Regucalcin is a multi-functional protein having roles in calcium homeostasis as well as in anti-apoptotic, anti-prolific and anti-oxidative functions. Recently, it has been reported from the male reproductive tract, but its role in male reproduction needs further investigation; for which the native regucalcin of reproductive origin will be more appropriate. The gel exclusion chromatography followed by diethyl aminoethane cellulose chromatography and two-dimentional cellulose acetate membrane electrophoresis used for its purification are time consuming and less specific. Here, the regucalcin gene from buffalo testis has been cloned, expressed and purified in recombinant form, and subsequently used for raising hyper-immune serum. The Western blot of seminal vesicular fluid probed with anti-regucalcin polyclonal and monoclonal antibodies showed the presence of 28 and 34 kDa bands specific to regucalcin. Further, an affinity matrix has been prepared using anti-regucalcin polyclonal antibodies. An immuno-affinity chromatography method has been standardized to isolate regucalcin from seminal vesicular fluid. The initial complexity of the protein mixture in the seminal vesicular fluid has been reduced by a heat coagulation step. The purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band at 68 kDa that has been further confirmed as regucalcin by Liquid chromatography-mass spectrometry/mass spectrometry. The RGN purified from seminal vesicular fluid will be more appropriate for studying its possible role in male reproduction, especially sperm cell capacitation, hyperactivation, acrosome reaction and cryopreservation. The study can be applied in purifying regucalcin from different tissues or species with minor modifications in the methodology.

Regucalcin is a multi-functional, calcium-binding protein with roles in calcium homeostasis, apoptosis, cell proliferation, and free radical neutralization. Regucalcin is broadly expressed in the male reproductive organs of rat and bovine; here, we report its expression in the reproductive tract of male buffalo-especially in testis, epididymis, seminal vesicle, prostate, and bulbourethral gland of buffalo-as analyzed by real-time PCR, Western blot, and immunolocalization. Regucalcin degradation in seminal plasma, despite its high abundance in vesicular fluid, was demonstrated using recombinant regucalcin co-incubated with buffalo seminal plasma. This depletion of regucalcin appears to be related to its suppressive effect on in vitro sperm capacitation, observed using the chlortetracycline assay after treating buffalo spermatozoa with recombinant protein. Indeed, addition of recombinant regucalcin to capacitating media significantly reduced (P < 0.05) the percentage of capacitated spermatozoa to 6.1 ± 0.6 from 36.4 ± 1.8 in the untreated group. Taken together, the wide distribution of regucalcin in male buffaloes, versus its degradation in the seminal plasma and suppressive effects on in vitro capacitation of spermatozoa, indicate its possible anti-capacitation role in the reproductive tract. Mol. Reprod. Dev. 84: 212-221, 2017. © 2016 Wiley Periodicals, Inc.

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.

Regucalcin is a calcium regulating multifunctional protein reported to have many important functions like calcium homeostasis, anti-oxidative, anti-apoptotic and anti-cancerous functions. Although it is demonstrated as a calcium regulating protein, the calcium binding ability of regucalcin is still a controversy. The main reason for the controversy is that it lacks a typical EF hand motif which is common to most of the calcium binding proteins. Even though many studies reported regucalcin as a calcium binding protein, there are some studies reporting regucalcin as non-calcium binding also. In the present study, we investigated the calcium binding ability of recombinant buffalo regucalcin by assessing the secondary structural changes of the protein using circular dichroism spectroscopy after adding Ca to the protein solution. Two types of calcium binding studies were done, one with different concentration of calcium chloride (0.5 mM CaCl, 1 mM CaCl, 2 mM CaCl) and other at different time interval (no incubation and 10 min incubation) after addition of calcium chloride. Significant structural changes were observed in both studies which prove the calcium binding ability of recombinant regucalcin. A constant increase in the α-helix (1.1% with 0.5 mM CaCl, 1.4% with 1 mM CaCl, 3.5% with 2 mM CaCl) and a decrease in β-sheets (78.5% with 0.5 mM CaCl, 77.4% with 1 mM CaCl, 75.7% with 2 mM CaCl) were observed with the increase in calcium chloride concentration. There was a rapid increase in α-helix and decrease in β-sheets immediately after addition of calcium chloride, which subsides after 10 min incubation.

Aim:The experiment was conducted to study the total seminal plasma protein (TSPP) and heparin-binding proteins (HBPs) in relation to initial semen quality of buffalo bull. Materials and Methods:Semen from two Murrah buffalo bulls (bull no. 605 and 790) with mass motility of ≥3+ were used for the study and categorized into three groups (Group I-Mass motility 3+, Group II-Mass motility 4+ and Group III-Mass motility 5+). Seminal plasma from semen was separated by centrifugation. HBPs was isolated and purified from heparin-agarose affinity column by modified elution buffer. TSPP and isolated HBPs concentration was estimated by Lowry's method. The purified HBPs were resolved on Sodium dodecyl sulfate polyacrylamide gel electrophoresis to check the protein profile of two bulls. Conclusion: TSPP and HBPs shows variation in concentration with respect to initial semen quality. Furthermore, presence of fertility related 31 kDa HBPs in one of the bull may be an indication of high fertility of a bull. In future, in-vivo and in-vitro correlative study on larger basis is needed for the establishment of fertility-related HBPs in semen which might establish criteria for selection of buffalo bull with high fertility potential. Results

Regucalcin is a multifunctional protein having an important role in calcium homeostasis, L-ascorbic acid biosynthesis, anti-prolific, anti-apoptotic functions as well as detoxification of chemical warfare nerve agents. Recently, it has been localized to male reproductive tract of rat and human, and identified as an androgen-target gene. The literature suggests a possible role of regucalcin in male fertility. However, no detailed studies have been conducted on its role in male reproductive organs of domestic animals. As an initial step, we had cloned and expressed regucalcin in Pichia pastoris. The sequence analysis showed 100% homology with regucalcin of Bos tours both at nucleotide and amino acid level. The SDS-PAGE and Western blot studies of recombinant protein probed with anti-regucalcin monoclonal antibody showed a higher molecular weight (56 kDa) than the expected (35.5 kDa) that could be due to hyperglycosylation. The recombinant regucalcin and its antibodies can be used to study the detailed role of the protein in male reproduction.

A study was conducted to assess the effect of buffalo seminal PDC-109 on its cauda epididymal spermatozoa. PDC-109 protein was purified by applying two-step chromatography procedures and included into epididymal spermatozoa. Epididymal ejaculates were splited into four groups as Gr 1: Control (without PDC), Gr 2: 20 μg/mL PDC-109, Gr 3: 40 μg/mL PDC-109 and Gr 4: 80 μg/mL PDC-109. Semen quality parameters (SQPs) and in vitro fertility assay were evaluated. Significant improvement in post-thaw SQPs was observed in Gr 2 than in Gr 3, however, Gr 1 had significantly higher value than other groups. It is concluded that PDC-109 has dose dependent effect as increased dose causes detrimental effect in buffalo cauda epididymal sperm.

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