Sperm, Progesterone, and CatSper Ion Channel. Polina V. Lishko, Ph.D. University of California, Berkeley

Transcription

1 Sperm, Progesterone, and CatSper Ion Channel Polina V. Lishko, Ph.D. University of California, Berkeley

2 Human contraception: 1. Arrest of spermato- and/or spermiogenesis or inhibition of the oocyte maturation. 2. Prevention of sperm delivery into female reproductive tract (vasectomy, condoms) 3. Inhibition of sperm motility and its ability to find the oocyte 4. Inhibition of the acrosome reaction

3 How spermatozoa reach the egg and overcome its protective barriers? ph i, [Ca 2+ ] i Cumulus cells Zona pellucida Sperm capacitation develops during first 3-5 hours in the fallopian tubes

4 Proton channel Hv1 and calcium channel CatSper: two channels, one goal Hv1 H + g d Ca 2+ CatSper out b in [ph] i [Ca 2+ ] i ph sensor Motility hyperactivation

5 Cytoplasmic droplet makes sperm patch-clamp possible The patch-clamp technique can unveil the signaling pathways that control male fertility Lishko et al., Cell, 2010

6 CatSper is a flagellar Ca 2+ channel required for male fertility Ca 2+ Q: Does human CatSper behave the same way? d g b out in A: No! His Putative ph-sensor CatSper1- CatSper4 CatSperb and CatSperg +10 0m pA/pF 200ms Mouse wild type -10 CatSper -/- 100pA/pF 200ms CatSper baseline CatSper baseline Kirichok et al., Nature, 2006 Lishko et al., Cell, 2010

7 Progesterone is the principal activator of human sperm Cumulus cells secrete progesteronec Progesterone 10 mm Ca 2+ influx Hyperactivation Identity of sperm progesterone receptor/ion channel has long remained a key unresolved question of reproductive biology

8 Normalized current increase Progesterone dramatically potentiates CatSper channel Progesterone 500nM EC 50 = 7.7±1.8 nm 0.2 s Baseline CatSper 100pA 0.0 1E-4 1E Progesterone, nm Lishko et al., Nature, 2011

9 CatSper can be inhibited by NNC nM P -8 control 2 mm NNC baseline 50pA 0.25s 2 mm NNC baseline IC 50 values of 7 μm and > 100 μm for inhibition of Cav3.1 T-type channels and HVA currents respectively Lishko et al., Nature, 2011

10 CatSper is directly activated by progesterone Whole sperm 8-8 G/G max 1.0 Capacitated + P V 1/2 = 30 ± 1 mv Baseline CatSper +Progesterone 500nM ATP, GTP or Ca Incapacitated + 500nM P V 1/2 = 52 ± 1 mv V (mv) Incapacitated V 1/2 = 85 ± 0.3 mv 100pA Tail 0.2 s Lishko et al., Nature, 2011

11 CatSper-associated progesterone binding site is external P-3-BSA 500nM nM P +8 50pA Control control 0.1 s Baseline 500nM Estradiol baseline 50pA 0.1 s BSA Progesterone-3-BSA Progesterone (P) Estradiol Lishko et al., Nature, 2011

12 CatSper-associated and nuclear progesterone receptors have different binding sites RU486 (Selective antagonist of nuclear progesterone and glucocorticoid receptors ) 50pA 0.1 s 11 baseline control + Progesterone 500nM + Progesterone 500nM +RU nM 3 Progesterone Lishko et al., Nature, 2011

13 Mouse CatSper IS NOT progesterone sensitive Human control 500nM P baseline 0.2 s 100pA Mouse control baseline 500nM P Lishko et al., Nature, 2011

14 Conclusions: 1. Female hormone progesterone activates sperm calcium channel CatSper by stabilizing its open state within sperm physiological membrane potentials. 2. Since the action of progesterone is fast and does not depend on the presence of secondary messengers, CatSper channel likely binds progesterone directly and serves as a nongenomic progesterone receptor of sperm. 3. Progesterone-binding site of human CatSper is external. 4. Progesterone- binding site of human CatSper does not resemble ligand binding domain of nuclear progesterone receptor and therefore represents an attractive target for development of new non-hormonal contraceptives g d Ca 2+ CatSper out b in

15 Take- Home Message: 1. Key molecular regulators of human and mouse sperm cells are different. 2. If we want to understand the molecular mechanisms of human fertilization and develop successful contraceptives we have to pay attention to the difference between human spermatozoa and spermatozoa from other species Hs Ion channels: Mouse Human Hv1 (proton channel) absent present Mm CatSper (calcium channel) Regulated by internal ph Regulated by ph AND progesterone Mm Hs

16 Kirichok Lab UCSF, Department of Physiology Yuriy Kirichok, Ph.D. Inna Botchkina, M.S. UCSF, Department of Urology James F. Smith, M.D., M.S. Lishko Lab UC Berkeley, MCB Olga Syritsyna Program for Breakthrough Biomedical Research (UCSF) NIH/NICHD R01HD068914