Extracellular vesicles (EV),nanoscale vesicles encapsulated by phospholipid bilayers,are rich in biological molecules such as nucleic acids,metabolites,proteins,and lipids derived from parental cells.They are mainly involved in intercellular communication,signal transmission,and material transport and affect the functions of target cells.Ovulation disorders account for a higher proportion in the factors causing infertility which demonstrates increasing incidence year by year.Non-coding RNAs participate in a series of physiological and pathological processes of follicular development,playing a key role in female infertility.This review systematically introduces the types and biological roles of EV and elaborates on the regulation of follicular development from the effects of EV and non-coding RNAs on granulosa cell function,oocyte maturation,ovulation,luteal formation,and steroid hormone synthesis,providing a new idea and a breakthrough point for the diagnosis and treatment of infertility.
Female ; Humans ; Oogenesis/physiology* ; Granulosa Cells ; Extracellular Vesicles/physiology* ; Cell Communication ; RNA, Untranslated ; Infertility

Melatonin,an endocrine hormone synthesized by the pineal gland,plays an important role in the reproduction.The growth and development of follicles is the basis of female mammalian fertility.Follicles have a high concentration of melatonin.Melatonin receptors exist on ovarian granulosa cells,follicle cells,and oocytes.It regulates the growth and development of these cells and the maturation and atresia of follicles,affecting female fertility.This paper reviews the protective effects and regulatory mechanisms of melatonin on the development of ovarian follicles,granulosa cells,and oocytes and makes an outlook on the therapeutic potential of melatonin for ovarian injury,underpinning the clinical application of melatonin in the future.
Animals ; Female ; Melatonin/pharmacology* ; Ovarian Follicle ; Oocytes ; Granulosa Cells/physiology* ; Mammals

BACKGROUNDPremature ovarian failure (POF) is a disease that affects female fertility but has few effective treatments. Ovarian reserve function plays an important role in female fertility. Recent studies have reported that hydrogen can protect male fertility. Therefore, we explored the potential protective effect of hydrogen-rich water on ovarian reserve function through a mouse immune POF model.

METHODSTo set up immune POF model, fifty female BALB/c mice were randomly divided into four groups: Control (mice consumed normal water, n = 10), hydrogen (mice consumed hydrogen-rich water, n = 10), model (mice were immunized with zona pellucida glycoprotein 3 [ZP3] and consumed normal water, n = 15), and model-hydrogen (mice were immunized with ZP3 and consumed hydrogen-rich water, n = 15) groups. After 5 weeks, mice were sacrificed. Serum anti-Müllerian hormone (AMH) levels, granulosa cell (GC) apoptotic index (AI), B-cell leukemia/lymphoma 2 (Bcl-2), and BCL2-associated X protein (Bax) expression were examined. Analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) software.

RESULTSImmune POF model, model group exhibited markedly reduced serum AMH levels compared with those of the control group (5.41 ± 0.91 ng/ml vs. 16.23 ± 1.97 ng/ml, P = 0.033) and the hydrogen group (19.65 ± 7.82 ng/ml, P = 0.006). The model-hydrogen group displayed significantly higher AMH concentrations compared with that of the model group (15.03 ± 2.75 ng/ml vs. 5.41 ± 0.91 ng/ml, P = 0.021). The GC AI was significantly higher in the model group (21.30 ± 1.74%) than those in the control (7.06 ± 0.27%), hydrogen (5.17 ± 0.41%), and model-hydrogen groups (11.24 ± 0.58%) (all P < 0.001). The GC AI was significantly higher in the model-hydrogen group compared with that of the hydrogen group (11.24 ± 0.58% vs. 5.17 ± 0.41%, P = 0.021). Compared with those of the model group, ovarian tissue Bcl-2 levels increased (2.18 ± 0.30 vs. 3.01 ± 0.33, P = 0.045) and the Bax/Bcl-2 ratio decreased in the model-hydrogen group.

CONCLUSIONSHydrogen-rich water may improve serum AMH levels and reduce ovarian GC apoptosis in a mouse immune POF model induced by ZP3.

Animals ; Anti-Mullerian Hormone ; blood ; Apoptosis ; drug effects ; Female ; Granulosa Cells ; cytology ; Hydrogen ; chemistry ; pharmacology ; Mice ; Mice, Inbred BALB C ; Ovarian Reserve ; drug effects ; physiology ; Ovary ; drug effects ; metabolism ; Primary Ovarian Insufficiency ; blood ; metabolism ; prevention & control ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Water ; administration & dosage ; chemistry ; pharmacology ; Zona Pellucida ; drug effects ; physiology ; bcl-2-Associated X Protein ; metabolism

Melatonin (N-acetyl-5-methoxytryptamine, MT) is a hormone synthesized and secreted by the pineal gland. Recent studies show that melatonin plays an essential role in the pathogenesis of many reproductive processes. High-concentration melatonin exists in human preovulatory follicular fluid and melatonin receptors are present in ovarian granulosa cells, which indicates the direct effects of melatonin on ovarian function. Reactive oxygen species are involved in a number of reproductive events, including folliculogenesis, follicular atresia, ovulation, oocyte maturation, and corpus luteum formation. Melatonin and its metabolites, as powerful antioxidants and free radical scavengers, can potentially inhibit premature ovarian failure. Literature published in recent years shows the essential roles of melatonin in improving human ovarian function and oocyte quality as well as in the management of infertility. Researches on the action mechanisms of melatonin may provide a theoretical basis for the prevention and treatment of some clinical diseases.
Female ; Granulosa Cells ; metabolism ; physiology ; Humans ; Melatonin ; metabolism ; physiology ; Ovarian Follicle ; growth & development ; metabolism ; Ovary ; physiology ; Reactive Oxygen Species ; metabolism

In mammals, ovarian follicle is made of an oocyte with its surrounding granulosa cells and theca cells. Follicular growth and development is a highly coordinated programmable process, which guarantees the normal oocyte maturation and makes it having the fertilizing capacity. The paracrine and autocrine between oocytes and granulosa cells are essential for the follicular development to provide a suitable microenvironment. Phosphatidylinositol-3 kinase /protein kinase B is one of these important regulatory signaling pathways during this developmental process, and bone morphogenetic protein-15 an oocyte-specific secreted signal molecule, which regulates the follicular development by paracrine in the mammalian ovary. The present article overviewed the role of phosphatidylinositol-3 kinase / protein kinase B signaling during the follicular development based on our previous investigation about protein kinase B /forkhead transcription factor forkhead family of transcription factors -3a, and then focused on the regulatory effects of bone morphogenetic protein-15, as a downstream signal molecule of phosphatidylinositol-3 kinase / forkhead family of transcription factors -3a pathway, on ovarian follicular development, which helped to further understand the molecular mechanism regulating the follicular development and to treat ovarian diseases like infertility.
Animals ; Bone Morphogenetic Protein 15 ; physiology ; Female ; Granulosa Cells ; physiology ; Humans ; Mammals ; Ovarian Follicle ; growth & development ; Ovary ; growth & development ; Phosphatidylinositol 3-Kinase ; physiology ; Proto-Oncogene Proteins c-akt ; physiology ; Signal Transduction

Echinomycin is a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity, which plays a crucial role in ovarian ovulation in mammalians. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1alpha-mediated endothelin (ET)-2 expressions contributed to ovarian ovulation in response to human chorionic gonadotropin (hCG) during gonadotropin-induced superuvulation. By real-time RT-PCR analysis, ET-2 mRNA level was found to significantly decrease in the ovaries after echinomycin treatment, while HIF-1alpha mRNA and protein expression was not obviously changed. Further analysis also showed that these changes of ET-2 mRNA were consistent with HIF-1 activity in the ovaires, which is similar with HIF-1alpha and ET-2 expression in the granulosa cells with gonadotropin and echinomycin treatments. The results of HIF-1alpha and ET-2 expression in the granulosa cells transfected with cis-element oligodeoxynucleotide (dsODN) under gonadotropin treatment further indicated HIF-1alpha directly mediated the transcriptional activation of ET-2 during gonadotropin-induced superuvulation. Taken together, these results demonstrated that HIF-1alpha-mediated ET-2 transcriptional activation is one of the important mechanisms regulating gonadotropin-induced mammalian ovulatory precess in vivo.
Animals ; Cells, Cultured ; Chorionic Gonadotropin/*pharmacology ; Echinomycin/*pharmacology ; Endothelin-2/genetics/*metabolism ; Female ; Gonadotropins, Equine/*pharmacology ; Granulosa Cells/drug effects/metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*antagonists & inhibitors/genetics/metabolism/physiology ; Oligonucleotides/genetics ; Ovary/cytology/drug effects/physiology ; Rats ; Rats, Sprague-Dawley ; Superovulation/*drug effects ; Transcriptional Activation

OBJECTIVETo study the role of extracellular signal-regulated protein kinase 5 (ERK5) during the biosynthesis of follicle-stimulating hormone (FSH)-mediated progesterone in primary granulosa cells.

METHODSThe expressions of phosphorylated and general forms of ERKS in primary granulosa cells after the treatment of FSH were detected by Western blot analysis. The subcellular localization of ERK5 was observed by confocal microscopy. The effect of ERK5 on FSH-mediated progesterone biosynthesis in primary granulosa cells was analyzed using recombinant adenovirus vectors.

RESULTSERK5 activation was induced by FSH in a time-dependent manner in primary cultured granulosa cells, although the general ERK5 protein level decreased also in a time-dependent manner. The treatment of FSH showed no remarkable effect on the subcellular distribution of endogenous ERK5, which was mainly in the cytoplasm of granulosa cells. The co-infection of Ad-caMEK5 and Ad-wtERK5 increased the progesterone production and StAR expression in primary cultured granulosa cells, whereas inhibition of ERK5 activation suppressed the FSH-stimulated progesterone production.

CONCLUSIONERK5 may stimulate FSH-mediated progesterone production in primary cultured granulosa cells.

Animals ; Cells, Cultured ; Female ; Follicle Stimulating Hormone ; pharmacology ; Granulosa Cells ; drug effects ; metabolism ; Mitogen-Activated Protein Kinase 7 ; metabolism ; physiology ; Progesterone ; biosynthesis ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To examine the association among malondialdehyde (MDA), superoxide dismutase (SOD) and incidence of apoptosis of granulosa cells in follicular fluid with the outcome of in vitro fertilization-embryo transfer (IVF-ET). METHODS: We recruited 51 women undergoing an IVF-ET programme for tubal factor infertility. The women with endometriosis or endocrine diseases and those with male factor infertility were excluded. All the 51 patients underwent a long gonadotropin-releasing hormone (GnRH) agonist protocol for pituitary downregulation followed by controlled ovarian hyperstimulation with rFSH. Granulosa cells were isolated from all aspirated follicular fluid using gradient centrifugation at oocyte retrieval. The level of MDA and the activity of the SOD were measured by the thiobarbituric acid(TBA)and the chemiluminescence method, respectively. The apoptosis was studied by flow cytometry using propidium iodide. RESULTS: Twenty-six out of the 51 patients (51.0%) were pregnant after IVF-ET. Non-pregnant patients showed significantly higher MDA level [(1.7±0.72) nmol/(g × prot) vs. (1.1±0.56) nmol/(g × prot), P<0.05)], higher incidence of apoptosis (24.8%±6.57% vs.19.0%±5.59%, P<0.05) and lower SOD level [(3.5±1.08)×10(3)NU/(g × prot) vs. (4.4±0.99)×10(3)NU/(g × prot), P<0.05)] in the granulose cells and lower good-embryo rate (54.9±20.22% vs. 65.9±16.16%, P<0.05) compared with the pregnant patients. No correlation was detected among SOD and the number of retrieved oocytes, oocyte maturity, embryo quality, fertilization, or cleavage. A significant negative correlation was detected between MDA and fertilization rate (r=-0.425, P=0.002). No significant correlation was detected between MDA and age, the number of retrieved oocytes, oocyte maturity, cleavage, or good-embryo rate. A significant negative correlation was detected between the incidence of apoptosis and the number of retrieved oocytes (r=-0.286, P=0.042), mature oocytes (r=-0.330, P=0.020) and good-embryo rate (r=-0.311, P=0.026). There was significant negative correlation between MDA and SOD levels (r=-0.471, P<0.001); and significant positive correlation between MDA level and incidence of apoptosis (r=0.475, P<0.001). CONCLUSION Oxidative stress may induce apoptosis in granulose cells and subsequently lower oocyte quality and lead to poor outcome of IVF-ET.
Adult ; Apoptosis ; physiology ; Embryo Transfer ; Female ; Fertilization in Vitro ; Granulosa Cells ; metabolism ; pathology ; Humans ; Malondialdehyde ; metabolism ; Ovary ; cytology ; metabolism ; Oxidative Stress ; Pregnancy ; Pregnancy Rate ; Superoxide Dismutase ; metabolism ; Treatment Outcome

OBJECTIVE: To determine the copy number of granular cell mitochondria DNA (mtDNA) and deletion of 4 977 bp in patients with diminished ovarian reserve (DOR) to primarily study the structural integrity of granular cell mtDNA. METHODS: We selected 50 DOR patients and 50 patients with normal ovarian reserve (NOR). Granular cells in liquor folliculi of these patients were collected at ovum pick-up day. DNA was extracted from the granular cells. The mtDNA 4 977 bp deletion of granular cells was detected by PCR and the number of granular cells mtDNA copies was detected by real-time PCR. RESULTS: No 4 977 bp deletion of ovary granular cell mitochondria DNA in the 100 patients was detected. There was no significant difference in the relative quantity of granular cell mitochondria DNA in the DOR group and the NOR group. CONCLUSION The structure of granular cells mtDNA in DOR patients is complete and granular cells may be used as donor cells for DOR patients plasma autologous transplants mitochondorial.
Adult ; Case-Control Studies ; DNA, Mitochondrial ; genetics ; Female ; Fertilization ; Granulosa Cells ; metabolism ; Humans ; Infertility, Female ; genetics ; Oocytes ; physiology ; Ovarian Diseases ; genetics ; Ovarian Follicle ; cytology ; Ovary ; physiopathology ; Sequence Deletion

AIMTo study the expression and control of telomerase in rat preantral ovary.

METHODSAdded different factors to the preantral ovarian granulosa cells, then using TRAP-ELISA to analyze the expression of telomerase.

RESULTSTelomerase activation was detected in granulosa cells. Telomerase activation could be significantly enhanced by hcG, FSH, verapamil and dbcAMP, and it was significantly reduced by antisense-c-myb ODN treatment. We also used radioimmunoassay to determine proterone and estrogen in these cell culture mediums. It was found that these two hormones' secretion were raised under verapamil and FSH; no change under hcG and dbcAMllted with telomerase activity. In MTT assay, the antisense hTERT ODN could significantly inhibited the proliferation of ovarian granulosa cells.

CONCLUSIONIt is suggested that telomerase activation is present in ovary antral granulosa cell. Anti-c-myb might inhibit telomerase activation, while FSH, hcG, verapamil, dbcAMP might enhance the telomerase activation.

Animals ; Cells, Cultured ; Chorionic Gonadotropin ; pharmacology ; Estradiol ; physiology ; Female ; Follicle Stimulating Hormone ; pharmacology ; Granulosa Cells ; cytology ; enzymology ; Ovary ; cytology ; enzymology ; Rats ; Rats, Sprague-Dawley ; Telomerase ; metabolism ; physiology ; Verapamil ; pharmacology