Quantitative RT-PCR for inhibin/activin subunits: measurements of rat hypothalamic and ovarian inhibin/activin subunit mRNAs during the estrous cycle.
Inhibins (alpha-beta(A) and alpha-beta(B)) and activins (beta(A)-beta(A), beta(A)-beta(B) and beta(B)-beta(B)) were originally isolated from ovarian follicular fluids as FSH secretion modifiers. Inhibin/activin subunits, alpha, beta(A) and beta(B), are widely distributed in several tissues, including gonads and brain, and inhibins and activins have been reported to be involved in ovarian or hypothalamic functions. In this study, we established and employed a competitive RT-PCR assay system for rat inhibin/activin subunits by capillary electrophoresis to determine rat hypothalamic and ovarian inhibin/activin subunit mRNA levels during the estrous cycle. Linearity of standards for alpha, beta(A), and beta(B) subunit assays were between 0.01-0.3 amol, 0.003-0.09 amol and 0.002-0.02 amol of each fragment DNA as a standard, respectively. Hypothalamic beta(A) subunit mRNA during the estrous morning (1000 h) tended to be increased compared with that of the proestrous evening (1700 h), although they were not significantly different. Ovarian alpha subunit mRNA levels tended to be increased during the proestrous morning (1000 h) and were significantly increased in the proestrous evening (1700 h), compared with diestrus and estrus (P < 0.05). Ovarian beta(A) subunit mRNA was also significantly higher in the proestrous evening, compared with diestrus and estrus (P < 0.05), but in the case of beta(B) subunit mRNA there was no difference among diestrus, proestrus and estrus. We thus established a sensitive competitive RT-PCR system for the measurement of inhibin/activin alpha, beta(A) and beta(B) subunits, and this assay system would be helpful for the study of inhibin/activin action in brain and other tissues where these factors are expressed at low levels.
Murata
T
T
Torii Nutrient-stasis Project, Exploratory Research for Advanced Technology, Research Development Corporation of Japan, Yokohama, Japan.
Takizawa
T
T
Funaba
M
M
Fujimura
H
H
Murata
E
E
Takahashi
M
M
Torii
K
K
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D019075:Electrophoresis, Capillary; D004971:Estrus / Q000235:genetics*; D005260:Female; D005453:Fluorescence; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D007031:Hypothalamus / Q000737:chemistry*; D007265:Inhibins / Q000235:genetics*; D010053:Ovary / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar
D017931:DNA Primers; D012333:RNA, Messenger; D007265:Inhibins; D004247:DNA