ZHANG Liang,HAN Qian,YI Yanxiao,JI Shaoyang,XIN Mingwei

ZHANG Liang,Department of Clinical Laboratory Medicine,Beijing Hospital of Integrated Traditional Chinese and Western Medicine,Beijing 100039,China

HAN Qian,Beijing Obstetrics and Gynecology Hospital,Capital Medical University,Beijing Maternal and Child Health Care Hospital,Beijing 100026,China

YI Yanxiao,Beijing Obstetrics and Gynecology Hospital,Capital Medical University,Beijing Maternal and Child Health Care Hospital,Beijing 100026,China

JI Shaoyang,Institute of Process Engineering,Chinese Academy of Sciences,Beijing 100190,China

XIN Mingwei,Beijing Obstetrics and Gynecology Hospital,Capital Medical University,Beijing Maternal and Child Health Care Hospital,Beijing 100026,China

Abstract OBJECTIVE:To primarily explore the effect and mechanism of Wenshen Yangxue decoction (温肾养血方)in promoting follicular development in elderly female mice.METHODS:Fifty Institute of Cancer Research mice were randomly divided into blank,controlled ovarian hyperstimulation (COH),low-dose Wenshen Yangxue decoction,medium-dose Wenshen Yangxue decoction,and high-dose Wenshen Yangxue decoction groups,with 10 mice in each group.The number of ovulations,number of fertilizations,mitochondrial adenosine triphosphate(ATP) level,and mitochondrial DNA (mtDNA) of oocytes in each group were compared.Reverse transcription- polymerase chain reaction and Western blotting were used to detect the mRNA and protein expression levels of silent information regulator 3 (Sirt3) and forkhead transcription factor O1 3a (FOXO3a).RESULTS:Wenshen Yangxue decoction significantly increased the number of ovulations in mice (P <0.05) and promoted the formation of fertilized eggs.The ATP level and mtDNA copy number of mice oocytes in the highdose groups were significantly higher than those in the COH group (P <0.05).Wenshen Yangxue decoction significantly increased the mRNA and protein levels of Sirt3 and FOXO3a in mouse oocytes.CONCLUSION:Wenshen Yangxue decoction promoted the development of follicles in elderly female mice,increased the number of ovulations and improved fertility.Its mechanism may be related to increased mitochondrial energy metabolism and regulation of the Sirt3/FOXO3a pathway.

Keywords:aging;silent information regulator 3;forkhead transcription factors;oocytes;Wenshen Yangxue decoction

1.INTRODUCTION

Egg aging can lead to low egg development potential,which affects the egg’s fertilization rate,cleavage rate,and embryonic development quality,leading to a significant decrease in pregnancy rate.Eggaging is a preapoptotic state.Oocyte aging is manifested as mitochondrial swelling,reduced the number of cristae,decreased intimal membrane potential,increased matrix density,decreased adenosine triphosphate (ATP)production,increased mitochondrial DNA (mtDNA)mutations,and decreased expression of adenylate transferase on the inner mitochondrial membrane.1Silent information regulator 3 (Sirt3) is mainly expressed in the mitochondria and is involved in the regulation of mitochondrial metabolism,ATP production,and oxidative stress.Decreased Sirt3 expression induces a decrease in the uptake of stereospecific DNA copies by oocytes and biogenesis,leading to a decrease in oocyte quality.2Forkhead transcription factor O1 3a (FOXO3a)regulates mitochondrial function and helps inhibit follicle activation during the early stages of follicle growth.3FOXO3a acts as a substrate for Sirt3 and is closely associated with oocyte growth,development,and maturation.4

Wenshen Yangxue decoction (温肾养血方) has exhibited significant effects in the treatment of infertility;these effects have been verified by a large number of clinical studies for more than 30 years.5In a clinical study,we found that the Wenshen Yangxue decoction reduced the levels of basic follicle stimulating hormone (bFSH),the bFSH/basic luteinizing hormone (bLH) ratio,and the integrals of Traditional Chinese Medicine (TCM)syndrome in elderly patients with poor ovarian response to in vitro fertilization and embryo transfer (IVF-ET).It also increases the antral follicle number (AFC),anti-Müllerian hormone (AMH) level,the number of eggs obtained,the number of fertilized eggs,and Estradiol (E2)level on human chorionic gonadotropin (HCG) day (P<0.01).6In another study,Wenshen Yangxue decoction improved the ovarian reserve function of rats with diminished ovarian reserve (DOR).The histomorphology of the ovary was significantly improved,the follicle count was significantly increased,the number of atretic follicles was significantly decreased,the levels of serum FSH and LH were significantly decreased,and the levels of E2,AMH,and inhibin B (INHB) were significantly increased (P <0.01).7The decline of oocyte quality is closely related to the mitochondria.In this study,to study the relationship between mitochondria and egg aging,the effects of Wenshen Yangxue decoction were investigated on the number of follicles,fertilization rate,cleavage rate,blastocyst formation rate,abnormal fertilized egg rate,and other factors related to oocyte quality and embryonic development potential of elderly Institute of Cancer Research (ICR) mice.The results of this study provide an experimental basis for the clinical application of Wenshen Yangxue decoction that the Chinese medicine theory that the kidney governs reproduction.

2.MATERIALS AND METHODS

2.1.Drugs and reagents

Components of the Chinese herbal medicine — Wenshen Yangxue decoction — were purchased from Beijing Tongrentang Co.,Ltd.;it contains Chaihu (Radix Bupleuri Chinensis) 6 g,Xiangfu (Rhizoma Cyperi) 10 g,Muxiang (Radix Aucklandiae) 6 g,Chishao (Radix Paeoniae Rubra) 10 g,Zelan (Herba Lycopi Hirti) 10 g,Yimucao (Herba Leonuri Japonici) 12 g,Niuxi (Radix Achyranthis Bidentatae) 10 g,Jixueteng (Caulis Spatholobi) 15 g,Honghua (Flos Carthami) 10 g,Danshen (Radix Salviae Miltiorrhizae) 10 g,Danggui(Radix Angelicae Sinensis) 10 g,Chuanxiong (Rhizoma Chuanxiong) 10 g,Puhuang (Pollen Typhae) 10 g,Dihuang (Radix Rehmanniae) 10 g,Yinyanghuo (Herba Epimedii Brevicornus) 10 g,Shanzhuyu (Fructus Corni)10 g,Tusizi (Semen Cuscutae) 10 g,Gouqizi (Fructus Lycii) 10 g,Fupenzi (Fructus Rubi Chingii) 10 g,Lujiao(Cornu Cervi Elaphi) 15 g,Qianghuo (Rhizoma et Radix Notopterygii) 6 g,Xixin (Herba Asari Mandshurici) 3 g,and Roucongrong (Herba Cistanches Deserticolae) 10 g.Appropriate proportions of the Chinese medicine components were weighed and mixed.Subsequently,all crude herbs were soaked in distilled water for 1 h.Lujiao(Cornu Cervi Elaphi) was decocted by boiling for 40 min first.Lujiao (Cornu Cervi Elaphi) mixed with other crude herbs the mixture was boiled for another 20 min.Following filtration using a gauze,the drugs were decocted for 15min with an equal volume of distilled water.The filtrate after two rounds of filtration was mixed and concentrated on evaporation to 1.68,3.33,and 6.76 g/mL for the low-dose,medium-dose,and highdose groups,respectively.The Chinese medicine was prepared in the Chinese Medicine Preparation Laboratory of the School of Traditional Chinese Medicine,Capital Medical University;concentrated into a medicinal solution after decoction;and stored in a refrigerator at 4 ℃for later use.Pregnant horse serum gonadotropin (pregnant mare serum gonadotropin,PMSG) and HCG (human chorionic gonadotrophin,HCG) were purchased from Prospec (East Brunswick,NJ,USA).

2.2.Test grouping

In this study,the mouse model was developed as per the methods described in previous studies.8,9Fifty 8-monthold ICR mice [Beijing Huafukang Biotechnology Co.,Ltd.,experimental animal license number:Scxk (Beijing)(2020-0004)],were randomly divided into five groups,with 10 mice in each group:Group A,blank group (Ctrl group);group B,controlled ovarian hyperstimulation(COH);group C,low-dose Wenshen Yangxue decoction group (16.75 g/kg,low group);group D,medium-dose Wenshen Yangxue decoction group (33.5 g/kg,medium group);and group E,high-dose Wenshen Yangxue decoction group (67 g/kg,high group);groups C,D,and E received the same ovarian hyperstimulation as group B.Groups A and B were given normal saline by gavage every day.Groups C,D,and E were given corresponding Chinese medicine suspensions.Mice in each group were reared in separate cages with food and water providedad libitum.The temperature of the rearing room was maintained at (20 ± 1) ℃,the relative humidity was approximately 50%,and a photoperiod of 14 L:10 D was adopted.

2.3.Administration and collection of oocytes

Continuous daily (6 pm) intragastric administration was performed for 10 d.On the 11th day,the female mice in groups B,C,D,and E were injected at the same time with PMSG (10 IU per mouse).The female mice in group A were injected simultaneously with 0.9% (mass fraction)sodium chloride.Forty-eight hours later,HCG (10 IU per mouse) was injected into the abdominal cavity.Fourteen hours later,the mouse ovaries and fallopian tubes were removed by laparotomy,and the ampulla of the fallopian tube was gently cut with a sterile TB needle to collect the cumulus complex.The cumulus complex was treated with plasminase.Then,the cumulus cells around the oocytes were removed;after repeated pipetting with a suction tube,the oocytes removed with granular cells were counted under a microscope.Oocyte morphology was observed under the microscope,and the number of superovulated mice were counted.

2.4.Fertilization and embryo culture

This experiment was performed on 8-9-week-old male ICR mice,who had under gone opening of the scrotum,and removal of the epididymis and vas deferens.These organs were torn in culture medium containing 10%nHTF to allow the sperm to swim out,and the sperms were then incubated at 37 ℃ and 5% CO2in an incubator for 1-1.5 h to obtain energy and reserve.(a) The mature oocytes were transferred into the fertilization drop and fertilized with 2-5×106live sperm per milliliter for 4-6 h.(b) To remove the granular cells around the fertilized egg,100 IU/mL hyaluronidase was added.(c) The fertilized eggs were then observed under a microscope.Without the formation of a pronucleus and the discharge of the second polar body,the fertilization rate was calculated.(d) The fertilized eggs were washed in liquid culture medium and cultured for 96-120 h.Then,the embryo development was observed,and the fertilization rate,2-cell rate,abnormal egg rate,and blastocyst formation rate were calculated.

2.5.Reverse transcription-polymerase chain reaction(RT-PCR) detection

TRIzol (15596018,Ambion,Austin,TX,USA) reagent was used to extract total RNA from the oocytes.RNA was reverse transcribed into cDNA using the reverse transcription kit (KR103,Tiangen,Beijing,China) as per the manufacturer’s instructions.Gene expression was verified by a fluorescent quantitative PCR kit (FP205,Tiangen) in a real-time fluorescent quantitative PCR instrument (Lightcycler96,Roche,Basel,Switzerland).Glyceraldehyde-3phosphate dehydrogenase (GAPDH)was used as the internal reference control for Sirt3 and FOXO3a.The relative expression of genes was calculated by the 2-△△ctmethod.The primer sequences are shown in Table 1.

Table 1 Primer sequence

2.6.Western blotting

A protein extraction kit (P0013,Beyotime,Shanghai,China) was used to extract total protein from the mouse oocytes.A bicinchoninic acid (BCA) detection kit(P0012,Beyotime,Shanghai,China) was used to measure the concentration of the extracted protein.The samples were separated via 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane.Diluted Sirt3 (1:1000,ab246522,abcam,Beijing,China),FOXO3a (1:1000,ab154786,abcam,Beijing,China),and GAPDH (1:2500,ab8245,abcam,Beijing,China)primary antibodies were added to the membrane,and then,the membrane was incubated overnight at 4 ℃.The membrane was washed 3 times in TBS containing 0.1%Tween 20 (TBS-T) and then incubated for 1 h at 22-24 ℃.Western blots were prepared using Ultra High Sensitivity ECL Kit (MA0186,Meilunbio,Dalian,China);they were exposed to a gel imaging system (Bio-Rad,Hercules,CA,USA) for visualization.Quantitative analysis of the bands was done using the ImageJ software(v1.51,National Institutes of Health,Bethesda,MD,USA).

2.7.Measurement of mitochondrial energy metabolism in oocytes

The amount of ATP was measured using the ATP detection kit (S0026,Beyotime,Shanghai,China) as per the manufacturer’s instructions,and luminescence was measured using a microplate reader (Molecular Devices Corp.,Sunnyvale,CA,USA).To calculate the final concentration,the following formula was used:ATP(μmol/g mass)=0.625 × ΔA (absorbance value) ÷ ΔA standard ÷ W (mass).

2.8.Statistical analysis

Data are expressed as the mean ± standard deviation.SPSS 22.0 (IBM Corp.,Armonk,NY,USA) software was used to analyze the experimental data,and differences among samples were evaluated using analysis of variance and Duncan’s multiple comparison test,cardinality test for unclassified data,and rank sum test for classified data.P<0.05 was considered significant.

3.RESULTS

3.1.Effect of Wenshen Yangxue decoction on the number of ovulations and fertilization ability in mice

Statistical analysis revealed that compared to that in the COH group,significantly more ovulations were observed in the low-dose group (P<0.05).However,no significant changes were observed in the medium-dose and high-dose groups compared to those in the control group (Figure 1A);The fertilization rate of the mice in the Wenshen Yangxue decoction groups was increased to a certain extent,and a significant difference was observed between the medium-dose group and the COH group (P<0.01) (Figure 1B).Wenshen Yangxue decoction significantly decreased the proportion of abnormally fertilized eggs in mice,a significant difference was observed between the low-dose group and the COH group (P<0.01),and between the mediumdose group and the COH group (P<0.05).The proportion of abnormal eggs in the high-dose group was also lower than that in the control group,but the difference was not significant (P>0.05) (Figure 1C).The cleavage rate in mouse oocytes in the low-dose and medium-dose administration groups was significantly increased (P<0.05),although there was no significant change in the high-dose group (P>0.05) (Figure 1D).The blastocyst formation rate of mice in the mediumdose group was higher than that of the COH group and the control group (P<0.05).The low-dose group and the high-dose group had slightly higher values than the COH group,but the difference was not significant (P>0.05)(Figure 1E).

Figure 1 Effect of Wenshen Yangxue decoction on ovulation number and fertilization ability in older ICR mice

Figure 2 Effect of Wenshen Yangxue decoction on mitochondria on the oocytes of older ICR mice

Figure 3 Effect of Wenshen Yangxue decoction on the expression of mitochondria-related genes

3.2.Effect of Wenshen Yangxue decoction on the energy metabolism of oocyte mitochondria

Different doses of Wenshen Yangxue decoction were administered to older ICR mice,and the ATP and mtDNA copy numbers in mouse oocytes were measured.The test results showed that compared to that of the blank control group (i.e.,the natural ovulation group,Ctrl group),the amount of ATP in mouse oocytes in the COH group was significantly decreased (P<0.05),while the low-dose administration group and the high-dose administration group exhibited recovery;there was a significant difference between the high-dose group and the COH group (P<0.05).Compared to that of the control group,the copy number of mitochondrial mtDNA in mouse oocytes in the COH group was significantly reduced (P<0.05),and the copy number of mtDNA in the Wenshen Yangxue decoction groups was significantly increased.A significant difference was observed between the high-dose group and the COH group (P<0.05).These results suggest that COH affects the mitochondrial function of the oocytes of older ICR mice and Wenshen Yangxue decoction can enhance mitochondrial energy metabolism in the oocytes of older ICR mice.

3.3.Effect of Wenshen Yangxue decoction on the expression of mitochondria-related genes

Sirt3 and FOXO3a were detected in the oocytes of ICR mice after gavage with different doses of Wenshen Yangxue decoction.RT-PCR and Western blotting analyses revealed that compared to that of the Ctrl group,the relative expression of Sirt3 and FOXO3a mRNA and protein in the COH group were decreased,but the difference was not significant between the two groups(P>0.05).Sirt3 and FOXO3a mRNA and protein expression in the low-dose and high-dose Wenshen Yangxue decoction groups increased and was significantly higher than those of the COH group (P<0.05).

4.DISCUSSION

As per the TCM,decreasing ovarian reserve in the elderly women is related to the kidneys.The kidneys store essence that is funneled into reproduction:“Two essences interact and form together.” The formation of embryos depends on the innate essence of both men and women.10The main reason for the obvious decrease in fertility in elderly women is that the number and the quality of oocytes are significantly decreased due to the decline in ovarian function.This decline in oocyte quality is usually referred to as egg aging.11Egg aging can lead to low egg development potential,which negatively affects the fertilization rate,cleavage rate,and embryonic development quality of the egg,thereby leading to a significant decrease in the pregnancy rate.12Assisted reproductive technology can solve the fertility problems of patients including ovulation difficulties,endocrine abnormalities,oligospermia,and asthenospermia.However,the outcome of assisted conception for elderly women has been poor,and the key problem is that the eggs of elderly women are aging.Aging is a preapoptotic state.Oocyte aging is manifested as mitochondrial swelling,reduced cristae,decreased intimal membrane potential,increased matrix density,decreased ATP production,increased mtDNA mutations,and decreased expression of adenylate transferase on the inner mitochondrial membrane.13In the current study,the Wenshen Yangxue decoction warmed the kidneys to fill the essence,and invigorate and nourish the blood.Dihuang (Radix Rehmanniae),Roucongrong (Herba Cistanches Deserticolae),Lujiao(Cornu Cervi Elaphi),and Yinyanghuo (Herba Epimedii Brevicornus) nourish the kidneyYin,and tonethe kidney Yang.Tusizi (Semen Cuscutae),Gouqizi (Fructus Lycii),Fupenzi (Fructus Rubi Chingii),Shanzhuyu (Fructus Corni),and other liver and kidney supplements have effects similar to those of some other TCM,such as Chaihu (Radix Bupleuri Chinensis),Xiangfu (Rhizoma Cyperi),Honghua (Flos Carthami),Danshen (Radix SalviaeMiltiorrhizae),Chuanxiong (Rhizoma Chuanxiong),Danggui (Radix Angelicae Sinensis),Chishao (Radix Paeoniae Rubra),Yimucao (Herba Leonuri Japonici),Zelan (Herba Lycopi Hirti),Jixueteng (Caulis Spatholobi),and Puhuang (Pollen Typhae),to sooth the liver,regulateQi,nourishing and activating blood.Qiand blood were enhanced so that the solid blood was inflated,theQiwas smooth,and the promotion of the blood contributed to the development of follicles,thereby improving ovarian function.14

Wenshen Yangxue decoction increased the ovulation number and the fertilization rate in older ICR mice;moreover,it also reduced the proportion of abnormally fertilized eggs in older ICR mice.The effect of low-dose Wenshen Yangxue decoction was slightly better than that of the medium-dose Wenshen Yangxue decoction.Wenshen Yangxue decoction also promoted the development of 2PN fertilized eggs of older ICR mice to the 2-cell stage and increased the potential of embryonic development.It also promoted the development of 2PN fertilized eggs of older ICR mice toward blastocyst formation,thereby affecting the number of mice born.However,these results were dependent on the drug concentration.The optimal drug concentration is the key to ensuring optimal clinical treatment effect.A low drug concentration may not be sufficient to achieve significant drug effects,and an overly high drug concentration may inhibit the development of fertilized eggs.

Mitochondria are important organelles of eukaryotic cells,and they are involved in cell growth,differentiation,proliferation,apoptosis,signal transduction,and other cell activities.15Mitochondria are the most abundant organelles in oocytes and are the factories that provide energy for egg maturation,fertilization,and transcription and translation during embryonic development.The morphology and distribution also produce significant changes during the development of oocytes.16Therefore,various parameters of mitochondria are used to measure the cytoplasmic maturation of oocytes.17

Because the mitochondria in mammals are maternally inherited,the mitochondria contained in the sperm disappear during fertilization.18The oocytes of the MII stage and the fertilized eggs contain the same set of mtDNA,and therefore,the quality and quantity of the mitochondria in the oocytes will have an important impact on the quality of early embryos.Mitochondria regulate the production of ATP through changes in its distribution in oocytes,and under the condition of limiting the level of reactive oxygen species (ROS),they can quickly and effectively provide cells with a “fixedpoint” to provide energy.19

Mitochondrial energy deficiency significantly reduces nuclear maturation rate,ROS generation,spindle/chromosome structural integrity rate,and blastocyst formation rate of the egg.20Studies have shown that the functional maturation of mitochondria affects the developmental potential of early embryos.21Mitochondrial parameters to measure the quality of oocytes and embryonic development potential include mtDNA copy number,mitochondrial structure and distribution,mitochondrial transmembrane potential,ATP level,and ROS level.22

Sirt3is a tumor suppressor gene,which encodes a highfidelity protein of mitochondria.It monitors cell metabolism and plays an important role in maintaining the integrity of the mitochondria.23Sirt3 is the basic deacetylase of mitochondria,and 65% of mitochondrial proteins are acetylated lysine,suggesting that Sirt3 plays an important regulatory role in molecular modification.24Sirt3 can regulate many aspects of mitochondrial function,including metabolism,ATP production,and regulation of oxidative stress.It is a key player in the regulation of mitochondrial function and is a central regulator of metabolic function and cell survival.25

Loss of Sirt3 expression can affect the activity of mitochondrial SOD2 and ROS levels,generating oxidative stress and genomic instability and leading to a series of decrease in ovarian reserve and an increase in embryonic development disorders.2,26Moreover,the expression of Sirt3 is positively correlated with the number of primordial follicles,and therefore,it is inferred that Sirt3 is an indicator of ovarian aging.27,28FOXO3a plays an important role in cell proliferation,differentiation,growth,development of the body,and cell cycle apoptosis.Our results show that Wenshen Yangxue decoction significantly increased the expression of Sirt3 and FOXO3a mRNA and protein in the oocytes of older ICR mice.Additionally,the Wenshen Yangxue decoction regulated the Sirt3/FOXO3a pathway.

In summary,we showed that Wenshen Yangxue decoction promotes follicular development in elderly female mice,increases the number of ovulations,and improved fertility.Its mechanism may be related to increasing mitochondrial energy metabolism and regulating the Sirt3/FOXO3a pathway.

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