ABSTRACT
The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity relies on the utilization of [γ-32P] ATP and the Kemptide substrate. This methodology presents several major drawbacks, including high-costs and health risks derived from the manipulation of radioactive isotopes. In this work we introduce an enhanced non-radioactive assay for quantifying PKA activity, termed KiMSA which relies on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that the KiMSA assay is suitable for purified PKA, and also to address both basal and capacitation induced PKA activity in mouse sperm cells. Furthermore, the assay enables monitoring the inhibition of PKA with inhibitors such as sPKI and H-89 in live cells. Therefore, the experimental and optimal assay conditions are set so that the KiMSA assay can be used to either assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways.
ABSTRACT
Sperm capacitation, crucial for fertilization, occurs in the female reproductive tract and can be replicated in vitro using a medium rich in bicarbonate, calcium, and albumin. These components trigger the cAMP-PKA signaling cascade, proposed to promote hyperpolarization of the mouse sperm plasma membrane through activation of SLO3 K+ channel. Hyperpolarization is a hallmark of capacitation: proper membrane hyperpolarization renders higher in vitro fertilizing ability, while Slo3 KO mice are infertile. However, the precise regulation of SLO3 opening remains elusive. Our study challenges the involvement of PKA in this event and reveals the role of Na+/H+ exchangers. During capacitation, calcium increase through CatSper channels activates NHE1, while cAMP directly stimulates the sperm-specific NHE, collectively promoting the alkalinization threshold needed for SLO3 opening. Hyperpolarization then feeds back Na+/H+ activity. Our work is supported by pharmacology, and a plethora of KO mouse models, and proposes a novel pathway leading to hyperpolarization.
ABSTRACT
The exclusive expression of CatSper in sperm and its critical role in sperm function makes this channel an attractive target for contraception. The strategy of blocking CatSper as a male, non-hormonal contraceptive has not been fully explored due to the lack of robust screening methods to discover novel and specific inhibitors. The reason for this lack of appropriate methodology is the structural and functional complexity of this channel. We have developed a high-throughput method to screen drugs with the capacity to block CatSper in mammalian sperm. The assay is based on removing external free divalent cations by chelation, inducing CatSper to efficiently conduct monovalent cations. Since Na+ is highly concentrated in the extracellular milieu, a sudden influx depolarizes the cell. Using CatSper1 KO sperm we demonstrated that this depolarization depends on CatSper function. A membrane potential (Em) assay was combined with fluorescent cell barcoding (FCB), enabling higher throughput flow cytometry based on unique fluorescent signatures of different sperm samples. These differentially labeled samples incubated in distinct experimental conditions can be combined into one tube for simultaneous acquisition. In this way, acquisition times are highly reduced, which is essential to perform larger screening experiments for drug discovery using live cells. Altogether, a simple strategy for assessing CatSper was validated, and this assay was used to develop a high-throughput drug screening for new CatSper blockers.
