Capacitation is a series of physiological, biochemical, and metabolic changes experienced by mammalian spermatozoa. These changes enable them to fertilize eggs. The capacitation prepares the spermatozoa to undergo the acrosomal reaction and hyperactivated motility. Several mechanisms that regulate capacitation are known, although they have not been fully disclosed; among them, reactive oxygen species (ROS) play an essential role in the normal development of capacitation. NADPH oxidases (NOXs) are a family of enzymes responsible for ROS production. Although their presence in mammalian sperm is known, little is known about their participation in sperm physiology. This work aimed to identify the NOXs related to the production of ROS in guinea pig and mouse spermatozoa and define their participation in capacitation, acrosomal reaction, and motility. Additionally, a mechanism for NOXs’ activation during capacitation was established. The results show that guinea pig and mouse spermatozoa express NOX2 and NOX4, which initiate ROS production during capacitation. NOXs inhibition by VAS2870 led to an early increase in the capacitation and intracellular concentration of Ca²⁺ in such a way that the spermatozoa also presented an early acrosome reaction. In addition, the inhibition of NOX2 and NOX4 reduced progressive motility and hyperactive motility. NOX2 and NOX4 were found to interact with each other prior to capacitation. This interaction was interrupted during capacitation and correlated with the increase in ROS. Interestingly, the association between NOX2-NOX4 and their activation depends on calpain activation, since the inhibition of this Ca²⁺-dependent protease prevents NOX2-NOX4 from dissociating and ROS production. The results indicate that NOX2 and NOX4 could be the most important ROS producers during guinea pig and mouse sperm capacitation and that their activation depends on calpain.

Mammalian spermatozoa undergo a series of physiological and biochemical changes in the oviduct that lead them to acquire the ability to fertilize eggs. During their transit in the oviduct, spermatozoa face a series of environmental changes that can affect sperm viability. A series of ion channels and transporters, as well as the sperm cytoskeleton, allow spermatozoa to remain viable and functional. Cl⁻ channels such as TMEM16A (calcium-activated chloride channel), CFTR (cystic fibrosis transmembrane conductance regulator), and ClC3 (chloride voltage-gated channel 3) are some of the ion transporters involved in maintaining cellular homeostasis. They are expressed in mammalian spermatozoa and are associated with capacitation, acrosomal reaction, and motility. However, little is known about their role in maintaining sperm volume. Therefore, this study aimed to determine the mechanism through which TMEM16A maintains sperm volume during capacitation. The effects of TMEM16A were compared to those of CFTR and ClC3. Spermatozoa were capacitated in the presence of specific TMEM16A, CFTR, and ClC3 inhibitors, and the results showed that only TMEM16A inhibition increased acrosomal volume, leading to changes within the acrosome. Similarly, only TMEM16A inhibition prevented actin polymerization during capacitation. Further analysis showed that TMEM16A inhibition also prevented ERK1/2 and RhoA activation. On the other hand, TMEM16A and CFTR inhibition affected both capacitation and spontaneous acrosomal reaction, whereas ClC3 inhibition only affected the spontaneous acrosomal reaction. In conclusion, during capacitation, TMEM16A activity regulates acrosomal structure through actin polymerization and by regulating ERK1/2 and RhoA activities.

While most lizard species are polygamous, dominant males typically have greater access to females and copulate more frequently than submissive ones. Several studies suggest that applying assisted reproduction techniques to this taxon could be an effective tool to enhance conservation programs. Therefore, the proper selection of males for assisted breeding programs is crucial for their overall success. In this study, we report for the first time data on semen and sperm quality from dominant males of the Mexican lizard Sceloporus torquatus using techniques commonly employed in assessing mammalian fertility. The semen and sperm characteristics were as follows: volume, 14.0 µL; sperm concentration, 125.7 × 10⁶ cells/mL; wave motion, 2.7; total motility, 87.8%; sperm viability, 89.0%; normal morphology, 88.8%; plasma membrane integrity, 87.7%; low plasma membrane fluidity, 94.9%; capacitation status: uncapacitated (F), 90.5%; capacitated (B), 7.2%; sperm with acrosome reaction (AR), 2.3%; and, acrosome integrity, 88.8%. These results suggest that, according to mammalian fertility parameters, dominant males of S. torquatus are fertile. This series of tests provides a valuable tool for conducting a comprehensive analysis of the functional changes that may occur in sperm handling during assisted processes, and that may contribute—along with the proper selection of individuals—to reproductive success.

Isthmus is the region of the oviduct considered a reservoir for spermatozoa, where they are retained and then released synchronously with ovulation. Integrins mediate this interaction, and it is suggested that they regulate the viability and capacitation of spermatozoa. Spermatozoa retained in the oviductal epithelial cells show specific characteristics: normal morphology, intact acrosome and plasma membrane, no DNA fragmentation, and low levels of intracellular Ca²⁺ and protein phosphorylation at Tyr. This work aimed to define spermatozoa’s ability to adhere to an immobilized fibronectin matrix and its effects on their viability and capacitation. We found that guinea pig spermatozoa showed a high affinity for adhering to an immobilized fibronectin matrix but not to those made up of type 1 collagen or laminin. This interaction was mediated by integrins that recognize the RGD domain. Spermatozoa adhered to an immobilized fibronectin matrix were maintained in a state of low capacitation: low levels of intracellular concentration of Ca²⁺, protein phosphorylation in Tyr and F-actin. Also, spermatozoa kept their plasma membrane and acrosome intact, flagellum beating, and showed low activation of caspases 3/7. The spermatozoa adhered to the immobilized fibronectin matrix, gradually detached, forming rosettes and did not undergo a spontaneous acrosomal reaction but were capable of experiencing a progesterone-induced acrosomal reaction. In conclusion, the adhesion of spermatozoa to an immobilized fibronectin matrix alters the physiology of the spermatozoa, keeping them in a steady state of capacitation, increasing their viability in a similar way to what was reported for spermatozoa adhered to oviductal epithelial cells.

Temephos is a larvicide used in health campaigns against disease vectors, classified as slightly toxic. However, it has adverse effects on sperm function, the mechanisms of which are unknown. During fertilization, sperm are capacitated and proteins undergo tyrosine phosphorylation (p-Tyr), followed by actin cytoskeleton remodeling, which is important for the acrosome reaction (AR). Finally, IZUMO1 relocation during the AR is essential for gamete fusion. This study aimed to investigate the mechanism by which temephos decreases fertilization in male rats exposed to 10 or 100 mg/kg/day/5 days (WHO-NOAEL and LOAEL, respectively) and sacrificed 1-day after treatment. Sperm cells were collected from the cauda epididymis-vas deferens. No effects were observed with 10 mg/kg/day/5 days. However, at 100 mg/kg/day/5 days, the fertilization rate decreased (22%) and the chlortetracycline assay showed a decrease in Ca²⁺ ionophore-induced AR (49%), suggesting that functional AR was impaired. This was confirmed by the decrease in p-Tyr and actin polymerization, which reduced IZUMO1 relocation. Furthermore, significant increases in protein serine and threonine phosphorylation (p-Ser and p-Thr) were observed, which may contribute to these effects. These results suggest that temephos exposure reduced the fertilization rate by decreasing sperm capacitation and actin polymerization, thereby affecting AR and IZUMO1 migration.