Anti-Aging Effect Of Urolithin A On Bovine Oocytes In Vitro

Please contact oscar.xiao@wecistanche.com for more information

Abstract: Oxidative stress and mitochondrial dysfunction have been associated with the age-related decline of oocyte quality and strategies for their prevention are currently quested. Urolithin A (UA) is a natural metabolite with pro-apoptotic and antioxidant effects, capable of preventing the accumulation of dysfunctional mitochondria in different aged cells. UA has never been tested in bovine oocytes. Our aim was to study the effect of UA on the developmental potential of cumulus-oocyte-complexes (COCs) and granulosa cells (GCs) expression of important genes related to reproductive competence. Nuclear maturation progression, mitochondrial membrane potential (MMP), and developmental competence of physiologically mature (22 h) and in vitro aged oocytes (30 h of IVM) obtained from prepubertal and adult females, either supplemented with UA or not were assessed. Additionally, the amount of mRNA of several genes(NFE2L2, NQO1, and mt-DN5)and the number of mt-ND5 DNA copies were quantified in cultured GCs from prepubertal and adult females, either supplemented with UA or not. Our study confirmed the harmful effect of oocyte aging on nuclear maturation progression, MMP, developmental competence, and gene expression levels. UA treatment during in vitro maturation enhanced (p<0.05) the maturation rate and subsequent developmental capacity of aged oocytes. A positive effect (p< 0.05) of UA on physiological maturation, MMP, and embryonic development was also identified. UA also interfered with the expression profile of NFE2L2 and NQO1 genes in GCs cultures. Our findings demonstrate that UA supplementation is an effective way to prevent oocyte aging and improves the subsequent bovine embryonic development.

KSL01

Please click here to know more

Keywords: oocyte; aging; Urolithin A; assisted reproductive technologies

1. Introduction

The decline of female reproductive ability is one of the first physiological functions adversely affected by aging and is thus considered an emerging health problem worldwide [1,2]. In addition to several pathological problems, the age-associated decrease in female fertility is largely attributed to a decline in the ovarian reserve of oocytes [1,3-5]allied to a time-dependent deterioration of their quality [2]. This deterioration process can occur due to the exposure of oocytes to an aged ovarian microenvironment before ovulation. In addition, the female gamete is often subjected to post-ovulatory aging when the fertilization process does not occur within the best optimal span period, and the unfertilized oocyte remains in the oviduct or in vitroprior to insemination for extended periods [6]The impairment of oocyte quality is a critical factor associated to the failure of assisted reproductive technologies(ART) since its quality is the main determinant for the embryo's developmental potential after fertilization [7,8]. Ovulation asynchrony and aged oocytes were often reported to impair the success of artificial insemination and embryo production programs in the mare, cattle, and sheep implying important economic losses[1,9,10]. Therefore, it is of primordial importance to study the mechanisms underlying oocyte aging, in order to design better therapeutic approaches to rescue fertility in several species, including humans, and also as a tool for genetic improvement in livestock. cistanche wirkung Particular attention must be devoted to improving the developmental capacity of oocytes from prepubertal cattle, which are often used to accelerate genetic gain and shorten generation intervals.

One of the major causes of impaired developmental competence in aged oocytes is the increase in oxidative stress, which induces mitochondrial dysfunction, DNA damage, and spindle formation errors, influencing the oocyte quality [1]. It is well established that increased production of free radicals is a cause of cellular aging in several chronic diseases and also in reproductive biology, resulting in poor fertility outcomes [12]. The ovarian microenvironment, which includes oocytes and granulosa cells (GCs), provides an antioxidant defense mechanism able to regulate oxidative conditions and maintain the oxidant/antioxidant balance [13]. However, during the aging process, the efficiency of antioxidant defenses to neutralize reactive oxygen species (ROS) is attenuated, thus increasing the level of oxidative stress. The Nuclear factor-E2-related factor2(Nrf2 or NFE2L2), also known as Nrf2/Kelch-like ECH-associated protein 1 (Keap1) pathway, is a dominant response cascade activated by oxidative stress[14]. This pathway is a cellular defense mechanism that cells have developed to cope with the deleterious effects of oxidative stress. Under normal conditions, Nrf2 is negatively regulated by Keap1, held in the cytoplasm, and maintained at low levels. When exposed to oxidants, Nrf2 is dissociated from Keapl, allowing its translocation in the nucleus where it binds to specific DNA sequences. These sequences, named antioxidant response elements(ARE), lead to the transcriptional activation of cytoprotective genes, such as NAD(P)H:quinone-oxidoreductase-1 (NQO1), heme oxygenase-1 (HMOX1), and glutamate-cysteine ligase catalytic subunit (GCLC)[15,16]. Previous studies showed that the activation of the Nrf2-Keapl signaling pathway decreases oxidative stress damage by elevating antioxidant levels in human GCs and mouse ovaries[17,18]. However, its role in the female gamete aging process remains elusive, although it is clearly established that mitochondria are the main production site for ROS during this process [9,10].

KSL02

Cistanche can anti-aging

Conversely, mitochondria are involved in several critical cellular functions and are fundamental for meeting the demand of energy production required during oocyte maturation and subsequent embryonic development [19]. Competent mitochondrial activity has been highly associated with higher contents of mitochondrial DNA (mt-DNA) and ATP generation [20], higher mitochondrial membrane potential [21], and maintenance of mitochondria quality and quantity through mitophagy [22]. Moreover, mitochondrial activity has been suggested to be directly correlated with embryo viability and better fertility outcomes [23]. Increasing evidence supports that age-related mitochondrial alterations drive to ovarian aging and subsequently reduced embryo viability and implantation potential. These alterations include decreased mt-DNA copy number, decreased ATP generation [20], alterations in mitochondrial gene expression [24,mt-DNA damage [25], and reduced mitochondrial membrane potential [26].

KSL03

Mitochondria-targeted therapeutic approaches prompted a huge interest in several pathologies associated with aging due to their great potential in enhancing mitochondrial function [27]. Within this framework, Urolithin A (UA)—a natural metabolite obtained after the ingestion of food such as pomegranates followed by its conversion by the gut microbiota—has been demonstrated to prevent the accumulation of dysfunctional mitochondria with age, inducing mitophagy and also maintaining mitochondrial biogenesis and respiratory capacity [28,29]. UA has been applied as a promising therapeutic drug to prevent some cancers, such as colorectal and prostate cancer[30,31]. Additionally, UA also has anti-inflammatory [32],anti-obesity [33],antioxidant [34] and anti-aging properties [35]. citrus bioflavonoids A recent study has highlighted the effects of UA supplementation to senescent human skin fibroblasts on the activation of the Nrf2-Keapl pathway enhancing their antioxidant capacity. This activation of the Nrf2-Keapl pathway effectively mitigates the ROS level, through the upregulation of the expression of Nrf2 downstream ARE-response genes (SOD, NQO1, GCLC, and HMOX1), indicating a promising anti-aging effect [35].

KSL04

Several studies have been performed with the goal of delaying ovarian aging, consequently improving oocyte quality and fertility outcomes. Due to the contribution of oxidative stress to the ovarian aging process, as well as to mitochondrial dysfunction, supplementation with antioxidants has appeared as a promising therapy [36,37]. However, it is unknown whether Urolithin A supplementation may restore the damage that occurs during ovarian aging contributing to preventing infertility problems. Therefore, the aim of this study was:(1) to demonstrate that aging could alter cumulus-oocyte complexes(COC)developmental potential and GCs' expression of important genes involved in the Nrf2 signaling pathway;(2) to determine whether UA can rescue female fertility demonstrating an anti-aging effect in aged COCs and GCs; and (3) to evaluate UA effect on the expression level of genes involved in the Nrf2 signaling pathway as well as on oocyte quality.

2. Materials and Methods

2.1. Experimental Design

This study was approved by the Animal Care Committee of the National Veterinary Authority (N°08965DGAV), following European Union guidelines (no.86/609/EEC). To investigate the effect of aging on the alteration of oocyte quality and the potential anti-aging effect of UA, a model using COCs collected from prepubertal and adult cows submitted to in Vitro aging (30 h of maturation) or to the physiological maturation (22 h) processes were applied.

2.1.1.Previous Assay—Dose-Response Study

A previous assay to determine the concentration of UA that should be used during the bovine COCs maturation process was performed based on a dose-response study in four sessions. Since UA has never been tested in bovine oocytes, previous doses successfully applied for the prevention and mitigation of some cancers and to demonstrate the anti-aging effect of UA in different cell lines were used [28,39]. COCs obtained from prepubertal and mature adult cows (n=978) were selected and then randomly divided into five groups to test different doses of UA: control,1,10,25, and 50 uM during physiological in vitro maturation. After the maturation period, some oocytes (n = 154) were stained to determine the chromosomal configuration and maturation stages. The remaining matured oocytes were submitted to in vitro insemination with frozen/thawed semen. Presumptive zygotes were cultured, and cleavage and blastocyst rates were determined at day 2 and day 7 of culture, respectively. Based on the obtained results, namely the absence of harmful effects and the promotion of maturation and blastocyst development, the concentration of 1 uM of UA was selected.

2.1.2.Experiment1

In this experiment, carried out in six sessions, both COCs from prepubertal (mean age = 9 months,n=660) and adult(mean age =39months,n=674)cows were collected to assess the oocyte quality and developmental potential of aged and physiologically matured oocytes as well as UA effect to rescue female fertility. COCs were randomly divided into 8 groups: (1) control prepubertal group, COCs from prepubertal calves matured for 22h (n=148);(2)UA prepubertal group, COCs from prepubertal calves matured in medium supplemented with 1 uM of UA for 22 h (n = 155);(3)control aged 30 h prepubertal group, COCs from prepubertal calves aged through 30h of in oitro maturation (n=149);(4)UA aged 30 h prepubertal group, COCs from prepubertal calves aged in vitro for 30h in maturation medium supplemented with 1 uM of UA(n=144);(5) control adult group, COCs from adult cows matured for22h (n=155);(6) UA adult group, COCs from adult cows matured in medium supplemented with 1 uM of UA for 22 h (n=129);(7) control aged 30 h adult group, COCs from adult cows aged through 30 h of in oitro maturation (n = 148); and (8) UA aged 30 h adult group, COCs from adult cows aged in oitro for 30 h in maturation medium supplemented with 1 uM of UA(n=138). cynomorium benefits After the respective in vitro maturation periods, oocytes were inseminated with thawed capacitated bull semen. Subsequently, embryonic development was assessed, evaluating both the rate of cleaved and produced embryos, as well as their quality.

Additionally, in this experiment, COCs from each group were retrieved to assess their nuclear maturation stage (control prepubertal group,n=7;UA prepubertal group, n =7; control aged 30 h prepubertal group, n=10; UA aged 30 h prepubertal group, n=6; control adult group,n=16;UA adult group,n=21;control aged 30 h adult group, n=16; UA aged 30 h adult group,n=18).The mitochondrial membrane potential (MP)of COCs was also evaluated (control prepubertal group, n=10; UA prepubertal group, n=9;control aged 30 h prepubertal group,n=9;UA aged 30 h prepubertal group,n=9;control adult group, n=15; UA adult group, n=13; control aged 30h adult group,n=10;UA aged 30 h adult group,n = 14).

2.1.3.Experiment2

As the GCs play an essential role in follicular growth and oocyte development, a second experiment was performed in five sessions to further study the effect of age and UA on the expression of NFE2L2, NQO1, and mt-ND5. The number of copies of the mt-ND5 gene was also evaluated. GCs were obtained after centrifugation of the follicular fluid aspirated from ovaries of prepubertal (mean age = 10 months) and adult cows (mean age =62months). These cells were cultured in the following conditions:(1)prepubertal control, culture of GCs of prepubertal calves; (2) prepubertal UA, culture of GCs of prepubertal calves supplemented with 1 uM UA;(3)adult control, culture of GCs of adult cows; and (4)adult UA, the culture of GCs of adult cows supplemented with 1 uM UA. After GCs confluence on the 5th day of culture, they were snap frozen in liquid nitrogen, and later the DNA and RNA were extracted, allowing the subsequent quantification of NFE2L2, NQO1, and mt-ND5 mRNA transcripts and also mt-ND5 copies number.

2.2. Oocyte Collection and In Vitro Maturation

Ovaries from adult and prepubertal cows (previous assay, n = 978 and exp. 1, n=1334) were collected at a local slaughterhouse, and kept at 35-37 ℃, in a phosphate-buffered saline (PBS)supplemented with 0.15% of bovine serum albumin (w/v, BSA)supplemented with 0.05 mg mL-l of kanamycin. At the laboratory, ovarian follicles of 2-8 mm in diameter were aspirated with a 19-gauge needle. Only COCs with at least three layers of compact cumulus cells and a homogeneous ooplasm were washed and selected for maturation according to the experimental design. desert hyacinth Maturation was accomplished in an incubator at 38.8 ℃,5% CO2 in humidified air for 22 or 30 h in a maturation medium composed of tissue culture medium 199 (TCM) with 10% of fetal bovine serum,0.2mM sodium pyruvate，10 ng mL-l of epidermal growth factor， and 10 μL mL-l of gentamicin【40】.

2.3.Granulosa Cells Collection and Culture

Granulosa cells were obtained from the recovered follicular fluid after centrifugation for 10 min at 200x g[41]. The pellet was suspended in 1 mL of culture medium (TCM199 +10% serum) to perform another centrifugation for 5 min. The new pellet was resuspended in 1 mL of culture medium either supplemented with 1 uM of UA or not according to the experimental design and homogenized with a syringe attached to a 19G-needle, at least 30 times to detach the cells. After evaluation of GC viability (trypan blue dye,0.4% w/v), cells were seeded at a concentration of 2×105 viable cells mL-1 and cultured for five days at 38.8 ℃,5% CO2 in a humidified atmosphere until confluence. At every 48 h, the culture medium was discharged and refreshed with a new one. For DNA and RNA extraction, GCs were collected and washed by centrifugation at 200x g for 10 min. Cell pellets were resuspended in 1 mL of PBS, immediately snap frozen in liquid nitrogen, and stored at -80°C.

2.4. Oocyte Nuclear Maturation

Nuclear maturation stages were assessed following the 22h or 30 h period of in Vitro maturation. flavonoid extraction method pdf Denuded oocytes were fixed in an acetic acid/ethanol (1:3, v/v) solution, and maintained at 4℃ for 48 h. Then oocytes were stained with 1% aceto-lacmoid solution, mounted in a Neubauer chamber, and observed under a phase contrast microscope (Olympus BX41). Oocytes were classified as follows: Germinal Vesicle(GV), Condensing Chromo-somes I (CCI), Condensing Chromosomes II(CCII), Diakinesis, Anaphase-I/Telophase-I (AI/TI), and MII (Metaphase-II). Only oocytes with visible chromatin staining were taken into account [42].

2.5. Assessment of Mitochondrial Membrane Potential

To measure the mitochondrial membrane potential (MP), an indicator of mitochondrial activity, mitochondria were stained with 5, 6, 6'-tetrachloro-1, 1', 3, 3'tetraethylbenzimidazolcarbocyanine iodide (JC-1, Invitrogen, Waltham, MA, USA). Denuded oocytes were incubated with 5 ug mL-l of JC-1 [37] in a maturation medium for 30 min at 38.8 ℃and 5% CO2 in humidified air in the dark. Oocytes were washed twice in PBS and immediately transferred to a pre-heated slide glass and observed under a fluorescence microscope (Olympus BX51) using the blue fluorescence filter(BP 470-490 objective UPlanFI 20×/0.50). Mitochondrial membrane potential was then calculated as the ratio of the measured red/green fluorescence using the Image] software (National Institute of Health, Bethesda, MD, USA). 2.6.In Vitro Fertilization and Embryo Culture

In vitro fertilization was performed with frozen-thawed sperm of a Holstein-Frisian bull, previously submitted to capacitation using the Percoll gradient (45 and 90) method, at a concentration of 2 x 10°spermatozoa mL-4.COCs and sperm were co-incubated for 20 h at 38.8 ℃and 5% CO2 in humidified air. Presumptive zygotes were then transferred to droplets of synthetic oviductal fluid (SOF) medium supplemented with BME and MEM amino acids, glutamine, glutathione, and BSA [40]. After 48 h of the insemination, the cleavage rate (cleaved embryos per total inseminated oocytes) was passed, and cleaved embryos were maintained in SOF supplemented with BSA and 10% of fetal bovine serum (FBS). Embryos were cultured for 12 days [41,43] to assess the blastocyst development rate (at days7,9, and 12; D7 embryos per cleaved embryos) and hatched embryo rate (hatched embryos per D7 embryos) and their quality [43]. Day 7 embryos were classified as grade 1 (good quality),2(fair quality), and grade 3(bad quality)[4].

2.7.DNA and RNA Extraction and Quantification

Total DNA and RNA were isolated from GCs using the High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland) and PureLinkTM RNA Mini Kit (InvitrogenTM, Waltham, MA, USA), respectively, according to the manufacturer's instructions. Those protocols included the use of spin columns used to isolate high-quality total DNA and RNA and DNase as a treatment to remove genomic DNA from RNA[40]. After extraction, the samples were stored at -80 ℃. The concentration and quality of DNA and RNA were determined using a NanoDropTM One/OneC Spectrophotometer (ThermoFisher ScientificTM, Waltham, MA, USA).

2.7.1. Complementary DNA Synthesis

Synthesis of complementary DNA (cDNA) from RNA isolates was performed using the Xpert cDNA Synthesis Mastermix kit (GRiSP, Porto, Portugal, according to the manufacturer's instructions. RNA was reverse transcribed using 500 ng of extracted RNA from each sample to perform cDNA synthesis, which was carried out using a thermocycler (T100 Thermal Cycler, Bio-Rad, Hercules,CA, USA).The resultant cDNAs were stored at-20℃until use for further assays. 2.7.2.Primer Design

For this study, primers for the targeted (NFE2L2 and NQO1) and an endogenous control gene (6-actin) were designed using the Primer-BLAST software of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/tools/primer-blast/,accessed on 6 March 2020). Sequences of primers for the reference genes and the target genes are depicted in Table 1. Additionally, the mt-ND5 gene was used in this work, and details about mt-ND5 primers were retrieved from a previous study [45].

2.7.3.Quantitative Reverse-Transcription Polymerase Chain Reaction

Real-time PCR analyses were performed using the Xpert Fast SYBR Green Mastermix 2X with ROX in a QuantStudio 3 thermocycler(ThermoFisher ScientificTM, Waltham, MA, USA), using cDNA at a concentration of 25 ng uL-l. The assessment of the mitochondrial DNA (mt-DNA) copies number of the ND5 gene was carried out by qPCR through the previously extracted DNA, using the same equipment as for the quantification of genes. Each optimized reaction was performed, consisting of Xpert Fast SYBR Green Mastermix 2X with ROX, primer (Forward and Reverse) of each target gene, sample (cDNA/DNA), and RNase-free water making up a total volume of 10 μL. The samples were analyzed in duplicate and reactions containing water instead of template were included as negative controls. The samples were subjected to an amplification protocol that consisted of an initial cycle at 95°C for 2 min of denaturation phase, followed by 40 denaturation cycles at 95℃for55s,40 annealing cycles for 30 s at 60℃(depending on the melting temperature of primer sequences), and extension phase at 72℃ for 30 s and, lastly, a final extension period at 72℃ for 10 min.

For the gene expression quantification, the relative quantification method was used. This method of relative quantification of gene expression was carried out with the expression levels of the target genes under study, which were normalized with the housekeeping genes, by the CT comparative method. As the amplification by RT-qPCR was performed in duplicate, the mean CT values for each gene were determined and the expression levels were calculated using the following formula:

where △Ct=Ct target gene -Ct endogenous control gene.

For the quantification of the mt-DNA number of copies, the relative quantification normalized against the unit mass method was used. This method was carried out with the CT values of GCs treated with UA (named as a test), which were normalized with control samples (currently designated as calibrator). As the mt-ND5 copy number was assessed by qPCR and performed in duplicate, the mean CT values for the tests and calibrators samples were determined and the ratios were calculated using the following formula: