscle, to the ampullary-isthmic junction.
Uncoupling (proton leak) is a normal feature of mitochondrial electron transport. In whole organisms, the major function of the proton leak is thought to be heat production, whereas at the cellular level it may provide a means of limiting free radical production by mitochondria (Brand, 2005). Proton leak was measured by determining the oxygen consumption of spermatozoa in the presence and absence of oligomycin. It was determined that 0.2 µg/mL oligomycin was sufficient to inhibit oxygen consumption due to ATP-forming processes. A significant proportion (16%–43%) of oxygen consumption was insensitive to oligomycin, and therefore was due to proton leak (Figure 4). Furthermore, within the beef bulls (Simmental, Holstein, and Belgian blue), the proportion of oxygen consumption due to the proton leak appeared to be negatively associated with NRR, suggesting that bulls of lower fertility "waste energy" because they require more energy to maintain the mitochondrial proton gradient than bulls with higher fertility. The oxygen consumption due to proton leak contributes 20% to 25% of the basal metabolic rate in rats (Rolfe and Brand, 1996). Similarly, the proton leak in rat hepatocytes is 20% to 26% and in rat muscle 35% to 50% of respiration (contracting and resting, respectively; Rolfe et al, 1999).
Nonmitochondrial oxygen consumption could not be detected in the present study supporting the work of Richer and Ford (2001), who concluded that human spermatozoa did not possess significant NADPH oxidase activity, an enzyme shown to be responsible for nonmitochondrial oxygen consumption in phagocytes (Rossi et al, 1985).
The oxygen consumption of frozen/thawed spermatozoa appeared to be higher than that of the fresh spermatozoa, although the difference was only significant for bull 3 (Figure 2). During the freezing process, it has been reported that changes in the spermatozoon membrane take place similar to those that occur during capacitation (Thomas et al, 2006), and it is possible that mitochondrial membranes are damaged during spermatozoa freezing. This damage could cause the membranes to become more "leaky" to protons, thereby increasing the oxygen consumption required for maintenance of the proton gradient across the mitochondrial membrane and ATP synthesis. It is equally possible that the increase in oxygen consumption is as a result of the spermatozoa becoming hyperactivated on account of the capacitation-like changes induced by freeze/thawing. Hyperactivated spermatozoa require that more ATP be supplied to the dynein ATPase than for normal motility (Ho et al, 2002). Moreover, in the present study, the bull with the highest NRR (bull 1) had the smallest difference in oxygen consumption between fresh and frozen/thawed spermatozoa, perhaps indicative of less sublethal injury caused by freezing to spermatozoa from this bull.
Cows inseminated with semen from a beef bull are less likely to be rebred by AI if they fail to conceive than cows inseminated with semen from a dairy bull. To allow for this difference, the NRRs of beef and dairy bulls are ranked separately. The ZO2 of frozen/thawed spermatozoa from 20 bulls was found to be positively correlated to their 49-day NRR (P = .01). When the beef breeds were considered separately, the correlation approached significance (P = .057). Bulls from the dairy breed had a very high correlation (r = 0.884), but this was not significant (P = .116), probably because of the low number of replicate bulls (n = 4). However, in statistical terms, the significance of correlation is not a good guide to the true significance of the relationship. It is better to use r2 as an indicator of the real significance, since this value indicates the amount of variation in one variable explained by the other (Dytham, 2003). Using the r2 values, the highest correlation between NRR and ZO2 was in the dairy bulls (r2 = 0.781; Table 2). There was only a slight correlation between total ATP formation and NRR (r2 = 0.233). However, when the dairy bull was excluded from the analysis (for the reason stated previously), r2 reached the remarkably high value of 0.912 (Table 3). This suggests that a strong correlation could exist between ATP formation and NRR; however, more measurements are required to test this trend.
Increased ATP formation could allow the spermatozoa to detach more easily from the oviductal reservoir and/or increase the chance of spermatozoa penetrating the egg vestments. In addition, the ATP could speed up the movement of the oocyte through the oviduct (Leese et al, 2001; Barrera et al, 2004). Spermatozoa have a limited lifespan once they become capaciated, and if an oocyte is transported rapidly to the site of fertilization after insemination, a greater number of viable spermatozoa will be available when the oocyte arrives.
There was an apparent contradiction in the results, in that spermatozoa from a more fertile bull produce more ATP than their less fertile counterparts; however, frozen/thawed spermatozoa, which are known to have compromised fertility (Pelaez et al, 2006), appeared to have a higher rate of oxygen consumption than fresh spermatozoa from the same bull. Further investigation is needed to determine whether the increased oxygen consumption is "useful oxygen consumption" used to form ATP or whether it is being used to maintain the proton gradient in the mitochondria.
The oxygen and ATP consumption was shown to vary between the different bulls examined in these experiments (Figures 2, 4, and 6), and it should be noted that the bulls were from different breeds. Hence, it is possible that these differences were due to interbreed variation rather than interbull variation. However, Figure 6 shows that individuals from each breed are not grouped together but are spread across the graph; it is therefore likely that differences shown in Figures 2, 4, and 6 are due to interbull variation.
In conclusion, the correlation between bull fertility and sperm oxygen consumption obtained in the present study was higher than that obtained previously for motility (Farrell et al, 1998; Januskauskas et al, 2003). The correlation is strengthened by the fact that in the present study, values were derived from a series of bulls with little variation in fertility. Measurement of total ATP formation correlated better with NRR than oxygen consumption, and could form the basis of a test for bull fertilizing ability after freeze/thawing. However, more individuals need to be studied to test this hypothesis.
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Acknowledgments |
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Footnotes |
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