Effect of naringenin regulating RIP1-RIP3-MLKL signaling pathway on apoptosis of ovarian granulosa cells in rats with polycystic ovary syndrome / 中国药理学通报

Aim Based on the apoptotic pathway mediated by receptor interacting protein kinase(RIP)1-RIP3-mixed spectrum kinase domain like protein(MLKL), to explore the effects of naringenin on ovarian granulosa cell apoptosis in rats with polycystic ovary syndrome(PCOS). Methods SD rats were randomly assigned into normal control group, model group, naringenin group, RIP1 inhibitor(Nec-1)group, RIP1-RIP3-MLKL necrosis signal activator(Z-VAD-fmk)group, naringenin+Z-VAD-fmk group, 15 rats per group. ELISA method was performed to measure the levels of IL-1β and TNF-α in ovarian tissue. HE method was performed to observe the shape of the ovary. Granular cells were isolated from ovarian tissue, and flow cytometry was performed to measure apoptosis rate and necrosis rate. Immunohistochemistry was performed to measure the positive expression of p-RIP1 in ovarian tissue. Western blot was employed to detect the expression of RIP1-RIP3-MLKL pathway. Results RIP1 specific inhibitor Nec-1 and naringenin could block the phosphorylation and activation of RIP1, inhibit the RIP1-RIP3-MLKL signaling pathway, reduce the inflammation level in PCOS rats, and alleviate the necrosis and apoptosis of ovarian granulosa cells(P<0.05). Z-VAD-fmk could promote the activation of RIP1-RIP3-MLKL pathway, aggravate the apoptosis of ovarian granulosa cells, and partially weaken the anti-apoptosis effect of naringenin(P<0.05). Conclusions Naringenin may inhibit the apoptosis of ovarian granulosa cells in PCOS rats by blocking the activation of the necrotic apoptotic pathway mediated by RIP1-RIP3-MLKL.

Xinjia Congrong Tusizi Decoction Regulates Ferroptosis of Granulosa Cells in Rat Model of Premature Ovarian Insufficiency via p53/Nrf2 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Xinjia Congrong Tusizi decoction （XJCTD） on ovarian functions in the rat model of premature ovarian insufficiency （POI） and decipher the mechanism of regulating the tumor suppressor protein （p53）/nuclear factor E2-related factor 2 （Nrf2） pathway to attenuate granulosa cell ferroptosis. MethodForty-eight SPF-grade female SD rats were randomized into control， model， low-， medium-， and high-dose （1.1， 2.2， 4.4 g·kg-1） XJCTD， and Western medicine （coenzyme Q10， 0.002 7 g·kg-1） groups， with eight rats in each group. The rat model of POI was established by gavage of triptolide （TP）， and after successful modeling， each group was administrated with the corresponding drugs by gavage for 14 d. The body weight and ovarian weight of each rat were weighed and the ovarian index was calculated. The morphology of the ovarian tissue was observed by hematoxylin-eosin staining， and the proportions of growing follicles and atretic follicles were calculated. The serum levels of anti-Müllerian hormone （AMM）， estradiol （E2）， and follicle-stimulating hormone （FSH） were measured by enzyme-linked immunosorbent assay （ELISA）. The DCFH-DA fluorescent probe was used to measure the reactive oxygen species （ROS） content in granulosa cells. The content of cellular Ferrous ion （Fe2+）， lipid peroxide （LPO）， malondialdehyde （MDA）， glutathione （GSH）， and superoxide dismutase （SOD） was detected by colorimetry. The expression of the tumor suppressor protein p53，Nrf2， solute carrier family 7 member 11 （SLC7A11）， and glutathione peroxidase 4 （GPX4） was determined by immunohistochemistry and Western blot. ResultCompared with the control group， the model group showed decreased ovarian weight， body weight， and ovarian index （P<0.01）， reduced ovarian tissue volume and proportion of growing follicles （P<0.01）， increased proportion of atretic follicles （P<0.01）， lowered AMH and E2 levels and elevated FSH level in the serum （P<0.01）， and elevated levels of Fe2+， ROS， LPO， and MDA （P<0.01） and lowered levels of GSH and SOD in granulosa cells （P<0.01）. Moreover， the modeling up-regulated the expression of p53 （P<0.01） and down-regulated the expression of Nrf2， SLC7A11， and GPX4 （P<0.05， P<0.01） in the ovarian tissue. Compared with the model group， XJCTD increased the body weight， ovarian weight， and ovarian index （P<0.01）， alleviated the pathological changes in the ovarian tissue， increased the proportion of growing follicles （P<0.01）， decreased the proportion of atretic follicles （P<0.01）， and reduced the content of ROS in granulosa cells （P<0.05， P<0.01）. In addition， medium- and high-dose XJCTD lowered the FSH level （P<0.01） and raised E2 and AMH levels （P<0.01） in the serum， reduced the Fe2+ content （P<0.05， P<0.01）， and increased the SOD content （P<0.01） in granulosa cells. High-dose XJCTD reduced the LPO and MDA content （P<0.01） and increased the SOD content （P<0.01） in the granulosa cells， down-regulated the expression of p53 （P<0.05）， and up-regulated the expression of Nrf2， SLC7A11， and GPX4 in the ovarian tissue （P<0.05， P<0.01）. ConclusionXJCTD may protect the ovarian function in the rat model of POI by regulating the p53/Nrf2 signaling pathway to attenuate the ferroptosis of ovarian granulosa cells.

Effect of follicular fluid-derived exosomal mi RNAs on follicular dysplasia mediated via glycolysis of granulosa cells in patients with polycystic ovary syndrome and its mechanism / 中国生物制品学杂志

@#Objective To evaluate the effect of follicular fluid(FF)exosomal miRNAs on follicular dysplasia in patients with polycystic ovary syndrome(PCOS)mediated by glycolysis pathway of granulosa cells(GCs),and to explore the mechanism. Methods Three PCOS infertile patients and three non-PCOS infertile patients were recruited. The baseline hormone levels of the two groups were measured before ovulation induction. The bilateral FF was obtained by puncture after short-acting and long-term ovulation induction,and the exosomes were collected by ultracentrifugation and identified by transmission electron microscopy. The total exosomal RNA was extracted by Trizol method to construct the library,which was compared to the reference genome GRCh38 for statistical analysis after miRNA sequencing and quality control processing. Clustering Profiler R package was used to implement GO annotation analysis and KEGG pathway analysis of the differentially expressed genes(DEGs),and Omnipath software for miRNAs interaction analysis. A total of 16 miRNA were randomly selected and detected by qPCR to verify the accuracy of the miRNA sequencing results. Results Compared with the non-PCOS group,luteinizing hormone(LH),anti-Muerian hormone(AMH),testosterone and antral follicle counts in PCOS group increased significantly(t = 2. 479 ～ 9. 163,each P < 0. 05). The exosomes of FF in both groups showed the cup-shaped vesicles with clear edge and light staining in the center,with the diameters of 100 — 150 nm and intact structure,and the concentration was about 8 × 1010particles/mL. A total of 928 miRNAs were detected by miRNA sequencing. Compared with the non-PCOS group,59 differentially expressed miRNA(DEmiRNA)were screened out in exosomes of POCS group,of which 31 were up-regulated and 28 were down-regulated. The differential trend of gene expression detected by qPCR was highly similar to that of miRNA sequencing. In FF exosomes of PCOS patients,the glycolysis efficiency and apoptosis of GCs were significantly changed by miRNA regulating mRNA. PKM,PFKL and HK2 were the key target genes for miRNA to regulate GCs glycolysis,and SLC2A1 was the key target gene for miRNA to regulate GCs apoptosis. Conclusion The miRNAs in FF exosomes of PCOS patients can weaken the glycolysis of GCs while accelerate the apoptosis,thus reducing the production of ATP and lactic acid,resulting in follicular dysplasia.

Regulatory Mechanism of Berberine in Inhibiting Apoptosis and Autophagy in Ovarian Granulosa Cells Based on SIRT1/FoxO1 Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect and regulatory mechanism of berberine （BBR） against the senescence of ovarian granulosa cells. MethodA cell senescence model in the human ovarian granulosa-like tumor （KGN） cell line was induced by H2O2. A control group， a model group， and high-dose （1 μmol·L-1） and low-dose （0.5 μmol·L-1） BBR groups were set up. The cells in the model group and the BBR groups were incubated with 10 μmol·L-1 H2O2 for 40 min. The effect of BBR on KGN cell proliferation was detected by cell counting kit-8 （CCK-8） assay. The effect of BBR on the senescence of KGN cells was detected by β-galactosidase staining. The effects of BBR on the apoptosis and ROS content of KGN cells were detected by flow cytometry. The effects of BBR on the mRNA expression of B-cell lymphoma-2 （Bcl-2）/Bcl-2-associated X protein （Bax）， cysteinyl aspartate-specific protease-3 （Caspase-3）， forkhead transcription factor O1 （FoxO1）， and catalase （CAT） was detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Western blot was used to detect the effects of BBR on protein expression of silent information regulator1 （SIRT1）， superoxide dismutase 2 （SOD2）， c-Jun N-terminal kinase （JNK）， FoxO1， autophagy-associated protein microtubule-associated protein light chain 3Ⅱ （LC3BⅡ）， mammalian ortholog of yeast Atg6 （Beclin-1）， and ubiquitin-binding protein p62. ResultAfter H2O2 induction for 40 min， the cell proliferation rate of the model group decreased compared with that of the control group （P<0.01）， and the cell proliferation rates of the BBR groups increased compared with that of the model group （P<0.05）. The results of β-galactosidase staining showed that the cells of the model group showed significant senescence compared with those of the control group （P<0.01）， and the cellular senescence in the BBR groups was reduced compared with that of the model group （P<0.01）. As revealed by flow cytometry， compared with the control group， the model group showed increased apoptosis rate （P<0.01）， and compared with the model group， BBR groups showed decreased apoptosis rates （P<0.05）. Meanwhile， the ROS content in the model group increased compared with that in the control group （P<0.01）， and compared with the model group， the BBR groups showed reduced cellular ROS content （P<0.01）. The Real-time PCR results showed that compared with the control group， the model group showed decreased mRNA expression of CAT and Bcl-2/Bax in KGN cells and increased mRNA expression of Caspase-3 and FoxO1 （P<0.05）， and compared with the model group， the BBR groups showed increased mRNA expression of CAT and Bcl-2/Bax （P<0.05） and reduced mRNA expression of Caspase-3 and FoxO1 in KGN cells （P<0.05）. As revealed by Western blot results， SIRT1， SOD2， and p62 protein levels decreased in the model group compared with those in the control group （P<0.01）， and JNK FoxO1， LC3BⅡ， and Beclin-1 protein levels increased （P<0.05）. After BBR intervention， SIRT1， SOD2， and p62 protein levels increased （P<0.01）， and JNK， FoxO1， LC3BⅡ， and Beclin-1 protein levels decreased compared with those in the model group （P<0.05）. ConclusionBBR has an inhibitory effect on ovarian granulosa cell senescence， and the mechanism is related to the inhibition of apoptosis and autophagy mediated by the SIRT1/FoxO1 pathway.

mRNA Expression of Aquaporins in Human Luteinized Granulosa Cells from Different Diameter Follicles / 中山大学学报(医学科学版)

ObjectiveTo investigate the mRNA expression levels of various aquaporins （AQPs） in luteinized granulosa cells from follicles of different diameters. MethodsFrom March 25， 2022 to September 23， 2022 in our reproductive medicine center， 48 women undergoing in-vitro fertilization （IVF） were enrolled and divided into the antagonist group and the agonist group according to the ovarian stimulation protocol. Follicular fluid samples were collected on the day of oocyte pick-up and granulosa cells were extracted from follicles of different diameters： small （<13 mm）， medium （13~18 mm） and large （≥18 mm）. After RNA quantification， 22 cases （66 samples） were included for analysis and mRNA expression levels of AQPs were compared among the three follicle groups. ResultsThe mRNA expression of aquaporin 2 （AQP2） in luteinized granulosa cells increased with the increase of follicle diameter （linear trend P = 0.004） and the difference was statistically significant between two groups of large and small follicles （P = 0.017）. Statistical difference was found in the antagonist group （P = 0.049 6）， but not in the agonist group （P = 0.108）. ConclusionThe mRNA level of AQP2 in luteinized granulosa cells increases with the increase of follicle diameter and its expression is related to the ovarian stimulation protocol， suggesting that AQP2 may play a role in follicle growth and follicular fluid formation， and its mRNA expression level may be regulated by follicle stimulating hormone （FSH） and luteinizing hormone （LH）.

Mechanism of Liuwei Dihuang Pills in treatment of mice with diminished ovarian reserve based on proteomics / 中国中药杂志

This study aims to investigate the efficacy and possible mechanism of Liuwei Dihuang Pills in the treatment of diminished ovarian reserve(DOR) by using proteomic techniques. Firstly, cyclophosphamide(60 mg·kg~(-1)) combined with busulfan(6 mg·kg~(-1)) was injected intraperitoneally to establish the mouse model of DOR. After drug injection, the mice were continuously observed and the success of modeling was evaluated by the disturbance of the estrous cycle. After successful modeling, the mice were administrated with the suspension of Liuwei Dihuang Pills by gavage for 28 days. At the end of the gavage, four female mice were selected and caged together with males at a ratio of 2∶1 for the determination of the pregnancy rate. Blood and ovary samples were collected from the remaining mice on the next day after the end of gavage. Hematoxylin-eosin(HE) staining and transmission electron microscopy(TEM) were then employed to observe the morphological and ultrastructural changes in the ovaries. The serum levels of hormones and oxidation indicators were measured by enzyme-linked immunosorbent assay. Quantitative proteomics techniques were used to compare the ovarian protein expression before and after modeling and before and after the intervention with Liuwei Dihuang Pills. The results showed that Liuwei Dihuang Pills regulated the estrous cycle of DOR mice, elevated the serum levels of hormones and anti-oxidation indicators, promoted follicle development, protected the mitochondrial morphology of ovarian granulosa cells, and increased the litter size and survival of DOR mice. Furthermore, Liuwei Dihuang Pills negatively regulated the expression of 12 differentially expressed proteins associated with DOR, which were mainly involved in lipid catabolism, inflammatory response, immune regulation, and coenzyme biosynthesis. These differentially expressed proteins were significantly enriched in sphingolipid metabolism, arachidonic acid metabolism, ribosomes, ferroptosis, and cGMP-PKG signaling pathway. In summary, the occurrence of DOR and the treatment of DOR with Liuwei Dihuang Pills are associated with multiple biological pathways, mainly including oxidative stress response, inflammatory response, and immune regulation. "Mitochondria-oxidative stress-apoptosis" is the key to the treatment of DOR by Liuwei Dihuang Pills. YY1 and CYP4F3 may be the key upstream targets that trigger mitochondrial dysfunction and ROS accumulation, and the metabolism of arachidonic acid is the main signaling pathway of drug action.

Effect of Qigongwan on Quality of Oocytes and Embryonic Development Potential in Patients with PCOS Infertility with Phlegm-dampness Syndrome / 中国实验方剂学杂志

ObjectiveTo observe the regulation of Qigongwan on the expression of proliferation and apoptosis-related factors programmed cell death 4 （PDCD4） and proliferating cell nuclear antigen （PCNA） in ovarian granulosa cells （GCs） in patients with polycystie ovarian syndrome （PCOS） infertility with phlegm-dampness syndrome， and to explore the effect of Qigongwan on the quality of oocytes and embryonic development potential. MethodSixty-six patients with PCOS with phlegm-dampness syndrome who underwent in vitro fertilization and embryo transfer （IVF-ET） were randomly selected and divided into an observation group （Qigongwan + western medicine） and a control group （western medicine）， with 33 patients in each group. Antagonist regimen was used to promote ovulation in the two groups. The observation group was given Qigongwan one cycle before IVF based on the treatment of conventional western medicine， while the control group was not given Chinese medicine. The improvement of phlegm and dampness syndrome， the dosage and the number of days of using gonadotropins （Gn）， the levels of luteinizing hormone （LH）， estradiol （E2）， and progesterone （P） on the day of human chorionic gonadotropin（HCG） injection， the 2PN fertilization rate， and the high-quality embryo rate of patients in the two groups were compared. Real-time polymerase chain reaction （Real-time PCR） and Western blot technology were used to detect the expression of PCNA and PDCD4 in GCs. ResultAs compared with groups before treatment， the score of phlegm-dampness syndrome in both groups was significantly lower （P<0.01）. The score of phlegm and dampness syndrome in the observation group was significantly lower than that of the control group （P<0.01）. As compared with the control group， the levels of LH， E2， and P in the observation group was higher， but only the difference in the level of E2 was statistically significant （P<0.01）. The 2PN fertilization rate ［82.25% （556/676） vs 69.92% （365/522）， χ2=25.172， P<0.01］ and high-quality embryo rate ［44.19% （190/430） vs 34.23% （102/298）， χ2=7.266， P<0.01］ in the observation group were significantly higher than that of the control group （P<0.01）. As compared with the control group， the mRNA and protein expression of PDCD4 in ovarian GCs was down-regulated in the observation group and that of PCNA was up-regulated （P<0.05）. ConclusionBy down-regulating the expression of PDCD4 and up-regulating the expression of PCNA， Qigongwan may interfere with follicle development， adjust hormone levels， improve the symptomatic manifestations of patients with PCOS with phlegm-dampness syndrome， inhibit the apoptosis of GCs， and promote growth， thus improving the quality of oocytes and embryonic development potential.

Research Progress in the Regulation of Follicle Development by Melatonin / 中国医学科学院学报

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.

Melatonin protects against ovarian damage by inhibiting autophagy in granulosa cells in rats

Abstract Objectives: This study sought to further verify the protective mechanism of Melatonin (MT) against ovarian damage through animal model experiments and to lay a theoretical and experimental foundation for exploring new approaches for ovarian damage treatment. Method: The wet weight and ovarian index of rat ovaries were weighted, and the morphology of ovarian tissues and the number of follicles in the pathological sections of collected ovarian tissues were recorded. And the serum sex hormone levels, the key proteins of the autophagy pathway (PI3K, AKT, mTOR, LC3II, LC3I, and Agt5) in rat ovarian tissues, as well as the viability and mortality of ovarian granulosa cells in each group were measured by ELISA, western blotting, CCK8 kit and LDH kit, respectively. Results: The results showed that MT increased ovarian weight and improved the ovarian index in ovarian damage rats. Also, MT could improve autophagy-induced ovarian tissue injury, increase the number of primordial follicles, primary follicles, and sinus follicles, and decrease the number of atretic follicles. Furthermore, MT upregulated serum AMH, INH-B, and E2 levels downregulated serum FSH and LH levels in ovarian damage rats and activated the PI3K/AKT/mTOR signaling pathway. Besides, MT inhibited autophagic apoptosis of ovarian granulosa cells and repressed the expression of key proteins in the autophagic pathway and reduced the expression levels of Agt5 and LC3II/I. Conclusions: MT inhibits granulosa cell autophagy by activating the PI3K/Akt/mTOR signaling pathway, thereby exerting a protective effect against ovarian damage.

Morphology and Biochemistry of Ovulation Morfologia e bioquímica da ovulação

Abstract The process of ovulation involves multiple and iterrelated genetic, biochemical, and morphological events: cessation of the proliferation of granulosa cells, resumption of oocyte meiosis, expansion of cumulus cell-oocyte complexes, digestion of the follicle wall, and extrusion of the metaphase-II oocyte. The present narrative review examines these interrelated steps in detail. The combined or isolated roles of the folliclestimulating hormone (FSH) and luteinizing hormone (LH) are highlighted. Genes indiced by the FSH genes are relevant in the cumulus expansion, and LH-induced genes are critical for the resumption ofmeiosis and digestion of the follicle wall. A nonhuman model for follicle-wall digestion and oocyte release was provided.

Resumo O processo de ovulação envolve modificações genéticas, bioquímicas e morfológicas múltiplas e interrelacionadas: suspensão da proliferação das células da granulosa, reinício da meiose do oócito, expansão das células do complexo cumulus-oócito, digestão da parede folicular, e extrusão do oócito. Esta revisão narrativa examina em detalhes cada um desses eventos e os principais genes e proteínas envolvidos. Mais importante, a ação combinada ou isolada do hormônio folículo-estimulante (HFE) e do hormônio luteinizante (HL) é destacada. Detalha-se o papel do HFE na expansão do cumulus e do HL na digestão da parede folicular, permitindo a extrusão do oócito na superfície ovariana. Proveu-se um modelo não humano para explicar a digestão da parede folicular.

Transforming growth factor β signaling pathway regulating the function of follicle stimulating hormone in ovarian granulosa cells / 解剖学报

Objective To explore the interaction between follicle stimulating hormone (FSH) and transforming growth factor Beta (TGF-β) signaling pathway in rat ovarian granulosa cells. Methods The granulosa cells isolated from the follicles of 21 days SD rats. The experiments were divided into three groups: control group, FSH treated group and transforming growth factor beta receptor II (TGF-β R II) neutralizated group. Immunocytochemistry (ICC) and Western blotting were then used to locate and detect the expression level of TGF-β R II and proliferating cell nuclear antigen (PCNA). The proliferation index (PI), cell cycle and percentage of apoptotic cells were assessed by flow cytometry (FCM), and the level of estradiol (E2) was determined by ELISA. Results FSH increased the expression of PCNA and PI, changed the cell cycle and inhibited apoptosis of GCs, and these actions were reduced significantly when TGF- βsignaling pathway was inhibited (P0.05). Conclusion The effects of FSH on ovarian granulosa cells are partly affected by the TGF-β signaling pathway.

Effect of Guizhi Fulingwan on Autophagy of Ovarian Granulosa Cells in Mice with Polycystic Ovary Syndrome / 中国实验方剂学杂志

Objective:To investigate the effect of Guizhi Fulingwan on autophagy of ovarian granulosa cells in mice with polycystic ovary syndrome （PCOS）. Method:Twenty SD mice were randomized into a normal group （<italic>n</italic>=10） and a PCOS model group （<italic>n</italic>=10）， followed by PCOS modeling and <italic>in vitro</italic> culture of extracted ovarian granulosa cells. The ovarian granulosa cells of normal mice were classified into the control group and treated with 10% blank serum while those of PCOS mice into the experimental groups and with 10% Guizhi Fulingwan-containing serum at different concentrations （17.6， 35.1， 70.2 mg·kg^-1） and 10% metformin-containing serum （25 mg·kg^-1）， respectively， for 72 h. During the modeling， the changes in mouse body weight were measured. After modeling， the ovarian morphology was observed by microscopy， and the fasting blood glucose （FBG） was measured by Roche glucometer. Following the detection of fasting insulin （FI） and testosterone （T） levels by radioimmunoassay， the proliferation of ovarian granulosa cells was determined using cell counting kit-8 （CCK-8） to figure out the maximal dose of drug-containing serum that did not obviously affect the cell viability for subsequent assay. The autophagy of ovarian granulosa cells was examined by flow cytometry， and the protein expression levels of intracellular microtubule-associated protein 1 light chain 3Ⅰ （LC3Ⅰ）， LC3Ⅱ， Beclin1， and p62 were assayed by Western blott. Result:Compared with the blank group， the model group showed increased body weight and elevated FI， FBG， and T levels （<italic>P</italic><0.05，<italic>P</italic><0.01）， indicating the successful modeling of PCOS mice. Flow cytometric assay proved that the incubation with 10% Guizhi Fulingwan serum-containing medium resulted in a decline of autophagy （<italic>P</italic><0.05）. As demonstrated by Western blot assay results， the protein expression levels of Beclin1 and LC3 Ⅱ/Ⅰ in the model group increased significantly as compared with those of the blank group， whereas the expression level of p62 decreased significantly （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with the model group， the medium- and high-dose Guizhi Fulingwan groups exhibited significantly down-regulated Beclin1 and LC3 Ⅱ/Ⅰ levels but remarkably up-regulated p62 （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:Guizhi Fulingwan inhibits the autophagy of ovarian granulosa cells by down-regulating the protein expression levels of Beclin1 and LC3 Ⅱ/Ⅰ.

Effect of Cangfu Daotan Pill Containing Serum on Autophagy of Ovarian Granulosa Cells of Rats / 中国药房

OBJECTIVE：To study the effect of Cangfu daotan pill （CDP）containing serum on autophagy of ovarian granulosa cells（GCs）of rat. METHODS ：Three-month-old SD rat were divided into normal saline group （normal saline ，ig），FSH injection group（10.71 IU/kg，ih），CDP irrigation high-dose ，medium-dose and low-dse groups [ 0.5，1，2 mg/g（by crude drug ），ig]，with 6 rats in each group. They were given relevant medicine subcutaneously/intragastrically ，once a day ，for consecutive 3 days. After last medication ，blood sample was collected from the abdominal aorta to obtain drug-containing serum. GCs of rat were divided into blank control group ，model group ，FSH group （positive control ）and CDP high-dose ，medium-dose and low-dose groups. The autophagy model was induced by giving testosterone propionate ，except that the blank control group was directly added with 100 μL serum of normal saline group. Then model group was given 100 μL serum of normal saline group，and administration groups were given 100 μL drug-containing serum of corresponding drug group. The contents of estradiol（E2）and progesterone （P）in supernatant of cells were determined by ELISA. Western blot assay was used to detect protein expression of PI 3K，Akt and mTOR in cells. The mRNA expression of PI 3K，Akt，mTOR，Beclin 1，LC3Ⅰ，LC3Ⅱ and p 62 were detected by RT-PCR. RESULTS ： Compared with blank control group ，the content of E 2 in supernatant ，relative mRNA and protein expression of PI 3K，Akt and mTOR were decreased significantly in model group （P＜0.01），while relative mRNA expression of Beclin 1，LC3Ⅰ and LC 3Ⅱ were increased significantly （P＜0.01）. Compared with model group ，the content of E 2 in supernatant were significantly increased in FSH group ，CDP medium-dose and high-dose groups ，while relative mRNA expression of Beclin 1，LC3Ⅰ，LC3Ⅱ and p 62 were decreased significantly （P＜0.05 or P＜0.01）；relative mRNA and protein expression of PI 3K，Akt and mTOR were increased significantly in administration groups （P＜0.05 or P＜0.01）. CONCLUSIONS ：CDP can inhibit autophagy of GCs by activating related protein and mRNA expression of PI 3K/Akt/ mTOR signaling pathway.

Effects of bisphenol A on apoptosis of ovarian preantral follicular granulosa cells and ovarian development in mice / 南方医科大学学报

OBJECTIVE@#To investigate the effect of environmental estrogen bisphenol A (BPA) exposure on apoptosis of mouse ovarian preantral follicular granulosa cells and ovarian development and explore the underlying mechanism.@*METHODS@#Mouse ovarian preantral follicular granulosa cells were isolated from female ICR mice at postnatal day (PND) 10 and cultured @*RESULTS@#Compared with the control cells group, the isolated cells exposed to a low concentration of BPA (50 μmol/L) showed a significantly lowered apoptosis rate, increased mitochondrial membrane potential, and enhanced cellular proliferation (@*CONCLUSIONS@#BPA can concentration-dependently regulate the function of ovarian preantral follicular granulosa cells in mice and potentially affects both the pregnant mice and the offspring female mice in light of early ovarian development.

Effect of Electro-acupuncture on Expression of IRS-1/PI3K/GLUT4 Pathway in Ovarian Granulosa Cells of Infertile Patients with Polycystic Ovary Syndrome-Insulin Resistance of Phlegm-Dampness Syndrome / 中国结合医学杂志

OBJECTIVE@#To evaluate the effect of electro-acupuncture (EA) in infertile patients with phlegm-dampness polycystic ovary syndrome-insulin resistance (PCOS-IR).@*METHODS@#Seventy-six PCOS-IR patients who underwnet in vitro fertilization and embryo transfer (IVF-ET) were equally assigned to two groups according to a random digital table: the EA group and the control group, with 38 cases in each group. Before undergoing IVF, the two groups were treated with EA or pseudo-acupuncture, respectively, for 3 menstrual cycles. The intervention was 25 min twice a week until the day of oocyte collection. The selected acupoints were Zhongwan (RN 12), Tianshu (ST 25), Daheng (SP 15), Daimai (GB 26), Qihai (CV 6), Guanyuan (CV 4), and bilateral points including Xuehai (SP 10), Fenglong (ST 40), Zusanli (ST 36), and Yinlingquan (SP 9). Evaluation of phlegm-dampness syndrome score and IR score were carried out before and after treatment. Additionally, the number of oocytes retrieved, transplantable embryo rate, high-quality embryo rate, clinical pregnancy rate and live birth rate were compared between the two groups. Real-time polymerase chain reaction analysis was used to monitor the mRNA expression of the insulin receptor substrate (IRS-1), phosphatidylinositiol 3-kinase (PI3K) and glucose transport factor 4 (GLUT4) in ovarian granulosa cells.@*RESULTS@#EA treatment reduced the phlegm-dampness syndrome score as well as the IR scores compared with the control group (P0.05). Moreover, the transplantable embryo rate [49.0% (284/580) vs. 41.9% (273/652)], high-quality embryo rate [36.6% (104/284) vs. 27.8% (76/273)], and live birth rate [50% (19/38) vs. 26.3% (10/38)] in the EA group were significantly higher than in the control group (P<0.05). Gene expression analyses revealed significantly elevated IRS-1, PI3K and GLUT4 mRNA in ovarian granulosa cells of the EA group compared with the control group (P<0.05).@*CONCLUSIONS@#EA may ameliorate the effects of phlegm-dampness syndrome and ovarian IR in PCOS-IR patients. Mechanistically, this effect might be through an upregulation of the IRS-1/PI3K/GLUT4 signaling pathway, which may result in improved oocyte quality and embryonic development potential. (Registration No. ChiCTR1800015453).

Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Cathepsin D (CTSD), the major lysosomal aspartic protease that is widely expressed in different tissues, potentially regulates the biological behaviors of various cells. Follicular granulosa cells are responsive to the increase of ovulation number, hence indirectly influencing litter size. However, the mechanism underlying the effect of CTSD on the behaviors of goat granulosa cells has not been fully elucidated. This study used immunohistochemistry to analyze CTSD localization in goat ovarian tissues. Moreover, western blotting was applied to examine the differential expression of CTSD in the ovarian tissues of monotocous and polytocous goats. Subsequently, the effects of CTSD knockdown on cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific traits, including bone morphogenetic protein receptor IB (

Tumor de células de la granulosa de tipo juvenil en ovario izquierdo / Juvenile granulosa - cells tumor in the left ovary

RESUMEN Se presentó el caso de una adolescente de 14 años, en el servicio de Cirugía Pediátrica del Hospital Pediátrico Docente "Pedro Agustín Pérez" de Guantánamo, que refirió aumento de volumen de todo el abdomen y dolor abdominal difuso. Al examinarla se constató una tumoración visible y palpable en hemiabdomen inferior. Los estudios complementarios imagenológicos mostraron una masa ecogénica, heterogénea que ocupaba hipogastrio, más lateralizada hacia la izquierda. Tras discusión colectiva multidisciplinaria se le realizó oforectomía izquierda y los estudios anatomopatológicos confirmaron la presencia de tumor de células de la granulosa de tipo juvenil en ovario izquierdo. Técnicas quirúrgicas que permitan preservar la capacidad reproductiva a niñas con neoplasias malignas, resultan usadas ahora con frecuencia y garantizan una mayor calidad de vida.

ABSTRACT A 14-year-old girl presented to the pediatric surgery department at the Pediatric Teaching Hospital "Pedro Agustín Pérez" in Guantanamo. She reported a difuse abdominal pain and distention. The examination revealed a visible and palpable tumor in the lower hemiabdomen. Complementary imaging studies showed an ecogenic and heterogeneous mass situated in the left hypogastrium. After a multidisciplinary team meeting, an ophthalmectomy was performed, and anatomopathological studies confirmed the presence of juvenile granulosa cells tumor on the left ovary. Surgical techniques that allow girls with malignant neoplasms to preserve their reproductive capacity are now frequently used and they guarantee higher life quality.

Investigation of ADAMTS-1, BAX and IL-10 expressions in granulosa cells determinanting fertilization success in IVF / Investigación de las expresiones ADAMTS-1, BAX e IL-10 en célula de la granulosa determinando el éxito de fertilización en FIV

Granulosa cells (GCs) are essential components of follicles and play a role in regulating follicle development. The aim of this study was to investigate certain cellular components involved in the proliferation, differentiation and functional characteristics of granulosa cells in the success of fertilization of human oocytes during invitro fertilization (IVF) via immunohistochemical techniques. In this study, 30 patients who were diagnosed as primary or secondary infertility, polycystic ovary syndrome in the IVF center of Memorial Hospital, Department of Obstetrics and Gynecology were included. The amount of Anti Müllerian Hormone (AMH) in blood and granulosa cell diameter and cell core diameter were measured in 20 cells collected from each patient. In addition, degeneration scoring and BAX, ADAMTS-1, IL-10 expressions in granulosa cells were evaluated (p <0.01). It was thought that apoptosis induced by human GCs might be an indicator of egg quality. Moderate expression of ADAMTS-1 was thought to be related to failure of ovulation, deterioration of oocyte quality and decreased fertilization rate. This decrease in AMH levels may be associated with defects in granulosa cells. Therefore, significantly lower AMH secretion and increase in IL10 expression levels in healthy people can be explained by the increase of granulocyte cells.

Las células de la granulosa (GC) son componentes esenciales de los folículos y tienen un papel en la regulación del desarrollo de éste. El objetivo del estudio fue investigar ciertos componentes celulares involucrados en la proliferación, diferenciación y características funcionales de las células de la granulosa en el éxito de la fertilización de los ovocitos humanos durante la fertilización in vitro (FIV) a través de técnicas inmunohistoquímicas. En este estudio, se incluyeron 30 pacientes diagnosticados con infertilidad primaria o secundaria, síndrome de ovario poliquístico en el centro de FIV del Departamento de Obstetricia y Ginecología del Hospital Memorial. La cantidad de Hormona Anti Mülleriana (AMH) en la sangre, el diámetro de las células de la granulosa y el diámetro del núcleo celular se midieron en 20 células obtenidas de cada paciente. Además, se evaluó la puntuación de degeneración y las expresiones BAX, ADAMTS-1, IL-10 en células de granulosa (p <0,01). Se estimó que la apoptosis inducida por los GC humanos podría ser un indicador de la calidad del huevo. Se estimó que la expresión moderada de ADAMTS-1 estaba relacionada con el fracaso de la ovulación, el deterioro de la calidad de los ovocitos y la disminución de la tasa de fertilización. La disminución en los niveles de AMH puede estar asociada con defectos en las células de la granulosa. Por lo tanto, el aumento de las células de granulocitos puede explicar la disminución significativa de la secreción de AMH y el aumento de los niveles de expresión de IL10 en personas sanas.

miR-106a regulates the proliferation of ovarian granulosa cells by targeting TIMP-2 / 中国临床药理学与治疗学

AIM: To investigate the regulation function of miR-106a on the proliferation of human ovarian granulosa cells, and to explore its possible target.METHODS: The expression of miR-106a in granulosa cells was regulated by cell transfection, and its expression level was detected by RT-PCR. The MTT assay was used to detect the cell proliferation activities of cells. Bioinformatics methods were used to predicted the possible target genes of miR-106a, which were verified them by double-luciferase assay. The expression of target protein was detected by Western blot. RESULTS:The expression of miR-106a in KGN cells was significantly higher than that of normal ovarian epithelial cell (IOSE80), the difference was statistically significant (P<0.05). The proliferation activity of KGN cells was significantly decreased after inhibiting the expression of miR-106a (P<0.05). The results of dual luciferase assay showed that miR-106a could directly target TIMP-2 gene. Western blot results showed that the expression level of TIMP-2 protein was significantly decreased after overexpression of miR-106a (P<0.05). CONCLUSION: miR-106a can promote the proliferation of KGN cell; The mechanism is related to the targeted reduction of TIMP-2 expression level.

Inhibitory effect of heat shock pretreated rat bone marrow mesenchymal stem cells on chemotherapy-induced ovarian granulocyte apoptosis / 医学研究生学报

ObjectiveBone marrow-derived mesenchymal stem cells (MSCs) can promote ovarian angiogenesis, improve ovarian insufficiency caused by chemotherapy, and repair ovarian function, while heat shock pretreatment can reduce the apoptosis rate of stem cells and improve the therapeutic effect of stem cells. This study aims to investigate the effect of heat shock pretreatment on MSCs, and further study the effect of heat shock pretreated mesenchymal stem cells on chemotherapy-induced apoptosis of ovarian granulosa cells.Methods1. The bone marrow-derived MSCs of rats were isolated, cultured and identified, and pretreated within a 42 °C water bath for one hour. 2. Cisplatin (5 mg/L) was added to MSCs to simulate the local microenvironment of chemotherapy. MSCs were divided into four groups: blank control group, heat shock control group, model group, and heat shock model group. The effects of heat shock pretreatment on the proliferation, apoptosis and survival rate of MSCs were investigated by CCK-8 method, Hoechst33342/PI, and flow cytometry. 3. We isolate and culture rat ovarian granulosa cells (GCs) to establish an in vitro model of GCs injury under the induction of cisplatin (5 mg/L). The experiment was carried out in four groups: a control group, model group, MSCs model group, HS-MSCs model group. The apoptosis and survival rate were detected by Hoechst33342/PI and flow cytometry, respectively.Results1. The proliferation level and survival rate of MSCs in the heat shock control group were significantly higher than those in the other three groups, and the apoptosis rate was significantly lower than the other three groups (P<0.05). Compared to the model group, the proliferation level of the heat shock model group was significantly increased, and the apoptosis rate was significantly decreased (P<0.05), and the cell survival rate increased; 2. The apoptosis rate of GCs in the HS-MSCs model group was significantly lower than that in the other three groups. Compared to the MSCs model group, the apoptosis rate of GCs in the HS-MSCs model group was significantly decreased (P<0.05).ConclusionHeat shock pretreatment can increase the proliferation level and survival rate of MSCs, and reduce its apoptosis rate. Heat shock pretreated stem cells can effectively inhibit chemotherapy-induced apoptosis of ovarian granulosa cells.