Implantation and Pregnancy
Session: Poster Session C
Suzuno Senda
Master Student
Nagoya university
Nagoya, Aichi, Japan
Suzuno Senda1, Rei Ichikawa1, Naoki Yamada1, Sho Nakamura1, Satoshi Ohkura1, Kaede Ito2, Yosuke Sugino2, Koji Kimura2, Shuichi Matsuyama1
1 Laboratory of Animal Production Science, Graduate School of Bioagricultural Sciences, Nagoya University, Aichi, Japan
2 Laboratory of Reproductive Physiology, Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan
Conception rate of multiparous cows is generally lower than that of heifers, possibly due to improper uterine recovery after calving. Senescent cells appear in the uterus during pregnancy and are gradually eliminated after calving. Poorclearance of these cells after calving adversely affects the subsequent pregnancy outcomes in mice. Therefore, improper removal of accumulated senescent cells after calving possibly contributes to the low conception rate of multiparous cows. To verify this, we investigated the effect of bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a selective senescent cell-removing drug, on the removal of senescent cells from the bovine endometrium after calving and assessed the underlying mechanisms in this study. To determine the effect of BPTES on senescent cell removal in vitro, we firstly examined the induction method of cellular senescence using cultured bovine endometrial stromal cells. Cellular senescence was induced by adding bleomycin (30 μg/mL), 5‑bromo‑2′‑deoxyuridine (8,000 μM), and doxorubicin (100 ng/mL) to the culture medium of bovine endometrial stromal cells. Subsequently, RNA was extracted from these cells and bovine endometrial tissues showing high accumulation of senescent cells 10 d post-partum, and gene expression patterns were compared via RNA-sequencing analysis. Gene Ontology analysis of the genes expressed in the drug-treated cells and endometrial tissues after calving revealed that doxorubicin-induced senescent cells exhibited similar characteristics as the cells remaining in the endometrium d 10 after calving. Next, bovine endometrial stromal cells were cultured with doxorubicin (100 ng/mL) to induce cellular senescence and treated with different BPTES concentrations (0, 10, and 50 µM) for 24 h. Senescence-associated β-galactosidase (SA-β-gal) staining intensity, senescent cell marker gene (p16, p21, and p53) levels, intracellular pH, and number of apoptotic cells were measured. The proportion of SA-β-gal positive cells among the cultured bovine endometrial stromal cells was decreased after the addition of 10 µM and 50 µM BPTES. Moreover, p16 mRNA levels were significantly decreased (p < 0.05) by 50 µM BPTES. Intracellular pH was also decreased by 50 µM BPTES (p = 0.09), whereas apoptotic cell proportion was significantly increased (p < 0.05) by 50 µM BPTES. Overall, these results suggest that BPTES eliminates the senescent bovine endometrial stromal cells by decreasing intracellular pH and inducing apoptosis. However, further studies are necessary to determine the optimal administration protocol for BPTES in postpartum cows.