Developmental failure of hybrid embryos generated by in vitro fertilization of water buffalo (Bubalus bubalis) oocyte with bovine spermatozoa

The developmental potential of inter-species hybrid embryos produced by in vitro fertilization of in vitro matured buffalo oocytes with bovine spermatozoa was studied with a view to investigate pre-implantation embryo development and its gross morphology, early embryonic gene expression, and embryonic genome activation. Fertilization events with both buffalo and cattle spermatozoa were almost similar. Overall fertilization rate with cattle spermatozoa was 78.4% was not significantly different from that of buffalo spermatozoa (80.2%). Initial cleavage rate between buffalo and hybrid embryo was also similar, and there was no significant difference in their developmental rate till 8-cell stage (26.0 +/- 4.1 vs. 24.3 +/- 4.8). However, only 5.3% of hybrid embryos developed into blastocyst stage compared to 21.7% in buffalo. mRNA phenotyping of insulin-like growth factor family (Insulin, insulin receptor, IGF-I, IGF-I receptor, IGF-II, and IGF-II receptor) and glucose transporter isoforms (GLUT-I, II, III, IV) in hybrid embryos clearly showed that these molecules were not expressed after 8-cell stage onward. Similarly, as observed in buffalo embryos, incorporation of (35)S-methionine and (3)H-uridine could not be observed in hybrid embryos from 8-cell stage onward. This suggests that the maternal-zygotic genome activation did not occur in hybrid embryos. Differential staining also showed that the blastomere stopped dividing after 8-cell stage. Collectively, these parameters clearly showed that there was developmental failure of hybrid embryos.     

Regulation of apoptosis in the bovine blastocyst by insulin and the insulin-like growth factor (IGF) superfamily

Insulin and the insulin-like growth factors, IGF-I and IGF-II, have been reported to exert a mitogenic effect on the preimplantation mammalian embryo. Furthermore, it has been proposed that loss of imprinting of the insulin-like growth factor II receptor gene and the consequent over-production of IGF-II may be involved in the aetiology of the Enlarged Offspring Syndrome, which occurs as an artefact of in vitro embryo production. We have previously shown that apoptosis occurs in the preimplantation bovine embryo and is influenced by in vitro culture conditions. We have therefore sought to establish the effects of insulin, IGF-I and IGF-II on apoptosis and cell proliferation in bovine blastocysts in vitro. Zygotes, obtained by in vitro maturation and fertilization of follicular oocytes, were cultured to blastocysts, with or without exogenous growth factors. Embryos were stained with propidium iodide to label all nuclei and by TUNEL to label apoptotic nuclei and analyzed by epifluorescent and confocal microscopy. IGF-I and IGF-II, but not insulin, were found to increase the proportion of embryos which formed blastocysts. Insulin decreased the incidence of apoptosis without affecting blastocyst cell number. IGF-I acted to decrease apoptosis and increase total cell number and IGF-II increased cell number alone. These data suggest roles for insulin and the IGFs as mitogens and/or apoptotic survival factors during early bovine development. Perturbation of IGF-II regulated growth may be involved in fetal oversize.     

Stimulation of the development of bovine embryos by insulin and insulin-like growth factor-I (IGF-I) is mediated through the IGF-I receptor

To study the effects of insulin and insulin-like growth factor-I (IGF-I) on the development of bovine embryos, fertilized bovine embryos in vitro were cultured in a chemically defined, protein-free medium: modified synthetic oviduct fluid (mSOF) supplemented with 1 mg/ml polyvinyl alcohol. Dose-response studies showed that insulin (0.5 to 10 microg/ml) and IGF-I (2 to 200 ng/ml) stimulated the development of bovine embryos to the morula stage 5 d after in vitro fertilization. The addition of 0.5 microg/ml insulin or 2 ng/ml IGF-I to the mSOF had beneficial effects on embryonic development to the morula stage in the presence of amino acids, but insulin and IGF-I did not affect the development of bovine embryos to the morula stage in the absence of amino acids. The antiIGF-I receptor antibody (alphaIR-3) completely blocked the stimulation of development to the morula stage by insulin and IGF-I. These findings suggest that the stimulation of embryonic development by insulin and IGF-I is mediated through the IGF-I receptor.     

Insulin-like growth factor (IGF) family genes are aberrantly expressed in bovine conceptuses produced in vitro or by nuclear transfer

Embryos produced through somatic cell nuclear transfer (NT) or in vitro production (IVP) are often associated with increased abortion and abnormalities thought to arise from disruptions in normal gene expression. The insulin-like growth factor (IGF) family has a major influence on embryonic, fetal and placental development; differences in IGF expression in NT- and IVP-derived embryos may account for embryonic losses during placental attachment. In the present study, expression of IGF-I, IGF-II, IGF-I receptor (IGF-IR), and IGF-IIR mRNAs was quantitated in Day 7 and 25 bovine embryos produced in vivo, by NT, IVP, or parthenogenesis, to further understand divergent changes occurring during development. Expression of the IGF-I gene was not detected in Day 7 blastocysts for any treatment. However, there were no differences (P>0.10) among Day 7 treatments in the amounts of IGF-IR, IGF-II, and IGF-IIR mRNA. For Day 25 conceptuses, there was higher expression of IGF-I mRNA for NT and IVP embryonic tissues than for in vivo embryonic tissues (P<0.05). Furthermore, embryonic tissues from NT-derived embryos had higher expression of IGF-II mRNA than IVP embryonic tissues (P<0.05). Placental expression of IGF-IIR mRNA was greater for NT-derived than in vivo-derived embryos (P<0.05). There were no differences in IGF-IR mRNA across all treatments and tissues (P>0.10). In conclusion, these differences in growth factor gene expression during early placental attachment and rapid embryonic growth may directly or indirectly contribute to increased losses and abnormalities in IVP- and NT-derived embryos.     

Functional foles of insulin and insulinlike growth factors in preimplantation mouse embryo development

Summary Growth factors are known to play important roles in cellular proliferation and differentiation. However, little information is available concerning their roles in the earliest stages of mammalian development. The effect of physiologic levels of insulin, insulinlike growth factor-I, and insulinlike growth factor II (IGF-I and-II) on DNA, RNA, and protein synthesis in preimplantation stages of the mouse are described in this study. Quantitative studies of the incorporation of labeled thymidine, uridine, and methionine into trichloroacetic acid-insoluble material by different developmental stages of preimplantation mouse embryos labeled in vitro, indicate that physiologic levels of insulin stimulated DNA, RNA, and protein synthesis with significant effects observed first at the morula stage of development. In contrast, neither IGF-I nor IGF-II stimulated DNA, RNA, or protein synthesis to a significant degree under the same experimental conditions. These results suggest a functional role for insulin at the earliest stages of mammalian embryogenesis. This work was supported by grant HD 23511 from the National Institutes of Health, Bethesda, MD.