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The importance of the ovarian extracellular environment and tissue rigidity on follicle survival and development has gained attention in recent years. Our laboratory has anecdotally observed differences in the rigidity of domestic cat and dog ovarian cortical tissues, which have been postulated to underlie the differences in in vitro culture responses between the species, wherein cat ovarian tissues display higher survival in extended incubation. Here, the tensile strengths of cat and dog ovarian cortical tissues were compared via micropipette aspiration. The underlying collagen patterns, including fiber length, thickness, alignment, curvature, branch points and end points, and overall tissue lacunary and high-density matrix (HDM) were quantified via picrosirius red staining and TWOMBLI analysis. Finally, we explored the potential of MMP (−1 and −9) and TIMP1 supplementation in modulating tissue rigidity, collagen structure, and follicle activation in vitro. No differences in stiffness were observed between cat or dog cortical tissues, or pre- versus post-pubertal status. Cat ovarian collagen was characterized by an increased number of branch points, thinner fibers, and lower HDM compared with dog ovarian collagen, and cat tissues exposed to MMP9 in vitro displayed a reduced Young's modulus. Yet, MMP exposure had a minor impact on follicle development in vitro in either species. This study contributes to our growing understanding of the interactions among the physical properties of the ovarian microenvironment, collagen patterns, and follicle development in vitro. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (A low ; 5 mg/L induction, 1 mg/L maintenance), high-dose alfaxalone (A high ; 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 A low or A high 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 A low 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.