Medizin - Open Access LMU - Teil 08/22 Archiv

We have used a recently characterized rabbit antiserum against basic fibroblast growth factor (bFGF), which recognizes various forms of bFGF, to examine the presence and localization of bFGF in the testes of adult rats and mice and the 5-day-old rat. In Western blots of testicular homogenates of adult rats and mice and immature rats, immunoreactive single bands at approximately 30 kDa were detected. Immunocytochemistry revealed specific staining restricted to the tubular compartment. In 5-day-old rat testes, prespermatogonia were immunoreactive. The cytoplasm of pachytene spermatocytes was heavily stained in the adult testes of both species. Staining of these cells became evident around stage IV/V, was prominent in stage VII through IX and declined about stage XII/XIII (rat) or X-XI (mouse). Staining was seen in type A spermatogonia and in elongating spermatids in their cytoplasmatic lobes and along their flagellae. Sertoli cells were unstained. We propose that the pluripotential growth factor bFGF could be involved in the regulation of germ cell proliferation and differentiation in the adult and immature testis.

In the adult mammalian ovary morphogenesis and differentiation processes are under hormonal control and, thus, occur in a highly regulated way during the sexual cycle. Cell-cell interactions, such as cell adhesion and cell separation, are crucial during these events. Here we show that the ovarian endocrine cells, which are prototypes of steroid-producing cells, express neural cell adhesion molecules (NCAMs). The combined use of in situ hybridization histochemistry, immunocytochemistry at the light and electron microscope levels, S1 nuclease protection assays, and Western blotting revealed that in the ovary of the adult rat during the estrus cycle and pregnancy, NCAM mRNA and the 140-kDa isoform of this protein are expressed mainly in granulosa cells of growing preantral and antral follicles and in corpora lutea. Since the granulosa cells lining the forming antrum and the antral fluid were strongly immunoreactive, a role for NCAM in the formation of the follicular antrum is proposed. The expression of NCAM was also associated with luteal cells of the active corpus luteum, indicating a role for NCAM in the morphogenesis of this endocrine compartment. Moreover, thecal cells of large follicles and hypertrophic thecal cells of atretic follicles expressed NCAM, as did interstitial cells, which are derived from thecal cells of atretic follicles. We propose that the adhesion molecule, NCAM, is an important factor involved in the recognition and intercellular interaction of ovarian endocrine cells and, thus, participates in the regulation of the cyclic remodeling processes of the ovarian endocrine compartments

The effects of Ca2+ and GTP on the release of Ca2+ from the inositol 1,4,5-trisphosphate (IP3) sensitive Ca2+ compartment were investigated with digitonin permeabilized rat pancreatic acinar cells. The amount of Ca2+ released due to IP3 directly correlated with the amount of stored Ca2+ and was found to be inversely proportional to the medium free Ca2+ concentration. Ca2+ release induced by 0.18 μM IP3 was half maximally inhibited at 0.5 μM free Ca2+, i.e. at concentrations observed in the cytosol of pancreatic acinar cells. GTP did not cause Ca2+ release on its own, but a single addition of GTP (20 μM) abolished the apparent desensitization of the Ca2+ release which was observed during repeated IP3 applications. This effect of GTP was reversible. GTPγS could not replace GTP. Desensitization still occurred when GTPγS was added prior to GTP. The reported data indicate that GTP, stored Ca2+ and cytosolic free Ca2+ modulate the IP3 induced Ca2+ release. EGTA, Ethylene-glycol-bis (2-aminoethylether)-N,N,N′,N′- tetra acetic acid; GTPγS, Guanosine 5′-O-[3-thio]triphosphate; GDPβS, Guanosine 5′-O-[2-thio]diphosphate; IP3, Inositol 1,4,5-trisphosphate; IP2, Inositol 1,4-bisphosphate; IP4, Inositol 1,3,4,5-tetrakisphosphate; MOPS, Morpholinopropane sulfonic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; pHMB, Parahydroxymercuribenzoate

The development of the spinal cord involves the proliferation of neurons, their migration to well-defined areas, fiber outgrowth and synapse formation. The present study was designed to correlate the spatiotemporal pattern of expression of synaptophysin, an integral membrane protein of small synaptic vesicles, with these basic processes occurring during the embryonic development of the rat spinal cord. Thoracic segments of spinal cords from embryonic days 12, 14, 16, 18, 20 and of adult spinal cords were studied. S1 nuclease protection assays and immunoblots revealed minute amounts of specific mRNA and synaptophysin at embryonic day 12. There was a steep increase of mRNA between embryonic days 14 and 16, after which levels reached a plateau. A rise in the amount of synaptophysin in the spinal cord occurred between embryonic days 12 and 14, and the levels changed only slightly until the end of embryonic development. Even higher levels of synaptophysin, found in the adult spinal cord, may indicate that its biosynthesis continued after birth. In situ hybridization histochemistry revealed the localization of specific synaptophysin mRNA in the neuroepithelium. However, immunocytochemistry failed to detect synaptophysin in the neuroepithelial cells. Following migration of the neuroblasts, synaptophysin was found in neurons concomitantly with the onset of fiber outgrowth. Thus, already at embryonic day 12, outgrowing fibers of the dorsal root sensory neurons and of motoneurons were synaptophysin positive. From embryonic day 14 throughout the prenatal period, strong synaptophysin immunoreactivity was seen in the ventrolateral and dorsal parts of the marginal layer. Most likely this staining pattern indicates transient functional synaptic contacts because, in the adult spinal cord, the corresponding region, the white matter, exhibited only faint synaptophysin immunoreactivity. In the intermediate layer of the embryonic spinal cord, which corresponds to the gray matter of the adult spinal cord, synaptophysin-positive fibers were observed prior to the formation of functional synapses. The latter are most likely permanent, since synaptophysin in the adult spinal cord is mainly confined to the gray matter. Our data (i) show transcription and translation of synaptophysin within the neurons of the spinal cord and correlate these processes with proliferation, migration, fiber outgrowth and the formation of transient or permanent synapses, and (ii) prove that synaptophysin is a marker for fiber outgrowth in addition to synapse formation.

The carcinoembryonic antigen (CEA) gene family belongs to the immunoglobulin supergene family and can be divided into two main subgroups based on sequence comparisons. In humans it is clustered on the long arm of chromosome 19 and consists of approximately 20 genes. The CEA subgroup genes code for CEA and its classical crossreacting antigens, which are mainly membrane-bound, whereas the other subgroup genes encode the pregnancy-specific glycoproteins (PSG), which are secreted. Splice variants of individual genes and differential post-translational modifications of the resulting proteins, e.g., by glycosylation, indicate a high complexity in the number of putative CEA-related molecules. So far, only a limited number of CEA-related antigens in humans have been unequivocally assigned to a specific gene. Rodent CEA-related genes reveal a high sequence divergence and, in part, a completely different domain organization than the human CEA gene family, making it difficult to determine individual gene counterparts. However, rodent CEA-related genes can be assigned to human subgroups based on similarity of expression patterns, which is characteristic for the subgroups. Various functions have been determined for members of the CEA subgroup in vitro, including cell adhesion, bacterial binding, an accessory role for collagen binding or ecto-ATPases activity. Based on all that is known so far on its biology, the clinical outlook for the CEA family has been reassessed.