Serge Weinman

Immunocytochemical and biochemical evidence for the presence of calmodulin in bull sperm flagellum isolation and characterization of sperm calmodulin

Upon fluorescent staining with a goat antibody anti-ram testis calmodulin, washed bull sperm appears to contain calmodulin in the acrosome, in the post acrosomal region, in the neck region probably associated with the implantation plates and thin laminated fibers, and in a sheath around the upper part of the flagellum. Heads and midpieces + tails were separated by elutriation of sonicated sperm. Immunofluorescent labeling of fragments confirms the presence of calmodulin in implantation plates, where sonication disrupted heads from midpieces, and in a sheath around the midpiece and the upper part of the principal piece. These results were confirmed by electrophoretic and radioenzymatic assays of calmodulin in the fragments, using calmodulin-deficient Ca2+/calmodulin-dependent myosin light chain kinase. Small but significant amounts (approx. 3 micrograms per 10 (10) sperm) are found in midpieces + tails vs. approx. 280 micrograms in the same number of heads. These results are in agreement with a recent report from Jones et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2772-2776. Sperm calmodulin was purified from a whole sperm 1 M KCl extract and found to exhibit the same characteristics as other mammalian calmodulins isolated so far in terms of ultraviolet absorption spectrum and amino acid composition, including one residue of epsilon-N-trimethyllysine. Its behavior upon SDS-polyacrylamide gel electrophoresis was dependent on the presence or absence of Ca2+. The high performance liquid chromatography tryptic peptide maps were similar, if not identical, to mammalian calmodulin maps (Autric et al. (1980) Biochim. Biophys. Acta 631, 139-147). Sperm calmodulin is therefore probably identical to the somatic cell protein.

Annexins in rat enterocyte and hepatocyte: an immunogold electron-microscope study.

In the present study, immunogold labeling of ultrathin sections of rat small intestine and liver has been used to obtain insights into the ultrastructural localization and possible functions of annexins. In enterocytes, annexins II, IV, and VI are found at the periphery of the core of each microvillus and of the rootlets, but are absent from the interrootlet space. Annexins II, IV, and VI are also observed close to the interdigitated plasma membrane. In hepatocytes, only annexin VI is found to be concentrated within the microvilli in the bile canaliculi, on the inner face of the sinusoidal cell surface, particularly in the space of Disse, and all along the plasma membrane. Annexin VI is also detected in mitochondria of enterocytes and hepatocytes. These localizations are in agreement with the concept of a close calcium-dependent association of annexins with membranes and cytoskeletal proteins, particularly with actin. Moreover, they support the hypothesis of an involvement of annexins in exocytotic and endocytotic processes, which take place in epithelial cells.

Annexin expressions are temporally and spatially regulated during rat hepatocyte differentiation

Annexin (Anx) 1, 2, 5, and 6 expressions were determined at the transcriptional and translational levels in the rat hepatocytes from gestational day 15 to postnatal day 17. Dramatic shifts were observed in Anx 1 and 2 levels, which peaked at day 1 and gestational day 20, respectively, and reached low levels thereafter. However, Anx 5 and 6 rates were more constant. Prenatal administration of dexamethasone (dex) resulted in a decrease of Anx 1 mRNA levels, and a strong increase in Anx 2 mRNA contents. In adult hepatocytes cultured in the presence of EGF or HGF, Anx 1 and 2 expressions resumed. By immunohistochemistry, Anx 1 was detected only in the cytoplasm of hepatocytes of 1‐ to 3‐day‐old rats, Anx 2 and 6 both exhibited a redistribution from the cytoplasm toward the plasma membrane, and Anx 5 was present in the nucleus, cytoplasm, and plasma membrane. Thus, Anx 1, 2, 5, and 6 have individual modes of expression and localization in the differentiating hepatocytes, where they might play unique roles at well defined phases of liver ontogeny.

Characterization and ultrastructural localization of annexin VI from mitochondria

Annexin VI, a member of a family of related intracellular proteins that associate reversibly with membrane phospholipids in a Ca2+‐dependent manner, has been purified from bovine liver mitochondria and characterized. Moreover, biochemical and immunocytochemical lines of evidence are presented which strongly suggest that annexin VI is closely associated with the cristae in the inner membrane of mitochondria. These findings are consistent with a calcium channel activity of annexin VI in mitochondria.

Evolution of Ca2+- and cAMP-dependent regulatory mechanisms during ram spermatogenesis

Calmodulin level and cAMP-dependent protein kinase activity of ram germ cells at different stages of spermatogenesis have been determined. Calmodulin levels decrease during maturation. Simultaneously, calmodulin localization changes during cell differentiation. In round, elongating, and elongated spermatids, calmodulin is closely associated with the developing acrosome; in spermatozoa, it becomes present in the postacrosome, the neck region and the tail. Protein kinase activity is relatively low in testicular cells but increases dramatically during epididymal maturation of spermatozoa. A concerted regulation by cAMP and Ca2+ of biochemical events in spermatogenic cells and spermatozoa is suggested.

Calmodulin level and cAMP-dependent protein kinase activity in rat spermatogenic cells and hormonal control of spermatogenesis.

The changes in intracellular calmodulin levels and cAMP-dependent protein kinase activities have been studied in the testis of normally developing and hypophysectomized rats. It appears that the onset of spermatogenesis which occurs on the first days of the postnatal development, is associated with a major (over fivefold) increase in the calmodulin level and enhancement of the cAMP-dependent protein kinase activity. On the contrary, hypophysectomy of adult animals is associated with a progressive decline in the calmodulin level and a rapid and regular decrease in the cAMP-dependent protein kinase activity. Moreover, measurements of the intracellular calmodulin level and cAMP-dependent kinase activity of isolated testicular germ cells or epididymal spermatozoa have shown that testosterone, administered to hypophysectomized rats as subcutaneous implants, maintains the concentration of these regulatory proteins to normal values.

Changes in calmodulin level after fertilization and during first cleavage in the egg of the urodelan amphibian Pleurodeles waltlii

We report three significant calmodulin rises related to Pleurodeles waltlii egg fertilization and following developmental events. These elevations are correlated to the major obvious Ca2+-dependent events: Na+-H+ exchange, activation of NAD kinase, triggering of cortical reaction, resumption of meiotic division II, initiation of DNA synthesis and regulation of cell division. Therefore, it is suggested that alterations in calmodulin level in fertilized egg may be part of the Ca2+-dependent regulatory mechanisms which turn on metabolisms, initiate development and govern cell cleavages.

Calmodulin in Zajdela hepatoma cell growth

Calmodulin levels were measured in Zajdela hepatoma cells growing both in vivo and in culture, with respect to the distribution of the cells into G1 and S+G2 phases of the cell cycle and growth conditions. These levels, expressed on a per-microgram of protein basis, were significantly elevated at the G1-S boundary and maintained throughout the remainder of the cell cycle. This elevation of calmodulin took place independently of the culture conditions. Taken together with previous observations, these data suggest that a threshold concentration for calmodulin is required for progression through the cell cycle, DNA synthesis and cell division.

Synthesis and infrared spectra of some 3α-hydroxy Δ5-steroids

Abstract A generally applicable method for the preparation of 3 α-hydroxy Δ 5 -steroids is used to obtain 3 α-hydroxyandrost-5-ene, 3α-hydroxypregn-5-ene, 3 α-hydroxypregn-5-ene-20-one and their corresponding acetates. The infrared spectra of these compounds and of 3 α-hydroxycholest-5-ene and 3 α-hydroxyandrost-5-ene-17-one, known steroids, are measured in the range of 3700–3900 cm −1 and the characteristic group frequencies reported.

Calmodulin in rat enterocyte: an immunogold electron-microscope study

Immunogold labeling of ultrathin sections of the epithelium of rat small intestine has been used to obtain insights into the ultrastructural localization and possible function of calmodulin in the enterocyte. Calmodulin is found mainly overlying the periphery of the microvillous core, in agreement with the location of the 110-kDa calmodulin complex. Extremely small amounts of calmodulin can be detected along the interdigitating basolateral membrane. This immunogold electron-microscope study suggests that calmodulin plays an important role in regulating the mechanochemical activity of myosin I but not in processes associated with the basolateral membrane of rat enterocyte.