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Objective: Endogenous opioid peptides were reported to be involved in the regulation of reproductive physiology and their precursors and receptors were described in many of the male and female reproductive tissues. Mu opioid receptor (MOR) was described in human endometrial cells and its expression and localization changed during the menstrual cycle. However, there is no data from the distribution of the other opioid receptors: Delta (DOR) and Kappa (KOR). The objective of the present work was to analyze the dynamics of expression and localization of DOR and KOR in human endometrium throughout the menstrual cycle., Study Design: Human endometrial samples from different menstrual cycle phases were analyzed by immunohistochemistry., Results: DOR and KOR were present in all samples analyzed and the protein expression and localization changed throughout the menstrual cycle. Both receptor expression increased during the late proliferative phase and decreased during the late secretory-one, especially in the luminal epithelium. DOR expression was generally higher than KOR expression in all cell compartments., Conclusions: The presence of DOR and KOR in human endometrium and their dynamic changes during the menstrual cycle join the results previously obtained in MOR suggesting a possible role of opioids in reproduction events related to the human endometrium., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

During preimplantational embryo development, PI3K/AKT regulates cell proliferation and differentiation and nobiletin modulates this pathway to promote cell survival. Therefore, we aimed to establish whether, when the AKT cascade is inhibited using inhibitors III and IV, nobiletin supplementation to in vitro culture media during the minor (2- to 8-cell stage, MNEGA) or major (8- to 16-cell stage, MJEGA) phases of EGA is able to modulate the development and quality of bovine embryos. In vitro zygotes were cultured during MNEGA or MJEGA phase in SOF + 5% FCS or supplemented with: 15 μM AKT-InhIII; 10 μM AKT-InhIV; 10 μM nobiletin; nobiletin + AKT-InhIII; nobiletin + AKT-InhIV; 0.03% DMSO. Embryo development was lower in treatments with AKT inhibitors, while combination of nobiletin with AKT inhibitors was able to recover their adverse developmental effect and also increase blastocyst cell number. The mRNA abundance of GPX1, NFE2L2, and POU5F1 was partially increased in 8- and 16-cell embryos from nobiletin with AKT inhibitors. Besides, nobiletin increased the p-rpS6 level whether or not AKT inhibitors were present. In conclusion, nobiletin promotes bovine embryo development and quality and partially recovers the adverse developmental effect of AKT inhibitors, which infers that nobiletin probably uses another signaling cascade that PI3K/AKT during early embryo development in bovine., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (C DMSO ) during minor (2 to 8-cell stage; MN EGA ) or major (8 to 16-cell stage; MJ EGA ) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MN EGA phase and in Nob10 (61.0 ± 0.8%) for MJ EGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.

The cannabinoid (CB) system has been involved in many aspects of reproduction and it is known that the systemic chronic use of exogenous CBs are deleterious to reproductive processes. Even so, it is not known what happens in relation to the physiology of the ovary when CB receptors are absent. The present study investigated the effect of the lack of CB1 and CB2 receptors in mice ovarian morphology, folliculogenesis, oocyte retrieval, and oocyte maturation and evaluated the use of Δ9-tetrahydrocannabinol (THC) on oocyte in vitro maturation (IVM) by comparing classical IVM and two-step IVM by analyzing the meiotic competence of the oocytes and their evolution toward embryos. Thus, when CB1 and CB2 receptors were missed, the ovary area and volume was significantly less and the action of the equine chorionic gonadotropin (eCG) hormone was diminished. In addition, the mutant genotypes had fewer ovarian follicles and they were less competent after eCG administration compared with wild-type mice, and this lack of CB receptors showed a mismatch of oocyte maturation. However, the in vitro use of THC showed improvements in oocytes IVM after a Pre-IVM step for 48 hr, as those oocytes reached a significantly higher polar body rate, a larger diameter and the best result on blastocysts rate was achieved when THC was used during the IVM step., (© 2020 Wiley Periodicals, Inc.)

Background and Purpose: Protective mechanisms of the endogenous cannabinoid system against drug-induced liver injury (DILI) are actively being investigated regarding the differential regulatory role of the cannabinoid CB 1 and CB 2 receptors in liver fibrogenesis and inflammation., Experimental Approach: The 2-arachidonoylglycerol (2-AG)-related signalling receptors and enzymatic machinery, and inflammatory/fibrogenic factors were investigated in the liver of a mouse model of hepatotoxicity induced by acute and repeated overdoses (750 mg·kg -1 ·day -1 ) of paracetamol (acetaminophen), previously treated with selective CB 1 (ACEA) and CB 2 (JWH015) agonists (10 mg·kg -1 ), or lacking CB 1 and CB 2 receptors., Key Results: Acute paracetamol increased the expression of CB 2 , ABHD6 and COX-2, while repeated paracetamol increased that of CB 1 and COX-2 and decreased that of DAGLβ. Both acute paracetamol and repeated paracetamol decreased the liver content of acylglycerols (2-AG, 2-LG and 2-OG). Human liver samples from a patient suffering APAP hepatotoxicity confirmed CB 1 and CB 2 increments. Acute paracetamol-exposed CB 2 KO mice had higher expression of the fibrogenic αSMA and the cytokine IL-6 and lower apoptotic cleaved caspase 3. CB 1 deficiency enhanced the repeated APAP-induced increases in αSMA and cleaved caspase 3 and blocked those of CYP2E1, TNF-α, the chemokine CCL2 and the circulating γ-glutamyltransferase (γGT). Although JWH015 reduced the expression of αSMA and TNF-α in acute paracetamol, ACEA increased the expression of cleaved caspase 3 and CCL2 in repeated paracetamol., Conclusion and Implications: The differential role of CB 1 versus CB 2 receptors on inflammatory/fibrogenic factors related to paracetamol-induced hepatotoxicity should be considered for designing alternative therapies against DILI., (© 2020 The British Pharmacological Society.)

The endogenous opioid peptides have been reported to be involved in the regulation of reproductive physiology. Many of the studies conclude with sentences around the harmful effect of opioids in male fertility but, actually, there is only one study regarding the real fertility potential of spermatozoa that have been exposed to mu specific opioids. The aim of the present study was to see if the modulation of delta (OPRD1) and kappa (OPRK1) opioid receptors in mouse sperm during capacitation was able to vary the embryo production after in vitro fertilization (IVF). The presence of OPRD1 and OPRK1 in mouse mature spermatozoa was analyzed by RT-PCR and immunofluorescence. Incubating the sperm with, on one hand, the delta specific agonist DPDPE and/or antagonist naltrindole, and, on the other hand, the kappa specific agonist U-50488 and antagonist nor-binaltorphimine, we analyzed the involvement of OPRD1 and OPRK1 on IVF and preimplantational embryo development. We verified the presence of OPRD1 and OPRK1 in mouse mature spermatozoa, not only at the mRNA level but also at protein level. Moreover, the sperm incubation with DPDPE, before the IVF, had an effect on the fertilization rate of sperm and reduced the number of reached blastocysts, which was reverted by naltrindole. Instead, the use of the kappa agonist U-50488 and the antagonist nor-binaltophimine did not have any effect on the amount and the quality of the achieved blastocysts. Although nowadays the pure delta or kappa opioid ligands are not used for the clinic, clinical trials are being conducted to be used in the near future, so it would be interesting to know if the modulation of these receptors in sperm would generate any consequence in relation to fertilization capacity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Rennin-angiotensin system (RAS) has been involved in sperm function, even so, little is known about the implication of one of the RAS axis formed by Ang-(1-7) (angiotensin-(1-7)) and MAS receptor. Hence, in the present work, we focused on elucidating the function of the MAS receptor in human spermatozoa. We analyzed the expression and localization of MAS receptor in human spermatozoa and we observed if its activation is able to modulate the sperm motility of normal motility and/or asthenozoospermic patients, as well as, the acrosome reaction of the spermatozoa. MAS receptor is present in human mature spermatozoa, not only at the mRNA level but also at protein level. MAS is localized at the acrosome region, as well as, in the tail of spermatozoa. The sperm incubation with MAS agonist Ang-(1-7) activates at dose-dependent manner the PI3K/AKT pathway (P < 0.01 vs control) and improves the motility of asthenozoospermic patients (P < 0.01 vs control), which is blocked by the specific antagonist (A779) (P < 0.01), but it do not modulate the acrosome reaction. These findings suggest that the ACE2/Ang-(1-7)/Mas axis may be a useful biochemical tool for the treatment of male infertility related to sperm mobility.

The endogenous opioid peptides are reported to be involved in the regulation of reproductive physiology. Many of the studies conclude with statements on the harmful effect of opioids on male fertility but, in fact, there are no studies regarding the real fertilisation potential of spermatozoa that have been exposed to opioids. The aim of the present study was to examine if modulation of mu opioid receptor (OPRM1) in murine spermatozoa during capacitation influenced embryo production after IVF. The presence of OPRM1 in murine mature spermatozoa was analysed by reverse transcription-polymerase chain reaction and immunofluorescence. We analysed the involvement of OPRM1 on IVF and pre-implantational embryo development by incubating the spermatozoa with the opioid agonist morphine and/or antagonist naloxone. We verified the presence of OPRM1 in murine mature spermatozoa, not only at the mRNA level but also the protein level. Moreover, incubation of the spermatozoa with morphine, before IVF, had an effect on the fertilisation rate of the spermatozoa and reduced the numbers of blastocysts, which was reversed by naloxone. Considering that opioids are widely used clinically, it is important to take into account their effect, via OPRM1, on the fertility of patients.

Oocyte maturation is the process by which immature oocytes acquire all the necessary characteristics for successful fertilization. The endogenous opioid peptides have been suggested to have a role modulating this process. However, little is known about its implication and the effect of exposing oocyte maturation to opioids on the subsequent fertilization and embryo development. Hence, in the present work, we focused on elucidating the function of the mu opioid receptor (OPRM1) in the modulation of the oocyte maturation. We analyzed the expression and localization of OPRM1 in mice oocytes and granulosa cells by reverse-transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. To observe the activity of the OPRM1, immature oocytes were incubated with morphine agonist and/or naloxone antagonist and we evaluated the PI3K/Akt and MAPK pathways, as well as the effect on the subsequent fertilization and embryo development. OPRM1 was present in mice oocytes and granulosa cells, changing its expression pattern depending on the maturation stage. Moreover, morphine, modulating PI3K/Akt and MAPK pathways, helped oocytes to reach blastocyst stage, which was reverted by naloxone. These results propose the OPRM1 as a possible therapeutic target for in vitro maturation culture medium, as it could improve the blastocyst rates obtained in the actual reproduction assisted techniques., (© 2019 Wiley Periodicals, Inc.)

Background/aims: Among the assisted reproductive techniques, the in vitro maturation of oocytes (IVM) is less developed than other techniques, but its implementation would entail a qualitative advance. This technique consists in the extraction of immature oocytes from antral ovarian follicles with the patient under low hormone stimulation or without hormone to mature exogenously in culture media supplemented with different molecules to promote maturation. In this sense, we are interested in the role that cannabinoids could have as IVM promoters because cannabinoid's molecular pathway is similar to the one by which oocyte's meiosis resumption is activated. With the intention of advancing in the possible use of cannabinoids as supplements for the media for in vitro maturation of oocytes, we intend to deepen the study of the function of the phytocannabinoid Δ-9-tetrahydrocannabinol (THC) in the IVM process., Methods: By immunocytochemistry, we detected the location pattern of cannabinoid receptor type 1 (CB1) and type 2 (CB2) during oocyte maturation in presence or absence of THC, as well as, the staining pattern of p-AKT and p-ERK. We used a genetic/ pharmacological approach generating knockout oocytes for CB1 and/or CB2 and they were incubated with THC during the oocyte maturation to visualize the physiological effects of THC, observing the rate of blastocyst achieved by oocyte., Results: This study confirms that the incubation of oocytes with THC during IVM accelerated some events of that process like the phosphorylation pattern of ERK and AKT and was able to increase the blastocyst rate in response to IVF. Moreover, it seems that both CB1 and CB2 are necessary to maintain a healthy oocyte maturation., Conclusion: Our data suggest that THC may be useful IVM supplements in clinic as is more feasible and reliable than any synthetic cannabinoid., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Endocannabinoids have been recognized as mediators of practically all reproductive events in mammals. However, little is known about the role of this system in oocyte maturation. In a mouse model, we observed that activation of cannabinoid receptor 1 (CB 1 ) during in vitro oocyte maturation modulated the phosphorylation status of Akt and ERK1/2 and enhanced the subsequent embryo production. In the absence of CB 1 , in vivo oocyte maturation was impaired and embryo development delayed. Cannabinoid receptor 2 (CB 2 ) was unable to rescue these effects. Finally, we confirmed abnormal oocyte maturation rather than impaired embryonic transport through the oviduct in CB 1 knockouts. Our data suggest that cannabinoid agonists may be useful in vitro maturation supplements. For in vitro fertilization patients intolerant to gonadotropins, this could be a promising and only option.-López-Cardona, A. P., Pérez-Cerezales, S., Fernández-González, R., Laguna-Barraza, R., Pericuesta, E., Agirregoitia, N., Gutiérrez-Adán, A., Agirregoitia, E. CB 1 cannabinoid receptor drives oocyte maturation and embryo development via PI3K/Akt and MAPK pathways., (© FASEB.)

The tubulin cofactors TBCD and TBCE play an essential role in regulation of the microtubule dynamics in a wide variety of somatic cells, but little information is known about the expression of these cofactors in human sperm and oocytes. In this study, we focused on the investigation of the presence of, and the differential distribution of, the tubulin cofactors TBCD and TBCE in human sperm and during human oocyte maturation. We performed expression assays for TBCD and TBCE by reverse transcription-polymerase chain reaction (RT-PCR), western blot and immunofluorescence and verified the presence of both cofactors in human gametes. TBCD and TBCE were located mainly in the middle region and in the tail of the sperm while in the oocyte the localization was cytosolic. The mRNA of both tubulin cofactors were present in the human oocytes but not in sperm cells. This finding gives a first insight into where TBCD and TBCE could carry out their function in the continuous changes that the cytoskeleton experiences during gametogenesis and also prior to fertilization.

Objective: To study the dynamics of the expression and localization of the mu opioid receptor (MOR) in human endometrium throughout the menstrual cycle., Design: Analysis of human endometrial samples from different menstrual cycle phases (menstrual, early/midproliferative, late proliferative/early secretory, midsecretory, and late secretory) by reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry., Setting: Academic research laboratory., Patient(s): Women from the Human Reproduction Unit of the Cruces University Hospital, fulfilling the following criteria: normal uterine vaginal ultrasound; absence of endometriosis, polycystic ovary syndrome, implantation failure, or recurrent miscarriage; and no history of opioid drug use., Intervention(s): Endometrial samples of 86 women categorized into groups for the menstrual cycle phases: 12 menstrual, 21 early/midproliferative, 16 late proliferative/early secretory, 17 midsecretory, and 20 late secretory., Main Outcome Measure(s): MOR gene and protein expression and localization in the different compartments of the human endometrium at different stages of the menstrual cycle., Result(s): The expression of MOR mRNA and protein changed throughout the cycle in human endometrium. MOR expression increased during the proliferative phase and decreased during the secretory one. Lower values were found at menstruation, and maximum values around the time of ovulation. Small variations for each endometrial compartment were found., Conclusion(s): The presence of MOR in human endometrium and the dynamic changes during the menstrual cycle suggest a possible role for opioids in reproduction events related to the human endometrium or endometriosis., (Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Cannabinoid receptors are able to couple to different families of G proteins when activated by an agonist drug. It has been suggested that different intracellular responses may be activated depending on the ligand. The goal of the present study was to characterize the pattern of G protein subunit stimulation triggered by three different cannabinoid ligands, Δ 9 -THC, WIN55212-2, and ACEA in mouse brain cortex. Stimulation of the [ 35 S]GTPγS binding coupled to specific immunoprecipitation with antibodies against different subtypes of G proteins (Gα i1 , Gα i2 , Gα i3 , Gα o , Gα z , Gα s , Gα q/11 , and Gα 12/13 ), in the presence of Δ 9 -THC, WIN55212-2 and ACEA (submaximal concentration 10 μM) was determined by scintillation proximity assay (SPA) technique in mouse cortex of wild type, CB 1 knock-out, CB 2 knock-out and CB 1 /CB 2 double knock-out mice. Results show that, in mouse brain cortex, cannabinoid agonists are able to significantly stimulate not only the classical inhibitory Gα i/o subunits but also other G subunits like Gα z , Gα q/11 , and Gα 12/13 . Moreover, the specific pattern of G protein subunit activation is different depending on the ligand. In conclusion, our results demonstrate that, in mice brain native tissue, different exogenous cannabinoid ligands are able to selectively activate different inhibitory and non-inhibitory Gα protein subtypes, through the activation of CB 1 and/or CB 2 receptors. Results of the present study may help to understand the specific molecular pathways involved in the pharmacological effects of cannabinoid-derived drugs.

The endogenous cannabinoid system has been characterized in some female reproductive organs but little is known about the expression and localization pattern of cannabinoid-degrading enzymes in relation to the CB1 cannabinoid receptor in human oocytes. In this study, we focus on the investigation of the presence and differential distribution of fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGLL) in relation to CB1 during the maturation of human oocytes. We used a total of 290 human oocytes not suitable for in vitro fertilization/intracytoplasmic sperm injection (ICSI): germinal-vesicle (GV) and metaphase-I (MI) stages and metaphase-II (MII) oocytes that had not developed into an embryo after ICSI. Cannabinoid-degrading enzymes and the cannabinoid CB1 receptor were present in human oocytes. Specifically, FAAH was detected in the periphery of the oocyte from the GV to MI stage and co-localized with CB1. Later, by the MII stage, FAAH was spread within the oocyte, whereas MGLL immunostaining was homogeneous across the oocyte at all stages of maturation and only overlapped with CB1 at the GV stage. This coordinated redistribution of cannabinoid system proteins suggests a role for this system in the maturation of the female gamete.

Objective: To describe the expression of cannabinoid receptors CB1 and CB2 and cannabinoid-degrading enzymes fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGLL) in human granulosa cells and to investigate their differential distribution with respect to CB1 at various stages during the nuclear maturation of the oocyte., Design: Analysis of granulosa cells from germinal vesicle (GV), metaphase I (MI), and MII oocytes by quantitative reverse transcriptase-polymerase chain reaction, Western blot, and indirect immunofluorescence assays., Setting: Academic research laboratory., Patient(s): Patients from the Human Reproduction Unit of Cruces University Hospital undergoing intracytoplasmic sperm injection., Intervention(s): We analyzed the granulosa cells of 300 oocytes from 53 patients. The oocyte maturation stages were 75 at GV stage, 51 at MI, and 174 at MII., Main Outcome Measure(s): The mRNA and protein expression of CB1, CB2, FAAH, and MGLL and localization in granulosa cells at each oocyte maturation stage., Result(s): CB1, FAAH, and MGLL are present in human granulosa cells during oocyte maturation, but the presence of CB2 receptor is not entirely clear in those cells. CB1 and FAAH were detected in the periphery of the granulosa cells from the GV to the MII oocytes, and they colocalized in some portions of the cell membrane. On the other hand, MGLL immunostaining was more homogeneous across the cell and overlapped with CB1 only weakly., Conclusion(s): The presence of the cannabinoid system in granulosa cells suggests a possible role of this system in the nuclear maturation of the oocyte., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To verify the presence of protein precursor pro-enkephalin (PENK) and met-enkephalin in human spermatozoa and to characterize the effects of exogenous and endogenous enkephalins on sperm motility., Design: We carried out expression assays for met-enkephalin and its protein precursor PENK by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunofluorescence techniques in sperm cells and motility analysis after incubation of semen samples with met-enkephlin enzyme inhibitors and the opioid receptor antagonist naloxone. Met-enkephalin secretion was analyzed by flow cytometry., Setting: Assisted reproduction unit and academic research laboratory., Patient(s): Semen from 50 normozoospermic healthy human donors., Intervention(s): Spermatozoa isolated from semen on discontinuous Percoll gradient (40%-80%) followed by a swim-up was used for all techniques., Main Outcome Measure(s): Immunoblotting blots, indirect immunofluorescence antibody assays, RT-PCR blots, flow cytometry plots, and percentage of motile sperm., Result(s): We found by RT-PCR and immunofluorescence that met-enkephalin and its protein precursor PENK are present in the head of human sperm cells. Endogenous met-enkephalin increased sperm motility, whereas the addition of exogenous met-enkephalin had a biphasic effect on motility, likely due to the activation of distinct receptor subtypes., Conclusion(s): We provide evidence for a new role of met-enkephalin as an endogenous mediator of sperm motility. This autocrine regulation of sperm function by the opioid system represents a new mechanism of regulation of male factor fertility and could be useful as an emerging target for male contraception., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The endogenous opioid system has been characterized in some female reproductive organs, but little is known about the expression of these receptors in human oocytes. This study investigated the presence and differential distribution of the opioid receptors during the maturation of human oocytes. A total of 821 human oocytes from an intracytoplasmic sperm injection (ICSI) programme were studied including 213 at germinal-vesicle (GV) stage and 164 at metaphase-I (MI) stage and 444 failed fertilization metaphase-II (MII) oocytes. Additionally 31 MII oocytes corresponding to cases where ICSI was not attempted and 50 failed fertilization MII oocytes from the IVF programme were included. Western blot analysis revealed the presence of the delta (OPRD1), kappa (OPRK1) and mu (OPRM1) opioid receptors in human oocytes. The OPRK1 and OPRM1 immunostaining patterns changed during the maturation of the oocyte, while the OPRD1 pattern was the same throughout. In particular, OPRD1 were detected in peripheral tissue from the GV to the MII stage. OPRK1 were found peripherally at the GV stage, more internally at MI and homogeneously at MII. Finally, OPRM1 were located peripherally at the GV stage and homogeneously in MI and MII oocytes. Opioids may have a role in oocyte maturation, acting via receptors. The opioid system has been well characterized in the central nervous system, but it is now known that opioids also act in reproductive organs. However, little is known about the presence and function of this system in human oocytes and its role in their maturation. In this study, we investigated the presence and differential distribution of three (delta, kappa and mu) opioid receptors (proteins which bind the opioids) during the maturation of human oocytes. A total of 821 human oocytes (from 253 patients) not suitable for intracytoplasmic sperm injection (ICSI) or which did not develop into an embryo after ICSI were studied. Thus, we have verified the presence of the delta, kappa and mu opioid receptors in human oocytes. The kappa and mu localization changed during the maturation of the oocyte, while the Delta localization was the same throughout. In particular, the delta receptor was detected in the periphery of the oocyte. On the other hand, the kappa receptor was found peripherally at the beginning, more internally during maturation and homogeneously at the end of maturation. Finally, the Mu receptor was located peripherally at the beginning of maturation and homogeneously in the rest of the maturation stages. This finding suggests a possible role for opioids, acting via receptors, in the maturation of the oocyte., (Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Aminopeptidase N (APN/CD13) and neutral endopeptidase (NEP/CD10) are enzymes present in human sperm cells and involved in regulation of sperm motility of noncapacitated spermatozoa. We investigated the involvement of APN/CD13 and NEP/CD10 in motility and in kinematic parameters of human capacitated spermatozoa. Sperm cells isolated by a discontinuous Percoll gradient (40%-80%) followed up by swim-up techniques were incubated with the APN/CD13-specific inhibitor, leuhistin (100 μmol L(-1)), and the NEP/CD10-specific inhibitor, thiorphan (1 μmol L(-1)). The complete inhibition of both APN/CD13 and NEP/CD10 improved sperm motility. Spermatozoa incubated with the APN/CD13-specific inhibitor leuhistin showed asymmetrical trajectories, whereas sperm trajectories were more regular after treatment with the NEP/CD10-specific inhibitor thiorphan. In conclusion, APN/CD13 and NEP/CD10 modulate the motility of capacitated spermatozoa, although each of the enzymes seems to participate in the control of different aspects of sperm motility. Therefore, both inhibitors may be useful for sperm activation at different functional stages of spermatozoa.

Objective: To analyze the expression and distribution of cannabinoid receptors in human sperm cells and evaluate the effects of activation of receptors by specific agonists and antagonists, with a special emphasis on the CB(2) receptor., Design: We performed expression assays for CB(1) and CB(2) by reverse transcriptase PCR, Western blot, and immunofluorescence techniques in spermatozoa and performed motility analysis after incubation of semen samples with cannabinoid agonists and CB(2) antagonist SR144528., Setting: Academic research laboratory., Patient(s): Semen from 50 normozoospermic, healthy human donors., Intervention(s): Spermatozoa isolated from semen by two consecutive swim-ups were used for all techniques., Main Outcome Measure(s): Reverse transcriptase PCR amplification gels, immunoblots, indirect immunofluorescence antibody assays, and percentage of motile sperm., Result(s): We have verified the presence of CB(1) and CB(2) receptors in human spermatozoa. The distribution of both of these receptors was distinct. Incubation with selective cannabinoid receptor agonists induced a significant reduction in the proportion of rapidly progressive motile spermatozoa, and whereas the CB(1) agonist increased the proportion of immobile sperm cells, the CB(2) receptor agonist increased the slow/sluggish progressive sperm cell population. The effect of the CB(2) agonist was antagonized by the CB(2)-specific antagonist., Conclusion(s): The functional CB(2) cannabinoid receptor is present in human spermatozoa and regulates the sperm motility in a more distinct manner than CB(1)., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the presence of two enkephalin-degrading enzymes (aminopeptidase N [APN] and neutral endopeptidase 24.11 [NEP]) in different fractions of human semen, their distribution in sperm cells, and their effect on sperm motility., Design: We performed expression assays for APN and NEP by real-time polymerase chain reaction, Western blot, and immunofluorescence techniques in sperm cells and performed motility analysis after incubation of semen samples with enzyme inhibitors and the opioid receptor antagonist naloxone., Setting: Assisted reproduction unit and academic research laboratory., Patient(s): Semen from 50 normozoospermic healthy human donors., Intervention(s): Spermatozoa isolated from semen on a discontinuous Percoll gradient (40%-80%), followed by swim-up, were used for all techniques except for sperm motility analysis, for which fresh semen was used., Main Outcome Measure(s): Immunoblotting blots, indirect immunofluorescence antibody assays, cycle threshold values for real-time polymerase chain reaction, and percentage of motile sperm., Result(s): We found APN in the equatorial segment of the upper post-acrosomal region of the sperm head, in the neck and along the tail of spermatozoa, in prostasomes, and in seminal fluid, whereas NEP was present in a very restricted area of a few sperm cells and in prostasomes. Messenger RNA of both enzymes was detected in spermatozoa. The inhibition of enkephalin-degrading enzymes attenuated the time-dependent decrease of sperm motility; this effect was reversed by naloxone., Conclusion(s): Enkephalin-degrading enzymes are present in human semen and may be involved in the control of sperm motility, mainly by the regulation of endogenous opioid peptides.

Renal cell carcinomas (RCCs) are neoplasias with high prevalence and mortality. We previously reported that several peptidases may be involved in the pathophysiology of clear cell renal cell carcinoma (CCRCC). Now, to gain insight into the reasons that lead the various RCC types to behave very differently with regard to aggressiveness and response to anticancer treatments, we analyzed subsets of chromophobe renal cell carcinoma (ChRCC), and renal oncocytoma (RO), a benign tumor; as well as different grades and stages of CCRCCs. Particulate APN, APB, and APA activities were decreased in both ChRCC and RO (tumor vs. nontumor tissues). Interestingly, activities were downregulated in a tumor-type specific way and the intensities of the decreases were stronger in the benign tumor than in the malignant type. Moreover, when two key histopathological parameters for tumor prognosis (high vs. low stage and grade) were analyzed, increases of activity were also observed in several of these cell surface peptidases (APN, APB). Some soluble activities (APB, Asp-AP) were also downregulated in the RCCs. With respect to genetic expression, PSA and APN were in a positive correlation related to their activities in both ChRCC and RO; but not APB, Asp-AP, APA, and PGI. These results may suggest an involvement of several peptidases in the pathophysiology of renal cancer, since they presented different patterns of activity and expression in tumors with different behaviors.

The involvement of peptidases in carcinogenetic processes of several tumor types has been researched in recent years. Although kidney is one of the major tissues known to express cystinyl-aminopeptidase (CAP), little is known about its role in renal neoplasia. This study analyzes fluorimetrically membrane-bound and soluble CAP activity in the three main renal cancers: clear cell (CCRCC), papillary (PRCC), and chromophobe (ChRCC) renal cell carcinomas. Overall, a marked decrease of membrane-bound CAP activity in all the three renal cell carcinomas was detected when compared with their respective surrounding non-tumor tissues. So, the tumor vs. non-tumor CAP ratios (units of peptidase per mg of protein) was as follows: 926+/-111 vs. 3778+/-276 for CCRCCs, 737+/-181 vs. 4351+/-950 for PRCCs, and 592+/-118 vs. 4905+/-935 for ChRCCs. In contrast, the soluble fraction of this enzyme displayed minor and non-significant changes when comparing tumor and non-tumor CAP activities in the whole series. After stratification by stage and grade, CCRCCs displayed significant differences: pT3 category had significantly higher levels of membrane-bound activity than pT1, and high grade cases (G3-4) had higher soluble CAP activity than low grade ones (G1-2). These data may open additional possibilities in the study of renal cell carcinoma with regard to the prognosis of patients.

Peptides play important roles in cell regulation and signaling in many tissues and are regulated by peptidases, most of which are highly expressed in the kidney. Several peptide convertases have a function in different tumor stages, and some have been clearly characterized as diagnostic and prognostic markers for solid tumors, including renal cancer; however, little is known about their in vivo role in kidney tumors. The present study compares the activity of a range of peptidases in human tumor samples and nontumor tissue obtained from clear cell renal cell carcinoma (CCRCC) patients. To cover the complete spectrum and subcellular distribution of peptide-converting activity, acid, neutral, basic, and omega activities were selected. CCRCC displays a selective and restricted pattern of peptidase activities. Puromycin-sensitive aminopeptidase activity in the tumor increases [tumor (t) = 10,775 vs. nontumor (n) = 7,635 units of peptidase (UP)/mg protein; P < 0.05], whereas aminopeptidase N decreases (t = 6,664 vs. n = 33,381 UP/mg protein; P < 0.001). Aminopeptidase B activity of the particulate fraction in tumors decreases (t = 2,399 vs. n = 13,536 UP/mg protein; P < 0.001) compared with nontumor tissues, and aspartyl-aminopeptidase activity decreases significantly in CCRCC (t = 137 vs. n = 223 UP/mg protein; P < 0.05). Soluble and particulate pyroglutamyl peptidase I activities, aminopeptidase A activity, and soluble aminopeptidase B activity do not vary in renal cancer. The relative expression for the aforementioned peptidases, assayed using quantitative RT-PCR, increases in CCRCC for aminopeptidases B (1.5-fold) and A (19-fold), aspartyl-aminopeptidase (3.9-fold), puromycin-sensitive aminopeptidase (2.5-fold), and pyroglutamyl peptidase I (7.6-fold). Only aminopeptidase N expression decreases in tumors (1.3-fold). This peptidase activity profile in the neoplastic kidney suggests a specific role for the studied convertases and the possible involvement of an intracrine renin-angiotensin system in the pathogenesis of CCRCC.

Context: Endogenous opioid peptides signal through delta-, kappa-, and mu-opioid receptors. Some of these peptides such as endorphins and enkephalins are present in the male reproductive tract, but the presence of the corresponding receptors in human sperm cells has not yet been reported., Objective: Our objective was to study the expression and localization of delta-, kappa-, and mu-opioid receptors on human spermatozoa and the implication in sperm motility., Methods: The expression of receptors was studied by RT-PCR, Western blot, and immunofluorescence techniques. We evaluated the effects of activation of each opioid receptor by specific agonist and antagonist., Results: Human spermatozoa express delta-, kappa-, and mu-opioid receptors. These receptors were located in different parts of the head, in the middle region, and in the tail of the sperm. Progressive motility of spermatozoa, an important parameter to evaluate male fertility, was found to be significantly reduced after incubation with the mu-receptor agonist morphine, whereas this effect was antagonized in the presence of the corresponding antagonist naloxone. The delta-receptor antagonist naltrindole significantly reduced progressive motility immediately after its addition. However, the delta-receptor agonist DPDPE had no significant effect. Finally, neither the kappa-receptor agonist U50488 nor its antagonist nor-binaltorphimine significantly affected the progressive motility of human spermatozoa., Conclusion: We report for first time the presence of functional delta-, kappa-, and mu-opioid receptors in human sperm membranes. These findings are indicative of a role for the opioid system in the regulation of sperm physiology.

Opioid peptides have been reported to have important functions in human reproduction. Indeed, very high concentrations of enkephalins and their degrading enzymes have been reported in human semen. In the present paper, we compare the activity of two enkephalin-degrading enzymes, aminopeptidase N and neutral endopeptidase 24.11, in different fractions of semen from normozoospermic, fertile men and from subfertile patients with different abnormalities revealed by spermiogram analysis (asthenozoospermia, necrozoospermia, and teratozoospermia). High levels of activity of aminopeptidase N were found in the soluble and particulate sperm fractions of semen from patients presenting asthenozoospermia with necrozoospermia. In contrast, lower aminopeptidase N activity was measured in the soluble sperm fraction of asthenozoospermic semen. The percentage of dead spermatozoa was positively correlated with aminopeptidase N activity in both soluble and particulate sperm fractions. In contrast, the percentage of immobile spermatozoa was negatively correlated with aminopeptidase activity in soluble and particulate sperm, and in prostasome fractions. Levels of activity of neutral endopeptidase were found to be unaltered among the different conditions. In summary, the results of the present study indicate that alterations in the activity of aminopeptidase N may be one of the molecular components that contribute to male human subfertility.