Melatonin Protects Bovine Spermatozoa by Reinforcing Their Antioxidant Defenses.

Cryopreserved semen is widely used in assisted reproductive techniques. Post-thawing spermatozoa endure oxidative stress due to the high levels of reactive oxygen and nitrogen species, which are produced during the freezing/thawing process, and the depletion of antioxidants. To counteract this depletion, supplementation of sperm preparation medium with antioxidants has been widely applied. Melatonin is a hormone with diverse biological roles and a potent antioxidant, with an ameliorative effect on spermatozoa. In the present study, we assessed the effect of melatonin on thawed bovine spermatozoa during their handling. Cryopreserved bovine spermatozoa were thawed and incubated for 60 min in the presence or absence of 100 µΜ melatonin. Also, the effect of melatonin was assessed on spermatozoa further challenged by the addition of 100 µΜ hydrogen peroxide. Spermatozoa were evaluated in terms of kinematic parameters (CASA), viability (trypan blue staining) and antioxidant capacity (glutathione and NBT assay, determination of iNOS levels by Western blot analysis). In the presence of melatonin, spermatozoa presented better kinematic parameters, as the percentage of motile and rapid spermatozoa was higher in the melatonin group. They also presented higher viability and antioxidant status, as determined by the increased cellular glutathione levels and the decreased iNOS protein levels.

A review of the use of antioxidants in bovine sperm preparation protocols.

Reactive oxygen species (ROS) and oxidative stress (OS), the imbalance between the production of free radicals and the cellular antioxidant defenses, are discussed in relation to their role in bovine sperm physiology. Oxidative stress has been associated to male infertility and low fertility rates in Assisted Reproductive Techniques (ART). Antioxidant supplementation is an interesting approach to overcome OS-related infertility and assisted reproduction drawbacks. Several studies have been conducted to identify the potential sources of ROS in a typical ART setting and the impact of antioxidant supplementation on semen quality and pregnancy outcome. Procedures such as freezing and thawing, centrifugation and incubation are thought to produce significant amounts of ROS with a negative impact on sperm quality parameters and reproductive competence. Given the important role of ROS in sperm function, the addition of antioxidants in sperm media to prevent OS and to improve the reproductive outcome requires attention. Currently, there is limited evidence to support the ameliorative effect of antioxidant supplementation on fertilization and embryo development in farm animals. This review summarizes the different types and concentrations of antioxidants used in sperm preparation media of bovine species and their effectiveness in neutralizing excessive ROS production while preserving physiological sperm function.

The addition of crocin in the freezing medium extender improves post-thaw semen quality.

Semen cryopreservation is arguably the most important method or technique contributing to the advancement of modern animal production. However, the quality of sperm after thawing is still highly variable. The addition of antioxidant compounds to the freezing medium has been used customarily to counteract the harmful effects of Reactive Oxygen Species (ROS) that are produced during the freeze/thaw process. Crocin, a potent antioxidant, improves the fertilizing capacity of spermatozoa. In this study, we evaluated the potential of crocin (0, 0.5 and 1 mM) as an extender additive to diminish the damaging effects of cryopreservation on bovine spermatozoa. Post-thaw semen quality was assessed by means of motility, viability and lipid peroxidation (LPO). We further investigated the effect of crocin supplementation upon freezing on sperm quality parameters during their incubation at 37°C for up to 2 hr. Overall, the data assessment indicates that crocin facilitated a general improvement of the quality of freeze/thawed spermatozoa, under the present experimental conditions. Crocin (1 mM) maintained a higher percentage of alive spermatozoa with intact acrosome with rapid and progressive motility, compared to the control extender. Moreover, the spermatozoa cryopreserved in the presence of crocin exhibited higher values in CASA kinematic parameters (VCL, VSL, VAP, ALH) immediately after thawing. Furthermore, the positive effect of crocin on motility parameters was also sustained over a period of 2 hr incubation at 37°C. This effect of crocin may be attributed to the observed inhibition of LPO during the incubation period. Thus, the results indicate that the addition of crocin (especially at a final concentration of 1 mM) in the freezing extender medium may benefit the preservation of the quality parameters of spermatozoa that are compromised by the freeze/thaw heat shock and the stress during handling for IVF or artificial insemination.

Oleuropein is a natural inhibitor of PAI-1-mediated proliferation in human ER-/PR- breast cancer cells.

PURPOSE: Elevated expression of PAI-1 has been widely linked with adverse outcomes in a variety of human cancers, such as breast, gastric and ovarian cancers, rendering PAI-1 a prognostic biomarker. As a result, several chemical inhibitors are currently being developed against PAI-1; however, the clinical setting where they might confer survival benefits has not yet been elucidated. METHODS: RNA sequencing data analysis from the TCGA/GTEx cancer portals (n = 3607 samples). In silico molecular docking analyses to predict functional macromolecule interactions. ER-/PR- (MDA-MB-231) and ER+/PR+ (MCF-7) breast cancer cell lines implemented to assess the effect of oleuropein as a natural inhibitor of PAI-1-mediated oncogenic proliferation. RESULTS: We show that high PAI-1 levels inversely correlate with ER and PR expressions in a wide panel of estrogen/progesterone-responsive human malignancies. By implementing an in silico molecular docking analysis, we identify oleuropein, a phenolic component of olive oil, as a potent PAI-1-binding molecule displaying increased affinity compared to the other olive oil constituents. We demonstrate that EVOO or oleuropein treatment alone may act as a natural PAI-1 inhibitor by incrementally destabilising PAI-1 levels selectively in ER-/PR- breast cancer cells, accompanied by downstream caspase activation and cell growth inhibition. In contrast, ER+/PR+ breast cancer cells, where PAI-1 expression is absent or low, do not adequately respond to treatment. CONCLUSIONS: Our study demonstrates an inverse correlation between PAI-1 and ESR1/PGR levels, as well as overall patient survival in estrogen/progesterone-responsive human tumours. With a focus on breast cancer, our data identify oleuropein as a natural PAI-1 inhibitor and suggest that oleuropein-mediated PAI-1 destabilisation may confer clinical benefit only in ER-/PR- tumours.

CCl4 induces tissue-type plasminogen activator in rat brain; protective effects of oregano, rosemary or vitamin E.

The high metabolic rate and relatively low antioxidant defenses of the lipid-rich brain tissue render it highly susceptible to reactive oxygen species (ROS) and oxidative stress, whereas the implication of ROS in the pathogenesis of several diseases in the central nervous system is well-established. The plasminogen activator (PA) system is a key modulator of extracellular proteolysis, extracellular matrix remodeling and neuronal cell signaling and has been implicated in the pathogenesis of these diseases. This study evaluates the role of tissue-type PA (t-PA) in oxidative stress and the protective role of dietary antioxidants in the rat brain. We used the CCl4 experimental model of ROS-induced lipid peroxidation and evaluated the antioxidant effect of oregano, rosemary or vitamin E. CCl4-treated Wistar rats exhibited elevated brain t-PA activity, which was decreased upon long-term administration of oregano, rosemary or vitamin E. PA inhibitor-1 (PAI-1) activity was also slightly elevated by CCl4, but this increase was not affected by the antioxidants. We hypothesize that the CCl4-induced t-PA activity indicates extracellular proteolytic activity that may be linked to neuronal cell death and brain damage. Vitamin E or antioxidants present in oregano or rosemary are effective in inhibiting t-PA elevation and can be considered as a potential protection against neuronal damage.

Endosulfan-induced lipid peroxidation in rat brain and its effect on t-PA and PAI-1: ameliorating effect of vitamins C and E.

Endosulfan provokes systemic toxicity in mammals and induces reactive oxygen species (ROS) and lipid peroxidation (LPO). The brain is susceptible to LPO and several studies implicate ROS and LPO in CNS diseases. Tissue plasminogen activator (t-PA) has been accredited with plasminogen-dependent roles in the CNS, as well as plasminogen-independent functions. The aim of this study was to investigate the activities of t-PA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1) in the adult rat brain, after subchronic endosulfan treatment. Furthermore, the potency of vitamins C and E to attenuate these effects was explored. Endosulfan was administered in Wistar rats either alone or with vitamin C and/or vitamin E. The induced oxidative stress was manifested by induction of LPO as determined by higher malondialdehyde levels. This was accompanied by elevation of t-PA and PAI-1 activities. Vitamins E and C, both well-known for their antioxidant properties, substantially acted in a preventive way and protected the brain from these effects.