Characterization of proteases in the seminal plasma and spermatozoa of llama

Camelids' semen has peculiar characteristics that differentiate it from other species, including the highly viscous aspect of seminal plasma that greatly difficult sperm manipulation and the development of techniques such as cryopreservation, artificial insemination, and/or in vitro fertilization. The presence of proteases in the seminal plasma is responsible for semen liquefaction, and sperm functionality to achieve fertilization. The enzymatic and molecular composition of the semen of llama remains unknown. Therefore, the goal of the study was to characterize the protease activity and composition of the seminal plasma and sperm of llama semen. The proteolytic activity was performed using gelatine zymography and the composition by mass-spectrometry. Metallo-proteases were the major source of gelatinolytic activity in seminal plasma, while serine-peptidases were the main enzymes of sperm cells. Matrix Metalloproteinase 2 (MMP2) was the most prominent metallo-protease of llama seminal plasma characterized under the exposure of different inhibitors (EDTA and benzamidine) and by a specific immunodetection. Moreover, the prostate and epididymis were identified as potential sites of its synthesis and secretion. Outstandingly, this metalloproteinase was undetectable in llama sperm. Regarding, the molecular composition of semen by mass-spectrometry, 4 metallo-, 9 serine-, 8 threonine-, and 1 aspartic-peptidases were identified alongside 15 regulators in the sperm cell; where 24 were directly or indirectly interacting. Whereas 6 metallo-, 12 serine-, 3 cysteine-, and 1 aspartic-peptidases were identified, besides 7 inhibitors and 5 regulators in llama seminal plasma where 30 of them were directly or indirectly interconnected. This is the first study describing a partial degradome of llama seminal plasma and spermatozoa suggesting significant differences especially the absence of MMP2 in spermatozoa in contrast to data observed in other species. The characterization of proteases in llama semen will provide a better understanding of the molecular mechanisms involved in the in vivo or in vitro fertilization process in this species.