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The effect of granulosa cell (GC) apoptosis and follicle size on the competence of bovine oocytes were studied using a well-in-drop (WID) oocyte/embryo culture system, which allows identification of follicular origin. Hatching rates of blastocysts did not differ (P>0.05) between oocytes cultured in the WID system (13%) and those cultured in the conventional group system (16%). Hatching rates of blastocysts were higher (P<0.05) in early atretic (17%) than in non-atretic (8%) and late atretic follicles (10%) of the same size (4-8mm), and in 6-8mm (22%) than in 4-5mm follicles (15%) at the early atretic stage. More oocytes (P<0.05) from late atretic (17%) than from non-atreteic (7%) or early atretic follicles (9%) of the same size (4-8mm) were arrested at Grade 1 cumulus expansion (only cells in the peripheral two layers began to expand). Similarly, more oocytes from 2 to 3mm follicles (30%) than from 6 to 8mm follicles (21%) at the same (late) atretic stage had Grade 1 cumulus expansion (P<0.05). Hatching blastocyst percentages of oocytes with Grade 3 (all layers of the cumulus except corona radiate cells expanded) or Grade 4 (full) cumulus expansion were higher in early atretic (20%) than in non-atretic (13%) or late atretic follicles (12%). Hatching blastocyst percentages of oocytes from follicles at the early atretic stage increased as cumulus expanded from Grade 2 (9%) to Grade 4 (27%). Regardless of the degree of follicle atresia, 72-76% of the floating cells in the follicular fluid (FF) were undergoing apoptosis. The floating cell density in FF was highly (r=0.6-0.7) correlated with oocyte developmental potency. In conclusion, the WID culture system was as efficient as group culture and allowed identification of follicular origin. Furthermore, the developmental potential of oocytes was affected by GC apoptosis, follicle size and cumulus expansion, and the floating cell density in FF could be used as a simple and non-invasive marker of oocyte quality.

Parthenogenetic activation is a major hurdle to be cleared for the examination of the human sperm chromosome after intracytoplasmic injection (ICSI) into golden hamster oocytes. Various factors that affect spontaneous activation of hamster oocytes were, therefore, investigated in this study. We collected cumulus-oocyte complexes (COC) from the oviducts of superovulated females and washed them thoroughly with Ca2+-containing or Ca2+-free TALP-HEPES medium (handling media). We cultured oocytes with intact cumulus or those without cumulus (removed by previous hyaluronidase treatment) in Ca2+-containing or -free m-TALP-3 for 6 or 12 h before examining for their activation. Among the oocytes recovered 17 h post-hCG, 92-94% were parthenogenetically activated by 6 h of in vitro culture. Activation rate in the oocytes collected at 13.5 h post-hCG (53%) was significantly (P < 0.05) lower than that in the oocytes collected 17 h post-hCG (92%), indicating that the spontaneous activation rate increased as the oocytes became older. Both cumulus-intact and cumulus-free oocytes had similar (P > 0.05) activation rates when cultured in vitro, suggesting that hyaluronidase treatment had no effect on the rate of oocyte activation. Omission of Ca2+ from the handling medium also had no effect on the activation of the oocytes. The rate of spontaneous activation of the oocytes cultured in calcium-free medium for 6 (9%) and 12 h (16%) was significantly (P < 0.01) lower than that (94%) of the control oocytes cultured in Ca2+-containing medium, implying a positive influence of Ca2+ on in vitro activation of hamster oocytes. When we cultured the oocytes first in calcium-free medium for 6 h, and then in calcium-containing medium for 6 h, 94% were activated, which is comparable to the rate for oocytes continuously cultured in Ca2+-containing medium. This indicates that the inhibition of hamster oocyte activation in Ca2+-free medium is reversible and can be used to control spontaneous activation of golden hamster oocytes.

Nuclear transplantation in the pig is more difficult than in other domestic animals and only one embryonic nuclear transplantation (NT) pig has been born to date. In this study, reconstituted porcine embryos were produced by electrofusion of blastomeres from in vivo four-cell embryos to enucleated in vivo or in vitro matured (IVM) oocytes. Nuclear transfer using cumulus cells as nuclear donors was also conducted. When blastomeres were used as donors, the electrofusion rate was significantly higher in oocytes matured in vivo (91.5%) than in those matured in vitro (66.1%) (p < 0.01). After fusion, the NT embryos reconstituted from in vivo matured oocytes developed to blastocysts at a rate of 10.3% after culture in rabbit oviducts for up to 5 days, while only 5.9% of the NT embryos reconstructed from in vitro matured oocytes developed to blastocyst stage. Electrofusion rate of cumulus cell nuclei with enucleated IVM oocytes was lower (47.6%) and only 1.5% (2/136) of the reconstituted eggs developed in vitro to morula stage, and 1.9% developed to blastocysts when cultured in the ligated rabbit oviducts. Transfer of 94 embryos reconstructed by blastomere NT with in vivo matured oocytes to five synchronous recipients resulted in the birth of two cloned piglets. No piglet was born following transfer to two recipients of embryos (n = 39) derived from NT with in vitro matured oocytes. The results demonstrate that in vivo matured oocytes are better recipients than those matured in vitro for pig cloning.

The methods of intracytoplasmic sperm injection (ICSI) and subzonal injection (SUZI) were used to study heterologous fertilization and embryonic development between the mouse and the rabbit. Results were as follows: 1. The mouse sperm nuclei decondensed and formed pronuclei following microinjection into cytoplasm and perivitelline space (PVS) of rabbit oocytes; 2. The hybrid embryos developed to the stage of 8-cell when cultured in vitro; 3. The karyotype analysis showed a normal complement of rabbit oocyte and mouse sperm chromosomes in the 4-cell hybrid embryos; 4. The ultrastructure of 4-cell hybrid embryos was similar to that of normal 4-cell rabbit embryos; 5. The fertilization rate (32.4%) and cleavage rate (22.2%) when 5-10 mouse spermatozoa were injected were higher than those of injection of a single spermatozoon into PVS of the rabbit oocyte, but the difference was not significant (P > 0.05). The fertilization rate (42.3%) and cleavage rate (30.8%) in rabbit oocytes in vitro matured for 11-12 h were higher than those in the oocytes which were in vitro matured for 24-25 h following microinjection of 1-2 mouse spermatozoa into PVS, but the difference was not significant (P > 0.05).

The tolerance of the oocyte plasma membrane (oolemma) to electrical pulses (TEP) was investigated for oocytes, zygotes, and embryos at the early and late 2-cell stage. The oocyte survival rate after two electrical pulses (1.4 kV/cm for 640 microns each) was used as an indicator of the TEP of the oolemma. Survival rate of mid-pronuclear zygotes (94.3% +/- 2.3%) was significantly (p < 0.05) higher than that of recently ovulated (2.1% +/- 1.9%) and in vivo aged (25.1% +/- 2.6%) oocytes; survival rate of in vivo aged oocytes was also significantly higher than that of recently ovulated oocytes. Soon after fertilisation, the survival rate of the oocytes markedly increased, up to 94% at the mid-pronuclear stage. Survival rate dropped thereafter. These results suggest that the characteristics of the oocyte plasma membrane (oolemma) change after fertilisation.