AbstractThe biochemical state of human sperm is critical for fertilisation potential, yet no convenient or reliable methods are available to assess essential second messengers. Cyclic nucleotides (cAMP and cGMP) play a crucial role as signalling molecules for capacitation, motility, and acrosome reaction in mammalian sperm. Given their importance in motility and fertilising ability, defective nucleotide function could be a cause of male infertility.
This work was undertaken to establish a method for the extraction and simultaneous quantification of cyclic nucleotides levels in human spermatozoa using HPLC. This study has demonstrated that HPLC can be used to detect basal cyclic nucleotide levels in healthy human spermatozoa, assess the changes upon compound manipulation and comparatively in patient sperm. The development of this method has aided progression towards a reference range for basal cyclic nucleotide levels in donorsperm and the quantification of cyclic nucleotide levels in patient sperm. Patients attending the assisted conception unit in Dundee for IVF treatment were screened for their basal cyclic nucleotide levels and motility parameters. This study found that a positive correlation exists between basal cyclic nucleotide levels, sperm hyperactivation and fertilisation rates at IVF.
Furthermore, this study aimed to apply the HPLC method in conjunction with a high throughput screening strategy and established in vitro tests to identify and characterise the action of compounds that improve sperm motility. Trequinsin hydrochloride, a compound that increased sperm motility in a preliminary screen was selected for detailed molecular (plate reader assays, electrophysiology and cyclic nucleotide measurement) and functional (motility and acrosome reaction) testing in sperm from healthy volunteer donors and, where possible, patients. Fluorometric analysis identified Trequinsin as an efficacious agonist of intracellular calcium. Functionally, Trequinsin significantly increased sperm hyperactivation and penetration into viscous medium in donor samples and sperm hyperactivation in patient samples. The Trequinsin‐induced intracellular calcium response was cross‐desensitised consistently by prostaglandin E1 but not with P4. Whole‐cell patch-clamp electrophysiology confirmed that Trequinsin activates CatSper and partially inhibits potassium channel activity. Trequinsin also increases intracellular cGMP. Trequinsin exhibits a novel pharmacological profile in human sperm and may be a suitable lead compound for the development of new pharmaceuticals to improve patient sperm function and fertilisation potential.
Similarly, this study targeted a series of compounds that have been demonstrated by Tardif et al., (2014) to enhance donor and patient sperm function (in vitro). It is proposed that these compounds promote cyclic nucleotide levels via their inhibition of phosphodiesterase, however this study demonstrates that improvements in sperm function is via diverse modes of action.
The heterogeneity of sperm intracellular response to these compounds is of significant interest as it is well known that not all patients respond identically to drug treatment. The utilisation of HPLC to aid the selection of compounds that increase cyclic nucleotides levels could open a new paradigm of assessment and identify novel therapeutics that promote sperm function.
|Date of Award||28 May 2020|
|Supervisor||Sean Brown (Supervisor) & Joanna Fraser (Supervisor)|
- Male infertility
- Cell sgnalling
- Cyclic nucleotides