NB ovary is characterized by heterogenous group of structures including germ cell clusters and primordial follicles. It is during this stage of ovarian development that many oocytes within the cluster are eliminated to yield a final pool of primordial follicles (Pangas and Rajkovic, 2006
). A number of germ cell specific RNA binding proteins have been identified such as MSY2, DAZLA, and NANOS3, and their respective knockouts show the importance of such proteins and RNA metabolism in the female gonadal development (Ruggiu et al., 1997
; McNeilly et al., 2000
; Tsuda et al., 2003
; Yang et al., 2006
). Conditional knockouts of Dicer1 and Argonaute2 in growing mouse oocytes (Murchison et al., 2007
; Kaneda et al., 2009
), two important enzymes in miRNA biogenesis, show normal ovarian development, but post-ovulation oocyte maturation is defective likely due to perturbed completion of meiosis I. The role of miRNAs during NB ovary development is unclear. We have previously shown that a number of germ cell specific transcriptional regulators are highly expressed in the NB ovary (Choi and Rajkovic, 2006
). We therefore examined the population of small RNAs in the NB ovary. We used massive parallel sequencing to determine the small RNA transcriptome in the 18–30 nucleotide range in the NB mouse ovary, and identified 398 known miRNAs and 30 novel small RNAs predicted to be miRNAs.
We also identified a significant expression of piRNAs, likely originating from the oocytes within the NB ovaries, and snoRNAs. piRNAs interact with PIWI proteins and knockouts of individual PIWI family members result in male sterility; however, female fertility is not affected by such knockouts (Deng and Lin, 2002
; Kuramochi-Miyagawa et al., 2004
; Carmell et al., 2007
). The significance of piRNAs in the NB ovaries is unclear, although recent evidence suggest that piRNAs are expressed in the Drosophila and mouse female germlines (Chambeyron et al., 2008
; Tam et al., 2008
) and important in germline silencing of transposable elements. Whether redundant, piRNA-independent pathways exist in the female germline and suppress transposable elements remains to be seen. It is perhaps of interest that almost 522 transcripts matched un-annotated sites in the genome, one of which showed preferential expression in the gonads. The role of such transcripts in the general nucleic acid metabolism of the germ cells remains to be established.
We showed that the total number of miRNA sequences mapped to the X chromosome more than any other chromosome, followed by chr2, chr1 and chr10. The number of miRNA genes is highest on chromosomes 2, chrX and chr12, and thus perhaps it is not unexpected that chrX and chr2 express more miRNA transcripts than other chromosomes. However, correlation between gene density on a particular chromosome, and expression is not uniform. For example, more reads mapped to chr10 than chr12, despite six times as many miRNA genes on chr12 rather than chr10. The significance of the preferential expression of miRNAs from certain chromosomes in the NB ovary is unclear.
We discovered a total of 30 potential novel miRNAs using methodologies described previously (Morin et al., 2008
). The number of novel small RNA species mapped in our study is much higher than previously described in the adult ovaries (Ro et al., 2007
; Mishima et al., 2008
) and is largely due to the use of massive parallel sequencing technology. For example, Mishima et al. (2008
) only sequenced 10 000 clones from the adult ovary and mapped them to only 154 known miRNA genes, whereas we obtained more than 4 000 000 reads that mapped to 398 known miRNA genes. There are also significant differences between NB and adult ovaries. RNA contribution in adult ovaries is mostly derived from the somatic cell component, whereas NB ovaries are highly enriched in the germ cell component. The novel miRNAs we discovered were not very abundant based on the sequence count, with total number of reads varying between 12 and 1100. Most of these miRNAs were expressed ubiquitously, although few did show preferential expression in the gonads. The isomiRs expressed in the NB ovary were not abundant with read counts numbering less than 100 for most of them. It is interesting to note that the top two expressed isomiRs, mmu-mir-135a-2-3p and mmu-mir-871-3p, were both preferentially expressed in gonads, suggesting that gonadal milieu plays a role in processing of these transcripts. The role of any of these miRNAs in ovarian follicular development, either novel or isomiRs, is unknown. Moreover, it is unclear whether these miRNAs are redundant in their function, as has been suggested for other miRNAs (Miska et al., 2007
). Future studies ablating specific miRNAs using transgenic technologies will help us better understand the role of these clusters as well as individual miRNAs in gonadal development and ovarian folliculogenesis.