Caged Progesterone - a new tool to resolve a 20-year-old puzzle

Steroid hormones act via a slow signaling pathway that triggers long lasting changes in cells: the de novo synthesis of proteins takes between minutes and days. This slow signaling pathway, which is also referred to as "genomic" and "classical", was discovered back in the 1960s. However, not all progesterone actions on cells can be explained by the genomic signaling pathway: for example, the progesterone action on human sperm. Protein synthesis is switched off in mature sperm; therefore, a genomic signaling pathway is out of the question here. Figure 1 shows Ca2+ signals in human sperm evoked by stimulation with various progesterone concentrations. The Ca2+ concentration increases with virtually no delay (latency), thus providing another clear indication of a non-genomic signaling path in sperm.

Figure 1: Progesterone-induced Ca2+ signals in human sperm. Human sperm were rapidly mixed with progesterone in a stopped-flow apparatus. Changes in the intracellular Ca2+ concentration were monitored with a Ca2+-sensitive fluorescent dye (?F = fluorescence). Sperm were stimulated with 15 nM (black), 50 nM (red), and 1.25 µM (green) progesterone. The stimulation occurred at t=0. The inset shows the first five seconds after stimulation.

Probably, this rapid Ca2+ response is important for the fertilization of the egg. It has been proposed that progesterone acts as a chemoattractant for sperm. Moreover, high micromolar progesterone concentrations elict the release of an enzymatic cocktail from the sperm head (acrosome reaction). The enzymes digest the egg coat and enable sperm to penetrate the gelatinous sheath of the egg. Although it has been known for over 20 years that progesterone triggers a Ca2+ response and the acrosome reaction in sperm, the underlying signaling pathway remains to be determined. Progesterone also triggers rapid Ca2+ signals in other cell types, e.g. liver cells and certain neurons.
To facilitate the detailed study of the effect of progesterone on sperm, neurons, and other cells, we developed a "caged" form of progesterone in co-operation with chemists from the Leibniz-Institut für Molekulare Pharmakologie in Berlin. To this end, a chemical group, named a "cage", was added to the progesterone (Figure 2). The sperm are approximately 500-1000 times less sensitive for the caged progesterone compared to normal progesterone. In short: the caged progesterone is biologically inactive, it is "invisible" to the sperm. Ultraviolet (UV) light cleaves the bond (photolysis) between the cage and the progesterone, thereby releasing "active" progesterone (Figure 2).

Figure 2: Caged Progesterone. A chemical group (cage) was added to the progesterone. The cagedprogesterone molecule is biologically inactive. The bond between the progesterone and thecagecan be cleaved by UV light (h?) and biologically active progesterone is released within microseconds.

Figure 3 shows a Ca2+ signal in sperm triggered by the photolytic release of progesterone. The higher the intensity of the UV flash, the more progesterone is released, and the larger the Ca2+ signals become. As the progesterone release by photolysis takes just a few microseconds, the latency of the Ca2+ signals can be determined precisely.

Figure 3:Ca2+ signals in human sperm evoked by photolytic release of progesterone from caged progesterone. Human sperm were mixed with caged progesterone (7.5 µM) in a stopped-flow apparatus. UV flashes were then applied to the cuvette at a relative intensity of 100% (blue), 50% (green) and 25% (red), thus releasing different concentrations of progesterone (black = no UV flash). Changes in the intracellular Ca2+ concentration were monitored using a Ca2+-sensitive fluorescent dye (?F = fluorescence). The UV flashes occurred at t=0. The inset shows the first five seconds after the progesterone release. The flash artifacts on the fluorescence signal indicate the time of the progesterone release.

The caged progesterone is a powerful tool to study behavioural changes in freely swimming sperm. The swimming behaviour of sperm can be observed in a recording chamber under the microscope. When sperm swim in a solution containing caged progesterone, progesterone can be released locally by a UV flash; in this way it is possible to simulate an artificial egg releasing its chemoattractant. If sperm are indeed attracted by progesterone, they should swim to the site where the progesterone was released. However, the analysis of behavioural experiments on sperm is complicated, because at a given time only a small fraction (~10%) of a sperm population reacts to the attractant. We are developing software tools that will help us to reliably and quantitatively analyze the swimming behaviour of human sperm.
The cagedprogesterone also allows the study of Ca2+ signals in other cells, and even in intact tissue. Experiments with tissues are often carried out under the microscope in special recording chambers that only allow a rather slow perfusion of the preparation with progesterone solutions. Rapid cellular responses to progesterone cannot be recorded under such conditions. These preparations, however, can be superfused with solutions containing caged progesterone. The progesterone can then be released rapidly in restricted areas and the response of the cells can be studied. We hope that this new tool will help to elucidate the non-genomic signaling pathway of progesterone.

Publication

Kilic F., Kashikar N.D., Schmidt R., Alvarez L., Dai L., Weyand I., Wiesner B., Goodwin N., Hagen V., Kaupp U.B., „Caged progesterone: a new tool for studying rapid nongenomic actions of progesterone“ J. Am. Chem. Soc. 131 (2009) 4027-4030