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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.

Recent studies by Buhi et al. have demonstrated that estrogen (E2) is responsible for the induction of de novo synthesis and secretion of certain oviductal secretory proteins (OSP) and inhibition of other OSP in porcine oviductal explant cultures. The present work was undertaken to evaluate the effect of E2-treated oviductal epithelial cell coculture on the development of early porcine embryos derived from in vitro matured and fertilized oocytes. In vitro synthesis of secretory proteins by E2-treated oviductal cells used for coculture was also investigated by one-dimensional (1D) and two-dimensional (2D) sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). The results showed that the cleavage rate was significantly enhanced by coculturing fertilized eggs with E2-treated oviductal epithelial cells. The in vitro protein synthetic pattern of oviductal secretory proteins was influenced by E2 treatment. These variations included the disappearance of one protein (82,000 M(r)) and the appearance of another (33,000 M(r)) in the E2-treated group as assessed by 1D-SDS-PAGE. Additional proteins of M(r) 97,000 and an M(r) 36,000-45,000 complex were increased in abundance by the E2 treatment. Analyses by 2D-SDS-PAGE revealed three major E2-dependent proteins, of M(r) 45,000 (pI 5.5), 43,000 (pI 5.5) and a 36,000-45,000 M(r) (pI 4.8) protein complex, whereas polypeptides of M(r) 97,000 (pI 5.1), 36,000 (pI 8.0) and 25,000 (pI 6.8) were inhibited by E2 treatment. The results demonstrated that porcine epithelial cell protein synthetic patterns are influenced by E2 treatment and that estradiol treatment of oviductal cells may increase the rate of zygote cleavage during early development in vitro in pigs.

We have investigated the patterns of expression and cellular localization of polypeptides and mRNAs encoding IGF-I and IGF-II in intact bovine oviduct and two bovine oviductal primary cultures (monolayers and vesicles) which are utilized for supporting development in vitro. IGF-I and IGF-II polypeptides were localized by immunocytochemistry in intact oviduct and in both primary cultures for an 8-day culture interval, but IGF-II polypeptide displayed a more restricted distribution in day 8 monolayer cultures. IGF-I and IGF-II mRNAs were localized in both oviductal cell cultures as assessed by in situ hybridization. We were unable to detect IGF-I and IGF-II mRNAs in intact oviduct by in situ hybridization; however, transcripts encoding IGF-I and IGF-II mRNAs were detected in intact oviduct cell preparations and all primary culture samples by reverse transcription-PCR methods. The origin and phenotypic stability of these cultures was assessed by immunostaining with antibodies raised against vimentin (mesenchymal cell marker) and cytokeratin (epithelial cell marker). Over the culture period, the proportion of vimentin-immunoreactive cells increased in the monolayer cultures but remained at a low level in the vesicle cultures which were predominantly composed of cytokeratin-positive cells. The results suggest that oviductal cell co-culture may facilitate early mammalian development, in part, by the establishment of paracrine growth factor circuits.

The roles of medium composition, serum source, embryo coculture, and culture under low O2 conditions on the development of in vitro-matured and in vitro-fertilized (IVMF) ovine zygotes were investigated in three separate experiments. In the first experiment, the proportion of cocultured IVMF zygotes developing to the blastocyst stage was significantly higher (38.0% vs. 3.5%; p < 0.05) than that of non-cocultured zygotes treated within three embryo culture media (TCM-199 + 10% fetal bovine serum [FBS]; bicarbonate-buffered, glucose-free synthetic oviduct fluid medium [mod-SOFM] + 10% FBS; and bicarbonate-buffered BSA-free Tyrode's salt solution [mod-TALP] + 10% FBS) under a 5% CO2 atmosphere in air. In a second experiment, a significantly higher (p < 0.05) proportion of cocultured zygotes placed in TCM-199 medium survived to the blastocyst stage (37.4% blastocysts vs. 23.4% in mod-SOFM). No significant effect of serum (FBS vs. human serum [HS]) was observed on embryonic development, but coculture was confirmed to exert a significant influence on development to the blastocyst stage. In the final experiment, survival of the embryo under a reduced oxygen (5% CO2:5% O2:90% N2) atmosphere was investigated. In contrast to results in the initial experiments, embryonic survival was significantly higher (p < 0.05) in the non-cocultured treatment groups (21.9% blastocysts vs. 0.4% for cocultured zygotes). Serum source also had a significant (p < 0.05) influence upon the development of non-cocultured zygotes: 32.3% of zygotes cultured with HS progressed to the blastocyst stage vs. 11.5% of zygotes cultured in FBS-supplemented medium. These results have characterized two distinct culture environments, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro.

The reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the patterns of expression for several growth factor ligand and receptor genes during ovine preimplantation development. Transcripts for insulin-like growth factor (IGF)-I, IGF-II, and the receptors for insulin and IGF-I were detected throughout ovine preimplantation development from the 1-cell to the blastocyst stage. Transforming growth factor alpha (TGF alpha) transcripts were also detected throughout ovine preimplantation development. The mRNAs encoding basic fibroblast growth factor (bFGF) were detected in all stages of the ovine preimplantation embryo, although the relative abundance of this transcript consistently decreased from the 1-cell to the blastocyst stage, suggesting that it may represent a maternal transcript in early sheep embryos. Transcripts encoding ovine trophoblast protein (oTP) were detected only within blastocyst-stage embryos. Primary ovine oviduct cell cultures express the transcripts for IGF-II, IGF-I, TGF alpha, bFGF, TGF beta 1, and the receptors for insulin and IGF-I, suggesting that paracrine growth factor circuits may exist between the oviduct epithelium and the early ovine embryo. Transcripts for insulin, epidermal growth factor (EGF), and nerve growth factor (NGF) were not detected in any stage of the ovine preimplantation embryo or within the oviduct cell preparations. The expression of growth factor transcripts very early in mammalian development would predict that these molecules fulfil a necessary role(s) in supporting the progression of early embryos through the preimplantation interval. Our future efforts will be directed to understanding the nature of these putative regulatory pathways.