Obese heifers with hyperinsulinemia produce fewer transferable embryos than normal heifers, and lean heifers produce more unfertilized ova (Kadokawa et al. 2008). Oviducts synthesize granulocyte macrophage colony stimulating factor (GMCSF) and macrophage migration inhibitory factor (MIF) to promote fertilization and embryogenesis. This study evaluated the hypothesis that moderate, but not low and high, level of insulin stimulates GMCSF or MIF expression level in bovine oviduct epithelial cells (BOECs). In first, we confirmed the expression of insulin receptor (InsR) in both ampullae and isthmuses utilizing real-time PCR and fluorescent immunohistochemistry (FIHC). The primary site of InsR expression in ampullae and isthmuses is tunica mucosa. In next, both ampullar and isthmic BOECs from Japanese Black heifers were prepared, cultured, and passaged. Then, the cultured BOECs were treated with insulin (final concentrations were 0, 1, 20, and 5,000 ng/mL) for 24 hours. GMCSF or MIF mRNA and protein in BOECs were measured by real-time PCR and western blot, respectively. The cultured BOECs expressed InsR, GMCSF, and MIF. Both GMCSF and MIF expression were higher in ampullar or isthmic BOECs treated with 20 ng/mL insulin than those treated with 0, 1, or 5,000 ng/mL (P < 0.05). The 1 and 5,000 ng/mL insulin had no significant effect on the expression compared to 0 ng/ml insulin. In conclusion, moderate level of insulin stimulated both GMCSF and MIF expression in oviducts, whereas low and high level of insulin had weak stimulation, suggesting insulin may regulate GMCSF and MIF expression level in bovine oviducts also in vivo.

Kisspeptin, NKB and dynorphin are co-expressed in KNDy neurons in the hypothalamus. We recently reported that granulosa cells are also KNDy cells and the expression of those peptides is stimulated by preovulatory LH surge, but relationship between these peptides is unknown. In the present study, mutual effect of these peptides on respective mRNA expression was investigated. Granulosa cells were prepared from eCG treated immature female rats. Cells were treated with hCG 0.01 IU/ml and/or kiss-10 (10^–11M), NKB (10^–6M) or dynorphin (10^–6M) after 24 hours of preincubation. 1) The expression of GnRH, kisspeptin, dynorphin and NKB were all augmented by hCG. 2) Dynorphin and hCG additively augmented both NKB and kisspeptin expression. Kiss-10 with hCG also stimulated both dynorphin and NKB expression, while NKB inhibited hCG stimulated kisspeptin and dynorphin expression. 3) Kiss-10 showed no effect on GnRH expression, while GnRH stimulated annexin A5 expression. Surprisingly GnRH agonist enhanced kisspeptin expression. 4) In vivo gene expression showed acute increase of kisspeptin and dynorphin after hCG injection, while NKB expression was delayed and sustained. 5) hCG treatment was demonstrated to proceed luteinization by p21, p27, LH receptor and FOXO1 expression and progesterone production. These data demonstrate that kisspeptin and dynorphin are stimulated their expression by hCG and augment each other in granulosa cells. NKB seems to cease the enhancement of kisspeptin and dynorphin by LH. The novel sequence of events found in this study induced by hCG/LH is hypothesized to be beneficial for luteinization of granulosa cells.

L-amino acid oxidase (LAO) is an enzyme which converts particular L-amino acids into keto acids, ammonia, and H₂O₂. We found that LAO was presented in acrosomes of sperm, and LAO knockout male, not female, mice revealed decrease of their litter size, which indicated that LAO plays an important role in displaying sperm function of reproductive male mice. Sperm collected from LAO KO mice showed high malformation rate and low viability in in vitro culture. In addition, medium cultured with LAO KO sperm contained low concentration of H₂O₂ than that of WT sperm. Though there was no difference in the fertilization ratio of oocytes via in vitro fertilization, Western blot and immunofluorescence analysis showed that the level of phosphorylated tyrosine proteins, which is associated with sperm capacitation, was lower in LAO KO sperm than WT sperm. When the LAO KO sperms were cultured with H₂O₂ supplement, the level of tyrosine protein phosphorylation was increased with a dose dependent manner and the sperm viability was also increased after H₂O₂ treatment. Those results suggested that sperm LAO provides H₂O₂ to increase the sperm viability and to induce the capacitation by stimulating the tyrosine phosphorylation levels. Thus, sperm expressing LAO have more chance to reach oocyte at oviduct, which influence the litter size in mice. Moreover, the positive correlation between LAO expression and sperm tyrosine phosphorylation were also found in pig sperm and high expression of LAO were also found in the cow sperm with high fertilization. In summary, LAO may serve as one novel marker for sperm function in different species.

The silk protein sericin has been identified as a potent antioxidant in mammalian cells. In this study, we investigated the potential effects of sericin supplementation during in vitro culture (IVC) medium on subsequent embryonic development and quality of bovine embryos exposed to heat stress (HS). After in vitro fertilization, putative zygotes were cultured in medium supplemented with sericin and subjected for 6 h heat stress at 40.5°C on day 2 or day 7 followed by 38.5°C until day 8. After IVC, the developmental rate to blastocysts, total cell number, and the percentage of apoptotic cells in blastocysts were evaluated on day 8 (day 0, IVF day). Intracellular reactive oxygen species (ROS) level of embryos were detected just after heat stress on day 2 and day 7. The developmental rate to blastocysts and total cell number of blastocysts were significantly (P < 0.05) reduced in HS-group. In addition, the number of TUNEL-positive cells and ROS level of embryos in HS-group were significantly (P < 0.01) increased compared with those of control group. On the other hand, supplementation of 0.1 % sericin significantly (P < 0.05) increased the developmental rate and quality of heat stressed embryos by decreasing the ROS levels and TUNEL-positive cells. Our findings indicate that sericin application effectively protects the embryos from HS and increases the developmental competence of preimplantation bovine embryos.