Introduction
Polycystic ovary syndrome (PCOS) is a complex, common endocrine condition and metabolic disease affecting reproductive aged women with a reported prevalence of 8–13% in the world, and PCOS is the main cause of primary infertility [
1]. The clinical manifestations of PCOS include hyperandrogenism (hirsutism, acne), irregular menstruation, ovulatory dysfunction, reproductive endocrine hormone disorder, insulin resistance and dyslipidemia. Dysfunction of the hypothalamic–pituitary–ovarian axis has been regarded as the main cause of PCOS [
2]. In PCOS women, the gonadotropin-releasing hormone pulse frequency is increased, which favors increased luteinizing hormone (LH) secretion over that of follicle-stimulating hormone (FSH). However, there is relative deficit in FSH secretion, which often results in impaired and arrested follicular development and reduced aromatase activity, thereby resulting in ovarian follicular atresia and hyperandrogenemia in PCOS women [
3]. In addition, high insulin levels in PCOS patients could increase the expression of LH, which could promote the secretion of androgen from the ovary and adrenal glands. The interaction between increased levels of insulin and LH can cause an increase in atresia follicles in the ovary, which may lead to development of PCOS [
4]. Diane-35 (composed of 2 mg cyproterone acetate and 35 μg ethinyl estradiol) plus metformin are the two commonly used drugs in clinical to treat PCOS, which have the advantages of improving glucose metabolism, modifying the insulin resistance state, reducing testosterone levels in serum and restoring ovulation of the ovary in patients with PCOS [
5]. However, the current research on the treatment of PCOS patients is at its preliminary stage; while the mechanism of how to improve folliculogenesis is still unclear.
There is growing evidence showing that granulosa cell energy metabolism disorder is an important cause of abnormal follicles and fertility decline in females. The main energy metabolism of normal folliculogenesis is that granulosa cells take up glucose from surrounding tissues via the glucose transporter (GLUT) on their cell membranes, then glucose producing pyruvic acid and lactic acid through the glycolysis pathway [
6,
7]. A large number of studies has shown that pyruvic acid and lactic acid are important factors in maintaining follicular growth and development [
8]. Harris et al. found that follicles in patients with PCOS require more pyruvate to maintain follicle growth than normal female [
9]. In vitro results show that testosterone and insulin stimulation can reduce lactic acid content in granulosa cells [
10]. The above studies indicate that the follicles of patients with PCOS require more pyruvate and lactic acid to stimulate follicle to develop normally on the one hand, and on the other hand, high androgen and insulin reduce the content of lactic acid. However, whether Diane-35 plus metformin treatment could restore the dysfunction of granulosa cell energy metabolism in PCOS development remain elusive.
In this study, we investigated the effects of Diane-35 combined with metformin treatment on ovulation in a PCOS rat model. Further studies were performed to determine the effects of the combined treatments on energy metabolism in the ovary. In addition, the ovarian glycolysis-related rate-limiting enzymes and sirtuin 1 (SIRT1) expression was determined to elucidate the mechanistic actions of the combined treatment in restoring the dysfunction of energy metabolism in the PCOS ovary. The present study could provide a new insight into understanding the mechanistic actions underlying therapeutic effects of Diane-35 combined with metformin treatment on PCOS.
Materials and methods
Animals and treatments
A total of 40 female Sprague Dawley (SD) rats (5 weeks old and 130–140 g) were purchased from the Laboratory Animal Center of the Third Military Medical University (Chongqing, China). The animals were randomly divided into a control group (10 animals) and a PCOS group (30 animals). The PCOS group was intragastric administration with letrozole [
11] (Heng Rui Pharmaceutical Factory, Lianyungang, Jiangsu, China) dissolved in 1% (w/w) CMC and fed with high fat diet. Vaginal smear of SD rats was monitored after 1 week of modeling, and the establishment of the model was preliminarily judged to be successful or not according to the estrous cycle. Two rats with successful initial model were randomly selected for ovarian morphology and serum reproductive hormone analysis for verification. We divided SD rats successfully modeled into PCOS group (10 animals) and PCOS + Diane-35 plus metformin (DM) group (10 animals). The PCOS + DM group was treated as follows: Diane-35 and metformin were intragastric administration to SD rats up to 21 days. The PCOS group was drenched with 1% CMC up to 21 days. All the animal experiments were approved by the Animal Ethics Committee of First Affiliated Hospital of Guilin Medical University (No.GLMC202003153).
Vaginal cytology
Vaginal smears were performed respectively for 14 d before the end of modeling and treatment. Briefly, the vagina was flushed with 35 μL normal saline (0.9% NaCl) for 2–3 times. The vaginal fluid (10–20 μL) was collected onto the glass slide. After air dry at room temperature, the slides were fixed with methanol followed by staining with the Wright’s–Gimsa (Solarbio, Beijing, China) according to the manufacturer’s protocol. The changes of vaginal epithelial cells were evaluated under a light microscope to determine the estrous cycle.
Serum collection and measurement of hormone levels
At the end of the experiment, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.2 ml/100 g). Blood was collected by cardiac puncture, and the serum was isolated by centrifuging at 2500 rpm for 20 min. The serum levels of testosterone and LH were determined by corresponding commercial enzyme-linked immunosorbent assay kits (Abcam, Cambridge, USA).
Morphological analysis of ovary
The ovarian tissues were dissected and fixed in 4% paraformaldehyde overnight at 4 °C, embedded in paraffin, sectioned into 5-μm thick slices, deparaffinized and stained with hematoxylin and eosin. The morphology of the ovarian tissues was evaluated under a light microscope.
Immunohistochemistry analysis of proliferating cell nuclear antigen (PCNA), lactate dehydrogenase a (LDH-A), pyruvate kinase isozyme M2 (PKM2) and SIRT1
Ovaries were fixed in 4% formaldehyde and embedded in paraffin; 5-μm thick slices were sectioned. The sections were permeabilized with 1% Triton X-100 in phosphate buffered saline (PBS) for 30 min at room temperature, boiled in 100 mM sodium citrate (pH 6.0) three times for 6 min each at 5-min intervals for antigen retrieval, and then incubated with 3% hydrogen peroxide for 30 min to remove endogenous peroxidase followed by blocking in 5% bovine serum albumin at room temperature for 1 h. The sections were then incubated overnight at 4 °C with primary goat polyclonal PCNA, LDH-A, PKM2 and SIRT1 antibodies in the blocking solution. Following three washes with 0.1% Tween-20 in PBS, the samples were incubated with rabbit anti-goat biotin-SP-conjugated antibody (1:100; SA00004–4, Protein Tech Group Inc.) in the blocking solution at room temperature for 45 min. The stained PCNA, LDH-A, PKM2 and SIRT1 proteins were visualized using the 3, 3-diaminobenzidine chromogen. The primary antibody replaced with normal goat IgG was served as a negative control. The stained sections were evaluated under a light microscope.
Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick end-labeling (TUNEL) analysis
Detection of the apoptotic cells in the ovarian granular cells was determined by TUNEL assay (Roche Diagnostic Systems, Branchburg, USA) by following the manufacturer’s instructions with modifications [
12]. In brief, sections were dewaxed and rehydrated in water, and then treated with proteinase K and 3% hydrogen peroxide followed by incubating with the TUNEL reaction mixture in a humidified chamber at 37 °C. After that, the sections were incubated with peroxidase-conjugated anti-biotin antibody. The apoptotic cells were visualized using the 3, 3-diaminobenzidine chromogen. Haematoxylin was used as counterstaining.
Quantitative real-time PCR (qPCR)
RNA extraction from the tissues was performed using TRIzol reagent (Takara, Dalian, China). Synthesis of cDNA was performed using the TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix kit (Transgen Biotech, Beijing, China) according to manufacturer’s protocol. Real-time PCR analyses for the gene expression level were performing on the Applied Biosystems 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). GAPDH was used as the reference control, and gene expression levels were calculated using comparative Ct method. The primer sequences were shown in Table
1.
Table 1Primer sequences of the qRT-PCR analysis
PCNA | F: GCTCCATCCTGAAGAAGGT | 55 °C | 121 | NM-022381.3 |
| R: TGCACTAAGGAGACGTGAGA | | | |
Bax | F: GAGACACCTGAGCTGACCTT | 55 °C | 104 | NM-017059.2 |
| R: TCCATGTTGTTGTCCAGTTC | | | |
Bcl-2 | F: AGTACCTGAACCGGCATCT | 55 °C | 120 | NM-016993.1 |
| R: CCGGTTACTATTCCTGGAGA | | | |
Caspase-3 | F: CCGGTTACTATTCCTGGAGA | 55 °C | 117 | NM-017008.4 |
| R: TAACACGAGTGAGGATGTGC | | | |
MCT4 | F: CCTTCCTTCTCACCATCCT | 55 °C | 136 | BC168146.1 |
R: TCAGTGAAGCCATTGAAGAA |
MCT2 | F: GGATTGGGATTTGGAAGTAT | 55 °C | 119 | NM-003676.8 |
R: AGAACTGGACAACACTCCAC |
GLUT1 | F: TGGCTCCTCATGTCAGAGA | 55 °C | 125 | AJ245935.1 |
R: AGGATCTCCATGATGCTGTT |
HK | F: AGAGGCTACGGACAGAGATG | 55 °C | 121 | NM-012734.1 |
R: AGGAAGTCACCGTGTTCAGT |
PFK | F: GGCGTGTGTTCATTGTAGAG | 55 °C | 125 | NM-017008.4 |
R: CTTCAAGTCGTGGATGTTGA |
PKM | F: ACATCCTGTGGCTGGACTAT | 55 °C | 130 | NM-053297.2 |
R: TCCACTTCTGTCACCAGGTA |
LDH | F: GGTTGACAGTGCATACGAAT | 55 °C | 106 | NM-017025.1 |
R: CCGCCTAAGGTTCTTCATTA |
GAPDH | F: CCTCAAGATTGTCAGCAATG | 55 °C | 134 | NM-017008.4 |
R: CAGTCTTCTGAGTGGCAGTG |
Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis of ovarian metabolites
A total of 40 mg rat ovarian tissue for each sample was homogenized in 200 μL pre-cooled ultrapure water. The homogenized samples were incubated with 800 μL pre-cooled methanol/acetonitrile (1:1, v/v) by vortex mixing with ultrasound in ice bath for 20 min, followed by incubation for 1 h at − 20 °C and centrifuge at 14000 r/min for 4 min at 4 °C. The supernatant was collected and dried in the vacuo. For mass spectrometry, 100 μL of acetonitrile-water solution (1:1, v/v) was reconstituted, centrifuging at 14000 r/min for 4 min at 4 °C, and the supernatant was taken for analysis. Samples were separated by using an Agilent 1290 Infinity LC Ultra Performance LC System. Mobile phase: liquid A was 10 mM aqueous ammonium acetate solution, and liquid B was acetonitrile. The sample was placed in a 4 °C autosampler at a column temperature of 45 °C with a flow rate of 300 μL/min and an injection volume of 2 μL. The relevant liquid phase gradient was as follows: 0–18 min, B liquid linearly changes from 90 to 40%; 18–18.1 min, B liquid linearly changes from 40 to 90%; 18.1–23 min, B liquid is maintained at 90%. A quality control sample is set up for each experimental sample in the sample queue for the detection and evaluation of the stability and repeatability of the system; a standard mixture of energy metabolites is set in the sample queue for the correction of chromatographic retention time. Mass spectrometry was performed in negative ion mode by using a 5500 QTRAP mass spectrometer (AB SCIEX). The 5500 QTRAP ESI source conditions were as follows: source temperature 450 °C, ion Source Gas 1: 45, Ion Source Gas 2: 45, Curtain gas: 30, ion Spray Voltage Floating- 4500 V; multi-response monitoring mode detects the pair of ions to be tested. The peak area and retention time were extracted by using Multiquant software. Standardization of energy metabolites was used to correct retention time for metabolite identification.
Statistical analysis
Data were analysed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). The experimental data are presented as the mean ± standard deviation. The unpaired Student’s t-test was used to analyze the comparison between the two groups. One-way ANOVA followed by Bonferroni’s multiple comparison tests was used for comparison among multiple groups. A P value less than 0.05 was considered to indicate a statistically significant result.
Discussion
Currently, high insulin resistance in PCOS patients is a hot topic in the field of reproductive endocrinology and insulin resistance appears to be the fundamental key factor within the pathophysiology of PCOS [
13]. In order to fully understand the pathogenesis of PCOS in vivo studies, it is important to establish an animal model that is close to human polycystic ovarian disease changes. Although letrozole treated SD rats exhibited many metabolic features of human PCOS and have been commonly used as an animal model for PCOS, there was no change in insulin sensitivity or lipid metabolism [
11]. Thus, we established the PCOS-IR rat model by treating the animals with letrozole and high-fat diets. The PCOS-IR rats showed increased body weight, elevated levels of luteinizing hormone and testosterone, and increased HOMA-IR; the The ovaries of PCOS-IR rats demonstrated a PCOS-like appearance, which contained large follicular cysts with poorly developed granulosa cells and many atretic follicles, which were similar to the pathological changes of human PCOS-IR [
14]. Therefore, the rat model of letrozole for 30 days with high-fat diet can not only study polycystic changes of PCOS ovary, but also study metabolic diseases such as insulin resistance, which is an ideal model for studying the pathogenesis of PCOS-IR.
Diane-35 is composed of 2-mg cyproterone acetate and 35-μg ethinyl estradiol, which is widely used for the management of PCOS patients with hyperandrogenism [
15]. It works by blocking the effects of androgens such as testosterone and by activating the progesterone receptor. The function of metformin is commonly described for the modulation of insulin sensitivity and glucose metabolism in PCOS women [
16]. Combination of Diane-35 and metformin treatment could decrease the BMI and LU levels, improve the condition of hyperandrogenemia and insulin resistance in PCOS patients [
5]. In the dihydrotestosterone-induced PCOS rats, Diane-35 was found to be effective to restore the reproductive functions [
17]. Consistently, in the present study, we found that combination of Diane-35 and metformin treatment reduced body weight, decreased the levels of testosterone and luteinizing hormone and insulin resistance, and also restored the estrous cycle and ovulation in the PCOS rats, indicating that the combined treatment improved the PCOS complications.
In a further study, we found that there is a decrease in the granulosa cell proliferation (as determined by the measurement of PCNA expression) and an increase in the granulosa cell apoptosis (as determined by the TUNEL assay) in the ovarian tissues from PCOS rats. In fact, studies have found that LH can inhibit the proliferation of granulosa cells, which would damage the growth and development of follicles [
18]. Granulosa cells in the ovaries have decreased cell proliferation with degenerated granulosa cell layers in the testosterone-induced PCOS rats and human chorionic gonadotropin (hCG) plus
l-norgestrel-induced PCOS rats [
19], and an increased number of TUNEL (+) granulosa cells were found in dehydroepiandrosterone-induced PCOS rats [
20]. In addition, studies also showed that the decreased expression of bcl-2 gene and increased expression of BAX gene and caspase-3 gene were detected in the PCOS rats [
21]. The changes in the cell proliferation and apoptosis in the PCOS rats from our studies were consistent with previous studies. After the Diane-35 plus metformin treatment, the expression of PCNA was enhanced and the number of TUNEL-positive cells were reduced, implying that Diane-35 plus metformin could improve the ovarian function possibly via increasing granulosa cell proliferation and inhibiting granulomas cell apoptosis.
The normal development of follicles relies mainly on the uptake of glucose from surrounding tissues by granulosa cells through the GLUT on their cell membranes, and the production of pyruvate and lactic acid via the glycolysis pathway to provide energy for follicular development [
22,
23]. Studies have found that the glycolytic activity was increased in developing follicles and as the diameter of the follicle increased, the rate of lactic acid production in the follicular fluid increased significantly [
24]. In addition, the lactic acid content in the follicular fluid of PCOS patients was significantly lower than that of normal women [
25]. Our study showed that the expression of GLUT1 was significantly increased in the ovary of PCOS rats, and the expression of HK and PFK was significantly decreased, suggesting that the utilization of glucose in PCOS rats was decreased. Moreover, the expression of MCT2/4 was down-regulated, and the transport rate of pyruvate and lactic acid decreased. ATP level and lactic acid concentration in the ovary of PCOS rats was significantly reduced, suggesting the disordered energy metabolism in the PCOS ovary. Importantly, combination of Diane-35 and metformin treatment significantly restored above changes in the PCOS rats. In the human studies, Diane-35 could increase the triglycerides levels, but had no relevant negative effects in the metabolic system in the PCOS patients, while Diane-35 plus metformin was effective in improving the metabolic profiles of PCOS patients [
26]. Collectively, these results indicated that Diane-35 plus metformin can improve the energy metabolism of the ovary via regulating the glycolysis pathway.
In the glycolysis pathway, PKM2 and LDH-A are two key glycolysis-related rate-limiting enzymes. PKM2 can catalyze the irreversible transphosphorylation between phosphoenolpyruvate (PEP) and adenosine diphosphate, which produces pyruvate and ATP [
27]. LDH-A can convert pyruvate into lactic acid by using NAD+ and NADH as coenzymes, rapidly regenerating NAD+ to maintain high-speed glycolysis [
28]. Studies have demonstrated that increased glycolytic activity was associated with the up-regulation of PKM2 and LDH-A in the cumulus granulosa cells [
29]. The abnormal expression of PKM2 can promote the proliferation, migration and invasion of tumor cells and other malignant biological behaviors [
30]. In the ovary cancer, knockdown of PKM2 resulted in inhibition of tumor cell proliferation [
31]. Recent studies by Wang et al., showed that PKM2 was down-regulated in the endometrial tissues from PCOS patients with hyperplasia, which was attenuated by metformin treatment [
32]. Our data showed that PKM2 was down-regulated in the ovarian tissues from PCOS rats, which was attenuated by the Diane-35 plus metformin treatment. The results suggest that PKM2 plays an important role in the glycolysis of follicular energy metabolism and Diane-35 plus metformin could improve the PCOS glycolysis process. Studies have shown that the expression of LDH-A in granulosa cells is higher than that in oocytes [
33], and the expression of LDH-A in COCs was remarkably increased in the late stage of follicular development [
34]. Androgen could inhibit LDH-A expression to reduce lactic acid production, which has been suggested to be associated with PCOS follicular developmental disorders [
35]. More importantly, excessive nerve growth factor in follicular fluid of PCOS patients can significantly reduce the expression of LDH-A, impair communication between granulosa cells and oocytes, and reduce oocyte developmental capacity [
36]. Metformin treatment could increase the LDH-A expression in the endometrial tissues from PCOS patients with hyperplasia [
32]. Furthermore, the interaction between LDH-A and SIRT1 could facilitate metabolic channeling and the subsequent epigenetic modification in the nucleus [
37]. SIRT1 is a key NAD
+-dependent deacetylase, which is regulated by nuclear levels of NADH, could regulate cell proliferation and apoptosis [
38]. SIRT1 could regulate the gluconeogenic/glycolytic pathways in liver in response to maintain blood glucose levels [
39], and activation of SIRT1 can significantly inhibit the ROS and NO production, and improve the oxidative stress-related insulin resistance [
40]. More importantly, recent studies found that the expression of SIRT1 in the ovaries of PCOS rats was significantly decreased, which could be improved by metformin treatment [
41,
42], and consistently, our data showed that SIRT1 was down-regulated in the ovary of POCS rats, which was restored the Diane-35 plus metformin treatment. Collectively, the therapeutic effects of Diane-35 plus metformin treatment in the PCOS rats may be associated with the regulation of glycolysis-related mediators (PKM2 and LDH-A) and SIRT1.
The present study for the first time demonstrated the beneficial effects of Diane-35 plus metformin treatment in PCOS rats, and more importantly, mechanistic studies revealed that several key mediators that related with glycolysis and energy metabolism could be modulated by the Diane-35 plus metformin treatment. However, the investigation is still limited to its preliminary stage, and further functional studies of these mediators in future studies should be investigated. As Diane-35 and metformin has been suggested to treatment PCOS, further clinical studies may be performed to determine the expression of these key mediators in the PCOS patients after the treatment, and to evaluate the clinical significance of these mediators.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.