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The autophagy-related protein LC3 is processed in stallion spermatozoa during short-and long-term storage and the related stressful conditions

Published online by Cambridge University Press:  02 March 2016

I. M. Aparicio ,
P. Martin Muñoz ,
G. M. Salido ,
F. J. Peña  and
J. A. Tapia
I. M. Aparicio*
Affiliation:
Cell Physiology Research Group, Department of Physiology, University of Extremadura, Caceres 10003, Spain
P. Martin Muñoz
Affiliation:
Veterinary Teaching Hospital, Laboratory of Spermatology, University of Extremadura, Caceres 10003, Spain
G. M. Salido
Affiliation:
Cell Physiology Research Group, Department of Physiology, University of Extremadura, Caceres 10003, Spain
F. J. Peña
Affiliation:
Veterinary Teaching Hospital, Laboratory of Spermatology, University of Extremadura, Caceres 10003, Spain
J. A. Tapia
Affiliation:
Cell Physiology Research Group, Department of Physiology, University of Extremadura, Caceres 10003, Spain
*
†E-mail: imad@unex.es
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Abstract

Use of cooled and frozen semen is becoming increasingly prevalent in the equine industry. However, these procedures cause harmful effects in the sperm cell resulting in reduced cell lifespan and fertility rates. Apoptosis and necrosis-related events are increased during semen cryopreservation. However, a third type of cell death, named autophagy, has not been studied during equine semen storage. Light chain (LC)3 protein is a key component of the autophagy pathway. Under autophagy activation, LC3-I is lipidated and converted to LC3-II. The ratio of LC3-II/LC3-I is widely used as a marker of autophagy activation. The main objective of this study was to investigate whether LC3 is processed during cooling, freezing and the stressful conditions associated with these technologies. A secondary objective was to determine if LC3 processing can be modulated and if that may improve the quality of cryopreserved semen. LC3 processing was studied by Western blot with a specific antibody that recognized both LC3-I and LC3-II. Viability was assessed by flow cytometry. Modulation of LC3-I to LC3-II was studied with known autophagy activators (STF-62247 and rapamycin) or inhibitors (chloroquine and 3-MA) used in somatic cells. The results showed that conversion of LC3-I to LC3-II increased significantly during cooling at 4°C, freezing/thawing and each of the stressful conditions tested (UV radiation, oxidative stress, osmotic stress and changes in temperature). STF-62247 and rapamycin increased the LC3-II/LC3-I ratio and decreased the viability of equine sperm, whereas chloroquine and 3-MA inhibited LC3 processing and maintained the percentage of viable cells after 2 h of incubation at 37°C. Finally, refrigeration at 4°C for 96 h and freezing at −196°C in the presence of chloroquine and 3-MA resulted in higher percentages of viable cells. In conclusion, results showed that an ‘autophagy-like’ mechanism may be involved in the regulation of sperm viability during equine semen cryopreservation. Modulation of autophagy during these reproductive technologies may result in an improvement of semen quality and therefore in higher fertility rates.

Keywords

cryopreservationrefrigerationviabilityautophagysperm
Type
Research Article
Information
animal , Volume 10 , Issue 7 , July 2016 , pp. 1182 - 1191
Copyright
© The Animal Consortium 2016 

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References

References (Since 2010)

Anbalagan, S, Pires, IM, Blick, C, Hill, MA, Ferguson, DJ, Chan, DA and Hammond, EM 2012. Radiosensitization of renal cell carcinoma in vitro through the induction of autophagy. Radiotherapy and Oncology 103, 388–393.Google Scholar
Bolanos, JM, Moran, AM, da Silva, CM, Davila, MP, Munoz, PM, Aparicio, IM, Tapia, JA, Ferrusola, CO and Pena, FJ 2014. During cooled storage the extender influences processed autophagy marker light chain 3 (LC3B) of stallion spermatozoa. Animal Reproduction Science 145, 40–46.Google Scholar
Burnaugh, L, Ball, BA, Sabeur, K, Thomas, AD and Meyers, SA 2010. Osmotic stress stimulates generation of superoxide anion by spermatozoa in horses. Animal Reproduction Science 117, 249–260.Google Scholar
Caselles, AB, Miro-Moran, A, Morillo Rodriguez, A, Gallardo Bolanos, JM, Ortega-Ferrusola, C, Salido, GM, Pena, FJ, Tapia, JA and Aparicio, IM 2014. Identification of apoptotic bodies in equine semen. Reproduction in Domestic Animals 49, 254–262.Google Scholar
Cordova, A, Strobel, P, Vallejo, A, Valenzuela, P, Ulloa, O, Burgos, RA, Menarim, B, Rodriguez-Gil, JE, Ratto, M and Ramirez-Reveco, A 2014. Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: presence and sensitivity of 5’ AMP-activated protein kinase (AMPK). Cryobiology 69, 473–481.CrossRefGoogle ScholarPubMed
Freitas-Dell’Aqua, CdP, Monteiro, GA, Júnior, JADA and Papa, FO 2012. The effects of refrigeration temperature and storage time on apoptotic markers in equine semen. Journal of Equine Veterinary Science 33, 27–30.Google Scholar
Gallardo Bolanos, JM, Miro Moran, A, Balao da Silva, CM, Morillo Rodriguez, A, Plaza Davila, M, Aparicio, IM, Tapia, JA, Ortega Ferrusola, C and Pena, FJ 2012. Autophagy and apoptosis have a role in the survival or death of stallion spermatozoa during conservation in refrigeration. PLoS One 7, e30688.Google Scholar
Jung, CH, Ro, SH, Cao, J, Otto, NM and Kim, DH 2010. mTOR regulation of autophagy. FEBS Letters 584, 1287–1295.Google Scholar
Karimfar, MH, Niazvand, F, Haghani, K, Ghafourian, S, Shirazi, R and Bakhtiyari, S 2015. The protective effects of melatonin against cryopreservation-induced oxidative stress in human sperm. International Journal of Immunopathology and Pharmacology 28, 69–76.Google Scholar
Kraft, C and Martens, S 2012. Mechanisms and regulation of autophagosome formation. Current Opinion in Cell Biology 24, 496–501.Google Scholar
Kroemer, G, Marino, G and Levine, B 2010. Autophagy and the integrated stress response. Molecular Cell 40, 280–293.Google Scholar
Lagares, MA, Martins, HS, Carvalho, IA, Oliveira, CA Jr, Souza, MR, Penna, CF, Cruz, BC, Stahlberg, R and Henry, MR 2012. Caseinate protects stallion sperm during semen cooling and freezing. CryoLetters 33, 214–219.Google Scholar
Li, M, Hou, Y, Wang, J, Chen, X, Shao, ZM and Yin, XM 2011. Kinetics comparisons of mammalian Atg4 homologues indicate selective preferences toward diverse Atg8 substrates. The Journal of Biological Chemistry 286, 7327–7338.Google Scholar
Long, L, Yang, X, Southwood, M, Lu, J, Marciniak, SJ, Dunmore, BJ and Morrell, NW 2013. Chloroquine prevents progression of experimental pulmonary hypertension via inhibition of autophagy and lysosomal bone morphogenetic protein type II receptor degradation. Circulation Research 112, 1159–1170.Google Scholar
Morillo-Rodriguez, A, Macias-Garcia, B, Tapia, JA, Ortega-Ferrusola, C and Pena, FJ 2012. Consequences of butylated hydroxytoluene in the freezing extender on post-thaw characteristics of stallion spermatozoa in vitro . Andrologia 44 (suppl. 1), 688–695.Google Scholar
Nikoletopoulou, V, Markaki, M, Palikaras, K and Tavernarakis, N 2013. Crosstalk between apoptosis, necrosis and autophagy. Biochimica et Biophysica Acta 1833, 3448–3459.Google Scholar
O’Farrell, F, Rusten, TE and Stenmark, H 2013. Phosphoinositide 3-kinases as accelerators and brakes of autophagy. FEBS Journal 280, 6322–6337.Google Scholar
Ortega Ferrusola, C, Gonzalez Fernandez, L, Salazar Sandoval, C, Macias Garcia, B, Rodriguez Martinez, H, Tapia, JA and Pena, FJ 2010. Inhibition of the mitochondrial permeability transition pore reduces ‘apoptosis like’ changes during cryopreservation of stallion spermatozoa. Theriogenology 74, 458–465.Google Scholar
Pena, FJ, Garcia, BM, Samper, JC, Aparicio, IM, Tapia, JA and Ferrusola, CO 2011. Dissecting the molecular damage to stallion spermatozoa: the way to improve current cryopreservation protocols? Theriogenology 76, 1177–1186.CrossRefGoogle ScholarPubMed
Rodriguez, AM, Ferrusola, CO, Garcia, BM, Morrell, JM, Martinez, HR, Tapia, JA and Pena, FJ 2011. Freezing stallion semen with the new Caceres extender improves post thaw sperm quality and diminishes stallion-to-stallion variability. Animal Reproduction Science 127, 78–83.CrossRefGoogle ScholarPubMed
Rovegno, M, Feitosa, WB, Rocha, AM, Mendes, CM, Visintin, JA and D’Avila Assumpcao, ME 2013. Assessment of post-thawed ram sperm viability after incubation with seminal plasma. Cell and Tissue Banking 14, 333–339.CrossRefGoogle ScholarPubMed
Said, TM, Gaglani, A and Agarwal, A 2010. Implication of apoptosis in sperm cryoinjury. Reproductive Biomedicine Online 21, 456–462.Google Scholar
Shang, L and Wang, X 2011. AMPK and mTOR coordinate the regulation of Ulk1 and mammalian autophagy initiation. Autophagy 7, 924–926.CrossRefGoogle ScholarPubMed
Zeng, C, Tang, K, He, L, Peng, W, Ding, L, Fang, D and Zhang, Y 2014. Effects of glycerol on apoptotic signaling pathways during boar spermatozoa cryopreservation. Cryobiology 68, 395–404.Google Scholar
Zhang, M, Jiang, M, Bi, Y, Zhu, H, Zhou, Z and Sha, J 2012. Autophagy and apoptosis act as partners to induce germ cell death after heat stress in mice. PLoS One 7, e41412.Google Scholar
Zhou, H, Luo, Y and Huang, S 2010. Updates of mTOR inhibitors. Anti-Cancer Agents in Medicinal Chemistry 10, 571–581.CrossRefGoogle ScholarPubMed
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