A genetically targeted sensor reveals spatial and temporal dynamics of acrosomal calcium and sperm acrosome exocytosis.

Secretion of the acrosome, a single vesicle located rostrally in the head of a mammalian sperm, through a process known as "acrosome exocytosis" (AE), is essential for fertilization. However, the mechanisms leading to and regulating this complex process are controversial. In particular, poor understanding of Ca2+ dynamics between sperm subcellular compartments and regulation of membrane fusion mechanisms have led to competing models of AE. Here, we developed a transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE) to investigate the spatial and temporal Ca2+ dynamics in AE in live sperm. AcroSensE combines a genetically encoded Ca2+ indicator (GCaMP) fused with an mCherry indicator to spatiotemporally resolve acrosomal Ca2+ rise (ACR) and membrane fusion events, enabling real-time study of AE. We found that ACR is dependent on extracellular Ca2+ and that ACR precedes AE. In addition, we show that there are intermediate steps in ACR and that AE correlates better with the ACR rate rather than absolute Ca2+ amount. Finally, we demonstrate that ACR and membrane fusion progression kinetics and spatial patterns differ with different stimuli and that sites of initiation of ACR and sites of membrane fusion do not always correspond. These findings support a model involving functionally redundant pathways that enable a highly regulated, multistep AE in heterogeneous sperm populations, unlike the previously proposed "acrosome reaction" model.

Comparison of Alfaxalone and Tricaine Methanesulfonate Immersion Anesthesia And Alfaxalone Residue Clearance In Rainbow Trout (Oncorhynchus Mykiss).

Alfaxalone, a synthetic neuroactive steroid, has been tested as an immersion anesthetic in ornamental fish, but its safety and efficacy in sport fish have not been investigated. In the current study, we compared the physiologic and behavioral effects of alfaxalone with those of tricaine methanesulfonate (MS222) for anesthesia of rainbow trout (Oncorhynchus mykiss) via water immersion. We also analyzed alfaxalone-exposed tissues to determine residue clearance times. Fish were anesthetized for 10 min by immersion in low-dose alfaxalone (Alow; 5 mg/L induction, 1 mg/L maintenance), high-dose alfaxalone (Ahigh; 5 mg/L induction, 2 mg/L maintenance), or MS222 (MS; 150 mg/L induction, 100 mg/L maintenance). Fish received all 3 treatments, separated by a washout period of at least 18 d in a blinded, complete crossover design. We hypothesized that immersion in Alow or Ahigh would provide a stable plane of anesthesia in rainbow trout, with dose-dependent time to recovery, and that opercular rates and depths of anesthesia would be equivalent to that of MS222. The time to anesthesia induction was longer for alfaxalone than MS222 but averaged less than 100 s. The time to recovery from anesthesia was also longer for alfaxalone than MS222, with significantly shorter recovery time for Alow than for Ahigh. All treatments decreased opercular rate and response to noxious stimuli. Alfaxalone residue clearance was greater than 80% from all tissues within 1 h, greater than 99% from muscle within 4 h, and 100% from all tissues within 36 h after exposure. We conclude that alfaxalone immersion at 5 mg/L for induction and 2 mg/L for maintenance provides a safe, viable alternative to MS222 for the anesthesia of rainbow trout.