Follicular development and ovary aging: single-cell studies.

Follicular development is a critical process in reproductive biology that determines the number of oocytes and interacts with various cells within the follicle (such as oocytes, granulosa cells, cumulus cells and theca cells, etc.), and plays a vital role in fertility and reproductive health due to the dogma of a limited number of oogonia. Dysregulation of follicular development can lead to infertility problems and other reproductive disorders. To explore the physiological and pathological mechanisms of follicular development, immunology-based methods, microarrays, and next-generation sequencing have traditionally been used for characterization at the tissue level. However, with the proliferation of single-cell sequencing techniques, research has uncovered unique molecular mechanisms in individual cells that have been masked by previous holistic analyses. In this review, we briefly summarize the achievements and limitations of traditional methods in the study of follicular development. Simultaneously, we focus on how to understand the physiological process of follicular development at the single-cell level and reveal the relevant mechanisms leading to the pathology of follicular development and intervention targets. Moreover, we also summarize the limitations and application prospects of single cell sequencing in follicular development research.

Impact of NAD+ metabolism on ovarian aging.

Nicotinamide adenine dinucleotide (NAD+), a crucial coenzyme in cellular redox reactions, is closely associated with age-related functional degeneration and metabolic diseases. NAD exerts direct and indirect influences on many crucial cellular functions, including metabolic pathways, DNA repair, chromatin remodeling, cellular senescence, and immune cell functionality. These cellular processes and functions are essential for maintaining tissue and metabolic homeostasis, as well as healthy aging. Causality has been elucidated between a decline in NAD levels and multiple age-related diseases, which has been confirmed by various strategies aimed at increasing NAD levels in the preclinical setting. Ovarian aging is recognized as a natural process characterized by a decline in follicle number and function, resulting in decreased estrogen production and menopause. In this regard, it is necessary to address the many factors involved in this complicated procedure, which could improve fertility in women of advanced maternal age. Concerning the decrease in NAD+ levels as ovarian aging progresses, promising and exciting results are presented for strategies using NAD+ precursors to promote NAD+ biosynthesis, which could substantially improve oocyte quality and alleviate ovarian aging. Hence, to acquire further insights into NAD+ metabolism and biology, this review aims to probe the factors affecting ovarian aging, the characteristics of NAD+ precursors, and the current research status of NAD+ supplementation in ovarian aging. Specifically, by gaining a comprehensive understanding of these aspects, we are optimistic about the prominent progress that will be made in both research and therapy related to ovarian aging.

Establishment of a Mouse Model of Premature Ovarian Failure Using Consecutive Superovulation.

BACKGROUND/AIMS: This study investigated the effect of consecutive superovulation on the ovaries and established a premature ovarian failure (POF) model in mice. METHODS: The mouse POF model was induced by 5-15 consecutive superovulation treatments with pregnant mare serum gonadotropin (PMSG), human chorionic gonadotropin (HCG) and prostaglandin F2α (PGF2α). Normal adult mice were compared with mice displaying natural ovarian aging. The following serum biochemical parameters were measured: including follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P), estradiol (E2), inhibin B (INH B), malondialdehyde (MDA), total superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels. Follicles were counted using H&E staining. Levels of 8-hydroxyguanosine (8-OhdG), 4-hydroxynonenal (4-HNE), nitrotyrosine (NTY), anti-Mullerian hormone (AMH) and CDKN2A/ p16 (p16) were detected using immunohistochemical staining. Reactive oxygen species (ROS) levels were measured using dihydroethidium (DHE) staining. Cell apoptosis was detected using an in situ TUNEL fluorescence staining assay. Levels of proteins involved in ROS-related pathways and the p16 protein were detected using Western blotting. Sod1, Sod2 and Sod3 mRNA levels were detected using quantitative polymerase chain reaction (Q-PCR). Oocyte quality was evaluated using in vitro fertilization (IVF) and zygote culture. RESULTS: Consecutive superovulation groups presented lower P, E2, SOD, GSH-Px and INH B levels, significantly higher FSH, LH, MDA and ROS levels, and significantly fewer primordial follicles compared with the control group. Consecutive superovulation groups presented significantly increased levels of Sod2, 8-OhdG, 4-HNE, NTY, significantly increased levels of the SIRT1 and FOXO1 proteins, significantly increased levels of the senescence-associated protein p16, as well as decreased AMH, Sod1 and Sod3 levels and increased granulosa cell apoptosis compared with the control group. CONCLUSION: Consecutive superovulation significantly decreased ovarian function and oocyte quality and increased oxidative stress and apoptosis in the ovary via a mechanism involving the p16 and SIRT1/FOXO1 signaling pathways. These findings suggest that consecutive superovulation may be used to establish a mouse model of ovarian aging.

Ovarian Aging and Osteoporosis.

Osteoporosis is the most common bone metabolic disease with a very high morbidity, and women usually got a higher risk of osteoporosis than men. The high incidence rate of osteoporosis in women was mainly caused by (1) women having fewer skeletons and bone mass, (2) pregnancy consuming a large amount of calcium and other nutrients, and most importantly (3) the cease of estrogen secretion by ovaries after menopause. Along with ovarian aging, the follicle pool gradually declines and the oocyte quality reduced, accompanied with decline in serum estrogen. Estrogen deficiency plays an important role in the pathogenesis of postmenopausal osteoporosis; it is mainly a result of the recognition that estrogen regulates bone remodeling by modulating the production of cytokines and growth factors from bone marrow and bone cells. This review will summarize current knowledge concerning ovarian aging and postmenopause osteoporosis and also discuss clinical treatment and new ideas of drug development for osteoporosis.

Impact of Menopause on Quality of Life in Community-based Women in China: 1 Year Follow-up.

Quality of life (QOL) throughout menopause has become an outcome variable requiring measurement in clinical care. Staff nurses can provide earlier nursing during the menopausal transition (MT) stage. The purpose of this study was to describe the changes of QOL in different stages of the MT according to The Stages of Reproductive Aging Workshop (STRAW) in Chinese women in community settings. Prospective longitudinal study design was used to analyze QOL of 327 community women age 30-65years old. They were followed up at 1-year. An instrument including the Chinese version of the Menopause-Specific Quality of Life Questionnaire was used to obtain data. A gradual decline in QOL was seen from premenopausal to menopausal transition (MT) and in postmenopausal women. Significant differences were observed in vasomotor, physical and sexual scores at baseline and follow-up (P<0.05). Significant differences in vasomotor scores were observed between baseline and follow-up for women in the premenopausal and Late MT stages (P<0.05). There were significant differences in psychosocial and physical scores between baseline and follow-up in the Late MT stage (P<0.05). Menopause might have a negative impact on QOL independent of age in community-based women in China. There seemed to be a potential model of the relationship of menopause status to change in QOL, but this needs supporting evidence from longer longitudinal studies.

Mitophagy in mammalian follicle development and health.

Mitophagy, the cellular process that removes damaged mitochondria, plays a crucial role in maintaining normal cell functions. It is deeply involved in the entire process of follicle development and is associated with various ovarian diseases. This review aims to provide a comprehensive overview of mitophagy regulation, emphasizing its role at different stages of follicular development. Additionally, the study illuminates the relationship between mitophagy and ovarian diseases, including ovary aging (OA), primary ovarian insufficiency (POI), and polycystic ovary syndrome (PCOS). A detailed understanding of mitophagy could reveal valuable insights and novel strategies for managing female ovarian reproductive health.

Adverse effects of 2-Methoxyestradiol on mouse oocytes during reproductive aging.

2-Methoxyestradiol (2-ME2) is a metabolite of 17ß-estradiol and is currently in clinical trials as an antitumor agent. Here we found 2-ME2 level remains stable in the local environment of ovaries but declines in serum in aging mice, and exogenous 2-ME2 impacts the meiotic maturation of mouse oocytes in dose-dependent manner. In vitro 2-ME2 application arrested oocytes at metaphase I (MI), with abnormal spindle structure and chromosome alignment. 2-ME2 exposure induced excessive production of reactive oxygen species (ROS) and malondialdehyde, as well as accelerated apoptosis progression. 2-ME2 unbalanced mitochondrial dynamics by increasing DRP1 and MFN1 while decreasing Opa1. Similar phenotypes were also observed in oocytes from mice injected intraperitoneally with 2-ME2. Taken together, this study indicates 2-ME2 exposure impairs oocyte meiotic maturation through inducing mitochondrial imbalance, oxidative stress and apoptosis. The gradual decline in oocyte quality and quantity may be associated with the stable 2-ME2 in ovaries during female reproductive aging.

Sirt3 deficiency accelerates ovarian senescence without affecting spermatogenesis in aging mice.

Sirtuin-3 (SIRT3), the main deacetylase in the mitochondria, maintains cellular energy metabolism and redox balance by deacetylating mitochondrial proteins in a NAD+-dependent manner. Growing evidence indicates that decreased Sirt3 expression is involved in various age-related maladies. However, the role of Sirt3 in ovarian and testicular senescence remains unclear. In this study, we observed that sirt3 expression showed age-dependent decreases in the ovary but not the testis. We generated Sirt3 null mice via CRISPR/Cas9-mediated genome editing. We observed that Sirt3 deletion accelerated ovarian aging, as shown by a decrease in offspring sizes, the follicle reserve and oocytes markers (Bmp15 and Gdf9) as well as increased expression of aging and inflammation-related genes (p16, p21, Il-1α, and Il-1ß). Sirt3 deficiency led to an accumulation of superoxide and disruption of spindle assembly accompanied by mitochondrial dysfunction (uneven mitochondria distribution, decreased mitochondrial potential as well as reduced mitochondrial DNA content) in aging oocytes. Meanwhile, in ovaries of Sirt3 null mice, the impaired mitochondrial functions were shown by decreases in mitochondrial respiratory complexes, along with lower levels of mitochondrial fusion (OPA1, MFN2) and fission (DRP1, FIS1) proteins. er levels of mitochondrial fusion (OPA1, MFN2) and fission (DRP1, FIS1) proteins. Interestingly, Sirt3-/- male mice exhibited no changes on the testicular histology, serum testosterone levels, germ-cell proliferation, and differentiation of spermatogonia. Meiotic prophase I spermatocytes were also normal. Levels of superoxide, mitochondrial potential as well as expression of mitochondrially-encoded genes were unaltered in Sirt3-/- testes. Collectively, the results indicated that SIRT3 plays a critical role in maintaining the ovarian follicle reserve and oocyte quality in aging mice, suggesting its important role in controlling ovarian senescence.

Fetal programming: in utero exposure to acrylamide leads to intergenerational disrupted ovarian function and accelerated ovarian aging.

In this study we investigated the effects of multigenerational exposures to acrylamide (ACR) on ovarian function. Fifty-day-old Wistar albino female rats were divided into the control and ACR-treated groups (2.5, 10, and 20 mg/kg/day) from day 6 of pregnancy until delivery. The obtained females of the first (AF1) and second generation (AF2) were euthanized at 4 weeks of age, and plasma and ovary samples were collected. We found that in utero multigenerational exposure to ACR reduced fertility and ovarian function in AF1 through inducing histopathological changes as evidenced by the appearance of cysts and degenerating follicles, oocyte vacuolization, and pyknosis in granulosa cells. TMR red positive cells confirmed by TUNEL assay were mostly detected in the stroma of the treated groups. Estradiol and IGF-1 concentrations significantly decreased as a result of decreased CYP19 gene and its protein expression. However, ACR exposure in AF2 led to early ovarian aging as evidenced by high estradiol and progesterone levels among all treated groups compared to control group, corresponding to the upregulation of the CYP19 gene and protein expression. The apoptotic cells of the stroma were greatly detected compared to that in the control group, whereas no significant difference was reported in ESR1 and ESR2 gene expression. This study confirms the developmental adverse effects of ACR on ovarian function and fertility in at least two consecutive generations. It emphasizes the need for more effective strategies during pregnancy, such as eating healthy foods and avoiding consumption of ACR-rich products, including fried foods and coffee.

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.

Increasing ovarian NAD+ levels improve mitochondrial functions and reverse ovarian aging.

Loss of follicles together with decreased oocyte quality and quantity contribute to age-associated ovarian senescence and infertility. Although underlying mechanisms for ovarian senescence are still unknown, mitochondrial dysfunctions have been reported. Here, we showed age-dependent decreases in ovarian Nicotinamide Adenine Dinucleotide (NAD+) levels in mice whereas supplementing aging mice with nicotinamide riboside (NR), an NAD+ precursor, increased ovarian NAD+ content. We found that increases in ovarian NAD+ levels in aging mice led to increased number of ovarian follicles and ovulatory potential as well as increased live birth rate. NR supplementation also reduced levels of reactive oxygen species and decreased spindle anomalies in aging oocytes, together with increased mitochondrial membrane potential (ΔΨm) and decreased mitochondrial clustering. In addition, NR supplementation improved ovarian mitochondrial energy metabolism. Our data suggested that supplementation with NAD+ precursors in vivo and in vitro could be potential therapeutic approaches for treating age-related ovarian infertility.

Advanced maternal age alters expression of maternal effect genes that are essential for human oocyte quality.

To investigate the effects of maternal age on the quality of oocytes, we used single-cell RNA sequencing to detect global gene transcriptome and identify key genes affected by advanced age in human mature oocytes. We isolated mRNA from mature oocytes obtained from IVF or ICSI patients (three oocytes from younger (≤30 years) and three oocytes from older (≥40 years) patients for scRNA-seq. We identified 357 genes differentially expressed between matured oocytes from older and younger women's. The up-regulated genes were significantly enriched with annotations related to transcriptional activation, oxidative stress and immune function, while down-regulated genes were enriched with catalytic activity. The key candidate gene TOP2B was found by protein interaction network analysis, and knockdown verification on younger mouse matured oocytes showed that TOP2B was a key gene affecting the oocyte quality and early embryo development. These results will contribute new knowledge on the molecular mechanisms of female ovary aging and establish a criterion to evaluate the quality of oocytes in women with advanced maternal age.

Curcumin exerts a protective effect against premature ovarian failure in mice.

This study was designed to investigate the protective effect of curcumin against d-galactose (d-gal)-induced premature ovarian failure (POF) in mice. A mouse POF model was induced by subcutaneous injection of d-gal (200 mg/kg/day) daily for 42 days. Mice in the curcumin group received both d-gal treatment and intraperitoneal injection of curcumin (100 mg/kg/day) for 42 days. Ovarian function, oxidative stress and apoptosis were evaluated. The P, E2 and SOD levels were higher, and the FSH, LH and MDA levels were significantly lower in the curcumin group than those in the d-gal group. The proportion of primordial follicles was also significantly higher in the curcumin group than that in the d-gal group. In addition, curcumin treatment after d-gal administration resulted in significantly lower Sod2, Cat, 8-OhdG, 4-HNE, NTY and senescence-associated protein P16 expression levels, higher Amh expression levels and less apoptosis in granulosa cells than was observed in the d-gal group. Moreover, the p-Akt, Nrf2 and HO-1 protein expression levels were significantly higher and the apoptosis-related cleaved caspase-3 and -9 protein expression levels were markedly lower in the curcumin group than in the d-gal group. In conclusion, curcumin effectively inhibited d-gal-induced oxidative stress, apoptosis and ovarian injury via a mechanism involving the Nrf2/HO-1 and PI3K/Akt signaling pathways, suggesting that curcumin is a potential protective agent against POF.

Regulatory factors and modulation mechanisms of functions of ovarian germline stem cell niche / 上海交通大学学报（医学版）

With increasing age in women, the ovarian function declines, which leads to decreased follicle generation, declined female fertility and age-related diseases ultimately. Female germline stem cells are epithelial cells existing on the ovarian surface, which can divide into new stem cells symmetrically and differentiate into germ cells and granulosa cells asymmetrically. The discovery of female germline stem cells brings much hope for the post-natal renewal of oocytes and solving female infertility problems. Ovarian germline stem cell niche in which female germline stem cells live is the surrounding microenvironment which plays an essential role in maintaining the function of female germline stem cells. Many factors including nutrition supply, protein, cytokines and signaling pathways can control the biological characters of female germline stem cells, and also influence their proliferation and differentiation. This paper reviewed the knowledge about the influencing factors and regulatory mechanisms of the function of ovarian germline stem cell niche.

Regulatory factors and modulation mechanisms of functions of ovarian germline stem cell niche / 上海交通大学学报（医学版）

With increasing age in women, the ovarian function declines, which leads to decreased follicle generation, declined female fertility and age-related diseases ultimately. Female germline stem cells are epithelial cells existing on the ovarian surface, which can divide into new stem cells symmetrically and differentiate into germ cells and granulosa cells asymmetrically. The discovery of female germline stem cells brings much hope for the post-natal renewal of oocytes and solving female infertility problems. Ovarian germline stem cell niche in which female germline stem cells live is the surrounding microenvironment which plays an essential role in maintaining the function of female germline stem cells. Many factors including nutrition supply, protein, cytokines and signaling pathways can control the biological characters of female germline stem cells, and also influence their proliferation and differentiation. This paper reviewed the knowledge about the influencing factors and regulatory mechanisms of the function of ovarian germline stem cell niche.

Fisiología reproductiva y cambios evolutivos con la edad de la mujer / Reproductive physiology and evolutive changes with women age

Los cambios fisiológicos con la edad, son consecuencia de cambios sistémicos generales producidos por el envejecimiento, entre los que se destacan cambios a nivel de los mecanismos de defensa relacionados con las especies reactivas del oxígeno (ROS) y alteraciones de la microcirculación. Los cambios evolutivos ováricos con la edad, se deben esencialmente a una depleción de la población folicular, la cual al disminuir por debajo de 1000 folículos, hace perder la capacidad del ovario de ciclar en forma normal, con la consiguiente disminución en los niveles de esteroides circulantes y sus efectos secundarios en los órganos efectores. Asociado a la disminución de la población folicular, se producen cambios en la calidad ovocitaria, los cuales determinan la disminución progresiva de la fertilidad en mujeres mayores de 35 años. Entre los cambios más frecuentes se observan aumento de aneuploidias, disfunciones mitocondriales, cambios de la microcirculación y disminución de la capacidad defensiva sobrelas ROS, entre otros.

The physiological changes with age are the consequence of systemic general changes produced by aging, where changes of the defense mechanisms related to ROS and microcirculation alterations are highlighted. The evolutive changes produced by age in the ovary are related to the follicular depletion. When the ovarian follicle population decrease below 1000 follicles, the ovary loses its capacityto cycle normally. As a consequence, the levels of circulating steroids diminished, producing negatives effects on secondary steroidal organs. In association with the follicular depletion, there are changes in oocyte quality, which determine the progressive diminution of fertility in women older than 35 years. The most frequent changes observed are an increase of the aneuploidies, mitochondrial dysfunction, microcirculation changes and a diminution of the defense capacity to ROS among others.