The distribution pattern of proliferating cell nuclear antigen (PCNA) and Ki-67 protein was studied in adult bovine seminiferous epithelium by means of immunohistochemistry using monoclonal antibodies. Tailoring the methodological protocol for each of the two proliferation markers was a necessary prerequisite for obtaining optimal results in tubular sections and whole-mounts. A-, I- and B-spermatogonia displayed PCNA-positive nuclei, except during meta-, ana- and telophases of mitosis. PCNA-negative nuclei in the basal tubular compartment, excluding those from non-cycling Sertoli cells, belonged to the spermatogonia precursor cell line. However, only about 30%, 45% and 47% of the respective A-, I-, B-spermatogonia had positive nuclei after exposure to the MIB-1 antibody directed against the Ki-67 protein. Spermatogonia with MIB-1-negative nuclei represented cells in the G1-phase. Both antibodies reacted intensely with the nuclei of preleptotene primary spermatocytes. PCNA reactivity was also present during leptotene through pachytene. Ki-67 protein expression was absent during leptotene and zygotene but was again encountered during pachytene and meiosis I and II. Anti-PCNA/anti-protein gene product 9.5 double-labelling indicated that the transition from spermatogonia precursor cells into A1-spermatogonia is not strictly synchronized in a given tubular segment, a possible reason for the flexibility in A-spermatogonial propagation seen in bovine seminiferous tubules.

Extracts from Petasites hybridus were found to inhibit peptido-leukotriene biosynthesis in isolated peritoneal macrophages. Chemical analysis by gas chromatography coupled with mass and infrared spectroscopy facilitated the identification of three isomeric oxopetasan esters, petasin, and isopetasin as the main compounds of these extracts. Fractionations obtained by column chromatography of the most effective extract indicated a correlation between peptido-leukotriene biosynthesis inhibition and the content of isopetasin, a sesquiterpene ester of isopetasol and angelic acid, as well as the isomeric oxopetasan esters. Petasin, a structural isomer of isopetasin, however, was found to be inactive. It may even reduce the peptido-leukotrine inhibitory effect of isopetasin. It is concluded that isopetasin and the oxopetasan esters in Petasites hybridus inhibit the biosynthesis of the vasoconstrictive peptido-leukotrienes. This effect may contribute to some of the medicinal properties of Petasites hybridus extracts such as, e.g., gastroprotection and spasmolytic activity.

The configuration and distribution of bovine spermatogonia, preleptotene primary spermatocytes and Sertoli cells in the basal seminiferous tubular compartment have been studied by means of whole-mount preparations, immunohistochemistry and quantitative morphology. Three types of spermatogonia (Sg) can be identified. Large A-spermatogonia are irregularly distributed in the tubular periphery. Following the period of propagation of the A-spermatogonia, an interconnected meshwork of medium-sized spermatogonia with different cytogenetic potency is observed. Although the majority of the medium-sized spermatogonia are kinetically of the I type and divide to produce small B-spermatogonia, some members of the medium-sized population are seen in a growth phase and differentiate into large A-spermatogonia. These mark the beginning of a new round of spermatocytogenesis. Only one generation of B-spermatogonia divides into preleptotene primary spermatocytes. The architectural arrangement of multiplying spermatogonia in circles or rows is primarily the result of the distribution of the Sertoli cells. Spermatogonial multiplication is not strictly coordinated with the stages of the seminiferous epithelial cycle. Spermatogonial degeneration amounts on average to 3.6% and has therefore no decisive impact on the yield of primary spermatocytes.

The distribution of F-actin, vimentin and α-tubulin was studied immunohistochemically in bovine seminiferous and straight testicular tubules, rete testis and intertubular tissue during postnatal development. Sites of antigenicity were detected by ABC immunoperoxidase technique and visualized by metal-enhanced deposition of diaminobenzidine. Within the seminiferous epithelium, F-actin appears at 20 weeks and is found in adult Sertoli cells as part of specialized cell contacts. In peritubular cells, F-actin increases gradually from 4 to 30 weeks when the adult concentration is achieved. After 20 weeks, subepithelial fibro-blasts of the mediastinum testis start to express F-actin and at 52 weeks, a thick layer of positive myofibroblasts is seen beneath the epithelia of rete testis and straight testicular tubules. Testicular macrophages and light intercalated cells (LIC) are also characteristically decorated following F-actin immunoreaction. Vimentin is localized in perinuclear position in pre-Sertoli cells of 4-20 weeks and in adult Sertoli cells. During the period of transformation from pre-Sertoli to Sertoli cells, the perinuclear vimentin coat is absent. The epithelia of rete testis and straight tubules exhibit a strong vimentin immunoreaction in their basal parts. This specific pattern does not change from 4 weeks to adulthood. α-Tubulin is absent in 4-week-old seminiferous tubules. At 8 weeks, the perinuclear area of pre-Sertoli cells reacts positive. The α-tubulin content increases in these cells continuously, and from 30 weeks on nearly the entire supranuclear cytoplasm of Sertoli cells is heavily decorated. The epithelial of rete and straight tubules display a growing number of α-tubulin-positive cells from 4 to 40 weeks. From then on, nearly all epithelial cells contain α-tubulin, particularly in a narrow zone beneath their lateral cell borders.

Extracts from Petasites hybridus are in therapeutic use for more than 2000 years. They have been claimed to improve gastrointestinal pain, lung-diseases such as asthma and cough, as well as spasms of the urogenital-tract. We have investigated these claims in animal models of gastro-intestinal ulcers and confirmed that alcoholic extracts of Petasites hybridus block the ethanol-induced gastric damage and reduce small intestinal ulcerations induced in rats by indomethacin. Searching for a possible mode of action, we found that ethanolic extracts of Petasites hybridus inhibit the peptido-leukotriene biosynthesis in mouse peritoneal macrophages but leave prostaglandin biosynthesis unimpaired. We conclude that the active principle could be useful in the treatment of gastro-intestinal human diseases and that Petasites hybridus may contribute to the inhibition of calcium-dependent processes such as leukotriene biosynthesis.