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Capacitation involves tyrosine phosphorylation (TP) as a key marker. Lifestyle-related factors, such as obesity and smoking, are recognized for their adverse effects on semen quality and male fertility, yet the underlying mechanisms, including their potential impact on TP, remain unclear. Moreover, the effect of sperm cryopreservation on TP at the human sperm population level is unexplored. Flow cytometry analysis of global TP was performed on pre-capacitated, post-capacitated and 1- and 3-hours' incubated fresh and frozen-thawed samples from sperm donors ( n = 40). Neither being overweight nor smoking (or both) significantly affected the percentage of sperm showing TP. However, elevated BMI and smoking intensity correlated with heightened basal TP levels (r = 0.226, p = 0.003) and heightened increase in TP after 3 h of incubation (r = 0.185, p = 0.017), respectively. Cryopreservation resulted in increased global TP levels after capacitation but not immediately after thawing. Nonetheless, most donors' thawed samples showed increased TP levels before and after capacitation as well as after incubation. Additionally, phosphorylation patterns in fresh and frozen-thawed samples were similar, indicating consistent sample response to capacitation stimuli despite differences in TP levels. Overall, this study sheds light on the potential impacts of lifestyle factors and cryopreservation on the dynamics of global TP levels during capacitation.

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 Ca 2+ 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.

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., (© 2022. Society for Reproductive Investigation.)

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., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Infertility has become a global health issue, with approximately 50% of infertility cases generated by disorders in male reproduction. Spermatozoa are conveyed towards female genital tracts in a safe surrounding provided by the seminal plasma. Interestingly, this dynamically changing medium is a rich source of proteins, essential not only for sperm transport, but also for its protection and maturation. Most of the seminal proteins are acquired by spermatozoa in transit through exosomes (epididymosomes and prostasomes). The high number of seminal proteins, the increasing knowledge of their origins and biological functions and their differential expression in the case of azoospermia, asthenozoospermia, oligozoospermia and teratozoospermia or other conditions of male infertility have allowed the identification of a wide variety of biomarker candidates and their involvement in biological pathways, thus to strongly suggest that the proteomic landscape of seminal plasma may be a potential indicator of sperm dysfunction. This review summarizes the current knowledge in seminal plasma proteomics and its potentiality as a diagnostic tool in different degrees of male infertility.

G-protein coupled receptors (GPCRs) belong to the seven transmembrane receptor superfamily that transduce signals via G proteins in response to external stimuli to initiate different intracellular signaling pathways which culminate in specific cellular responses. The expression of diverse GPCRs at the plasma membrane of human spermatozoa suggests their involvement in the regulation of sperm fertility. However, the signaling events downstream of many GPCRs in spermatozoa remain uncharacterized. Here, we selected the kappa-opioid receptor (KOR) as a study model and applied phosphoproteomic approach based on TMT labeling and LC-MS/MS analyses. Quantitative coverage of more than 5000 proteins with over 3500 phosphorylation sites revealed changes in the phosphorylation levels of sperm-specific proteins involved in the regulation of the sperm fertility in response to a specific agonist of KOR, U50488H. Further functional studies indicate that KOR could be involved in the regulation of sperm fertile capacity by modulation of calcium channels. Our findings suggest that human spermatozoa possess unique features in the molecular mechanisms downstream of GPCRs which could be key regulators of sperm fertility and improved knowledge of these specific processes may contribute to the development of useful biochemical tools for diagnosis and treatment of male infertility., (© 2019 Urizar-Arenaza et al.)

Purpose: The neurokinin B (NKB)/NK 3 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.

The aim of this study was to evaluate the possible beneficial effects of diet supplementation with a highly concentrated and purified docosahexaenoic acid (DHA) formula on human sperm function. We performed a prospective, randomized, double blind, placebo-controlled intervention study. One-hundred eighty human semen samples from sixty infertile patients recruited in a private assisted reproduction center were included. All samples were examined according to World Health Organization guidelines. We analyzed macroscopic and microscopic sperm parameters, oxidative stress, apoptosis, lipid peroxidation, mitochondrial membrane potential and DNA fragmentation before and after supplementation with different DHA daily doses (0.5, 1 and 2 g) or placebo for 1 and 3 months. No differences were found in traditional sperm parameters except for progressive sperm motility, with a significant increase after DHA ingestion after the first month with 1 or 2 g doses and after 3 months with 0.5 g of DHA. This effect was more evident in asthenozoospermic patients. No differences were found in any molecular semen parameter except oxidative stress, in which a slight benefit was observed after DHA treatment. In conclusion, this study support previous indications that highlight the importance of DHA supplementation as a means of improving sperm quality in asthenozoospermic men., (Copyright © 2018 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Objective: To explore the relationship between iron and infertility by investigating iron-related gene expression in granulosa and uterine cervical cells., Design: Case-control study., Setting: Two tertiary hospitals., Patient(s): Two independent cohorts of fertile (n = 18 and n = 17) and infertile (n = 31 and n = 35) women., Intervention(s): In vitro fertilization., Main Outcome Measure(s): Gene expression levels of ferritin light chain (FTL), ferritin heavy chain (FTH), transferrin receptor (TFRC), and ferroportin (SLC40A1) mRNA were analyzed in granulosa and cervical cells., Result(s): In the first cohort, fertile and infertile women were similar in body mass index. Ferroportin mRNA levels were decreased in granulosa cells from infertile women in parallel with increased serum hepcidin levels. A positive association between ferroportin and TFRC mRNA, a gene associated with intracellular iron deficiency, was observed only in granulosa cells from fertile women. The major findings were replicated in a second independent cohort., Conclusion(s): Ferroportin mRNAs and circulating hepcidin identify a subset of infertile women and may constitute a target for therapy., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To study the expression levels of tachykinins and tachykinin receptors in uterine leiomyomas and matched myometrium., Design: Laboratory study., Setting: University research laboratories and academic hospital., Patient(s): Women undergoing hysterectomy for symptomatic leiomyomas., Intervention(s): Quantitative polymerase chain reaction, immunohistochemistry and Western blot., Main Outcome Measure(s): Expression and tissue immunostaining of substance P, neurokinin A, hemokinin-1, neurokinin 1 receptor full-length (NK1R-Fl) and truncated (NK1R-Tr) isoforms, and neurokinin 2 receptor (NK2R) in paired samples of leiomyoma and adjacent normal myometrium., Result(s): TAC1 messenger RNA (mRNA) was significantly up-regulated in leiomyomas, whereas intense immunoreaction for the three peptides was particularly abundant in connective tissue cells. Differential regulation of TACR1 mRNA was observed, and at the protein level there was a significant increased expression of NK1R short isoform (NK1R-Tr). TACR2 mRNA was significantly up-regulated in leiomyomas, although levels of NK2R protein were similar in normal and tumor cells., Conclusion(s): These and our previous data demonstrate that the whole tachykinin system is differentially regulated in leiomyomas. The increased expression of NK1R-Tr might stimulate leiomyoma growth in a similar way to that observed in other steroid-dependent tumors., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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., (© 2016 by the Society for the Study of Reproduction, Inc.)

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., (© 2015 by the Society for the Study of Reproduction, Inc.)

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.

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.

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.)

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., (Copyright © 2011 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.

Since tachykinins appear to be involved in the pathogenesis of allergic asthma, we investigated a possible association between 28 single nucleotide polymorphisms of the tachykinin genes TAC1, TAC3 and TAC4, and asthma susceptibility. A case-control study was conducted on 102 patients and 100 healthy subjects from the Canary Islands (Spain). A significant association with asthma was observed for two SNPs: rs2291855 in the TAC3 gene conferring asthma protection (Odds ratio [OR]: 0.46; 95% Confidence Interval [CI]: 0.22-0.97; P=0.038), and rs4794068 in the TAC4 gene associated with an increased risk for asthma (OR: 1.94; 95% CI: 1.06-3.54; P=0.03). The present study represents a preliminary step in elucidating the association between tachykinin gene polymorphisms and asthma susceptibility., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: We examined the presence and function of tachykinins and the tachykinin-degrading enzymes neprilysin (NEP) and neprilysin-2 (NEP2) in human spermatozoa., Methods: Freshly ejaculated semen was collected from forty-eight normozoospermic human donors. We analyzed the expression of substance P, neurokinin A, neurokinin B, hemokinin-1, NEP and NEP2 in sperm cells by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot and immunocytochemistry assays and evaluated the effects of the neprilysin and neprilysin-2 inhibitor phosphoramidon on sperm motility in the absence and presence of tachykinin receptor-selective antagonists. Sperm motility was measured using WHO procedures or computer-assisted sperm analysis (CASA)., Results: The mRNAs of the genes that encode substance P/neurokinin A (TAC1), neurokinin B (TAC3), hemokinin-1 (TAC4), neprilysin (MME) and neprilysin-2 (MMEL1) were expressed in human sperm. Immunocytochemistry studies revealed that tachykinin and neprilysin proteins were present in spermatozoa and show specific and differential distributions. Phosphoramidon increased sperm progressive motility and its effects were reduced in the presence of the tachykinin receptor antagonists SR140333 (NK1 receptor-selective) and SR48968 (NK2 receptor-selective) but unmodified in the presence of SR142801 (NK3 receptor-selective)., Conclusion: These data show that tachykinins are present in human spermatozoa and participate in the regulation of sperm motility. Tachykinin activity is regulated, at least in part, by neprilysins.

Background: In the mouse uterus, pregnancy is accompanied by changes in tachykinin and tachykinin receptor gene expression and in the uterotonic effects of endogenous tachykinins. In this study we have investigated whether changes in tachykinin expression and responses are a result of changes in ovarian steroid levels., Methods: We quantified the mRNAs of tachykinins and tachykinin receptors in uteri from ovariectomized mice and studied their regulation in response to estrogen and progesterone using real-time quantitative RT-PCR. Early (3 h) and late (24 h) responses to estrogen were evaluated and the participation of the estrogen receptors (ER), ERalpha and ERbeta, was analyzed by treating mice with propylpyrazole triol, a selective ERalpha agonist, or diarylpropionitrile, a selective agonist of ERbeta., Results: All genes encoding tachykinins (Tac1, Tac2 and Tac4) and tachykinin receptors (Tacr1, Tacr2 and Tacr3) were expressed in uteri from ovariectomized mice. Estrogen increased Tac1 and Tacr1 mRNA after 3 h and decreased Tac1 and Tac4 expression after 24 h. Tac2 and Tacr3 mRNA levels were decreased by estrogen at both 3 and 24 h. Most effects of estrogen were also observed in animals treated with propylpyrazole triol. Progesterone treatment increased the levels of Tac2., Conclusion: These results show that the expression of tachykinins and their receptors in the mouse uterus is tightly and differentially regulated by ovarian steroids. Estrogen effects are mainly mediated by ERalpha supporting an essential role for this estrogen receptor in the regulation of the tachykinergic system in the mouse uterus.

Background: We have investigated the expression of voltage-gated sodium channels in human spermatozoa and characterized their role in sperm motility., Methods: Freshly ejaculated semen was collected from thirty normozoospermic human donors, with each donor supplying 2 different samples. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence techniques were used to detect the mRNAs and proteins of interest. Sperm motility was measured by a computer-assisted sperm analysis system (CASA). Cytosolic free calcium was determined by fluorimetry in cells loaded with the fluorescent calcium indicator Fura-2., Results: The mRNAs that encode the different Nav alpha subunits (Nav1.1-1.9) were all expressed in capacitated human spermatozoa. The mRNAs of the auxiliary subunits beta1, beta3 and beta4 were also present. Immunofluorescence studies showed that, with the exception of Nav1.1 and Nav1.3, the Nav channel proteins were present in sperm cells and show specific and different sites of localization. Veratridine, a voltage-gated sodium channel activator, caused time- and concentration-dependent increases in progressive sperm motility. In sperm suspensions loaded with Fura-2, veratridine did not modify intracellular free calcium levels., Conclusion: This research shows the presence of voltage-gated sodium channels in human sperm and supports a role for these channels in the regulation of mature sperm function.

The preprotachykinin gene Tac4 expressed in murine uterus and placenta is thought to encode a peptide RSRTRQFYGLM-NH(2), mouse hemokinin 1. We have examined the uterotonic effects of mouse hemokinin 1 and its N-terminally truncated analogue, mouse hemokinin 1(2-11) on mouse uterus. Mouse hemokinin 1(2-11) was equieffective with but slightly less potent than substance P in tissues from non-pregnant Swiss mice. On myometrium from Balb C mice primed with oestrogen the positions of concentration-response curves to substance P and the mouse hemokinins were similar to those of neurokinin A, but the maximum responses were lower. The tachykinin NK(1) receptor antagonist, 1-{2-(3, 4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl) piperidin-3-yl]ethyl}-4phenyl-1-azonia-bicyclo[2.2.2]octane (SR 140333), reduced the effects of the agonists in tissues from both groups of mice. In myometria from late pregnant (Days 17-18) Balb C mice the responses to mouse hemokinin 1(2-11) were less potent than in those from oestrogen-primed mice. Human hemokinin 1, the human orthologue of mouse hemokinin 1, was more effective than mouse hemokinin 1(2-11), while endokinin D was inactive. Mouse hemokinin 1 effects were blocked by SR 140333 alone and in combination with ((S)-N-methyl-N[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide (SR 48968) but not by SR 48968 alone. Thus the mouse hemokinins are tachykinin NK(1) receptor-preferring uterotonic agonists in non-pregnant mice but lack action at the myometrial tachykinin NK(2) receptors present in late pregnant mice.

We investigated the function and expression of voltage-gated Na(+) channels (VGSC) in the uteri of nonpregnant rats using organ bath techniques, intracellular [Ca(2+)] fluorescence measurements, and RT-PCR. In longitudinally arranged whole-tissue uterine strips, veratridine, a VGSC activator, caused the rapid appearance of phasic contractions of irregular frequency and amplitude. After 50-60 min in the continuous presence of veratridine, rhythmic contractions of very regular frequency and slightly increasing amplitude occurred and were sustained for up to 12 h. Both the early and late components of the contractile response to veratridine were inhibited in a concentration-dependent manner by tetrodotoxin (TTX). In small strips dissected from the uterine longitudinal smooth muscle layer and loaded with Fura-2, veratridine also caused rhythmic contractions, accompanied by transient increases in [Ca(2+)](i), which were abolished by treatment with 0.1 microM TTX. Using end-point and real-time quantitative RT-PCR, we detected the presence of the VGSC alpha subunits Scn2a1, Scn3a, Scn5a, and Scn8a in the cDNA from longitudinal muscle. The mRNAs of the auxiliary beta subunits Scbn1b, Scbn2b, Scbn4b, and traces of Scn3b were also present. These data show for the first time that Scn2a1, Scn3a, Scn5a, and Scn8a, as well as all VGSC beta subunits are expressed in the longitudinal smooth muscle layer of the rat myometrium. In addition, our data show that TTX-sensitive VGSC are able to mediate phasic contractions maintained over long periods of time in the uteri of nonpregnant rats.

Voltage-gated Na+ channels are composed of one alpha subunit and one or more auxiliary beta subunits. A reverse transcription-polymerase chain reaction assay was used to analyse the expression of the nine known alpha subunits (Na(v)1.1-Na(v)1.9) in 20 different human tissues. The mRNA expression of the currently known beta subunits (beta1, beta2, beta3 and beta4) was also assessed. The mRNAs of voltage-gated Na+ channel alpha and beta subunits were found in a wide variety of human tissues assayed and were present in neuronal and non-neuronal types of cells. These data suggest that, in addition to its well-established role in skeletal muscle, cardiac cells and neurons, voltage-gated Na+ channels might play important, still undetermined local roles in the regulation of cellular functions. These channels could emerge in the next future as potential, new therapeutic targets in the treatment of visceral diseases.

Tachykinins (TKs) are a family of peptides involved in the central and peripheral regulation of urogenital functions through the stimulation of TK NK1, NK2 and NK3 receptors. At the urinary system level, TKs locally stimulate smooth muscle tone, ureteric peristalsis and bladder contractions, initiate neurogenic inflammation and trigger local and spinal reflexes aimed to maintain organ functions in emergency conditions. At the genital level, TKs are involved in smooth muscle contraction, in inflammation and in the modulation of steroid secretion by the testes and ovaries. TKs produce vasodilatation of maternal and fetal placental vascular beds and appear to be involved in reproductive function, stress-induced abortion, and pre-eclampsia. The current data suggest that the genitourinary tract is a primary site of action of the tachykininergic system.

The aims of this study were to determine the cysteinyl leukotriene (CysLT) receptors expressed in the human saphenous vein, to examine contractile response to LTC4 and LTD4, to evaluate antagonist activity of montelukast, a specific CysLT1 receptor antagonist used in asthma, and to characterize the CysLT receptors involved in the contractile response. The analysis by reverse-transcriptase polymerase chain reaction indicated that CysLT1 and CysLT2 receptors are expressed by saphenous veins. In varicose vein rings, the potencies (pD2) of LTC4 and LTD4 were similar: 7.4 +/- 0.2 and 7.4 +/- 0.1, respectively. Pretreatment with acivicin, a gamma-glutamyl transpeptidase (gamma-GT) inhibitor, to prevent potential metabolism of LTC4 to LTD4, did not alter the response to LTC4. In nondistended vein rings from patients undergoing arterial bypass, the LTC4 pD2 was 7.8 +/- 0.1, and pretreatment with S-hexyl-GSH, a potent gamma-GT inhibitor, caused a fourfold rightward shift of the LTC4 concentration-response curve. In varicose and nondistended saphenous vein rings, montelukast (10(-8) and 10(-7) M) exerted a potent activity against LTD4 and LTC4, in the presence or absence of gamma-GT inhibitors. In varicose vein rings, the two active metabolites of montelukast also exerted antagonist activities with potencies similar to montelukast. BAY u9773 (CysLT2 agonist/dual CysLT1/CysLT2 antagonist) did not cause contraction and inhibited the LTC4- and LTD4-induced contractions. In conclusion, human saphenous veins express CysLT1 and CysLT2 receptors, but only CysLT1 receptors are implicated in the contraction.

The effects of neurokinins and neurokinin receptor selective agonists have been investigated on human intralobar pulmonary vessels. Substance P (SP) and [Sar(9) Met(O(2)) ]SP(11), a selective NK(1) receptor agonist, induced concentration-dependent relaxation of pulmonary vessels precontracted with phenylephrine. The mean negative log (M) EC (50) values for SP and [Sar (9) Met(O2))]SP(11) were 8.6 and 8.9, respectively, on arterial preparations and 8.9 and 8.6, respectively, on venous preparations. Relaxations to [Sar(9) Met(O(2) ) ]SP were abolished by the NK receptor antagonist SR140333. The relaxations to a second application of [Sar(9) Met(O (2)) ]SP were markedly reduced, suggesting a rapid desensitization of the NK(1) receptor. Such desensitization was not observed with acetylcholine. The selective NK receptor agonist, [Nle(10)]NKA, and the selective NK (3) receptor agonist, [MePhe(7)]NKB, caused neither contractions nor relaxations of pulmonary vessels. The NK(1) receptor-mediated relaxations were abolished by removing the endothelium or by a combination of -nitro-L-arginine and indomethacin, whereas each compound exerted a partial inhibitory effect. Similar results were observed with acetylcholine. Positive immunostaining for NK(1) receptors was only found in the endothelium. Reverse transcription-polymerase chain reaction detected messenger RNA for NK(1) receptors without any detection of messenger RNA for NK(2) or NK(3) receptors. In conclusion, human pulmonary arteries and veins express endothelial NK(1) receptors that mediate relaxation through a combination of cyclooxygenase and nitric oxide activities and are subjected to rapid tachyphylaxis.

The contractile effect of okadaic acid (OA), a highly selective inhibitor of protein serine/threonine phosphatases, was analyzed in the rat uterus during the estrous cycle and during the course of pregnancy. Contractile effects were related to circulating levels of estrogen and progesterone and to mRNA levels of myosin light chain kinase (MLCK) and of myosin light chain protein phosphatase catalytic (PP1-delta) and larger regulatory subunit (MYPT). Both in nonpregnant and pregnant uteri, OA (20 microM) induced a transient contraction, which after plateauing, slowly decreased. In the nonpregnant uterus, the amplitude of this contraction varied at different stages of the estrous cycle, being higher at proestrus and lower at diestrus. In the pregnant uterus, the contraction to OA increased significantly during the course of pregnancy, reaching a maximum in day 21 pregnant rats, and declined after delivery. Whatever the day of pregnancy, the amplitude of the contraction to OA was not significantly modified when obtained in Ca(2+)-free solution. The magnitude of the OA-induced contraction in spontaneously cycling and pregnant rats was positively correlated to the ratio of estrogen/progesterone serum levels. Reverse transcription-polymerase chain reaction assays on myometrial tissue demonstrated that mRNA expression of PP1-delta and MYPT was higher at early (day 3) than at late (day 21) pregnancy. MLCK mRNA levels were similar in day 3 and day 21 pregnant rats. These data suggest that changes in the expression and activity of myosin phosphatase may contribute to modulating the level of uterine contractile force during the estrous cycle, pregnancy, and labor.

The contractile effect of okadaic acid (OA) and its derivatives was investigated in the rat uterus. OA (20 microM) induced a transient contraction which, after plateauing, slowly decreased. The structurally related compound okadanol (20 microM) failed to induce any significant contraction. Conversely, the synthetic compound methyl okadaate (20 microM) and the naturally occurring ester 7'-hydroxy-4'-methyl-2'-methylen-hept-4'(E)-enyl okadaate (20 microM) were as active as the free acid. The OA-induced contraction was unaffected in the presence of neomycin (5 mM), mepacrine (30 microM), 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperaz ine (10 microM), calphostin C (3 microM) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (30 microM). The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (100 microM) did not modify the amplitude of the OA-induced contraction but significantly increased the rate of tension decay. The myosin light chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (1 mM) significantly reduced the peak amplitude of the contraction. Staurosporine (0.03-0.1 microM) did not modify the contractile component of the OA-induced response but inhibited the subsequent decrease in tension. In freshly dispersed myometral cells loaded with the fluorescent Ca++ indicator indo 1, OA did not produce any significant increase in [Ca++]i. OA (5- to 90-min contact) also failed to modify the intracellular levels of arachidonic acid, compared with basal values. These data suggest that in the rat uterus 1) the contractile effect of OA (20 microM) is specifically mediated by inhibition of protein phosphatases type 1 and/or 2A and is related to a direct interaction with the contractile machinery; 2) the decreasing phase of the OA-induced mechanical response could be mediated by a staurosporine-sensitive protein kinase different from protein kinase C.

The mechanism of relaxant activity of six benzylisoquinolines was examined in order to determine the minimal structural requirements that enable these compounds to have either a non-specific action like papaverine or an inhibitory activity on calcium entry via potential-operated channels. All the alkaloids tested totally or partially relaxed KCl-depolarized rat uterus and inhibited oxytocin-induced rhythmic contractions. Only glaucine and laudanosine inhibited K(+)-induced uterine contractions more than oxytocin-induced uterine contractions. In Ca(+)-free medium, sustained contractions induced by oxytocin or vanadate were relaxed by the alkaloids tested except for glaucine and laudanosine indicating no inhibitory effect on intracellular calcium release. Those alkaloids containing an unsaturated heterocyclic ring (papaverine, papaverinol, papaveraldine, N-methylpapaverine and dehydropapaverine) exhibited a more specific activity than those with a tetrahydroisoquinoline ring.

The effects of nifedipine, papaverine and four benzylisoquinoline alkaloids (cularine, cularidine, celtisine and isocrasifoline) were studied in isolated rat uterus in order to clarify the mechanism of their relaxant action. All the compounds tested completely relaxed KCl-induced contractions and totally or partially inhibited oxytocin-induced rhythmic contractions. Only papaverine acted intracellularly, promoting relaxation of contractile responses induced by oxytocin or vanadate in a Ca(2+)-free medium. In spite of the structural relationship between papaverine and the other alkaloids, the mechanism of their relaxant action is not the same. The activities of cularine derivatives and of isocrasifoline were similar to that of nifedipine.