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Abd-Elmaksoud, Ahmed (2005): Morphological, Glycohistochemical, and Immunohistochemical Studies on the Embryonic and Adult Bovine Testis. Dissertation, LMU München: Tierärztliche Fakultät



In the present study, the testes of 32 bovine embryos with different crown-rump length (2.5- 90 cm CRL) and of 15 sexually mature bulls (Deutsches Fleckvieh) were investigated using light- and electron microscope as well as glycohistochemical and immunohistochemical methods. The gestation period was divided into 3 stages; early, mid, and late gestation. Developmental changes in the testicular morphogenesis were therefore analyzed in details during these phases. Generally, embryonic development of bovine testis involves the same mechanism described in other mammals. At the first stage of this study (2.5 cm CRL/43 dpc), the anlage of the testes protruded to the coelomic cavity as paired bean-shaped structures on either side of the dorsal mesentery medial to the mesonephros. It consists of primitive testicular cords, interstitium, and rete testis blastema. Proceeding with fetal age, these basic testicular structures are further differentiated. The tunica albuginea is separated into two layers: an outer fibrous layer (tunica fibrosa) with some mesenchymal cells, numerous fibroblast, and much fibrous content and an inner cellular layer with several blood vessels (tunica vasculosa). The testicular cords are surrounded by a marked basal lamina and peritubular cells and lined by two types of cells: a large number of dark polygonal cells with irregular nuclei, pre-Sertoli cells and small number of large light round cells with relatively round nuclei, the prespermatogonia. The average number of the germ cells per cross section of cord increases, particularly form 3.5 to 14 cm CRL, resulting in a germ cell maximum at the end of this stage (14 cm CRL). Although most of the germ cells are located toward the periphery of the cord, some are also found in the center. Pre-Sertoli cells form a complete layer at the periphery of the cords. Generally, these cells are irregular in shape and numerous but considerably smaller than the germ cells. Unlike prespermatogonia, mitotic figures are seen in pre-Sertoli cells during the whole embryonic life. As a consequence of the expansion in the interstitium, the seminiferous cords are progressively separated from each other. The testicular interstitium is rapidly differentiated and is composed of several islets or clusters of polygonal Leydig cells, peritubular flattened cells surrounding the testicular cords, connective tissue cells, and numerous blood vessels. In the present study, fetal Leydig cells were first recognized at 3.5 cm CRL. Thereafter, the average number of these cells is rapidly increased to attain their maximum with the end of the first gestation period (14 cm CRL). This generation of Leydig cells however dedifferentiates progressively with developmental age. A continuous system of basal lamina joins the testicular cords with rete strands from 10 cm CRL and onwards. This system establishes the first connection between these two testicular components via ill-developed uncanalized straight tubules (tubuli recti). Rete testis channels are lined by simple layer of cuboidal epithelium with round nuclei occupying most of the cytoplasm and enclosed by well-defined basal lamina. The adult bovine testis is enclosed by a connective tissue capsule, tunica albuginea, composed predominantly of collagen fibers and few elastic fibers. Most of the testicular parenchyma is made up of the convoluted seminiferous tubules (tubuli seminiferi contorti), two-ended convoluted loops, with both ends opening into the rete testis via specialized terminal segments. The seminiferous tubules of sexually mature bulls are enclosed by a distinct lamina propria and are lined by two cell populations, non-proliferating Sertoli cells and highly proliferating spermatogenic cells. The bovine lamina propria consists of basal lamina, collagen and elastic fibers, and 3-5 layers of partially overlapping myofibroblasts. Additionally, fibrocytes, collagen fibrils, and fibroblasts-like cells form the outermost border of the tubulus. Sertoli cells are easily identifiable elements of the seminiferous epithelium. Adult Sertoli cells are large irregularly shaped cells with their broad bases resting on the basal lamina while the remaining cytoplasmic processes extend upward to the tubular lumen. They are characterized by round or oval euchromatin-rich nuclei situating in the basal portion near the basal lamina of the seminiferous tubules. Adult bovine germ cells are present in four morphologically different groups, i.e., spermatogonia, spermatocytes, spermatids, and spermatozoa. The seminiferous cycle stages are identified using changes in the germ cell nuclei as well as location and shape of spermatids. According to this method, eight stages are defined in the seminiferous epithelium of bovine. The interstitial or intertubular tissue of adult bovine testis consists of Leydig cells, macrophages, scattered lymphocytes and plasma cells, and contains numerous blood and lymph vessels. Not all Leydig cells have contact to blood or lymph capillaries. The excurrent duct system of the adult bovine testis consists of terminal segment of the convoluted seminiferous tubules, straight tubules, and rete testis. The terminal segment can be further subdivided into a proximal (transitional) region, middle portion, and distal part (terminal plug). The proximal region is lined by typical Sertoli cells while the last two parts are lined by modified Sertoli cells. The tubulus rectus of adult bovine testis is composed of three morphologically different regions: a proximal cup-shaped region, a middle narrow stalk, and a distal festooned portion. The rete testis is a complicated centrally positioned meshwork of intercommunicating channels that lies within the mediastinum testis parallel to the long axis of epididymis. The simple cuboidal epithelium of straight tubules and rete testis is shown to contain some lymphocytes and macrophages. The cellular distribution of glycoconjugates within the fetal and adult bovine testis was investigated using thirteen (ConA, PSA, LCA, PNA, GSA-I, ECA, DBA, SBA, HPA, VVA, WGA, UEA-I, LTA) different fluorescein isothiocyanate (FITC) conjugated lectins. In fetal testes, detection of sugar moieties by lectins was carried out on Bouin õ s-fixed paraffin-embedded sections while in adult it was performed on both Bouin õ s-fixed paraffin-embedded and acetone-fixed frozen sections. Only five lectins (PSA, PNA, GSA-I, DBA, WGA) showed a positive reaction in the embryonic testes. PNA, GSA-I, DBA, and WGA were detected in the germ cells whereas PSA, DBA and WGA labeled the fetal Leydig cells. None of the lectins used was observed in the pre-Sertoli cells. Further on, some lectins were seen in tunica albuginea (PSA, PNA, GSA-I, WGA), basal lamina of testicular cords (PSA, WGA), interstitial blood vessels (PSA, GSA-I, WGA), mediastinum testis (PSA, PNA, WGA) and rete testis epithelium (PNA). In adult animals, spermatogonia and spermatocytes were positively stained with PSA, LCA, DBA, SBA, and VVA. All the lectins investigated except that of the fucose-binding lectin (UEA-I and LTA) were definitely detected in the acrosome of round and elongated spermatids. These results indicate a role for carbohydrates in spermiogenesis. Apical Sertoli cells processes and Leydig cells were weakly stained with PSA and LCA as well. DBA binding sites were also seen in the Leydig cells. Immunohistochemical studies were performed using the Avidin-Biotin-Peroxidase Complex (ABC) method for localization of fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), S-100, laminin, alpha-smooth muscle actin (á -SMA), vascular endothelial growth factor (VEGF), connexin 43 (Cx43), CD4, CD8, CD68, angiotensin-converting enzyme (ACE), and galactosyltransferase (GalTase) in the bovine testis. The expression of FGF-1 and FGF-2 was further investigated in the adult bovine testis using in situ hybridization and PCR. Immunohistochemically, FGF-1 was seen in the Sertoli cells, Leydig cells, endothelium of the blood vessels, and epithelium of straight tubules and rete testis of fetal and adult testis. It was additionally detected in spermatogonia and spermatids of sexual mature animals. FGF-2 exhibited a striking positive reaction in fetal (from 6 to 30 cm CRL) and adult Leydig cells. Moreover, it showed marked reaction in the endothelium of blood vessels and in the epithelium of tubulus rectus and rete testis. FGF-2 was also localized in some spermatogonia, and myofibroblasts. By means of in situ hybridization, FGF-1 and FGF-2 mRNA were found in Leydig and Sertoli cells as well as in the modified Sertoli cells of the terminal segment. FGF-1 transcripts were additionally recognized in the straight tubules and rete testis epithelium. Distinct S100 immunostaining was observed in the Sertoli cells, endothelium of blood vessels and in the rete testis epithelium of fetal and adult testis. Laminin was localized to the basal lamina of seminiferous tubules, blood vessels, myofibroblasts, and rete testis. Although á -SMA was detected in smooth muscle cells of the blood vessels, no immunoreactivity was seen in the peritubular cells during the whole gestation period. The myofibroblasts surrounding the seminiferous tubules and rete testis showed intense positive reaction for á -SMA in the adult testis. VEGF was detected in the acrosomes of the elongating spermatids. Connexin 43 was localized to gap junctions between Leydig cells in the fetal and adult life as well as to the seminiferous epithelium apical to spermatogonia and basal to spermatocytes, a position correlating with Sertoli-Sertoli cell junctions. The detection of cells positive for CD4, CD8, CD68 within the adult testis interstitium clearly indicate the presence of lymphocytes and macrophages within this testicular compartment. GalTase showed striking positive reaction in the Golgi complex of Sertoli cells, Leydig cells, and some spermatocytes as well as at the cell membrane of elongating spermatids and in the simple cuboidal epithelium of rete testis. ACE positive reaction was found in the prespermatogonia (only at 6-10 cm CRL) and in fetal and adult testicular blood vessels. The functional significance of these immunocytochemically-demonstrated proteins is discussed.