Lipid peroxides, spermatozoa quality and activity of glutathione peroxidase in bull semen

In bull semen spontaneous lipid peroxidation measured by the level of endogenous lipid peroxides and the consequences of this process for morphological and biochemical changes was studied. Glutathione peroxidase activity as protective enzyme against peroxidative damage was also determined. Obtained results showed that approximately two thirds of GSH-Px activity in bull semen was non Se-dependent glutathione peroxidase activity. Malonaldehyde (MDA) level was negative correlated with selenium-dependent GSH-Px activity (r = = -0.38, P less than 0.01). Spermatozoa with acrosome entirely lost appeared to increase as the MDA level increased (r = 0.18, P less than 0.05). The negative correlation between Se GSH-Px activity and spermatozoa with acrosome separation from head (r = -0.28, P less than 0.01) and entirely lost (r = -0.21, P less than 0.05) suggest that selenium-dependent GSH-Px plays role in protecting the acrosome against disruption of the acrosomal membrane. The total glutathione peroxidase activity was unrelated to studied variables of bull semen.     

Activity, substrate detection and immunolocalization of glutathione peroxidase (GPx) in bovine reproductive organs and semen

The purpose of the current study was to further investigate the role of the antioxidant selenium-dependent enzyme glutathione peroxidase (GPx) in reproductive organs and semen from bulls. To this end a fast and convenient combined method for immune detection and substrate localization was adapted, which allows the assessment of both molecular weight and peroxidase activity of proteins on one and the same SDS-PAGE gel plate. After routine semen analysis of ejaculates, a spectrophotometrical assay of GPx activity in bovine semen was performed. For the immunological analyses performed, a rabbit polyclonal monospecific antibody against GPx was raised. Substrate detection and immunolocalization of GPx in lysates from bovine testis, epididymis, spermatozoa, and seminal plasma was performed. In order to determine the localization of GPx in spermatozoa, immunofluorescence analysis was performed. A positive correlation was established between GPx activity in semen and the number of motile spermatozoa. A negative correlation was observed between GPx activity and the number of immotile spermatozoa. The combined method for immunodetection and substrate localization was tested and proved reliable. Both tetramer and monomer forms of GPx were detected in lysates from testis, epididymis, and spermatozoa. We found no GPx activity in seminal plasma. Immunofluorescence shows the presence of GPx chiefly in the mitochondrial and in the acrosome regions of spermatozoa. GPx activity remained stable under the extreme experimental conditions.     

Localization of the epidermal growth factor (EGF) in the epididymis and accessory genital glands of the boar and functional effects on spermatozoa

The epidermal growth factor (EGF) family plays an important role in reproductive function regulation. The aim of this work was to investigate the localization of EGF, transforming growth factor alpha (TGFalpha) and epidermal growth factor receptor (EGFr) in boar epididymis and accessory genital glands, as well as study the presence of EGFr and the effects of EGF on boar spermatozoa. In the epididymis, prostate and vesicular glands EGF, TGFalpha and EGFr were detected in the pseudostratified epithelium. None of them were observed in the bulbourethral glands. Epidermal growth factor receptor was detected by immunofluorescence in non-capacitated, capacitated and acrosome reacted spermatozoa. Confocal microscopy revealed different staining patterns over the head, midpiece and/or flagellum whereas, flow cytometry analysis showed that the population of positive spermatozoa did not exceed 58% and did not depend on the functional state. To study the effects of EGF, spermatozoa were capacitated in Tyrodes medium containing 0, 10 and 100ng/ml EGF. Acrosome status, membrane integrity and motility patterns were evaluated after capacitation and after the acrosome reaction (AR). Capacitation in the presence of 100ng/ml EGF significantly improved the quality of movement (P<0.01) after the AR. These findings suggest that EGF and TGFalpha are produced in the reproductive tract of the boar where they may act locally and/or on a population of spermatozoa, improving the quality of movement after the AR.     

Separation, characterization and identification of boar seminal plasma proteins

Methods used for the isolation, separation and characterization of boar seminal plasma proteins are discussed, as well as techniques applied to study their binding properties. Attention is paid to interactions of these proteins with different types of saccharides and glycoconjugates, with membrane phospholipids, and to interactions between proteins. Boar seminal plasma contains different types of proteins: spermadhesins of the AQN and AWN families; DQH and PSP proteins belong to the most abundant. Some of these proteins are bound to the sperm surface during ejaculation and thus protein-coating layers of sperm are formed. Sperms coated with proteins participate in different types of interactions occurring in the course of the reproduction process, e.g. formation of the oviductal sperm reservoir, sperm capacitation, oocyte recognition and sperm binding to the oocyte.     

Selenium and glutathione peroxidase in seminal plasma of men and bulls

High levels of selenium and glutathione peroxidase (GSH-Px) were found in bull seminal plasma but low concentrations in human seminal plasma. In man the seminal plasma selenium was associated with two macromolecules separable by gel filtration, but no GSH-Px was found in the same fractions. Selenium in bull seminal plasma was associated with two proteins, which could be separated by gel filtration and anion exchange chromatography. Both macromolecules coeluted with GSH-Px activity and had identical optima at pH 7.0. Their responses to thermal treatment, however, differed. Seminal vesicle secretory fluid in the bull contained both these proteins, while the larger molecule was also found in fractionations of ampulla, prostate and Cowper's glands. The larger enzyme form is evidently a tetramer of the smaller one. Both enzyme forms were extremely sensitive to heavy metals and some divalent metal ions. GSH caused an activation while other reducing agents were suppressive. Triton X-100 had no effect, while sodium deoxycholate was inhibitory. These properties are typical for a phospholipid hydroperoxide GSH-Px. It is concluded that this selenium-dependent enzyme may be important in the protection of bovine spermatozoa against damage caused by oxygen radicals, while in man such a mechanism is not functional.     

In vitro evaluation of glutathione peroxidase (GPx)-like activity and antioxidant properties of some Ebselen analogues

Four analogues of Ebselen were synthesized and their glutathione peroxidase activity and antioxidant property evaluated and compared to Ebselen. Among the studied compounds, only diselenide [3] exhibited both glutathione peroxidase activity and radical-scavenging capability. Compounds [3] and [4] showed a strong inhibitory effect (53% and 43%, respectively) on the lipid peroxidation of linoleic acid compared to Ebselen and selenide derivatives ([1] and [2]) which were less active (28%, 26% and 18% inhibition, respectively). A concentration-dependent inhibitory effect was also found in the model of the formation of ABTS*+ radical cation: 65% and 89% inhibition for compound [3] at 10(-4) M and 5 x 10(-5) M, respectively, and 68% and 90% for compound [4], compared to 14% and 52% inhibition for Ebselen and the diselenides [1] and [2] (29%, 46% and 45%, 68%, respectively). By EPR spin trapping technique, the following inhibitory profile of the Ebselen analogues was observed towards the formation of thiyl radicals: Ebselen = [3]>[1]>[2]>[4]. Studies with compound [3] are in progress on oxidative stress cell models.     

Catalase activity in human spermatozoa and seminal plasma

Catalase activity was determined in human semen by measuring the oxygen burst with a Clark electrode, after H₂O₂ addition. Significant catalase activities (mean ± SD) were found in migrated, motile spermatozoa (44 ± 17 nmoles O₂/min/10⁸ cells) and in seminal plasma of normozoospermic men (129 ± 59 nmoles O₂/min/ml). It has been demonstrated that seminal catalase originated from prostate; however, its activity was not correlated with the usual prostatic markers (such as citric acid and zinc). Our data suggest a multiglandular function secreted by this organ. The catalase activities measured in seminal samples from asthenozo-ospermic, infertile men were found lower than those from normozoospermic subjects. The understanding of the relative contribution of the different enzyme systems against O₂ toxicity (superoxide dismutase, catalase, glutathione peroxidase) seem to be a priority area of research to understand disturbances of sperm function.     

Comparative aspects of the accessory sex glands and seminal biochemistry of mammals

• 1.1. Despite large gaps in the literature of semen biochemistry, especially of wild species, recent studies of the carbohydrate biochemistry of marsupial semen allow an approach to the question of the origin and evolution of mammalian seminal plasma.
• 2.2. The marsupials are a very important part of an overall picture of seminal plasma evolution because they are the end product of an alternative line of mammalian evolution genetically separated from the eutherians for over 100 million years.
• 3.3. The present paper discusses the accessory sex glands and seminal sugars of mammals from a comparative point of view in the light of present knowledge of evolutionary relationships and suggests possible schemes of evolution of mammalian seminal plasma.

     

Biological significance of phospholipid hydroperoxide glutathione peroxidase (PHGPx,GPx4) in mammalian cells

Reactive oxygen species (ROS) are known mediators of intracellular signal cascades. Excessive production of ROS may lead to oxidative stress, loss of cell function, and cell death by apoptosis or necrosis. Lipid hydroperoxides are one type of ROS whose biological function has not yet been clarified. Phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a unique antioxidant enzyme that can directly reduce phospholipid hydroperoxide in mammalian cells. This contrasts with most antioxidant enzymes, which cannot reduce intracellular phospholipid hydroperoxides directly. In this review, we focus on the structure and biological functions of PHGPx in mammalian cells. Recently, molecular techniques have allowed overexpression of PHGPx in mammalian cell lines, from which it has become clear that lipid hydroperoxides also have an important function as activators of lipoxygenase and cyclooxygenase, participate in inflammation, and act as signal molecules for apoptotic cell death and receptor-mediated signal transduction at the cellular level.     

Activation of the glutathione peroxidase 2 (GPx2) promoter by beta-catenin

GPx2, formerly named gastrointestinal glutathione peroxidase, is highly expressed in the proliferative area of the intestinal crypt-to-villus axis and in Paneth cells. Additionally, GPx2 is transiently up-regulated during development of gastrointestinal adenocarcinomas. Because both normal proliferation and differentiation of intestinal epithelial cells as well as carcinogenesis are regulated by the Wnt pathway, it was tested whether GPx2 may be a target of the beta-catenin/TCF complex which transfers Wnt signals. The GPx2 promoter contains five putative beta-catenin/TCF binding sites. Accordingly, the promoter was active in two cell lines with a constitutively active Wnt pathway, HepG2 and SW480, but not in BHK-21 cells in which the pathway is silent. Overexpression of beta-catenin/TCF activated the GPx2 promoter in all three cell lines. Overexpression of wild-type adenomatous polyposis coli (APC) in SW480 cells which harbor a mutated APC gene decreased basal GPx2 promoter activity. Truncation of the promoter identified one beta-catenin/TCF binding site that was sufficient for activation. Mutation of this site reduced the response to beta-catenin/TCF by more than 50%. These findings suggest a function of GPx2 in the maintenance of normal renewal of the intestinal epithelium. Whether up-regulation of GPx2 during carcinogenesis supports tumor growth or can rather be considered as a counteracting effect remains to be investigated.     

Morphological characterization of female accessory sex glands of Eupolybothrus fasciatus (Newport) (Chilopoda Lithobiomorpha)

The female reproductive system of Eupolybothrus fasciatus (Newport) (Chilopoda Lithobiomorpha) includes three types of well-developed accessory glands, viz. large glands, small glands, and the periatrial gland. External morphology and the ultrastructural organization of these glands have been investigated by light and electron microscopy. The small and large glands are paired and have coiled ducts that open, respectively, into and externally to the genital atrium. By contrast, the periatrial gland is unpaired and is located on the ventral wall of the atrium into which it opens via several small canals. Ultrastructural features show that all three glands consist of two different types of cells: secretory cells and ductule cells. The secretary cells contain prominent secretory granules and are similar to a class of insect epidermal gland cells (class 3) organized as acini surrounding an extracellular lumen into which microvilli project. The granules, which have different morphological features in each gland, could be responsible for important differential functions such as producing a sexual attractant, providing a coating material that protects eggs laid on the ground, and contributing to a fluid that digests spermatophores. © 1996 Wiley-Liss, Inc.     

Antioxidant effect of vitamin E and glutathione on lipid peroxidation in boar semen plasma

The protective effect of vitamin E and reduced glutathione (GSH) against lipid peroxidation in boar semen plasma was studied. The lipid peroxidation, measured by the test for thiobarbituric acid reactive substances (TBARS), doubled in the presence of the lipid peroxidation Fe²⁺-sodium ascorbate-inducing system. The ascorbate-induced TBARS were inhibited by about 62% through the water-soluble vitamin E analog (TROLOX) and about 57% by GSH. In the in vivo experiments, 7 wk of oraldl-α-tocopherol acetate (1000 IU/d/animal) administration caused a significant fall in the level of the semen plasma TBARS, from 2.2±0.09 to 1.2±0.13 nmol MDA/mL. The semen plasma superoxide dismutase (SOD) and GSSG tended to increase with the time of vitamin E administration, but the increment did not reach a significant level by the seventh week. The vitamin E supplementation significantly increased the number of spermatozoa per 1 cm³ of ejaculate. The protective role of vitamin E and GSH with respect to boar semen against fatty acid peroxidation and a positive influence of vitamin E supplementation on semen quality have been evidenced.     

Purification and characterization of a protein tyrosine acid phosphatase from boar seminal vesicle glands

A protein tyrosine phosphatase (PTPase) with acid phosphatase activity was purified (500-fold) from the fluid of boar seminal vesicles. Preparative purification was performed with a 3-step procedure, employing FPLC S-Sepharose Fast Flow, Mono Q and Superdex 75 column. Protein tyrosine acid phosphatase (PTAPase) was homogeneous by polyacrylamide gel electrophoresis (PAGE, SDS-PAGE). PTAPase is a glycoprotein which has a molecular weight of about 41-42 kDa. This enzyme was maximally active at pH 5.5, and its thermostability was less than 80 degrees C. The K(m) value for p-nitrophenylphosphate, a specific synthetic substrate, was 0.87 x 10(-3)M, however, higher substrate specificity was shown when phosphotyrosine (K(m)=0.37 x 10(-3)M) and protein fragments, such as gastrin (K(m)=0.0032 x 10(-3)M) and hirudin (K(m)=0.0075 x 10(-3)M), were used as substrates. Activity of PTAPase was inhibited by dephostatin, molybdate and orthovanadate by 100, 95 and 70%, respectively, when phosphotyrosine was used as the substrate. Immunofluorescence study has shown that the seminal vesicles are the only source of PTAPase in boar seminal plasma.