Proteomic strategy to identify the cell-surface progesterone-receptor mediating nongenomic steroid-signaling in human sperm cells

For a human sperm cell to successfully fertilize a human egg, the functions of the sperm cell must be precisely regulated during its journey through the female reproductive tract. Many of these functions are controlled via intracellular Ca2+ concentration, e.g., sperm motility, and acrosome reaction. Interestingly, all channel-mediated Ca2+ influx in human sperm cells occurs via CatSper, the calcium/cation channel that is exclusively expressed in sperm cells. It has been known for decades that the steroid progesterone induces a rapid Ca2+ influx in the transcriptionally silent human sperm cell. While it has been revealed that the binding site of progesterone in sperm cells must be located in close proximity of the CatSper channel in the plasma membrane, the actual receptor is yet to be identified. As CatSper is exclusively expressed in sperm cells, it, and its main endogenous ligand progesterone, present a promising specific and safe pharmacological target for both novel on-demand non-hormonal unisex contraceptives and for novel treatments to enhance male fertility.

Here we employed a strategy based on a combination of state-of-the-art mass spectrometry-based proteomic techniques to identify and characterize the progesterone receptor. We used a combination of Thermal Proteome Profiling, Limited Proteolysis coupled with Mass Spectrometry, and crosslinking combined with a biotin tagged progesterone for Mass affinity purification coupled with Mass spectrometry. Using offline high-pH (HpH) reversed-phase peptide fractionation and short online liquid chromatography (LC) gradients, we profiled the sperm proteome analyzing the head and the tail separately and identified more than 9000 proteins. This is an increase of 40% in protein coverage compared to previous attempts. . Using the aforementioned techniques, we created a shortlist of potential cell-surface progesterone-receptor candidates, which will be validated through human sperm cell-based assays to determine perturbation of the progesterone-induced Ca2+ signals upon inhibition of these proteins.