The Expression of Alamandine Receptor MrgD in Clear Cell Renal Cell Carcinoma Is Associated with a Worse Prognosis and Unfavorable Response to Antiangiogenic Therapy.

Renal cell carcinoma (RCC) ranks among the most prevalent malignancies in Western countries, marked by its notable heterogeneity, which contributes to an unpredictable clinical trajectory. The insufficiency of dependable biomarkers adds complexity to assessing this tumor progression. Imbalances of several components of the intrarenal renin-angiotensin system (iRAS) significantly impact patient prognoses and responses to first-line immunotherapies. In this study, we analyzed the immunohistochemical expression of the Mas-related G-protein-coupled receptor D (MrgD), which recognizes the novel RAS peptide alamandine (ALA), in a series of 87 clear cell renal cell (CCRCCs), 19 papillary (PRCC), 7 chromophobe (ChRCC) renal cell carcinomas, and 11 renal oncocytomas (RO). MrgD was expressed in all the renal tumor subtypes, with a higher mean staining intensity in the PRCCs, ChRCCs, and ROs. A high expression of MrgD at the tumor center and at the infiltrative front of CCRCC tissues was significantly associated with a high histological grade, large tumor diameter, local invasion, and locoregional node and distant metastasis. Patients with worse 5-year cancer-specific survival and a poorer response to antiangiogenic tyrosine-kinase inhibitors (TKIs) showed higher MrgD expression at the center of their primary tumors. These findings suggest a possible role of MrgD in renal carcinogenetic processes. Further studies are necessary to unveil its potential as a novel biomarker for CCRCC prognosis and response to frontline therapies.

Fibroblast Activation Protein-α (FAP) Identifies Stromal Invasion in Colorectal Neoplasia.

The increasing detection of colorectal adenomas and early adenocarcinomas (ADCs) in the context of nationwide screening programs has led to a significant increase in the incidence of inconclusive diagnoses in which histopathologic analysis of endoscopic biopsies does not allow pathologists to provide a reliable diagnosis of stromal invasion. The objective of this study was to analyze the discriminative capacity of the immunohistochemical expression of fibroblast activation protein-α (FAP) in distinguishing colorectal adenomas with low-grade dysplasia (LGD) and high-grade dysplasia (HGD) from invasive intestinal-type ADCs. The study analyzed the first endoscopic biopsies from a series of patients classified as inconclusive or conclusive for stromal invasion based on the pathologic report. In total, 30 ADCs, 52 HGDs, and 15 LGDs were included in the study. FAP expression was detected in 23/30 ADCs and was negative in all adenomas with either LGD or HGD features (100% specificity and 76.7% sensitivity, area under the curve=0.883, CI=0.79-0.98). Considering these findings, we conclude that FAP is a potentially useful tool for helping pathologists identify invasive lesions in colorectal endoscopic biopsies, avoiding unnecessary biopsy repetitions.

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.

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 CB(2) cannabinoid receptor regulates human sperm cell motility.

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).

Expression of enkephalin-degrading enzymes in human semen and implications for sperm motility.

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.

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.

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.

Enkephalin-degrading enzymes in normal and subfertile human semen.

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.

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.