Immunoreactivity for ferritin in Leydig cells of human testis

The aim of the present study was to demonstrate the immunocytochemical localization of ferritin in human Leydig cells. Testes from patients orchidectomized for carcinoma of the prostate were used. The immunoreactivity for ferritin was visualized in the Leydig cells by amplification immunocytochemical technique. The Sertoli cells and some of the germ cells show moderate immunoreactivity for the examined antigen. Our result represent an immunocytochemical verification for presence of ferritin in the human Leydig cells (also in other cellular components) and suggest the role of this factor in the local auto- and/or paracrine control of the testicular functions.     

Bcl-2/Bax ratio, mitochondrial membranes and aerobic enzyme activity in cardiomyocytes of rats after submaximal training

INTRODUCTION: Mitochondria are an active and continuous source of reactive oxygen species (ROS) during respiration. The ROS increased production during endurance training is a result of an augmented electron transport through the respiratory chains, making in this way the mitochondria a potential target for oxidative damage. The Bcl-2 protein family plays a central role in the transition of apoptotic signals towards the mitochondria in stress-induced apoptosis. AIM: The present work studied the effect of endurance training on the expression of the apoptotic proteins Bcl-2 and Bax in rat cardiomyocytes, as well as the concomitant changes in the ultrastructure of the mitochondria and activity of some enzymes residing there. MATERIAL AND METHODS: Two groups of male Wistar rats were used. One was the control and the other was trained on treadmill with submaximal loading for eight weeks. At the end of the trial, samples of the myocardium of all the experimental animals were obtained. Immunohistochemical reactions for Bcl-2 and Bax and enzymehistochemical reactions for succinate dehydrogenase and NADH2-cytochrome C-reductase were done. The results were analyzed using specialized software. Transmission electron microscopical study was carried out too. RESULTS: In the myocardium of the trained animals the expression of Bcl-2 and Bcl-2/Bax ratio were significantly higher compared to the controls. The mitochondria had intact outer and inner membranes, with no signs of swelling. Mitochondria with denser packed cristae were found predominantly. No significant differences were found in the activity of the investigated enzymes in the cardiomyocytes of the animals from both groups. CONCLUSIONS: In the myocardium of the experimental animals endurance training for eight weeks does not lead to activation of apoptotic processes via the mitochondrial pathway. This type of exercise training could be used for cardioprotection in order to elevate apoptotic threshold of cardiomyocytes.     

Cloning and characterization of KlCOX18, a gene required for activity of cytochrome oxidase in Kluyveromyces lactis

We describe the isolation and initial characterization of KlCOX18, a gene that is essential for the assembly of a functional cytochrome oxidase in the yeast Kluyveromyces lactis. Cells carrying a recessive nuclear mutation in this gene are respiratory deficient and contain reduced levels of cytochromes a and a ₃ . The KlCOX18 gene has been cloned by complementation of the respective nuclear mutation, sequenced, and disrupted. KlCOX18 is located on chromosome II and contains an open reading frame of 939 base pairs. The corresponding protein exhibits 70.4% similarity to the Cox18p of Saccharomyces cerevisiae. It contains three possible membrane-spanning domains and a putative amino-terminal mitochondrial import sequence. The strain carrying a null mutation in KlCOX18 does not grow on non-fermentable carbon sources and is deficient in both cytochrome c oxidase and respiratory activity. It is proposed that KlCox18p, like its S. cerevisiae counterpart, provides an important function at a later step of the cytochrome oxidase assembly pathway. Received: 25 March / 3 July 1997     

Identification and functional analysis of a Kluyveromyces lactis homologue of the SPT4 gene of Saccharomyces cerevisiae

Sequence analysis of a DNA fragment containing the KlCOX18 gene originating from chromosome II of the yeast Kluyveromyces lactis revealed the presence of an adjacent open reading frame (ORF) for a protein exhibiting 78.4% identity with the Saccharomyces cerevisiae Spt4p. Based on the identical length (102 aa) and the conservation of the zinc-finger motif found in Spt4p we named this ORF KlSPT4. When expressed in S. cerevisiae the KlSPT4 gene complemented all spt4 mutant phenotypes. It is proposed that KlSpt4p, like its S. cerevisiae counterpart is a protein involved in the establishment or maintenance of the chromatin structure that influences the expression of many yeast genes. Received: 15 June / 31 August 1998     

Isolation and molecular analysis of the gene for cytochrome c1 from Kluyveromyces lactis

By ethyl methanesulphonate mutagenesis of the yeast Kluyveromyces lactis we have isolated five nuclear mutants that were unable to grow on non-fermentable carbon sources. The mutations were found to belong to three complementation groups. After functional complementation of the mutation in one of these mutants we have cloned the structural gene for cytochrome c ₁, named KlCYT1. This gene has been assigned to chromosome VI and its nucleotide sequence exhibited 74.3% identity to the homologous gene of S. cerevisiae. Received: 6 February 1996 / 2 April 1996     

Isolation,heterological cloning and sequencing of the RPL28 gene in Kluyveromyces lactis

By virtue of heterologous functional complementation of the Saccharomyces cerevisiae Delta pdr5 mutant strain, using a Kluyveromyces lactis genomic library, three different K. lactis chromosomal inserts were obtained. Transformation of the S. cerevisiae Delta pdr1 Delta pdr3 mutant strain, hypersensitive to drugs, with isolated plasmids resulted in resistance to cycloheximide and fluconazole. Transformation of K. lactis host strains, using the cloned chromosomal fragments, led to an increased level of resistance to some mitochondrial inhibitors and azole antifungals. The nucleotide sequence of the cloned inserts revealed that two of them contain the drug efflux transporter gene Kl-PDR5 and the third contains a DNA segment homologous to chromosome VII of S. cerevisiae. Along with three novel ORFs, encoding two proteins of unknown molecular function and one putative hexose transporter, this segment also contained the Kl-RPL28 gene, found to be responsible for the cycloheximide resistance of heterologous transformants. This gene codes for the large subunit ribosomal protein (149 amino acids) that shares 89.9% identity with its S. cerevisiae counterpart. The coding region of Kl-RPL28 was found to be interrupted with one intron near the 5' end. The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the accession number AF493565.     

INSLF3-LGR8 ligand-receptor system in testes of mature rats after exposure to ethane dimethanesulphonate (short communication)

The cytotoxic agent ethane-1,2-dimethanesulphonate (EDS) specifically destroys the Leydig cells (LC) in the adult testis, followed by a complete regeneration. The process of LC renewal after exposure to EDS shows homology to the development of the adult-type LC population in prepubertal testis. INSL3, also known as Leydig insulin-like peptide or relaxin-like factor, is a peptide hormone, a novel member of the insulin/relaxin family, and seems to be localized predominantly in the gonadal tissues. INSL3 mRNA is expressed in the LC in a constitutive fashion and INSL3 thus seems to be a useful marker of LC differentiation status. The present study was aimed at establishing the chronology and dynamic of expression of INSL3 and its specific receptor LGR8 in the LC repopulation after exposure to mature rats to EDS. As material, testes of mature Wistar rats that received single intraperitoneal injection of EDS (75 mg/kg body weight) were used. The animals were killed 1, 7, 14, 21 and 35 days after the initial treatment. The pattern of INSL3-LGR8 expression in newly formed LC after EDS administration was established using a high sensitive immunohistochemical polymer detection kit. After treatment with EDS, the immunoreactivity for INSL3 and LGR8 disappeared from the testis and reappeared again at the time of regeneration of the first LC, 14 days after EDS. The INSL3-LGR8 positive cells grew in number concomitantly with the increase of the LC repopulation. Thirty-five days after EDS destruction a larger number of immunopositive LC were seen in form of clusters corresponding with the regeneration of adult type LC population. The present findings support the hypothesis that EDS-treated rats can serve as a model for studying the LC development in the prepubertal testis and indicate a specific role of hormonal factors like INSL3 in this process.     

Dexamethasone and oxytocin effects on rat Leydig cells--morphological and enzyme-histochemical characteristics

In the present study the fine structure of rat Leydig cells was examined by electron microscopy. Oxytocin and dexamethasone induced changes in the activities of 3beta hydroxysteroid dehydrogenase, NADH2 cytochrome-C-reductase and glucose-6-phosphate dehydrogenase in these cells were studied in an in vivo experiment. Two groups of male Wistar rats were used to test the effects of oxytocin--rats from group I received a single injection of oxytocin; rats from group II were given a 10-day course of oxytocin. Pregnant female rats were injected with dexamethasone on day 17 and 18 post conception. The testes of 19 and 20-day old embryos were removed. It was established that both short and long term courses of oxytocin increased the activities of the above-mentioned enzymes. On the contrary, prenatal administration of dexamethasone decreased enzymatic activity in Leydig cells. Electron microscopy revealed clusters of fetal Leydig cells. Our results indicated the role of oxytocin in the local regulation of steroidogenesis and the importance of glucocorticoids in the differentiation and activity of Leydig cells.