[Regulating effects of whole-body vibration on protein expression of p-GSK3ß in bone marrow cells of ovariectomized osteoporosis rats].

The aim of this study was to investigate the effects of whole-body vibration on Wnt/ß-catenin signaling pathway in bone marrow cells of ovariectomized osteoporosis rats. Thirty-six healthy 3-month old female Sprague Dawley (SD) rats were randomly divided into the following three groups by body weight: sham-operation (Sham), ovariectomized (OVX), and OVX whole-body vibration (WBV) groups. Ten weeks after ovariectomization, the rats of WBV group received vibration treatment (90 Hz, 15 min) twice per day. At the end of 8-week vibration, the whole-body bone mineral density (BMD) and body composition were detected by dual energy X-ray absorptiometry (DEXA) in vivo. The protein expressions of ß-catenin and p-GSK3ß in both bone marrow cells and bone marrow stromal cells were detected by Western blot. The results showed that, compared with OVX group, WBV group showed decreased fat mass and fat mass content, as well as increased lean body mass content. The BMD of the proximal tibia in WBV group was significantly higher than that in OVX group, however, there was no difference of BMD in whole-body and other positions between the two groups. The ß-catenin expression in bone marrow stromal cells showed no difference between OVX and WBV groups. The p-GSK3ß expression of bone marrow cells was increased in WBV group compared with that in OVX group, whereas bone marrow stromal cells from two groups did not exhibit the difference of the p-GSK3ß expression. These results suggest that whole body vibration can stimulate the protein expression of p-GSK3ß in bone marrow cells of ovariectomized osteoporosis rats, which could improve the bone loss induced by ovariectomization.

Treadmill training regulates ß-catenin signaling through phosphorylation of GSK-3ß in lumbar vertebrae of ovariectomized rats.

Postmenopausal osteoporosis is associated with high level of adipogenesis within the bone marrow at the expense of osteoblast population. The mechanical effect on ß-catenin through phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) is critical for inhibition of adipogenesis in mesenchymal stem cells in vitro. In present study, we hypothesized that treadmill training could regulate the ß-catenin signaling through phosphorylation of GSK-3ß in the lumbar vertebrae of ovariectomized (OVX) rats. 3-month-old female Sprague-Dawley rats were divided randomly into the following four groups: (a) Sham, (b) OVX, (c) OVX exercised (EX), and (d) OVX estrogen replacement (E(2)). At the end of the experiment, the serum levels of estradiol (E(2)) and luteinizing hormone (LH), the ultimate lumbar vertebra strength, as well as the protein expression for peroxisome proliferators-activated receptor γ (PPARγ), ß-catenin, P-GSK-3ß, and osterix (Osx) in lumbar vertebrae were analyzed. Moreover, the protein expression for ß-catenin and P-GSK-3ß were also examined in the uterus. The EX group had lower protein level of PPARγ, higher ultimate lumbar vertebral strength, and higher protein levels of ß-catenin, and P-GSK-3ß in lumbar vertebral bodies compared with sedentary OVX group. The effects of EX treatment on the protein levels of ß-catenin and P-GSK-3ß in bones were not reproducible in the uterus. Moreover, exercise treatment produced no estrogenic effect as evidenced by serum level of LH. In conclusion, this study suggested that treadmill training could activate the GSK-3ß/ß-catenin signaling and inhibit the production of PPARγ in lumbar vertebrae of OVX rats, which may contribute to the prevention of bone loss in OVX rats.

Treadmill training prevents bone loss by inhibition of PPARγ expression but not promoting of Runx2 expression in ovariectomized rats.

Exercise training has been reported to prevent bone loss in ovariectomized (OVX) rats and postmenopausal women. We hypothesized that treadmill training inhibited adipogenesis and enhanced osteogenesis through the regulation of adipocyte differentiation factor peroxisome proliferators-activated receptor gamma (PPARγ) and the osteogenic factor runt-related transcription factor 2 (Runx2) in a model of OVX-induced osteoporosis. To test this hypothesis, 3-month-old female Sprague-Dawley rats were divided randomly into the following groups: Sham, OVX, OVX exercised (EX), and OVX estrogen replacement (E(2)). At the end of the experiment, the bone mineral density (BMD) was detected using DEXA and the morphology change of bone tissues and uterus was observed by HE staining. The protein expression for PPARγ and Runx2 were measured by immunohistochemistry and western blot and the bone triacylglycerol (TG) was extracted by methanol/chloroform. OVX dramatically increased the number of fat vacuoles, protein levels for PPARγ and Runx2 as well as the TG level in tibiae and lumbar vertebrate. In contrast, the serum level of E(2), the lumbar vertebrate BMD as well as the proximal and distal femur BMD was significantly decreased in the OVX group. All changes induced by OVX were significantly reversed by exercise treatment except for the protein expression level of Runx2. Moreover, exercise treatment produced no estrogenic effects on uterus as evidenced by the uterus wet weight and histology. Treadmill training could prevent bone loss induced by OVX through the inhibition of adipocyte differentiation factor PPARγ rather than promoting osteogenic factor Runx2.

Effects of treadmill exercise training on liver fat accumulation and estrogen receptor alpha expression in intact and ovariectomized rats with or without estrogen replacement treatment.

To explore the mechanism(s) of exercise training on ovariectomized (OVX)-induced liver lipid disorder, we observed effects of treadmill training on liver fat accumulation and ER alpha expression in intact and ovariectomized rats. Sixty female rats were randomly assigned to six groups: Sham sedentary (S-S), Sham exercised (S-EX), ovariectomized sedentary (O-S), ovariectomized exercised (O-EX), ovariectomized injected subcutaneously with 17beta-estradiol (E(2)) (O-E(2)), and ovariectomized treated with E(2) and exercise (O-E(2)-EX). Twelve weeks after intervention, OVX resulted in significantly higher body weight gain, intra-abdominal fat mass, serum levels of total cholesterol (TC), and liver triacylglycerol (TAG) concentrations and ER alpha expression than S-S group, while the relative uterus and liver mass, serum levels of E(2), TAG, and the ratio of high density lipoprotein (HDL) to TC were markedly lower in O-S group. All of these changes were decreased in O-S rats after treatment with E(2) alone with the exception of serum TC and HDL-C levels and liver ER alpha expression. Exercise alone significantly reversed the effect of OVX on serum E(2), the ratio of HDL-C to TC and the liver and intra-abdominal fat accumulation in OVX rats. The addition of E(2) to exercise induced the same uterus and lipid profile as E(2) alone. Moreover, an additive effect of exercise and E(2) was observed on liver ER alpha expression in Sham or OVX rats. In conclusion, treadmill training alone could prevent liver fat accumulation in OVX rats and the regulation of exercise on liver ER alpha expression in both OVX and Sham rats needs the presence of physical estrogen levels.

[Changing trends of the expression of TIMP-4 in mouse ovary during pregnant and postpartum period].

AIM: The changes of tissue inhibitor of metalloproteinase-4 (TIMP-4) expression in mouse ovary during pregnant and postpartum period were studied to investigate the role of TIMP-4 in corpus luteum (CL). METHODS: RT-PCR was used to deter mine the change of TIMP-4 mRNA and indirect immunofluorescence was used to observe the change of TIMP-4 protein. The expression of TIMP-4 mRNA was observed in various periods throughout the stage of pregnancy and postpartum day 1. RESULTS: The expression of TIMP-4 was gradually enhanced from day 1 to day 8, reached a maximal expression at day 8, while decreased at day 11 and to the lowest level at postpartum day 1. Indirect immunofluorescence results further indicated that TIMP-4 protein was localized to CL and theca-intera cells in various periods throughout the pregnancy and postpartum day 1. In addition, the change pattern of TIMP-4 protein agreed with that of the TIMP-4 mRNA in pregnancy CL. CONCLUSION: The expression of TIMP-4 in mouse ovary during pregnancy and postpartum is in spatio-temporal pattern and it may be involved in the formation and function maintain of CL during pregnancy in mice.

Developmental and hormonal regulation of meltrin beta (ADAM19) expression in mouse testes during embryonic and postnatal life.

More than half of ADAM (a disintegrin and metalloprotease) family members are expressed in mammalian male reproductive organs such as testis and epididymis. The ADAM19 gene identified in mouse is a member of the ADAM family and is highly enriched in testes of a newborn mouse. The present study was performed to determine its expression pattern in whole mouse testes in vivo as well as its in vitro action and regulation in testis cells from 2-day-old mice. Reverse transcriptase polymerase chain reaction (RT-PCR) detected ADAM19 mRNA from 15.5 days postcoitum (dpc) to 21 days postpartum (dpp), with high expression during the perinatal period. Immunohistochemistry demonstrated ADAM19 protein localization to the seminiferous cords at both embryonic and postnatal ages examined (from 15.5-19.5 dpc to 2 dpp). In particular, we obtained new evidence that a neutralizing antibody to ADAM19 had no influence on the proliferation of 2 dpp testis cells cultured in serum-free medium when compared to controls. Interestingly, it inhibited the 2 dpp testis cell proliferation elicited by stimulation with 10% FCS (P<0.01) or FSH (P<0.05). Lastly, using a model of 2 dpp testis cell cultures and RT-PCR procedures, we demonstrated that follicle stimulating-hormone (FSH) reduced the levels of ADAM19 mRNA in a time-dependent manner. Taken together, these results indicate that the expression of ADAM19 may be subject to regulation by FSH during mouse testis development. Furthermore, ADAM19 can act to regulate the proliferation of perinatal testis cells in the perinatal period.

Localization and temporal regulation of tissue inhibitor of metalloproteinases-4 in mouse ovary.

Tissue inhibitors of metalloproteinases (TIMPs) are potential regulators of tissue remodeling in the ovary. The aim of the present study was to examine the localization and temporal regulation of TIMP-4 protein in the mouse ovary. An induced superovulation model (eCG/hCG) was employed in immature mice to evaluate TIMP-4 protein expression profiles in ovaries collected during the follicular phase, the pre ovulatory period, and the luteal lifespan. Immunofluorescence results indicated that TIMP-4 protein was localized to theca of both antral and preovulatory follicles and adjacent ovarian stroma. After the initiation of luteinization with hCG, TIMP-4 was observed within the luteinizing granulosa cells and persisted throughout the lifespan of the corpus luteum. In the cycling ovary, TIMP-4 signaling localized to corpus luteum from previous estrous cycles, the theca of preovulatory follicles, and appeared to be lower in newly forming corpus luteum. Western analysis further showed that the levels of TIMP-4 increased significantly during the luteinization process of granulosa cells, but no significant change was found among all corpus luteum stages. A putative regulatory mechanism of TIMP-4 expression was identified utilizing an in vitro model. Treatment of cultured granulosa cells with hCG significantly augmented TIMP-4 protein expression levels. Together our data indicate that the luteinization process of granulosa cells is associated with up-regulation of TIMP-4 and that TIMP-4 might play an essential role in maintenance of the luteal function during the whole lifespan of corpus luteum.

Nitric oxide influences the maturation of cumulus cell-enclosed mouse oocytes cultured in spontaneous maturation medium and hypoxanthine-supplemented medium through different signaling pathways.

Nitric oxide (NO) has been recently shown to act with a dual action in mouse oocyte meiotic maturation depending on its concentration, but the mechanism(s) through which it influences oocyte maturation has not been fully clarified to date. The purpose of this study was to test the hypothesis that different signaling mechanisms exist for NO-stimulated and NO-inhibited in vitro maturation of meiosis in cumulus cell-enclosed oocytes (CEOs) from PMSG-primed immature female mice. CEOs were cultured in both spontaneous maturation model and hypoxanthine (HX) arrested model to investigate the mechanism(s). Sodium nitroprusside (SNP, an NO donor) at a concentration of 1mM delayed significantly germinal vesical breakdown (GVBD) during the first 5 h of incubation period and further inhibited the formation of first polar body (PB1) at the end of 24 h of incubation. While SNP, at a concentration of 10 microM, stimulated significantly the meiotic maturation of oocytes by overcoming the inhibition of HX. Methinine blue (MB, 10 microM) or 1-H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 microM)), two soluble guanylate cyclase (sGC) inhibitors, could reverse SNP-inhibited spontaneous oocyte maturation, but had no effect on SNP-stimulated meiotic maturation in the presence of HX. 8-Br-cGMP (1mM), a cell-permeating cGMP analogue, demonstrated a significant inhibitory effect on both spontaneous meiotic maturation and HX-arrested meiotic maturation. The delay effect of SNP on GVBD occurrence was similar to that of forskolin (6 microM, an adenylate cyclase stimulator) and rolipram (250 microM, a phosphodiesterase 4 inhibitor), two cAMP elevating reagents. Both forskolin and rolipram reversed significantly the SNP-stimulated meiotic maturation, but did not reverse the SNP-inhibited spontaneous meiotic maturation. Cilostamide (1 microM), the selective inhibitor of phosphodiestrase 3 (PDE3), could mimic the inhibitory effect of HX on the spontaneous meiotic maturation in CEOs and this inhibitory effect could also be reversed by SNP (10 microM). Moreover, sphingosine (3 microM), a protein kinase C (PKC) inhibitor, blocked the SNP-inhibited spontaneous meiotic maturation, but did not block the SNP-stimulated meiotic maturation. Clearly, these results suggest that pathway differences are present between SNP-inhibited spontaneous meiotic maturation and SNP-stimulated meiotic maturation of mouse oocytes.

[The suppression of melatonin on mouse oocyte in vitro maturation of mouse].

AIM: To study whether melatonin has effect on oocyte maturation of mouse in vitro. METHODS: Mouse oocytes were cultured in maturation medium, HX-medium, or HX-medium supplemented with FSH, and the effects of MT on meiotic maturation of mouse oocyte were examined. RESULTS: (1) MT at all doses of 0.1 g/L, 0.02 g/L, 0.4 g/L or 0.8 g/L inhibited the formation of PB1 in CEO cultured in maturation medium and had no effect on GVBD. (2) MT could delay GVBD and the extrusion of PB1 in CEOs of mouse oocytes by dynamic curves. In contrast to the control, GVBD and PB1 extrusion of oocytes in the treated groups had been delayed by 8-10 hours and 3-4 hours respectively. (3) MT inhibited the effect of FSH on resumption of meiosis, but no effect on the formation of PB1. (4) MT and HX had cooperation effects on spontaneous oocyte maturation in CEO, but not in DO. CONCLUSION: MT is able to affect mouse oocyte maturation and the regulation mechanisms may be related to cumulus cells.

Dual effects of nitric oxide on meiotic maturation of mouse cumulus cell-enclosed oocytes in vitro.

The present experiment used cultured mouse cumulus cell-enclosed oocytes (CEOs) and denuded oocytes (DOs) to study the function of nitric oxide (NO) in mouse oocyte meiotic maturation. Either positive or negative actions of NO on meiotic maturation has been observed when CEOs or DOs were cultured for 24 h in a medium containing 4 mM hypoxanthine (HX) to maintain meiotic arrest, or in maturation medium (without HX) supplemented with different doses of sodium nitroprusside (SNP, a NO donor), N-omega-nitro-L-arginine methyl ester (L-NAME) or N(w)-nitro-L-arginine (L-NNA) (two inhibitors of NO synthase, NOS), and L-arginine (the only substrate of NOS). Both NOS inhibitors suppressed the formation of first polar body (PB1) of the oocytes in CEOs in a dose dependent manner, but no effect on germinal vesicle break down (GVBD) was observed. An optimal inhibitory effect was observed with either 10(-3) M L-NAME (P<0.01) or 10(-3) M L-NNA (P<0.01) and the inhibition could be reversed by the addition of SNP (10(-5) M). The above mentioned optimal concentration of L-NAME or L-NNA on CEOs exhibited no effect on oocyte meiotic maturation of DOs. Treatments of low concentrations of SNP (10(-7), 10(-6), 10(-5) M) stimulated significantly the oocyte meiotic maturation of CEOs which were inhibited with HX, but had no effect on DOs in the same culture medium. While, the treatment with high concentrations of SNP (0.1-4 mM) during the CEOs cultured in maturation medium resulted in a lower percentage of oocytes at PB1 stage and a higher percentage of atypical oocytes in a dose dependent manner compared with control. A dose of SNP at 1 mM exhibited significant inhibitory effect on the formation of PB1, but without effect on the number of atypical oocytes compared with control, while, this SNP dosage not only inhibited the oocyte PB1 formation but also increased the percentage of dead oocytes in DOs. Although oocytes of all groups underwent GVBD at the end of the culture in the spontaneous maturation medium, the results of the kinetics showed that the treatment of the optimal concentration of SNP (1 mM) could significantly delay GVBD during the first 5 h culture period. The concomitant addition of L-NAME with SNP did not reverse the inhibitory effect of SNP on CEOs. Similarly, neither pre-incubation nor illumination by ultraviolet ray could balance the inhibitory effect of SNP. Finally, when added alone at a concentration of 4 mM, L-arg caused extensive death of both CEOs and DOs. While, administration of 4 mM L-arg and 1 mM L-NAME to both CEOs and DOs simultaneously resulted in markedly reduced CEOs death percentage as compared with L-arg treatment alone, but not in DOs. These data support the idea that NO could act with a dual action (stimulation or inhibition) in mouse meiotic maturation depending on its concentration.