3D in situ imaging of the female reproductive tract reveals molecular signatures of fertilizing spermatozoa in mice.

Out of millions of ejaculated sperm, a few reach the fertilization site in mammals. Flagellar Ca2+ signaling nanodomains, organized by multi-subunit CatSper calcium channel complexes, are pivotal for sperm migration in the female tract, implicating CatSper-dependent mechanisms in sperm selection. Here using biochemical and pharmacological studies, we demonstrate that CatSper1 is an O-linked glycosylated protein, undergoing capacitation-induced processing dependent on Ca2+ and phosphorylation cascades. CatSper1 processing correlates with protein tyrosine phosphorylation (pY) development in sperm cells capacitated in vitro and in vivo. Using 3D in situ molecular imaging and ANN-based automatic detection of sperm distributed along the cleared female tract, we demonstrate that spermatozoa past the utero-tubal junction possess the intact CatSper1 signals. Together, we reveal that fertilizing mouse spermatozoa in situ are characterized by intact CatSper channel, lack of pY, and reacted acrosomes. These findings provide molecular insight into sperm selection for successful fertilization in the female reproductive tract.

Ded L ，Hwang JY ，Miki K ，Shi HF ，Chung JJ ... -  《eLife》

Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa.

Zeng XH ，Navarro B ，Xia XM ，Clapham DE ，Lingle CJ ... -  《-》

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.

Tamburrino L ，Marchiani S ，Vicini E ，Muciaccia B ，Cambi M ，Pellegrini S ，Forti G ，Muratori M ，Baldi E ... -  《-》

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

Tamburrino L ，Marchiani S ，Minetti F ，Forti G ，Muratori M ，Baldi E ... -  《-》

CatSper channels are regulated by protein kinase A.

Mammalian sperm must undergo capacitation as a preparation for entering into hyperactivated motility, undergoing the acrosome reaction, and acquiring fertilizing ability. One of the initial capacitation events occurs when sperm encounter an elevated HCO3- concentration. This anion activates the atypical adenylyl cyclase Adcy10, increases intracellular cAMP, and stimulates protein kinase A (PKA). Moreover, an increase in intracellular Ca2+ concentration ([Ca2+] i ) is essential for sperm capacitation. Although a cross-talk between cAMP-dependent pathways and Ca2+ clearly plays an essential role in sperm capacitation, the connection between these signaling events is incompletely understood. Here, using three different approaches, we found that CatSper, the main sperm Ca2+ channel characterized to date, is up-regulated by a cAMP-dependent activation of PKA in mouse sperm. First, HCO3- and the PKA-activating permeable compound 8-Br-cAMP induced an increase in [Ca2+] i , which was blocked by the PKA peptide inhibitor PKI, and H89, another PKA inhibitor, also abrogated the 8-Br-cAMP response. Second, HCO3- increased the membrane depolarization induced upon divalent cation removal by promoting influx of monovalent cations through CatSper channels, which was inhibited by PKI, H89, and the CatSper blocker HC-056456. Third, electrophysiological patch clamp, whole-cell recordings revealed that CatSper activity is up-regulated by HCO3- and by direct cAMP injection through the patch-clamp pipette. The activation by HCO3- and cAMP was also blocked by PKI, H89, Rp-cAMPS, and HC-056456, and electrophysiological recordings in sperm from CatSper-KO mice confirmed CatSper's role in these activation modes. Our results strongly suggest that PKA-dependent phosphorylation regulates [Ca2+] i homeostasis by activating CatSper channel complexes.

Orta G ，de la Vega-Beltran JL ，Martín-Hidalgo D ，Santi CM ，Visconti PE ，Darszon A ... -  《-》