Parturition in baboons (PAPIO SPP.).

The Old World non-human primates (NHP) - baboons (Papio spp.) share similarities with humans regarding fetal and placental development and some pregnancy-related complications. Information about the mechanism of birth and complications arising during parturition in these species is relatively sparse. In this manuscript, we add information from a series of pathological and observational cases to highlight insights and selected complications of birth in Papio spp, based on video-recording of the delivery process, X-ray, MRI, and ultrasound evaluations in pregnant baboons. Additionally, we abstracted pathology records obtained from perinatal loss in a large baboon colony during a 17 year period. The presented cases provide important information for the management of pregnancy and delivery in Papio spp.

Effect of vaginal distention on elastic fiber synthesis and matrix degradation in the vaginal wall: potential role in the pathogenesis of pelvic organ prolapse.

Matrix metalloprotease (MMP) activity is increased in the postpartum vagina of wild-type (WT) animals. This degradative activity is also accompanied by a burst in elastic fiber synthesis and assembly. The mechanisms that precipitate these changes are unclear. The goals of this study were to determine how vaginal distention (such as in parturition) affects elastic fiber homeostasis in the vaginal wall and the potential significance of these changes in the pathogenesis of pelvic organ prolapse. Vaginal distention with a balloon simulating parturition resulted in increased MMP-2 and MMP-9 activity in the vaginal wall of nonpregnant and pregnant animals. This was accompanied by visible fragmented and disrupted elastic fibers in the vaginal wall. In nonpregnant animals, the abundant amounts of tropoelastin and fibulin-5 in the vagina were not increased further by distention. In contrast, in pregnant animals, the suppressed levels of both proteins were increased 3-fold after vaginal distention. Distention performed in fibulin-5-deficient (Fbln5(-/-)) mice with defective elastic fiber synthesis and assembly induced accelerated pelvic organ prolapse, which never recovered. We conclude that, in pregnant mice, vaginal distention results in increased protease activity in the vaginal wall but also increased synthesis of proteins important for elastic fiber assembly. Distention may thereby contribute to the burst of elastic fiber synthesis in the postpartum vagina. The finding that distention results in accelerated pelvic organ prolapse in Fbln5(-/-) animals, but not in WT, indicates that elastic fiber synthesis is crucial for recovery of the vaginal wall from distention-induced increases in vaginal protease activity.

Effect of periurethral denervation on function of the female urethra.

OBJECTIVE: The purpose of this study was to determine the effect of periurethral denervation on contractile function of the female rat urethra. STUDY DESIGN: Periurethral nerve transection or sham operation was performed in 16 young female rats. After 2 weeks, contractile function of the external urethra sphincter (EUS) and longitudinal smooth muscle was determined. Inhibitors of nitric oxide (NO) signaling were used to evaluate the role of nitric oxide in nerve-mediated relaxation. Statistical comparisons were conducted by Student t test. RESULTS: Periurethral nerve transection resulted in gross evidence of urinary retention and bladder distention. In normal and sham-operated rats, nerve-mediated relaxation of urethral smooth muscle was inhibited by L-nitroarginine and oxadiazolo quinoxalin-1-one (ODQ), and this relaxation response was impaired significantly after periurethral nerve injury. Relaxation responses to the NO donor sodium nitroprusside remained intact. Contractile function of the EUS was not altered by periurethral nerve injury. CONCLUSION: Neurons surrounding the urethra contain NO and innervate smooth muscle of the inner urethra. Periurethral denervation results in impaired urethral smooth muscle relaxation with no appreciable effect on contractility of the external striated sphincter.

Smooth muscle myosin heavy chain and caldesmon expression in the anterior vaginal wall of women with and without pelvic organ prolapse.

OBJECTIVE: The aim of this study was to quantify the expression of smooth muscle myosin heavy chain (SM-MHC) and caldesmon in the anterior vaginal wall of women with and without pelvic organ prolapse. STUDY DESIGN: Immunoblot analysis was conducted on protein extracts from the vaginal muscularis of 15 women with and 11 women without pelvic organ prolapse by using specific antibodies for SM-MHC, nonmuscle MHC-B, and caldesmon. The fraction of muscularis containing smooth muscle was determined by morphometric analysis of histologic cross sections. Reverse transcriptase-polymerase chain reaction was used to amplify SM-MHC isoforms produced by alternative splicing in the myosin head. RESULTS: Whereas the expression of SM-MHC was increased modestly (2-fold), expression of smooth muscle caldesmon was increased 6- to 7-fold in vaginal muscularis from women with prolapse. The relative distribution of SM-MHC isoforms was similar in both groups. CONCLUSIONS: Caldesmon is increased substantially in vaginal smooth muscle of women with pelvic organ prolapse. Caldesmon inhibits actin-activated myosin magnesium adenosine triphosphatase activity and inhibits the maintenance of contractile force. Thus, this disproportionate increase in caldesmon, relative to myosin, may result in inhibition of vaginal smooth muscle contractility and force maintenance.

Regulation of uterine 5 alpha-reductase type 1 in mice.

A null mutation in the murine gene encoding steroid 5 alpha-reductase type 1 (5 alpha R1) leads to failure of normal parturition at term. This observation, together with the finding that mRNA levels of uterine 5 alpha R1 increase significantly at term in normal pregnant animals, indicates that 5 alpha R1 plays an important role in murine parturition. The current studies were conducted to elucidate the regulation of 5 alpha R1 in uterine tissues of nonpregnant and pregnant animals. Nonpregnant, ovariectomized ICR mice were treated with vehicle (control), 17 beta-estradiol (E(2)), progesterone (P(4) ), or E(2)+P(4) for 3 days. Thereafter, uterine tissues were obtained for histology, quantification of 5 alpha R1 specific activity, and Northern blot analysis of 5 alpha R1 mRNA expression. The 5 alpha R1 enzyme activity was significantly increased in animals treated with E(2)+P(4). However, activity was much less in uterine tissues from E(2)+P(4)-treated animals than in uterine tissues from pregnant animals near term. To evaluate further the regulation of 5 alpha R1 during gestation, mice underwent unilateral tubal ligation before timed matings. The 5 alpha R1 activity increased eightfold in uterine tissues from the fetal horn from Gestational Days 12 to 18. This temporal pattern in 5 alpha R1 activity paralleled marked increases in uterine diameter. Taken together, these studies indicate that expression of 5 alpha R1 is regulated by E(2)+P(4) in uterine tissues. Whereas E(2) alone is insufficient to induce enzyme activity, E(2) may be required to increase P(4) receptors and, thereby, mediate the effects of P(4) on 5 alpha R1 gene expression. Further increases in enzyme activity during late gestation are mediated by fetal occupancy, possibly through stretch-induced increases in endometrial growth. Thus, like other genes involved in parturition, expression of 5 alpha R1 is regulated by both hormonal and fetal-derived signaling pathways.

Reorganization of myofilament proteins and decreased cGMP-dependent protein kinase in the human uterus during pregnancy.

Excessive or premature contractions of uterine smooth muscle may contribute to preterm labor. Contractile stimuli induce myosin and actin filament interactions through calcium-dependent myosin phosphorylation. The mechanisms that maintain myometrial quiescence until term are not well established, but may include control of calcium levels by nitric oxide and cGMP signaling and thin filament (caldesmon and calponin) regulation. Previously, we reported that myometrial tissues from pregnant rats are not responsive to cGMP due to decreases in cGMP-dependent protein kinase. Considering the well documented differences in the endocrinology of parturition among species, this study was conducted to test the hypothesis that the levels and subcellular distribution of caldesmon, calponin, and cGMP-dependent protein kinase are regulated with the hormonal milieu of human pregnancy. Whereas cGMP-dependent protein kinase was significantly reduced in the human uterus during pregnancy, caldesmon expression was significantly increased, and both caldesmon and calponin were redistributed to a readily extractable subcellular pool. These data suggest that cGMP-dependent protein kinase does not mediate gestational quiescence. Redistribution of thin filament-associated proteins, however, may alter uterine smooth muscle tone or the cytoskeletal framework of myocytes to maintain gestation despite the substantial distention that accompanies all intrauterine pregnancies.

Interleukin-1beta induces expression of neuropeptide Y in primary astrocyte cultures in a cytokine-specific manner: induction in human but not rat astrocytes.

Previous studies have demonstrated that astrocyte cultures express neuropeptide Y (NPY) in a regulated manner, namely, phorbol ester leads to an increase in proNPY-mRNA and NPY production. In this respect, the behavior of astrocytes derived from the human fetal or rat neonatal brain is similar (Regul. Pept. 75 (1998) 293). Since astrocytes can be exposed to high levels of IL-1beta, we addressed the question: Does IL-1beta regulate NPY expression by the astrocytes? Primary astrocytes derived from the human fetal or rat neonatal cortex were cultured in serum-free medium. IL-1beta, but not IL-6 or TNF-alpha, led to an increase in NPY production dose-dependently. IL-1beta action manifested in the human but not in the rat astrocytes and it was completely abolished by IL-1 receptor antagonist. The responsiveness to IL-1beta did not diminish upon sub-culture of the astrocytes (five passages). In addition, IL-1beta led to an increase in the abundance of proNPY-mRNA, which was preceded by a rapid and transient increase in cFos-mRNA and a rapid and sustained increase in JunB-mRNA. In contrast to cFos/JunB, IL-1beta did not alter the abundance of cJun-mRNA. In summary, we demonstrate that IL-1beta induction of NPY expression in astrocytes is species- and cytokine-specific and that IL-1 receptor is involved. Moreover, induction of NPY expression is preceded by a rapid increase in the expression of two transcription factors (cFos, JunB) that have been previously (Oncogene 9 (1994) 2369; J. Neurochem. 70 (1998) 1887) implicated in transcriptional regulation of the human NPY gene.

Regulation of cyclic guanosine monophosphate-dependent protein kinase in human uterine tissues during the menstrual cycle.

Contractility of uterine smooth muscle is essential for the cyclic shedding of the endometrial lining and also for expulsion of the fetus during parturition. The nitric oxide (NO)-cGMP signaling pathway is involved in smooth muscle relaxation. The downstream target of this pathway essential for decreasing cytoplasmic calcium and muscle tone is the cGMP-dependent protein kinase (PKG). The present study was undertaken to localize expression of PKG in tissues of the female reproductive tract and to test the hypothesis that uterine smooth muscle PKG levels vary with the human menstrual cycle. Immunohistochemistry was used to localize PKG in myometrium, cervix, and endometrium obtained during proliferative and secretory phases. The PKG was localized to uterine and vascular smooth muscle cells in myometrium, stromal cells in endometrium, and a small percentage of cervical stromal cells. Using Western blot analysis and protein kinase activity assays, the expression of PKG was reduced significantly in progesterone-dominated uteri compared with myometrium from postmenopausal women or women in the proliferative phase. These findings support a role for PKG in the control of uterine and vascular smooth muscle contractility during the menstrual cycle.

The parturition defect in steroid 5alpha-reductase type 1 knockout mice is due to impaired cervical ripening.

Successful delivery of the fetus (parturition) depends on coordinate interactions between the uterus and cervix. A majority (70%) of mice deficient in the type 1 isozyme of steroid 5alpha-reductase fail to deliver their young at term and thus manifest a parturition defect. Using in vitro and in vivo measurements we show here that rhythmic contractions of the uterus occur normally in these mutant mice at the end of gestation. In contrast, the cervix of the mutant animal fails to ripen at term as judged by biomechanical, histological, and endocrinological assays. Impaired metabolism of progesterone in the cervix of the mutant mice in late gestation leads to an accumulation of this steroid in the tissue. We conclude that a failure of cervical ripening underlies the parturition defect in mice lacking steroid 5alpha-reductase type 1 and that this enzyme normally plays an essential role in cervical progesterone catabolism at the end of pregnancy.

Effect of progesterone on intracellular Ca2+ homeostasis in human myometrial smooth muscle cells.

Although it is well known that progesterone alters uterine contractility and plays an important role in maintenance of pregnancy, the biochemical mechanisms by which progesterone alters uterine contractility in human gestation are less clear. In this investigation we sought to identify progesterone-induced adaptations in human myometrial smooth muscle cells that may alter Ca2+ signaling in response to contractile agents. Cells were treated with vehicle or the progesterone analog medroxyprogesterone acetate (MPA) for 5 days, and intracellular free Ca2+ concentration ([Ca2+]i) was quantified after treatment with oxytocin (OX) or endothelin (ET)-1. OX- and ET-1-induced increases in [Ca2+]i were significantly attenuated in cells pretreated with MPA in a dose-dependent manner. Progesterone receptor antagonists prevented the attenuated Ca2+ transients induced by MPA. ETA and ETB receptor subtypes were expressed in myometrial cells, and treatment with MPA resulted in significant downregulation of ETA and ETB receptor binding. MPA did not alter ionomycin-stimulated increases in [Ca2+]i and had no effect on inositol trisphosphate-dependent or -independent release of Ca2+ from internal Ca2+ stores. We conclude that adaptations of Ca2+ homeostasis in myometrial cells during pregnancy may include progesterone-induced modification of receptor-mediated increases in [Ca2+]i.

A 310-bp minimal promoter mediates smooth muscle cell-specific expression of telokin.

A cell-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene directs transcription of telokin exclusively in smooth muscle cells. Transgenic mice were generated in which a 310-bp rabbit telokin promoter fragment, extending from -163 to +147, was used to drive expression of simian virus 40 large T antigen. Smooth muscle-specific expression of the T-antigen transgene paralleled that of the endogenous telokin gene in all smooth muscle tissues except uterus. The 310-bp promoter fragment resulted in very low levels of transgene expression in uterus; in contrast, a transgene driven by a 2.4-kb fragment (-2250 to +147) resulted in high levels of transgene expression in uterine smooth muscle. Telokin expression levels correlate with the estrogen status of human myometrial tissues, suggesting that deletion of an estrogen response element (ERE) may account for the low levels of transgene expression driven by the 310-bp rabbit telokin promoter in uterine smooth muscle. Experiments in A10 smooth muscle cells directly showed that reporter gene expression driven by the 2.4-kb, but not 310-bp, promoter fragment could be stimulated two- to threefold by estrogen. This stimulation was mediated through an ERE located between -1447 and -1474. Addition of the ERE to the 310-bp fragment restored estrogen responsiveness in A10 cells. These data demonstrate that in addition to a minimal 310-bp proximal promoter at least one distal cis-acting regulatory element is required for telokin expression in uterine smooth muscle. The distal element may include an ERE between -1447 and -1474.

Regulation of cGMP-induced relaxation and cGMP-dependent protein kinase in rat myometrium during pregnancy.

Increases in guanosine 3',5'-cyclic monophosphate (cGMP) induced by nitric oxide (NO), nitrovasodilators, and atrial peptides correlate with relaxation of vascular smooth muscle. Relaxation of myometrial smooth muscle by increases in cGMP, however, has required unusually high concentrations of the cyclic nucleotide. We tested the hypothesis that the sensitivity of myometrium to relaxation by cGMP is increased during pregnancy. Aortic smooth muscle was more sensitive to relaxation by cGMP than myometrial tissues, and, contrary to our hypothesis, myometrium from pregnant rats was least sensitive. Although levels of cGMP were elevated after treatment with the NO donor, S-nitroso-N-acetylpenicillamine, relaxation of myometrial tissues obtained from pregnant rats occurred only at extraordinarily high concentrations. The levels of cGMP-dependent protein kinase (PKG) were significantly decreased in myometrium from pregnant rats compared with myometrium from nonpregnant cycling animals or aortic smooth muscle. Administration of estradiol to ovariectomized rats increased myometrial PKG expression, and progesterone antagonized this response. We conclude that 1) myometrial tissues from pregnant rats are not sensitive to relaxation by cGMP and 2) this insensitivity to cGMP is accompanied by progesterone-mediated decreases in the level of PKG expression.

Human ovarian expression of 17 beta-hydroxysteroid dehydrogenase types 1, 2, and 3.

Three isozymes of 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) have been cloned and characterized as distinct gene products (17 beta HSD1, 17 beta HSD2, and 17 beta HSD3). The presence and location of these isozymes in the human ovary have not been defined. In this study, we utilized Northern analysis and RT-PCR to examine transcripts for the three isozymes of 17 beta HSD. RNA was isolated from ovarian cortex, stroma (pre- and postmenopausal), hilum, follicles, and corpora lutea obtained from adult women, as well as whole fetal ovaries. By Northern analysis, high levels of 17 beta HSD1 messenger RNA were found in follicles, corpora lutea, and cortex, whereas low levels were detected in the postmenopausal stroma and in fetal ovaries by RT-PCR. 17 beta HSD1 messenger RNA was not detected in hilar tissue by either Northern analysis or RT-PCR. Utilizing RT-PCR, transcripts for 17 beta HSD2 were not detectable in cortex, stroma, (pre-or postmenopausal), hilum, or follicles, but were present in RNA derived from the corpora lutea and fetal ovary. The androgenic isozyme 17 beta HSD3 was not detectable in any of the ovarian compartments examined by either Northern analysis or RT-PCR. These data provide additional insight into the mechanism of testosterone and estradiol synthesis within the ovary. Specifically, the high level of 17 beta HSD1 is clearly localized to follicles and corpora lutea indicating involvement in the synthesis of estradiol. Secondly, androgenic 17 beta HSD3 is not expressed in the human ovary. Thus testosterone production within the human ovary, occurring under physiological conditions, arises from either the 17 beta HSD1 or an uncharacterized isozyme.

Human ovarian tumor cells: a potential model for thecal cell steroidogenesis.

Ovarian thecal cell production of C19 steroids (i.e. dehydroepiandrosterone, androstenedione, and testosterone) is necessary to provide substrate for granulosa cell biosynthesis of estrogen; however, excessive production of C19 steroids can lead to disorders associated with androgen excess. Because of difficulties in obtaining adequate numbers of thecal cells, the biomolecular regulation of C19 steroid production and expression of steroidogenic enzymes is not well defined. We have overcome this obstacle by developing a highly dependable and unique human ovarian thecal-like tumor (HOTT) cell culture model system from an ovarian tumor found to produce excessive amounts of C19 steroids. Aliquots of freshly dispersed tumor cells were frozen for future use. Once placed in monolayer culture, HOTT cells proliferated and could be maintained for extended periods. Acutely, cultured HOTT cells increased progesterone and cAMP production in response to 2 h of forskolin treatment. These cells were, however, unresponsive to treatment with LH. Steroid hormone production continued in cells that were maintained in culture for up to 2 months. Analysis of the steroids produced by HOTT cells was accomplished using RIA and high performance liquid chromatography. Under basal conditions, HOTT cells produced mainly 17 alpha-hydroxyprogesterone and progesterone. Treatment with forskolin or dibutyryl cAMP (dbcAMP) increased the production of progesterone and 17 alpha-hydroxyprogesterone as well as C19 steroids. Treatment of monolayer cultures of HOTT cells with forskolin (0.01 to 20 mumol/L) or dbcAMP (0.01 to 1 mmol/L) for 48 h increased the production of androstenedione (8- to 15-fold) and progesterone (2- to 5-fold). In HOTT cells chronically treated with forskolin or dbcAMP (up to 72 h), progesterone production was observed to plateau, although the amount of androstenedione continued to increase. The enzymatic activities of both 3 beta-hydroxysteroid dehydrogenase (6-fold), and 17 alpha-hydroxylase P450 (P450c17; 9-fold) were also increased by activation of the protein kinase A messenger pathway. Treatment of HOTT cells with forskolin caused a time-dependent induction of the messenger RNAs for cholesterol side-chain cleavage P450, 3 beta-hydroxysteroid dehydrogenase, and P450c17. No changes in steroidogenic enzyme expression were observed following treatment with LH. In conclusion, these data demonstrate that certain ovarian tumor cells may serve well as appropriate models to study the molecular mechanisms regulating human ovarian thecal cell C19 steroidogenesis and the expression of steroid-metabolizing enzymes.

Angiotensin II receptor characteristics and subtype expression in uterine arteries and myometrium during pregnancy.

The uteroplacental vasculature is less sensitive to angiotensin II (ANG II)-induced vasoconstriction than the systemic vasculature. Although the mechanism(s) responsible is unclear, uterine arteries (Ua) may demonstrate ANG II receptor (AT receptors) down-regulation or expression of AT2 receptors, which do not mediate vasoconstriction. We determined AT receptor binding characteristics and subtype expression in Ua from normotensive pregnant (n = 14; 38 +/- 0.5 weeks gestation) and nonpregnant (n = 28) women. Comparative studies were performed with myometrium, a nonvascular smooth muscle. We measured binding density (Bmax) and affinity (Kd) in plasma membrane preparations employing radioligand binding. Receptor subtypes were assessed by inhibiting [125I]ANG II binding with specific antagonists. During pregnancy, the Ua Bmax and Kd were unchanged (P > 0.1; 221 +/- 36 vs. 159 +/- 27 fmol/mg protein and 0.8 +/- 0.1 vs. 0.9 +/- 0.1 nmol/L, respectively). However, myometrial Bmax decreased 92% (580 +/- 129 vs. 44 +/- 7 fmol/mg protein; P < 0.001), and Kd rose 4-fold (1.5 +/- 0.4 to 6.0 +/- 0.6 nmol/L; P < 0.001). AT1/AT2 expression averaged 15%/85% in Ua from nonpregnant and pregnant women, whereas in myometrium, values were 10%/90% and 60%/40%, respectively. In myometrium from laboring women (n = 8), force (1.38 +/- 0.14 to 1.59 +/- 0.12 x 10(4) N/m2; P < 0.04) and contractile frequency (0.038 +/- 0.05 to 0.116 +/- 0.014 contractions/min; P < 0.001) increased with 10(-5) mol/L ANG II and were abolished by AT1 receptor inhibition. Myometrium from nonpregnant women (n = 3) was unresponsive, and AT2 inhibition did not alter responses. In nonpregnant women, AT2 receptors predominate in Ua and myometrium. Although Ua AT receptors are unaltered during pregnancy, myometrial Bmax decreases, reflecting decreases in the expression of AT2 >> AT1 receptors and differential receptor regulation.

Potassium negatively regulates angiotensin II type 1 receptor expression in human adrenocortical H295R cells.

We have previously shown that the human adrenocortical H295R cell line expresses the type 1 angiotensin II receptor (AT1-R) and that expression of this receptor is downregulated at the level of mRNA by forskolin or dibutyryl-cAMP as well as by angiotensin II (Ang II). In this study we examine the effects of K+ on both AT1-R mRNA and receptors, as monitored through 125I-Ang II binding in the presence of PD 123319. After treatment with a maximal stimulatory steroidogenic dose of K+ (14 mmol/L), H295R cells showed an increase in cytosolic free Ca2+ from 113 to 212 nmol/L. Unlike the effects of Ang II, this increase could be abolished by pretreatment with the Ca2+ channel antagonist nifedipine (1 mumol/L). AT1-R mRNA levels also fell in response to elevated extracellular K+ in a dose-dependent (Kd, 9 mmol/L; maximal fall in message at 12 mmol/L) and time-dependent (maximum 50% at 12 hours) manner. The change in AT1-R mRNA level was less rapid than that in response to activation of phosphoinositidase C by Ang II or adenylyl cyclase by forskolin or by dibutyryl-cAMP. Unlike the action of Ang II but similar to the action of forskolin or dibutyryl-cAMP, the action of K+ was sustained. Changes in mRNA level in response to treatment with K+, Ang II, or dibutyryl-cAMP were also paralleled by changes in 125I-Ang II binding in each case.(ABSTRACT TRUNCATED AT 250 WORDS)

Myosin phosphorylation and the control of myometrial contraction/relaxation.

In summary, phosphorylation of the regulatory light chain of myosin by Ca2+/CaM-dependent MLCK plays an important role in smooth muscle contraction. Although there have been major advances in our understanding of the regulation and physiological functions of contractile proteins in smooth muscle in recent years, very little information exists on the functional status of these proteins in human myometrium during pregnancy. The simple view that contractile force in smooth muscle is proportionate to cytoplasmic Ca2+ concentrations (Ca2+i) and myosin light chain phosphorylation is now more complex as more experiments provide insights into mechanisms of regulation of the contractile elements. MLCK can be phosphorylated, which desensitizes its activation by Ca2+/CaM, and protein phosphatase activity toward myosin may also be regulated. Examples in smooth muscle tissue are sparse, and the different mechanisms by which these processes may be adapted in uterine smooth muscle during pregnancy are not well-defined. Much research is needed to define further the cellular, biochemical, and molecular basis for these physiological processes involved in the regulation of uterine smooth muscle contraction and relaxation.

Immunohistochemical and biochemical analysis of a human Sertoli-Leydig cell tumor: autonomous steroid production characteristic of ovarian theca cells.

OBJECTIVE: To ascertain the steroidogenic profile and location of steroidogenic enzymes in a steroid-secreting Sertoli-Leydig cell tumor of the ovary. METHODS: Steroid levels from peripheral, left ovarian (tumor), and right ovarian venous blood were measured. Tumor tissue was examined for the steroidogenic enzymes 17 alpha-hydroxylase (P450c17) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) by immunohistochemistry. Tumor cells were isolated and incubated in serum-free media. Thereafter, media were analyzed for steroid production, steroidogenic response to effectors, and metabolism of radiolabeled pregnenolone and androstenedione. RESULTS: Levels of C19 steroids and 17-hydroxyprogesterone (17OHP) were elevated in peripheral blood. The majority (80%) of steroids in serum from the left ovarian vein (tumor) were C19 steroids (dehydroepiandrosterone [DHEA], 45%; androstenedione, 27%, testosterone, 7%, with 13% 17OHP, 7% progesterone, and less than 1% estradiol (E2). Immunoreactivity for both P450c17 and 3 beta HSD was identified in clusters of large cells surrounded by nonimmunoreactive cells composing cord-like structures. Of the steroids that accumulated in the incubation medium of unstimulated, freshly isolated tumor cells, 84% were C19 steroids (DHEA, 44%; androstenedione, 36%; testosterone 2%, with 16% 17OHP and less than 1% progesterone and E2. Basal steroid production was not stimulated by LH or FSH. However, treatment with forskolin (10 mumol/L), dibutyryl cAMP (1 mmol/L), or steroid precursors (22-hydroxycholesterol, 1 mumol/L; pregnenolone, 1 mumol/L) increased the production of all steroids measured. Forskolin treatment increased androstenedione (fivefold), DHEA (tenfold), and 17OHP (40-fold) compared with basal levels. Incubation of freshly isolated cells with [3H]pregnenolone demonstrated the ability of these cells to metabolize this C21 steroid precursor to androstenedione, DHEA, and 17OHP. However, [3H]androstenedione was not readily metabolized by these cells to either estrone or testosterone. CONCLUSIONS: The steroidogenic properties of a steroid hormone-producing tumor were described. Cells isolated from this tumor produced steroids similar to those secreted by ovarian theca cells. These properties suggest that certain ovarian steroidogenic tumor cells may be an appropriate model for ovarian theca cells and could be used to develop steroid-secreting cell lines.

Calbindin-D9K gene expression in human myometrium during pregnancy and labor.

The 9-kilodalton vitamin D-dependent calcium-binding protein (CaBP9K) is expressed in the intestine and uterus of mammals. In this study, we demonstrated the intracellular location of CaBP9K and quantified its expression in human myometrial tissues from nonpregnant and pregnant women (before and after the onset of labor). By Western blot analysis, we found that antiserum raised against bovine intestinal CaBP9K was specific for CaBP9K in human myometrium. By Northern blot analysis, with an oligodeoxynucleotide probe specific for human CaBP9K, we identified a single 0.7-kilobase messenger ribonucleic acid (mRNA) species in myometrial tissues from pregnant women before and after the onset of labor. CaBP9K mRNA and immunoreactive protein were localized within myometrial smooth muscle cells by in situ hybridization and immunohistochemistry. The highest levels of CaBP9K mRNA and immunoreactive protein were found in myometrial tissues obtained from pregnant women at term before the onset of labor. CaBP9K mRNA and immunoreactive levels of CaBP9K were decreased significantly in myometrial tissues obtained after the onset of labor (2- and 8-fold, respectively). These findings demonstrated for the first time that CaBP9K was present in human myometrium and suggested that it may play a role in regulating uterine smooth muscle function during pregnancy.

Activation properties of myosin light chain kinase during contraction/relaxation cycles of tonic and phasic smooth muscles.

In intact smooth muscle, myosin light chain kinase (MLCK) is phosphorylated at its regulatory site by Ca2+/calmodulin-dependent protein kinase II resulting in an increase in the concentration of Ca2+/calmodulin required for half-maximal activation of the enzyme (KCaM). We investigated the physiological significance of MLCK phosphorylation during cycles of contraction and relaxation in tonic (tracheal) and phasic (uterine) smooth muscles. MLCK phosphorylation and dephosphorylation occurred at rates sufficient to modulate the Ca2+ sensitivity of light chain phosphorylation. In contractions of both smooth muscles (though using different sources of activating Ca2+), increases in [Ca2+]i preceded light chain phosphorylation; but, the rate of increase in light chain phosphorylation was significantly greater than the rate of increase in [Ca2+]i. The onset of MLCK phosphorylation with the resultant increase in KCaM coincided with the diminished rate of light chain phosphorylation. During spontaneous contractions of uterine smooth muscle, the Ca2+ transient was characterized by an initial rapid increase, a sustained plateau, and rapid decline. During the sustained phase of the Ca2+ transient, MLCK phosphorylation increased and coincided with dephosphorylation of light chain and relaxation. These results indicate that MLCK is sensitive to small increases in intracellular Ca2+ during the initiation of contraction and that the enzyme subsequently becomes desensitized to Ca2+/calmodulin, thereby limiting the extent of light chain phosphorylation.