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Hardening of equine zona pellucida after incubation in vitro either in a mares' oviduct or in culture medium
Theriogenology
388
HARDENING OF EQUINE ZONA PELLUCIDA AFTER INCUBATION I N VITRO EITHER IN A MARES' OVIDUCT OR IN CULTURE MEDIUM A. Ok61ski, W. Mlodawska and I. Polak Department of Animal Reproduction, University of Agriculture, AI. Mickiewicza 24/28, 30-059 Krak6w, Poland A consequence of maturation, fertilization or parthenogenetic activation of mammalian eggs is an alteration of the properties of the zonae pellucidae (ZP). In the literature, there is still lack of information regarding hardening of the equine ZP. The objective of this study was to determine whether exposure of equine oocytes to culture medium (TCM) or mares' oviduct explants would affect hardening of ZP. The basic assay used to determine ZP differences, involved the incubation of individual oocytes in 50 ~tl of Ringer-Krebs solution containing 0.1% pronase. ZP were considered digested when it disappeared. Mares' ovaries and oviducts were collected at slaughterhouse and transported to the laboratory in a thermos at 25-30°C. Oocytes were recovered by aspiration from follicles within 4h after slaughter. The oviducts were isolated, dissected free of excess connective tissue, ligated at both ends and kept in Petri dishes containing TCM Hepes + 10% FCS at 38.5°C. In Exp. I ZP digestion was performed immediately after the recovery of oocytes from follicles (n=29), following incubation in vitro in PBS + 20% FCS for 4 h (n=lS) or 6 h (n=19) as well as after incubation in vitro for 4h (n=30) or 6h (n=47) in mares' oviduct explants immersed in TCM + 10% FCS. In one oviduct were incubated 6-8 oocytes. In Exp. II ZP digestion was performed also immediately after the recovery of oocytes from follicles (n=39) or after 30-32 h of incubation in TCM + 20% FCS (n=19). Before the digestion test, cumulus and corona cells were removed from the oocytes by aspiration through a fine bore pipet. Data were analysed using one-way AOV and Scheffe's pairwise comparisons of means. The average digestion time (mean + SD) of ZP was 202 i 62 s for oocytes before incubation, 181 ± 56 s for oocyte incubated 4 hr in PBS, 266 ± 45 s for oocytes incubated 6 hr in PBS and 245 ± 47 s for oocytes incubated 4h in mares' oviduct. The longest digestion time of ZP was in oocytes incubated 6 hr in mares oviducts. It was 392 ± 65 s, significantly longer (p<0,01) than in all other groups of oocytes. We found that digestion time was 226 + 57, 181 _+43, 199 _+53, 229 + 47 and 246 _+41 s for oocytes originated from follicles with diameter <_10, 11-15, 16-20, 21-30 and >_ 31mm, respectively. While comparing the digestion time of ZP between the oocytes which were cultured in vitro for 30-32 h in TCM + 20% FCS (194 _+ 17 s) and those without culture (212 _+ 56 s) we did not find the differences. It is of interest that the old degenerated oocytes (n=42) left in the oviducts displayed very short ZP digestion time (123 _+5 s). Our study indicated that equine oocyte incubation in the oviduct explants increases, almost doubles the duration of ZP digestion time compared to that required by oocytes uncultured. A moderate hardening of ZP in the oocytes in oviduct may be of significant importance in the mechanism of penetration spermatozoa during fertilization. This research is supported by KBN Grant Nr 5 P06D 019 11.
388
HARDENING OF EQUINE ZONA PELLUCIDA AFTER INCUBATION I N VITRO EITHER IN A MARES' OVIDUCT OR IN CULTURE MEDIUM A. Ok61ski, W. Mlodawska and I. Polak Department of Animal Reproduction, University of Agriculture, AI. Mickiewicza 24/28, 30-059 Krak6w, Poland A consequence of maturation, fertilization or parthenogenetic activation of mammalian eggs is an alteration of the properties of the zonae pellucidae (ZP). In the literature, there is still lack of information regarding hardening of the equine ZP. The objective of this study was to determine whether exposure of equine oocytes to culture medium (TCM) or mares' oviduct explants would affect hardening of ZP. The basic assay used to determine ZP differences, involved the incubation of individual oocytes in 50 ~tl of Ringer-Krebs solution containing 0.1% pronase. ZP were considered digested when it disappeared. Mares' ovaries and oviducts were collected at slaughterhouse and transported to the laboratory in a thermos at 25-30°C. Oocytes were recovered by aspiration from follicles within 4h after slaughter. The oviducts were isolated, dissected free of excess connective tissue, ligated at both ends and kept in Petri dishes containing TCM Hepes + 10% FCS at 38.5°C. In Exp. I ZP digestion was performed immediately after the recovery of oocytes from follicles (n=29), following incubation in vitro in PBS + 20% FCS for 4 h (n=lS) or 6 h (n=19) as well as after incubation in vitro for 4h (n=30) or 6h (n=47) in mares' oviduct explants immersed in TCM + 10% FCS. In one oviduct were incubated 6-8 oocytes. In Exp. II ZP digestion was performed also immediately after the recovery of oocytes from follicles (n=39) or after 30-32 h of incubation in TCM + 20% FCS (n=19). Before the digestion test, cumulus and corona cells were removed from the oocytes by aspiration through a fine bore pipet. Data were analysed using one-way AOV and Scheffe's pairwise comparisons of means. The average digestion time (mean + SD) of ZP was 202 i 62 s for oocytes before incubation, 181 ± 56 s for oocyte incubated 4 hr in PBS, 266 ± 45 s for oocytes incubated 6 hr in PBS and 245 ± 47 s for oocytes incubated 4h in mares' oviduct. The longest digestion time of ZP was in oocytes incubated 6 hr in mares oviducts. It was 392 ± 65 s, significantly longer (p<0,01) than in all other groups of oocytes. We found that digestion time was 226 + 57, 181 _+43, 199 _+53, 229 + 47 and 246 _+41 s for oocytes originated from follicles with diameter <_10, 11-15, 16-20, 21-30 and >_ 31mm, respectively. While comparing the digestion time of ZP between the oocytes which were cultured in vitro for 30-32 h in TCM + 20% FCS (194 _+ 17 s) and those without culture (212 _+ 56 s) we did not find the differences. It is of interest that the old degenerated oocytes (n=42) left in the oviducts displayed very short ZP digestion time (123 _+5 s). Our study indicated that equine oocyte incubation in the oviduct explants increases, almost doubles the duration of ZP digestion time compared to that required by oocytes uncultured. A moderate hardening of ZP in the oocytes in oviduct may be of significant importance in the mechanism of penetration spermatozoa during fertilization. This research is supported by KBN Grant Nr 5 P06D 019 11.