The Role of Sperm Membrane Potential and Ion Channels in Regulating Sperm Function.

During the last seventy years, studies on mammalian sperm cells have demonstrated the essential role of capacitation, hyperactivation and the acrosome reaction in the acquisition of fertilization ability. These studies revealed the important biochemical and physiological changes that sperm undergo in their travel throughout the female genital tract, including changes in membrane fluidity, the activation of soluble adenylate cyclase, increases in intracellular pH and Ca2+ and the development of motility. Sperm are highly polarized cells, with a resting membrane potential of about -40 mV, which must rapidly adapt to the ionic changes occurring through the sperm membrane. This review summarizes the current knowledge about the relationship between variations in the sperm potential membrane, including depolarization and hyperpolarization, and their correlation with changes in sperm motility and capacitation to further lead to the acrosome reaction, a calcium-dependent exocytosis process. We also review the functionality of different ion channels that are present in spermatozoa in order to understand their association with human infertility.

Female Infertility Is Associated with an Altered Expression Profile of Different Members of the Tachykinin Family in Human Granulosa Cells.

Neurokinin B (NKB) and its cognate receptor, NK3R, play a key role in the regulation of reproduction. NKB belongs to the family of tachykinins, which also includes substance P and neurokinin A, both encoded by the by the gene TAC1, and hemokinin-1, encoded by the TAC4 gene. In addition to NK3R, tachykinin effects are mediated by NK1R and NK2R, encoded by the genes TACR1 and TACR2, respectively. The role of these other tachykinins and receptors in the regulation of women infertility is mainly unknown. We have analyzed the expression profile of TAC1, TAC4, TACR1, and TACR2 in mural granulosa and cumulus cells from women presenting different infertility etiologies, including polycystic ovarian syndrome, advanced maternal age, low ovarian response, and endometriosis. We also studied the expression of MME, the gene encoding neprilysin, the most important enzyme involved in tachykinin degradation. Our data show that TAC1, TAC4, TACR1, TACR2, and MME expression is dysregulated in a different manner depending on the etiology of women infertility. The abnormal expression of these tachykinins and their receptors might be involved in the decreased fertility of these patients, offering a new insight regarding the diagnosis and treatment of women infertility.

Female infertility is associated with an altered expression of the neurokinin B/neurokinin B receptor and kisspeptin/kisspeptin receptor systems in ovarian granulosa and cumulus cells.

OBJECTIVE: To analyze and compare the expression profile of TAC3, TACR3, KISS1, and KISS1R in mural granulosa and cumulus cells from healthy oocyte donors and patients with different infertility etiologies, including advanced maternal age, endometriosis, and low ovarian response. DESIGN: Genetic association study. SETTING: Private fertility clinic and public research laboratory. PATIENT(S): Healthy oocyte donors and infertile women undergoing in vitro fertilization (IVF) treatment. INTERVENTION(S): IVF. MAIN OUTCOME MEASURE(S): Gene expression levels of KISS1, KISS1R, TAC3, and TACR3 in human mural granulosa and cumulus cells. RESULT(S): Infertile women showed statistically significantly altered expression levels of KISS1 (-2.57 ± 2.30 vs. -1.37 ± 2.11), TAC3 (-1.21 ± 1.40 vs. -1.49 ± 1.98), and TACR3 (-0.77 ± 1.36 vs. -0.03 ± 0.56) when compared with healthy oocyte donors. Advanced maternal age patients, endometriosis patients, and low responders showed specific and altered expression profiles in comparison with oocyte donors. CONCLUSION(S): Abnormal expression levels of KISS1/KISS1R and TAC3/TACR3 systems in granulosa cells might be involved in the decreased fertility associated to advanced maternal age, endometriosis, and low ovarian response.

Tachykinins and Kisspeptins in the Regulation of Human Male Fertility.

Infertility is a global disease affecting one out of six couples of reproductive age in the world, with a male factor involved in half the cases. There is still much to know about the regulation of human male fertility and thus we decided to focus on two peptide families that seem to play a key role in this function: tachykinins and kisspeptins. With this aim, we conducted an exhaustive review in order to describe the role of tachykinins and kisspeptins in human fertility and their possible implications in infertility etiopathogenesis. Many advances have been made to elucidate the roles of these two families in infertility, and multiple animal species have been studied, including humans. All of this knowledge could lead to new advances in male infertility diagnosis and treatment, but further research is needed to clarify all the implications of tachykinins and kisspeptins in fertility.

Altered expression of the kisspeptin/KISS1R and neurokinin B/NK3R systems in mural granulosa and cumulus cells of patients with polycystic ovarian syndrome.

PURPOSE: The neurokinin B (NKB)/NK3 receptor (NK3R) and kisspeptin (KISS1)/kisspeptin receptor (KISS1R), two systems essential for reproduction, are present in human granulosa cells (GCs) of healthy women and contribute to the control of fertility, at least partially, by acting on the gonads. However, little is known about the expression of these systems in GCs of women with polycystic ovarian syndrome (PCOS). The aim of this study was to analyze the expression of NKB/NK3R and KISS1/KISS1R in mural granulosa (MGCs) and cumulus cells (CCs) of PCOS women. METHODS: A cross-sectional study was performed in 46 healthy women and 43 PCOS women undergoing controlled ovarian stimulation. MGCs and CCs were collected from pre-ovulatory follicles after transvaginal ultrasound-guided oocyte retrieval and the expression of the genes encoding NKB (TAC3), NK3R (TACR3), KISS1, and its receptor (KISS1R) was analyzed using real-time quantitative RT-PCR. RESULTS: TAC3, TACR3, and KISS1 mRNA levels were decreased in MGCs and CCs of PCOS women. TAC3 positively correlated with KISS1 in MGCs of healthy women and TACR3 was positively associated with KISS1R in CCs from healthy women. These associations were not observed in PCOS women. CONCLUSION: The NKB/NK3R and KISS1/KISS1R systems are dysregulated in MGCs and CCs of PCOS women. The lower expression of these systems in GCs could contribute to the abnormal follicle development and defective ovulation that characterize the pathogenesis of PCOS.

Expression of Tachykinins and Tachykinin Receptors and Interaction with Kisspeptin in Human Granulosa and Cumulus Cells.

The neurokinin B/NK3 receptor (NK3R) and kisspeptin/kisspeptin receptor (KISS1R), two systems which are essential for reproduction, are coexpressed in human mural granulosa (MGC) and cumulus cells (CCs). However, little is known about the presence of other members of the tachykinin family in the human ovary. In the present study, we analyzed the expression of substance P (SP), hemokinin-1 (HK-1), NK1 receptor (NK1R), and NK2 receptor (NK2R) in MGCs and CCs collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval. RT-PCR, quantitative RT-PCR, immunocytochemistry, and Western blotting were used to investigate the patterns of expression of tachykinin and tachykinin receptor mRNAs and proteins and the possible interaction between the tachykinin family and kisspeptin. Intracellular free Ca(2+) levels ([Ca(2+)]i) in MGCs after exposure to SP or kisspeptin in the presence of SP were also measured. We found that SP, HK-1, the truncated NK1R isoform NK1R-Tr, and NK2R were all expressed in MGCs and CCs. NK1R-Tr mRNA and NK2R mRNA and protein levels were higher in MGCs than in CCs from the same patients. Treatment of cells with kisspeptin modulated the expression of HK-1, NK3R, and KISS1R mRNAs, whereas treatment with SP regulated kisspeptin mRNA levels and reduced the [Ca(2+)]i response produced by kisspeptin. These data demonstrate that the whole tachykinin system is expressed and acts in coordination with kisspeptin to regulate granulosa cell function in the human ovary.

Analysis of the Expression of Tachykinins and Tachykinin Receptors in the Rat Uterus During Early Pregnancy.

The peptides of the tachykinin family participate in the regulation of reproductive function acting at both central and peripheral levels. Our previous data showed that treatment of rats with a tachykinin NK3R antagonist caused a reduction of litter size. In the present study, we analyzed the expression of tachykinins and tachykinin receptors in the rat uterus during early pregnancy. Uterine samples were obtained from early pregnant rats (Days 1-9 of pregnancy) and from nonpregnant rats during the proestrus stage of the ovarian cycle, and real-time quantitative RT-PCR, immunohistochemistry, and Western blot studies were used to investigate the pattern of expression of tachykinins and tachykinin receptors. We found that all tachykinins and tachykinin receptors were locally synthesized in the uterus of early pregnant rats. The expression of substance P, neurokinin B, and the tachykinin receptors NK1R and NK3R mRNAs and proteins underwent major changes during the days around implantation and they were widely distributed in implantation sites, being particularly abundant in decidual cells. These findings support the involvement of the tachykinin system in the series of uterine events that occur around embryo implantation in the rat.

Altered Activity and Expression of Cytosolic Peptidases in Colorectal Cancer.

BACKGROUND AND OBJECTIVE: The role of peptidases in carcinogenic processes and their potential usefulness as tumor markers in colorectal cancer (CRC) have been classically attributed to cell-surface enzymes. The objective of the present study was to analyze the activity and mRNA expression of three cytosolic peptidases in the CRC and to correlate the obtained results with classic histopathological parameters for tumor prognosis and survival. METHODS: The activity and mRNA levels of puromycin-sensitive aminopeptidase (PSA), aminopeptidase B (APB) and pyroglutamyl-peptidase I (PGI) were measured by fluorimetric and quantitative RT-PCR methods in colorectal mucosa and tumor tissues and plasma samples from CRC patients (n=81). RESULTS: 1) PSA and APB activity was higher in adenomas and carcinomas than in the uninvolved mucosa. 2) mRNA levels of PSA and PGI was lower in tumors. 3) PGI activity in CRC tissue correlated negatively with histological grade, tumor size and 5-year overall survival of CRC patients. 4) Higher plasmatic APB activity was independently associated with better 5-year overall survival. CONCLUSIONS: Data suggest that cytosolic peptidases may be involved in colorectal carcinogenesis and point to the determination of this enzymes as a valuable method in the determination of CRC prognosis.

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 voltage-gated sodium channel nav1.8 is expressed in human sperm.

The role of Na(+) fluxes through voltage-gated sodium channels in the regulation of sperm cell function remains poorly understood. Previously, we reported that several genes encoding voltage-gated Na(+) channels were expressed in human testis and mature spermatozoa. In this study, we analyzed the presence and function of the TTX-resistant VGSC α subunit Nav1.8 in human capacitated sperm cells. Using an RT-PCR assay, we found that the mRNA of the gene SCN10A, that encode Na v1.8, was abundantly and specifically expressed in human testis and ejaculated spermatozoa. The Na v1.8 protein was detected in capacitated sperm cells using three different specific antibodies against this channel. Positive immunoreactivity was mainly located in the neck and the principal piece of the flagellum. The presence of Na v1.8 in sperm cells was confirmed by Western blot. Functional studies demonstrated that the increases in progressive motility produced by veratridine, a voltage-gated sodium channel activator, were reduced in sperm cells preincubated with TTX (10 µM), the Na v1.8 antagonist A-803467, or a specific Na v1.8 antibody. Veratridine elicited similar percentage increases in progressive motility in sperm cells maintained in Ca(2+)-containing or Ca(2+)-free solution and did not induce hyperactivation or the acrosome reaction. Veratridine caused a rise in sperm intracellular Na(+), [Na(+)]i, and the sustained phase of the response was inhibited in the presence of A-803467. These results verify that the Na(+) channel Na v1.8 is present in human sperm cells and demonstrate that this channel participates in the regulation of sperm function.

Clinical impact of aspartyl aminopeptidase expression and activity in colorectal cancer.

Aspartyl aminopeptidase (ASP; EC 3.4.11.21) is a widely distributed and abundant cytosolic enzyme that regulates bioactive peptides such as angiotensin II. It has been demonstrated that the expression and activity of this enzyme is modified in tissue and serum of patients with several types of cancer. However, the involvement of ASP in the neoplastic development and survival of patients with colorectal cancer (CRC) has not been analyzed to date. The activity and messenger RNA expression of ASP in tumor tissue (n = 71) and plasma (n = 40) of patients with CRC was analyzed prospectively using fluorometric and quantitative real-time polymerase chain reaction methods. Data obtained from tumor tissue were compared with those from the surrounding normal mucosa. Classic pathologic parameters (grade, stage, nodal invasion, distant metastases and perineural, lymphatic, and vascular invasion) were stratified following ASP data and analyzed for 5-year survival. ASP was upregulated in CRC tissues, and greater activity correlated significantly with the absence of lymph node metastases and with better overall survival. Inversely, greater plasmatic ASP activity was associated with worse overall and disease-free survival. Data suggest that ASP is involved in colorectal neoplasia and point to this enzyme as a potential useful diagnostic tool in clinical practice.

Differentially regulated expression of neurokinin B (NKB)/NK3 receptor system in uterine leiomyomata.

STUDY QUESTION: Are the vasoactive peptide neurokinin B (NKB) and its preferred NK3 receptor (NK3R) differentially expressed in leiomyomas compared with normal myometrium? SUMMARY ANSWER: In leiomyomas, NKB is up-regulated and delocalized, while its preferred NK3R is also differentially regulated. WHAT IS KNOWN ALREADY: The expression of NKB/NK3R in the central nervous system is essential for proper function of the human reproductive axis. Additionally, this system is also widely expressed throughout the female genital tract. Leiomyomas impair fertility and are a major source of abnormal uterine bleeding. The aberrant synthesis of local factors can contribute to the pathological symptoms observed in women with leiomyomata. NKB could be one of these factors, since a vasoactive role of this peptide at a peripheral level has been observed in different systems and species, including humans. NK3R is strongly regulated by estrogens and its activation leads to nuclear translocation affecting chromatin structure and gene expression. STUDY DESIGN, SIZE, DURATION: Samples were obtained between 2006 and 2012 from 28 women of reproductive age at different stages of the menstrual cycle by hysterectomy. Leiomyomas and matched macroscopically normal myometrium from each woman were analysed in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: RT-PCR, quantitative real time, immunohistochemistry and in situ hybridization were used to investigate the pattern of expression of NKB/NK3R in tissue samples. MAIN RESULTS AND THE ROLE OF CHANCE: Expression of the gene encoding NKB (TAC3) was up-regulated 20-fold in leiomyomas, compared with matched myometrium (P = 0.0008). In tumour tissue, not only connective cells, but also myometrial, endothelial and vascular smooth muscle cells express TAC3 mRNA. Immunoreactivity to NKB was preferentially located in the smooth muscle cell nuclei from normal myometrium in the secretory phase, unlike matched leiomyoma, which showed a predominant cytoplasmic expression pattern. In the normal myometrium, TACR3 mRNA showed variable expression throughout the menstrual phases, with samples showing strong, reduced or no amplification. In leiomyoma, TACR3 was significantly up-regulated compared with matched myometrium (P = 0.0349). LIMITATIONS, REASONS FOR CAUTION: This study is descriptive and although we observed clear differential regulation of the NKB/NK3R system at mRNA and immunohistochemical staining levels in leiomyoma, future functional studies are needed to determine the precise role of NKB in the myometrium in normal and pathological conditions. In addition, further analysis (e.g. in cell culture models) will be required to determine the role of NKB in the nucleus of normal smooth muscle cells, whether nuclear translocation is mediated by NK3R and the consequences of the cytoplasmic expression of NKB in tumour cells. WIDER IMPLICATIONS OF THE FINDINGS: The NKB/NK3R system dysregulation observed in leiomyoma may contribute to the pathological symptoms observed in women with leiomyomata.

Cannabinoid CB1 receptor is expressed in chromophobe renal cell carcinoma and renal oncocytoma.

OBJECTIVE: To analyze the mRNA and protein expression of cannabinoid receptors CB1 and CB2 in chromophobe renal cell carcinoma (ChRCC) and renal oncocytoma (RO). DESIGN AND METHODS: Fresh and formalin-fixed tissue samples of ChRCC and RO were analyzed by using real-time quantitative RT-PCR and immunohistochemical techniques (n=40). RESULTS: Quantitative RT-PCR analysis showed that CB1 mRNA was underexpressed by 12-fold in ChRCC and had a variable expression in RO. CB1 protein showed intense positive immunostaining in both neoplasms. Both CB2 mRNA and protein were not expressed in tumor and non tumor renal tissue. CONCLUSION: This distinct immunoprofile may eventually be used as an additional tool with practical interest in the differential diagnosis of renal tumors.

Autocrine regulation of human sperm motility by the met-enkephalin opioid peptide.

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.

Analysis of the expression of neurokinin B, kisspeptin, and their cognate receptors NK3R and KISS1R in the human female genital tract.

OBJECTIVE: To investigate the presence of neurokinin B (NKB)/NK(3) receptor (NK(3)R) and kisspeptin/KISS1 receptor (KISS1R) messenger RNA (mRNA) and proteins throughout the human female genital tract. DESIGN: In vitro study. SETTING: Academic research laboratories and academic hospitals. PATIENT(S): Fifteen reproductive-age women and 16 postmenopausal women provided fresh samples of uterus, ovary, or oviduct, and 12 women provided archival samples of endometrium or oviduct. INTERVENTION(S): Fresh and archival samples of uterus, ovary, and oviduct obtained from reproductive-age and postmenopausal women. MAIN OUTCOME MEASURE(S): Results of reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry to investigate the pattern of expression of NKB/NK(3)R and kisspeptin/KISS1R in target tissues. RESULT(S): Expression of the genes encoding NKB (TAC3) and NK(3)R (TACR3), and kisspeptin (KISS1) and its receptor (KISS1R) was found in the uterus, ovary, and oviduct. Both NKB and NK(3)R immunoreactivity was detected in the endometrium, the oviduct, and the ovary, with marked expression in endometrial and oviductal epithelial cells, where intense coexpression of kisspeptin and KISS1R was also detected. Positive staining for NKB and NK(3)R was found in the myometrium where, in contrast, kisspeptin and KISS1R were absent. CONCLUSION(S): NKB/NK(3)R and kisspeptin/KISS1R are present in female peripheral reproductive tissues with colocalization of both systems in some non-neuronal cell populations of the human female genital tract. Our findings are compatible with a potential modulatory role of NKB and kisspeptin at peripheral reproductive tissues.

Airway response to acute mechanical stress in a human bronchial model of stretch.

INTRODUCTION: Lung inflation may have deleterious effects on the alveoli during mechanical ventilation. However, the consequences of stretch during excessive lung inflation on basal tone and responsiveness of human bronchi are unknown. This study was undertaken to devise an experimental model of acute mechanical stretch in isolated human bronchi and to investigate its effect on airway tone and responsiveness. METHODS: Bronchi were removed from 48 thoracic surgery patients. After preparation and equilibration in an organ bath, bronchial rings were stretched for 5 min using a force (2.5 × basal tone) that corresponded to airway-inflation pressure > 30 cm H2O. The consequences of stretch were examined by using functional experiments, analysis of organ-bath fluid, and ribonucleic acid (RNA) isolation from tissue samples. RESULTS: Following removal of the applied force the airways immediately developed an increase in basal tone (P < 0.0001 vs. paired controls) that was sustained and it did so without significantly increasing responsiveness to acetylcholine. The spontaneous tone was abolished with a Rho-kinase inhibitor and epithelium removal, a leukotriene antagonist or nitric oxide synthase inhibitors reduced it, whereas indomethacin, sensory nerve inhibitors or antagonists for muscarinic, endothelin and histamine receptors had no effect. Stretch enhanced leukotriene-E4 production during the immediate spontaneous contraction of human bronchi (P < 0.05). Moreover, stretch up-regulated the early mRNA expression of genes involved in wingless-type mouse mammary tumor virus integration-site family (WNT)-signaling and Rho-kinase pathways. CONCLUSIONS: Stretching human bronchi for only 5 min induces epithelial leukotriene release via nitric oxide synthase activation and provokes a myogenic response dependent on Rho-kinase and WNT-signaling pathways. From a clinical perspective, these findings highlight the response of human airway to acute mechanical stress during excessive pulmonary inflation.

Smooth muscle neurokinin-2 receptors mediate contraction in human saphenous veins.

Substance P (SP) and neurokinin A (NKA) are members of the tachykinin peptides family. SP causes endothelial-dependant relaxation but the contractile response to tachykinins in human vessels remains unknown. The objective was to assess the expression and the contractile effects of tachykinins and their receptors in human saphenous veins (SV). Tachykinin expression was assessed with RT-PCR, tachykinin receptors expression with RT-PCR and immunohistochemistry, and functional studies were performed in organ bath. Transcripts of all tachykinin and tachykinin receptor genes were found in SV. NK(1)-receptors were localized in both endothelial and smooth muscle layers of undistended SV, whereas they were only found in smooth muscle layers of varicose SV. The expression of NK(2)- and NK(3)-receptors was limited to the smooth muscle in both preparations. NKA induced concentration-dependent contractions in about half the varicose SV. Maximum effect reached 27.6±5.5% of 90 mM KCl and the pD(2) value was 7.3±0.2. NKA also induced the contraction of undistended veins from bypass and did not cause the relaxation of these vessels after precontraction. The NK(2)-receptor antagonist SR48968 abolished the contraction induced by NKA, and a rapid desensitization of the NK(2)-receptor was observed. In varicose SV, the agonists specific to NK(1)- or NK(3)-receptors did not cause either contraction or relaxation. The stimulation of smooth muscle NK(2)-receptors can induce the contraction of human SV. As SV is richly innervated, tachykinins may participate in the regulation of the tone in this portion of the low pressure vascular system.

Control of APN/CD13 and NEP/CD10 on sperm motility.

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.

Cannabinoid CB1 receptor is downregulated in clear cell renal cell carcinoma.

Several studies in cell cultures and in animal models have demonstrated that cannabinoids have important antitumoral properties. Because many of these effects are mediated through cannabinoid (CB) receptors CB1 and CB2, the study of their expression in human neoplasms has become of great interest in recent years. Fresh and formalin-fixed tissue samples of 20 consecutive clear cell renal cell carcinomas (CCRCCs) were collected prospectively and analyzed for the expression of both CB receptors by using RT-PCR, Western blot (WB), and immunohistochemical techniques. RT-PCR assays demonstrated the expression of mRNA encoding the CB1 in tumor tissue and in adjacent non-neoplastic kidney. Conversely, WB and IHC revealed a marked downregulation of CB1 protein in tumor tissue; CB2 was not expressed. The obtained data suggest a possible implication of the endocannabinoid system in renal carcinogenesis. A posttranscriptional downregulation of CB1 and the absence of expression of CB2 characterize CCRCC.

Angiotensin-converting enzymes (ACE and ACE2) are downregulated in renal tumors.

The angiotensin-converting enzymes (ACE and ACE2) are highly expressed in renal tubules and play an important role in the regulation of renal function by the intrarenal renin-angiotensin system (iRAS). Dysregulation of these cell-surface peptidases has been associated with renal injury. Most of these studies, however, have focused on non-neoplastic kidney diseases. In the present study, ACE and ACE2 activity and protein and mRNA expression were analysed in a subset of clear-cell (CCRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Enzyme activity was measured by spectrofluorometric (ACE2) and spectrophotometric assays (ACE), and protein and mRNA expression were determined by immunohistochemistry and qRT-PCR assays, respectively. The enzyme activities and immunohistochemistry showed that both enzymes are mainly downregulated in these neoplasms. qRT-PCR studies in CCRCC showed no positive correlation between ACE and ACE2 activity/protein expression and mRNA levels, whereas downregulation of ACE2 mRNA levels was observed in tumors from the distal nephron (ChRCC and RO). These findings suggest a metabolic imbalance in iRAS and a role of this system in renal neoplastic diseases, and point to ACE and ACE2 as potential prognostic/diagnostic markers.