Identificação de peptídeos do veneno de Hadruroides lunatus com atividade antiastenospermia

Abstract

The cryopreservation of horse semen plays a crucial role in genetic improvement by allowing the wide use of high-quality breeders. However, studies show that sperm motility decreases after cryopreservation, hindering their ability to fertilize oocytes, especially in animals that already have asthenospermia, a condition characterized by reduced or absence of motility in recent ejaculate. The search for effective drugs that can increase sperm motility plays a crucial role in assisted reproduction techniques. It is necessary to improve the success rates of fertilization and also in the processes of cryopreservation from semen samples. In this context, the study aimed to use in silico approaches to develop four peptide molecules capable of improving sperm motility. The proposed peptides comprise conserved regions between Spermaurin, a protein capable of increasing sperm motility, and the Hadruroides lunatus protein, similar to Spermaurin. The peptides, called Conf_1, Conf_2, Linear_1 and Linear_2 were synthesized and validated using in vitro methodologies. The four peptides were hand- designed based on the identified key amino acids. The chemical synthesis of the peptides was performed using the Fmoc method. Next, evaluation of the ability of engineered peptides to improve sperm motility was conducted through computer-assisted analysis of sperm from stallions. In addition, analyzes were carried out to assess whether the peptides affected sperm morphology, plasma membrane functionality and chromatin compaction. Spermaurin and H. lunatus protein showed 52% and 72% identity and similarity, respectively. For protein modeling, ColabFold obtained the best scores in relation to SWISS-MODEL, satisfying the requirements that indicate a good quality of in silico models. The four peptides drawn are water soluble. The linear peptides were larger compared to the conformational ones, with about 10 more amino acids. In the in vitro assays, the results in cooled semen samples showed an improvement in motility, especially in the group treated with the peptide Conf_2 at a concentration of 200 nM. For cryopreserved samples, no peptide capable of increasing motility parameters was observed. However, further investigations are needed to confirm the effective increase in motility parameter values and the use of fresh semen for a more robust validation.