RESEARCH ARTICLE Cyclic AMP in oocytes controls meiotic prophase I and primordial folliculogenesis in the perinatal mouse ovary Yijing Wang 1 , Zhen Teng 1 , Ge Li 1,2 , Xinyi Mu 1,3 , Zhengpin Wang 1 , Lizhao Feng 1 , Wanbao Niu 1 , Kun Huang 1 , Xi Xiang 1 , Chao Wang 1 , Hua Zhang 4, * and Guoliang Xia 1, * ABSTRACT In mammalian ovaries, a fixed population of primordial follicles forms during the perinatal stage and the oocytes contained within are arrested at the dictyate stage of meiotic prophase I. In the current study, we provide evidence that the level of cyclic AMP (cAMP) in oocytes regulates oocyte meiotic prophase I and primordial folliculogenesis in the perinatal mouse ovary. Our results show that the early meiotic development of oocytes is closely correlated with increased levels of intra-oocyte cAMP. Inhibiting cAMP synthesis in fetal ovaries delayed oocyte meiotic progression and inhibited the disassembly and degradation of synaptonemal complex protein 1. In addition, inhibiting cAMP synthesis in in vitro cultured fetal ovaries prevented primordial follicle formation. Finally, using an in situ oocyte chromosome analysis approach, we found that the dictyate arrest of oocytes is essential for primordial follicle formation under physiological conditions. Taken together, these results suggest a role for cAMP in early meiotic development and primordial follicle formation in the mouse ovary. KEY WORDS: cAMP, Meiotic prophase I, Oocytes, Primordial follicle formation INTRODUCTION In mammals, the oocytes that are generated early in life represent the entirety of female reproductive potential over the life span (Faddy et al., 1992; Kezele et al., 2002). To maintain an immature oocyte in a dormant state, it is enclosed by several flattened pregranulosa cells to establish a functional unit called the primordial follicle, which forms in the fetal or neonatal ovary (Pepling, 2012). Proper oogenesis and folliculogenesis in the perinatal ovary are essential for fertility; however, the mechanisms regulating these processes remain unclear. In mice, primordial germ cells (PGCs), which are oocyte progenitors, migrate to the genital ridge and form the germline cyst by rapidly dividing during embryonic development (Ginsburg et al., 1990; Edson et al., 2009; Pepling, 2012). Retinoic acid then stimulates the PGCs in the ovary to enter meiosis at ∼13.5 days post coitum (dpc) (Bowles et al., 2006; Bowles and Koopman, 2007). The female germ cells are then referred to as oocytes (Pepling, 2006). The oocytes then progress through the leptotene, zygotene, pachytene and diplotene stages of meiotic prophase I, and arrest at the dictyate stage in the neonatal ovary (Slizynski, 1957; Borum, 1961). It is crucial for fertility that oocytes undergo the correct meiotic progression in the perinatal ovary. Forcing oocytes to undergo abnormal meiosis by deleting meiosis-related genes, such as Dazl, Spo11, Dmc1, Atm, Msh4 and Msh5, leads to infertility in mice (Pepling, 2006; Edson et al., 2009). However, the upstream signaling mechanism that regulates early oocyte meiosis in the perinatal ovary remains unknown. Cyclic AMP (cAMP) is a well-characterized intracellular second messenger that is involved in many biological processes, including oogenesis. In mammals, the concentration of intra-oocyte cAMP plays a pivotal role in controlling the arrest and resumption of meiosis in oocytes in the adult ovary (Mehlmann et al., 2004; Zhang and Xia, 2012). A high concentration of cAMP produced by both oocytes and cumulus cells maintains the meiotic arrest of immature oocytes, whereas a decrease in cAMP concentration in oocytes leads to the resumption of meiosis (Conti et al., 2012). Although the function of cAMP in regulating late stages of meiosis in activated oocytes has been studied extensively, it is not known whether cAMP contributes to early oocyte meiosis and what the mechanism of its involvement might be. Recently, a series of studies identified several cellular factors and pathways that are crucial for regulating the formation of primordial follicles (Pepling, 2012). For example, the Notch pathway has been shown to regulate the breakdown of germline cysts and the assembly of primordial follicles in mice (Trombly et al., 2009; Guo et al., 2012; Manosalva et al., 2013; Vanorny et al., 2014), and overactive KIT signaling has been shown to accelerate cyst breakdown in cultured fetal mouse ovaries (Jones and Pepling, 2013). Progesterone (P4) and estradiol (E2) have also been shown to have roles in controlling the formation of primordial follicles in several mammalian species (Chen et al., 2007; Dutta et al., 2014). A number of additional growth factors, such as nerve growth factor (NGF) (Dissen et al., 2001; Abir et al., 2005; Chaves et al., 2013) and connective tissue growth factor (CTGF) (Schindler et al., 2010), have also been reported to participate in primordial follicle formation and development (Pepling, 2012). Less is known about the relationship between early meiosis and primordial follicle formation. The premature loss of synaptonemal complex protein 1 (SYCP1) has been reported to increase the number of oocytes entering the diplotene stage and accelerate primordial follicle formation in rodents (Paredes et al., 2005), suggesting that the progress of oocyte meiosis is correlated with the formation of primordial follicles. However, the relationship between oocyte meiosis and primordial follicle formation under physiological conditions is not well understood. In this study, we investigated the function of cAMP in regulating early meiosis and the formation of primordial follicles in the perinatal mouse ovary. We show that cAMP produced by the oocyte Received 18 May 2014; Accepted 13 November 2014 1 State Key Laboratory of Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing 100193, China. 2 Guangdong Laboratory Animals Monitoring Institute, Guangzhou 510663, China. 3 Department of Histology and Embryology, Chongqing Medical University, Chongqing 400016, China. 4 Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg SE-405 30, Sweden. *Authors for correspondence (hua.zhang@gu.se; glxiachina@sohu.com) 1 © 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 1-9 doi:10.1242/dev.112755 DEVELOPMENT Development ePress. Posted online 11 December 2014 http://dev.biologists.org/lookup/doi/10.1242/dev.112755 Access the most recent version at Development Advance Online Articles. First posted online on 11 December 2014 as 10.1242/dev.112755