The Illumina deep sequencing platform is efficient for miRNA discovery and is widely used to generate small RNA profiles in various organisms. In the present study, we obtained detailed miRNA profiles of sexually mature (162-d) and immature (42-d) chicken ovaries using this method. To our knowledge, this is the first report of miRNA expression profiles of sexually immature and mature ovaries in the chicken. The sequence analysis showed that the dominant size of small RNAs in chicken ovary was 22 nt followed by 23 and 21 nt sequences. This result is typical of Dicer-processed small RNA products and was consistent with the known 19–24 nt range for miRNAs. Our data are consistent with previous findings in mouse  and pig  testis and ovary as well as chicken skeletal muscle  and somites . However, in Holstein Cattle testis and ovary, the 20 nt size is the most abundant, followed by 22 nt , while another study in bovine ovary indicated that 21 nt is the predominant size . This is likely caused by difference in species or developmental stages.
In the sexually mature chicken ovary library, gga-miR-10a and gga-miR-21 were the two most frequently sequenced miRNAs, and the let-7 miRNA family was another abundant cluster with let-7a being the most abundantly expressed miRNA. The let-7 miRNA family was also expressed abundantly in ovary and oocyte of bovines [28, 29, 38, 39], as well as in murine ovaries and testis . Furthermore, gga-miR-101, gga-miR-1a, gga-miR-146c, gga-miR-148a, gga-miR-126, gga-miR-26a and gga-miR-30d were abundant in our sequencing libraries, as has been shown in other animal gonads [25, 27, 28]. These miRNAs may have important roles in female reproductive physiology. A previous study reported that miR-21 was significantly up-regulated in murine granulosa cells before and 4 h after the hCG/LH surge and that it plays a role in preventing apoptosis in periovulatory granulosa cells as they transit to luteal cells. Knockdown of miR-21 in granulosa cells resulted in increased apoptosis and was associated with a reduced ovulation rate [41, 42]. Some studies have shown that miR-21 was down-regulated by estradiol in MCF7 cancer cells [43, 44]. In the present study, gga-miR-21 exhibited a significant 2.39-fold increase in a sexually mature ovary compared with an immature ovary, and the highest level was in F1 follicles, implying that gga-miR-21 plays an important role in sexual maturation in hens.
In this study, gga-let-7b is up-regulated while gga-let-7 g, gga-miR-17-3p gga-miR-30d, gga-miR-29a, gga-miR-26a and gga-miR-181a are all down-regulated in the mature ovary compared with the immature ovary. It has also been reported that miR-30c, let-7a, let-7b and let-7c were up-regulated, while miR-30d, miR-29a and miR-26a were down-regulated in rat granulosa cells treated with FSH for 12 h, which suggests that these miRNAs participate in FSH-mediated progesterone biosynthesis of granulosa cells . Another study showed that a lack of miR17-5p and let-7b resulted in corpus luteum insufficiency and infertility in mice and that exogenous administration of the two miRNAs could prevent this phenotype . A recent study showed that the activated estrogen receptor can suppress the expression of BAX by up-regulating a group of miRNAs including hsa-let-7 family members in endometrial adenocarcinoma and precancerous lesions . In the same study, miR-181a and miR-30d are significantly down-regulated after estradiol treatment.
miR-26a is reported to have anti-apoptotic effects in many cancers [48–51]. In MCF7 cancer cells, estradiol can repress miR-26a expression to regulate numerous genes associated with cell growth and proliferation . miR-26a was also found to play a role in normal tissue growth and development and to have an impact on cell proliferation and differentiation. One study showed that, during myogenesis, overexpression of miR-26a targets the enhancer of Zeste homolog 2 (Ezh2), which normally suppresses skeletal muscle differentiation . In osteogenesis, miR-26a was found to regulate osteoblast cell growth and differentiation in human adipose tissue-derived stem cells . In this study, the highest level of gga-miR-26a was found in SF follicles. SF were to be chosen from pre-hierarchical follicles (<8 mm) to pre-ovulatory hierarchy follicles (>10 mm), therefore, gga-miR-26a is likely associated with the mechanism of recruitment of dominant follicle in chicken.
Studies indicated that gga-miR-31, gga-miR-101, gga-miR-202 and gga-miR-202* may be involved in regulating gonadal differentiation in embryonic chicken gonads [54–56]. Reduced miR-202* expression is correlated with reduced expression of the testis-associated genes DMRT1 and SOX9, and up-regulation of the ovary-associated genes FOXL2 and CYP19A1 (aromatase) . FOXL2 is a critical factor required for ovarian growth and differentiation whose functions include regulating aromatase enzyme expression, inhibins and follistatin gene expression, and granulosa cell development [57, 58]. In our study, gga-miR-202* was down-regulated by 3.2-fold and gga-miR-31 was down-regulated by 1.68-fold in sexually mature ovaries compared with immature ovaries, suggesting they are also involved in the sexual maturity of ovary.
Bioinformatic analysis shows that miR-101 targets TGIF1 (TGFB-induced factor homeobox 1), ZEB2 (zinc finger E-box binding homeobox 2) and BMPR1B, which participate in the regulation of TGF-β signaling . TGF-β signaling is critical to folliculogenesis and oogenesis in mammalian ovaries . The increase in female miR-101 expression in differentiating ovaries can ease repression of TGF-β signaling . In this study, gga-miR-101 was abundantly expression in chicken ovary and 123 putative target genes were involved in TGF-β signaling pathway. Study has shown that overexpression of miR-375 suppressed glucose-induced insulin secretion, and conversely, inhibition of endogenous miR-375 function enhanced insulin secretion . In this study, gga-miR-375 is the largest down-regulated by 11-fold in the mature ovary and 191 putative target genes were involved in insulin signaling pathway.