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Makanji Y, Tagler D, Pahnke J, Shea LD, Woodruff TK. Hypoxia-mediated carbohydrate metabolism and transport promote early-stage murine follicle growth and survival. Am J Physiol Endocrinol Metab 306: E893-E903, 2014. First published February 25, 2014 doi:10.1152/ajpendo.00484.2013.-Oxygen tension is critical for follicle growth and metabolism, especially for early-stage follicles, where vascularity is limited. Its role and underlying mechanism in the in vitro activation and maturation of immature to ovulatory follicles is largely unknown. In this study, early secondary (110 m) murine follicles were isolated and encapsulated in alginate hydrogels to replicate the in vivo environment of the growing/maturing follicle. Encapsulated follicles were cultured for 8 days at either 2.5 or 20% O2. Survival (2.6-fold) and growth (1.2-fold) were significantly higher for follicles cultured at 2.5% compared with 20% O2. Using a mouse hypoxia-signaling pathway qRT-PCR array and GeneGo Metacore analysis, we found that direct target genes of the hypoxia-activated HIF1-complex were significantly upregulated in follicles cultured for 8 days at 2.5% compared with 20% O2, including the carbohydrate transport and metabolism genes Slc2a3, Vegfa, Slc2a1, Edn1, Pgk1, Ldha, and Hmox1. Other upregulated genes included carbohydrate transporters (Slc2a1, Slc2a3, and Slc16a3) and enzymes essential for glycolysis (Pgk1, Hmox1, Hk2, Gpi1, Pfkl, Pfkp, Aldoa, Gapdh, Pgam1, Eno1, Pkm2, and Ldha). For follicles cultured at 2.5% O2, a 7.2-fold upregulation of Vegfa correlated to an 18-fold increase in VEGFA levels, and a 3.2-fold upregulation of Ldha correlated to a 4.8-fold increase in lactate levels. Both VEGFA and lactate levels were significantly higher in follicles cultured at 2.5% compared with 20% O2. Therefore, enhanced hypoxia-mediated glycolysis is essential for growth and survival of early secondary follicles and provides vital insights into improving in vitro culture conditions. hypoxia; ovarian; follicle metabolism; glycolysis; oxygen GROWTH AND MATURATION OF OVARIAN FOLLICLES requires coordinated activity of various growth factors and hormones that interact with the physical environment of the follicle, including pH, temperature, and oxygen (O 2 ) tension. O 2 is critical for aerobic ATP production, in which pyruvate is converted to energy; however, excess O 2 generates free radicals and reactive oxygen species, which can damage the oocyte and its precious DNA cargo within the follicle (44). By design, ovarian follicles operate in a moderate hypoxic environment rather than an anoxic one (16). Granulosa cells use relatively less O 2 and convert glucose to pyruvate and lactate via the glycolytic pathway; the pyruvate is shunted to oocyte, which uses it and O 2 to create ATP via the TCA cycle (8, 13).The ability to culture follicles in vitro to produce mature, fertilizable oocytes has potential clinical applications in fertility preservation, and yet recapitulating the complexity of follicle growth and metabolism in vitro has been chall...

Primordial follicles dictate a female’s reproductive lifespan and therefore are central to fertility preservation for both endangered species as well as for individuals with fertility threatening conditions. Ovarian tissue containing primordial follicles can be cryopreserved and later thawed and transplanted back into individuals to restore both endocrine function and fertility. Importantly, increasing numbers of human live births have been reported following ovarian tissue cryopreservation and transplantation. A current limitation of this technology is patient access to sites that are approved or equipped to process and cryopreserve ovarian tissue – especially in larger countries or low resource settings. Here we review empirical evidence from both animal models and human studies that suggest that ovarian tissue can be transported at cold temperatures for several hours while still maintaining the integrity and reproductive potential of the primordial follicles within the tissue. In fact, several human live births have been reported in European countries using tissue that was transported at cold temperatures for up to 20 hours prior to cryopreservation and transplantation. Ovarian tissue transport, if implemented widely in clinical practice, could therefore expand both patient and provider access to emerging fertility preservation options.