Desarrollo y Caracterización de un prototipo de hormona folículo estimulante bovina de simple cadena (BscFSH), para reproducción asistida en rumiantes.
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Date
2023
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Universidad de Concepción
Abstract
La transferencia de embriones permite la diseminación de características genéticas desde animales de alto rendimiento hacia los rebaños bovinos de producción, tanto lecheros como de carne. Para su uso se implementan protocolos de superovulación, que actualmente, utilizan diversas glicoproteínas que no son especie específicas, siendo la hormona folículo estimulante (FSH) extraída de pituitaria de cerdo, la más utilizada actualmente. Sin embargo, esta FSH porcina tiene varias limitantes. Entre ellas, una vida media en circulación de 5 h, contaminación de hormona luteinizante y otras proteínas del extracto, se realizan 8 frecuencias de aplicación en 4 días, lo que precisa un mayor manejo animal y genera estrés, incidiendo significativamente en la respuesta ovárica y la obtención de embriones transferibles. En este trabajo se diseñó y caracterizó una variante recombinante de cadena única, derivada de la hormona folículo estimulante bovina (bscFSH). Esta variante se diseñó considerando las secuencias aminoacídicas de las cadenas alfa y beta de la FSH nativa de Bos taurus, covalentemente unidas mediante un espaciador de 33 aminoácidos y conteniendo un total de 6 sitios potenciales de N-glicosilación, con estructuras altamente sialiladas. La hormona se produjo en un clon de células CHO genéticamente transformadas y se purificó en un único paso de cromatografía de afinidad, obteniéndose con un 97% de pureza. La bscFSH presentó un tiempo medio de residencia de 66,44 h y un tiempo de vida media en circulación de 44,16 h, administrada por vía intramuscular. Esto nos permitió desarrollar protocolos de superovulación en bovinos con la mitad de la frecuencia de aplicaciones (4 administraciones) utilizada actualmente con las FSH derivadas de pituitaria de cerdo, y obteniendo una mayor cantidad de embriones transferibles. La nueva hormona desarrollada induce una respuesta ovárica eficiente, permite un manejo más amigable, tiene un alto nivel de pureza y está libre de contaminantes animales, evitando posibles zoonosis, y disminuyendo la respuesta inmune y estrés animal.
Embryo transfer allows the dissemination of genetic characteristics from highyielding animals to production cattle herds, both dairy and beef. For its use, superovulation protocols are implemented, which currently use various glycoproteins that are not specific, being the follicle-stimulating hormone (FSH) extracted from pig pituitary, the most currently used. However, this porcine FSH has several limitations. Among them, a circulating half-life of 5 h, contamination of luteinizing hormone and other extract proteins, 8 application frequencies are carried out in 4 days, which requires greater animal handling and generates stress, significantly influencing the ovarian response and the obtaining of transferable embryos. In this work, a single-chain recombinant variant derived from bovine follicle stimulating hormone (bscFSH) was developed and characterized. This variant was designed by considering the amino acid sequences of the alpha and beta chains of native Bos taurus FSH, covalently linked by a 33-amino acid spacer and containing a total of 6 potential N-glycosylation sites, with highly sialylated structures. The hormone was produced in a clone of genetically transformed CHO cells and was purified in a single affinity chromatography step, obtaining 97% purity. bscFSH had a mean residence time of 66.44 h and a circulating half-life of 44,16 h when administered intramuscularly. This allowed us to develop bovine superovulation protocols with half the frequency of applications (4 administrations) currently used with pig pituitary-derived FSH, and obtaining a greater number of transferable embryos. The newly developed hormone induces an efficient ovarian response, allows friendlier handling, has a high level of purity and is free of animal contaminants, avoiding possible zoonoses, and reducing the immune response and animal stress.
Embryo transfer allows the dissemination of genetic characteristics from highyielding animals to production cattle herds, both dairy and beef. For its use, superovulation protocols are implemented, which currently use various glycoproteins that are not specific, being the follicle-stimulating hormone (FSH) extracted from pig pituitary, the most currently used. However, this porcine FSH has several limitations. Among them, a circulating half-life of 5 h, contamination of luteinizing hormone and other extract proteins, 8 application frequencies are carried out in 4 days, which requires greater animal handling and generates stress, significantly influencing the ovarian response and the obtaining of transferable embryos. In this work, a single-chain recombinant variant derived from bovine follicle stimulating hormone (bscFSH) was developed and characterized. This variant was designed by considering the amino acid sequences of the alpha and beta chains of native Bos taurus FSH, covalently linked by a 33-amino acid spacer and containing a total of 6 potential N-glycosylation sites, with highly sialylated structures. The hormone was produced in a clone of genetically transformed CHO cells and was purified in a single affinity chromatography step, obtaining 97% purity. bscFSH had a mean residence time of 66.44 h and a circulating half-life of 44,16 h when administered intramuscularly. This allowed us to develop bovine superovulation protocols with half the frequency of applications (4 administrations) currently used with pig pituitary-derived FSH, and obtaining a greater number of transferable embryos. The newly developed hormone induces an efficient ovarian response, allows friendlier handling, has a high level of purity and is free of animal contaminants, avoiding possible zoonoses, and reducing the immune response and animal stress.
Description
Tesis presentada para optar al grado de Doctor en Biotecnología Molecular