The influence of experimental anterior knee pain during running on electromyography and articular cartilage metabolism.

OBJECTIVE: To determine whether anterior knee pain (AKP), during running, acutely affects lower-extremity electromyography (EMG) and articular cartilage metabolism. METHODS: A within-subjects design was used. Each of 12 able-bodied subjects ran on a treadmill for 30 min for three different sessions: control (no infusion), sham (0.9% NaCl infusion into the involved-leg infrapatellar fat pad), and pain (5.0% NaCl infusion into the involved-leg infrapatellar fat pad). Bilateral surface EMG was monitored for the vastus medialis (VM), vastus lateralis (VL), and gastrocnemius (GA). Serum cartilage oligomeric matrix protein (COMP) concentration was determined before, after, and 60 min after the run. A functional analysis approach was used to compare EMG amplitude, across the entire stance phase, between sessions and legs. Mixed-model analysis of covariance was used to compare serum COMP concentration between sessions, across time. RESULTS: Relative to the uninvolved leg, greater involved-leg VL and GA EMG amplitude existed during midstance for the sham and control sessions (P < 0.01). During the painful session, however, involved-leg VM, VL, and GA EMG amplitude was 5-10% less than for the uninvolved leg. COMP concentration immediately post-run was 14% and 21% greater than pre-run (P = 0.01) and 60 min post-run concentrations (P < 0.01), respectively. Session, however, did not significantly influence COMP. CONCLUSION: During a 30-min run, AKP acutely alters midstance VM, VL, and GA EMG amplitude. AKP during a 30-min run does not, however, acutely influence articular cartilage metabolism.

Identification and developmental expression of the estrogen receptor alpha and beta in the baboon fetal adrenal gland.

We have previously shown that estrogen regulates the development and function of the fetal and definitive/transitional zones of the primate fetal adrenal gland. Thus, during baboon pregnancy estrogen acts directly on the fetal zone to suppress ACTH-stimulated dehydroepiandrosterone (DHA) formation, potentially to modulate C19-steroid production and consequently placental estrogen synthesis. It is proposed that this action of estrogen is mediated by the estrogen receptor. Therefore, in the present study a developmental approach was used to determine whether the messenger RNA (mRNA) and protein for the estrogen receptor were expressed in the fetal and definitive/transitional zones ofthe baboon fetal adrenal gland at mid (day 100) and late (day 170) gestation (term = 184 days). Estrogen receptor alpha mRNA levels, determined by competitive RT-PCR, were approximately 7-fold greater (P < 0.02) in the fetal adrenal of late (187.8+/-40.3 attomoles/microg RNA) compared with mid (27.4+/-5.4 attomoles/microg RNA) gestation. Moreover, estrogen receptor alpha mRNA expression, determined by quantitative in situ hybridization, was approximately 2.5-fold greater (P < 0.05) in the definitive/transitional zones (21.6+/-0.5 silver grains/0.025 mm2) than in the fetal zone (8.3+/-1.5 grains/0.025 mm2) late in gestation. The mRNA for the beta-isoform of the estrogen receptor was also expressed in the baboon fetal adrenal cortex. There was a gradient of immunocytochemical staining for the estrogen receptor alpha and beta proteins, with extensive immunoreactivity for both isoforms in the definitive zone and lower staining in the transitional zone and the fetal zone. In summary, the results of the present study show that estrogen receptor alpha and beta were expressed in the fetal and definitive/transitional zones of the baboon fetal adrenal cortex at mid and late gestation. The presence of the estrogen receptor provides a mechanism for mediating the action of estrogen in modulating ACTH-dependent and cortical zone-specific development and function of the primate fetal adrenal gland.

Development of the baboon fetal adrenal gland: regulation of the ontogenesis of the definitive and transitional zones by adrenocorticotropin.

Throughout gestation, the primate fetal adrenal gland is comprised of the fetal zone, which expresses the P-450 17alpha-hydroxylase-C17,20 lyase (P-450c17) enzyme that catalyzes the synthesis of C19 steroids used for placental estrogen production. The development of the transitional zone comprised of cortical cells that express the P-450c17 and the 3beta-hydroxysteroid dehydrogenase-isomerase (3betaHSD) enzymes for cortisol production, and the definitive zone, which expresses 3betaHSD, but not P-450c17, for mineralocorticoid synthesis, does not occur until relatively late in gestation. Although ACTH is considered essential to fetal adrenal growth and function, the role that ACTH has in the development of the transitional and definitive zones, is less clear. To answer this question, the width of these zones was determined by immunocytochemical expression of P-450c17 and/or 3betaHSD in fetal adrenal glands obtained on day 100 (mid) of gestation (term = day 184) from baboons in which ACTH was administered to the fetus on days 95-99 of gestation or on day 165 (late) of gestation from baboons in which fetal ACTH was suppressed by treatment of the mother and fetus with betamethasone on days 150-164 of gestation. At midgestation, the fetal adrenal was comprised almost exclusively of fetal zone cells and a small definitive zone (38 +/- 2 microm in width), but was essentially devoid of a transitional zone (7 +/- 2 microm). Treatment with ACTH enhanced (P < 0.05) the width of the transitional zone (67 +/- 4 microm), but not the size of the definitive zone (10 +/- 4 microm). In late gestation, the width of the definitive zone, although 2-fold greater than that on day 100, was smaller (P < 0.05) than that of the transitional zone (120 +/- 15 microm), which greatly exceeded that at midgestation. Treatment with betamethasone in late gestation eliminated the transitional zone, but had no effect on the size of the definitive zone (120 +/- 8 microm). These findings indicate that the development of the baboon fetal adrenal transitional zone late in gestation is dependent on fetal pituitary ACTH. In contrast, the ontogenesis of the definitive zone at midgestation and its growth in late gestation occur in the relative absence of ACTH.

Functional capacity of fetal zone cells of the baboon fetal adrenal gland: a major source of alpha-inhibin.

We have shown that ACTH receptor mRNA expression and steroidogenesis were increased in the transitional zone and decreased in the fetal zone of the baboon fetal adrenal in the second half of gestation. Thus, we proposed that there is a divergence in ACTH receptor-mediated zone-specific steroidogenesis within the fetal adrenal during mid to late gestation. We have also demonstrated that fetal serum alpha-inhibin levels decline with advancing development. It is possible, therefore, that the alpha subunit of inhibin provides a good marker of fetal zone cellular function and that the changes in circulating fetal alpha-inhibin with advancing pregnancy reflect ontogenetic changes in fetal adrenal cortical zone-specific cell function. However, it remains to be determined whether the fetal adrenal is a major source of circulating alpha-inhibin in the fetus and whether alpha-inhibin is expressed in the fetal, definitive, and/or transitional zones. Therefore, the current study compared fetal serum alpha-inhibin levels with immunocytochemical localization of alpha-inhibin in baboon fetal adrenals obtained on Days 60 (early), 100 (mid), and 165 or 182 (late) of gestation (term averages Day 184) from animals untreated or treated with betamethasone, which we previously demonstrated suppressed fetal pituitary ACTH and adrenal weight. Fetal serum alpha-inhibin levels (mean +/- SE) were greater (p < 0.05) at mid (5863 +/- 730 microliter eq/ml) than at late (3246 +/- 379) gestation and were reduced (p < 0. 05) by betamethasone. The inhibin alpha subunit was expressed in abundant quantities in the fetal adrenal cortex, but not in medulla, throughout gestation. At mid and late gestation, alpha-inhibin was expressed throughout the fetal adrenal cortex but most intensely in the innermost area of fetal zone cells. By late gestation, the fetal adrenal exhibited a gradient of alpha-inhibin expression. Thus, the outermost definitive zone cells were devoid of alpha-inhibin, the transitional zone exhibited a relatively low alpha-inhibin content, and fetal zone cells continued to exhibit extensive expression of alpha-inhibin. Betamethasone diminished the intensity of alpha-inhibin expression throughout the fetal adrenal cortex. These results indicate that the fetal adrenal fetal zone is a significant source of circulating alpha-inhibin in the baboon fetus and that alpha-inhibin provides a good marker to study the developmental regulation of fetal zone-specific adrenocortical function.

Effect of maternal betamethasone administration at midgestation on baboon fetal adrenal gland development and adrenocorticotropin receptor messenger ribonucleic acid expression.

Although fetal pituitary ACTH is important to fetal adrenal growth and steroidogenesis in the second half of primate pregnancy, its role in adrenal development and function has not been established in vivo in the first half of gestation. In the present study, therefore, baboons were treated at midgestation with betamethasone to determine the effect of fetal pituitary ACTH on fetal adrenal growth, development, and ACTH receptor and P-450 enzyme messenger ribonucleic acid (mRNA) levels. The administration of betamethasone to baboon mothers on days 60-99 of gestation (term = 184 days) decreased fetal pituitary POMC mRNA levels by 54% (P < 0.01) and fetal serum ACTH levels to undetectable values (P < 0.05). The decline in ACTH was associated with decreases in fetal adrenal weight (P < 0.001), cortical cell size (P < 0.05), appearance of apoptosis and cellular disorganization, and a loss of immunocytochemically demonstrable definitive zone-specific delta5-3beta-hydroxysteroid dehydrogenase expression. The concomitant administration of ACTH and betamethasone restored these aspects of adrenal integrity to normal. Moreover, there was approximately a 95% decrease (P < 0.01) in fetal adrenal expression of ACTH receptor, P-450 cholesterol side-chain cleavage, and P-450 17alpha-hydroxylase 17/20-lyase mRNA levels after betamethasone administration. We conclude that fetal pituitary ACTH is necessary for the growth and development of fetal and definitive cortical zones and the marked coordinated increase in ACTH receptor and maintenance of P-450 cholesterol side-chain cleavage/P-450 17alpha-hydroxylase 17/20-lyase expression in the baboon fetal adrenal gland during the first half of gestation.

Inhibition of fetal adrenal adrenocorticotropin receptor messenger ribonucleic acid expression by betamethasone administration to the baboon fetus in late gestation.

Throughout the majority of intrauterine development, the primate fetal adrenal gland is comprised primarily of fetal zone cells and only late in gestation do definitive zone cells, which express the enzyme delta5-3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) emerge to produce cortisol. The present study was designed to determine whether the induction of definitive zone ACTH receptor messenger RNA (mRNA) levels and components of the steroidogenic pathway known to be expressed specifically in the definitive zone, e.g. the 3beta-HSD enzyme, are dependent upon fetal pituitary ACTH. Fetal pituitaries and adrenal glands were obtained on day 165 (term = day 184) from untreated controls (n = 7) and from baboons in which betamethasone was administered im to the fetus (0.6 mg/100 microl; n = 4) or to the fetus (0.6 mg) and mother (6 mg/ml; n = 4) every other day between days 150 and 164 of gestation. Although fetal pituitary weight was not altered by betamethasone, POMC mRNA levels determined by in situ hybridization were lower (P < 0.05) in betamethasone-treated (0.34 +/- 0.07 arbitrary densitometric units) than in untreated controls (0.63 +/- 0.04). Associated with this decline in pituitary POMC, levels of the major 3.4-kb mRNA transcript for the ACTH receptor expressed as a ratio of beta-actin were approximately 80% lower (P < 0.05) in fetal adrenals of betamethasone-treated baboons (0.12 +/- 0.02) than in untreated controls (0.84 +/- 0.05). In situ hybridization indicated that ACTH receptor mRNA expression in the definitive zone exceeded that in the fetal zone and was reduced by betamethasone. Associated with the decrease in ACTH receptor expression, fetal adrenal weight was suppressed (P < 0.05) by 50% and reflected a marked reduction (P < 0.05) in the size of the cells of the definitive and fetal zones. Betamethasone treatment also induced a decrease (P < 0.05) in the width (microm) of the definitive zone (183 +/- 14 vs. 128 +/- 7; determined by immunohistochemical expression of 3beta-HSD), as well as the levels of the mRNA and protein for 3beta-HSD. Levels of the mRNA for the LDL-receptor and the enzymes 17alpha-hydroxylase-C(17,20) lyase and P450 cholesterol side chain cleavage were also suppressed in adrenals of betamethasone-treated baboons. These findings indicate that treatment of the baboon fetus with betamethasone in late gestation suppressed fetal pituitary POMC mRNA expression and ACTH receptor mRNA levels in the fetal adrenal gland, as well as the hypertrophy and ACTH receptor mRNA and 3beta-HSD mRNA/protein levels in the cells comprising the newly emerging definitive zone. We conclude that ACTH is necessary for the up-regulation of the mRNAs for the ACTH receptor and steroidogenic enzymes in the definitive zone of the primate fetal adrenal gland in late gestation.

Developmental increase in expression of the messenger ribonucleic acid and protein levels of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in the baboon placenta.

Cortisol-cortisone interconversion is catalyzed by the NADP/NADPH-dependent oxido-reductase, 11beta-hydroxysteroid dehydrogenase-1 (11betaHSD-1) and the NAD-dependent oxidase, 11betaHSD-2. Because of the importance of placental corticosteroid metabolism in dictating the amount of cortisol arriving in the fetus to regulate fetal pituitary-adrenocortical function, the present study determined whether there was a developmental change in the expression of 11betaHSD-1 and/or -2 in placental syncytiotrophoblast, the site of maternal:fetal exchange. A syncytiotrophoblast-enriched (>95%) cell fraction was isolated from baboon placentas obtained at early (day 60), mid (day 100), and late (day 165) gestation (term = day 184), and 11betaHSD-1 and -2 messenger RNA (mRNA) and protein levels were determined by Northern and Western blots. The levels (mean +/- SE) of the single 1.6-kilobase (kb) mRNA for 11betaHSD-1, expressed as a ratio to beta-actin, increased (P < 0.05) between early (0.36 +/- 0.16; n = 4) and mid (0.95 +/- 0.21; n = 11) gestation and further increased (P < 0.05) by late gestation (1.82 +/- 0.29; n = 13). Similarly, the levels of the single 1.9-kb mRNA for 11betaHSD-2 in late gestation (2.46 +/- 0.35; n = 8) were greater (P < 0.05) than respective values at mid (1.36 +/- 0.22; n = 8) and early (0.64; n = 2) gestation. The levels of 11betaHSD-1 (arbitrary densitometric units), detected as a dominant band of 34 kDa, were greater (P < 0.05) in late gestation (2.6 +/- 0.2; n = 4) than at early (1.2 +/- 0.1; n = 4) or mid (1.9 +/- 0.3; n = 4) gestation. In contrast, 11betaHSD-2 was not detected by Western blot in syncytiotrophoblast isolated by collagenase dispersion. However, immunocytochemistry revealed that 11betaHSD-2 was present in and localized to the syncytiotrophoblast layer of the baboon placenta and that expression in late gestation (n = 4) appeared to exceed that in placentas of early (n = 4) and mid (n = 4) gestation. These results indicate that both 11betaHSD-1 and 11betaHSD-2 were expressed in syncytiotrophoblasts of the baboon placenta and that the mRNA and protein levels of these two 11betaHSD enzymes increased with advancing gestation. However, because 11betaHSD-2 was not detected in syncytiotrophoblast isolated by collagenase dispersion, we suggest that the 11betaHSD-1 and -2 reside in different membrane fractions of the syncytiotrophoblast. Consequently, the estrogen-regulated change in transplacental cortisol metabolism with advancing gestation may result in a developmental change in the expression and location of the two 11betaHSD enzymes controlling cortisol-cortisone metabolism and transfer into the fetus, resulting in activation of the fetal pituitary adrenocortical system.

Fertilization and mouse embryo development in the presence of midazolam.

Mouse embryo in vitro development elucidates the effect of a pharmacologic agent on cellular differentiation. Midazolam provides conscious sedation for patients undergoing egg retrieval for in vitro fertilization and is found in patient follicular fluid. Mouse preimplantation embryo formation and development were evaluated in the presence of midazolam. Midazolam was cocultured with two-cell mouse preimplantation embryos over 72 h and injected systemically just before ovulation and coitus. Concentrations to 12.5 micrograms/mL displayed no significant toxic effects on in vitro two-cell-to-blastocyst development. Doses to 35.0 mg/kg did not prevent or impair in vivo fertilization. Midazolam has no adverse effect on in vitro development of two-cell-to-blastocyst-stage embryos nor on in vivo fertilization and cell division at concentrations approximating and exceeding those that ova are exposed to during clinical anesthesia. Midazolam is recommended for use to induce sedation in human in vitro fertilization where association with gametes and zygotes is probable.