ABSTRACT
OBJECTIVE: The aim of this study was to evaluate the effects on murine fetal ovarian gene expression of prenatal exposure to 1.765 GHz of microwave irradiation. METHODS:Ten pregnant ICR mice were divided into two groups. At 5th days after mating, dam mice were exposed to microwave (SAR: 0.38~1.71 W/kg) in the insulated cage for 8 hours each day. The remaining mice were treated in the same way. Neonatal ovaries were removed for study 7 days after delivery. Microarray analysis was performed using total RNA extracted from the removed ovaries. We investigated the differences in ovarian gene expression between the groups. SPSS 12.0 was used for statistical analysis. P<0.05 was considered to be statistically significant. RESULTS: The mean birth weight of the offspring in the irradiated group was significantly lower than that in the sham group (1.54+/-.22 g vs. 1.60+/-.21 g, P=0.012). The mean number of offspring per pregnancy in the irradiated group was significantly higher than in the sham group (13.60+/-.70 vs. 11.40+/-.17, P=0.009). We detected that in the irradiated ovaries, 14 genes were expressed at levels 2-fold higher than in the sham ovaries and 74 genes were expressed at levels 2-fold lower than in the sham ovaries. CONCLUSION: We found differences in fetal ovarian gene expression between the irradiated and sham groups. In the irradiated group, the Tnfaip8, TNFsf 12, Cfd, CCL 11, and Zfp74 genes were down-regulated and the Brd 3 gene was up-regulated.
Subject(s)
Animals , Female , Mice , Pregnancy , Birth Weight , Gene Expression , Mice, Inbred ICR , Microarray Analysis , Microwaves , Ovary , RNA , SalicylamidesABSTRACT
OBJECTIVE: The aim of this study was to evaluate the effects on murine fetal ovarian gene expression of prenatal exposure to 1.765 GHz of microwave irradiation. METHODS:Ten pregnant ICR mice were divided into two groups. At 5th days after mating, dam mice were exposed to microwave (SAR: 0.38~1.71 W/kg) in the insulated cage for 8 hours each day. The remaining mice were treated in the same way. Neonatal ovaries were removed for study 7 days after delivery. Microarray analysis was performed using total RNA extracted from the removed ovaries. We investigated the differences in ovarian gene expression between the groups. SPSS 12.0 was used for statistical analysis. P<0.05 was considered to be statistically significant. RESULTS: The mean birth weight of the offspring in the irradiated group was significantly lower than that in the sham group (1.54+/-.22 g vs. 1.60+/-.21 g, P=0.012). The mean number of offspring per pregnancy in the irradiated group was significantly higher than in the sham group (13.60+/-.70 vs. 11.40+/-.17, P=0.009). We detected that in the irradiated ovaries, 14 genes were expressed at levels 2-fold higher than in the sham ovaries and 74 genes were expressed at levels 2-fold lower than in the sham ovaries. CONCLUSION: We found differences in fetal ovarian gene expression between the irradiated and sham groups. In the irradiated group, the Tnfaip8, TNFsf 12, Cfd, CCL 11, and Zfp74 genes were down-regulated and the Brd 3 gene was up-regulated.
Subject(s)
Animals , Female , Mice , Pregnancy , Birth Weight , Gene Expression , Mice, Inbred ICR , Microarray Analysis , Microwaves , Ovary , RNA , SalicylamidesABSTRACT
OBJECTIVE: The purposes of the present study were to investigate the effect of gamma-radiation on the expression of inhibin-alpha proteins and genes for inhibin alpha, betaA, and betain the ovary. METHODS: Immature mice were whole-body gamma-irradiated with 25% of a lethal dose. At time 0, 3, 6, 12, and 24 hours after the irradiation,the ovaries were collected and used for immunohistochemistry for inhibin-alpha, and RT_PCR for inhibin-alpha, betaA, and betaB. RESULTS: The expression of the immunoreactive inhibins-alpha was maintained at 12 hours post-irradiation and reduced thereafter. The expression of inhibin-alpha mRNA was significantly increased with the time after the irradiation. However there were no significant changes in the expression of betaA and betaB mRNAs. CONCLUSION: It might be thought that inhibin acts as one of the regulatory factors in the gamma-radiation-induced follicular atresia in mice
Subject(s)
Animals , Female , Mice , Follicular Atresia , Immunohistochemistry , Inhibins , Ovary , RNA, MessengerABSTRACT
OBJECTIVES: Bok, Bcl-2-related ovarian killer, is a proapoptotic Bcl-2 family protein identified in the ovary based on its dimerization with the antiapoptotic protein Mcl-1. The present study examined the hormonal regulation and localization of Bok messenger RNA levels in the mouse ovary during the follicle development. METHODS: The animals were implanted subcutaneously with Silastic brand capsules containing the synthetic estrogen, DES at 21~23 days of age. Ovaries were collected 1~3 days after implantation for RNA analysis and in situ hybridization. Some mice were removed capsule for 1~2 days to induce ovarian follicle apoptosis. Ovaries were also collected from 26 day-old immature mice at various times after treatment with 10 iU PMSG. Some mice received a single intraperitoneal injection of 10 iU hCG to induce ovulation, and ovaries were obtained at different time intervals for Northern blot and in situ hybridization analysis, respectively. RESULTS: Treatment of immature mice with diethylstilbestrol (DES) for 24~48 h increased ovarian Bok mRNA levels. Bok mRNA was remained the same levels in mice removed DES for 24~48 h to induce apoptosis. High signals of Bok mRNA after DES treatment were detected in granulosa cells of early antral follicles. Treatment of immature mice with PMSG for 12 h increased markedly ovarian Bok mRNA expression which was detected mainly in preantral and atretic follicles. interestingly, low levels of Bok mRNA were also expressed in granulosa cells of preovulatory follicles. Treatment of PMSGprimed mice with hCG stimulated strongly ovarian Bok mRNA expression at 6~9 h. At that time, Bok mRNA was expressed in granulosa cells of atretic and small growing follicles. CONCLUSION: These results demonstrate that Bok is one of proapoptotic Bcl-2 members expressed in early growing and atretic follicles during the ovarian follicular development. Gonadotropins induce a transient increase of Bok gene expression in granulosa cells of preantral and preovulatory follicles indicating some role in the ovulatory process.
Subject(s)
Animals , Female , Humans , Mice , Apoptosis , Blotting, Northern , Capsules , Diethylstilbestrol , Dimerization , Estrogens , Gene Expression , Gonadotropins , Granulosa Cells , In Situ Hybridization , Injections, Intraperitoneal , Ovarian Follicle , Ovary , Ovulation , RNA , RNA, MessengerABSTRACT
OBJECTIVE: Recently, microdissection of tissue sections has been used increasingly for the isolation of morphologically identified homogeneous cell populations, thus overcoming the obstacle of tissue complexity for the analysis cell-specific expression of macromolecules. The aim of the present study was to establish the minimal conditions required for the RNA extraction and amplification from the cells captured by the laser captured microdissection. METHODS: Mouse ovaries were fixed and cut into serial sections (7 micrometer thickness). Oocytes were captured by laser captured microdissection (LCM) method by using PixCell IITM system. The frozen sections were fixed in 70% ethanol and stained with hematoxylin and eosin, while the paraffin sections were stained with Multiple stain. Sections were dehydrated in graded alcohols followed by xylene and air-dried for 20 min prior to LCM. All reactions were performed in ribonuclease free solutions to prevent RNA degradation. After LCM, total RNA extraction from the captured oocytes was performed using the guanidinium isothiocyanate (GITC) solution, and subsequently evaluated by reverse transcriptase -polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). RESULTS: With the frozen sections, detection of the GAPDH mRNA expression in the number of captured 25 oocytes were not repeatable, but the expression was always detectable from 50 oocytes. With 25 oocytes, at least 27 PCR cycles were required, whereas with 50 oocytes, 21 cycles were enough to detect GAPDH expression. Amount of the primary cDNA required for RT-PCR was reduced down to at least 0.25 microl with 50 oocytes, thus the resting 19.75 microl cDNA can be used for the testing other interested gene expression. Tissue-to-slide, tissue-to-tissue forces were very high in the paraffin sections, thus the greater number of cell procurement was required than the frozen sections. CONCLUSION: We have described a method for analyzing gene expression at the RNA level with the homogeneously microdissected cells from the small amount of tissues with complexity. We found that LCM coupled with RT-PCR could detect housekeeping gene expression in 50 oocytes captured. This technique can be easily applied for the study of gene expression with the small amount of tissues.