Zinc dynamics regulate early ovarian follicle development

Abstract

Zinc fluctuations regulate key steps in late oocyte and preimplantation embryo development; however, roles for zinc in preceding stages in early ovarian follicle development, when cooperative interactions exist between the oocyte and somatic cells, are unknown. To understand the roles of zinc during early follicle development, we applied single cell X-ray fluorescence microscopy, a radioactive zinc tracer, and a labile zinc probe to measure zinc in individual mouse oocytes and associated somatic cells within early follicles. Here, we report a significant stage-specific increase and compartmental redistribution in oocyte zinc content upon the initiation of early follicle growth. The increase in zinc correlates with the increased expression of specific zinc transporters, including two that are essential in oocyte maturation. While oocytes in follicles exhibit high tolerance to pronounced changes in zinc availability, somatic survival and proliferation are significantly more sensitive to zinc chelation or supplementation. Finally, transcriptomic, proteomic, and zinc loading analyses reveal enrichment of zinc targets in the ubiquitination pathway. Overall, these results demonstrate that distinct cell type–specific zinc regulations are required for follicle growth and indicate that physiological fluctuation in the localization and availability of this inorganic cofactor has fundamental functions in early gamete development. Zinc fluctuations regulate key steps in late oocyte and preimplantation embryo development; however, roles for zinc in preceding stages in early ovarian follicle development, when cooperative interactions exist between the oocyte and somatic cells, are unknown. To understand the roles of zinc during early follicle development, we applied single cell X-ray fluorescence microscopy, a radioactive zinc tracer, and a labile zinc probe to measure zinc in individual mouse oocytes and associated somatic cells within early follicles. Here, we report a significant stage-specific increase and compartmental redistribution in oocyte zinc content upon the initiation of early follicle growth. The increase in zinc correlates with the increased expression of specific zinc transporters, including two that are essential in oocyte maturation. While oocytes in follicles exhibit high tolerance to pronounced changes in zinc availability, somatic survival and proliferation are significantly more sensitive to zinc chelation or supplementation. Finally, transcriptomic, proteomic, and zinc loading analyses reveal enrichment of zinc targets in the ubiquitination pathway. Overall, these results demonstrate that distinct cell type–specific zinc regulations are required for follicle growth and indicate that physiological fluctuation in the localization and availability of this inorganic cofactor has fundamental functions in early gamete development. The ovarian follicle, consisting of the oocyte surrounded by supporting somatic cells, progresses through well-defined developmental stages in the ovary (1Wassarman P.M. Albertini D.F. The mammalian ovum.in: Knobil E. Neill J.D. The Physiology of Reproduction. Raven Press, New York, NY1994: 79-122Google Scholar). Follicle development, or folliculogenesis, begins with the activation of selective dormant primordial follicles. These primordial follicles are formed in the first few days of life in the mouse and are characterized by a nongrowing oocyte arrested at prophase of meiosis I, surrounded by one layer of mitotically arrested, squamous pre-granulosa cells (also known as primordial follicle granulosa cells; pfGCs). Through a process that is not fully understood, primordial follicle activation initiates oocyte growth, and squamous pfGCs re-enter the mitotic cycle and differentiate into cuboidal granulosa cells (GCs), forming a primary follicle with an oocyte surrounded by a single layer of GCs. As the follicle unit continues to mature, the GCs proliferate to form multiple layers and the oocyte produces an extracellular glycoprotein coat termed the zona pellucida (ZP). This is now known as a secondary follicle with specialized filopodia spanning the ZP and governing the communication between the oocyte and the GCs (2El-Hayek oocytes to the for Scholar). 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