Quantitation of spatial and temporal variability of biomarkers for Barrett's Esophagus.

Chemoprevention and risk-stratification studies in Barrett's esophagus (BE) rely on biomarkers but the variability in their temporal and spatial expression is unknown. If such variability exists, it will impact sampling techniques and sample size calculations. Specimens from three levels of biopsies over two serial endoscopies in nondysplastic BE patients were analyzed for aneuploidy, proliferation markers (Ki67, Mcm2), and cell cycle markers (cyclin A and cyclin D1). A modification of the image cytometry technique, where cytokeratin staining automatically distinguished epithelial and stromal cells, measured aneuploidy on whole tissue sections. Other biomarkers were studied by immunohistochemistry. Coefficient of variability (SD/mean) was calculated; a value <10% indicated low variability. A total of 120 specimens (20 subjects each with three biopsy levels at two time points) from nondysplastic BE patients (71 ± 8.8 years, all Caucasian, 90% males, C5.1M7.5 ± 3.4 cm) were analyzed. The mean interval between endoscopies was 32.8 ± 8.4 months. Aneuploidy had a spatial variability of 6.8% at visit 1 (mean diploid index: 1.1 ± 0.09) and 7.9% at visit 2 (mean diploid index: 1.1 ± 0.06) and a temporal variability of 7.0-8.1% for the three levels. For other biomarkers, the spatial variability ranged from ∼5 to 30% at visit 1 and 11-92% at visit 2 and the temporal variability ranged from 0 to 77%. To conclude, of all the biomarkers, only aneuploidy had both spatial and temporal variability of <10%. Spatial and temporal variability were biomarker dependent and could be as high as 90% even without progression. These data will be useful to design chemoprevention and risk-stratification studies in BE.

Feasibility of mcroRNAs as biomarkers for Barrett's Esophagus progression: a pilot cross-sectional, phase 2 biomarker study.

OBJECTIVES: Risk stratification of Barrett's esophagus (BE) using biomarkers remains an important goal. We evaluated feasibility and clinical accuracy of novel microRNA (miRNA) biomarkers for prediction of BE dysplasia. METHODS: Paired fresh-frozen and hematoxylin/eosin specimens from a prospective tissue repository where only biopsies with the lesion of interest (i.e., intestinal metaplasia (IM) or high-grade dysplasia (HGD)/esophageal adenocarcinoma (EAC)) occupying >50% of biopsy area were included. Tissue miRNA expression was determined by microarrays and validated by quantitative reverse transcription-PCR (qRT-PCR). Three groups were compared-group A, IM tissues from BE patients without dysplasia; group B, IM tissues from group C patients; and group C, dysplastic tissues from BE patients with HGD/EAC. RESULTS: Overall, 22 BE patients, 11 with and without dysplasia (mean age 64 ± 8.2 and 63 ± 11.6 years, respectively, all Caucasian males) were evaluated. Nine miRNAs were identified by high-throughout analysis (miR-15b, -21, -192, -205, 486-5p, -584, -1246, let-7a, and -7d) and qRT-PCR confirmed expression of miR-15b, -21, 486-5p, and let-7a. Two of 4 miRNAs (miR-145 and -203, but not -196a and -375) previously described in BE patients also exhibited differential expression. Sensitivity and specificity of miRNAs for HGD/EAC were miR-15b: 87 and 80%, miR-21: 93 and 70%, miR-203: 87 and 90%, miR-486-5p: 82 and 55%, and miR-let-7a: 88 and 70%. MiRNA-15b, -21, and -203 exhibited field effects (i.e., groups A and B tissues while histologically similar yet exhibited different miRNA expression). CONCLUSIONS: This pilot study demonstrates feasibility of miRNAs to discriminate BE patients with and without dysplasia with reasonable clinical accuracy. However, the specific miRNAs need to be evaluated further in future prospective trials.

Differentiation-induced post-transcriptional control of B7-H1 in human trophoblast cells.

Trophoblast expression of immunomodulatory proteins in the human placenta is among the mechanisms that are critical for ensuring lymphocyte tolerance to the semi-allogeneic fetus. High levels of B7-H1 on trophoblast cells together with the known role of this protein in establishment of peripheral tolerance suggest that B7-H1 mediates immunological protection of the placenta during gestation. In this study, we investigated the molecular mechanisms of regulation of B7-H1 in trophoblast cells by epidermal growth factor (EGF), a key regulator of trophoblast cell differentiation. EGF increased B7-H1 protein levels within 24 h and mRNA levels within 4h of the initiation of treatment; by 24 h B7-H1 mRNA levels were similar between control and EGF-treated cells. Analysis of two different potential promoter regions revealed strong promoter activity in response to IFN-gamma. In contrast, no promoter activity could be induced by EGF, suggesting that this cytokine regulates B7-H1 expression post-transcriptionally in trophoblast cells. EGF-induced B7-H1 protein expression was completely blocked in the presence of inhibitors of the PI3Kinase/Akt/mTOR pathway, a pathway known to regulate gene expression at the translational level. Finally, analysis of monosomal and polysomal mRNA fractions of untreated and EGF-treated term trophoblast cells revealed that EGF induces a shift towards the translatable fractions and away from the untranslated fractions. These results highlight a novel mechanism for regulation of B7 family proteins in the placenta.

Steroidogenic acute regulatory protein (StAR) and the intramitochondrial translocation of cholesterol.

The steroidogenic acute regulatory (StAR) protein regulates the rate limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. Insight into the structure and function of StAR was attained through molecular genetic studies of congenital lipoid adrenal hyperplasia, a rare disease caused by mutations in the StAR gene. Subsequent functional analysis defined two major domains within the StAR protein, the N-terminal mitochondrial targeting sequence and the C-terminus, which promotes the translocation of cholesterol between the two mitochondrial membranes. Two models of StAR's mechanism of action, (1) stimulation of cholesterol desorption from the outer mitochondrial membrane and (2) an intermembrane shuttle hypothesis, are discussed with respect to the known biochemical and biophysical events associated with the process of steroidogenesis and the structure of StAR. StAR gene expression is regulated primarily at the transcriptional level, and the roles of transcription factors that govern basal and cAMP-dependent StAR expression including SF-1, C/EBP beta, Sp1 and GATA-4 are reviewed.

Proliferation of microvascular endothelial cells in the primate corpus luteum during the menstrual cycle and simulated early pregnancy.

The objective of this study was to evaluate endothelial vs. steroidogenic cell proliferation throughout the lifespan of the primate corpus luteum during the menstrual cycle and simulated early pregnancy (CG treatment). Tissues were collected from rhesus monkeys (Macaca mulatta; n = 3/day) on days 3-4, 7, 10, 12, and 14 of the of the luteal phase and at menses during spontaneous menstrual cycles and after 1, 3, 6, or 9 days of hCG treatment beginning on day 9 of the luteal phase. Corpora lutea were snap-frozen in mounting medium for immunocytochemical and histochemical evaluation. The labeling index (percentage of positive to total nuclei) for Ki-67 antigen, a cell proliferation marker, was determined in conjunction with cell-specific markers. Immunolocalization of platelet/endothelial cell adhesion molecule-1 and von Willebrand factor in addition to histochemical staining for the Ulex europaeus agglutinin-1 (i.e. lectin)-binding site were used to identify endothelial cells. Histochemical detection of 3 beta-hydroxysteroid dehydrogenase activity was used to identify steroidogenic cells. Progesterone secretion was high on days 3-10 of the luteal phase and then declined progressively (P < 0.05) on days 12 and 14 and at menses; luteal weight followed a similar pattern, declining 2 days (i.e. day 14) after progesterone secretion. In contrast, after hCG treatment, luteal progesterone production increased (P < 0.05) 3-fold, and luteal weight was maintained. The cell proliferation index was greatest (44.5 +/- 1.9%) on days 3-4 of the luteal phase and remained high on days 7 and 10 (34.6 +/- 0.3% and 27.1 +/- 3.4%), but this was followed by a sharp decline on day 12 (9.6 +/- 2.3%), which was sustained on day 14 and at menses. After 1 day of hCG treatment, cell proliferation was less than that observed on the equivalent day of the luteal phase (day 10), but thereafter, it was similar on days 3, 6, and 9 of simulated early pregnancy to those in the late luteal phase of the menstrual cycle (i.e. day 12 to menses). Dual label immunocytochemistry indicated that more than 85% of cells staining positively for the Ki-67 antigen costained for platelet/endothelial cell adhesion molecule-1. No cells staining positively for both 3 beta-hydroxysteroid dehydrogenase activity and the Ki-67 antigen were noted. Thus, the level of cell proliferation within the primate corpus luteum varies during the luteal lifespan in the menstrual cycle, and endothelial cells comprised the vast majority of proliferative cells, whereas steroidogenically active cells were not proliferating. Further, the elevated progesterone secretion and sustained luteal weight that occurred during CG exposure simulating early pregnancy were not associated with an increase or maintenance of cellular proliferation.

Aberrant cholesterol transport and impaired steroidogenesis in Leydig cells lacking estrogen sulfotransferase.

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfoconjugation and inactivation of estrogens. It is expressed abundantly in the mammalian testes in which it may modulate the activity of locally produced estrogen. We demonstrate here that testicular Leydig cells from mice rendered deficient in EST expression by targeted gene deletion acquire a phenotype of increased cholesterol ester accumulation and impaired steroidogenesis with natural aging or in response to estrogen challenge. Abnormal accumulation of cholesterol ester in the mutant Leydig cells correlated with induced expression of the scavenger receptor type B class I, and cultured EST-deficient but not wild-type Leydig cells avidly uptook high-density lipoprotein cholesterol ester ex vivo. EST-deficient Leydig cells in culture produced 50-70% less testosterone than wild-type cells. This deficiency was reversed by androstenedione but not progesterone supplementation, indicating that reduced activities of 17-alpha-hydroxylase-17, 20-lyase were responsible. This conclusion was corroborated by decreased expression levels of 17-alpha-hydroxylase-17, 20-lyase but not of other key steroidogenic enzymes in the mutant cells. These results suggest that EST plays a physiologic role in protecting Leydig cells from estrogen-induced biochemical lesions and provide an example of critical regulation of tissue estrogen sensitivity by a ligand-transformation enzyme rather than through estrogen receptors.

Immunocytochemical localization of cytochromes P450 17 alpha-hydroxylase and aromatase in embryonic cell layers of elongating porcine blastocysts.

Localized expression of cytochromes P450 17 alpha-hydroxylase (P450c17) and aromatase (P450arom) was investigated in embryonic cell layers of elongating porcine blastocysts by immunocytochemistry. Blastocysts were flushed from the uterus on day 12 of pregnancy, fixed in paraformaldehyde, embedded in paraffin, sectioned, and stained using immunogold- and peroxidase-based techniques. Staining for both P450c17 and P450arom was intense in spherical 7- to 10-mm blastocysts, but was absent in earlier stage 2- to 4-mm blastocysts and less intense or absent in later stage 20-mm and filamentous embryos. Cytochrome P450c17 was limited to the trophoblast of all blastocysts expressing the enzyme, and in spherical 7- to 10-mm blastocysts, essentially all cells of the trophoblast layer stained positively for P450c17. However, as elongation became apparent in 10-mm blastocysts, the cells of the trophoblast became flattened, and the expression of P450c17 declined particularly in those trophoblast cells adjacent to the embryonic disc where mesoderm outgrowth was occurring. In fact, two distinct populations of trophoblast cells became obvious: one that maintained P450c17 expression, and one that did not. Moreover, those trophoblast cells expressing P450c17 were less flattened than neighboring cells in which P450c17 expression was absent. These two morphologically and functionally distinct trophoblastic cell populations were most obvious in areas furthest from the embryonic disc. Cytochrome P450arom was expressed in the trophoblast as well as the hypoblast under the embryonic disc. Neither P450c17 nor P450arom appeared to be expressed in the embryonic disc or the mesoderm of the expanding blastocyst. These functional and structural changes in the embryonic cell layers of the elongating conceptus may be associated with the transient synthesis and secretion of estrogen that occur at the time of maternal recognition of pregnancy in the pig.

Immunocytochemical localization of cytochromes P450 17 alpha-hydroxylase and aromatase in embryonic cell layers of elongating porcine blastocysts.

Localized expression of cytochromes P450 17 alpha-hydroxylase (P450c17) and aromatase (P450arom) was investigated in embryonic cell layers of elongating porcine blastocysts by immunocytochemistry. Blastocysts were flushed from the uterus on day 12 of pregnancy, fixed in paraformaldehyde, embedded in paraffin, sectioned, and stained using immunogold- and peroxidase-based techniques. Staining for both P450c17 and P450arom was intense in spherical 7- to 10-mm blastocysts, but was absent in earlier stage 2- to 4-mm blastocysts and less intense or absent in later stage 20-mm and filamentous embryos. Cytochrome P450c17 was limited to the trophoblast of all blastocysts expressing the enzyme, and in spherical 7- to 10-mm blastocysts, essentially all cells of the trophoblast layer stained positively for P450c17. However, as elongation became apparent in 10-mm blastocysts, the cells of the trophoblast became flattened, and the expression of P450c17 declined particularly in those trophoblast cells adjacent to the embryonic disc where mesoderm outgrowth was occurring. In fact, two distinct populations of trophoblast cells became obvious: one that maintained P450c17 expression, and one that did not. Moreover, those trophoblast cells expressing P450c17 were less flattened than neighboring cells in which P450c17 expression was absent. These two morphologically and functionally distinct trophoblastic cell populations were most obvious in areas furthest from the embryonic disc. Cytochrome P450arom was expressed in the trophoblast as well as the hypoblast under the embryonic disc. Neither P450c17 nor P450arom appeared to be expressed in the embryonic disc or the mesoderm of the expanding blastocyst. These functional and structural changes in the embryonic cell layers of the elongating conceptus may be associated with the transient synthesis and secretion of estrogen that occur at the time of maternal recognition of pregnancy in the pig.

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.

Follicle-stimulating hormone and luteinizing hormone/chorionic gonadotropin stimulation of vascular endothelial growth factor production by macaque granulosa cells from pre- and periovulatory follicles.

Granulosa cells in the ovulatory follicle express messenger ribonucleic acid encoding vascular endothelial growth factor (VEGF), an agent that may mediate the neovascularization of the developing corpus luteum, but it is not known whether luteinizing granulosa cells synthesize and secrete VEGF during the periovulatory interval. Studies were designed to evaluate the effects of an in vivo gonadotropin surge on VEGF production by macaque granulosa cells (study 1) and to test the hypothesis that gonadotropins act directly on granulosa cells to regulate VEGF production (study 2). Monkeys received a regimen of exogenous gonadotropins to promote the development of multiple preovulatory follicles. Nonluteinized granulosa cells (i.e. preovulatory; NLGC) and luteinized granulosa cells (i.e. periovulatory; LGC) were aspirated from follicles before and 27 h after an ovulatory gonadotropin bolus, respectively. Cells were either incubated for 24 h in medium with or without 100 ng/mL hCG (study 1) or cultured for 6 days in medium with or without 100 ng/mL hCG or 0.1, 1, 10, and 100 ng/mL of recombinant human LH (r-hLH) or r-hFSH (study 2). Culture medium was assayed for VEGF and progesterone. In study 1, LGC produced 8-fold greater levels of VEGF than NLGC (899 +/- 471 vs. 111 +/- 26 pg/mL, mean +/- SEM; P < 0.05). In vitro treatment with hCG increased (P < 0.05) VEGF production by NLGC to levels that were not different from the LGC incubated under control conditions. In vivo bolus doses of r-hCG (100 and 1000 IU) and r-hFSH (2500 IU) were equally effective in elevating granulosa cell VEGF production. In study 2, in vitro treatment with r-hFSH, r-hLH, and hCG markedly increased (P < 0.05) VEGF and progesterone production by the NLGC in a dose- and time-dependent manner. By comparison, the three gonadotropins (100 ng/mL dose) only modestly increased VEGF and progesterone production by LGC. These experiments demonstrate a novel role for the midcycle surge of gonadotropin (LH/CG or FSH) in primates to promote VEGF production by granulosa cells in the periovulatory follicle. Further, the data demonstrate that FSH-like as well as LH-like gonadotropins directly stimulate VEGF synthesis by granulosa cells.

Expression of steroidogenic acute regulatory protein in the human corpus luteum throughout the luteal phase.

The expression of the steroidogenic acute regulatory protein (StAR) in the human corpus luteum (CL) was examined throughout the luteal phase. The primary 1.6-kb StAR transcript was in greater abundance in early (3.1-fold) and mid (2.2-fold) luteal phase CL compared with late luteal phase CL. The larger StAR transcript (4.4 kb) was found in early and midluteal phase CL, but was not detected in late luteal phase specimens. Mature StAR protein (30 kDa) was present in lower amounts within late CL compared with early and midluteal phase CL. The StAR preprotein (37 kDa) was also detected in greater abundance in early and midluteal CL. Immunohistochemistry revealed that StAR staining was most prominent in thecal-lutein cells throughout the luteal phase. The intensity of the signal for StAR exhibited significant changes throughout the luteal phase, being most intense during the midluteal phase and least during the late luteal phase. Plasma progesterone concentrations were highly correlated (r = 0.73 and r = 0.79) with luteal expression of the preprotein and mature StAR isoforms, respectively, throughout the luteal phase. To examine the LH dependency of StAR expression, the GnRH antagonist, Cetrorelix, was administered during the midluteal phase. Cetrorelix caused a decline in serum LH levels within 2 h, which, in turn, caused a pronounced decline in plasma progesterone within 6 h. The StAR 4.4-kb transcript was not detectable, and the 1.6-kb transcript was reduced by approximately 50% within 24 h of Cetrorelix treatment. The mature 30-kDa StAR protein level declined approximately 30% after Cetrorelix treatment. We conclude that 1) StAR mRNA and protein are highly expressed in early and midluteal phase CL; 2) StAR protein is present in both thecal-lutein and granulosa-lutein cells throughout the luteal phase; 3) StAR protein levels in the CL are highly correlated with plasma progesterone levels; 4) declining StAR mRNA and protein levels are characteristic of late luteal phase CL; and 5) suppression of LH levels during the midluteal phase results in a marked decline in plasma progesterone and a diminished abundance of StAR transcripts in the CL without a corresponding significant decline in StAR protein. Collectively, these data are consistent with the idea that StAR gene expression is a key determinant of luteal progesterone during the normal menstrual cycle. However, the pharmacologically induced withdrawal in the midluteal phase of LH support diminishes luteal progesterone output by mechanisms others than reduced StAR protein levels.

Allelic variants of the follistatin gene in polycystic ovary syndrome.

In an earlier study of 37 candidate genes for polycystic ovary syndrome (PCOS), the strongest evidence for genetic linkage was found with the region of the follistatin gene. We have now carried out studies to detect variation in the follistatin gene and assess its relevance to PCOS. By sequencing the gene in 85 members of 19 families of PCOS patients, we found sequence variants at 17 sites. Of these, 16 sites have variants that are too rare to make a major contribution to susceptibility; the only common variant is a single base pair change in the last exon at a site that is not translated. In our sample of 249 families, the evidence for linkage between PCOS and this variant is weak. We also examined the expression of the follistatin gene; messenger RNA levels in cultured fibroblasts from PCOS and control women did not differ appreciably. We conclude that contributions to the etiology of PCOS from the follistatin gene, if any, are likely to be small.

Unveiling the mechanism of action and regulation of the steroidogenic acute regulatory protein.

Stimulation of steroid-producing cells of the gonads and adrenals with trophic hormone (LH, and ACTH, respectively) produces a marked increase in steroid hormone synthesis within minutes. The rate-limiting step in this acute steroidogenic response is the transport of cholesterol from the outer to the inner mitochondrial membrane, where the first committed step in steroid synthesis is performed by the side-chain cleavage enzyme system (P450scc), resulting in the production of pregnenolone. This process of cholesterol translocation is blocked by inhibitors of protein synthesis (i.e. cycloheximide) indicating that the effect of trophic hormones, acting through the intermediacy of cAMP, most likely involves the de novo synthesis of a protein that is rapidly inactivated. The recently identified steroidogenic acute regulatory protein (StAR) appears to be the most likely candidate for the labile protein: (1) StAR is synthesized in response to cAMP and the StAR preprotein disappears rapidly in the presence of inhibitors of protein synthesis; (2) StAR has an N-terminal targeting sequence that directs the protein to the mitochondria; and (3) StAR protein is expressed almost exclusively in steroid-producing cells, its presence is correlated with steroid hormone production, and lack of functional StAR causes the autosomal recessive disease congenital lipoid adrenal hyperplasia (lipoid CAH), characterized by markedly impaired gonadal and adrenal steroid hormone synthesis. We have demonstrated that StAR is a target for serine phosphorylation mediated by protein kinase A (PKA), a process that is essential to maximizing StAR activity. StAR import by mitochondria is not essential to its steroidogenesis enhancing activity, and more likely, represents a means of rapidly inactivating StAR. Truncation mutations and site-directed mutations in StAR demonstrated that the C-terminus of the protein contains the functionally important domains. Further, we have demonstrated potent steroidogenic activity of recombinant StAR protein on isolated mitochondria from bovine corpus luteum using protein that lacks the mitochondrial targeting sequence. These observations confirm that StAR import is not essential for its steroidogenic activity and suggest that StAR acts directly on the outer mitochondrial membrane in the absence of intermediary cytosolic factors. More recently, we have found that StAR functions as a cholesterol transfer protein that does not require a protein receptor or co-factor, suggesting that StAR acts directly on lipids of the outer mitochondrial membrane to promote cholesterol translocation.

Vascular endothelial growth factor levels in serum and follicular fluid of patients undergoing in vitro fertilization.

OBJECTIVE: To define the relationship between serum and follicular fluid (FF) levels of vascular endothelial growth factor (VEGF), E2, and P in patients undergoing IVF; to quantify the effects of hCG on serum levels of VEGF during early pregnancy, and to report serial measurements of serum and ascites fluid levels of VEGF in a patient with severe ovarian hyperstimulation syndrome (OHSS). DESIGN: Prospective observational study. SETTING: University IVF program. PATIENTS(S): Women undergoing conventional IVF, receiving donated oocytes or spontaneously conceiving. One patient hospitalized with severe OHSS. MAIN OUTCOME MEASURE(S): Concentrations of VEGF, E2, and P in serum, FF, or peritoneal fluid. RESULT(S): At the time of egg retrieval, FF VEGF concentrations were positively correlated with serum and FF P concentrations and with patient age. At 11 to 14 days after ET, pregnant recipients of autologous fresh embryos had higher serum VEGF levels than both nonpregnant recipients of autologous fresh embryos and pregnant recipients of donor eggs. Elevated serum VEGF levels in a patient with severe OHSS coincided with the clinical onset and recurrence of symptoms. CONCLUSION(S): In patients undergoing IVF, FF VEGF levels at the time of egg retrieval correlated with the degree of follicular luteinization. There is a significant ovarian contribution to circulating VEGF levels during early gestation. Elevated serum VEGF levels may be a factor in the etiology of OHSS symptoms.

Steroidogenic acute regulatory protein: an update on its regulation and mechanism of action.

Steroidogenic acute regulatory (StAR) protein controls the rate-limiting step in steroidogenesis: the transport of cholesterol from the outer to the inner mitochondrial membrane. Early studies indicated that rate of transcription of the StAR gene is a primary determinant of steroidogenesis. The transcription factors that govern basal and cAMP-dependent StAR expression are reviewed, as are new findings regarding chromatin modifications associated with activation of the StAR promoter. Molecular genetic studies of congenital lipoid adrenal hyperplasia, a rare disease caused by mutations in the StAR gene, and structure-function studies defined two major domains within the StAR protein, the N-terminal mitochondrial targeting sequence and the C-terminal StAR-related lipid transfer (START) domain, which promotes the translocation of cholesterol between the two mitochondrial membranes. Several models of StAR's mechanism of action have been proposed based on a combination of structure/function studies or on the crystal structure of a related START domain. The models-intermembrane shuttle hypothesis, and cholesterol desorption hypothesis-are discussed with respect to the known biochemical and biophysical events associated with steroidogenesis and the structure of StAR.

Evaluation of biochemical and structural changes in individual porcine corpora lutea during prostaglandin F2 alpha-induced luteolysis with an in vivo implant system.

To date, no in vitro system has been devised to allow the study of both the functional and the structural regression of luteal cells in response to prostaglandin (PG) F2 alpha. This study describes the use of a novel intraluteal PGF2 alpha implant system that results in the death of individual corpora lutea (CL), while surrounding CL on the same ovary remain fully functional. By this technique, it was possible to study both the functional and the structural regression of individual CL in vivo, without the confounding effects resulting from the systemic injection of PGF2 alpha. Biochemical measurements of individual CL included progesterone concentration, protein kinase C activity, and diacylglycerol levels. Structural measurements included luteal weight and the protein:DNA ratio, which was used to estimate cell size. Further, the determination of large luteal cell size was accomplished directly via light microscopy. Nonpregnant gilts were injected with 5 mg of estradiol benzoate every 12 hr from 8:00 a.m. on Day 11 to 8:00 a.m. on Day 13 to prevent uterine PGF2 alpha secretion. At 7:00 a.m. on Day 13, CL on one ovary were selected at random to receive PGF2 alpha-implants (n = 4) or implant material only (n = 4), whereas the remaining CL on that ovary served as unimplanted controls. The other ovary was removed at the point, and the CL on that ovary served as 0-hr controls. Gilts were relaparotomized at 3, 6, 12, and 24 hr after CL implantation, the PGF2 alpha-implanted ovary was removed, and individual CL were evaluated. PGF2 alpha-implanted CL exhibited a decline (P < 0.05) in progesterone concentrations at 12 and 24 hr and a decline (P < 0.05) in weight at 24 hr when compared with control CL (implant-only, unimplanted, and 0-hr control CL). Furthermore, the protein:DNA ratio was reduced (P < 0.10) in the PGF2 alpha-treated CL at 12 and 24 hr. Moreover, this change in the protein:DNA ratio (cell size) was consistent with the reduced diameter (P < 0.05) of the large luteal cell in the PGF2 alpha-treated CL. Protein kinase C activity and diacylglycerol concentrations did not change (P > 0.10) and therefore appear to be unassociated with either functional or structural changes in the PGF2 alpha-treated CL. Contrary to in vitro culture studies, the results of our in vivo study demonstrate no clear role for protein kinase C in the PGF2 alpha-induced luteolytic process.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of ingestion of ponderosa pine needles by late-pregnant cows on uterine blood flow and steroid secretion.

Consumption of Ponderosa pine needles by late-pregnant beef cows results in the premature delivery of a viable calf. We have demonstrated the presence of a factor(s) in plasma from cows fed pine needles that specifically increased uterine arterial tone (i.e., decreased arterial diameter) in vitro. This study was designed to investigate changes in uterine blood flow and steroid secretion/uptake by the gravid uterus of cows fed pine needles to induce premature parturition. Sixteen beef cows were laparotomized on d 240 of gestation, and an electromagnetic blood-flow probe was placed around the uterine artery supplying the gravid horn. Cows were randomly assigned on d 250 of gestation to a control (n = 8; 8.2 kg/d of alfalfa hay) or pine needle (n = 8; 2.7 kg/d of pine needles + 5.5 kg/d of alfalfa hay) diet. Uterine blood flow was monitored, and systemic blood (uterine arterial and[or] jugular venous) and uterine venous blood samples were collected daily between 0630 and 0800, just before feeding. Five of eight cows fed pine needles calved prematurely (average day of gestation = 260.2 +/- .6) compared with cows fed the control diet, which calved on 287.6 +/- 3.4 d of gestation. Uterine blood flow in the control cows remained constant from d 250 through the day of parturition. In contrast, uterine blood flow of cows fed pine needles that calved early decreased progressively (P less than .01), declining to 25.2% of its original value by the day of parturition.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigations into the control of litter size in swine: III. A reciprocal embryo transfer study of early conceptus development.

A reciprocal embryo transfer study with Meishan and Yorkshire pigs was conducted to examine the influence of embryonic genotype and uterine environment on preimplantation embryonic growth and development. Embryos were collected from gilts on d 2 of a spontaneous estrous cycle (d 0 = onset of estrus), transferred to synchronous recipients, and collected from recipients on d 12 to obtain measurements of embryonic diameter (size) and embryonic DNA, protein, and estrogen content. No difference was detected between Meishan and Yorkshire donors for number of ovulations, number of embryos recovered, or number of cells per embryo. Embryonic genotype affected (P < .01) d-12 embryonic characteristics; Meishan embryos (n = 101) were smaller (4.7 mm diameter) and contained less DNA (4.5 micrograms) and protein (104 micrograms) than Yorkshire embryos (n = 85; 5.9 mm, 6.1 micrograms, and 149 micrograms, respectively). Embryos (n = 80) transferred into a Meishan uterus were reduced (P < .001) in diameter (4.2 vs 6.4 mm) and in DNA (3.2 vs 7.2 micrograms), protein (103.8 vs 149 micrograms), and estrogen (352 vs 1,643 pg) content compared with embryos (n = 106) transferred into a Yorkshire uterus. These data indicate that the increased prolificacy of the Meishan breed may be due to an increased embryonic survival resulting from slower growing embryos and a suppressive effect of the uterus on embryonic growth rate and estrogen secretion.

Effects of Ponderosa pine needle ingestion of uterine vascular function in late-gestation beef cows.

Consumption of Ponderosa pine (Pinus ponderosa) needles (PN) by beef cows during late gestation results in premature delivery in association with profound constriction of the caruncular arterial bed. Further, PN extracts and plasma from PN-fed cows increase uterine arterial tone in vitro. Uterine arterial tone is a measure of the arterial resistance to stretch and controls the baseline rate of flow through the vascular bed. Uterine arterial tone results from the uptake of extracellular Ca2+ into smooth muscle cells through specific membrane channels called potential sensitive channels. Functional potential sensitive channels remain open for prolonged periods after activation, allowing a continuous uptake of Ca2+ and the maintenance of uterine arterial tone. Recent evidence from our laboratory has demonstrated that a group of estrogen metabolites produced by the placenta and(or) endometrium, called catechol estrogens, inhibits Ca2+ uptake through the potential sensitive channels. During gestation, progressive decreases in uterine arterial tone are observed, with resultant increases in uterine arterial blood flow. Thus, the continuous production of catechol estrogens may be necessary to maintain the pronounced uterine vasodilation that is required for fetal survival. Ponderosa pine needle extracts exhibit antiestrogenic activity, as evidenced by their inhibition of estrogen-induced uterine hyperemia. Data from our laboratory show that after consumption of PN by beef cows during late gestation, uterine arterial blood flow progressively decreased to less than 50% of prefeeding rates before premature delivery of a live calf.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific effects of blood plasma from beef cows fed pine needles during late pregnancy on increasing tone of caruncular arteries in vitro.

Consumption of Ponderosa pine needles by cows during late pregnancy results in the premature delivery of viable calves. Previous observations made at necropsy suggested that vascular insufficiency in the caruncular bed was associated with this induced parturition. The present studies used the perfused caruncular arterial bed of an isolated bovine placentome to determine whether blood plasma collected from cows fed pine needles contained a vasoactive factor(s). Changes in caruncular arterial tone (i.e., long-term changes in vessel diameter) and phasic contractility (i.e., transient reductions in vessel diameter) were evaluated. Tone was quantified as the pressure exerted against a constant intraluminal flow and as the force of contraction during K(+)-stimulated depolarization of membranes. Phenylephrine, an alpha 1-adrenergic agonist, was used to stimulate a phasic contractile response. Jugular blood was collected from beef cows fed pine needles (n = 4) or a control diet (n = 4), during the last 3 d of gestation, and plasma was used for two studies. In Study 1, placentomes were perfused with decreasing dilutions (1:150 to 1:40) of plasma pooled over the last 3 d of pregnancy from cows fed pine needle or control diets. Caruncular arterial perfusion pressures and responses to K+ progressively increased (P less than .05) with decreasing dilutions of plasma from cows fed pine needles, and responses to phenylephrine remained constant. Caruncular arterial perfusion pressures and responses to K+ and phenylephrine remained constant during perfusion of plasma from cows fed the control diet. In Study 2, the changes in vasoactivity of plasma (1:60 dilution) from cows fed pine needles was evaluated during the last 3 d of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)