Regulation of ovarian function by the germ cell specific DAZL gene
Brown, Yvonne A. R.
The RNA binding protein DAZL (Deleted in Azoospermia) is essential for germ cell survival and subsequent fertility. The transgenic mouse DAZL model has confirmed that knockout (KO) females are infertile as a direct consequence of complete postnatal oocyte ablation. Interestingly, the heterozygous (Het) DAZL females have increased fertility giving rise to significantly more viable offspring, accompanied by significantly reduced plasma FSH and increased inhibin B compared to levels observed in the wildtype (Wt) females. Recent studies to identify putative DAZL mRNA targets suggest that DAZL may have multiple functions and mRNA targets throughout germ cell development. However, how this protein functions within the oocyte and how functional copy number gives rise to increased fertility remains to be fully elucidated. The studies in this thesis sought to identify putative DAZL mRNA targets in addition to molecular mechanisms which may be either affected direct or indirectly as a result of the functional copy number of DAZL (Wt or Het) within the oocyte or follicular unit. Oocytes from Wt and Het were evaluated for their expression of selected oocyte genes and comparative analysis suggests that oocyte gene expression is significantly altered between the genotypes. Genes of interest include Oosp1 and H1foo, both of which are down-regulated in mRNA expression in Het d21 oocytes and d10 ovaries compared to the Wt. Furthermore, an in silico bioinformatics approach was utilised to identify putative DAZL mRNA targets using a consensus DAZL binding sequence. One candidate target, PDCD4, previously identified as a tumour suppressor gene was selected for further investigation. Despite PDCD4 mRNA and protein being highly expressed within the ovary, no difference in mRNA levels between Het and Wt was observed. However, although not ruling out the possibility of being a DAZL target we now have evidence that PDCD4 can function within the steroidogenic cells of the corpus luteum in relation to functional luteolysis. Indirect actions of DAZL upon local regulation and response of follicle growth in culture were evaluated to investigate follicles at the gonadotrophin dependent stage of growth. Individual follicles from Wt and Het d21 mice were cultured in the presence of FSH at 1iu, 0.5iu, 0.1iu and 0.01iu for a six day period. Final follicle size/morphology did not differ between genotypes at 1iu, 0.5iu and 0.1iu of FSH, but by d3 at 0.01iu FSH growth of Wt follicles was significantly (P<0.001) perturbed compared to the Het. Despite no difference in final size between 1iu, 0.5iu, 0.1iu FSH treatments, mRNA analysis of individual follicles demonstrated a significant up-regulation of FSH receptor (P<0.05), aromatase (P<0.05) and inhibin βA (P<0.01) and a significant down-regulation in inhibin βB (P<0.01) expression in the Het follicles compared to the Wt, suggesting an increase in follicle maturity, sensitivity and hence suitability for selection as viable pre-ovulatory follicles. Furthermore, atresia rates from cultured follicles were significantly lower (P<0.05 (1iu, 0.1iu FSH); P<0.01(0.01iu FSH)) in the Het compared to the Wt. These studies provide strong evidence that multiple mechanisms within the oocyte/follicle are directly and indirectly affected as a result of functional copy number of DAZL. Although direct in vivo targets remain to be identified it is clear that DAZL protein potentially targets multiple mRNAs at different stages of development, pre-programming the oocyte to increase the sensitivity of follicle and/or the functioning within a transcription complex regulating development. In conclusion, the beneficial consequences of increased fertility in the Het females is accompanied by a possible acceleration in oocyte and follicle maturation, an increased sensitivity to FSH in vitro with evidence of advanced stages of growth and, a reduction in follicle atresia. These differences support the suggestion that DAZL is having systemic effects on the paracrine communication within the follicle unit between the oocyte and somatic cells altering regulation and subsequent selection, and affecting final ovulation rate and litter size.