Abstract
In female mammals, reproductive potential is determined during fetal life by the formation of a non-renewable pool of primordial follicles. Initiation of meiosis is one of the defining features of germ cell differentiation and is well established to commence in response to retinoic acid. WIN 18,446 inhibits the conversion of retinol to retinoic acid, and therefore it was used to explore the impact of reduced retinoic acid synthesis on meiotic progression and thus germ cell development and subsequent primordial follicle formation. e13.5 mouse fetal ovaries were cultured in vitro and treated with WIN 18,446 for the first 3 days of a total of up to 12 days. Doses as low as 0.01 µM reduced transcript levels of the retinoic acid response genes Stra8 and Rarβ without affecting germ cell number. Higher doses resulted in germ cell loss, rescued with the addition of retinoic acid. WIN 18,446 significantly accelerated the progression of prophase I; this was seen as early as 48 h post treatment using meiotic chromosome spreads and was still evident after 12 days of culture using Tra98/Msy2 immunostaining. Furthermore, ovaries treated with WIN 18,446 at e13.5 but not at P0 had a higher proportion of growing follicles compared to vehicle controls, thus showing evidence of increased follicle activation. These data therefore indicate that retinoic acid is not necessary for meiotic progression but may have a role in the regulation of its progression and germ cell survival at that time and provide evidence for a link between meiotic arrest and follicle growth initiation.
Highlights
Female reproductive senescence results from the depletion of a finite ovarian follicle pool that is assembled during fetal life
Using a mouse fetal ovary culture model, where ovaries are dissected at e13.5 once meiosis has been initiated, we have shown that inhibition of retinoic acid synthesis with WIN 18,446 resulted in an acceleration of progression through prophase I and a subsequent increase in primordial follicle growth activation, suggesting a potential link between meiotic progression and primordial follicle formation and growth initiation
Culture with the lowest dose of WIN 18,446 (0.01μM) resulted in approximately 50% reduction in the transcript levels of Stra8 (P = 0.008), and a clear dose response was observed with the 5 μM dose, causing approximately 97% reduction in Stra8 expression (Fig. 1A). To demonstrate that this reduction was caused by reduced retinoic acid synthesis, fetal ovaries were cultured with 0.7 μM of retinoic acid in combination with 2 μM of WIN 18,446; this stimulated Stra8 expression at least 20-fold when compared to ovaries cultured with 2 μM of WIN 18,446 alone (P = 0.016)
Summary
Female reproductive senescence results from the depletion of a finite ovarian follicle pool that is assembled during fetal life. Nests of interconnected germ cells break down to release individual oocytes which associate with somatic pre-granulosa cells forming primordial follicles; this takes place during early postnatal life in rodents (Pepling & Spradling 2001) and in humans begins around 15 weeks gestation (Motta et al 1997, Bendsen et al 2006) These primordial follicles remain in a dormant state within the ovarian cortex until they receive signals for activation and are recruited to grow. Using a mouse fetal ovary culture model, where ovaries are dissected at e13.5 once meiosis has been initiated, we have shown that inhibition of retinoic acid synthesis with WIN 18,446 resulted in an acceleration of progression through prophase I and a subsequent increase in primordial follicle growth activation, suggesting a potential link between meiotic progression and primordial follicle formation and growth initiation
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