ADHESION OF STALLION SPERMATOZOA ON POLY(ACRYLAMIDE)-BASED HYDROGELS: EFFECT OF HYDROGEL IONIC CHARGE

EBEL, FRANCISCA; LIAUDAT, ANA CECILIA; BLOIS, DAMIAN A.; RODRIGUEZ, NANCY; RIVAROLA, CLAUDIA R.; BOSCH, PABLO

Congreso; IV Joint Meeting of The Biology Societies of Argentina; 2021

Polymeric hydrogels are soft materials used in several biotechnological applications. We demonstrated that poly(acrylamide) (PAAm) based hydrogels are devices for equine sperm selection; however, the molecular mechanisms responsible for this interaction are largely unknown. We hypothesized that the net ionic charge of the hydrogel surface may be involved in the sperm attachment. Therefore, we copolymerized acrylamide with 10% in moles of (3-acrylamidopropyl) trimethylammonium chloride (10%APTA, cationic monomer), N-[tris(hydroxymethyl)methyl] acrylamide (10% HMA, neutral monomer) and acrylic acid (10% AA, anionic monomer at pH > 4) to achieve different ionic charges on the hydrogel surface. Then, we performed the sperm binding experiments in presence or absence of bovine serum albumin (BSA), a protein that is negatively charged at pH 7.4. For this purpose, each hydrogel was incubated in a culture dish containing sperm-TALP medium (pH 7.4) with and without BSA (6 mg/mL) at 37°C in 5% CO2 for 30 min. Finally, an aliquot of raw stallion sperm suspension was added (1×106 sperm/dish) and incubated at the same conditions mentioned before. Sperm incubated in culture dishes without hydrogels served as controls. The percentage of sperm attached to the surfaces was determined as the difference between the number of sperm initially added into the culture dish and the recovered non-bound cells after incubation. Data (mean ± SD) were analyzed by two-way ANOVA/Bonferroni post-test; a P < 0.05 was considered to be significant. The results evidenced that the percentage of equine spermatozoa attached to the hydrogel surface varied according to hydrogel physicochemical characteristics and BSA supplementation (P < 0.05). Thus, for hydrogels cultured in BSA free-medium, the percentage of attached spermatozoa was higher in 10% APTA surface than in 10% AA and 10% HMA surfaces; and also respect to the control (10% APTA: 74.5 ± 8.7% > 10% AA: 37.2 ± 16.3% > 10% HMA: 22.6 ± 2.3% > control: 7.1 ± 1.1%; P < 0.05). Interestingly, for hydrogels cultured with BSA supplementation, the spermatozoa attached mainly to 10% HMA and 10% AA surfaces and only few sperm bonded to 10% APTA surface (10% HMA: 57.1 ± 9.6% ≈ 10% AA: 48.6 ± 3.3% > 10% APTA: 8.1 ± 1.7% and control: 15.0 ± 3.1%; P < 0.05). In fact, the percentage of sperm bound to 10% APTA surface was similar to that in the control (P > 0.05). Additionally, in all experiments independent of BSA supplementation, spermatozoa attached to 10% AA and 10% HMA surfaces remained motile; whereas spermatozoa bound to 10% APTA surfaces were immotile. In conclusion, the ability of bonding between copolymeric hydrogels based on PAAm and spermatozoa is influenced by the hydrogel net ionic charge. The supplementation with BSA modifies the hydrogel superficial property and the ability of equine spermatozoa to bind on hydrogels without affecting the motility of bounded spermatozoa. This information might contribute to assisted reproduction in stallions with low reproductive efficiency.