Abstract

   The Ames dwarf (df/df) mice have extended longevity and can preserve
   the ovarian reserve longer than Normal (N) mice. Based on this, the aim
   of our study was to evaluate the ovarian microRNA (miRNA) profile in
   young and aged df/df and N mice. Ovarian tissue was collected at 5–6
   months and at 21–22 months of age for miRNA sequencing. We detected a
   total of 404 miRNAs in the ovarian samples, from which the abundance of
   22 and 33 miRNAs changed with age in N and df/df mice, respectively. Of
   these, only three miRNAs were commonly regulated with age between N and
   df/df mice, indicating a very divergent miRNA profile between
   genotypes. We also detected that 46 miRNAs were regulated between N and
   df/df mice, of which 23 were regulated exclusively in young mice, 12
   exclusively in old mice and 12 commonly regulated at young and old
   ages. Many genes likely to be targeted by these miRNAs are involved in
   the FoxO, mTOR, PI3k/Akt and insulin signaling pathways. These results
   suggest that the aging process has a differential impact on the ovarian
   miRNA profile in df/df mice, and suggest that these miRNAs can be
   central players in the maintenance of a younger ovarian phenotype.

Introduction

   Regulation of gene expression plays a key role in follicle development
   and aging within the ovary [[36]1]. Some key cellular processes,
   including mRNA transcription and stability, are regulated by small
   non-coding RNAs (sncRNAs), which includes short sequences of about 20
   nucleotides known as microRNAs (miRNAs) [[37]2]. When miRNAs are
   exported to the cytoplasm, they modulate gene expression
   post-transcriptionally by interacting with the Argonaute proteins,
   forming an RNA Induced Silencing Complex (RISC) that binds to the 3’
   untranslated region (UTR) and cleaves target mRNAs [[38]3]. In addition
   to that, miRNAs can be secreted to the extracellular space [[39]4] and
   have a role in intercellular communication in a hormone-like pattern
   between different cell types [[40]5]. Therefore, miRNAs play an
   essential role in ovarian follicle development [[41]6, [42]7], although
   little is known about the how miRNAs are regulated during the ovarian
   aging process [[43]8, [44]9].

   Oocytes enclosed in primordial follicles constitute the ovarian reserve
   and remain dormant until activated to grow during the female
   reproductive life [[45]10]. During this period there is a progressive
   decline and depletion of the ovarian primordial follicle reserve
   culminating in the menopause [[46]11]. In the female mouse the size of
   the ovarian reserve reduces 10 fold from six to 18 months of age
   [[47]12]. Some miRNAs are known to be involved in premature ovarian
   failure in the rat [[48]13] and were observed to be differentially
   regulated in the ovaries of ageing women [[49]8, [50]9]. This evidence
   suggests that there is an association between miRNA expression and
   regulation of ovarian aging. However, no specific studies have been
   performed using unbiased, transcriptome-wide methods to determine miRNA
   changes during normal aging in mice with parallel comparison of these
   alterations in long-living animals such as the Ames dwarf (Prop-1^df;
   df/df) mice.

   The df/df mice have a defective Prop1 (Prophet of Pit1) gene, impairing
   anterior pituitary gland development, and resulting in deficient
   secretion of growth hormone (GH), thyroid-stimulating hormone (TSH) and
   prolactin [[51]14]. These mice are characterized by severely reduced
   circulating IGF-I and adult body size [[52]14]. The df/df mice are
   considered excellent models for the study of aging, since they can live
   35–75% longer than their normal littermates [[53]14], and share several
   characteristics with GH receptor gene disrupted mice and mice subjected
   to caloric restriction [[54]15]. Although female df/df mice have normal
   ovarian cyclicity and can maintain pregnancy under hormonal
   stimulation, they have delayed puberty [[55]16]. We previously reported
   that Forkhead Box O3a (Foxo3a) phosphorylation, one of the main
   pathways implicated in irreversible primordial follicle activation
   [[56]17, [57]18], is reduced in oocytes enclosed in primordial
   follicles from df/df mice, preventing primordial follicle activation
   [[58]19, [59]20]. As a result df/df mice have a delayed ovarian aging
   and an increased number of primordial follicles compared to old Normal
   (N) mice [[60]19, [61]20]. Activation of primordial follicle growth is
   also impaired in GH receptor disrupted mice [[62]21] and mice subjected
   to caloric restriction [[63]22], also culminating in delayed ovarian
   aging and a greater number of primordial follicles in the quiescent
   stage. On the other hand, transgenic mice overexpressing GH have
   increased activation of Foxo3a and accelerated loss of primordial
   follicles [[64]19], evidencing the role of GH/IGF-I in this process.

   microRNAs are well known for their roles in cell growth and
   proliferation, regulating pathways which are also essential to cancer
   development [[65]23], and there is an extensive characterization of the
   role of microRNAs in the etiology of ovarian cancer [[66]24].
   Epithelial ovarian cancers represent nearly 90% of all ovarian cancers
   and develop mainly in postmenopausal women [[67]25]. In parallel with
   an increase in life expectancy, the rate of female reproductive cancer
   incidence has nearly doubled in the past 30 years [[68]26]. The current
   hypothesis, supported by epidemiological and experimental evidence,
   suggests that repetitive ovulation and wounding of the ovarian surface
   during reproductive life can increase the risk of epithelial ovarian
   cancers [[69]25], and most ovarian cancers usually develop immediately
   after menopause [[70]27]. Therefore, a better understanding of ovarian
   aging and its links to known ovarian cancer markers can benefit healthy
   aging for women, since the current evidence supports that miRNAs may
   play a key role in both processes.

   It was previously demonstrated, using the same mice analyzed in the
   present study, that df/df mice have a characteristic circulating miRNA
   signature associated with aging [[71]28]. However, very little is known
   about the miRNA profile associated with ovarian aging, since most mouse
   models for ovarian aging (knockouts for PTEN and FoxO3 for example)
   have complete ovarian failure early after puberty [[72]17, [73]29].
   df/df mice can maintain the ovarian reserve for a longer period than N
   mice [[74]19, [75]20], which allows for comparison of expression
   profiles at different stages of life, making it a very valuable model
   of ovarian aging. Therefore, the aim of the current work is to evaluate
   the ovarian miRNA profile in young and aged df/df and N mice in order
   to establish miRNAs potentially involved in ovarian aging.

Materials and Methods

   Ames dwarf mice (df/df; n = 10) Normal control littermates (N; n = 10)
   (all females) were bred and maintained under temperature- and
   light-controlled conditions (22 ± 2°C, 12 hour light/12 hour dark
   cycle) [[76]30]. Mice were subjected to overnight fasting, and
   isofluorane was used for anesthesia and after blood collection by
   cardiac puncture mice were euthanized by performing cervical
   dislocation. Mice were dissected and the pair of ovaries was collected
   and stored at -80°C. The ovarian collection was performed at 5–6 months
   of age (young; 5 N and 5 df/df mice) and at 21–22 months of age (old; 5
   N and 5 df/df mice). All animal procedures employed in our presented
   work were approved by and performed in accordance to the guidelines
   from the Laboratory Animal Care and Use Committee (LACUC) at the
   Southern Illinois University School of Medicine (Springfield, IL).

   The pair of ovaries was removed from the -80°C and homogenized with
   Qiazol (Qiagen, Valencia, CA, USA) using 0.5 mm zirconium oxide beads
   in the Bullet Blender 24 (Next Advance, Averill Park, NY, USA). Total
   RNA was extracted using a commercial column purification system
   (miRNeasy Mini Kit, Qiagen) and on-column DNase treatment (RNase-free
   DNase Set, Qiagen) following manufacturer's instructions. The quantity
   and quality of RNA samples was determined using BioAnalyzer and RNA
   Nano Lab Chip Kit (Agilent Technologies, Santa Clara, CA, USA) and only
   samples with RNA Integrity Number higher than 7.0 were selected for
   further analysis (n = 20).

   MicroRNA libraries were prepared using the TruSeq Small RNA Sample Prep
   Kit (Illumina Inc., San Diego, CA, USA) following the manufacturer's
   instructions according to Matkovich, Hu [[77]31]. Briefly, small RNAs
   from 1 μg of total RNA were ligated with 3′ and 5′ adapters, followed
   by reverse transcription to produce single stranded cDNAs. Samples were
   amplified by PCR in 14 cycles using indexes to allow all 20 individual
   libraries to be processed in a single flowcell lane during sequencing.
   The amplified libraries were size-selected and purified in a 6%
   acrilamide gel, combined in a single microtube and submitted to
   sequencing on a HiSeq 2500 instrument (Illumina Inc.) at the Washington
   University Genomic Technology Access Center (GTAC). Alignment and
   quantification of miRNA libraries was performed using sRNAtoolbox as
   described before [[78]32]. Briefly, sequences in the fasta format were
   adaptor trimmed, the sequences were aligned to the mouse genome (mm10)
   and known miRNA sequences confirmed by miRBase, and the output read
   count was expressed as normalized reads per million (rpm).

   Statistical analyses of differentially expressed miRNAs was performed
   using EdgeR [[79]33] between each pair of groups and miRNAs with a
   FDR<0.02 and FC>1.5 were considered as up or down-regulated. The
   unsupervised hierarchical clustering heatmap from the variance
   stabilization transformed data of the top 50 most expressed miRNAs
   ordered by average count was performed using the software R (3.2.2) and
   the Bioconductor package DESeq (1.2.0) [[80]34]. miRNA gene families
   were identified using miRBase [[81]35]. The mirPath tool (version 3.0)
   was used to predict target genes of the differentially regulated miRNAs
   using the microT-CDS v.5.0 database [[82]36] and for retrieving gene
   ontology (GO) terms (biological processes) and KEGG molecular pathways
   [[83]37, [84]38], considering a corrected P value lower than 0.05 as
   significant for pathway enrichment.

   All miRNA-Seq data are available at the Gene Expression Omnibus (GEO)
   at NCBI under accession number [85]GSE87518.

Results

   A total of 404 miRNAs were detected in the ovaries of N and df/df mice
   (present in at least 50% of the samples with more than 5 reads per
   million). On average 8,648,485 raw reads per sample were obtained, and
   from these 6,603,534 adaptor cleaned unique reads were used per sample,
   and 43.9% of those were aligned to the mouse genome. The overall
   percentage of the aligned reads mapped to different categories of
   sncRNA was: 57.9% for miRNAs, 28.7% for repeated sequences, 4,3% for
   snRNA, 4.2% for rRNA and 0.1% for snoRNA. About 10% of the detected
   miRNAs had more than 5,000 rpm, 13% between 1,000 and 5,000 rpm, 30%
   between 100 and 1,000 rpm, 37% between 10 and 100 rpm and 8% between 5
   and 10 rpm.

   The 50 highest expressed miRNAs in the mice ovaries are represented in
   a heatmap in [86]Fig 1 and for miRNA expression levels in [87]Fig 2.
   The top five most expressed miRNAs in the ovary (all samples combined)
   were mmu-miR-92a-3p (miR-25 family), mmu-let-7c-5p (let-7 family),
   mmu-miR-143-3p (miR-143 family), mmu-miR-26a-5p (miR-26 family) and
   mmu-miR-145a-5p (miR-145 family). Pathway enrichment analysis indicated
   that these top five miRNAs target genes that are likely to be involved
   in important ovarian signaling pathways (FoxO, MAPK, AMPK, mTOR,
   PI3K/Akt, TGF-β, Estrogen and GnRH; for further details see [88]S1
   Table) and biological processes ([89]S1 Table).

Fig 1. Unsupervised hierarchical clustering ordered by the adjusted level of
miRNA expression for the top 50 most expressed miRNAs in ovaries of df/df (n
= 10; Young–DY and Old–DO) and N mice (n = 10; Young–NY and Old–NO).

   [90]Fig 1
   [91]Open in a new tab

Fig 2. Abundance of the top 50 most expressed miRNAs in ovarian samples.

   [92]Fig 2
   [93]Open in a new tab

   The values are an average reads per million (rpm) from Ames dwarf
   (df/df) and Normal (N) mice at 6 and 22 months of age.

   There were 11 down-regulated and 11 up-regulated miRNAs in N mice with
   age (N young vs. N old) (FDR<0.02). In df/df mice, 31 miRNAs were
   down-regulated with age, while two were up-regulated (FDR<0.02;
   [94]Table 1). When comparing miRNA expression between df/df and N mice,
   12 miRNAs were commonly down-regulated at both young and old ages (five
   down- and seven up-regulated; FDR<0.02; [95]Table 2), 23 were regulated
   only in young mice (13 down- and 10 up-regulated; FDR<0.02; [96]Table
   2) and 12 miRNAs were regulated exclusively in old mice (4 down- and 8
   up-regulated; FDR<0.05; [97]Table 2). A Venn diagram with the mutually
   regulated miRNAs between different categories is presented in [98]Fig
   3A and the number of regulated miRNA families is represented in [99]Fig
   3B.

Table 1. MicroRNAs differentially expressed during aging in Normal and Ames
dwarf mice.

   miRNA name miRNA gene family FC[100]^1 RPM[101]^2 PValue[102]^3
   FDR[103]^3 miRNA name miRNA gene family FC[104]^1 RPM[105]^2
   PValue[106]^3 FDR[107]^3
   Normal mice Ames dwarf mice
   Commonly down-regulated Commonly down-regulated
   mmu-miR-217-5p miR-217 23.83 10.53 9.36E-04 1.75E-02 mmu-miR-217-5p
   miR-217 4.93 10.53 1.05E-04 2.50E-03
   mmu-miR-410-3p miR-154 2.45 69.50 6.12E-04 1.37E-02 mmu-miR-410-3p
   miR-154 3.65 69.50 7.48E-07 5.00E-05
   mmu-miR-673-5p miR-673 2.35 42.02 2.13E-04 7.16E-03 mmu-miR-673-5p
   miR-673 3.00 42.02 9.89E-07 5.00E-05
   Down-regulated only in Normal mice Down-regulated only in Ames dwarf
   mice
   mmu-miR-6538 - 19.43 92.01 5.83E-05 2.94E-03 mmu-miR-298-5p miR-298
   12.01 40.51 1.22E-09 4.94E-07
   mmu-miR-450b-5p miR-450 7.37 8.46 5.86E-04 1.37E-02 mmu-miR-296-3p
   miR-296 7.60 18.12 9.88E-07 5.00E-05
   mmu-miR-370-3p miR-370 6.34 9.62 1.00E-05 8.08E-04 mmu-miR-296-5p
   miR-296 6.37 88.46 4.88E-08 8.97E-06
   mmu-miR-5126 - 5.85 34.48 2.82E-04 8.46E-03 mmu-miR-130b-3p miR-130
   5.55 101.52 6.66E-08 8.97E-06
   mmu-miR-503-5p miR-503 4.08 52.67 2.93E-04 8.46E-03 mmu-miR-130b-5p
   miR-130 5.44 20.57 1.57E-06 7.04E-05
   mmu-miR-351-3p miR-351 3.24 98.17 1.57E-04 5.78E-03 mmu-miR-301b-3p
   miR-130 4.94 69.68 6.49E-06 2.62E-04
   mmu-miR-485-3p miR-485 3.06 21.31 3.42E-04 9.21E-03 mmu-miR-486b-3p
   miR-486 4.61 8.83 4.85E-04 7.75E-03
   mmu-miR-3102-3p miR-3102 2.25 104.61 9.01E-04 1.75E-02 mmu-miR-486a-3p
   miR-486 4.61 8.83 4.82E-04 7.75E-03
   mmu-miR-381-3p miR-154 4.46 9.14 9.67E-05 2.44E-03
   Up-regulated only in Normal mice mmu-miR-17-5p miR-17 4.16 552.57
   2.03E-07 2.05E-05
   mmu-miR-215-5p miR-192 21.95 191.47 3.04E-06 3.07E-04 mmu-miR-20a-5p
   miR-17 3.91 342.08 9.89E-07 5.00E-05
   mmu-miR-142a-3p miR-142 7.79 586.99 1.04E-06 1.40E-04 mmu-miR-323-3p
   miR-154 3.17 19.08 2.57E-04 5.42E-03
   mmu-miR-205-5p miR-205 6.47 541.62 2.82E-05 1.63E-03 mmu-miR-299b-5p
   miR-299 3.03 33.57 7.97E-05 2.15E-03
   mmu-miR-187-3p miR-187 6.13 147.95 1.74E-05 1.17E-03 mmu-miR-299a-5p
   miR-299 3.03 33.57 7.98E-05 2.15E-03
   mmu-miR-146a-5p miR-146 4.80 1783.78 4.45E-07 8.98E-05 mmu-miR-409-3p
   miR-154 3.00 66.85 1.63E-05 5.50E-04
   mmu-miR-142a-5p miR-142 4.43 258.92 9.59E-05 4.31E-03 mmu-miR-494-3p
   miR-154 2.87 158.07 4.99E-04 7.75E-03
   mmu-miR-150-5p miR-150 4.32 9575.94 2.43E-07 8.98E-05 mmu-miR-341-3p
   miR-341 2.78 114.55 2.12E-05 6.59E-04
   mmu-miR-146b-5p miR-146 3.06 992.32 8.33E-04 1.75E-02 mmu-miR-329-3p
   miR-329 2.77 15.86 9.24E-04 1.20E-02
   mmu-miR-223-5p miR-223 2.38 36.30 9.51E-04 1.75E-02 mmu-miR-667-3p
   miR-667 2.70 144.89 2.95E-04 5.67E-03
   mmu-miR-29c-5p miR-29 2.32 114.74 1.16E-04 4.67E-03 mmu-miR-376a-5p
   miR-368 2.47 46.24 2.68E-04 5.42E-03
   mmu-miR-29c-3p miR-29 2.29 1613.93 3.86E-04 9.76E-03 mmu-miR-434-3p
   miR-434 2.34 7570.42 1.62E-05 5.50E-04
   mmu-miR-434-5p miR-434 2.18 42.45 8.37E-04 1.18E-02
   mmu-miR-93-3p miR-17 2.16 32.23 8.80E-04 1.18E-02
   mmu-miR-17-3p miR-17 2.15 82.36 8.67E-04 1.18E-02
   mmu-miR-181c-5p miR-181 2.12 212.90 4.11E-04 7.22E-03
   mmu-miR-379-3p miR-379 2.10 73.53 8.19E-04 1.18E-02
   mmu-miR-181c-3p miR-181 2.07 1108.31 3.31E-04 6.08E-03
   mmu-miR-93-5p miR-17 1.99 3218.94 1.35E-04 3.03E-03
   Up-regulated only in Ames dwarf mice
   mmu-miR-592-5p miR-592 3.97 16.28 1.02E-03 1.28E-02
   mmu-miR-10b-3p miR-10 1.93 70.41 1.38E-03 1.69E-02
   [108]Open in a new tab

   ^1Fold change between groups

   ^2Average reads per million (RPM) for each miRNA for the groups being
   compared

   ^3P Value and False discovery rate. miRNAs with FDR<0.05 were
   considered as differentially expressed between groups

Table 2. MicroRNAs differentially expressed between Ames dwarf and Normal
mice at young (6 months) and old (22 months) ages.

   miRNA name miRNA gene family FC[109]^1 RPM[110]^2 PValue[111]^3
   FDR[112]^3 miRNA name miRNA gene family FC[113]^1 RPM[114]^2
   PValue[115]^3 FDR[116]^3
   Young mice (6 months old) Old mice (22 months old)
   Commonly down-regulated Commonly down-regulated
   mmu-miR-212-3p miR-132 12.16 69.74 1.10E-12 2.22E-10 mmu-miR-212-3p
   miR-132 11.96 69.74 1.62E-11 2.18E-09
   mmu-miR-132-5p miR-132 9.32 10.64 4.62E-07 2.67E-05 mmu-miR-132-5p
   miR-132 8.16 10.64 1.25E-05 6.29E-04
   mmu-miR-132-3p miR-132 6.87 2055.67 2.39E-09 2.41E-07 mmu-miR-132-3p
   miR-132 7.38 2055.67 7.27E-10 7.34E-08
   mmu-miR-21a-3p miR-21 4.17 380.77 1.78E-07 1.44E-05 mmu-miR-21a-3p
   miR-21 5.26 380.77 1.87E-09 1.51E-07
   mmu-miR-21a-5p miR-21 3.75 14917.59 2.15E-11 2.89E-09 mmu-miR-21a-5p
   miR-21 4.33 14917.59 1.72E-13 3.47E-11
   Commonly up-regulated Commonly up-regulated
   mmu-miR-7a-2-3p miR-7 17.79 24.83 3.40E-13 1.37E-10 mmu-miR-7a-2-3p
   miR-7 52.52 24.83 9.07E-20 3.66E-17
   mmu-miR-217-5p miR-217 10.03 10.53 3.72E-07 2.50E-05 mmu-miR-217-5p
   miR-217 49.58 10.53 5.57E-06 3.21E-04
   mmu-miR-465c-5p miR-465 8.06 30.26 6.17E-05 2.49E-03 mmu-miR-465c-5p
   miR-465 7.37 30.26 2.39E-04 6.12E-03
   mmu-miR-470-5p miR-743 6.59 15.76 1.56E-03 1.86E-02 mmu-miR-470-5p
   miR-743 11.99 15.76 2.42E-04 6.12E-03
   mmu-miR-871-5p miR-743 5.30 23.38 3.58E-04 9.64E-03 mmu-miR-871-5p
   miR-743 8.75 23.38 2.28E-05 1.02E-03
   mmu-miR-7a-5p miR-7 3.33 56.07 4.58E-04 9.73E-03 mmu-miR-7a-5p miR-7
   3.62 56.07 1.00E-04 3.38E-03
   mmu-miR-107-3p miR-103 2.15 2359.91 7.75E-04 1.29E-02 mmu-miR-107-3p
   miR-103 3.19 2359.91 5.55E-07 3.74E-05
   Down-regulated in young mice only Down-regulated in old mice only
   mmu-miR-133b-3p miR-133 5.19 48.09 6.00E-05 2.49E-03 mmu-miR-215-5p
   miR-192 8.45 191.47 5.96E-04 1.09E-02
   mmu-miR-501-3p miR-500 4.70 20.78 1.27E-03 1.70E-02 mmu-miR-142a-3p
   miR-142 5.25 586.99 5.41E-05 2.18E-03
   mmu-miR-133a-3p miR-133 4.01 513.58 3.57E-04 9.64E-03 mmu-miR-296-5p
   miR-296 3.02 88.46 8.98E-04 1.58E-02
   mmu-miR-3102-3p.2-3p miR-3102 3.17 51.46 2.74E-06 1.38E-04
   mmu-miR-101b-3p miR-101 2.26 1990.35 4.73E-04 1.01E-02
   mmu-miR-582-5p miR-582 3.14 16.45 1.43E-03 1.75E-02
   mmu-miR-330-3p miR-330 2.90 26.73 5.95E-04 1.05E-02 Up-regulated in old
   mice only
   mmu-let-7a-1-3p let-7 2.75 162.24 5.30E-04 1.03E-02 mmu-miR-370-3p
   miR-370 5.43 9.62 9.03E-05 3.32E-03
   mmu-miR-148b-3p miR-148 2.54 715.73 1.27E-03 1.70E-02 mmu-miR-547-3p
   miR-547 4.36 413.17 3.27E-04 7.35E-03
   mmu-miR-3102-3p miR-3102 2.38 104.61 4.30E-04 9.66E-03 mmu-miR-201-5p
   miR-743 4.35 43.39 1.14E-04 3.55E-03
   mmu-miR-28a-5p miR-28 2.11 436.17 1.29E-03 1.70E-02 mmu-miR-201-3p
   miR-743 3.80 40.54 5.81E-04 1.09E-02
   mmu-miR-28c miR-28 2.11 436.17 1.31E-03 1.70E-02 mmu-miR-1249-3p
   miR-1249 2.42 609.50 5.35E-04 1.08E-02
   mmu-miR-34a-5p miR-34 2.04 247.19 7.98E-04 1.29E-02 mmu-miR-1247-5p
   miR-1247 2.39 665.42 1.09E-03 1.84E-02
   mmu-miR-28a-3p miR-28 2.00 79.11 1.20E-03 1.70E-02 mmu-miR-652-3p
   miR-652 2.18 3266.88 1.46E-04 4.21E-03
   mmu-miR-181b-5p miR-181 2.06 7681.97 2.93E-04 6.97E-03
   Up-regulated in young mice only
   mmu-miR-215-5p miR-192 9.63 191.47 3.13E-04 9.64E-03
   mmu-miR-465abc-3p miR-465 7.33 7.41 2.79E-04 9.40E-03
   mmu-miR-463-5p miR-463 5.96 11.26 2.17E-04 7.98E-03
   mmu-miR-181a-2-3p miR-181 2.73 2361.59 1.41E-03 1.75E-02
   mmu-miR-92a-1-5p miR-25 2.61 32.62 4.12E-04 9.66E-03
   mmu-miR-150-5p miR-150 2.45 9575.94 1.21E-03 1.70E-02
   mmu-miR-434-3p miR-434 1.97 7570.42 5.37E-04 1.03E-02
   mmu-miR-128-3p miR-128 1.93 166.76 1.63E-03 1.88E-02
   mmu-miR-92a-3p miR-25 1.89 97651.91 4.30E-04 9.66E-03
   mmu-miR-130a-3p miR-130 1.88 6489.13 5.94E-04 1.05E-02
   [117]Open in a new tab

   ^1Fold change between groups

   ^2Average reads per million (RPM) for each miRNA for the groups being
   compared

   ^3P Value and False discovery rate. miRNAs with FDR<0.05 were
   considered as differentially expressed between groups

Fig 3.

   [118]Fig 3
   [119]Open in a new tab

   Venn diagram indicating individual miRNAs (A) and miRNAs gene families
   (B), based on miRBase classification of highly similar miRNAs, that
   were significantly regulated between Ames dwarf (df/df) mice with
   aging, and between young (6 months old) and aged (22 months old) df/df
   and Normal (N) mice.

   The analysis of enriched pathways and gene ontology (GO) terms of
   target genes from the differentially regulated miRNAs affected by aging
   (young vs. old) or genotype (N vs. df/df) is presented in
   [120]S2–[121]S5 Tables. Venn diagram analysis of enriched pathways and
   GO terms pointed to 16 biological processes commonly regulated among
   all categories, including: cell differentiation, developmental
   maturation, anatomical structure development, cell death, growth, cell
   motility, cytoskeleton organization, cell division, cell morphogenesis,
   chromosome organization, circulatory system process, and cell cycle.
   Among the commonly regulated pathways between categories there was
   FoxO, mTOR, MAPK, PI3K-Akt, AMPK signaling pathways and
   Phosphatidylinositol signaling system. In addition, the enriched
   pathways and GO terms for the target genes of the 12 miRNAs commonly
   regulated between N and df/df mice at young and old ages is presented
   in [122]Table 3. The main signaling pathways target by the miRNAs
   regulated between N and df/df mice at both young and old ages are also
   summarized in [123]Fig 4. The insulin (Fig A in [124]S1 Fig), Pi3k/Akt
   (Fig B in [125]S1 Fig), FoxO (Fig C in [126]S1 Fig) and mTOR (Fig D in
   [127]S1 Fig) signaling pathways are represented in more details in the
   [128]S1 Fig provided.

Table 3. Enriched KEGG pathways and GO Terms for biological processes for the
genes targeted by miRNA differentially expressed between Ames dwarf and
Normal mice at both young and older ages.

   Pathways and GO Terms P value Genes miRNAs
   KEGG pathways
   Pathways in cancer 0.008 77 11
   PI3K-Akt signaling pathway 0.021 66 11
   MAPK signaling pathway 0.036 50 11
   Regulation of actin cytoskeleton 0.024 45 11
   Proteoglycans in cancer 0.002 44 11
   Ras signaling pathway 0.007 42 11
   Rap1 signaling pathway 0.030 42 11
   cAMP signaling pathway 0.023 41 11
   cGMP-PKG signaling pathway 0.023 37 11
   Axon guidance 0.004 33 11
   FoxO signaling pathway 0.007 32 11
   AMPK signaling pathway 0.007 31 11
   Signaling pathways regulating pluripotency of stem cells 0.008 31 11
   Insulin signaling pathway 0.036 31 11
   Adrenergic signaling in cardiomyocytes 0.036 30 11
   Hippo signaling pathway 0.014 26 11
   Prostate cancer 0.005 24 11
   mTOR signaling pathway 2.39E-04 22 11
   Melanoma 0.043 17 11
   Glioma 0.007 16 11
   Protein processing in endoplasmic reticulum 0.023 37 10
   Glutamatergic synapse 0.008 23 10
   Estrogen signaling pathway 0.021 22 10
   Adherens junction 0.030 15 10
   Acute myeloid leukemia 0.031 14 10
   Morphine addiction 4.58E-05 26 9
   GABAergic synapse 1.75E-07 20 9
   Hedgehog signaling pathway 0.041 14 8
   Long-term depression 0.031 14 7
   Other types of O-glycan biosynthesis 0.021 7 6
   GO Terms Biological Processes
   Anatomical structure development 1.16E-140 769 12
   Embryo development 2.48E-43 220 12
   Anatomical structure formation involved in morphogenesis 4.79E-27 180
   12
   Cellular nitrogen compound metabolic process 1.74E-15 701 11
   Biosynthetic process 2.20E-10 595 11
   Cell differentiation 5.68E-75 565 11
   Cellular protein modification process 8.65E-12 375 11
   Cellular component assembly 9.90E-06 207 11
   Cell cycle 2.64E-04 171 11
   Cell morphogenesis 9.09E-26 163 11
   Homeostatic process 8.60E-09 161 11
   Cell death 0.002 144 11
   Chromosome organization 2.08E-22 134 11
   Cytoskeleton organization 7.25E-07 133 11
   Cell motility 8.02E-11 126 11
   Cell-cell signaling 0.007 105 11
   Cell division 5.37E-07 99 11
   Growth 1.42E-06 86 11
   Developmental maturation 1.55E-11 46 11
   Circulatory system process 0.001 35 11
   Regulation of neural precursor cell proliferation 0.039 9 8
   Vasculogenesis involved in coronary vascular morphogenesis 0.002 7 7
   [129]Open in a new tab

Fig 4. Schematic representation of the main target genes of the microRNAs
differentially regulated between Normal (N) and Ames dwarf (df/df) mice at
both ages (6 and 22 months).

   [130]Fig 4
   [131]Open in a new tab

   Green–target gene of a regulated miRNA; Blue–non regulated target gene.

Discussion

   In the current study we observed that the abundance of 22 and 33 of the
   404 identified ovarian miRNAs changed with age in N and df/df mice,
   respectively. Of these, only three miRNAs were commonly regulated with
   age between N and df/df mice, indicating a very divergent profile of
   ovarian miRNAs as both mice genotypes age. It was previously
   demonstrated that the size of the primordial follicle reserve decreases
   by approximately 90% from the ages of 0.5 to 1.5 years in N female mice
   [[132]12], and we demonstrated before that the ovarian reserve of
   primordial follicles in df/df mice is three times larger than in N mice
   [[133]19, [134]20]. Therefore, our finding indicates that changes in
   the ovarian reserve with aging are reflected in changes in the ovarian
   miRNA profile of N and df/df mice. We also demonstrated that 46
   different miRNAs are regulated between N and df/df mice at young and
   old ages, of which 23 are exclusively regulated in young mice, 12
   exclusively in old mice and 12 commonly regulated at young and old
   ages. It can be hypothesized that differences in the expression of
   miRNAs between df/df and N mice and the differential impact of the
   aging process on their expression could be an important contributing
   factor for the preservation of a younger ovarian phenotype in df/df
   mice. Ovarian aging does not just affect the size of the ovarian
   follicular reserve but also affect tissue composition, including
   stromal fibrosis, changes in vascularization and infiltration of immune
   cells [[135]39, [136]40]. Therefore, the changes detected in the miRNA
   profile with aging and between genotypes are also a reflection of
   changes in ovarian tissue composition. As mentioned before, miRNAs can
   also be secreted to the extracellular space [[137]4] and have a role in
   intercellular communication in a hormone-like pattern between different
   cell types [[138]5]. Consequently, independent of its source, the
   ovarian detected miRNAs of this study can have an overall impact on the
   ovarian physiology, affecting antral follicle development, oocyte
   quality and primordial follicular activation, which are well known
   hallmarks of ovarian aging [[139]41].

   The top five most expressed ovarian miRNAs detected in the current
   study target several pathways involved in the regulation of cell growth
   and apoptosis. Specifically, the FoxO, MAPK, AMPK, mTOR, PI3K/Akt and
   TGF-β signaling pathways were targeted significantly by these miRNAs,
   suggesting that abundant miRNAs in the ovary serve as endogenous
   suppressors of key growth and differentiation pathways. In fact, cell
   growth, division, differentiation and death were amongst the enriched
   GO terms for these miRNAs. Interestingly, FoxO, mTOR, PI3k/Akt and
   insulin signaling pathways were also the main targets of the miRNAs
   differentially expressed between N and df/df mice, suggesting that its
   regulation by these miRNAs may be associated to the maintenance of a
   younger ovarian phenotype in older df/df mice. The PI3K/Akt, FoxO and
   mTOR signaling pathways are essential for the maintenance of the
   quiescent stage and initiation of primordial follicle growth [[140]17,
   [141]18, [142]29] and can be regarded as regulators of the rate of
   ovarian aging. In agreement with that, we have previously observed
   using the same ovarian RNA samples as the ones employed in the current
   study for RNASeq transcriptome evaluation and observed that ovarian
   expression of the Pi3k/Akt pathway is one of the main regulated
   pathways in df/df mice compared to N mice [[143]20]. In addition,
   FoxO3a phosphorylation was reduced in primordial follicles of
   GH-deficient df/df mice [[144]19, [145]42], suggesting that inhibition
   of this pathway (insulin/PI3k/Akt/FoxO) is essential for maintenance of
   primordial follicle quiescence. The PI3k, MAPK and mTOR pathways are
   considered survival pathways and their deregulation is frequently
   associated to abnormal cellular proliferation and inhibition of
   apoptosis [[146]43]. Aberrant expression of genes in these pathways is
   observed in several types of cancers, including ovarian cancers
   [[147]44], and not surprisingly these same pathways are well known for
   being involved in cellular senescence and aging [[148]43].

   The miR-17 family (mmu-miR-17, mmu-miR-20a and mmu-miR-93) was highly
   expressed within the ovaries and down-regulated with age exclusively in
   df/df mice. The miR-17 gene family is located at the same cluster (< 10
   kb spaced on mouse chromosome 14) with mmu-miR-92a, the highest
   expressed ovarian miRNA in our samples, which was up-regulated in young
   df/df in comparison to young N mice. The miR-17 gene family is involved
   in the regulation of stem cell differentiation [[149]45], and indeed
   signaling pathways regulating pluripotency in stem cells was one of the
   enriched pathways observed to be differentially regulated with aging in
   df/df mice. The miRNAs located in the miR-17/92 cluster are considered
   oncogenes and are frequently over-expressed in malignant cells, having
   the phosphatase and tensin homologue (PTEN) as one of their main
   targets [[150]46]. Despite that, PTEN gene expression was not changed
   with age or between genotypes in the RNASeq experiment using the same
   set of samples [[151]20]. Specifically, miR-20a overexpression is
   associated with increased proliferation and invasion in ovarian cancer
   cells [[152]47], and cell division, motility and differentiation were
   among the enriched GO terms for miRNAs regulated with age in df/df
   mice, further suggesting this. In addition, several of the identified
   pathways are related to cancer development in various tissues. Both
   miR-17 and miR-20a also directly target TGFBRII [[153]46], a gene
   well-known to be directly involved in the regulation of primordial
   follicle activation and ovarian aging [[154]7]. Interestingly, miR-145,
   the fifth most expressed ovarian miRNA in our study, also targets
   TGFBRII, and is a known regulator of primordial follicle activation
   [[155]7]. TGFBRII was up-regulated with age in N mice and
   down-regulated between old df/df and old N mice in the RNASeq
   experiment using the same set of samples [[156]20], further suggesting
   the importance of these miRNAs in the ovarian aging.

   The miR-21 family members were also among the most expressed miRNAs in
   the ovaries. Interestingly, these miRNAs were strongly down-regulated
   in ovaries of both young and older df/df mice compared to N mice. Both
   the -5p and -3p arms of miR-21 were downregulated to a similar extent,
   which suggests regulation at the level of initial transcription of the
   miR-21 precursor. In fact, this same pattern of regulation was observed
   for other seven miRNAs (mmu-miR-29c, mmu-miR-296, mmu-miR-130b,
   mmu-miR-17, mmu-miR-434, mmu-miR-181c, mmu-miR-132), which further
   confirms the validity of our analysis and suggests miRNA regulation at
   the gene expression level [[157]48]. Although it is found at much lower
   levels, it has been suggested that the 3p arm can also possess
   regulatory activity [[158]49]. miR-21 is considered an oncogene and is
   the most commonly over-expressed miRNA in cancerous tissues [[159]23].
   Overexpression of miR-21 was detected in ovarian epithelial carcinoma
   [[160]50], and was inversely correlated with drug sensitivity and
   progression-free survival [[161]51]. miR-21 is known for regulating
   cell growth and proliferation by targeting PTEN, and therefore its
   over-expression is associated with the activation of the Pi3k/Akt
   pathway [[162]52]. The Pi3k/Akt signaling pathway was one of the main
   targets of differentially expressed miRNA between young and old N and
   df/df mice. In addition, among the enriched GO terms for biological
   processes it was observed that cell death, motility, proliferation, and
   differentiation are represented and are terms associated to the
   development of neoplasic lesions. As mentioned before, activation of
   the Pi3k/Akt pathway is also a key process in the initiation of
   primordial follicle growth [[163]18], and PTEN knockout mice exhibit
   premature ovarian failure due to over-activation of the primordial
   follicle pool [[164]29]. This evidence points to miR-21 as a strong
   candidate for regulating ovarian aging in df/df mice, through
   regulation of the Pi3k/Akt pathway. In agreement with this, we
   previously demonstrated a reduced FoxO3a phosphorylation in oocytes
   contained in primordial follicles of df/df mice [[165]42], which is one
   of the main transcription factor targeted by the Pi3k/Akt pathway. As
   mentioned before, the Pi3k/Akt pathway component genes are highly
   regulated when evaluated by RNASeq in the ovaries of the same df/df and
   N mice [[166]20]. Additionally, miR-21-5p was up-regulated in cumulus
   cells from oocytes of poor-responder older women undergoing IVF
   procedures [[167]53], indicating that the down-regulation of miR-21-5p
   observed in df/df mice can be also associated to increased fertility
   potential.

   miR-132 and -212 are both members of the miR-132 gene family and were
   also strongly down-regulated in young and old df/df mice in comparison
   to N mice. Both miRNAs are known for being up-regulated in murine
   granulosa cells following LH/hCG induction [[168]54]. Therefore, our
   findings are not unexpected. Ames dwarf mice have an underdeveloped
   pituitary and decreased serum levels of gonadotropins (luteinizing and
   follicle stimulating hormones, LH and FSH) in comparison to N mice
   [[169]55]. The miR-132/mir-212 cluster can also regulate hematopoietic
   stem cells survival during aging by regulating FoxO3 expression
   [[170]56]. Deletion of miR-132/212 in hematopoietic cell lines
   increases their survival by preventing abnormalities during aging in
   response to nutritional stress [[171]56]. miR-132 and -212 are
   important regulators of cell cycling and renewal as well as survival
   and autophagy [[172]57]. Therefore, since FoxO3 is also directly
   involved in primordial follicle activation and maintenance of the
   dormancy status [[173]17], we can expect that the down-regulation of
   these miRNA observed in df/df mice can be also involved with the
   delayed ovarian aging in df/df mice. In agreement with this, we
   previously observed in this same set of samples that ovarian Foxo3a
   mRNA expression was higher in old df/df than N mice, although not
   different between young df/df and N mice [[174]42]

   miR-434-3p was one the most expressed miRNAs in ovarian samples from
   the current study and was observed to be down-regulated with age in
   df/df mice. In addition, miR-434-3p expression was also up-regulated in
   young df/df compared to young N mice. A previous study has shown that
   miR-434 up-regulation in the skeletal muscle tissue of aged mice is
   prevented by calorie restriction [[175]58]. There is very little
   published data on the mRNAs targeted by miR-434, but it is known for
   targeting a retrotransposon-like gene (RTL1) which has a role in
   genomic imprinting of parental origin [[176]59], although not changed
   between ages or genotypes in the RNASeq evaluation [[177]20].
   Furthermore, miR-181b-5p was also among the most expressed ovarian
   miRNAs and its variants were up-regulated in young (mmu-miR-181a-2-3p)
   and aged df/df mice (mmu-miR-181b-5p). Likewise, miR-181c-5p and -3p
   were down-regulated with age exclusively in df/df mice. The mir-181
   family has a role in differentiation of hematopoietic cells [[178]60],
   and was shown to be induced by TGF-β ligands [[179]61]. One of the main
   targets of miR-181 is the ATM mRNA, an important cell cycle checkpoint
   kinase [[180]61] that has role in repairing double strand DNA breaks
   [[181]62]. Overexpression of miR-181a in hepatocytes can suppress SIRT1
   at the protein level and therefore reduce insulin sensitivity
   [[182]63]. Neither, Atm or Sirt1 gene expression was regulated in
   between ages or genotype in these same samples [[183]20]. We observed
   that the insulin signaling pathway was among the main predicted
   enriched pathways for df/df mice with age. In astrocytes, the reduction
   of miR-181a expression was associated with reduced cell death, reduced
   oxidative stress, and increased mitochondrial function [[184]64]. These
   processes can play an essential role in protecting the oocyte from
   environmental damage, therefore reducing potential embryonic
   abnormalities and preserving fertility. In the ovary, double stranded
   DNA breaks have been shown to increase with age and were associated
   with increased rate of primordial follicle loss [[185]65].

   Several members of the miR-465 and miR-743 families were up-regulated
   in both young and aged df/df mice, although they were expressed at
   lower levels than the previously discussed miRNAs. miR-465 was
   previously detected as one of the most highly expressed miRNAs in the
   newborn mouse ovary, despite being undetectable in the adult ovary
   [[186]6]. The mir-465 cluster is located in the X chromosome, and is
   strongly expressed in the testis, although not detected in other
   tissues [[187]6]. Despite the low levels detected in the ovaries of N
   mice at young and older ages, expression of members of this cluster was
   up-regulated in the ovaries of df/df mice. This finding suggests that
   the preservation of a high expression of this exclusively newborn miRNA
   could be related to the preservation of the younger ovarian phenotype
   previously observed in df/df mice [[188]66]. While little is known
   about miR-743, one study demonstrated that miR-743 is down-regulated by
   increased oxidative stress, which in turn increases translation of
   genes involved in cellular protection mechanisms [[189]67].

   The serum from the same mice used in the current study was also
   submitted to miRNA sequencing and the results suggest a specific serum
   miRNA signature associated with healthy aging in df/df mice [[190]28].
   When we overlap these results with our current ovarian regulated miRNAs
   in df/df and N mice, we observed that miR-146a-5p was about 5 times
   more expressed in old than young N mice in both serum and ovarian
   samples, although its expression did not change in df/df mice in both
   studies [[191]28]. In addition, serum miR-592-5p was increased in df/df
   mice with age but did not change in N mice [[192]28]. We also observed
   an increase of miR-592-5p with age in df/df but not in N mice. Serum
   miRNAs have been used as a tool for diagnosis of a variety of
   conditions. In this sense, the serum miRNA profile of woman with
   ovarian epithelial cancer revealed several differentially regulated
   miRNAs that can be potentially used as markers for early diagnosis
   [[193]68]. Serum miR-205 was higher in ovarian cancer patients and had
   the best diagnostic accuracy [[194]68]. In our study miR-205-5p
   expression was six times higher in old than young N mice, although it
   did not change in df/df mice with age. This further point to an
   association between aging and ovarian cancer, and to miRNAs as
   important biomarkers of both conditions. miR-34a was also up-regulated
   in the serum of cancer patients [[195]68] and we observed that it was
   less expressed in the ovaries of young df/df mice in comparison to N
   mice. Therefore, this evidence suggests that a specific serum and
   ovarian miRNA profile is associated with ovarian aging and cancer
   development, and can be involved directly in the pathogenesis of these
   conditions or serve as useful biomarkers for early diagnosis.

   Overall, in the present study we found 54 different miRNA families
   differentially expressed between young and old N and df/df mice,
   providing the basis for understanding how aging can regulate the
   ovarian miRNA network. Many genes likely to be targeted by these
   differentially expressed miRNAs are involved in the FoxO, mTOR,
   Pi3k/Akt and insulin signaling pathways, which were shown to be
   regulated at the mRNA level evaluating the same ovaries from this mice
   in a RNASeq experiment [[196]20]. These pathways are well known to be
   involved in maintenance of primordial follicle quiescent stage,
   cellular senescence and ovarian cancer development. Therefore, they are
   interesting targets as biomarkers for diagnosis of these conditions, as
   well as possible central players in the maintenance of the younger
   ovarian phenotype in df/df mice. However, these conclusions must be
   considered as tentative. Although these miRNAs have been shown to
   regulate specific mRNAs and its translation in several cell types, it
   does not necessarily mean that the same will occur in the different
   ovarian compartments. Thus, extensive work validating these
   interactions in the ovary is necessary and must be the goal of future
   studies.

Supporting Information

   S1 Fig

   Fig A–Schematic representation of the insulin signaling pathway and the
   target genes of the microRNAs differentially regulated between Normal
   (N) and Ames dwarf (df/df) mice at both ages (6 and 22 months). Yellow
   box–target gene of one down-regulated miRNA; Orange box–target gene of
   two or more down-regulated miRNA. Fig B–Schematic representation of the
   Pi3k/Akt signaling pathway and the target genes of the microRNAs
   differentially regulated between Normal (N) and Ames dwarf (df/df) mice
   at both ages (6 and 22 months). Yellow box–target gene of one
   down-regulated miRNA; Orange box–target gene of two or more
   down-regulated miRNA. Fig C–Schematic representation of the FoxO
   signaling pathway and the target genes of the microRNAs differentially
   regulated between Normal (N) and Ames dwarf (df/df) mice at both ages
   (6 and 22 months). Yellow box–target gene of one down-regulated miRNA;
   Orange box–target gene of two or more down-regulated miRNA. Fig
   D–Schematic representation of the mTOR signaling pathway and the target
   genes of the microRNAs differentially regulated between Normal (N) and
   Ames dwarf (df/df) mice at both ages (6 and 22 months). Yellow
   box–target gene of one down-regulated miRNA; Orange box–target gene of
   two or more down-regulated miRNA.

   (DOC)
   [197]Click here for additional data file.^ (291.5KB, doc)
   S1 Table. Enriched KEGG pathways and GO Terms for biological process
   for the genes targeted by the top five most expressed miRNA in mice
   ovaries.

   (DOC)
   [198]Click here for additional data file.^ (250KB, doc)
   S2 Table. Enriched KEGG pathways and GO Terms for biological process
   for the genes targeted by the regulated miRNA during aging in Normal
   mice.

   (DOC)
   [199]Click here for additional data file.^ (273.5KB, doc)
   S3 Table. Enriched KEGG pathways and GO Terms for biological process
   for the genes targeted by the regulated miRNA during aging in Ames
   dwarf mice.

   (DOC)
   [200]Click here for additional data file.^ (288.5KB, doc)
   S4 Table. Enriched KEEG pathways and GO Terms for biological process
   for the genes targeted by miRNA differentially expressed between Ames
   dwarf and Normal mice at young (6 months) age.

   (DOC)
   [201]Click here for additional data file.^ (289KB, doc)
   S5 Table. Enriched KEEG pathways and GO Terms for biological process
   for the genes targeted by miRNA differentially expressed between Ames
   dwarf and Normal mice at old (22 months) age.

   (DOC)
   [202]Click here for additional data file.^ (271KB, doc)

Acknowledgments