Integrated analyses of the single-cell ATAC-seq and RNA-seq reveal the epigenetic landscape of human ovarian aging

Studying the molecular mechanisms of ovarian aging is crucial for understanding the age-related fertility issues in females. Recently, a single-cell transcriptomic roadmap of ovarian aging based on non-human primates revealed the molecular signatures of the oocytes at different developmental stages. Herein, we present the first epigenetic landscape of human ovarian aging, through an integrated analysis of the single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) and single-cell RNA-seq. We depicted the transcriptional profiles and chromatin accessibility of the ovarian tissues isolated from old (n=4) and young (n=2) donors. The unsupervised clustering of data revealed seven distinct cell populations in the ovarian tissues and six subtypes of oocytes, which could be distinguished by age difference. Further analysis of the scATAC-seq data from the young and old oocytes revealed that the interaction between the Notch signaling pathway and AP-1 family transcription factors may crucially determine oocyte aging. Finally, a machine-learning algorithm was applied to calculate the optimal model based on the single-cell dataset for predicting oocyte aging, which exhibited excellent accuracy with a cross-validated area under the receiver operating characteristics score of 0.99. In summary, this study provides a comprehensive understanding of human ovarian aging at both the transcriptomic and epigenetic levels, based on an integrated analysis of large-scale single-cell datasets. We believe our results will shed light on the discovery of potential therapeutic targets or diagnostic markers for age-related ovarian disorders.

Secoisolariciresinol Diglucoside Improves Ovarian Reserve in Aging Mouse by Inhibiting Oxidative Stress

Ovarian reserve is a key factor in the reproductive function of the ovaries. Ovarian aging is characterized by a gradual decline in the quantity and quality of follicles. The underlying mechanism of ovarian aging is complex and age-related oxidative stress is considered one of the most likely factors. Secoisolariciresinol diglucoside (SDG) has been shown to have good scavenging ability against reactive oxygen species (ROS) which slowly accumulates in ovarian tissues. However, it is unknown whether SDG had beneficial effects on aging ovaries. In this study, we used 37-week-old female C57BL/6J mouse as a natural reproductive aging model to evaluate the role of SDG in ovarian aging. SDG (7 and 70 mg/kg) intragastric administration was performed in the mice daily. After 8 weeks, the effects of SDG on aging ovaries were evaluated by counting the number of follicles and the expression of follicle-stimulating hormone receptors (FSHR) in the ovary. The mechanism of SDG on the aging ovaries was further explored through ovarian metabolomics. It was found that SDG can effectively increase the number of growing follicles and increase the expression of the FSHR protein. The metabolomics results showed that the ovaries in the SDG intervention group achieved better uptake and transport of nutrients, including amino acids and glucose that are necessary for the development of oocytes. At the same time, the ovaries of the SDG intervention group showed that the drug reduced ROS generation. Additionally, we found that ovarian telomere length and ovarian mitochondrial DNA copy number that are highly susceptible to ROS damage and are also related to aging. The results showed that SDG can significantly increase mitochondrial DNA copy number and slow down the process of telomere shortening. These data indicate that SDG improves ovarian reserve by inhibiting oxidative stress.

Physiological Ovarian Aging Is Associated with Altered Expression of Post-Translational Modifications in Mice

Post-translational modifications (PTMs) have been confirmed to be involved in multiple female reproductive events, but their role in physiological ovarian aging is far from elucidated. In this study, mice aged 3, 12 or 17 months (3M, 12M, 17M) were selected as physiological ovarian aging models. The expression of female reproductive function-related genes, the global profiles of PTMs, and the level of histone modifications and related regulatory enzymes were examined during physiological ovarian aging in the mice by quantitative real-time PCR and western blot, respectively. The results showed that the global protein expression of Kbhb (lysineβ-hydroxybutyryllysine), Khib (lysine 2-hydroxyisobutyryllysine), Kglu (lysineglutaryllysine), Kmal (lysinemalonyllysine), Ksucc (lysinesuccinyllysine), Kcr (lysinecrotonyllysine), Kbu (lysinebutyryllysine), Kpr (lysinepropionyllysine), SUMO1 (SUMO1 modification), ub (ubiquitination), P-Typ (phosphorylation), and 3-nitro-Tyr (nitro-tyrosine) increased significantly as mice aged. Moreover, the modification level of Kme2 (lysinedi-methyllysine) and Kac (lysineacetyllysine) was the highest in the 3M mice and the lowest in 12M mice. In addition, only trimethylation of histone lysine was up-regulated progressively and significantly with increasing age (p < 0.001), H4 ubiquitination was obviously higher in the 12M and 17M mice than 3M (p < 0.001), whereas the modification of Kpr (lysinepropionylation) and O-GlcNA in 17M was significantly decreased compared with the level in 3M mice (p < 0.05, p < 0.01). Furthermore, the expression levels of the TIP60, P300, PRDM9, KMT5B, and KMT5C genes encoding PTM regulators were up-regulated in 17M compared to 3M female mice (p < 0.05). These findings indicate that altered related regulatory enzymes and PTMs are associated with physiological ovarian aging in mice, which is expected to provide useful insights for the delay of ovarian aging and the diagnosis and treatment of female infertility.

Ovarian Aging: Role of Pituitary-Ovarian Axis Hormones and ncRNAs in Regulating Ovarian Mitochondrial Activity

The number of mitochondria in the oocyte along with their functions (e.g., energy production, scavenger activity) decline with age progression. Such multifaceted functions support several processes during oocyte maturation, ranging from energy supply to synthesis of the steroid hormones. Hence, it is hardly surprising that their impairment has been reported in both physiological and premature ovarian aging, wherein they are crucial players in the apoptotic processes that arise in aged ovaries. In any form, ovarian aging implies the progressive damage of the mitochondrial structure and activities as regards to ovarian germ and somatic cells. The imbalance in the circulating hormones and peptides (e.g., gonadotropins, estrogens, AMH, activins, and inhibins), active along the pituitary-ovarian axis, represents the biochemical sign of ovarian aging. Despite the progress accomplished in determining the key role of the mitochondria in preserving ovarian follicular number and health, their modulation by the hormonal signalling pathways involved in ovarian aging has been poorly and randomly explored. Yet characterizing this mechanism is pivotal to molecularly define the implication of mitochondrial dysfunction in physiological and premature ovarian aging, respectively. However, it is fairly difficult considering that the pathways associated with ovarian aging might affect mitochondria directly or by altering the activity, stability and localization of proteins controlling mitochondrial dynamics and functions, either unbalancing other cellular mediators, released by the mitochondria, such as non-coding RNAs (ncRNAs). We will focus on the mitochondrial ncRNAs (i.e., mitomiRs and mtlncRNAs), that retranslocate from the mitochondria to the nucleus, as active players in aging and describe their role in the nuclear-mitochondrial crosstalk and its modulation by the pituitary-ovarian hormone dependent pathways. In this review, we will illustrate mitochondria as targets of the signaling pathways dependent on hormones and peptides active along the pituitary/ovarian axis and as transducers, with a particular focus on the molecules retrieved in the mitochondria, mainly ncRNAs. Given their regulatory function in cellular activities we propose them as potential diagnostic markers and/or therapeutic targets.

Theaflavin 3, 3 ′ -Digallate Delays Ovarian Aging by Improving Oocyte Quality and Regulating Granulosa Cell Function

Ovarian aging refers to the gradual decline of ovarian function with increasing physiological age, manifested as decreased ovarian reserve, elevated aging-related markers, and reduced oocyte quality. With a declining female fertility and a growing aging population, it is urgent to delay ovarian aging to maintain fertility and improve the life quality of women. Theaflavin 3, 3 ′ -digallate (TF3) is a naturally bioactive polyphenol compound extracted from black tea, and its antioxidant properties play an important role in maintaining human health and delaying aging; however, the effects of TF3 on female reproduction and ovarian function are not yet clear. Here, we show that TF3 can preserve primordial follicle pool, partially restore the estrous cycle, and increase the offspring number of aged mice. Meanwhile, TF3 gavage increased the number of oocytes retrieved, decreased the level of reactive oxygen species, increased the level of glutathione, and decreased the abnormal rate of oocyte spindle after ovulation induction. Moreover, TF3 inhibited human granulosa cell apoptosis and improved their antioxidative stress ability. High-throughput sequencing and small-molecule-targeted pharmacological prediction show that TF3 affects multiple pathways and gene expression levels, mainly involved in reproductive and developmental processes. It may also affect cellular function by targeting mTOR to regulate the autophagic pathway, thereby delaying the process of ovarian aging. This study shows that TF3 can be used as a potential dietary supplement to protect ovary function from aging and thereby improving the life quality of advanced-age women.

Increased Systemic Antioxidant Power Ameliorates the Aging-Related Reduction in Oocyte Competence in Mice

Ovarian aging is associated with elevated oxidative stress and diminished oocyte developmental competence. We aimed to determine the impact of systemic antioxidant treatment in aged mice. Female outbred CF-1 mice were aged for 9 months prior to an 8-week 45 mg Euterpe oleracea (açaí) daily supplement. The açaí treatment induced a threefold increase in serum antioxidant power (FRAP) compared to both young and aged mice (p < 0.0001). Compared to young mice, aged mice had fewer oocytes and reduced blastocyst development (p < 0.0001); açaí did not affect the oocyte numbers, but improved blastocyst formation (p < 0.05). Additionally, açaí alleviated the aging-related decrease in implantation potential (p < 0.01). The aged mice showed evidence of elevated ovarian ER stress (increased whole-ovary PDIA4 expression, granulosa cell and oocyte GRP78 expression, and oocyte PDIA4 protein), reduced oocyte mitochondrial quality (higher PRKN activation and mitochondrial DNA oxidative damage), and dysregulated uterine glandular epithelium. Antioxidant intervention was sufficient to lessen these effects of ovarian aging, likely in part by the upregulation of NRF2. We conclude that açaí treatment is a promising strategy to improve ER and mitochondrial function in the ovaries, thereby ameliorating the decreased oocyte competence that occurs with ovarian aging.

Integrated analyses of the single-cell ATAC-seq and RNA-seq reveal the epigenetic landscape of human ovarian aging

Studying the molecular mechanisms of ovarian aging is crucial for understanding the age-related fertility issues in females. Recently, a single-cell transcriptomic roadmap of ovarian aging based on non-human primates revealed the molecular signatures of the oocytes at different developmental stages. Herein, we present the first epigenetic landscape of human ovarian aging, through an integrated analysis of the single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) and single-cell RNA-seq. We depicted the transcriptional profiles and chromatin accessibility of the ovarian tissues isolated from old (n=4) and young (n=2) donors. The unsupervised clustering of data revealed seven distinct cell populations in the ovarian tissues and six subtypes of oocytes, which could be distinguished by age difference. Further analysis of the scATAC-seq data from the young and old oocytes revealed that the interaction between the Notch signaling pathway and AP-1 family transcription factors may crucially determine oocyte aging. Finally, a machine-learning algorithm was applied to calculate the optimal model based on the single-cell dataset for predicting oocyte aging, which exhibited excellent accuracy with a cross-validated area under the receiver operating characteristics score of 0.99. In summary, this study provides a comprehensive understanding of human ovarian aging at both the transcriptomic and epigenetic levels, based on an integrated analysis of large-scale single-cell datasets. We believe our results will shed light on the discovery of potential therapeutic targets or diagnostic markers for age-related ovarian disorders.

Influence of Age and Breed on Bovine Ovarian Capillary Blood Supply, Ovarian Mitochondria and Telomere Length

Worldwide, dairy cows of the type of high-producing cattle (HPC) suffer from health and fertility problems at a young age and therefore lose productivity after an average of only three lactations. It is still contentious whether these problems are primarily due to genetics, management, feeding or other factors. Vascularization plays a fundamental role in the cyclic processes of reproductive organs, as well as in the regeneration of tissues. In a previous study, HPC were shown to have a greater ovarian corpus luteum vascularization compared to dual-purpose breeds. We hypothesize that this activated angiogenesis could likely lead to an early exhaustion of HPC′s regenerative capacity and thus to premature reproductive senescence. The objective of this study was to investigate if a HPC breed (Holstein-Friesian, HF) exhibits higher ovarian angiogenesis than a dual-purpose breed (Polish Red cow, PR) and if this is related to early ovarian aging and finally reproductive failure. For this purpose, we assessed the degree of vascularization by means of ovarian blood vessel characterization using light microscopy. As indicators for aging, we measured ovarian mitochondrial size and telomere length in peripheral leukocytes. We report in this study that in both breeds the distance between capillaries became smaller with increasing age and that the mean telomere length decreased with increasing age. The only difference between the two breeds was that PR developed larger capillaries than HF. Neither a relationship between telomere length, nor the morphology of the mitochondrial apparatus and nor angiogenesis in HF was proven. Although the data trends indicated that the proportion of shortened telomeres in HF was higher than in the PR, no significant difference between the two breeds was detected.