TY - JOUR
AU - Wang,, Hai-Long
AB - Abstract Women with polycystic ovary syndrome (PCOS) are characterized by endocrine disorders accompanied by a decline in oocyte quality. In this study, we generated a PCOS mice model by hypodermic injection of dehydroepiandrosterone, and metformin was used as a positive control drug to study the effect of pachymic acid (PA) on endocrine and oocyte quality in PCOS mice. Compared with the model group, the mice treated with PA showed the following changes (slower weight gain, improved abnormal metabolism; increased development potential of GV oocytes, reduced number of abnormal MII oocytes, and damaged embryos; lower expression of ovarian-related genes in ovarian tissue and pro-inflammatory cytokines in adipose tissue). All these aspects show similar effects on metformin. Most notably, PA is superior to metformin in improving inflammation of adipose tissue and mitochondrial abnormalities. It is suggested that PA has the similar effect with metformin, which can improve the endocrine environment and oocyte quality of PCOS mice. These findings suggest that PA has the similar effect with metformin, which can improve the endocrine environment and oocyte quality of PCOS mice. Introduction Polycystic ovary syndrome (PCOS) is a common endocrine-metabolic disorder characterized by anovulation, increased production of ovarian androgen, and polycystic ovaries, affecting up to 10% of reproductive-aged women worldwide [1]. In traditional Chinese medicine (TCM), there is no disease named PCOS, but associated symptoms have long been described, such as abdominal mass, obesity, infertility, menorrhagia, menstrual amenorrhea, and metrorrhagia. In Chinese medicine, the impact of obesity on reproduction and the use of relevant drugs have been documented since ancient times. Poria provides a common ingredient in some Chinese medicine, such as Dao Tan Wan used to treat obese infertile women (described in “Ye Tianshi gynecological secret recipe” Volume 1), Qi Gong Wan used to treat obese infertile women with excessive sputum (described in “TCM prescription collection and annotation”), as well as Ding Jing Tang, a TCM decoction used to treat women with menstrual disorders (described in “Fu Qingzhu Nüke”). Pachymic acid (PA) is one of the main components of Poria, and considering these TCM records, it may have a therapeutic role in PCOS. In addition, modern studies have found that PA exhibits various pharmacological effects. For example, PA has anti-inflammatory, anti-oxidation, and insulin-like effects [2, 3]. Abnormalities in extra- and intra-ovarian factors, such as inflammatory cytokines, reactive oxygen species, and hyperinsulinemia that impact the ovarian microenvironment, oocyte maturation, and embryonic developmental competence, are common in PCOS [4, 5]. So, modern research suggests that PA has a relationship with the treatment of PCOS. Yet, no study has reported the use of PA to treat PCOS. Metformin is a regular drug for patients with PCOS, however, long-term use may lead to anemia, gastrointestinal upset, and other side effects [6, 7]. In comparison, the clinical application of Poria cocos has provided a high level of curative effect, and no side effects have been reported so far [8, 9]. A new drug is required, such as PA, that can alleviate the symptoms of PCOS, including disrupted reproductive function. In the current work, we investigated the therapeutic effect of PA on impaired oocyte developmental quality in a PCOS mice model [10, 11], and explored the underlying mechanism. The results of this study provide new knowledge for the future clinical treatment with minimal adverse effects for PCOS women. Materials and methods All chemicals used in this study were bought from Sigma Chemical unless otherwise indicated. This study used 4-week-old female ICR mice according to strict regulations of the Animal Research Committee of Xiamen University, China (approval ID: XMUMC 2011-10-08). Experimental design As shown in Figure 1, female mice weighing 20 g–22 g were randomly divided into four groups (30–40 per group; control, dehydroepiandrosterone (DHEA), DHEA+PA, and DHEA+metformin). Mice in the control were subcutaneously injected with soybean oil (Emerging (Tie ling) pharmaceutical Limited by Share Ltd.) and given physiological saline by gavage once a day for 21 consecutive days. The DHEA-treated groups were subcutaneously injected daily with DHEA (6 mg/100 g body weight; Wuhan Xinxin Jiali Biotechnology Co., Ltd.) dissolved in soybean oil and given physiological saline daily by gavage. The PA-treated and metformin (Met)-treated groups were given suspensions by gavage containing PA (5 mg/100 g body weight; Shanghai Shi Feng Biological Technology Co., Ltd.) or metformin (50 mg/100 g body weight; Wuhan Xinxin Jiali Biotechnology Co., Ltd.), respectively, dissolved in physiological saline [9, 12]. All mice were maintained under controlled temperature (23°C) and lighting conditions (simulated natural light/dark cycle of 12 h) and supplied adequate food and water. Figure 1 Open in new tabDownload slide The experimental design. Figure 1 Open in new tabDownload slide The experimental design. During the experimental process, female mice were weighed once every 2 days, and vaginal smears were taken daily beginning 14 days from the end of the experiment. The PCOS model was considered successful in mice whose vaginal epithelial cells exhibited keratosis for eight consecutive days. Some mice (approximately 15/group) were used for collecting blood samples, retroperitoneal white adipose tissue, ovarian tissue, and germinal vesicle (GV) stage oocytes for in vitro maturation (IVM). Collected serum samples and tissues were quickly processed in 5 min for follow-up experiments. For the remaining mice, approximately 10 per group were fasted for the oral glucose tolerance test (OGTT) and then used to collect MII oocytes, and the remaining were mated for blastocyst collection. Estrous cycle determination The estrous cycle was determined by microscopic examination of vaginal cells, which were collected by fresh vaginal lavage using physiological saline and then stained with methylene blue (0.1%). Stages of the estrous cycle were determined by vaginal cytology. Oral glucose tolerance test (OGTT) Female mice were orally administered D-glucose (2 g/kg body weight) in saline after 8 h of fasting. Blood samples were collected from the tail vein at zero (before oral glucose), 30 min, 60 min, 90 min, and 120 min after glucose administration and immediately used for the measurement of blood glucose levels by the OneTouch Ultra glucometer. Data were recorded as absolute values of blood glucose concentrations. The whole area under the curve (AUC) of the glucose response was determined using Graph Pad Prism 5.0 software. Figure 2 Open in new tabDownload slide Effect of pachymic acid (PA) on body weight, glucose tolerance, and estrous cycle in polycystic ovary syndrome mice. (A) Body weight growth curves of mice in the control (green), DHEA (red), DHEA+PA (blue), DHEA+Met (black) groups (n = 10/group). (B) The oral glucose tolerance test (OGTT). (C) Area under the curve (AUC) for glucose in the four groups for 21 days of treatment. (D) Estrous cycle of the four groups (n = 3/group, from day 14 to 21, monitored for eight consecutive days; M: metestrus，E: estrus，P: proestrus，D: diestrus). DHEA, dehydroepiandrosterone; Met, metformin. Figure 2 Open in new tabDownload slide Effect of pachymic acid (PA) on body weight, glucose tolerance, and estrous cycle in polycystic ovary syndrome mice. (A) Body weight growth curves of mice in the control (green), DHEA (red), DHEA+PA (blue), DHEA+Met (black) groups (n = 10/group). (B) The oral glucose tolerance test (OGTT). (C) Area under the curve (AUC) for glucose in the four groups for 21 days of treatment. (D) Estrous cycle of the four groups (n = 3/group, from day 14 to 21, monitored for eight consecutive days; M: metestrus，E: estrus，P: proestrus，D: diestrus). DHEA, dehydroepiandrosterone; Met, metformin. Analysis of biochemical indicators in serum Blood samples were collected from mice fasted for 8 h, and then anesthetized by isoflurane inhalation and immediately subjected to eyeball removal for blood collection. The serum was immediately separated and stored at −80°C for subsequent hormone measurements. The level of anti-Müllerian hormone (AMH), estradiol (E2), insulin (INS), leptin (LEP), IL-6, testosterone (T), and TNF-α were measured using ELISA at Hunan Fengrui Biotechnology Co., Ltd. The cholesterol (CHO), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels were measured by Kits (Nanjing Institute of Bioengineering). Ovarian histology and follicle counting After collecting blood samples, the ovaries from three mice per group were quickly removed, fixed in 4% paraformaldehyde, and then following conventional histological procedures were embedded in paraffin. One half of each ovary was sectioned as 5-μm sheets, which were mounted on glass slides for ovarian morphology assessment. The other side of each ovary was serially sectioned (8-μm sheets) for follicle counts. The sections were stained with hematoxylin and eosin for observation under light microscopy. Referring to previous studies, the number of resting (primordial) and growing (primary, secondary) and mature follicles per ovary was determined. Briefly, only follicles with a visible nucleus in a section were counted to avoid an overestimation of the larger follicle numbers, and the number of follicles at each level of a slice was recorded every five slices, and the number of follicles at each level of the slice was counted, which was the true value of the follicles at each level of each ovary. Transmission electron microscopy (TEM) Three mice per group were sacrificed according to the above method, and one half of each collected ovary was preserved in Trizol at −80°C for RNA isolation. The other side of each ovary was dissected into two parts on a wax plate and immediately fixed in 2.5% glutaraldehyde at 4°C for at least 8 h. Subsequently, the postfixation tissue was incubated with osmium tetroxide, then dehydrated, and followed by penetration and embedding procedures. Finally, ultra-TEM (JEM-2100, TEM) was used to observe the ultrathin sections stained with 3% uranyl acetate-lead citrate. The results of ovarian electron microscopy include many abnormalities, such as abnormal mitochondria and other ultrastructures [13, 14]. RNA isolation and quantitative RT-PCR Total ovarian RNA was isolated using Trizol and RNA concentration was determined using a spectrophotometer (260/280 nm). RNA was reverse transcribed to cDNA using the Superscript III One-Step reverse-transcriptase polymerase chain reaction (RT-PCR) kit. cDNA was diluted in RNase-free water and amplified using an Eppendorf PCR Master cycler. Primers sequences were designed by Primer 3 and are listed in Table 2. The specific parameters of qPCR are 95°C 30 s, 95°C 3 s, and 60°C 30 s (one cycle is completed). There are 40 cycles, followed by 95°C 15 s, 60°C 1 min, 95°C 15 s. Table 1 Comparison of biochemical indicators in serum from control, DHEA, DHEA+PA, and DHEA+Met groups (N = 8). The effects of pachymic acid and metformin on metabolic parameters and serum homone levels . Serum variable . Control(n = 8) . DHEA(n = 8) . DHEA+PA(n = 8) . DHEA+Met(n = 8) . E2(pmol/L) 98.03 ± 23.835 125.11 ± 47.558↑ 101.53 ± 6.267 102.03 ± 27.006 T(ng/mL) 51.50 ± 5.264 57.45 ± 14.768↑ 51.31 ± 3.766 53.39 ± 4.501 AMH(pg/mL) 5507.51 ± 450.302 6365.12 ± 2310.262↑ 5485.47 ± 305.119 5829.64 ± 632.930 INS(mIU/L) 42.14 ± 4.401 50.56 ± 17.020↑ 44.06 ± 6.381 42.06 ± 4.879 LEP(ng/mL) 11.28 ± 0.791 13.55 ± 1.885↑△△ 11.70 ± 1.477* 12.02 ± 1.874* CHO(mmol/L) 2.25 ± 0.370 2.99 ± 0.306↑△△ 2.25 ± 0.301** 2.16 ± 0.329** TG(mmol/L) 2.17 ± 0.305 3.15 ± 0.664↑△△ 1.78 ± 0.577** 2.01 ± 0.537** HDL(mmol/L) 2.41 ± 0.574 1.93 ± 0.211↓△△ 2.12 ± 0.343 2.05 ± 0.289 LDL(mmol/L) 0.187 ± 0.021 0.208 ± 0.025↑△△ 0.192 ± 0.015 0.190 ± 0.020 IL-6(pg/mL) 189.97 ± 17.969 181.61 ± 8.019↓ 189.59 ± 18.611 177.66 ± 19.823 TNF-α(pg/mL) 1231.11 ± 228.463 1163.82 ± 226.497↓ 1031.05 ± 129.551↓△ 1186.06 ± 188.255 The effects of pachymic acid and metformin on metabolic parameters and serum homone levels . Serum variable . Control(n = 8) . DHEA(n = 8) . DHEA+PA(n = 8) . DHEA+Met(n = 8) . E2(pmol/L) 98.03 ± 23.835 125.11 ± 47.558↑ 101.53 ± 6.267 102.03 ± 27.006 T(ng/mL) 51.50 ± 5.264 57.45 ± 14.768↑ 51.31 ± 3.766 53.39 ± 4.501 AMH(pg/mL) 5507.51 ± 450.302 6365.12 ± 2310.262↑ 5485.47 ± 305.119 5829.64 ± 632.930 INS(mIU/L) 42.14 ± 4.401 50.56 ± 17.020↑ 44.06 ± 6.381 42.06 ± 4.879 LEP(ng/mL) 11.28 ± 0.791 13.55 ± 1.885↑△△ 11.70 ± 1.477* 12.02 ± 1.874* CHO(mmol/L) 2.25 ± 0.370 2.99 ± 0.306↑△△ 2.25 ± 0.301** 2.16 ± 0.329** TG(mmol/L) 2.17 ± 0.305 3.15 ± 0.664↑△△ 1.78 ± 0.577** 2.01 ± 0.537** HDL(mmol/L) 2.41 ± 0.574 1.93 ± 0.211↓△△ 2.12 ± 0.343 2.05 ± 0.289 LDL(mmol/L) 0.187 ± 0.021 0.208 ± 0.025↑△△ 0.192 ± 0.015 0.190 ± 0.020 IL-6(pg/mL) 189.97 ± 17.969 181.61 ± 8.019↓ 189.59 ± 18.611 177.66 ± 19.823 TNF-α(pg/mL) 1231.11 ± 228.463 1163.82 ± 226.497↓ 1031.05 ± 129.551↓△ 1186.06 ± 188.255 Open in new tab Table 1 Comparison of biochemical indicators in serum from control, DHEA, DHEA+PA, and DHEA+Met groups (N = 8). The effects of pachymic acid and metformin on metabolic parameters and serum homone levels . Serum variable . Control(n = 8) . DHEA(n = 8) . DHEA+PA(n = 8) . DHEA+Met(n = 8) . E2(pmol/L) 98.03 ± 23.835 125.11 ± 47.558↑ 101.53 ± 6.267 102.03 ± 27.006 T(ng/mL) 51.50 ± 5.264 57.45 ± 14.768↑ 51.31 ± 3.766 53.39 ± 4.501 AMH(pg/mL) 5507.51 ± 450.302 6365.12 ± 2310.262↑ 5485.47 ± 305.119 5829.64 ± 632.930 INS(mIU/L) 42.14 ± 4.401 50.56 ± 17.020↑ 44.06 ± 6.381 42.06 ± 4.879 LEP(ng/mL) 11.28 ± 0.791 13.55 ± 1.885↑△△ 11.70 ± 1.477* 12.02 ± 1.874* CHO(mmol/L) 2.25 ± 0.370 2.99 ± 0.306↑△△ 2.25 ± 0.301** 2.16 ± 0.329** TG(mmol/L) 2.17 ± 0.305 3.15 ± 0.664↑△△ 1.78 ± 0.577** 2.01 ± 0.537** HDL(mmol/L) 2.41 ± 0.574 1.93 ± 0.211↓△△ 2.12 ± 0.343 2.05 ± 0.289 LDL(mmol/L) 0.187 ± 0.021 0.208 ± 0.025↑△△ 0.192 ± 0.015 0.190 ± 0.020 IL-6(pg/mL) 189.97 ± 17.969 181.61 ± 8.019↓ 189.59 ± 18.611 177.66 ± 19.823 TNF-α(pg/mL) 1231.11 ± 228.463 1163.82 ± 226.497↓ 1031.05 ± 129.551↓△ 1186.06 ± 188.255 The effects of pachymic acid and metformin on metabolic parameters and serum homone levels . Serum variable . Control(n = 8) . DHEA(n = 8) . DHEA+PA(n = 8) . DHEA+Met(n = 8) . E2(pmol/L) 98.03 ± 23.835 125.11 ± 47.558↑ 101.53 ± 6.267 102.03 ± 27.006 T(ng/mL) 51.50 ± 5.264 57.45 ± 14.768↑ 51.31 ± 3.766 53.39 ± 4.501 AMH(pg/mL) 5507.51 ± 450.302 6365.12 ± 2310.262↑ 5485.47 ± 305.119 5829.64 ± 632.930 INS(mIU/L) 42.14 ± 4.401 50.56 ± 17.020↑ 44.06 ± 6.381 42.06 ± 4.879 LEP(ng/mL) 11.28 ± 0.791 13.55 ± 1.885↑△△ 11.70 ± 1.477* 12.02 ± 1.874* CHO(mmol/L) 2.25 ± 0.370 2.99 ± 0.306↑△△ 2.25 ± 0.301** 2.16 ± 0.329** TG(mmol/L) 2.17 ± 0.305 3.15 ± 0.664↑△△ 1.78 ± 0.577** 2.01 ± 0.537** HDL(mmol/L) 2.41 ± 0.574 1.93 ± 0.211↓△△ 2.12 ± 0.343 2.05 ± 0.289 LDL(mmol/L) 0.187 ± 0.021 0.208 ± 0.025↑△△ 0.192 ± 0.015 0.190 ± 0.020 IL-6(pg/mL) 189.97 ± 17.969 181.61 ± 8.019↓ 189.59 ± 18.611 177.66 ± 19.823 TNF-α(pg/mL) 1231.11 ± 228.463 1163.82 ± 226.497↓ 1031.05 ± 129.551↓△ 1186.06 ± 188.255 Open in new tab Western blotting for IL-6 and TNF-α in adipose tissue The proteins were isolated from retroperitoneal white adipose tissue using Tris-HCl, as described previously. First, protein samples from each group were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes. The membranes were then blocked with 5% bovine serum albumin and incubated with primary antibodies (anti-β-actin, 1:10 000, Abcam ab8226; anti-IL-6, 1:1000, Proteintech 21865-1-AP; anti-TNF-α, 1:1000, Proteintech 17590-1-AP) overnight at 4°C. After washing three times in phosphate buffered solution (PBST) (10 min each), membranes were incubated with secondary antibody for 1 h at room temperature. Finally, membrane bands were detected by Western Lightning ECL profession detection reagent. GV oocyte collection and IVM For collection of GV oocytes, we first killed mice by cervical dislocation, and separated the bilateral ovaries quickly. Then the ovaries were fully minced in a dish with a blade, with M2 medium added to release oocytes. Next, under the stereomicroscope, GV stage oocytes with full shape, complete GV and without granular cells around are selected by hand-drawn thin glass tube (Beijing zhengtianyi science and Trade Co., Ltd.). The separated GV oocytes were cultured in M2 medium at 37°C with 5% CO2 in a live cell station. The number of GV breakdown (GVBD) oocytes was determined after 2 h, and the number of first polar body (PB1) extruded oocytes was recorded after 12 h. Then calculate the GVBD rate and PB1 rate according to the following formula: $$ \mathrm{GVBD}\left(\%\right)=\mathrm{oocytes}\ \mathrm{with}\ \mathrm{ruptured}\ \mathrm{germinal}\ \mathrm{vesicle}/\mathrm{all}\ \mathrm{oocytes}; $$ $$ \mathrm{PB}1\left(\%\right)=\mathrm{Oocytes}\ \mathrm{with}\ \mathrm{excreted}\ \mathrm{first}\ \mathrm{polar}\ \mathrm{body}/\mathrm{oocytes}\ \mathrm{with} $$ $$ \!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\mathrm{ruptured}\ \mathrm{germinal}\ \mathrm{vesicle} $$ MII oocyte and embryo collection and morphological assessment To collect MII oocytes, mice were superovulated by intraperitoneal injection of 5 IU PMSG, followed by 5 IU hCG 48 h later. Cumulus-oocyte complexes were collected from oviduct ampulla, then cumulus cells were removed in 0.2% hyaluronidase. Denuded oocytes were collected and observed under stereomicroscopy. Finally, healthy MII oocytes were collected for spindle immunofluorescence. Table 2 Sense and antisense primers used for amplification. Gene names and primer pair sequences used for qPCR . Gene name . Primer pair sequences . PI-3K(mouse) F 5’-AGCCAACAACAGCATGAACA-3’; R5’-AAGGTCCCATCAGCAGTGTC-3’ GLUT-4 F 5’-TCTCAATGGTTGGGAAGGAA-3’; R5’-GAGGAACCGTCCAAGAATGA-3’ IRS-1 F 5’-AGCCTGTTGTGGACTTGGTC-3’; R5’-ACTCGAGCCTGTGCATTCTT-3’ CYP-17 F 5’-TGGTCATATGCATGCCAACT-3’; R5’-CCCTTCTTCACGAGCACTTC-3’ GSK-3β F 5’-TTCCTTTGGAATCTGCCATC-3’; R5’-TGAAACATTGGGCTCTCCTC-3’ IL-6 F 5’-AGTTGCCTTCTTGGGACTGA-3’; R5’-CCTCCGACTTGTGAAGTGGT-3’ TNF-α F 5’-ACGGCATGGATCTCAAAGAC-3’; R5’-GTGGGTGAGGAGCACGTAGT-3’ Gene names and primer pair sequences used for qPCR . Gene name . Primer pair sequences . PI-3K(mouse) F 5’-AGCCAACAACAGCATGAACA-3’; R5’-AAGGTCCCATCAGCAGTGTC-3’ GLUT-4 F 5’-TCTCAATGGTTGGGAAGGAA-3’; R5’-GAGGAACCGTCCAAGAATGA-3’ IRS-1 F 5’-AGCCTGTTGTGGACTTGGTC-3’; R5’-ACTCGAGCCTGTGCATTCTT-3’ CYP-17 F 5’-TGGTCATATGCATGCCAACT-3’; R5’-CCCTTCTTCACGAGCACTTC-3’ GSK-3β F 5’-TTCCTTTGGAATCTGCCATC-3’; R5’-TGAAACATTGGGCTCTCCTC-3’ IL-6 F 5’-AGTTGCCTTCTTGGGACTGA-3’; R5’-CCTCCGACTTGTGAAGTGGT-3’ TNF-α F 5’-ACGGCATGGATCTCAAAGAC-3’; R5’-GTGGGTGAGGAGCACGTAGT-3’ Open in new tab Table 2 Sense and antisense primers used for amplification. Gene names and primer pair sequences used for qPCR . Gene name . Primer pair sequences . PI-3K(mouse) F 5’-AGCCAACAACAGCATGAACA-3’; R5’-AAGGTCCCATCAGCAGTGTC-3’ GLUT-4 F 5’-TCTCAATGGTTGGGAAGGAA-3’; R5’-GAGGAACCGTCCAAGAATGA-3’ IRS-1 F 5’-AGCCTGTTGTGGACTTGGTC-3’; R5’-ACTCGAGCCTGTGCATTCTT-3’ CYP-17 F 5’-TGGTCATATGCATGCCAACT-3’; R5’-CCCTTCTTCACGAGCACTTC-3’ GSK-3β F 5’-TTCCTTTGGAATCTGCCATC-3’; R5’-TGAAACATTGGGCTCTCCTC-3’ IL-6 F 5’-AGTTGCCTTCTTGGGACTGA-3’; R5’-CCTCCGACTTGTGAAGTGGT-3’ TNF-α F 5’-ACGGCATGGATCTCAAAGAC-3’; R5’-GTGGGTGAGGAGCACGTAGT-3’ Gene names and primer pair sequences used for qPCR . Gene name . Primer pair sequences . PI-3K(mouse) F 5’-AGCCAACAACAGCATGAACA-3’; R5’-AAGGTCCCATCAGCAGTGTC-3’ GLUT-4 F 5’-TCTCAATGGTTGGGAAGGAA-3’; R5’-GAGGAACCGTCCAAGAATGA-3’ IRS-1 F 5’-AGCCTGTTGTGGACTTGGTC-3’; R5’-ACTCGAGCCTGTGCATTCTT-3’ CYP-17 F 5’-TGGTCATATGCATGCCAACT-3’; R5’-CCCTTCTTCACGAGCACTTC-3’ GSK-3β F 5’-TTCCTTTGGAATCTGCCATC-3’; R5’-TGAAACATTGGGCTCTCCTC-3’ IL-6 F 5’-AGTTGCCTTCTTGGGACTGA-3’; R5’-CCTCCGACTTGTGAAGTGGT-3’ TNF-α F 5’-ACGGCATGGATCTCAAAGAC-3’; R5’-GTGGGTGAGGAGCACGTAGT-3’ Open in new tab For the collection of embryos, the above-superovulated mice were mated with fertile ICR males. Successfully mated females were sacrificed 4 days later by cervical dislocation and uteri were carefully removed. The uteri were washed with M2 medium in a syringe and embryos were harvested under a stereomicroscope. Figure 3 Open in new tabDownload slide Early apoptosis of germinal vesicle (GV) stage oocytes and oocyte developmental potential in control, DHEA, DHEA + PA, and DHEA + met groups. (A) Representative pictures of normal (a) and apoptotic oocytes (b) after annexin-V labeling; arrows indicate zona or oocyte membranes; Bar = 10 μm. The percentage of GV oocytes displaying early stage apoptosis in the four groups. (B) Effects of pachymic acid (PA) on oocyte maturation, number of oocytes, oocyte death, and number of GV breakdown (GVBD) and polar body extruded (PB1) oocytes. N shows the number of mice. Figure 3 Open in new tabDownload slide Early apoptosis of germinal vesicle (GV) stage oocytes and oocyte developmental potential in control, DHEA, DHEA + PA, and DHEA + met groups. (A) Representative pictures of normal (a) and apoptotic oocytes (b) after annexin-V labeling; arrows indicate zona or oocyte membranes; Bar = 10 μm. The percentage of GV oocytes displaying early stage apoptosis in the four groups. (B) Effects of pachymic acid (PA) on oocyte maturation, number of oocytes, oocyte death, and number of GV breakdown (GVBD) and polar body extruded (PB1) oocytes. N shows the number of mice. Immunofluorescence To examine apoptosis, fixed ovary slices from different groups were treated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) reaction mixture (Keygene Biotech, China), counterstained with DAPI (Vector, Switzerland) and mounted in mounting medium. TUNEL-positive cells and cell nuclei were observed and counted with a FV1000 confocal laser scanning microscope (Olympus, Japan). To evaluate oocyte apoptosis, an annexin-V probe (Beyotime, China) was used according to the manufacturer’s instructions. Oocytes were cultured in M2 medium supplemented with the annexin-V probe in a humidified atmosphere at 37°C with 5% CO2 for 30 min and then washed three times. Stained oocytes were fixed and analyzed with a FV1000 confocal microscope. For evaluating spindles and chromosomes of MII oocytes, we fixed oocytes in 4% paraformaldehyde for 30 min at room temperature and then perforated membranes using a permeabilization solution (0.5% Triton X-100) for 30 min. After incubation in 1% bull serum albumin-supplemented PBS for 1 h, oocytes were incubated with primary antibody (mouse anti-α-tubulin-FITC antibody, 1:200; Sigma, USA) at 4°C overnight. After three washes in PBS containing 0.1% Tween 20 and 0.01% Triton X-100 (5 min each), oocytes were transferred to the 2.5ul DAPI (Vector, Switzerland) predropped on the clean slide, gently mixed with a glass needle, covered with clean coverslips and pressed; finally, the cover slide was sealed with adhesive to prevent water loss. The final staining results were observed using a FV1000 confocal microscope. The detection of apoptosis and octamer-binding transcription factor 4 (Oct4) in embryos was performed using TUNEL reaction mixture and anti-Oct4 polyclonal antibody (1:100, Proteintech, 11263-1-AP), respectively. Briefly, embryos were fixed in 4% paraformaldehyde for 30 min at room temperature, permeabilized, incubated with antibody, then labeled with a fluorescence secondary antibody (Anti-mouse IgG AF546, ZSGB-BIO, ZF-0511). Fixed embryos were then incubated with TUNEL reaction mixture. Embryos were co-stained with DAPI and mounted on glass slides. Oct4-positive and TUNEL-positive cells, and cell nuclei were visualized and counted using a FV1000 confocal microscope. Statistical analysis Data were expressed as mean ± SEM and analyzed by one-way ANOVA, with a Bonferroni’s post hoc test using SPSS software (IBM Corp, USA). P < 0.05 was considered statistically significant. Values are shown as the mean ± SEM; n shows the number of mice; ↑ and ↓, P = X, ΔP < 0.05, ΔΔP < 0.01 vs. Control; *P < 0.05, **P < 0.01 vs. DHEA; one-way ANOVA, with LSD post hoc test. Results PA reduced the increased body weight and impaired glucose tolerance in PCOS mice PA significantly inhibited the weight gain of PCOS mice. As shown in Figure 2A, the body weights of mice in all groups were similar at the beginning of treatments. From days 11 to 21 of treatment, the DHEA group exhibited significantly more weight gain than the control group (P < 0.01). The weight of PA-treated and Met-treated groups increased more slowly and the weight of the PA-treated group was closer to that of the control group. The OGTT showed that PA improved glucose tolerance in PCOS mice. As shown in Figure 2B, fasting glucose levels were similar among the groups. The PA- and Met-treated groups had markedly decreased serum glucose levels 30 min and 60 min after glucose administration compared with the DHEA group. The pattern of the glucose response was similar in the PA-treated and control groups. Furthermore, there was a significant decrease in the 0 to 120 min AUC value for the PA- and Met-treated groups compared with the DHEA group (Figure 2C). Figure 4 Open in new tabDownload slide Morphological evaluation and internal structure of MII-stage oocytes isolated from control, DHEA, DHEA + PA, and DHEA + Met groups. (A) Images of MII-stage oocytes, (N, normal appearance; a, degraded polar body; b, enlarged perivitelline space; c, fragmented cytoplasm indicated by red box); percentage of different MII oocytes (n = number of oocytes; Bar = 10 μm). (B) Representative images of spindles (stained with FITC-α-Tubulin) and chromosomes (stained with DAPI) in normal and abnormal MII oocytes (a, b, normal spindle and aligned chromosomes; c, multipolar spindle apparatus and aligned chromosomes; d, abnormal spindle shapes and lagging chromosomes; e, disintegrated spindle poles and irregularly scattered chromosomes). The proportion of MII oocytes with abnormal spindles and misaligned chromosomes in the four groups. Figure 4 Open in new tabDownload slide Morphological evaluation and internal structure of MII-stage oocytes isolated from control, DHEA, DHEA + PA, and DHEA + Met groups. (A) Images of MII-stage oocytes, (N, normal appearance; a, degraded polar body; b, enlarged perivitelline space; c, fragmented cytoplasm indicated by red box); percentage of different MII oocytes (n = number of oocytes; Bar = 10 μm). (B) Representative images of spindles (stained with FITC-α-Tubulin) and chromosomes (stained with DAPI) in normal and abnormal MII oocytes (a, b, normal spindle and aligned chromosomes; c, multipolar spindle apparatus and aligned chromosomes; d, abnormal spindle shapes and lagging chromosomes; e, disintegrated spindle poles and irregularly scattered chromosomes). The proportion of MII oocytes with abnormal spindles and misaligned chromosomes in the four groups. Figure 5 Open in new tabDownload slide Effect of pachymic acid (PA) on early embryo development in polycystic ovary syndrome mice. (A) Representative images showing the morphology of embryos (4-day embryo in vivo); (BI = blastocyst; Ly = lysed embryo; Mo = morula). Representative immuno-fluorescence images for Oct4 (red) and TUNEL (green) staining in embryos from the four groups, with nuclei stained with DAPI (blue). Scale bar = 20 μm. (B) Percentage of blastocysts (n shows the number of embryos), cell number per blastocyst, TUNEL-positive cells per blastocyst, Oct4-positive cells per blastocyst (blastocyst indicated by one point), and percent of apoptotic blastocysts. Figure 5 Open in new tabDownload slide Effect of pachymic acid (PA) on early embryo development in polycystic ovary syndrome mice. (A) Representative images showing the morphology of embryos (4-day embryo in vivo); (BI = blastocyst; Ly = lysed embryo; Mo = morula). Representative immuno-fluorescence images for Oct4 (red) and TUNEL (green) staining in embryos from the four groups, with nuclei stained with DAPI (blue). Scale bar = 20 μm. (B) Percentage of blastocysts (n shows the number of embryos), cell number per blastocyst, TUNEL-positive cells per blastocyst, Oct4-positive cells per blastocyst (blastocyst indicated by one point), and percent of apoptotic blastocysts. PA had no obvious effect on the disordered estrous cycle inPCOS mice. The estrus cycle of normal mice follows the regular changes of proestrus, estrus, metestrus, and diestrus. Compared with the control group, mice in the DHEA group showed irregular estrous cycles, including the disappearance of the diestrus or proestrus period, or the prolongation of the estrous period (Figure 2D). Neither PA nor metformin treatment could fully restore the estrous cycle of PCOS mice, which may be related to the treatment time. It may take a longer process to restore the normal estrous cycle. PA improved the abnormality of serum hormones and inflammatory factors in PCOS mice Hormonal disturbance involving AMH, E2, INS, LEP, and T is a most common feature of PCOS. As shown in Table 1, all these hormone levels were increased in the DHEA group (E2, P = 0.08; T, P = 0.16; AMH, P = 0.17; INS, P = 0.09; LEP, P = 0.007) compared with the control group. However, after treatment with PA or Met, all hormone levels were reduced by different degrees. In addition to the above indicators, serum total CHOL and TG, and HDL and LDL levels were determined. As shown in Table 1, the levels of CHO, TG, and LDL increased in the DHEA groups, and PA and Met treatment reduced all of these levels. Conversely, the level of HDL increased. These results suggested that PA, like Met, regulated lipid metabolism. Surprisingly, serum concentrations of IL-6, TNF-α were reduced in the DHEA and therapy groups. The TNF-α levels were lower in the PA-treated group compared with the DHEA group, and the concentration of IL-6 in the PA-treated group was closest to the control group. These data illustrate that PA, like Met, can partially improve the metabolic abnormalities induced by DHEA in PCOS mice. PA improved the developmental potential of GV oocytes by inhibiting early apoptosis in PCOS mice To investigate the effects of various treatments on the developmental potential of GV oocytes, we examined the incidence of GVBD, PB1, and early apoptosis in GV oocytes. As shown in Figure 3B, the quantity and quality of GV oocytes were significantly decreased in the DHEA group (P < 0.01) compared with the control group. Compared with the DHEA group, the oocyte death rate in the PA group was decreased, while the GVBD rate and PB1 rate increased (2.2% vs. 10.3%, 91.0% vs. 83.6%, 69.6% vs. 55.3%, respectively), and similar effects were observed in Met group. However, there was no significant difference in the number of recovered oocytes from the PA-treated, Met-treated, and DHEA groups. The rate of apoptosis of GV oocytes (by Annexin-V labeling) was significantly decreased in the PA- and Met-treated groups compared with the DHEA group. As shown in Figure 3Ab, GV oocytes with early apoptosis were characterized by a green signal in the oocyte membrane, whereas oocytes with green fluorescence signals only at the zona pellucid (Figure 3Aa, arrow) were not. As shown in Figure 3A, the early apoptotic rate of GV oocytes from the DHEA-treated group (37.1%, n = 116) was significantly higher than the control group (9.4%, n = 138). However, the early apoptotic rate of GV oocytes was significantly reduced in the PA- (12.6%, n = 132) and met-treated groups (12.6%, n = 159). These results suggested that PA improved GV oocyte quality by inhibiting early apoptosis. PA decreased the number of abnormal MII oocytes induced by DHEA in vivo To explore the effects of DHEA, PA, and Met on mature oocytes in vivo, the MII oocytes from fallopian tubes were isolated for analysis. Morphological abnormalities in MII oocytes were examined by stereomicroscopy. We found most of the MII oocytes in the control group exhibited a normal morphological appearance (Figure 4A). The number of abnormal MII oocytes (exhibiting degraded polar bodies, an enlarged perivitelline space, or fragmented cytoplasm) was significantly lower in the PA- and Met-treated groups than the DHEA group (Figure 4A). We observed barrel-shaped spindles and well-aligned chromosomes in normal MII oocytes (Figure 4Ba and b). As shown in Figure 4B, 27.5%, 13.7%, and 12.1% of MII oocytes exhibited defective spindles (disintegrated spindle poles, additional asters, and abnormal spindle shapes) in the DHEA, PA- and Met-treated groups, respectively (Figure 4Bc, d and e). The number of oocytes with severe defects in chromosome arrangement was significantly lower in the PA- (5.5%) and the met-treated (6.1%) groups compared with the DHEA (16.3%) group. These results showed that the chromosomal and spindle development in MII oocytes in vivo can be protected by PA. Figure 6 Open in new tabDownload slide Ovarian morphology and follicular development in the control, DHEA, DHEA + PA, and DHEA + met groups (N = 3 mice/group). (A) Representative H&E staining of ovarian sections from each group. Micrographs were taken at a magnification of ×40, ×100, and ×400. The boxed areas are shown at higher magnifications. (*, cystic follicle; HC, hemorrhagic cyst; arrows indicate granulosa layer; scale bar = 200 μm). (B) Effects of pachymic acid and metformin on follicular development in dehydroepiandrosterone-treated mice (atretic, primordial, primary, secondary, preovulatory follicles). Figure 6 Open in new tabDownload slide Ovarian morphology and follicular development in the control, DHEA, DHEA + PA, and DHEA + met groups (N = 3 mice/group). (A) Representative H&E staining of ovarian sections from each group. Micrographs were taken at a magnification of ×40, ×100, and ×400. The boxed areas are shown at higher magnifications. (*, cystic follicle; HC, hemorrhagic cyst; arrows indicate granulosa layer; scale bar = 200 μm). (B) Effects of pachymic acid and metformin on follicular development in dehydroepiandrosterone-treated mice (atretic, primordial, primary, secondary, preovulatory follicles). PA partially prevented the reduction in embryo quality induced by DHEA in vivo To further evaluate the effect of PA on DHEA-induced abnormalities in cleavage and early embryo development in mice, we compared morphological characteristics, apoptosis, and Oct4 expression in randomly chosen embryos. As shown in Figure 5A, most of the embryos in the control group were blastocysts. Abnormal embryos, including lysed embryos (LY) and morula (MO), were observed in PA-treated (LY, 11.1%; MO, 36.1%; n = 72), Met-treated (LY, 14.7%; MO, 22.4%; n = 89) and the DHEA groups (LY, 26.9%; MO, 34.2%; n = 85). We examined total cell numbers, apoptosis, and Oct4 expression in embryos by confocal microscopy. As shown in Figure 5B and C, the total number of cells was significantly increased in the PA- (37 ± 1.4; P < 0.01) and Met-treated groups (43 ± 1.8; P < 0.01) compared with the DHEA group (34 ± 1.9). Furthermore, apoptosis was reduced in the PA- (4 ± 0.6; P < 0.01) and Met-treated (7 ± 0.8; P < 0 .05) groups compared with the DHEA (10 ± 0.8) group, and the rate of apoptosis was significantly decreased (P < 0.01) in the PA-treated (36%) compared with the DHEA (81%) group. These data show that DHEA reduced cleavage and blastocyst development, and led to aberrant Oct4 expression and increased apoptosis in developing early embryos, which was partially prevented by PA treatment. Effect of PA on follicular development We explored the changes in ovarian morphology and follicular development. As shown in Figure 6A, ovarian follicles at different stages of development, and the theca cells and granulosa cell layers appeared normal in the control group. However, the ovaries from the DHEA-treated group had multiple cystic follicles, abnormal ovarian morphology, and granulosa cells detachment (Figure 6A, ×40 and ×100). The same abnormal structures also appeared in the PA- and met-treated groups. However, the granular cell layer was thicker in these two groups than that in the DHEA group (arrows, Figure 6A, ×400), suggesting that PA and Met reduced apoptosis of granulosa cells. Next, we investigated the effects of various treatments upon follicular formation by counting follicle numbers of at different stages of development. As shown in Figure 6B, there was no statistical difference in the number of secondary follicles and preovulatory follicles between all groups. However, the number of primordial and primary follicles was reduced in the DHEA-treated group compared with the control group, and the number of atretic follicles increased in the DHEA-treated versus control group. However, those effects were reduced by PA and Met treatment. These results suggested that PA impacts on follicular development associated with granulosa cells. PA reduced apoptosis of granulosa cells in growing follicles of PCOS mice and decreased mitochondrial damage We investigated apoptosis of granulosa cells using TUNEL and detected ultrastructural changes by TEM to examine changes in organelles of various types of cells in the growing follicle. As shown in Figure 7Ab, panel 2, apoptotic granulosa and stromal cells were found in the ovaries of DHEA-treated females, and the number of apoptotic cells was reduced in the PA-treated compared with the Met-treated group. Figure 7 Open in new tabDownload slide Effects of pachymic acid (PA) on ovarian apoptosis and oocyte mitochondrial damage induced by DHEA. (A) Immunofluorescence images of TUNEL (green) in ovaries. DNA stained with DAPI (blue). Scale bar = 100 μm. (B) Density of mitochondrial and the rate of abnormal mitochondria in oocytes from the four groups. In each group, six fields of magnification (×5000) were randomly taken and the total number of mitochondria and abnormal mitochondria within these fields of view were counted to calculate the abnormality rate. Figure 7 Open in new tabDownload slide Effects of pachymic acid (PA) on ovarian apoptosis and oocyte mitochondrial damage induced by DHEA. (A) Immunofluorescence images of TUNEL (green) in ovaries. DNA stained with DAPI (blue). Scale bar = 100 μm. (B) Density of mitochondrial and the rate of abnormal mitochondria in oocytes from the four groups. In each group, six fields of magnification (×5000) were randomly taken and the total number of mitochondria and abnormal mitochondria within these fields of view were counted to calculate the abnormality rate. Figure 8 Open in new tabDownload slide Transmission electron microscopic analysis of follicles from control, DHEA, DHEA + PA, and DHEA + Met groups (N = 3 mice/group). Representative images of growing follicles from three ovary samples per group (a1–f1; Scale bar = 10 μm). Oocyte (a2–f2), granulosa cell (a4–f4), theca cell (a6–f6), and ovary stroma cell (a8–f8) from growing follicles (scale bar = 2 μm). The red boxed areas are shown at higher magnifications (a3–f3 from the oocyte image; a5–f5 from the granulosa cell image; a7–f7 from the theca cell image; a9–f9 from the ovary stroma cell image; scale bar = 0.5 μm). Normal mitochondria in terms of size and the appearance of cristae are shown for the control (a), DHEA+PA (e), and DHEA+Met (f) groups (green ovals). Mitochondrion with a “vacuolated” appearance (red ovals); “dark/ghost like” appearance (yellow ovals); and “tubular/honeycombs” cristae in cross section (orange ovals) are shown for the DHEA (b, c, d and the DHEA+PA (e) and DHEA+Met (f) groups. The cytolipin vacuole (yellow*) are shown in the four groups. Mitochondrial apoptosis/necrosis is shown (d8, red*). DHEA, dehydroepiandrosterone; Met, metformin. Figure 8 Open in new tabDownload slide Transmission electron microscopic analysis of follicles from control, DHEA, DHEA + PA, and DHEA + Met groups (N = 3 mice/group). Representative images of growing follicles from three ovary samples per group (a1–f1; Scale bar = 10 μm). Oocyte (a2–f2), granulosa cell (a4–f4), theca cell (a6–f6), and ovary stroma cell (a8–f8) from growing follicles (scale bar = 2 μm). The red boxed areas are shown at higher magnifications (a3–f3 from the oocyte image; a5–f5 from the granulosa cell image; a7–f7 from the theca cell image; a9–f9 from the ovary stroma cell image; scale bar = 0.5 μm). Normal mitochondria in terms of size and the appearance of cristae are shown for the control (a), DHEA+PA (e), and DHEA+Met (f) groups (green ovals). Mitochondrion with a “vacuolated” appearance (red ovals); “dark/ghost like” appearance (yellow ovals); and “tubular/honeycombs” cristae in cross section (orange ovals) are shown for the DHEA (b, c, d and the DHEA+PA (e) and DHEA+Met (f) groups. The cytolipin vacuole (yellow*) are shown in the four groups. Mitochondrial apoptosis/necrosis is shown (d8, red*). DHEA, dehydroepiandrosterone; Met, metformin. Figure 9 Open in new tabDownload slide Comparison of mRNA expression in ovarian tissue from the different groups using reverse transcription quantitative PCR (N = 3). The factors involved in the PI3K signaling pathway and inflammation included PI3-K, GLUT-4, GSK-3β, IRS-1, CYP-17, and TNF-α. Figure 9 Open in new tabDownload slide Comparison of mRNA expression in ovarian tissue from the different groups using reverse transcription quantitative PCR (N = 3). The factors involved in the PI3K signaling pathway and inflammation included PI3-K, GLUT-4, GSK-3β, IRS-1, CYP-17, and TNF-α. Figure 10 Open in new tabDownload slide Effect of pachymic acid (PA) on adipose tissue levels of IL-6 and TNF-α in the DHEA-treated mouse model (N = 3). (A) Western blot of IL-6 and TNF-α expression in adipose tissue after different treatments. (B) Densitometric analysis of the protein levels of IL-6 and TNF-α, normalized for β-actin. A representative western blots of one experiment is shown. (C) mRNA expression of the pro-inflammatory cytokines IL-6 and TNF-α in adipose tissue using reverse transcription quantitative PCR. Figure 10 Open in new tabDownload slide Effect of pachymic acid (PA) on adipose tissue levels of IL-6 and TNF-α in the DHEA-treated mouse model (N = 3). (A) Western blot of IL-6 and TNF-α expression in adipose tissue after different treatments. (B) Densitometric analysis of the protein levels of IL-6 and TNF-α, normalized for β-actin. A representative western blots of one experiment is shown. (C) mRNA expression of the pro-inflammatory cytokines IL-6 and TNF-α in adipose tissue using reverse transcription quantitative PCR. We examined the TEM photomicrographs of various types of cells surrounding growing follicles, including oocytes, granulosa, internal theca, and stroma cells. As shown in Figure 8, the overall membrane structure was considerably damaged in the DHEA group compared with the control group, and was less damaged in the PA- and Met-treated groups compared with the DHEA group. The ultrastructural examination of oocytes showed that the number and appearance of mitochondria were different in the various groups. Oocytes from the control group displayed normal mitochondria, with the organized appearance of normal mitochondrial cristae with many parallel membrane layers (blue, Figure 8a3, a5, a7, a9). However, oocytes from the DHEA groups exhibited more mitochondria with vary sparse/absent cristae, and mitochondria with a “vacuolated” (red, Figure 8c, d, e, f) and “dark/ghost like” appearance (yellow, Figure 8c5, f5, c7, b9). All of the above examples of abnormal mitochondrial morphology were rarely present in the PA-treated and Met-treated groups. Furthermore, the number of mitochondria was higher in the PA-treated group than in the other groups. As shown in Figure 7B, the mitochondrial damage in oocytes was higher in the DHEA group than in the control group; however, the PA reduced this damage (P < 0.01). In addition, apoptotic granulosa and theca cells were observed in the DHEA group (Figure 7Ab), with mitochondrial abnormalities and expansion of the endoplasmic reticulum. Interestingly, a new kind of abnormal mitochondria was found in the DHEA and met-treated groups, referred to as “tubular/honeycombs” (orange, Figure 8 f7, c9, d9, f9) exhibiting thicker and distended mitochondrial cristae. Ultrastructural observations of stroma cells showed a larger volume and significantly increased density of lipid droplets in the DHEA group compared with the control group, and mitochondria also displayed “honeycomb” cristae. All of these findings suggested that various cells of growing follicles had been damaged in the DHEA group, and was associated with mitochondrial and liposomal abnormalities. The PA and Met treatments effectively reduced this damage, and PA significantly prevented the mitochondrial damage. Effects of PA on insulin and glucose metabolism-related genes in ovarian tissue Quantitative real-time PCR (q-PCR) is used to quantify important ovarian genes in groups after various treatments. We selected six genes that encoded proteins involved in either glucose uptake or insulin signaling pathways. Expression was normalized using Gapdh expression. Figure 9 shows that compared with the control group, the expression of insulin receptor substrate 1 (IRS-1), Glucose Transporter 4 (GLUT4) decreased, and the expression of 17 hydroxylase (CYP17) increased in DHEA group, indicating that the ovaries of PCOS mice have obvious insulin resistance. At the same time, the increased expression of TNF-α also indicates that the ovary is in an inflammatory state. In the PA group, the expression of IRS-1 and GLUT4 increased significantly (P < 0.01) compared with the DHEA group, with the expression of CYP17 decreased significantly (P < 0.01), revealing that PA can effectively improve the insulin resistance of PCOS mice ovary. In addition, after PA treatment, the expression of TNF-α also decreased, although it did not reach a statistical difference. PA reduced pro-inflammatory cytokines in adipose tissue The mRNA levels of TNF-α and IL-6 in adipose tissue were measured by q-PCR, and protein levels were observed by western blotting. As shown in Figure 10C, the mRNA levels of TNF-α and IL-6 were significantly higher in the DHEA-treated group compared with the control group. The RNA levels of these pro-inflammatory cytokines were significantly reduced to near-normal levels in the PA-treated group (TNF-α, P = 0.09; IL-6, P < 0.01), but not in the Met-treated group (TNF-α, P = 0.9; IL-6, P = 1), compared with the DHEA group. Interestingly, the expression of these cytokine proteins was decreased in the PA- and Met-treated groups compared with the DHEA group, but the reductions were not statistically significant (Figure 10A, B). Discussion In the present study, we successfully established a PCOS mice model exhibiting characteristics of obesity, insulin resistance, and chronic low-grade inflammation through the subcutaneous injection of DHEA. Using this model, we explored the effects of PA on PCOS mice and found that it reduced various PCOS-like characteristics. Furthermore, we observed changes in ovarian microenvironment in DHEA-induced PCOS mice with or without PA treatment by measuring inflammation endpoints and hormone levels. We also examined the potential therapeutic effects of PA on the developmental quality of oocytes and embryos. This study found that PA improved the developmental quality of oocytes in the PCOS mouse model in part by improving the ovarian microenvironment. The alteration of many factors may impair the ovarian microenvironment. It is well known that hormonal disorders contribute to the altered ovarian microenvironment of PCOS patients, including high levels of AMH, E2, and T [15–17]. In our study, PA-treatment decreased the high levels of serum hormones found in the PCOS mouse model. The aggravated inflammatory signaling was also associated with PCOS. These inflammatory factors can affect the production of reactive oxygen species and oxidative stress [18], which in turn may severely damage the ovarian environment and cause cell apoptosis. In our study, the serum level of TNF-α was reduced in the PA-treated group. In addition, compared with the model group, the protein expression of TNF-α and IL-6 in the PA-treated and Met-treated groups were reduced, indicating the improvement of inflammation in PCOS model. Moreover, a previous study showed that the insulin-like activities of PA stimulated glucose uptake [2, 3], and we found that the level of circulating insulin was reduced after PA treatment, with an improved OGTT in the DHEA-induced PCOS model. In addition, we selected six genes that encoded proteins involved in either glucose uptake or insulin signaling pathways. It was found that in the DHEA group, the expression of IRS-1, CYP-17 and TNF-α were significantly increased, and the expression of glucose transporter 4 (GLUT-4) and glycogen synthase kinase 3β (GSK-3β) were reduced, suggesting that PCOS mice have the characteristics of chronic inflammation and insulin resistance. After the PA treatment, it reversed the high expression of IRS-1 and CYP-17, along with the increased expression of GLUT-4, which improved insulin sensitivity. All these may involve the intracellular signaling cascade of glucose uptake, related to leptin-related AMPK pathway and PI3K signaling [19–21]. Next, we studied the effects of PA on the quality of oocytes in PCOS mice at various stages. Our study found elevated numbers of vesicular and atretic follicles, accompanied with higher rates of apoptotic granulosa cells, after DHEA treatment, consistent with previous reports [12]. We observed that PA improved the quality of oocyte development in DHEA-induced PCOS mice, reflected by an increased number of GV oocytes, and increased rate of GVBD and PB1 in vitro. For oocytes that developed to MII stage in vivo, the abnormal oocyte numbers and spindle morphology induced by DHEA were reduced by co-treatment with DHEA. Abnormal MII oocytes are considered the main cause of frequent miscarriages and congenital defects [22]. We studied the quality of early embryos, an indicator of suboptimal fetal and neonatal outcomes, and found marked apoptosis and very low expression of Oct4 in the PCOS mouse model [23]. These DHEA-induced parameters were partially reversed by co-treatment with PA. All of these observations may be due to PA-induced improvements in the ovarian microenvironment, including changes to hormone levels, insulin resistance and inflammation. According to recent studies, PA has several biological effects, including anticancer, anti-inflammatory, and antioxidant activity, as well as sedative effects. Researchers found that women with PCOS often suffer from psychological illness, and depression is commonly diagnosed [24]. Previous work showed PA has an antidepressant effect relating to its sedative actions. Oxidative stress caused by inflammation or self-induced oxidative damage plays an important role in pathological processes, including apoptosis and aging [25, 26]. The anti-oxidization effects of PA could reduce such damage. In conclusion, the therapeutic effect of PA in this PCOS model may involve multiple pathways, including the inhibition of inflammation and reduction of insulin resistance. Recently, metformin has been extensively studied and used to restore normal endocrine and other clinical parameters in patients with PCOS [27]. In this experiment, metformin was used as a positive control drug, to compare the effects of PA on PCOS. We found that PA has a similar effect to metformin, and prevented DHEA-induced damage in PCOS mice by reducing insulin resistance. In addition, PA had more pronounced anti-inflammatory effects than metformin. Importantly, according to the likely use of PA as a main active ingredient in TCM, PA may have fewer side effects (such as gastrointestinal stimulation, depression) and more long-term therapeutic potential in preventing metabolic disorders than metformin. Therefore, we believe that PA can be used as a potential candidate to improve the endocrine environment and oocyte quality of PCOS patients. We suggest that the combination of PA and metformin can be considered for clinical patients to improve the therapeutic effect. Conclusion In summary, PA protected oocytes and embryos from DHEA-induced damage by improving indicators in the ovarian environment associated with IR and inflammation. Future research should explore the mechanisms of PA in improving DHEA-induced damage, including identification of drug targets and cell signal transduction pathways, as well as carefully investigating potential side effects. Acknowledgments This work was supported by research grants from the National Natural Science Foundation of China (No. 31941006, 31772407, and 31672248). This study was also supported by grants from The National Key Research and Development Program of China under Grant No.2017YFD0501905. Author Contributions Hai-Long Wang and Chang-Long Xu designed the study and applied for Research Ethics Board approval. Xian-Pei Fu, Lin Xu, and Bin-Bin Fu collected and analyzed the data and prepared draft figures and tables. Kang-Na Wei, Yu Liu, and Bao-Qiong Liao prepared the manuscript draft with important intellectual input from Shu-Wen He, Ya-Long Wang, and Ming-Huang Chen. All authors approved the final manuscript. Yan-Hong Lin, Fei-Ping Li, Zi-Wei Hong, and Xiao-Hua Huang had complete access to the study data. Conflict of interest The authors have declared that no competing interest exists. Footnotes †Grant Support: The National Natural Science Foundation of China (No. 31941006, 31772407, and 31672248). The National Key Research and Development Program of China under Grant No.2017YFD0501905. References 1. Lee YS , Kim WS, Kim KH, Yoon MJ, Cho HJ, Shen Y, Ye JM, Lee CH, Oh WK, Kim CT, Hohnen-Behrens C, Gosby A et al. Berberine, a natural plant product, activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states . Diabetes 2006 ; 55 : 2256 – 2264 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Li TH , Hou CC, Chang CLT, Yang WC. Anti-Hyperglycemic properties of crude extract and triterpenes from Poria cocos . Evid Based Complement Alternat Med 2011 ; 2011 :128402. Google Scholar OpenURL Placeholder Text WorldCat 3. Shah VK , Choi JJ, Han JY, Lee MK, Hong JT, Oh KW. Pachymic acid enhances pentobarbital-induced sleeping behaviors via GABAA-ergic systems in mice . Biomol Ther 2014 ; 22 : 314 – 320 . Google Scholar Crossref Search ADS WorldCat 4. Hirshfield AN . Development of follicles in the mammalian ovary . Int Rev Cytol 1991 ; 124 : 43 – 101 . Google Scholar Crossref Search ADS PubMed WorldCat 5. Qiao J , Feng HL. Extra- and intra-ovarian factors in polycystic ovary syndrome: impact on oocyte maturation and embryo developmental competence . Hum Reprod Update 2011 ; 17 : 17 – 33 . Google Scholar Crossref Search ADS PubMed WorldCat 6. Aghahosseini M , Aleyaseen A, Safdarian L, Moddaress-Hashemi S, Mofid B, Kashani L. Metformin 2,500 mg/day in the treatment of obese women with polycystic ovary syndrome and its effect on weight, hormones, and lipid profile . Arch Gynecol Obstet 2010 ; 282 : 691 – 694 . Google Scholar Crossref Search ADS PubMed WorldCat 7. Huang Y , Sun J, Wang X, Tao X, Wang H, Tan W. Asymptomatic chronic gastritis decreases metformin tolerance in patients with type 2 diabetes . J Clin Pharm Ther 2015 ; 40 : 461 – 465 . Google Scholar Crossref Search ADS PubMed WorldCat 8. Ma J , Liu J, Lu CW, Cai DF. Pachymic acid induces apoptosis via activating ROS-dependent JNK and ER stress pathways in lung cancer cells . Cancer Cell Int 2015 ; 15 . Google Scholar OpenURL Placeholder Text WorldCat 9. Wang FY , Lv WS, Han L. Determination and pharmacokinetic study of Pachymic acid by LC-MS/MS . Biol Pharm Bull 2015 ; 38 : 1337 – 1344 . Google Scholar Crossref Search ADS PubMed WorldCat 10. Dowling AR , Nedorezov LB, Qiu XL, Marino JS, Hill JW. Genetic factors modulate the impact of pubertal androgen excess on insulin sensitivity and fertility . Plos One 2013 ; 8 . Google Scholar OpenURL Placeholder Text WorldCat 11. Luchetti CG , Solano ME, Sander V, Arcos MLB, Gonzalez C, Di Girolamo G, Chiocchio S, Cremaschi G, Motta AB. Effects of dehydroepiandrosterone on ovarian cystogenesis and immune function . J Reprod Immunol 2004 ; 64 : 59 – 74 . Google Scholar Crossref Search ADS PubMed WorldCat 12. Huang Y , Yu Y, Gao JM, Li R, Zhang CM, Zhao HC, Zhao Y, Qiao J. Impaired oocyte quality induced by Dehydroepiandrosterone is partially rescued by metformin treatment . Plos One 2015 ; 10 :e0122370. Google Scholar OpenURL Placeholder Text WorldCat 13. Dioguardi CC , Uslu B, Haynes M, Kurus M, Gul M, Miao DQ, De Santis L, Ferrari M, Bellone S, Santin A, Giulivi C, Hoffman G et al. Granulosa cell and oocyte mitochondrial abnormalities in a mouse model of fragile X primary ovarian insufficiency . Mol Hum Reprod 2016 ; 22 : 384 – 396 . Google Scholar Crossref Search ADS PubMed WorldCat 14. Ma WW , Xiao J, Song YF, Ding JH, Tan XJ, Song KK, Zhang MM. Effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation implantation dysfunction . J Huazhong Univ Sci Technol Med Sci 2017 ; 37 : 401 – 406 . Google Scholar Crossref Search ADS WorldCat 15. Dumesic DA , Padmanabhan V, Abbott DH. Polycystic ovary syndrome and oocyte developmental competence . Obstet Gynecol Surv 2008 ; 63 : 39 – 48 . Google Scholar Crossref Search ADS PubMed WorldCat 16. Hardy K , Robinson FM, Paraschos T, Wicks R, Franks S, RML W. Normal development and metabolic-activity of preimplantation embryos in-vitro from patients with polycystic ovaries . Hum Reprod 1995 ; 10 : 2125 – 2135 . Google Scholar Crossref Search ADS PubMed WorldCat 17. Patel SS , Carr BR. Oocyte quality in adult polycystic ovary syndrome . Semin Reprod Med 2008 ; 26 : 196 – 203 . Google Scholar Crossref Search ADS PubMed WorldCat 18. Nteeba J , Ganesan S, Keating AF. Progressive obesity alters ovarian Folliculogenesis with impacts on pro-inflammatory and steroidogenic Signaling in female mice . Biol Reprod 2014 ; 91 . Google Scholar OpenURL Placeholder Text WorldCat 19. McDonald CA , Millena AC, Reddy S, Finlay S, Vizcarra J, Khan SA, Davis JS. Follicle-stimulating hormone-induced aromatase in immature rat sertoli cells requires an active phosphatidylinositol 3-kinase pathway and is inhibited via the mitogen-activated protein kinase signaling pathway . Mol Endocrinol 2006 ; 20 : 608 – 618 . Google Scholar Crossref Search ADS PubMed WorldCat 20. McGee EA , Hsueh AJW. Initial and cyclic recruitment of ovarian follicles . Endocr Rev 2000 ; 21 : 200 – 214 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 21. Reddy P , Zheng WJ, Liu K. Mechanisms maintaining the dormancy and survival of mammalian primordial follicles . Trends Endocrinol Metab 2010 ; 21 : 96 – 103 . Google Scholar Crossref Search ADS PubMed WorldCat 22. Bennabi I , Terret ME, Verlhac MH. Meiotic spindle assembly and chromosome segregation in oocytes . J Cell Biol 2016 ; 215 : 611 – 619 . Google Scholar Crossref Search ADS PubMed WorldCat 23. Liu L , Czerwiec E, Keefe DL. Effect of ploidy and parental genome composition on expression of Oct-4 protein in mouse embryos . Gene Expr Patterns 2004 ; 4 : 433 – 441 . Google Scholar Crossref Search ADS PubMed WorldCat 24. Yu QX , Hao SX, Wang HM, Song X, Shen QY, Kang JH. Depression-like behavior in a Dehydroepiandrosterone-induced mouse model of polycystic ovary syndrome . Biol Reprod 2016 ; 95 :79. Google Scholar OpenURL Placeholder Text WorldCat 25. Agarwal A , Gupta S, Sharma R. Oxidative stress and its implications in female infertility - a clinician's perspective . Reprod Biomed Online 2005 ; 11 : 641 – 650 . Google Scholar Crossref Search ADS PubMed WorldCat 26. Tripathi A , Khatun S, Pandey AN, Mishra SK, Chaube R, Shrivastav TG, Chaube SK. Intracellular levels of hydrogen peroxide and nitric oxide in oocytes at various stages of meiotic cell cycle and apoptosis . Free Radic Res 2009 ; 43 : 287 – 294 . Google Scholar Crossref Search ADS PubMed WorldCat 27. Duleba AJ . Medical management of metabolic dysfunction in PCOS . Steroids 2012 ; 77 : 306 – 311 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Xian-Pei Fu and Lin Xu contributed equally to this work. © The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Pachymic acid protects oocyte by improving the ovarian microenvironment in polycystic ovary syndrome mice
JF - Biology of Reproduction
DO - 10.1093/biolre/ioaa141
DA - 2020-10-29
UR - https://www.deepdyve.com/lp/oxford-university-press/pachymic-acid-protects-oocyte-by-improving-the-ovarian-MGZSEncJCM
SP - 1085
EP - 1098
VL - 103
IS - 5
DP - DeepDyve
ER -