Tomi Ohkawa

Determination of Salivary Cortisol by ELISA and Its Application to the Assessment of the Circadian Rhythm in Children

In 35 young children, circadian rhythms of salivary cortisol levels were determined by ELISA using a commercially available kit with a minor modification. The concentration of labeled cortisol in the serum kit was reduced in order to measure cortisol in 10 microliters of saliva. Intra- and interassay coefficients of variation for salivary cortisol ranged from 2.4 to 9.9 and 3.2 to 8.9%, respectively. Recovery of salivary cortisol was 82.9-107.0%. There was a highly significant correlation between cortisol levels in saliva and serum in adults (r = 0.857). Salivary cortisol levels ranged from 0.01 to 2.252 micrograms/100 ml and showed significant diurnal variation in the children. Our ELISA is a precise, simple, noninvasive and useful method for clinical practice and study in infants and children.

The Involvement of Dopamine in the Regulation of Steroidogenesis in Rat Ovarian Cells

To investigate a role for dopamine (DA) in steroidogenesis in the rat ovary, ovarian cells of pregnant-mare-serum gonadotropin (PMSG)-treated rats were incubated with DA agonists, antagonists, adrenergic drugs and beta-blocker for 1 h. DA, norepinephrine (NE) and isoproterenol (Iso) increased the level of progesterone (P4) and cAMP in the conditioned medium. D1 agonists (SKF38393, SKF82526J, CY208-243) increased P4 secretion, while the D2 agonist, bromocriptine, showed no significant effect on P4 secretion. The effect of NE or Iso was inhibited by the beta-blocker propranolol (Pro), but was not suppressed by the D2 antagonist, domperidone. The effects of D1 agonists were suppressed by bulbocapnine (Bul), while neither Pro nor the D2 antagonist, domperidone, affected the levels of P4. The D1 receptor was demonstrated in the PMSG-treated rat ovary, and its Bmax was 1.33 fmol/mg tissue and the Kd was 0.357 nM. These results suggest that DA has a direct stimulatory effect on P4 secretion in PMSG-treated rat ovarian cells through they D1 receptor. The observed action may indicate a physiological role for DA in the regulation of ovarian functions in rats.

Effects of Gonadotropin-Releasing Hormone Agonist on Steroidogenesis in the Rat Ovary

To assess the regulatory roles of gonadotropin-releasing hormone (GnRH) in ovarian function, the kinetics of the ovarian GnRH receptor and the effects of the GnRH superagonist buserelin on steroidogenesis in ovarian cell culture were examined. Scatchard analysis of buserelin-binding to crude ovarian cell membrane revealed a specific high-affinity GnRH receptor. Buserelin together with follicle-stimulating hormone stimulated estradiol (E2) production in immature follicles in hypophysectomized and DES-treated rats. On the other hand, applied to developing follicles of rats treated with pregnant-mare-serum gonadotropin buserelin suppressed E2 production to terminate follicle maturation and simultaneously stimulated progesterone (P4) production to induce luteinization. With ovarian cells luteinized by human chorionic gonadotropin in vitro, buserelin suppressed production of both P4 and E2, leading to luteolysis. Buserelin affected steroid production by modulating activities of key enzymes in steroid synthesis. These findings indicate that buserelin action depended on the gonadotropin priming of ovarian cells, and suggest the possible involvement of GnRH in the regulation of steroidogenesis throughout the ovulatory cycle.

A new radioimmunoassay for serum 16α-hydroxyandrost-4-ene-3,17-dione with specific antiserum

Abstract A radioimmunoassay system for serum 16α-hydroxyandrost-4-ene-3,17-dione was developed with the use of rabbit antiserum against 16α-hydroxyandrost-4-ene-3,17-dione-3-(O-carboxymethyl)oxime which was conjugated with bovine serum albumin. The antiserum was highly specific for 16α-hydroxyandrost-4-ene-3,17-dion, with cross reactions to other steroids being less than 0.8% except for androst-4-ene-3,17-dione(3.4% cross reaction). Use of LH-20 column chromatography, however, clearly separated these two steroids. Pregnancy sera were measured with this assay system after an addition of labelled internal standard, extraction and separation by column chromatography. The lower limit of detection for 16α-hydroxyandrost-4-ene-3,17-dione was 2pg/tube. The mean recovery rate of the added standard was 98.3 ± 8.8% (mean ± SE). Intra- and inter-assay coefficients of variation were 8.6% (n = 6) and 12.1% (n =7), respectively.