Developmental potential of human spermatogenic cells co-cultured with Sertoli cells.
BACKGROUND
Development of an in-vitro culture system capable of supporting human early germ cell differentiation would be important for treatment of azoospermic patients.


METHODS
Sertoli cells, spermatogonia and spermatocytes were isolated from testicular biopsies of 61 non-obstructive azoospermic patients, and co-cultured using Vero cell conditioned medium only or supplemented with recombinant (r)FSH or rFSH plus testosterone. Germ cell purity was checked by fluorescent in-situ hybridization (FISH) analysis.


RESULTS
Best results were achieved with both hormones, which elicited 6.9% of meiosis index and 22.7% of differentiation into normal late spermatids after 2-3 weeks of culture. In-vitro matured spermatids were microinjected into oocytes to study their developmental potential. Round spermatids elicited 37.5% of fertilization and 28.6% blastocyst rates. Abnormal elongating and elongated spermatids enabled 8.3 and 27.3% fertilization rates respectively, but none achieved the blastocyst stage. Normal elongating and elongated spermatids elicited 30.5% fertilization and 42.9% of blastocyst rates. FISH analysis showed sex chromosome anomalies in all embryos, except in the case of morulae from normal late spermatids.


CONCLUSIONS
Results suggest that meiosis and spermiogenesis can be resumed in vitro, with normal differentiated spermatids showing a low fertilization potential but regular rates of blastocyst formation. However, most of the embryos did not reach the morula stage and showed major sex chromosome abnormalities.
Sousa
Mário
M
Department of Medical Genetics, Faculty of Medicine, State University of Porto, Porto, Portugal. msousa@icbas.up.pt
Cremades
Nieves
N
Alves
Cláudia
C
Silva
Joaquina
J
Barros
Alberto
A
D000818:Animals; D001706:Biopsy; D002454:Cell Differentiation; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D004622:Embryo, Mammalian / Q000648:ultrastructure; D005260:Female; D005307:Fertilization in Vitro; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008540:Meiosis; D009845:Oligospermia / Q000473:pathology; D011994:Recombinant Proteins / Q000494:pharmacology; D012708:Sertoli Cells / Q000502:physiology*; D020554:Sperm Injections, Intracytoplasmic; D013087:Spermatids / Q000502:physiology; D013091:Spermatogenesis; D013093:Spermatogonia / Q000502:physiology*; D013094:Spermatozoa / Q000502:physiology*; D013737:Testis / Q000473:pathology; D013739:Testosterone / Q000494:pharmacology; D020858:Tissue and Organ Harvesting; D014709:Vero Cells; D014960:X Chromosome
D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D013739:Testosterone; D005640:Follicle Stimulating Hormone