(71) Hormonal Regulation of Lactate Secretion by Bovine Uterine Epithelial Cells and Embryo-Maternal Cross Talk by Lactate and Extracellular Vesicles.

Muhammad Ashir Nabeel^a, Lyda Yuliana P. Forero^a, Keely Mogge^a Matthew Dean^ab, Romana A. Nowak^ab

^aDepartment of Animal Sciences, College of Agricultural, Consumer and Environmental Sciences, University of Illinois, Urbana, IL, USA

^bDivision of Nutritional Science, College of Agricultural, Consumer and Environmental Sciences, University of Illinois, Urbana, IL, USA

Abstract Text:

Basigin, a highly glycosylated transmembrane protein, is a protein of interest due to its involvement in processes crucial to reproduction including uterine remodeling and implantation. Basigin interacts with monocarboxylate transporters (MCTs) and facilitates their proper expression, localization, and function in the cell membrane.  Lactate is transported across the cell membrane in and out of the cell through MCTs 1 and 4 respectively. Lactate is an important energy source for developing embryos and is thought to also help prepare the uterine environment for embryo implantation. Our previous studies showed that basigin expression in a bovine uterine epithelial (BUTE) cell line was upregulated by progesterone (P4) and it is released in extracellular vesicle (EVs) released by these cells. The goals of this study were to determine whether P4 regulates the expression of MCTs and/or lactate secretion by BUTE cells, determine the expression of basigin and MCTs in preimplantation bovine embryos, and confirm internalization of EVs from BUTE cells into bovine embryos. BUTE cells were treated with 0, 30, 300, and 3000 nM P4 for 24 hours to determine its effects on the expression of MCTs and lactate secretion. The total protein in the cell lysates was measured via BCA assay. Immunoblotting of cell lysates was carried out to quantify the expression of basigin and MCTs. The conditioned medium was collected after 24 hours of treatment and a lactate assay carried out to quantify lactate secretion by the BUTE cells. The expression of basigin and MCTs in pre-implantation bovine embryos was determined by immunofluorescence (IF) staining at the 8-cell, morula, and blastocyst stages. BUTE cell-derived EVs were stained with the fluorescent marker PKH67 and co-cultured with bovine embryos at day 5 after fertilization for 24 hours and IF was performed to determine their internalization by bovine embryos.  MCT4 expression was upregulated by 300 and 3000 nM of P4 and lactate secretion by BUTE cells was upregulated in response to 3000 nM P4. This hormone-dependent regulation of MCT4 and lactate secretion indicates that lactate may serve as an important metabolic substrate or signaling molecule supporting the uterine environment necessary for embryo implantation and development. We determined the expression of basigin as well as MCT1 and MCT4 across multiple stages of pre-implantation embryo development, including the 8-cell, morula, and blastocyst stages. The expression of basigin, MCT1, and MCT4 across the various pre-implantation stages supports that these proteins may facilitate the transport of lactate and other metabolic substrates important for the energy needs of developing embryos. Furthermore, we showed that EVs from BUTE cells were successfully internalized by bovine embryos. Our study provides insight into the hormonal regulation of basigin and MCT4 expression and lactate secretion in BUTE cells, highlighting the impact of P4 on basigin and its role as a chaperone for MCTs in these cells. The expression of basigin and MCTs at the various stages of pre-implantation bovine embryo development supports that these proteins may play an important role in meeting the metabolic needs of the developing embryo. The observed internalization of BUTE cell EVs by bovine embryos highlights their potential involvement in intercellular communication during early embryonic development.

Key words: Basigin, Monocarboxylate transporters, Lactate, Steroid hormones, Bovine embryo, Western blot

Financial support: This work was supported by a Future Interdisciplinary Research Exploration Grant from the College of ACES, University of Illinois (RAN)