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Mammalian caput and cauda epididymidal spermatozoa exhibit diverse stages of maturation, and their plasma membrane shows diverse composition and stability levels, thus enabling these spermatozoa to undergo the acrosomal reaction after transit through the epididymis. As a result, the study of antiperoxidative mechanisms is quite relevant, since epididymal spermatozoa must be properly protected against agents such as reactive oxygen species, which can impair the complex maturation process. We considered activities of certain enzymes (glutathione peroxidase [GPx], phospholipid hydroperoxide glutathione peroxidase [PHGPx], glutathione reductase [GR], superoxide dismutase [SOD], and catalase [CAT]) and the vitamin E content in isolated rat caput and cauda epididymidal spermatozoa. The results indicate that caput epididymidal sperm have significantly greater PHGPx (3.5x), GPx (2.4x), and SOD (1.7x) activities, as well as a greater amount of vitamin E (3.8x). There were no detectable differences in the GR and CAT activities of caput and cauda epididymidal spermatozoa. The substantial drop in PHGPx activity during epididymal transit is discussed in relation to an additional function of this enzyme: the use of caput sperm protamines as a sulfhydryl substrate. In vitro peroxidation of the two sperm populations by the free radical generator (azo-initiator) 2,2'-azobis(2-amidinopropane) dihydrochloride revealed that only about 13% of the vitamin E content of the caput epididymidal spermatozoa was consumed, which contrasts with the greater consumption (about 70%) of the vitamin in cauda epididymidal spermatozoa. Selective inhibition of PHGPx, SOD, or CAT did not change this picture. The higher susceptibility of cauda epididymidal spermatozoa to radicals is discussed in relation to the diverse enzymatic activities, vitamin E content, and peroxidative response. These factors are correlated with the different stages of sperm cell maturation, which are characterized-from caput to cauda epididymidis-by progressive destabilization of the plasma and acrosomal membranes.

These results demonstrate that the MR appearance of articular cartilage correlates with the collagen content, but not with that of PGs, of the different zones. Other matrix components might, however, influence the MR appearance by contributing to the macromolecular organization of the tissue.

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx, EC 1.11.1.12) is present, in both free and membrane-bound form, in several mammalian tissues. It utilizes thiols such as glutathione to specifically scavenge phospholipid hydroperoxides. The testis exhibits the highest PHGPx-specific activity so far measured, and interest in the presence and function of the enzyme in this tissue has recently grown. Here we report the localization of PHGPx in rat epididymal spermatozoa and its distribution in subfractions obtained by sucrose density gradient centrifugation. Immunochemical evidence and enzymatic activity revealed for the first time that PHGPx is present in sperm heads and tail midpiece mitochondria. The binding of the enzyme to spermatozoa, head, and mitochondria was barely affected by ionic strength or thiols or detergents, as compared to the detachment of PHGPx obtained from testis nuclei. Moreover, we demonstrated that pure PHGPx exhibits a higher thiol-oxidase activity toward isolated epididymal caput protamines than toward protamines from epididymal cauda. These results suggest a role for the enzyme in the maturation of spermatozoa through the metabolism of hydroperoxides and sperm thiol oxidation, in addition to its serving as an antioxidant protector.

In this work, evidence for the presence of ferritins in plant mitochondria is supplied. Mitochondria were isolated from etiolated pea stems and Arabidopsis thaliana cell cultures. The proteins were separated by SDS/PAGE. A protein, with an apparent molecular mass of approximately 25-26 kDa (corresponding to that of ferritin), was cross-reacted with an antibody raised against pea seed ferritin. The mitochondrial ferritin from pea stems was also purified by immunoprecipitation. The purified protein was analyzed by MALDI-TOF mass spectrometry and the results of both mass finger print and peptide fragmentation by post source decay assign the polypeptide sequence to the pea ferritin (P < 0.05). The mitochondrial localization of ferritin was also confirmed by immunocytochemistry experiments on isolated mitochondria and cross-sections of pea stem cells. The possible role of ferritin in oxidative stress of plant mitochondria is discussed.

Several embryonic stages (nuclear multiplication stage up to blastoderm) were incubated at 37°C. The 84,000 dalton heat-shock protein was always present in the heat-shocked embryos at all these stages. This protein was also found in nonheat-shocked controls. Different amounts of two other heat-shock proteins, the 70,000 and 68,000 daltons, were synthesized depending upon the embryonic stage. A massive synthesis of these two later proteins was observed in embryos which were able to recover from the heat-shock.It has been reported that prior to nuclear migration, Drosophila melanogaster embryos incorporate very little uridine (Zalokar, '76) and that at the blastoderm stage the synthesis of RNA is significantly activated (Lamb and Laird, '76; McKnight and Miller, '76; Anderson and Lengyel, '79). These results suggest that during nuclear multiplication the zygotic genome is mainly, if not entirely, silent. This interpretation is also supported by the shortness of interkinesis: 4 minutes a t 24°C (Rabinowitz, '41). During this period the DNA is engaged in replicating 200 million base pairs and presumably there is little time for possible transcription.Transcription and translation of specific messages can be experimentally stimulated by incubation at 37°C in many tissues, cell cultures, and organs (Tissieres et al., '74; McKenzie et al., '75) We used heat-shock as a tool to investigate whether the corresponding genes can be activated, during the earliest stages of embryonic development. MATERIALS AND METHODS Flies maintenance and egg harvestingRoughly 10,000 flies (Oregon-R strain) were kept in a population cage at 24°C. Eggs were collected on food plates left in the cage for L 1 0 minutes. Details on egg harvesting were reported by Graziosi et al. ('79). Labelling of eggs and heat-shock treatmentThe eggs were dechorionated in 50% bleach for 2 minutes, permeabilized, and incubated as described by Limbourg and Zalokar ('73). WMethionine was added to the incubation medium to obtain an activity of 1 mCi/ml (The Radiochemical Centre, Hamersham, United Kingdom, 600 Ciimmol). The heat-shock was carried out in a water bath at 37°C for 15 minutes and the eggs were kept at this temperature further for 45 minutes in radioactive medium. ElectrophoresisThe SDS gels were run according to the method of Laemmli ('70). The O'Farrell ('75) technique was used for the two-dimensional gel electrophoresis. All the two-dimensional gels underwent fluorography as described by Bonner and Laskey ('74). RESULTSSeveral embryonic stages were considered: from early nuclear multiplication (20-to 30-minute average age) up to blastoderm (3 hours). The survival rate of embryos incubated at 37°C for 30 minutes and then transferred to 24°C without any further treatment was recorded. A large proportion of eggs 20 to 30-minUtes-old failed to hatch (about 20% survival rate) while 63-83% of embryos treated at 150 minutes (syncytial blastoderm) hatched as larvae. Treatment at 3 hours did not cause any mortality. A few hundred embryos were obser...

The uptake of some flavonoids into the vascular endothelium occurs via the bilirubin-specific membrane transporter bilitranslocase. This offers new insights into the vascular effects of both flavonoids and bilirubin.

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