title: An in vitro, short-term culture method for mammalian haploid round spermatids amenable for molecular manipulation.
abstract: Extensive chromatin remodeling is a characteristic feature of mammalian spermiogenesis. To date, methods for the molecular manipulation of haploid spermatids are not available as there is a lack of a well-established culture system. Biochemical experiments and knockout studies reveal only the final outcome; studying the incremental details of the intricate mechanisms involved is still a challenge. We have established an in vitro culture system for pure haploid round spermatids isolated from rat testes that can be maintained with good viability for up to 72 hr. Changes in cell morphology and flagellar growth were also studied in the cultured spermatids. Further, we have demonstrated that upon treatment of cells with specific histone deacetylase inhibitors, sodium butyrate and trichostatin A, there is an increase in the hyperacetylation status of histone H4, mimicking an important event characteristic of histone replacement process that occurs during later stages of spermiogenesis. We have also tried various methods for introducing DNA and protein into these round spermatids in culture, and report that while DNA transfection is still a challenging task, protein transfection could be achieved using Chariot™ peptide as a transfection reagent. Thus, the method described here sets a stage to study the molecular roles of spermatid-specific proteins and chromatin remodelers in the cellular context.
authors.affiliation: Chromatin Biology Lab, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India.
authors.forename: Tushna
authors.initials: T
authors.lastname: Dehnugara
authors.forename: Surbhi
authors.initials: S
authors.lastname: Dhar
authors.forename: M R Satyanarayana
authors.initials: MR
authors.lastname: Rao
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D042002:Chromatin Assembly and Disassembly / Q000187:drug effects; D003543:Cysteamine / Q000008:administration & dosage / Q000031:analogs & derivatives; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006238:Haploidy; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006657:Histones / Q000378:metabolism; D006877:Hydroxamic Acids / Q000494:pharmacology; D058610:Isobutyrates / Q000494:pharmacology; D008297:Male; D010455:Peptides / Q000008:administration & dosage / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D051381:Rats; D013087:Spermatids / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013737:Testis / Q000502:physiology; D014162:Transfection / Q000379:methods; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D058610:Isobutyrates; C503924:Pep-1 peptide; D010455:Peptides; D049452:Green Fluorescent Proteins; C012589:trichostatin A; D003543:Cysteamine; C020380:isobutyric acid; D001616:beta-Galactosidase