title: In vitro production of haploid sperm cells from male germ cells of foetal cattle.
abstract: The purpose of this study was to isolate the foetal cattle male germ cells (mGCs) and then induce them into sperm cells. The mGCs were purified and enriched by a two-step plating method based on the different adherence velocities of mGCs and somatic cells. The percentage of the vasa and the c-kit positive cells were 95.34+/-2.25% and 53.3+/-1.03% by using flow cytometry analysis (FCA), respectively. In feeder-free culture system, the half-suspending cells appeared and formed a 16-cell rosary in medium after the mGCs were cultured for 6-8 days. On immunocytochemical staining during the second passage, some single cells adhering to the plate appeared to be both Oct-4 and alpha6-integrin positive. During the third passage, the mGCs were induced for 48 h by retinol acid (RA) on Sertoli cell-feeder layer, followed by 5-7 days culture in an RA-free medium. Some elongated sperm-like cells appeared in the medium at this stage. We found that the most effective concentration of RA for the inducement was 10(-7)moll(-1) (P<0.01). The haploid cells in suspension were identified by FCA. The elongated sperm-like cells showed proacrosome-like structure and the flagellum with fibre construct under electron microscopy. The mRNA of outer dense fibre-3 (ODF-3) and transcription protein-1 (TP-1) could be detected in the suspended cells by using reverse transcription polymerase chain reaction (RT-PCR). About 23.1% bovine oocytes could be activated to perform cleavage by intracytoplasmic injection with the sperm-like cells, but embryos did not further develop. Our investigation further demonstrated that foetal cattle mGCs could be induced in vitro into haploid sperm in the short term.
authors.affiliation: Institute of Stem Cell Engineering & Technology, Northwest A&F University, Yangling 712100, People's Republic of China. wzdong@sibs.ac.cn
authors.forename: Wu-Zi
authors.initials: WZ
authors.lastname: Dong
authors.forename: Jin-Lian
authors.initials: JL
authors.lastname: Hua
authors.forename: Wen-Zheng
authors.initials: WZ
authors.lastname: Shen
authors.forename: Zhong-Ying
authors.initials: ZY
authors.lastname: Dou
mesh_terms: D000818:Animals; D002417:Cattle / Q000196:embryology*; D002469:Cell Separation / Q000662:veterinary; D002478:Cells, Cultured; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary; D006238:Haploidy; D008297:Male; D020554:Sperm Injections, Intracytoplasmic / Q000662:veterinary; D013091:Spermatogenesis / Q000187:drug effects; D013094:Spermatozoa / Q000166:cytology* / Q000502:physiology; D013737:Testis / Q000166:cytology / Q000196:embryology; D020858:Tissue and Organ Harvesting / Q000662:veterinary; D014212:Tretinoin / Q000494:pharmacology; D015053:Zygote / Q000254:growth & development
chemical_list: D014212:Tretinoin