Higher temperatures lead to an increase of testicular metabolism that results in spermatic damage. Oxidative stress is the main factor responsible for testicular damage caused by heat stress. The aim of this study was to evaluate lasting effects of heat stress on ejaculated sperm and immediate or long-term effects of heat stress on epididymal sperm. We observed decrease in motility and mass motility of ejaculated sperm, as well as an increase in the percentages of sperm showing major and minor defects, damaged plasma and acrosome membranes, and a decrease in the percentage of sperm with high mitochondrial membrane potential in the treated group until one spermatic cycle. An increased enzymatic activity of glutathione peroxidase and an increase of stressed cells were observed in ejaculated sperm of the treated group. A decrease in the percentage of epididymal sperm with high mitochondrial membrane potential was observed in the treated group. However, when comparing immediate and long-term effects, we observed an increase in the percentage of sperm with low mitochondrial membrane potential. In conclusion, testicular heat stress induced oxidative stress that led to rescuable alterations after one spermatic cycle in ejaculated sperm and also after 30 days in epididymal sperm.

Our study aimed to assess the impact of sperm oxidative stress on embryo development by means of a dose-dependent model. In experiment 1, straws from five bulls were subjected to incubation with increasing H2O2 doses (0, 12.5, 25, and 50 μM). Motility parameters were evaluated by Computed Assisted System Analysis (CASA). Experiment 2 was designed to study a high (50 μM) and low dose (12.5 μM) of H2O2 compared to a control (0 μM). Samples were incubated and further used for in vitro fertilization. Analyses of motility (CASA), oxidative status (CellROX green and 2'-7' dichlorofluorescein diacetate), mitochondrial potential (JC-1), chromatin integrity (AO), and sperm capacitation status (chlortetracycline) were performed. Embryos were evaluated based on fast cleavage (30 h.p.i.), cleavage (D = 3), development (D = 5), and blastocyst rates (D = 8). We observed a dose-dependent deleterious effect of H2O2 on motility and increase on the percentages of positive cells for CellROX green, capacitated sperm, and AO. A decrease on cleavage and blastocyst rates was observed as H2O2 increased. Also, we detected a blockage on embryo development. We concluded that sperm when exposed to oxidative environment presents impaired motility traits, prooxidative status, and premature capacitation; such alterations resulting in embryo development fail.

SUMMARYMelatonin is a ubiquitous molecule found in a wide range of fluids, one of them being ram seminal plasma, in which it can reach higher concentrations than those found in blood, suggesting an extrapineal secretion by the reproductive tract. In order to identify the source of the melatonin found in ram seminal plasma, we first tried to determine whether the melatonin levels were maintained during the day. For this purpose, melatonin concentrations were measured in seminal plasma obtained from first ejaculates of six rams at 6:00 a.m. in total darkness, at 10:00 a.m. and at 14:00 p.m. The melatonin concentration was higher (p < 0.05) in ejaculates collected at 6:00 a.m. than at 10:00 and 14:00. There was no statistical difference between the latter. To further corroborate an extrapineal secretion of melatonin, the presence of the two key enzymes involved in melatonin synthesis, arylalkylamine-N-acetyltransferase (AANAT) and Nacetylserotonin-O-methyltransferase (ASMT) was analyzed by RT-PCR, q-PCR and Western-blot in ram testes, epididymis, and accessory glands. The RT-PCR showed the presence of the m-RNA codifying both AANAT and ASTM in all the tissues under study, but the q-PCR and Western-blot revealed that gene expression of these enzymes was significantly higher in the testis (p < 0.05). Immunohistochemistry confirmed the presence of AANAT and ASMT in the testis and revealed that they were found in the Leydig cells, spermatocytes, and spermatids. Also, measurable levels of melatonin were found in testicular tissue and the tail of the epididymis. In conclusion, our study indicates that the testes are one of the likely sources of the high levels of melatonin found in ram seminal plasma, at least during the day.

Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period. Sperm analysis (motility, mass motility, abnormalities, membrane and acrosome status, mitochondrial potential, DNA fragmentation, lipid peroxidation and intracellular free radicals production); protamine deficiency; PRM1, TNP1 and TNP2 gene expression; and determination of glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) and superoxide dismutase activity and immunodetection in seminal plasma were performed. Samples were distributed into four groups according to the sperm susceptibility to lipid peroxidation after induction with ascorbate and ferrous sulfate (low, medium, high and very high). The results were analyzed by GLM test and post hoc least significant difference. We observed an increase in native GPx activity and CAT immunodetection in groups with high susceptibility to induced lipid peroxidation. We also found an increase in total sperm defects, acrosome and membrane damages in the group with the highest susceptibility to induced lipid peroxidation. Additionally, the low mitochondrial membrane potential, susceptible to chromatin fragmentation and the PRM1 mRNA were increased in the group showing higher susceptibility to lipid peroxidation. Ram sperm susceptibility to lipid peroxidation may compromise sperm quality and interfere with the oxidative homeostasis by oxidative stress, which may be the main cause of chromatin damage in ram sperm.

Sperm DNA fragmentation is considered one of the main causes of male infertility. The most accepted causes of sperm DNA damage are deleterious actions of reactive oxygen species (ROS), defects in protamination, and apoptosis. Ram sperm are highly prone to those damages due to the high susceptibility to ROS and to oxidative stress caused by heat stress. We aimed to evaluate the effects of heat stress on the chromatin of ejaculated and epididymal sperm and the activation of apoptotic pathways in different cell types in ram testis. We observed higher percentages of ejaculated sperm with increased chromatin fragmentation in the heat stress group; a fact that was unexpectedly not observed in epididymal sperm. Heat stress group presented a higher percentage of spermatozoa with DNA fragmentation and increased number of mRNA copies of transitional protein 1. Epididymal sperm presented greater gene expression of protamine 1 on the 30th day of the spermatic cycle; however, no differences in protamine protein levels were observed in ejaculated sperm and testis. Localization of proapoptotic protein BAX or BCL2 in testis was not different. In conclusion, testicular heat stress increases ram sperm DNA fragmentation without changes in protamination and apoptotic patterns.

ContentsMelatonin is a ubiquitous molecule, present in a wide range of organisms, and involved in multiple functions. Melatonin relays the information about the photoperiod to the tissues that express melatonin-binding sites in both central and peripheral nervous systems. This hormone has a complex mechanism of action. It can cross the cell plasma membrane and exert its actions in all cells of the body. Certain melatonin actions are mediated by receptors that belong to the superfamily of G-protein-coupled receptors (GPCRs), the MT 1 and MT 2 membrane. Melatonin can also bind to calmodulin as well as to nuclear receptors of the retinoic acid receptor family, RORa1, RORa2 and RZRb. The purpose of this review is to report on recent developments in the physiological role of melatonin and its receptors. Specific issues concerning the biological function of melatonin in mammalian seasonal reproduction and spermatozoa are considered. The significance of the continuous presence of melatonin in seminal plasma with a fairly constant concentration is also discussed.

SummarySperm DNA fragmentation is referred to as one of the main causes of male infertility. Failures in the protamination process, apoptosis and action of reactive oxygen species (ROS) are considered the most important causes of DNA fragmentation. Action of ROS or changes in sperm protamination would increase the susceptibility of sperm DNA to fragmentation. Routine semen analysis is unable to estimate sperm chromatin damage. Sperm DNA integrity influences sperm functional capability, therefore tests that measure sperm DNA fragmentation are important to assess fertility disorders. Actually, there is a considerable number of methods for assessing sperm DNA fragmentation and chromatin integrity, sperm chromatin stability assay (SCSA modified), sperm chromatin dispersion (SCD), comet assay, transferase dUTP nick end labelling (TUNEL); and protamine evaluation in sperm chromatin assay, such as toluidine blue, CMA3, protamine expression and evaluation of cysteine radicals. This review aims to describe the main causes of sperm DNA fragmentation and the tests commonly used to evaluate sperm DNA fragmentation.

Some melatonin functions in mammals are exerted through MT1 and MT2 receptors. However, there are no reports of their presence in the reproductive tract of the ram, a seasonal species. Thus, we have investigated their existence in the ram testis, epididymis, accessory glands and ductus deferens. Real-time polymerase chain reaction (qPCR) revealed higher levels of m-RNA for both receptors in the testis, ampulla, seminal vesicles, and vas deferens, than in the other organs of the reproductive tract (p < 0.05). Western blot analyses showed protein bands compatible with the MT1 in the testis and cauda epididymis, and for the MT2 in the cauda epididymis and deferent duct. Immunohistochemistry analyses revealed the presence of MT1 receptors in spermatogonias, spermatocytes, and spermatids, and MT2 receptors in the newly-formed spermatozoa in the testis, whereas both receptors were located in the epithelial cells of the ampulla, seminal vesicles, and ductus deferens. Indirect immunofluorescence showed significant differences in the immunolocation of both receptors in spermatozoa during their transit in the epididymis. In conclusion, it was demonstrated that melatonin receptors are present in the ram reproductive tract. These results open the way for new studies on the molecular mechanism of melatonin and the biological significance of its receptors.