Single layer centrifugation improves the quality of fresh donkey semen and modifies the sperm ability to interact with polymorphonuclear neutrophils

This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 °C for 180 min. Integrity and lipid disorder of sperm plasma membrane, mitochondrial membrane potential, and intracellular levels of calcium and reactive oxygen species were evaluated at 0, 60, 120, and 180 min. In the second experiment, polymorphonuclear neutrophils (PMN) isolated from jennies blood were mixed with selected and unselected spermatozoa. Interaction between spermatozoa and PMN was evaluated after 0, 60, 120, and 180 min of co-incubation at 38 °C. SLC-selection increased the proportions of spermatozoa with an intact plasma membrane and low lipid disorder, of spermatozoa with high mitochondrial membrane potential and with high calcium levels, and of progressively motile spermatozoa. In addition, selection through SLC augmented the proportion of phagocytosed spermatozoa, which supported the modulating role of seminal plasma proteins on sperm-PMN interaction. In conclusion, SLC of fresh donkey semen increases the proportions of functionally intact and motile spermatozoa, and appears to remove the seminal plasma proteins that inhibit sperm-PMN binding

Effects of cryopreservation on the mitochondrial bioenergetics of bovine sperm

This study evaluated the bioenergetic map of mitochondria metabolism in cryopreserved bovine sperm. The detected oligomycin-sensitive basal respiration supported ATP production; frozen-thawed spermatozoa were found to have a coupling efficiency higher than 0.80. Cell respiration, however, was not stimulated by the protonophoric action of FCCP, as its titration with 1, 2, 4 and 6 mu M did not stimulate the uncoupling activity on oxidative phosphorylation as highlighted by unresponsive oxygen consumption. The unusual effect on the stimulation of maximal respiration was not related to fibronectin- or PDL-coated plates used for cellular metabolism analysis. Conversely, irradiation of frozen-thawed bovine sperm with the red light improved mitochondrial parameters. In effect, the maximal respiration of red-light-stimulated sperm in PDL-coated plates was higher than the non-irradiated. In spite of this, red-light irradiation had no impact on membrane integrity and mitochondrial activity evaluated by epifluorescence microscopy

Extracellular Reactive Oxygen Species (ROS) Production in Fresh Donkey Sperm Exposed to Reductive Stress, Oxidative Stress and NETosis

Altres ajuts: Generalitat de Catalunya 2017-SGR-1229Jenny shows a large endometrial reaction after semen influx to the uterus with a large amount of polymorphonuclear neutrophils (PMN) migrating into the uterine lumen. PMN act as a sperm selection mechanism through phagocytosis and NETosis (DNA extrudes and, together with proteins, trap spermatozoa). While a reduced percentage of spermatozoa are phagocytosed by PMN, most are found to be attached to neutrophil extracellular traps (NETs). This selection process together with sperm metabolism produces a large amount of reactive oxygen species (ROS) that influence the reproductive success. The present study aimed to determine the extracellular ROS production in both sperm and PMN. With this purpose, (1) donkey sperm were exposed to reductive and oxidative stresses, through adding different concentrations of reduced glutathione (GSH) and hydrogen peroxide (HO), respectively; and (2) PMN were subjected to NETosis in the presence of the whole semen, sperm, seminal plasma (SP) or other activators such as formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular ROS production (measured as HO levels) was determined with the Amplex ® Red Hydrogen Peroxide/Peroxidase Assay Kit. Donkey sperm showed more resilience to oxidative stress than to the reductive one, and GSH treatments led to greater HO extracellular production. Moreover, not only did SP appear to be the main inducer of NETosis in PMN, but it was also able to maintain the extracellular HO levels produced by sperm and NETosis

Involvement of extracellular vesicle-encapsulated miRNAs in human reproductive disorders: a systematic review

In recent years, extracellular vesicles (EVs) have emerged as essential players in cell-to-cell communication, particularly having an active regulating role in biological systems. Because reproductive-associated processes are not exempt of this communication, multiple studies have been devoted to this realm, focusing on gamete maturation, embryo implantation or fetal development. The aim of the present review was to comprehensively and systematically collect evidence about the function of the microRNA (miRNA) encapsulated in EVs isolated from different reproductive tissues or fluids in reproductive-related diseases. Following PRISMA guidelines, we conducted a systematic search of the literature published in MEDLINE-PubMed until the end of February 2021. After selection, 32 studies were included in the qualitative review comparing the miRNA expression profile in EVs between different pathological disorders. Most reports showed the potential of the miRNAs carried by EVs to be used as putative biomarkers of reproductive disorders, including pregnancy affections, disease progression and quality of preimplantation embryos. The most relevant miRNAs were found to be highly heterogeneous among studies, with some conflicting results. Further research is thus warranted to address whether cofounding factors, such as the methods to isolate EVs and miRNAs, the subset of EVs, the criteria of patient selection, the timing of sample retrieval, or any other factor, may explain the inconsistencies between studies

Inflammatory Markers in Uterine Lavage Fluids of Pregnant, Non-Pregnant, and Intrauterine Device Implanted Mares on Days 10 and 15 Post Ovulation

Intrauterine devices (IUDs) are used in mares to suppress oestrous behaviour, but the underlying mechanism is yet to be elucidated. The presence of an embryo or an IUD prevents cyclooxygenase-2 (COX-2) and, subsequently, prostaglandin (PG) release and luteolysis. However, inflammation may also be involved. Endometrial inflammatory markers in uterine lavage fluid were measured on Day 10 (EXP 1, n = 25) and Day 15 (EXP 2, n = 27) after ovulation in inseminated mares, non-pregnant or pregnant, and in mares in which a small plastic sphere had been inserted into the uterus 4 (EXP 1) or 3 days (EXP 2) after ovulation. Uterine lavage fluid samples were analysed for nitric oxide (NO), prostaglandin E2 (PGE2) (only EXP 1), prostaglandin F2α (PGF2α), inhibin A and cytokines, and blood samples for progesterone and oestradiol. On Day 10, the concentration of PGF2α was lower (p < 0.05) in the IUD group than in pregnant mares. The concentration of the modulatory cytokine IL-10 was significantly higher in the IUD group in comparison to non-pregnant mares, and inhibin A was significantly higher in IUD mares than in the pregnant counterparts on Day 15. The results suggest that the presence of IUD causes endometrial inflammation which is at a resolution stage on Day 15

Aquaporins 7 and 11 in boar spermatozoa : detection, localisation and relationship with sperm quality

Aquaporins (AQPs) are integral membrane water channels that allow transport of water and/or small solutes across cell membranes. Although water permeability is known to play a critical role in mammalian cells, including spermatozoa, little is known about their localisation in boar spermatozoa. Against this background, two different aquaporins, AQP7 and AQP11, were identified in boar spermatozoa by Wwestern blotting and localised through immunocytochemistry analyses. WOur western blot results showed that boar spermatozoa present AQP7 (25KDa) and AQP11 (50KDa). Immunocytochemistry analyses demonstrated that AQP7 is localised at the connecting piece of boar spermatozoa, while AQP11 was found in the head and in the midpiece, and a diffuse labelling was also seen along the tail. Despite differences in AQP7- and AQP11-content being seen between boar ejaculates, these differences were not found to be correlated with sperm quality in the case of AQP7. Conversely, AQP11-content showed a significant correlation (P<0.05) with sperm quality parameters, including sperm membrane integrity and fluidity, and sperm motility. In conclusion, boar spermatozoa present AQP7 and AQP11,. Additionally, and the amounts of the latterAQP11 but not of AQP7 the former are correlated with sperm motility and membrane integrity

Inflammatory Markers in Uterine Lavage Fluids of Pregnant, Non-Pregnant, and Intrauterine Device Implanted Mares on Days 10 and 15 Post Ovulation

Intrauterine devices (IUDs) are used in mares to suppress oestrous behaviour, but the underlying mechanism is yet to be elucidated. The presence of an embryo or an IUD prevents cyclooxygenase-2 (COX-2) and, subsequently, prostaglandin (PG) release and luteolysis. However, inflammation may also be involved. Endometrial inflammatory markers in uterine lavage fluid were measured on Day 10 (EXP 1, n = 25) and Day 15 (EXP 2, n = 27) after ovulation in inseminated mares, non-pregnant or pregnant, and in mares in which a small plastic sphere had been inserted into the uterus 4 (EXP 1) or 3 days (EXP 2) after ovulation. Uterine lavage fluid samples were analysed for nitric oxide (NO), prostaglandin E2 (PGE2) (only EXP 1), prostaglandin F2α (PGF2α), inhibin A and cytokines, and blood samples for progesterone and oestradiol. On Day 10, the concentration of PGF2α was lower (p < 0.05) in the IUD group than in pregnant mares. The concentration of the modulatory cytokine IL-10 was significantly higher in the IUD group in comparison to non-pregnant mares, and inhibin A was significantly higher in IUD mares than in the pregnant counterparts on Day 15. The results suggest that the presence of IUD causes endometrial inflammation which is at a resolution stage on Day 15