Characterization of different oligomeric forms of CRISP2 in the perinuclear theca versus the fibrous tail structures of boar spermatozoa

Mammalian sperm carry a variety of highly condensed insoluble protein structures such as the perinuclear theca, the fibrous sheath and the outer dense fibers, which are essential to sperm function. We studied the role of cysteine rich secretory protein 2 (CRISP2); a known inducer of non-pathological protein amyloids, in pig sperm with a variety of techniques. CRISP2, which is synthesized during spermatogenesis, was localized by confocal immunofluorescent imaging in the tail and in the post-acrosomal region of the sperm head. High resolution localization by immunogold labeling electron microscopy (EM) of ultrathin cryosections revealed that CRISP2 was present in the perinuclear theca and neck region of the sperm head, as well as in the outer dense fibers and the fibrous sheath of the sperm tail. Interestingly, we found that under native, non-reducing conditions CRISP2 formed oligomers both in the tail and the head but with different molecular weights and different biochemical properties. The tail oligomers were insensitive to reducing conditions but nearly complete dissociated into monomers under 8 M urea treatment, while the head 250 kDa CRISP2 positive oligomer completely dissociated into CRISP2 monomers under reducing conditions. The head specific dissociation of CRISP2 oligomer is likely a result of the reduction of various sulfhydryl groups in the cysteine rich domain of this protein. The sperm head CRISP2 shared typical solubilization characteristics with other perinuclear theca proteins as was shown with sequential detergent and salt treatments. Thus, CRISP2 is likely to participate in the formation of functional protein complexes in both the sperm tail and sperm head, but with differing oligomeric organization and biochemical properties. Future studies will be devoted to the understand the role of CRISP2 in sperm protein complexes formation and how this contributes to the fertilization processes.

Early signs of toxicity in testes and sperm of rats exposed to low cadmium doses

The cadmium (Cd) concentration in the environment has increased as a consequence of anthropogenic activity. The objective of this study was to determine early signs of Cd toxicity in testes and sperm as possible biomarkers. The dose orally administered to Wistar rats was within the range where chronic toxicity can appear. At the light microscopic level, gonads presented preserved cytoarchitecture throughout treatment; however, after the second month, transmission electron microscopy (TEM) revealed disruption of the blood–testis barrier. The study of sperm with light microscopy showed defects in gamete morphology after 2 months of treatment. Another parameter that revealed alteration was sperm motility after 3 months of treatment. TEM was used to analyze the flagellum, which in the midpiece showed aberrant mitochondria and displacement of outer dense fibers in relation to the central axoneme after 2 months. The data obtained were associated with Cd concentration in the testes, an increase in its levels being observed in a time-dependent manner. The results provided in this study demonstrated that early signs of Cd toxicity were observed in gonads and gametes during the second month of the treatment, generating morphological and functional alterations in the sperm that could lead to infertility.

Intraflagellar transport is essential for mammalian spermiogenesis but is absent in mature sperm

Drosophila sperm are unusual in that they do not require the intraflagellar transport (IFT) system for assembly of their flagella. In the mouse, the IFT proteins are very abundant in testis, but we here show that mature sperm are completely devoid of them, making the importance of IFT to mammalian sperm development unclear. To address this question, we characterized spermiogenesis and fertility in the Ift88Tg737Rpw mouse. This mouse has a hypomorphic mutation in the gene encoding the IFT88 subunit of the IFT particle. This mutation is highly disruptive to ciliary assembly in other organs. Ift88−/− mice are completely sterile. They produce ∼350-fold fewer sperm than wild-type mice, and the remaining sperm completely lack or have very short flagella. The short flagella rarely have axonemes but assemble ectopic microtubules and outer dense fibers and accumulate improperly assembled fibrous sheath proteins. Thus IFT is essential for the formation but not the maintenance of mammalian sperm flagella.