👤 C A Suarez-Quian

The objective of this study was to compare the expression of Col1a1, Col1a2, and procollagen I in the seminiferous tubules of immature and adult mice and to characterize the cellular expression pattern of procollagen I in germ cells during spermatogenesis in order to provide necessary groundwork for further functional studies in the process of spermatogenesis. Microarray analysis demonstrated that Col1a1 and Col1a2 were abundantly expressed in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice, and the expression levels of Col1a1 and Col1a2 mRNA were validated using a semi-quantitative RT-PCR assay. Western blot analysis further confirmed that procollagen I was expressed at a higher level in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice. Immunohistochemical analysis revealed that type A spermatogonia were positive for procollagen I in the testis of 6-day-old mice, whereas Sertoli cells were negative for this protein. The in vivo procollagen I staining in type A spermatogonia was corroborated in spermatogonia exhibiting a high potential for proliferation and the ability to form germ cell colonies in in vitro culture. Moreover, procollagen I was also detected in type A spermatogonia, intermediate spermatogonia, type B spermatogonia, and preleptotene spermatocytes in the adult mouse testes, but positive staining disappeared in more differentiated germ cell lineages detaching from the basement membrane, including leptotene spermatocytes, pachytene spermatocytes, round spermatids and elongated spermatids. These data suggest that Col1a1, Col1a2 and procollagen I are associated with type A spermatogonia and play a potential role in mediating the detachment and migration of germ cells during spermatogenesis. Show less

Sertoli cell nuclei are characterized by deep invaginations and, in addition, the orientation of the nuclei with respect to the wall of the seminiferous tubules varies during the cycle of the seminiferous epithelium. These events may be the result of cytoplasmic filaments acting at the level of the nuclear capsule and may represent significant changes in Sertoli cell activity. Thus, a study was performed to characterize the nature of the perinuclear filaments of Sertoli cells in vivo and in vitro. In Sertoli cells in vivo, microtubules and microfilaments were often detected in the perinuclear cytoplasm, and these cytoskeletal components were observed to course either parallel to, or abut at, the nuclear capsule. In Sertoli cells in vitro, the nuclear infoldings are retained and the perinuclear cytoskeleton was shown to contain microtubules, f-actin, and intermediate filaments. A fixation-permeabilization protocol employing tannic acid-saponin was used and it significantly enhanced the preservation of cytoskeletal components. The presence of f-actin was demonstrated by using the S1 fragment of muscle myosin to decorate the microfilaments. Treatment of the cultured cells with either microtubule or f-actin depolymerizing agents had no effect on nuclear shape. Thus, at present, the function of the prominent perinuclear cytoskeletal components remains unknown. Show less

A morphological and immunocytochemical study of the Golgi apparatus in pachytene spermatocytes was performed in an effort to correlate the structure and function of this organelle during meiotic prophase. In stages I-III of the cycle, the Golgi complex of pachytene spermatocytes is a flattened discoid, 0.5-1 microns in diameter, composed of vesicles interspersed with classically described Golgi cisternae. During subsequent maturation of pachytene spermatocytes (stages IV-XIII), the size of the Golgi complex increases significantly, attaining a size of 2-3 microns. However, unlike pachytene spermatocytes of stages I-III, the majority of the Golgi complex of more mature spermatocytes is characterized by an abundance of distinct stacks of cisternae interspersed with numerous vesicles and tubules. The composition of the Golgi complex was also studied by using two monoclonal antibodies that recognize either the cis or the trans Golgi cisternae, respectively, and employing biotin-streptavidin-peroxidase immunocytochemistry in 5 micron frozen sections of testes. Immunodetection of the distinct cisternae revealed that the increase in size of the Golgi complex during maturation of pachytene spermatocytes was due predominantly to an accumulation of trans Golgi; the amount of cis Golgi remained unchanged. The morphological data presented in this study are consistent with an heightened secretory activity of pachytene spermatocytes during their maturation. In addition, the increase in size of the Golgi apparatus during the extensive prophase of pachytene spermatocytes may suggest that the mechanism employed by germ cells to partition the Golgi complex during the first division of meiosis varies significantly from that of somatic cells undergoing mitosis. Show less

Epidermal growth factors receptor (EGFR) was localized immunocytochemically in the testes of mature and immature rats and immature monkeys. One polyclonal antibody, recognizing the intracellular domain (RK2) of the receptor, was used to carry out the EGFR immunodetection. The RK2 antibody revealed the presence of the EGFR predominantly in Sertoli cells of mature and immature rats and of immature monkeys, although limited interstitial localization of the EGFR was also discerned in the mature rat. In cultured Sertoli cells of immature rats, grown in the absence of epidermal growth factor (EGF), the EGFR was randomly distributed at the cell surface, whereas after the addition of EGF the receptor became aggregated into distinct focal regions. In addition, EGFR of cultured Sertoli cells exhibited autophosphorylation activity upon stimulation with EGF, but failed to transcytose iodinated EGF across a permeability barrier formed by the cultured cells. Instead, all of the added iodinated EGF was internalized and degraded. Show less