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Research Article
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Alterations of Domains in the Plasmatic Membrane Due to Damages of the Perinuclear Theca of Pig Preserved Spermatozoa |
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Maria Guadalupe Orozco Benitez,
Clemente Lemus Flores,
Juan Antonio Hernandez Ballesteros,
Raul Navarrete Mendez
and
Maria de Lourdes Juarez-Mosqueda
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ABSTRACT
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Samples of semen from 12 pigs, three from Yorkshire,
Landrace, Duroc and Mexican Hairless each where obtained to study cryopreservation
methods. Three stages of boar semen cryopreservation were evaluated: none
(fresh stage), cooling at 5 °C and freezing at -196 °C then thawing
to 56 °C for 12 sec. Perinuclear theca damage and domain alterations
were selected as indices of seminal quality, as measured by electronic
and fluorescence microcopy, respectively according to two lineal models
considering by separately the effect of semen preservation and breed.
Integrity and absence of perinuclear theca significantly (p<0.001)
decreased and increased, respectively according to a decrease in temperature
of cryopreservation, from 87.4 to 58.8% and from 0.8 to 26.2%, respectively.
This same significant (p<0.001) effect was found for acrosomal and
post-acrosomal membrane distribution of domains, from 92.1 to 76.8% and
from 3.1 to 13.1% in this same order. Slight but highly significant (p<0.001)
differences were observed when theca integrity was evaluated as affected
by breed, with highest and lowest values for Yorkshire and Pelón
Mexicano pigs, respectively. No breed effect was encountered for presence
of acrosomal domains. A strong interdependence was found between perinuclear
theca damage and domain distribution. In this connection, a highly significant
(p<0.001) positive, interdependence was observed between the theca
damage and acrosomal domain (r = 0.87), while this same relationship was
although highly significant (p<0.001), negative in nature for equatorial
and post-acrosonal domains (r = -0.77 and -0.85, respectively). This experiment
confirmed that cryopreservation methods may severely affect semen quality
of pigs and that genotype may further influence these same indices. More
research is needed for improving methods of preservation of pig semen
quality, from the point of view of perinuclear theca and domain characteristics
of spermatozoa.
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How
to cite this article:
Maria Guadalupe Orozco Benitez, Clemente Lemus Flores, Juan Antonio Hernandez Ballesteros, Raul Navarrete Mendez and Maria de Lourdes Juarez-Mosqueda, 2008. Alterations of Domains in the Plasmatic Membrane Due to Damages of the Perinuclear Theca of Pig Preserved Spermatozoa. Pakistan Journal of Biological Sciences, 11: 1360-1364. DOI: 10.3923/pjbs.2008.1360.1364 URL: https://scialert.net/abstract/?doi=pjbs.2008.1360.1364
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INTRODUCTION
The use of frozen pig semen is far from being placed in a relevant
position (Gilian et al., 2004; Wongtawan et al., 2006) and
it is at least partially due to the spermatozoid susceptibility to damage
provoked by the freezing-thawing process (Wevar et al., 1997; Johnson
et al., 2000; Guthrie and Welch, 2005; Roca et al., 2006).
This fact leads to the use of higher cell concentrations per dosage during
insemination (Cerolini et al., 2001). However, freezing of pig
semen shows interesting possibilities for applying not only in breeding
or commercial pig herd improving, but in programs of local breed conservation
and research in reproduction (Curry, 2000; Holt, 2000).
The real problem that cryopreservation experiences is not the ability
degree of the spermatic cell to be kept viable during storage at -196°C,
but it is in fact the combination of negative effects that freezing-thawing
process determine on sperm physiology and morphology (Watson, 2000; Córdova
and Gutierrez, 2002). Overall, cell damage is reflected in a decrease
in motility and ultrastructural membrane damage (Johnson et al.,
2000; Tienthai et al., 2004). In this connection, membranes of
pig spermatozoid are more sensible to damage due to freezing than those
of other domestic species, due to its lipid composition (Cerolini et
al., 2001). This membrane is considered as a puzzle of domains where
every one does have specific characteristics. The domains are established
during spermiogenesis and they experiment changes during cell life (Mújica
et al., 2003).
The domains are organized as mitochondrial leaf, flagellum, acrosoma
and perinuclear theca (Watson, 2000).
Among these domains, the perinuclear theca exerts diverse functions such
as protection of the spermatozoid nucleus and other processes involved
in fecundation, by favouring the penetration of structure of ovocites
during fusion, activation of this cell and decondensation of the genetic
material after fertilization (Sutovsky et al., 1997; Sutovsky and
Schatten, 2000; Juárez, 2000; Mújica et al., 2003).
The aim of the present investigation was to study the effect of different
cryopreservation processes of pig semen on alterations of domains in the
plasmatic membrane, due to damages of the perinuclear theca.
MATERIALS AND METHODS
This investigation was undertaken in 2007, at the Laboratory of
Histology, Faculty of Veterinary Medicine of the National Autonomous University
of Mexico (UNAM in Spanish) and at the Laboratory of Reproduction, Faculty
of Veterinary Medicine of the Autonomous University of Nayarit (UAN in
Spanish). This investigation was part of a Mexican, national project concerning
the study, rescue and conservation the Pelón Mexicano pig through
cryopreservation of boar spermatozoa for futurein animal improvement.
Three boars of approximately two years old of three commercial breeds,
Landrace, Yorkshire and Duroc and the creole breed of Mexican Hairless
(Pelón Mexicano) pigs were used. The animals were trained for semen
collection by the method of the gloved hand. Two ejaculates per boar were
evaluated. Following sample collection, the techniques of volume, temperature
and color estimation were conducted in the pig sperm immediately for quality
evaluation, together with other microscopic procedures: motility, concentration
and alive to dead cell ratio in the pig semen. Only those ejaculates exhibiting
a motility of 80% and morphoanomalies lower than 15% and a cell concentration
of 300 millions mL-1 were packed in 0.5 mL plastic straws and
sealed. The modified procedure of Westendorf for semen freezing was used
(Bwanga, 1990). All determinations were conducted in triplicate.
Semen samples at either fresh state, at 35°C in cooling curves at
5°C or those at -196°C, then thawed in water bath at 56°C
during 12 sec, were processed for evaluating semen quality. The status
of perinuclear theca was established by transmission electronic and fluorescent
microscopy, using two types of lectins, agglutinin ex-Triticum vulgare
and concanavanine ex-Canavalia ensiformis, conjugated with flourescein
isothiocyanate. Agglutinin identifies the N-acetyl glucosamine residues
and sialic acid and whereas concanavanine A binds mannose and glucose
residues. Surface domains of protoplasmatic membrane localization were
observed in the spermatozoa. For both above referred techniques, a concentration
of 3x106 cells mL-1 was employed. In order to expose
the perinuclear theca surface, the spermatozoids were suspended in aqueous
solution of Brij 36-T at 10% and then fixed by the Karnovsky technique.
After this, the samples were adsorbed in copper meshes of 200 mesh, covered
with membranes of collodion-carbon support, dyed with 0.02% phosphotungstic
acid and placed in a grid support for its observation in an electronic
microscope.
The identification of surface domains of spermatozoids was conducted
by cell fixation in 3% formaldehyde (in volume) for 20 min. afterwards
centrifuged and then discarding the supernatant, for re-suspension of
the sediment in NH4Cl 50 mM for 10 min.
Thereafter, the new supernatant was discarded again and the sediment
was suspended again in PBS at pH 7.4 for preparation of frotis in a defatted
glass slide. After air drying, reagents containing the two types of lectins
(Vide supra), were added in parallel, at a concentration of 10
μL lectin mL-1 PBS sample solution. Control samples were
only treated with the PBS reagent, then incubated during 30 min in a humid
chamber at 37°C in the darkness and finally washed four times with
the PBS reagent and dried. A glycerol/PBS (9:1) mixture was applied and
thereafter protected from light until its examination by fluorescence
microscopy provided of a video camera.
Damages of perinuclear theca were identified by electronic microscopy,
using as morphologic marker the appearance of the sub-structure of the
post-acrosomal leaf. The status of this structure was classified as intact,
altered or absent. Surface domain distribution was identified by fluorescence
microscopy, according to florescence patterns of the acrosomal, aequatorial
and post-acrosomal regions of spermatozoa, corresponding to the main,
central and post-head regions. In both types of examination, counting
at random of 50 sperm cells per sample was carried out.
Perinuclear theca damage and domain alterations were selected as indices
of seminal quality, as measured by electronic and fluorescence microcopy,
respectively according to two lineal models considering by separately
the effect of semen preservation and breed. For measurements carried out
by electronic microscopy of the perinuclear theca, three samples per animals
were observed by duplicate (n = 72) and for membrane domain distribution,
the same procedure was adopted, except that no duplicates were evaluated
(n = 36). When pig genotypes were examined, 24 and 12 observations were
conducted for theca integrity and domain distribution, respectively. All
indices were expressed in percentage. When the analysis of variance revealed
significant differences (p<0.05), means were separated by the Tukey
test. In addition correlation analysis was conducted in the necessary
cases (SAS, 1995).
RESULTSAND DISCUSSION
The electronic microphotography evidence clearly shows different status
of the perinuclear theca of pig spermatozoa heads, either intact, altered
or absent. These identifications were clearly delimited and there were
no difficulties for identification of the different degrees of perinuclear
theca modifications, caused by the cryopreservation methods assayed in
the current investigation.
The Inmunofluorescence microscopy evidence clearly shows of different
distribution of domains, acrosomal, aequatorial and post-acrosomal. In
this connection, fluorescence determined with the selected lectins in
this experiment did not revealed breed differences. This fluorescence
as determined with WGA mainly marked the surface surrounding the head
and tail of spermatozoa, whereas concanavanine A slightly limited head
membrane and the middle of spermatozoa in fresh spermatozoa of the examined
samples.
Integrity and absence of perinuclear theca significantly (p<0.001)
decreased and increased, respectively according to a decrease in temperature
of cryopreservation, from 87.4 to 58.8% and from 0.8 to 26.2%, respectively
(Table 1). This same significant (p<0.001) effect
was found for acrosomal and post-acrosomal membrane distribution of domains,
from 92.1 to 76.8% and from 3.1 to 13.1% in this same order. Overall,
a notorious deterioration in integrity of the perinuclear theca of pig
spermatozoa was evident.
Slight but highly significant (p<0.001) differences were observed
when theca integrity was evaluated as affected by breed, with highest
and lowest values for Yorkshire and Pelón Mexicano pigs, respectively
(Table 2). In fact these two breeds showed extreme values
for all the examined indices determined in the current investigation.
On the other hand, no breed effect was encountered for presence of acrosomal
domains.
The interdependence found between theca damage and domain distribution.
A highly significant (p<0.001) positive, interdependence was observed
between the perinuclear theca damage and acrosomal domain (r, 0.87), while
this same relationship was although highly
Table 1: |
Changes in pig spermatozoa status as affect by method
of semen cryopreservation |
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1Fresh state (environmental temperature):
2Samples examined after thawing at 56°C for 16 sec.
*** p<0.001, abcMeans without letter in common in the
same row differ significantly (p<0.05) according to the Tukey test |
Table 2: |
Perinuclear theca integrity and presence of membrane
domains in pig spermatozoa as affected by breed according to method
of processing |
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1Fresh state (environmental temperature),
2Samples examined after thawing at 56°C for 12 sec.***
p<0.001, abcMeans without letter in common in the same
column differ significantly (p<0.05) according to the Tukey test |
significant (p<0.001), negative in nature for aequatorial and post-acrosonal
domains (r, -0.77 and -0.85, respectively).
Results of the present experiment indicate that the freezing-thawing
process originates alterations in the perinuclear theca of the several
studied breeds. These damages are in agreement with those of others (Tovich
et al., 2004; MartÃnez et al., 2006), who reported
a similar effect after freezing bovine spermatozoids and even more, suggested
that an increase in the percentage of sperm cell containing no perinuclear
theca determined a decrease in live cells. On the other hand, Arancibia
et al. (2007) found damages of importance in the perinuclear theca
during freezing pig spermatozoids, possibly due to a destabilization of
actin, which would provoke an alteration or even a total lost of this
cell sub-structure. In this connection, it is worthy to note that in other
types of cells, during the freezing-thawing process actin F can experience
polymerization and depolymerization, depending on temperature (Delgado
et al., 1999; Watson, 2000).
The observed changes in the present investigation concerning the perinuclear
theca are of importance, since this theca is the main cytoskeletal structure
in the spermatic head, to what it has been attributed functions of protection,
keeping of membrane domains and participation in the processes of fusion
and decondensation of spermatic cells after fertilization (Juárez
and Mújica, 1999). In this connection, it has been shown that this
structure also suffers alterations after thawing (Hernández et
al., 2007; MartÃnez et al., 2006). On the other hand
Petrunkina et al. (2004, 2005) reported that cytoskeleton proteins
which support plasmatic and acrosomal membranes have a depolymerization
and repolymerization depending on temperature, which is reflected in the
accelerated changes in volume occurring in the spermatic cells during
the freezing process.
It has been shown that the plasmatic membrane is affected by cryopreservation
(Holt, 2000; Watson, 2000; Roca et al., 2006) and that there is
a re-distribution of components in the domains of this membrane. In the
current investigation, it was observed that fresh semen contained the
highest number of spermatozoid with the fluorescence pattern in the acrosomal
region and that when the temperature decreased during freezing up to 5°C,
there was an increase in the percentage of cell sperms with a fluorescence
pattern in the aequatorial region. This same shift of fluorescence pattern,
but towards the post-acrosomal region, was observed in thawed samples
(Frits et al., 1998; Jiménez et al., 2003).
In this study, both agglutinin and concanavanine A linked to glycoconjugates
present in the acrosomal region of the plasmatic membrane of fresh spermatozoa,
in accordance to that reported by others (Frits et al., 1998; Jiménez
et al., 2003; Marini and Cabada, 2003), who employed the same types
of lectins in pig spermatozoids.In fact, it has been observed (Frits et
al., 1998; Jiménez et al., 2003; Marini and Cabada,
2003) that N-acetyl glucosamine, sialic acid, mannose and glucose are
widely distributed in the spermatozoid membrane of the pig, including
tail and acrosomal membrane.
Furthermore, these authors found that after agglutinin treatment of capacitated
spermatozoa, there was a decrease in the site of linkage and this compound
was concentrated in the acrosomal region. These results confirm that the
presence of N-acetyl glucosamine and sialic acid in the plasmatic membrane
of pig spermatozoa, in fact indicate that this cells were not capacitated
yet (Jiménez et al., 2003).
After the freezing-thawing process, it was observed a major frequency
of fluorescence in the domain of the aequatorial region of spermatozoids
marked with agglutinine, whereas this phenomenum occurred in the post-acrosomal
domain when concanavanine A was the marker substance. There are several
investigations where changes in carbohydrates of the plasmatic membrane
surface are described during capacitation and acrosomal reaction (Frits
et al., 1998; Jiménez et al., 2003; Marini and Cabada,
2003). According to these authors, there is an increase in fluorescence
of the acrosomal portion of the plasmatic membrane in capacitated pig
spermatozoa, after treatment with concanavanine A. It has also been reported
that the perinuclear theca experiment changes during acrosomal reaction
(Juárez and Mújica, 1999) where actin F is involved too.
As it is well known, spermatozoa capacitation implies changes in the plasmatic
membrane.
CONCLUSION
This experiment confirmed that cryopreservation methods may severely
affect semen quality of pigs and that genotype may further influence these
same indices. More research is needed for improving methods of preservation
of pig semen quality, from the point of view of perinuclear theca characteristics
of spermatozoa.
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