M.L. Marcante

Morphological, histochemical and biochemical studies on germ cell mitochondria of normal rats

SummaryMorphological changes in rat germ cell mitochondria are described. In diplotene and secondary spermatocytes and in the spermatids of the Golgi, cap and acrosomal phases, the mitochondria take on a rounded appearance with the inner space containing the matrix flattened against the outer membrane and the intracristal spaces considerably swollen (“condensed” mitochondria).Functional studies on “condensed” mitochondria isolated from the germ cells of normal rats have been performed. The following parameters have been evaluated: ADP/O ratio, respiratory control ratio (RCR) and ADP affinity. The ADP/O values found in the presence of various substrates are in agreement with the theoretical figures. The RCR is remarkably high. Moreover, the ADP affinity of these mitochondria is very high, as demonstrated by the low values of the “apparent Km”. These biochemical findings, which demonstrate a high oxidative capacity coupled with a marked phosphorylation, suggest that the “condensed” appearance of germ cell mitochondria is the expression of an active functional state.

Morphological and biochemical modifications of rat germ cell mitochondria induced by new antispermatogenic compounds: Studies in vivo and in vitro☆

Abstract The morphological changes induced in rat germ cell mitochondria by chlorobenzyl-1 H -indazol-3-carboxylic acid (AF 1312/TS) and lonidamine (AF 1980), two antispermatogenic compounds, are described. Twenty-four hours after treatment, numerous dumbbell-shaped mitochondria appear in the Golgi, cap, and acrosomic spermatids, whereas after 48 hrs severe signs of mitochondrial degeneration are visible in the maturation phase spermatids. Biochemical studies performed on isolated germ cells and their mitochondria, harvested from the testes of normal and treated rats, are also described. In the normal rats, the cells and mitochondria were incubated with the antispermatogenic compounds. Aerobic and anaerobic glycolysis was evaluated on isolated germ cells, whereas the following parameters were evaluated in the mitochondria: ADP O ratio, respiratory control ratio (RCR), and ADP affinity. The results obtained demonstrate that the antispermatogenic agents induce, both in vivo and in vitro , similar changes in energy metabolism; respiration, RCR, and ADP affinity are significantly reduced, while aerobic glycolysis is increased.

The effect of the association of Gossypol and Lonidamine on the energy metabolism of Ehrlich ascites tumor cells

The effect of the association of Gossypol and Lonidamine on the energy metabolism of Ehrlich ascites tumor cells has been investigated. The action of the drug on tumor cells was studied by addition of the drugs to cells harvested from Swiss male mice. The results may be summarized as follows: (1) Low concentrations of Gossypol increase the rate of oxygen consumption by uncoupling oxidative phosphorylation. High concentrations result in an inhibition of oxygen consumption with a mechanism that must be regarded as not directly related to the uncoupling activity. (2) Gossypol, at concentrations at which it exerts an uncoupling activity, stimulates mitochondrial ATPase which in turn increases the aerobic and anaerobic rates of lactate production. The decrease of glycolysis at high concentrations of Gossypol does not depend on the inhibition of enzymes of the glycolytic pathway, but must be ascribed to cell death. (3) The association of a low concentration of Gossypol with Lonidamine brings about a further inhibition of oxygen consumption. Moreover, Lonidamine abolishes the stimulation of glycolysis induced by Gossypol and lowers lactate production to values that are quite similar to those found with Lonidamine alone. (4) It may be concluded that the association of Gossypol and Lonidamine results in a very effective decrease of the energy requirements of cancer cells.

Effect of lonidamine on protein synthesis in neoplastic cells

The action of lonidamine, 1,(2,4 dichlorobenzyl)-1H-indazol-3-carboxylic acid, on protein synthesis of neoplastic cells growing both in vivo and in vitro has been investigated. Lonidamine decreases amino acid incorporation in all cells tested, although the inhibition is partially relieved by glucose. The inhibition of labeled precursors into acid-insoluble material cannot be ascribed to an impairment of amino acid uptake which, on the contrary, is enhanced by the drug. Tests on cell-free systems showed that lonidamine does not inhibit the tobacco mosaic virus (TMV)-mRNA-directed in vitro protein synthesis, thus indicating that protein synthetic machinery per se is not affected. The inhibition of the rate of protein synthesis achieved by lonidamine must be ascribed to an effect on energy-yielding processes with a mechanism similar to that observed in other metabolic inhibitors. Lonidamine, however, because of its capacity to inhibit both respiration and glycolysis in neoplastic cells, is effective at 10 to 20 times lower concentrations. DNP and oligomycin potentiate the inhibitory effect of lonidamine on the rate of protein synthesis. This finding substantiates the idea that neoplastic cells, including those growing in ascitic form, utilize mitochondrial oxidative phosphorylation as the main source of ATP for their biosynthetic processes.

Energy metabolism of normal and lonidamine-treated sertoli cells of rats

The effect of lonidamine on oxygen consumption, aerobic lactate production, and [U-14C]glucose metabolism of rat Sertoli cells was investigated. The results may be summarized as follows: (1) Sertoli cells show well-developed energy metabolism both in vitro and in vivo. (2) The rate of aerobic lactate production is markedly higher than in other cell types, either normal differentiated or neoplastic, such as Ehrlich ascites tumor cells. (3) Lonidamine does not affect respiration and aerobic glycolysis of Sertoli cells. This finding is consistent with previous data which demonstrated that the antispermatogenic effect can be mainly ascribed to an irreversible alteration of germ cell mitochondria induced by 1-substituted indazole-3-carboxylic acids of which lonidamine represents one of the most potent derivatives. (4) The functional impairment induced by lonidamine on rat Sertoli cells cannot be ascribed to an action on the energy metabolism even if, up to date, the biochemical target is still unclear.

Sertoli cells of adult rats “in vitro”

SummaryA cell line obtained from isolated seminiferous tubules of adult rat testis has been studied “in vitro” over a period of 35 days.Light and electron microscopic studies performed from hour 2 to the end of culture have shown the presence of a monomorphic cell population. After 5–6 days of culture the cells formed a monolayer. The cytoplasm of the cells contained numerous lipid bodies and produced numerous projections. The nucleus showed several indentations and one or more nucleoli. From the 9th to the 15th day of culture the cells developed a large amount of endoplasmic reticulum, Golgi apparatus and aggregates of electron dense granules. From the 20th to 40th day the cell cultures progressively degenerated.Immunochemical analysis of the culture medium revealed the presence of estradiol-17β, which reached its maximum production rate from the 8th day to the 18th day of culture. Corresponding to cell involution estradiol concentration underwent a rapid decrease.On the basis of morphological and biochemical data the cells could be considered Sertoli cells.This work was supported by Grants n.∘ 74.00155.04 and n.∘ 75.01224.04 from the Consiglio Nazionale delle Ricerche (C.N.R.), Rome, Italy, and by Istituto di Ricerca F. Angelini, Rome, Italy

Studies on the structure of α1-glycoprotein isolated from Yoshida ascites tumor: I. Enzymic and acid degradation of the carbohydrate unit

Abstract The structure of carbohydrate moiety of acid glycoprotein isolated from Yoshida ascites tumor and normal rat blood serum has been studied after the removal of neuraminic acid by neuraminidase. It has been observed that this enzyme splits off 15 residues of neuraminic acid from the tumoral protein and 18 residues from the normal one. The neuraminic acid-freed glycoprotein has been subjected to mild acid hydrolysis, and the time-course appearance of hexoses and hexosamine has been investigated by means of chemical and chromatographic analysis. It has been shown that after the removal of neuraminic acid, the most labile grouping in both glycoproteins is that involving galactose. Mannose, glucosamine, and galactosamine are the other components of the pentasaccharide which constitute the carbohydrate portion of both tumoral and normal glycoproteins.

The effect of gossypol and Lonidamine on electron transport in Ehrlich ascites tumor mitochondria.

The effect of the association of gossypol and Lonidamine on the electron transport in Ehrlich ascites tumor mitochondria has been investigated by addition of drugs to isolated mitochondria. The results may be summarized as follows. (1) Low concentrations of gossypol increase the rate of oxygen consumption at the level of three energy-conserving sites of the respiratory chain. Higher concentrations result in an inhibition of oxygen consumption at (or near) both energy-conserving sites 1 and 2, while energy-conserving site 3 is unaffected. (2) Gossypol, at concentrations at which it exerts its uncoupling effect, stimulates ATPase activity. Higher concentrations inhibit the enzyme activity. (3) The addition of gossypol to mitochondria respiring on pyruvate plus malate or succinate induces a more oxidized state of NAD+ and cytochrome b, respectively. (4) Gossypol enhances the effect of Lonidamine on oxygen consumption. Lonidamine does not affect state 4 respiration, but in the presence of gossypol, it determines a marked decrease in the rate of oxygen consumption. The inhibition of oxidation of NAD-linked substrates is greater than that of FAD-linked substrates. (5) It may be concluded that gossypol is very effective in potentiating the effect of Lonidamine. Moreover, it may be suggested that the antitumor activity of Lonidamine is enhanced if it is used in combination with other drugs and/or treatments, such as hyperthermia, which modify the energy status of mitochondria.

The disulphide bridges and immunochemical properties of Yoshida ascites tumor and normal serum glycoprotein

Abstract 1. 1.|Different procedures have been applied for the partial and full reduction of the glycoproteins isolated from Yoshida ascites tumor and from normal serum. 2. 2.|Neither glycoprotein possesses free reactive or unreactive SH groups. They are therefore disulphide proteins. 3. 3.|Reduction with mercaptoethanol, even if extensively carried out and followed by alkylation, was never complete, when the lower number of titrated SH groups was used as criterion. 4. 4.|The quantitative reduction of disulphide bonds has been achieved by reaction with sulphite, and the sulphydryl groups have been spectrophotometrically titrated with p -hydroxymercuribenzoate. The presence of 4 disulphide bridges in the Yoshida glycoprotein and 10 in the normal serum glycoprotein has been ascertained. 5. 5.|The relation between the reductive cleavage of S:S bonds and the antigenicity of both glycoproteins has been studied. Both molecules lost their antibody-precipitating capacity after the opening of more than one disulphide bridge.

Effect of lonidamine on the aerobic glycolysis of normal and phytohemagglutinin-stimulated human peripheral blood lymphocytes

The effect of lonidamine on the aerobic glycolysis and on the ultrastructure of normal and phytohemagglutinin-stimulated human peripheral blood lymphocytes has been investigated. In quiescent lymphocytes lonidamine does not affect aerobic lactate production and does not induce ultrastructural modifications. On the contrary, lymphocytes stimulated with phytohemagglutinin become susceptible to lonidamine inhibition. The rate of lactate production is decreased by 50% and mitochondria appear swollen with rarified matrix and disrupted cristae. The different effect of lonidamine can be ascribed both to the biochemical modifications induced by phytohemagglutinin and to the mechanism of lonidamine itself. Phytohemagglutinin increases the activity of hexokinase and phosphofructokinase and determines a shift of cytoplasmatic hexokinase toward the mitochondria-bound form which is responsible for the increased lactate production. This interpretation is supported by the finding that lonidamine, which specifically inhibits mitochondria-bound hexokinase only when mitochondria are in a condensed state, decreases lactate production to a value similar to that found in unstimulated cells. The inability of lonidamine to affect the aerobic glycolysis of quiescent lymphocytes can be interpreted along the same line. On this basis it is suggested that the inhibition of mitochondria-bound hexokinase might be ascribed to marked changes in membrane conformation that affect the activity of membrane-associated enzymes, rather than to a direct effect of the enzyme itself.