Pre-treatment of cattle sperm and/or oocyte with antibody to lipocalin type prostaglandin D synthase inhibits in vitro fertilization and increases sperm-oocyte binding.
The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO(2) in air) for 1h in the following treatments either 500 microL of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1h either FM or FM with alpha L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) alpha L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) alpha L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 x 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha L-PGDS: (1) 26.4+/-3.0; (2) 25.6+/-3.0; (3) 59.7+/-3.0; (4) 56.4+/-3.0; and (5) 57.1+/-3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P<0.05) compared with the control: (1) 89.2+/-2.0%; (2) 87.5+/-2.0%; (3) 19.4+/-2.0%; (4) 27.2+/-3.1%; and (5) 14.1+/-3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization.
Gonçalves
R F
RF
Department of Animal Reproduction, College of Veterinary Medicine and Animal Science, São Paulo University, Cidade Universitária, SP, Brazil. ona@usp.br
Barnabe
V H
VH
Killian
G J
GJ
D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D002417:Cattle / Q000502:physiology*; D005260:Female; D005307:Fertilization in Vitro / Q000187:drug effects / Q000662:veterinary*; D007074:Immunoglobulin G / Q000494:pharmacology; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000276:immunology* / Q000502:physiology; D054834:Lipocalins / Q000737:chemistry / Q000276:immunology* / Q000502:physiology; D008297:Male; D009865:Oocytes / Q000187:drug effects*; D013084:Sperm-Ovum Interactions / Q000187:drug effects*; D013094:Spermatozoa / Q000187:drug effects*; D013997:Time Factors
D000906:Antibodies; D007074:Immunoglobulin G; D054834:Lipocalins; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase