P-077 Human sperm acrosome becomes alkalinized during capacitation
Authors: Carrasquel Martínez G , Aldana A , Carneiro J , Treviño C , Darszon A 
Published: June 2022

Abstract

Study question
Does during the human sperm capacitation the acrosomal pH (pHa) increases naturally and how it change can be modulate?

Summary answer
Sperm acrosome is progressively alkalinized during capacitation in a HCO3- and Ca2+ dependent manner. pHa is regulated by V-ATPase and involving the sAC-PKA signaling pathway.

What is known already
Capacitation involves biochemical and physiological modifications undergone by sperm as they travel through the female reproductive tract. These modifications prepare the sperm to enact the acrosome reaction (AR), an essential step for egg fertilization. Capacitation requires [Ca2+]i and pHi increases. Mouse sperm capacitation is accompanied by acrosomal alkalinization and artificial elevation of the pHa is sufficient to trigger the AR in mouse and human sperm, but it is unknown if the pHa increases physiologically during human sperm capacitation.

Study design, size, duration
In this work we followed pHa changes during human sperm capacitation (6 h) and evaluated the mechanisms of its regulation using pharmacological tools. Sperm were stained with LysoSensor green and the relative fluorescence measurements for pHa kinetics during capacitation were performed at 15 min of incubation (initial time: 0 h) and after 3 and 6 h of incubation. Independent experiments of at least 3 donors were performed per condition.

Participants/materials, setting, methods
We used semen samples from 11 healthy donors fulfilled the requirements defined by the OMS (OMS, 2010). Sperm recovered by swim-up were incubated in a capacitating or non-capacitating HTF medium. LysoSensor green fluorescence dye was used to evaluate pHa dynamic during capacitation using single cell epifluorescence microscopy and image-based flow cytometry. Additionally, pHa was evaluated in the presence of specific inhibitors to assess regulatory mechanisms. A linear model was used to study the pHa kinetics.

Main results and the role of chance
Human sperm pHa is alkalinized progressively at 3 and 6 h of the capacitation (p &lt; 0.05).
V-ATPase activity mainly responsible for the acrosome acidity and can modulate at least in part acrosome alkalinization during capacitation (***p&lt;0.001, *p&lt;0.05). V-ATPase is immunolocalized to the acrosome and equatorial segment of human sperm.
Bicarbonate (HCO3-) was essential for the pHa increase observed during capacitation and its absence inhibits completely the acrosome alkalinization (**p&lt;0.01, *p&lt;0.05). Inhibition of the Cystic Fibrosis Transmembrane Regulator (CFTR) or the Na+/HCO3- Cotransporter (NBC) partially blocked the pHa changes during capacitation.
Extracelular Ca2+ is involved in pHa regulation during capacitation. Under low external Ca2+ the pHa regulation is altered and occurred delay in acrosome alkalinization (*p &lt; 0.05 and **p &lt; 0.001).
Finally, we tested a pharmacological blockade of sAC and PKA during capacitation. sAC inhibition did not prevent acrosome alkalinization, but it significantly reduced the rate of the pHa changes as compared to the capacitation control (***p=0.0009), revealing a certain influence. On the other hand, PKA inhibition induced acrosomal acidification at the start of capacitation and accelerated significantly the alkalinization (*p&lt;0.05). Together these results allow us to assume that a signaling pathway mediated to sAC is participating in the pHa regulation.

Limitations, reasons for caution
Experiments were performed in vitro and due to model limitations is difficult to establish strategies that allow us to know more directly, other molecular mechanisms that regulate the pHa.

Wider implications of the findings
Our findings show that pHa is subject of regulation during human sperm capacitation. The results provide new insights regarding the molecular mechanisms involved in human sperm capacitation and this knowledge results interesting for the study of specific molecules that inhibit capacitation or AR.

Trial registration number
Not applicable