Ginkgolide B effectively mitigates neuropathic pain by suppressing the activation of the NLRP3 inflammasome through the induction of mitophagy in rats.

Neuropathic pain is a pathological state induced by the aberrant generation of pain signals within the nervous system. Ginkgolide B(GB), an active component found of Ginkgo. biloba leaves, has neuroprotective properties. This study aimed to explore the effects of GB on neuropathic pain and its underlying mechanisms. In the in vivo study, we adopted the rat chronic constriction injury model, and the results showed that GB(4 mg/kg) treatment effectively reduced pain sensation in rats and decreased the expressions of Iba-1 (a microglia marker), NLRP3 inflammasome, and inflammatory factors, such as interleukin (IL)-1ß, in the spinal cord 7 days post-surgery. In the in vitro study, we induced microglial inflammation using lipopolysaccharide (500 ng/mL) / adenosine triphosphate (5 mM) and treated it with GB (10, 20, and 40 µM). GB upregulated the expression of mitophagy proteins, such as PINK1, Parkin, LC3 II/I, Tom20, and Beclin1, and decreased the cellular production of reactive oxygen species. Moreover, it lowered the expression of inflammation-related proteins, such as Caspase-1, IL-1ß, and NLRP3 in microglia. However, this effect was reversed by Parkin shRNA/siRNA or the autophagy inhibitor 3-methyladenine (5 mM). These findings reveal that GB alleviates neuropathic pain by mitigating neuroinflammation through the activation of PINK1-Parkin-mediated mitophagy.

Effects of chitooligosaccharide-zinc on the ovarian function of mice with premature ovarian failure via the SESN2/NRF2 signaling pathway.

Chitooligosaccharide-zinc (COS·Zn) is a powerful anti-oxidant and anti-aging scavenger, whose anti-oxidative ability immensely exceeds vitamin C. Therefore, this study was aimed to investigate the protective effects of COS·Zn against premature ovarian failure (POF) and potential mechanisms. Female KM adult mice were divided into the following groups: a treatment group (150 mg·kg-1·d-1 COS·Zn), a treatment group (300 mg·kg-1·d-1 COS·Zn), a prevention group, two control groups and two CY/BUS groups. COS·Zn (150, 300 mg·kg-1·d-1) and COS·Zn (300 mg·kg-1·d-1) were therapeutically and preventatively administered to POF mice in the treatment and prevention studies, respectively. All the groups were administered for 21 days. Fewer primary and secondary follicles were observed in the COS·Zn-treated groups (including the treatment and prevention groups) than those of the control groups. Meanwhile, the ovarian index and the levels of FSH and LH notably increased in the treatment and prevention groups compared with those in the CY/BUS group. The levels of MVH, OCT4 and PCNA in the treatment group (300·kg-1·d-1 COS·Zn) and MVH in the prevention group remarkably increased compared with those in the CY/BUS groups. Meanwhile, the levels of P53 and P16 protein were down-regulated in the treatment and prevention groups compared with those in the CY/BUS groups. Additionally, the amounts of Sestrin2 (SESN2) and SOD2 protein were obviously higher in the treatment group (150 mg·kg-1·d-1 COS·Zn) than those in the CY/BUS groups. Similarly, the amounts of NRF2 and SESN2 protein were up-regulated in the prevention group. Besides, an increased GSH level was observed in the two treatment groups, compared with that in the CY/BUS groups, and the same trend was also present in the prevention group. Taken together, COS·Zn improves the ovarian and follicular development through regulating the SESN2/NRF2 signaling pathway. These results suggest the role of COS·Zn as a novel agent for the treatment and prevention of POF.

Effects of shikonin on the development of ovarian follicles and female germline stem cells.

OBJECTIVE: To investigate the effects and potential mechanism of action of shikonin (SHK) on the development of ovarian follicles and female germline stem cells (FGSCs). METHODS: Female Kunming adult mice were administered SHK (0, 20 and 50 mg/kg) by oral gavage. Cultures of FGSCs were treated with SHK 32 µmol/l for 24 h. The ovarian index in mouse ovaries was calculated. Numbers of primordial, primary and atretic follicles were counted. Germline stem cell markers and apoptosis were examined. Levels of glutathione (GSH), superoxide dismutase (SOD) and reactive oxygen species (ROS) were measured. RESULTS: Both doses of SHK significantly decreased the ovarian index, the numbers of primordial follicles, primary follicles and antral follicles in mice. SHK significantly increased the numbers of atretic follicles and atretic corpora lutea. SHK promoted apoptosis in vivo and in vitro. SHK significantly decreased the levels of the germline stem cell markers. SHK significantly lowered GSH levels and the activity of SOD in the peripheral blood from mice, whereas SHK significantly elevated cellular ROS content in FGSCs. CONCLUSIONS: These current results suggested that follicular development and FGSCs were suppressed by SHK through the induction of apoptosis and oxidative stress might be involved in this pathological process.

[Expression relationship of Hippo signaling molecules and ovarian germline stem cell markers in the ovarian aging process of women and mice].

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.

Hippo pathway functions as a downstream effector of AKT signaling to regulate the activation of primordial follicles in mice.

Clarifying the molecular mechanisms by which primordial follicles are initiated is crucial for the prevention and treatment of female infertility and ovarian dysfunction. The Hippo pathway has been proven to have a spatiotemporal correlation with the size of the primordial follicle pool in mice in our previous work. But the role and underlying mechanisms of the Hippo pathway in primordial follicle activation remain unclear. Here, the localization and expression of the core components were examined in primordial follicles before and after activation. And the effects of the Hippo pathway on primordial follicle activation were determined by genetically manipulating yes-associated protein 1 (Yap1), the key transcriptional effector. Furthermore, an AKT specific inhibitor (MK2206) was added to determine the interaction between the Hippo pathway and AKT, an important signaling regulator of ovarian function. Results showed that the core components of the Hippo pathway were localized in both primordial and primary follicles and the expression levels of them changed significantly during the initiation of primordial follicles. Yap1 knockdown suppressed primordial follicle activation, while its overexpression led to the opposite trend. MK2206 downregulated the ratio of P-MST/MST1 and upregulated the ratio of P-YAP1/YAP1 significantly, whereas Yap1-treatment had no influence on AKT. In addition, YAP1 upregulation partially rescued the suppression of the primordial follicle activation induced by MK2206. Our findings revealed that the Hippo-YAP1 regulates primordial follicular activation, which is mediated by AKT signaling in mice, thus providing direct and new evidence to highlight the role of Hippo signaling in regulating ovarian follicles development.

[Effects of COS on promoting the pathological ovarian aging mice ovarian function by regulating immune function].

OBJECTIVE: To determine whether the immunopotentiator chitosan oligosaccharide(COS)can recover the reproductive functions of pathological ovarian recession mice and improvetheir immunity. METHODS: Forty-three fertile female mice (at around 2 months),in addition to a normal control group (n=8), injected intraperitoneally with busulfan and cyclophosphamide to construct premature ovarian failure mod-els. Three of them were used to test whether the infertility model was constructed successfully by HE staining. Then the models were randomly divided into four groups (n=8) and treated with different dosages of COS by gavage, after which compared different groups' organ ratios (the weight of immune organs and ovary/body weight), ovarian follicles and peritoneal macrophages' phagocytosis as well as estragon(E2) and pro-gesterone(P) levels in peripheral blood. In addition, we measured the expression dynamics of the ovarian protein reproductive cell marker mouse vasa homolog(MVH), germ stem cell marker OCT-4 in ovarian surface epithelium (OSE) and part of immune factors including tumor necrosis factor (TNF-α),interleukin-2(IL-2)as well as IL-6 to analyze the correlativity between germline stem cells marker dynamics and im-mune factors expression changes. RESULTS: With increasing dosages of COS, organ ratios of ovaries, thymus and spleen both went up syn-chronously; The whole number of follicles and every stages of follicles are all presenting with progressive tendency; E2 level in peripheral blood ascends, however, progesterone level declined relatively; Neutral red experiment revealed the phagocytosis ability of peritoneal macrophages became stronger with increasing dosages of COS; the results of Western blot had shown that no matter the expression level of germ stem cells marker or immune factors were all presenting increasing tendency, which means that the expression level dynamics of germ stem cell marker has a positive correlation with immune factors expression changes. The results were statistically significant. CONCLUSIONS: COS could improve the immunity of mice with pathological ovarian recession and at the same time it would promote the proliferation and differentiation of female germ line stem cells (FGSCs), and then helped saving ovarian functionsto some extent.

Ghrelin receptor agonist, GHRP-2, produces antinociceptive effects at the supraspinal level via the opioid receptor in mice.

GHRP-2 is a synthetic agonist of ghrelin receptor. GHRP-2 has similar physiological functions with ghrelin. In our previous study, ghrelin (i.c.v.) could induce analgesic effect through an interaction with GHS-R1α and with the central opioid system in the acute pain in mice. To date, the function of GHRP-2 in pain processing was not understood. Therefore the aim of this study was to investigate the effects of GHRP-2 on pain modulation at supraspinal level in mice using the tail immersion test. Intracerebroventricular (i.c.v.) administration of GHRP-2 (0.1, 0.3, 1, 3 and 10 nmol/L) produced a concentration- and time-related antinociceptive effect. This effect could be fully antagonized by GHS-R1α antagonist [d-Lys(3)]-GHRP-6, indicating that the analgesic effect induced by GHRP-2 is mediated through the activation of GHS-R1α. Interestingly, naloxone, naltrindole and nor-binaltorphimine, but not ß-funaltrexamine, could also block the analgesic effect markedly, suggesting that δ- and κ-opioid receptor is involved in the analgesic response evoked by GHRP-2. Moreover, i.c.v. administration of GHRP-2 potentiated the analgesic effect induced by morphine (i.c.v., 1 nmol/L) and this potentiated effect could not be reversed by [d-Lys(3)]-GHRP-6. Thus these findings may be a new strategy on investigating the interaction between ghrelin system and opioids on pain modulation. Furthermore, GHRP-2 may be a promising peptide for developing new analgesic drugs.

RNA interference mediated pten knock-down inhibit the formation of polycystic ovary.

Pten (phosphatase and tensin homolog deleted on chromosome 10), a kind of tumor suppressor gene, plays important roles in female reproductive system. But its expression and roles in the formation of polycystic ovaries are yet to be known. In this study, we constructed a rat model of PCOS using norethindrone and HCG injections and found the expressions of pten mRNA and PTEN protein increased significantly in the polycystic ovary tissue by immunohistochemistry, RT-PCR, and western blot. Furthermore, the results showed that in vivo ovaries could be effectively transfected by lentiviral vectors through the ovarian microinjection method and indicated that pten shRNA may inhibit the formation of polycystic ovaries by pten down-regulation. Our study provides new information regarding the role of PTEN in female reproductive disorders, such as polycystic ovary syndrome.

The proto-oncogene c-src is involved in primordial follicle activation through the PI3K, PKC and MAPK signaling pathways.

BACKGROUND: C-src is an evolutionarily conserved proto-oncogene that regulates cell proliferation, differentiation and apoptosis. In our previous studies, we have reported that another proto-oncogene, c-erbB2, plays an important role in primordial follicle activation and development. We also found that c-src was expressed in mammalian ovaries, but its functions in primordial follicle activation remain unclear. The objective of this study is to investigate the role and mechanism of c-src during the growth of primordial follicles. METHODS: Ovaries from 2-day-old rats were cultured in vitro for 8 days. Three c-src-targeting and one negative control siRNA were designed and used in the present study. PCR, Western blotting and primordial follicle development were assessed for the silencing efficiency of the lentivirus c-src siRNA and its effect on primordial follicle onset. The expression of c-src mRNA and protein in primordial follicle growth were examined using the PCR method and immunohistochemical staining. Furthermore, the MAPK inhibitor PD98059, the PKC inhibitor Calphostin and the PI3K inhibitor LY294002 were used to explore the possible signaling pathways of c-src in primordial folliculogenesis. RESULTS: The results showed that Src protein was distributed in the ooplasmic membrane and the granulosa cell membrane in the primordial follicles, and c-src expression level increased with the growth of primordial follicle. The c-src -targeting lentivirus siRNAs had a silencing effect on c-src mRNA and protein expression. Eight days after transfection of rat ovaries with c-src siRNA, the GFP fluorescence in frozen ovarian sections was clearly discernible under a fluorescence microscope, and its relative expression level was 5-fold higher than that in the control group. Furthermore, the c-src-targeting lentivirus siRNAs lowered its relative expression level 1.96 times. We also found that the development of cultured primordial follicles was completely arrested after c-src siRNA knockdown of c-src expression. Furthermore, our studies demonstrated that folliculogenesis onset was inhibited by Calphostin, PD98059 or LY294002 treatment,but none of them down-regulated c-src expression. In contrast, the expression levels of p-PKC, p-ERK1/2 and p-PI3K in the follicles were clearly decreased by c-src siRNA transfection. Correspondingly, both Calphostin and LY294002 treatment resulted in a decrease in the p-PKC level in follicles, but no change was observed in the PD98059 group. Finally, LY294002 treatment decreased the p-PI3K expression level in the follicles, but no changes were observed in the PD98059 and Calphostin groups. CONCLUSIONS: C-src plays an important role in regulating primordial follicle activation and growth via the PI3K-PKC- ERK1/2 pathway.

c-erbB2 and c-myb induce mouse oocyte maturation involving activation of maturation promoting factor.

Proto-oncogenes are involved in cell growth, proliferation, and differentiation. In the present study, we investigated the roles and mediating pathways of proto-oncogenes c-erbB(2) and c-myb in mouse oocyte maturation by RT-PCR, real-time quantitative PCR, western blot, and recombinant proto-oncogene protein microinjection. Results showed that both c-erbB(2) and c-myb antisense oligodeoxynucleotides (c-erbB(2) ASODN and c-myb ASODN) inhibited germinal vesicle breakdown and the first polar body extrusion in a dose-dependent manner. However, microinjection of recombinant c-erbB(2) or c-myb protein into germinal vesicle stage oocytes stimulated oocyte meiotic maturation. In addition, the expression of c-erbB(2) and c-myb mRNA was detected in oocytes; and c-erbB(2) ASODN and c-myb ASODN inhibited c-erbB(2) mRNA and c-myb mRNA expression, respectively. Maturation promoting factor (MPF) inhibitor roscovitine did not affect the expression of c-erbB(2) mRNA and c-myb mRNA, but blocked the effects of recombinant c-erbB(2) and c-myb protein-induced oocyte maturation. Further, cyclin B1 protein expression in oocytes was remarkably inhibited by c-erbB(2) ASODN, c-myb ASODN, and roscovitine. Nonsense tat ODN had no effect on the expression of c-erbB(2), c-myb, and cyclin B1. These results suggest that c-erbB(2) and c-myb may induce oocyte maturation through mediating a pathway involving the activation of MPF.

[Preliminary study about the role of c-src in the initiation of primordial follicle in rat ovary].

OBJECTIVE: To explore the role of c-src on the initiation of primordial follicles. METHODS: 2-days-old female SD rats' ovaries were cultured in Waymouth culture system and were used HE staining and immunohistochemy to observe the number of follicles after 0, 4, 8 days cultured. Use chemically synthesized small interference RNA (siRNA) transfected into ovarian tissue in cultured for RNA interference, and use HE staining and RT-PCR to detect the best siRNA and packaging it by lentiviruses to test the interference effect. RESULTS: With the increase of culturing days, the nummber of the primordial follicles in ovarian gradually reduced. We packed the best siRNA by lentiviruses to doing RNA interference and found comparing with the blank control group and blank vector group, c-src mRNA of the best interference group were significantly decreased. The total number of primordial follicles was relatively greater and the development of primordial folliculars was inhibited. CONCLUSION: c-src plays an important role in primordial follicle development and folliculogenesis initiation.

[Roles of proto-oncogene c-erbB2 during the initiation growth of rat primordial follicles].

OBJECTIVE: To study the expression and possible roles of proto-oncogene c-erbB2 during the initiation growth of primordial follicles. METHODS: Ovaries were collected from 2-day-old SD rats and cultured in the Waymouth culture system. In-situ hybridization, RT-PCR and immunohistochemistry were performed to assess the expressions of c-erbB2 mRNA and protein during the initiation growth of primordial follicles and after the effect of EGF. Western blot was used to observe the PCNA, p-ERK1/2 contents and correlation analysis was used to study the correlation relationship between contents of p-ERK1/2 and expressions of c-erbB2 mRNA at the same time of the primordial follicles growth. RESULTS: PCNA protein levels appeared to be more intense during the initiation growth of primordial follicles, EGF could promote the proliferation and differentiation of the primordial follicles. c-erbB2 mRNA existed in the oocytes endochylema and ErbB2 existed in the oocytes membrane, the expressions of c-erbB2 mRNA and ErbB2 appeared to be more intense when primordial follicles were cultured for 8 d than cultured for 0 d in the Waymouth culture system and were further increased with 50 ng/ml EGF for 4 d and 8 d. The same results were observed by RT-PCR, too. p-ERK1/2 protein levels were consistent with the changes of c-erbB2 mRNA and protein. Furthermore, Spearman rank correlation analysis showed there was a significant positive correlation relationship between the changes of p-ERK1/2 and the changes of c-erbB2 mRNA during the primordial follicles growth and after the effect of EGF (rs = 0.900, P < 0.05). CONCLUSION: It was suggested that proto-oncogene c-erbB2 may be play an important role during the initiation growth of primordial follicles with EGF, and it is indirectly suggested that c-erbB2 promotes the development of the primordial follicles via ERK-MAPK signal transduction.

[The expression of proto-oncogene c-erbB2 and its role in the initiation of primordial follicle growth in rat ovary].

Little is known about the factors that control the initiation of growth of primordial follicles. The objective of the present study was to investigate the effect of c-erbB2 on the onset of primordial follicle development, and whether c-erbB2 mediates the effect of epidermal growth factor (EGF) in this process. We synthesized three pairs of siRNAs targeting the c-erbB2 mRNA and transferred them into the newborn rat ovary cultured in vitro with Metafectene. After siRNAs transfection, the efficiency of siRNAs was tested by examining c-erbB2 mRNA and protein levels. The level of c-erbB2 mRNA was reduced by 49.6%, 46.7% and 82.6% respectively after transfecting siRNA1, siRNA2 and siRNA3, and the level of ErbB2 protein also reduced remarkably after siRNA3 transfection. c-erbB2 siRNA3 significantly inhibited the primordial follicle initiation and development; EGF augmented primordial follicles formation, but the effect was abolished by c-erbB2 siRNA3. All of these results suggest that c-erbB2 plays an important role in primordial follicle development and folliculogenesis initiation, and mediates the effect of EGF on primordial follicle development.

[Role of MAPK and PKC signaling pathways in the regulation of c-erbB2 in the primordial follicles onset].

Our previous studies showed that the proto-oncogene c-erbB2 played an important role in primordial follicles growth. The present study was conducted to investigate the role of MAPK and PKC signaling pathways in the primordial follicle onset in neonatal rats, and the relationship between c-erbB2 and MAPK/PKC signaling pathways. Ovaries collected from 2-day-old Sprague-Dawley rats were cultured in the Waymouth culture system in vitro. Ovaries were transfected with c-erbB2 siRNA, or treated with PD98059 (50 mumol/L) or Calphostin (0.5 mumol/L) in the culture medium. RT-PCR was performed to measure the expression of c-erbB2 mRNA, and Western blot analysis was performed to measure the expression of ErbB2, MAPK and PKC protein after the neonatal rat ovaries were cultured for 8 d. The quantities of every-stage follicles of ovaries cultured for 8 d were obtained in histological section stained with hematoxylin eosin. The results showed that c-erbB2 siRNA reduced the levels of c-erbB2 mRNA (P<0.01) and the levels of ErbB2, MAPK and PKC protein (P<0.01) significantly. But the levels of c-erbB2 mRNA and ErbB2 protein exhibited no change (P>0.05) in the ovaries cultured with PD98059 or Calphostin. After the ovaries were transfected with c-erbB2 siRNA or cultured with PD98059 or Calphostin for 8 d, the quantities of primary follicles and second follicles were lower than those in the control group (P<0.05 or P<0.01), but the quantity of the primordial follicles was higher than that in the control group (P<0.01). These results suggest that proto-oncogene c-erbB2 promotes the initiation of primordial follicle growth through the MAPK and PKC signal transduction, and c-erbB2 is possibly the upstream of PKC and MAPK signaling pathway in the regulation of primordial follicle onset.

[The effects of protooncogene on oocyte maturation mediated by cytokines].

AIM: The mechanisms of cytokines in regulating oocyte maturation is still little known. The present study attempt to investigate whether the protooncogene of c-erbB2, c-myb are involved in introducing of cytokines to regulate oocyte maturation. METHODS: This research used mouse GV stage oocyte culture model in vitro and RT-PCR, Western blotting method to explore the effect of EGF, TNFalpha, ET-1 and NO on oocyte maturation; to analyze the c-erbB2 mRNA and c-myb mRNA expression and the phosphorylation of MAPK and cyclinB1 expression in oocytes affected by above cytokines. RESULTS: EGF(10 microg/L) stimulated meiosis of oocytes significantly, the level of c-erbB2 mRNA, c-myb mRNA were increased, and promoted the phosphorylation of MAPK and cyclinB1 expression; TNFalpha (1 microg/L) and ET-1 ((10(-1) mol/L) had the results to EGF. Low dose of SNP (10(-5)mol/L) had no effect on oocyte maturation, but could significantly reverse the suppression of dbcAMP on oocyte maturation. CONCLUSION: c-erbB2 and c-myb were involved in introducing of cytokines to regulate oocyte maturation, might be the middle link in connection of the cytokines with MAPK and MPF in regulation oocyte maturation.

[c-erbB(2) and c-myb induce oocyte maturation via activation of mitogen-activated protein kinase and maturation promoting factor].

It is important to study the mechanism of oocyte maturation because oocyte maturation is essential for the female procreation. The present study was designed to observe the effects of protooncogenes c-erbB(2) and c-myb on oocyte maturation and the upstream and downstream relationship with mitogen-activated protein kinase (MAPK) and maturation promoting factor (MPF). The investigation was designed as follows: (1) In order to explore the effects of protooncogenes on oocyte maturation, the dose- and time-dependent effects of c-erbB(2) antisense oligodeoxynucleotide (ASODN) and c-myb ASODN on oocyte maturation were examined, and the effects of oocyte microinjection with recombinant c-erbB(2) and c-myb proteins on oocyte maturation were investigated; (2) In order to study the upstream and downstream relationship among protooncogenes of c-erbB(2), c-myb and protein kinases of MAPK and MPF in regulating oocyte maturation, mouse oocytes were cultured in the medium treated with c-erbB(2) ASODN, c-myb ASODN, PD98059 (the MAPK inhibitor) or roscovitine (the MPF inhibitor) for 8 h, respectively, and the expressions of c-erbB(2) mRNA, c-myb mRNA, MAPK and MPF were examined. The results showed that both c-erbB(2) ASODN and c-myb ASODN inhibited the rate of germinal vesicle breakdown (GVBD) and the first polar (PB1) extrusion of denuded oocytes (DOs) in a dose- and time-dependent way, and delayed their maturation time significantly. When recombinant c-erbB(2) and c-myb proteins were microinjected into cytoplasm of germinal vesicle stage oocyte, we found that the GVBD rate increased by 23.1% (P<0.05) and 32.2% (P<0.05), respectively, for 6-hour culture, and the PB1 extrusion rate increased by 17.3% (P<0.05) and 23.5% (P<0.05), respectively, for 12-hour culture. RT-PCR showed that the mRNA expressions of c-erbB(2) and c-myb were detected in oocytes; c c-erbB(2) ASODN inhibited c-erbB(2) mRNA and c-myb mRNA expressions; c-myb ASODN inhibited c-myb mRNA expression but had no effect on c-erbB(2) mRNA expression. Nonsense tat ODN had no effects on the expressions of c-erbB(2) mRNA and c-myb mRNA. Neither PD98059 nor roscovitine changed the expressions of c-erbB(2) mRNA and c-myb mRNA though both of them inhibited recombinant c-erbB(2) and c-myb proteins-induced oocyte maturation. Furthermore, MAPK phosphorylation and cyclin B1 synthesis in oocytes were inhibited remarkably when oocytes were treated with c-erbB(2) ASODN, c-myb ASODN, PD98059 and roscovitine. Nonsense tat ODN had no effects on MAPK phosphorylation and cyclinB1 content. The results suggest that protooncogenes c-erbB(2) and c-myb play an important role in oocyte maturation; the effects of c-erbB(2) and c-myb depend upon the action of MAPK and MPF, and their activation is the event that occurs downstream of c-erbB(2) and c-myb in the maturation signal pathway.

[The expression and control of telomerase in preantral ovary].

AIM: To study the expression and control of telomerase in rat preantral ovary. METHODS: Added different factors to the preantral ovarian granulosa cells, then using TRAP-ELISA to analyze the expression of telomerase. RESULTS: Telomerase 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. CONCLUSION: It 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.

[Effect of protooncogene c-myb on progesterone-induced mouse germinal vesicle stage oocyte maturation in vitro].

AIM: To investigate the effects of c-myb on progesterone-induced mouse germinal vesicle(GV) stage denuded oocyte (DO) maturation in vitro. METHODS: We used mouse GV stage oocyte cultured with special concentration progesterone, or/and antisense c-myb ODN, or/and db-cAMP, or/and heparin for 24 h, and observed oocyte maturation and analysed the relationship among them. RESULTS: We cultured DO in the medium 199 for 24 h, and found 10 micromol/L progesterone had more significant effect than 5 micromol/L progesterone (2 h GVBD% P < 0.05, 8 h PB 1% P < 0.05), but had not more significant effect than 20 micromol/L progesterone. We found that 16 micromol/L antisense c-myb ODN significantly inhibited progesterone (10 micromol/L)-induced mouse germinal vesicle stage oocyte maturation in vitro (2 h GVBD% P < 0.05, 8 h PBI% P < 0.01). 1 x 10(-4) micromol/L dbcAMP, 100 microg/ml heparin could single significantly inhibited progesterone-induced mouse GV stage oocyte maturation in vitro (2 h PBI% all P < 0.01, 8 h PBI% all P < 0.01), and could enhanced the inhibition of 16 micromol/L antisense c-myb ODN (2 h GVBD% all P < 0.01, 8h PBI% all P < 0.01). CONCLUSION: Progesterone, protooncogene c-myb,cAMP and calcium all pay important role in regulating oocyte maturation and the mechanism of progesterone, cAMP and calcium in regulating oocyte maturation may be through the expression of protooncogene c-myb.

[Telomerase: the expression and regulatory mechanisms in preovulatory ovarian granulosa cells].

The objective of this study was to analyze the expression of telomerase in granulosa cells and the influential factors in telomerase expression. TRAP-ELISA (telomeric repeat amplification protocol-enzyme linked immunoadsordent assay) method was used to study the expression and control of telomerase in rat preovulatory ovary. We also used radioimmunoassay (RIA) to determine the expression of estradiol (E(2)) and progesterone (P(0)). Furthermore we used MTT to study the proliferation of ovarian granulosa cells. Telomerase activity was significantly enhanced by human chorionic gonadotropin (HCG), follicle-stimulating hormone (FSH), verapamil and dbcAMP, and was significantly reduced by antisense-c-myb oligodeoxynucleotide (anti-c-myb ODN) treatment. RIA was used to determine the secretion of P(0) and E(2) in all these cell culture media. We found that the secretion of these two hormones was increased when verapamil and FSH were added; no change after adding HCG and dbcAMP; and reduced when anti-c-myb was added. In MTT assay, we found that the antisense hTERT ODN significantly inhibited the proliferation of ovarian granulosa cells. These results demonstrate that telomerase activity is present in ovary antral granulosa cells and its activity is controlled by FSH, HCG, verapamil and anti-c-myb, and is directly related with the function of proliferation.

[Study of effect and mechanism of c-myb on the fertilization in mouse].

AIM: To investigate the distribution of c-myb, an oncoprotein, in mouse oocytes-cumulus cell complex and sperm immunohistochemically. METHODS: To study the effect of c-myb on mouse fertilization in vitro, various concentration of c-myb antisense-oligodeoxynucleotides (c-myb ASODNs) were incubated with sperms and oocytes during fertilization. To explore the possible mechanism involved in fertilization, the relationship between c-myb ASODNs and GABA or dbcAMP or Verapamil or Progesterone in fertilization was also observed by immunohistochemical methods. RESULTS: c-myb oncoprotein was observed on the nucleus of cumulus cell and head of sperm. c-myb ASODNs inhibited the rate of fertilization in vitro in a dose-dependent way. The fertilization rates of the control group, low (5 micromol/L), medium (10 micromol/L), high (20 micromol/L) concentration c-myb ASODNs groups and nonsense tat oligodeoxynucleotides (20 micromol/L) group were 34.97%, 30.89%, 20.14%, 16.68%, 34.47%, respectively. All of GABA, Progesterone and dbcAMP inversed the c-myb ASODNs inhibition effects on fertilization rate, but neither of them showed significant effect on the percentages of immunohistochemical stain of Myb on sperm and cumulus cells. By contrast, Verapamil inhibited the fertilization rate. Co-treated with c-myb ASODNs, Verapamil showed synergic inhibiting effects on the fertilization with c-myb ASODNs. Verapamil also inhibited the expression of Myb on head of sperm. The fertilization rates of the control group, medium (10 micromol/L) concentration c-myb ASODNs group, GABA group, P4 group, Verapamil group, dbcAMP group were 34.81%, 22.96%, 40.83%, 39.12%, 7.46%, 40.61%, respectively. CONCLUSION: c-myb ASODNs is closely correlated with fertilization. Verapamil can inhibit fertilization in vitro through regulating Myb expression of sperm, while GABA, dbcAMP and Verapamil may affect the process of fertilization through the way other than Myb expression.