Time-Lapse Flow Cytometry: A Robust Tool to Assess Physiological Parameters Related to the Fertilizing Capability of Human Sperm

Plasma membrane (PM) hyperpolarization, increased intracellular pH (pHi), and changes in intracellular calcium concentration ([Ca2+]i) are physiological events that occur during human sperm capacitation. These parameters are potential predictors of successful outcomes for men undergoing artificial reproduction techniques (ARTs), but methods currently available for their determination pose various technical challenges and limitations. Here, we developed a novel strategy employing time-lapse flow cytometry (TLFC) to determine capacitation-related membrane potential (Em) and pHi changes, and progesterone-induced [Ca2+]i increases. Our results show that TLFC is a robust method to measure absolute Em and pHi values and to qualitatively evaluate [Ca2+]i changes. To support the usefulness of our methodology, we used sperm from two types of normozoospermic donors: known paternity (subjects with self-reported paternity) and no-known paternity (subjects without self-reported paternity and no known fertility problems). We found relevant differences between them. The incidences of membrane hyperpolarization, pHi alkalinization, and increased [Ca2+]i were consistently high among known paternity samples (100%, 100%, and 86%, respectively), while they varied widely among no-known paternity samples (44%, 17%, and 45%, respectively). Our results indicate that TLFC is a powerful tool to analyze key physiological parameters of human sperm, which pending clinical validation, could potentially be employed as fertility predictors.

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Membrane hyperpolarization abolishes calcium oscillations that prevent induced acrosomal exocytosis in human sperm

The FASEB Journal ◽

10.1096/fj.202002333rr ◽

2021 ◽

Vol 35 (6) ◽

Author(s):

Paula A. Balestrini ◽

Claudia Sanchez‐Cardenas ◽

Guillermina M. Luque ◽

Carolina Baro Graf ◽

Jessica M. Sierra ◽

...

Keyword(s):

Human Sperm ◽

Calcium Oscillations ◽

Membrane Hyperpolarization ◽

Acrosomal Exocytosis

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Assessment of human sperm acrosome reaction by flow cytometry: validation and evaluation of the method by fluorescence-activated cell sorting**Presented in part at the 48th Annual Meeting of The American Fertility Society, New Orleans, Louisiana, November 2 to 5, 1992.

Fertility and Sterility ◽

10.1016/s0015-0282(16)56413-9 ◽

1993 ◽

Vol 60 (6) ◽

pp. 1076-1081 ◽

Cited By ~ 11

Author(s):

Meike L. Uhler ◽

Andrew Leung ◽

Steven Y.W. Chan ◽

Ingrid Schmid ◽

Christina Wang

Keyword(s):

Flow Cytometry ◽

New Orleans ◽

Annual Meeting ◽

Cell Sorting ◽

Acrosome Reaction ◽

Human Sperm ◽

Fluorescence Activated Cell Sorting ◽

American Fertility Society ◽

Sperm Acrosome Reaction ◽

Sperm Acrosome

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Zidovudine-Mediated Autophagy Inhibition Enhances Mitochondrial Toxicity in Muscle Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01443-18 ◽

2018 ◽

Vol 63 (1) ◽

Cited By ~ 2

Author(s):

H. Lin ◽

M. V. Stankov ◽

J. Hegermann ◽

R. Budida ◽

D. Panayotova-Dimitrova ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Viability ◽

Therapeutic Drug ◽

Ros Generation ◽

Mtor Inhibition ◽

Membrane Hyperpolarization ◽

Thymidine Analogue ◽

Drug Concentrations ◽

Mitochondrial Functions ◽

The Impact

ABSTRACT Nucleoside reverse transcriptase inhibitors (NRTI), such as zidovudine (AZT), are constituents of HIV-1 therapy and are used for the prevention of mother-to-child transmission. Prolonged thymidine analogue exposure has been associated with mitochondrial toxicities to heart, liver, and skeletal muscle. We hypothesized that the thymidine analogue AZT might interfere with autophagy in myocytes, a lysosomal degradation pathway implicated in the regulation of mitochondrial recycling, cell survival, and the pathogenesis of myodegenerative diseases. The impact of AZT and lamivudine (3TC) on C2C12 myocyte autophagy was studied using various methods based on LC3-green fluorescent protein overexpression or LC3 staining in combination with Western blotting, flow cytometry, and confocal and electron microscopy. Lysosomal and mitochondrial functions were studied using appropriate staining for lysosomal mass, acidity, cathepsin activity, as well as mitochondrial mass and membrane potential in combination with flow cytometry and confocal microscopy. AZT, but not 3TC, exerted a significant dose- and time-dependent inhibitory effect on late stages of autophagosome maturation, which was reversible upon mTOR inhibition. Inhibition of late autophagy at therapeutic drug concentrations led to dysfunctional mitochondrial accumulation with membrane hyperpolarization and increased reactive oxygen species (ROS) generation and, ultimately, compromised cell viability. These AZT effects could be readily replicated by pharmacological and genetic inhibition of myocyte autophagy and, most importantly, could be rescued by pharmacological stimulation of autophagolysosomal biogenesis. Our data suggest that the thymidine analogue AZT inhibits autophagy in myocytes, which in turn leads to the accumulation of dysfunctional mitochondria with increased ROS generation and compromised cell viability. This novel mechanism could contribute to our understanding of the long-term side effects of antiviral agents.

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