Attenuated expression of MTR in both prenatally androgenized mice and women with the hyperandrogenic phenotype of PCOS
Attenuated expression of MTR in both prenatally androgenized mice and women with the hyperandrogenic phenotype of PCOS
Lei Lei 0 1 2
Lijun Ding 0 2
Jing Su 0 2
Mengyuan Liu 0 2
Qingqing Shi 0 2
Jianjun Zhou 0 2
Haixiang Sun 0 1 2
Guijun Yan 0 2
0 Funding: This study was funded by the National Nature Science Foundation of China (81590391
1 Center for Reproductive Medicine, Drum Tower Clinic Medical College of Nanjing Medical University , Nanjing, Jiangsu , China , 2 Department of Obstetrics and Gynecology, Nanjing First Hospital, Nanjing Medical University , Nanjing, Jiangsu , China , 3 Center for Reproductive Medicine, Department of Obstetrics and Gynecology, the Affiliated Drum Tower Hospital of Nanjing University Medical School , Nanjing, Jiangsu , China
2 Editor: Meijia Zhang, China Agricultural University , CHINA
Polycystic ovary syndrome (PCOS) is a common endocrine, metabolic and heterogeneous disorder in women of reproductive age, the exact etiology of which remains unknown. To unravel the molecular mechanisms underlying the hyperandrogenic phenotype of PCOS, prenatally androgenized (PNA) mice were used to mimic this phenotype in women with PCOS. Using microarray analysis, 1188 differentially expressed genes, including 671 upregulated and 517 downregulated genes, were identified in ovaries from PNA mice. Five differentially expressed genes (Aldh1a7, Bhmt, Mtr, Nrcam, Ptprg) were validated, and decreased MTR expression was shown in ovaries of PNA mice. In addition, results from qRT-PCR showed decreased MTR expression in granulosa cells (GCs) from women with the hyperandrogenic phenotype of PCOS. Serum levels of S-adenosyl methionine (SAM), the downstream product of MTR, were also decreased in PNA mice and women with the hyperandrogenic phenotype of PCOS. Our study provides evidence that the hyperandrogenic phenotype of PCOS is linked to abnormal folate one-carbon metabolism.
Polycystic ovary syndrome (PCOS) is one of the most common endocrine and metabolic
disorders, affecting about 5%-15% of women of reproductive age [
]. Symptoms of PCOS include
amenorrhea or oligomenorrhea, hyperandrogenism, and polycystic ovarian morphology. As a
heterogeneous disorder, PCOS shows evidence of a genetic predisposition among patients, but
the exact etiology remains unknown [
Previous studies have been conducted on many candidate genes for PCOS, principally
related to reproductive hormones, insulin resistance, and chronic inflammation, including
follicle-stimulating hormone receptor (FSHR) [
], insulin receptor (INSR) [
], and tumor
necrosis factor (TNF) [
]. Microarraywas also used to screen for differentially expressed genes in
31590189, and 30900847), the Health Department
of Jiangsu Province (LJ201102), the Natural
Science Foundation of Jiangsu Province
(BK20141087 and BL2014003), and the Nanjing
Medical Science Development Project (JQ2014004
and ZKX16042), China.
PCOS using ovaries [
], theca cells , oocytes , cumulus cells [
], and even skeletal muscle
] and adipose tissue [
]. The first genome-wide association studies (GWAS) and
subsequent follow-ups were performed in Han Chinese women, and investigators identified the
following PCOS candidate loci: DENND1A, INSR, YAP1, C9orf3, RAB5B, HMGA2, TOX3,
SUMO1P1/ZNF217, THADA, FSHR, and LHCGR [
]; DENND1A was validated by another
Unfortunately, susceptibility genes for PCOS were often controversial in nature in
previously reported studies. The controversy is partly due to ethnic differences, but different PCOS
phenotypes could also be another reason [
]. Animal models may help to investigate the
pathophysiologic mechanisms in a certain phenotype of PCOS. As an important feature of PCOS,
hyperandrogenism is also one of the diagnostic criteria for this disease, a feature distinct from
metabolic dysfunction. Therefore, to investigate the etiology of the hyperandrogenic
phenotype of PCOS, a prenatally androgenized (PNA) mouse model was validated and used for
microarray analysis. Differentially expressed genes (1188) were identified in ovaries from PNA
mice, and five of these (Aldh1a7, Bhmt, Mtr, Nrcam, Ptprg) were validated by qRT-PCR; MTR
expression was then further evaluated by western immunoblotting analysis and
immunohistochemistry. MTR is the only mammalian enzyme that metabolizes N5-MeTHF to regenerate
the active cofactor tetrahydrofolate (THF)[
]. The overall reaction converts
5-methyltetrahydrofolate (N5-MeTHF) into THF while transferring a methyl group to homocysteine(Hcy) to
form methionine(Met). Met is further used to synthesize S-Adenosyl methionine(SAM), the
main methyl donor of DNA methylation, under ATP action. Decreased MTR expression in
granulosa cells (GCs) from women with the hyperandrogenic phenotype of PCOS was also
validated by qRT-PCR. Additionally, serum levels of SAM, the downstream product of MTR,
were decreased in both PNA mice and the hyperandrogenic phenotype of women with PCOS.
The present study, therefore, provides novel basic information on the relationship between
MTR and the hyperandrogenic phenotype of PCOS.
Materials and methods
All experimental procedures were performed in accordance with the guidelines of the
Experimental Animals Management Committee (Jiangsu Province, China) and were approved
by Nanjing Drum Tower Hospital Experimental Animals Welfare &Ethical committee
(20150302).Adult ICR mice (females, 6 weeks of age, n = 50; males, 10 weeks of age, n = 10)
were purchased from the Animal Experimental Center of Yangzhou University (Jiangsu
Province, China), and housed in the Drum Tower Hospital Animal Experimental Center (Jiangsu
Province, China) at 22ÊC, on a 12 h light/12 h dark cycle with lights on at 07:00 am, and with
ad libitum access to chow and water. Females were mated with males and checked for
copulatory plugs daily. The date of the plug was considered day 1 of gestation. Pregnant dams were
injected daily s.c. with 70 μl of sesame oil containing 350 μg of DHT (521-18-6, Sigma, USA)or
sesame oil vehicle on days 16±18 of gestation, and female offspring were studied. The mice
were euthanized through anesthesia with chloral hydrate. Tissues and blood were harvested
from all animals post euthanization.
Assessment of estrous cyclicity and fertility
The body weights of PNA and control mice were recorded, starting at 21 days of age. Vaginal
smears were obtained daily in all adult mice from 2 months of age for 3 weeks or those
showing consecutive estrous cycles. The fertility of adult mice (n = 6 each group) was tested by
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mating with proven fertile ICR males (1: 1) for 3 months. The numbers of litters and pups per
litter were recorded.
Testosterone and S-adenosyl methionine measurements in mice
The mice were anesthetized with chloral hydrateon diestrus, and blood was collected from the
posterior orbital venous plexus. The blood samples were then centrifuged and the serum was
frozen at -80ÊC for hormonal analysis. The concentration of testosterone (T) was measured
using an ELISA kit (YANYU, Shanghai, China), and the serum levels of SAM were also
detected using an ELISA kit (CEG414Ge, Cloud-Clone Corp, Wuhan, China).
Ovarian histology and follicle counting
After blood was collected from mice, ovaries were removed, weighted and then fixed in
Bouin's solution. Ovarian index was calculated using ovarian weight 1000/body weight 100
(%). After fixation for 4±6 h, the samples were dehydrated and embedded in paraffin, sections
cut at 5 μm, and every fifth section was stained with hematoxylin and eosin (H&E). Follicle
counting was performed using an unbiased stereologic method. Only follicles containing an
oocyte nucleus were counted. The total number of follicles was then multiplied by five.
Follicles were classified according to the classification system in a previous report [
The microarray was conducted by Genechem Co., Ltd. (Shanghai, China). The GeneChip1
Mouse Genome 430 2.0 Array (900496, Affymetrix) was used for the experiment, and 2 groups
of samples were tested, with each group containing 5 mouse ovaries. RNA was extracted using
Trizol reagents, and qualified with a NanoDrop 2000 and Agilent Bioanalyzer 2100. The
quality control standards were as follows:1.7 < A260/A280 < 2.2 (ThermoNanoDrop 2000),
RIN 7.0 and 28S/18S > 0.7 (Agilent 2100 Bioanalyzer). The qualifying samples were entered
into the microarray experiment.
Quantitative real-time PCR (qRT-PCR)
RNA was extracted using Trizol reagents and measured with spectrometry for OD260/280.
cDNA was prepared with 5× All-In-One RT MasterMix (G490, ABM, USA) according to the
manufacturer's recommendations. Real-time PCR was performed with a SYBR-Green Mixture
(Bio-Rad, USA). Primers were searched from https://pga.mgh.harvard.edu/primerbank/. The
sequences of specific PCR primers in this study are listed in S1 Table. Primers were synthesized
by Sangon (Sangon Biotech, Shanghai, China).Each sample was run in triplicate as follows:
2 μl cDNA, 1 μl primer, 7 μldd H2O, and 10 μl SYBR Green Master Mix in a total volume of
20 μl; and 18s rRNA was measured as an internal control. The PCR procedure was as follows:
initial denaturation at 95ÊCfor 1 min followed by 40 cycles of amplification (denaturation at
95ÊC for 15 sec, annealing at 60ÊC for 1 min), and then primer template extension at 72ÊC for
3 min. Melting curve analysis was performed to confirm the specificity of amplification, and
the relative MTR level was determined using 2−ΔΔCT.
The expression of MTR was also confirmed by immunohistochemistry. Sections were baked at
65ÊC, dehydrated, and incubated in 3% H2O2. Sections were subjected to antigen retrieval by
boiling in 10 mM citrate buffer (pH 6.0), and rinsed in TBS. We added solution A (Biotin
block), washed with TBS, added solution B (Biotin block), and we again washed sections with
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TBS. Sections were incubated in normal goat serum for 1 h at 4ÊC, incubated with anti-MTR
(1:200, ab66039, Abcam, UK) in 3% BSA overnight at 4ÊC, incubated in biotinylated goat
antirabbit IgG (1:200 dilution) for 2 h at room temperature, and then washed with PBS. We added
reagent SABC (12E02A, BOSTER, Wuhan, China) and incubated sections at 37ÊC for 20 min,
washed with PBST, visualized binding with DAB, and terminated the incubation with distilled
water. Slides were stained with hematoxylin, dehydrated, and mounted. The average integrated
optical density (IOD) was measured for each sample by Image-pro Plus 6.0 (Media
Western blot(WB) analysis
Ovarian lysates from mice were separated by SDS/PAGE and transferred onto nitrocellulose
membranes. Membranes were probed with polyclonal MTR antibodies (1:2000, ab66039,
Abcam, UK), and blots were visualized by using peroxidase-conjugated second antibody and
an ECL detection kit (Amersham Pharmacia Biosciences). Western blot data were quantified
and normalized to GAPDH (1:10000, Bio-Rad, USA).
Women with the hyperandrogenic phenotype of PCOS and age-matched controls were
recruited from the Center for Reproductive Medicine, Nanjing Drum Tower Hospital, from
January to December 2016. All subjects were unrelated Han Chines eand were recruited
after providing informed written consent. The study was approved by the Medical Ethical
Committee of Nanjing Drum Tower Hospital. The diagnosis of PCOS was based upon the
2003 Rotterdam Diagnostic Criteria [
].All patients with PCOS in this study had
oligomenorrhea, hyperandrogenism, and a clear diagnosis of polycystic ovaries by ultrasonography. The
controls were fertile women undergoing IUI or IVF for male factor infertility; women with
ovarian factor or complications affecting ovulation (e.g., hypothyroidism, diabetes mellitus,
endometriosis, hyperprolactinemia) were excluded.
Hormonal and S-adenosyl methionine measurements in women
On cycle days 2 or 3, venous blood samples were collected; and serum hormone levels,
including follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), estradiol
(E2) and testosterone (T), were measured using ELISA Kits (FSH, 33520; LH, 33510; PRL,
33530; E2, 33540; T, 33560; Beckman Coulter, Inc, USA). Blood samples were also collected
and centrifuged on the day before HCG was injected, and serum was frozen at -80ÊC for SAM
analysis (CEG414Ge, Cloud-Clone Corp, Wuhan, China). Ultrasonography was performed on
cycle day 4 or 5 to measure the thickness of endometrium, count the number of antral follicles
and confirm the absence of corpus luteum.
Isolation of human granulosa cells
All women were injected with gonadotropin-releasing hormone (GnRH) agonist at the
beginning of the midluteal phase, and then ultrasonographic scans and serum estradiol assays were
performed to monitor follicular size. When 3 or more follicles with a mean diameter of 16 mm
were observed, 5000±10,000 IU human chorionic gonadotropin (HCG) was injected.
Ultrasound-guided oocyte retrieval was performed 36 hours later. The granulosa cells (GCs) around
oocytes were collected and washed twice with Dulbecco's modified Eagle's medium (DMEM)
after removal of the oocyte, and kept in TRIzol (15596±018, Invitrogen, USA) at -80ÊC for
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Statistical analysis was performed using SPSS 19.0 (IBM, USA). The means ± SD of the data
were calculated. Either Student's t-test or t’-test was used to determine the significance between
the two groups. A P value < 0.05 was considered to be statistically significant.
Hyperandrogenism disrupts estrous cyclicity, impairs fertility and increases serumtestosterone levels in PNA mice
PNA mice exhibited prolonged estrous cycles (8.79 ± 2.58 days, n = 15) compared with
controls (4.5 ± 0.46 days, n = 15) (P< 0.001),which was mainly due to the increased durations of
estrus and metestrus. An absence of proestrus was also be observed in the PNA mice (Fig 1A,
S1A and S1B Fig). Adult PNA mice had fewer litters and smaller litter sizes (1.67 ± 0.52 litters,
4.83 ± 1.25 pups/litter) compared with the controls (3.33 ± 0.52 litters, 14.65 ± 0.91 pups/litter)
during a 3- month period (P< 0.001) (S1C and S1D Fig).
To examine the endocrine factor contributing to abnormal cyclicity in PNA mice, serumT
was measured. T levels at 3 weeks of age showed no difference between the two groups (P>
0.05), but PNA mice had increased T levels at 2 months of age (253.14 ± 90.60 nmol/L, n = 21
vs. 202.79 ± 30.32 nmol/L, n = 15; P = 0.026) and 3 months of age (218.80 ± 41.05 nmol/L,
n = 21 vs.170.24 ± 23.21 nmol/L, n = 13; P< 0.001) (Fig 1B). These data suggest that PNA
treatment elevated T levels in adult PNA mice, but not before puberty. It was also found that T
Fig 1. Estrous cycle, serum testosterone levels, ovarian morphology and follicles counting of mouse.
(A) Representative estrous cycles in control (Upper) and PNA mice (Lower) (M, metestrus; E, estrus; D,
diestrus; P, proestrus). (B) T levels in diestrus mice. (C) (D) HE stained ovarian section of control mouse at 3
months. (E) (F) HE stained ovarian section of PNA mouse at 3 months. (G) Follicle counting at 3 months,
numbers represent total counting of every fifth section from serially sectioned ovaries. (H) Proportion of
follicles in each stage.(*: P< 0.05).
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levels at 3 months decreased compared with levels at 3 weeks in the control group, which was
not found in the PNA mice.
Hyperandrogenism alters ovarian morphology and folliculogenesis in
Body weight of PNA mice (13.91 ± 2.84 g, n = 18) was increased compared with the control
group (12.01 ± 2.46 g, n = 29) at 3 weeks of age (P = 0.020), but there was no difference
between the two groups after puberty (S1E Fig). Ovarian weight of the PNA mice (33.36 ± 8.61
×10−4 g, n = 18) was increased compared to the control group (26.45 ± 7.67 ×10−4 g, n = 29)
(P = 0.006), but the ovarian index was not significantly different between the two groups at 3
weeks of age. At 2 months, the ovarian weight and ovarian index of the PNA mice were both
significantly decreased compared to the control group (52.03 ± 21.04 ×10−4 g, 35.38 ± 10.60%,
n = 14 vs.77.67 ± 21.04 ×10−4 g, 50.54 ± 16.27%, n = 12; P = 0.008, P = 0.009). At 3 months of
age, there was no statistical difference in ovarian weight or index between the two groups (S1F
and S1G Fig).
Compared with the controls, ovaries in the adult PNA mice (3 months old) exhibited a
greater number of small antral follicles, fewer corpora lutea, but contained cyst-like structures
(Fig 1C±1F). Although the total number of antral follicles was increased, the number of
preovulatory follicles was decreased in the ovaries of adult PNA mice. In addition, antral follicles
of the PNA mice exhibited a thinner granulosa cell layer.
Follicle counting (n = 10 each group) showed that PNA mice possessed fewer primordial
follicles (1126 ± 352.79 vs. 2318.5 ± 491.95, P< 0.001) and more antral follicles than controls
(433 ± 200.52 vs. 204 ± 55.26, P = 0.006). There was no difference in the number of total
follicles, or in primary or secondary follicle number (P> 0.05) (Fig 1G).However, when we
analyzed the proportion of follicles at each stage, the PNA mice exhibited a lower percentage of
primordial follicles (31.92 ±2.50% vs. 57.95 ± 4.98%, P< 0.001) and a higher percentage of the
other three follicular stages (primary, 37.33 ± 2.76% vs. 24.55 ± 4.10%, P< 0.001; secondary,
18.65 ± 3.26% vs. 12.389 ± 1.85%, P< 0.001; antral,12.10 ± 2.70% vs. 5.12 ± 1.11%,P< 0.001)
(Fig 1H). These results suggested that follicles were overly activated in the PNA mice.
Novel differentially expressed genes in PNA mice
RNA extracted from ovaries was qualified using a NanoDrop 2000 and Agilent Bioanalyzer
2100 (S2 Fig). Over 39,000 transcripts were analyzed with the GeneChip1 Mouse Genome 430
2.0 Array by using the selection criteria of fold change 1.5, and a total of 671 gene transcripts
were activated while 517 others were repressed in PNA mouse ovaries (Fig 2A). Genes
involved in androgen biosynthesis and LH secretion (Hsd11b1, Hsd17b2, Hsd17b7, Cyp11a1,
Cyp17a1, Cyp19a1, Cyp2d22, Cyp39a1, Cyp4f18 and Lhcgr), ovarian function and
folliculogenesis (Pten, Amh and Nppc), and inflammation (CCL2, CCL5, CCL7, CXCL1, CXCL9, CXCL10,
and CCR7) were among those with differential expression.
The differentially expressed genes were enriched and analyzed according to the gene
information of all pathways in KEGG and BIOCARTA. The top 3 pathways were cancer,
cytokinecytokine receptor interaction, and chemokine signaling pathway; the top 3 under biological
processes were signal transduction, multicellular organismal development and anatomical
structure development; and the top 3 molecular functions were receptor binding, receptor
activity and enzyme regulator activity (S2±S4 Tables).
To validate the expression changes identified by microarray analysis, the expression levels
of 5 differentially expressed genes (Aldh1a7, Bhmt, Mtr, Nrcam, Ptprg) were assessed by
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Fig 2. Microarrayanalysis and validation. (A) Heatmap of microarray analysis. (B) -(F) Five differentially
expressed genes were validated by qRT-PCR(B, Mtr; C, Aldh1a7; D, Bhmt; E, Nrcam; F, Ptprg).(*: P< 0.05).
qRT-PCR (Fig 2B±2F). Results showed that the 5 selected genes displayed a trend similar to
that of the microarray data, thus confirming the validity of the microarray results.
By IHC, we found that MTR was mainly expressed in granulosa cells, and rarely expressed
in theca cells (Fig 3C and 3D).Subcellular localization of MTR showed the greatest expression
in the cytoplast and was not appreciably observed in the nucleus (Fig 3B±3D). The expression
of MTR in the ovaries of adult PNA mice (at 3 months of age) was significantly decreased
compared with the controls (P = 0.014; Fig 3E).
Western-blot analysis also confirmed the above results. The expression of MTR in the
ovaries of PNA mice was decreased at both 3 weeks and 3 months of age (Fig 3F and 3G).
Although we also observed for adult mice in the control group an attenuated expression of
MTR compared with mice before puberty, this change was not obvious in PNA mice (S3 Fig).
These results indicated that the expression of MTR was decreased in the ovaries of PNA
mice even prior to puberty.
Hyperandrogenism decreases serum SAM levels in PNA mice
SAM is the downstream product of MTR, and ELISA showed that serum SAM levels were
significantly decreased in PNA mice (21.41 ± 9.79 ng/ml) compared with the control group
(15.20 ± 5.62 ng/ml)(n = 21 in each group, P = 0.016; Fig 4A).
Women with PCOS have attenuated expression of MTR in GCs and lowered SAM levels in serum
Characteristics of patients are shown in Table 1. The control groups and patients showing the
hyperandrogenic phenotype of PCOS were matched for age. Serum E2 and PRL were not
significantly different between the two groups. However, BMI, Mean arterial pressure (MAP),
ovarian volume, serum LH, T and the LH/FSH ratio of the patients with PCOS were higher than the
controls (BMI, 23.38 ± 3.87 kg/m2 vs. 21.71 ± 3.07 kg/m2; MAP, 90.90 ± 11.33mmHg vs. 85.41±
9.01mmHg; ovarian volume, 8.33 ± 3.79 cm3 vs. 4.82 ± 2.04cm3; LH,10.19 IU/L ± 7.66 vs.
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Fig 3. Differentially expressed MTR in mice. (A)±(D) Immunohistochemistry. (A) Negative control (ovary).
(B) Positive control (pancreas). (C) Ovary of control mouse. (D) Ovary of PNA mouse. (E) IOD of MTR was
significantly lower in PNA mice. (*: P< 0.05). (F) WB showed expression of MTR in ovaries of PNA mice
decreased at 3 months age. (G) WB showed expression of MTR in ovaries of PNA mice decreased at 3 weeks
4.28 ± 1.61 IU/L; T, 0.67 ± 0.29 vs. 0.42 ± 0.14; LH/FSH, 1.70 ± 1.18 vs. 0.63 ± 0.23; P< 0.05).
Additionally, more oocytes were retrieved from PCOS patients (15.19 ± 5.00) compared to
controls (12.18 ± 4.19) (P< 0.001). These results showed that many physiologic differences were
typically observed between PCOS patients and controls.
The expression of MTR in GCs from women with the hyperandrogenic phenotype of PCOS
was significantly decreased compared with controls (P = 0.036; Fig 4B). Furthermore, serum
SAM levels were significantly lower in PCOS patients (50.01 ± 19.21 ng/ml) compared to
controls (68.26 ± 29.29 ng/ml) (n = 42 in each group, P = 0.001; Fig 4C).
Fig 4. Differentially expressed MTR in women, serum SAM levels in mouse and women. (A) PNA
decreases serum SAM levels in mouse. (B) Women with PCOS have low- expressed MTR in GCs. (C)
lowSAM level in serum of women with PCOS.(*: P< 0.05).
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It is exceedingly difficult to create a model that reflects all phenotypes of PCOS because of the
syndrome's heterogeneity. In the present study, we demonstrated that prenatal
androgenization of the ICR mouse with DHT can induce many characteristics of PCOS similar to those
observed in previous studies using the C57/BL6 mouse [18±21]. The PNA mouse model
exhibits disrupted estrous cyclicity, impaired fertility, elevated serum testosterone levels, and
increased numbers of small antral follicles. Using this model, we have identified novel
candidate genes for the hyperandrogenic phenotype of PCOS, including MTR. In the PNA mice and
hyperandrogenic phenotype of women with PCOS, the expression of MTR was decreased and
levels of its target, SAM, were also decreased in serum compared with the normal controls.
MTR is crucial to one-carbon metabolism and the folate one-carbon pool pathway, while
SAM is the main methyl donor of DNA methylation. Thus the present study indicates that the
hyperandrogenic phenotype of PCOS is linked to dysregulated one-carbon metabolism and
will likely affect DNA methylation. This may provide new clues in our understanding and
treatment of the hyperandrogenic phenotype of PCOS.
Our study shows that the PNA mouse is an ideal model for emulating the hyperandrogenic
phenotype of PCOS in women. Disrupted estrous cycles in PNA mice are mainly due to an
absence of proestrus, increased duration of estrus and metestrus, and even a continuous
switching between estrus and metestrus. Previous studies were controversial with respect to
the extended duration of diestrus[
], estrus , and metestrus; but an absence of
proestrus was representative of this phenotype. Disrupted estrous cyclicity indicates abnormal
ovulation in PNA mice, and this is similar to the abnormal menstrual patterns seen in patients
with PCOS. Fewer mouse litters and smaller litter sizes in the fertility test paralleled
observations for human infertility in the hyperandrongenic phenotype of PCOS.
About 70%-90% of PCOS women with hyperandrogenism were defined either clinically or
]. Previous studies indicated that in-utero androgen exposure in humans
can cause PCOS in adulthood, and PNA treatment in mice was used to mimic the process
of in-utero androgen exposure. PNA mice showed increased androgen levels in adulthood
rather than before puberty, indicating that hyperandrogenism in PCOS is due to altered
function of the hypothalamicÐpituitaryÐgonadal axis. LH hypersecretion is a common feature of
PCOS, where LH is believed to enhance ovarian androgen production. Meanwhile,
hyperandrogenism can further elevate LH level through diminishing negative feedback, forming a
]. Hyperandrongenism can also promote the development of PCOS, and affect
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hypothalamicÐpituitary function, resulting in abnormal ovarian function and folliculogenesis.
In the present study, genes involved in androgen biosynthesis and LH secretion, ovarian
function, and folliculogenesis were found to be abnormally expressed using microarray analysis.
For example, expression of Nppc was increased in PNA mice. CNP (C-type natriuretic
peptide), encoded by NPPC, is downstream of FSH in the regulation of follicle development, and
is secreted by granulosa cells of secondary and antral follicles to stimulate follicular
]. In addition, CNP is expressed in cumulus cells of antral and preovulatory follicles,
so as to suppress oocyte maturation [
]. These results are consistent with increased antral
follicle number and lack of mature follicles in PCOS. Chronic low-grade inflammation is also a
common feature of PCOS according to previous studies [
], in light of which many genes
encoding cytokines, especially chemokines, were abnormally increased in PNA mice,
suggesting that the hyperandrogenic phenotype of PCOS is potentially associated with inflammation.
It appears to be paradoxical that overly activated follicles and infertility would coincide in
PCOS, but a defect in follicle maturation is certainly a key factor. The reason for an increase in
antral follicles leading to decreased follicular maturation in PCOS may be explained by
nutritional factors. It is possible that nutrients are inadequate to support excessive antral follicle
development and maturation. Previous studies have demonstrated that PCOS patients have
altered plasma amino acid levels, including reduced Met level[
]. Met plays an important
role in the one-carbon metabolism pathway. SAM, the active form of Met, is the universal
substrate for all protein and DNA methylation reactions, and is also involved in the synthesis of
purines and pyrimidines. In this study, we found that Bhmt and Mtr, which encode enzymes
that generate Met, were downregulated.
BHMT is an important gene in the one-carbon metabolism pathway, and its
downregulation could lead to hyperhomocysteinemia[
]. BHMT was demonstrated to be upregulated in
a previous microarray analysis for androgen receptor-deficient mice at 8 weeks of age [
Consistently, the PNA mice in our study have decreased expression of BHMT and increased
In this study, we demonstrate for the first time that the expression of MTR is decreased in
both PNA mice and women with the hyperandrogenic phenotype of PCOS. A previous study
indicated that a single nucleotide polymorphism (SNP) was present in the MTR gene (MTR
2756 A>G)among south Asian women with similar frequency of minor alleles between
women with and without PCOS[
]; Despite the small sample size (N = 21:9), the study
interestingly showed that women in the control group had significantly lower hemoglobin and
even manifested mild-to-moderate anemia (91, 85±101 vs. 123,105±129); the study, however,
did not distinguish among different phenotype of PCOS [
]. In our study, we found that
MTR expressionwas decreased in PNA mice starting from 3 weeks of age, whileT levels
remained the same at 3weeks and was higher in PNA mice at 2 months of age. It appears that
the inhibited MTR expression precedes increased circulating T, indicating a potential causal
relationship between MTR and T levels. Nevertheless, further investigation is needed to
confirm the causality.
MTR is known to play a key role in the folate one-carbon pool pathway and one-carbon
metabolism pathway, and the main purpose of MTR is to regenerate Met in the SAM cycle; it
also serves an essential function by allowing the SAM cycle to perpetuate without a constant
influx of Met. The overall reaction converts 5-methyltetrahydrofolate into THF while
transferring a methyl group to Hcy to form Met, while Met is further used to synthesize SAM under
ATP action. MTR is the only mammalian enzyme that metabolizes N5-MeTHF to regenerate
the active cofactor THF [
]. Therefore, a defect in MTR would lead to
hyperhomocysteinemia, and low levels of Met and THF. Herein, we showed that serum SAM levels were
significantly lower in PNA mice and the hyperandrogenic phenotype of women with PCOS. This
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suggests that down-regulation of MTR may reduce methionine levels and result in decreased
SAM levels with PCOS.
Hcy is a non-essential amino acid formed by demethylation of Met, and previous studies
showed that patients with PCOS exhibited elevated Hcy concentrations in their serum [
and follicular fluid. Elevated Hcy levels in follicular fluid are now associated with poor-quality
oocytes and embryos in PCOS patients undergoing assisted reproduction [
investigators have demonstrated that defective homocysteine re-methylation was found in MTR
heterozygous knockout mice [
]. Our study showed that MTR was downregulated in GCs of PCOS
patients, and that Mtr and Bhmt were downregulated in ovaries of PNA mice, revealing a
reason for the elevated Hcy levels in PCOS patients.
Decreased SAM levels and hyperhomocysteinemia further indicates dysregulation of MTR
with the hyperandrogenic phenotype of PCOS. Thus replenishing SAM or MTR could
potentially benefit the hyperandrogenic phenotype of PCOS. Previous studies indicated that folate
therapy reduced serum Hcy levels in patients with coronary artery disease [
], and the same
phenomenon was observed in patients with PCOS [
]. It was also reported that folate
supplementation in patients with PCOS had beneficial effects on metabolic profiles [
], and on
inflammatory factors and biomarkers of oxidative stress [
].Furthermore, preconception folic
acid supplementation was associated with suppression of the inflammatory pathway and
upregulation of the HDL pathway in human follicular fluid[
]. It appears that folate may benefit
patients with PCOS by compensating for the low expression of MTR.
Epigenetic mechanisms serve to elucidate the etiology of PCOS in the context of specific
environmental factors before and after birth [
].Studies have shown genome-wide DNA
methylation difference between PCOS and normal ovaries . In the present study, we
demonstrated that levels of SAM, the main methyl donor of DNA methylation, were decreased
with the hyperandrogenic phenotype of PCOS. This notion may explain the abnormal
methylation patterns observed in PCOS.
It should be noted that because human ovarian tissue is extraordinarily difficult to obtain,
we used GCs to validate the data generated from the PNA mouse. Results from this study
should be interpreted with caution considering the relatively small number of samples, which
could potentially introduce bias error. This study entailed an animal model and cells from
women with the hyperandrogenic phenotype of PCOS; thus, whether these same results would
be observed in PCOS women without hyperandrogenism would certainly require further
Prenatal androgenization of the ICR mouse with DHT can replicate most of the common
clinical features of PCOS, especially the hyperandrogenic phenotype of PCOS. By using this mouse
model we demonstrate that PCOS is characterized by decreased MTR expression and serum
SAM, which can potentially explain the dysregulated DNA methylation patterns in PCOS
patients. Our evidence for the first time shows that an abnormal one-carbon metabolism
pathway is linked to the hyperandrogenic phenotype of PCOS, the causality of which will be
examined in our ongoing research.
S1 Table. Primer sequences used for qRT-PCR.
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S2 Table. Pathway enrichment of different expressed genes.
S3 Table. Biological process of different expressed genes.
S4 Table. Molecular function of different expressed genes.
S1 Fig. Estrous cycle, fertility and follicles counting of mouse. (A) PNA mice exhibited
prolonged estrous cycles. (B) Percent of days spent in each estrous cycle stage.(C) (D) PNA mice
produced fewer litters and smaller litter sizes. (F) T levels in adult diestrus mice.(E) Body
weight of mice. (F) Ovarian weight of mice. (G) Ovarian index of mice.( : P< 0.05).
S2 Fig. Sample quality control information. (A) Results summary table. (B) (C) Result of
Agilent 2100 Bioanalyzer.
S3 Fig. Different expressed MTR in 3 weeks and 3 months mice. (A) Expression of MTR in
ovaries decreased in 3 months control mice. (B) Expression of MTR in ovaries did not change
in PNA mice.
We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this
Conceptualization: Haixiang Sun.
Data curation: Lei Lei, Lijun Ding.
Formal analysis: Lei Lei, Jianjun Zhou.
Funding acquisition: Haixiang Sun.
Investigation: Lei Lei.
Methodology: Lei Lei, Lijun Ding, Jing Su.
Project administration: Haixiang Sun, Guijun Yan.
Resources: Qingqing Shi, Jianjun Zhou.
Supervision: Guijun Yan.
Validation: Lijun Ding, Mengyuan Liu.
Writing ± original draft: Lei Lei.
Writing ± review & editing: Haixiang Sun, Guijun Yan.
12 / 15
13 / 15
14 / 15
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