Role of Angiotensin-(1-7) via MAS receptor in human sperm motility and acrosome reaction.

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 epigenetic regulation of the opioid system: new individualized prompt prevention and treatment strategies.

The most well-known physiological effect associated with opiod system is their efficacy in pain reduction or analgesia, although their effect on a variety of other physiological and physiophological functions has become apparent in recent years. This review is an attempt to clarify in more detail the epigenetic regulation of opioid system to understand with more precision their transcriptional and posttranscriptional regulation in multiple pyisiological and pharmacological contexts. The opioid receptors show an epigenetic regulation and opioid peptide precursors by methylation, chromatin remodeling and microRNA. Although the opioid receptor promoters have similarity between them, they use different epigenetic regulation forms and they exhibit different pattern of expression during the cell differentiation. DNA methylation is also confirmed in opioid peptide precursors, being important for gene expression and tissue specificity. Understanding the epigenetic basis of those physiological and physiopathological procesess is essential for the development of individualized prompt prevention and treatment strategies.

Altered glutamyl-aminopeptidase activity and expression in renal neoplasms.

BACKGROUND: Advances in the knowledge of renal neoplasms have demonstrated the implication of several proteases in their genesis, growth and dissemination. Glutamyl-aminopeptidase (GAP) (EC. 3.4.11.7) is a zinc metallopeptidase with angiotensinase activity highly expressed in kidney tissues and its expression and activity have been associated wtih tumour development. METHODS: In this prospective study, GAP spectrofluorometric activity and immunohistochemical expression were analysed in clear-cell (CCRCC), papillary (PRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Data obtained in tumour tissue were compared with those from the surrounding uninvolved kidney tissue. In CCRCC, classic pathological parameters such as grade, stage and tumour size were stratified following GAP data and analyzed for 5-year survival. RESULTS: GAP activity in both the membrane-bound and soluble fractions was sharply decreased and its immunohistochemical expression showed mild staining in the four histological types of renal tumours. Soluble and membrane-bound GAP activities correlated with tumour grade and size in CCRCCs. CONCLUSIONS: This study suggests a role for GAP in the neoplastic development of renal tumours and provides additional data for considering the activity and expression of this enzyme of interest in the diagnosis and prognosis of renal neoplasms.

The impact of peptidase activity on clear cell renal cell carcinoma survival.

Several studies have proposed that protease expression and activity may have a predictive value in the survival of clear cell renal cell carcinoma (CCRCC). Most efforts on this issue have been focused on the analysis of matrix metalloproteinases (MMP) and very little on the role of other proteases, such as peptidases. The catalytic activity of 9 peptidases (APN, APB, ASP, CAP, DPP-IV, NEP/CD10, PEP, PGI, and PSA) was quantified by fluorometric methods in a series of 79 CCRCC patients, and the results obtained were analyzed for survival (Kaplan-Meier curves, log-rank test, and Cox multivariate analysis). CCRCC patients with higher activity levels of membrane-bound APN and soluble APN, DPP-IV, and CAP had significantly shorter 5-yr survival rates than those with lower levels. By contrast, higher soluble APB activity significantly correlated with longer survival. Our data suggest the involvement of peptidases in the biological aggressiveness of CCRCC and support the usefulness of measuring these proteases to assess the prognosis of patients with CCRCC.

Regulation of male fertility by the opioid system.

Endogenous opioid peptides are substances involved in cell communication. They are present in various organs and tissues of the male and female reproductive tract, suggesting that they may regulate some of the processes involved in reproductive function. In fact, the opioid system that operates as a multi-messenger system can participate in the regulation of reproductive physiology at multiple levels, for example, at the levels of the central nervous system, at the testes level and at sperm level. A better understanding of the implication of the opioid system in reproductive processes may contribute to clarifying the etiology of many cases of infertility and the effect of opiate abuse on fertility. Indeed, a novel biochemical tool for the diagnosis and treatment of male infertility could be based upon components of the opioid system. The presence of the opioid system in sperm cells also represents a novel opportunity for reproductive management, for either enhancing the probability of fertilization or reducing it through the development of novel targeted contraceptives.

Synovial fluid peptidase activity as a biomarker for knee osteoarthritis clinical progression.

AIMS: To analyze the potential role of synovial fluid peptidase activity as a measure of disease burden and predictive biomarker of progression in knee osteoarthritis (KOA). METHODS: A cross-sectional study of 39 patients (women 71.8%, men 28.2%; mean age of 72.03 years (SD 1.15) with advanced KOA (Ahlbäck grade ≥ 3 and clinical indications for arthrocentesis) recruited through the (Orthopaedic Department at the Complejo Asistencial Universitario de León, Spain (CAULE)), measuring synovial fluid levels of puromycin-sensitive aminopeptidase (PSA), neutral aminopeptidase (NAP), aminopeptidase B (APB), prolyl endopeptidase (PEP), aspartate aminopeptidase (ASP), glutamyl aminopeptidase (GLU) and pyroglutamyl aminopeptidase (PGAP). RESULTS: Synovial fluid peptidase activity varied significantly as a function of clinical signs, with differences in levels of PEP (p = 0.020), ASP (p < 0.001), and PGAP (p = 0. 003) associated with knee locking, PEP (p = 0.006), ASP (p = 0.001), GLU (p = 0.037), and PGAP (p = 0.000) with knee failure, and PEP (p = 0.006), ASP (p = 0.001), GLU (p = 0.037), and PGAP (p < 0.001) with knee effusion. Further, patients with the greatest functional impairment had significantly higher levels of APB (p = 0.005), PEP (p = 0.005), ASP (p = 0.006), GLU (p = 0.020), and PGAP (p < 0.001) activity, though not of NAP or PSA, indicating local alterations in the renin-angiotensin system. A binary logistic regression model showed that PSA was protective (p = 0.005; Exp (B) 0.949), whereas PEP (p = 0.005) and GLU were risk factors (p = 0.012). CONCLUSION: These results suggest synovial fluid peptidase activity could play a role as a measure of disease burden and predictive biomarker of progression in KOA. Cite this article: Bone Joint Res 2020;9(11):789-797.

Brain endocannabinoid system is involved in fluoxetine-induced anorexia.

In order to describe the effects of chronic fluoxetine administration on the brain endocannabinoid system in lean and obese Zucker rats, brain immunostaining for the CB1 and CB1-phosphorylated cannabinoid receptors was carried out. Obese Zucker rats showed significantly increased the numbers of neural cells positively immunostained for the CB1-phosphorylated receptor in the striatum, compared to their lean litter-mates. Chronic fluoxetine administration decreased the number of neural cells immunostained for CB1-phosphorylated receptor in several striatal and hippocampal regions of obese Zucker rats, compared to controls treated with saline. In contrast, no change in CB1-phosphorylated receptor immunostaining was observed in fluoxetine-treated lean rats, with respect to controls. Taken together, these results suggest the involvement of the hippocampal and striatal endocannabinoid receptor system in fluoxetine-induced anorexia in lean and obese Zucker rats.

Human sperm testicular angiotensin-converting enzyme helps determine human embryo quality.

Angiotensin-converting enzyme functions in the male reproductive system, but the extent of its function in reproduction is not fully understood. The primary objective of this work was to investigate the relationship between the testicular isoform of angiotensin-converting enzyme present in human spermatozoa and semen parameters, human embryo quality, and assisted reproduction success. A total of 81 semen samples and 635 embryos from couples undergoing oocyte donation cycles at the IVI Bilbao Clinic were analyzed. Semen parameters, embryos quality, and blastocyst development were examined according to the World Health Organization standards and the Spanish Association of Reproduction Biology Studies criteria. The percentage of testicular angiotensin-converting enzyme-positive spermatozoa and the number of molecules per spermatozoon were analyzed by flow cytometry. Both parameters were inversely correlated with human sperm motility. Higher percentages of testicular angiotensin-converting enzyme-positive spermatozoa together with fewer enzyme molecules per spermatozoon were positively correlated with better embryo quality and development. Our results suggest that embryos with a higher implantation potential come from semen samples with higher percentages of testicular angiotensin-converting enzyme-positive cells and fewer enzyme molecules per spermatozoon. Based on these findings, we propose that testicular angiotensin-converting enzyme could be used to aid embryologists in selecting better semen samples for obtaining high-quality blastocysts during in vitro fertilization procedures.

Associations of Anthropometric Characteristics, Dietary Habits, and Aerobic Capacity With Cardiovascular Risk Factors of Health-Science Students.

The aim of this cross-sectional study was to study the relative importance of dietary habits and aerobic capacity in parameters related to cardiovascular risk in 271 female and 95 male health-science students (mean age = 19.1 ± 1.4 years). In females, fatty-meat consumption predicted triglycerides (ß = .649, p < .001) and high-density lipoprotein (HDL; ß = -.242, p = .001) and low-density lipoprotein (LDL; ß = .373, p < .001) cholesterol levels. Consumption of nuts, legumes, and complex carbohydrates predicted triglyceride (ß = -.099, p = .074), HDL (ß = .231, p = .001), and LDL (ß = -.155, p = .025) levels, respectively. Aerobic capacity (ß = -.245, p < .001) and fatty-meat intake (ß = .230, p < .001) predicted diastolic blood pressure (BP); body mass index (BMI) predicted systolic BP (ß = .340, p < .001). In males, body fat percentage was the strongest predictor of triglycerides (ß = .348, p = .004), cholesterol (ß = .366, p = .006), HDL (ß = -.378, p = .004), and LDL (ß = .271, p = .043) levels. Aerobic capacity (ß = -.263, p = .013) and fatty-meat consumption (ß = .334, p = .005) independently predicted triglyceride levels. Nut (ß = -.286, p = .013) and fatty-meat intake (ß = .361, p = .002) predicted systolic BP, while BMI predicted diastolic BP (ß = .209, p = .045). As health sciences students, these participants are future health professionals; targeting such populations is important for chronic disease prevention.

Ontogeny of prolyl endopeptidase and pyroglutamyl peptidase I in rat tissues.

Prolyl endopeptidase and pyroglutamyl peptidase I are enzymes which participate in the degradation of thyrotropin-releasing hormone (TRH), a hormone which is thought to play an important role in the development of organs and tissues. Here, we have characterized the ontogeny of TRH degrading enzyme activity in the brain cortex, lung, heart, kidney and liver. Overall, prolyl endopeptidase activity was found to be 2 to 5 fold higher in newborn vs. adult rat tissues, with the exception of the soluble form in the liver and the particulate form in the lung. In contrast, the developmental profile of pyroglutamyl peptidase I activity was found to be more variable and tissue dependent. These results corroborate the idea that both enzymes play important, tissue-specific roles during the development and maturation of rat organs.

Cystinyl aminopeptidase activity is decreased in renal cell carcinomas.

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.

Expression and localization of delta-, kappa-, and mu-opioid receptors in human spermatozoa and implications for sperm motility.

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.

Expression and Localization of Opioid Receptors in Male Germ Cells and the Implication for Mouse Spermatogenesis.

The presence of endogenous opioid peptides in different testicular cell types has been extensively characterized and provides evidence for the participation of the opioid system in the regulation of testicular function. However, the exact role of the opioid system during the spermatogenesis has remained controversial since the presence of the mu-, delta- and kappa-opioid receptors in spermatogenic cells was yet to be demonstrated. Through a combination of quantitative real-time PCR, immunofluorescence, immunohistochemistry and flow cytometry approaches, we report for the first time the presence of active mu-, delta- and kappa-opioid receptors in mouse male germ cells. They show an exposition time-dependent response to opioid agonist, hence suggesting their active involvement in spermatogenesis. Our results contribute to understanding the role of the opioid receptors in the spermatogenesis and could help to develop new strategies to employ the opioid system as a biochemical tool for the diagnosis and treatment of male infertility.

Effect of the antidepressant nefazodone on the density of cells expressing mu-opioid receptors in discrete brain areas processing sensory and affective dimensions of pain.

RATIONALE: The principal use of antidepressants is in the treatment of depression and affective disorders. Antidepressants have also been used as an adjuvant to analgesics in pain treatment. However, in chronic treatment, their antinociceptive and antidepressive effects coexist simultaneously. Antidepressants can interact with the opioid system, which is also involved in regulating nociceptive processing and affective state. Chronic antidepressants could act by increasing mu-opioid receptor expression in many brain areas involved in the regulation of nociception and affective state. OBJECTIVES: The aim of this study was to evaluate the antinociceptive and antidepressant-like effects and the possible variations in mu-opioid receptor expression induced by a chronic nefazodone treatment in brain areas related to pain and affective state. METHODS: Wistar rats were chronically treated with nefazodone (10 and 25 mg/kg IP, twice a day, for 14 days). Twelve hours after the last day 14 dose of nefazodone, a tail-flick test was performed. After the administration of a daily dose of nefazodone, Porsolt's test was carried out 12 h after last dose. Two hours after completion of 14 days treatment, other animals were processed for mu-opioid receptor immunocytochemistry using polyclonal antisera raised in rabbits. Several brain regions were analyzed: the frontal and cingulate cortex, the dorsal raphe nucleus and the periaqueductal gray. RESULTS: Chronic nefazodone treatment induced a significant increase in tail-flick latency and a significant decrease in immobility time at total doses of 20 and 50 mg/kg per day ( P<0.05). In treated animals, the density of neural cells immunostained for mu-opioid receptor in the frontal and cingulate cortices, dorsal raphe nucleus and periaqueductal gray had increased after chronic nefazodone compared to controls. CONCLUSION: Therefore, chronic nefazodone induces antinociceptive and antidepressant-like effects in rats and increases mu-opioid receptor expression in brain areas related to pain and affective state. These results suggest that antidepressants could be effective on somatic and affective dimensions of pain and this action could be related to its influence on the opioid system.

Distribution of prolyl endopeptidase activities in rat and human brain.

Prolyl endopeptidase is a proteolytic enzyme which could have a neuropeptide catabolising role in the central nervous system. Although prolyl endopeptidase has been described as a cytosolic enzyme, it has become clear that it can also be found in particulate form. The regional and subcellular distribution of this enzyme was evaluated in rat and human brain. The activity of the enzyme was higher in the human than in the rat brain. In the human brain, the activity levels of both soluble and particulate prolyl endopeptidase were the highest in frontal, parietal and occipital cortices and the lowest in the cerebellum. In the rat brain, the regional distribution of the enzyme was more homogeneous. The activity in all the areas of the central nervous system is higher than in peripheral tissues. Subcellular distribution of the enzyme in the brain indicates that prolyl endopeptidase was higher in the cytosolic fraction than in the particulate fractions. The particulate form was enriched in the synaptosomal and the myelinic membranes. The high activity of prolyl endopeptidase in the human cortex suggests that prolyl endopeptidase could play a role in the functions of this brain area.

Effect of aging on rat tissue peptidase activities.

The process of aging is known to involve alterations in the activity of peptidases and proteases. However, the precise changes in the activity of many peptidases in aged tissues have not yet been fully characterized, and both decreases and increases in both peptidase activity and peptide levels have been reported to occur during the aging process. In the present study, we measured the activity of several peptidases in selected tissues (brain cortex, brain stem, liver, kidney, heart, and lung) of the young adult (3 months old) and aged (18 months old and 22 months old) rat. The activities of prolyl endopeptidase, pyroglutamyl peptidase I, puromycin sensitive aminopeptidase, and aminopeptidase N were assayed using beta-naphthylamine aminoacidic derivatives as substrates. The activity of the soluble fractions of prolyl endopeptidase was found to be reduced in the lungs of aged animals, while reduced activity of soluble pyroglutamyl peptidase I and also aminopeptidase N was measured in the aged kidney and heart, respectively. In contrast, increased activity of particulate prolyl endopeptidase was measured in the brain stem of older animals. Since most of these changes can be correlated with known alterations in the levels of peptides controlled by each enzyme, the results of the present study indicate that the studied peptidases may play an important role in regulating tissue peptide levels during aging.

Peptidase activities in human semen.

Enkephalins are one of the opioids present in human semen and to date their function in this tissue remains unknown. The present work studies enkephalin-degrading enzyme activities, puromycin-sensitive alanyl aminopeptidase (AAP-S), puromycin-insensitive alanyl aminopeptidase N (Ap N) and neprilysin (NEP) in human seminal fractions. AAP-S activity was not detected in any fractions, whereas Ap N appeared in soluble and particulate sperm fractions in seminal fluid and in prostasome fraction. With regard to NEP activity, this was exclusively located in prostasome membranes. The high activity values observed in the prostasome fraction suggested that these peptidases and their substrates could be involved in seminal physiology.

Aminopeptidases in visceral organs during alterations in body fluid volume and osmolality.

Enzymatic cleavage of some peptides in the local environment could be included among the mechanisms related to the regulation of hydrosaline balance. In order to examine this hypothesis, we measured representative aminopeptidase activities in visceral organs of rats after applying certain hydrosaline challenges. Decreased levels (about 30%) of particulate puromycin-insensitive-neutral aminopeptidase in the renal medulla and of soluble acid aminopeptidase in the lung were observed under hyperosmolality and hypovolemia. Decreased levels (more than 45%) of particulate type-I-pyroglutamyl aminopeptidase in the heart were observed under altered volemia. These results indicate that aminopeptidases at these anatomical locations might be involved in the regulation of body fluid volume and osmolality.

Effects of morphine administration and its withdrawal on rat brain aminopeptidase activities.

The endogenous opioid neuropeptide system seems to be involved in the neural processes which underlie drug addiction. Several studies have reported that the administration of morphine induces changes in the levels and/or activity of endogenous opioid peptides (enkephalin, dynorphin) and their precursors in specific brain regions of the adult CNS. The aim of this work was to study the effects of chronic morphine exposure and its withdrawal on certain aminopeptidases capable of degrading opioid peptides in brain areas including the amygdala, hypothalamus, hippocampus, striatum and brain cortices. In animals treated with morphine, aminopeptidase N presented higher enzyme activity levels in the striatum, the hypothalamus and the amygdala compared to control animals, although statistically significant differences were observed only in the case of the striatum. In addition, the activity of soluble puromycin-sensitive aminopeptidase (PSA) was found to be higher in the frontal cortex of these rats. In contrast, rats experiencing withdrawal symptoms presented decreased levels of aminopeptidase activity in certain brain areas. Thus, the activity of aminopeptidase N in the hippocampus and soluble puromycin-sensitive aminopeptidase in the frontal cortex were found to be lower in rats experiencing naloxone precipitated withdrawal symptoms, compared to the corresponding controls. Finally, the activity of the three studied aminopeptidases in vitro was unaltered by incubation with morphine, suggesting that the observed effects are not due to a direct action of this opioid upon the aminopeptidases. The results of the present report indicate that aminopeptidases may play an important role in the processes of tolerance and withdrawal associated with morphine administration.

Pyroglutamyl peptidase I and prolyl endopeptidase in human semen: increased activity in necrozoospermia.

Thyrotropin-releasing hormone (TRH) and its analogues have been reported to have important functions in human semen. In the present paper, we have characterized the activity of the TRH-degrading enzymes pyroglutamyl peptidase I and prolyl endopeptidase in the fluid and prostasomes of human semen and in subcellular fractions of the corresponding sperm. Enzymatic activities were measured fluorimetrically using beta-naphthylamine derivatives as substrate. Activity associated with both enzymes was detected in seminal fluid and in the prostasome fraction, as well as in soluble and particulate sperm subcellular fractions. Pyroglutamyl-peptidase I activity presented highest levels in the particulate sperm fraction, whereas the activity of prolyl endopeptidase was maximal in the soluble sperm fraction. In addition, we compared the activity of both enzymes in different seminal fractions in normozoospermic, fertile men and in subfertile patients with different abnormalities revealed by spermiogram analysis (astenozoospermia, necrozoospermia and teratozoospermia). The activities of pyroglutamyl peptidase I and prolyl endopeptidase in necrozoospermia were found to be higher in the corresponding soluble and particulate sperm fractions, respectively, with respect to those measured in normozoospermic semen. The results of the present study indicate that these enzymes may participate in regulating the levels of seminal TRH analogues and in mediating sperm death associated with necrozoospermia.