Ca2+ signalling in regulation and therapeutic manipulation of human sperm behaviour

Then ability of sperm to swim is vital for human fertilization. Not only must sperm be motile, they must also be able to perform different swimming 'styles' with different functions and employ them as needed. As the sperm migrates form the vagina to the oviduct, where it will fertilise an egg, it must progress through very different types of fluid and gel, must navigate through complex, 3-dimensional environments and must then penetrate two 'coats' that surround the egg, first a thick layer of small cells embedded in jelly (the cumulus cells) and inside this a tough, protein shell (the zona pellucida). To succeed at all these different stages of its journey the sperm must, at each stage, select correctly from its various swimming styles or even use them in combination, alternating between two. This sophisticated process is orchestrated by cues provided by the female tract but also depends on events inside the sperm. The cues cause a change in the concentration of calcium ions (Ca2+) inside the sperm cell, which in turn acts a as a signal to select the type of swimming to use.
Our initial experiments have shown us that there are at least two different methods by which the sperm can control the concentration of Ca2+ inside the cell and that when we stimulate the cells with drugs selectively to activate one or other of these two Ca2+ 'pathways', we can cause the sperm to swim in very different styles according to which method we turn on. In this study we will measure sperm Ca2+ concentration and sperm swimming style when they are provided with cues that they would meet as they travel through the female tract and meet the egg. We will investigate the types of Ca2+ 'signal' that are turned on (which Ca2+ pathway is used), the characteristics of the signal produced and how the different cues provided by the female tract can select different types of Ca2+ signal and thus how they are able to control what kind of swimming the sperm does. The understanding that we gain from these experiments will be used to study sperm from subfertile men. We will investigate how these processes go wrong in men whose sperm cannot regulate their motility and swimming style - an important cause of male infertility. Finally, we will use this information on how sperm swimming is controlled to test drugs that will potentially be used to 'activate' sperm that normally fail to swim properly and therefore cannot reach the egg and/or cannot fertilise it when they get there. For men where this problem significantly affects their fertility, correcting or bypassing the problem in this way will provide a route for simple, low cost treatment without needing to use the highly invasive, potentially traumatic and very expensive techniques of in vitro assisted reproduction.

To ascend the female tract, penetrate the layers that surround the oocyte and fertilise it a sperm must employ diverse motility patterns, switching between behaviours in response to cues. [Ca2+]i signaling, the central regulator of sperm motility, mediates these behavioural choices. Impaired Ca2+ signaling renders the sperm infertile. This study addresses regulation of human sperm motility and how it may be pharmacologically enhanced in subfertile men. Activation of CatSper channels and release of Ca2+ from a store at the sperm neck are both implicated in motility regulation. We have shown that, while both raise [Ca2+]i, they elicit functionally different behaviours in human sperm. CatSper activation causes subtle changes in flagellar beat that enhance penetration into viscous medium whereas mobilisation of stored Ca2+ induces robust hyperactivation. Since CatSper is activated by diverse stimuli (pHi, hormones, prostaglandins, Em) whereas the Ca2+ store is sensitive primarily to Ca2+-induced-Ca2+-release, we propose that (a) CatSper functions as a multimodal Ca2+-signaling node that secondarily recruits release of the stored Ca2+ and (b) availability of Ca2+ store release (and the behaviours it regulates) is regulated by capacitation and by stimuli encountered in the female tract such as NO. Using advanced imaging, Ca2+ uncaging, patch clamp and sperm tracking to assess signaling and behavioural regulation in human sperm we will (i) characterise generation/regulation of Ca2+ signals mediated by CatSper, stored Ca2+ and putative non-CatSper conductances in response to endogenous stimuli (ii) elucidate the diversity of sperm behaviours, their functional significance and their 'selection' by Ca2+ signals induced by stimuli encountered in the female tract and (iii) characterise incidence and types of Ca2+-signaling lesions in men with motility-based subfertility and identify agents for targeted manipulation of Ca2+ signaling components to reverse/bypass these lesions.

Our collaboration has a focus on understanding sperm function in humans (rather than in cells from model animals) and relating laboratory findings to lesions in and observations on infertile and subfertile men. Our aim is translation of basic research. By elucidating the role(s) of the different components of the sperm's Ca2+-signalling apparatus, their regulation by the female tract (capacitation, internal cues from the female tract and cumulus-oocyte complex) and the malfunctioning of stores and membrane Ca2+ channels in subfertile men, the proposed work will (i) provide key information on the occurrence, nature and effects of Ca2+-signalling malfunction in human sperm and (ii) identify targets and potential compounds for the development of pharmacological interventions that could modulate sperm function in vitro both negatively and positively. The outcomes of this study will be of direct benefit to companies wanting to develop compounds to enhance the fertilisation capacity of the spermatozoon and therefore to the nations's wealth. A particularly exciting possibility offered by our model (and the data on which it is based) is that sperm from men with CatSper deficits may be treatable by targeting Ca2+ stores, bypassing the lesion. The second group of beneficiaries from this work will be infertile and subfertile patients. Although in vivo treatment of sperm dysfunction is ~10 years from clinical application, the use of in vitro compounds to add to the cell to correct/improve sperm function is a tangible goal. Overcoming functionally debilitating defects in calcium signalling using in vitro compounds could reduce the need of some patients to have more invasive ART treatment e.g. IVF and opt for more cost effective less invasive treatment such as intra-uterine insemination, contributing directly to improvement of the nation's health, wealth and quality of life. Though the focus of our proposal is the understanding of and pharmacological improvement of human sperm function, the other potential and very powerful use of knowledge is the development of new contraceptive approaches. By interfering with their regulatory mechanisms we can impair key aspects of human sperm motility, rendering them infertile. With the world population exceeding 9 billion and the number of pregnancies being terminated in many areas continually increasing, there is an urgent and pressing requirement to develop new contraceptive approaches for men. Ca2+ signaling
components, particularly ion channels, have proved to be ideal targets for manipulation in other systems (e.g. cardiovascular physiology). Our work will provide a strong platform for these studies but the end goal would be a more distant prospect.
For the two PDRAs in Birmingham and Dundee this programme of work will provide excellent training in application of advanced physiological techniques to human spermatozoa. Our laboratories are world leaders in this area. The importance and impact of this field of study is growing and there is a demand for highly trained and able scientists with these skills.