1 Soy Lecithin Interferes with Mitochondrial Function in Frozen-thawed Ram Spermatozoa I. Del Valle 1, , A. Gómez-Durán 1,2 , W.V. Holt 3 , T. Muiño-Blanco 1 , and J.A. Cebrián- 5 Pérez 1, . 1 Departamento de Bioquímica y Biología Molecular y Celular, Instituto Universitario de Investigación en Ciencias Ambientales de Aragón (IUCA), 2 Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Facultad de Veterinaria, Zaragoza, Spain. 3 Institute 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
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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
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
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.
20
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.