33 results on '"Perez Martinez S"'
Search Results
2. Fibronectin induces capacitation-associated events through the endocannabinoid system in bull sperm
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Osycka-Salut, C.E., Martínez-León, E., Gervasi, M.G., Castellano, L., Davio, C., Chiarante, N., Franchi, A.M., Ribeiro, M.L., Díaz, E.S., and Perez-Martinez, S.
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- 2020
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3. Characterisation and partition of valuable metals from WEEE in weathered municipal solid waste incineration bottom ash, with a view to recovering
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Pérez-Martínez, S., Giro-Paloma, J., Maldonado-Alameda, A., Formosa, J., Queralt, I., and Chimenos, J.M.
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- 2019
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4. Lysophosphatidic acid increases the production of pivotal mediators of decidualization and vascularization in the rat uterus
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Beltrame, J.S., Sordelli, M.S., Cella, M., Perez Martinez, S., Franchi, A.M., and Ribeiro, M.L.
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- 2013
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5. Characterization of human sperm N-acetylglucosaminidase
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Perez Martinez, S. L., Menendez Helman, R. J., Zitta, K. S., Brandelli, A., and Miranda, P. V.
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- 2008
6. Nitric oxide inhibits prostanoid synthesis in the rat oviduct
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Perez Martinez, S., Farina, M., Ogando, D., Ribeiro, M.L., Gimeno, M., and Franchi, A.M.
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- 2000
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7. The effect of epidermal growth factor on prostaglandin synthesis of oestrogenized rat uterus is mediated by nitric oxide
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Ribeiro, M.L., Perez Martinez, S., Farina, M., Ogando, D., Gimeno, M., and Franchi, A.
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- 1999
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8. Involvement of cAMP efflux, through MRP4 transporter, in the acquisition of fertilization ability of mouse spermatozoa.
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Alonso, C. A. I., Lottero Leconte, R., Luque, G. M., Vernaz, Z., Di Siervi, N., Gervasi, M. G., Buffone, M. G., Davio, C., and Perez Martinez, S.
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SPERMATOZOA ,FERTILIZATION in vitro ,EXTRACELLULAR space ,CAMPS ,KNOCKOUT mice ,MULTIDRUG resistance - Abstract
Mammalian spermatozoa must undergo biochemical and structural changes to acquire fertilizing ability, in a process known as capacitation. Activation of PKA is essential for capacitation, and thus cAMP levels are tightly regulated during this process. Previously, we demonstrated that during capacitation, bovine spermatozoa extrude cAMP through the Multidrug Resistance Protein 4 (MRP4) which regulates intracellular levels of the nucleotide and provides cAMP to the extracellular space. Here, we report the presence of a functional MRP4 in murine spermatozoa, since its pharmacological inhibition with MK571 decreased levels of extracellular cAMP. This also produced a sudden increase in PKA activity, with decreased tyrosine phosphorylation (pY) at the end of capacitation. Blockade of MRP4 inhibited induced-acrosome reaction, hyperactivation and in vitro fertilization. Moreover, MRP4 inhibition induced an increase in Ca2+ levels mediated by PKA and depletion of Ca2+ salts from the medium prevented the loss of motility and pY inhibition produced by MK571. These results were supported using spermatozoa from CatSper Ca2+-channel knockout mice. Altogether, these results suggest that cAMP efflux, through MRP4, plays an essential role in mouse sperm capacitation. [ABSTRACT FROM AUTHOR]
- Published
- 2019
9. Sperm Release From the Oviductal Epithelium Depends on Ca2+ Influx Upon Activation of CB1 and TRPV1 by Anandamide.
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Gervasi, M.G., Osycka‐Salut, C., Sanchez, T., Alonso, C.A.I., Llados, C., Castellano, L., Franchi, A.M., Villalón, M., and Perez‐Martinez, S.
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- 2016
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10. Lysophosphatidic acid increases the production of pivotal mediators of decidualization and vascularization.
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Beltrame, J.S., Sordelli, M.S., Szmelc, A., Cella, M., Perez Martinez, S., Franchi, A.M., and Ribeiro, M.L.
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- 2015
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11. The nitric oxide synthase of mouse spermatozoa
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Herrero, M.B, Goin, J.C, Boquet, M, Canteros, M.G, Franchi, A.M, Perez Martinez, S, Polak, J.M, Viggiano, J.M, and Gimeno, M.A.F
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- 1997
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12. Effect of bicarbonate and polyvinyl alcohol on in vitro capacitation and fertilization ability of cryopreserved equine spermatozoa.
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Arroyo-Salvo C, Río S, Bogetti ME, Plaza J, Miragaya M, Yaneff A, Davio C, Fissore R, Gervasi MG, Gambini A, and Perez-Martinez S
- Abstract
Background: Factors contributing to the limited success of in vitro fertilization in horses remain to be studied. In this work, we elucidated the effect of different essential capacitation media components, bicarbonate, and bovine serum albumin or polyvinyl-alcohol, and the incubation microenvironment on sperm parameters associated with capacitation, acrosome reaction, and their ability to activate oocytes via heterologous intracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa., Methods: Frozen-thawed spermatozoa underwent incubation at different time intervals in either Tyrode's albumin lactate pyruvate medium (non-capacitating; NC) or Tyrode's albumin lactate pyruvate supplemented with bicarbonate, bicarbonate and polyvinyl-alcohol, bicarbonate and bovine serum albumin, polyvinyl-alcohol and bovine serum albumin alone. Protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels, sperm motility, and acrosome reaction percentages were evaluated. After determining the best condition media (capacitating; CAP), heterologous intracytoplasmic spermatozoa injection on pig oocytes was performed and the phospholipase C zeta sperm localization pattern was evaluated., Results: Incubation of frozen-thawed equine spermatozoa with bicarbonate and polyvinyl-alcohol in atmospheric air for 45 min induced an increase in protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels compared to NC condition. Sperm incubation in bicarbonate and polyvinyl-alcohol medium showed an increase in total motility and progressive motility with respect to NC (p ≤ 0.05). Interestingly, three parameters associated with sperm hyperactivation were modulated under bicarbonate and polyvinyl-alcohol conditions. The kinematic parameters curvilinear velocity and amplitude of lateral head displacement significantly increased, while straightness significantly diminished (curvilinear velocity: bicarbonate and polyvinyl-alcohol = 120.9 ± 2.9 vs. NC = 76.91 ± 6.9 µm/s) (amplitude of lateral head displacement: bicarbonate and polyvinyl-alcohol = 1.15 ± 0.02 vs. NC = 0.77 ± 0.03 µm) (straightness: bicarbonate and polyvinyl-alcohol = 0.76 ± 0.01 vs. NC = 0.87 ± 0.02) (p ≤ 0.05). Moreover, the spontaneous acrosome reaction significantly increased in spermatozoa incubated in this condition. Finally, bicarbonate and polyvinyl-alcohol medium was established as CAP medium. Although no differences were found in phospholipase C zeta localization pattern in spermatozoa incubated under CAP, equine spermatozoa pre-incubated in CAP condition for 45 min showed higher fertilization rates when injected into matured pig oocytes (NC: 47.6% vs. CAP 76.5%; p ≤ 0.05)., Conclusion: These findings underscore the importance of bicarbonate and polyvinyl-alcohol in supporting critical events associated with in vitro sperm capacitation in the horse, resulting in higher oocyte activation percentages following heterologous intracytoplasmic spermatozoa injection. This protocol could have an impact on reproductive efficiency in the equine breeding industry., (© 2024 American Society of Andrology and European Academy of Andrology.)
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- 2024
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13. Deep Learning-Based Predictive Model for Revascularization of Chronic Total Occlusions on Angiographic Imaging.
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Perez-Martinez S, Fernandez-Cisnal A, Perez-Pelegri M, Garcia-Blas S, Minana G, Valero E, Sanchis J, and Moratal D
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- Humans, Treatment Outcome, Coronary Angiography, Percutaneous Coronary Intervention methods, Deep Learning, Coronary Occlusion diagnostic imaging, Coronary Occlusion surgery
- Abstract
Revascularization of chronic total occlusions (CTO) is currently one of the most complex procedures in percutaneous coronary intervention (PCI), requiring the use of specific devices and a high level of experience to obtain good results. Once the clinical indication for extensive ischemia or angina uncontrolled with medical treatment has been established, the decision to perform coronary intervention is not simple, since this procedure has a higher rate of complications than non-PCI percutaneous intervention, higher ionizing radiation doses and a lower success rate. However, CTO revascularization has been shown to be helpful in symptomatic improvement of angina, reduction of ischemic burden, or improvement of ejection fraction. The aim of this work is to determine whether a model developed using deep learning techniques, and trained with angiography images, can better predict the likelihood of a successful revascularization procedure for a patient with a chronic total occlusion (CTO) lesion in their coronary artery (measured as procedure success and the duration of time during which X-ray imaging technology is used to perform a medical procedure) than the scales traditionally used. As a preliminary approach, patients with right coronary artery CTO will be included since they present standard angiographic projections that are performed in all patients and present less technical variability (duration, projection angle, image similarity) among them.The ultimate objective is to develop a predictive model to help the clinician in the decision to intervene and to analyze the performance in terms of predicting the success of the technique for the revascularization of chronic occlusions.Clinical Relevance- The development of a deep learning model based on the angiography images could potentially overcome the gold standard and help interventional cardiologists in the treatment decision for percutaneous coronary intervention, maximizing the success rate of coronary intervention.
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- 2023
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14. Evaluation of sperm integrin α5β1 as a potential marker of fertility in humans.
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Vernaz ZJ, Lottero-Leconte RM, Alonso CAI, Rio S, Morales MF, Arroyo-Salvo C, Valiente CC, Lovaglio Diez M, Bogetti ME, Arenas G, Rey-Valzacchi G, and Perez-Martinez S
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- Animals, Biomarkers, Cattle, Female, Fertilization in Vitro, Humans, Male, Spermatozoa, Integrin alpha5beta1, Semen
- Abstract
Sperm selection for assisted reproduction techniques is generally based on basic parameters, while key aspects of sperm competence and its journey from the deposition site to the fertilization site are overlooked. Consequently, identifying molecular markers in spermatozoa that can efficiently predict the fertility of a semen sample could be of great interest, particularly in cases of idiopathic male infertility. When spermatozoa reach the female reproductive tract, it provides to them the cellular and molecular microenvironment needed to acquire fertilizing ability. In this sense, considering the role that integrin α5β1 of spermatozoa plays in reproduction-related events, we investigated the correlation between the subcellular localization of sperm integrin α5β1 and early embryo development outcome after in vitro fertilization (IVF) procedures in human. Twenty-four semen samples from normozoospermic men and metaphase II (MII) oocytes from healthy women aged under 38 years, from couples who underwent IVF cycles, were used in this work. Sperm α5β1 localization was evaluated by immunofluorescence assay using an antibody against integrin α5 subunit. Integrin α5β1 was mainly localized in the sperm acrosomal region (45.33±7.89%) or the equatorial segment (30.12±7.43%). The early embryo development rate (data obtained from the Fertility Center) correlated positively with the localization of α5β1 in the acrosomal region (number of usable embryos / inseminated oocytes: ρ = 0.75; p<0.01 and number of usable embryos/total number of two pronuclear zygotes: ρ = 0.80; p<0.01). However, this correlation was not significant when the equatorial segment mark was evaluated. In addition, human sperm released from co-culture with bovine oviductal epithelial cells (BOEC) showed a significant enrichment in the acrosomal localization pattern of α5β1 compared to those sperm that were not co-cultured with BOEC (85.20±5.35% vs 35.00±17.09%, respectively, p<0.05). In conclusion, the evaluation of sperm integrin α5β1 immunolocalization could be a useful tool to select sperm with fertilizing ability from human semen samples before IVF procedures., Competing Interests: The authors have declared that no competing interests exist.
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- 2022
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15. How, where and when is SPINK3 bound and removed from mouse sperm?
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Nicolli AR, Alonso CAI, Otamendi C, Cerletti M, Poetsch A, Sharma V, Zalazar L, Perez-Martinez S, and Cesari A
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- Acrosome, Animals, Female, Fertilization, Humans, Male, Mammals, Mice, Sperm Capacitation, Serine Proteinase Inhibitors, Spermatozoa metabolism
- Abstract
Sperm capacitation in mammals is a fundamental requirement to acquire their fertilizing capacity. Little is known about the action mechanism of the molecules that prevent capacitation from occurring prematurely. These molecules are known as decapacitation factors (DFs) and they must be removed from the sperm surface for capacitation to occur successfully. Serine protease inhibitor Kazal type 3 (SPINK3) has been proposed as one of these DFs. Here, we evaluate how this protein binds to mouse sperm and its removal kinetics. We describe that SPINK3 is capable of binding to the membrane of mature epididymal sperm through protein-lipid interactions, specifically to lipid rafts subcellular fraction. Moreover, cholera toxin subunit b (CTB) avoids SPINK3 binding. We observe that SPINK3 is removed from the sperm under in vitro capacitating conditions and by the uterine fluid from estrus females. Our ex vivo studies show the removal kinetics of this protein within the female tract, losing SPINK3 formerly from the apical region of the sperm in the uterus and later from the flagellar region within the oviduct. The presence of acrosome-reacted sperm in the female duct concurs with the absence of SPINK3 over its surface.
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- 2022
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16. Cyclic AMP efflux through MRP4 regulates actin dynamics signalling pathway and sperm motility in bovines.
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Chiarante N, Alonso CAI, Plaza J, Lottero-Leconte R, Arroyo-Salvo C, Yaneff A, Osycka-Salut CE, Davio C, Miragaya M, and Perez-Martinez S
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- Animals, Cattle, Male, Phosphorylation, Signal Transduction, Acrosome physiology, Actins physiology, Cyclic AMP metabolism, Multidrug Resistance-Associated Proteins metabolism, Sperm Capacitation, Sperm Motility physiology
- Abstract
Previously we demonstrated that multidrug resistance-associated protein 4 transporter (MRP4) mediates cAMP efflux in bovine spermatozoa and that extracellular cAMP (ecAMP) triggers events associated to capacitation. Here, we deepen the study of the role of MRP4 in bovine sperm function by using MK571, an MRP4 inhibitor. The incubation of spermatozoa with MK571 during 45 min inhibited capacitation-associated events. MRP4 was localized in post-acrosomal region and mid-piece at 15 min capacitation, while at 45 min it was mainly located in the acrosome. After 15 min, MK571 decreased total sperm motility (TM), progressive motility (PM) and several kinematic parameters. The addition of ecAMP rescued MK571 effect and ecAMP alone increased the percentage of motile sperm and kinematics parameters. Since actin cytoskeleton plays essential roles in the regulation of sperm motility, we investigated if MRP4 activity might affect actin polymerization. After 15 min capacitation, an increase in F-actin was observed, which was inhibited by MK571. This effect was reverted by the addition of ecAMP. Furthermore, ecAMP alone increased F-actin levels while no F-actin was detected with ecAMP in the presence of PKA inhibitors. Our results support the importance of cAMP efflux through MRP4 in sperm capacitation and suggest its involvement in the regulation of actin polymerization and motility.
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- 2020
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17. MRP4-mediated cAMP efflux is essential for mouse spermatozoa capacitation.
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Alonso CAI, Lottero-Leconte R, Luque GM, Vernaz ZJ, Di Siervi N, Gervasi MG, Buffone MG, Davio C, and Perez-Martinez S
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- Animals, Calcium metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Male, Mice, Inbred C57BL, Models, Biological, Phosphorylation drug effects, Phosphotyrosine metabolism, Propionates pharmacology, Quinolines pharmacology, Sperm Capacitation drug effects, Sperm Motility drug effects, Spermatozoa drug effects, Spermatozoa metabolism, Cyclic AMP metabolism, Multidrug Resistance-Associated Proteins metabolism, Sperm Capacitation physiology
- Abstract
Mammalian spermatozoa must undergo biochemical and structural changes to acquire the capacity for fertilization, in a process known as capacitation. Activation of PKA enzymes is essential for capacitation, and thus cAMP levels are tightly regulated during this process. Previously, we demonstrated that during capacitation, bovine spermatozoa extrude cAMP through multidrug resistance-associated protein 4 (MRP4, also known as ABCC4), which regulates intracellular levels of the nucleotide and provides cAMP to the extracellular space. Here, we report the presence of functional MRP4 in murine spermatozoa, since its pharmacological inhibition with MK571 decreased levels of extracellular cAMP. This also produced a sudden increase in PKA activity, with decreased tyrosine phosphorylation at the end of capacitation. Blockade of MRP4 inhibited induction of acrosome reaction, hyperactivation and in vitro fertilization. Moreover, MRP4 inhibition generated an increase in Ca
2+ levels mediated by PKA, and depletion of Ca2+ salts from the medium prevented the loss of motility and phosphotyrosine inhibition produced by MK571. These results were supported using spermatozoa from CatSper Ca2+ channel knockout mice. Taken together, these results suggest that cAMP efflux via MRP4 plays an essential role in mouse sperm capacitation.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)- Published
- 2019
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18. Steroid hormones induce in vitro human first trimester trophoblast tubulogenesis by the lysophosphatidic acid pathway.
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Beltrame JS, Sordelli MS, Cañumil VA, Alonso CAI, Perez Martinez S, and Ribeiro ML
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- Cell Line, Estradiol pharmacology, Female, Humans, Neovascularization, Physiologic drug effects, Phosphoric Diester Hydrolases genetics, Phosphoric Diester Hydrolases metabolism, Pregnancy, Progesterone pharmacology, RNA, Messenger genetics, RNA, Messenger metabolism, Trophoblasts drug effects, Lysophospholipids metabolism, Pregnancy Trimester, First metabolism, Steroids pharmacology, Trophoblasts cytology, Trophoblasts metabolism
- Abstract
Successful implantation and placentation requires that extravillous cytotrophoblast acquires an endovascular phenotype and remodels uterine spiral arteries. Progesterone (P4) and estradiol (E2) control many of the placental functions, but their role in vascular remodeling remains controversial. Here, we investigated whether P4 and E2 regulate the acquisition of the human first trimester trophoblast endovascular phenotype, and the participation of the lysophosphatidic acid pathway. For this purpose, human first trimester HTR-8/SVneo cells were seeded on Geltrex and assayed for capillary-like tube formation. P4 and E2 increased HTR-8/SVneo tube formation in a concentration-dependent manner and this effect is mediated by the LPA3 receptor. Moreover, sex steroids increased the mRNA levels of the main enzyme that produce lysophosphatidic acid (lysophospholipase-D) but did not regulate LPA3 mRNA levels. Overall, we demonstrate that steroid hormones regulate HTR-8/SVneo trophoblast capillary-like structures formation and we propose that this process could be modulated directly or indirectly by mechanisms associated to the LPA/LPA3 pathway., (Copyright © 2018 Elsevier B.V. All rights reserved.)
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- 2018
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19. Fibronectin From Oviductal Cells Fluctuates During the Estrous Cycle and Contributes to Sperm-Oviduct Interaction in Cattle.
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Osycka-Salut CE, Castellano L, Fornes D, Beltrame JS, Alonso CAI, Jawerbaum A, Franchi A, Díaz ES, and Perez Martinez S
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- Animals, Cattle, Epithelial Cells cytology, Female, Male, Oviducts cytology, Spermatozoa cytology, Cell Communication physiology, Epithelial Cells metabolism, Estrous Cycle physiology, Fibronectins metabolism, Oviducts metabolism, Spermatozoa metabolism
- Abstract
During the passage of sperm through the oviduct, spermatozoa bind to the oviductal epithelium and form the oviductal reservoir. This interaction keeps the fertilizing capacity of sperm until ovulation-associated signals induce sperm release from the oviductal epithelium, allowing the transit of spermatozoa to the fertilization site. Fibronectin is a glycoprotein from the extracellular matrix that binds to α5β1 receptors. Fibronectin has been found to be expressed in the oviduct, whereas α5β1 has been found to be expressed in the sperm of different species. Fibronectin is involved through α5β1 in sperm functions. The aim of this work was to study the participation of oviductal fibronectin in the regulation of the sperm-oviduct interaction in cattle. We found that oviductal epithelial cells differentially expressed all mRNA splice variants of fibronectin during the estrous cycle. Fibronectin was localized in the apical region of oviductal epithelial cells and fibronectin levels in the oviductal fluid fluctuated during the estrous cycle. Also, bovine spermatozoa expressed α5β1. Using in vitro sperm-oviduct co-cultures, we found that spermatozoa were attached to the oviductal epithelium through α5β1. The incubation of co-cultures with fibronectin induced sperm release from the oviductal cells through α5β1. The sperm population released from oviductal cells by fibronectin was enriched in motile and capacitated spermatozoa. Based on our in vitro culture system results, we propose that fibronectin and α5β1 are involved in the sperm-oviduct interaction. Also, an increase in fibronectin levels in the oviductal fluid during the pre-ovulatory period may promote sperm release from the oviductal epithelium in cattle. J. Cell. Biochem. 118: 4095-4108, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)
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- 2017
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20. Extracellular cAMP activates molecular signalling pathways associated with sperm capacitation in bovines.
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Alonso CAI, Osycka-Salut CE, Castellano L, Cesari A, Di Siervi N, Mutto A, Johannisson A, Morrell JM, Davio C, and Perez-Martinez S
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- Animals, Calcium metabolism, Cattle, Cells, Cultured, Cyclic AMP-Dependent Protein Kinases metabolism, Enzyme Inhibitors pharmacology, Fertility, MAP Kinase Signaling System drug effects, Male, Multidrug Resistance-Associated Proteins metabolism, Type C Phospholipases antagonists & inhibitors, Type C Phospholipases metabolism, Cyclic AMP metabolism, Signal Transduction drug effects, Sperm Capacitation drug effects
- Abstract
Study Question: Is extracellular cAMP involved in the regulation of signalling pathways in bovine sperm capacitation?, Summary Answer: Extracellular cAMP induces sperm capacitation through the activation of different signalling pathways that involve phospholipase C (PLC), PKC/ERK1-2 signalling and an increase in sperm Ca2+ levels, as well as soluble AC and cAMP/protein kinase A (PKA) signalling., What Is Known Already: In order to fertilize the oocyte, ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, known as capacitation. This correlates with a number of membrane and metabolic modifications that include an increased influx of bicarbonate and Ca2+, activation of a soluble adenylyl cyclase (sAC) to produce cAMP, PKA activation, protein tyrosine phosphorylation and the development of hyperactivated motility. We previously reported that cAMP efflux by Multidrug Resistance Protein 4 (MRP4) occurs during sperm capacitation and the pharmacological blockade of this inhibits the process. Moreover, the supplementation of incubation media with cAMP abolishes the inhibition and leads to sperm capacitation, suggesting that extracellular cAMP regulates crucial signalling cascades involved in this process., Study Design, Size, Duration: Bovine sperm were selected by the wool glass column method, and washed by centrifugation in BSA-Free Tyrode's Albumin Lactate Pyruvate (sp-TALP). Pellets were resuspended then diluted for each treatment. For in vitro capacitation, 10 to 15 × 106 SPZ/ml were incubated in 0.3% BSA sp-TALP at 38.5°C for 45 min under different experimental conditions. To evaluate the role of extracellular cAMP on different events associated with sperm capacitation, 10 nM cAMP was added to the incubation medium as well as different inhibitors of enzymes associated with signalling transduction pathways: U73122 (PLC inhibitor, 10 μM), Gö6983 (PKC inhibitor, 10 μM), PD98059 (ERK-1/2 inhibitor, 30 μM), H89 and KT (PKA inhibitors, 50 μM and 100 nM, respectively), KH7 (sAC inhibitor, 10 μM), BAPTA-AM (intracellular Ca2+ chelator, 50 μM), EGTA (10 μM) and Probenecid (MRPs general inhibitor, 500 μM). In addition, assays for binding to oviductal epithelial cells and IVF were carried out to test the effect of cAMP compared with other known capacitant agents such as heparin (60 μg/ml) and bicarbonate (40 mM)., Participants/materials, Setting, Methods: Straws of frozen bovine semen (20-25 × 106 spermatozoa/ml) were kindly provided by Las Lilas, CIALE and CIAVT Artificial Insemination Centers. The methods used in this work include western blot, immunohistochemistry, flow cytometry, computer-assisted semen analysis, live imaging of Ca2+ and fluorescence scanning. At least three independent assays with bull samples of proven fertility were carried., Main Results and the Role of Chance: In the present study, we elucidate the molecular events induced by extracellular cAMP. Our results showed that external cAMP induces sperm capacitation, depending upon the action of PLC. Downstream, this enzyme increased ERK1-2 activation through PKC and elicited a rise in sperm Ca2+ levels (P < 0.01). Moreover, extracellular cAMP-induced capacitation also depended on the activity of sAC and PKA, and increased tyrosine phosphorylation, indicating that the nucleotide exerts a broad range of responses. In addition, extracellular cAMP-induced sperm hyperactivation and concomitantly increased the proportion of spermatozoa with high mitochondrial activity (P < 0.01). Finally, cAMP increased the in vitro fertilization rate compared to control conditions (P < 0.001)., Large Scale Data: None., Limitations, Reasons for Caution: This is an in vitro study performed with bovine cryopreserved spermatozoa. Studies in other species and with fresh samples are needed to extrapolate these data., Wider Implications of the Findings: These findings strongly suggest an important role of extracellular cAMP in the regulation of the signalling pathways involved in the acquisition of bull sperm fertilizing capability. The data presented here indicate that not only a rise, but also a regulation of cAMP levels is necessary to ensure sperm fertilizing ability. Thus, exclusion of the nucleotide to the extracellular space might be essential to guarantee the achievement of a cAMP tone, needed for all capacitation-associated events to take place. Moreover, the ability of cAMP to trigger such broad and complex signalling events allows us to hypothesize that cAMP is a self-produced autocrine/paracrine factor, and supports the emerging paradigm that spermatozoa do not compete but, in fact, communicate with each other. A precise understanding of the functional competence of mammalian spermatozoa is essential to generate clinical advances in the treatment of infertility and the development of novel contraceptive strategies., Study Funding and Competing Interest(s): This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas [PIP0 496 to S.P.-M.], Agencia Nacional de Promoción Científica y Tecológica [PICT 2012-1195 and PICT2014-2325 to S.P.-M., and PICT 2013-2050 to C.D.], Boehringer Ingelheim Funds, and the Swedish Farmers Foundation [SLF-H13300339 to J.M.]. The authors declare there are no conflicts of interests., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)
- Published
- 2017
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21. Sperm Release From the Oviductal Epithelium Depends on Ca(2+) Influx Upon Activation of CB1 and TRPV1 by Anandamide.
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Gervasi MG, Osycka-Salut C, Sanchez T, Alonso CA, Llados C, Castellano L, Franchi AM, Villalón M, and Perez-Martinez S
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- Animals, Calcium Signaling, Cattle, Cells, Cultured, Coculture Techniques, Female, Male, Oviducts cytology, Sperm Capacitation, Arachidonic Acids pharmacology, Cannabinoid Receptor Agonists pharmacology, Endocannabinoids pharmacology, Oviducts metabolism, Polyunsaturated Alkamides pharmacology, Receptor, Cannabinoid, CB1 metabolism, TRPV Cation Channels metabolism
- Abstract
The oviduct acts as a functional sperm reservoir in many mammalian species. Both binding and release of spermatozoa from the oviductal epithelium are mainly modulated by sperm capacitation. Several molecules from oviductal fluid are involved in the regulation of sperm function. Anandamide is a lipid mediator involved in reproductive physiology. Previously, we demonstrated that anandamide, through activation of the cannabinoid receptor type 1 (CB1), promotes sperm release from bovine oviductal epithelial cells, and through CB1 and the transient receptor potential vanilloid 1 (TRPV1), induces sperm capacitation. Herein we investigate co-activation between CB1 and TRPV1, and Ca(2+) influx as part of the mechanism of action of anandamide during sperm release from oviductal cells. Our results indicate that in the absence of Ca(2+) anandamide failed to release spermatozoa from oviductal epithelial cells. Additionally, sperm release promoted by cannabinoid and vanilloid agonists was abolished when the spermatozoa were preloaded with BAPTA-AM, a Ca(2+) chelator. We also determined Ca(2+) levels in spermatozoa preloaded with FURA2-AM co-cultured with oviductal cells and incubated with different cannabinoid and vanilloid agonists. The incubation with different agonists induced Ca(2+) influx, which was abolished by CB1 or TRPV1 antagonists. Our results also suggest that a phospholypase C (PLC) might mediate the activation of CB1 and TRPV1 in sperm release from the bovine oviduct. Therefore, our findings indicate that anandamide, through CB1 and TRPV1 activation, is involved in sperm release from the oviductal reservoir. An increase of sperm Ca(2+) levels and the PLC activation might be involved in anandamide signaling pathway., (© 2015 Wiley Periodicals, Inc.)
- Published
- 2016
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22. Cyclic AMP efflux, via MRPs and A1 adenosine receptors, is critical for bovine sperm capacitation.
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Osycka-Salut C, Diez F, Burdet J, Gervasi MG, Franchi A, Bianciotti LG, Davio C, and Perez-Martinez S
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- 1-Methyl-3-isobutylxanthine pharmacology, Adenosine chemistry, Adenosine A1 Receptor Antagonists pharmacology, Adenylyl Cyclase Inhibitors, Animals, Bicarbonates pharmacology, Biological Transport, Cattle, Humans, Male, Phosphodiesterase Inhibitors pharmacology, Probenecid pharmacology, Sperm Motility, Xanthines pharmacology, Cyclic AMP metabolism, Multidrug Resistance-Associated Proteins metabolism, Receptor, Adenosine A1 metabolism, Sperm Capacitation drug effects, Spermatozoa metabolism
- Abstract
Sperm capacitation has been largely associated with an increase in cAMP, although its relevance in the underlying mechanisms of this maturation process remains elusive. Increasing evidence shows that the extrusion of cAMP through multidrug resistance associated protein 4 (MRP4) regulates cell homeostasis not only in physiological but also in pathophysiological situations and studies from our laboratory strongly support this assumption. In the present work we sought to establish the role of cAMP efflux in the regulation of sperm capacitation. Sperm capacitation was performed in vitro by exposing bovine spermatozoa to bicarbonate 40 and 70 mM; cAMP; probenecid (a MRPs general inhibitor) and an adenosine type 1 receptor (A1 adenosine receptor) selective antagonist (DPCPX). Capacitation was assessed by chlortetracycline assay and lysophosphatidylcholine-induced acrosome reaction assessed by PSA-FITC staining. Intracellular and extracellular cAMP was measured by radiobinding the regulatory subunit of PKA under the same experimental conditions. MRP4 was detected by western blot and immunohistochemistry assays. Results showed that the inhibition of soluble adenylyl cyclase significantly inhibited bicarbonate-induced sperm capacitation. Furthermore, in the presence of 40 and 70 mM bicarbonate bovine spermatozoa synthesized and extruded cAMP. Interestingly, in the absence of IBMX (a PDEs inhibitor) cAMP efflux still operated in sperm cells, suggesting that cAMP extrusion would be a physiological process in the spermatozoa complementary to the action of PDE. Blockade of MRPs by probenecid abolished the efflux of the cyclic nucleotide resulting not only in the accumulation of intracellular cAMP but also in the inhibition of bicarbonate-induced sperm capacitation. The effect of probenecid was abolished by exposing sperm cells to cAMP. The high-affinity efflux pump for cAMP, MRP4 was expressed in bovine spermatozoa and localized to the midpiece of the tail as previously reported for soluble adenylyl cyclase and A1 adenosine receptor. Additionally, blockade of A1 adenosine receptor abolished not only bicarbonate-induced sperm capacitation but also that stimulated by cAMP. Present findings strongly support that cAMP efflux, presumably through MRP4, and the activation of A1 adenosine receptor regulate some events associated with bicarbonate-induced sperm capacitation, and further suggest a paracrine and/or autocrine role for cAMP.
- Published
- 2014
- Full Text
- View/download PDF
23. Anandamide levels fluctuate in the bovine oviduct during the oestrous cycle.
- Author
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Gervasi MG, Marczylo TH, Lam PM, Rana S, Franchi AM, Konje JC, and Perez-Martinez S
- Subjects
- Amidohydrolases metabolism, Animals, Body Fluids metabolism, Cattle, Epithelial Cells metabolism, Ethanolamines metabolism, Female, Gene Expression Regulation, Intracellular Space metabolism, Ovarian Follicle metabolism, Oviducts cytology, Phosphatidylethanolamines metabolism, Phospholipase D genetics, Phospholipase D metabolism, Protein Transport, RNA, Messenger genetics, RNA, Messenger metabolism, Arachidonic Acids metabolism, Endocannabinoids metabolism, Estrous Cycle, Oviducts metabolism, Polyunsaturated Alkamides metabolism
- Abstract
Mammalian oviduct acts as a reservoir for spermatozoa and provides an environment in which they may compete for the opportunity to fertilize the oocyte. Whilst in the oviduct spermatozoa undergo capacitation essential for fertilization. Sperm-oviduct interaction is essential for sperm capacitation and is a tightly regulated process influenced by the local microenvironment. Previously we reported that the endocannabinoid anandamide (AEA) regulates sperm release from epithelial oviductal cells by promoting sperm capacitation. The aims of this work were to measure the AEA content and to characterize the main AEA metabolic pathway in the bovine oviduct and determine how these change through the oestrous cycle. In this study, the levels of AEA and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in bovine oviduct collected during different stages of oestrous cycle by ultra high performance liquid chromatography tandem mass spectrometry. Results indicated that intracellular oviductal epithelial levels of all three N-acylethanolamines fluctuate during oestrous cycle. Anandamide from oviductal fluid also varied during oestrous cycle, with the highest values detected during the periovulatory period. Endocannabinoid levels from ipsilateral oviduct to ovulation were higher than those detected in the contralateral one, suggesting that levels of oviductal AEA may be regulated by ovarian hormones. The expression and localization of N-acylethanolamines metabolizing enzymes in bovine oviduct were also determined by RT-PCR, Western blot, and immunohistochemistry but no change was found during the oestrous cycle. Furthermore, nanomolar levels of AEA were detected in follicular fluids, suggesting that during ovulation the mature follicle may contribute to oviductal AEA levels to create an endocannabinoid gradient conducive to the regulation of sperm function for successful fertilization.
- Published
- 2013
- Full Text
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24. Anandamide induces sperm release from oviductal epithelia through nitric oxide pathway in bovines.
- Author
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Osycka-Salut C, Gervasi MG, Pereyra E, Cella M, Ribeiro ML, Franchi AM, and Perez-Martinez S
- Subjects
- Animals, Cattle, Cell Communication drug effects, Endocannabinoids, Epithelial Cells cytology, Epithelial Cells drug effects, Epithelial Cells metabolism, Fallopian Tubes drug effects, Female, Hemoglobins pharmacology, Male, NG-Nitroarginine Methyl Ester pharmacology, Nitric Oxide Donors pharmacology, Nitric Oxide Synthase Type I metabolism, Nitric Oxide Synthase Type III metabolism, Receptor, Cannabinoid, CB1 metabolism, Spermatozoa drug effects, Spermatozoa enzymology, TRPV Cation Channels metabolism, Arachidonic Acids pharmacology, Fallopian Tubes cytology, Fallopian Tubes metabolism, Nitric Oxide metabolism, Polyunsaturated Alkamides pharmacology, Signal Transduction drug effects, Spermatozoa cytology
- Abstract
Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 µM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 µM L-NAME, a NO synthase inhibitor, or 30 µg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation.
- Published
- 2012
- Full Text
- View/download PDF
25. Interaction between lysophosphatidic acid, prostaglandins and the endocannabinoid system during the window of implantation in the rat uterus.
- Author
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Sordelli MS, Beltrame JS, Cella M, Gervasi MG, Perez Martinez S, Burdet J, Zotta E, Franchi AM, and Ribeiro ML
- Subjects
- Amidohydrolases metabolism, Animals, Cyclooxygenase 2 metabolism, Dinoprostone metabolism, Female, Phosphoric Diester Hydrolases analysis, Phosphoric Diester Hydrolases metabolism, Pregnancy, Rats, Rats, Wistar, Receptors, Lysophosphatidic Acid analysis, Receptors, Lysophosphatidic Acid metabolism, Uterus blood supply, Uterus metabolism, Embryo Implantation, Endocannabinoids metabolism, Lysophospholipids metabolism, Prostaglandins metabolism
- Abstract
Bioactive lipid molecules as lysophosphatidic acid (LPA), prostaglandins (PG) and endocannabinoids are important mediators of embryo implantation. Based on previous published data we became interested in studying the interaction between these three groups of lipid derivatives in the rat uterus during the window of implantation. Thus, we adopted a pharmacological approach in vitro using LPA, DGPP (a selective antagonist of LPA3, an LPA receptor), endocannabinoids' receptor selective antagonists (AM251 and AM630) and non selective (indomethacin) and selective (NS-398) inhibitors of cyclooxygenase-1 and 2 enzymes. Cyclooxygenase isoforms participate in prostaglandins' synthesis. The incubation of the uterus from rats pregnant on day 5 of gestation (implantation window) with LPA augmented the activity and the expression of fatty acid amide hydrolase, the main enzyme involved in the degradation of endocannabinoids in the rodent uteri, suggesting that LPA decreased endocannabinoids' levels during embryo implantation. It has been reported that high endocannabinoids are deleterious for implantation. Also, LPA increased PGE2 production and cyclooxygenase-2 expression. The incubation of LPA with indomethacin or NS-398 reversed the increment in PGE2 production, suggesting that cyclooxygenase-2 was the isoform involved in LPA effect. PGs are important mediators of decidualization and vascularization at the implantation sites. All these effects were mediated by LPA3, as the incubation with DGPP completely reversed LPA stimulatory actions. Besides, we also observed that endocannabinoids mediated the stimulatory effect of LPA on cyclooxygenase-2 derived PGE2 production, as the incubation of LPA with AM251 or AM630 completely reversed LPA effect. Also, LPA augmented via LPA3 decidualization and vascularization markers. Overall, the results presented here demonstrate the participation of LPA3 in the process of implantation through the interaction with other groups of lipid molecules, prostaglandins and endocannabinoids, which prepare the uterine milieu for embryo invasion during the window of implantation.
- Published
- 2012
- Full Text
- View/download PDF
26. Anandamide capacitates bull spermatozoa through CB1 and TRPV1 activation.
- Author
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Gervasi MG, Osycka-Salut C, Caballero J, Vazquez-Levin M, Pereyra E, Billi S, Franchi A, and Perez-Martinez S
- Subjects
- Acrosome Reaction drug effects, Animals, Cattle, Dose-Response Relationship, Drug, Endocannabinoids, Gene Expression Regulation drug effects, Heparin pharmacology, Male, Protein Transport drug effects, Receptor, Cannabinoid, CB1 antagonists & inhibitors, Receptor, Cannabinoid, CB2 antagonists & inhibitors, Receptor, Cannabinoid, CB2 metabolism, Spermatozoa cytology, TRPV Cation Channels antagonists & inhibitors, Arachidonic Acids pharmacology, Cannabinoid Receptor Modulators pharmacology, Polyunsaturated Alkamides pharmacology, Receptor, Cannabinoid, CB1 metabolism, Spermatozoa drug effects, Spermatozoa metabolism, TRPV Cation Channels metabolism
- Abstract
Anandamide (AEA), a major endocannabinoid, binds to cannabinoid and vanilloid receptors (CB1, CB2 and TRPV1) and affects many reproductive functions. Nanomolar levels of anandamide are found in reproductive fluids including mid-cycle oviductal fluid. Previously, we found that R(+)-methanandamide, an anandamide analogue, induces sperm releasing from bovine oviductal epithelium and the CB1 antagonist, SR141716A, reversed this effect. Since sperm detachment may be due to surface remodeling brought about by capacitation, the aim of this paper was to investigate whether anandamide at physiological concentrations could act as a capacitating agent in bull spermatozoa. We demonstrated that at nanomolar concentrations R(+)-methanandamide or anandamide induced bull sperm capacitation, whereas SR141716A and capsazepine (a TRPV1 antagonist) inhibited this induction. Previous studies indicate that mammalian spermatozoa possess the enzymatic machinery to produce and degrade their own AEA via the actions of the AEA-synthesizing phospholipase D and the fatty acid amide hydrolase (FAAH) respectively. Our results indicated that, URB597, a potent inhibitor of the FAAH, produced effects on bovine sperm capacitation similar to those elicited by exogenous AEA suggesting that this process is normally regulated by an endogenous tone. We also investigated whether anandamide is involved in bovine heparin-capacitated spermatozoa, since heparin is a known capacitating agent of bovine sperm. When the spermatozoa were incubated in the presence of R(+)-methanandamide and heparin, the percentage of capacitated spermatozoa was similar to that in the presence of R(+)-methanandamide alone. The pre-incubation with CB1 or TRPV1 antagonists inhibited heparin-induced sperm capacitation; moreover the activity of FAAH was 30% lower in heparin-capacitated spermatozoa as compared to control conditions. This suggests that heparin may increase endogenous anandamide levels. Our findings indicate that anandamide induces sperm capacitation through the activation of CB1 and TRPV1 receptors and could be involved in the same molecular pathway as heparin in bovines.
- Published
- 2011
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27. The endocannabinoid system in bull sperm and bovine oviductal epithelium: role of anandamide in sperm-oviduct interaction.
- Author
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Gervasi MG, Rapanelli M, Ribeiro ML, Farina M, Billi S, Franchi AM, and Perez Martinez S
- Subjects
- Amidohydrolases antagonists & inhibitors, Amidohydrolases metabolism, Animals, Arachidonic Acids pharmacology, Benzamides pharmacology, Blotting, Western methods, Camphanes pharmacology, Carbamates pharmacology, Cattle, Endocannabinoids, Epithelium metabolism, Female, Immunohistochemistry, Male, Microscopy, Fluorescence, Piperidines pharmacology, Polyunsaturated Alkamides pharmacology, Pyrazoles pharmacology, Receptor, Cannabinoid, CB1 antagonists & inhibitors, Receptor, Cannabinoid, CB1 metabolism, Receptor, Cannabinoid, CB2 antagonists & inhibitors, Receptor, Cannabinoid, CB2 metabolism, Sperm Motility drug effects, Sperm-Ovum Interactions drug effects, Spermatozoa drug effects, Arachidonic Acids physiology, Fallopian Tubes metabolism, Sperm-Ovum Interactions physiology, Spermatozoa metabolism
- Abstract
Anandamide binds to cannabinoid receptors and plays several central and peripheral functions. The aim of this work was to study the possible role for this endocannabinoid in controlling sperm-oviduct interaction in mammals. We observed that bull sperm and bovine oviductal epithelial cells express cannabinoid receptors, CB1 and CB2, and fatty acid amide hydrolase, the enzyme that controls intracellular anandamide levels. A quantitative assay to determine whether anandamide was involved in bovine sperm-oviduct interaction was developed. R(+)-methanandamide, a non-hydrolysable anandamide analog, inhibited sperm binding to and induced sperm release from oviductal epithelia. Selective CB1 antagonists (SR141716A or AM251) completely blocked R(+)-methanandamide effects. However, SR144528, a selective CB2 antagonist, did not exert any effect, indicating that only CB1 was involved in R(+)-methanandamide effect. This effect was not caused by inhibition of the sperm progressive motility or by induction of the acrosome reaction. Overall, our findings indicate for the first time that the endocannabinoid system is present in bovine sperm and oviductal epithelium and that anandamide modulates the sperm-oviduct interaction, by inhibition of sperm binding and induction of sperm release from oviductal epithelial cells, probably by activating CB1 receptors.
- Published
- 2009
- Full Text
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28. Steroid hormones augment nitric oxide synthase activity and expression in rat uterus.
- Author
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Ogando D, Farina M, Ribeiro ML, Perez Martinez S, Cella M, Rettori V, and Franchi A
- Subjects
- Animals, Blotting, Western, Calcium, Enzyme Inhibitors pharmacology, Estradiol physiology, Female, Gonadal Steroid Hormones physiology, Guanidines pharmacology, Isoenzymes analysis, Isoenzymes antagonists & inhibitors, Isoenzymes metabolism, Nitric Oxide Synthase analysis, Nitric Oxide Synthase antagonists & inhibitors, Ovariectomy, Progesterone physiology, Rats, Rats, Wistar, Up-Regulation, Uterus enzymology, Estradiol pharmacology, Gonadal Steroid Hormones pharmacology, Nitric Oxide Synthase metabolism, Progesterone pharmacology, Uterus drug effects
- Abstract
Nitric oxide (NO) is synthesized in a variety of tissues, including rat uterus, from L-arginine by NO synthase (NOS), of which there are three isoforms, namely neuronal, endothelial and inducible NOS (nNOS, eNOS and iNOS, respectively). Nitric oxide is an important regulator of the biology and physiology of the organs of the reproductive system, including the uterus. Some studies have shown increased variation in NO production and NOS expression during the oestrous cycle. However, the factors that regulate NO production in the uterus remain unclear. Therefore, in the present study, we investigated the effect of sex steroids on NOS expression and activity in the ovariectomized rat uterus. Ovariectomized rats received progesterone (4 mg per rat) or 17beta-oestradiol (1 microg per rat). All rats were killed 18 h after treatment. Both progesterone and oestradiol were able to augment NOS activity. The effect of oestradiol was abolished by pre-incubation with 500 micro M aminoguanidine, an iNOS inhibitor, or by coadministration of oestradiol with 3 mg kg(-1) dexamethasone, but the effect of progesterone was not affected by these treatments. Uterine nNOS, eNOS and iNOS protein levels were assessed using Western blots. Ovariectomized rat uteri expressed iNOS and eNOS. Progesterone increased the expression of eNOS and iNOS, whereas oestradiol increased iNOS expression only. These results suggest that oestradiol and progesterone are involved in the regulation of NOS expression and activity during pregnancy and implantation in the rat.
- Published
- 2003
- Full Text
- View/download PDF
29. Regulation of lipid peroxidation by nitric oxide and PGF2alpha during luteal regression in rats.
- Author
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Motta AB, Estevez A, Franchi A, Perez-Martinez S, Farina M, Ribeiro ML, Lasserre A, and Gimeno MF
- Subjects
- Animals, Blotting, Western, Cyclooxygenase Inhibitors pharmacology, Enzyme Inhibitors pharmacology, Female, Glutathione analysis, Indomethacin pharmacology, Models, Animal, NG-Nitroarginine Methyl Ester pharmacology, Nitric Oxide Synthase antagonists & inhibitors, Ovary drug effects, Pseudopregnancy, Rats, Rats, Wistar, Thiobarbituric Acid Reactive Substances analysis, Dinoprost pharmacology, Lipid Peroxidation drug effects, Luteolysis physiology, Nitric Oxide pharmacology, Ovary metabolism
- Abstract
Corpus luteum regression is related to an increased generation of reactive oxygen species. Although several studies indicate that PGF(2alpha) is involved in regression of the corpus luteum in mammalian species through an increase in reactive oxygen species, the exact mechanism remains unknown. In the present study, the relationship between nitric oxide and PGF(2alpha) in regulation of lipid peroxidation was studied. Ovarian tissue from pseudopregnant rats at mid- (day 5) or late phase or at the time of regression (day 9 of pseudopregnancy) of corpus luteum development was used. Thiobarbituric acid reactants, used as a lipid peroxidation index, were higher on day 9 of pseudopregnancy than on day 5. In contrast, glutathione content (an antioxidant metabolite) was lower on day 9 than on day 5 of pseudopregnancy. These results indicate that there was an enhanced oxidative status in ovarian tissue during luteolysis. Administration of N(omega)-nitro-L-arginine methyl ester (L-NAME: 600 micromol l(-1)), a competitive nitric oxide synthase (NOS) inhibitor, led to a decrease in basal thiobarbituric acid reactant content in ovarian tissue from rats on day 9 of pseudopregnancy only, indicating that during regression of the corpus luteum, NO could act as intermediary in ovarian lipid peroxidation. Administration of a luteolytic dose (3 microg kg(-1) body weight i.p.) of a synthetic PGF(2alpha) increased thiobarbituric acid reactant content in ovaries from rats on day 9 of pseudopregnancy. As this effect was reversed partially by L-NAME, it is proposed that during regression of corpora lutea, PGF(2alpha) and NO are involved in regulation of lipid peroxidation. As this effect was only reversed partially, it is possible that there is another mechanism involving PGF(2alpha) (but not the NO-NOS pathway) in regulation of ovarian lipid peroxidation. Furthermore, the administration of PGF(2alpha) enhanced ovarian NOS activity, whereas cyclooxygenase inhibition (by indomethacin treatment in vivo) reduced it. As western blotting of ovarian homogenates obtained from PGF(2alpha)-injected rats increased inducible NOS (iNOS) content, it is concluded that PGF(2alpha) enhances both activity and synthesis of NO in rat ovarian tissues during luteolysis. Taken together, these results indicate that in ovaries with regressing corpora lutea, both NO and PGF(2alpha) are involved in part in regulation of lipid peroxidation.
- Published
- 2001
- Full Text
- View/download PDF
30. Effect of nitric oxide synthase inhibitors on ovum transport and oviductal smooth muscle activity in the rat oviduct.
- Author
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Perez Martinez S, Viggiano M, Franchi AM, Herrero MB, Ortiz ME, Gimeno MF, and Villalón M
- Subjects
- Analysis of Variance, Animals, Cell Count, Dose-Response Relationship, Drug, Enzyme Inhibitors pharmacology, Female, Microspheres, Muscle, Smooth drug effects, Nitric Oxide Donors pharmacology, Pregnancy, Rats, Rats, Wistar, Spermine pharmacology, Fallopian Tubes drug effects, Muscle Contraction drug effects, NG-Nitroarginine Methyl Ester pharmacology, Nitric Oxide Synthase antagonists & inhibitors, Ovum Transport drug effects, omega-N-Methylarginine pharmacology
- Abstract
The effect of the inhibition of nitric oxide synthase (NOS) on ovum transport and oviductal motility in rats was investigated. Three different NOS inhibitors were injected into the ovarian bursa at oestrus or day 3 of pregnancy. Oviducts and uteri were flushed 24 h later and the presence of ova was recorded. In oestrous and pregnant rats, treatment resulted in accelerated egg transport, as shown by a decrease in the number of ova present in the oviducts. In cyclic rats, intrabursal injection of 1 mg kg-1 of either N-monomethyl-L-arginine (L-NMMA) or N omega nitro-L-arginine methyl ester (L-NAME) elicited a 30% reduction in the number of ova present in the oviducts, whereas in pregnant animals, the same dose of L-NMMA produced a reduction of 40%. Simultaneous administration of the NO donor spermine NONOate (5 mg kg-1) completely reversed the effect of L-NMMA. Tubal motility was assessed by microsphere displacement analysis within the oviduct. Surrogate ova were transferred to the oviductal lumen at oestrus and 24 h later the effect of intraoviductal injection of 1 microgram L-NMMA or vehicle was assessed. The microspheres in the isthmus showed an oscillating motion, and periods in which movement was not detectable. However, L-NMMA treatment produced a 3.6-fold increase in the maximum instant velocities and a significant reduction in the resting periods of the microspheres compared with the control group (P < 0.001). These results provide evidence that NO inhibition increases tubal motility that results in accelerated ovum transport, and indicate that NO could act as a paracrine signal between different layers of the oviductal wall, providing a role for endogenous NO in regulation of tubal function.
- Published
- 2000
- Full Text
- View/download PDF
31. Effect of prostaglandin F2 alpha (PGF2 alpha) on oviductal nitric oxide synthase (NOS) activity: possible role of endogenous NO on PGF2 alpha-induced contractions in rat oviduct.
- Author
-
Perez Martinez S, Franchi AM, Viggiano JM, Herrero MB, and Gimeno M
- Subjects
- Animals, Arginine metabolism, Calcium pharmacology, Chelating Agents, Citrulline metabolism, Egtazic Acid pharmacology, Enzyme Inhibitors pharmacology, Fallopian Tubes enzymology, Female, Guanidines pharmacology, Muscle, Smooth physiology, NG-Nitroarginine Methyl Ester pharmacology, Nitric Oxide Synthase antagonists & inhibitors, Rats, Rats, Wistar, Dinoprost pharmacology, Fallopian Tubes physiology, Muscle Contraction drug effects, Nitric Oxide physiology, Nitric Oxide Synthase metabolism
- Abstract
In this paper, we evaluated the hypothesis that prostaglandin F2 alpha (PGF2 alpha) regulates NOS activity and we also investigated, by means of nitric oxide inhibitors (N-monomethyl-L-arginine monoacetate, L-NMMA) the role of endogenous NO on PGF2 alpha-induced contractions in rat oviduct. NOS activity was determined by measuring the conversion of 14[C]-L-arginine to 14[C]-L-citrulline on oviductal homogenates from estrogenized rats (1 microgram/rat). The presence of PGF2 alpha (10(-8) M) in the incubation medium produced an increase in NOS activity (p < or = 0.05). The effect of the prostanoid was blocked completely by the presence of two NOS inhibitors: N-nitro-L-arginine methyl ester (L-NAME, 0.6 mM) and aminoguanidine (Ag, 0.5 mM). These results suggested that PGF2 alpha could be modulating the Ca(2+)-independent NOS activity. We determined NOS activity using 1 mM EGTA, a chelator of Ca2+, in a free Ca2+ medium. These results indicated that PGF2 alpha produces an increase in Ca(2+)-independent NOS activity (p < or = 0.05). PGF2 alpha induces contraction of the oviductal smooth muscle in a concentration dependent manner. L-NMMA enhanced PGF2 alpha induced contraction of the oviduct, providing indirect evidence that there is a basal release of NO in the oviduct, which may reduce and/or modulate the contractile effects of PGF2 alpha.
- Published
- 1998
- Full Text
- View/download PDF
32. Potential contraceptive use of epididymal proteins: evidence for the participation of specific antibodies against rat epididymal protein DE in male and female fertility inhibition.
- Author
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Perez Martinez S, Conesa D, and Cuasnicú PS
- Subjects
- Animals, Blotting, Western, Epididymal Secretory Proteins, Female, Immune Sera, Immunization, Isoantibodies biosynthesis, Isoantibodies immunology, Male, Metalloproteins immunology, Rats, Rats, Inbred Lew, Rats, Wistar, Sperm Agglutination, Sperm Capacitation, Sperm Motility, Testicular Hormones immunology, Contraception, Immunologic, Epididymis immunology, Isoantibodies pharmacology, Metalloproteins physiology, Sperm-Ovum Interactions drug effects, Testicular Hormones physiology
- Abstract
Previous studies in our laboratory indicated that immunization of male and female Wistar and Lewis rats with epididymal protein DE, resulted in the development of anti-DE antibodies in over 90% of the animals, with a significant and reversible reduction of fertility. In the present study, ELISA assays performed to analyze the evolution of the immune response indicated that antibody levels in the sera of immunized animals reached a maximum at 8 weeks after the initial injection and then gradually decreased, returning to control values by the end of the sixth month. Western blot experiments demonstrated that the immune sera specifically recognized DE in epididymal sperm extracts and epididymal cytosol, while no reaction was observed with different reproductive and essential organs. The immune sera were also capable of recognizing DE on the surface of both fresh and capacitated sperm as indicated by indirect immunofluorescence experiments. Finally, the exposure of sperm to immune sera prior to uterine insemination resulted in a significant (P < 0.05) reduction in the percentage of fertilized eggs compared to controls, with no effect on sperm motility and viability, nor on their ability to undergo capacitation. Together, these results support the participation of the raised antibodies as mediators of the antifertility effect and suggest a specific interference at the sperm-egg interaction level.
- Published
- 1995
- Full Text
- View/download PDF
33. [Mucoid cysts of the skin].
- Author
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Vigil Suarez J and Perez Martinez S
- Subjects
- Aged, Female, Humans, Male, Middle Aged, Mucus, Cysts, Skin Diseases
- Published
- 1970
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