Biomedical subjects

M Tetsuka

Publications and source records attributed to M Tetsuka.

At least 19 recordsLinked to original sources

PubMed2006

Insulin-like growth factor-I (IGF-I) system during follicle development in the bovine ovary: relationship among IGF-I, type 1 IGF receptor (IGFR-1) and pregnancy-associated plasma protein-A (PAPP-A).

Insulin-like growth factor-I (IGF-I) system that is exerted mainly through the type 1 IGF receptor (IGFR-1) and releasing of free IGF-I is regulated by the proteases of IGF-binding proteins (IGFBPs), an important factor in follicle development of bovine ovary. The aims of the present study were to examine the mRNA expressions of IGF-I, IGFR-1 and pregnancy-associated plasma protein-A (PAPP-A) in granulosa cells and theca tissues during bovine follicular development and the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of these genes in cultured bovine granulosa cells. Follicles were classified into four groups such as small follicle (SF), estrogen inactive dominant follicle (EID), estrogen active dominant follicle (EAD) and preovulatory follicle (POF). The concentration of free IGF-I in follicular fluid of POF was significantly higher than those in EID, whereas the total IGF-I in follicular fluid did not change at all developmental stages. The expression of IGF-I mRNA was not detected in the granulosa cells at all at any developmental stages but the expression was detected in the theca tissues. The amount of IGFR-1 mRNA in granulosa cell showed the constant level at all developmental stages except EID. The expressions of IGFR-1 and PAPP-A in cultured bovine granulosa cells were stimulated with FSH but not with E2. The PAPP-A mRNA expression was stimulated by FSH in presence of 1 ng/ml E2. These results indicate that IGF-I in follicular fluid is mainly derived from the circulation and that FSH is an inducer for the expression of IGFR-1 and PAPP-A genes in granulosa cells. Therefore, we suggest that PAPP-A stimulated with FSH play a crucial role for IGF-I system in bovine follicular development.

Animals↗

PubMed2006

Gene expression of glucose transporter (GLUT) 1, 3 and 4 in bovine follicle and corpus luteum.

Glucose is the main energy substrate in the bovine ovary, and a sufficient supply of it is necessary to sustain the ovarian activity. Glucose cannot permeate the plasma membrane, and its uptake is mediated by a number of glucose transporters (GLUT). In the present study, we investigated the gene expression of GLUT1, 3 and 4 in the bovine follicle and corpus luteum (CL). Ovaries were obtained from Holstein x Japanese Black F1 heifers. Granulosa cells and theca interna layers were harvested from follicles classified into five categories by their physiologic status: follicular size (>or= 8.5 mm: dominant; < 8.5 mm: subordinate), ratio of estradiol (E(2)) to progesterone in follicular fluid (>or= 1: E(2) active;<1: E(2) inactive), and stage of estrous cycle (luteal phase, follicular phase). CL were also classified by the stage of estrous cycle. Expression levels of GLUT1, 3 and 4 mRNA were quantified by a real-time PCR. The mRNA for GLUT1 and 3 were detected in the bovine follicle and CL at comparable levels to those in classic GLUT-expressing organs such as brain and heart. Much lower but appreciable levels of GLUT4 were also detected in these tissues. The gene expression of these GLUT showed tissue- and stage-specific patterns. Despite considerable differences in physiologic conditions, similar levels of GLUT1, 3 and 4 mRNA were expressed in subordinate follicles as well as dominant E(2)-active follicles in both luteal and follicular phases, whereas a notable increase in the gene expression of these GLUT was observed in dominant E(2)-inactive follicles undergoing the atretic process. In these follicles, highly significant negative correlations were observed between the concentrations of glucose in follicular fluid and the levels of GLUT1 and 3 mRNA in granulosa cells, implying that the local glucose environment affects glucose uptake of follicles. These results indicate that GLUT1 and 3 act as major transporters of glucose while GLUT4 may play a supporting role in the bovine follicle and CL.

Animals↗

PubMed2004

Local interaction of prostaglandin F 2alpha with endothelin-1 and tumor necrosis factor-alpha on the release of progesterone and oxytocin in ovine corpora lutea in vivo: a possible implication for a luteolytic cascade.

Endothelin-1 (ET-1) and tumor necrosis factor-alpha (TNFalpha) participate in the cascade of luteolysis. Thus, in the present study the interactions of ET-1 and TNFalpha with prostaglandin F(2alpha) (PGF(2alpha)) on the release of progesterone and oxytocin (OT) within the corpus luteum (CL) were investigated. A microdialysis system (MDS) was surgically implanted in ovine CL (one MDS line/CL; 5-10 lines/ewe) formed after super-ovulation. A 4-h perfusion with PGF(2alpha) (0.01-1 micromol l (-1)) induced no clear effect on progesterone release, but acutely stimulated OT release in a dose-dependent manner. A perfusion of PGF(2alpha) (1 micromol l (-1)) increased ET-1 release over a period of 12 h. Two perfusions of ET-1 (0.1 micromol l(-1)) or a perfusion of ET-1 followed by TNFalpha (200 ng ml(-1)) decreased progesterone release (56-64% at 36-48 h). When the CL were pre-perfused with PGF(2alpha) (1 micromol l(-1)), two consecutive perfusions of ET-1 decreased progesterone release more rapidly. Similarly, a pre-perfusion with PGF(2alpha) followed by consecutive perfusions of ET-1 and then TNFalpha rapidly decreased progesterone release, with the inhibition most pronounced (35%) at 36-48 h. The simultaneous infusion of ET-1 with PGF(2alpha) induced a rapid decrease in progesterone release (36% at 36-48 h). In a further study, the possible second messenger systems involved in PGF(2alpha) action on the release of progesterone, OT and ET-1 were investigated. A perfusion with 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 10 micromol l(-1)), A23187 (10 micromol l(-1)), or PGF(2alpha) + A23187 increased progesterone release during infusion, but decreased it after perfusion. All treatments induced a massive release of OT during infusion, and increased ET-1 release after infusion. These results show that ET-1 is capable of suppressing progesterone release in the PGF(2alpha)-primed ovine CL in vivo and thus ET-1 works as a local luteolysin together with PGF(2alpha) during the process of functional luteolysis. During structural luteolysis, TNFalpha may interact with PGF(2alpha) and ET-1 to cause a rapid drop in progesterone release and accelerate the process of luteolysis. This result supports the contention that ET-1 and TNFalpha interact with PGF(2alpha) as local luteolytic mediators in the ewe as previously suggested.

Animals↗

PubMed2003

Expression of 11beta-hydroxysteroid dehydrogenases in bovine follicle and corpus luteum.

In glucocorticoid target organs, local concentrations of active glucocorticoid are determined by the relative expression of two 11beta-hydroxysteroid dehydrogenases (HSDs): bi-directional 11beta-HSD type1 (11HSD1) that mainly activates cortisone to cortisol, and dehydrogenase 11beta-HSD type2 (11HSD2) that inactivates cortisol to cortisone. In this study, we examined the expression of mRNA encoding these two 11beta-HSDs in bovine granulosa cells harvested from preovulatory follicles and corpora lutea (CL). Ovaries were obtained from Holstein cows at a local slaughterhouse. Follicles larger than 10 mm in diameter and CL were dissected and follicular fluid and granulosa cells were taken. Corpora lutea were weighed and their stages were morphologically assessed (stage I, days 1-4; stage II, days 5-10; stage III, days 11-17; stage IV, days 8-20). Follicles were classified into four groups according to their hormonal status (oestradiol (E(2)): progesterone (P(4))>1: oestrogen active; E(2):P(4)<1: oestrogen inactive) and stage of the oestrous cycle (luteal or follicular phase). Total RNA was extracted with phenol-chloroform and subjected to a semi-quantitative RT-PCR for 11HSD1, 11HSD2 and beta-actin. Concentrations of steroids in follicular fluid were determined by an enzyme immunoassay. In granulosa cells, only 11HSD1 mRNA was detected. There was a negative correlation between the expression of 11HSD1 and the concentration of cortisol in follicular fluid (P<0.05), indicating 11HSD1 may act as a dehydrogenase in the bovine follicle. Both types of 11beta-HSDs were expressed in CL. The levels of mRNA for both isozymes were high in stage I and II, and were decreased in stage III CL. In stage IV CL, the expression of 11HSD2 but not 11HSD1 mRNA increased. These results indicate that the bovine granulosa cells and CL express 11HSD1 and 11HSD2, and they may play an important physiological role in the bovine ovary through modulating the local glucocorticoid environment.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed2001

Improvement on in vitro maturation, fertilization and development of minke whale (Balaenoptera acutorostrata) oocytes.

The aims of the present study were to improve in vitro maturation, fertilization and subsequent development of minke whale oocytes. We investigated the effects of different concentrations (0, 10 and 20%) of fetal whale serum (FWS) in maturation medium on nuclear maturation, morphological grade (A or B) of cumulus-oocyte complexes (COC) obtained from prepubertal and adult minke whales. Grade A (> or = 5 layers of cumulus cells) COC collected from the adult whales and cultured in the medium with 20% FWS had a higher (P < 0.05) maturation rate (31.8%) than those in the medium without FWS (0%). Adding FWS to the maturation medium significantly (P < 0.01) improved the proportion of oocytes at Metaphase II (M-II): without FWS (7.9%), with 10% (19.4%) and 20% (21.4%) FWS. However, sexual maturity of whales and COC grades were not significantly affected by M-II oocytes. When in vitro fertilization of matured oocytes was performed in the presence of 20% FWS or 0.6% BSA in the fertilization medium, the proportions of sperm penetration and two-pronuclei formation in matured oocytes were not significantly different. Grade A COC cultured in a culture medium supplemented with 10% FWS cleaved at a higher rate (15.4%, P < 0.05) than did Grade A and B COCs cultured in the medium without FWS (0%). Neither Grade A nor B COCs cleaved when the medium was without FWS. The proportions of cleaved oocytes increased (P < 0.05) with FWS supplementation (6.9% and 8.1% for 1.0% FWS and 20% FWS, respectively). Grade A COC was significantly (P < 0.05) superior in its ability to cleave (14.5%) and develop to morula (4.2%) compared with that of the oocytes from Grade B COC (2.5% and 0%). Coculture with granulosa cells during in vitro culture did not significantly affect cleavage and development to the morula stage. These results indicate that FWS addition in the maturation medium improved the rate of in vitro maturation and cleavage after insemination of minke whale oocytes. The BSA supplementation in fertilization medium was as effective as FWS supplementation for in vitro fertilization of matured oocytes. In vitro embryo production beyond the morula stage of minke whale oocytes could be possible, if Grade A COC was selected and cultured in the maturation medium supplemented with 10% or 20% FWS.

Animals↗

PubMed2001

Plasma and pituitary concentrations of gonadotropins (FSH and LH) in minke whales (Balaenoptera acutorostrata) during the feeding season.

This study investigated plasma and pituitary concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and steroid hormones (progesterone: P4, testosterone:T, estradiol-17beta: E2) by enzyme-immunoassay (EIA) in minke whales (Balaenoptera acutorostrata) captured during the feeding season (December to March) in the Antarctic Ocean. Plasma FSH and LH levels in female minke whales were higher (P <0.05) than in male whales. Although the pituitary weight was not significantly different between male and female whales, pituitary FSH and LH levels were higher in females than in males (P<0.01) and mature whales than immature whales (P<0.05). Plasma levels of FSH, T and E2 were not significantly different between immature and mature male whales, but plasma LH and pituitary FSH and LH levels were higher (P<0.05) in mature than in immature whales. In both immature and mature whales regardless of gender, pituitary FSH and LH levels were correlated significantly (r=0.69: P<0.01). In mature male whales, plasma T and E2 levels (r=0.60: P<0.01), and testis weight and plasma T levels (r=0.46: P <0.05) were correlated. In immature female whales, plasma FSH and LH levels were highly correlated (r=0.68: P<0.001), but were not for mature female whales. The results show that gender and maturity influence gonadal and pituitary function of minke whales during the feeding season.

Age Factors↗

PubMed2001

An attempt at intracytoplasmic sperm injection of frozen-thawed minke whale (Balaenoptera bonaerensis) oocytes.

Little is known about the characteristics of fertilisation events in minke whales. Cryopreserved minke whale oocytes and spermatozoa do not fertilise in a standard IVF. This study was conducted to investigate the pronucleus formation ability of cryopreserved minke whale oocytes and their subsequent development following intracytoplasmic sperm injection (ICSI). In experiment 1, frozen-thawed minke whale immature oocytes were cultured for in vitro maturation (IVM) in a maturation medium (TCM199) supplemented with either porcine follicle stimulating hormone (pFSH)/estradiol-17beta (E2) or pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG). After 120 h of IVM, oocyte survival was examined before ICSI, and showed no significant difference in morphological normality (24-36%) between the two IVM media. Two-cell embryos (two oocytes from 21 sperm-injected oocytes) were obtained when the maturation medium was supplemented with pFSH/E2 or PMSG/hCG. In experiment 2, cryopreserved maturing oocytes were investigated for the effects of repeat-culture (2 h or 24 h) on survival before ICSI. Pronuclear formation and development were examined for the effects of sperm pretreatment with dithiothreitol (DTT) and oocyte activation with ethanol at ICSI. A frequency of 49-69% of frozen-thawed maturing oocytes was used for ICSI. Although oocyte activation did not produce a significant difference in survival, pronucleus formation and embryonic development, 2- and 4-cell cleaved oocytes were observed after injection of sperm pretreated with DTT.

Animals↗

PubMed1999

GABA(B) receptors in the nucleus tractus solitarii modulate the carotid chemoreceptor reflex in rats.

In urethane-chloralose anesthetized rats, the role of GABA(B) receptor in the commissural subnucleus of the nucleus tractus solitarii (commNTS) on the carotid chemoreceptor reflex was investigated. Microinjection of a GABA(B) agonist baclofen into the commNTS did not have any effects on arterial blood pressure (BP) or respiration (RP), while it attenuated the increases in BP and RP elicited by carotid chemoreceptor stimulation. These effects were blocked by microinjection of a GABA(B) antagonist 2-OH-saclofen into the same site. Prior microinjection of 2-OH-saclofen did not have any effects on the chemoreflex or on resting BP or RP, while the effects of baclofen on the chemoreflex were completely blocked. These results suggest that GABAB receptors are present in the carotid chemoreceptor reflex pathway in commNTS and modulate the chemoreceptor reflex.

Animals↗

PubMed1999

Expression of 11beta-hydroxysteroid dehydrogenase, glucocorticoid receptor, and mineralocorticoid receptor genes in rat ovary.

A new concept in reproductive endocrinology is that the status of the ovary as a glucocorticoid target organ alters with follicular development. Evidence for a physiological role of glucocorticoids in the regulation of ovarian folliculogenesis has been strengthened by the discovery that 11beta-hydroxysteroid dehydrogenase (11betaHSD) mRNA expression in human granulosa cells is developmentally regulated. In this study, we quantified the pattern of expression and investigated the cellular location of 11betaHSD type 1 (11betaHSD1), 11betaHSD type 2 (11betaHSD2), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) mRNAs during follicular maturation in rat ovary. Immature female rats received treatment with eCG to induce preovulatory follicular development or eCG followed by hCG to induce luteinization. 11betaHSD1, 11betaHSD2, GR, and MR mRNAs were all detectable by ribonuclease protection assay in ovarian total RNA. Treatment with eCG alone caused an approximately 8-fold increase in the ovarian level of 11betaHSD1 mRNA, which rose to approximately 30-fold after additional treatment with hCG. Equine CG alone did not measurably affect the ovarian 11betaHSD2 mRNA level, but additional treatment with hCG reduced it to 34% of the control level. Expression of GR mRNA was unchanged by any gonadotropin treatment, while MR mRNA was down-regulated. A similar pattern of 11betaHSD1, 11betaHSD2, GR, and MR mRNA expression was observed in isolated granulosa cells. These results provide direct experimental evidence that 11betaHSD genes are gonadotropically regulated in the rat ovary, including granulosa cells, and are consistent with a shift in glucocorticoid metabolism from inactivation (due to oxidation by 11betaHSD2) to activation (reduction by 11betaHSD1) during hCG-induced granulosa cell luteinization.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1999

Regulation of 11beta-hydroxysteroid dehydrogenase type 1 gene expression by LH and interleukin-1beta in cultured rat granulosa cells.

Granulosa cells from preovulatory follicles show increased expression of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) at the time of ovulation. As ovulation may be an inflammatory process, this may be a mechanism of local enhancement of the activity of anti-inflammatory glucocorticoids. In this study, we examined direct effects of LH, the proinflammatory cytokine, interleukin-1beta (IL-1beta), and pharmacological activators of protein kinase A (PKA) (forskolin and dibutyryl (db) cAMP) and PKC (LH-releasing hormone and phorbol 12-myristate 13-acetate (PMA)) signalling on the expression of 11betaHSD1 mRNA in vitro. Granulosa cells from immature female rat ovaries were cultured (pretreatment) in serum-free medium 199 containing recombinant human (rh) FSH (1 ng/ml) for 48 h to induce responsiveness to LH. Cell monolayers were then washed and cultured (test treatment) for a further 12 h in the presence of rhLH (0-100 ng/ml), IL-1beta (0-50 ng/ml), or both. Total RNA was extracted from granulosa cell monolayers and taken for quantitative ribonuclease protection analysis of 11betaHSD1 mRNA. The low level of 11betaHSD1 mRNA detectable in unstimulated (control) cultures was increased approximately twofold by the 48-h pretreatment with rhFSH. Subsequent exposure to rhLH (1-100 ng/ml) for a further 12 h dose-dependently increased 11betaHSD1 mRNA expression by an additional two- to threefold. Forskolin (10 microM), db-cAMP (2 mM), LH-releasing hormone (LHRH; 1 microM) and PMA (200 nM) were also stimulatory. IL-1beta (0.05-50 ng/ml) stimulated 11betaHSD1 mRNA expression in a dose-related manner, both in the absence and in the presence of rhLH (3 ng/ml). The interaction between IL-1beta (5 ng/ml) and rhLH (3 ng/ml) was additive. Co-treatment with a 50-fold excess of IL-1 receptor antagonist fully reversed the action of IL-1beta. We conclude that 11betaHSD1 mRNA expression in functionally mature granulosa cells is directly stimulated by gonadotrophins and IL-1beta in vitro, potentially involving post-receptor signalling via PKA- and PKC-mediated pathways. Thus both LH and IL-1beta may serve physiological roles in the upregulation of 11betaHSD1 gene expression by granulosa cells in ovulatory follicles.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1999

Regulation of 11beta-hydroxysteroid dehydrogenase isoforms and glucocorticoid receptor gene expression in the rat uterus.

Glucocorticoids are known to have diverse effects on the uterus, generally believed to be mediated by the glucocorticoid receptor (GR). To date, two isoforms of the enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD) have been identified, namely 11betaHSD1 and 11betaHSD2, which interconvert active and inactive glucocorticoids and regulate local levels of hormones available to the GR in target tissues. The aim of the present study was to examine the uterine expression of 11betaHSD and GR mRNA. The interplay of these parameters is probably an important factor in determining actions of glucocorticoids on the uterus. Using Northern analysis we investigated the uterine expression of 11betaHSD1, 11betaHSD2 and GR mRNA in relation to serum levels of sex steroid hormones and uterine progesterone receptor mRNA expression in an animal model. Immature female rats were treated with 10 IU pregnant mare serum gonadotrophin (PMSG) followed by 10 IU human chorionic gonadotrophin (hCG) 48 h afterwards, and then killed at 0, 3, 6, 9, 12 and 24 h and 5 days after the hCG injection. Expression of both 11betaHSD1 and 11betaHSD2 mRNA in total uterine RNA was found to be up-regulated by more than 50% at 48 h after PMSG injection when oestradiol levels were also high. Following hCG treatment the expression of 11betaHSD1 and 11betaHSD2 further increased to reach maximal levels at 24 and 12 h respectively. GR mRNA expression was down-regulated by more than 50% by PMSG but gradually recovered after hCG injection. The results show that mRNA expression of 11betaHSD1, 11betaHSD2 and GR in the uterus is developmentally regulated, suggesting that these key determinants of glucocorticoid action may play an important role in uterine function.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1998

Expression of oestrogen receptor isoforms in relation to enzymes of oestrogen synthesis in rat ovary.

Oestrogen acts as a local regulator of follicular development, mediated by specific nuclear oestrogen receptors (ER). The aim of this study was to examine the gene expression of two ER isoforms, ERalpha and beta, in relation to oestrogenic enzymes, P450aromatase (P450arom) and 17betaHSD type1 (17betaHSD1) during follicular maturation and luteinization. Ovaries were obtained from immature rats treated with PMSG (10 iu for 48 h) followed by hCG (10 iu). Expression of ERalpha and beta was down-regulated by the treatment with PMSG. Following the hCG injection, further down-regulation of both ERs occurred. Conversely, expression of P450arom and 17betaHSD1 was initially up-regulated by PMSG and then rapidly down-regulated following injection of hCG. In isolated granulosa cell, ERbeta was the predominant ER while ERalpha was mainly expressed in residual ovary and corpora lutea (CL). These results indicate that ERalpha and ERbeta mRNAs are expressed in different cell types and are both down-regulated during gonadotrophin stimulated follicular maturation and luteinization in the rat ovary. Down-regulation of ER is accompanied by down-regulation of oestrogenic enzymes during luteinization. Thus both oestrogen production and reception are shut down as ovulation approaches.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1998

An anti-inflammatory role for glucocorticoids in the ovaries?

Roles of glucocorticoids in the direct regulation of ovarian function are poorly understood. This opinion paper highlights: (1) the inflammatory nature of the ovulatory process; (2) the contributions of cytokines and prostaglandins (hence inflammation) to ovulation; (3) the development-related pattern of 11beta-hydroxysteroid (11beta HSD) isoform expression (hence glucocorticoid metabolism) that occurs in ovarian follicles; and (4) the attribution of general anti-inflammatory properties of glucocorticoids to their interference with prostaglandin synthesis. We interpret the evidence cited to hypothesise that corticosteroids serve an anti-inflammatory role during ovulation, thereby promoting rapid healing of the wound left by follicular rupture. Other possible levels of glucocorticoid action in the ovaries are also considered.

Animals↗

PubMed1998

Corticosteroid metabolism in human granulosa-lutein cells.

OBJECTIVE: The aims of this study were to determine the type and level of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) in human granulosa-leutein cells (GLE) shortly before ovulation and to correlate activity with the outcome of treatment in patients undergoing in vitro fertilization and embryo transfer (IVF/ET). DESIGN: GLC from 32 patients undergoing IVF/ET were tested for type and level of 11 beta HSD activity in relation to treatment outcome. PATIENTS: Periovulatory follicles were aspirated by ultrasound guided transvaginal puncture following a standard controlled ovarian stimulation protocol, approximately 36 h after administering an ovulation-inducing dose of human chorionic gonadotrophin (HCG). GLC were separated from follicular fluid by density-gradient centrifugation and taken for measurement of 11 beta HSD activity in vitro; oocytes were used for IVF/ET. MEASUREMENTS: Interconversion of cortisol (F) and cortisone (E), and dexamethasone (D) and 11-dehydrodexamethasone (DHD) was measured in standardized assays comprising incubation of GLC with 3H-labelled substrate, with separation of substrate and product by thin-layer radiochromatography. RESULTS: Conversion of F to E varied from 10.5 to 30.9% while that of E to F was between 2.4 and 44.6%. In the GLC of 25 patients in whom both activities were measured, dehydrogenase (F to E) activity predominated in 13 and reductase (E to F) in 12. By contrast, D (substrate for 11 beta HSD2 but not 11 beta HSD1) showed less than 1% metabolism in this system while DHD (substrate for 11 beta HSD1 and 11 beta HSD2) was converted significantly (65.6-90.5%) to D in the four patients tested. There was no significant difference in the interconversion of F and E between patients who became pregnant and those who did not. CONCLUSIONS: The dehydrogenase and oxoreductase reactions catalysed by 11 beta HSD both occur in granulosa-lutein cells at the time of follicular rupture, probably due to 11 beta HSD1. A lack of measurable conversion of dexamethasone to 11-dehydrodexamethasone suggests that dehydrogenation due to 11 beta HSD2 is low or absent. Neither type nor level of 11 beta HSD activity measured under the present assay conditions correlates with IVF outcome.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1998

Expression of oestrogen receptor alpha and beta in cultured human ovarian surface epithelial cells.

Ovarian surface epithelial (OSE) cells participate in the formation of the ovarian cortex and are potential targets of oestrogen action. Oestrogens typically act through nuclear oestrogen receptors (ER) of which there are two known subtypes: ERalpha and ERbeta. In view of the potential importance of oestrogen as a local regulator of OSE cell function, we screened for ERalpha and ERbeta mRNA in primary OSE cell cultures by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, and used freshly isolated granulosa cells (GC) and granulosa-lutein cells (GLC) as positive controls. OSE cells, scraped from the ovarian surface of women undergoing laparotomy for benign gynaecological conditions, were cultured for up to 21 days to obtain enough cells for mRNA extraction. GC were obtained from spontaneously cyclic women undergoing total hysterectomy; while GLC were obtained from follicular aspirates of gonadotrophin-stimulated in-vitro fertilization patients. Total RNA (1 microg) was reverse transcribed into single-stranded cDNA for PCR (30 cycles) using primers selected to give specific ERalpha and ERbeta products. The ERalpha and ERbeta PCR products, authenticated by cloning and sequencing, were both weakly detectable by Southern analysis in cultured OSE cells and readily detectable in GC and GLC. These results show that cultured human OSE express both ERalpha and ERbeta mRNA, consistent with a role for oestrogen in the regulation of OSE cell function in vivo.

Adult↗

PubMed1997

Decrease in cerebral free magnesium concentration following closed head injury and effects of VA-045 in rats.

1. We examined the alterations in cerebral free Mg2+ concentration in closed head injury (CHI) in rats and the effects of VA-045, a novel apovincaminic acid derivative, on them with in vivo 31P-NMR. 2. Free Mg2+ decreased by about 30% within 20 min after head impact and, afterward, it gradually decreased further to reach about 60% of the control level after 3 hr. VA-045 inhibited the decrease. 3. In nonimpacted rats, VA-045 did not alter the free Mg2+ level. 4. The decrease in cerebral free Mg2+ following CHI may be a critical factor in the development of irreversible tissue injury, and VA-045 may prevent it.

Animals↗

PubMed1997

Role of androgens in follicle maturation and atresia.

Androgens are products of progestogen metabolism, intermediates in oestrogen biosynthesis and local regulators of ovarian function. Current understanding of intraovarian androgen formation, metabolism and action is reviewed, highlighting the contribution of androgens to the paracrine regulation of follicular maturation and atresia. Any factor that alters intracellular cAMP levels is a potential modulator of granulosa cell differentiation, and hence follicular development. Androgen appears to modulate gonadotrophin action on granulosa cells through amplification of cAMP-mediated post-receptor signalling. Here it is argued that during intermediate stages of follicular development, locally produced androgen acts via granulosa cell androgen receptors (AR) to promote follicle-stimulating hormone (FSH)-induced granulosa cell differentiation through amplifying cAMP-mediated post-receptor signalling. During late pre-ovulatory follicular development, higher concentrations of cAMP caused by stimulation with luteinizing hormone (LH) suppress granulosa cell proliferation and down-regulate some of the genes induced by FSH at earlier stages of pre-ovulatory development, including aromatase activity. Other granulosa cell functions, including progesterone synthesis, are enhanced by the high concentrations of cAMP induced by LH. There is experimental evidence from studies of rat and non-human primate (common marmoset) ovaries that AR levels in granulosa cells decline during pre-ovulatory follicular maturation. Since androgens augment FSH-induced cAMP formation and action, loss of AR could be a means of avoiding inappropriately high cAMP levels and hence avoiding premature activation of 'high-tone' cAMP-response genes that lead to atresia. Negative regulation of the granulosa cell AR could be part of the intra-ovarian mechanism that determines which follicle(s) becomes dominant and secretes oestrogen in the normal menstrual cycle.

Androgens↗

PubMed1997

Location and developmental regulation of androgen receptor in primate ovary.

Locally produced androgens act via granulosa cell androgen receptors to modulate follicular responsiveness to gonadotrophins and thereby contribute to the paracrine regulation of ovarian function. We used quantitative androgen receptor immunocytochemistry to assess androgen receptor distribution in relation to pre-ovulatory follicular development in the common marmoset (Callithrix jacchus), a New World primate that ovulates two to four follicles in each approximately 28 day ovarian cycle. Ovaries from four adult females in the late follicular phase and from four in the luteal phase were fixed in 4% paraformaldehyde and subjected to an immunocytochemical analysis using a polyclonal androgen receptor antibody with detection by a standard avidin-biotin-peroxidase technique for alkaline phosphatase. Specific androgen receptor immunostaining occurred mainly in granulosa cell nuclei, with little or no specific staining in theca, stroma or oocytes. Granulosa cell androgen receptor immunostaining was most abundant in healthy preantral/early antral follicles, being low or absent from pre-ovulatory follicles and corpora lutea. Differences in granulosa cell androgen receptor immunostaining between immature (0.1-1.0 mm diameter) and pre-ovulatory (> or = 2.0 mm diameter) follicles were quantified using a videodensitometric analysis of grey-scale values. Readings were taken from the granulosa cell layers of 53 immature follicles and 10 pre-ovulatory follicles in late follicular phase ovaries. The average androgen receptor level in granulosa cells of immature follicles proved to be 4.2-fold higher (P < 0.01) than that in granulosa cells of pre-ovulatory follicles. Because other evidence suggests that paracrine androgen action in granulosa cells converts from stimulation to inhibition as follicles mature, we speculate that a development-related reduction in androgen receptor numbers serves to "protect' granulosa cells against the inhibitory action of androgen, thereby promoting pre-ovulatory follicular dominance in primate ovarian cycles.

Animals↗