Efficacy of robotic versus open transversus abdominis release in a porcine model.

PURPOSE: Transversus abdominis muscle release (TAR) combines retromuscular mesh placement with posterior component separation and muscle release. TAR is usually an open technique for abdominal wall reconstruction; however, several centers have performed this operation robotically and claim better clinical outcomes when compared to open surgery. We sought to compare robotic versus open TAR utilizing a porcine model. METHODS: Animals were randomized to open versus robotic TAR with mesh placement, survived for 4 weeks, then underwent diagnostic laparoscopy to assess adhesive burden and adhesion tenacity. T-peel testing was utilized to assess mesh ingrowth. The primary outcome was adhesive burden; secondary outcomes included mesh incorporation, contraction, and operative time. RESULTS: Nine robotic and eight open TARs were performed. Mean operative time was significantly shorter for the open cases compared to robotic cases (88.6 ± 12.9 min versus 228.3 ± 46.2, p < 0.01). Operative time in the robotic arm of the study decreased over time, from 300 to 165 min. No difference was seen in the mean adhesion area between the two groups. Adhesion tenacity and mesh flatness were similar. The work required to peel the mesh off surrounding tissue was significantly higher in the open TAR than in the robotic TAR group: 52.6 ± 15.5 and 32.9 ± 10.6 mJ/cm2, respectively (p < 0.01). CONCLUSIONS: There were no differences in adhesions between the robotic and open approaches, but greater mesh contraction and ingrowth was observed in the open TAR group. Though operative time was longer in the robotic group, time dropped by about 40% from the first case to the last.

The biochemical basis for increased testosterone production in theca cells propagated from patients with polycystic ovary syndrome.

Ovarian theca cells propagated from patients with polycystic ovary syndrome (PCOS) convert steroid precursors into T more efficiently than normal theca cells. To identify the basis for increased T production by PCOS theca cells, we examined type I-V 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) isoform expression in long-term cultures of theca and granulosa cells isolated from normal and PCOS ovaries. RT-PCR analysis demonstrated that theca cells express type V 17 beta HSD a member of the aldo-keto reductase (AKR) superfamily (17 beta HSDV, AKR1C3), whereas expression of type I, II, and IV 17 beta HSD, which are members of the short-chain dehydrogenase/reductase superfamily, was limited to granulosa cells. Type III 17 beta HSD, the testicular isoform, was not detected in either granulosa or theca cells. Northern and real-time PCR analyses demonstrated that 17 beta HSDV transcripts were not significantly increased in PCOS theca cells compared with normal theca cells. RT-PCR analysis revealed that theca cells also express another AKR, 20 alpha HSD (AKR1C1). Both basal and forskolin-stimulated 20 alpha HSD mRNA levels were increased in PCOS theca cells compared with normal theca cells. However, 17 beta HSD enzyme activity per theca cell was not significantly increased in PCOS, suggesting that neither AKR1C3 nor AKR1C1 contributes to the formation of T in this condition. In contrast, 17 alpha-hydroxylase/C17,20 lyase and 3 beta HSD enzyme activities were elevated in PCOS theca cells, driving increased production of T precursors. These findings indicate that 1) increased T production in PCOS theca cells does not result from dysregulation of "androgenic" 17 beta HSD activity or altered expression of AKRs that may express 17 beta HSD activity; and 2) increased synthesis of T precursors is the primary factor driving enhanced T secretion in PCOS.

Conditional response of the human steroidogenic acute regulatory protein gene promoter to sterol regulatory element binding protein-1a.

The steroidogenic acute regulatory protein (StAR) gene controls the rate-limiting step in the biogenesis of steroid hormones, delivery of cholesterol to the cholesterol side-chain cleavage enzyme on the inner mitochondrial membrane. We determined whether the human StAR promoter is responsive to sterol regulatory element-binding proteins (SREBPs). Expression of SREBP-1a stimulated StAR promoter activity in the context of COS-1 cells and human granulosa-lutein cells. In contrast, expression of SREBP-2 produced only a modest stimulation of StAR promoter activity. One of the SREBP-1a response elements in the StAR promoter was mapped in deletion constructs and by site-directed mutagenesis between nucleotides -81 to -70 from the transcription start site. This motif bound recombinant SREBPs in electrophoretic mobility shift assays, but with lesser affinity than a low density lipoprotein receptor SREBP-binding site. An additional binding site for the transcriptional modulator, yin yang 1 (YY1), was observed within the SREBP-binding site (nucleotides -73 to -70). Mutation of the YY1-binding site increased the responsiveness of the StAR promoter to exogenous SREBP-1a, but did not alter the affinity for SREBP-1a binding in electrophoretic mobility gel shift assays. Manipulations that altered endogenous mature SREBP-1a levels (e.g. culture in lipoprotein-deficient medium and addition of 27-hydroxycholesterol) did not affect StAR promoter function, but influenced low density lipoprotein receptor promoter activity. We conclude that 1) the human StAR promoter is conditionally responsive to SREBP-1a such that promoter activity is up-regulated in the presence of high levels of SREBP-1a, but is unaffected when mature SREBP levels are suppressed; and 2) the human StAR promoter is selectively responsive to SREBP-1a.

Differential activity of the cytochrome P450 17alpha-hydroxylase and steroidogenic acute regulatory protein gene promoters in normal and polycystic ovary syndrome theca cells.

17alpha-Hydroxylase (CYP17) expression in propagated theca cells isolated from the ovaries of women with polycystic ovary syndrome (PCOS) is persistently elevated, compared with theca cells isolated from normal ovaries. To investigate the mechanism for increased CYP17 messenger RNA accumulation in PCOS theca cells, we examined CYP17 and steroidogenic acute regulatory protein (StAR) promoter activities in normal and PCOS theca cells. Conditions were established to transiently transfect human theca cells with reporter gene constructs containing 5' truncations of the human CYP17 and StAR promoters. Cotransfection of a steroidogenic factor-1 expression plasmid was found to be required for detection of basal and forskolin-stimulated CYP17 promoter activity but not for StAR promoter activity. However, cotransfection with a steroidogenic factor-1 expression plasmid augmented both basal and forskolin-stimulated StAR promoter activity. CYP17 reporter activity was compared in theca cells isolated from normal and PCOS patients. Basal and forskolin-stimulated CYP17 promoter activity was 4-fold greater in PCOS cells than in theca cells isolated from normal ovaries. In contrast, StAR promoter activity, and the activity of a reporter construct containing three copies of a cAMP response element (3xCRE), were similar in normal and PCOS theca cells. The results of these studies document: 1) that basal and cAMP-dependent CYP17 gene transcription is increased in PCOS theca cells; 2) that there is differential regulation of promoters of genes required for steroidogenesis in PCOS theca cells; and 3) that passaged normal and PCOS theca cells provide a model system for studying tissue-specific regulation of genes encoding steroidogenic enzymes and identifying the molecular mechanisms involved in increased androgen production in PCOS.

Insulin and insulin-like growth factor-I and -II modulate human granulosa-lutein cell steroidogenesis: enhancement of steroidogenic acute regulatory protein (StAR) expression.

Insulin and insulin-like growth factors (IGF)-I and -II stimulate granulosa cell steroidogenesis. Since steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroid hormone biosynthesis, the ability of insulin and IGF to modulate StAR protein and mRNA expression was examined in two human granulosa cell culture systems: (i) proliferating granulosa-lutein cells and (ii) luteinized-granulosa cells derived during in-vitro fertilization (IVF). In proliferating granulosa-lutein cells, IGF-I and IGF-II increased StAR protein approximately 4-5-fold, while insulin and 8-bromoadenosine 3',5'-cAMP (8-Br-cAMP) increased amounts of StAR protein 2.5- and 6-fold respectively. A combination of IGFs/insulin and 8-Br-cAMP increased StAR 7-9-fold. Luteinized granulosa cells also had increased StAR expression after treatment with IGF-I (2. 8-fold), IGF-II (3-fold), insulin (2.5-fold) and 8-Br-cAMP (4. 5-fold). Progesterone production generally followed a pattern similar to StAR protein in both cell systems. In proliferating granulosa-lutein cells, both IGF-I and insulin increased StAR mRNA (3-fold) and 8-Br-cAMP increased StAR mRNA 4-fold, whereas a combination of IGF-I and 8-Br-cAMP had an additive effect on StAR mRNA expression (7-fold). Transient transfection of proliferating granulosa-lutein cells with StAR promoter-luciferase reporter constructs demonstrated that IGF-I, IGF-II, and insulin had no effect on the StAR promoter activity, while 8-Br-cAMP stimulated StAR promoter function. The results indicate that: (i) IGFs and insulin stimulate StAR mRNA and protein expression in human granulosa-lutein cells; (ii) IGF-I and 8-Br-cAMP have an additive effect on StAR gene expression; and (iii) IGF-I increases StAR mRNA and protein by a mechanism that does not involve activation of the proximal StAR gene promoter.

CCAAT/enhancer-binding proteins regulate expression of the human steroidogenic acute regulatory protein (StAR) gene.

Two putative CCAAT/enhancer-binding protein (C/EBP) response elements were identified in the proximal promoter of the human steroidogenic acute regulatory protein (StAR) gene, which encodes a key protein-regulating steroid hormone synthesis. Expression of C/EBPalpha and -beta increased StAR promoter activity in COS-1 and HepG2 cells. Cotransfection of C/EBPalpha or -beta and steroidogenic factor 1, a transcription factor required for cAMP regulation of StAR expression, into COS-1 augmented 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP)-stimulated promoter activity. When the putative C/EBP response elements were mutated, individually or together, a pronounced decline in basal StAR promoter activity in human granulosa-lutein cells resulted, but the fold stimulation of promoter activity by 8-Br-cAMP was unaffected. Recombinant C/EBPalpha and -beta bound to the two identified sequences but not the mutated elements. Human granulosa-lutein cell nuclear extracts also bound these elements but not the mutated sequences. An antibody to C/EBPbeta, but not C/EBPalpha, supershifted the nuclear protein complex associated with the more distal element. The complex formed by nuclear extracts with the proximal element was not supershifted by either antibody. Western blot analysis revealed the presence of C/EBPalpha and C/EBPbeta in human granulosa-lutein cell nuclear extracts. C/EBPbeta levels were up-regulated 3-fold by 8-Br-cAMP treatment. Our studies demonstrate a role for C/EBPbeta as well as yet to be identified proteins, which can bind to C/EBP response elements, in the regulation of StAR gene expression and suggest a mechanism by which C/EBPbeta participates in the cAMP regulation of StAR gene transcription.

Augmented androgen production is a stable steroidogenic phenotype of propagated theca cells from polycystic ovaries.

To test the hypothesis that the hyperandrogenemia associated with polycystic ovary syndrome (PCOS) results from an intrinsic abnormality in ovarian theca cell steroidogenesis, we examined steroid hormone production, steroidogenic enzyme activity, and mRNA expression in normal and PCOS theca cells propagated in long-term culture. Progesterone (P4), 17alpha-hydroxyprogesterone (17OHP4), and testosterone (T) production per cell were markedly increased in PCOS theca cell cultures. Moreover, basal and forskolin-stimulated pregnenolone, P4, and dehydroepiandrosterone metabolism were increased dramatically in PCOS theca cells. PCOS theca cells were capable of substantial metabolism of precursors into T, reflecting expression of an androgenic 17beta-hydroxysteroid dehydrogenase. Forskolin-stimulated cholesterol side chain cleavage enzyme (CYP11A) and 17alpha-hydroxylase/17,20-desmolase (CYP17) expression were augmented in PCOS theca cells compared with normal cells, whereas no differences were found in steroidogenic acute regulatory protein mRNA expression. Collectively, these observations establish that increased CYP11A and CYP17 mRNA expression, as well as increased CYP17, 3beta-hydroxysteroid dehydrogenase, and 17beta-hydroxysteroid dehydrogenase enzyme activity per theca cell, and consequently increased production of P4, 17OHP4, and T, are stable properties of PCOS theca cells. These findings are consistent with the notion that there is an intrinsic alteration in the steroidogenic activity of PCOS thecal cells that encompasses multiple steps in the biosynthetic pathway.

Oxysterol regulation of steroidogenic acute regulatory protein gene expression. Structural specificity and transcriptional and posttranscriptional actions.

Oxysterols exert a major influence over cellular cholesterol homeostasis. We examined the effects of oxysterols on the expression of steroidogenic acute regulatory protein (StAR), which increases the delivery of cholesterol to sterol-metabolizing P450s in the mitochondria. 22(R)-hydroxycholesterol (22(R)-OHC), 25-OHC, and 27-OHC each increased steroidogenic factor-1 (SF-1)-mediated StAR gene transactivation by approximately 2-fold in CV-1 cells. In contrast, cholesterol, progesterone, and the 27-OHC metabolites, 27-OHC-5beta-3-one and 7alpha,27-OHC, had no effect. Unlike our findings in CV-1 cells, SF-1-dependent StAR promoter activity was not augmented by 27-OHC in COS-1 cells, Y-1 cells, BeWo choriocarcinoma cells, Chinese hamster ovary (CHO) cells, and human granulosa cells. Studies examining the metabolism of 27-OHC indicated that CV-1 cells formed a single polar metabolite, 3beta-OH-5-cholestenoic acid from radiolabeled 27-OHC. However, this metabolite inhibited StAR promoter activity in CV-1, COS-1 and CHO cells. Because 7alpha,27-OHC was unable to increase SF-1-dependent StAR promoter activity, we examined 27-OHC 7alpha-hydroxylase in COS-1 and CHO cells. COS-1 cells contained high 7alpha-hydroxylase activity, whereas the enzyme was undetectable in CHO cells. The hypothesis that oxysterols act in CV-1 cells to increase StAR promoter activity by reducing nuclear levels of sterol regulatory element binding protein was tested. This notion was refuted when it was discovered that sterol regulatory element binding protein-1a is a potent activator of the StAR promoter in CV-1, COS-1, and human granulosa cells. Human granulosa and theca cells, which express endogenous SF-1, contained more than 5-fold more StAR protein following addition of 27-OHC, whereas StAR mRNA levels remained unchanged. We conclude that 1) there are cell-specific effects of oxysterols on SF-1-dependent transactivation; 2) the ability to increase transactivation is limited to certain oxysterols; 3) there are cell-specific pathways of oxysterol metabolism; and 4) oxysterols elevate StAR protein levels through posttranscriptional actions.

The homeodomain protein Pbx1 is involved in cAMP-dependent transcription of human CYP17.

Pbx1 is a homeodomain transcription factor involved in cAMP-dependent transcriptional regulation of the bovine CYP17 gene. In this study, we have investigated the involvement of Pbx1 in the transcriptional regulation of the human CYP17 gene. Although a sequence identical to previously determined Pbx-binding sites is not present in the promoter region of the human CYP17 gene, three putative Pbx-binding sites are identified by sequence similarity analysis. Coexpression of Pbx1 and a catalytic subunit of protein kinase A (PKA) greatly enhances reporter gene transcription via the 5'-flanking region of the human CYP17 gene. Upon gel shift analysis utilizing nuclear extracts from human adrenal H295R cells, one of the three putative Pbx1-binding sites, -250/-241 bp, shows the typical intense doublet observed with other Pbx-binding sites. 5'-Deletion analyses of the reporter construct containing this Pbx-binding site showed approximately sixfold induction by coexpression of Pbx1 and PKA compared to the basal transcription, suggesting that Pbx1 binds the -250/-241 bp sequence and participates in cAMP-dependent regulation of the human CYP17 gene.

Multiple steroidogenic factor 1 binding elements in the human steroidogenic acute regulatory protein gene 5'-flanking region are required for maximal promoter activity and cyclic AMP responsiveness.

A proximal element from the human StAR gene promoter, containing the sequence (-105)TATCCTTGAC(-95), was shown to confer responsiveness to 8-Br-cAMP in the presence of steroidogenic factor 1 (SF-1) when placed behind a minimal thymidine kinase promoter or an SV40 promoter and transfected into BeWo cells which normally lack StAR and SF-1. This element was also transactivated by SF-1 in a yeast one-hybrid system. The -105 to -95 sequence was protected by SF-1 in footprint analysis and a double-stranded oligonucleotide containing the element bound SF-1 specifically in electrophoretic mobility shift assays. Another SF-1-binding sequence 35 bp upstream of the transcription start site ((-42)CAGCCTTC(-35)) was identified in the DNase 1 footprint analysis and, when mutated, markedly reduced SF-1-dependent and 8-Br-cAMP-stimulated StAR promoter activity in BeWo cells. The two proximal SF-1 response elements were shown to be critical for StAR promoter function in human granulosalutein cells, which express SF-1 and respond to cAMP with increased transcription of the StAR gene. Mutation of either element substantially reduced basal and forskolin-stimulated promoter activity, although mutation of the -105 to -95 element had more pronounced effects. Mutation of a third, more distal, SF-1-binding site at -926 to -918 also reduced basal but not forskolin-stimulated promoter activity in the granulosa-lutein cells. These findings demonstrate that multiple SF-1 response elements are required for maximal StAR promoter activity and regulation by cAMP.

Regulation of expression of the steroidogenic acute regulatory protein (StAR) gene: a central role for steroidogenic factor 1.

Steroidogenic acute regulatory protein (StAR) plays a critical role in regulating the rate-limiting step in steroid hormone synthesis, cholesterol side-chain cleavage. StAR gene expression is transcriptionally controlled in the gonads by gonadotropic hormones via a cAMP second message. We have begun to analyze factors responsible for the transcriptional activation of the StAR gene. The human StAR gene promoter has at least two cis elements that govern basal and cAMP-regulated gene expression. One of these elements (the distal element) is a consensus binding sequence for the orphan nuclear receptor transcription factor, steroidogenic factor 1 (SF-1); the other (the proximal element) is a related motif. The human StAR promoter is not active in BeWo choriocarcinoma cells, but is functional and cAMP-responsive in murine Y1 adrenal cortical tumor cells. Cotransfection of a plasmid expressing SF-1 allows a StAR promoter construct to function in BeWo cells. Other orphan nuclear transcription factors do not support StAR promoter function in BeWo cell hosts. Deletion or mutation of the distal and proximal cis elements individually substantially reduces SF-1-supported StAR promoter activity. The distal site binds SF-1 with high affinity, whereas the proximal site binds SF-1 with lower affinities. These findings demonstrate a requirement for SF-1 for human StAR gene expression.

Expression of steroidogenic acute regulatory protein (StAR) in the human ovary.

Expression of steroidogenic acute regulatory protein (StAR) messenger ribonucleic acid (mRNA) in human ovary and cultured proliferating granulosa-lutein cells, theca interna cells, and luteinized granulosa cells was examined. The StAR transcripts were restricted in situ to theca of preovulatory follicles and luteinized granulosa and thecal cells of the corpus luteum. The cyclic nucleotide analog, 8-bromo-cAMP (8-Br-cAMP), increased StAR mRNA in all cell types studied by a process requiring on-going RNA and protein synthesis. Phorbol myristate acetate prevented the stimulatory effects of 8-Br-cAMP. In proliferating granulosa-lutein cells, 8-Br-cAMP increased StAR gene transcription and did not significantly affect StAR mRNA stability. Forskolin treatment was also found to increase the expression of a human StAR proximal promoter-luciferase fusion gene transfected into the proliferating granulosa-lutein cells. We conclude that 1) the StAR gene is expressed in the most steroidogenic compartments of the human ovary; 2) induction of StAR gene transcription by cAMP, produced in response to the LH surge, accounts for the appearance of StAR transcripts in luteinized granulosa cells; and 3) the effects of cAMP are antagonized by activators of protein kinase C.

Radiation keratosis associated with exposure to a gold ring.

A radiation keratosis occurred in a woman who wore a radioactive gold ring forty-three years ago. Clinicians should be aware that not all "warty lesions" on the hands are actinic keratosis, seborrheic keratosis, or warts. Radioactive gold rings still exist. Diagnosis requires a high index of suspicion, since patients may no longer be wearing the offending ring.

Functional, long-term human theca and granulosa cell cultures from polycystic ovaries.

Reproducible culture conditions for obtaining large numbers of functional PCOS theca interna and granulosalutein cells will be indispensable in studies focussing on the molecular basis for androgen overproduction by ovarian cells of patients with polycystic ovarian syndrome (PCOS). The objective of the present study was to determine if granuiosa and theca interna cells obtained from ovarian follicles of patients with PCOS could be passaged with maintenance of inducible steroidogenic activity. PCOS theca interna and granuiosa cells were obtained from individual follicles of polycystic ovaries containing multiple cystic follicles with characteristic hypertrophied theca interna. Utilizing conditions for growing normal ovarian cells, both cell types were passaged successively and conditions for cell freezing, storing and thawing were established. In granulosa-lutein cultures grown and passed for successive passages, and transferred into serum-free medium, forskolin stimulated aromatase activity increased 3-10-fold over control non-stimulated values. Concurrent treatment with IGF-I (50 ng/mL) enhanced forskolin-stimulated aromatase activity in PCOS granulosa-lutein cultures. In passaged PCOS theca interna cells, forskolinstimulated 17α-hydroxyprogesterone production was increased 4-25-fold over control values. Treatment of PCOS theca interna cells with insulin (50 ng/mL) enhanced forskolin-stimulated 17α-hydroxyprogesterone biosynthesis. The effects of various growth factors and phorbol esters on 17α-hydroxylase activity in cultured PCOS theca interna cells was also investigated. Treatment of PCOS theca cells with EGF, FGF, TGFß and TPA resulted in the inhibition of forskolin-stimulated 17α-hydroxyprogesterone production. These data suggest that PCOS theca interna and granuiosa cells respond to insulin and to the growth factors similarly to cells obtained from normal cycling ovaries.

The effects of growth factors and phorbol esters on steroid biosynthesis in isolated human theca interna and granulosa-lutein cells in long term culture.

In this report we examined the effects of growth factors and phorbol esters on steroid hydroxylase activity in cultured human thecal and granulosa-lutein cells. Treatment of thecal cells with epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor-beta (TGF beta), and tetradecanoyl phorbol acetate (TPA) resulted in the inhibition of forskolin- and dibutyryl cAMP-stimulated 17 alpha-hydroxylase activity and 17 alpha-hydroxyprogesterone and dehydroepiandrosterone production. In contrast, cAMP-stimulated 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity was enhanced by FGF and TGF beta, and treatment with EGF enhanced cAMP-stimulated progesterone production. cAMP stimulated 3 beta HSD activity was unaffected by TPA (10 nmol/L) treatment, yet TPA inhibited cAMP-stimulated progesterone production. Basal 3 beta HSD activity and progesterone production were inhibited by TPA. In contrast to the inhibitory actions of EGF, FGF, and TGF beta on 17 alpha-hydroxylase expression, insulin and insulin-like growth factor-I enhanced forskolin-stimulated 17 alpha-hydroxylase activity. In granulosa-lutein cells, forskolin-stimulated aromatase activity was suppressed by EGF, FGF, and TPA. TGF beta had no effect on forskolin-stimulated aromatase activity. EGF, FGF, and TGF beta did not affect forskolin-stimulated progesterone production, whereas treatment with TPA inhibited cAMP-stimulated progesterone secretion. These data suggest that growth factors may differentially regulate cAMP-dependent processes in human thecal and granulosa cells of the developing follicle.

Human intraovarian interleukin-1 (IL-1) system: highly compartmentalized and hormonally dependent regulation of the genes encoding IL-1, its receptor, and its receptor antagonist.

To delineate the scope of the human intraovarian IL-1 system we used a solution hybridization/RNase protection assay to test for expression of the genes encoding IL-1, its type I receptor (IL-1R), and its receptor antagonist (IL-1RA). IL-1 transcripts were not detected in whole ovarian material from days 4 or 12 of an unstimulated menstrual cycle but transcripts (IL-1 beta much greater than IL-11 alpha) were detected in preovulatory follicular aspirates from gonadotropin-stimulated cycles. Concurrently obtained peripheral monocytes did not contain IL-1 beta transcripts but macrophage-depleted follicular aspirates did, thus implicating the granulosa cells as the site of IL-1 expression. IL-1R transcripts were detected in RNA from whole ovaries and follicular aspirates but not in RNA from peripheral monocytes. IL-1RA transcripts were detected in whole ovarian material as well as in macrophage-free follicular aspirates. Cultured human granulosa and theca cells did not contain mRNA for IL-1 beta or IL-1RA but did contain mRNA for IL-1R. Treatment of cell cultures with forskolin (25 microM) induced IL-1 beta transcripts in granulosa but not theca cells. Forskolin also increased the basal levels of IL-1R transcripts in both granulosa and theca cells but did not induce IL-RA transcripts in either cell type. Taken together, these findings reveal the existence of a complete, highly compartmentalized, hormonally dependent intraovarian IL-1 system replete with ligands, receptor, and receptor antagonist.

Regulation of corticotropin responsiveness in human fetal adrenal cells.

The human fetal adrenal gland exhibits a high rate of steroidogenesis during fetal development and produces the majority of steroids used by the placenta for estrogen synthesis. Corticotropin appears to be the principal hormonal regulator of steroidogenesis in the fetal adrenal gland. However, little is known concerning the regulation of corticotropin receptors. In this study we examined the long-term regulation of corticotropin responsiveness as measured by the ability of human fetal adrenal gland cells to produce cyclic adenosine monophosphate after corticotropin treatment for 3 hours. We also examined the regulation of corticotropin receptors as determined by iodine 125-labeled corticotropin binding to fetal adrenal cells. Fetal adrenal glands were obtained from second-trimester abortuses. The two distinct zones of the fetal adrenal gland, the definitive zone and the fetal zone, were separated and the tissue mechanically dispersed. Freshly isolated cells responded to corticotropin with a sevenfold to tenfold increase in the production of cyclic adenosine monophosphate, indicating a functional corticotropin receptor-adenylate cyclase coupling. However, when either fetal zone or definitive zone cells were grown and passed in monolayer culture, corticotropin stimulation of cyclic adenosine monophosphate production dropped to only twofold. The loss of corticotropin stimulation of cyclic adenosine monophosphate production occurred with a loss of the steroid-metabolizing enzyme 17 alpha-hydroxylase (P-450(17 alpha]. Because P-450(17 alpha) expression can be stimulated after treatment of fetal adrenal gland cells with corticotropin or forskolin, we attempted to increase the ability of corticotropin to stimulate cyclic adenosine monophosphate production in a similar manner. After cells were pretreated with corticotropin (0.1 to 100 nmol/L) or forskolin (0.1 to 100 mumol/L) for 4 days, their ability to produce cyclic adenosine monophosphate in response to corticotropin was examined. Pretreatment with both corticotropin and forskolin caused a dose-dependent increase in the ability of corticotropin to stimulate the production of cyclic adenosine monophosphate. Cells stimulated with corticotropin after pretreatment with forskolin exhibited a 35- to 50-fold increase in cyclic adenosine monophosphate production compared with nontreated cells (approximately twofold). Corticotropin pretreatment increased responsiveness to a lesser extent than forskolin pretreatment. The increase in corticotropin responsiveness occurred along with an induction of P-450(17 alpha) enzyme levels. The effect of pretreatment with corticotropin and forskolin on the binding of iodine 125-labeled corticotropin to definitive zone cells was also investigated. Corticotropin pretreatment increased corticotropin receptor binding 2.8 times; forskolin pretreatment increased corticotropin binding by seven times.(ABSTRACT TRUNCATED AT 400 WORDS)

Proliferating human granulosa-lutein cells in long term monolayer culture: expression of aromatase, cholesterol side-chain cleavage, and 3 beta-hydroxysteroid dehydrogenase.

The development of long term culture conditions with which to study the regulation of expression of aromatase, cholesterol side-chain cleavage enzyme, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human granulosa-lutein cells is described in this report. Conditions have been established for the dispersal, growth, freezing, and storage of functional human granulosa cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Optimal growth conditions for human granulosa-lutein cells were determined by plating cells at a low density and testing the capacity of a variety of culture conditions to support growth. A combination of fetal bovine serum (FBS), horse serum, and the serum substitute UltroSer G was found to increase cell number to maximal levels, 8- to 10-fold higher than with sera alone. Human granulosa-lutein cells grown under these conditions had a doubling rate of 36-40 h and were morphologically distinct from human theca interna cells grown under similar conditions. Human granulosa-lutein cells treated with forskolin retracted and rounded up, whereas cultures of human ovarian theca interna cells or human fibroblasts treated similarly did not retract. Human granulosa-lutein cells were grown for successive passages and transferred to serum-free medium containing forskolin, LH, hCG, or cholera toxin. Addition of these agents resulted in a time- and dose-dependent increase in aromatase activity and progesterone secretion. In these studies FSH treatment was found not to increase aromatase activity. In a study of the time course of 3 beta HSD activity in the absence of forskolin under serum-free conditions, it was found that 3 beta HSD activity increased 3-fold during the 72-h treatment period. Forskolin-stimulated 3 beta HSD activity also increased in a time-dependent manner, with levels in treated cells 3-fold higher than those in control cells. Northern analysis performed on total RNA obtained from forskolin- or hCG-stimulated granulosa-lutein cells confirmed that the increase in aromatase activity was associated with a corresponding increase in levels of mRNA specific for aromatase cytochrome P-450. Levels of mRNA encoding cholesterol side-chain cleavage cytochrome P-450 were similarly increased in cells treated with forskolin compared with unstimulated values at each of the time points investigated. Under serum-free conditions in the absence of stimulation, the 3.4-kilobase band of aromatase cytochrome P-450 mRNA was detectable.(ABSTRACT TRUNCATED AT 400 WORDS)