203 PRETREATMENT OF BOVINE SPERM OR OOCYTE WITH ANTIBODY TO LIPOCALIN-TYPE PROSTAGLANDIN D SYNTHASE INHIBITS FERTILIZATION

Abstract

Lipocalin-type prostaglandin D synthase (L-PGDS) has been identified in cow uterine tube fluid (UTF), and as fertility-associated protein in the Holstein bull seminal plasma, but its function is unclear. A previous study demonstrated that L-PGDS is associated with the bovine zona pellucida, and that antibody incubated with UTF decreased embryo development in vitro. This study was conducted to determine whether IVF of bovine oocytes would be affected by pretreating either the sperm or oocytes, or both, with L-PGDS antibody. In vitro-matured bovine oocytes were incubated for 1 h in IVF TALP medium supplemented with penicillamine, hypotaurine, epinephrine, and heparin containing (a) no antibody, or (b) a rabbit polyclonal antibody against recombinant bovine L-PGDS (α L-PGDS; 1:2000). Frozen–thawed spermatozoa were washed by a 45:90% layered Percoll gradient centrifugation and incubated for 1 h in IVF TALP with (a) no antibody, or (b) α L-PGDS. For this study we had 4 different treatments: (1) no antibody (control), (2) α L-PGDS at fertilization time, (3) α L-PGDS-treated oocytes, or (4) α L-PGDS-treated sperm. Oocytes were inseminated with 10 × 10⁴ washed spermatozoa in 4-well culture dishes. After 18 h (39°C, 5% CO₂ in air), oocytes were vortexed to remove cumulus cells and accessory spermatozoa, and fixed in 3.7% paraformaldehyde and 10% Triton X–100 for 1 h. Oocytes were washed and transferred to a solution with PBS, 0.3% BSA, and 1% Triton for 1 h, stained with Hoechst 33342, and observed in the presence of 2 pronuclei in the cytoplasm. There were 4 replicates of 200 to 250 oocytes for fertilization assays. Data were analyzed by SAS. Addition of α L-PGDS with sperm, oocytes, or both significantly decreased fertilization (P < 0.05) compared with the control: (1) 89.2 ± 2.0%; (2) 19.4 ± 2.0%; (3) 27.2 ± 3.1%; or (4) 14.1 ± 3.4%. These studies demonstrated that a rabbit polyclonal antibody against recombinant bovine L-PGDS reacts with both oocytes and spermatozoa, resulting in inhibition of fertilization in vitro, and has a possible role in bovine fertilization.

This study was supported by FAPESP #2007/00363-5 and #2006/06008-0, Brazil.