ABSTRACT. We analyzed the expression of mRNAs for growth factor [epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF) and platelet-derived growth factor A chain (PDGF-A)] and their receptor (R) by reverse transcriptionpolymerase chain reaction in bovine ova during oocyte maturation in vitro (0-21 hr) and after fertilization in vitro (6-144 hr: zygotes to blastocysts). Transcripts for EGF were not found before fertilization. Transcripts for IGF-I were present in immature oocytes immediately after collection and in embryos from the 2-cell stage onward. Transcripts for bFGF were present in all stages of oocyte maturation and after fertilization up to the 16-cell stage. Transcripts for PDGF-A were present in all stages of oocyte maturation and after fertilization up to the 2-cell stage. Transcripts for ErbB3 (a member of the EGF-R subfamily), and bFGF-R were present in all stages of oocyte maturation, after fertilization up to the 2-cell stage, and the blastocyst stage. Transcripts for IGF-I-R and PDGF-Rα were present in all stages of oocyte maturation and embryo development. The results of this study showed that eight different messages for growth factor and their receptor were detectable in bovine ova during oocyte maturation and/or after fertilization in vitro and their expression patterns were the gene-specific rather than the developmental stage of bovine ova. -KEY WORDS: bovine ova, growth factor, growth factor receptor, mRNA, RT-PCR.

Repeat breeder cattle do not become pregnant until after three or more breeding attempts; this represents a critical reproductive disorder. Embryo transfer (ET) following artificial insemination (AI) in repeat breeder cattle reportedly improves pregnancy rate, leading to speculation that interferon tau (IFNT) is associated with this phenomenon. However, the reason why the conception rate improves remains unknown. We investigated the effect of ET following AI on repeat breeder cattle in field tests, and determined whether adding an embryo affects the maternal immune cells detected by interferon-stimulated genes (ISGs), marker genes of IFN response. In total, 1122 repeat breeder cattle were implanted with in vitro fertilization (IVF) embryos after previous AI. ET following AI resulted in pregnancy rates of 46.9% in repeat breeder dairy cattle. In basic in vivo tests, to investigate the effect of adding embryos, ISGs mRNA expression levels were significantly higher in the AI + ET group than in the AI + sham group (transfer of only embryonic cryopreservation solution). Then, we examined the effect of cultured conditioned media (CM) of IVF embryos on splenic immune cells and Madin-Darby bovine kidney (MDBK) cells with stably introduced ISG15 promoter-reporter constructs. These cells exhibited a specific increase in ISG15 mRNA expression and promoter activity when treated with the CM of IVF embryos, suggesting that IVF embryos have the potential to produce and release IFNT. In conclusion, ET following AI is beneficial for improving conception in repeat breeder cattle. Added embryos may produce and secrete IFNT, resulting in the increased expression of ISGs.

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Repeat breeding is a reproductive disorder in cattle. Embryo transfer following artificial insemination (AI) improves pregnancy rate by replenishing interferon tau (IFNT), but it results in a notably higher rate of twin occurrence. This study hypothesized that parthenogenetic (PA) embryo transfer following AI (AI + PA) could improve the conception rate because that PA embryo become as a supplemental source of IFNT without twins. PA embryos showed higher IFNT mRNA expression than in vitro fertilization (IVF) embryos. An examination of the effect of the cultured conditioned media (CM) of PA or IVF embryos on Madin-Darby bovine kidney cells with stably introduced promoter-reporter constructs of interferonstimulated gene 15 (ISG15, marker of IFN response) showed higher stimulation levels of ISG15 promoter activity with PA than with IVF embryo. We investigated in vivo the effect of AI + PA on healthy Japanese Black cattle. Cattle transferred with PA embryo alone were non-fertile, but those that underwent AI + PA showed a pregnancy rate of 53.3%, the similar as that with AI alone (60%). In pregnant cattle in AI + PA group, adding the PA embryo upregulated the expression of ISGs and plasma progesterone concentration. No twin were generated in AI only and AI + PA groups. Using repeat breeding Holstein cows that did not become pregnant with 4-9 times of AI, transfer of PA embryo following AI resulted in a higher pregnancy rate than that of control (AI only). We suggest that AI + PA may be beneficial for improving maternal pregnancy recognition in repeat breeder cattle while avoiding twin generation.

The zona pellucida (ZP) of mammalian ova plays an important role during maturation, fertilization, and early embryonic development. The hardness of ZP of mammalian ova has been mainly evaluated by a biochemical method, such as a difference in ova dissolution speed with an enzyme or an acid reagent treatment. However, the physical hardness of ZP in bovine embryos is largely unknown. Recently, we developed a system measuring the physical hardness of ZP of mammalian ova with a micro bio-sensor (MBS). The objective of this study was to examine the physical ZP hardness of in vivo- and in vitro-derived bovine embryos using an MBS. In the measurement system, the thin glass needle was connected at the tip of a piezo-electric ceramic tactile sensor, and the sample was pushed into a sensor at fixed speed under the computer-controlled micromanipulation system. The change in frequency at the time of displacement of the sample into the sensor was recorded using the computer program. Measurement of gelatin samples of known concentration (4 to 8%) was used to determine the basic characteristic of a sensor and to make a standard curve. In vivo-generated embryos were recovered from 6 superovulated Japanese Black cows with multiple injection of FSH. On Day 7 after insemination, morulae (M), early blastocysts (EB), and blastocysts (BL) were recovered by nonsurgical flushing of uterine horns. In vitro-generated embryos were produced as described earlier (Yoshida et al. 1998 J. Vet. Med. Sci. 60, 549–554). The M, EB, and BL at Days 5 to 7 of post-insemination in vitro were used for measurement of ZP hardness. When a sensor made contact with a harder gelatin sample, the change in frequency was large; the change in frequency has the characteristic of being small for a softer gelatin sample. By comparison with a standard curve, ZP hardness converted into gelatin concentration for each stage of bovine embryos generated in vivo was 3.95% (M: n = 9), 4.14% (EB: n = 32), and 3.92% (BL: n = 14), respectively. On the other hand, ZP hardness of bovine embryos generated in vitro was 3.42% (M: n = 56), 3.33% (EB: n = 36), and 3.25% (BL: n = 23), respectively. There was a significant difference (P < 0.01) in the hardness of ZP between in vivo- and in vitro-generated bovine embryos.