Recombinant peptide reverses cryo-capacitation in ram sperm and improves in vitro fertilization

Abstract

Semen cryopreservation is a very important technique for assisted reproduction; however, the cryopreservation process is harmful because it results in a reduction in sperm motility and viability, and leads to premature signals of capacitation, resulting in lesser than desirable fertility rates after artificial insemination. A fraction of seminal plasma, enriched in proteins that contain type II fibronectin domains (FNII) can reverse molecular indicators of cryo-capacitation. The beneficial effects of these proteins, however, depend on the relative abundance in seminal plasma. To create a safe additive for improving frozen sperm functionality, in the present study there was cloning and expression of a recombinant peptide containing four FNII domains (named TrxA-FNIIx4-His6) and evaluation of its effect after addition to frozen/thawed ram sperm. The cDNA for this protein was expressed in E. coli and after denaturation and re-naturalization of the protein, toxicity and binding capacity were assessed. By fluorescent labelling assessment, there was binding of the protein to the thawed sperm. At the two doses used (0.15 and 0.3 µM), TrxA-FNIIx4-His6 had the capacity to reverse the molecular indicators of cryo-capacitation as indicated by the reduction on phosphorylated substrates of PKA. Furthermore, the supplementation with this protein resulted in a normal capacitation process as evidenced by the increase in the in vitro fertilization rate when the greatest concentration of the protein was evaluated (73.25 ± 2.95; 40.13 ± 11.82 for 0.3 µM and control, respectively). There was no effect of protein supplementation on sperm objective motility compared to untreated sperm. In conclusion, the use of TrxA-FNIIx4-His6 is a promising biotechnological approach for cryopreserving ram sperm and maintaining sperm viability.

Sperm Cryodamage in Ruminants: Understanding the Molecular Changes Induced by the Cryopreservation Process to Optimize Sperm Quality

“…Proteomics studies on seminal plasma have greatly contributed to identifying those proteins with beneficial effects on sperm cryopreservation, facilitating the generation of recombinant proteins as a promising strategy for sperm cryopreservation. Recently, supplementation of the extender with recombinant seminal plasma proteins such as regucalcin (RGN), a recombinant peptide containing four FNII domains (TrxA-FNIIx4-His 6 ) and serine protease inhibitor kazal-type 3 (SPINK3) have been shown to exert a cryoprotective effect on sperm [148][149][150]. A greater number of cryopreserved buffalo sperm were attached to zona pellucida when the freezing medium was supplemented with the recombinant RGN due to its positive effect on post-thaw sperm motility and acrosome integrity [148].…”

“…The beneficial effects of RGN may rely on its calcium-regulating and antioxidant functions. In frozen-thawed ram sperm, TrxA-FNIIx4-His 6 has been shown to reduce cryo-capacitation, enhancing in vitro fertilization rates [149]. Another protein from seminal plasma that prevents and reverts cryo-capacitation is SPINK3.…”

The current state of the problem of in vitrogene pool preservation in poultry

“…Therefore, the addition of some proteins normalizes the process of condensation and accelerates fertilization in vitro. For example, use of TrxA-FNIIx4-His6 is a promising biotechnological approach for cryopreservation of ram semen and maintenance of spermatozoon viability (Ledesma et al, 2019).…”

Ram epididymal sperm frozen in an extender containing ethylene glycol have higher post‐thaw longevity and in vitro fertility

“…21 Moreover, the frozen-thawed sperm undergo a capacitation-like process known as cryo-capacitation 22,23 which may further reduce the longevity of cryopreserved sperm within the female reproductive tract, decreasing the likelihood of typical oviductalsperm interactions, or affecting the fertilization process. 24 The in vitro assessment of the functional activity of sperm plasma membrane is mainly carried out by measuring its reactivity to hypo-osmotic swelling test (HOST), 21 and the proportion of HOST-responsive sperm was identified as a significant predictor of the in vitro fertility of frozenthawed sperm. 25 Additionally, in vitro evaluation of the cryopreserved ram sperm fertility is considered as a useful tool for predicting their in vivo fertility.…”

“…Only those spermatozoa with intact functional plasma membranes can undergo the complex changes in the female reproductive tract necessary for fertilizing the oocyte 21 . Moreover, the frozen‐thawed sperm undergo a capacitation‐like process known as cryo‐capacitation 22,23 which may further reduce the longevity of cryopreserved sperm within the female reproductive tract, decreasing the likelihood of typical oviductal–sperm interactions, or affecting the fertilization process 24 . The in vitro assessment of the functional activity of sperm plasma membrane is mainly carried out by measuring its reactivity to hypo‐osmotic swelling test (HOST), 21 and the proportion of HOST‐responsive sperm was identified as a significant predictor of the in vitro fertility of frozen‐thawed sperm 25 .…”