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From the Department of Physiology, School of Veterinary, University of Murcia, Spain.
| Correspondence to: Prof Dr Joaquín Gadea, Department of Physiology. Facultad de Veterinaria. Universidad de Murcia, 30100 Murcia, Spain (e-mail: jgadea{at}um.es). |
| Received for publication March 23, 2005; accepted for publication June 8, 2005. |
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Abstract |
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Key words: Pig spermatozoa, oxidative stress, IVF, capacitation status
-glutamyl-L-cysteinylglycine; GSH) is a tripeptide
ubiquitously distributed in living cells, and it plays an important role as an
intracellular defense mechanism against oxidative stress
(Irvine, 1996). The process of
freezing is associated with a significant reduction in GSH content in porcine
(Gadea et al, 2004), bovine
(Bilodeau et al, 2000) and
human sperm (Molla and Gadea, unpublished data). Sperm freezing has also been
reported to result in a reduction in sperm viability, changes in sperm
function, lipid composition, and organization of the sperm plasma membrane
(Buhr et al, 1994), as well as
changes in sulfhydryl group content in membrane proteins
(Chatterjee et al, 2001). We have previously reported the effect of addition of GSH to the freezing and thawing extender on sperm cryosurvival (Gadea et al, 2004, 2005). However, few studies have investigated the precise mechanism by which GSH could mediate this effect (Nishimura and Morii, 1993; Funahashi and Sano, 2005). Therefore, more thorough studies are needed to elucidate what changes in sperm function take place during cryopreservation and the mechanisms by which GSH exerts its effects. To answer these questions, we incorporated a new set of functional sperm tests. These included tests of sperm motility assayed by computer-assisted semen analysis (CASA), changes in sulfhydryl group content in the membrane protein, capacitation status, free radical production (ROS generation) and sperm chromatin condensation by flow cytometry, and finally the in vitro penetrability of immature oocytes.
CASA has provided an objective and accurate means of evaluating overall sperm motility (Verstegen et al, 2002). Likewise, flow cytometry has also been very helpful in evaluating sperm quality by providing a specific, objective, accurate, and reproducible method compared to traditional microscopy-based methods (Graham, 2001).
Sperm capacitation and acrosome reaction are two key steps in the fertilization process. Thus, evaluation of these processes would be of paramount importance in assessing sperm fertilizing ability (Harrison, 1997). The measurements of the ROS intracellular generation could be decisive when evaluating the balance between free-radical generating and scavenging systems. Although levels of ROS are commonly evaluated by chemiluminescence assay (Sikka, 2004), it is possible to use flow cytometry for accurate and low-cost measurements, using fluorochromes for evaluating intracellular ROS production as the dichloro-dihydrofluorescein diacetate.
Previous studies have shown greater boar sperm chromatin condensation after freezing-thawing procedures (Hamamah et al, 1990; Cordova et al, 2002). In addition, changes in the status of nuclear chromatin structure have been proposed as a possible cause of infertility (Evenson et al, 1994; Fernandez et al, 2003; Alvarez et al, 2004). Thus, evaluation of chromatin condensation could be a valuable tool for evaluation of cryopreserved boar spermatozoa.
However, the binding and penetration of the zona pellucida is one of the most important barriers that spermatozoa must overcome to fertilize the egg. Also, sperm interaction with the oocyte plasma membrane appears to explain much of the variability in sperm fertilizing potential observed among fertile boars (Berger et al, 1996). Therefore, tests that measure gamete interaction may be more predictive of male fertility than routine semen analysis (Rodriguez-Martinez, 2003; Gadea, 2005). Moreover, we have previously shown that in vitro fertilization (IVF) has a high predictive value in evaluating boar semen fertility in both refrigerated and frozen-thawed semen (Gadea et al, 1998; Sellés et al, 2003). Therefore, IVF could also be helpful in identifying changes in sperm function that standard assays fail to detect (Rodriguez-Martinez, 2003).
The main objective of this study was to evaluate the effect of GSH supplementation of the thawing extender on boar sperm functionality measured by different assays.
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Materials and Methods |
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Semen Collection and Handling
Semen was routinely collected from eight mature fertile boars using the
manual method and a dummy. The sperm-rich fraction was collected in a
prewarmed thermo flask, and the gel fraction was held on a gauze tissue
covering the thermo opening. The semen was then diluted with isothermal
Beltsville Thawing Solution extender (BTS;
Pursel and Johnson, 1975).
Freezing and Thawing Protocol
Semen samples were processed using the straw-freezing procedure described
by Westendorf et al (1975)
with minor modifications, as indicated below. Diluted semen was placed at
15°C for 2 hours and centrifuged at 800 x g for 10 minutes.
The supernatant was discarded, and the semen pellet was resuspended with
lactose egg yolk (LEY) extender (80 mL of 11% lactose and 20 mL egg yolk) to
provide 1.5 x 109 spermatozoa/mL. After further cooling to
5°C for 120 minutes, two parts of LEY-extender semen were mixed with LEY
extender with 1.5% Orvus Es Paste (Equex-Paste, Minitüb, Tiefenbach,
Germany) and 9% glycerol. The final concentration of semen to be frozen was 1
x 109 spermatozoa/mL and 3% glycerol. The diluted and cooled
semen was loaded into 0.5-mL straws (Minitüb), sealed, and transferred to
a programmable freezer (Icecube 1800; Minitüb) and frozen horizontally in
racks. The freezing rate was 1°C/min from 5°C to -4.5°C, 1 minute
at -4.5°C, and then 30°C/min from -4.5°C to -180°C. The straws
were then stored in liquid nitrogen until thawing.
Thawing was carried out by immersing the straws in a circulating water bath at 52°C for 12 seconds (Sellés et al, 2003). Immediately after thawing, the semen was diluted in the thawing media (BTS with or without GSH addition) at 37°C and maintained 30 minutes in these media before being assayed.
Analysis of Seminal Parameters by Microscopy
Percentage motility and progression were determined by placing two sample
aliquots on warm glass slides (37°C) and examining them under light
microscopy (magnification x100). The percentage of motile sperm was
estimated to the nearest 10% (MOT) and the forward progressive motility, using
an arbitrary scale from 0 to 5.
Motion Parameters
Motion parameters were determined using a CASA system (Sperm Class
Analyzer, Microptic, Barcelona, Spain). The CASA-derived motility
characteristics studied were curvilinear velocity (VCL, µm/s),
straight-line velocity (VSL, µm/s), average path velocity (VAP, µm/s),
linearity of the curvilinear trajectory (LIN, ratio of VSL/VCL, %),
straightness (STR, ratio of VSL/VAP, %), amplitude of lateral head
displacement (ALH, µm), wobble of the curvilinear trajectory (WOB, ratio of
VAP/VCL, %), and beat cross-frequency (BCF, Hz).
A 7-µL drop of the sample was placed on a warmed (37°C) slide and covered with a 24 x 24 mm cover slip. The setting parameters were: 25 frames, of which spermatozoa had to be present in at least 15 to be counted; images were obtained at x200 magnification in a contrast phase microscope. Spermatozoa with a VAP less than 20 µm/s were considered immotile. A minimum of five fields per sample was evaluated, counting a minimum of 200 spermatozoa per subsample.
Analysis of Seminal Parameters by Flow Cytometry
Flow cytometric analyses were performed on a Coulter Epics XL cytometer
(Beckman Coulter Inc, Miami, Fla). A 15-mW argon ion laser operating at 488 nm
excited the fluorophores. Data from 10,000 events per sample were collected in
list mode, and four measures per sample were recorded. Flow cytometric data
were analyzed using the program Expo32ADC (Beckman Coulter Inc) using a gate
in forward and side scatter to exclude eventual remaining debris and
aggregates from the analysis.
Assessment of Sperm Capacitation
To detect increase in plasma membrane lipid-packing disorder, sperm samples
were stained with merocyanine 540 (M540) and Yo-Pro 1
(Harrison et al, 1996). Stock
solutions of M540 (1 mM) and Yo-Pro 1 (25 µM, Molecular Probes, Eugene,
Ore) in DMSO were prepared. For each 1 mL diluted semen sample (containing
5-10 x 106 cells), 2.7 µL M540 stock solution (final
concentration of 2.7 µM) and 1 µL of Yo-Pro (25 nM final concentration)
were added. M540 fluorescence was collected with a FL2 sensor, using a 575-nm
band-pass filter, and Yo-Pro 1 with a FL1 sensor, using a 525-nm band-pass
filter. Cells were classified in three categories: low merocyanine
fluorescence (viable, incapacitated), high merocyanine fluorescence (viable,
capacitated), or Yo-Pro-1 positive (dead).
Sulfhydryl Group Content of Proteins from the Sperm Surface
The sulfhydryl group content of proteins from the sperm surface were
evaluated with a fluorescent staining 5-iodoacetamidofluoresceine (5-IAF).
Seminal samples (1 mL of semen with 5-10 x 106 cells) were
incubated with 5 µL of 5-IAF stock solution (500 µM), final solution 2.5
µM, and 5 µL propidium iodide (PI) stock solution (500 mg/mL) at room
temperature for 10 minutes. Green 5-IAF fluorescence was collected with a FL1
sensor using a 525-nm band-pass filter, and red PI fluorescence was collected
with a FL3 sensor using a 650-nm band-pass filter. Cells were classified in
three categories: low 5-IAF fluorescence (viable, intact proteins), high 5-IAF
fluorescence (viable, altered proteins), or PI positive (dead).
Production of Reactive Oxygen Species
Production of ROS was measured by incubating the spermatozoa in thawing
media (BTS with and without the addition of GSH) in the presence of
2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA)
(0.5 µM) during 30, 60, and 90 minutes at 37°C. This dye is a
fluorogenic probe commonly used to detect cellular ROS production.
H2DCFDA is a stable cell-permeable nonfluorescent probe. It is
deesterified intracellularly and turns to highly fluorescent
2',7'-dichlorofluorescin on oxidation. Green 5-IAF fluorescence
was collected with a FL1 sensor using a 525-nm band-pass filter. Measurements
were expressed as the mean green intensity fluorescence units (mean channel in
the FL1), and it was used as index of ROS generation.
Determination of Chromatin Condensation
Sperm chromatin was stained with propidium iodide for the determination of
sperm chromatin condensation (Molina et
al, 1995; Cordova et al,
2002).
Thawed samples were centrifuged (1200 x g for 3 minutes), and the pellet resuspended in a solution of ethanol and Dulbecco's phosphate-buffered saline (PBSD) (70/30 v/v) for 30 minutes for the sperm membranes' permeabilization. After that, the samples were centrifuged, the supernatant discarded, and the pellet resuspended in a propidium iodide solution (PI, 10 mg/mL) in PBSD. Samples were maintained in the darkness for 1 hour before flow cytometric analysis. Red PI fluorescence was collected with a FL3 sensor using a 650-nm band-pass filter. Measurements were expressed as the mean red intensity fluorescence units (mean channel in the FL3), and they were used as an index of the state of the chromatin condensation, as this is directly related to the PI uptake by DNA.
In Vitro Penetration
Sperm in vitro penetration ability was assessed using immature oocytes, as
previously described (Gadea et al,
1998) with minor modifications. In brief, porcine oocytes were
collected from fresh ovaries from prepubertal gilts weighing approximately 95
kg, just after slaughter at a local abattoir, and transported to the
laboratory within 30 minutes in saline (0.9% w/v NaCl) containing 100 mg
mL-1 kanamycin at 37°C. Cumulus-oocyte complexes were collected
from nonatretic follicles (3-6 mm in diameter) by slicing and were washed
twice in modified Dulbecco's phosphate-buffered saline supplemented with 1 mg
mL-1 polyvinyl alcohol. Only oocytes with a homogeneous cytoplasm
and a complete and dense cumulus oophorus were used. The selected complexes
were then again washed twice in fertilization medium, previously equilibrated
for a minimum of 3 hours at 38.5°C under 5% CO2 in air.
The sperm samples (diluted in BTS with or without GSH) were maintained at 37°C for 30 minutes, washed (1200 x g, 3 minutes), and then resuspended in the corresponding IVF medium. Each group of 15 immature oocytes was coincubated with spermatozoa (107 cells/mL) for 18 hours in a Petri dish containing 2 mL of fertilization medium modified Tyrode's Albumin-Lactate-Pyruvate (Rath et al, 1999) at 38.5°C under 5% CO2 in air. At the end of the coincubation period, oocytes were stripped of cumulus cells and attached spermatozoa, mounted on slides, and fixed for a minimum of 24 hours with ethanol:acetic acid (3:1 v/v). They were later stained with 1% lacmoid and examined for evidence of sperm penetration under a phase contrast microscope (magnification x400). Immature oocytes were considered to be penetrated when spermatozoa heads and their corresponding tails were found in the vitellus.
Experimental Design
To examine the effect of GSH supplementation during the thawing process,
spermatozoa from five freezing batches of pooled ejaculates from three boars
were processed without addition of GSH (control) and with addition of 1 or 5
mM GSH to the BTS thawing extender, and maintained 30 minutes in these media
before assayed.
Effect of the Addition of GSH to the Thawing Media on Sperm Function
Seminal samples were evaluated for percentage motility and forward
progressive motility by microscopic observation, motion parameters by CASA,
capacitation status by merocyanine 540 and plasma membrane integrity by Yo-Pro
1, sulfhydryl group content in membrane protein by 5-IAF and plasma membrane
integrity by propidium iodide, reactive oxygen formation by H2DCFDA
staining, and chromatin condensation by propidium iodide staining.
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Effect of the Addition of GSH to the Thawing Media on Sperm In Vitro Penetration Ability of Immature Oocytes |
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In vitro penetration rate data (categorical data) were modeled according to the binomial model of parameters and were analyzed by ANOVA.
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Results |
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TopAbstractMaterials and MethodsEffect of the Addition...ResultsDiscussionReferences |
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The addition of GSH to the thawing media reduced the percentage of capacitated viable sperm in a dose-dependent manner (P = .003, Table 3) and reduced the percentage of viable spermatozoa with changes in the sulfhydryl groups in membrane proteins (P < .04, Table 4). In both cases, no significant differences were observed with regard to the percentage of dead spermatozoa, and a significant batch effect was observed (P < .01, Tables 3 and 4).
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The generation of ROS increased as a function of incubation time (30, 60, and 90 minutes), and ROS levels were significantly reduced following addition of GSH to the thawing media (P < .0001, Table 5). Mean values of ROS generation for the GSH groups were close to 50% of the values of the control group (control: 140.45 vs. 1 mM GSH: 74.64 and 5 mM GSH: 66.32 fluorescence units).
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Chromatin condensation was equally affected by the addition of GSH (P = .0013). When GSH was added, a lower chromatin condensation was observed, as reflected by higher red fluorescence intensity and higher PI uptake (control: 78.11 ± 2.32 vs. 1 mM GSH: 89.24 ± 2.28 and 5 mM GSH: 92.85 ± 3.42 fluorescence units).
Effect of the Addition of GSH to the Thawing Media on Sperm In Vitro Penetration Ability
The data from the in vitro penetration assays showed that addition of GSH
to the thawing media had a positive effect on the parameters studied in a
dose-dependent manner (P < .001,
Table 6). For the penetration
rate and mean number of sperm per penetrated oocyte, a significant effect of
the interaction between GSH addition and batches was detected (P <
.05). When GSH was added to the thawing extender, a higher proportion of
decondensed sperm heads was observed inside the oocyte (18.52 vs 24.20 and
34.52%; P = .002, Table
6).
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Discussion |
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TopAbstractMaterials and MethodsEffect of the Addition...ResultsDiscussionReferences |
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In this study, we evaluated the effects of GSH supplementation of the thawing extender on sperm function to compensate the observed decrease in GSH content produced during the sperm freezing. The main findings emerging from this study indicate that addition of GSH to the thawing media resulted in a lower number of capacitated viable spermatozoa, a decrease in the number of spermatozoa with changes in sulfhydryl groups in membrane proteins, a reduction in ROS generation, lower chromatin condensation, and a higher oocyte penetration rate in vitro and a higher proportion of decondensated sperm heads inside the oocyte. This protective effect on sperm function was more pronounced with 5 mM GSH than with 1 mM GSH.
Addition of GSH to the freezing and thawing extenders would be expected to improve the quality and fertilizing ability of frozen-thawed boar spermatozoa (Nishimura and Morii, 1993; Gadea et al, 2004, 2005), as addition of GSH has been shown to help to maintain sperm motility (Lindemann et al, 1988; Bilodeau et al, 2001, Foote et al, 2002; Funahashi and Sano, 2005) and to protect sperm against oxidative damage (Alvarez and Storey, 1989). In this study, no significant effect on percentage motility and motion parameters was found after addition of GSH to the thawing medium. In contrast, motility significantly improved when GSH was added to the freezing media (Gadea et al, 2005). This observation could be related to the contact time of GSH with the sperm cells. When GSH was added to the freezing media, exposure time was longer (at least 90 minutes) than when it was added to the thawing media (30 minutes). Perhaps 30 minutes was an insufficient contact time to produce a significant effect on the motility pattern. The other possible explanation could be related to the variability associated to the different batches used in this study that could mask the GSH effect.
The results of this study indicate that GSH probably affects plasma membrane lipid packing and sulfhydryl group content in membrane proteins in sperm, as reflected by the lower percentage of capacitated sperm and the lower number of crosslinked proteins observed when GSH was added to the thawing media. Similar results were found when GSH was added to the freezing media (Gadea et al, 2005).
The initiation of the sperm capacitation process is related to an alteration in the redox balance between ROS generation and the activity of the antioxidant defense mechanisms (Aitken et al, 1989; Griveau and Le Lannou, 1997). GSH could be an important regulator of the scavenging system and one of the most important nonenzymatic antioxidants in sperm. In this study, a decreased ROS generation was found when GSH was present in the thawing media, indicating that it may be responsible, at least in part, for the lower disruption of lipid packing and the lower changes in membrane proteins. Nevertheless, studies on these cellular changes affect sperm function, and whether these changes could be reversed by antioxidants is open to further investigations (reviewed by Sikka, 2004).
One of the main findings emerging from this study is that the addition of GSH to the thawing extender significantly improves sperm's in vitro oocyte penetration ability, as we previously hypothesized (Gadea et al, 2004). Therefore, cryopreservation-induced oxidative stress in boar sperm affects a sperm property that is related to both sperm binding and penetration of the zona pellucida, sperm-oocyte membrane fusion, oocyte activation, or pronuclear formation. In this sense, the membrane fusion events involved in binding with the oolema and the acrosome reaction appear to be more vulnerable to ROS-induced damage than overall motility (Aitken et al, 1989). Also, we have previously shown that IVF is a highly valuable tool to assess boar semen fertilizing ability in both refrigerated and frozen-thawed semen (Selles et al, 2003; Gadea et al, 1998).
Another important observation is the higher proportion of decondensed sperm heads found inside the oocytes when GSH was added to the thawing extender. We had previously reported improved male pronuclear formation ratios in an IVM/IVF system when oocytes matured in vitro were used (Gadea et al, 2004). In this way, up to now, the main factors associated with sperm head decondensation were related to the enzymatic mechanisms present in the oocyte cytosol (Funahashi et al, 1995). However, GSH also participates in the decondensation of the paternal genome after oocyte penetration (Calvin et al, 1986; Perreault et al, 1988) and may alter spindle micro-tubule formation in the ovum (Sikka, 2004). These results indicate that pretreatment of sperm with GSH could have an additional effect on sperm decondensation and male pronuclear formation inside the oocyte. Also, Boquest et al (1999) reported a higher embryo development rate when GSH was present during the gamete incubation. Further studies are needed to clarify these observations.
With regard to the sperm chromatin condensation/de-condensation status, it is well known that sperm freezing induces chromatin hypercondensation (Hamamah et al, 1990; Cordova et al, 2002). In this study, a lower level of sperm chromatin condensation was observed when GSH was added. This protective effect of GSH could be the result of two different mechanisms: an indirect one, by reducing oxidative stress, thiol oxidation, and hyper-condensation of sperm chromatin, and a direct one, by inducing sperm decondensation, as it normally occurs in the cytosol of the oocyte after sperm penetration. In addition, GSH may also be acting by reducing oxidative stress-induced DNA oxidation and DNA fragmentation (reviewed by Agarwal and Said, 2003).
In any case, important questions still remain to be solved. Is GSH content in raw semen a good predictor of semen quality following the freeze-thaw cycle? In a recent paper Meseguer et al (2004) found a negative correlation between postthaw motile sperm recovery rate and GSH concentration in raw human semen. In boar sperm, a direct correlation was found between acrosome status in frozen-thawed spermatozoa and GSH content in fresh semen (r = 0.36, P = .02), whereas no significant correlation was found for motility and viability (Gadea, unpublished data).
Other interesting questions are: How is GSH produced by the sperm cell? How
is GSH metabolized in this highly specialized cell? In other type of cells,
intracellular GSH levels are maintained indirectly by two tightly coupled
enzymatic processes involving -glutamyl transpeptidase and
membrane-bound dipeptidases that supply amino acids for GSH and protein
biosynthesis. -glutamyl transpeptidase is widely expressed in many
mammalian tissues, and it is essential for catalyzing secreted GSH into
cysteinyl-glycine and -glutamic acid. After cleavage of cysteinyl-glycine by a
dipeptidase, the amino acids are reabsorbed and used to synthesize GSH, a
process known as the -glutamyl cycle. The generation of GSH is crucial
for the protection of cells against oxidative stress and other forms of
cellular injury. Although GSH metabolic pathways are known, little is known
about GSH metabolism in sperm. Glutamyl transpeptidase is present in the
mid-piece and acrosomal regions of spermatozoa
(Funahashi et al, 1996;
Boilart et al, 2002), and it also has been detected in seminal fluid
(Tate and Meister, 1981;
Zalata et al, 1995). In fact,
it has been used for semen identification in forensic samples
(Abe et al, 1998).
In conclusion, GSH appears to play an important role in sperm antioxidant defense strategy. The addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen boar spermatozoa.
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Acknowledgments |
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Footnotes |
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