Generation of progeny via ICSI following enrichment of elongated spermatids from mouse testis by flow-cytometric cell sorting.
BACKGROUND
Although ICSI is a useful technique, low elongated spermatid numbers frequently causes technical difficulties, especially in the case of azoospermic patients. Enrichment of elongated spermatids from the testis prior to ICSI may solve this problem.


METHODS
To determine whether elongated spermatids had a characteristic phenotype suitable for purification, testicular cells prepared from 25-day-old mice (from spermatogonia to round spermatids) and adult mice (from spermatogonia to elongated spermatids) were compared by flow cytometry. After flow-cytometric cell sorting (FCS) based on their side (SSC) and forward scatter (FSC), purity of the elongated spermatids in the fractionated population was microscopically examined, and functional ability of purified elongated spermatids was assessed by ICSI.


RESULTS
Elongated spermatids in testicular cells showed characteristic SSC and FSC phenotypes. In the purified population, approximately 70-80% of the cells were morphologically determined as elongated spermatids, in contrast to only 10% before sorting. Using ICSI, purified elongated spermatids supported full-term development similar to that of unsorted elongated spermatids. Furthermore, we succeeded in enriching the elongated spermatids from the infertile testis model by approximately 10-fold.


CONCLUSIONS
Elongated spermatids with normal developmental ability can be efficiently purified by FCS based on SSC and FSC characteristics.
Ohta
Hiroshi
H
Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, Chuo-ku, Kobe 650-0047, Japan. ohta@cdb.riken.go.jp
Sakaide
Yuko
Y
Wakayama
Teruhiko
T
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D005260:Female; D005434:Flow Cytometry; D007248:Infertility, Male; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D020554:Sperm Injections, Intracytoplasmic; D013087:Spermatids / Q000166:cytology*; D013737:Testis / Q000166:cytology*