Identification, isolation, and RT-PCR analysis of single stage-specific spermatogenetic cells obtained from portions of seminiferous tubules classified by transillumination microscopy.
The protocol here described allows the analysis of gene expression in single specific mouse spermatogenetic cells. Germ cells were singularly isolated by microdissection of portions of seminiferous tubules classified, based on their transillumination pattern, into four distinct zones along their length. Single portions of a seminiferous tubule, corresponding to specific zones, were mechanically disaggregated into single cells that were (1) identified as spermatogonia, spermatocytes, round or elongated spermatids, (2) isolated using a micromanipulator, and (3) singularly transferred into a test tube for retro-transcription PCR analysis. On each single isolated cell, we have determined the quantitative profile of expression of Gapdh, an endogenous housekeeping gene known to be expressed throughout spermatogenesis. The protocol described allows an accurate analysis of the temporal and quantitative profile of gene expression throughout the whole male gamete differentiation process which so far has mainly been performed on enriched population of cells.
Vasco
Chiara
C
Dipartimento di Biologia Animale, Università degli Studi di Pavia, Pavia, PV 27100, Italy.
Zuccotti
Maurizio
M
Redi
Carlo Alberto
CA
Garagna
Silvia
S
D000818:Animals; D008297:Male; D051379:Mice; D008853:Microscopy / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012671:Seminiferous Tubules / Q000166:cytology*; D013087:Spermatids / Q000166:cytology / Q000502:physiology; D013090:Spermatocytes / Q000166:cytology / Q000502:physiology; D013091:Spermatogenesis / Q000502:physiology*; D013093:Spermatogonia / Q000166:cytology / Q000502:physiology; D014172:Transillumination