Shoichi Okinaga

Hypothalamic and hypophysial progesterone receptors: Estrogen-priming effect, differential localization, 5α-dihydroprogesterone binding, and nuclear receptors

Abstract Specific progesterone receptor of 7 S which can be isolated from rat female hypothalamic and hypophysial cytosols was further investigated in terms of its induction effects by estrogen-priming, intracerebral localization, 5α-dihydroprogesterone (DHP) binding, and nuclear receptors. Estrogen-priming was necessary for the appearance of the cytosol receptors. The 7 S hypothalamic and hypophysial receptors were increased in a dose dependent fashion by estradiol injections. The maximal effective doses of estradiol benzoate for both tissues were 1 and 10 μg, respectively. The receptors were mostly localized in the median eminence, preoptic-anterior hypothalamus and anterior hypophysis of estrogen-primed immature and mature rats, but little or no 7 S binding was detected in the cerebral cortex, reticular formation, amygdaloid complex and posterior hypophysis. This differential localization of progesterone receptors resembles that of estrogen receptors, suggesting possible induction of progesterone receptors in these specific brain regions by estrogen. Progesterone receptor complexes of 5 S were isolated from “purified” nuclei of anterior pituitaries from estrogen-primed adult female rats. These results on the cytosol and nuclear receptors suggest that progesterone can directly act on the brain through its interaction with specific progesterone receptors in the hypothalamus and hypophysis. It is noteworthy that 5α-DHP can bind the progesterone receptors in both tissues, suggesting its direct feedback action on the brain through the receptors.

A sensitive bridge heterologous enzyme immunoassay of progesterone using geometrical isomers

A sensitive bridge heterologous enzyme immunoassay of progesterone using geometrical isomers of progesterone 3(O-carboxymethyl)oxime(E/Z) was developed. Isomers were separated by synthesis of N-hydroxysuccinimide esters. Progesterone 3(Z)(O-carboxymethyl)oxime N-hydroxysuccinimide ester bound with beta-galactosidase in an appropriate molar ratio provided a conjugate suitable for an enzyme immunoassay. The antiserum was raised in rabbits by immunizing the animals with the progesterone 3(E)(O-carboxymethyl)oxime-bovine serum albumin conjugate. This bridge heterologous enzyme immunoassay proved to have sufficient sensitivity equivalent to radioimmunoassay and excellent specificity.

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.

Adrenocorticotropic hormone in human fetal blood at delivery.

Adrenocorticotropic hormone (ACTH) concentrations in umbilical arterial and venous plasma were measured in newborn infants delivered both vaginally and by cesarean section. Mean ACTH was significantly higher in the arterial (602.0 pg. per milliliter) than venous (261.5 pg. per milliliter) plasma in vaginal deliveries (pless than 0.01). Arterial plasma of babies born by elective cesarean section before initiation of labor contained less ACTH (384.7 pg. per milliliter) when compared to those delivered after labor had started (698.7 pg. per milliliter). Serial determinations of ACTH in pooled fetal blood obtained during labor showed that the average hormone titers increased significantly from first stage (295.2 pg. per milliliter) to second stage of labor (440.5 pg. per milliliter), and reached a peak at delivery (645.4 pg. per milliliter). The pituitary gland of the fetus at midtrimester contained ACTH. Possible mechanisms for fetal secretion of ACTH at delivery are discussed.

Calreticulin is present in the acrosome of spermatids of rat testis

We have already succeeded in purifying a calcium-binding protein (CalBP) from rat spermatogenic cells [Nakamura et al., Biochem. Biophys. Res. Commun., 176 (1991) 1358]. In this study, the location of this protein within rat testis was examined, using a rabbit antisera for this protein. The antigen was localized on the developing acrosomes during spermiogenesis. The NH2-terminal amino acid sequence obtained for rat CalBP was identical to that of calreticulin obtained for the skeletal muscle of mice and closely resembled that for rabbit calreticulin. On the immunoblot analysis, the purified rat CalBP reacted with an antibody raised against rabbit skeletal muscle calreticulin. The results indicate that calreticulin is present in the acrosome of spermatids of rat testes.

Antisera to calreticulin inhibits sperm motility in mice

Mouse sperm were rapidly immobilized when exposed to rabbit antisera against rat calreticulin. The inhibition of sperm motility was concentration dependent at dilutions 1:50-350. The velocity of sperm did not change significantly as long as they were motile. Neither motility nor velocity of sperm was affected by adding sheep antisera to bovine calmodulin. The antisera to calreticulin also inhibited in vitro fertilization of mouse eggs. Using the avidin-biotin-peroxidase complex method, the antigen was found to be localized in the acrosome of sperm. The results indicate that calreticulin is present in the acrosome of mouse sperm and may play an important role in sperm motility.

Immunohistochemical localization of pregnancy-associated plasma protein A (PAPP-A) in placentae from normal and pre-eclamptic pregnancies.

An immunohistochemical method was used to locate pregnancy-associated plasma protein A (PAPP-A) in the placenta and uterus. In addition to 10 placentae and basal plates from normal pregnancies, ranging in gestational age from 37 to 40 weeks, the following specimens were studied: three uteri obtained by hysterectomy during early pregnancy; and three placentae from patients with severe hypertensive pre-eclampsia. In early gestation, PAPP-A was localized in the villous cytotrophoblastic cell layer and the endometrial glands but was not found in the villous syncytiotrophoblast, the cytotrophoblastic cell columns or the decidual cells. On histochemical examination of placentae from cases of pre-eclampsia with hypertension, an increased number of villous cytotrophoblastic cells and so-called X-cells was observed. The monospecific antiserum to PAPP-A reacted strongly and evenly with the cytoplasm of these cells. The present study strongly suggests that the active production sites of PAPP-A are the villous cytotrophoblastic cells and the X-cells.

Metabolism of Round Spermatids: A Possible Regulation of Hexose Transport

Round spermatids (steps 1–8) were isolated from rat testes and glucose transport into the cells was examined. The exposure of spermatids to glucose resulted in an extremely low level of ATP. In contrast, the level of ATP remained constant in the presence of pyruvate. Transport of a glucose analogue, 2‐deoxy‐D‐[3H]glucose ([3H]dGlc) into spermatids was correlated with intracellular levels of ATP and was much greater in cells with higher rather than lower levels of ATP. [3H]dGlc transport into spermatids with low levels of ATP was partially reversed when the cells were incubated with pyruvate. Inhibition of [3H]dGlc transport was exerted on Vmax and not on Km. Moreover, glucose acted as a competitive inhibitor of [3H]dGlc uptake (Km increased; Vmax unaltered). These results suggest that glucose transport into spermatids is active in vitro and probably regulated by the intracellular level of ATP.

Influence of Temperature on Lactate Utilization by Round Spermatids from Rat Testes

Rotenone‐sensitive 14CO2 formation from [14C]lactate and oxygen consumption by round spermatids were found to be greater at elevated temperatures than at 34°C. More than 96% of the total radioactivity of the metabolized [14C]lactate was recovered in the released CO2 and the acid soluble fraction of the cells. There was practically no incorporation of [14C]latctate into the lipid, nucleic acid, and protein fractions. Intracellular level of ATP in spermatids was enhanced in the presence of lactate (20 mM) at 34°C (scrotal temperature), whereas it was decrease at 37°C (body temperature). However, this was reversible when the cells were transferred from the elevated temperature to 34°C. It was also found that oxygen consumption and CO2 production were increased at 34°C by 2, 4‐dinitrophenol (DNP), but decreased by oligomycin. On the other hand, oligomycin and DNP had no effect on oxygen consumption and 14CO2 formation at the elevated temperature.

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.