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In vitro culture of epithelial cells from the caput, corpus, and cauda epididymis of Sus domesticus

This work describes a protocol to culture epididymal epithelial cells from the caput, corpus, and cauda regions of Sus domesticus. Epididymal epithelial fragments were obtained by dissection and enzymatic digestion with collagenase. About 30 epididymal fragments from each epididymal region were cultured in 24-well culture plates with supplemented RPMI-1640 medium at 37 8C, 5% CO2 in air, and 100% humidity. A confluent monolayer of polygonal and tightly packed epithelioid cells from the three epididymal regions was obtained after 12–16 days in culture and maintained in vitro for more than 60 days. The proportion of epididymal epithelial cells in these cultures was assessed by immunofluorescent staining for cytokeratins. Throughout the 2 months of culture, about 80% of the cells were cytokeratin-positive. Electron microscopy observations indicated that cultured cells from caput, corpus, and cauda epididymal regions were tightly adhered to each other by junctional complexes and that stereocilia were present in their apical membranes. Moreover, the presence of an extensive rough endoplasmic reticulum, Golgi apparatus and numerous vesicles in the cytoplasm suggested that cultured cells maintained secretory and absorptive activities. These results show that the epididymal epithelial cells in culture from S. domesticus retain some fundamental features that characterize the epididymal epithelium in the intact organ. This system might be a valuable tool for studying the mechanism of sperm maturation in vitro, including epididymal cell secretions and the analysis of regional differences

Grant support: Projects AGL 2002-01924 (GAN-ACU) of Ministerio de Ciencia y Tecnología (MCYT), entitled ‘‘Development of a methodology for the in vitro maturation of spermatozoa from Pie´train boar’’ and RZ02-013 of Ministerio de Ciencia y Tecnología (MCYT) and Instituto Nacional de Investigaciones Agrarias (INIA), entitled ‘‘Improvement of sperm quality and fertility ability of immature sperm from Iberic boars after in vitro coculture with epididymal epithelial cells"

Elsevier

Manager: Ministerio de Ciencia y Tecnología (Espanya)
Author: Bassols Casadevall, Judit
Kádár García, Elisabeth
Briz González, Maria Dolors
Pinart Nadal, Elisabeth
Sancho Badell, Sílvia
Garcia Gil, Núria
Badia Brea, Maria Elena
Pruneda Sais, Anna
Bussalleu Muntada, Eva
Yeste Oliveras, Marc
Bonet, Sergi
Date: 2004 September 1
Abstract: This work describes a protocol to culture epididymal epithelial cells from the caput, corpus, and cauda regions of Sus domesticus. Epididymal epithelial fragments were obtained by dissection and enzymatic digestion with collagenase. About 30 epididymal fragments from each epididymal region were cultured in 24-well culture plates with supplemented RPMI-1640 medium at 37 8C, 5% CO2 in air, and 100% humidity. A confluent monolayer of polygonal and tightly packed epithelioid cells from the three epididymal regions was obtained after 12–16 days in culture and maintained in vitro for more than 60 days. The proportion of epididymal epithelial cells in these cultures was assessed by immunofluorescent staining for cytokeratins. Throughout the 2 months of culture, about 80% of the cells were cytokeratin-positive. Electron microscopy observations indicated that cultured cells from caput, corpus, and cauda epididymal regions were tightly adhered to each other by junctional complexes and that stereocilia were present in their apical membranes. Moreover, the presence of an extensive rough endoplasmic reticulum, Golgi apparatus and numerous vesicles in the cytoplasm suggested that cultured cells maintained secretory and absorptive activities. These results show that the epididymal epithelial cells in culture from S. domesticus retain some fundamental features that characterize the epididymal epithelium in the intact organ. This system might be a valuable tool for studying the mechanism of sperm maturation in vitro, including epididymal cell secretions and the analysis of regional differences
Grant support: Projects AGL 2002-01924 (GAN-ACU) of Ministerio de Ciencia y Tecnología (MCYT), entitled ‘‘Development of a methodology for the in vitro maturation of spermatozoa from Pie´train boar’’ and RZ02-013 of Ministerio de Ciencia y Tecnología (MCYT) and Instituto Nacional de Investigaciones Agrarias (INIA), entitled ‘‘Improvement of sperm quality and fertility ability of immature sperm from Iberic boars after in vitro coculture with epididymal epithelial cells"
Format: application/pdf
Document access: http://hdl.handle.net/10256/12391
Language: eng
Publisher: Elsevier
Collection: info:eu-repo/semantics/altIdentifier/doi/10.1016/j.theriogenology.2003.12.015
info:eu-repo/semantics/altIdentifier/issn/0093-691X
info:eu-repo/semantics/altIdentifier/eissn/1879-3231
MICYT/2002-2005/RZ02-013
Rights: Tots els drets reservats
Subject: Porcs -- Inseminació artificial
Swine -- Artificial insemination
Porcs -- Espermatozoides -- Investigació
Swine -- Spermatozoa -- Research
Porcs -- Espermatozoides -- Anàlisi
Swine -- Spermatozoa -- Analysis
Title: In vitro culture of epithelial cells from the caput, corpus, and cauda epididymis of Sus domesticus
Type: info:eu-repo/semantics/article
Repository: DUGiDocs

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