Colonización intraluminal de los túbulos seminíferos de ratones post

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Dedico este trabajo de Investigación a mis padres que con su apoyo en cada paso que andaba en
el mundo siempre estuvieron apoyándome, y a todas las personas que confiaron en mi.
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Quiero agradecer a mis padres por inculcarme la lucha por los sueños desde el comienzo de mi
vida, su apoyo durante toda mi carrera como estudiante y en el desarrollo de esta tesis. A mi
hermana, cuyo amor fraternal siento que crece cada día. A mi familia entera por su amor y
buenos deseos.
A Guillermo Llerena Cano que como jefe del Laboratorio de Reproducción Asistida y
Genética del Instituto de Ginecología y Reproducción y Jefe del Laboratorio de Andrología y
Genética de CONCEBIR, quien confió en mi persona al permitirme desarrollar la presente
investigación, asesorando y prestando para ello todo el apoyo necesario para el desarrollo de la
presente investigación.
A Rosmary Lopez Sam con su apoyo en la parte experimental estuvo siempre presente en
los estresantes y largos procedimientos, de igual manera, agradezco a mis compañeros y amigos
del equipo de trabajo del laboratorio de Reproducción y Biología del Desarrollo.
A Víctor Benavides Soto, quien me aconsejó y compartió con dedicación sus conocimientos.
Quiero agradecer al Profesor José Luis Pino Gaviño por aceptar ser mi asesor y por su apoyo
para la sustentación de esta tesis. A los profesores Fernando Retuerto y Margarita Velásquez por
las facilidades para la realización de la tesis.
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El propósito primario de cualquier organismo superior con reproducción sexual es pasar sus
genes a la siguiente generación vía sus células germinales primordiales (PGCs), células troncales
del sistema reproductor. Al transplantar intraespecíficamente las células germinales, éstas
colonizan la membrana basal de los túbulos seminíferos del animal receptor en busca del nicho
donde se desarrollan las células madre. Es así que la técnica de transplante de PGCs permite que
las células madres de origen fetal sean conservadas y divididas en su estado multipotente por
largos periodos de tiempo en el animal receptor, preservando el material genético. Así, el
presente trabajo de tesis tiene por objetivo evaluar la colonización intraluminal de una
suspensión cruda de PGCs de fetos de ratón de la cepa Balb C en los túbulos seminíferos de
ratones adultos. Para ello se aislaron las PGCs desde las crestas genitales de fetos machos con
dos pasos de digestiones enzimáticas (Tripsina 0.25% y colagenasa tipo IV, 1 mg/ml),
transplantándose en los túbulos seminíferos del animal receptor vía rete testis, a los ratones
receptores previamente se les disminuyó drásticamente su línea germinal con tratamiento
químico con Busulfán (40 mg/kg). Los animales receptores fueron mantenidos por 54 días en un
bioterio con 14 horas luz y 10 horas oscuridad. Luego de este periodo los animales fueron
sacrificados por dislocación cervical, se les extrajo los testículos y se realizó cortes histológicos
seriados de 10 µm de espesor, coloreados con hematoxilina y eosina Y. La evaluación evidenció
una colonización intraluminal en 1 de cada 100 túbulos seminíferos evaluados. Lo que
demostraría que las PGCs de fetos de 14.5 días post coitum (dpc) si tienen la capacidad de
colonizar los túbulos seminíferos de individuos adultos de la misma especie.
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-The aim function of a organism with sexual reproduction is to transmit their genes to the next
generation throught their primordial germ cell (PGCs), stem cells of the reproductor system, they
colonized basal membrane of seminiferous tubule in the receptor animal, search their nich where
they can differentiate to sperms. In the Transplant PGCs Technique, we could preserved and
multiplicated cells for long time in their multipotent state in the receptor animal, conserved in
their multipotent state. The present Thesis objective is to evaluate the intraluminal colonization
of a suspension of PGCs of fetus mice Balb C in semiferous tubule of adult mice. In my
research, PGCs were isolated from fetus male mice with two enzimatic digestion steps (Tripsin
0.25% and Colagenase Type IV 1 mg/ml), PGCs were transplanted into the rete testis of the
receptor animal that previously were injected with Busulfán (40 mg/kg) to decrease their own
spermatogenesis. Mice receptor were maintained in bioterio conditions with 14 hours light and
10 hours dark for fifty four days, they were sacrificed by cervical dislocation, their testis were
extracted for and histology procedures of 10 µm, colored with hematoxilin and Y eosin. In this
research the intraluminal colonization was identified in 1 of 100 seminiferous tubule. In
conclusion, transplantation of PGCs of foetus from 14.5 dpc can colonized the seminiferous
tubule.
Palabras clave: Transplante, Células Germinales Primordiales, Espermatogénesis.
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