1 soy lecithin interferes with mitochondrial function in frozen
TRANSCRIPT
1
Soy Lecithin Interferes with Mitochondrial Function in Frozen-thawed Ram
Spermatozoa
I. Del Valle1,, A. Gómez-Durán1,2, W.V. Holt3, T. Muiño-Blanco1�, and J.A. Cebrián-5
Pérez1,. 1Departamento de Bioquímica y Biología Molecular y Celular, Instituto Universitario
de Investigación en Ciencias Ambientales de Aragón (IUCA), 2Centro de
Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Facultad
de Veterinaria, Zaragoza, Spain. 3Institute of Zoology, Zoological Society of London, 10
Regent’s Park, London NW1 4RY, UK
Running Title: Mitochondrial Function of Ram Spermatozoa
15 Keywords: Sperm cryopreservation, egg yolk, motility. 20
*Corresponding author: T Muiño-Blanco, Department of Biochemistry and 25
Molecular and Cell Biology, University of Zaragoza, Miguel Servet, 177, 50013
Zaragoza, Spain. E-mail: [email protected]
Published-Ahead-of-Print on December 1, 2011 by Journal of Andrology
Copyright 2011 by The American Society of Andrology
2
Abstract 30
Egg yolk and milk are the two major membrane cryoprotectants commonly used
in freezing media for the long-term preservation of semen (alone or in combination
with others). However, in recent years there have been increasing arguments
against the presence of egg yolk or milk, due to the risk of introducing diseases
through the use of cryopreserved semen. In this study, we have analysed the 35
protective effect of lecithin as an alternative to egg-yolk for the cryopreservation of
ram semen, using a range of functional markers for sperm viability, motility,
apoptosis, mitochondrial functionality analyses (mitochondrial inner membrane
surface, MIMS; mitochondrial inner membrane potential, MIMP; and cell membrane
potential, CMP) as methods of assessment in samples diluted in 3 different media: 40
Tris-citrate-glucose as control, and two media supplemented with soy lecithin or egg
yolk. The results showed that lecithin is able to effectively protect certain sperm
quality characteristics against freezing–induced damage. However, lecithin induced
loss of mitochondrial membrane potential or mitochondrial loss that, in fresh semen,
was not reflected by modifications in sperm motility. MIMS and MIMP values 45
decreased in thawed lecithin-treated samples, concomitant with a lower (p<0.05)
percentage of total and progressively motile cells than those in egg yolk-containing
samples. Further incubation of thawed samples revealed changes in motility and
mitochondrial functionality that, otherwise, would not have been detected. These
results indicate that lecithin may have affected the inner mitochondrial membrane in 50
frozen-thawed spermatozoa, and confirm that sublethal damages that seriously
affect sperm functionality, not detected by classical sperm quality analyses, can be
evidenced by changes in the inner mitochondrial membrane surface. These findings
strengthen the relationship between mitochondrial membrane potential and motility,
and show that the mitochondrial alterations induced by the cryopreservation 55
process could be specific targets for the improvement of semen cryopreservation
protocols.
Introduction
Among the basic components of diluents for freezing semen, sources of lipoprotein, 60
proteins or lipids have usually been used to prevent cold shock; these include egg
yolk, milk, or purified soy lecithin (L-α-phosphatidylcholine), a normal constituent of
3
the sperm plasma membrane (Parks and Lynch, 1992). Soya beans extracts have
been widely used since the first half of the twentieth century in the former Soviet
Union as a component of freezing diluents for ram semen (Salamon and Maxwell, 65
2000), and two soviet researchers, Milovanov and Golubj (1973) proposed the
suitability of phospholipid extract from soya beans as replacements for egg yolk in
semen diluents (Salamon and Maxwell, 1995a).
For many years, egg yolk has been a common component of ram semen diluents
for freezing (Salamon and Maxwell, 1995a) although its mechanism of action 70
remains unclear (Bergeron and Manjunath, 2006). However, no ‘artificial’ or
chemically defined component, except soy lecithin, has been reported to result in
comparably high sperm recovery. In recent decades, the use of soy lecithin has
been demonstrated to be safer than egg yolk in terms of biosecurity (Bousseau, et
al., 1998), and it has been used for sperm cryopreservation in species such as eel 75
(Tanaka, et al., 2002), bull (Aires, et al., 2003), stallion (Aurich, et al., 2007), boar
(Zhang, et al., 2009), human (Reed, et al., 2009) and, particularly, in ram (de Paz,
et al., 2010, Forouzanfar, et al., 2010, Gil, et al., 2003). Lecithin has been reported
to have neither a cytotoxic effect (Report on the Safety Assessment of Lecithin and
Hydrogenated Lecithin, 2001) nor a negative effect on sperm motility (Hong, et al., 80
1986), while lysophosphatidylcholine (LPC) and other fatty acids have inhibitory
effects on sperm motility and also induce acrosomal damage. The activation of
phospholipase A2 (PLA2), an enzyme that is expressed in mammalian
spermatozoa, results in the breakdown of phosphatidylcholine and the formation of
LPC and free fatty acids. 85
Mitochondrial functionality has been associated with human sperm quality (for
review see Ramalho-Santos et al., 2009), and mitochondrial membrane potential
(MMP) has been considered a good indicator of sperm functionality that can be
assessed using specific fluorescent markers (Amaral and Ramalho-Santos, 2010).
Furthermore, enzymatic activities of the electron transfer chain complexes, and 90
expression of their subunits, have been correlated with classic sperm quality
parameters (Ruiz-Pesini, et al., 1998); moreover inhibitors of the electron transfer
chain are known to induce a rapid decrease in sperm motility (Ruiz-Pesini, et al.,
2000).
Freezing and thawing processes induce dramatic changes in the sperm structure 95
unless preventative measures are taken. Some of these changes are closely related
4
to an apoptotic like process. Mitochondria undergo two major changes during early
apoptosis; changes in mitochondrial inner membrane function are accompanied by
a decrease in the inner membrane transmembrane potential (Castedo, et al., 2002)
and an increase in outer membrane permeability. These lead to the release of 100
soluble intermembrane proteins, including cytochrome c and apoptosis-inducing
factor (AIF), which activate apoptogenic caspases (cysteinylaspartate-specific
proteases) and DNases, respectively (Kluck, et al., 1997, Liu, et al., 1996, 1999,
Susin, et al., 1996, Yang, et al., 1997, Zamzami, et al., 1996). Therefore, it is
interesting to consider whether sperm cryoinjury inadvertently activates some of the 105
mitochondrial changes that lead to apoptosis. Although spermatozoa do not
possess the full range of cellular components that lead to controlled DNA
degradation, it seems likely that mitochondrial damage could potentiate a cascade
of changes resulting in the loss of sperm quality. On that basis we propose that the
targeted protection of mitochondrial function might be beneficial in the development 110
of novel cryopreservation methods. In the present study, we investigate the actions
of lecithin as a cryopreservation agent and focus on mitochondria in order to see
whether it is mediated via mitochondrial protection.
Materials and Methods 115
Reagents and Preparation of Diluents
Stains (CFDA and propidium iodide) and other chemicals were purchased from
Sigma Chemical Co. (Madrid, Spain). MitoTracker Deep Red, Yo-Pro-1, DiOC6 and
nonylacridine orange (NAO) were acquired from Invitrogen S.A. (Barcelona, Spain).
The medium used was the one described by Salamon (1977) containing 299.75 120
mM Tris, 27.75 mM glucose, 103.37 mM monohydrate citric acid and 5% glycerol,
pH 6.5 with (1) glycerol alone as the control treatment and (2) egg yolk (15% v/v) or
(3) soy lecithin (3.5% w/v) as additives. Egg yolk was centrifuged at 1000 xg for 1h
and the supernatant was used. Lecithin was first diluted, centrifuged at 1000 g for
20 min and filtered through a PVDF 22 µm Millex HV filter (Millipore Iberica S.A.U., 125
Madrid, Spain). Tris-Citrate-Fructose medium (TCFm; 3.634 g Tris, 1 g fructose and
1.99 g citric acid monohydrate; glass-distilled water to 100 ml) was used as the
thawing solution.
130
5
Semen Collection and Freeze-thawing Procedure
All the experiments were performed with fresh semen taken from nine mature Rasa
Aragonesa rams using an artificial vagina. All the rams belonged to the National
Association of Rasa Aragonesa Breeding (ANGRA) and were 2-4 years old. They 135
were housed under uniform nutritional conditions at the Experimental Farm of the
University of Zaragoza in compliance with the requirements of the European Union
for Scientific Procedure Establishments. All experimental procedures were
performed under the supervision of the Ethics Committee of the University of
Zaragoza. The sires were kept apart, divided into two groups, and two successive 140
ejaculates were collected every third day to avoid deterioration of spermatozoa
(Ollero et al., 1996). For every experiment, the second ejaculates from each group
(four rams) were pooled and used for each assay in order to eliminate individual
differences. Under these conditions, pooled ejaculates provide a good quality
uniform sperm sample suitable for representative studies of ram semen (Ollero, et 145
al., 1996).
Semen was diluted with each medium (1:5 at 33 ºC) within 30 min after
collection. Then, diluted semen was assessed for motility and other sperm quality
parameters (controls), cooled slowly using a programmable water bath
(Polysciences, MiniTub Iberica SL) from 33 ºC to 5 ºC at -0.2 ºC/min (2 h 20 min), 150
and frozen by the pellet method described by Evans and Maxwell (1987). Frozen
semen pellets were obtained by placing 200 µl droplets of the cooled diluted semen
directly onto small holes made on dry ice for 2 min, and then transferred to liquid
nitrogen until use when they were thawed in dry glass tubes (1 min and 30 sec at 37
ºC) and immediately diluted 1:1 in TCFm. Sperm quality was assessed immediately 155
after thawing (0 h) and after 3 hours of incubation at 37 ºC in a water bath. Each
experiment was replicated 6 times.
Assessment of Motility
Kinetic parameters of motility were captured with a computerized system using the 160
CASA system (ISAS 1.0.4, Proiser SL, Valencia, Spain). The hardware was a
Basler A312f camera connected to a Nikon eclipse 50i microscope with a 10x
negative phase contrast objective.
6
Aliquots of 8 µl of diluted spermatozoa (1/100) were placed on a pre-warmed
slide (37 ºC), covered with a 0.86 x 0.86 inches cover slip, and images were 165
recorded for 1.5 s (25 images/s; at least 200 cells of each slide). The percentage of
total motile and progressive motile spermatozoa, curvilinear velocity (VCL), straight-
line velocity (VSL), average path velocity (VAP), linearity (LIN) and straightness
(STR) were recorded and used for analysis. A total of more than 40000 cells were
measured. 170
Flow cytometry
All the measurements were performed on a Beckman Coulter FC 500 (IZASA,
Barcelona, Spain) with CXP software, equipped with two lasers of excitation (Argon
ion laser 488 nm and solid state laser 633 nm) and 5 filters of absorbance (FL1-525 175
± 5 nm band pass filter, FL2-575 ± 5 nm band pass filter, FL3-610 ± 5 nm band
pass filter, FL4-675 ± 5 nm band pass filter and FL5-755 ± 5 nm band pass filter).
The side and forward light scatter parameters were gated so that only those cells
possessing the light scatter characteristics of spermatozoa were analysed for
fluorescence. A minimum of 20000 events was counted in all the experiments. 180
Viability: Two µl of each stain [(CFDA, 1 mM) and propidium iodide (PI, 0.75 mM)]
were added to 200 µl of diluted semen (6 x 106 cells/ml) (modified from Harrison
and Vickers, (1990). Samples were incubated at room temperature in darkness for
15 min. The argon laser and filters of 525 and 675 nm were used to avoid
overlapping. Monitored parameters were FS log, SS log, FL1 (CFDA) and FL4 (PI). 185
Non apoptotic-like cells/Mitochondrial inner membrane potential (MIMP): A triple
stain method was performed using Yo-Pro-1 (1 mM in DMSO), a DNA dye used as
an apoptotic marker because it only permeates into cells that are beginning to
undergo apoptosis); PI (0.75 mM) and MitoTracker Deep red (MT, which passively
diffuses across the plasma membrane and accumulates in active mitochondria, 10 190
µM in DMSO) to evaluate mitochondrial membrane potential (∆Ψm) of viable cells.
Two µl of each dye were added to 200 µl of diluted semen, as indicated for the
viability assessment. Samples were incubated at room temperature in darkness for
15 min. YoPro1 emissions were collected with 525 nm filter, PI with 620 nm and
MitoTracker with 755 nm to avoid spectral overlap. Monitored parameters were FS 195
log, SS log, FL1 (YoPro1), FL3 (PI) and FL5 (MitoTracker) and calibration of
overlapping fluorescence was done.
7
Three different sperm populations were classified according to YoPro1/PI
staining: YP-/PI-, intact, considered viable; YP+/PI-, apoptotic; andYP+/PI +,
necrotic cells, those that have lost the integrity of their plasmalemma (Peña, et al., 200
2005). Data for MitoTracker were analysed as suggested by Hallap et al. (2005)
considering spermatozoa with high fluorescence as cells with high ∆Ψm; most of
them from the group of intact dells (YoPro1-/IP-).
Cell membrane potential (CMP): measured by DiOC6. Final stain concentration was
20 nM as suggested by Castedo et al. (2002) with slight modification, and combined 205
with PI (7.5 µM) to excluded non-viable cells. Samples were incubated at room
temperature in darkness for 15 min. Monitored parameters were FS log, SS log,
FL1 (DiOC6) and FL4 (PI). Previously, we carried out control experiments in the
presence of MitoTracker, DiOC6 and carbonyl cyanide m-chlorophenylhydrazone,
an uncoupling agent that causes a complete disruption of the ∆Ψm (CCCP; Sigma 210
Chemical Co., Madrid, Spain, 5 μM) for 30 min at 37 °C. We proved that CCCP
caused mitochondrial depolarization without changes in the fluorescence signal of
DiOC6; hence, only plasma membrane potential was measured (data not shown).
Inner Mitochondrial membrane surface, cardiolipin content (MIMS): Nonyl acridine
orange (NAO) is preferably incorporated into the inner mitochondrial membrane 215
(Maftah, et al., 1989) where it binds to cardiolipin. A linear relationship between the
cardiolipin content of membranes and the incorporated nonyl acridine orange (NAO)
was demonstrated by Petit et al. (1992). In this study, we combined NAO (1 µM)
with PI (7.5 µM) to exclude non-viable cells, modified from Petit et al. (1994).
Samples were incubated at room temperature in darkness for 15 min. Controls with 220
di-nitro phenol (DNP) were made to ensure that mitochondrial uncoupling was
reflected by the staining. Monitored parameters were FS log, SS log, FL1 (NAO)
and FL4 (PI).
Cluster and Statistical Analyses 225
Each experiment was replicated 6 times and was analysed with the repeated
measures model followed by LSD analysis (Statistix 8.0). In order to analyse
whether the treatment differences might be associated with specific sperm
populations, a grouping study was carried out to define functionally distinguishable
sperm clusters. Cluster analysis was undertaken on a total of 40,000 cells, using 230
VCL, VSL, VAP, LIN and STR as input variables. Three different clusters were
8
considered, of low, medium and high quality cells, which were recognized as
showing (1) slow, non-linear movement, (2) moderate and non-linear movement,
and (3) fast-linear motion, respectively. The results presented here come from the
high quality cluster only. Cluster analysis was performed for motility parameters 235
followed by a simple ANOVA of the cell percentage in the high quality cluster. Fresh
and thawed semen samples were analysed separately. Data are presented as
means ± S.E. Values of p<0.05 were regarded as statistically significant by LSD
test. A principal component analysis was performed using all variables in the
dataset; these were standardised prior to analysis. 240
Multivariate analyses of sperm motion parameters were carried out using the
computer program PATN (Belbin, 1987, 1991, 1993). The program uses a series of
procedures to analyse and compare the motility parameter values associated with
each spermatozoon so as to identify sub-groups within the sperm population
(“patterns”). The identification of the sub-groups and their hierarchical classification 245
is carried out by the program independently of the investigator, who is simply
required to judge to what degree subgroups may be combined to yield a sufficiently
small number of groups to allow practical interpretation. In the experiments
described here the PATN software identified three sperm subpopulations. A more
complete description and illustration of the use of PATN analysis to identify 250
subpopulations within boar sperm samples is given by Abaigar et al. (Abaigar et al.
1999).
It is worth mentioning that PATN analysis was performed using data from all
individual spermatozoa within a single experiment and the data need not be
normally distributed or standardized. Any zero values in the dataset were 255
transformed to 0.1. Upon completion of the PATN analysis, each individual
spermatozoon was categorised as belonging to one of the small number of groups,
or subpopulations, described above. In this study the 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 260
interpretation of the sperm motion behaviour that each group represents.
Multivariate group centroids were calculated to assist with this interpretation.
Once the sub-populations had been identified, the relative frequencies of
spermatozoa within each experimental sample, and belonging to each group, were
compared by ANOVA using Statistica for Windows (Statsoft UK, Letchworth, UK). 265
9
Replicated experiments were evaluated by combining frequency data (percentages)
across replicates, then using ANOVAs for further analysis. Data expressed as
percentages were subjected to arcsine transformation prior to ANOVA.
Results 270
As a wide variety of measurements were used in this study, a principal component
analysis (PCA) was carried out to explore their inter-relationships and to see
whether any were correlated. The first 2 factors explained 70.2% of the variance. As
illustrated in Fig. 1 three groups of variables could be recognised: (1) a set of
viability parameters involved the proportion of non-apoptotic cells; (2) the second 275
set of variables indicated that motility characteristics were correlated with the MIMP
response (inner membrane potential, MitoTracker) and motility parameters (total
and progressive motile cells and high quality cluster), and (3) the third independent
variable consisted of MIMS (inner membrane surface, NAO) and the CMP (DiOC6)
response. A correlation matrix helped to confirm these results (Table 1). High 280
correlation between viability and non-apoptotic state as in PCA was found while
MIMS was negatively correlated with this first group.
Both additives, egg-yolk and lecithin, increased the proportion of viable and non-
apoptotic spermatozoa in fresh semen, without significant differences between them
(Fig. 2). Furthermore, significant differences were found in the percentage of non-285
apoptotic cells between control and both cryoprotectant-containing samples
(p<0.05).
However, the addition of lecithin significantly diminished the cell membrane
potential (CMP) and the mitochondrial inner membrane potential (MIMP) of fresh
semen, compared with egg-yolk-containing samples (Table 2). Samples with egg 290
yolk scored the highest percentage of cells with the three mitochondrial parameters
assessed (p<0.05).
Nevertheless, neither the addition of lecithin nor egg yolk influenced the motility
of fresh semen (Fig. 3). Likewise, no significant differences (p>0.05) were found in
the proportion of cells in the high quality cluster (HQ population). 295
After freezing-thawing, the assessment of viability and non-apoptotic
spermatozoa showed that both lecithin and egg-yolk effectively protected
spermatozoa against cryoinjury. The addition of each compound accounted for a
significant increase in sperm membrane integrity, and no difference was found
10
between the effect of lecithin and egg yolk three hours after thawing (Fig. 4). 300
Regarding the apoptotic state (YoPro1 staining), the addition of each cryoprotector
resulted in a higher proportion (p<0.05) of non-apoptotic spermatozoa after thawing
and even after further incubation (Fig. 4).
Nevertheless, the mitochondrial functionality parameters of frozen semen
evolved in different ways after thawing because MIMP was dramatically lowered by 305
the presence of lecithin after 3 h of incubation (p<0.05), and CMP was also lower in
the presence of lecithin or egg yolk (Table 2).
Regarding total and progressive motility, the addition of soy lecithin and egg yolk
significantly improved both kinetic parameters (p<0.05) at 0 h and following
incubation after thawing (Fig. 5) However, differences between lecithin- and egg 310
yolk-containing samples increased following incubation at 3 h.
To analyse whether these differences might be associated with any specific
sperm population, a cluster analysis was carried out over 40,000 cells, taking into
account VCL, VSL, VAP, LIN and STR. The results obtained from the high quality
cluster are shown as percentages of the total motile cell population (Fig. 5) and 315
revealed that egg yolk appeared to be a superior cryoprotectant immediately after
thawing and following further incubation.
Discussion
The improvement of semen cryopreservation protocols implies the need to study, 320
along with classical sperm quality parameters, alternative markers that provide a
better understanding of cell cryoinjury. In this study, we have analysed the
protective effect of lecithin as an alternative to egg-yolk for the cryopreservation of
ram semen, assessing mitochondrial functionality parameters and undertaking
detailed computerized motility analyses. 325
It is well known that changes in mitochondrial inner membrane function are
related to an increase in the outer membrane permeability, leading to the release of
soluble intermembrane proteins and apoptotic factors, that might activate
apoptogenic metabolic pathways (Castedo, et al., 2002, Susin, et al., 1996).
To the best of our knowledge, mitochondrial disorders have not been studied in 330
detail in ram spermatozoa, although several reports have shown that mitochondria
is involved in the fertilization success in human (Frank and Hurst, 1996, Rajender,
et al., 2010, Ruiz-Pesini, et al., 1998). Likewise, the mechanism by which
11
mitochondria may play a role in the energy maintenance needed for sperm motility,
one of the major parameters related to fertility, has also been evidenced 335
(Mackenna, 1995, Pascual, et al., 1996, Ruiz-Pesini, et al., 2007, Ruiz-Pesini, et al.,
2000), and the role of mitochondria in sperm physiology and pathology has been
briefly reviewed (Peña, et al., 2009).
Not all mitochondrial probes have either the same specificity or the same
sensitivity (Amaral and Ramalho-Santos, 2010), although, in principle, there should 340
be a high correlation between them (Garner, et al., 1997). In particular, viable cells
with high mitochondrial membrane potential correlate with high motility (Rajender et
al., 2010) and high fertility (Kasai, et al., 2002) in human. Mitochondrial enzyme
activities have been correlated with not only sperm motility in human (Ruiz-Pesini,
et al., 2000) and bull (Soderquist, et al., 1991) but also vitality and cell concentration 345
in different species (Hrudka, 1987), which suggests an association between
mitochondrial functionality and the overall sperm quality. However, sperm motility
might be relatively independent on mitochondrial activity in several species. Thus,
spermatozoa from bull, but not from mouse (Aitken, et al., 2004, Mukai and Okuno,
2004), depend on the Krebs cycle to maintain sperm motility. Defective oxidative 350
phosphorylation has been shown not to inhibit sperm motility in mouse (Escalier,
2006, Mukai and Okuno, 2004). Likewise, boar spermatozoa incorporate a very
small amount of the produced lactate into the Krebs cycle (Marin, et al., 2003), the
mitochondrial membrane potential of bull spermatozoa may drop temporally in
response to stress and recover after a return to physiological conditions (Martin, et 355
al., 2007), and motility is largely independent of high mitochondrial membrane
potential in deer (Martinez-Pastor, et al., 2008).
Our results showed that lecithin is able to effectively protect spermatozoa
against freezing-induced cryoinjury, because its addition resulted in increased
proportions of viable and non-apoptotic spermatozoa in fresh and frozen-thawed 360
semen reaching egg yolk levels. But the values for mitochondrial function (MIMS,
MIMP and CMP) obtained in lecithin-diluted fresh semen strongly suggest that
lecithin induces mitochondrial membrane alterations that are not reflected in sperm
motility modifications. The addition of egg-yolk prevented in part the decrease of
MIMP and CMP after thawing and incubation. However, lecithin induced a 365
decrease in MIMS at 0 h and in MIMS and MIMP 3 h after thawing. MIMS,
determined by NAO staining, indicates the content in cardiolipin (Maftah, et al.,
12
1989, Petit, et al., 1992), which is the main acidic phospholipid present in the inner
mitochondrial membrane (Cheneval, et al., 1985, Krebs, et al., 1979). Therefore,
the obtained results indicate that lecithin may have affected the inner mitochondrial 370
membrane in frozen-thawed spermatozoa, possibly by displacement of cardiolipin,
a finding that is reflected in the low motility values obtained 3 h after thawing,
compared to those in egg yolk-containing samples. These results indicate that
sublethal damages that seriously affect sperm functionality can be evidenced by
changes in the cardiolipin content, although they are not detected by classical 375
sperm quality analyses. Further incubation of thawed samples revealed changes in
motility and mitochondrial functionality that, otherwise, would not have been
detected.
It is worth noting that the proportion of motile and progressive spermatozoa is
higher than the proportion of spermatozoa with high MIMP in most cases, which 380
suggests that many spermatozoa are motile despite they have low mitochondrial
activity, and they are still motile after 3 h of incubation. These results support the
idea that sperm motility may be relatively independent on mitochondrial activity as
already proposed in several species (Escalier, 2006, Marin, et al., 2003, Martin, et
al., 2007, Martinez-Pastor, et al., 2008, Mukai and Okuno, 2004). Furthermore, 385
although the obtained differences are statistically significant, the fact that our
results represent percentages of cells must be pointed out. The analysis of
variance was not originally developed for its use with percentages, although it is
usually used in this type of studies.
In order to analyse whether the observed differences might be associated with a 390
specific sperm population, a grouping study was carried out to define clusters
related to the sperm determined characteristics. Taking into account motility
parameters, we defined three clusters of low, medium and high quality
spermatozoa. The high quality cluster (HQ) showed a significantly higher quality in
all the studied situations. The principal components analysis identified two groups of 395
factors which together explain 70.2% of variance, considering three groups of
parameters; one contained viability and non-apoptotic cells, another including
MIMP, high quality, progressive and total motility, and the third was represented by
CMP and MIMS.
Our results indicate that lecithin actively induces a specific form of mitochondrial 400
damage, which might be due to a decrease in cardiolipin content, given that the
13
mitochondrial membrane surface area is reduced (Maftah, et al., 1989, Petit, et al.,
1992). Some authors have found differences between quality parameters and
motility, suggesting that there may be differential protective effects of additives,
particularly cryoprotectants, on the sperm tail and acrosome (Celeghini, et al., 2008, 405
Salamon and Maxwell, 1995b). Between these two agents, lecithin and egg yolk,
purified L-α-phosphatidylcholine (soy lecithin) might work only via the acrosome
through interactions with the plasma membrane, while egg yolk, which is composed
not only of L-α-phosphatidylcholine but also lipoproteins, might have better ability to
protect mitochondria as it is a more complex and versatile macromolecule. The 410
results suggest that prevention of the mitochondrial alterations induced by the
cryopreservation process could be specific targets for the improvement of semen
cryopreservation protocols.
In conclusion, lecithin induces serious mitochondrial damage that clearly affects
the inner mitochondrial membrane and, consequently, sperm motility. These 415
alterations become more evident along with thawing time and, therefore, they may
negatively affect the sperm fertilizing capacity.
Acknowledgments
Supported by grant AGL2010-18975, DGA 2011 and the scholarship BES-2006-420
12340 of the Ministry of Investigation, Science and New Technologies of Spain. The
authors thank ANGRA for supplying the sires and S. Morales for the collection of
semen in Zaragoza.
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605
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Table 1. Correlations between 8 different variables after freezing-thawing.
Viability Non-
apoptotic CMP MIMP Total Motile
Progress. Motile
High quality
Non-apoptotic 0.8071
CMP -0.6139 -0.5583
MIMP 0.0440 0.1493 0.2177
Total Motile 0.3699 0.4260 -0.2915 0.4580
Progressive 0.3099 0.3279 -0.2248 0.3697 0.9433
High quality 0.3222 0.4543 -0.3179 0.2142 0.6507 0.5939
MIMS -0.5509 -0.4782 0.7451 0.2401 -0.0279 0.0769 -0.0705
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Table 2. Fresh and frozen-thawed semen mitochondrial quality parameters. Values calculated as proportions of obtained fluorescence in viable cells.
Fresh semen Thawed 0 h Thawed 3 h
MIMS MIMP CMP MIMS MIMP CMP MIMS MIMP CMP Control 0.07 b 45.2 b 0.08 ab 0.17 a 4.0 b 0.14 a 0.16 a 16.3 b 0.12a Soy lecithin 0.04b 40.0 b 0.03 b 0.05 b 11.2 0.03 b 0.03 b 3.3 c 0.03b
Egg yolk 0.14 a 64.3 a 0.11 a 0.17 a 13.5 a 0.08 0.17 a 28.8 a 0.09 S.E. 0.03 7.23 0.03 0.02 2.84 0.01 0.02 2.84 0.01 Within a column, values with different superscripts indicate significant differences (p<0.05). MIMS: mitochondrial inner membrane surface; MIMP: mitochondrial inner membrane potential; CMP: cellular membrane potential. Different SE was calculated for fresh and frozen-thawed samples.
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FIGURE LEGENDS
Fig. 1. Principal component plot of Factor 1 and 2, which explains 70.2% of variance. Fig. 2. Percentage of viable (CFDA+/PI-) and non-apoptotic (YoPro1-/PI-) sperm in fresh semen. Different pairs of letters indicate significant difference (p<0.05). Viability, SE 4.92; Non-apoptotic cells, SE 3.72. Ctrl: control sample; lecit: lecithin; yolk: egg-yolk. Fig. 3. Motility parameters (percentage) in fresh semen. No significant differences were found (p>0.05). Total motile SE 4.25; Progressive motile SE 5.30; High quality cluster SE 1.32. Ctrl: control sample; lecit: lecithin; yolk: egg-yolk. Fig. 4. Viable cells (%) stained with CFDA/PI, and non-apoptotic like cells (YoPro1/PI) in frozen-thawed semen. Different pairs of letters means significant difference within the same parameter and time (p<0.05). Viability SE 2.09; Non-apoptotic like SE 1.28. Ctrl: control sample; lecit: lecithin; yolk: egg- yolk. Fig. 5. Lecithin and egg-yolk effect on sperm motility parameters in frozen-thawed ram semen. Different pair of letters means significant difference within the same parameter and time (p<0.05). Total motile SE 4.81; Progressive motile SE 2.93; High quality cluster SE 1.92. Ctrl: control sample; lecit: lecithin; yolk: egg-yolk.
