No abstract

Previously, we showed that epididymal sperm binding protein 1 (ELSPBP1) characterizes spermatozoa already dead before ejaculation in bovine. In this study, we investigated the presence of ELSPBP1 in bull genital tract as well as its acquisition by spermatozoa during epididymal transit. As assessed by real-time RT-PCR, ELSPBP1 was highly expressed in the caput and the corpus epididymis but was present in lower expression levels in the testis and the cauda epididymis. Immunohistochemistry revealed the same expression pattern. However, Western blot on tissue homogenates showed some discrepancies, as ELSPBP1 was found in a comparable concentration all along the epididymis. This difference was due to the presence of ELSPBP1 in the epididymal fluid. In both caput and cauda epididymal fluid, ELSPBP1 was associated with the epididymosomes, small membranous vesicles secreted by epithelial cells of the epididymis and implicated in the transfer of proteins to spermatozoa. As assessed by immunocytometry, ELSPBP1 was found on a subset of dead spermatozoa in caput epididymis but was found on all dead spermatozoa in cauda epididymis. To assess ELSPBP1 acquisition by spermatozoa, caput epididymal spermatozoa were incubated with cauda epididymosomes under various conditions. ELSPBP1 detection by immunocytometry assay revealed that only spermatozoa already dead before incubation were receptive to ELSPBP1 transfer by epididymosomes. This receptivity was enhanced by the presence of zinc in the incubation medium. This specificity for a sperm subpopulation suggests that an underlying mechanism is involved and that ELSPBP1 could be a tag for the recognition of dead spermatozoa during epididymal transit.

Delivery of recombinant proteins to therapeutic cells is limited by a lack of efficient methods. This hinders the use of transcription factors or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleoproteins to develop cell therapies. Here, we report a soluble peptide designed for the direct delivery of proteins to mammalian cells including human stem cells, hard-to-modify primary natural killer (NK) cells, and cancer cell models. This peptide is composed of a 6x histidine-rich domain fused to the endosomolytic peptide CM18 and the cell penetrating peptide PTD4. A less than two-minute co-incubation of 6His-CM18-PTD4 peptide with spCas9 and/or asCpf1 CRISPR ribonucleoproteins achieves robust gene editing. The same procedure, co-incubating with the transcription factor HoxB4, achieves transcriptional regulation. The broad applicability and flexibility of this DNA- and chemical-free method across different cell types, particularly hard-to-transfect cells, opens the way for a direct use of proteins for biomedical research and cell therapy manufacturing.

An increase in protein tyrosine phosphorylation occurs during sperm capacitation in numerous species. The involvement of Src-related tyrosine kinases in this phenomenon has been demonstrated using different inhibitors specifically targeting this family of enzymes. In mammals, this group of nonreceptor tyrosine kinases is made up of 8 members with similar SRC homology domain 3 (SH3) and SH2 domains. Although some members of this group of enzymes can compensate for one another, showing some redundancy, each is unique and may perform specific functions during male germ cell development. To further characterize the importance of Src-related tyrosine kinases in the events leading to proper sperm formation, and because no inhibitor affecting a single gene product exists, expression of Src, Yes1, Fyn, Lyn, Lck, Hck, Blk, and Fgr was assessed by real-time polymerase chain reaction in developing mouse testes and in enriched populations of mouse spermatogenic cells, revealing distinct expression profiles for each kinase during testis development and in isolated male germ cells. Immunolocalization of SRC, LYN, and HCK in adult mouse testes as well as in mature spermatozoa further confirmed differential localization of these kinases during spermatogenesis. Although mRNA levels of these latter kinases were higher in spermatogonia and spermatocytes than in spermatids, protein levels were highest in spermatids, suggesting delayed transcript translation. Taken together, these results clearly show an uneven expression of each kinase in different spermatogenic cells, indicating that each member may play a different role during spermatogenesis, in addition to highlighting the complexity of Src-related kinase expression regulation in male germ cells. Furthermore, differential localization of these tyrosine kinases in mature spermatozoa also suggests a specific role for each member in sperm function and integrity.