To evaluate mechanisms of cell proliferation in the fetal female rat reproductive tract, diethylstilbestrol (DES) effects on cell division and estrogen receptor (ER), epidermal growth factor (EGF) and EGF receptor (EGF-R) expressions were determined from gestational day (GD) 15.5 to 21.5. Reproductive tracts were evaluated within three regions along the Mullerian duct axis; these were proximal, middle and caudal, which differentiate into oviduct, uterus and upper vagina respectively. In fetuses from non-treated dams, epithelial and mesenchymal proliferation, as evaluated by 5-bromo-2'-deoxyuridine incorporation, was decreased with development in all regions of the Mullerian duct. EGF levels were determined by immunohistochemistry. Mullerian epithelial EGF immunoreactivity was intense in the proximal and middle regions on GDs 15.5 and 17.5. EGF staining remained intense only in the proximal epithelia by GD 19.5 and was weak in the caudal epithelium, but substantially reduced throughout epithelia in all regions by GD 21.5. Thus, decreased cell proliferation correlated with decreased EGF expression in the developing Mullerian duct. DES (100 microg/kg body weight) was injected from GD 15 to 19 and female fetuses were collected on GD 19.5. DES increased Mullerian duct cell proliferation in the proximal epithelium and mesenchyme but decreased it in the caudal epithelium compared with oil-treated controls. No proliferative DES effect was observed in any cell type in the middle region. Mullerian duct EGF immunoreactivity was suppressed by DES compared with oil. Competitive RT-PCR indicated DES also decreased mRNAs for EGF, ERbeta1 and ERbeta2, but not ERalpha and EGF-R. These results indicate EGF may be an important regulatory factor of Mullerian duct cell proliferation, and that DES may alter cell proliferation by disrupting normal EGF, ERbeta1 and ERbeta2 expression in the developing female rat reproductive tract.
A Okada, T Sato, Y Ohta, DL Buchanan and T Iguchi
K Toda, T Saibara, T Okada, S Onishi and Y Shizuta
Aromatase P450 (CYP19) is an enzyme responsible for conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeting disruption of the CYP19 gene and observed that the ArKO males exhibited a complete loss of aggressive behaviour against intruder mice when examined using a resident-intruder paradigm. The defect in the behaviour of ArKO males was reinstated when the mice received supplements of 17beta-oestradiol soon after birth. Nevertheless, the cumulative duration of the behaviour displayed by the treated mice during the test period of 15 min was 19+/-10 s, which was much shorter than that displayed by wild-type males, 90+/-17 s. When the supplementation was started at 7 days after birth, the defect was not restored. These findings illustrate an absolute requirement for oestrogen during the neonatal stage of a male's life for the development of the potential for aggression observed in adulthood. Furthermore, the present study demonstrates that ArKO males are a useful model in which to investigate the neural mechanisms by which aggressive behaviour is controlled.
Y Tsuboi, S Ishikawa, G Fujisawa, K Okada and T Saito
The present study was undertaken to determine whether a non-peptide arginine vasopressin (AVP) antagonist (5-dimethylamino - 1- [4-(2-methylbenzoylamino)benzoyl]-2,3,4,5-tetrahydro-1H-benzazepine; OPC-31260) antagonizes the antidiuretic action of endogenous and exogenous AVP in conscious rats. OPC-31260, given orally at a dose of 5 mg/kg or higher, increased urinary volume (UV) and reduced urinary osmolality (Uosm) in a dose-dependent manner, in rats acutely denied access to water. Minimal Uosm was obtained 1–2 h after oral administration of OPC-31260. OPC-31260 caused sustained water diuresis for more than 12 h when water was available ad libitum since OPC-31260 (30 mg/kg) reduced Uosm to less than 230 mOsmol/kg H2O, significantly less than the control value of 600 mOsmol/kg H2O. Water deprivation for 24 h increased plasma AVP levels to 7·2 pmol/l and increased Uosm to 2160 mOsmol/kg H2O. In such water-deprived rats, oral administration of OPC-31260 at 100 mg/kg was diuretic; it markedly increased free water clearance and decreased Uosm to 202 mOsmol/kg H2O.
In homozygous Brattleboro rats (with inherited AVP deficiency), given free access to water, subcutaneous infusion of the V2 agonist 1-deamino-8-D-AVP (dDAVP) at a rate of 1 ng/h markedly decreased UV to 12.6 from 148·7 ml/day and increased Uosm to 1762 from 231 mOsmol/kg H2O. OPC-31260 (30 mg/kg) promptly increased UV and reduced Uosm to levels similar to those before the administration of dDAVP; repeated OPC-31260 treatment had sustained effects. These results indicate that OPC-31260 is an orally effective non-peptide AVP antagonist to the antidiuretic action of AVP in the conscious rat.
Journal of Endocrinology (1994) 143, 227–234
A Okada, Y Ohta, SL Brody and T Iguchi
Expression of transcription factors binding to the activating protein-1 (AP-1) site is induced by estrogens in association with epithelial proliferation in the uterus, but, in the oviduct, the relationship between cell proliferation and differentiation and AP-1 transcription factors is not well understood. In the developing rat oviduct, we found that proliferation and differentiation of epithelial cells were region-dependently regulated by 17beta-estradiol (E2). To determine the role of AP-1 transcription factors in the development of rat oviduct, we performed immunohistochemistry for epithelial c-jun and c-fos proteins in E2-untreated and -treated newborn rats. E2 increased the expression of c-jun and c-fos during proliferation of undifferentiated epithelial cells, but diminished both proteins during accelerated differentiation of ciliated epithelial cells. A pure estrogen receptor (ER) antagonist, ICI 182,780, inhibited changes in their expression during both cell proliferation and differentiation. Importantly, no reduction of c-jun was noted in the epithelial cells of the foxj1-deficient oviduct, which lacks cilia development. This study shows that c-jun and c-fos are regulated during epithelial cell proliferation and differentiation in a region-specific manner. This provides critical information for understanding the molecular and cellular mechanisms of the development of the neonatal oviduct.
E. Ohmura, M. Okada, Y. Ohba, N. Onoda, T. Sano, T. Tsushima and K. Shizume
The effect of phorbol ester pretreatment on rat (r) GH release induced by GH-releasing factor (GRF) or 8-bromo-cyclic (c)AMP was investigated using rat pituitary cells cultured in monolayers. Pretreatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 3 h significantly suppressed the rGH release induced by GRF, but not that by 8-bromo-cAMP 20 h later; this suppressive effect of TPA was concentration-dependent from 8 to 160 nmol/l, and complete suppression was observed after pretreatment with 80–160 nmol TPA/l. Production of cAMP by pituitary cells stimulated with GRF was similarly attenuated in TPA-pretreated cells. The rGH responsiveness to GRF of these cells was fully recovered on prolonged culture (40 h), suggesting that the inhibitory effect of TPA is reversible.
In contrast, pretreatment with GRF (5 nmol/l) resulted in suppression of the rGH response to subsequent exposure to GRF (5 nmol/l) or 8-bromo-cAMP (10 mmol/l), but not to TPA. These observations suggest that pretreatment with TPA modifies the rGH response to GRF at steps before the formation of cAMP.
J. Endocr. (1988) 118, 423–428
T. Tamura, J. Kitawaki, T. Yamamoto, Y. Osawa, S. Kominami, S. Takemorit and H. Okada
Immunohistochemical localization of 17α-hydroxylase/C17-20 lyase (P-45017α,lyase) and aromatase cytochrome P-450 (P-450arom) in polycystic ovary (PCO) syndrome was studied using specific polyclonal antibodies which had been raised against the corresponding enzymes. In the majority of follicles that were atretic and smaller than 7 mm in diameter, theca interna cells showed high P-45017α,lyase immunoreaction, while small numbers of granulosa cells showed little P-450arom immunoreaction. In some atretic follicles that were larger than 11 mm in diameter, the hyperplastic theca interna cell layer showed high immunoreaction to P-45017α,lyase, while the poorly proliferated granulosa cell layer showed a mixture of weak and negative immunoreaction to P-450arom. No immunoreaction to P-45017α,lyase or P-450arom was recognized in PCO stroma. These findings suggest that the theca interna cells and the granulosa cells from PCOs show abnormal steroidogenic function, while the localization of P-45017α,lyase and P-450arom in PCOs was essentially identical to that in the normal ovary. Theca interna cells in PCO atretic follicles are the main site of excess androgen production.
Journal of Endocrinology (1993) 139, 503–509
T. Tamura, J. Kitawaki, T. Yamamoto, Y. Osawa, S. Kominami, S. Takemori and H. Okada
Immunohistochemical localization of 17α-hydroxylase/C17–20 lyase (P-45017α,lyase) and aromatase cytochrome P-450 (P-450arom) in normal human ovaries during the menstrual cycle was studied using specific polyclonal antibodies which were raised against corresponding enzymes. In the follicular phase of matured follicles, P-45017α,lyase was localized in theca interna cells and P-450arom in granulosa cells. P-45017α,lyase was expressed in theca interna cells before P-450arom was expressed in granulosa cells. The corpus luteum showed immunoreactivity to both enzymes and, after menstruation, immunoreactivity decreased gradually until it could not be detected in the corpus albicans. In corpus luteum graviditatis the immunoreactivity continued to be expressed strongly. In some atretic follicles, P-45017α,lyase and/or P-450arom continued to be expressed. In the stromal layer, P-45017α,lyase was detected in secondary interstitial cells, which originated from the theca interna of atretic follicles, and P-450arom was detected in hilar cells. Immunoreactivity to both enzymes was also detected in oocytes of developing follicles. These results are consistent with the two cell theory in the human ovary. They also suggest that androgens and oestrogens are produced not only by follicles and corpora lutea but also by stroma and oocytes.
Journal of Endocrinology (1992) 135, 589–595
K Toda, T Okada, K Takeda, S Akira, T Saibara, M Shiraishi, S Onishi and Y Shizuta
Aromatase P450 (CYP19) is an enzyme responsible for the conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeted disruption of Cyp19 and studied the role of oestrogens in male reproductive ability. Approximately 85% of ArKO males were unable to sire offspring. However, no obvious difference was found in testicular and epididymal weights, numbers of sperm in the epididymis or the ability of sperm to fertilize eggs in vitro between wild-type and ArKO males. An examination of mating behaviour demonstrated that ArKO males showed an impairment in mounting behaviour against sexually mature females. The inability of more than 90% of ArKO males to sire offspring was reversed by repeated subcutaneous injections of 17beta-oestradiol when initiated on the day of birth. The effects of 17beta-oestradiol on reproduction were concentration dependent and evident when supplementation was initiated on day 7, but not on day 15 after birth. These findings suggest that oestrogens acting during neonatal life are required for normal mating behaviour in adulthood.
K Toda, K Takeda, T Okada, S Akira, T Saibara, T Kaname, K Yamamura, S Onishi and Y Shizuta
Aromatase P450 (CYP19) is an enzyme catalysing the conversion of androgens into oestrogens. We generated mice lacking aromatase activity (ArKO) by targeted disruption of Cyp19 and report the characteristic features of the ArKO ovaries and uteri as revealed by histological and biochemical analyses. ArKO females were totally infertile but there were as many developing follicles in their ovaries at 8 weeks of age as in wild-type ovaries. Nevertheless, no typical corpus luteum was observed in the ArKO ovaries. Electron microscopy revealed the presence of well-developed smooth endoplasmic reticulum, few lipid droplets and mitochondria with less organized tubular structures in the ArKO luteinized interstitial cells. These ultrastructural features were different from those of the wild-type interstitial cells, where there are many lipid droplets and mitochondria with well-developed tubular structures, characteristic of steroid-producing cells. When ArKO mice were supplemented with 17beta-oestradiol (E(2); 15 microg/mouse) every fourth day from 4 weeks of age for 1 month, increased numbers of follicles were observed in the ovaries as compared with those of untreated ArKO mice, although no typical corpus luteum was detectable. Ultrastructural analysis revealed the disappearance of the accumulated smooth endoplasmic reticulum in the luteinized interstitial cells after E(2 )supplementation. Transcripts of pro-apoptotic genes such as p53 and Bax genes were markedly elevated in the ArKO ovaries as compared with those of wild-type mice. Although E(2) supplementation did not cause suppression of the elevated expression of p53 and Bax mRNAs, it caused marked enhancement of expression levels of lactoferrin and progesterone receptor mRNAs in the uteri as well as increases in uterine wet weight. At 8 months of age, ArKO mice developed haemorrhages in the ovaries, in which follicles were nearly depleted, while age-matched wild-type females still had many ovarian follicles. Furthermore, macrophage-like cells were occasionally observed in the ArKO ovarian follicles. These results suggested that targeted disruption of Cyp19 caused anovulation and precocious depletion of ovarian follicles. Additionally, analysis of mice supplemented with E(2) demonstrated that E(2) apparently supports development of ovarian follicles, although it did not restore the defect in ovulation.
H Kishi, T Okada, M Otsuka, G Watanabe, K Taya and S Sasamoto
The present study was conducted to study the effect of immunoneutralization against endogenous inhibin on FSH, LH, oestradiol-17β and progesterone secretion and to investigate the effect of removal of endogenous inhibin on subsequent follicular development in the hamster. After treatment with anti-inhibin serum (inhibin-AS) at 1100 h on day 2 of the oestrous cycle (day 1=day of ovulation), a marked increase in plasma levels of FSH and a slight increase in plasma levels of LH were noted and pituitary contents of FSH, but not LH, were also increased. In the group treated with inhibin-AS, superovulation occurred on day 1 of the following cycle. Plasma levels of oestradiol-17β markedly increased with the increase in the number of ovulations induced by human chorionic gonadotrophin (hCG) as compared with those in control animals. In the second cycle, plasma concentrations and pituitary contents of FSH in the animals given 200 μl inhibin-AS still showed high values as compared with those in the animals treated with control serum, although superovulation did not occur on day 1 of the third cycle. Plasma concentrations and pituitary contents of LH in the hamster given 200 μl inhibin-AS tended to decrease as compared with those in control animals during the second cycle. Plasma concentrations of oestradiol-17β in the animals treated with 200 μl inhibin-AS changed in a similar way to controls. A marked increase in plasma concentrations of progesterone was noted on days 1 and 2 of the second cycle in the group receiving inhibin-AS. The twice daily injection of 1 IU hCG during the second cycle to the animals given 200 μl inhibin-AS induced superovulation on day 1 of the third cycle.
These results indicate that circulating inhibin may be an important indicator of the number of developing follicles and may maintain the species-specific number of developing follicles through suppression of FSH secretion in the cyclic hamster. They also suggest that high levels of inhibin slightly suppress plasma levels of LH, indicating that plasma LH may also regulate follicular development in the cyclic hamster.
Journal of Endocrinology (1996) 151, 65–75