Effect of recombinant and native buffalo OVGP1 on sperm functions and in vitro embryo development: a comparative study

Abstract

Background: An oviduct- specific glycoprotein, OVGP1, is synthesized and secreted by non-ciliated epithelial cells of the mammalian oviduct which provides an essential milieu for reproductive functions. The present study reports the effects of recombinant buffalo OVGP1 that lacks post-translational modifications, and native Buffalo OVGP1 isolated from oviductal tissue, on frozen- thawed sperm functions and in vitro embryo development.

Results: The proportion of viable sperms was greater (P < 0.05) in the recombinant OVGP1-treated group compared to the native OVGP1-treated group at 2 h, 3 h, and 4 h of incubation. The proportion of motile sperms at 3 h and 4 h of incubation; and membrane- intact sperms at 4 h was greater (P < 0.05) in the native OVGP1-treated group compared to the control and recombinant OVGP1-treated groups. The proportion of capacitated and acrosome- reacted sperms was greater (P < 0.05) in the native OVGP1-treated group compared to the recombinant OVGP1 group at 4 h. The rates of cleavage of embryos and their development to the blastocyst stage were greater (P < 0.05) in the presence of either native or recombinant OVGP1 in comparison to control at 10 μg/mL concentration as compared to 5 or 20 μg/mL.

Conclusions: The study suggests that both native and recombinant OVGP1 impart a positive effect on various sperm features and in vitro embryo development. However, native OVGP1 was found to have a more pronounced effect in comparison to recombinant non-glycosylated OVGP1 on various sperm functions except viability. Hence, our current findings infer that glycosylation of OVGP1 might be essential in sustaining the sperm functions but not the in vitro embryo development.

Keywords: Blastocyst; Capacitation; Fertilization; Glycoprotein; In vitro fertilization; Oestrus; Semen; Sperm.

Fig. 1

a SDS-PAGE analysis showing expression of recombinant OVGP1. Lane 1: Uninduced, lane 2: Molecular weight marker, lane 3: Induced, lane 4: Pellet, lane 5: Soluble fraction; (b) SDS-PAGE analysis of His-tag affinity purified recombinant OVGP1 (indicated by arrow). Lane 1: Molecular weight marker, lane 2–5: Eluted fractions for partially purified His- tagged recombinant OVGP1; (c) Anion- exchange purification of recombinant OVGP1 (indicated by arrow). Lane 1: Unbound fraction, lane 2: Molecular weight marker, lane 3–9: Eluted fractions for partially purified His- tagged recombinant OVGP1; and (d) Gel filtration purification representing single band (indicated by arrow) for purified His- tagged recombinant OVGP1, lane 1: Unstained molecular weight marker, lane 2: purified His- tagged recombinant OVGP1; (e) Western blot analysis of purified His-tagged recombinant OVGP1. Lane 1: Prestained molecular weight marker, lane 2: purified His- tagged recombinant OVGP1

Fig. 2

Sequential purification steps of recombinant OVGP1 (R_OVGP1). Chromatogram of (a) IMAC based affinity purification: purification of recombinant OVGP1 through IMAC resulted in a single peak (indicated by arrow) after applying a gradient of 0–100% (300 mmol/L imidazole) in 30 mL. The peak fractions contained partially purified recombinant OVGP1. b Ion-Exchange Chromatography: Purification step through IEX showing elution of three merged peaks (indicated by arrows) during a stepwise gradient run. The latter peak contained recombinant OVGP1 with a few remaining contaminating proteins. c Size-Exclusion Chromatography: Purification of recombinant OVGP1 through the step resulted in elution of a peak (indicated by arrow) containing purified recombinant OVGP1

Fig. 3

Purification of native OVGP1and its Western blot confirmation: (a) Lane 1: Total protein lysate, lane 2: Unbound fraction, lane 3: Molecular weight marker, lane 4: Eluted protein fraction from WGA affinity column, lane 5: Eluted protein fraction from Mono Q anion- exchange column representing three major protein bands (~60- ~95 kDa) for different glycosylated forms of native OVGP1. b Western blot confirmation, lane 1: Molecular weight marker, lane 2: Purified native OVGP1

Fig. 4

Percentage of motile sperms during incubation with native and recombinant OVGP1as compared to control (without native and recombinant OVGP1). Cryopreserved sperms were incubated with native and recombinant OVGP1 for 4 h at 38 °C, 5% CO₂. (* significant P < 0.05)

Fig. 5

Percentage of live sperms during incubation with native and recombinant OVGP1 as compared to control (without native and recombinant OVGP1). Cryopreserved sperms were incubated with native and recombinant OVGP1 for 4 h at 38 °C, 5% CO₂. Sperm viability was assessed using SYBR14/PI staining. Superscripts a, b & c indicate the significant differences in the control, native and recombinant OVGP1-treated groups during different hours of incubation (P < 0.05)

Fig. 6

Percentage of membrane intact sperms during incubation with native and recombinant OVGP1 as compared to control (without native and recombinant OVGP1). Cryopreserved sperms were incubated with native and recombinant OVGP1 for 4 h at 38 °C, 5% CO₂. Membrane integrity was assessed using CFDA/PI staining. (*significant P < 0.05)

Fig. 7

Percentage of uncapacitated (a), capacitated (b), and acrosome reacted (c) sperms during incubation with native and recombinant OVGP1 as compared to control (without native and recombinant OVGP1). Cryopreserved sperms were incubated with native and recombinant OVGP1 for 4 h at 38 °C, 5% CO₂. Sperm capacitation status was estimated using CTC staining. Superscripts a, b & c indicate the significant differences in the control, native and recombinant OVGP1-treated groups during different hours of incubation (P < 0.05)

Fig. 8

Effect of native (a) and recombinant (b) OVGP1 on cleavage rate and blastocyst production rate in buffalo. (*, * * and * * * shows the significance of variation among the groups with P < 0.05 from three trials of each treatment