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.

Keywords: Cryopreservation; Fibronectin (FNII) domains; Ram; Recombinant peptide; Seminal plasma.

Fig. 1.

Bovine Epididymis cDNA library construction; Bioanalyzer electropherograms of total RNA, mRNA and cDNA fractions (A, B and C respectively); Panel D. depicts the range of inserts in 24 random clones from library after excision from the pDONR plasmid using the BsrG I restriction enzyme; Samples were processed using a 1.5% agarose gel compared to 1Kb ladder (Invitrogen)

Fig. 2.

Cloning, gene expression and purification of TrxA-FNIIx4-HiS₆; A. cDNA encoding four tandem FNII repeats from Epididymal Sperm Binding Protein 1 precursor (ELSPBP1) was amplified from a bovine epididymal cDNA library and cloned into the expression vector pET-32 Ek/LIC (Novagen, 5.917 bp); Construction included a thioredoxin tag (TrxA) at the N-terminal and a 6 Histidine tag (Hisx6) at the C-terminal; B. E. coli Rosetta cells were transformed with the construction FNIIx4-pET-32 Ek; Electrophoretic profile using SDS–PAGE indicates the soluble (S) and insoluble (I) fractions of E. coli cells after 16 h induction (+IPTG T16) with 0.1 mM IPTG at 18 °C; A soluble fraction of cells without induction is depicted (control); TrxA-FNIIx4-HiS₆ protein solubilized from inclusion bodies was refolded and purified using a HiTrap nickel affinity column (Purification); Molecular weight marker is depicted at left; Arrow indicates the expected size for the protein (38 kDa)

Fig. 3.

Interaction between FITC-TrxA-FNIIx4-HiS₆ and thawed ram sperm using fluorescence confocal microscopy; Thawed ram sperm (20 x 10⁶ cells/mL) were incubated with 25 μg 0.37μM FITC-TrxA-FNIIx4-HiS₆ during 60 min at room temperature and fixed; Left panel: Spermatozoa incubated with FITC-TrxA-FNIIx4-HiS₆ (TrxA-FNIIx4-HiS₆) or FITC without protein (control) were visualized using a microscope (60x); Right panel: The graph depicts the distribution fluorescence intensity from the apical region to the flagella of each sperm; Upper line corresponds to spermatozoa incubated with FITC-TrxA-FNIIx4-HiS₆ and bottom line corresponds to control

Fig. 4.

Phosphorylated PKA substrates pattern in frozen/thawed ram sperm; A. Phosphorylation was detected by western blot using an antibody anti-PKA on total proteins obtained after 60 min incubation of frozen/thawed ram sperm with 0.15 μM (Lane 1), 0.3 μM TrxA-FNIIx4-His₆ (Lane 2) and contrasted with control without protein (Lane 3); Experiment was performed three times and a representative western blot is included; B. Quantification of signal intensity detected for each protein band (1-4) was normalized to the corresponding tubulin densities; Mean values ± SEM of each phosphorylation signal band (area x intensity); For each protein band, different superscripts letters indicate differences between treatments

Fig. 5.

Motility analysis on frozen/thawed ram sperm treated with TrxA-FNIIx4-HiS₆; Thawed spermatozoa were incubated with or without 0.15 μM, 0.3 μM of TrxA-FNIIx4-HiS₆ at 37 °C in PBS for 30, 60, 90 and 120 min; Values for kinetic variables VSL (A), VCL (B), VAP (C), ALH (D), BCF (E), LIN (F) and STR (G) were determined using a CASA system; *Value differences compared to control samples (P< 0.05); VAP: Average Path Velocity, VSL: Straight-Line Velocity, VCL: Curvilinear Velocity, ALH: Amplitude of Lateral Head Displacement, BCF: Beat Cross Frequency, LIN: linearity coefficient, STR: straightness coefficient; Four fields containing among 700 and 2000 cells were analyzed for each treatment, time and replicate (n = 3)

Fig. 6.

Heterologous in vitro fertilization rate of frozen/thawed ram sperm treated with 0.15 μM or 0.3 μM TrxA-FNIIx4-HiS₆; Fertilization rate was assessed by the presence of cleaved oocytes (two to eight cells) and/or the presence of two or more nuclei after 40 h insemination; Mean values ± SEM; Different letters indicate differences between treatments (n = 3)