Expression of testis-specific histone genes during the development of rat spermatogenic cells in vitro

We have used two radiolabeled oligonucleotide probes (TH2B and Hlt), Northern blotting, two-dimensional gel electrophoresis, and autoradiography to study the temporal expres- sion of TH2B and Hlt testis-specific histone genes during the development of rat spermatogenic cells in vitro. These studies were carried out to deter- mine whether meiotic prophase spermatocytes, known to synthesize in vivo THBB and Hlt his- tones among other histones, are capable of ex- pressing these testis-specific genes in vitro during an extended period of time. We have found abun- dant TH2B and Hlt mRNA steady state levels as well as newly-synthesized TH2B and Hlt histones after 5 days of coculture. Northern blots reprobed with Hlt-specific oligonucleotide showed that Hlt mRNA remained prominent when TH2B mRNA started to decline after 8-12 days of coculture. Phase-contrast and transmission electron micros- copy studies carried out throughout the course of the experiments demonstrated that the number of viable spermatogonia and meiotic prophase sper- matocytes was relatively constant during 12 days of coculture. Spermatocytes, in a clone-like ar- rangement, remained attached to Sertoli cell surfaces and displayed subcellular features con- sistent with those observed in the intact seminif- erous epithelium. Spermatogonia formed long, branching chains of interconnected cells. Results of this study indicate that spermatogenic cells in coculture with Sertoli cells express testis-specific histone genes for an extended period of time. Tes- tis-specific histone gene expression in vitro should facilitate further studies for understanding the role of these histones in chromatin structure, tran- scription, and genetic recombination during male meiotic prophase.