Mammalian sperm must undergo a functionally defined process called capacitation to be able to fertilize oocytes. They become capacitated in vivo by interacting with the female reproductive tract or in vitro in a defined capacitation medium that contains bovine serum albumin, calcium (Ca ), and bicarbonate (HCO ). In this work, sperm were double stained with propidium iodide and the Ca dye Fluo-4 AM and analyzed by flow cytometry to determine changes in intracellular Ca concentration ([Ca ] ) in individual live sperm. An increase in [Ca ] was observed in a subpopulation of capacitated live sperm when compared with noncapacitated ones. Sperm exposed to the capacitating medium displayed a rapid increase in [Ca ] within 1 min of incubation, which remained sustained for 90 min. These rise in [Ca ] after 90 min of incubation in the capacitating medium was evidenced by an increase in the normalized median fluorescence intensity. This increase was dependent on the presence of extracellular Ca and, at least in part, reflected the contribution of a new subpopulation of sperm with higher [Ca ] . In addition, it was determined that the capacitation-associated [Ca ] increase was dependent of CatSper channels, as sperm derived from CatSper knockout (CatSper KO) or incubated in the presence of CatSper inhibitors failed to increase [Ca ] . Surprisingly, a minimum increase in [Ca ] was also observed in CatSper KO sperm suggesting the existence of other Ca transport systems. Altogether, these results indicate that a subpopulation of sperm increases [Ca ] very rapidly during capacitation mainly due to a CatSper-mediated influx of extracellular Ca .
Acrosomal exocytosis (AR) is a critical process that sperm need to undergo to fertilize an egg. The evaluation of the presence or absence of the acrosome is usually performed by using lectins or dyes in fixed cells. With this approach, it is neither possible to monitor the dynamic process of exocytosis and related molecular events while discriminating between live and dead cells, nor to evaluate the acrosomal status while sperm reside in the female reproductive tract. However, over the last two decades, several new methodologies have been used to assess the occurrence of AR in living cells allowing different groups to obtain information that was not possible in the past. These techniques have revolutionized the whole study of this process. This review summarizes current methods available to analyze AR in living cells as well as the important information that emerged from studies using these approaches.
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.
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