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We have previously reported that a heterodimeric protein secreted by rat Sertoli cells is antigenically related to a protein associated with outer dense fibers of the sperm tail. Therefore, we have explored the possibility that Sertoli and spermatogenic cells express a similar gene encoding a homologous protein. A Sertoli cell heterodimeric protein cDNA probe recognizes specific mRNA in pachytene and round spermatids fractionated by centrifugal elutriation; however, this specific mRNA was less prominent than in cultured Sertoli cells. In agreement with these observations, in situ hybridization experiments show that Sertoli cells are predominantly engaged in active heterodimeric protein mRNA synthesis, while meiotic prophase spermatocytes and spermatids also show significant but less abundant specific mRNA. Immunoblotting experiments demonstrate that, while Sertoli cells synthesize a heterodimeric protein consisting of two disulfidelinked components with molecular masses of 45 and 35 kD, both primary spermatocytes and round spermatids synthesize single 30 kD monomers not associated by disulfide linkage but recognized by antisera to Sertoli cell heterodimeric protein Immunoblotting and immunogold electron microscopic studies show that antisera to Sertoli cell heterodimeric protein recognize a protein associated with outer dense fibers. This immunoreactivity was abolished by a 5-min pronase treatment, without affecting the integrity of outer dense fibers. Results of this study and previous studies demonstrate that both Sertoli and spermatogenic cells express a similar gene and that an antigenically related product encoded by this gene becomes associated with outer dense fibers during their assembly at spermiogenesis. © 1992 Wiley-Liss, Inc.

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.

Examination of major biochemical classes during the spermato- genic cycle provides a definition of the changing chemical microenvironment influencing germinal cells and also suggests temporal relationships among successive spermatogenic events. Testes from sea stars collected throughout the year were homogenized and lyophilized, and aliquots were assayed for DNA, RNA, total protein, free amino acids, total lipids, glycogen, and other carbohydrates; spermatogenic stage was determined by examination of paraf- fin sections. The resulting data, expressed as 1) mglg dry mass, 2) mghg DNA, and 3) total content, were analyzed by weighted periodic regression and circu- lar-linear correlation with spermatogenic stage and time of year. With the exception of lipid/DNA, all components show a significant regres- sion against time. DNA concentration increases during the proliferative and differentiative phases, attaining maximal values late in the differentiative phase; DNA synthesis appears to be essentially complete by the end of March. RNA concentration increases in the late aspermatogenic phase, as testicular cells begin preparations for proliferation, and remains high until late in the differentiative phase. Protein accumulates during proliferation, preceding the accumulation of DNA, but then gradually declines during differentiation. Free amino acids, simple sugars, and glycogen all increase during the aspermato- genic phase, possibly as a result of nutritive input. The observed patterns of change correlate closely with cytological variations observed within the germinal epithelium. Biochemical data thus complement existing data on the cytology of the germinal epithelium. Asteroid testes are a useful model system for the study of mechanisms regulating sper- matogenesis. Because fertilization is exter- nal in

We have established the patterns of ornithine decarboxylase activity (an enzyme related to cell growth, differentiation, and proliferation) during rat testicular development and studied the effect of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-type beta (TGF-beta), and a serum-free, hormone/growth factor-supplemented medium (TKM) on ornithine decarboxylase (ODC) activity in Sertoli-spermatogenic cell cocultures and cultured seminiferous peritubular cells prepared from sexually immature rats (20-22 days old). Results were correlated with timing of ODC activities during rat testicular development. We have found that: (1) although EGF, alone or combined with PDGF and TGF-beta, and TKM stimulated ODC activity in Sertoli-spermatogenic cell cocultures after 6 and 24 h of stimulation, PDGF exerted an inhibitory effect, and (2) cultured peritubular cells stimulated with EGF, PDGF, TGF-beta (and their combinations), and TKM displayed an increase in ODC activity after 6 h of stimulation, but ODC activities for most of these treatments declined considerably 24 h after stimulation. Light microscopic autoradiographic studies of [3H]thymidine labeled samples demonstrated that (1) clones of spermatogenic cells traverse S phase synchronously, (2) Sertoli cells are not significantly radiolabeled, probably because of contact inhibition achieved by high cell plating density, and (3) peritubular cells are significantly [3H]thymidine labeled in the presence of TKM, a culture medium that facilitates spermatogenic cell long-term viability and differentiation. We conclude that TKM and EGF have stimulatory effects on the biochemical pathway that precedes synchronous DNA synthesis in spermatogonia and preleptotene spermatocytes, and that ODC activity is a sensitive marker for monitoring these events.