Flow cytometry isolation and reverse transcriptase-polymerase chain reaction characterization of human round spermatids in infertile patients

Flow cytometry coupled to cell sorting is proposed as a method to isolate round spermatids from testicular biopsies in obstructive azoospermic patients. The cells were separated on the basis of their size and density only. We obtained homogenous populations of alive round spermatids free of lymphocy...

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Bibliographic Details
Published in:Human reproduction (Oxford) Vol. 14; no. 2; p. 379
Main Authors: Ziyyat, A, Lassalle, B, Testart, J, Briot, P, Amar, E, Finaz, C, Lefèvre, A
Format: Journal Article
Language:English
Published: England 01.02.1999
Subjects:
Adult
Antigens, Neoplasm
Biopsy
CD3 Complex - metabolism
Cell Separation
Ejaculation - physiology
Flow Cytometry
Gene Expression - physiology
Humans
Infertility, Male - genetics
Infertility, Male - metabolism
Infertility, Male - pathology
Isomerism
Male
Melanoma-Specific Antigens
Middle Aged
Neoplasm Proteins - metabolism
Protamines - genetics
Reverse Transcriptase Polymerase Chain Reaction
RNA, Messenger - metabolism
Spermatids - metabolism
Spermatids - pathology
Spermatids - physiology
Testis - pathology
ISSN:0268-1161
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Summary:Flow cytometry coupled to cell sorting is proposed as a method to isolate round spermatids from testicular biopsies in obstructive azoospermic patients. The cells were separated on the basis of their size and density only. We obtained homogenous populations of alive round spermatids free of lymphocytes and diploid germ cells. The detection of protamine 1 gene (PRM1) and PRM2 expression in the sorted cells proves that these cells are round spermatids. On the contrary, neither the expression of CD3-delta, which is specific to lymphoid cells, nor that of MAGE1, which has been demonstrated in diploid germ cells, could be observed in the round spermatid population even after using a nested polymerase chain reaction (PCR) assay. The flow cytometry procedure failed to isolate round spermatids from ejaculates in non-obstructive azoospermic patients. In > 39 ejaculates tested by reverse transcriptase-PCR, only nine revealed the presence of some round spermatids, as demonstrated by the expression of PRM1. However, these round spermatids did not express PRM2.
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ISSN:0268-1161
DOI:10.1093/humrep/14.2.379