Influence of temperature, serum, and gonadotropin supplementation in short- and long-term organotypic culture of human immature testicular tissue

Fecha de publicación: 01/11/2018

Autores de IIS La Fe

• Jose Vicente Medrano Plaza

  Autor

• Teresa Vilanova Perez

  Autor

• Victoria Fornés Ferrer

  Autor

• María Luisa Martínez Triguero

  Autor

• Sergio García Muñoz

  Autor

• Javier Gómez-Chacón Vilalba

  Autor

• Ivan Povo Martin

  Autor

• Antonio Pellicer Martínez

  Autor

• Mercedes Andres Martinez

  Autor

• Edurne Novella Maestre

  Autor

Participantes ajenos a IIS La Fe

• Navarro-Gomezlechon, A

Grupos

• Medicina Reproductiva

  

  Investigador Principal

  José Morales Roselló

Abstract

Objective: To study how temperature, serum, and gonadotropin supplementation affect the organotypic culture of human immature testicular tissue (ITT) in vitro. Design: Experimental basic science study. Setting: Reproductive biology laboratory. Patient(s): ITT from 4 boys with cancer that had testicular tissue cryopreserved as part of their fertility preservation treatment. Intervention(s): In vitro organotypic culture of ITT, exposed to different temperatures (37 degrees C vs. 34 degrees C), serum (fetal bovine serum [FBS] vs. Knockout Serum Replacement [KOS]), and gonadotropin supplementation (with and without FSH and LH). Main Outcome Measure(s): Characterization of the tissue was performed at days 0, 14, and 70 with the use of reverse-transcription quantitative polymerase chain reaction, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, histologic analysis by means of hematoxylin-eosin staining, and immunohistochemical staining. Hormonal secretion was determined at days 3, 14, 28, and 70 by means of immunofluorescent assay. Result(s): The 37 degrees C conditions showed an accelerated loss of tubular morphology and higher intratubular apoptosis. KOS supplementation triggered the up-regulation of STAR, SOX9, DAZL, DDX4, PLZF, and UTF1, the percentage of SOX9+/androgen receptor (AR)-positive mature Sertoli cells at day 14, and testosterone secretion. Gonadotropin supplementation increased the numbers of both undifferentiated UTF1+ spermatogonia and premeiotic VASA+/SYCP3+ spermatogonia at day 14, and the number of SOX9+ Sertoli cells at day 70. The low SOX9+/AR+ colocalization, the disorganized pattern of ZO-1, and the progressive decrease of antimullerian hormone secretion indicated inefficient Sertoli cell maturation in vitro. Conclusion(s): The 34 degrees C condition in KOS showed the best results for the survival of both spermatogonia and Sertoli cells. FSH/LH supplementation also improved long-term survival of Sertoli cells and the maturation of spermatogonia up to meiotic initiation in short-term culture. (C) 2018 by American Society for Reproductive Medicine.

Keywords

• Fertility preservation; human immature testicular tissue; organotypic culture; in vitro spermatogenesis