Emilia Franchi

Evidence that a Ca2+ chelator and a calmodulin blocker interfere with the structure of inter-Sertoli junctions

Ca2+ dependence of tight junction structure has been well documented in cultured epithelial tissues, and regulatory mechanisms have been identified. To analyse the possible control exerted on inter-Sertoli junctions, we exposed guinea-pig seminiferous tubules to the presence of a Ca2+ chelator (EGTA) and to a calmodulin blocker (Trifluoperazine, TFP) in vitro, for times ranging from 30 to 120 min. We observed the morphology of junctional complexes and the basal cytoplasmic regions in sections and replicas. Sertoli cell response to Ca2+ depletion involved several events: retraction of cells toward the base of the tubule and a consequent stretching of the points of fusion, augmented density of the cytoplasm, and destabilization of the array of intramembrane particles. Exposure of tubules to TFP resulted in disruption of the interactions between actin filaments and membrane junctional specialization, as well as a disorganization of other cytoskeletal elements. Thus, in vitro, junction integrity appears to be related to Ca2+ level, and Ca2+ depletion apparently interferes with Ca2+ distribution inside the cell and on microfilaments involved in junction regulation. Our results do not provide direct evidence for any particular mechanism of action of TFP, but a multiple effect is evident. TFP, which affects Ca2+ regulation and membrane fluidity, probably acts indirectly on junction-associated filaments. Both the experimental conditions tested suggest a Ca2+-mediated regulatory role of microfilaments of this complex junction.

Morphological evidence of a permeability barrier in urodele testis.

The testis of Triturus cristatus is multilobular and each lobe is composed of individual transient cysts in which Sertoli cells develop parallelly to the maturation of germ cells. The junctions between Sertoli cells in immature and mature cysts were studied with lanthanum used as a tracer and surface replicas. Immature cysts appeared as an “open” compartment, as they were penetrated by the tracer, and both sections and replicas showed that Sertoli junctions were differentiating. Mature cysts appeared as a “tight” compartment, as the tracer never reached the lumen and was stopped within inter-Sertoli specializations. Replicas evidenced ridges and grooves, coincident across face transition and continuous over a long distance around the surface of Sertoli membranes. These findings reinforce the idea that a barrier exists, regardless of the general organization of the gonad, and its establishment is related to the differentiation of the haploid male cells.

Dynamic aspect of inter-sertoli junctions in monkeys

The extensive tight junctions that link Sertoli cells were studied in testes of monkeys (Cercopithecus aetiops) in conventional thin sections, lathanum-perfused specimens, and free-fracture replicas. The results demonstrated that tight junctions linked Sertoli cells from the base, and in conventional sections they were identifiable as punctate points of occlusions. Colloidal lanthanum penetrated many of these points. Freeze-fracture replicas showed a large number of strands and displayed various degrees of complexity; this variability was associated with the exposed area. Areas with less organized and regularly spaced strands, breakdown of strands, and organizing and disorganizing particles were related to the dynamic aspect of these junctions, which continuously disassembled and reorganized to allow the transfer of germ cells from the basal to the adluminal compartment. This report hypothesizes that the blood—testis barrier may result from the summation of the resistances contributed by a large number of moderately leaky structures arranged in series. The coexistence of septate with tight junctions was never observed, since all strands were of the tight junction type. These were frequently associated with communicating junctions.

Morphological evidence for calcium stores at Sertoli-Sertoli and Sertoli-spermatid interrelations

Potassium pyroantimonate technique has been employed to localize calcium ultrastructurally at Sertoli-Sertoli and Sertoli-spermatid junctional specializations. Identification of Ca++ as the major cation precipitated was performed by EGTA sensitivity and X-ray microprobe analysis. Ca++ deposits have been demonstrated in the endoplasmic reticulum cisternae underlying junctional complexes and along the plasma membranes of both Sertoli and germ cells.

Ultrastructure of acrosomal malformations in men with obstructive azoospermia.

Testicular biopsies from ten infertile men with obstructive azoospermia were evaluated by light and electron microscopy. Light microscopic analysis revealed normal testicular pattern with active spermatogenesis and many spermatozoa. At ultrastructural level the majority of early spermatids presented acrosomal abnormalities that become evident during the approach of the acrosomal vesicle to the anterior pole of the nucleus, and in subsequent maturational stages; these spermatids continue their development until mature spermatozoa with malformed acrosomes. The type of malformation was very similar in all cases observed and was associated with the same type of seminiferous tubule pattern in patients with the same type of azoospermia. The abnormalities at acrosomal level were related to an abnormal differentiation rather than to degenerative changes. Acrosomal disturbance did not influence nucleus morphology or chromatin condensation.

Thick filaments and paramyosin of annelid muscles.

Thick filaments from annelid muscle were observed by positive and negative staining of the glycerinated sample, which was homogenized in a relaxing medium containing ATP. The preservation of myosin on the filament surface was strictly related to very high concentration of ATP (12 m M .). The 144 A periodicity was scarcely visible. When the ATP in the homogenate was at low concentration (less than 3 m M ), the underlyng periodic structure of paramyosin appeared. Purified paramyosin cores (at low pH and high ionic strength) presented a striation with a 144 A pitch. The Bear-Selby net pattern was also present, even in a small number of filaments. Paramyosin was extracted and purified from fresh muscles of Lumbricus terrestris and Sipunculus nudus . The purified protein exibited a single peak in the ultracentrifuge, and the intrinsic sedimentation constant was 3.29 S for Lumbricus and 3.19 S for Sipunculus . After dialysis with divalent cations, paracrystals were obtained, most of which showed a 144 A periodicity; sometimes, paracrystals with a different band pattern appeared. In all tactoids observed a 48 A subperiod was evident, as in the native isolated cores.

Ultrastructure of Leydig cells in the African green monkey.

Active Leydig cells of Cercopithecus aetiops occur singly or in clusters in the intertubular areas. They are characterized by a polymorphic smooth endoplasmic reticulum (SER), constituted of tubules, whorls of cisternae, and concentric rings surrounding lipid droplets. Some aspects of the endoplasmic reticulum, described in other mammals during nonbreeding periods, demonstrate that the variable forms of SER are not necessarily linked to cell activity. Mitochondria are variable in form and associated with the elements of SER usually observed in steroid-producing cells. The presence of thin filaments between lipid droplets and mitochondria may represent the morphologic aspect of their hypothesized function of cholesterol transport to mitochondria. Golgi system-derived vesicles appear much more numerous and easy to recognize in replicas than in sections. This observation may confirm a secretory process, as suggested by previous observations.

Ultrastructural investigation of spermiogenesis in Peripatopsis capensis (Onychophora)

Early spermatids of the onychophoran Peripatopsis capensis are spherical cells with a centrally located nucleus, numerous mitochondria, Golgi complexes, microtubules and two centrioles. During spermiogenesis, Golgi vesicles migrate to one side of the cell where they form a tight aggregate, which is later shed. The mature spermatozoon has no acrosome. Several mitochondria fuse to form a middle piece containing three large mitochondria. Nucleus and middle‐piece elongate, presumably under the influence of helically twisted microtubules. Outside this set of microtubules a continuous layer of endoplasmic reticulum cisternae is formed which separates the interior portion of the cell from an external cytoplasmic rim, which is later shed. Outside the 9 + 2 complex, the tail presents nine accessory microtubules, and a peripheral layer of microtubules beneath the plasma membrane. The enforcement of the tail structure may be related to the fertilization biology of this animal, which is by “hypodermal” impregnation.

Testicular Biopsy of Secretory Azoospermia: Electron and Light Microscopic Analysis

The histological and ultrastructural features of testicular cells were examined in testes with secretory azoospermia in seven patients with germinal cell arrest, two with Sertoli-cell-only; and one with tubular hyalinization. Germinal cell arrest was characterized by the presence of spermatogonia AD, AP, B, and some primary spermatocytes: these cells had similar ultrastructural features typical of the adult normal testis. Sertoli cells contained large number of various lipid inclusions and lipofuscin bodies. The multilayered peritubular wall presented increased collagen fibers. The cytoplasm of Leydig cells was filled with dilated vesicles of smooth endoplasmic reticulum and precursors of Reinke crystalloids. A blockage in the seminiferous tubules occurred after puberty, when tubular components, peritubular wall and Leydig cells had reached maturity.

Ultrastructure of spermiogenesis in men with congenital absence of the vasa deferentia

Spermiogenesis has been investigated in four cases of agenesia of vasa deferentia. During acrosome formation various anomalies gave rise to late spermatids with deformed heads. Chromatin condensation proceeded normally, but completion of this process appeared to be delayed. Redundant nuclear membranes frequently persisted at the basal region of the nuclei in mature spermatozoa, which occupied niches within Sertoli cells, as the tubules had no lumen. Flagellar structure was normal. These findings support the view that the altered local milieu and variations in acrosome formation may induce the observed anomalies.