Abstract
Biochemical as well as immunochemical studies were carried out to quantitatively and qualitatively evaluate the hormonal regulation of nuclear cAMP-dependent protein kinase subunits in ovaries from estrogen-treated hypophysectomized rats. Photoaffinity labeling of nuclear extracts with 8-azido-[32P]cAMP and electrophoretic analysis showed the existence of three variants of the regulatory subunit RI and of a 52,000-dalton RII variant (RII-52) in ovarian nuclei of estrogen-primed hypophysectomized rats. After follicle-stimulating hormone (FSH) stimulation, an additional variant of RII (RII-51, Mr = 51,000) was detected in nuclei. The cytosolic RII-54 variant (Mr = 54,000) could not be identified in nuclei by photoaffinity labeling. The FSH-mediated appearance of the nuclear RII-51 variant was accompanied by an approximate 2-fold increase of nuclear catalytic subunit activity. Using quantitation by enzyme-linked immunosorbent assay, we identified a marked FSH-mediated increase of nuclear RII variant(s) and confirmed the increase of nuclear catalytic subunit levels. Furthermore, morphometric analysis of nuclear and cytoplasmic antigen density by immunogold electron microscopy demonstrated a cell-specific modulation by FSH of RII and C subunit density. In granulosa cells, both nuclear as well as cytoplasmic RII density was increased by FSH, whereas catalytic subunit density was increased in the nuclear area only. In thecal cells, FSH increased only the nuclear catalytic subunit density. These results provide biochemical as well as immunochemical evidence for a cell-specific FSH regulation of nuclear RII and catalytic subunit levels which may be involved in the molecular events responsible for the FSH-mediated differentiation of the rat ovary.
Highlights
Biochemical as well as immunochemical studies were natal development (Lamprecht et al, 1973; DeAngelo et al, carried out to quantitatively and qualitatively evalua1t9e75) and in response to gonadotropins administered to neothe hormonal regulation of nuclear CAMP-dependent natal rats or adult hypophysectomized rats
Sorbent assay, we identified a marked FSH-mediated In the ragtranulosa cell, the level of the cytosolicregulatory increase of nuclear RII variant(s) and confirmed the subunit RII increases as preantral follicles differentiate into increase of nuclear catalytic subunit levels
Terns (Richards et al, 1983;Halpren-Ruder et al, 1980).Since both nuclear as well as cytoplasmic RII density was at least some of these CAMP-mediatedevents canbe expected increased by FSH, whereas catalytic subunit density to involve a nuclear action of cAMP and CAMP-dependent was increased in the nuclear aroenaly
Summary
Subunits gold particles (GAR 20) was purchased from Janssen Pharmaceuti- of pure nuclei and quantitative recovery of nuclear CAMP-. Animals-Immature female rats (Charles River, Sprague-Dawley), hypophysectomized at the ageof 21 days, were purchased from the dependent protein kinase subunits in ovarian nuclear NaCl extracts. Nuclear isolation procedurwe hich involves homogenizationof hormone treatments were initiated as follows: Rats wereinjected tissue in buffer containing high molarity sucrose (Laks et al, subcutaneouslyoncedailyfor 4 dayswith1.5 mgof 17P-estradiol 1981). Undertheseconditions, loss of nuclearproteinis dissolved in 0.2 ml of propylene glycoland twice daily for days minimized (Yu, 1975), and the isolatednuclei are not signifiwith 7.5 pg of ovine FSH (in 0.1 ml of phosphate-buffered saline)or cantly contaminated with non-nuclear materialas judged by only with 0.1ml of saline. Undertheseconditions, loss of nuclearproteinis dissolved in 0.2 ml of propylene glycoland twice daily for days minimized (Yu, 1975), and the isolatednuclei are not signifiwith 7.5 pg of ovine FSH (in 0.1 ml of phosphate-buffered saline)or cantly contaminated with non-nuclear materialas judged by only with 0.1ml of saline. h after the last injection
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