Immunohistochemistry and in situ hybridization of P-45017 alpha (17 alpha-hydroxylase/17,20-lyase).

Cytochrome P-45017 alpha catalyzes both 17 alpha-hydroxylation and 17,20-side-chain cleavage in steroidogenesis and lies at a key branch point in the pathways of steroid hormone biosynthesis. To obtain information on the precise localization of P-45017 alpha in swine testis, ovary, and adrenal, we undertook the simultaneous detection of P-45017 alpha mRNA and protein by combining immunohistochemistry with in situ hybridization. In situ hybridization was performed on 4% paraformaldehyde-fixed, paraffin-embedded sections by employing either a 39-base oligomer or a cDNA insert (1.7 KB) of porcine testis P-45017 alpha as DNA probe. Immunohistochemical study was performed by employing anti-P-45017 alpha. Hybridization signals were obtained in Leydig cells of the testis, theca interna of the ovarian follicle, and zona fasciculata reticularis cells of the adrenal cortex. Oligonucleotide probing yielded lower background signal than the cDNA probe. No specific signals were obtained in seminiferous tubules of the testis, medulla, and zona glomerulosa of the adrenal, and in membrana granulosa and interstitial cells of the ovary. Hybridization signals were obtained in the cells where immunoreactivity of the enzyme was observed by immunohistochemistry, except for some Leydig cells of the testis and theca interna cells of the ovary in which only immunoreactivity but not hybridization signal was observed. The present study provided detailed information about the precise cellular localization of P-45017 alpha expression at both the protein and mRNA levels in swine adrenal glands and gonads. This approach of simultaneous immunohistochemistry and in situ hybridization analysis of steroidogenic enzymes can be applied in the future to tissues exhibiting abnormal steroid metabolism and should contribute to a better understanding of steroidogenesis.     

Direct effect of Pa(CO2) on respiratory sinus arrhythmia in conscious humans

Respiratory sinus arrhythmia (RSA) may improve the efficiency of pulmonary gas exchange by matching the pulmonary blood flow to lung volume during each respiratory cycle. If so, an increased demand for pulmonary gas exchange may enhance RSA magnitude. We therefore tested the hypothesis that CO2 directly affects RSA in conscious humans even when changes in tidal volume (V(T)) and breathing frequency (F(B)), which indirectly affect RSA, are prevented. In seven healthy subjects, we adjusted end-tidal PCO2 (PET(CO2)) to 30, 40, or 50 mmHg in random order at constant V(T) and F(B). The mean amplitude of the high-frequency component of R-R interval variation was used as a quantitative assessment of RSA magnitude. RSA magnitude increased progressively with PET(CO2) (P < 0.001). Mean R-R interval did not differ at PET(CO2) of 40 and 50 mmHg but was less at 30 mmHg (P < 0.05). Because V(T) and F(B) were constant, these results support our hypothesis that increased CO2 directly increases RSA magnitude, probably via a direct effect on medullary mechanisms generating RSA.     

Developmental roles of the steroidogenic acute regulatory protein (StAR) as revealed by StAR knockout mice

Steroidogenic acute regulatory protein (StAR) is essential for adrenal and gonadal steroidogenesis, stimulating the translocation of cholesterol to the inner mitochondrial membrane where steroidogenesis commences. StAR mutations in humans cause congenital lipoid adrenal hyperplasia (lipoid CAH), an autosomal recessive condition with severe deficiencies of all classes of steroid hormones. We previously described StAR knockout mice that mimic many features of lipoid CAH patients. By keeping StAR knockout mice alive with corticosteroid replacement, we now examine the temporal effects of StAR deficiency on the structure and function of steroidogenic tissues. The adrenal glands, affected most severely at birth, exhibited progressive increases in lipid deposits with aging. The testes of newborn StAR knockout mice contained scattered lipid deposits in the interstitial region, presumably in remnants of fetal Leydig cells. By 8 weeks of age, the interstitial lipid deposits worsened considerably and were associated with Leydig cell hyperplasia. Despite these changes, germ cells in the seminiferous tubules appeared intact histologically, suggesting that the StAR knockout mice retained some capacity for androgen biosynthesis. Sperm maturation was delayed, and the germ cells exhibited histological features of apoptosis, consistent with suboptimal androgen production. Immediately after birth, the ovaries of StAR knockout mice appeared normal. After the time of normal puberty, however, prominent lipid deposits accumulated in the interstitial region, accompanied by marked luteinization of stromal cells and incomplete follicular maturation that ultimately culminated in premature ovarian failure. These studies provide the first systematic evaluation of the developmental consequences of StAR deficiency in the various steroidogenic organs.     

Immunohistochemical localization of type I collagen, fibronectin and tenascin C during embryonic osteogenesis in the dentary of mandibles and tibias in rats

Type I collagen, fibronectin and tenascin C play an important role in regulating early osteoblast differentiation, but the temporal and spatial relationship of their localization during embryonic osteogenesis in vivo is not known. The present study was designed to localize these three molecules in the dentary of mandibles and tibias in rat embryos using immunohistochemistry. Serial paraffin sections were cut and adjacent sections were processed for von Kossa staining or immunohistochemistry for type I collagen, fibronectin and tenascin C. In the dentary, tenascin C was localized within and around the mesenchymal cell condensation in embryos at 14 days in utero. The bone matrix at 15 days showed immunoreactivity for both type I collagen and fibronectin. The immunoreactivity of type I collagen was persistent, whereas that of fibronectin decreased with age of embryos. In tibias, tenascin C was localized in the perichondral mesenchymal tissue at 17 days. Immunoreactivity for type I collagen was persistent in the bone matrix, whereas the tibial bone showed little immunoreactivity for fibronectin at any embryonic age examined. The present study demonstrated characteristic localization of type I collagen, fibronectin and tenascin C during embryonic osteogenesis in the dentary of mandibles and tibias.     

Breast cancer aromatase expression evaluated by the novel antibody 677: Correlations to intra-tumor estrogen levels and hormone receptor status

Breast cancer tissue estrogen levels on an average exceed plasma as well as benign breast tissue levels. To evaluate the contribution of intra-tumor aromatization to individual tumor estrogen levels (estradiol, E₂; estrone, E₁; estrone sulfate, E₁S), breast cancer tissue sections obtained during mastectomy in 28 postmenopausal breast cancer patients were stained for aromatase protein expression using the aromatase antibody 677. The findings were correlated to intra-tumor estrogen levels determined with a highly sensitive HPLC-RIA. Staining with 677 alone (irrespective of the hormone receptor status) revealed no difference in tumor E₂ levels comparing 677+ versus 677? tumors, although a non-significant trend towards higher tumor E₁ and E₁S levels was observed in 677+ breast cancers. In contrast, tumor levels of E₂ were significantly higher in ER+ tumors compared to ER? tumors (P < 0.001) and to benign breast tissue from the same breast (P < 0.001). Analysing the additional effect of positive staining with the aromatase antibody 677 on tumor estrogen levels in the subgroup of ER+ tumors, revealed significantly higher tumor levels of E₂ (mean level of 544.7 versus 197.1 fmol/g tissue) as well as a non-significant trend concerning tumor E₁ (mean level of 296.9 versus 102.1 fmol/g tissue). The mean tumor tissue E₁S level was observed somewhat lower in ER+677+ (103.5 fmol/g) versus ER+677? tumors (190.1 fmol/g). In the subgroup of ER+PgR+ tumors, tissue levels of E₂ were also found to be significantly higher among 677+ compared to 677? tumors: 873.2 fmol/g (95% CI 395.9–1925.6) versus 217.9 fmol/g (95% CI 88.8–534.9) (P = 0.015).In conclusion, our results indicate a moderate effect of aromatase enzyme expression evaluated by IHC using the antibody 677 on intra-tumor estrogen levels among ER+ breast cancers. A substantial interindividual variation in the ratios between the individual estrogen fractions suggests additional effects, like alterations in other enzymes to be involved in the intra-tumor estrogen homeostasis.     

Controversies of aromatase localization in human breast cancer--stromal versus parenchymal cells

Aromatase is a key enzyme of estrogen production through conversion from serum androgens in estrogen-dependent postmenopausal breast cancer. Aromatase has been reported to be predominantly located in intratumoral stromal cells and adipocytes but not in parenchymal or carcinoma cells in breast cancer tissue. It is, however, true that there have been controversies regarding intratumoral localization of aromatase in human breast carcinoma, especially whether intratumoral production of estrogens through aromatase occurs in parenchymal or stromal cells. Results of several studies suggested that aromatase present in parenchymal carcinoma cells plays more important roles in the growth and invasion of breast carcinomas than that in stromal cells through providing higher levels of estrogens to carcinoma cells. Aromatase inhibitors are increasingly being used in place of tamoxifen after results of various clinical trials demonstrated that aromatase inhibitors are more effective in increasing survival and recurrence of estrogen-dependent breast cancer patients. Therefore, it is important to clarify the estrogen supplying pathway by aromatase inside of breast carcinoma tissues in order to evaluate the possible efficacy of aromatase inhibitor treatment. In this review, the controversies regarding these intratumoral localization patterns in human breast carcinoma will be briefly summarized.     

Glucagon signalling in the dorsal vagal complex is sufficient and necessary for high‐protein feeding to regulate glucose homeostasis in vivo

High‐protein feeding acutely lowers postprandial glucose concentration compared to low‐protein feeding, despite a dichotomous rise of circulating glucagon levels. The physiological role of this glucagon rise has been largely overlooked. We here first report that glucagon signalling in the dorsal vagal complex (DVC) of the brain is sufficient to lower glucose production by activating a Gcgr–PKA–ERK–K_ATP channel signalling cascade in the DVC of rats in vivo. We further demonstrate that direct blockade of DVC Gcgr signalling negates the acute ability of high‐ vs. low‐protein feeding to reduce plasma glucose concentration, indicating that the elevated circulating glucagon during high‐protein feeding acts in the brain to lower plasma glucose levels. These data revise the physiological role of glucagon and argue that brain glucagon signalling contributes to glucose homeostasis during dietary protein intake.