Asthenozoospermia is a leading cause of male infertility, but the development of pharmaceuticals to improve sperm motility has been hindered by the lack of effective screening platforms and knowledge of suitable molecular targets. We have demonstrated that a high throughput screening (HTS) strategy in conjunction with established in vitro tests can identify and characterise the action of compounds that improve sperm motility. The study aimed to apply HTS to identify new compounds from a novel small molecule library that increase intracellular calcium, [Ca2+]I, promote human sperm cell motility and systemically determine the mechanism of action.
A validated HTS fluorometric [Ca2+]i assay was used to screen an in-house library of compounds. Trequinsin hydrochloride (a phosphodiesterase 3 inhibitor) 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.
The fluorometric analysis identified Trequinsin as an efficacious agonist of [Ca2+]i, although less potent than progesterone (P4). Functionally, Trequinsin significantly increased cell hyperactivation and penetration into viscous medium in all donor sperm samples and cell hyperactivation in 22/25 (88%) patient sperm samples. The Trequinsin-induced [Ca2+]i 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.
Conclusion and Implications
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.
28 May 2020
Supervisor: Brown, S. (Supervisor) & Fraser, J. (Supervisor)
Student thesis: Doctoral Thesis