In the present study the distribution of various sugar residues in the cells of the male gonad during postnatal organogenesis was examined employing eight lectin-horseradish peroxidase conjugates (BS-I, ConA, DBA, PNA, RCA-I, SBA, UEA-I, WGA) on paraffin-embedded testicular tissue. The tissue was obtained from bull calves and young bulls of recorded age (4, 8, 16, 20, 25, 30, 40 and 52 weeks) and two adult bulls. During the whole observation period, lectin affinity in the developing testicular tubules was restricted to the germ cell line, while the Sertoli cells and their precursors remained completely unstained. DBA, a lectin with specific affinity to alpha-D-GalNAc, served as a selective marker for prespermatogonia (PSG), the only precursors of bovine spermatogonia until the onset of spermatogenesis at week 30. alpha-D-GalNAc, detected in the PSG Golgi zone and its vicinity, seems to play an important role during PSG proliferation and migration in the prepuberal testis. Concomitant with the differentiation of PSG into spermatogonia, the binding intensity of DBA to the Golgi zone of these cells decreased. After the gradual onset of spermatogenesis, the lectins revealed staining of Golgi complexes of most germ cell stages. Glycosylation of the cell components takes place in the Golgi complex, which explains the strong affinity of the lectins to this cell compartment. Inner and outer membrane of the acrosomal complex of spermatids, especially during Golgi and cap phase of spermiogenesis, were intensely stained with PNA, RCA-I and SBA. This staining disappeared in the maturation phase at the latest and indicates a role of terminal D-Gal-(beta 1----3)-D-GalNAc, D-Gal and D-GalNAc during the formation of the sperm head and intraepithelial orientation of the spermatid. Other parts of the spermatid, such as the anulus and the cytoplasmic droplet, exhibited D-Gal, D-GlcNAc or sialic acid and D-GalNAc. In the intertubular tissue BS-I, RCA-I and UEA-I bound to vascular endothelia. Components of the intertubular extracellular matrix were stained with ConA (alpha-D-Man), RCA-I (D-Gal), UEA-I (alpha-L-Fuc) and WGA (D-GlcNAc or sialic acid).

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.

The bovine male germ cell population was studied over the entire period from testicular differentiation in the embryo through onset of spermatogenesis in the pubertal calf. Germ cells were identified by protein gene product 9.5 immunohistochemistry and characterized by their ultrastructure. The proliferation pattern of germ cells was studied with immunohistochemical anti-Ki 67 and anti-proliferating cell nuclear antigen reactions. Germ cells with a high proliferation rate are observed from day 50 p.c. to day 80 p.c. These cells are in transition from primordial germ cells to prespermatogonia. From day 80 p.c. until approximately the 15th postnatal week the germ cells present are identified as prespermatogonia. From day 80 p.c. to day 200 p.c. germ cell multiplication decreases continuously; then the prespermatogonia enter a phase of relative mitotical quiescence that lasts until the 4th postnatal week. Between the 4th and the 15th postnatal week, testicular tubular diameters grow from 40 to 80 microm and the prespermatogonia resume their proliferation. In seminiferous tubules with diameters between 80 and 120 microm, found in animals between 18 and 27 weeks of age, a central lumen is normally still absent. During this period germ cell proliferation reaches a second maximum. The cells involved represent the members of the spermatogonia stem and precursor cell line kinetically interpolated between the prespermatogonia and the first differentiating A-spermatogonia. This second phase of prepubertal germ cell multiplication coincides with the period when the pre-Sertoli cells transform into adult-type Sertoli cells and enter the G0-phase for the rest of life.

The distribution pattern of actin, desmin, vimentin and tubulin in the ovine testis during postnatal development was investigated by means of immunohistochemical methods. The postnatal development of the ovine testis can be divided into five phases. Phases I through III represent the prepubertal period, phase IV puberty and phase V the postpubertal adult stage. In peritubular cells alpha-smooth muscle actin is present, its amount increasing with advancing age of the animals. Structural F-actin is localized in peritubular myoid cells and Sertoli cells, of the adult testis. In Sertoli cells structural F-actin-positive material is observed at the level of the Sertoli-Sertoli junctions, at contact sites of Sertoli cells with primary spermatocytes and in the immediate vicinity of elongating spermatid heads during the acrosome phase of spermiogenesis. Desmin is present in intertubular and peritubular cells during the early prepubertal period, but vanishes completely as soon as the animals reach puberty. Vimentin is present in the cytoplasm of prespermatogonia I, but disappears when these change into prespermatogonia II. In prepubertal supporting cells the vimentin content increases, and in the adult the positive filament bundles create a flame-like pattern around the unstained nucleus. Cyclical variations during the seminiferous epithelial cycle are not observed. Expression of alpha-tubulin is found in the cytoplasm of prespermatogonia I and to a lesser extent in prespermatogonia II and spermatogonia. The immunoreaction is also seen in the microtubules of the axonema and manchette of elongating spermatids. The histochemical demonstration of the high alpha-tubulin concentration in supporting and Sertoli cells is an excellent method for studying changes of cellular shape and size during ontogenesis as well as during the seminiferous epithelial cycle.

The bovine tubouterine junction is composed of three parts (terminal tubal segment, transition region proper, uterine apex) and follows a sigmoidal course displaying a tubal and an uterine curvature. In the terminal tubal segment, 4-8 primary longitudinal folds and a system of lower secondary folds, ridges and chords project into the centrally located lumen. The transition region proper possesses a slit-like lumen because of the existence of a thick mucosal pad containing the first uterine glands. The longitudinal primary folds of the tube broaden, flatten and start to diverge when they reach the transition region proper. The mucosal pad and broadened folds are heavily vascularized. A system of lateral outpocketings with blind ends pointing in an ampullary direction develops between the primary and secondary folds, the ridges and chords of the terminal tubal segment and transition region proper. From the bottom of these outpocketings, short tubulo-alveolar crypts originate. The mucosa of the uterine apex forms low transversal ridges. The musculature of the bovine tubouterine junction is divided into a continuous circular or spiral intermediate layer, flanked by inner and outer longitudinal layers. The outer longitudinal layer is incomplete in the terminal tubal segment but increases in thickness to form a continuous stratum in the uterine apex. An inner longitudinal layer occurs only in the terminal tubal segment where it is best developed in the bases of the primary longitudinal folds. The simple columnar surface epithelium of the tubouterine junction contains ciliated and non-ciliated cells. The former undergo cyclical changes, and increase during estrus and postestrus. During proestrus, groups of non-ciliated cells display bulbous apical protrusions. During proestrus and estrus, circumscribed epithelial lesions expose the underlying basal lamina.