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From the * Veterinary Teaching Hospital,
Laboratory of Spermatology, and the Department
of Physiology, Faculty of Veterinary Medicine, University of Extremadura,
Cáceres, Spain.
| Correspondence to: Dr F. J. Peña, Section of Reproduction and Obstetrics, Department of Herd Health and Medicine, Faculty of Veterinary Medicine, Avd de la Universidad, s/n 10071 Cáceres, Spain (e-mail: fjuanpvega{at}unex.es). |
| Received for publication July 17, 2007; accepted for publication October 22, 2007. |
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Abstract |
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Key words: JC-1, YO-PRO-1, caspases, flow cytometry
The major function of mitochondria is supplying cellular energy, but the second major function of the mitochondria is the regulation of cell death (Ott et al, 2007). In addition, this subcellular structure is the major source of reactive oxygen species (ROS), which are mainly generated at complexes I and III of the respiratory chain. Mitochondria-generated ROS play an important role in the release of cytochrome c and other proapoptotic proteins, which can trigger caspase activation and apoptosis. In relation to this, mitochondria have been identified as the most sensitive sperm structure to cryopreservation (Peña et al, 2003b), and active caspases have been identified in canine and bovine spermatozoa after thawing (Peña et al, 2006; Martin et al, 2007).
All of these changes result in reduced longevity of the cryopreserved spermatozoa within the female reproductive tract. This fact is especially problematic in species such as horses, which have long estrus periods, requiring more careful diagnosis of timing of ovulation and insemination. In horses, frequent ultrasound examinations of the mare are required, and ovulation induction is necessary (Samper et al, 2007).
These methods increase the labor requirements in the equine industry, and so research aimed at identifying factors involved in this premature aging of sperm—that could lead to the development of methods to increase the life span of the spermatozoa within the female reproductive tract—would have a tremendous impact on the equine industry. We therefore studied specific apoptotic parameters during the main steps of the cryopreservation process (fresh semen, after centrifugation and equilibration, and after thawing) in equine spermatozoa and studied the expression of additional markers before cryopreservation.
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Material and Methods |
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TopAbstractMaterial and MethodsResultsDiscussionReferences |
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Semen Collection and Processing
Semen (3 ejaculates per stallion) was obtained from 6 Andalusian horses
individually housed at the Veterinary Teaching Hospital of the University of
Extremadura. Stallions were maintained according to the institutional and
European regulations. Semen was collected with the use of a Missouri model
artificial vagina with an in-line filter lubricated and warmed to 45°C to
50°C. The semen was transported immediately to the laboratory for
evaluation and processing. Filtered semen was diluted 1:1 in INRA 96 (IMV
Technologies, l'Aigle, France) extender and centrifuged at 600 x
g for 10 minutes. Then the sperm pellet was dissolved in the freezing
medium (Ghent; Minitüb Ibérica SL, Tarragona, Spain) to a final
concentration of 100 x 106 spermatozoa/mL. The semen was
slowly cooled to 4°C for 1 hour, loaded in 0.5-mL straws, frozen
horizontally in racks placed 4 cm above the surface of liquid N2
for 10 minutes, and then plunged directly in liquid N2. After 4
weeks of storage, straws were thawed in a water bath at 37°C for 30
seconds.
Experimental Design
For the evaluation of changes along the cryopreservation process, complete
semen analysis was performed during 3 steps of the procedure. Three ejaculates
per stallion were evaluated: 1) shortly after collection, 2) after
centrifugation and cooling to 4°C, and 3) after thawing.
Cytofluorometric Assessment of Activated Caspases— The caspase FITC-VAD-FMK in situ marker was used to detect active caspases. This cell-permeable caspase inhibitor peptide conjugated to FITC covalently binds to activated caspases, serving as an in situ marker for apoptosis (Martin et al, 2004). In 1 mL of 1 x 106 spermatozoa in phosphate-buffered saline (PBS), 1 µL of FITC-VAD-FMK (5 mM) was added, and then the mixture was incubated at room temperature in the dark for 20 minutes. After incubation, spermatozoa were washed in PBS and resuspended to the initial cell concentration, and then 1 µL of Eth (1.167 mM) was added and flow cytometry and fluorescence microscopy were performed within 10 minutes. With this technique, 4 sperm subpopulations were detected. Events in the lower-left quadrant represented live (no apoptotic) spermatozoa (ZVAD–/Eth–), events in the lower-right quadrant represented live spermatozoa with active caspases (ZVAD+/Eth–), events in the upper-right quadrant represented dead spermatozoa showing caspase activity (ZVAD+/Eth+), and events in the upper-left quadrant represented dead spermatozoa (ZVAD–/Eth+).
Evaluation of Mitochondrial Membrane Potential (
m)—
JC-1 possesses the unique ability to differentially label mitochondria with
low and high m. In mitochondria with high m, JC-1
forms multimeric aggregates that emit in the high orange wavelength of 590 nm
when excited at 488 nm. In mitochondria with low m, JC-1 forms
monomers; these monomers emit in the green wavelength (525–530 nm) when
excited at 488 nm. For staining, a 3 mM stock solution of JC-1 in
dimethylsulfoxide (DMSO) was prepared. From each sperm sample, 1 mL of a sperm
solution in PBS containing 5 x 106 cells/mL was stained with
0.5 µl of JC-1 stock solution. The samples were incubated at 38°C in
the dark for 40 minutes before flow cytometric analysis. In this way, 3 sperm
subpopulations were identified: 1) events in region E (upper left) represented
spermatozoa with high m (orange fluorescence), events in region F
(lower right) represented spermatozoa with m (green fluorescence),
and events in region G represented spermatozoa having heterogeneous
mitochondria, with high and low m within the same spermatozoa
(orange and green fluorescence simultaneously).
Assessment of Subtle Membrane Changes and Viability— Early membrane changes and sperm viability were determined as described in Peña et al (2005) with modifications for adaptation to equine semen. In brief, the following stock solutions in DMSO were prepared: YO-PRO-1 (25 µM) and Eth (1.167 mM). Then 1 mL of a sperm suspension containing 5 x 106 spermatozoa/mL was stained with 3 µL of YO-PRO-1 and 1 µL of ethidium homodimer. After thorough mixing, the sperm suspension was incubated at 37°C in the dark for 16 minutes. This staining distinguished 4 sperm subpopulations. The first was the subpopulation of unstained spermatozoa. These spermatozoa are considered alive without any membrane alteration. Another subpopulation was the YO-PRO-1+ cells emitting green fluorescence. It has been demonstrated that in early stages of apoptosis, there is modification of membrane permeability that selectively allows entry of some nonpermeable DNA-binding molecules (Ormerod et al, 1993; Wronsky et al, 2002). In this subpopulation, the spermatozoa may show early damage or a shift to another physiologic state because membranes become slightly permeable during the first steps of cryoinjury, enabling YO-PRO-1 but not Eth to penetrate the plasma membrane (Idziorek et al, 1995; Wronsky et al, 2002). None of these probes enters intact cells. Finally, 2 subpopulations of cryoinjury-induced necrotic spermatozoa were easily detected: early necrotic spermatozoa stained with both YO-PRO-1 and Eth (emitting both green and red fluorescence), and late necrotic spermatozoa stained only with Eth (emitting red fluorescence).
Flow Cytometry— Flow cytometric analyses were carried out with an EPICS XL (Coulter Corporation Inc, Miami, Fla) flow cytometer equipped with standard optics, an argon-ion laser (Cyonics; Coherent, Santa Clara, Calif) emitting 15 mW at 488 nm, and the EXPO 2000 software. Subpopulations were divided by quadrants, and the frequency of each subpopulation was quantified. Nonsperm events (debris) were gated out based on the forward scatter and side scatter dot plots by drawing a region enclosing the cell population of interest. Events with scatter characteristics similar to sperm cells but without reasonable DNA content were also gated out. Forward and sideway light scatter were recorded for a total of 10 000 events per sample (YO-PRO-1 and caspases) or 30 000 events for JC-1. Samples were measured at a flow rate of approximately 200 to 300 cells/s. Green fluorescence was detected in FL1, red fluorescence was detected in FL3, and orange fluorescence in FL2.
Western Blotting— Stallion semen was centrifuged at 600 x g for 10 minutes and washed twice with PBS. After the last centrifugation, supernatant was removed and pelleted cells were sonicated for 5 seconds at 4°C in 100 µL of lysis buffer consisting of 50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 1 mM EGTA, 0.4 mM EDTA, protease inhibitor cocktail (complete, EDTA free), and 0.2 mM Na3VO4. The homogenates were clarified by centrifugation at 10 000 x g for 15 minutes at 4°C, and the supernatant was used for analysis of protein concentration followed by dilution with 4X SDS sample buffer. Proteins (25 µg/well) from stallion sperm lysates were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 12% polyacrylamide gels and were transferred to nitrocellulose membranes. Membranes were blocked for 1 hour at room temperature using blocking buffer (5% nonfat dried milk in a solution containing 50 mM Tris/HCl [pH 8.0], 2 mM CaCl2, 80 mM NaCl, and 0.05% [v/v] Tween-20) and incubated overnight at 4°C with anti–caspase-3 (1:1000), anti–caspase-7, or anti–caspase-9 polyclonal antibodies. After incubation with the primary antibody, membranes were washed twice for 4 minutes each with blocking buffer and incubated for 45 minutes at 25°C with horseradish peroxidase–conjugated anti-rabbit IgG. Membranes were then washed with washing buffer (50 mM Tris/HCl [pH 8.0], 2 mM CaCl2, 80 mM NaCl, 0.05% [v/v)] Tween-20), incubated for 5 minutes with ECL detection reagents (Pierce), and finally exposed to ECL Hyperfilm (Amersham Biosciences). The intensity and molecular weight of bands appearing on the membranes were quantified using the software Scion Image for Windows (version 4.02; Scion Corp, Frederick, Md).
Immunocytochemistry— Spermatozoa were washed and suspended in PBS, with an adjustment of the cell concentration to 1 x 106 cells/mL. Fifteen microliters of the sperm suspension were spread on poly-L-lysine–coated slides and allowed to attach for 10 minutes. Cells were then fixed with 3% formaldehyde in PBS for 15 minutes at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 5 minutes. Slides were washed 3 times for 10 minutes each with PBS and incubated in PBS supplemented with 5% bovine serum albumin (BSA) (w/v) for 90 minutes to block nonspecific sites. After blocking, slides were incubated overnight at 4°C with anti–caspase-3 (1:1000), anti–caspase-7 (1:1000), or anti–caspase-9 (1:1000) polyclonal antibody diluted in PBS containing 5% BSA (w/v). On the next day, samples were extensively washed with PBS and further incubated with an Alexa 488-conjugated goat anti-rabbit antibody for 45 minutes at room temperature. Finally, slides were washed with PBS and examined with a Bio-Rad MRC1024 confocal microscope with a 60x objective in oil immersion. Samples were excited at 488 nm with an argon laser, and emission was recorded using a 515-nm longpass filter set. Samples without any primary antibody were assessed to confirm the absence of nonspecific staining.
Statistical Analysis
The data were first examined using the Kolmogorov-Smirnov test to determine
their distribution. In view of the non-Gaussian distribution of most of the
data, multivariate analysis of variance was performed; when significant
differences were found, the nonparametric Mann-Whitney U test was
used to directly compare pairs of values. All analyses were performed using
SPSS for Windows (version 11.0; SPSS Inc, Chicago, Ill). Significance was set
at P 
.05.
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Results |
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Caspase Activity— In all of the stallions except stallion 2, there was a high percentage of spermatozoa showing caspase activity (Table 2) in fresh semen. Caspase activity was detected both in live (Eth–) and dead (Eth+) spermatozoa. Representative cytograms are shown in Figure 2.
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Identification and Subcellular Localization of Caspases-3, -7 and -9—
Results showed the presence in the lysates of stallion sperm (fresh and
frozen-thawed) of full-length (
34 kd) and cleaved (23 kd) caspase-3,
full-length (37 kd) and cleaved (24 kd) caspase-7, and full-length
(47 kd) and cleaved (38 kd) caspase-9
(Figure 3). The subcellular
distribution of caspases in fixed and permeabilized stallion sperm was
assessed by immunocytochemistry with anti-caspase antibodies following the
procedures indicated in "Materials and Methods." The
immunoreactivity obtained with anti–caspase-3 antibody was mainly
distributed in the acrosome and the midpiece. Caspase-7 immunoreactivity was
mainly located in the midpiece and showed a weaker signal in the head and rest
of the tail. Finally, caspase-9 immunoreactivity was mainly detected in the
tail, showing little or no signal in the head
(Figure 4).
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m—
Representative cytograms are shown in
Figure 5. In fresh samples, 3
sperm subpopulations were easily detected: spermatozoa showing orange
florescence (high m), those depicting green fluorescence (low
m, apoptotic spermatozoa) and sperm showing simultaneously green
and red fluorescence (spermatozoa initiating an apoptotic changes). Results
are summarized in Table 3.
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Effect of Cooling and Freezing-Thawing on the Expression of Apoptotic Markers in Spermatozoa Increased Membrane Permeability— Whereas the effect of centrifugation and cooling was subtle, not affecting the percentage of live sperm, freezing-thawing caused, as expected, a significant decrease in the percentage of live spermatozoa. This effect was stallion dependent, with decreases in the percentage of live spermatozoa ranging from 20.5% to 30.4% (Table 1). The increase in permeability of the sperm membrane ranged from 0% to 19.4%. The percentage of dead sperm in 2 groups of Eth+ spermatozoa also increased significantly after freezing-thawing.
Caspase Activity— The percentage of spermatozoa with active caspases was not modified by centrifugation and cooling to 4°C. However, freezing-thawing caused marked effects on the percentage of spermatozoa showing caspase activity (Table 3). Change varied significantly among stallions—in some, the change was from ZVAD–/Eth– (live sperm with no caspase activity) to ZVAD+/Eth– (live sperm with caspase activity). In other stallions, there was a shift from ZVAD+/Eth– to ZVAD+/Eth+ (dead sperm with caspase activity).
m—
Of all the of apoptotic markers studied, m was the one most
significantly affected by the cryopreservation procedure. Both centrifugation
and cooling and freezing-thawing caused significant decreases in m
(Table 3).
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Discussion |
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Recent studies in human and bovine sperm (Kemal Duru et al, 2001; Martin et al, 2004, 2007; Paasch et al, 2004a,b, 2005; Wündrich et al, 2006) show that freezing-thawing induces an "apoptotic-like phenomenon" that may explain the reduced life span of the surviving population. The identification of the cellular and molecular mechanisms responsible for this reduced life span and its control would have a tremendous impact on the equine industry. Frozen-thawed sperm with increased life spans within the female genitalia would reduce the labor requirements (frequent ultrasound examinations of the mare) associated with artificial insemination with frozen-thawed sperm.
Apoptosis is a complex phenomenon regulating cellular proliferation that
can be divided into 3 phases: induction, execution, and degradation. After
induction of apoptosis, mitochondrial pores are opened, leading to decreased
m. Opening of mitochondrial pores causes the release of
proapoptotic factors into the cytoplasm where they are activated, leading to
the degradation phase. During this phase, changes such as increases in sperm
membrane permeability and externalization of phosphatidylserine (which
triggers noninflammatory recognition of apoptotic cells by phagocytic cells)
occur (Desagher and Martinou,
2000). Mitochondria play a major role in the control of apoptosis
(Rasola and Bernardi, 2007).
Mitochondria also have a crucial role in diverse cellular functions such as
energy production, modulation of redox status, osmotic regulation, and
Ca2+ homeostasis. They orchestrate a wide number of signals to
determine cell commitment to cell death or survival
(Rasola and Bernardi, 2007).
Because oxidative and osmotic insults, together with changes in intracellular
Ca2+ concentrations, have been largely recognized as changes
related to cryopreservation (Watson,
2000; Rodríguez
Martínez, 2003), it is plausible to think that the sperm
mitochondria may play a major role in the subtle damages related to
cryoinjury. In fresh and frozen-thawed equine sperm, procaspase-9 and active
caspase-9 were detected. Caspase-9 is involved in the mitochondrial pathway of
apoptosis. Similarly, this caspase was identified in a bovine study
(Martin et al, 2007). However,
in contrast to our detection of both caspases-3 and -7, caspase-3 was not
detected in the bovine study. Also, caspases-3 and -7 have recently been
detected in ram semen (Martin et al,
2007). In humans, caspases-1, -3, -8, and -9 are activated as a
result of cryopreservation (Weng et al,
2002; Paasch et al,
2004a,b,
2005;
Wündrich et al, 2006).
These findings confirm that an apoptotic mechanism is involved in the damage
induced by cryopreservation.
We identified caspases-3, -7, and -9 in equine spermatozoa for the first
time. Moreover, we detected, for the first time, active caspases both in
fresh, cooled, and frozen-thawed equine spermatozoa. Interestingly,
significant correlations were observed between spermatozoa with low
m and apoptotic sperm. Also, significant correlations were
observed between late apoptotic spermatozoa (YO-PRO-1+/Eth+) and Eth+
spermatozoa showing caspase activity (data not shown). These findings suggest
that sperm mitochondria are at the origin of the molecular damage related to
cryoinjury. In addition, a number of reports suggest that sperm mitochondria
are the most sensitive cellular structure to cryopreservation (Peña et
al, 2003). In our study, changes in m were observed after cooling
to 4°C, whereas other apoptotic markers only varied significantly after
cryopreservation. Similar findings have recently been described in a bovine
study (Martin et al, 2007).
This supports the theory of a mitochondrial origin of subtle sperm damage
after cooling and rewarming. Indirectly, the hypothesis is also supported by
taking in account that the main location of caspases
(Figure 4) is the midpiece and
to a less extent the tail and acrosome.
The presence of a high percentage of spermatozoa showing caspase activity in fresh semen is not easy to interpret. Caspase activity has previously been described in fresh human and bovine sperm (Weng et al, 2002; Martin et al, 2004, 2007). However, the percentage of sperm with caspase activity was much higher in our study. This can be explained through 2 hypotheses. First, equine sperm is known to induce an immunologic response in the mare uterus after breeding. It is plausible that because the fate of most of the spermatozoa is to be phagocytosed, an apoptotic mechanism may be involved in the attraction of phagocytes, as occurs in the final steps of apoptosis in somatic cells, to remove the bulk ejaculate from the mare genitalia. The other explanation may be related to the microbial flora of the equine ejaculate. A human study has demonstrated that the presence of bacteria induces sperm apoptosis (Villegas, 2005).
In short, apoptotic markers are present in ejaculated equine sperm, and these markers change in different ways during the cryopreservation process. In addition, mitochondria appear to be the most sensitive cellular structure to cooling and rewarming; thus, research into the protection of mitochondria during cooling and rewarming may be a successful approach to increase the quality of equine cryopreserved sperm.
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Footnotes |
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References |
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