Seeing is believing: Current methods to observe sperm acrosomal exocytosis in real time

Balestrini, Paula A.; Jabloñski, Martina; Schiavi‐Ehrenhaus, Liza J.; Marı́n-Briggiler, Clara I.; Sánchez-Cárdenas, Claudia; Darszon, Alberto; Krapf, Darío; Buffone, Mariano G.

doi:10.1002/mrd.23431

Cited by 16 publications

(10 citation statements)

References 79 publications

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“…4B) spermatozoa. Consistent with this finding, we observed that NEN and BAM15 had no effect on other ATP-dependent human sperm physiological processes, including the acrosome reaction, a necessary pre-fertilization exocytotic event (29,30) that exposes key fertilization receptors (31,32) (Fig. S4A – C), and human sperm hyperactivated motility (33) (Fig.…”

Section: Resultssupporting

confidence: 82%

Mitochondrial uncouplers impair human sperm motility without altering ATP content

Skinner

Petersen

Unger

et al. 2022

Preprint

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Sperm motility is necessary for successful fertilization, but there remains controversy about whether human sperm motility is primarily powered by glycolysis or oxidative phosphorylation. To evaluate the plausibility of reducing human sperm mitochondrial ATP production as an avenue for contraceptive development, we treated human sperm with small-molecule mitochondrial uncouplers, which reduce mitochondrial membrane potential by inducing passive proton flow, and evaluated the effects on a variety of physiological processes that are critical for fertilization. We also sought to clarify the subcellular localization of Adenosine Nucleotide Translocator 4 (ANT4), a gamete-specific protein that has been suggested as a contraceptive target. We determined that ANT4 is mitochondrially localized, that induced mitochondrial uncoupling can be partially mediated by the ANT family, and that two uncouplers, Niclosamide Ethanolamine and BAM15, significantly decreased sperm progressive motility. However, these uncouplers did not reduce sperm ATP content or impair other physiological processes, implying that human sperm can rely on glycolysis for ATP production in the absence of functional mitochondria. Thus, since certain mitochondrial uncouplers impair motility through ATP-independent mechanisms, they could be useful ingredients in on-demand, vaginally-applied contraceptives. However, systemically delivered contraceptives that target sperm mitochondria to reduce their ATP production would need to be paired with sperm-specific glycolysis inhibitors.

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Section: Resultssupporting

confidence: 82%

Mitochondrial uncouplers impair human sperm motility without altering ATP content

Skinner

Petersen

Unger

et al. 2022

Preprint

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“…As mentioned before, this co-staining advantage of TLFC opens up the possibility of combining additional fluorescent dyes to further explore other physiological parameters in sperm. For instance, the simultaneous assessment of [Ca 2+ ] i changes and the sperm’s Pg-induced acrosome exocytosis could be performed using the appropriate combination of fluorescent labels (e.g., Fluo3 and a fluorescent-labeled lectin, respectively) [ 61 ]. This evaluation is of special interest, since the level of Pg-triggered acrosome exocytosis is yet another parameter associated with capacitation and fertility [ 30 , 62 ].…”

Section: Discussionmentioning

confidence: 99%

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

Matamoros-Volante

Castillo-Viveros

Torres-Rodríguez

et al. 2020

IJMS

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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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“…For this goal, epididymis was collected from all groups and was incubated in 1 mL PBS at 37°C for 15 min. Then, they were resuspended in a total volume of 1.8 mL, after adding 800 µL of 10% formalin buffer solution, and then stored on ice until analysis with a ow cytometry under illumination in the range of the 360-400 nm that is related to eGFP uorescence (32). A four-color FACS Calibur ow cytometer (BD bioscience) was used to collect the data and data were analyzed using the CellQuest Pro software package (BD bioscience).…”

Section: Flow Cytometry Of Enhanced Green Uorescent Protein Expression (Egfp)mentioning

confidence: 99%

Restoration of spermatogenesis in azoospermic mice by bone marrow mesenchymal stromal/stem cells conditioned medium

Zhankina

Afshar

Farrar

et al. 2022

Preprint

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Background One of the main cause of male infertility is non-obstructive azoospermia, which is not manageable medically. The first aim of the current research was to show the effect of extracellular vesicle-contained conditioned media (CM) instead of mesenchymal stromal/stem cells (MSCs) for treatment of non-obstructive azoospermia. In the next step, we aimed to study the differentiation potential of MSCs into spermatocytes after injection of MSCs in mice seminiferous tubules. This study has provided an applied treatment for busulfan-induced azoospermia using adipose tissue-derived (AT-MSCs) and bone marrow-derived MSCs (BM-MSCs) and bone marrow CM (BMCM) in animal models. Methods Thirty male adult Balb/C mice (30±5 g) and two female eGFP+/+Balb/C mice (30±5 g) were used to design experimental groups and to culture stem cells, respectively. Then, six groups including intact control, azoospermia, AT-MSC therapy, BM-MSC therapy, BMCM therapy, and spontaneous healing groups were considered. All groups except intact control were induced azoospermia by injecting two doses of busulfan (10 mg/kg) with 21 days’ interval. Testes of all mice were removed and studied through histomorphometry and flow cytometry analysis 60 days after treatment. Results Histomorphometry and flow cytometry evaluation of testes showed normal morphology of most of the seminiferous tubules of therapy groups as well as successful recovery of spermatogenesis, but spermatogenesis was not observed in the azoospermia group. It is worth notable that the results of the BM-MSC therapy group were more favorable than other therapy groups. Conclusions AT-MSC, BM-MSC and BMCM can be strongly suggested as candidates in the therapy of azoospermia.

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Seeing is believing: Current methods to observe sperm acrosomal exocytosis in real time

Cited by 16 publications

References 79 publications

Mitochondrial uncouplers impair human sperm motility without altering ATP content

Mitochondrial uncouplers impair human sperm motility without altering ATP content

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

Restoration of spermatogenesis in azoospermic mice by bone marrow mesenchymal stromal/stem cells conditioned medium

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