Effect of supplementation of recombinant Regucalcin in extender on cryopreservation of spermatozoa of water buffalo (Bubalus bubalis)

Abstract

Elevated intracellular calcium concentration and oxidative damage are two major factors contributing to the poor fertility of cryopreserved spermatozoa. Regucalcin (RGN), also known as Senescence marker protein-30 (SMP-30), is a calcium-binding protein with multiple roles that include calcium homeostasis, anti-oxidative, anti-apoptosis, and anti-proliferation. In Drosophila, RGN is reportedly a putative cold-tolerance gene and a cytoprotective role for RGN against intracellular calcium elevation and oxidative stress was reported in P19 cell lines. Given that RGN has anticapacitatory effect and abundant in the male reproductive tract, we hypothesized that it may play a cryoprotective role for spermatozoa. We investigated this by including RGN, at three different concentrations (20, 40, and 60 μg/ml), as a supplement for Tris-egg yolk-based semen extender. Post-thaw metrics of progressive motility, acrosome integrity, and zona pellucida binding of spermatozoa were evaluated for three ejaculates of three clinically normal, breeding Murrah buffaloes. A concentration of 40 μg/ml of recombinant RGN supplemented during sperm freezing resulted in significant increases in the post-thaw progressive motility of spermatozoa (50.6 ± 3.5% vs 40.6 ± 2.6%; p < 0.01), acrosome integrity (53.3 ± 7.4 vs 75.6 ± 6.8; p < 0.05), and zona pellucida binding (31.6 ± 14.0 vs 191.9 ± 12.3 bound spermatozoa; p < 0.01) compared to control conditions without RGN. Thus, ∼1 μM recombinant RGN, which retains the ability to bind calcium, has a cryoprotective effect for buffalo spermatozoa in extender.

Proteome and microbiota analysis reveals alterations of liver-gut axis under different stocking density of Peking ducks

“…In the proteomic analysis of this study, regucalcin and catalase expression were reduced in high density group. Catalases are involved in protecting cells from the damaging effects of ROS [ 22 ], while regucalcin is a calcium-binding protein with multiple roles that include calcium homeostasis, anti-oxidative, anti-apoptosis, and anti-proliferation [ 23 ]. Decreases in these proteins indicate that high stocking density may reduce duck anti-oxidant capacity.…”

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].…”

Improved quality and fertilizability of cryopreserved buffalo spermatozoa with the supplementation of methionine sulfoxide reductase A

“…It is observed that the supplementation of various enzymatic and non‐enzymatic antioxidants to the semen extender like catalase, 80 α‐tocopherol, 81 thiols, 57 ascorbate, 82 SOD, 83 regucalcin 84 and also the partial deoxygenation of semen extender 85 improved the cryopreserved sperm quality. Further, the regucalcin showed a suppressive effect on capacitation 29 .…”