UNIVERSIDAD COMPLUTENSE DE MADRID

UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE VETERINARIA DEPARTAMENTO DE FISIOLOGÍA (FISIOLOGÍA ANIMAL) TESIS DOCTORAL SEX RATIO CONTROL BEFORE FERTILIZATION AND PREIMPLANTATIONAL SEXUAL DIMORPHISM IN BOVINE CONTROL DE LA PROPORCIÓN DE SEXOS ANTERIOR A LA FECUNDACIÓN Y DIMORFISMO SEXUAL PREIMPLANTACIONAL EN LA ESPECIE BOVINA TESIS DOCTORAL MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Pablo Bermejo Álvarez Directores: Alfonso Gutiérrez Adán, Pedro Luis Lorenzo González, Dimitrios Rizos Madrid, 2010 ISBN: 978-84-693-8306-3 Pablo Bermejo Álvarez, 2010

SEX RATIO CONTROL BEFORE FERTILIZATION AND PREIMPLANTATIONAL SEXUAL DIMORPHISM IN BOVINE CONTROL DE LA PROPORCIÓN DE SEXOS ANTERIOR A LA FECUNDACIÓN Y DIMORFISMO SEXUAL PREIMPLANTACIONAL EN LA ESPECIE BOVINA Pablo Bermejo Álvarez Directors/Directores: Alfonso Gutiérrez Adán Pedro Luis Lorenzo González Dimitrios Rizos

Los doctores Alfonso Gutiérrez Adán y Dimitrios Rizos, Investigadores Titulares del Departamento de Reproducción Animal y Conservación de Recursos Zoogenéticos del Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), y el doctor Pedro Luis Lorenzo González, Proferor Titular del Departamento de Fisiología (Fisiología Animal) de la Facultad de Veterinaria de la Universidad Complutense de Madrid, hacen constar: Que la memoria presentada por el Licenciado en Veterinaria Pablo Bermejo Álvarez, con el título: Sex ratio control before fertilization and preimplantational sexual dimorphism in bovine/control de la proporción de sexos anterior a la fecundación y dimorfismo sexual preimplantacional en la especie bovina, ha sido realizada bajo nuestra dirección y que tras su revisión consideramos que tiene la debida calidad para su presentación y defensa. Madrid, Febrero de 2010. Fdo: D. Alfonso Gutiérrez Adán Fdo: D. Pedro Luis Lorenzo González Fdo: D. Dimitrios Rizos

EUROPEAN DOCTORATE MENTION This thesis has been proposed for the European doctorate mention by virtue of the following European research stays and thesis reports: Research stays: Lyons Research Farm, University College Dublin (Ireland). Dr. Pat Lonergan laboratory. 5 months in three stays during 2007-2008. School of Biosciences, University of Nottingham (United Kingdom). Dr. Keith Campbell laboratory. 4 months in 2009. Thesis reports ( very good grading): Isabelle Donnay, Unité des Sciences vétérinaires, Institut des Sciences de la Vie, Université Catholique de Louvain, Belgium. Giovanna Lazzari. AVANTEA, Laboratorio di Tecnologie della Riproduzione, Cremona, Italy. Pilar Coy Fuster. Facultad de Veterinaria, Universidad de Murcia, Spain. Rosa Ana Picazo González. Facultad de Veterinaria, Universidad Complutense de Madrid, Spain.

Changes in testosterone or temperature during the in vitro oocyte culture do not alter the sex ratio of bovine embryos Díez C, Bermejo-Álvarez P, Trigal B, Caamaño JN, Muñoz M, Molina I, Gutiérrez-Adán A, Carrocera S, Martín D, Gómez E. Journal of Experimental Zoology. Part A, Ecological Genetics and Physiology; 311(6):448-452. 2009. Biological differences between in vitro produced bovine embryos and parthenotes Gómez E, Gutiérrez-Adán A, Díez C, Bermejo-Álvarez P, Muñoz M, Rodríguez A, Otero J, Álvarez-Viejo M, Carrocera S, Martín D, Caamaño J. Reproduction; 137(2):285-295. 2009. Consequences of in vitro culture conditions on embryo development and quality Rizos D, Clemente M, Bermejo-Alvarez P, de La Fuente J, Lonergan P, Gutiérrez-Adán A. Reproduction of Domestic Animals; 43(S4):44-50. 2008. Effect of duration of oocyte maturation on the kinetics of cleavage, embryo yield and sex ratio in cattle Rizos D, Bermejo-Álvarez P, Gutiérrez-Adán A, Lonergan P. Reproduction, Fertility and Development; 20(6):734-740. 2008. Can bovine in vitro-matured oocytes selectively process X- or Y-sorted sperm differentially? Bermejo-Álvarez P, Rizos D, Rath D, Lonergan P, Gutiérrez-Adán A. Biology of Reproduction; 79(4):594-597. 2008 Long-term effects of mouse intracytoplasmic sperm injection with DNAfragmented sperm on health and behavior of adult offspring Fernández- González R, Moreira P, Pérez-Crespo M, Sánchez-Martín M, Ramírez MA, Pericuesta E, Bilbao A, Bermejo-Alvarez P, Hourcade JD, Rodríguez de Fonseca F, Gutiérrez-Adán A. Biology of Reproduction; 78(4):761-772. 2008. Epigenetic differences between male and female blastocyst produced in vitro Bermejo-Álvarez P, Rizos D, Rath D, Lonergan P, Gutiérrez-Adán A. Physiological Genomics; 32(2):264-272. 2008. Other publications Telomere lengthening from oocyte to embryonic stem cell Chapter of the book Telomeres: Function, Shortening and Lenghtening Ed. Nova Publishers. 2009. Telomere lengthening during preimplantation development Pericuesta E, Ramírez MA, Fernández-González R, Pérez-Crespo M, Hourcade JD, Bermejo P, Rizos D, Gutiérrez-Adán A. Association Europeenne de transfert embryonnaire A.E.T.E. Newsletter; 29:10-14. 2007. Research stays Kleberg Center, University of Texas A&M (USA). Dr. Fuller Bazer laboratory. 2 months, July-September 2005. Research focussed on pregnancy signalling in ovine. Lyons Research Farm, University College Dublin (Ireland). Dr. Pat Lonergan laboratory. 4 months, August-December 2007, plus 1 month in March and December 2008. Research focussed on bovine in vitro and in vivo embryo production. 174

Center for Regenerative Biology, University of Connecticut (USA). Dr. Cindy Tian laboratory. 4 months, June-October 2008. Research focussed on SCNT in mouse and bovine. School of Biosciences, University of Nottingham (United Kingdom). Dr. Keith Campbell laboratory. 4 months, July-October 2009. Research focussed on SCNT in ovine. Oral communications in international scientific meetings Identification of five genes expressed preferentially from the paternal X chromosome Bermejo-Alvarez P, Rizos D, Lonergan P, Gutiérrez-Adán. 36 th Annual Conference of the International Embryo Transfer Society (IETS). Córdoba, Argentina, USA. January 2010. Micro array analysis reveals that one third of the genes actively expressed are differentially expressed between male and female bovine blastocysts. Bermejo-Álvarez P, Rizos D, Rath D, Lonergan P, Gutiérrez-Adán A. 42 nd Annual Meeting of the Society for the Study of Reproduction (SSR). Pittsburgh, USA. July 2009. Effect of oxygen tension during in vitro maturation and fertilization on bovine embryo production and cumulus-oocyte-complex mrna abundance. Bermejo-Álvarez, Rizos D, Lonergan P, Gutiérrez-Adán A. 35 th Annual Conference of the International Embryo Transfer Society (IETS). San Diego, USA. January 2009. Differences in methylation status between male and female bovine blastocysts produced in vitro Bermejo-Álvarez P, Rizos D, Rath D, Lonergan P, Gutiérrez-Adán A. 23 rd Scientific Meeting of the European Embryo Transfer Society (AETE). Alghero, Italy. September 2007. Poster presentations in international scientific meetings as first author mtdna copy number during bovine preimplantation development. Bermejo-Álvarez P, Rizos D, Lonergan P, Gutiérrez-Adán A. Cost Action Gemini Maternal Interactions with Gametes and Embryos. Alghero, Italy. October 2009. Bovine sex ratio is not altered following three different modifications in IVF protocol. Bermejo-Álvarez P, Rizos D, Rath D, Lonergan P, Gutiérrez-Adán A. 25 th Scientific Meeting of the European Embryo Transfer Society (AETE). Poznan, Poland. September 2009. Sex ratio in bovine preimplantation embryos produced either in vitro or in vivo. Bermejo-Álvarez P, Rizos D, Rath D, Lonergan P, Gutiérrez-Adán A. Cost Action Gemini Maternal Interactions with Gametes and Embryos. Lansk, Poland. May 2009. Effects of oxygen tension during in vitro maturation and fertilization in bovine embryo production Bermejo-Álvarez P, Rizos D, Lonergan P, Gutiérrez-Adán A 1 st International Congress of the Spanish Society for Animal Reproduction (AERA) and British Andrology Society (BAS). Gijón, Spain. October 2008. Developmental kinetics of male and female bovine embryos produced in vitro with sex-sorted sperm Bermejo-Álvarez P, Lonergan D, Rath A, 175

Gutiérrez-Adán A, Rizos D 24 th Scientific Meeting of the European Embryo Transfer Society (AETE). Pau, France. September 2008. Can bovine in vitro matured oocytes process differentially X- or Y-bearing spermatozoa? Bermejo-Álvarez P, Rizos D, Rath D, Lonergan P, Gutiérrez- Adán A. 41 st Annual Meeting of the Society for the Study of Reproduction (SSR). Lailua-Lona, USA. May 2008. Does duration of bovine oocyte maturation affect the speed of embryo development in in vitro and sex ratio at the two-cell or blastocyst stage? Bermejo-Álvarez P, Gutiérrez-Adán A, Lonergan P, Rizos D. 34 th Annual Conference of the International Embryo Transfer Society (IETS). Denver, USA. January 2008 Other contributions in international meetings Poster: Sex-dependent metabolic differences of bovine preimplantation embryos Sturmey RG, Bermejo-Álvarez P, Gutiérrez-Adán A, Rizos D, Leese HJ, Lonergan P. 36 th Annual Conference of the International Embryo Transfer Society (IETS). Córdoba, Argentina, USA. January 2010. Poster: Relative mrna abundance in ovine blastocysts produced in vivo or in vitro in different culture media. Sanna D, Bermejo-Álvarez P, Mara L, Rizos D, Gutiérrez-Adán A, Dattena M. 25 th Scientific Meeting of the European Embryo Transfer Society (AETE). Poznan, Poland. September 2009. Oral communication: Relationship between follicle size, intrafollicular testosterone concentration and sex ratio in individually cultured bovine embryos. García-Herreros M, Bermejo-Álvarez P, Rizos D, Gutiérrez-Adán A, Fahey AH, Lonergan P. 25 th Scientific Meeting of the European Embryo Transfer Society (AETE). Poznan, Poland. September 2009. Poster: Testosterone in the oocyte culture does not alter sex-ratio of in vitro produced bovine embryos. Díez C, Bermejo-Álvarez P, Gutiérrez-Adán A, Caamaño JN, Muñoz M, Carrocera S, Martín D, Gómez E. 35 th Annual Conference of the International Embryo Transfer Society (IETS). San Diego, USA. January 2009. Poster: Sex ratio in bovine embryos is not regulated by temperature incubation of oocytes Díez C, Bermejo-Álvarez P, Gutiérrez-Adán A, Caamaño JN, Muñoz M, Carrocera S, Martin D, Gómez E. 1 st International Congress of the Spanish Society for Animal Reproduction (AERA) and British Andrology Society (BAS). Gijón, Spain. October 2008. Poster Timing of blastocysts expansion in bovine IVF embryos and parthenotes affects expression of genes involved in pluripotency, methylation, apoptosis and pregnancy recognition Caamaño JN, Muñoz M, Gutiérrez- Adán A, Bermejo-Álvarez P, Díez C, Otero J, Álvarez-Viejo M, Carrocera S, Martin D, Gómez E. 1 st International Congress of the Spanish Society for Animal Reproduction (AERA) and British Andrology Society (BAS). Gijón, Spain. October 2008. Poster: Expression of genes involved in compactation, blastulation and metabolism in bovine IVF embryos and parthenotes depends on timing of blastocysts expansion Gómez E, Bermejo-Álvarez P, Caamaño, JN, Muñoz M, Díez C, Carrocera S, Martin D, Rodríguez A, Gutiérrez-Adán A 24 th 176

Scientific Meeting of the European Embryo Transfer Society (AETE). Pau, France. September 2008. Poster: Effect of exogenous leptin on in vitro development of bovine embryos Arias-Álvarez M, Bermejo-Álvarez P, Rizos D, Lorenzo PL, Gutiérrez-Adán A, Lonergan P 24 th Scientific Meeting of the European Embryo Transfer Society (AETE). Pau, France. September 2008. Oral Communication: Use of Axinfu mutation model to analyze preimplantation epigenetic alterations produced by in vitro culture in mice Fernández-González R, Pérez-Crespo M, Bermejo P, De Dios J, Pintado B, Gutiérrez-Adán A. 2 nd International Meeting of Mammalian Embryogenomics. Paris, France. October 2007. Oral communication: Effects of BSA and hyaluronan in in vitro culture of ovine embryos and their quality in terms of cryotolerance, gene expression, lambing rate and birth weight Sanna S, Rizos D, Mara L, Bermejo-Álvarez P, Gutiérrez-Adán A, Dattena M. 23 rd Scientific Meeting of the European Embryo Transfer Society (AETE). Alghero, Italy. September 2007. Oral communication: Inheritance of epigenetic alterations produced by in vitro culture in mice Fernández-González R, Ramirez MA, Pérez-Crespo M, Pericuesta E, Bermejo P, Hourcade JD, Pintado B, Gutiérrez-Adán A. 23 rd Scientific Meeting of the European Embryo Transfer Society (AETE). Alghero, Italy. September 2007. Oral communication: Transgenerational epigenetic alterations in mice produced by in vitro culture Fernández-González R, Ramírez MA, Pérez- Crespo M, Pericuesta E, Bermejo P, Hourcade JD, Pintado B, Gutiérrez-Adán A. 40 th Annual Meeting of the Society for the Study of Reproduction (SSR). San Antonio, USA. July 2007. Poster: Unexpected severe abnormalities in mouse ROSI offspring Moreira PN, Fernández-González R, Pérez-Crespo M, Bermejo P, Hourcade JD, Rey R, Gutiérrez-Adán A. 33th Annual Conference of the International Embryo Transfer Society (IETS). Kyoto, Japón. January 2007. Poster: Effects of maturation stage of bovine oocytes on sex ratio and speed of embryo development Rizos D, Bermejo P, Gutiérrez-Adán A, Lonergan, P. 22 nd Scientific Meeting of the European Embryo Transfer Society (AETE). Zug, Suiza. September 2006. Poster: Progesterone levels during 20 days of pregnanty of rabbit treated for endometriosis or with anti-cd44 Illera MJ, Bermejo P, Natarajan A, Willingham C, Hernández J, González A, Silván G, Illera JC. Copenhague, Denmark. January 2005. Poster: The effect of anti-cd44 on embryo implantation in rabbits Illera MJ, Bermejo P, Hernández J, González A, Illera JC. Portland, USA. January 2004. Assistance to international meetings 36 th Annual Conference of the International Embryo Transfer Society (IETS). Travel grant. Córdoba, Argentina, USA. January 2010. COST-GEMINI Maternal interaction with gametes and embryos. Local Comitee Grant. Alguero, Italy. October 2009. 177

42nd Annual Meeting of the Society for the Study of Reproduction (SSR). Pittsburgh, USA. July 2009. COST-GEMINI Maternal interaction with gametes and embryos. Student Special Grant. Lansk, Poland. May 2009. 35th Annual Conference of the International Embryo Transfer Society (IETS). Travel grant. San Diego, USA. January 2009. 1 st International Congress of the Spanish Society for Animal Reproduction (AERA) and British Andrology Society (BAS). Gijón, Spain. October 2008. 23nd Scientific Meeting of the European Embryo Transfer Society (AETE). Alghero, Italy. September 2007. Contributions and assistance to National Meetings Efectos del anticd44 sobre la implantación y los niveles hormonales de la coneja. II Congreso Europeo de Investigación en Pregrado de Ciencias de la Salud. Madrid, 2005. Influencia de las combinaciones anestésicas ketamina-xilacina y ketaminaxilacina-buprenorfina sobre la función adrenal en el conejo ; Efectos de la ampliación de la UE sobre la política agraria española ; Niveles de esteroides sexuales durante la primera mitad de la gestación en la coneja endometriosa ; Efectos del anticd44 sobre la implantación embrionaria y la curva de progesterona en la coneja. IV Congreso de ciencias veterinarias y biomédicas. Madrid, 2005. Niveles hormonales de la coneja endometriosa durante el primer tercio de la gestación, Efectos del anticd44 sobre la implantación y los niveles hormonales en la coneja. III Congreso de Ciencias Veterinarias y Biomédicas. Madrid, 2004. Efectos de la anestesia intravenosa e inhalatoria sobre la función adrenal en conejos NZW. II Congreso de Ciencias Veterinarias y Afines. Madrid, 2003. Influencia del Estrés en la Reproducción Animal en Hembras I Congreso de Ciencias Veterinarias y Afines. Madrid, 2002. Awards Best oral communication in IV Congreso de Ciencias Veterinarias y Biomédicas Madrid, Spain. April 2005. First prize in Student Competition in 23nd Scientific Meeting of the European Embryo Transfer Society (AETE). Alghero, Italy. September 2007. Finalist in Student Competition in 35th Annual Conference of the International Embryo Transfer Society (IETS). San Diego, USA. January 2009. Finalist in Student Competition in 36th Annual Conference of the International Embryo Transfer Society (IETS). Córdoba, Argentina. January 2009. Teaching XXXII International Course of Animal Reproduction, INIA, Madrid, Spain. November 2009. XXXI International Course of Animal Reproduction, INIA, Madrid, Spain. November 2008. 178