Biology subjects

Buffone, M. G.

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

7 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↗

bioRxiv2025

Semenogelin-1 Inhibition of Mouse Sperm Hyperactivation Reveals Two Functional Domains Modulating CatSper Channel

Seminal plasma is essential for sperm survival and function after ejaculation. Semenogelin-1 (SEMG1), the predominant seminal plasma protein, transiently suppresses sperm motility and hyperactivation after ejaculation through epididymal protease inhibitor (EPPIN) binding. However, the molecular mechanism underlying SEMG1-mediated inhibition of hyperactivation remains unclear. Here, we tested the hypothesis that SEMG1 inhibits CatSper, a sperm-specific calcium channel crucial for hyperactivation. Full-length recombinant mouse SEMG1 (mSEMG1; Q32-G375) inhibited both progressive motility and hyperactivation; the latter was not recovered with NH4Cl-induced alkalinization, indicating an effect downstream of capacitation-associated intracellular alkalinization. Electrophysiological recordings revealed that mSEMG1 reduced CatSper currents at physiologically relevant concentrations. Truncated mSEMG1 fragments mSEMG1Q32-V118 and mSEMG1R98-G375, but not mSEMG1Y221-G375, inhibited sperm hyperactivation and CatSper currents to a similar extent as full- length mSEMG1. Notably, only mSEMG1R98-G375 retained full EPPIN-binding capacity. Together, our findings identify two functional domains within mSEMG1 (Q32-V118 and R98-S220) that inhibit sperm hyperactivation by suppressing CatSper activity and differ in their EPPIN-binding capacities. These functional domains represent promising prototypes for the design of spermiostatic molecules, offering additional avenues for non-hormonal male contraception. Summary statementWe report a novel mechanism by which SEMG1, a major seminal plasma protein, modulates sperm hyperactivation by inhibiting a key calcium channel, offering new insights into sperm function and non- hormonal male contraception.

physiology↗

bioRxiv2025

SPINK3-sperm interaction determines a stable sperm subpopulation with intact CatSper channel

Sperm capacitation involves proteolytic remodeling of membrane proteins, including components of the CatSper calcium channel, which is essential for hyperactivation and male fertility. Here, we identify the seminal protease inhibitor SPINK3, a known decapacitation factor that suppresses premature capacitation in the female tract, as the first physiological inhibitor of CATSPER1 processing. In mouse sperm, SPINK3 blocks capacitation-induced CATSPER1 cleavage, preserving a subpopulation with intact CatSper channels and lacking pTyr development in the flagellum. SPINK3 localizes to the outer surface of the sperm principal piece membrane in a CatSper-dependent but non-quadrilateral pattern, stabilizes membrane organization, and delays cholesterol efflux. These results reveal SPINK3 as a multifunctional regulator of capacitation, shaping sperm subpopulations in the female reproductive tract. SIGNIFICANCE STATEMENTThis study unveils a novel physiological regulator from the male reproductive tract, SPINK3, which extracellularly controls the processing of CATSPER1, a key subunit of the CatSper calcium channel essential for sperm capacitation in mice. We show that SPINK3 binds to the sperm surface and prevents the processing of CATSPER1 during capacitation. SPINK3-sperm interaction is dependent on the presence of the sperm-specific CatSper channel. Our findings reveal that SPINK3 stabilizes sperm membranes providing new insights into the molecular regulation of sperm function and fertility. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/672324v3_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@164c737org.highwire.dtl.DTLVardef@1531499org.highwire.dtl.DTLVardef@16d7abaorg.highwire.dtl.DTLVardef@945453_HPS_FORMAT_FIGEXP M_FIG C_FIG

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↗

bioRxiv2018

The actin cytoskeleton of the mouse sperm flagellum is organized in a helical structure

Conception of a new mammalian organism is determined by the fusion of a sperm cell with an oocyte during fertilization. Motility is one of the features of sperm that allows them to succeed in fertilization, and their flagellum is essential for this function. Longitudinally, the flagellum divides into the midpiece, the principal piece, and the end piece. A precise cytoskeletal architecture of the sperm tail is key for the acquisition of fertilization competence. It has been proposed that the actin cytoskeleton plays essential roles in the regulation of sperm motility, however, actin organization in sperm remains elusive. In the present work, we found different types of actin structures in the sperm tail, using stochastic optical reconstruction microscopy (STORM). In the principal piece, actin is radially distributed between the axoneme and the plasma membrane. The actin-associated proteins spectrin and adducin are also found in these structures. Strikingly, polymerized actin in the midpiece forms a double-helix that accompanies mitochondria. Our findings illustrate a novel specialized structure of actin filaments in a mammalian cell.

cell biology↗