Jackson Kirkman-Brown

Ca2+-stores in sperm: their identities and functions

Intracellular Ca2+ stores play a central role in the regulation of cellular [Ca2+](i) and the generation of complex [Ca2+] signals such as oscillations and waves. Ca2+ signalling is of particular significance in sperm cells, where it is a central regulator in many key activities (including capacitation, hyperactivation, chemotaxis and acrosome reaction) yet mature sperm lack endoplasmic reticulum and several other organelles that serve as Ca2+ stores in somatic cells. Here, we review i) the evidence for the expression in sperm of the molecular components (pumps and channels) which are functionally significant in the activity of Ca2+ stores of somatic cells and ii) the evidence for the existence of functional Ca2+ stores in sperm. This evidence supports the existence of at least two storage organelles in mammalian sperm, one in the acrosomal region and another in the region of the sperm neck and midpiece. We then go on to discuss the probable identity of these organelles and their discrete functions: regulation by the acrosome of its own secretion and regulation by membranous organelles at the sperm neck (and possibly by the mitochondria) of flagellar activity and hyperactivation. Finally, we consider the ability of the sperm discretely to control mobilisation of these stores and the functional interaction of stored Ca2+ at the sperm neck/midpiece with CatSper channels in the principal piece in regulation of the activities of mammalian sperm.

Human sperm accumulation near surfaces: a simulation study

A hybrid boundary integral/slender body algorithm for modelling flagellar cell motility is presented. The algorithm uses the boundary element method to represent the ‘wedge-shaped’ head of the human sperm cell and a slender body theory representation of the flagellum. The head morphology is specified carefully due to its significant effect on the force and torque balance and hence movement of the free-swimming cell. The technique is used to investigate the mechanisms for the accumulation of human spermatozoa near surfaces. Sperm swimming in an infinite fluid, and near a plane boundary, with prescribed planar and three-dimensional flagellar waveforms are simulated. Both planar and ‘elliptical helicoid’ beating cells are predicted to accumulate at distances of approximately 8.5–22 μm from surfaces, for flagellar beating with angular wavenumber of 3π to 4π. Planar beating cells with wavenumber of approximately 2.4π or greater are predicted to accumulate at a finite distance, while cells with wavenumber of approximately 2π or less are predicted to escape from the surface, likely due to the breakdown of the stable swimming configuration. In the stable swimming trajectory the cell has a small angle of inclination away from the surface, no greater than approximately 0.5°. The trapping effect need not depend on specialized non-planar components of the flagellar beat but rather is a consequence of force and torque balance and the physical effect of the image systems in a no-slip plane boundary. The effect is relatively weak, so that a cell initially one body length from the surface and inclined at an angle of 4°–6° towards the surface will not be trapped but will rather be deflected from the surface. Cells performing rolling motility, where the flagellum sweeps out a ‘conical envelope’, are predicted to align with the surface provided that they approach with sufficiently steep angle. However simulation of cells swimming against a surface in such a configuration is not possible in the present framework. Simulated human sperm cells performing a planar beat with inclination between the beat plane and the plane-of-flattening of the head were not predicted to glide along surfaces, as has been observed in mouse sperm. Instead, cells initially with the head approximately 1.5–3 μm from the surface were predicted to turn away and escape. The simulation model was also used to examine rolling motility due to elliptical helicoid flagellar beating. The head was found to rotate by approximately 240° over one beat cycle and due to the time-varying torques associated with the flagellar beat was found to exhibit ‘looping’ as has been observed in cells swimming against coverslips.

Bend Propagation in the Flagella of Migrating Human Sperm, and Its Modulation by Viscosity

A pre-requisite for sexual reproduction is successful unification of the male and female gametes; in externally-fertilising echinoderms the male gamete is brought into close proximity to the female gamete through chemotaxis, the associated signalling and flagellar beat changes being elegantly characterised in several species. In the human, sperm traverse a relatively high-viscosity mucus coating the tract surfaces, there being a tantalising possible role for chemotaxis. To understand human sperm migration and guidance, studies must therefore employ similar viscous in vitro environments. High frame rate digital imaging is used for the first time to characterise the flagellar movement of migrating sperm in low and high viscosities. While qualitative features have been reported previously, we show in precise spatial and temporal detail waveform evolution along the flagellum. In low viscosity the flagellum continuously moves out of the focal plane, compromising the measurement of true curvature, nonetheless the presence of torsion can be inferred. In high viscosities curvature can be accurately determined and we show how waves propagate at approximately constant speed. Progressing waves increase in curvature approximately linearly except for a sharper increase over a distance approximately 20-27 microm from the head/midpiece junction. Curvature modulation, likely influenced by the outer dense fibres, creates the characteristic waveforms of high viscosity swimming, with remarkably effective cell progression against greatly increased resistance, even in high viscosity liquids. Assessment of motility in physiological viscosities will be essential in future basic research, studies of chemotaxis and novel diagnostics.

Human spermatozoa migration in microchannels reveals boundary-following navigation

The migratory abilities of motile human spermatozoa in vivo are essential for natural fertility, but it remains a mystery what properties distinguish the tens of cells which find an egg from the millions of cells ejaculated. To reach the site of fertilization, sperm must traverse narrow and convoluted channels, filled with viscous fluids. To elucidate individual and group behaviors that may occur in the complex three-dimensional female tract environment, we examine the behavior of migrating sperm in assorted microchannel geometries. Cells rarely swim in the central part of the channel cross-section, instead traveling along the intersection of the channel walls (“channel corners”). When the channel turns sharply, cells leave the corner, continuing ahead until hitting the opposite wall of the channel, with a distribution of departure angles, the latter being modulated by fluid viscosity. If the channel bend is smooth, cells depart from the inner wall when the curvature radius is less than a threshold value close to 150 μm. Specific wall shapes are able to preferentially direct motile cells. As a consequence of swimming along the corners, the domain occupied by cells becomes essentially one-dimensional, leading to frequent collisions, and needs to be accounted for when modeling the behavior of populations of migratory cells and considering how sperm populate and navigate the female tract. The combined effect of viscosity and three-dimensional architecture should be accounted for in future in vitro studies of sperm chemoattraction.

Ca2+ signals generated by CatSper and Ca2+ stores regulate different behaviors in human sperm.

Background: Ca2+ signals, elicited by cues from the oocyte and female tract, regulate human sperm behavior. Results: CatSper channel activation (flagellum) and Ca2+ store mobilization (neck) caused similar [Ca2+]i elevation but induced functionally different behaviors. Conclusion: Sperm motility pattern is determined by the site of Ca2+ mobilization. Significance: Selection of Ca2+ signaling components and/or regulation of their availability for activation controls human sperm behavior. [Ca2+]i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca2+ signaling pathway used. Activation of CatSper (by raising pHi or stimulating with progesterone) caused sustained [Ca2+]i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca2+ caused sustained [Ca2+]i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca2+ stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca2+-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca2+]i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca2+ at the sperm neck can be mobilized by Ca2+-induced Ca2+ release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca2+ store, which may be regulated by capacitation and NO from the cumulus.

Counting sperm does not add up any more: time for a new equation?

Although sperm dysfunction is the single most common cause of infertility, we have poor methods of diagnosis and surprisingly no effective treatment (excluding assisted reproductive technology). In this review, we challenge the usefulness of a basic semen analysis and argue that a new paradigm is required immediately. We discuss the use of at-home screening to potentially improve the diagnosis of the male and to streamline the management of the sub-fertile couple. Additionally, we outline the recent progress in the field, for example, in proteomics, which will allow the development of new biomarkers of sperm function. This new knowledge will transform our understanding of the spermatozoon as a machine and is likely to lead to non-ART treatments for men with sperm dysfunction.

Pregnancy and perinatal outcomes after assisted reproduction: a comparative study

BackgroundIncreasing use of fertility therapy has elicited concerns regarding adverse effects for expectant mothers and the health of children thus conceived.AimsTo study the risk of adverse perinatal outcomes, birth defects and pregnancy complications following assisted reproductive technology (ART).MethodsQuestionnaire-based study involving 1,524 children and 1,182 pregnancies conceived following in vitro fertilisation (IVF) in two units. Outcomes were compared with the general population.ResultsIn the study group versus the general population; multi-foetal gestations, 26 versus 2%; singleton preterm delivery and low birth weight, 8.7 and 6.4 versus 4.3 and 4%, respectively; non-lethal congenital malformation rate, 2.6 versus 2.1%; placenta praevia, 2.8 versus 0.5%.ConclusionsMulti-foetal gestations remain the principal cause of adverse perinatal outcomes after ART. Singleton ART pregnancies have an increased risk of preterm delivery and low birth weight at term. Non-lethal congenital malformation rates are not increased following ART. Placenta praevia is increased following ART.

Slow calcium oscillations in human spermatozoa

We have used single-cell imaging to investigate intracellular Ca2+ signalling in human spermatozoa stimulated with progesterone (3 microM). In approx. 9% of cells progesterone caused the activation of slow repetitive [Ca2+]i (intracellular Ca2+ concentration) oscillations, with a period of 1-4 min, which persisted for the duration of recording (20-30 min). Pretreatment with nifedipine, which blocks T- and L-type voltage-operated Ca2+ channels in spermatogenic cells, did not modify the characteristics of the oscillations, but reduced the proportion of cells in which they were observed. Stimulation with Bay K 8644 or FPL64176 induced [Ca2+]i oscillations in 5-10% of cells, but their frequency was low (period, 4-5 min). Application of valinomycin (1 microM) to clamp membrane potential at E(K) (equilibrium potential for potassium) did not modify activity in oscillating cells, showing that plasma membrane potential and activation of voltage-operated conductances are not involved in the mechanism by which sperm [Ca2+]i oscillations are generated.

Mobilisation of Ca2+ stores and flagellar regulation in human sperm by S-nitrosylation: a role for NO synthesised in the female reproductive tract.

Generation of NO by nitric oxide synthase (NOS) is implicated in gamete interaction and fertilisation. Exposure of human spermatozoa to NO donors caused mobilisation of stored Ca2+ by a mechanism that did not require activation of guanylate cyclase but was mimicked by S-nitroso-glutathione (GSNO; an S-nitrosylating agent). Application of dithiothreitol, to reduce protein -SNO groups, rapidly reversed the actions of NO and GSNO on [Ca2+]i. The effects of NO, GSNO and dithiothreitol on sperm protein S-nitrosylation, assessed using the biotin switch method, closely paralleled their actions on [Ca2+]i. Immunofluorescent staining revealed constitutive and inducible NOS in human oviduct and cumulus (the cellular layer investing the oocyte). 4,5-diaminofluorescein (DAF) staining demonstrated production of NO by these tissues. Incubation of human sperm with oviduct explants induced sperm protein S-nitrosylation resembling that induced by NO donors and GSNO. Progesterone (a product of cumulus cells) also mobilises stored Ca2+ in human sperm. Pre-treatment of sperm with NO greatly enhanced the effect of progesterone on [Ca2+]i, resulting in a prolonged increase in flagellar excursion. We conclude that NO regulates mobilisation of stored Ca2+ in human sperm by protein S-nitrosylation, that this action is synergistic with that of progesterone and that this synergism is potentially highly significant in gamete interactions leading to fertilisation.

2-APB-potentiated channels amplify CatSper-induced Ca2+ signals in human sperm

Ca2+i signalling is pivotal to sperm function. Progesterone, the best-characterized agonist of human sperm Ca2+i signalling, stimulates a biphasic [Ca2+]i rise, comprising a transient and subsequent sustained phase. In accordance with recent reports that progesterone directly activates CatSper, the [Ca2+]i transient was detectable in the anterior flagellum (where CatSper is expressed) 1–2 s before responses in the head and neck. Pre-treatment with 5 μM 2-APB (2-aminoethoxydiphenyl borate), which enhances activity of store-operated channel proteins (Orai) by facilitating interaction with their activator [STIM (stromal interaction molecule)] ‘amplified’ progesterone-induced [Ca2+]i transients at the sperm neck/midpiece without modifying kinetics. The flagellar [Ca2+]i response was unchanged. 2-APB (5 μM) also enhanced the sustained response in the midpiece, possibly reflecting mitochondrial Ca2+ accumulation downstream of the potentiated [Ca2+]i transient. Pre-treatment with 50–100 μM 2-APB failed to potentiate the transient and suppressed sustained [Ca2+]i elevation. When applied during the [Ca2+]i plateau, 50–100 μM 2-APB caused a transient fall in [Ca2+]i, which then recovered despite the continued presence of 2-APB. Loperamide (a chemically different store-operated channel agonist) enhanced the progesterone-induced [Ca2+]i signal and potentiated progesterone-induced hyperactivated motility. Neither 2-APB nor loperamide raised pHi (which would activate CatSper) and both compounds inhibited CatSper currents. STIM and Orai were detected and localized primarily to the neck/midpiece and acrosome where Ca2+ stores are present and the effects of 2-APB are focussed, but store-operated currents could not be detected in human sperm. We propose that 2-APB-sensitive channels amplify [Ca2+]i elevation induced by progesterone (and other CatSper agonists), amplifying, propagating and providing spatio-temporal complexity in [Ca2+]i signals of human sperm.