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Results showed that reduced fluorescence intensity of the MitoTracker DeepRed FM was correlated with reduced mitochondrial ΔΨm. Induction of an electrochemical gradient across the inner mitochondrial membrane (ΔΨm) is a reliable marker of mitochondrial activity and is a vital step during the sperm maturation process [41]. A decrease in ΔΨm is a well-known consequence of capacitation, and is associated with the acrosome reaction [44]. This study documents that the use of a real-time method based on high-resolution fluorescence microscopy is the basis for an analysis of the capacitation-related events associated with the decrease in mitochondrial membrane potential, since the early mitochondrial depolarization described here was not associated with an acrosome reaction. Mitochondrial depolarization was reported here in real-time for the first time for sperm cells during capacitation. Such changes may be related to the activation of mitochondrial fission, which may result in dysfunctional mitochondria [65,66]. Alternatively, mitochondrial depolarization may be related to the increase in reactive oxygen species (ROS) production during capacitation [67,68]. ROS are produced by spermatozoa in response to capacitation-associated changes, and this may be associated with reduced sperm motility [44]. The decrease in mitochondrial membrane potential associated with the capacitation-associated increase in ROS production was already identified in the mid-1970s by S. Hatch, W. Gordon, and J. Phillips [68]. A change in mitochondrial membrane potential has also been reported in a previous model of human sperm capacitation in vitro [69]. This latter study used a different method to assess the mitochondrial membrane potential, namely time-lapse microscopy. The authors reported that sperm cells underwent a loss of mitochondrial membrane potential during the mid-to-late phase of capacitation. Additional confocal microscopy studies are required to verify the precise order of events occurring during the sperm maturation process and to determine the relationship between mitochondria and the sperm acrosome reaction, which is still obscure. Surprisingly, the mitochondrial depolarization observed here is not associated with an acrosome reaction. In fact, the percentage of sperm cells that underwent an acrosome reaction remained low over time.

Stardew Valley is one of those games that you can play forever. If you've completed the Community Center or the Joja route and reached max friendship with all of the villagers, you may think you've done most of what there is to do. However, that's just the start.

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