Bovine oviductal monolayer and vesicle primary cultures express insulin-like growth factor (IGF)-I and -II mRNAs and polypeptides. Early bovine embryos also express IGF-I, IGF-II, IGF-I receptor, IGF-II receptor, and insulin receptor mRNAs. This study reports the expression of IGF binding protein (IGFBP) mRNAs and polypeptides in bovine oviduct primary cultures and IGFBP mRNAs in preattachment embryos. Release of immunoreactive IGF-I and IGF-II by oviduct cultures and bovine blastocysts was also determined. IGFBP-2, -3, -4, and -5 transcripts were observed in oviduct primary cultures throughout an 8-day interval. IGFBP-1 and -6 mRNAs were consistently not detected in the oviduct. Messenger RNAs encoding IGFBPs -2, -3, and -4 were detected throughout bovine preattachment development, while transcripts encoding IGFBP-5 were detected only in blastocysts. IGFBP-1 and -6 transcripts were not detected in early embryos. Ligand blot analysis with 125I-labeled IGF-II revealed the presence of four prominent polypeptide bands of approximate molecular masses 24, 31, and 36 kDa, and a broad band extending from 46 to 53 kDa, in conditioned media samples prepared from oviduct primary cultures. Western immunoblot analysis confirmed the identity of the 24-kDa, 31-kDa, and 36-kDa species as IGFBP-4, -5, and -2, respectively. Levels of the release of IGF-II from oviductal vesicle cultures were significantly greater than levels observed for monolayer cultures (p < 0.005). No significant difference in the levels of IGF-I release between monolayer and vesicle cultures was observed. Pools of 10 blastocysts released on average 36.2 +/- 3.9 pg of IGF-II per embryo, while the release of embryonic IGF-I was below the levels of detection for our assay. The results suggest that maternally derived IGF may be regulated by IGFBPs to support bovine preattachment development.

The present study was conducted to examine the karyotypes of parthenogenetic bovine embryos arising from the application of standard oocyte activation and diploidization methods. Bovine cumulus–oocyte complexes were collected and matured in vitro for 24 hr prior to oocyte activation with either 5 μM ionomycin or 7% ethanol for 5 min. Groups of activated oocytes were further treated with 5 μg/ml cytochalasin D or 1.9 mM 6‐dimethylaminopurine (DMAP) for 6 hr. Cleavage varied significantly (P < .05) among the treatment groups with 68.0% of the ethanol‐ and DMAP‐treated oocytes dividing. Blastocyst development did not vary with 18.4 ± 2.5% of all treated oocytes progressing to this stage. Blastocyst development did not occur in groups subjected to oocyte activation alone. Blastocysts displayed haploid (2.3%), diploid (11.4%), tetraploid (40.9%), octaploid (4.5%), and mixoploid chromosomal complements (40.9%). Two‐cell stage parthenogenotes resulting from ethanol or ionomycin treatment alone displayed haploid (66.7%), diploid (16.7%), tetraploid (4.2%), and mixoploid (12.5%) complements. Our results demonstrate that diploid bovine parthenogenotes arising from these procedures are a minority, with the majority of parthenogenotes displaying polyploid and mixoploid chromosomal complements. The events contributing to these abnormal chromosomal complements occur as early as completion of the first cell cycle, possibly linking these events with the absence of a paternally supplied centrosome. Dev. Genet. 21:160–166, 1997. © 1997 Wiley‐Liss, Inc.

Two experiments, one comparing nuclear transfer (NT) embryo activation compounds, the other donor cell treatments, were conducted with a goal of identifying factors that improve the in vitro development of cloned bovine embryos. In experiment 1, 539 NT embryos were produced by combining serum starved bovine fetal fibroblasts with enucleated in vitro matured oocytes, activated with ionomycin, then randomly allocated to be incubated for 4 hours in either Butyrolactone-I (BL-I) or 6-dimethylaminopurine (DMAP). There was no significant difference in development to blastocyst or compact morula of fused embryos at Day 6.5 between BL-I and DMAP activated embryos (22.4% vs. 20.2%; p = 0.18). Karyotyping of 20 blastocysts and compact morula from each group determined that 65% of BL1 and 63% of DMAP embryos were diploid with the remainder mixoploid (2n + 4n). In Experiment 2, the development of 389 NT embryos reconstructed from either serum starved or serum fed fetal fibroblasts was assessed. More Day 7 blastocysts and compact morula developed in the serum starved group (34.5% vs. 18.8%; p = 0.008). To verify the viability of BL-I activated embryos, 10 blastocytes from experiment 2 were transferred into 4 recipient cows. Two morphologically normal fetuses, genetically identical to the original fetal cell line, were surgically recovered at day 45 of gestation. In summary, serum starvation of bovine fetal fibroblasts prior to NT significantly improved development to blastocyst. Additionally, we have shown that BL-I is a novel alternative compound for use in combination with ionomycin to activate NT embryos.

No abstract