Localization of Prohibitin in Isolated Germinal Vesicle Oocytes and In Vitro Fertilized and Cloned Embryos
The rat and mouse prohibitin genes are functional ho-mologues of human prohibitin. This gene is highly conserved, because comparative analysis of the amino acid sequences indicates a single amino acid substitution between these three species. We have determined that the protein expression patterns of prohibitin in rat ovarian follicles are similar to that observed in porcine follicles (data not shown).
Considering the ease of isolating large numbers of pig ova and the difficulty of the same in rats, the porcine model, to which both in vitro fertilization (IVF) and NT procedures have routinely and successfully been applied, was used in the present study to provide a rationale for future use of porcine model in studies of prohibitin during early embryo development. To gain further insight regarding the expression of prohi-bitin during normal and aberrant oogenesis and preimplantation embryonic development, standard immunofluorescence procedures on whole-cell mounts (as opposed to the processing of tissue sections) were conducted. Prohibitin was readily detectable in normal GV-stage oocytes (Fig. 5, A and D), albeit at low levels. A distinct, speckled pattern of prohibitin immunoreactivity was detected in the GV-stage oocytes that also showed abnormal condensation of chromatin, suggestive of atresia (Fig. 5, B and C). Association of prohibitin with oocyte chromatin was not seen in metaphase II oocytes after in vitro or in vivo maturation (Fig. 5D). Similarly, neither the male nor the female pronuclei of zygotes fertilized in vitro (Fig. 5E) displayed significant prohibitin immunoreactivity. However, the zygotes and embryos arrested at the 2-cell stage after the apoptosis-inducing heat shock treatment displayed the pattern of nuclear prohibitin accumulation similar to that seen in defective GV-stage oocytes (Fig. 5F). This nuclear expression pattern was more pronounced in the embryos that failed to develop normally after NT (Fig. 5, G and H). In contrast, low levels of nuclear immunoreactivity were observed in embryos that reached the blastocyst stage at Day 5-6 after NT (Fig. 5I) or IVF (not shown) and displayed an acceptable morphology.
FIG. 5. Immunofluorescence localization of prohibitin (red) in the isolated porcine follicular oocytes (A-D) and in the apoptotic IVF (E and F) and cloned (G-I) porcine embryos. A) Prohibitin is not readily detectable in the mitochondria or the GV of morphologically normal GV-stage oocytes. B and C) A speckled pattern of prohibitin immunoreactivity is observed in the GV of oocytes showing abnormal condensation of chromatin (arrows in B) and extreme-eccentric position of GV, which is suggestive of follicular atresia or oocyte aging. D) Association of prohibitin with oocyte chromatin is not seen in metaphase-II oocyte after in vitro maturation. E) Neither the male nor the female pronucleus (arrows) of a morphologically normal zygotes fertilized in vitro displays significant prohibitin immunoreactivity. F) An IVF embryo arrested at the 2-cell stage after the apoptosis-inducing heat shock (HS) treatment displays a distinct nuclear accumulation of prohibitin. G and H) Nuclear prohibitin is seen in the embryos that failed to develop normally after nuclear transfer. I) Low levels of nuclear immunoreactivity are observed in a morphologically normal blastocyst at day 6 after nuclear transfer. Corresponding differential interference contrast (DIC) images of cells in A-G are shown in A’-G’. DNA was counterstained with 4′,6′-diamidino-2-phenylindole (blue). Bar = 10 ^m.