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 Oospl and
Hlfoo, both of which are down-regulated in mRNA expression in Het d21 oocytes
and dlO 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.
Abstract
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 liu, 0.5iu, O.liu and O.Oliu for a six day period. Final follicle
size/morphology did not differ between genotypes at liu, 0.5iu and O.liu of FSH, but
by d3 at O.Oliu FSH growth o f Wt follicles was significantly (PO.OOl) perturbed
compared to the Het. Despite no difference in final size between liu, 0.5iu, O.liu
FSH treatments, mRNA analysis of individual follicles demonstrated a significant
up-regulation of FSH receptor (P<0.05), aromatase (P<0.05) and inhibin PA (P<0.01)
and a significant down-regulation in inhibin PB (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 (liu, O.liu
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