Metabolic signalling to the ovary

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Financial support is sought for the continuation of my NSERC research program with a long-term goal of understanding the mechanisms metabolic signaling to the ovary. The short-term goal for the present period of funding is to understand the role of insulin like growth factor 1 (IGF1) system in the regulation of ovarian function. Specific objectives are: 1) To explore the importance of the IGF1 of liver and granulosa cell origin; 2) to determine the mechanisms of IGF1 receptor in FSH signalling; and 3) to decipher molecular mechanisms by which gonadotropins regulate the expression of PAPPA in bovine granulosa cells RATIONALE: Infertility in lactating dairy cattle has been attributed to severe negative energy balance due to high metabolic demands of milk production. Abnormal levels of metabolic hormone such as insulin, leptin and IGF1 are the major feature negative energy balance. Our recent data demonstrate that circulating concentrations of IGF1 are dramatically reduced in lactating cows as compared to heifers. This abnormal endocrine milieu is reflected in the follicular fluid of the dominant follicles of lactating cows as compared to those at similar developmental stage in heifers. Reduced follicular fluid IGF1 concentration is associated with decreased expression of PAPPA in granulosa cells of the dominant follicle in lactating cows. As PAPPA is a protease of IGF1 binding proteins, these data indicate that the follicular microenvironment of lactating cows is severely deficient of bioactive IGF1. Thus, a better understanding of the molecular mechanisms of IGF1 in ovarian follicles will contribute toward development of novel treatment and/or management strategies to improve fertility in lactating dairy cows. For objective one, we propose to develop liver- and granulosa-specific Igf1 knockout mice using our expertise in cre-recombinase directed deletion of genes. For this we will breed mice carrying conditional allele of Igf1 with mice expressing Cre-recombinase specifically in the liver (Alb1-Cre) and granulosa cells (Cyp19-Cre), respectively. We will undertake extensive phenotyping approaches to determine consequences of loss of Igf1 production in the liver or granulosa cells on ovarian follicular dynamics, reproductive hormonal profile and fertility. For the second objective, we will use a well-established culture system of bovine primary granulosa cells to investigate the signaling pathways of IGF1R in the regulation of granulosa cell proliferation and steroidogenesis. We will employ gene knockdown and pharmacologic inhibition strategies to test signaling pathways of IGF1R in bovine granulosa cells. For the third objective, we will first employ an in vivo ultrasound guided follicular aspiration strategy to synchronize follicular wave and collect samples. We will systematically analyze expression pattern of PAPPA, its abundance in follicular fluid and its protease activity in follicles at specific developmental stages. We will then investigate the molecular mechanisms by which FSH regulates the expression of PAPPA in bovine granulosa cells using cutting edge molecular biology techniques. Our aim will be to determine signaling pathways and transcription factors involved in FSH-regulated expression of PAPPA. Overall, the results of the proposed research program should significantly enhance our understanding how IGF1 system regulates ovarian functions and thus female fertility.