Hypoxia inducible factor-1α (HIF-1α), as a hypoxia inducible factor, affects women's reproductive function by regulating the development and excretion of follicles. HIF-1α induces glycolysis and autophagy in the granule cells by promoting oocyte development, regulating the secretion of related angiogenic factors, and improving follicle maturity. In addition, HIF-1α promotes the process of luteinization of follicular vesicles, maintains luteal function, and finally completes physiological luteal atrophy through cumulative oxidative stress. Dysfunction of HIF-1α will cause a series of pathological consequences, such as angiogenesis defect, energy metabolism abnormality, excessive oxidative stress and dysregulated autophagy and apoptosis, resulting in ovulation problem and infertility. This article summarizes the previous studies on the regulation of follicle development and excretion and maintenance of luteal function and structural atrophy by HIF-1α. We also describe the effective intervention mechanism of related drugs or bioactive ingredients on follicular dysplasia and ovulation disorders through HIF-1α, in order to provide a systematic and in-depth insights for solving ovulation disorder infertility.
Female ; Humans ; Atrophy/metabolism* ; Hypoxia ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Infertility/metabolism* ; Ovarian Follicle ; Ovulation

This study aims to observe the effect and explore the mechanism of Qirong Tablets in the treatment of premature ovarian insufficiency(POI) in mice via the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/hypoxia inducible factor 1(HIF-1) signaling pathway. Sixty SPF female BALB/c mice were randomly divided into normal group, model group, positive control group, Qirong Tablets low-, medium-and high-dose group. The normal group was intraperitoneally injected with the same amount of normal saline, and the other groups were intraperitoneally injected with cyclophosphamide 120 mg·kg~(-1)·d~(-1) once to establish a POI animal model. After the model was successfully established, the low-, medium-and high-dose groups of Qirong Tablets were administered orally with 0.6, 1.2, 2.4 mg·kg~(-1)·d~(-1) respectively. The positive control group was given 0.22 mg·kg~(-1)·d~(-1) Clementine Tablets by intragastric administration, and the normal group and model group were given intragastric administration with the same amount of normal saline, and the treatment was 28 d as a course of treatment. After drug intervention, enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of estradiol(E_2), follicle-stimulating hormone(FSH), luteinizing hormone(LH), and anti-mullerian hormone(AMH) in peripheral blood, and hematoxylin-eosin(HE) staining to observe the ovarian tissue. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay was used to detect the apoptosis of granulosa cells, and Western blot to determine the expression levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, PI3K, Akt, and HIF-1. Compared with the normal group, the modeling of POI caused loose or destroyed ovarian tissue with vacuolar structures, edema and fibrosis in the ovarian interstitium, disordered or loose arrangement of granulosa cells, and reduced normal follicles. Compared with the model group, drug interventions restored the ovarian tissue and follicles at all the development stages and reduced atretic follicles. Compared with the normal group, the modeling of POI lowered the serum level of E_2 and AMH(P<0.01), and elevated the level of FSH and LH(P<0.01). Compared with the model group, high-dose Qirong Tablets elevated the levels of E_2 and AMH(P<0.05), and lowered the levels of FSH and LH(P<0.05). Compared with the normal group, the modeling of POI up-regulated the protein levels of PI3K, Akt, HIF-1, Bax, and caspase-3 and down-regulated the protein level of Bcl-2 in the ovarian tissue(P<0.01). Compared with the model group, low-, medium-, and high-dose Qirong Tablets down-regulated the protein levels of PI3K, Akt, HIF-1, Bax, and caspase-3 proteins and up-regulated the protein level of Bcl-2 in the ovarian tissue(P<0.05). In conclusion, Qirong Tablets can up-regulate the expression Bcl-2, down-regulate the expression of Bax and caspase-3 in POI mice. Qirong Tablets may inhibit the apoptosis of follicular granulosa cells in mice, thereby delaying ovarian aging, improving reproductive axis function, and strengthening ovarian reserve capacity, which may be associated with the inhibition of PI3K/Akt/HIF-1 pathway.
Humans ; Mice ; Female ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; bcl-2-Associated X Protein ; Phosphatidylinositol 3-Kinases/metabolism* ; Caspase 3/metabolism* ; Saline Solution/therapeutic use* ; Signal Transduction ; Granulosa Cells ; Primary Ovarian Insufficiency/drug therapy* ; Follicle Stimulating Hormone/therapeutic use* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis

OBJECTIVE: Moxibustion, a common therapy in traditional Chinese medicine, has potential benefits for treating decreased ovarian reserve (DOR). The present study investigates the protective effect of moxibustion in a rat model of DOR and explores the possible mechanisms. METHODS: Sixty-four female Sprague-Dawley rats were randomly divided into four groups: control, DOR, moxibustion (MOX), and hormone replacement therapy (HRT). The DOR rat model was established by intragastric administration of 50 mg/kg Tripterygium glycoside suspension (TGS), once daily for 14 days. MOX and HRT treatments were given from the day TGS administration was initiated. The ovarian reserve function was evaluated by monitoring the estrus cycle, morphological changes in ovaries, levels of serum estradiol (E₂), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and anti-Mullerian hormone (AMH), pregnancy rate and embryo numbers. Terminal-deoxynucleotidyl transferase-mediated nick-end-labeling staining was used to identify ovarian granulosa cell apoptosis, while the protein and mRNA expressions of Bax, B-cell lymphoma-2 (Bcl-2), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) in ovarian tissues were examined by immunohistochemistry, Western blot and quantitative reverse transcription-polymerase chain reaction. RESULTS: Compared with the DOR group, MOX improved the disordered estrous cycle, promoted follicular growth, reduced the number of atresia follicles, increased the concentrations of serum E₂ and AMH, and decreased serum FSH and LH concentrations. More importantly, the pregnancy rate and embryo numbers in DOR rats were both upregulated in the MOX treatment group, compared to the untreated DOR model. Further, we found that the MOX group had reduced apoptosis of ovarian granulosa cells, increased Bcl-2 expression and reduced expression of Bax. Furthermore, the PI3K/AKT signaling pathway was triggered by the moxibustion treatment. CONCLUSION Moxibustion improved ovarian function and suppressed apoptosis of ovarian granulosa cells in a rat model of DOR induced by TGS, and the mechanism may involve the PI3K/AKT signaling pathway.
Animals ; Female ; Follicle Stimulating Hormone ; Luteinizing Hormone ; Moxibustion ; Ovarian Reserve ; Phosphatidylinositol 3-Kinase/pharmacology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Pregnancy ; Proto-Oncogene Proteins c-akt/pharmacology* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; bcl-2-Associated X Protein/genetics*

This study aims to explore the molecular mechanism of Lycium barbarum polysaccharides(LBP) in alleviating premature ovarian failure(POF) in mice via the 5'-adenosine monophosphate activated protein kinase(AMPK)/silent information regulator 1(Sirt1) signaling pathway. The POF mouse model was established by D-galactose(D-gal) injection at the back. Six groups were set up, including a normal control group, a model group, a LBP group, a 3-MA(autophagy inhibitor 3-methyladenine) group, an AMPK inhibitor group, and a LBPAMPK inhibitor group, with 15 mice in each group. After 28 continuous days of administration, enzyme-linked immunosorbent assay(ELISA) was employed to determine the levels of sex hormones [estradiol(E_2), luteinizing hormone(LH), and follicle-stimulating hormone(FSH)] in serum. The ovarian mass coefficient was measured. Senescence-associated β-Galactosidase(SA-β-Gal) staining and hematoxylin-eosin(HE) staining were performed for observing the state of ovarian senescence and the morphological changes of the ovary. Immunohistochemical method was used to measure the expression of the autophagy marker LC3-Ⅱ in ovarian tissue. Western blot was employed to measure the expression levels of the senescence marker p16~(INK4 a), autophagy markers(LC3-Ⅱ and Beclin-1), the autophagy substrate p62, lysosome-associated membrane protein 2(LAMP2), and the proteins in the AMPK/Sirt1 pathway and mammalian target of rapamycin complex 1(mTORC1)/UNC-51-like kinase 1 Ser757 site(Ulk1 Ser757) pathway. Compared with the normal control group, the modeling of POF decreased the ovarian granulosa cells and follicles, led to the ovarian aging and severe sex hormone secretion disorders, weakened ovarian autophagy activity, and down-regulated the expression of proteins in the AMPK/Sirt1 pathway(P<0.05). Compared with the model group, the treatment with LBP increased ovarian granulosa cells and follicles, alleviated aging and sex hormone disorders, increased autophagy activity, and activated the AMPK/Sirt1 pathway(P<0.05). Both 3-MA and AMPK inhibitor can inhibit autophagy and aggravate ovarian damage and aging in mice. AMPK inhibitor can partially attenuate the role of LBP in promoting autophagy activation and alleviating aging and ovarian tissue damage(P<0.05). LBP can alleviate the symptoms of POF induced by D-gal by promoting the activation of AMPK/Sirt1 pathway.
Animals ; Female ; Humans ; Mice ; AMP-Activated Protein Kinases/metabolism* ; Autophagy/drug effects* ; Follicle Stimulating Hormone/blood* ; Lycium/chemistry* ; Polysaccharides/therapeutic use* ; Primary Ovarian Insufficiency/drug therapy* ; Sirtuin 1/metabolism*

OBJECTIVE: To explore the effect of curcumin on the insulin receptor substrate 1 (IRS1)/phosphatidylinositol-3-kinase (PI3K)/endometrial expression of glucose 4 (GLUT4) signalling pathway and its regulator, phosphatase and tensin homolog (PTEN), in a rat model of polycystic ovarian syndrome (PCOS). METHODS: PCOS model was induced by letrozole intragastric administration. Sprague-Dawley rats were randomized into 4 groups according to a random number table: (1) control group; (2) PCOS group, which was subjected to PCOS and received vehicle; (3) curcumin group, which was subjected to PCOS and treated with curcumin (200 mg/kg for 2 weeks); and (4) curcumin+LY294002 group, which was subjected to PCOS, and treated with curcumin and LY294002 (a specific PI3K inhibitor). Serum hormone levels (17 β-estradiol, follicle stimulating hormone, luteinizing hormone, progesterone, and testosterone) were measured by enzyme linked immunosorbent assay, and insulin resistance (IR) was assessed using the homeostasis model assessment of IR. Ovarian tissues were stained with haematoxylin and eosin for pathological and apoptosis examination. Expression levels of key transcriptional regulators and downstream targets, including IRS1, PI3K, protein kinase B (AKT), GLUT4, and PTEN, were measured via reverse transcription polymerase chain reaction and Western blot, respectively. RESULTS: The PCOS group showed impaired ovarian morphology and function. Compared with the PCOS group, curcumin treatment exerted ovarioprotective effects, down-regulated serum testosterone, restored IR, inhibited inflammatory cell infiltration in ovarian tissues, decreased IRS1, PI3K, and AKT expressions, and up-regulated GLUT4 and PTEN expressions in PCOS rats (P<0.05 or P<0.01). In contrast, IRS1, PI3K, AKT, and PTEN expression levels were not significantly different between PCOS and curcumin+LY294002 groups (P>0.05). CONCLUSION The beneficial effects of curcumin on PCOS rats included the alteration of serum hormone levels and recovery of morphological ovarian lesions, in which, PTEN, a new target, may play a role in regulating the IRS1/PI3K/GLUT4 pathway.
Animals ; Female ; Humans ; Rats ; Cell Proliferation ; Curcumin/therapeutic use* ; Follicle Stimulating Hormone ; Glucose ; Hyperandrogenism ; Insulin Receptor Substrate Proteins/metabolism* ; Insulin Resistance ; Ovarian Cysts ; Ovarian Neoplasms ; Phosphatidylinositol 3-Kinase/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Polycystic Ovary Syndrome/drug therapy* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats, Sprague-Dawley ; Testosterone

Polycystic ovary syndrome (PCOS) is a common disease caused by complex endocrine and metabolic abnormalities in women of childbearing age. Metformin is the most widely used oral hypoglycemic drug in clinic. In recent years, metformin has been used in the treatment of PCOS, but its mechanism is not clear. In this study, we aimed to investigate the effect of metformin on PCOS and its mechanism through PCOS mouse model. Female C57BL/6J mice aged 4-5 weeks were intragastrically given letrozole (1 mg/kg daily) combined with a high-fat diet (HFD) for 21 days to establish the PCOS model. After modeling, metformin (200 mg/kg daily) was intragastrically administered. One month later, the body weight and oral glucose tolerance test (OGTT) were measured. Hematoxylin eosin (H&E) staining was used to detect the pathological changes of ovary. The serum levels of anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), E₂ and testosterone (T) were measured by ELISA. The expression of DDX4/MVH was detected by immunohistochemistry. DDX4/MVH and PCNA were co-labeled by immunofluorescence. The protein levels of DDX4/MVH, PCNA, cyclin D2, AMPK and mTOR were detected by Western blot. The results showed that after metformin treatment, the body weights of PCOS mice were gradually returned to normal, glucose tolerance was significantly improved, serum E₂ levels were increased, while AMH, LH, T levels and LH/FSH ratio were decreased. Ovarian polycystic lesions were reduced with reduced atresia follicles. Furthermore, the number of proliferative female germline stem cells (FGSCs) and levels of proliferation related proteins (PCNA, cyclin D2) were significantly increased, and the p-mTOR and p-AMPK levels were markedly up-regulated. These results suggest that metformin treatment not only improves hyperandrogenemia, glucose intolerance and polycystic ovarian lesions in PCOS, but also activates the function of FGSCs. The underlying mechanism may be related to the phosphorylation of AMPK and mTOR. These findings provide new evidence to use metformin in the treatment of PCOS and follicular development disorder.
AMP-Activated Protein Kinases ; Animals ; Cyclin D2 ; Female ; Follicle Stimulating Hormone/therapeutic use* ; Humans ; Luteinizing Hormone/therapeutic use* ; Metformin/pharmacology* ; Mice ; Mice, Inbred C57BL ; Oogonial Stem Cells/metabolism* ; Ovarian Cysts/drug therapy* ; Ovarian Neoplasms ; Polycystic Ovary Syndrome/drug therapy* ; Proliferating Cell Nuclear Antigen/therapeutic use* ; TOR Serine-Threonine Kinases

The growth and development of follicles are regulated by genes,hormones and growth factors autocrined and paracrined from granulosa cells,theca cells,and oocytes.Products of glycolysis from granulosa cells such as pyruvate and lactate are one of the main energy sources,which play an important role during folliculogenesis and follicle maturity.Studies on the changes of the products and rate-limiting enzymes during granulosa cells' glycolysis help to clarify the molecular mechanism of energy demand in folliculogenesis and guide the clinical treatment of infertility due to abnormal follicular development.This article reviews recent research advances in the energy demand and regulatory mechanism in different states of folliculogenesis.
Energy Metabolism ; Female ; Glycolysis ; Granulosa Cells ; Humans ; Intercellular Signaling Peptides and Proteins ; Oocytes ; Ovarian Follicle ; growth & development ; Theca Cells

BACKGROUND: Vitamin is a well-known co-factor for many metabolic processes and its roles in fertility and follicular growth have been studied. Vitamin supplementation is frequently achieved by daily ingestion in the form of a complex capsule. However, the role of single and complex vitamins in in vitro maturation of murine follicles is not fully elucidated. METHODS: In this study, we evaluated the effects of two forms of vitamins. Pure L-ascorbic acid, and multi-vitamin (vitamin C+vitamin B complex) was treated at two different concentrations (50 and 100 µg/ml), to pre-puberty murine follicles during in vitro maturation. To determine the specific stage of growth that is affected by treatment with vitamins, the vitamins were treated from day 0, 4, 9, and 13. Growth of each follicle was assessed by measuring diameters of whole expanded area and of the granulosa cells. Expression of follicular and oocyte growth-related genes and the effect of vitamin on the viability of follicles was assessed using senescence associated β-galactosidase staining. RESULTS: Treatment with vitamins promoted the in vitro growth of murine follicles and the upregulated the expression of granulosa cell- and oocyte-specific genes such as BMP15, Fsh receptor, and GDF9. The proliferation of the granulosa cells was enhanced by the treatment of vitamin. Fifty µg/ml concentration vitamin showed greater effects compared to higher concentration. The viability of in vitro grown follicles was also significantly improved in vitamin-treated follicles. The effects of single L-ascorbic acid and complex vitamin were not significantly different to those of day 4 and day 9 follicles. Vitamins promoted murine follicle development in vitro with different effects on specific growth stage. CONCLUSION: Supplementation of vitamins during in vitro maturation of murine follicles is an efficient strategy for in vitro expansion of follicular cells. These results could be customized to the sophisticated culture of follicles retrieved from aged or cancer-survived female that contain smaller number of follicles with reduced potential to develop into mature follicles.
Aging ; Ascorbic Acid ; Eating ; Female ; Fertility ; Granulosa Cells ; Humans ; In Vitro Techniques ; Metabolism ; Oocytes ; Ovarian Follicle ; Receptors, FSH ; Vitamins

The present study was aimed to investigate the expression relationship of Hippo signaling molecules and ovarian germline stem cell (OGSC) markers in the development schedule of OGSCs during ovarian aging in women and mice. The ovaries of 2-month-old mature (normal control) and 12-month-old (physiological ovarian aging) KM mice were sampled, and the ovarian cortex samples of young (postpuberty to 35 years old), middle age (36-50 years old) and menopausal period (51-60 years old) women were obtained with consent. The mice model of pathological ovarian aging was established by intraperitoneal injection of cyclophosphamide/busulfan (CY/BUS). HE staining was used to detect the changes of follicles at different stages, and the localization and expression changes of Hippo signaling molecules and OGSCs related factors (MVH/OCT4) were detected by immunohistochemistry and immunofluorescence staining. Western blot was used to detect the protein expression levels of the major molecules in the Hippo signaling pathway and OGSCs related factors. The results showed that there were not any normal follicles, but a few atresia follicles in the ovaries from physiological and pathological ovarian aging mice. Compared with the normal control mice, both the physiological and pathological ovarian aging mice showed decreased protein expression levels of the main Hippo signaling molecules (pYAP1) and MVH/OCT4; Whereas only the pathological ovarian aging mice showed increased ratio of pYAP1/YAP1. In comparison with the young women, the middle age and menopausal women showed looser structure of ovarian surface epithelium (OSE) and less ovarian cortical cells. The protein expression level of LATS2 in the OSE was the highest in young women, MST1 expression was the lowest in the menopausal period women, and the expression levels of YAP1 and pYAP1 were the highest in middle age women. Compared with the young women, the middle age and menopausal period women exhibited significantly decreased ratio of OSE pYAP1/YAP1, whereas there was no significant difference between them. The expression level of MVH protein in OSE from the young women was significantly higher than those of the middle age and menopausal period women. These results indicate that there is an expression relationship between the main molecules of Hippo signaling pathway and OGSCs related factors, which suggests that Hippo signaling pathway may regulate the expression levels of OGSCs related factors, thus participating in the process of physiological and pathological degeneration of ovarian.
Adaptor Proteins, Signal Transducing ; metabolism ; Adult ; Aging ; Animals ; Epithelium ; Female ; Humans ; Mice ; Middle Aged ; Octamer Transcription Factor-3 ; metabolism ; Oogonial Stem Cells ; metabolism ; Ovarian Follicle ; Ovary ; Phosphoproteins ; metabolism ; Protein-Serine-Threonine Kinases ; metabolism ; Signal Transduction ; Tumor Suppressor Proteins ; metabolism

Primary ovarian insuffiency (POI), which accounts for female infertility, is characterized by amenorrhea before the age of 40 and high serum level of follicular stimulating hormone (>40 U/L) at two measurements taken at least one month apart. The disorder is believed to have a strong genetic component. A large number of candidate genes have been proposed, though few of them were extensively studied. With the rapid evolvement of genome sequencing technology, recent research raised the possibility that the genes involved in essential steps of meiosis such as chromosome synapsis and recombination play an important role in the pathogenesis of POI. Clarifying the genetic pathogenesis of POI not only can enhance understanding of the molecular mechanism of reproductive functions and infertility, but also provide accurate information for genetic counseling for such patients.
Female ; Follicle Stimulating Hormone ; metabolism ; Humans ; Infertility, Female ; genetics ; Meiosis ; Primary Ovarian Insufficiency ; genetics ; metabolism