Proper formation of ovarian follicles is the basis for establishing a reserve of oocytes that will serve a female for her reproductive lifespan. Correspondingly, an insufficient ovarian reserve is associated with premature ovarian failure and infertility (Nelson, 2009). In order to elucidate better methods for treating premature infertility or delaying the onset of female reproductive senescence it is critical to understand the mechanisms that contribute to the establishment and maintenance of this oocyte reserve.
The process of oocyte development and follicle formation begins in the fetus with the migration of primordial germ cells to the developing ovary (Molyneaux et al., 2001). The germ cells then undergo several rounds of mitosis, and during this time they are referred to as oogonia. Groups of oogonia, connected by intracellular bridges, are formed as the result of incomplete cytokinesis after each round of mitosis (Pepling and Spradling, 1998). These groups of oogonia are referred to as germ cell cysts and become oocytes when they enter meiosis (Bullejos and Koopman, 2004; Menke et al., 2003). Cysts first fragment into smaller cysts which then reassociate so that clusters contain some oocytes connected by intercellular bridges and other oocytes associated by aggregation (Lei and Spradling, 2013). During this time, the oocytes progress through the first stages of meiotic prophase I and become arrested at an extended diplotene stage called dictyate (Cohen et al., 2006; Dutta et al., 2016). Beginning at 17.5 days post coitum (dpc) the cells separate and individual oocytes become surrounded with pregranulosa cells (Pepling et al., 2010). This process is accompanied by apoptosis of several oocytes from each cyst (Pepling and Spradling, 2001). There is evidence that the oocytes that are lost serve to support or “nurse” the surviving oocytes (Lei and Spradling, 2016). Those that remain become enclosed by pregranulosa cells to make up the ovarian reserve consisting of diplotene arrested oocytes housed within primordial follicles (Kerr et al., 2013). Despite the significance of this process for female fertility, the precise mechanisms that regulate cyst breakdown and follicle formation in mammals remain poorly understood.
Estrogens are implicated in the regulation of cyst breakdown and primordial follicle formation. Work from our lab has demonstrated that estradiol and other estrogenic compounds inhibit cyst breakdown and primordial follicle formation in the ovaries of fetal mice (Chen et al., 2007; Karavan and Pepling, 2012). Fetal ovaries are likely exposed to estrogen from the maternal circulation It is thought that during pregnancy high levels of estrogen in the developing ovary maintains oocytes in cysts, and when levels decrease cysts break apart and granulosa cells envelope each oocyte. One question that remains is the relative importance of fetal and maternal sources of estrogen in maintaining oocytes in cysts prior to follicle formation.
There is some evidence supporting the idea that estrogen synthesized by fetal ovaries may play a role in maintaining oocytes in cysts. First, the fetal mouse ovary is capable of synthesizing its own estrogen. Estradiol was detected in the ovaries of fetal mice along with the enzymes aromatase and 3βHSD, which are required for the biosynthesis of estradiol (Dutta et al., 2014). Second, when fetal mouse ovaries were grown in culture in the absence of estrogen, cyst breakdown and follicle formation were accelerated as compared to in vivo (Chen et al., 2007). In addition, if the estrogen that inhibits cyst breakdown is derived from the maternal circulation, one would expect the initiation of cyst breakdown to coincide with a decrease in circulating maternal estrogen. In mice, the initiation of cyst breakdown begins at 17.5 dpc, however a rapid decrease in maternal estrogen is not observed until the day of birth (Dutta et al., 2014). Furthermore, the capacity for fetal ovaries to secrete estradiol has been observed in other species such as cattle (Yang and Fortune, 2008). However, when fetal ovaries were cultured during the time of cyst breakdown and follicle formation in the presence of letrozole, an aromatase inhibitor, to eliminate any production of estrogen by the fetal ovary, cyst breakdown and follicle formation was not affected (Dutta et al., 2014). These findings indicate that a source of estrogen apart from the fetal ovary regulates follicle formation.
Accurate measurement of steroid hormones such as estradiol has been problematic (Vesper et al., 2014). Instead, the enzyme aromatase can be used as a proxy of estradiol location and amount in the ovary. Aromatase is encoded by the Cyp19 gene (Nelson et al., 1993), and is responsible for converting androgens to estrogens through a pathway originating with cholesterol (Cui et al., 2013). Aromatase has been detected in fetal and adult rat ovaries (Stocco, 2008) and in fetal through neonatal stage ovaries of mice (Dutta et al., 2014).
In order to further investigate the potential role of fetal estrogen in cyst breakdown and follicle development an aromatase deficient (ArKO) mouse strain was acquired. This strain carries a neomycin disruptive insert, which replaces 163 base pairs encoding for amino acid residues 349–403 of exon 9 of Cyp19, the gene encoding aromatase (Fisher et al., 1998). Adult ovaries have been characterized by Britt and colleagues (Britt et al., 2000; Britt et al., 2001; Britt et al., 2004). Their findings indicate that at 10 weeks of age, ArKO ovaries have fewer total oocytes, follicles of all stages but no corpora lutea, and hemorrhagic cysts. However, the effects of aromatase knockout on the processes of cyst breakdown and primordial follicle formation, which begin during fetal development, have not been evaluated. Furthermore, the ovarian phenotype of young animals as folliculogenesis begins has not been reported. It was noted that these animals are infertile (Britt et al., 2000; Fisher et al., 1998), however there are no data available comparing litter sizes or survival rates with wild type females. The goal of this study was to elucidate the effects of estradiol deficiency on early follicle formation, follicle development and fertility by studying the ovaries of ArKO mice between the ages of 17.5 dpc and adulthood.