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From the * Institute of Zoology, Regent's Park,
London, United Kingdom; the Royal Veterinary
College, London, United Kingdom; and the Genus
Breeding Ltd, Ruthin, United Kingdom.
| Correspondence to: Nilendran S. Prathalingam, Newcastle Fertility Centre, Centre for Life, Newcastle Upon Tyne NE2 3BZ, United Kingdom (e-mail: n.prathalingam{at}ncl.ac.uk). |
| Received for publication September 1, 2006; accepted for publication December 4, 2006. |
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Abstract |
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Key words: Isthmus, ampulla, motility, sperm-oviduct interactions
In the present study, we investigated whether the presence of oviductal proteins during sperm cryopreservation had any beneficial effects, and if so, whether the isthmus-derived proteins were particularly effective. To examine these effects, we not only investigated sperm responses in terms of plasma membrane integrity and osmotic resistance, but also used a novel approach to bovine semen assessment, whereby sperm motility responses to the bicarbonate ion were evaluated. Following ejaculation, spermatozoa encounter a range of chemically altered environmental conditions. The most notable change is the increase in bicarbonate ion concentration that occurs from the epididymal plasma, seminal plasma, uterine fluids, and finally, the oviductal fluids. This increase in bicarbonate concentration has been reported in a variety of mammals (although to our knowledge, there are no reports in cattle), as reviewed by Zhou et al (2005). Since there are very limited data in the literature on the effects of bicarbonate on the modulation of bovine sperm motility, we initially undertook the present study in order to characterize these responses. In other species, such as the pig, enhancement of motility has previously been reported in the presence of bicarbonate (Satake et al, 2006; Holt & Harrison, 2002). This provides an objective means of assessing the physiologically relevant sperm-activation response, and is therefore more informative than carrying out motility assessments in cryopreservation media alone. Satake et al, (2006) have demonstrated that porcine sperm subpopulations are differentially activated by the addition of bicarbonate/CO2, and that this response is modulated by proteins derived from the oviductal epithelium. The presence of oviductal epithelial proteins suppresses the motility activation responses without altering bicarbonate uptake. In view of these results, we investigated whether any beneficial effects of oviductal epithelial proteins on sperm viability and osmotic resistance would modulate the sperm physiological motility response in the presence of bicarbonate.
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Materials and Methods |
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Preparation of Oviductal Cells
Oviducts were collected from freshly slaughtered cows at an abattoir and
transported on ice to the laboratory. Within 8 hours, the oviducts were
trimmed from the surrounding connective tissue and the isthmic and ampullar
regions were separated. A region of about 3 cm between the two regions was
discarded. The isthmic and ampullar tissues were washed three times in
phosphate-buffered saline (Dulbecco PBS; pH 7.2) and the oviductal epithelial
cells were extruded from the open end of the duct by applying a gentle
external constricting action along the length of the segment. The oviducts
were kept on ice throughout the whole period. Cells from about 20 oviducts
were suspended in PBS and centrifuged at 200 x g for 10
minutes; the supernatant was then removed. The cells were then reconstituted
in 20 mL Buffer 1 (60 mM mannitol, 5 mM EGTA, 1 µM
phenylmethylsulfonylfluoride [PMSF], in Tris Base [pH 7.4]) and frozen at
–80°C, until the apical plasma membrane (APM) proteins were
prepared.
Preparation of Apical Plasma Membrane Proteins From Oviductal Epithelial Cells
APM preparation from the oviduct was as described by Fazeli et al
(2003). Briefly, samples were
homogenized for 1 minute (Silverson, Waterside, United Kingdom), supplemented
with MgCl2 to a final concentration of 10 mM, and incubated on ice
for 30 minutes. The homogenate was centrifuged for 15 minutes at 4300 x
g, and the supernatant was removed and centrifuged for 30 minutes at
90 000 x g. The final pellet was resuspended in 20 mL of Buffer
2 (60 mM mannitol, 7 mM EGTA, 1 µmol PMSF, in Tris Base [pH 7.4]) with 10
strokes of a potter homogenizer, and supplemented with MgCl2 to a
final concentration of 10 mM. Samples were then incubated on ice for 30
minutes. The homogenate was centrifuged for 15 minutes at 4300 x
g and the supernatant was removed and centrifuged for 30 minutes at
90 000 x g. After centrifugation, the final pellet was
resuspended in 20 mL of Tyrode M medium (2 mM CaCl2, 3.1 mM KCl,
0.4 mM Mg.Cl26H2O, 100 mM NaCl, 0.3 mM
NaH2PO4.2H2O, 10 mM Hepes, 21.6 mM sodium
lactate, 1 mM sodium pyruvate [pH 7.4 and 300 mOsm]) with 10 strokes of the
potter homogenizer. The suspension was centrifuged for 30 minutes at 90 000
x g. The supernatant was discarded and the remaining pellet was
resuspended in 900 µL of Tyrode M medium using a 23G Yale needle (Becton
Dickinson, Oxford, United Kingdom).
Cryopreservation and Thawing
All egg yolk used in the cryopreservation media was powdered egg yolk
(Babyfood Grade; Mayjex Ltd, Buckinghamshire, United Kingdom), which was
dispersed in distilled water and centrifuged at 20 000 x g, to
remove the particles and retain the lipoprotein fraction; this was the active
fraction for cryopreservation (Watson and
Martin, 1973). The objective of this step was to simplify the
subsequent flow cytometry analysis and increase the accuracy of the results.
Each semen sample was diluted at room temperature to 120 x
106 sperm/mL in a freezing diluent (Tris/citrate/egg yolk without
glycerol) that contained either a control (APM absent) or APM isolated from
either the isthmic or ampullar regions at 4, 40, or 400 µg/mL. The diluted
samples were allowed to cool gradually to 4°C over a 1-hour period, prior
to being further diluted 1:1 with freezing diluent that contained 12% glycerol
(resulting in a final concentration of 6% glycerol), and a final dilution of
APM at 2, 20, or 200 µg/mL. The final concentration of spermatozoa was 60
x 106 sperm/ml. The diluted ejaculates were incubated for 1
hour at 4°C, loaded into 0.25-mL straws (IMV, L'Aigle, France) and frozen
horizontally in cold nitrogen vapor at 2.5 cm above liquid nitrogen for 7
minutes. The samples were stored in liquid nitrogen for a minimum of one week
prior to thawing. To thaw, the semen straws were plunged into a water bath at
39°C for 30 seconds. The cryopreserved semen samples were analyzed for
cell viability, osmotic resistance, and motility, as described below.
Cell Viability (Membrane Integrity)
Cell viability was measured immediately after thawing using a dual
fluorescent stain, which was supplied in a live/dead kit (Invitrogen, Paisley,
United Kingdom) that consisted of SYBR14 and propidium iodide (PI). Three
million spermatozoa were diluted in PBS (pH 7.2) to a final volume of 980
µL, after which 15 µL of SYBR14 (100 nM stock solution) and 5 µL of
PI (2.4 mM stock solution) were added, giving final concentrations of 1.5 nM
SYBR14 and 12 nM PI. The samples were incubated for 15 minutes at 38°C
prior to analysis on the flow cytometer with an argon laser (Epics XL; Beckman
Coulter, Fullerton, Calif) at an excitation wave-length of 488 nm. Events were
displayed by dot plots on log scales. Spermatozoa were distinguished from
other cellular debris on the basis of light scattering, and viable cells were
identified by their SYBR14-positive (emission wavelength 520 nm) and
PI-negative (emission wavelength 620 nm) characteristics.
Osmotic Resistance Test
The osmotic resistance test (ORT) to determine membrane stability was
carried out as described by Revell and Mrode
(1994). Briefly, 200 µL of
spermatozoa suspended in either Eqcellsire (IMV) or in the freezing diluent
after thawing was added to 1000 µL of ORT solution. The ORT solution was
150 mOsm (13.51 g fructose, 7.35 g trisodium citrate, 1000 mL distilled water)
for samples diluted in the long term extender or 100 mOsm for samples after
freeze-thaw (9.0 g fructose, 4.9 g trisodium citrate, 1000 mL distilled water
[100 mOsm]). Samples were incubated for 40 minutes at 38°C, prior to
incubating with SYBR14 and PI and measuring cell viability on the flow
cytometer, as described above.
Sperm motility
The assessment of sperm motility was similar to that described by Satake et
al (2006). Briefly, after
freezing and thawing, semen samples were diluted to approximately 2 x
106 spermatozoa/mL in 940 µL Tyrode M medium in the absence of
bicarbonate/CO2 (2 mM CaCl2, 3.1 mM KCl, 0.4 mM
Mg.Cl26H2O, 116 mM NaCl, 0.3 mM
NaH2PO4.2H2O, 10 mM Hepes, 21.6 mM sodium
lactate, 1 mM sodium pyruvate [pH 7.4 and 300 mOsm]), and the suspension was
incubated in a water bath at 38°C for 10 minutes. Half the sperm
suspension was transferred to an empty prewarmed tube and 15 mM NaCl (control
treatment) was added. In the first tube (in the absence of NaCl), 12 minutes
after the initiation of incubation, a bicarbonate/CO2 mixture was
added to the rest of the sample, to achieve a final bicarbonate concentration
of 15 mM. The bicarbonate medium was prepared in aliquots of 300 mM and
saturated with 100% CO2 prior to addition to the sperm sample.
Following the addition of bicarbonate to the sample and after each motility
reading, CO2 was gassed over the sperm suspension. The sperm
suspension incubated in NaCl was analyzed for motility 2 and 20 minutes after
the addition of NaCl and the bicarbonate-treated sample was analyzed 5, 10,
and 15 minutes after bicarbonate addition. Sperm motility was recorded using a
procedure similar to that described by Holt and Harrison
(2002). A 2-µL sample of
the sperm suspension was placed on a 20-µm depth Microcell slide
(Conception Technologies, San Diego, CA). Microscopy was performed for 3
minutes using an Olympus BH-2 microscope with a 10x negative high phase
contrast objective. Sperm video sequences were recorded on a VHS video
recorder (Hitachi, Tokyo, Japan).
Two hundred individual sperm trajectories were analyzed quantitatively for each of the treatment/time combinations using a Hobson sperm tracker (Hobson Tracking Systems, Sheffield, United Kingdom) operating at 50 Hz with an IBM-compatible Pentium computer. The search radius was set to 11.25 µm and the "minimum track point" setting was 50 frames.
Experimental Design
Experiment 1—
The effects of a long-term extender in the presence and absence of
bicarbonate on sperm viability and motility. In an initial experiment, one raw
ejaculate was collected from each of seven bulls at a commercial artificial
insemination centre (Genus Freezing Unit). A subsample of the ejaculate was
diluted 1:4 in either the standard Eqcellsire (IMV) long-term extender in the
presence of sodium bicarbonate (21 mM) or in the absence of bicarbonate
(replaced with an osmotic equivalent of NaCl). The samples were cooled to room
temperature and sent by overnight post to London. Samples were assessed for
cell viability 24 and 96 hours after dilution using the protocol described
previously.
Sperm motility was analyzed 24 hours after the ejaculation and dilution. Due to poor motility, 1 sample was removed from the study (n = 6). The assessment of motility was carried out using the same protocol as described above, with the exception that the sperm suspension incubated in NaCl was analyzed for motility 0 and 20 minutes after the addition of NaCl and the bicarbonate-treated sample was analyzed 5, 10, and 15 minutes after bicarbonate addition.
Experiment 2— The effect of apical plasma membrane proteins on sperm cryopreservation. Ten ejaculates were collected from seven bulls at a commercial artificial insemination centre (Genus Freezing Unit). A subsample of each ejaculate was diluted by adding 500 µL of the raw ejaculate to 2000 µL of the long-term extender (without bicarbonate), cooled to room temperature, and sent by overnight post to London. Samples were assessed for cell viability and osmotic resistance as outlined previously. Semen samples were then cryopreserved with 0 (control), 2, 20, or 200 µg/mL of apical plasma membrane (APM) preparations isolated from either the isthmic or ampullar regions of the oviduct. After thawing, one straw of each sample was analyzed for viability and osmotic resistance and a second straw was analyzed for the bicarbonate-induced motility response. Sperm motility assessment was carried out only for the treatment group with 20 µg/mL APM and the control, in order to economize on the use of APM. The large numbers of oviducts and intensive labor required to collect a small amount of the protein made it impractical to freeze several straws for each concentration.
Statistical analysis
All data are presented as the means ± SEM unless otherwise stated.
Statistical analyses for cell viability and ORT were carried out using Minitab
version 13.0 (State College, Pa) Cell viability and osmotic resistance between
treatments postthaw were compared using analysis of variance. All samples were
compared with the control.
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Prior to in-depth analysis of the motility data, the straight line velocity (VSL) and linearity (LIN) data were generated to identify the time-points at which the greatest difference occurred between the bicarbonate treatment and the control (in the absence of APM proteins). Since t = 10 minutes showed the largest difference between the treatments and the control, this time-point was used for further analysis. The median value for all the motility variables was initially analyzed using ANOVA, in order to determine the parameters required for a multivariate analysis. All the individual sperm data were pooled into a single data set for multivariate analysis, which was carried out using the PATN software (Belbin, 1993), which allowed all the data to be analyzed objectively. The PATN software generates estimates of association and then classifies these objects into groups, which allows for further analysis without any manual aid. Following exploratory analyses, we decided to use the following 4 motility variables for the PATN analysis: curvilinear velocity (VCL µm/s), average path velocity (VAP µm/s), straight line velocity (VSL µm/s), and linearity of track (LIN %). A full description of the analysis of spermatozoa using PATN is given by Abaigar et al (1999). Briefly, the PATN analysis was performed using data from individual spermatozoa and approximately 200 sperm trajectories were measured per treatment sample, resulting in a final dataset that contained many thousands of sperm trajectories. Groups were distinguished on the basis of multivariate combinations of motion descriptors, and qualitative interpretation of the group structure was therefore based on the descriptive interpretation of the sperm motion behavior that each group represented. Following the multivariate analysis using PATN to classify the sperm into 3 subpopulations, the treatment effects were analyzed using ANOVA.
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Results |
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Ejaculates were ranked according to the proportion of viable sperm, osmotic resistance, and motility parameters, and a regression analysis was carried out comparing the data for each of these measurements. In the absence of bicarbonate CO2, the regression was significant between osmotic resistance and VAP (r 2 = 60.8; P < .05). There was no significant effect of any of the other parameters measured.
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Discussion |
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The mechanism of bicarbonate-induced sperm motility activation probably depends upon adenylyl cyclase and protein kinase A activation, as demonstrated previously for boar (Holt & Harrison, 2002) and human spermatozoa (Luconi et al, 2005). Although the bicarbonate stimulation effect on bovine spermatozoa has not been studied in detail, support for the view that the general mechanism resembles that in other mammals has been provided by Garty and Salomon (1987), who have demonstrated bicarbonate-induced adenylyl cyclase activation. Several reports have demonstrated high bicarbonate concentrations in the female reproductive tract in a variety of species (Murdoch and White, 1968; Visconti et al, 1999; Vishwakarma, 1962), and it is likely that the in vivo effect is to stimulate maximal motility when appropriate. These observations suggest that the presence of bicarbonate in semen extenders is undesirable, probably subjecting spermatozoa to a constant degree of metabolic stimulation that results in substrate depletion and the potential generation of reactive oxygen species.
Having established that bicarbonate induces marked changes in motility, we decided that this stimulatory effect could permit more detailed and objective analyses of sperm motility for use in the subsequent experiments. Analysis of motility stimulation, together with a general assessment of sperm subpopulation structures within the samples may be useful strategies for the examination of this commonly used functional parameter.
One of the initial aims of Experiment 2 was to determine whether there were any beneficial effects on postthaw sperm quality to be gained by adding oviductal proteins to cryopreservation media. Although there was no detectable increase in sperm viability (membrane integrity), oviductal membrane proteins derived from the isthmus slightly but significantly increased the osmotic resistance of spermatozoa. This is encouraging, as osmotic resistance is highly correlated with fertility (nonreturn rates) in cattle (Revell and Mrode, 1994; r = 0.79). However, this effect was relatively small (4%) and should not be overinterpreted.
In the present experiment, APM was added to the required concentration at the final dilution once the spermatozoa had been cooled. This was done for technical expediency. However, it is likely that coincubation of spermatozoa with APM at physiological temperatures prior to cryopreservation enhances the osmotic resistance of the sperm plasma membranes. The beneficial response to proteins derived from the isthmic epithelium occurred at 2 µg/mL but did not increase in a dose-dependent manner with higher concentrations. This suggests that the relevant mechanism for inhibiting osmotic stress is already saturated at 2 µg/mL. There are several plausible explanations for the beneficial effect of isthmic proteins on the osmotic resistance of the spermatozoa. One is that oviductal proteins (in buffalo) reduce lipid peroxidation (Kumaresan et al, 2006) and that reactive oxygen species are generated during cryopreservation (Chatterjee and Gagnon, 2001). Brouwers and Gadella (2003) have demonstrated in cattle that a high proportion (61%) of viable cryopreserved spermatozoa suffer from peroxidative damage, and Strzezek et al (2004) have demonstrated in the boar that antiperoxidant activity in seminal plasma is associated with a decrease in osmotic stress. This leads to the plausible but not conclusive explanation that the increased sperm osmotic resistance may be attributable to the beneficial action arising from oviductal preparations. The explanation of the different results observed between the isthmic and ampullar protein treatments remains elusive, as previous studies have not investigated the differences in the inhibition of lipid peroxidation and proteins between the isthmus and the ampulla. Since the isthmus is where sperm reside prior to ovulation (Hunter, 1995; Yaniz et al, 2000), additional substances may be present that stabilize the sperm membranes.
In addition to the beneficial effects of the oviductal isthmus proteins during cryopreservation, an alternate hypothesis may be a negative effect of ampullar proteins on osmotic resistance. Although the data cannot be dissected to the extent necessary to verify this hypothesis, it is plausible that the negatively acting ampullar proteins interact with the spermatozoa during freezing and thawing to neutralize any beneficial effects conferred by the other proteins in the extract. When spermatozoa are incubated in capacitation medium in the presence of the ampullar and isthmic explants in vitro, a higher proportion become capacitated in the presence of the ampullar cells (Lefebvre and Suarez, 1996). Therefore, we hypothesize that rather than the absence of factors within the ampullar region making spermatozoa more susceptible to cryopreservation, it may be the presence of proteins that capacitates the spermatozoa. Spermatozoa undergo a capacitation-like reaction during cryopreservation (Watson, 1995), and the presence of ampullar proteins may have a cumulative effect that overcomes any positive effects of the ampulla. This hypothesis is supported by evidence that substrates, such as sulfated glycosaminoglycans, which promote capacitation, occur at higher concentrations in the ampulla, especially around the time of estrus (Bergqvist and Rodriguez-Martinez, 2006).
Previous studies of oviductal proteins on bull sperm motility have shown contrasting results. Lapointe et al (1996) demonstrated an increase in motility in response to oviduct proteins, whereas Boquest et al, (1999) recorded a reduction in motility. Our combined statistical and biochemical approach to the assessment of sperm motility allows for a more objective assessment by analyzing sperm subpopulations in either the presence of absence of bicarbonate/CO2. Numerous experiments have been carried out using motility to determine the fertility potential of spermatozoa, although very few of these studies have any physiological basis. In the presence of bicarbonate/CO2 in the boar, motility is repressed by apical plasma membrane proteins derived from the oviductal epithelium (Satake et al, 2006). Following freezing and thawing in the absence of bicarbonate, spermatozoa exposed to isthmic proteins were more progressively motile (ie, had a higher proportion of sperm in PATN Group 3) than either the control or the ampullar treatment. These results were initially surprising but were similar to those reported by Kumaresan et al (2005), who reported higher motility for buffalo spermatozoa exposed to isthmic rather than ampullar oviductal fluid. This interesting finding suggests that either the ampullar proteins do not invoke this response or may have stimulated and depleted the relevant substrates during the cryopreservation process. Following exposure to bicarbonate, the motility of spermatozoa in the presence of ampullar proteins revealed a higher proportion of cells in categories 1 and 2 (ie, decreased progressive motility) compared to the control and isthmic protein treatment. This supports our hypothesis that the motility response elucidated through the ampullar proteins acts through a similar mechanism as bicarbonate, thereby depleting the relevant substrates.
In conclusion, we have demonstrated that the absence of bicarbonate from a long-term extender increases the proportion of viable cells 96 hours after collection. Furthermore, the addition of bicarbonate 24 hours after collection and dilution in a bicarbonate-free medium elicits an increase in sperm motility. The presence of proteins derived from the isthmic epithelial cells had a beneficial effect on osmotic resistance of spermatozoa following cryopreservation. However, these proteins modulated the motility responses prior to and after the addition of bicarbonate.
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Footnotes |
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