Assembly of the fluorescent acrosomal matrix and its fate in fertilization in the water strider, Aquarius remigis

Abstract

Animal sperm show remarkable diversity in both morphology and molecular composition. Here we provide the first report of intense intrinsic fluorescence in an animal sperm. The sperm from a semi-aquatic insect, the water strider, Aquarius remigis, contains an intrinsically fluorescent molecule with properties consistent with those of Flavin Adenine Dinucleotid (FAD) which appears first in the acrosomal vesicle of round spermatids and persists in the acrosome throughout spermiogenesis. Fluorescence recovery after photobleaching reveals that the fluorescent molecule exhibits unrestricted mobility in the acrosomal vesicle of round spermatids, but is completely immobile in the acrosome of mature sperm. Fluorescence polarization microscopy shows a net alignment of the fluorescent molecules in the acrosome of the mature sperm but not in the acrosomal vesicle of round spermatids. These results suggest that acrosomal molecules are rearranged in the elongating acrosome and FAD is incorporated into the acrosomal matrix during its formation. Further, we followed the fate of the acrosomal matrix in fertilization utilizing the intrinsic fluorescence. The fluorescent acrosomal matrix was observed inside the fertilized egg and remained structurally intact even after gastrulation started. This observation suggests that FAD is not released from the acrosomal matrix during the fertilization process or early development and supports an idea that FAD is involved in the formation of the acrosomal matrix. The intrinsic fluorescence of the A. remigis acrosome will be a useful marker for following spermatogenesis and fertilization.

Polarized light microscopy in reproductive and developmental biology

“…The underlying grayscale image represents the intensity ratio between maximum and minimum fluorescence (black for ratio = 1, white for ratio = 4) measured at each location. Reprinted with permission from Miyata et al ().…”

“…The underlying grayscale image represents the intensity ratio between maximum and minimum fluorescence (black for ratio ¼ 1, white for ratio ¼ 4) measured at each location. Reprinted with permission from Miyata et al (2011). remarkable molecular choreography of septin assemblies involved in cell division was first discovered in the yeast bud neck (Vrabioiu and Mitchison, 2006) and then revealed in other cell types (DeMay et al, 2011a).…”

FRAP, FLIM, and FRET: Detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy

“…FRAP has also been used to monitor the incorporation of a flavin adenine dinucleotide (FAD)‐associated protein into the acrosomal matrix of sperm from the water strider, Aquarius remigis , during spermatogenesis and spermiogenesis (Fig. ) (Miyata et al, ). Along with fluorescence polarization microscopy, this study demonstrated that, following meiosis, the FAD‐containing protein rapidly accumulates into a rigid matrix that ultimately extends as a ∼2,500 μm helical structure from base to tip.…”

“…FRAP has been applied to many developmental studies, including the original studies monitoring the behavior of acetylcholine receptors in chick myotubes (Axelrod et al, 1976b); the extent of lipid mobility in gametes (Wolf and Voglmayr, 1984;Wolf et al, 1986Wolf et al, , 1988; the distribution and dynamics of galactosyltransferase on the surface of mouse sperm (Cardullo and Wolf, 1995) and ADAM1/ ADAM2 on guinea pig sperm (Hunnicutt et al, 2008); and the formation of an acrosomal matrix during spermatogenesis in water strider sperm ( Fig. 3) (Miyata et. al., 2011).…”

“…The recovery curves document that flavin adenine dinucleotide (FAD)-conjugated monomers are freely diffusing in round spermatids, but become immobilized into a rigid helical matrix in mature sperm. Reprinted with permission fromMiyata et al (2011).Molecular Reproduction & DevelopmentDE LOS SANTOS ET AL.…”