Abstract
Ghrelin, an orexigenic gut-derived peptide, is gaining increasing
attention due to its multifaceted role in a number of physiological
functions, including reproduction. Ghrelin exists in circulation
primarily as des-acylated and acylated ghrelin. Des-acyl ghrelin, until
recently considered to be an inactive form of ghrelin, is now known to
have independent physiological functionality. However, the relative
contribution of acyl and des-acyl ghrelin to reproductive development
and function is currently unknown. Here we used
ghrelin-O-acyltransferase (GOAT) knockout (KO) mice that have no
measurable levels of endogenous acyl ghrelin and chronically high
levels of des-acyl ghrelin, to characterize how the developmental and
life-long absence of acyl ghrelin affects ovarian development and
reproductive capacity. We combined the assessment of markers of
reproductive maturity and the capacity to breed with measures of
ovarian morphometry, as well as with ovarian RNA sequencing analysis.
Our data show that while GOAT KO mice retain the capacity to breed in
young adulthood, there is a diminished number of ovarian follicles (per
mm^3) in the juvenile and adult ovaries, due to a significant reduction
in the number of small follicles, particularly the primordial
follicles. We also show pronounced specific changes in the ovarian
transcriptome in the juvenile GOAT KO ovary, indicative of a potential
for premature ovarian development. Collectively, these findings
indicate that an absence of acyl ghrelin does not prevent reproductive
success but that appropriate levels of acyl and des-acyl ghrelin may be
necessary for optimal ovarian maturation.
Keywords: acyl ghrelin, des-acyl ghrelin, ovarian follicles,
reproductive success, RNA-seq
Introduction
Since its initial discovery and characterization ([29]1), ghrelin has
received increasing attention in metabolic, cardiovascular, stress,
motivation, and memory research as having multiple important roles in
these fields ([30]2–[31]10). Ghrelin has also been implicated in
supporting reproductive function, acting at all levels of the
hypothalamic-pituitary-gonadal (HPG) axis ([32]11–[33]14), including
both directly and indirectly on the mammalian ovary ([34]11,
[35]15–[36]17). In doing so it mediates changes in the metabolic state
and in the levels of stress and reward on puberty, fertility, and
fecundity ([37]18).
Ghrelin is a 28 amino acid peptide, produced in the gastrointestinal
tract. It is modified by n-octanoylation of serine at the third
position (Ser-3) by the enzyme ghrelin-O-acyltransferase (GOAT), to
form acylated ghrelin ([38]19, [39]20). Ghrelin and GOAT are highly
conserved in vertebrates ([40]21). In circulation, ghrelin exists in at
least two major bioactive forms: acylated and des-acylated ghrelin
([41]22). It is the acylated form that acts at the growth hormone
secretagogue receptor (GHSR). The receptor for des-acyl ghrelin has not
yet been identified, nevertheless, des-acyl ghrelin has been shown to
suppress the effects of acyl ghrelin ([42]23, [43]24) and to exert
independent biological effects on metabolism ([44]25–[45]27),
cardiovascular function ([46]9, [47]28), stress ([48]10), and
reproduction ([49]29–[50]31) and is thus an additional important target
for investigation.
Both high and low levels of ghrelin appear to be detrimental for
fertility, suggesting that a certain balance between circulating acyl
and des-acyl ghrelin is important for reproductive potential ([51]18).
As such, acute administration of acyl ghrelin in rats impairs
folliculogenesis, induces morphometric changes in the ovary, and
reduces ovarian volume ([52]32). Chronic administration of acyl or
des-acyl ghrelin, or the combination of both, delays follicle
maturation and reduces ovarian weight, suggesting the inhibitory
effects of ghrelin on the ovary may not be solely dependent on the
GHSR-mediated signaling pathway ([53]31). In mice, both administration
of a high dose of acyl ghrelin and GHSR antagonism during
peri-implantation and early gestation impair fertilization,
implantation, and embryo development ([54]33). Human data show that
while acyl ghrelin inhibits ovarian steroidogenesis ([55]16),
endometrial expression of the ghrelin gene and GHSR1a are decreased in
infertile women ([56]34), supporting the hypothesis that an adequate
balance within the ghrelin system is required to maintain healthy
reproductive function.
The focus on the physiological role of des-acyl ghrelin has only
recently begun to gain attention and a large number of studies report
the levels and the effects of either total or acyl ghrelin, with only a
limited number of studies assessing des-acyl ghrelin ([57]18, [58]35).
Given that both high and low concentrations of ghrelin exert negative
effects on fertility, and that some of these effects may be mediated
through GHSR-independent pathways, it is imperative to further our
understanding of the role des-acyl ghrelin plays in reproduction.
Genetic deletion of GOAT removes the capacity for ghrelin acylation and
results in undetectable concentrations of acyl and chronically high
levels of des-acyl ghrelin during development and throughout life
([59]36, [60]37). These GOAT KO mice have been used to investigate the
role of the GOAT-ghrelin system in metabolism and stress
([61]36–[62]39), and we used it here to test the hypothesis that acyl
ghrelin plays a significant role in the development and function of the
reproductive system. GOAT KO mice do not display developmental or overt
anatomical differences from WT ([63]40). They do, however, respond
differently to conditions of altered metabolic state and stress, as
evident from findings in males ([64]10, [65]37). Thus, we hypothesized
that the absence of GOAT (and so the absence of acyl ghrelin and high
levels of des-acyl ghrelin) would alter the development of the ovary,
leading to detrimental changes in the ovarian transcriptomic profile
and impaired development and function of the ovary. We also tested
whether these changes were maintained into adulthood and were reflected
in differences in the number of ovarian follicles per mm^3 of ovary or
in major reproductive endpoints, including puberty onset and fecundity.
Materials and Methods
Animals
In these experiments we used female mice. GOAT KO mice on a C57/Bl6
background were obtained from Regeneron Pharmaceuticals (Tarrytown, NY)
and bred (het × het; 1 male × 2 females) in the Monash Animal Services
to generate WT and KO littermates, as previously described ([66]10).
Mice were group-housed under standard laboratory conditions with ad
libitum access to food and water at 23°C in a 12 h light/dark cycle.
All procedures described here were in accordance with the National
Health and Medical Research Council Australia Code of Practice for the
Care of Experimental Animals and the Monash University Animal Ethics
Committee guidelines.
Ovarian Tissue Collection
To assess the effects of GOAT deletion on ovarian morphology and
transcriptome, we collected ovaries from GOAT KO and WT juvenile (3
weeks old) and adult (10 weeks old) mice. Mice were deeply anesthetized
by isoflurane inhalation and ovaries were excised. One ovary from each
animal was snap frozen in liquid nitrogen and stored at −80°C for gene
analysis, and one ovary was fixed in Bouin's solution (Sigma-Aldrich,
St Louis, MO, USA) overnight, rinsed four times in 70% ethanol and
stored in ethanol until processing.
Characterization of Ovarian Morphometry
Exogenous acyl ghrelin suppresses follicle maturation and reduces
ovarian volume in the prepubertal ovary ([67]31, [68]32). It also
disrupts granulosa cell steroidogenesis ([69]16). We therefore
investigated if the deletion of GOAT and thus a change in acyl and
des-acyl ghrelin concentrations would induce morphometric changes in
the GOAT KO ovary. We thus assessed the number of ovarian follicles in
juvenile and adult mice. As previously described ([70]41, [71]42),
fixed ovaries were dehydrated, embedded in paraffin and sectioned at 4
μm. For morphometric analysis, 20 sections on 10 slides, 36 μm apart,
were stained with haematoxylin-eosin (H&E). Two sections per slide were
assessed on the basis of an 8 μm distance between the sections,
allowing a complete assessment of primordial follicle counts at this
location, as per our previous publications ([72]42, [73]43). Follicles
were classified as: (a) primordial: an oocyte surrounded by a single
layer of flattened pregranulosa cells; (b) early primary: an oocyte
surrounded by a single layer of flattened pregranulosa cells with at
least two cuboidal granulosa cells; (c) primary: an oocyte surrounded
by cuboidal granulosa cells; (d) preantral: follicles with no antral
cavity and two or more layers of cuboidal granulosa cells; (e) antral:
an antral cavity visible, with at least two layers of cuboidal
granulosa cells. We scanned whole ovarian sections using an Olympus
VS120 slide scanner (Olympus, Tokyo, Japan). Only follicles with
visible nuclei and nucleoli were counted to prevent counting the same
follicle more than once. Area measurements were obtained using ImageJ
(National Institutes of Health, MD, USA). Follicle counts were adjusted
per total section volume, calculated as area multiplied by section
thickness, according to Bernal et al. ([74]44), Aiken et al. ([75]45,
[76]46), Tsoulis et al. ([77]47), Asadi-Azarbaijani et al. ([78]48),
and Chan et al. ([79]49), and presented as counts per mm^3 (n = 4–6
animals per group). Follicles were classified as atretic if they
presented with one or more of the following criteria: oocyte
degeneration; granulosa cell degeneration, disorganization and
retraction from the oocyte; appearance of pyknosis in more than 10% of
granulosa cells ([80]49–[81]51).
Immunohistochemistry
We used proliferating cell nuclear antigen (PCNA) immunolabeling to
visualize follicle activation and growth, as previously described
([82]42, [83]52). We de-waxed paraffin-embedded sections (4 μm) in
histolene and rehydrated them in ethanol washes. Antigen retrieval was
carried out by microwaving sections in sodium citrate buffer for 15 min
(10 mM sodium citrate, pH = 6). Slides were then cooled down to room
temperature (RT) and blocked in 3% bovine serum albumin (BSA)/0.03%
Triton X-100/ phosphate-buffered saline (PBS) for 1 h at RT. Sections
were then incubated overnight at 4°C with mouse monoclonal anti-PCNA
(1:200; #ab29, lot #[84]GR201287, Abcam, Cambridge, UK). We then washed
the slides in PBS/0.1% Triton X-100 and incubated them with Alexa Fluor
594 donkey anti-mouse IgG fluorescent-conjugated secondary antibody
(1:200; A21203 Thermo Scientific, Rockford, IL, USA). Sections were
then counterstained with 4′,6-diamidino-2-phenylindole (DAPI) using
Fluoroshield with DAPI mounting medium (Sigma-Aldrich, St Louis, MO,
USA) and viewed under an Olympus BX61 fluorescent microscope fitted
with a Nikon DS-Ri2 camera. A minimum of two randomly selected slides
were evaluated for each animal. We assessed fluorescence intensity in
PCNA-positive follicles with a visible nucleus using ImageJ (National
Institutes of Health, MD, USA). Mean fluorescence intensity was
calculated using a corrected total cell fluorescence (CTCF) formula
(CTCF = integrated density—(area of selected section × mean
fluorescence of background readings), as previously described ([85]53).
Data were normalized to the mean fluorescence intensity of the control
group and expressed in arbitrary units (AU) ([86]43, [87]54, [88]55).
For detection of follicular apoptosis, we de-paraffinised and
rehydrated paraffin-embedded sections as described above. We
pre-treated the sections with 20 μg/mL proteinase K for 15 min at RT
and performed analysis for terminal deoxynucleotidyl transferase dUTP
nick end labeling (TUNEL) using ApopTag Fluorescein In situ Apoptosis
Detection Kit (Merck Millipore, Burlington, MA, USA) according to the
manufacturer's instructions. We then counterstained the sections using
Fluoroshield with DAPI mounting medium (Sigma-Aldrich). Positive
controls consisted of sections treated with DNAse I (Qiagen, Carlsbad,
CA, USA) to induce non-specific DNA fragmentation, and negative control
staining was performed without active Terminal deoxynucleotidyl
Transferase (TdT) but including proteinase K digestion, to control for
non-specific incorporation of nucleotides. Slides were viewed under a
fluorescent microscope and follicles were classified as apoptotic if
they contained a TUNEL-positive oocyte and/or ≥4 TUNEL-positive
granulosa cells for primary, secondary and antral follicles, or >1
TUNEL-positive granulosa cells for primordial follicles ([89]42,
[90]56, [91]57).
RNA Isolation
We isolated total RNA using QIAzol reagent and RNeasy Mini Kits
(QIAGEN, Valencia, CA, USA). RNA concentrations were determined using a
spectrophotometer, NanoDrop 2000/2000c (Thermo Fisher Scientific,
Wilmington, DE USA) and 1 μg RNA was transcribed to cDNA using an
iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA),
according to the manufacturer's instructions.
Next-Generation Sequencing
Total RNA (1 μg) samples isolated from juvenile GOAT KO and WT mouse
ovaries (n = 4 per group) were submitted to the Australian Gene
Research Facility (AGRF; Melbourne, VIC, Australia) for processing and
bioinformatics analysis. The quality of the total RNA was assessed by a
Bioanalyser (RNA Integrity Number = 10 for all samples). The samples
were then sequenced on an Illumina HiSeq 2000 platform (Illumina, San
Diego, CA) generating 50 bp single-reads per lane. The primary
bioinformatics analysis involved de-multiplexing and quality control.
The per base sequence quality indicated that the Phred quality score
was above 30 for >96% of bases across all samples ([92]58, [93]59). The
reads were also screened for the presence of any Illumina
adaptor/overrepresented sequences and cross-species contamination. The
cleaned sequence reads were then aligned against the Mus musculus
genome (Build version mm10). The Tophat aligner (v2.0.14) was used to
map reads to the genomic sequences. The counts of reads mapping to each
known gene were summarized. The transcripts were assembled with the
Stringtie tool v1.0.4 ([94]http://ccb.jhu.edu/software/stringtie/)
utilizing the reads alignment with reference annotation based assembly
option (RABT). The GENCODE annotation containing both coding and
non-coding annotations for mouse genome version GRcm38 (Ensemble
release 81) was used as a guide.
To estimate differences in gene counts between groups, differential
expression analysis was undertaken using specialized R libraries from
Bioconductor version 3.2 ([95]http://www.bioconductor.org) ([96]60). A
multidimensional scaling plot revealed that two samples (one from each
of the GOAT KO and WT groups) did not cluster with the rest of the
samples from that group and were thus considered as outliers for
further analysis. The data filter was set to 0.5 < logFC < −0.5
difference and p < 0.05. A test for over-representation of Gene
Ontology (GO) terms was performed using the GOANA method
([97]https://www.bioconductor.org/packages/devel/bioc/manuals/limma/man
/limma.pdf). The clusterProfiler software package was used to analyse
and visualize functional profiles (GO and Kyoto Encyclopedia of Genes
and Genomes, KEGG) of gene and gene clusters
([98]http://www.bioconductor.org/packages/release/bioc/html/clusterProf
iler.html). The clusterProfiler supports enrichment analysis of
Reactome and KEGG with either hypergeometric test or Gene Set
Enrichment Analysis (GSEA) ([99]61).
In addition to these analyses, we also used the Ingenuity Pathway
Analysis (IPA; Qiagen Inc.,
[100]https://www.qiagenbioinformatics.com/products/ingenuity-pathway-an
alysis) platform to explore further downstream and upstream effects of
GOAT deletion in our dataset. The recommended set size for IPA core
analysis from gene expression data is 200–3,000 molecules, and
different fold change cut-offs are routinely used to allow inclusion of
more differentially expressed genes for meaningful pathway analyses
[e.g., ([101]62, [102]63)]. We therefore performed the core analysis at
0.3 < logFC < −0.3 difference and p < 0.05, resulting in 656
analysis-ready molecules. These cut-off criteria allowed us to predict
directionality of change in downstream functions and upstream
regulators, accounting for a potential dilution of information as a
result of whole ovary sequencing. Statistical significance was
calculated using the right-tailed Fisher's Exact Test. The activation
z-score, a statistical measure that assesses the match between observed
and predicted upstream or downstream regulation patterns based on
previous literature was also used to evaluate significance of effects
on diseases and biological functions, as well as the activation and
inhibition states of predicted upstream regulators [see ([103]64) for
further information on the IPA core analysis]. The data discussed in
this publication have been deposited in NCBI's Gene Expression Omnibus
and are accessible through GEO Series accession number [104]GSE106339.
Real-Time Quantitative PCR Array
We also used a Custom RT^2 PCR array (CLAM26350; Qiagen) designed to
specifically examine, in juvenile and adult ovaries, the changes in the
top ten over- and under-expressed genes (Table [105]1) that were
identified by RNA sequencing in the ovaries of juvenile GOAT KO mice.
Pseudogenes were excluded from the analysis. We also used this ovary to
confirm the absence of GOAT (Mboat4) in the ovaries of GOAT KO mice.
Total RNA, 400 ng, extracted as detailed above, was transcribed to cDNA
using the RT^2 First Strand Kit (Qiagen), according to the
manufacturer's instructions. Samples were then diluted as per
manufacturer's instructions in RT^2 SYBR Green Mastermix, loaded onto
384-well PCR array plates and amplified on the QuantStudio™ 7 Flex
Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA), including
an initial activation step at 95°C for 10 min followed by 40 cycles at
95°C for 15 s and 60°C for 1 min. Actb and Gapdh were used as
endogenous controls. The C(t) values for these genes were averaged and
used for the comparative threshold cycle (ΔΔ C(t)) calculations, where
C(t) is ≤ 40. Fold changes were then calculated using the 2^−ΔΔC(t)
Equation ([106]98).
Table 1.
Top 10 over- and under-expressed genes in GOAT KO mouse ovary (p <
0.05) and supporting literature.
Gene symbol Gene name NCBI Log[2]FC Summary of function
Gm10036 Predicted gene 10036 2.75 Ribosomal protein L11 pseudogene
([107]65).
Grik3 Glutamate receptor, ionotropic, kainate 3 1.74 Also known as
Glur7. Plays a role in neuroactive ligand-receptor interaction,
underlying glutamate-mediated excitatory synaptic transmission, and is
expressed in the ovary ([108]66, [109]67)
Spocd1 SPOC domain containing 1 1.72 Involved in negative regulation of
phosphatase activity. SPOC domain (Spen paralogue and ortholog C
terminal) plays a role in developmental signaling ([110]68, [111]69).
Grem1 Gremlin 1, DAN family BMP antagonist 1.60 Expressed in granulosa
cells. Regulates folliculogenesis and primordial to primary follicle
transition ([112]70, [113]71)
Cyp19a1 Cytochrome P450, family 19, subfamily a, polypeptide 1 1.40
Encodes aromatase, the key enzyme in estrogen biosynthesis.
Significantly increased during preovulatory follicle development
([114]72–[115]74).
Inhba Inhibin, beta A 1.37 Encodes activin β A subunit that negatively
regulates pituitary follicle-stimulating hormone (FSH) synthesis.
Prominently expressed in granulosa cells of preantral and antral
follicles. Deletion causes neonatal lethality, significant craniofacial
defects and abnormal folliculogenesis ([116]75–[117]77).
Hspb7 Heat shock protein family B (small) member 7 1.29 Potent
suppressor of protein aggregation, assists in the clearance of
stress-induced misfolded proteins ([118]78, [119]79).
Gm15421 Predicted gene 15421 1.17 Ribosomal protein L22 like 1
pseudogene ([120]80).
Sohlh1 Spermatogenesis and oogenesis specific basic helix-loop-helix 1
1.08 Expression is confined to primordial oocytes and is required for
their differentiation. In adult ovary transcript expression is
decreased as the oocytes are recruited to form primary and secondary
follicles ([121]81, [122]82).
Drd4 Dopamine receptor D4 1.05 Androgen-dependent gene
([123]83–[124]85). Strongly expressed in mouse adult ovary, with no
known function ([125]67).
Leprotl1 Leptin receptor overlapping transcript-like 1 −0.98 Negatively
regulates growth hormone (GH) receptor expression and is overexpressed
during fasting ([126]86). Transgenic mice overexpressing leprotl1 show
growth retardation ([127]87). Expressed in granulosa cells throughout
follicular development from small to ovulatory follicles and
significantly increased in corpus luteum, compared to small follicles
([128]88).
Dcdc2b Doublecortin domain containing 2b −1.02 Encodes a member of the
doublecortin family. The doublecortin domain binds tubulin and
increases microtubule polymerisation ([129]89).
Gm5620 Predicted gene 5620 −1.06 tubulin, alpha 1B pseudogene.
Non-protein coding.
Cited4 Cbp/p300-interacting transactivator, with Glu/Asp-rich
carboxy-terminal domain, 4 −1.07 Luteinising hormone (LH) target gene
during ovulation. The pre-ovulatory LH surge induces Cited4 expression
in cumulus and granulosa cells and this expression is required for
cumulus-oocyte complex expansion and ovulation. Regulates histone
acetylation in the promoters of the LH-induced target genes as a
histone acetyltransferase in mouse granulosa cells undergoing
luteinization after the ovulatory LH surge ([130]90).
Gm13152 Zinc finger protein 982 −1.09 Expressed in mouse fetal ovary in
meiotic prophase, with increasing expression between E12.5 to E16.5
([131]91). No specific function in adult ovary has been attributed.
Gm13103 Predicted gene 13103 −1.11 Highly expressed in adult mouse
ovary. Specifically expressed in fully grown oocytes ([132]92)
Rab3b RAB3B, member RAS oncogene family −1.15 A marker for regulated
secretion, expressed in cells with a high activity of regulated
exocytosis. In the pituitary, Rab3b is essential for GnRH-regulated
exocytosis in gonadotrophs ([133]93). In the sheep ovary, Rab3b has
been co-localized with oxytocin to the same luteal staining granules of
the corpus luteum during the luteal phase of the estrous cycle
([134]94)
Rab6b RAB6B, member RAS oncogene family −1.53 Controls retrograde
transport from the Golgi body to the endoplasmic reticulum and is
predominantly expressed in neuronal cells ([135]95, [136]96).
Gm6166 Predicted gene 6166 −1.81 Fatty acid-binding protein,
epidermal-like. Non-protein coding.
Myh6 Myosin, heavy polypeptide 6, cardiac muscle, alpha −2.05 Involved
in protein dimerization activity. Overexpressed in the ovaries of
5α-dihydrotestosterone treated rats, mimicking the hyperandrogenic
state in women with polycystic ovarian syndrome ([137]97).
[138]Open in a new tab
Onset of Puberty and Breeding Capacity
Changes in the availability of acyl ghrelin have been implicated in the
timing of puberty onset ([139]99, [140]100). We therefore examined if
the deletion of GOAT and thus an absence of acyl ghrelin affected the
onset of puberty in our study. Mice (n = 7–8 per group) were inspected
daily, beginning at postnatal day (P)25, to determine the day of
vaginal opening (a physical marker of puberty onset). When vaginal
opening occurred, mice were weighed and left undisturbed until
adulthood. It has been previously reported that GOAT KO mice are
capable of breeding normally ([141]37), but we wanted to test if this
capacity was affected in our housing facility. We therefore also
evaluated historic breeding records from Monash Animal Research
Platform of 15 WT and 16 GOAT KO females ranging between 2.5 and 6
months of age at first mating, that were continuously mated for 3–5
months with WT and GOAT KO male studs, respectively. Breeding success
was indicated by the mean number of pups per litter per dam, as well as
by the mean number of pups in the first litter, since C57/Bl6 dams have
been shown to have a higher pup mortality rate in their first than in
subsequent litters ([142]101).
Statistical Analysis
In addition to the analysis of RNA sequencing data described above, we
used Student's unpaired t-tests for the assessment of ovarian
morphometry, RT^2 PCR array, puberty onset, and breeding data between
GOAT KO and WT mice. We also used Pearson's correlation analysis to
assess the relationship between RT^2 PCR array and RNA sequencing fold
changes. Data are presented as the mean ± SEM. Statistical significance
was assumed when p ≤ 0.05.
Results
Juvenile GOAT KO Mice Have a Reduced Number of Ovarian Follicles (Per mm^3)
Body weights in the juvenile phase were not affected by the absence of
GOAT (data not shown). However, the juvenile GOAT KO mice had a
significant reduction in their small follicle numbers per mm^3 of
ovarian tissue, such that GOAT KO mice had a reduction of more than 50%
in the number of primordial [t[(]7) = 2.52, p = 0.039, Figures
[143]1A,D] and early primary follicles [t[(]7) = 2.72, p = 0.029,
Figures [144]1A,D; expressed per mm^3], compared to age-matched WT
controls. There were no differences in the numbers of large healthy
follicles or atretic follicles (Figures [145]1B,C; expressed per mm^3).
Figure 1.
[146]Figure 1
[147]Open in a new tab
Effects of GOAT deletion on ovarian follicles in juvenile mice. (A)
Small follicle; (B) Large follicle; and (C) Atretic follicle counts in
the ovary of juvenile GOAT KO and WT mice. Follicle numbers are
expressed per mm^3 of ovarian tissue. Panel (D) shows morphological
representation of H and E stained juvenile WT and GOAT KO ovary. Black
arrows in inserts at higher magnification indicate small follicles
(i.e., primordial, early primary and primary) in ovarian cortical
region. Scale bars = 20 μm in low and 10 μm in high magnification
images. (E) Number of TUNEL-positive follicles in the ovary of juvenile
GOAT KO and WT mice. (F) Representative images of TUNEL-positive
staining (green) in the juvenile ovary. (G) Normalized mean
fluorescence intensity of proliferating cell nuclear antigen (PCNA)
staining in juvenile GOAT KO and WT ovaries. (H) Representative images
of PCNA staining (red) in the juvenile ovary. Scale bars = 50 μm. Data
are expressed as mean ± SEM. *p < 0.05.
Consistent with a lack of differences in large healthy and atretic
follicles, there were no significant differences in the number of
TUNEL-positive follicles between WT and GOAT KO mice, in the juvenile
phase (Figures [148]1E,F). TUNEL-positive granulosa cells were
primarily expressed in large follicles (i.e., secondary and antral),
consistent with previous studies showing that the current commonly used
apoptotic markers are unable to detect primordial and primary follicle
atresia and therefore follicle counts provide the most accurate
assessment of primordial and primary follicle loss ([149]102,
[150]103). PCNA, a marker of follicle growth, was also highly expressed
in the granulosa cells and oocytes of large follicles and some primary
follicles, as previously described ([151]104), with no differences in
PCNA expression between juvenile WT and GOAT KO mice ovaries (Figures
[152]1G,H).
Juvenile GOAT KOs Have Differences in the Ovarian Transcriptome Compared to
WT Mouse Ovaries
Since reliable markers of apoptosis and proliferation in the small
follicle population remain to be developed, we assessed if the
reduction in the follicle numbers was associated with changes in
ovarian genes and pathways related to reproduction. We thus performed
RNA sequencing, characterizing the ovarian transcriptome of juvenile
GOAT KO compared to WT mice. The counts of reads mapped to each known
gene are summarized (Figure [153]2A). Differential expression analysis
for estimating differences in transcripts across groups identified
14,573 genes, with a significant difference in the expression of 252
genes of at least 1-fold change (−0.5 < logFC < 0.5), p < 0.05. A
summary of the RNA sequencing results and the distribution of
differentially expressed genes (DEGs) are presented in Figures
[154]2B,C. The top ten over- and under-expressed genes in the ovaries
of GOAT KO mice are presented in Table [155]1 along with a summary of
their known functions. These DEGs included several genes associated
with major biological processes and functions regulating reproductive
development. For instance, Grem1, Cyp19a, Inhba, and Sohlh1 play a
critical role in folliculogenesis. Grem1, regulates primordial to
primary follicle transition. Sohlh1, is required for oogenesis and is
essential for primordial follicle activation. Inhba expression is
associated with follicular growth, regulating cell proliferation, and
follicle stimulating hormone (FSH) action in the ovary. Cyp19a1,
encodes aromatase cytochrome P450, catalyzing a critical step in
ovarian estrogen biosynthesis ([156]71, [157]72, [158]81, [159]105).
Figure 2.
[160]Figure 2
[161]Open in a new tab
RNA sequencing analysis of ovaries from juvenile GOAT KO versus WT
mice. (A) Summary of number of reads and their mapping to genome. (B)
Summary of RNA sequencing. Total number of identified transcripts are
presented as black and differentially expressed genes (DEGs; −0.5 >
logFC > 0.5; p < 0.05) are presented as white. The red bar represents
the number of over-expressed transcripts and the green bar represents
the number of under-expressed transcripts in the ovaries of juvenile
GOAT KO mice. (C) Volcano plot for differentially expressed genes,
where the p-value (Y axis) is plotted against the log fold change (X
axis). p-value cutoff of 0.05 is represented by horizontal red line.
Log fold change filters are represented by vertical dashed lines. Red
dashed lines represent a filter set to ±0.5 that was used to test for
over-representation of Gene Ontology terms and for pathway enrichment
analysis. Green dashed lines represent a filter set to ±0.3, for the
analysis using the Ingenuity Pathway Analysis (IPA) platform.
DEGs were annotated by association with three GO term categories:
biological process, molecular function and cellular component. The top
22 GO terms for each category are presented in Figure [162]3. These
included biological processes regulating reproduction, particularly its
positive regulation; immune response (e.g., defense response,
regulation of immune system process, positive regulation of antigen
processing and presentation, myeloid leukocyte migration, innate immune
response); cell signaling and transport, and others (Figure [163]3A).
Figure 3.
[164]Figure 3
[165]Open in a new tab
Gene Ontology (GO) enrichment analysis of the differentially expressed
genes. GO terminology and the number of differentially expressed genes
(DEGs) associated with each of the three categories: (A) Biological
processes. (B) Cellular Component. (C) Molecular Function. p-value for
over-representation of GO terms in DEGs is represented by: red, p <
0.002; yellow, p < 0.004; and pink, p < 0.006.
In the ontology of cellular component, GO categories of extracellular
space, plasma membrane, trans-Golgi network, and major
histocompatibility complex (MHC) class II protein complex, were among
the most significant overrepresented GO terms in the DEGs (Figure
[166]3B). Molecular functions, such as retinoid binding, activin
receptor binding, endopeptidase/peptidase regulator and inhibitor
activity and growth factor activity were among the top 22 most
significant GO terms (Figure [167]3C). Using enrichment analysis for
Reactome and KEGG pathways we identified several pathways involved in
the immune response and cell signaling. Pathway enrichment results are
summarized in Table [168]2.
Table 2.
Pathway enrichment analysis according to Reactome and Kyoto
Encyclopedia of genes and Genomes (KEGG).
Pathway ID Statistics Annotated genes
(A) ENRICHED REACTOME PATHWAYS
Neuronal System 5604671 GeneRatio = 6/55; BgRatio = 237/6,598; p-value
= 0.013; p.adjust = 0.121 Kcnab3, Kcnn3, Camkk1, Gls2, Gabrb1, Kcnmb2
Immune System 5604803 GeneRatio = 14/55; BgRatio = 1007/6,598; p-value
= 0.033; p.adjust = 0.150 Prkar2b, C3, Sh3gl2, Fbxo44, Kif5a,
Ctss/H2-Ab1, Peli3, C8g, Trem2, Il6ra, Tlr7, Itgb2, Cd74
Adaptive Immune System 5604808 GeneRatio = 8/55; BgRatio = 552/6,598;
p-value = 0.085; p.adjust = 0.193 C3, Sh3gl2, Fbxo44, Kif5a, Ctss,
H2-Ab1, Itgb2, Cd74
Hemostasis 5605036 GeneRatio = 7/55; BgRatio = 460/6,598; p-value =
0.086; p.adjust = 0.193 Prkar2b, Igf2, Kif5a, H3f3a, Serpinc1, Itgb2,
Kcnmb2
Innate immune system 5604802 GeneRatio = 7/55; BgRatio = 487/6,598;
p-value = 0.108; p.adjust = 0.194 Prkar2b, C3, Ctss, C8g, Trem2, Tlr7,
Itgb2
(B) ENRICHED KEGG PATHWAYS
Drug metabolism—cytochrome P450 mmu00982 GeneRatio = 4/58; BgRatio =
29/3,997; p-value < 0.001; p.adjust < 0.05 Gsta2, Gsta4, Fmo1, Adh1
Retinol metabolism mmu00830 GeneRatio = 3/58; BgRatio = 15/3,997;
p-value < 0.01; p.adjust < 0.05 Adh1, Lrat, Cyp26b1
Complement and coagulation cascades mmu04610 GeneRatio = 4/58; BgRatio
= 35/3,997; p-value < 0.01; p.adjust < 0.05 Serpina5, C3, C8g, Serpinc1
Systemic lupus erythematosus mmu05322 GeneRatio = 4/58; BgRatio =
37/3,997; p-value < 0.01; p.adjust < 0.05 C3, H2-Ab1, H3f3a, C8g
Staphylococcus aureus infection mmu05150 GeneRatio = 3/58; BgRatio =
21/3,997; p-value < 0.01; p.adjust < 0.05 C3, H2-Ab1, Itgb2
Metabolism of xenobiotics by cytochrome P450 mmu00980 GeneRatio = 3/58;
BgRatio = 25/3,997; p-value < 0.01; p.adjust < 0.05 Gsta2, Gsta4, Adh1
[169]Open in a new tab
GeneRatio: ratio between the number of DEGs in the pathway and the
number of DEGs. BgRatio: ratio between the number of genes in the
pathway and the total examined background of genes. p.adjust: p-value
for hypergeometric test adjusted for Benjamini–Hochberg correction.
Ingenuity Pathway Analysis of DEGs From Juvenile GOAT KO and WT Mice
Using IPA, we found several canonical pathways were affected by GOAT
deletion. The most common of these pathways were those involved in the
immune response [Complement System, interleukin (IL)-6 Signaling,
Dendritic Cell Maturation, Acute Phase Response Signaling; Figure
[170]4, Supplementary Table [171]1], similar to the results of the
pathway enrichment analysis for Reactome and KEGG pathways, as
described above.
Figure 4.
[172]Figure 4
[173]Open in a new tab
Downstream effects and upstream regulators, as predicted by the
Ingenuity Pathway Analysis (IPA) platform. (A) Top canonical pathways
affected by GOAT deletion. (B) Regulator Effects analysis, featuring
the top predicted upstream regulator, Follicle Stimulating Hormone
(FSH), its connection to the differentially expressed genes (DEGs) in
the dataset and their influence on the top biological functions, likely
to be affected by GOAT deletion.
DEGs between the GOAT KO and WT juveniles were found to be mostly
related to diseases and disorders associated with inflammation
(Inflammatory Disease, Fisher's Exact Test p-value range = 1.28E-03 to
1.81E-10, 112 molecules; Inflammatory Response, p-value range 1.20E-03
to 1.37E-09, 133 molecules) and organismal injury (Organismal Injury
and Abnormalities, p-value range 1.32E-03 to 1.81E-10, 456 molecules;
Connective Tissue Disorders, p-value range 1.00E-03 to 1.81E-10, 111
molecules), as well as to contribute to Organismal Development (p-value
range 1.30E-03 to 2.90E-07, 166 molecules). This latter biological
process included 26 functions associated with Reproductive System
Development and Function. The functions in this category with an
absolute z-score of >1 are presented in Table [174]3.
Table 3.
Top functions associated with Reproductive System Development and
Function, identified by Ingenuity Pathway Analysis platform.
Diseases or function annotation p-value Activation z-score Genes and
their direction of change # Genes
Development of genital organ 0.001 2.226
[MATH: ↓ :MATH]
Bmp7,
[MATH: ↓ :MATH]
C14orf39,
[MATH: ↑ :MATH]
Cyp11a1,
[MATH: ↑ :MATH]
Cyp19a1,
[MATH: ↓ :MATH]
Cyp26b1,
[MATH: ↑ :MATH]
Dnah9,
[MATH: ↓ :MATH]
Dnajc19,
[MATH: ↑ :MATH]
Dnd1,
[MATH: ↑ :MATH]
Fads2,
[MATH: ↑ :MATH]
Fkbp6,
[MATH: ↑ :MATH]
Fshr,
[MATH: ↑ :MATH]
Fst,
[MATH: ↓ :MATH]
H3f3a,
[MATH: ↑ :MATH]
Inhba,
[MATH: ↑ :MATH]
Inhbb,
[MATH: ↑ :MATH]
Mael,
[MATH: ↑ :MATH]
Mcmdc2,
[MATH: ↑ :MATH]
Mov10l1,
[MATH: ↓ :MATH]
Ngf,
[MATH: ↑ :MATH]
Nppc,
[MATH: ↑ :MATH]
Piwil2,
[MATH: ↓ :MATH]
Serpina5,
[MATH: ↑ :MATH]
Smc1b,
[MATH: ↑ :MATH]
Sohlh1,
[MATH: ↓ :MATH]
Sox9,
[MATH: ↓ :MATH]
Stra6,
[MATH: ↑ :MATH]
Tarbp2,
[MATH: ↑ :MATH]
Tbpl2,
[MATH: ↑ :MATH]
Tk1,
[MATH: ↑ :MATH]
Ubb,
[MATH: ↓ :MATH]
Wt1,
[MATH: ↓ :MATH]
Zmynd15 32
Development of female reproductive tract 0.001 2.113
[MATH: ↑ :MATH]
Cyp11a1,
[MATH: ↑ :MATH]
Cyp19A1,
[MATH: ↑ :MATH]
Fads2,
[MATH: ↑ :MATH]
Fshr,
[MATH: ↑ :MATH]
Fst,
[MATH: ↑ :MATH]
Inhba,
[MATH: ↑ :MATH]
Inhbb,
[MATH: ↓ :MATH]
Ngf,
[MATH: ↑ :MATH]
Nppc,
[MATH: ↑ :MATH]
Sohlh1,
[MATH: ↓ :MATH]
Stra6,
[MATH: ↑ :MATH]
Tbpl2,
[MATH: ↑ :MATH]
Ubb 13
Quantity of antral follicle < 0.001 1.342
[MATH: ↓ :MATH]
Bmp7,
[MATH: ↑ :MATH]
Cyp19A1,
[MATH: ↑ :MATH]
Inhba,
[MATH: ↓ :MATH]
Ngf,
[MATH: ↓ :MATH]
Ngfr 5
Fertility < 0.001 1.280
[MATH: ↑ :MATH]
Ctfr,
[MATH: ↓ :MATH]
Chdh,
[MATH: ↑ :MATH]
Cyp19a1,
[MATH: ↓ :MATH]
Dio3,
[MATH: ↑ :MATH]
Dppa3,
[MATH: ↑ :MATH]
Fads2,
[MATH: ↑ :MATH]
Fshr,
[MATH: ↓ :MATH]
H3f3a,
[MATH: ↓ :MATH]
H2-Ab1,
[MATH: ↑ :MATH]
Hsd17b1,
[MATH: ↑ :MATH]
Inhba,
[MATH: ↑ :MATH]
Inhbb,
[MATH: ↑ :MATH]
Khdc3l,
[MATH: ↓ :MATH]
Kmt2c,
[MATH: ↑ :MATH]
Pip5k1b,
[MATH: ↓ :MATH]
Rarg,
[MATH: ↑ :MATH]
Scara5,
[MATH: ↓ :MATH]
Serpina5,
[MATH: ↑ :MATH]
Spink13,
[MATH: ↑ :MATH]
Srd5a1,
[MATH: ↑ :MATH]
Stc1,
[MATH: ↑ :MATH]
Tk1,
[MATH: ↓ :MATH]
Wt1 23
Quantity of gonad < 0.001 1.173
[MATH: ↓ :MATH]
Bmp7,
[MATH: ↑ :MATH]
Cdkn2d,
[MATH: ↑ :MATH]
Cftr,
[MATH: ↑ :MATH]
Cyp19a1,
[MATH: ↓ :MATH]
Cyp26b1,
[MATH: ↑ :MATH]
Fads2,
[MATH: ↑ :MATH]
Fshr,
[MATH: ↑ :MATH]
Hsd17b1,
[MATH: ↑ :MATH]
Igf2,
[MATH: ↓ :MATH]
Il6r,
[MATH: ↑ :MATH]
Inhba,
[MATH: ↑ :MATH]
Inhbb,
[MATH: ↑ :MATH]
Itpa,
[MATH: ↓ :MATH]
Ngf,
[MATH: ↓ :MATH]
Wt1 16
Quantity of ovarian follicle < 0.001 1.122
[MATH: ↓ :MATH]
Bmp7,
[MATH: ↑ :MATH]
Cftr,
[MATH: ↑ :MATH]
Cyp19a1,
[MATH: ↑ :MATH]
Fads2,
[MATH: ↑ :MATH]
Fshr,
[MATH: ↑ :MATH]
Hsd17b1,
[MATH: ↑ :MATH]
Inhba,
[MATH: ↑ :MATH]
Inhbb,
[MATH: ↓ :MATH]
Ngf,
[MATH: ↓ :MATH]
Ngfr 10
Estrous cycle 0.001 1
[MATH: ↑ :MATH]
C3,
[MATH: ↑ :MATH]
Cftr,
[MATH: ↑ :MATH]
Cyp19a1,
[MATH: ↑ :MATH]
Fads2,
[MATH: ↑ :MATH]
Fshr,
[MATH: ↑ :MATH]
Inhba,
[MATH: ↓ :MATH]
Ngf,
[MATH: ↓ :MATH]
Ngfr 8
Quantity of corpus luteum < 0.001 0.896
[MATH: ↑ :MATH]
Cftr,
[MATH: ↑ :MATH]
Cyp19a1,
[MATH: ↑ :MATH]
Fshr,
[MATH: ↑ :MATH]
Hsd17b1,
[MATH: ↑ :MATH]
Inhba,
[MATH: ↑ :MATH]
Inhbb 6
Quantity of primary ovarian follicle < 0.001 −1
[MATH: ↓ :MATH]
Bmp7,
[MATH: ↑ :MATH]
Cyp19a1,
[MATH: ↑ :MATH]
Fshr,
[MATH: ↓ :MATH]
Ngf 4
[175]Open in a new tab
The activation z-score makes predictions by using information about the
direction of gene regulation in the dataset. Positive z-score predicts
an increase in the biological process or disease, while a negative
z-score predicts a decrease (inhibition). −2 ≥ z-score ≥ 2 indicates a
significant change. A red up arrow indicates an increase in the
dataset; A green down arrow indicates a decrease in the dataset. p <
0.05 indicate a statistically significant, non-random association
between a set of genes in the dataset and a related function.
To gain further insight into the biological impact of DEGs in the
dataset, we performed a Regulator Effects analysis. The Regulator
Effects algorithm connects potential upstream regulators with DEGs in
and downstream functions that are affected in the dataset. This
algorithm thus aims to provide a hypothesis that may explain how an
upstream regulator affects the downstream gene expression and the
impact of this activation or inhibition on biological functions and
diseases ([176]64). Upstream regulators were limited to genes, RNAs and
proteins, while the diseases and functions category was limited to
include the previously identified diseases and disorders associated
with inflammation and organismal injury and development (Table [177]3).
A cut-off setting of p < 0.05 and an absolute z-score of > 2 were
applied. The analysis identified $10 potential regulators. FSH was
identified as the main potential regulator of several genes that are
likely to be involved in the development of female reproductive tract
and development of genital organ, the top biological functions that are
likely to be affected by GOAT deletion (See Figure [178]4B). This
networks summary is presented in Table [179]4.
Table 4.
Regulator Effects networks, identified by Ingenuity Pathway Analysis
platform.
Regulators Consistency score Target genes in the dataset Diseases and
functions Predicted relationship Known regulator/disease relationship
FSH 3
[MATH: ↑ :MATH]
Cyp11a1,
[MATH: ↑ :MATH]
Cyp19a1,
[MATH: ↑ :MATH]
Fshr,
[MATH: ↑ :MATH]
Fst,
[MATH: ↑ :MATH]
Inhba,
[MATH: ↑ :MATH]
Inhbb,
[MATH: ↑ :MATH]
Nppc,
[MATH: ↑ :MATH]
Tk1,
[MATH: ↓ :MATH]
Wt1 Development of female reproductive tract; Development of genital
organ Activation 2/2
[MATH: ↓ :MATH]
ELF4 1.155
[MATH: ↓ :MATH]
Cdkn1a,
[MATH: ↓ :MATH]
Cxcl2,
[MATH: ↓ :MATH]
Spp1 Cell movement of phagocytes Inhibition 0/1
[MATH: ↑ :MATH]
STAT1 −4.536
[MATH: ↓ :MATH]
Angpt2,
[MATH: ↓ :MATH]
Apoe,
[MATH: ↑ :MATH]
C3,
[MATH: ↓ :MATH]
Cdkn1a,
[MATH: ↓ :MATH]
Cxcl2,
[MATH: ↓ :MATH]
Edn1,
[MATH: ↓ :MATH]
Serpina3 Cell movement of phagocytes Inhibition 1/1
TNF −6.791
[MATH: ↓ :MATH]
Apoe,
[MATH: ↓ :MATH]
Bmper,
[MATH: ↓ :MATH]
Cdkn11a,
[MATH: ↑ :MATH]
Col1a1,
[MATH: ↓ :MATH]
Cyp26b1,
[MATH: ↓ :MATH]
Edn1,
[MATH: ↑ :MATH]
Frzb,
[MATH: ↑ :MATH]
Fst,
[MATH: ↓ :MATH]
Mndal,
[MATH: ↑ :MATH]
Igf2,
[MATH: ↑ :MATH]
Naglu,
[MATH: ↓ :MATH]
Ngfr,
[MATH: ↓ :MATH]
Sox9,
[MATH: ↑ :MATH]
Th,
[MATH: ↑ :MATH]
Thbs2,
[MATH: ↑ :MATH]
Tnfrsf1b,
[MATH: ↑ :MATH]
Vegfc Development of sensory organ Activation 1/1
IL1A −7.506
[MATH: ↓ :MATH]
Bcl3,
[MATH: ↑ :MATH]
Il18,
[MATH: ↑ :MATH]
Tac1 Inflammation of the limb Activation 0/1
CSF2 −8.89
[MATH: ↑ :MATH]
C3,
[MATH: ↓ :MATH]
Cd74,
[MATH: ↓ :MATH]
Cdkn1a,
[MATH: ↓ :MATH]
Cxcl2,
[MATH: ↓ :MATH]
Edn1,
[MATH: ↑ :MATH]
Inhba,
[MATH: ↓ :MATH]
Spp1,
[MATH: ↑ :MATH]
Tnfrsf1b Cell movement of phagocytes Inhibition 1/1
EPO −11.023
[MATH: ↓ :MATH]
Angpt2,
[MATH: ↓ :MATH]
Cdkn1a,
[MATH: ↓ :MATH]
Edn1,
[MATH: ↑ :MATH]
Il18,
[MATH: ↓ :MATH]
Spp1,
[MATH: ↑ :MATH]
Vegfc Cell movement of phagocytes Inhibition 1/1
LIF −11.431
[MATH: ↓ :MATH]
Cdkn1a,
[MATH: ↓ :MATH]
Cxcl2,
[MATH: ↑ :MATH]
Cyp19a1,
[MATH: ↓ :MATH]
Serpina3,
[MATH: ↓ :MATH]
Spp1,
[MATH: ↑ :MATH]
Tac1 Cell movement of phagocytes Inhibition 0/1
IFNα −11.839
[MATH: ↑ :MATH]
C3,
[MATH: ↓ :MATH]
Cdkn11a,
[MATH: ↓ :MATH]
Cxcl2,
[MATH: ↓ :MATH]
Il17ra,
[MATH: ↓ :MATH]
Il6r,
[MATH: ↑ :MATH]
Mmp28 Cell movement of phagocytes Inhibition 1/1
IFNγ −14.500
[MATH: ↓ :MATH]
Bcl3,
[MATH: ↑ :MATH]
Il18,
[MATH: ↓ :MATH]
Itgb2,
[MATH: ↑ :MATH]
Tac1 Inflammation of the limb Activation 1/1
[180]Open in a new tab
The networks are scaled by a consistency score, a measure of how
causally consistent and densely connected a network is. A red up arrow
indicates an increase in the dataset; A green down arrow indicates a
decrease in the dataset.
qRT-PCR Analysis of Key DEGs in Juvenile GOAT KO Mouse Ovaries
We next performed a qRT-PCR assessment of the top DEGs in juvenile WT
and GOAT KO mice, to more specifically identify individual genes that
might be influenced by the absence of GOAT. While expression of certain
ovarian genes can be influenced by estrous cycle stage, cyclicity was
not controlled in the study to avoid additional handling and stress
associated with vaginal smearing, and the clear role for ghrelin in
regulation of the stress response ([181]106). Gene expression analysis
confirmed the absence of Mboat4 transcript in the juvenile GOAT KO
mouse ovaries, in which no amplification was observed (data not shown).
In the juvenile ovary, there was no significant correlation between the
RNA sequencing and RT^2 PCR array in the fold changes of the six top
under-expressed genes assessed. This absence of correlation was
specifically due to a different direction of change in the expression
of Myh6, which was under-expressed in the RNA sequencing and
over-expressed in the RT^2 PCR array (Table [182]5). Leprotl1, a gene
closely involved in the negative regulation of growth hormone (GH)
receptor expression and intracellular protein trafficking ([183]86),
was under-expressed in both the RNA sequencing and the RT^2 PCR array.
Table 5.
RT^2-PCR array gene expression in juvenile and adult GOAT KO mice.
Gene symbol Gene name NCBI Juvenile ovary fold change (p-value) Adult
ovary fold change (p-value)
Grik3 Glutamate receptor, ionotropic, kainate 3 2.7 ([184]^*0.01) 1.30
(0.14)
Spocd1 SPOC domain containing 1 2.6 ([185]^*0.01) 0.74 (0.36)
Grem1 Gremlin 1, DAN family BMP antagonist 1 (0.97) 1.67 (0.53)
Cyp19a1 Cytochrome P450, family 19, subfamily a, polypeptide 1 0.6
(0.07) 0.81 (0.76)
Inhba Inhibin, beta A 0.8 (0.24) 0.9 (0.81)
Hspb7 Heat shock protein family B (small) member 7 1.1 (0.63) 2.85
(0.33)
Sohlh1 Spermatogenesis and oogenesis specific basic helix-loop-helix 1
1.7 ([186]^*0.05) 0.91 (0.66)
Drd4 Dopamine receptor D4 0.6 (0.14) 1.11 (0.73)
Leprotl1 Leptin receptor overlapping transcript-like 1 0.8
([187]^*0.02) 0.73 ([188]^*0.04)
Dcdc2b Doublecortin domain containing 2b 1 (0.90) 1.08 (0.68)
Cited4 Cbp/p300-interacting transactivator, with Glu/Asp-rich
carboxy-terminal domain, 4 0.8 (0.45) 0.97 (0.93)
Rab3b RAB3B, member RAS oncogene family 1 (0.92) 0.35 ([189]^*0.03)
Rab6b RAB6B, member RAS oncogene family 0.9 (0.63) 2.02 (0.09)
Myh6 Myosin, heavy polypeptide 6, cardiac muscle, alpha 3.5 ([190]^*
0.01) 0.35 (0.14)
[191]Open in a new tab
Gene expression changes in GOAT KO mice ovaries relative to WT
controls.
^*
p < 0.05.
There was a significant positive correlation between RNA sequencing and
RT^2 PCR array fold changes of the top over-expressed genes (r = 0.66,
p = 0.04). This was driven principally by significant increases in
Grik3, a glutamate ionotropic receptor encoding gene and an excitatory
target in the ovary ([192]66, [193]107), and Spocd1, a gene involved in
negative regulation of phosphatase activity ([194]68, [195]69).
Effects of GOAT on the Mature Ovary and the Capacity to Breed
Since GOAT KO mice had a pronounced reduction in the number of ovarian
follicles (per mm^3 of ovarian tissue) as juveniles, coupled with
changes in genes and gene pathways closely involved in the regulation
of reproductive development and function, we next examined if these
effects were likely to be carried through into adulthood and if the
breeding capacity of these mice was likely to be altered.
The reduction in the number of follicles seen in juvenile GOAT KO mice
persisted into adulthood, with significantly reduced numbers of
primordial [t[(]9) = 3.46, p = 0.007, Figure [196]5A; expressed per
mm^3] and primary follicles [t[(]9) = 2.39, p = 0.04, Figure [197]5A;
expressed per mm^3]. There were, again, no differences in the
populations of large healthy and atretic follicles (Figures [198]5B,C;
expressed per mm^3), and no effect of GOAT deletion on apoptosis
(TUNEL) or proliferation (PCNA) markers able to detect changes
primarily in large follicles (Figures [199]5D,E). Of all the top 10
over- and under-expressed DEGs identified in GOAT KO juvenile ovaries
by RNA sequencing, only two were also altered in adult GOAT KO ovaries:
Rab3b, involved in exocytosis ([200]93, [201]94), and Leprotl1, which
was also under-expressed in the juvenile phase and is involved in the
regulation of GH action ([202]86, [203]87).
Figure 5.
[204]Figure 5
[205]Open in a new tab
Effects of GOAT deletion on adult ovarian follicles, reproductive
development and breeding capacity. (A) Small follicle; (B) Large
follicle; and (C) Atretic follicle counts in the ovary of adult GOAT KO
and WT mice. Follicle numbers are expressed per mm^3 of ovarian tissue.
(D) Number of TUNEL-positive follicles in the ovary of adult GOAT KO
and WT mice. (E) Normalized mean fluorescence intensity of
proliferating cell nuclear antigen (PCNA) staining in adult GOAT KO and
WT ovaries. (F) Day of vaginal opening. (G) Mean number of pups born in
the first litter. (H) Mean number of pups born in all litters to GOAT
KO and WT mice. Data are expressed as mean ± SEM. *p < 0.05.
Despite these remaining subtle effects of GOAT deletion on the mature
ovary, the age at puberty onset was not affected in these mice (Figure
[206]5F). Encouragingly, there were also no differences between GOAT KO
and WT mice in the number of pups born in the dam's first litter
(Figure [207]5G) or in all litters (data not shown), suggesting
pregnancy was not compromised. There were also no differences in the
numbers of litters produced by continuously mated dams within a 3–5
month period (Figure [208]5H) or in the time between litters (data not
shown). These data suggest that despite acyl ghrelin's regulatory role
in reproductive development and pregnancy ([209]31, [210]33, [211]34),
it is not necessary for successful reproduction, at least not under
institutional breeding facility conditions during the peak of the
reproductive lifespan.
Discussion
Acylated and des-acylated ghrelin peptides regulate multiple
physiological functions, including reproduction [reviewed in
([212]18)]. While the function of des-acyl ghrelin is not yet fully
elucidated, and its receptor is currently unknown, there is now
substantial evidence to support its independent role in a number of
physiological conditions ([213]9, [214]10, [215]24, [216]108). Here, we
show that genetic deletion of GOAT, an enzyme responsible for the
acylation of ghrelin that thus leads to an absence of acyl and a
chronic increase in the levels of des-acyl ghrelin, resulted in
long-term changes in ovarian morphology, as well as changes in gene
pathways associated with reproductive development and function. These
changes were not reflected in the reproductive maturation timeline or
breeding capacity, suggesting that while GOAT KO mice do not have an
overt reproductive phenotype, some of their underlying biological
functionality is notably different from that in WT mice. These findings
therefore have important implications for future studies employing this
global knockout model, as well as for the greater understanding of
ghrelin's role in reproductive development. These data may also
indicate a degree of functional redundancy within the ghrelin system to
ensure reproductive success, similar to a substantial functional
redundancy that exists within the gonadotropin-releasing hormone (GnRH)
neuronal population, where the presence of only 12% of GnRH neurons is
sufficient for pulsatile gonadotropin release and puberty onset, and
12% to 34% are sufficient for the control of estrous cyclicity in the
mouse ([217]109). It may therefore be possible that while the presence
of GOAT and acyl ghrelin at certain levels is essential for optimal
development of the ovary, these may not be essential in maintaining
fertility. It is also important to consider that in GOAT KO mice a
certain compensation may occur in the context of life-long absence of
acyl ghrelin. These mice have significantly elevated levels of des-acyl
ghrelin, compared to WT controls ([218]37). While the receptor for
des-acyl ghrelin remains to be discovered, it is now acknowledged to
have an independent bioactivity, that alternatively counteracts or
mimics the actions of acyl ghrelin. As such, des-acyl ghrelin has been
shown to reduce the levels of acyl ghrelin ([219]24) and to normalize
acyl-ghrelin induced changes in insulin and glucose levels ([220]29),
while it does not affect acyl ghrelin-induced GH, prolactin or
adrenocorticotropic hormone production ([221]29, [222]30). It does,
however, mimic the inhibitory effects of acyl ghrelin on luteinising
hormone (LH) release ([223]30). It is plausible that at least to some
degree the elevated des-acyl ghrelin levels in GOAT KO mice exert
compensatory effects driven by the absence of acyl ghrelin, through
GHSR-independent pathways.
Our characterization of the ovarian transcriptome in juvenile mice
revealed that although the number of DEGs between the two genotypes
represented a relatively small subset of genes, these genes were
associated with several biological processes and functions regulating
reproductive development. As such, Grem1, Cyp19a1, Inhba, and Sohlh1,
that were among the top ten upregulated transcripts, play a critical
role in folliculogenesis. Grem1, expressed in the granulosa cells of
developing follicles ([224]110), regulates primordial to primary
follicle transition, by antagonizing the members of the bone
morphogenetic protein (BMP) family ([225]111), such as the
anti-Müllerian hormone (AMH) that controls the activation of primordial
follicles into the growing follicle pool ([226]71). Sohlh1, that is
required for oogenesis, is also expressed in postnatal ovary where it
is confined to primordial oocytes ([227]81, [228]112). Sohlh1
expression, together with other oocyte-specific transcription factors,
is essential for primordial follicle activation ([229]113), with its
absence leading to follicular arrest ([230]81). Inhba expression is
associated with follicular growth, regulating cell proliferation and
FSH action in the ovary ([231]76, [232]105, [233]114, [234]115).
Together with an aromatase-encoding gene, Cyp19a1, that was also
significantly upregulated in the ovaries of GOAT KOs, these transcripts
modulate endocrine signaling ([235]116).
Overall, increased expression of the above transcripts suggests the
GOAT KO juvenile ovary may exhibit advanced follicle maturation,
growth, and recruitment of primordial follicles into the growing pool.
When we assessed the numbers of ovarian follicles (per mm^3 of ovarian
tissue), we saw a significant reduction in the presence of small
follicles (primordial and early primary in juveniles, and primordial
and primary follicles in adults). Secondary and antral were not
affected by GOAT deletion at any age and this was further confirmed by
the absence of follicular atresia, apoptosis, and proliferation in
these follicle populations. These data suggest that by three weeks of
age the number of primordial follicles (at least when expressed per
mm^3 of ovarian tissue) are already significantly reduced in GOAT KO
mice, but these primordial follicles are not excessively recruited to
grow, at least not at this age. It remains to be established whether
the reduction in the number of primordial follicles in these mice is
driven by a reduction in the number of embryonic germ cells; by
excessive apoptosis during the mitotic-meiotic transition [embryonic
days (E) 13.5–15.5]; or during the nest breakdown and primordial
follicle pool formation (E17.5-P5), typically associated with a
significant wave of germ cell loss and oocyte death [reviewed in
([236]117)]; or whether this occurs later during postnatal development.
It would be also of interest to examine, in future studies, if any
alterations in gonadal development are also evident in male GOAT KO
mice. Nevertheless, the decline in the small follicle populations in
GOAT KO mice (per mm^3 of ovarian tissue), particularly primordial
follicles, is possibly indicative of an accelerated exhaustion of the
ovarian reserve and a shortened reproductive lifespan ([237]118). While
this also remains to be explored in future studies, the reduction in
the number of the primordial follicles was not associated with changes
in reproductive development and function. We found no differences in
the onset of puberty in GOAT KOs, as well as no changes to the
reproductive capacity of these mice, as also noted in initial studies
using this global knockout model ([238]37, [239]40). It is important to
note, however, that our assessments were conducted under standard
non-stressed laboratory housing conditions and that the mice were not
assessed into the period of expected reproductive senescence. We have
previously shown that GOAT KO mice are more anxious than WT animals,
under stressed conditions ([240]10). GOAT is also essential for
survival in a calorie-restricted environment ([241]37). It therefore
remains to be established whether the reproductive capacity of GOAT KOs
is affected in a suboptimal environment, and how the increased
depletion of the primordial follicles affects the timing of cessation
of the reproductive lifespan.
In addition to differences in ovary specific genes and processes in
juvenile GOAT KO mice, we observed significant changes in genes
contributing to cell signaling and immune pathways, as identified by
the pathway enrichment analysis, using the Reactome and KEGG databases,
as well as by the IPA platform. For instance, two top enriched
canonical pathways included EIF2 Signaling and Complement System. eIF2
(eukaryotic initiation factor-2) initiates protein translation and
synthesis in ribosomes. Phosphorylation of eIF2 is among the first
steps in response to cellular stress and apoptosis ([242]119), and the
EIF2 Signaling pathway is significantly enriched in human primordial
oocytes during the transition from primordial to primary stage
([243]120). The complement system integrates the interaction between
the innate and adaptive immune responses, and its major role is the
clearance of immune complexes and apoptotic cells ([244]121). An
upregulation of the Complement System pathway in xenobiotic-treated
neonatal mouse ovaries has been suggested to underlie
xenobiotic-induced ovotoxicity and primordial follicle apoptosis
([245]122), and may be associated with the reduction in the numbers of
small follicles in the GOAT KOs in our study.
Our use of the IPA platform, in addition to the Reactome and KEGG
databases of pathway enrichment analysis, allowed us to make
predictions for what potential upstream regulators may be modulating
the DEGs in the juvenile GOAT KO ovaries, and what downstream
biological functions they affect. In this analysis we focused on
downstream functions associated with inflammatory diseases and
disorders, as well as functions associated with organismal development,
as we identified these to be most reflective of the changes in our
dataset. As the result of this analysis, FSH was predicted as the main
upstream regulator, affecting the expression of several genes in the
dataset (Wt1, Tk1, Cyp11a1, Fst, Cyp19a1, Fshr, Nppc, Inhba, Inhbb),
subsequently driving the development of genital organ and the
development of female reproductive tract, the top biological functions
associated with Reproductive System Development and Function, as
identified by the IPA platform in our dataset. The overall predicted
activation state of these downstream biological functions is once again
suggestive of premature ovarian development in the GOAT KOs, which may
be the cause of the significant reduction in the number of primordial
follicles (per mm^3) in these mice. The number of primordial follicles
is a major predictor of the female reproductive lifespan. In the
mammalian ovary, the vast majority of germ cells are lost before the
primordial follicle formation. In the mouse ovary, the establishment of
primordial follicle pool is completed by P7 ([246]123). By P19,
approximately half of these follicles are already depleted and in the
post-pubertal ovary at P45, only a third of the initial population of
primordial follicles are left ([247]124). After this phenomenal loss,
only a small proportion of primordial follicles will be recruited into
the growing follicle pool and reach ovulation, while the remainder of
the primordial follicle pool continues to gradually decline during the
period of sexual maturity, until only ~4% of the primordial follicle
population is left at 12 months of age ([248]124). The period of
pubertal development therefore represents an important milestone of
primordial follicle depletion. This extensive depletion appears to be
gonadotropin driven, and while the exact mechanisms are unknown,
pubertal increases in the levels of FSH and LH that drive
folliculogenesis are also likely to indirectly drive primordial
follicle depletion, since GnRH antagonism during the peri-pubertal
period prevents the significant primordial follicle loss that typically
occurs during this time ([249]103, [250]125, [251]126). Our sequencing
data from the juvenile ovary did not reveal changes in the expression
of BCL-2 modifying factor (BMF), which has recently been identified as
a critical promoter of fetal oocyte and prepubertal primordial follicle
loss ([252]103, [253]127). However, differential expression of genes
driving reproductive development and their potential regulation by FSH,
as indicated by our upstream regulator analysis, warrant investigation
of the quantity and quality of the follicle pool in the fetal and early
postnatal GOAT KO ovary in future studies. Importantly, mutations in
several genes that had significant contribution to the pathway analyses
in our study, such as inhibin genes, including INHBA, as well as SOHLH1
and FSHR are associated with premature ovarian failure in humans, and
these genes are among the potential candidate genes responsible for
this condition ([254]128–[255]130).
In summary, here, for the first time, we have characterized the ovarian
gene and follicle profiles, as well as the reproductive potential of
female GOAT KO mice, a model that through a genetic deletion of GOAT
results in an absence of circulating acyl and high levels of des-acyl
ghrelin ([256]37). Our findings indicate that while the ovarian
transcriptome and follicles in these animals are affected by the global
deletion of GOAT, their reproductive capacity is unchanged. Although
global gene knockout may induce widespread developmental effects, these
data suggest that while a presence of acyl ghrelin supports ovarian
development, as is the case in WT mice, its absence is not detrimental
for successful reproduction. Our data also suggest that substantial
reduction in ovarian follicle numbers (per mm^3 of ovarian tissue) can
be sustained without overt detrimental effects on the ability to
reproduce at least not during the peak of the reproductive capacity.
Author Contributions
LS, ZA, and SS conceived of and designed the work. LS, JG, ZA, and SS
(i.e., all authors) made substantial contributions to the acquisition,
analysis and interpretation of data. LS and SS wrote the manuscript.
All authors critically revised it for important intellectual content.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.
Footnotes
Funding. LS is a recipient of an RMIT Vice-Chancellor's Postdoctoral
Fellowship. JG was a recipient of a funding from Conselho Nacional de
Desenvolvimento Cientifico e Tecnologico-CNPq Brazil. ZA is supported
by a Career Development Fellowship II from the National Health and
Medical Research Council of Australia to ZA (APP1084344). SS is a
recipient of a National Health and Medical Research Council Career
Development Fellowship II (APP1128646) and was also supported by a
Brain Foundation Research Gift.
Supplementary Material
The Supplementary Material for this article can be found online at:
[257]https://www.frontiersin.org/articles/10.3389/fendo.2018.00815/full
#supplementary-material
[258]Click here for additional data file.^ (60.3KB, pdf)
References