Cloned embryos from semen. Part 1: In vitro proliferation of epithelial cells on embryonic fibroblasts after isolation from semen by gradient centrifugation

Liesl Nel-Themaat, Martha C. Gómez, C. Earle Pope, Monica Lopez, Gemechu Wirtu, Alex Cole, Betsy L. Dresser, Leslie A Lyons, Kenneth R. Bondioli, Robert A. Godke

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Although epithelial-like somatic cells have been previously isolated from semen, cell proliferation rates were low. Culture of whole semen samples resulted in loss of potentially valuable spermatozoa. The aims of the present study were to (1) isolate somatic cells from semen, while preserving sperm viability, and (2) optimize in vitro culture conditions for semen-derived epithelial cells. Density gradient centrifugation of washed ejaculates of two rams (Ovis aries) (n = 24) and one eland bull (Taurotragus oryx) (n = 4) was performed using a three-layer discontinuous Percoll column consisting of 90% (P-90), 50% (P-50), and 20% (P-20) Percoll. In vitro culture and Trypan Blue staining indicated that live somatic cells settled in the P-20 layer. Nonmotile spermatozoa were recovered at the P-50 and P-90 interfaces, whereas motile spermatozoa were collected in the pellet from the P-90 layer. Subsequently, somatic cells isolated from the P-20 layer were plated either on inactivated 3T3 mouse embryonic fibroblast feeder layers, collagen-coated plates with 3T3 feeder cell inserts, or on collagen-coated plates. Initial somatic cell plating was similar among treatments, but proliferation significantly increased when cocultured with 3T3 cells (feeder or insert). Furthermore, two different types of epithelial cells were obtained. The exact origin of the cells in the male reproduction system is uncertain and probably variable. The present method of cell isolation and in vitro culture may be of value for preserving endangered species. Specifically, cells isolated and cultured from cryopreserved semen of nonliving males could be used for producing embryos by somatic cell nuclear transfer.

Original languageEnglish (US)
Pages (from-to)143-159
Number of pages17
JournalCloning and Stem Cells
Volume10
Issue number1
DOIs
StatePublished - Mar 1 2008

Fingerprint

Semen
Centrifugation
Embryonic Structures
Fibroblasts
Epithelial Cells
Spermatozoa
Feeder Cells
3T3 Cells
Collagen
Domestic Sheep
Endangered Species
Trypan Blue
Density Gradient Centrifugation
Cell Separation
In Vitro Techniques
Reproduction
Cultured Cells
Cell Proliferation
Staining and Labeling

ASJC Scopus subject areas

  • Biotechnology
  • Developmental Biology
  • Applied Microbiology and Biotechnology

Cite this

Cloned embryos from semen. Part 1 : In vitro proliferation of epithelial cells on embryonic fibroblasts after isolation from semen by gradient centrifugation. / Nel-Themaat, Liesl; Gómez, Martha C.; Pope, C. Earle; Lopez, Monica; Wirtu, Gemechu; Cole, Alex; Dresser, Betsy L.; Lyons, Leslie A; Bondioli, Kenneth R.; Godke, Robert A.

In: Cloning and Stem Cells, Vol. 10, No. 1, 01.03.2008, p. 143-159.

Research output: Contribution to journalArticle

Nel-Themaat, Liesl ; Gómez, Martha C. ; Pope, C. Earle ; Lopez, Monica ; Wirtu, Gemechu ; Cole, Alex ; Dresser, Betsy L. ; Lyons, Leslie A ; Bondioli, Kenneth R. ; Godke, Robert A. / Cloned embryos from semen. Part 1 : In vitro proliferation of epithelial cells on embryonic fibroblasts after isolation from semen by gradient centrifugation. In: Cloning and Stem Cells. 2008 ; Vol. 10, No. 1. pp. 143-159.
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AU - Lopez, Monica

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AU - Cole, Alex

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