Effect of oviductal and cumulus cells on zona pellucida and cortical granules of porcine oocytes fertilized in vitro with epididymal spermatozoa.
The objective of this study was to evaluate the effects of porcine oviductal epithelial cell (POEC) monolayers and cumulus cells on the zona pellucida (ZP) and cortical granules (CG) of in vitro matured porcine oocytes. Denuded and cumulus-enclosed oocytes were exposed to POEC before or during in vitro fertilization (IVF). The functional effects of the co-culture system were the tested on the ZP resistance, measured by the time necessary to dissolve the ZP with 0.1% pronase, and the distribution and density of the cortical granules. CG density in the equator and cortex of each oocyte was evaluated by confocal microscopy after staining with fluorescein isothiocyanate-labelled peanut agglutinin (FITC-PNA). Both variables were assessed immediately after an in vitro maturation period (IVM group), 3 and 6h after culture with or without (Control) oviductal cells (Experiment 1) and 3h after insemination with frozen-thawed epididymal spermatozoa in the presence or absence (Control) of oviductal cells (Experiment 2). The time to dissolve the ZP of oocytes from IVM group was 440.4 +/- 61.7 s and no difference was observed among groups in Experiment 1. In contrast, the density of CG was affected; oocytes pre-incubated for 6h had a higher density than those pre-incubated for 3 h (P <0.001). Oocytes fertilized in vitro in the presence of POEC (Experiment 2) had a similar ZP digestion time as control oocytes 3 h after insemination. The presence of POEC during IVF as well as the presence of cumulus cells had no effect on the density and distribution of CG. However, a significant decrease in the density of CG was observed in the fertilized oocytes compared to in vitro matured oocytes (P <0.001). It is concluded that under the conditions employed the oviductal and cumulus cells in the perifertilization period had no effect on ZP hardening and CG density. However, an increase in CG density was observed when oocytes were maintained in culture. In addition, no hardening of ZP was observed after IVF, and denuded and cumulus-enclosed oocytes showed similar cortical reactions after insemination with epididymal spermatozoa regardless of the presence of POEC.
Romar
R
R
Department of Physiology (Veterinary Physiology), Faculty of Veterinary Science, University of Murcia, 30071 Murcia, Spain. rromar@um.es
Coy
P
P
Gadea
J
J
Rath
D
D
D000818:Animals; D002478:Cells, Cultured; D018920:Coculture Techniques; D004822:Epididymis / Q000166:cytology; D004847:Epithelial Cells / Q000502:physiology; D005187:Fallopian Tubes / Q000166:cytology*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D008297:Male; D018613:Microscopy, Confocal; D009865:Oocytes / Q000502:physiology* / Q000648:ultrastructure; D006080:Ovarian Follicle / Q000166:cytology*; D011247:Pregnancy; D013094:Spermatozoa; D013552:Swine; D015044:Zona Pellucida / Q000502:physiology*