We commissioned the company (Si Wei Tech Bio Co., Ltd, Zhengzhou, Henan, China) to mix clove extract, Sophora flower bud extract and Chinese yam extract in a mass ratio of 15:6:10 to make DACHAO. The content of eugenol in clove extract was 90%, the content of quercetin in Sophora flower bud extract was 95%, and the content of diosgenin in Chinese yam extract was 15%. All the active contents were qualified by high performance liquid chromatography ( Figure 1A ). The chemical structural formulas of these bioactive compounds are displayed in Figures 1B–D . Thus, in every 100 mg of DACHAO, the content of eugenol is 43.55 mg, the content of quercetin is 18.39 mg, and the content of diosgenin is 4.84 mg. The ratio of this product is determined according to gavage dosage of each component per mouse. The dosage of each plant extract on mouse refers to the previous reports (30–33).

All mice were obtained from the Model Animal Research Center of Nanjing Medical University (Nanjing, Jiangsu, China) and housed in the animal facility at Nanjing Medical University. Mice were maintained on a 12-h light: 12-h darkness cycle and were provided with food and water ad libitum. Except for mice in mating trails, others were group housed with up to five mice per cage. Adult male B6D2F1 mice (10–14 weeks old) were chosen as sperm donors for IVF experiment. All experiments requiring the use of animals were approved by the Committee on the Ethics of Animal Experiments of Nanjing Medical University.

40W ICR female mice were randomly divided into control group and treatment group, with 60 mice in each group. In addition to the daily diet, mice were gavaged one time at 10:00 am every day. In treated group, each mouse was gavaged with 0.5 ml normal saline contained DACHAO (the plant extracts) at a dose of 310 mg/kg BW. In control group, mice were gavaged with the same volume of normal saline. All mice were treated for one month. At the end of the experiment, serum from 8 mice in the interphase were collected for relevant testing. A random selection of 5 mice were co-caged mating for fertility testing. A random selection of 8 mice were euthanized and the ovaries of them were collected for histomorphological and molecular experiments. The remaining mice were superovulated and euthanized in batches when needed, then oocytes were collected for experiments ( Figure 1E ). To test the biosafety of DACHAO, we additionally prepared ten 40W aged female ICR mice to gavage with 2 times the dose of DACHAO for one month. After the gavage, serum from 5 mice in the interphase were collected and detected the liver and kidney function and blood lipid-related indicators with the DACHAO group and the control group.

48 hours after intraperitoneal injection with eCG, the mice were intraperitoneal injected with hCG. 14 hours after the hCG injection, the mice were euthanized and the ovulated oocytes were collected in the ampulla of the fallopian tubes from both sides.

Ovarian proteins were extracted by radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime Institute of Biotechnology) containing protease inhibitor cocktails (M221, Amresco). Protein was quantified by using the BCA protein quantification kit (BI-WB005-500T, Biochannel, Nanjing, China). A total of 10 ug of proteins in each sample was loaded and separated by electrophoresis (165-8000, Bio-Rad, USA). Proteins were separated by electrophoresis and then electronically transferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% skimmed milk-TBST (TBS containing 0.1% Tween 20) for 60 min and incubated overnight at 4°C with specific antibodies. After washing with Tris-buffered saline with Tween 20 (TBST) (5 mL) three times, the horseradish peroxidase (HRP)-conjugated relative secondary antibodies were then used to detect proteins through enhanced chemiluminescence (RPN2232, GE Healthcare, USA) on the Tanon 5200 analysis system. Antibodies against PARP (9542, CST), cleaved-PARP (5625, CST), caspase-3 (9665, CST), cleaved-caspase-3 (9664, CST), CAT (D122036, Sangon Biotech, Shanghai, China), GSS (D154022, Sangon Biotech, Shanghai, China), SOD-1(Bs6057, Bioworld, Shanghai, China) were all rabbit antibodies. Antibodies against β-Actin (YFMA0052, YIFEIXUE, Nanjing, China) were all mouse antibodies.

Mouse ovaries were fixed in 10% buffered formalin for paraffin embedding and sectioning. After deparaffinization and rehydration, sections were processed for blocking of endogenous peroxidase activity and antigen retrieval pretreatment. To detect the expression of PCNA, immunohistochemical analyses were performed using a Histo stain Histostain Kit (856743, Invitrogen) with antibodies against PCNA (13110, CST) overnight at 4C. Diaminobenzidine (DAB) reagent was used for coloration on the second day. Non-immune immunoglobulin G (IgG) was applied as a negative control and antigen retrieval pretreatment.

To determine the ROS content, mitochondrial membrane potential and mitochondrial distribution of oocytes, the procedure was conducted according to the protocols with the ROS kit (50101ES01, Yeasen, Shanghai, China), the JC-1 assay kit (40706ES60, Yeasen, Shanghai, China) and the MitoTracker (40740ES50, Yeasen, Shanghai, China), respectively. First, MII oocytes were collected from ampulla of fallopian tube and immediately washed with M2 medium 3 times. Then oocytes were transferred into M2 medium containing staining solution and incubated at 37 C in the dark for 30 min. After that, oocytes were washed with M2 medium to remove the excess staining solution. All of the live oocytes were observed by a laser scanning confocal microscope (LSM800, Zeiss, Germany). The relative fluorescence intensity was calculated by software of ZEN.

For IVF studies, donor sperm was collected from B6D2F1 male mice into human tubal fluid (HTF) media (MR-070-D, Millipore, USA) and incubated under oil for 1 h at 37°C in 5% CO2 for capacitation. MII oocytes were collected from ampulla of fallopian tube and then placed into 200 mL of media with sperm (2–3 ×105/mL) for 8 h. After fertilization, zygotes with clear pronuclei were transferred into fresh HTF media overnight until the two-cell embryonic stage. Two-cell embryos were then cultured in small droplets of KSOM media (MR-020P-5F, Millipore, USA) to blastocyst stage.

Total RNAs from ovaries were purified by TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. RNA concentrations were measured by a spectrophotometer (NanoDrop 2000c, Thermo Scientific, USA). Subsequently, the FastQuant RT kit (Tiangen Biotech, China) was applied to produce cDNA from RNA. The target gene transcripts (Amhr, Fshr, Lhr, Star, SOD-1, CAT, GSS, IL-6, TNFα, iNOS) were detected using SYBR Green mix (Applied Biological Materials, Canada) through an ABI StepOnePlus platform (Thermo Scientific, USA) based on the manufacturer’s protocols. Quantification of various mRNAs was performed by using the actin amplification signal as control. The relative expression of target genes was measured by the 2–ΔΔCT method. All of the primer sequences used are listed in Table S1 .

Mouse ovaries were collected and fixed in 10% buffered formalin for 12 hours, embedded in paraffin, serially sectioned at a thickness of 5 μm, and were then stained with HE for quantification of follicles at each level. Follicle classification and counting are as follows: Follicles were classified as primordial if they contained an oocyte surrounded by a partial or complete layer of squamous granulosa cells. Primary follicles showed a single layer of cuboidal granulosa cells. Oocytes sur-rounded by more than one layer of cuboidal granulosa cells with novisible antrum were determined to be secondary follicles. Antral follicles possessed a clearly defined antral space and a cumulus granulosa cell layer. Corpora lutea were filled with lutein cells. Follicles were considered atretic if they contained either a degenerating oocyte, disorganized granulosa cells, pyknotic nuclei, shrunken granulosa cells, or apoptotic bodies (34). The results are reported as a percentage of follicles at each level counted per ovary.

After intraperitoneal injection of anesthesia into mice, about 1 ml of venous blood was withdrawn via orbital puncture, and left standing in an EP tube for more than one hour at room temperature. Then the venous blood was centrifuged at 3000 rpm for 10 minutes. The supernatant we collected was serum. The collected serum was divided into two parts. One part was measured by AMH Elisa kit according to the manufacturer’s instructions (YIFEIXUE, YFXEM00802, China). The other part was frozen at -80°C and sent to Beijing Northern Biotechnology Research Institute Co., Ltd to detect serum FSH and E2 hormone levels (35).

TUNEL immunofluorescence was done by standard kits according to manufacturer’s instructions(40307ES60, Yeasen, Shanghai, China) and were viewed under a laser scanning confocal microscope (LSM 800 META, Zeiss, Germany). TUNEL positive cells were counted by using Image-pro plus 6.0 at different microscopic fields (n=5) for n=3 sections.

GraphPad Prism 6.0 and SPSS 20.0 were used to perform the T test to evaluate differences between groups. Data are mean ± SEM. Statistical significance was set at a probability (p) value< 0.05.

To see the effect of DACHAO compound on the ovarian functions of aged mice. We established a mouse model with the gavage of DACHAO at 310 mg/kg BW to 10-month-old female mice for one month. After finishing the protocol, we found no statistical differences on the body weights between the two groups ( Figure S1A ). The mice in control and treated groups were then given PMSG/HCG for superovulation and mature oocytes were washed from the ampulla of the fallopian tube 14 hours after HCG injection. Ovulated oocyte counting and oocyte fragmentation rate were evaluated and the results showed not only the increased numbers of ovulated oocytes, but also the significant reduction of fragmented oocytes in DACHAO treated mice. In DACHAO treated group, the number of ovulated oocytes per mouse increased from 4.11 to 7.64, meanwhile the fragmentation rate of these oocytes reduced from 30.71 to 14.47 ( Figures 2A, B ). To further assess the ovulated oocyte quality, about oocytes in each group were then labeled with MitoTracker to check the distribution of mitochondria. We found that the ratio of oocytes with abnormal mitochondrial distribution (mitochondria over-concentrated around the nucleus) was reduced in the DAOCHAO-administered group, with the ratio decreasing from 60.19% to 34.40% ( Figures 2C, D ). In another set of experiments, a ROS green fluorescent probe was employed to label and detect the level of oxidative stress in about 30 oocytes of each group. We assessed the ROS green fluorescence intensity and performed statistical analysis. The results showed that DACHAO treatment reduced the senescence-induced increase in the ROS levels in the oocytes ( Figures 2E, F ). A JC-1 fluorescent probe was also used to label and detect the mitochondrial membrane potential in the oocytes, about 30 of each group. We determined the ratio of the red/green fluorescence in two groups of oocytes and DACHAO improved the aging-caused alterations on mitochondrial membrane potential in the oocyte ( Figures 2G, H ).

Next, IVF and embryo culture were performed to evaluate the developmental potential of oocytes in each group. About 30-50 mature oocytes with good morphology in each group were selected for IVF and the result showed no difference on the rate of 2-cell embryonic development, however, DACHAO treatment showed its beneficial effect on embryonic development which was shown by increased embryonic development at 4–8-cell, morula, and blastocyst stages, as the increasing ratio from 36.67% to 48.48% on the 4-8 cell stage; the increasing ratio from 23.33% to 40.15% on the morula stage; the increasing ratio from 23.33% to 48.48% on the blastocyst stage( Figures 3A, B ). We then collected blastocysts in each group for TUNEL analysis and Hoechst 33342 were counterstained to label nuclei for cell counting. The results revealed DACHAO treatment significantly decreased apoptosis and increased total cell number in blastocyst. In DACHAO treated group, the number of TUNEL positive cells reduced from 14.33 to 3.33 cells of each blastocyst and the total cell number increased from 14.33 to 25.00 cells of each blastocyst. ( Figures 3C–E ).

As compared with control group, we observed higher percentages of primordial follicles, growing follicles and corpus luteum of mice ovaries in DACHAO group (primordial follicles: 5.17% vs. 7.38%; growing follicles: 1.72% vs. 5.28%; corpus luteum: 4.26% vs. 6.95%; Ctrl vs. DACHAO) ( Figure 4A, B ). To see if the in vivo treatment of DACHAO has any effect on fertility of aged mice, the mice in each group were mated with fertile male mice, and their litter size were recorded ( Figures 4C ). As shown in Figures 4D, E , in control group, only 40% mice were mated and the average pups was 1.6 per female, whereas all mice treated DACHAO were pregnant with average 8.4 pups delivered. Moreover, the increased weaning weight of the offspring was also observed in DACHAO treated mice ( Figure 4F ). In conclusion, the DACHAO treatment improved the fertility of aged mice.

Since follicular developed was improve in DACHAO treated mice, PCNA immunostaining was then performed to examine cell proliferation in the two groups. We found that in the treated group, the number of PCNA positive signal points around the follicles was more than that in the control group at growing follicle stage ( Figure 5A ). The results suggest that DACHAO administration can increase the proliferation of granulosa cells of growing follicles. To detect cell apoptosis in control and treated ovaries, western blot was performed to check the expression of apoptosis related proteins and the results showed that the expression of cleaved-caspase-3 and cleaved-PARP in the ovaries of DACHAO-treated mice was much lower than that of the control group ( Figures 5B–D ). Consistent with the western blot results, TUNEL staining on the ovaries from the two groups of mice further demonstrated the apoptosis in DACHAO treated group was lower than that in the control group ( Figures 5E, F ). Pictures from negative control and positive control (DNase-treated) for TUNEL assay are shown in Figure S2 . This indicates that DACHAO administration can reduce the apoptotic levels in the aged ovaries. DACHAO enhanced the fertility by improving serum sex hormone levels and ovarian expression of hormone-related mRNAs.

Since better follicular development was observed after DACHAO treatment, we then randomly selected mice in each group whose estrous cycle was at the interphase and collected venous blood samples for the measurement of serum AMH, FSH, and E2 levels by ELISA. The results showed that DACHAO treatment increased the serum AMH and E2 hormone levels in aged mice, with the value of AMH level increasing from 376.69 ng/L to 580.61 ng/L and the value of E2 level increasing from 10.73 pg/ml to 19.33 pg/ml. Although a decreased trend of FSH hormone level was observed in DACHAO treated mice, there is no-statistical significance between the two groups ( Figures 6A–C ). To test the biosafety of DACHAO, we additionally prepared ten 40W aged female ICR mice to gavage with 2 times the dose of DACHAO for one month. After the gavage, we collected venous blood samples of these mice and detected the liver and kidney function and blood lipid-related indicators between the two groups. The results showed that normal dose and high dose of DACHAO treatment for one month had no adverse effects on liver and kidney function and blood lipid levels in mice ( Figure S1B ). We then performed qRT-PCR analysis of steroid hormone-related mRNA expressions. DACHAO treatment demonstrated the increase of Amhr and Fshr mRNAs in the ovary ( Figures 6D–G ). Thus, the results suggest the improvement of granulosa cell function after DACHAO treatment.

Due to the improvement of mitochondria function in DACHAO treated oocytes, the effect of DACHAO on ovarian oxidative stress was evaluated by qRT-PCR and we found the increased expression of some antioxidant factors SOD-1 (Superoxide dismutase 1), CAT (Catalase), and GSS (Glutathione synthetase) mRNAs in DACHAO treated ovaries ( Figures 7A–C ). Western blot further manifested the elevated protein levels of SOD-1, CAT, and GSS in the ovaries of DACHAO treated mice ( Figures 7D–G ). In addition, qRT-PCR also revealed the decreased expression of some inflammatory factors, such as IL-6, TNFα, and iNOS in DACHAO treated ovaries ( Figures 7H–J ). In conclusion, DACHAO treatment could alleviate the oxidative stress and the inflammatory environment in the aged ovary.