University of Birmingham Effects of pH manipulation, CatSper stimulation and Ca2+-store mobilisation on [Ca2+]i and behaviour of human sperm

19 Study question: How do the alkaline pH, progesterone and 4-aminopyridine interact in their effects 20 on human sperm? 21 Summary answer: Behaviour of human sperm (proportion of hyperactivated cells and motility 22 kinematics) were related directly to [Ca 2+ ]i irrespective of pH or the agonist applied. 23 What is known already? CatSper channels of human sperm, which are central to generation of 24 sperm [Ca 2+ ]i signals and induction of hyperactivated motility, are activated by intracellular 25 alkalinisation and progesterone. Progesterone (P4) is much less effective than 4-aminopyridine (4-AP) 26 (which mobilises stored Ca 2+ but also raises pHi) as an inducer of hyperactivation. 27 Study design, size, duration: This was a laboratory study, spanning approximately 18 months 28 that used 15 sperm donors and involved more than 100 separate experiments. 29 Participants/materials, setting, methods: Semen donors and patients were recruited in 30 accordance with local ethics approval (ERN_12-0570R). [Ca2+]i responses of suspended cell 31 populations were examined by fluorimetric recording and motility parameters assessed by computer32 assisted sperm analysis (CASA). 33 Main results and the role of chance: Increasing pHo from 7.4 to 8.5 raised pHi (from 6.9 to 7.2) 34 and significantly increased both [Ca 2+ ]i and the proportion of hyperactivated cells. Stimulation of cells 35 with P4 (1 nM-20 M) induced a biphasic (transient and plateau) increase in [Ca 2+ ]i. The [Ca 2+ ]i 36 increase was of similar amplitude and dose-dependency at pHo=7.4 and pHo= 8.5. 4-aminopyridine 37 (0.2-5 mM) induced a biphasic [Ca 2+ ]i increase that was dose-dependent across the entire range tested 38 and was strongly enhanced at pH 8.5. Motility was assessed 300 s post-stimulation, during the plateau 39 phase of the progesterone and 4-AP-induced [Ca 2+ ]i responses. Progesterone had only a small effect 40 on hyperactivated motility even at the highest dose used (20 M; <5% increase in the proportion of 41 cells classified as hyperactivated) which was insensitive to pHo. 4-aminopyridine potently stimulated 42 hyperactivated motility, this effect being dose-dependent and greatly enhanced at pHo=8.5. The 43 relationship between [Ca 2+ ]i (fluorescence of fluo4) and proportion of hyperactivated cells, 44 irrespective of pHo, agonist or dose, was fitted by a single curve (2 nd order polynomial; R 2 =0.96). 45 Similar analysis of curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) 46 showed a linear relationship to [Ca 2+ ]i (R 2 >0.9). 47 Limitations, reasons for caution: This was an in-vitro study and caution must be taken when 48 extrapolating these results to in vivo regulation of sperm. Though controls indicate that saturation of 49 fluo4 did not affect the findings, at the highest doses of progesterone the true amplitude of the [Ca 2+ ]i 50 transient may not have been reported by the dye. 51 Wider implications of the findings: These findings indicate that (i) activation of human sperm 52 CatSper by progesterone (and presumably other ligands that act similarly) and consequent acquisition 53 of hyperactivated motility is not significantly enhanced by intracellular alkalinisation; (ii) VCL, ALH 54 and hyperactivation are directly related to [Ca 2+ ]i, irrespective of the mechanism by which Ca 2+ is 55 mobilised, and the ability of stimuli to induce prolonged [Ca 2+ ]i elevation (as occurs upon 56 mobilisation of stored Ca 2+ ) determines the observed effect on cell behaviour. 57 Study funding/competing interest(s): CA was supported by the Nigerian government (Tertiary 58 Education Trust (TET) Fund). The authors have no conflicts of interest. 59