RESEARCH | RESEARCH ARTICLES surface-charge screening and inactivation of 18. B. Abbasi et al., J. Res. Med. Sci. 17,1161–1169 (2012). expert technical assistance and C. Cirelli, G. Tononi, W. Wang, the NALCN channel–dependent Na+-leak cur- 19. I. Slutsky et al., Neuron 65, 165–177 (2010). and C. Nicholson for comments on the manuscript. All authors – rent (20). High [Mg2+] in combination with 20. B. Lu et al., Neuron 68, 488 499 (2010). contributed to data collection, technical design, and writing. e 21. J. Schummers, H. Yu, M. Sur, Science 320, 1638–1643 (2008). hyperpolarization will reduce the likelihood of 22. H. Sontheimer, Glia 11, 156–172 (1994). SUPPLEMENTARY MATERIALS NMDA receptor activation during sleep, reducing 23. A. Torres et al., Sci. Signal. 5, ra8 (2012). the brain’s ability to undergo activity-dependent 24. A. S. Thrane et al., Proc. Natl. Acad. Sci. U.S.A. 109, www.sciencemag.org/content/352/6285/550/suppl/DC1 18974–18979 (2012). Materials and Methods changes in excitatory transmission (LTP). Ma- Figs. S1 to S5 + 2+ 2+ nipulation of [K ]e, [Ca ]e, and [Mg ]e in the ACKNOWLEDGMENTS Table S1 References (25–45) CSF bathing the brain drove behavioral shifts This study was supported by NIH (NS078167 and NS078304) between sleep and awake states (Fig. 5). Astro- and the Office of Naval Research/Department of the Navy. We 18 September 2015; accepted 24 March 2016 cytic Ca2+ signaling is also enhanced by lowering thank W. Song, R. Rasmussen, E. Nicholas, and W. Peng for 10.1126/science.aad4821 2+ 2+ of [Ca ]e and [Mg ]e (21–23) and is strongly inhibited by anesthesia (24).These observations suggest that regulation of extracellular ion ho- meostasis is sufficient to alter behavioral state REPRODUCTIVE BIOLOGY both locally and globally, providing a path for neuromodulators to exert consistent, stable shifts in neuronal and astrocytic activity across Unconventional endocannabinoid the brain. A myriad of electrophysiological slice studies have in the past taken advantage of minor mod- signaling governs sperm activation ifications in the ion composition of the bath solution to induce stable and highly reprodu- via the sex hormone progesterone cible changes in neural excitability. The observa- tions presented here suggest that the CNS itself Melissa R. Miller,1 Nadja Mannowetz,1 Anthony T. Iavarone,2 Rojin Safavi,1 uses the same trick to control state-dependent Elena O. Gracheva,3 James F. Smith,4 Rose Z. Hill,1 Diana M. Bautista,1 on June 15, 2016 changes in neuronal activity. One advantage of Yuriy Kirichok,5 Polina V. Lishko1* this “ionostatic control” of neural activity is to provide a backdrop for coordinating shifts in be- Steroids regulate cell proliferation, tissue development, and cell signaling via two havioral state through the widespread regulation pathways: a nuclear receptor mechanism and genome-independent signaling. of excitability without relying on complex recep- Sperm activation, egg maturation, and steroid-induced anesthesia are executed via the tor activation in diverse subclasses of neurons. latter pathway, the key components of which remain unknown. Here, we present The characteristic state-dependent pattern of characterization of the human sperm progesterone receptor that is conveyed by the EEG activity can, at least in part, be explained orphan enzyme a/b hydrolase domain–containing protein 2 (ABHD2). We show by differences in extracellular ion composition that ABHD2 is highly expressed in spermatozoa, binds progesterone, and acts as in sleep versus wakefulness. Our observations a progesterone-dependent lipid hydrolase by depleting the endocannabinoid add new and critical insight into understanding 2-arachidonoylglycerol (2AG) from plasma membrane. The 2AG inhibits the sperm what drives arousal, as well as the loss of con- calcium channel (CatSper), and its removal leads to calcium influx via CatSper sciousness during sleep and anesthesia. Future and ensures sperm activation. This study reveals that progesterone-activated http://science.sciencemag.org/ studies should define whether changes in extra- endocannabinoid depletion by ABHD2 is a general mechanism by which progesterone cellular ion concentrations are involved in dis- exerts its genome-independent action and primes sperm for fertilization. orders such as stupor and coma. REFERENCES AND NOTES ccording to the conventional model of ration (3) and human sperm cell activation (4, 5), 1. G. Tononi, C. Cirelli, Neuron 81,12–34 (2014). steroid signaling, steroid hormones act and P4 likely triggers anesthesia in rodents (6). 2. J. H. Benington, H. C. Heller, Prog. Neurobiol. 45, 347–360 through their corresponding genomic re- Many tissues have both nuclear and membrane Downloaded from (1995). ceptorstoregulategeneexpressioninde- progesterone receptors, which complicates iden- 3. V. Hinard et al., J. Neurosci. 32, 12506–12517 (2012). A 4. C. B. Saper, T. E. Scammell, J. Lu, Nature 437, 1257–1263 velopment, metabolism, and reproduction tification of the latter. Because sperm are tran- (2005). (1, 2). The time scale of these signaling events scriptionally silent cells and, therefore, lack the 5. J. O’Donnell, D. Zeppenfeld, E. McConnell, S. Pena, ranges from hours to days (2). However, there nuclear progesterone receptor effect, we used – M. Nedergaard, Neurochem. Res. 37, 2496 2512 is a much faster pathway that alters ion chan- human spermatozoa as a cellular model to iden- (2012). 6. M. Steriade, D. A. McCormick, T. J. Sejnowski, Science 262, nel activity through steroid activation of mem- tify the nongenomic progesterone receptor. 679–685 (1993). brane receptors. Nongenomic signaling of the Progesterone is a major component of follic- 7. G. G. Somjen, Ions in the Brain: Normal Function, Seizures, steroid hormone progesterone (P4) occurs on a ular fluid and is released by ovaries and cumu- and Stroke (Oxford Univ. Press, 2004). time scale of seconds and is required for dis- lus cells that surround the oocyte. Nanomolar 8. J. Seigneur, D. Kroeger, D. A. Nita, F. Amzica, Cereb. Cortex 16, 655–668 (2006). tinct physiological events, such as oocyte matu- concentrations of P4 act through the nongenomic 9. S. M. Rothman, Science 220, 536–537 (1983). P4 receptor pathway and cause robust elevation 2+ 10. L. Xie et al., Science 342, 373–377 (2013). of sperm cytoplasmic [Ca ](7–9) through the 11. M. Anděrová et al., Glia 35, 189–203 (2001). 1Department of Molecular and Cell Biology, University of activation of the cation channel, CatSper (10–14). 12. C. B. Saper, P. M. Fuller, N. P. Pedersen, J. Lu, T. E. Scammell, California, Berkeley, CA 94720, USA. 2QB3/Chemistry Mass This rise in intracellular [Ca2+]leadstochanges Neuron 68, 1023–1042 (2010). Spectrometry Facility, University of California, Berkeley, CA in sperm motility, known as hyperactivation, and 13. A. J. Hansen, Physiol. Rev. 65, 101–148 (1985). 3 94720, USA. Department of Cellular and Molecular 4 5 15 14. L. Sun et al., Magnes. Res. 22, 266–272 (2009). Physiology; Department of Neuroscience, Program in Cellular primes sperm for acrosomal exocytosis ( , , ). 15. A. M. Romani, Arch. Biochem. Biophys. 512,1–23 Neuroscience, Neurodegeneration, and Repair (CNNR), Yale Here, we show that the orphan enzyme a/b hy- (2011). School of Medicine, Yale University, New Haven, CT 06536, drolase domain–containing protein 2 (ABHD2) 4 16. W. W. Anderson, D. V. Lewis, H. S. Swartzwelder, W. A. Wilson, USA. Department of Urology, University of California, San serves as a sperm progesterone receptor. It func- Brain Res. 398, 215–219 (1986). Francisco, CA 94143, USA. 5Department of Physiology, 17. G. F. Gilestro, G. Tononi, C. Cirelli, Science 324, 109–112 University of California, San Francisco, CA 94158, USA. tionsasalipidhydrolasebyremovingendogenous (2009). *Corresponding author. Email: [email protected] CatSper inhibitors upon association with P4. Thus, SCIENCE sciencemag.org 29 APRIL 2016 • VOL 352 ISSUE 6285 555 RESEARCH | RESEARCH ARTICLES Fig. 1. Inhibition of sperm mSHs removes P4- dependent CatSper activation. (A) (Left) Rep- resentative ICatSper recorded from control human spermatozoa in the absence (black) and presence (red) of P4. (Right) ICatSper recorded from human spermatozoa pretreated with MAFP, an irreversible serine hydrolase inhibitor, for 30 min in the absence or presence of P4. (B) Fold increase of ICatSper in response to P4, PGE1, and MAFP. (C) Human capacitated spermatozoa do not hyperactivate in response to P4 if pretreated with MAFP,as recorded with computer-aided sperm analyses (CASA). (D) Arachidonoylglycerols are inhibitors of ICatSper. ICatSper densities are obtained at +80 mV (positive scale) or –80 mV (negative scale). (E) Representa- tive ICatSper in the absence (black) or presence of the corresponding endocannabinoid. Data are repre- sented as means ± SEM; numbers in parentheses indicate the number of experiments; and ****P < 0.0001, ***P <0.001,**P <0.005. on June 15, 2016 Fig. 2. Photoreactive progesterone analog P4* acti- vates ICatSper and associates with P4 receptor. (A)P4* consists of an active group (progesterone), a diazirine– UV-sensitive moiety, and a biotin tag that allows for pull- down of bound proteins. CMO, (O-carboxymethyl)oxime; PEG, polyethylene glycol. (B) Western blot from P4* binding experiments resolves biotinylated (P4*-bound) proteins. Lanes are labeled –, mock-irradiated cells; +, UV- treated cells; P4, P4 (20 mM); E, estrogen (0.5 mM); and Ru, Ru486 (0.5 mM). Acetylated a-tubulin (middle) stain- ing was used as the loading control. (Bottom) The blot http://science.sciencemag.org/ was probed with AbA. One experiment out of six inde- pendent pull-downs is shown. (C) ABHD2 is present in the human sperm transcriptome. mRNA sequencing (mRNA- seq) reads from human blood monocytes and sperm cells. FPKM, fragments per kilobase of transcript per million mapped reads. Red line indicates expected num- ber of sequencing reads for genes with similar expression Downloaded from levels in the two samples. CatSperG and AKAP4 indicate sperm-specific transcripts; CD14 and CD33 indicate monocyte-specific transcripts.Transcripts for FAAH and abhd2 are also shown.