METABOLIC AND MITOCHONDRIAL SIGNALS DURING OVULATION

Consistent with the objective of the Dual Purpose with Dual Benefit RFA this project utilizes an agriculturally important domestic animal model (beef cattle) to improve human health though advancement of biomedical research. As observed in women, fertility in cattle has declined significantly for several decades. Beef cattle production is the largest sector of animal agriculture in the United States with 711,827 farms and is 20% of all agriculture sales. Reproductive inefficiency is a limiting factor for the sustainability of beef cattle production systems that leads to financial losses to cattle producers; the cost to U.S. cattle producers was estimated to be over $1.06 billion annually. A major infertility problem in cattle is anovulation, which is the failure to ovulate an egg, or to display behavioral estrus and ovulate at the appropriate time. Ovulatory dysfunction in women (defined as a history of oligomenorrhea or amenorrhea or as luteal progesterone levels repeatedly less than 3 ng/mL, or both) accounts for a significant proportion of female infertility and up to one third of women with normal menstrual cycles are anovulatory. Often anovulation is due to impaired follicle development.Understanding the cellular events underlying ovulation and subsequent differentiation of ovarian follicular cells into a functional corpus luteum is critically important because these biological processes provide the foundation to regulate female fertility. The luteinizing hormone (LH) surge activates complex intercellular signaling pathways, which induce or activate specific pathways in granulosa cells of ovulatory follicles. Little is known about the LH surge-activated/induced signaling pathways and transcription factors in humans. The protein kinase A (PKA) and PKC/ERK signaling pathways are widely regarded as primary pathways mediating LH/hCG action in the preovulatory follicle. Although changes occurring at the transcriptome level have been characterized in some species, little is known about metabolic and energetic pathways required for ovarian follicular cell maturation and ovulation. The proposed research will fill a gap in our understanding of the molecular processes regulating follicular metabolism and mitochondrial biology in response to an ovulatory stimulus in vivo and during cellular differentiation in vitro. Our long-term goals are to understand the mechanism of action of LH and hCG.Specific Aim 1: Determine metabolic pathways associated with ovulation and ovarian follicular cell differentiation. We will test the hypothesis that a metabolic shift between ovarian follicular cells and luteal cells is fundamental to support the increasing demand for cell differentiation and progesterone synthesis. To test this hypothesis, we will determine: (1) the metabolic phenotype of ovarian follicle cells in pre- and post-ovulatory stages, (2) effects of PKA and ERK signaling on metabolic pathways associated with NADPH and acetyl-CoA synthesis, (3) effects of LHCGR signaling on metabolic pathways leading to NADPH and FA (Fatty Acid) synthesis during differentiation of ovarian follicular cells, and (4) how disruption of metabolic pathways that contribute to acetyl-CoA and NADPH production affects follicular differentiation and progesterone production.Specific AIM 2: Determine the molecular processes regulating mitochondrial biology during the differentiation of ovarian follicular cells. We will test the hypothesis that LHCGR signaling governs exquisite regulatory mechanisms to fine-tune the coordination of mitochondrial structure and function with metabolic demands via modulation of mitochondrial dynamics and quality control systems. To test this hypothesis, we will determine: (1) the effects of LHCGR signaling on mitochondria biology of pre-ovulatory follicular cells in vivo, (2) the effects of LHCGR signaling on pre- and post-ovulatory follicular cells mitochondria biology in vitro, and (3) the effects of LHCGR signaling on mitochondrial energetics and steroidogenic capacity in pre- and post-ovulatory follicular cells in vitro.Impact: Our ability to use a combination of unbiased methods such as metabolomics, lipidomics, and transcriptomics together with the measurement of cofactors such as ATP and NADPH, and mitochondrial structure/function biology will unveil the metabolic phenotypes of ovarian follicular cells as they respond to hGC and differentiate into luteal cells. Identification of metabolic and energetic pathways activated by LHCGR signaling will enhance our understanding of ovulation and differentiation of follicular cells and is expected to contribute to novel approaches for improving fertility due to anovulation in both the cow and women.