Project Summary / Abstract: Defective uterine function for embryo implantation is associated with two major public health problems in reproductive women, infertility and early pregnancy loss. How a uterus transiently transforms into a receptive state (uterine receptivity) for embryo implantation is the least well understood step during pregnancy. Uterine epithelium is the first maternal contact for an implanting embryo. Our novel finding of uterine epithelial lysosomal acidification upon embryo implantation led us to the investigation of lysosomes in the establishment of uterine receptivity. Lysosomal acidity regulates lysosomal activities in essential cellular processes, such as vesicular trafficking in endocytosis and exocytosis. Lysosomal acidity is maintained by V- ATPase and counter ion channels. Our preliminary data demonstrate prominent expression of TRPML1 (a counter ion channel encoded by MCOLN1) in the uterine epithelium and defective embryo implantation in 5 months old Mcoln1-/- mice, which have progesterone deficiency and uterine luminal fluid retention that could not be alleviated by exogenous progesterone treatment. Mutations of MCOLN1 in human and mouse cause mucolipidosis type IV, a severe lysosomal disorder, indicating a critical role of TRPML1 in lysosomal functions. The mechanisms of TRPML1 in uterine fluid resorption to facilitate uterine lumen closure and embryo implantation remain as a significant knowledge gap. Our long-term goal is to understand the mechanisms involved in the establishment of uterine receptivity thus help overcome infertility and early pregnancy loss associated with defective uterine receptivity. The defined objective of this R03 application is to determine a cellular mechanism of TRPML1 in uterine fluid resorption, a prerequisite for the establishment of uterine receptivity. Our central hypothesis is that TRPML1 regulates lysosomal function in endocytosis to absorb uterine luminal fluid for uterine lumen closure and subsequent embryo implantation. It will be tested in the following two specific aims using a Mcoln1-/- mouse model. Aim 1. Determine uterine epithelial lysosomal activity in preimplantation Mcoln1-/- uterus. Aim 2. Determine uterine epithelial endocytosis in preimplantation Mcoln1-/- uterus. Ovariectomy, ovarian hormone injection, tracer injection, immunofluorescence, and transmission electron microscopy are among the approaches that will be employed. The proposed work is significant for advancing our understanding of the mechanisms in the establishment of uterine receptivity as well as for providing knowledge to develop diagnostic and therapeutic approaches for infertility and early pregnancy loss, which is a high priority research area identified by NICHD.