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

We have used two radiolabeled oligonucleotide probes (TH2B and H1t), Northern blotting, two-dimensional gel electrophoresis, and autoradiography to study the temporal expression of TH2B and H1t testis-specific histone genes during the development of rat spermatogenic cells in vitro. These studies were carried out to determine whether meiotic prophase spermatocytes, known to synthesize in vivo TH2B and H1t histones among other histones, are capable of expressing these testis-specific genes in vitro during an extended period of time. We have found abundant TH2B and H1t mRNA steady state levels as well as newly-synthesized TH2B and H1t histones after 5 days of coculture. Northern blots reprobed with H1t-specific oligonucleotide showed that H1t mRNA remained prominent when TH2B mRNA started to decline after 8-12 days of coculture. Phase-contrast and transmission electron microscopy studies carried out throughout the course of the experiments demonstrated that the number of viable spermatogonia and meiotic prophase spermatocytes was relatively constant during 12 days of coculture. Spermatocytes, in a clone-like arrangement, remained attached to Sertoli cell surfaces and displayed subcellular features consistent with those observed in the intact seminiferous 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. Testis-specific histone gene expression in vitro should facilitate further studies for understanding the role of these histones in chromatin structure, transcription, and genetic recombination during male meiotic prophase.