Abstract
Deleted in azoospermia-like (DAZL) is a germ cell RNA-binding protein that is essential for entry and progression through meiosis. The phenotype of the Dazl knockout mouse has extensive germ cell loss because of incomplete meiosis. We have created a Dazl hypomorph model using short interfering RNA knockdown in mouse fetal ovary cultures, allowing investigation of Dazl function in germ cell maturation. Dazl hypomorph ovaries had a phenotype of impaired germ cell nest breakdown with a 66% reduction in total follicle number and an increase in the proportion of primordial follicles (PMFs), with smaller oocytes within these follicles. There was no significant early germ cell loss or meiotic delay. Immunostaining of intercellular bridge component testis-expressed protein (Tex)14 showed ∼59% reduction in foci number and size, without any change in Tex14 mRNA levels. TEX14 expression was also confirmed in the human fetal ovary across gestation. Using 3'UTR-luciferase reporter assays, translational regulation of TEX14 was demonstrated to be DAZL-dependant. Dazl is therefore essential for normal intercellular bridges within germ cell nests and their timely breakdown, with a major impact on subsequent assembly of PMFs.-Rosario, R., Crichton, J. H., Stewart, H. L., Childs, A. J., Adams, I. R., Anderson, R. A. Dazl determines primordial follicle formation through the translational regulation of Tex14.
Highlights
The oocytes in primordial follicles (PMFs) are thought to represent the entire pool of potential gametes available to a female throughout her life
Dazl mRNA and protein expression are reduced in fetal ovaries cultured for 3 d with short interfering RNA (siRNA)
Because many germ cells were still found in nest structures on d 12 of culture and had not formed PMFs in Dazl hypomorph ovaries, we investigated whether germ cell nest breakdown was affected through assessment of Tex14 expression, because Tex14 is an essential component of male and female intercellular bridges [25]
Summary
We sought to develop a model in which Dazl expression was reduced, but with minimized loss of germ cells, which would enable us to study the role of Dazl in germ cell development This is potentially significant to human reproduction because polymorphisms in DAZL may influence risk of premature ovarian insufficiency and age at menopause in women [26]. We have used our model, which uses short interfering RNA (siRNA), to knockdown Dazl expression in the mouse fetal ovary as meiosis commences, to investigate the role of Dazl once meiosis is underway, and to investigate the impact of Dazl in subsequent PMF formation With this model, we have demonstrated for the first time that Dazl is important in the formation and breakdown of germ cell nests as well as in subsequent PMF formation via the translational regulation of Tex
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