CatSper1 Expression Research Articles

Down Regulation of Catsper1 Expression by Calmodulin Inhibitor (Calmidazolium): Possible Implications for Fertility.

The CatSper channel localizes exclusively in the flagella of sperm cells. The Catsper1 protein, together with three pore units, is essential for the CatSper Channel formation, which produces flagellum hyperactivation and confers sperm fertility. Catsper1 expression is dependent on Sox transcription factors, which can recognize in vitro at least three Sox binding sites on the promoter. Sox transcription factors have calmodulin-binding domains for nuclear importation. Calmodulin (CaM) is affected by the specific inhibitor calmidazolium (CMZ), which prevents the nuclear transport of Sox factors. In this work, we assess the regulation of the Catsper1 promoter in vivo by Sox factors in the murine testis and evaluate the effects of the inhibitor calmidazolium on the expression of the Casper genes, and the motility and fertility of the sperm. Catsper1 promoter has significant transcriptional activity in vivo; on the contrary, three Sox site mutants in the Catsper1 promoter reduced transcriptional activity in the testis. CaM inhibition affects Sox factor nuclear transport and has notable implications in the expression and production of Catsper1, as well as in the motility and fertility capability of sperm. The molecular mechanism described here might conform to the basis of a male contraceptive strategy acting at the transcriptional level by affecting the production of the CatSper channel, a fundamental piece of male fertility.

SOX11-dependent CATSPER1 expression controls colon cancer cell growth through regulation the PI3K/AKT signaling pathway.

Colorectal cancer (CRC) is one of the most common malignant tumors and the fourth leading cause of cancer death worldwide. Constitutive activation of the PI3K/AKT signaling pathway is a hallmark of colon tumor growth. CATSPER1 gene encodes a pore-forming and pH-sensing subunit of the CatSper Ca2+-permeable channel, a sperm-specific calcium channel essential for hyperactivated motility and male fertility. However, the function of CATSPER1 outside the male reproductive system is unclear. This study was designed to explore whether CatSper exerted its functional role in the progress of CRC, and investigate the possible mechanisms. Microarray data (GSE146587) from 6 patients diagnosed with stage III CRC post-surgery was analyzed by Limma R package. The Kaplan Meier plotter (KM plotter) database was used to assess the relevance of CATSPER1 mRNA expression to the overall survival (OS) rates in CRC. Western blot, real-time PCR and luciferase reporter assays were used to determine the SOX11-CATSPER1 axis in CRC cells. Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing was used to generate CATSPER1 knockout (KO) CRC cells. The proliferation of CRC cells was determined by BrdU incorporation and colony formation assays. The effect of CATSPER1 on CRC tumor growth in vivo was investigated in a mice tumor xenograft model. Here, we show that CATSPER1 expression was significantly up-regulated in CRC and elevated CATSPER1 was associated with poor overall survival (OS). Moreover, the transcription factor SOX11 (SRY-related high-mobility-group (HMG) box 11) activated CATSPER1 transcription in CRC cells. Functionally, we showed that CATSPER1 promoted CRC cells proliferation both in vitro and in vivo. At the molecular level, we demonstrated that CATSPER1 might maintain CRC malignant process partly through the activation of the PI3K/AKT signaling pathway. Increased CATSPER1 expression facilitates CRC cells proliferation, suggesting that targeting CATSPER1 might represent a promising strategy for colon cancer treatment.

Regulation of CATSPER1 expression by the testis-determining gene SRY.

CATSPER1 gene encodes a pore-forming and pH-sensing subunit of the CatSper Ca2+- permeable channel, a protein in the flagellum essential for sperm hyperactivation. Previous studies have shown that the murine Catsper1 gene promoter is regulated by different Sox proteins. Likewise, it is acknowledged that the human CATSPER1 gene promoter sequence is enriched in potential interaction sites for the sex-determining region Y gene (SRY), which suggest a novel regulatory transcriptional mechanism for CatSper1 channel expression. Therefore, in this work, we sought to determine whether the human CATSPER1 gene expression is regulated by the SRY transcription factor. To this end, a series of deletions and mutations were introduced in the wild- type CATSPER1 gene promoter to eliminate the SRY sites, and the different constructs were tested for their ability to activate transcription in human embryonic kidney and murine spermatogonial germ cell lines (HEK-293 and GC1-spg, respectively) using luciferase assays. In addition, by using a strategy that combines electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) we investigated whether the CATSPER1 gene expression is regulated by the SRY transcription factor both in vitro and in vivo. Our results show that the transcriptional factor SRY specifically binds to different sites in the promoter sequence and has the ability to control CATSPER1 gene transcription.

Human CATSPER1 Promoter Is Regulated by CREB1 and CREMτ Transcriptional Factors In Vitro

The CATSPER1 gene encodes a CATSPER channel protein that selectively permeates Ca2+ ions, and CATSPER expression in sperm is essential for flagellum hyperactivation and, thus, male fertility. Little is known regarding the transcriptional regulation of CATSPER1, but previous studies have performed in silico analyses of transcription factor binding sites, including three CRE sites designated 0-2, in which CRE0 is located near the transcription start site. We investigate if overexpression of CREB-A and CREMτ transcription factors might regulate CATSPER1 expression. In this study, the transcriptional regulation of the CATSPER1 gene by CREB-A and CREMτ transcriptions factors was determined by dual-luciferase assays in HEK293 and GC1-spg cells, and important CRE sites were mutated and analyzed for transcriptional regulation. The deletion of the CRE1 site dramatically increased the transcriptional activity of the CATSPER1 promoter in HEK293 and GC1-spg cells. In HEK293cells, the CREB-A transcription factor positively regulated CATSPER1 gene expression, while the presence of CREB-A and CREMτ factors synergistically enhanced promoter activity in these cells. In contrast, deletion of CRE0 prevented any transcriptional activity of the CATSPER1 promoter in GC1-spg spermatogonial cells, but expression of either CREB-A or CREMτ restored such transcriptional activity. The human CATSPER1 promoter is positively regulated invitro by CREB-A in HEK293 and GC1-spg cells. Both lines showed differential transcriptional regulation, which was defined by the factors and coactivators present in each cell line as well as the context in which the CRE sites were found in the promoter.

Quantification of CatSper1 expression in human spermatozoa and relation to functional parameters.

Is CatSper1 expression in human spermatozoa related to semen parameter values and sperm functions? CatSper1 expression is positively related to progressive and hyperactivated (HA) motility, [Ca(2+)]i responsiveness to progesterone but not the acrosome reaction (AR). The role of cationic channel of sperm (CatSper) in sperm functions is clear in animal models but less defined in human sperm cells. Current knowledge is mostly based on low specificity CatSper inhibitors showing agonistic and toxic effects on human spermatozoa and is thus of little help in clarifying the role of the CatSper channel in human sperm functions. CatSper1 protein expression was evaluated in 115 men undergoing semen analysis for couple infertility. CatSper1 expression was related to routine semen parameters, motility kinematic parameters and basal and progesterone-stimulated [Ca(2+)]i and the AR. CatSper1 expression was evaluated (n = 85 normozoospermic, n = 30 asthenozoospermic patients) by immunofluorescence coupled to flow cytometry leading to quantitative measurement of the percentage of ejaculated sperm cells expressing the protein. Semen analysis was evaluated according to World Health Organization guidelines. Kinematic parameters were evaluated by a computer-aided sperm analysis system. [Ca(2+)]i was measured by a spectrofluorimetric method in fura-2-loaded spermatozoa. The AR was evaluated in live sperm cells by fluorescent-labeled lectin. CatSper1 protein expression in spermatozoa was reduced in asthenozoospermic men (mean ± SD: 53.0 ± 15.5%, n = 30 versus 67.9 ± 17.1% in normozoospermic, n = 85, P < 0.01) and was significantly correlated with progressive (r = 0.36, P < 0.001), total (r = 0.35, P < 0.001) and HA (r = 0.41, P < 0.005) motility. In addition to a higher percentage of spermatozoa not expressing CatSper1, asthenozoospermic men showed a large number of spermatozoa with immunofluorescent signal localized outside the principal piece compared with those in normozoospermia. A significant positive correlation was found between CatSper1 protein expression and the increase of [Ca(2+)]i in response to progesterone (r = 0.36, P < 0.05, n = 40) but not with basal [Ca(2+)]i. No correlation was found with the AR, either basal or in response to progesterone. The study is partly descriptive. Furthermore, we cannot rule out the possibility that some round cells remain after a single round of 40% density gradient centrifugation or that this step may have removed some defective or slow swimming sperm, and therefore this preparation may not be representative of the entire sperm sample. Although our data suggest that CatSper1 may be a useful marker for infertility, and a possible contraceptive target, any clinical application is limited without further research. Our results demonstrate an association of CatSper1 expression with human sperm progressive and HA motility and provide preliminary evidence that lack of expression or mislocalization of CatSper1 in spermatozoa may be involved in the pathogenesis of asthenozoospermia. However, mechanistic studies are needed to confirm that the correlations between CatSper1 expression and sperm functions are causative. Supported by grants from Ministry of University and Scientific Research (PRIN project to E.B. and FIRB project to S.M.) and by Regione Toscana (to G.F.). L.T. was recipient of a grant from Accademia dei Lincei (Rome, Italy). The authors have no conflicts of interest to declare.

OR10-4 * CORRELATION BETWEEN EXPRESSION OF CATSPER FAMILY AND SPERM PROFILES IN THE ADULT MOUSE TESTIS FOLLOWING IRANIAN KERACK ABUSE

Introduction. The aim was to investigate the effects of an Iranian illicit drug, Kerack, on sperm parameters and CatSper genes expression of mice. Method. 25 male mice were divided into five groups consisting of control, sham and three experimental groups. All animal in experimental groups were addicted to Kerack for 7 days. These experimental groups include experimental I which was given Kerack at a dose of 5 mg/kg, experimental II, 35 mg/kg and experimental III, 70 mg/kg, intraperitoneally twice a day for a period of 35 days. Spermatozoas were analyzed for count, motility, morphology (normal/abnormal) and viability. Testes were subjected to total mRNA extraction for using in qPCR. Result. Sperm parameters were decreased in experimental groups vs. control groups (p < 0.05). qPCR results showed that CatSper 2, 3, 4 genes expressions were reduced with 35 and 70 mg/kg injected Kerack when compared with control testes (p ≤ 0.05). However, CatSper1 expression was only reduced with high dose injected Kerack in comparison to control testes (p ≤ 0.05). Conclusion. This study shows the effects of Kerack used in Iran particularly on sperm morphology in adult mouse. It could down-regulate the expression of CatSper genes, resulting in depression of sperm motility.

The transcription factors Sox5 and Sox9 regulate Catsper1 gene expression

Catsper is a Ca2+permeable channel required for sperm hyperactivation. In spite of its central role in male fertility, the transcriptional mechanisms that regulate Catsper1 expression are ill defined. In this work, we describe the identification and characterization of important regulatory elements in the murine Catsper1 gene proximal promoter. Four transcription start sites and three functional Sox-binding sites were identified in the Catsper1 promoter. Interestingly, transcription factors Sox5 and Sox9 caused a significant increase in transactivation of the Catsper1 promoter in heterologous systems, and chromatin immunoprecipitation assays showed that both transcription factors interact with the Catsper1 promoter in vivo. These results provide new insights into the molecular mechanisms that control Catsper channel expression.

Correlation between expression of CatSper family and sperm profiles in the adult mouse testis following Iranian Kerack abuse

Illicit drug use can be an important cause of male infertility. The aim of this study was to investigate the effects of an Iranian illicit drug, Kerack, on sperm parameters, testicular structure and CatSper genes expression of mice. In this study, 25 male mice were divided into five groups consisting of control, sham and three experimental groups. All animal in experimental groups were addicted to Kerack for 7 days. These experimental groups include experimental I which was given Kerack at a dose of 5 mg/kg, experimental II, 35 mg/kg and experimental III, 70 mg/kg, intraperitoneally twice a day for a period of 35 days. Mice were then sacrificed and spermatozoas were removed from cauda epididymis and analyzed for count, motility, morphology (normal/abnormal) and viability. Right testes were removed, weighed and processed for light microscopic studies whereas left testes removed were subjected to total mRNA extraction for using in real-time PCR (RT-PCR). The results were analyzed by performing anova (Tukey's tests) and Pearson correlation coefficient. Sperm parameters and seminiferous epithelium thickness were decreased in experimental groups (dose-dependently) vs. sham and control groups (p < 0.05). RT-PCR results showed that CatSper 2, 3, 4 genes expressions were reduced with 35 and 70 mg/kg injected Kerack when compared with control testes (p ≤ 0.05). However, CatSper1 expression was only reduced with high dose injected Kerack (70 mg/kg) in comparison to control testes (p ≤ 0.05). This study shows the deleterious effects of Kerack used in Iran on testis structure and sperm parameters in general, and particularly sperm morphology in adult mouse. It could down-regulate the expression of CatSper genes, resulting in depression of sperm motility.

The CatSper calcium channel in human sperm: relation with motility and involvement in progesterone-induced acrosome reaction

Does CatSper have a role in the achievement of human sperm motility and in the Progesterone (P)-induced acrosome reaction (AR)? CatSper1 expression is associated with human sperm progressive motility and the P-induced AR; it may have a role in the pathogenesis of asthenozoospermia. Knockout mice for any of the Catsper family genes fail to acquire hyperactivated motility and are infertile. CatSper channels mediate P-induced Ca(2+) influx in human sperm. The role of CatSper in human sperm hyperactivated/activated motility and in asthenospermia is less clear. A few men with CatSper mutations have been described but the phenotype regarding sperm motility has not been well established. The effects of two Catsper inhibitors, NNC55-0396 (NNC, 10 and 20 µM) and Mibefradil (Mib, 30 and 40 µM), were tested on human sperm motility parameters and the P-induced AR. Catsper1 protein expression was evaluated in unselected and swim-up selected sperm samples and in sperm from normo- and astheno-zoospermic subjects. Semen sample kinematic parameters were analysed by a CASA system. A fluorescent-labelled lectin was used to evaluate P-induced AR in live sperm by fluorescence microscopy. CatSper1 protein expression was determined by western blot analysis and by flow cytometry. Intracellular calcium concentrations ([Ca(2+)]i) were evaluated by a spectrofluorimetric method following sperm loading with the calcium-sensitive probe fura 2/AM. CatSper1 protein was localized in the tail of human sperm. CatSperI expression was higher in swim up selected than unselected sperm both when measured by western blot or by flow cytometry (52.7 ± 15.8% versus 27.2 ± 9.01%, n = 7, P < 0.01). Basal and P-stimulated [Ca(2+)]i were significantly higher in swim-up selected compared with unselected sperm. CatSper1 expression (western blot analysis) was found to be decreased in sperm from asthenozoospermic (n = 10) compared with those from normozoospermic (n = 9) men (intensity values relative to β-actin: 244.4 ± 69.3 versus 385.8 ± 139.5, P < 0.01). A positive correlation was found between CatSper1 protein expression and the percentage of sperm with progressive motility (n = 19, r = 0.59, P = 0.007). NNC (10 µM) and Mib (30 µM) significantly reduced the percentage of sperm with progressive motility and several kinematic parameters but did not affect the percentage of hyperactivated sperm. Their effects were the same whether they were added to swim-up selected and capacitated sperm or were added to the swim-up medium. Mib was found to have a slight but significant effect on sperm viability at both concentrations tested. P-stimulated AR was significantly reduced by both inhibitors (P < 0.05). Overall, our results indicate that, in human sperm, CatSper channel expression and function are associated with progressive motility and may be involved in the pathogenesis of asthenozoospermia. In general, studies evaluating the effect of inhibitors have the limitation of the specificity of the molecules. We show here that Mib may have toxic effect on human sperm. Although most of the parameters have been evaluated in live sperm, the toxic effect could have contributed to the observed decreases. More studies are necessary to evaluate further the role of CatSper1 in asthenozoospermia. In view of the involvement in P-induced AR and of the evidence of a role in the pathogenesis of astenozoospermia, CatSper channels may represent a promising target for the development of new drugs for the treatment of male infertility and for non-hormonal contraception. This work was supported by grants from Ministry of University and Scientific Research (Prin project to E.B. and FIRB project to S.M) and Regione Toscana (to G.F.). The authors have no conflicts of interest to declare.

Down-regulation of CatSper1 channel in epididymal spermatozoa contributes to the pathogenesis of asthenozoospermia, whereas up-regulation of the channel by Sheng-Jing-San treatment improves the sperm motility of asthenozoospermia in rats

To determine the expression of CatSper1 channel in epididymal spermatozoa in a rat model of asthenozoospermia, induced by cyclophosphamide (CP), and further examine the effects of soluble granules of Sheng-Jing-San (SJS), a traditional Chinese medicine recipe, on CatSper1 expression and sperm motility in the CP-induced asthenozoospermic rats. Placebo-controlled, randomized trial. Neuroscience Research Institute, Peking University, China. Sexually mature male Sprague-Dawley rats (n = 60). In the CP group, CP at the dose of 35 mg/kg intraperitoneally injected into rats once a day for 7 days; in the normal saline (NS) group, 0.9% saline solution was injected as control. Sperm motility and count were evaluated by computer-assisted sperm assay (CASA); protein and mRNA expression of CatSper1 channel in epididymal spermatozoa was determined by Western blotting and quantitative real-time RT-PCR, respectively. The rats were randomly divided into five groups with 12 rats in each group: CP, normal saline (NS), CP + SJS, CP + NS, and treatment naïve. In the CP + SJS group, after the last injection of CP, SJS at a dose of 30 mg/kg was intragastrically administrated to rats once a day for 14 days; in CP + NS group, saline solution instead of SJS was administrated as control. In the treatment naïve group, rats were normally fed for 21 days as controls. We found a statistically significant reduction of the CatSper1 channel, which is associated with an impairment of sperm motility in the epididymal spermatozoa of CP-induced asthenozoospermic rats. Soluble granules of SJS could dramatically restore the CP-induced down-regulation of CatSper1 in epididymal spermatozoa, which greatly improved the sperm motility in the asthenozoospermic rats. Down-regulation of the CatSper1 channel in epididymal spermatozoa likely contributes to the pathogenesis of asthenozoospermia, whereas up-regulation of the channel by SJS improves sperm motility and thus can be used as an effective therapeutic strategy for the treatment of male infertility diagnosed with asthenozoospermia.

A Chinese herbal decoction can increase the intracellular Ca2+ concentration and CatSper1 expression in mouse sperm tails

The aim of this study was to investigate the impact of a Chinese herbal decoction on the intracellular calcium (Ca2+) concentration of sperm and the expression of the cation channel1 of sperm (CatSper1), which is a calcium-channel protein specific to sperm tail, in a murine model of asthenospermia induced with cyclophosphamide. After 34days of intragastric administration of Chinese herb decoction to the murine model used, routine analyses of the mouse sperm were conducted, the intracellular Ca2+ concentration of the sperm tails was measured using flow cytometry, and the expression of CatSper1 protein was detected using reverse transcription‑polymerase chain reaction (RT-PCR). Sperm concentration, percentage of gradeA and B sperm (i.e., sperm activity) and percentage of gradeA, B and C sperm (i.e., overall sperm motility) of the model group mice (MG) were markedly lower compared to the control murine group (CG) (one-way ANOVA, P<0.05). Subsequent to treatment, sperm concentration, percentage of sperm activity and overall sperm motility of the large dose of herbal medicine group murine (LG) were markedly increased compared to MG mice (P<0.05). Intracellular Ca2+ concentration in MG mice was markedly lower compared to CG mice (P<0.05). However, following therapy, a significant increase was observed in the intracellular Ca2+ concentration in LG mice as compared to MG mice (P<0.05). In addition, the expression of CatSper1 in LG mice was significantly higher compared to MG mice (P<0.05), while no statistically significant difference was observed for the CG mice. Intraperitoneal injection of cyclophosphamide reduced sperm concentration, percentage of sperm activity and overall sperm motility, intracellular Ca2+ concentration and CatSper1 expression. Large doses of this Chinese herbal decoction increased sperm intracellular Ca2+ concentration, sperm concentration, and percentages of sperm activity and overall sperm motility by upregulating CatSper1 expression. The findings of this study have demonstrated a therapeutic effect of this decoction on asthenospermia.