Differential expression and localization of TRPV channels in the mature sperm of Anas platyrhynchos.

Sperm cells perform precise chemotactic and thermotactic movement which is crucial for fertilization. However, the key molecules involved in detection of different chemical and physical stimuli which guide the sperm during navigation are not well understood. Ca2+ -signalling mediated by ion channels seem to play important role in motility and other fertility parameters. In this work, we explored the endogenous localization pattern of TRPV channels in the mature spermatozoa of avian species. Using sperm from white pekin duck (Anas platyrhynchos) as the representative avian model, we demonstrate that duck sperm endogenously express the thermosensitive channels TRPV1, TRPV2, TRPV3, TRPV4, and highly Ca2+ -selective channels TRPV5 and TRPV6 in specific yet differential locations. All of these TRPV channels are enriched in the sperm tail, indicating their relevance in sperm motility. Interestingly, the TRPV3 and TRPV4 channels are present in the mitochondrial region. Calcium selective TRPV5 channel is exclusively present in sperm tail and is most abundant among the TRPV channels. This is the first report describing the endogenous presence of TRPV2 and TRPV3 channels in the sperm of any species. Using confocal imaging and super-resolution imaging, we demonstrate that though the TRPV channels are evolutionarily closely related, they have distinct localization pattern in the duck sperm, which could impact their role in fertilization.

TRPA1 is selected as a semi-conserved channel during vertebrate evolution due to its involvement in spermatogenesis.

TRPA1 is a non-selective cation channel originated in invertebrates. The genomic locus containing TRPA1 gene remains highly conserved and retained in all vertebrates. TRPA1 gene is evolutionarily selected, yet maintained as a highly diverged protein. Throughout the vertebrate evolution, the extracellular loops of TRPA1 become most diverged indicating that TRPA1 may be involved in detecting large spectrum and uncertain stimulus which is critical for adaptive benefit. We tested the expression of TRPA1 in mature sperm from different vertebrates. This is the first report demonstrating that TRPA1 is expressed endogenously in mature spermatozoa of multiple species representing entire vertebrate phyla. However, its specific localization within sperm remains species-specific. Accordingly, we report that in rodents TRPA1 expression correlates with different stages of spermatogenesis. We propose that presence of endogenous TRPA1 in testes and in mature sperm provides reproductive benefit.

Light and electron microscopic study of mature spermatozoa from White Pekin duck (Anas platyrhynchos): an ultrastructural and molecular analysis.

The morphology, ultrastructure, and functions of mature avian spermatozoa have been of immense commercial and scientific interest for several reasons. This is mainly important in case of birds in poultry production, conservation, and in the use of sperm morphometry as phylogenetic evidence. Avian spermatozoa share complex or no correlation of sperm morphometry with respect to testis and/or body size as described before. In this work, we have isolated mature spermatozoa from White Pekin duck (Anas platyrhynchos) by non-invasive methods and performed several immunostaining analysis as well as cytochemical analysis using electron and light microscopes. Here, we report the presence of different post-translationally modified tubulin such as tyrosinated-, detyrosinated-, acetylated-, polyglutamylated-, and glycylated-tubulin in specific regions of the mature spermatozoa. By using field-emission scanning electron microscope, we confirm the presence of acrosome-like structure at the tip of the sperm head. However, this structure remains non-reactive to common lectins such as Peanut Agglutinin (PNA) and cholesterol-sensitive dyes such as Filipin. We report that this acrosomal structure is primarily made of lipid-based structures and is resistant to 0.1% Triton X100. Confocal microscopy and super resolution structured illumination microscopy study indicates that the nucleus is bifurcated at the tip region. By using specific markers, we report that the perforatorium structure present at the tip of the spermatozoa head contains specialized organelles that is similar to atypical mitochondria. We propose that these ultrastructural and molecular parameters can be used as species-specific features. The bifurcated nucleus and presence of atypical mitochondria within this structure may be relevant for the complex mitochondrial inheritance and mitochondrial heteroplasmy observed in case of avian population.