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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.

To determine whether the immunopotentiator chitosan oligosaccharide(COS)can recover the reproductive functions of pathological ovarian recession mice and improvetheir immunity.Forty-three fertile female mice (at around 2 months),in addition to a normal control group (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; ECOS 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.

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 β-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.

To explore the role of c-src on the initiation of primordial follicles.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.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.c-src plays an important role in primordial follicle development and folliculogenesis initiation.

The distribution of ErbB2 in mouse testis, epididymidis, ovaries, oocyte-cumulus cells-complexes in oviducts and sperms was investigated immunohistochemically. To study the effect of c-erbB2 on mouse fertilization in vitro, various concentrations of c-erbB2 antisense oligonucleotides (c-erbB2 ASODNs) were incubated with sperms and oocyte-cumulus cells-complexes during fertilization in vitro. To explore possible mechanisms involved in fertilization, the relationship between c-erbB2 ASODNs and GABA, or dbcAMP, or verapamil during fertilization in vitro was also observed.ErbB2 oncoprotein was observed in epithelial cells in epididymis, sperms and cumulus cells. C-erbB2 ASODNs inhibited the rate of fertilization in vitro in a dose-dependent way. The fertilization rate of the control group, low (5 micromol/L), medium (10 micromol/L), high (20 micromol/L) concentration c-erbB2 ASODNs group, and nonsense at oligonucleotides group (20 micromol/L) was 38.3%, 19.6%, 10.7%, 5.0%, and 33.8% respectively. Integral optical density immunostaining of ErbB2 in sperms was notably reduced. Medium and high concentration of c-erbB2 ASODNs notably inhibited cumulus cells adhering to inner wall of Petri dish. Treated alone with GABA or dbcAMP, the rate of fertilization was increased. Both GABA and dbcAMP partially inversed the ASODNs inhibition effect on fertilization rate, but neither of them showed significant effect on sperm integral optical density of ErbB2 immunostaining. In contrast, verapamil inhibited fertilization rate. Co-treated with c-erbB2 ASODN, verapamil showed synergic inhibiting effect on fertilization with c-erbB2 ASODN. Verapamil also inhibited the expression of c-erbB2 in sperms.It is suggested that c-erbB2 is closely correlated with fertilization. Ca2+ may inhibit fertilization in vitro through regulation the expression of c-erbB2 gene in sperm cells, while both of GABA and dbcAMP may affect the process of fertilization through the way other than c-erbB2 expression in sperm cells.

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.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.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.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.

To investigate the effects of c-myb on progesterone-induced mouse germinal vesicle(GV) stage denuded oocyte (DO) maturation in vitro.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.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% P0.05, 8 h PB 1% P0.05), 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% P0.05, 8 h PBI% P0.01). 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 P0.01, 8 h PBI% all P0.01), and could enhanced the inhibition of 16 micromol/L antisense c-myb ODN (2 h GVBD% all P0.01, 8h PBI% all P0.01).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.

The method of labeled streptavidin biotin was used to study the expression of c-fos in various functional state of rat ovaries and its relationship with the levels of serum estradiol and progesterone.(1) In the mature rat, c-fos expression was found higher in interstitial gland and stroma of proestrous ovaries and lower in estrous ovaries, and was found higher in luteal cells and stroma of pregnant ovaries and lower in diestrous ovaries. There was a positive correlation between the area and optical density of c-fos expression and the levels of serum E2 and P. (2) In the immature rats, c-fos expression was not found in the ovaries containing preantral follicles from DES-treated rats,and was found both in interstitial gland and stroma of the ovaries containing preovulatory follicles from PMSG-treated rats ,and the expression was higher in day 4 luteinized ovaries and lower in day 9 luteinized ovaries from PMSG with hCG treated rats. There also was a positive correlation between the area and optical density of c-fos expression and the levels of serum E2 and P.The results suggested in rat ovaries and might play an important role in follicles development, ovulation, luteum formation and regression.

To investigate the distribution of c-myb, an oncoprotein, in mouse oocytes-cumulus cell complex and sperm immunohistochemically.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.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.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.

To study the expression and control of telomerase in rat preantral ovary.Added different factors to the preantral ovarian granulosa cells, then using TRAP-ELISA to analyze the expression of telomerase.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.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.

To study the expression and possible roles of proto-oncogene c-erbB2 during the initiation growth of primordial follicles.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.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, P0.05).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.

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.

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 (P0.01) and the levels of ErbB₂, MAPK and PKC protein (P0.01) significantly. But the levels of c-erbB₂ mRNA and ErbB₂ protein exhibited no change (P0.05) 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 (P0.05 or P0.01), but the quantity of the primordial follicles was higher than that in the control group (P0.01). 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.

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% (P0.05) and 32.2% (P0.05), respectively, for 6-hour culture, and the PB1 extrusion rate increased by 17.3% (P0.05) and 23.5% (P0.05), 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 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.