Alteraciones morfológicas y fisiológicas en espermatozoides

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A mis padres que me dieron la vida y a mi hijo que la continuará...
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L a presente tesis fue financiada a través de un proyecto de la Dirección de Investigación
Científica y Tecnológica de la Universidad Católica del Norte (Coquimbo - Chile) del año 2000
y 2001 asignado al M. Sc. Enrique Dupré, quien me dio la oportunidad de participar en dicho
proyecto.
M i profundo agradecimiento a mi asesora y co-asesor de tesis, M. Sc. Martha Valdivia y
M. Sc. Enrique Dupré, por el aporte intelectual brindado; pero principalmente por su
comprensión, apoyo moral, paciencia y amistad.
A gradezco en forma especial a mi familia base, por ser mi consuelo y apoyo; a mis padres
Rufi y Tito, mi ejemplo de vida, a mis hermanas Gipsy y Zule, mis alegrías, a Tico, mi consejera.
A gradezco a mi nueva familia, Silvia y Carlos Antonio, mi esperanza y nueva razón de
vida.
F inalmente gracias a ti, porque en todo momento me hiciste merecedor de tanta dicha y
estuviste presente en los momentos más difíciles... Gracias Dios!
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AC: Acrosoma del espermatozoide.
AMFE: Agua de mar microfiltrada a 0,22 #m y esterilizada en un sistema de luz ultravioleta.
C: Muestra control. Espermatozoides sin tratamiento alguno.
CA: Cabeza del espermatozoide.
FL: Flagelo del espermatozoide.
IL: Espermatozoides ilesos.
MEB: Microscopía electrónica de barrido.
ME2SO: Dimetil sulfóxido. Compuesto utilizado como crioprotector penetrante.
PM: Pieza media del espermatozoide, el cual está compuesto por las mitocondrias.
POST: Muestra incubada en la solución crioprotectora, congelada y descongelada. También
llamada muestra POST-congelamiento.
PRE: Muestra incubada en la solución crioprotectora pero no congelada. También llamada
muestra PRE-congelamiento.
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El presente trabajo de tesis tiene como objetivo identificar y cuantificar las diferentes
alteraciones morfológicas de espermatozoides de Argopecten purpuratus, ocasionadas por el
proceso de criopreservación, y relacionarlas con su motilidad y capacidad de fecundación
post-descongelamiento.
Se utilizaron conchas de abanico maduras obtenidas de cultivos suspendidos de la Bahía la
Herradura de Guayacán, Coquimbo-Chile. Se disectó la gónada y la porción masculina se cortó
en trozos para liberar los espermatozoides en AMFE. Estos, fueron incubados en una solución
crioprotectora compuesta por ME2SO al 10%, 125 mM de sacarosa y 10% de vitelo del huevo de
gallina. Se congeló los espermatozoides a una tasa de 8,8 ºC/min. El descongelamiento se realizó
24 horas después mediante inmersión en un recipiente con agua a 50º C durante 20 seg y luego
en otro a temperatura ambiente. Posteriormente se cuantificó el número de espermatozoides
móviles en un microscopio de luz; también se cuantificó la cantidad de espermatozoides
lesionados en la cabeza, acrosoma, pieza media y flagelo en un microscopio electrónico de
barrido. Finalmente, se determinó la capacidad fecundante de los espermatozoides inseminando
ovocitos frescos.
La motilidad varió significativamente entre los tratamientos (C, PRE y POST) en cuanto a
porcentaje de espermatozoides móviles y tiempo de actividad.
Se observó distintas alteraciones en la morfología del espermatozoide
congelado-descongelado. Algunas tuvieron la cabeza deforme, hinchada o lisada y la membrana
celular plegada o rota. También fue observada, reacción acrosómica y mitocondrias fuera de su
posición normal o ausentes. Pero las alteraciones más comunes se encontraron en el flagelo, el
cual sufrió ruptura, rigidez y pérdida de su estructura lineal.
En C y PRE el porcentaje de espermatozoides ilesos fue mayor (87,7 ± 3,4 % y 79,0 ± 3,0 %
respectivamente), mientras que en POST fue 14,2 ± 2,8 %. La estructura lesionada en la mayor
cantidad de espermatozoides POST, fue el flagelo (77,0 ± 3,0 %); el porcentaje de
espermatozoides con lesión en la cabeza fue 55,1 ± 7,4 % y los que tuvieron el acrosoma
reaccionado fueron 28,7 ± 3,3 %. La pieza media fue afectada en el 23,9 ± 4,1 % de
espermatozoides.
Los porcentajes de fecundación fueron 68,3 ± 6,6 %, 67,9 ± 4,2 % y 58,2 ± 7,3 % para C,
PRE y POST respectivamente, no encontrándose diferencias significativas entre ellos.
Se correlacionó la motilidad total cuantificada a los 5 y a los 30 minutos, las lesiones
ocurridas en las diferentes estructuras espermáticas y los porcentajes de fecundación;
encontrándose mayor correlación entre las alteraciones y la motilidad que entre las alteraciones y
los porcentajes de fecundación. La correlación entre la motilidad y fecundación fue baja (0,650 y
0,668 con la motilidad a los 5 y 30 min respectivamente).
La incubación durante el tiempo de equilibrio en la solución crioprotectora utilizada, no
tiene un efecto tóxico que altere la motilidad o la morfología de los espermatozoides, sino más
bien, tiene un efecto activador de la motilidad. Esta propiedad de la solución crioprotectora, se
suma a la de protección, que les daría a los espermatozoides durante el
congelamiento-descongelamiento, que es la etapa del proceso de criopreservación durante la cual
los espermatozoide son afectado negativamente..
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Las cabezas destrozadas de los espermatozoides sería consecuencia de la lisis celular o del
crecimiento de cristales de hielo, todo esto como consecuencia de desórdenes osmóticos. La
rigidez de los flagelos podría ser por alteraciones en la estructura del axonema. Posiblemente, la
reacción acrosomal sería ocasionada por los cambios de polaridad ocurridos por el
congelamiento-descongelamiento. En general, las lesiones en cualquiera de las estructuras
espermáticas están relacionadas finalmente con la membrana celular.
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The present work identifies and quantifies the morphological external alterations of spermatozoa
of the scallop A. purpuratus due to long-term cryopreservation. Moreover, it relates the injuries
to motility and fertilization capacity.
Sexually mature scallops were collected from scallops farmed in La Herradura Bay,
Coquimbo - Chile. The male portion of the gonad was cut in pieces (5 mm), which were after
placed in a petri dish containing filtered sea water. Liberation of sperm was induced, which made
it possible to obtain a spermatic solution.
The sperm was equilibrated for 5 min in a crioprotective solution containing 10% ME2SO,
125 mM sucrose and 10% yolk of hen eggs diluted in sea water. Subsequently, the amples were
frozen at a rate of 8,8 ºC/min. After 24 hours, the samples were firstly thawed while immersed
for 20 seconds in water of 50º C, finally they were thawed while immersed in water at room
temperature. The percentage of motility, fertilization of fresh oocytes and injured spermatozoa
(in head, acrosome, middle piece and tail) were quantified with a light microscopy, stereoscopy
and scanned electron microscopy, respectively.
Both the freeze-thawing treatment and the post dilution time had significant effects on the
spermatic motility. Moreover, it was observed a significant interaction between the post-dilution
time and the freeze-thaw treatment (C, PRE o POST).
Spermatozoa exposed to the pre-freezing treatment (PRE) remained motile for a longer
while than the ones exposed to the control treatment (C). The motility in the spermatozoa in the
post-thaw treatment was always much lower than in the PRE and C treatments.
The morphology of the spermatozoids was affected in several ways by the freeze-thawing
treatment. Some had their head deformed or swollen, others had their cell membrane folded or
broken. Acrosome reaction, anomalous positions or absence of mitochondria were also observed.
Broken or stiff or lineal structure loosed of tail were the most frequent injuries.
The C and PRE had higher percentage of unhurt sperm (87,7 ± 3,4 % and 79,0 ± 3,0 %
respectively), while the PRE samples had 14,2 ± 2,8 % of unhurt sperm. The tail was the
spermatic structure that most commonly injured during the freezing-thawing process (77,0 ± 3,0
%). The percentage of sperm with head injury was 55,1 ± 7,4 % and with acrosome reaction was
28,7 ± 3,3 %. The middle piece was affected in 23,9 ± 4,1 % of the spermatozoa.
The percentage of fertilization was 68,3 ± 6,6 %, 67,9 ± 4,2 % and 58,2 ± 7,3 % for C, PRE
and POST respectively, which were not significantly different.
The injuries were correlated with motility (at 5 and 30 min) and with fertilization success.
There was a better correlation between injuries and motility than between injuries and
fertilization success. The correlation between motility and fertilization was low (0,605 and 0,668
with motility at 5 min and 30 min respectively).
The crioprotective solution did not have any toxic effects on the spermatozoa, on the
contrary, it rather had an activating effect on motility. Moreover, the crioprotective solution
provided protection during the freezing thawing process, which is a critical process in
cryopreservation in which spermatozoa suffer of decreased motility. The crioprotective solution
did not have a significant effect on the external morphology of the spermatozoa. Hence, again
the freezing thawing process is causing the injuries.
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The heads destroyed of the spermatozoa would be consequence of the accumulation of ice
crystals. The tails could be rigid due to alterations in the axonomic structure. Possibly the
acrosome reaction could be by the bipolarity changes due to the process of freezing-thawing. As
a rule, the injury in any structure of the spermatozoa is related to the cell membrane.
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Alberts, B.; D. Bray; J. Lewis; M. Raff; K. Roberts y J. Watson. 1994. Biología Molecular
de La Célula. Segunda edición., Barcelona, Ediciones Omega, 1300 pp.
Alberts, B.; D. Bray; J. Lewis; M. Raff; K. Roberts y J.D. Watson. 2000. Biología
molecular de la Célula. Tercera edición, Ediciones Omega, 1387 pp.
Alberts, B.; D. Bray; J. Lewis; M. Raff; K. Roberts y J.D. Watson. 2002. Biología
molecular de la Célula. Cuarta edición, Ediciones Omega, 1387 pp.
Alderson, R. y A.J. Macneil. 1984. Preliminary investigation of cryopreservation of milt
of Atlantic Salmon (Salmo salar) and its application to comercial farming.
Aquaculture, 43:351-354.
Anchordoguy, T. ; A. Rudolph ; J. Carpenter y J. Crowe. 1987. Modes of interaction of
cryoprotectants with membrane phospholipids during freezing. Cryobiology, 24:
324-331.
Babiak, I.; J. Glogowski; R. Kujawa; D. Kucharczk y A. Mamcarz. 1998.
Cryopreservation of sperm from asp Aspius aspius. The Progressive Fish-Culturist
60:146-148.
Bayyari, G. R.; J. R. Cook; G. C. Harris Jr; L. B. Macy; M. F. Slavik y J. K. Skeeles.
1990. Evaluation of chicken spermatozoa using Fluorescent Staining in a 96-well
Format. Poultry Science, 69:1602-1605.
Billard, R. 1983. Ultraestructure of trout spermatozoa: Changes after dilution and
0'% 12 )3 4 $5 ! " 0
8>
! "#$ "%$ $ "
"="== &'( )*)+, -. $ /0
deep-freezing. Cell Tissue Res., 228:205-218.
Bougrier, S. y L.D. Rabenomanana. 1986. Cryopreservation of spermatozoa of the
Japanese Oyster, Crassostrea gigas. Aquaculture, 58:277-280.
Bury, N. y P. Olive. 1993. Ultrastructural observation on membrane changes associated
with cryopreserved spermatozoa of two polychaete species and subsequent mobility
induced by quinacine. Invertebrate Reproduction and Development, 23(2-3):139-150.
Conget, P.; M. Fernandez; G. Herrera y J. Minguell. 1996. Cryopreservation of rainbow
trout (Oncorhynchus mykiss) spermatozoa using programable freezing. Aquaculture,
143:319-329.
Curry, M. y P. Watson. 1992. The role of osmotic stress in sperm cryosurvival. In: Low
temperature and Aquatic Organism preservation of lower plants. Society for low
temperature biology. K.J. Rana (Host). Stirling University 7-9, Sept. 1992.
Chambeyron, F. y Y. Zohar. 1990. A diluent for sperm cryopreservation of gilthead
seabream, Sparus aurata. Aquaculture, 90:345-352.
Cheryl, A.; D. Garner; J. Mel Dejarnette y C. Marshall. 1997. Fluorometric Assessments
of Acrosomal Integrity and Viability in cryopreserved Bovine Spermatozoa. Biology of
Reproduction 56:991-998.
Dupré, E.; C. Arqueros; J. Barraza y J.C. Maureira. 1999. Toxicidad de diferentes
crioprotectantes sobre espermatozoides y larvas de ostra Crassostrea gigas y del
ostión Argopecten purpuratus. Informe Final: Proyecto Dirección General de
Investigación y Cooperación Técnica (DGI-98), 1999.
Dupré, E.; M. Romero y M. Alhambra. 1997. Cryopreservation of spermatozoa and larva
of the scallop Argopecten purpuratus and the oyster Crassostrea gigas. In:
International Workshop: Cryopreservation, Reproduction and Genome Conservation,
12-14 Nov 1997, Reñaca-Chile; pp. 23-24.
Dupré, E. y C. Espinoza. 2000. Determinación de porcentajes de fecundación del ostión
del norte Argopecten purpuratus utilizando espermatozoides criopreservados.
Informe Final Proyecto DGI-1999 UCN.
Dupré, E. y C. Espinoza. 2003 Congelamiento de espermatozoides del ostión del norte
Argopecten purpuratus mediante un congelador mecánico. Investigaciones Marinas.
(en prensa).
Eastman, J. y A. De Vries. 1987. Peces antárticos. Investigación y Ciencia 124, 60-68.
Ericsson, S.; D. Garner; C. Thomas; T. Downing y C. Marshall. 1993. Interrelationships
among fluorometric analyses of spermatozoal function, classical semen quality
parameters and the fertility of frozen-thawed bovine spermatozoa. Theriogenology,
39:1009-1024.
Fernández, M., G. Font de Valdez y A. Disalvo. 2000. Changes in the surface potencial
of Lactobacillus acidophilus under freeze-thawing stress. Cryobiology41, 10-16.
Garner, D.; L. Johnson y H. Allen. 1988. Fluorometric evaluation of cryopreserved
bovine spermatozoa extended in egg yolk and milk. Theriogenology, 30(2):369-378.
Glogowski, J.; A. Ciereszko y K. Dabrowski . 1999. Cryopreservation of muskellunge
and yellow perch semen. North American Journal of Aquaculture 61:258-262.
Glogowski, J.; L. Babiak; K. Goryczko; S. Dobosz y H. Kuzminski. 1997. Properties and
9?
0'% 12 )3 4 $5 ! " 0
B
::8FB
cryopreservation of danube salmon (Hucho hucho) milt. Arc. Ryb. Pol. Arch. Pol.
Fish, 5(2):235-239.
Harvey, B. 1983. Cryopreservation of Sarotherodon mossambicus spermatozoa.
Aquaculture, 32:313-320.
Huges, J. 1973. An examination of eggs challenged with criopreserved spermatozoa of
the American Oyster, Crassostrea virginica. Cryobiology, 10:342-344.
Kurokura, H.; K. Namba y T. Ishikawa. 1990. Lesions of Spermatozoa by
Cryopreservation in Oyster Crassostrea gigas. Nippon Suisan Gakkaishi,
56(11):1803-1806.
Lahnsteiner, F.; T. Weismann y R. Ptzner. 1996. Cryopreservation of semen of the
grayling (Thymallus thymallus) and the Danube salmon (Hucho hucho). Aquaculture,
144:265-274.
Leibo, S. y P. Mazur. 1971. The role of cooling rates in low-temperature preservation.
Cryobiology, 8:447-452.
Leung, L. 1987. Cryopreservation of spermatozoa of the Barrumundi, Lates calcarifer
(Teleostei: Centropomidae). Aquaculture, 64:243-247.
Liu, X. y A. Robinson. 1996. Impact of cryoprotectants ME2SO, ethylene glicol,
methanol, glycerol, sucrose and polyvinylpyrrolidone on oyster (Crassostrea gigas)
eggs before freezing. Journal of Shellfish Research, 15(3):787.
Longo, F. 1987. Fertilization. Editorial Chapman and Hall, London, 183 pp.
Mazur, P. 1977. The role of intracellular Freezing in the death of cells cooled at
supraoptimal rates. Cryobiology, 14, 251-272.
Mazur, P. 1984. Freezing of living cells: Mechanisms and implications. Am. J. Physiol.
247 (Cell Physiol. 16): C125-C142.
Mazur, P. 1996. Principles of Medical Cryobiology: The Freezing of Living Cells,
Tissues, and Organs. In: Principles of Medical Biology, Vol 4, Cell Chemistry and
Physiology: Part IV, Chapter 10, pp 355-384.
Mazur P. y C. Koshimoto. 2002. Is intracellular ice formation the cause of death of
mouse sperm frozen at high cooling rates. Biology of Reproduction 66, 1485-1490.
Mazur, P.; S. Leibo y E. Chu. 1972. A two-factor hypothesis of freezing injury.
Experimental Cell Research, 71:345-355.
Mc Williams, R.; W. Gibbons y S. .Leibo. 1995. Osmotic and physiological responses of
mouse zygotes and human oocytes to mono- and di-saccharides. Human
ReproductiON, 10(5):1163-1171.
Mediavilla, M.; R. Álvarez y P. Herráez. 1995 . Estudio de la viabilidad espermática en
semen congelado de trucha común (Salmo trutta). Proc. V National Congress on
Aquaculture, pp392-397.
Meryman, H. 1971. Cryoprotective agents. Cryobiology, 8:173-183.
Morisawa, M. y Suzuki, K. 1980. Osmolality and potassium ion: their roles in initiation of
sperm motility in teleosts. Science, Vol. 210, 5:1145-1146.
Murali, C.; R. Yanagimachi y G. Cherr. 1994. In vivo and In Vitro Initiation of Sperm
Motility Using Fresh and Cryopreserved Gametes from the Pacific Herring, Clupea
0'% 12 )3 4 $5 ! " 0
96
! "#$ "%$ $ "
"="== &'( )*)+, -. $ /0
pallasi. The Journal of Experimental Zoology, 269:62-68
Ohta, H.; K. Ikeda y T. Izawa. 1997. Increases in concentrations of potassium and
bicarbonate ions promote acquisition of motility in vitro by japanese eel spermatozoa.
The Journal of Experimental Zoology, 277:171-180.
Oteme, Z.; J. Nunez; C. Kouassi; S. Hem y J. Agnese. 1996. Testicular structure,
spermatogenesis and sperm cryopreservation in the african clariid catfish
Heterobranchus longifilis (Valenciennes, 1840). Aquacult. Res. 27(11):805-813.
Piironen, J. 1993. Cryopreservation of sperm from brown trout (Salmo trutta m. lacustris
L.) and Artic charr (Salvelinus alpinus L.). Aquaculture, 116:275-285.
Rana, K. 1995. Preservation of gametes. In: Bromage, N.R., Roberts, R.J. (Eds.),
Broodstock Management and egg and larval quality. Blackwell, Oxford, 53-75.
Richardson, G.; C. Wilson; L. Crim y Z. Yao. 1999. Cryopreservation of yellowtail
flounder (Pleuronectes ferrugineus) semen in large straws. Aquaculture, 174:89-94.
Ritar, A. 1999. Artificial insemination with cryopreserved semen from striped trumpeter
(Latris lineata). Aquaculture, 180:177-187.
Scheerer, P. y H. Thorgaard. 1989. Androgenetic rainbow trout produced from inbred
and outbred sperm sources show similar survival. Aquaculture, 57:289-298.
Storey, K. y J. Storey. 1991. Congelados y vivos. Investigación y Ciencia, 173:58-64.
Sztein, J.; K. Nobel; J. Farley y L. Mobraaten. 2001. Comparison of permeating and
nonpermeating cryoprotectants for mouse sperm cryopreservation. Cryobiology,
41:28-39.
Taylor, R.; G. Adams; C. Boardman y R. Wallis. 1974. Cryoprotection-permeant vs
Nonpermeant Additives. Cryobiology, 11:430-438.
Terner, C. 1986. Evaluation of salmonid sperm motility for cryopreservation. The
Progresive Fish-Culturist, 48:230-232.
Usuki, H.; H. Masami y I. Hiroko. 1997. Long-term cryopreservation of Pacific oyster,
Crassostrea gigas, sperm. Bull. Nansei Natl. Fish. Res. Inst. Nanseisuikenho,
30:115-123.
Valdebenito, I. y M. Ratto. 1996. Efecto de la cafeína en la motilidad y fertilidad del
espermatozoide de trucha arcoiris (Oncorhynchus mykiss). Acuicultura en
Latinoamérica. IX Congreso Latinoamericano de Acuicultura. 2º Simposio Avances y
Perspectivas de la Acuacultura en Chile. pp. 370-373. UCN Coquimbo-Chile.
Wen, D. y R. Laursen. 1992. A model for binding of an antifreeze polypeptide to ice.
Biophysical Journal, 63:1659-1662.
Wheeler, P. y G. Thorgaard. 1991. Cryopreservation of rainbow trout semen in large
straws. Aquaculture, 93:95-100.
Yankson, K. y J. Moyse. 1991. Cryopreservation of the spermatozoa of Crassostrea
tulipa and three other oysters. Aquaculture, 97:259-267.
Zachariassen, K. y E. Kristiansen. 2000. Ice nucleation and Antinucleation in Nature.
Cryobiology 41, 257-279.
Zar, H. 1999. Biostatistical Analysis. 4º ed., Edit. Prentice Hall, New Jersey, 663 pp.
Zell, S.; M. Bamford y H. Hidu. 1979. cryopreservation of spermatozoa of the American
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