Reliable estimation and improvement of the developmental potential of in vitro production (IVP) embryos requires functional criteria of embryo quality. Antiapoptotic and mitogenic effects of insulin-like growth factor I (IGF-I), applied during bovine IVP, were studied. Day 6.5 blastocysts were fixed and processed for TUNEL to detect apoptotic cells, for immunocytochemical detection of proliferating cell nuclear antigen (PCNA), and for propidium iodide (PI) staining to detect all nuclei. Laser scanning confocal microscopy was used to determine apoptotic (TUNEL/PI) and proliferative (PCNA/PI) indices. Addition of IGF-I to the culture but not to the maturation medium increased the morula/blastocyst yield (P = 0.03), but the cleavage rate was not affected. During culture, IGF-I significantly lowered the apoptotic index by decreasing the number of apoptotic cells per embryo and elevated the total cell number of the blastocysts. The frequency of blastocysts with apoptotic cells was not affected. IGF-I increased the proportion of blastocysts with apoptotic cells in the inner cell mass area only by reducing apoptosis in the trophectoderm area. The PCNA index was not affected by IGF-I. A positive correlation observed between apoptotic and PCNA-positive cells was significant in groups stimulated with IGF-I during in vitro culture. Of TUNEL-positive cells, 30%-40% per embryo were also positive for PCNA. This colocalization may indirectly suggest an activation of DNA repair process in TUNEL-positive cells in response to DNA fragmentation. IGF-I reduces apoptosis in bovine IVP embryos. The requirement of IGF-I is more critical during embryo culture than during oocyte maturation. Our data suggest that an assay for TUNEL in conjunction with cell proliferation analysis can provide useful information about the quality of IVP embryos.

Transgenic rabbits provide a useful biological model for the study of the regulation of mammalian genes. However, transgene integration efficiency has generally been low. Here we present a first attempt to increase the integration rate of exogenous DNA into the rabbit genome, using a double pronuclei microinjection method. Pronuclear stage rabbit embryos were recovered from superovulated NZW females, 19-20 h after hCG injection. About 5 microg/mL of exogenous DNA solution was microinjected either into one pronucleus (single microinjection, SM) or into both pronuclei (double microinjected, DM). The transgene consisted of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb cDNA of the human clotting factor VIII (hFVIII), and 4.6 kb that of 3' flanking sequences of the mWAP gene. The in vitro survival of DM embryos to the blastocyst stage was lower than that of SM embryos (68 vs. 89%). Similar results were obtained using EGFP as a control gene construct. However, there was no difference in the percentage of embryos that developed into live offspring using DM (25%) vs. SM (26%). The integration frequency of mWAP-hFVIII into the genome of transgenic rabbits was 3.3% (1/30) upon SM and 8.1% (4/49) at DM (p < 0.05). All founders transmitted the transgene to their offspring in a Mendelian fashion. The SM founder female secreted 87.4 microg/mL rhFVIII in milk, with an activity of 0.594 IU/mL. The DM founder female produced 118 microg/mL rhFVIII, with activity values of 18 IU/ mL. This is the first report of transgenic rabbit production using a double microinjection technique. Our preliminary results suggest that this method can increase the efficiency of production of transgenic rabbit founders, giving a higher integration rate than single microinjection.

-The aim of our in vitro experiments was to study the role of growth factors and protein kinase A (PKA)-dependent intracellular mechanisms in the control of nuclear maturation of porcine oocytes. Oocytes were cultured with or without growth factors (IGF-I, IGF-II, EGF; 10 ng . mL -1 medium) and inhibitors of PKA (Rp-cAMPS or KT5720; 100 ng . mL -1 ). Stages of meiosis were determined from the structure of chromosomes after staining with Giemza. Intracellular levels of PKA were evaluated immunocytochemically using primary antisera against the PKA regulatory and catalytic subunits and by Western immunoblotting using primary antiserum against the PKA catalytic subunit. It was found that after 24 h culture the majority of oocytes had resumed nuclear maturation (they were at a stage of meiosis after diplotene) and that after 48 h culture the majority of cells had completed maturation (they had reached metaphase II of meiosis). Addition of IGF-I, IGF-II or EGF, or a combination of IGF-I and EGF, significantly increased the proportion of oocytes which resumed and completed meiosis. Immunocytochemistry demonstrated a significant increase in the proportion of cells containing catalytic and, in some cases, the regulatory subunits of PKA after addition of IGF-I, IGF-II and EGF. Immunoblotting showed the presence of 2 forms of the PKA catalytic subunit within the oocytes (MW approximately 52 and 40 kD). EGF, but not IGF-I or IGF-II, increased the content of both isoforms. Inhibitors of PKA, when given alone, did not substantially influence the proportion of oocytes which resumed or completed meiosis. However, Rp-cAMPS and KT5720 both prevented the stimulatory effects of IGF-I, IGF-II and EGF on the resumption and completion of oocyte maturation. The present observations suggest (1) that IGF-I, IGF-II and EGF are potent stimulators of both resumption and completion of porcine oocyte nuclear maturation, (2) that PKA is present in oocytes, and (3) that PKA-dependent intracellular mechanisms can mediate the action of growth factors on porcine oocytes.insulin-like growth factor / epidermal growth factor / protein kinase A / meiosis Reprod. Nutr. Dev. 40 (2000)

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