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Controlling fertilization and cAMP signaling in sperm by optogenetics
German scientists have published a report detailing their methods on how to control and study fertilization and cAMP signaling in sperm by optogenetics, a cutting-edge technique that allows control of cellular activity by light. Conducting this study presents another opportunity to examine how feasible the optogenetic control of second messengers is, which in turn reveals alternate ways to study cAMP signaling in a noninvasive manner, control behaviors of single cells, and replicate a fundamental biological progress like fertilization.
In this study, the scientists utilized a transgenic mouse model expressing a photoactivated adenylyl cyclase (bPAC) in sperm. The overarching goal was to prove that bPAC is able to replace endogenous adenylyl cyclase activity (originally modulated by soluble adenylyl cyclase, or SACY), which leads to cAMP level elevation and accelerated flagellar movement, indirectly controlling sperm behavior. However, since cAMP signaling is closely intertwined with calcium signaling in mammalian sperm, it was critical to determine that activating bPAC influenced cAMP levels independently from the calcium signaling pathway. To accomplish this, several experiments were conducted to prove that light/bPAC-stimulated cAMP synthesis did not affect intracellular calcium levels in any way. During these experiments, a red-shifted fluorescent calcium indicator was used to track calcium levels in bPAC-sperm, to enable separate activation of bPAC at 485 nm and excitation of the red-shifted indicator at 520 nm. For non-bPAC-sperm, Cal-520 was used to monitor changes in calcium levels of capacitated wild-type and CatSper-null sperm, demonstrating that cyclic nucleotide analogs to cAMP, like 8-Br-cGMP, 8-Br-cAMP, and db-cAMP could still affect calcium influx in bPAC and wild-type sperm but not CatSper-null sperm. This confirms past reports that cNMP-induced activation of calcium channel CatSper was necessary to affect calcium influx, and that bPAC operates separately from calcium.
Regarding the calcium detection approach for the experiments, the materials and methods section details the loading requirements for both the red-shifted calcium indicator and Cal-520. It was specified that the red-shifted indicator had to be loaded with bPAC-sperm at 20 µM in the presence of Pluronic F-127, while Cal-520 was loaded at only 5 µM for all other types of sperm. The experiment exhibits how using Cal-520 simplifies loading and reduces the amount of reagent required, which in higher concentrations may influence cell function. The compatibility of Cal-520 with non-bPAC sperm also shows how Cal-520 is a widely applicable tool for calcium detection.
In this study, the scientists utilized a transgenic mouse model expressing a photoactivated adenylyl cyclase (bPAC) in sperm. The overarching goal was to prove that bPAC is able to replace endogenous adenylyl cyclase activity (originally modulated by soluble adenylyl cyclase, or SACY), which leads to cAMP level elevation and accelerated flagellar movement, indirectly controlling sperm behavior. However, since cAMP signaling is closely intertwined with calcium signaling in mammalian sperm, it was critical to determine that activating bPAC influenced cAMP levels independently from the calcium signaling pathway. To accomplish this, several experiments were conducted to prove that light/bPAC-stimulated cAMP synthesis did not affect intracellular calcium levels in any way. During these experiments, a red-shifted fluorescent calcium indicator was used to track calcium levels in bPAC-sperm, to enable separate activation of bPAC at 485 nm and excitation of the red-shifted indicator at 520 nm. For non-bPAC-sperm, Cal-520 was used to monitor changes in calcium levels of capacitated wild-type and CatSper-null sperm, demonstrating that cyclic nucleotide analogs to cAMP, like 8-Br-cGMP, 8-Br-cAMP, and db-cAMP could still affect calcium influx in bPAC and wild-type sperm but not CatSper-null sperm. This confirms past reports that cNMP-induced activation of calcium channel CatSper was necessary to affect calcium influx, and that bPAC operates separately from calcium.
Regarding the calcium detection approach for the experiments, the materials and methods section details the loading requirements for both the red-shifted calcium indicator and Cal-520. It was specified that the red-shifted indicator had to be loaded with bPAC-sperm at 20 µM in the presence of Pluronic F-127, while Cal-520 was loaded at only 5 µM for all other types of sperm. The experiment exhibits how using Cal-520 simplifies loading and reduces the amount of reagent required, which in higher concentrations may influence cell function. The compatibility of Cal-520 with non-bPAC sperm also shows how Cal-520 is a widely applicable tool for calcium detection.
References
- Vera Jansen, Luis Alvarez, Melanie Balbach, Timo Strünker, Peter Hegemann, U Benjamin Kaupp, Dagmar Wachten. Controlling fertilization and cAMP signaling in sperm by optogenetics. eLife 2015; 4:e05161. DOI: 10.7554/eLife.05161
- Cal-520®, AM. AAT Bioquest, n.d. Web. 1 July 2016
Original created on November 1, 2016, last updated on November 1, 2016
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