Biology subjects

Luque, G. M.

Publications and source records attributed to Luque, G. M..

4 recordsLinked to original sources

bioRxiv2026

A high-throughput flow cytometry assay to evaluate CatSper-mediated Ca2+ influx triggered by high-K+/high-pH stimulation in mouse sperm

The sperm-specific Ca2+ channel CatSper is essential for male fertility, as it mediates Ca2+ influx required for sperm hyperactivation. Because of its restricted expression in sperm and crucial role in fertilization, CatSper represents a promising target for the development of non-hormonal male contraceptives. However, its structural complexity and the absence of functional heterologous expression systems have limited inhibitor discovery. Here, we developed a high-throughput flow cytometry assay to identify compounds capable of blocking CatSper activity in mammalian sperm. The method relies on the stimulation of CatSper by exposing live mouse sperm to a high-K+/high-pH solution (K8.6), which induces membrane depolarization and intracellular alkalinization, triggering Ca2+ entry through CatSper. Changes in intracellular Ca2+ levels were monitored using the fluorescent indicator Fluo-4. To increase throughput and minimize variability, this assay was combined with fluorescent cell barcoding, which assigns each sperm sample a unique fluorescent signature, enabling the simultaneous acquisition of multiple conditions within a single tube. This integration greatly reduces acquisition time and reagent consumption, thereby facilitating large-scale screening experiments. Moreover, we implemented a compound pooling strategy to further improve assay efficiency, allowing the rapid identification of active compounds within a library. The assay was validated using CatSper1 knockout sperm and known pharmacological inhibitors, confirming that both genetic deletion and pharmacological blockade of CatSper abolish the K8.6-induced Ca2+ response. Together, these results establish a robust, scalable, and high-throughput screening platform for identifying novel CatSper blockers and potential male contraceptive candidates.

cell biology↗

bioRxiv2024

The sodium-proton exchangers sNHE and NHE1 control plasma membrane hyperpolarization in mouse sperm.

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. TeaserAlkalinization of sperm cytoplasm activates potassium channels to hyperpolarize the plasma membrane in a PKA independent cascade.

cell biology↗

bioRxiv2023

Reorganization of the Flagellum Scaffolding Induces a Sperm Standstill Required for Fertilization

Mammalian sperm delve into the female reproductive tract to fertilize the female gamete. The available information about how sperm regulate their motility during the final journey to the fertilization site is extremely limited. In this work, we investigated the structural and functional changes in the sperm flagellum after AE and during the interaction with the eggs. The evidence demonstrates that the double helix actin network surrounding the mitochondrial sheath of the midpiece undergoes structural changes prior to the motility cessation. This structural modification is accompanied by a decrease in diameter of the midpiece and is driven by intracellular calcium changes that occur concomitant with a reorganization of the actin helicoidal cortex. Midpiece contraction occurs in a subset of cells that undergo AE, live-cell imaging during in vitro fertilization showed that the midpiece contraction is required for motility cessation after fusion is initiated. These findings provide the first evidence of the F-actin networks role in regulating sperm motility, adapting its function to meet specific cellular requirements during fertilization, and highlighting the broader significance of understanding sperm motility. Significant statementIn this work, we demonstrate that the helical structure of polymerized actin in the flagellum undergoes a rearrangement at the time of sperm-egg fusion. This process is driven by intracellular calcium and promotes a decrease in the sperm midpiece diameter as well as the arrest in motility, which is observed after the fusion process is initiated.

cell biology↗

bioRxiv2020

Cdc42 activity is essential for the interplay between cAMP/PKA pathway and CatSper function

Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+-dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by sAC, providing a new regulatory mechanism for the stimulation of CatSper by the cAMP/PKA-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.

cell biology↗