Snorc is a novel cartilage specific small membrane proteoglycan expressed in differentiating and articular chondrocytes.

OBJECTIVE: Maintenance of chondrocyte phenotype is a major issue in prevention of degeneration and repair of articular cartilage. Although the critical pathways in chondrocyte maturation and homeostasis have been revealed, the in-depth understanding is deficient and novel modifying components and interaction partners are still likely to be discovered. Our focus in this study was to characterize a novel cartilage specific gene that was identified in mouse limb cartilage during embryonic development. METHODS: Open access bioinformatics tools and databases were used to characterize the gene, predicted protein and orthologs in vertebrate species. Immunohistochemistry and mRNA expression methodology were used to study tissue specific expression. Fracture callus and limb bud micromass culture were utilized to study the effects of BMP-2 during experimental chondrogenesis. Fusion protein with C-terminal HA-tag was expressed in Cos7 cells, and the cell lysate was studied for putative glycosaminoglycan attachment by digestion with chondroitinase ABC and Western blotting. RESULTS: The predicted molecule is a small, 121 amino acids long type I single-pass transmembrane chondroitin sulfate proteoglycan, that contains ER signal peptide, lumenal/extracellular domain with several threonines/serines prone to O-N-acetylgalactosamine modification, and a cytoplasmic tail with a Yin-Yang site prone to phosphorylation or O-N-acetylglucosamine modification. It is highly conserved in mammals with orthologs in all vertebrate subgroups. Cartilage specific expression was highest in proliferating and prehypertrophic zones during development, and in adult articular cartilage, expression was restricted to the uncalcified zone, including chondrocyte clusters in human osteoarthritic cartilage. Studies with experimental chondrogenesis models demonstrated similar expression profiles with Sox9, Acan and Col2a1 and up-regulation by BMP-2. Based on its cartilage specific expression, the molecule was named Snorc, (Small NOvel Rich in Cartilage). CONCLUSION: A novel cartilage specific molecule was identified which marks the differentiating chondrocytes and adult articular chondrocytes with possible functions associated with development and maintenance of chondrocyte phenotype.

Calcium and vitamin D status in the adolescent: key roles for bone, body weight, glucose tolerance, and estrogen biosynthesis.

This review paper highlights a number of important public health issues related to calcium and vitamin D status in adolescents. Dietary calcium intake has declined dramatically over the past several decades among adolescents, and inadequate serum vitamin D levels have been documented in up to 54% of teens. A recent trend of decreasing consumption of dairy foods, especially milk, has contributed to this problem. Calcium and vitamin D are critically important for bone mineral accrual during adolescence, and altered calcium homeostasis can impact optimal bone acquisition. Serum and cellular calcium concentrations are controlled, in part, by the actions of vitamin D. Newer research seeks to clarify the potential functions of calcium and vitamin D in the regulation of body weight, glucose tolerance, and ovarian function. Numerous observational studies have noted an inverse association between body weight, percent body fat, and dietary calcium intake; however, clinical trials evaluating the affect of increased calcium on weight loss have been mixed. There is a reduced incidence of insulin resistance syndrome with increasing dairy intake in overweight individuals, and serum 25 hydroxyvitamin D levels are positively correlated with insulin sensitivity. Vitamin D receptor is expressed in all calcium-regulated tissues, including the ovary; thus, calcium and vitamin D appear to be necessary for full ovarian function. This review paper will examine the important role of vitamin D and calcium in the regulation of bone, weight, glucose tolerance, and estrogen biosynthesis.

Embryo development and establishment of pregnancy after embryo transfer in pigs: coping with limitations in the availability of viable embryos.

Embryo transfer and pregnancy maintenance strategies in pigs were evaluated with reference to situations in which limited numbers of viable embryos or micromanipulated embryos are available, such as pig cloning. Development of embryos with compromised zona pellucida was compared with development of embryos with intact zona pellucida. Micromanipulation had no effect on blastocyst production rates after development in vivo or in vitro, but development in vivo improved the number of embryos reaching the blastocyst stage. Transfer of embryos with compromised zona pellucida resulted in live piglets. Several hormone treatments to maintain pregnancy were tested in a model in which three embryos were transferred into unmated recipient gilts, compared with transfer of three embryos into mated recipients. None of the hormonal treatments resulted in pregnancy rates of more than 25% at term and no more than 9% of transferred embryos survived, in comparison with 50% of the mated recipients successfully carrying 25% of transferred embryos. Lastly, the developmental potential of parthenogenetic embryos was assessed and 62% of transferred embryos resulted in pregnancies, none of which continued beyond day 55 of gestation. After co-transfer of three fertilized embryos with 55-60 parthenogenetic embryos into each of six recipients, two live piglets were delivered. The results from the present study indicate that transfer of zona pellucida compromised embryos can yield litters of normal piglets. In addition, it was demonstrated in a model system involving the transfer of three fertilized embryos into mature gilts that hormonal pregnancy maintenance strategies support a low proportion of embryos to term. Lastly, the present study shows for the first time a comparably effective but novel alternative for pregnancy maintenance in the pig involving the co-transfer of parthenote embryos.

Evaluation of gestational deficiencies in cloned sheep fetuses and placentae.

Sheep fetal development at 35 days of gestation was examined following natural mating, in vitro production (IVP) of fertilized embryos, or somatic cell nuclear transfer (NT). Five crossbred (Blackface x Black Welsh) and four purebred (Black Welsh) fetuses and their associated placentae produced by natural mating were morphologically normal and consistent with each other. From 10 ewes receiving 21 IVP embryos, 17 fetuses (81%) were recovered, and 15 of these (88%) were normal. The NT fetuses were derived from two Black Welsh fetal fibroblast cell lines (BLW1 and 6). Transfer of 21 BLW1 and 22 BLW6 NT embryos into 12 and 11 ewes, respectively, yielded 7 (33%) and 8 (36%) fetuses, respectively. Only three (43%) BLW1 and two (25%) BLW6 NT fetuses were normal, with the rest being developmentally retarded. The NT fetal and placental deficiencies included liver enlargement, dermal hemorrhaging, and lack of placental vascular development reflected by reduced or absent cotyledonary structures. Fibroblasts isolated from normal and abnormal cloned fetuses did not differ in their karyotype from sexually conceived fetuses or nuclear donor cell lines. Our results demonstrate that within the first quarter of gestation, cloned fetuses are characterized by a high incidence of developmental retardation and placental insufficiency. These deficiencies are not linked to gross defects in chromosome number.

Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at -196 C.

We have previously demonstrated that ovarian function and fertility can be preserved in sheep after castration by autotransplantation of cryopreserved strips of ovarian cortex. In the current experiments we have investigated the long term survival of such grafts by detailed measurements of ovarian function for a period of nearly 2 yr after autotransplantation. After ovariectomy and transplantation of frozen/thawed grafts, the concentrations of FSH and LH rose to castrate levels for about 14 weeks before falling gradually to reach near-normal levels at about 60 weeks. In the breeding season from October 1994 to March 1995, all ewes had 5-10 estrous cycles that were similar in length to those in the 4 control ewes. Luteal function as indicated by the progesterone concentration was identical before and 11 months after transplantation. In contrast, the basal concentrations of FSH and LH were persistently raised throughout the luteal phase, but showed a normal decline during the follicular phase. The concentration of inhibin A in ovarian venous plasma measured at the end of the experiment 22 months after transplantation was significantly lower than that in control ewes (mean +/- SE, 409 +/- 118 vs. 1914 +/- 555 pg/ml; P < 0.004). Transplantation of frozen/thawed ovarian tissue to SCID mice demonstrated that about 28% of primordial follicles survived the procedure. All of the ovaries transplanted into sheep contained large antral follicles and/or cysts, but very few primordial oocytes when recovered at autopsy after 22 months. These results demonstrate that despite a drastic reduction in the total number of primordial follicles, cyclical ovarian function is preserved in sheep after autotransplantation of frozen/thawed ovarian tissue and provide experimental confirmation that such a technique could provide a means of preserving fertility in women undergoing chemo- or radiotherapy for malignant disease.

Direct effects of ovine follicular fluid on ovarian steroid secretion and expression of markers of cellular differentiation in sheep.

The objective of this study was to assess the effect of ovine follicular fluid (FF) treatment (with or without FSH replacement) during the late follicular phase on plasma concentrations of gonadotrophins and the development of the ovulatory follicle. Ovarian steroid secretion and expression of mRNA encoding inhibin alpha and beta A, beta B subunits, P450 aromatase and P450 17 alpha-hydroxylase were used as endpoints. After induction of luteolysis by injection of 100 micrograms cloprostenol on days 10-12, Scottish Blackface ewes were allocated to one of three groups: (1) control (n = 7): no further treatment; (2) FF (n = 9): subcutaneous injections of 3 ml steroid-free ovine follicular fluid at 9 h intervals, 18 and 27 h after cloprostenol injection; (3) FF + FSH (n = 8): injections of follicular fluid as above plus subcutaneous injections of 0.36 iu ovine FSH at 6 h intervals, 18, 24, and 30 h after cloprostenol injection. Jugular venous blood samples were obtained via indwelling cannulae at 6 h intervals from 0 to 36 h after cloprostenol injection, and at 10 min intervals from 12 to 18 h (control phase) and from 30 to 36 h after cloprostenol injection (treatment phase). At laparotomy, 36 h after cloprostenol injection, ovarian venous blood was collected and ovaries were removed and processed for in situ hybridization. Plasma concentrations of FSH, luteinizing hormone (LH) and oestradiol were determined by radioimmunoassay. Follicular fluid treatment resulted in a decrease (P < 0.001) in FSH concentrations associated with an acute decrease in ovarian steroid secretion (P < 0.01) and a specific depression in P450 aromatase, (P < 0.001), inhibin-activin beta B subunit (P < 0.05) and thecal LH receptor (P < 0.001) expression. Follicular fluid treatment had no effect on inhibin-activin alpha and beta A, subunit or P450 17 alpha-hydroxylase expression. FSH co-treatment with follicular fluid restored circulating FSH concentrations to normal values and reversed some of the effects of follicular fluid (androstenedione, testosterone and progesterone secretion, and inhibin beta B and thecal LH receptor expression) but not oestradiol secretion or P450 aromatase expression. It was concluded that the actions of follicular fluid are mediated via both central effects on pituitary FSH secretion and by direct ovarian effects on granulosa cell aromatase activity. The results indicate that follicular fluid contains a factor that inhibits aromatase activity of granulosa cells directly and may play a role in the selection of the dominant follicle.

Randomised controlled trial of sucrose by mouth for the relief of infant crying after immunisation.

OBJECTIVES: To evaluate the effect of sucrose solution given by mouth on infant crying times and measures of distress in the immunisation clinic. DESIGN: Randomised, double blind, placebo controlled trial of sucrose solution 75% wt/vol v sterile water as a control. SETTING: The immunisation clinic of the Women's and Children's Hospital, Adelaide. PATIENTS: A total of 107 healthy infants attending for 2, 4, or 6 month immunisations with polio by mouth (Sabin), intramuscular diphtheria, tetanus, and pertussis (DTP), and intramuscular Haemophilus influenzae type b were randomised to receive 2 ml 75% sucrose solution or sterile water by mouth before the two injections. METHODS: The duration of infant crying was recorded during and immediately after two intramuscular immunisations and infant distress was assessed by a visual analogue scale (Oucher scores) independently by a nurse and a parent. RESULTS: The administration of 2 ml 75% sucrose solution by mouth reduced the infant crying time and Oucher distress scores after immunisation with DTP/H influenzae type b. CONCLUSIONS: Infant immunisation by intramuscular injection is a distressing procedure for infants and parents. Sucrose solution at a high concentration reduces infant distress and is safe and clinically useful in this setting.

Morphological and molecular characteristics of living human fetuses between Carnegie stages 7 and 23: developmental stages in the post-implantation embryo.

Determination of embryonic age groups or stages has been based on the Carnegie Institute collection started in 1887. Improved technology has enabled the building of a new collection of embryos of < 9 weeks gestation; these were then used to compare with the original Carnegie collection. The results suggest that in providing definitive stages that are rigidly bound by developmental events, limitations are placed on categorizing the embryo. Allocation of embryos to a specific stage can assist in identifying post-ovulatory age but overlaps between stages could lead to classification into an incorrect stage.

Morphological and molecular characteristics of living human fetuses between Carnegie stages 7 and 23: ultrasound scanning and direct measurements.

The developmental age of an embryo in the first trimester of pregnancy is generally determined by ultrasound scanning and/or by calculation from menstrual age. In the original studies, validation of the estimate of gestational age by ultrasound was not possible as the exact date of conception was unknown. Variation in growth rates of identically aged fetuses has previously been reported after assisted conception and with the use of ultrasound scanning. As these pregnancies were ongoing the accuracy of the scanning results could not be determined. Comparison of scanning and direct measurements after termination of pregnancy and menstrual age were carried out to determine the accuracy in fetal dating. The results suggest that the use of ultrasound scanning to determine gestational age is of less use than previously thought, and that the use of menstrual age is severely limited.

Morphological and molecular characteristics of living human fetuses between Carnegie stages 7 and 23: immunolocalization of inhibin alpha and beta a subunits.

Transforming growth factor (TGF) is known to have the ability to modify mitogenic responses of tissues to other peptide growth factors and therefore may contribute to the rapid growth rate of an embryo. Throughout the TGF superfamily there is a similar fundamental molecular architecture. Included in this superfamily are inhibin A, activin A and activin B. It has been shown that activin is a powerful mesodermal inducing factor in the early embryo. The human embryo has shown localization of inhibin in the gonads after 16 weeks gestation but it has not been previously identified in earlier embryos. The inhibin-activin protein was found in a range of tissues including the liver stages 19-21 (alpha) and stages 19-22 (beta); oesophagus stages 19-22 (alpha and beta); stomach stages 21 and 22 (alpha and beta); gut stages 16-22 (alpha) and 21 and 22 (beta); pericardium stages 12-22 (alpha and beta); gonad stages 21 and 22 (beta) stage 22 (alpha); adrenal stages 19-22 (alpha and beta); urogenital system stages 21 and 22 (alpha and beta); yolk sac stage 12 (alpha and beta); mesenchyme stages 16-22 (alpha); surface ectoderm stages 13-22 (alpha) and stages 16-22 (beta a); notocord stages 13-22 (beta) and stages 21 and 22 (alpha); nasal, trachea and bronchi stages 19-22 (alpha and beta) leading to speculation of the role of both subunits.

Morphological and molecular characteristics of living human fetuses between Carnegie stages 7 and 23: localization of inhibin mRNA alpha and beta a subunits by in-situ hybridization.

Localization of the mRNA alpha and beta a subunits of inhibin has previously been reported in the human gonads during the second trimester. Adrenal inhibin has also been reported in the second trimester for the alpha, beta a and beta b subunits. Investigations showing localization by in-situ hybridization during the first trimester have not been reported. The results have shown hybridization of the alpha and beta a subunits, throughout the period of development studied, in a variety of tissues including the dorsal and thoracic aortas and pericardium stages 13-22 (beta a subunit); liver stages 19-21 (beta a) and stages 21-22 (alpha); mesonephos stages 21 and 22 (beta a); gonad stages (alpha and beta a); adrenal stages 19-22 (alpha); surface ectoderm stages 16-22 (beta a); mesenchyme stages 16-22 (beta a); amnion stages 13-16 (beta a); yolk sac stage 12 (alpha and beta a); cartilage stages 19-22 (beta a); and nasal proliferation stages 21 and 22 (beta a). When compared with distribution of the protein subunits it was noted that more immunostaining activity was found, suggesting that that probes were not sufficiently sensitive enough to detect all levels of mRNA expressed. It can be surmised, therefore, that the lack of visual hybridization of the mRNA cannot preclude the possibility that it is not being translated within the tissue even though hybridization was not apparent.

Expression of inhibin alpha- and beta A-subunit mRNA and protein in the fetal sheep ovary throughout gestation.

In adult sheep, inhibin expression in developing follicles appears to be associated with antrum formation. Our objective was to investigate using in situ hybridization and immunohistochemistry whether antral follicles present before birth in the sheep expressed mRNA or peptide for inhibin alpha- and beta A-subunits. At days 70 and 100 when only primordial and primary follicles were present, there was no detectable mRNA or peptide for either inhibin subunit. By days 130 and 140 (term = 145 days), many secondary follicles were present, a proportion of which (approximately 50%) expressed detectable levels of alpha-subunit mRNA but not peptide. A number of antral follicles were present by this stage, all of which expressed alpha-subunit mRNA and peptide. Expression of beta A-subunit mRNA and peptide was undetectable at all stages of gestation. Our results indicates that even in non-ovulatory follicles present before birth, expression of inhibin, at least the alpha-subunit, is developmentally linked with antrum formation.

Evaluation of maternal serum immunoreactive inhibin as a first trimester marker of Down's syndrome.

BACKGROUND AND OBJECTIVE: Maternal serum immunoreactive inhibin has been shown to be significantly elevated in Down's affected pregnancies in the second trimester, suggesting that it may be useful in prenatal diagnosis. We have investigated whether it is similarly elevated in the first trimester. DESIGN: Stored maternal sera from women with Down's affected pregnancies and chromosomally normal control pregnancies were retrieved for analysis. These sera had been collected prospectively at either 11 or 12 weeks gestation as a routine antenatal booking procedure. SUBJECTS: From records, 11 women were identified as having had a Down's pregnancy. For each of these, 4 controls matched for gestation and duration-of-storage were also identified. MEASUREMENTS: Two different inhibin immunoassays were evaluated, one using an antibody raised against 31 kDa bovine inhibin and the other, a commercial two-site assay, using two antibodies directed against two distinct alpha-subunit epitopes. RESULTS: Neither assay detected a significant effect of gestation on serum inhibin levels. After combining the data from both gestations, no significant difference between the Down's samples and controls for either assay was detected. However, analysis of the data for each gestation separately revealed that one assay detected a significant difference in inhibin levels between Down's affected and unaffected pregnancies at 11 weeks gestation (mean +/- SEM 3186 +/- 195 vs 2020 +/- 172 ng/l, P < 0.01) but not at 12 weeks. The other, commercial, assay did not detect a significant difference at either gestation. In addition, there was poor association between the results of the two assays. CONCLUSIONS: These data suggest that immunoreactive inhibin, as detected by these assays, will not be useful as a late first trimester marker for Down's syndrome and also that these two assays detect different inhibin species in pregnancy serum.

Midtrimester hemoperitoneum caused by placenta percreta in association with elevated maternal serum alpha-fetoprotein level.

We report a case of hemoperitoneum in the second trimester due to placenta percreta which was associated with an elevated maternal serum alpha-fetoprotein. A 29-year-old woman, gravida 4, para 1-0-2-1, was seen at 17 weeks' gestation with an acute abdomen. Maternal serum alpha-fetoprotein in a sample drawn 1 week previously revealed a value of 5.0 multiples of the median. At laparotomy, placenta percreta was discovered. This case of placenta percreta diagnosed in the second trimester was associated with an elevated maternal serum alpha-fetoprotein level. Physicians counseling patients with unexplained elevated maternal serum alpha-fetoprotein levels should include placenta accreta or percreta in the differential diagnosis and should maintain an awareness of its existence in patients with acute abdomen in pregnancy.

Cytogenetic analysis of unfertilized human oocytes.

Cytogenetic studies were carried out on 180 oocytes that appeared unfertilized after in-vitro fertilization. The majority of the 135 that were informative had grossly haploid second meiotic metaphases, two were grossly diploid, and five had a variety of different abnormalities. Twenty-one oocytes were abnormally fertilized and included prematurely condensed sperm chromosomes. The frequency of this phenomenon varied according to the stimulation protocol, those oocytes maturing longer in vivo showing less propensity to abnormal fertilizations. Thirteen per cent of the analysable haploid metaphases were hyperhaploid but none contained extra whole chromosomes. The extra components were a single chromatid (one case), or two single chromatids replacing a whole chromosome (four cases). The data suggest that the chromatids arose as a result of premature centromere division at meiosis I, and that this may be a major mechanism for trisomy formation rather than non-disjunction of whole bivalents at meiosis I, as generally believed.

Comparison of the accuracy of glucose reflectance meters in pregnant insulin-dependent diabetics.

Home monitoring of blood glucose by reflectance meters has been shown to be accurate in the nonpregnant diabetic and is currently used for outpatient glucose control in the pregnant diabetic as well. Beckman ASTRA glucose results from samples collected into sodium fluoride were used as the standard for this study. Comparisons were then made to four glucose reflectance meters: Accu-Check II, One Touch, DiaScan S, and ExacTech. Although the reflectance meters appeared to be useful for assessing blood glucose trends in the pregnant diabetic, the results obtained from these meters would be unacceptable in the laboratory setting. Unfortunately, because of the erratic combination of proportional and constant bias, correction factors are not easily ascertained. Laboratories and physicians should reconsider the use of these reflectance meters for inpatient evaluation and general population screening of pregnant women.

Elevation of urinary norepinephrine/cortisol ratio in posttraumatic stress disorder.

We have previously reported the unusual combination of low urinary free cortisol levels with high urinary norepinephrine excretion in posttraumatic stress disorder (PTSD) patients in comparison with four other patient groups: major depressive disorder, endogenous type; bipolar I, manic; paranoid schizophrenia; undifferentiated schizophrenia. Cortisol levels alone did not distinguish PTSD from paranoid schizophrenia patients and norepinephrine levels alone did not distinguish PTSD from bipolar I, manic, patients. In further consideration of these findings, we have found that combining the values for the two systems in a norepinephrine/cortisol (N/C) ratio provides a measure that significantly distinguishes PTSD from all the other patient groups throughout the hospitalization period. The N/C ratio was more than twice as high in the PTSD group than in all the other patient groups in the first sample following hospital admission, in the mean sample during hospitalization, and in the last sample before discharge. The mean N/C ratio for the PTSD group was 2.54, compared with a mean of .99 for the other four groups, which ranged from .81 to 1.18. The diagnostic sensitivity was 78% and the specificity was 94% for correct classification of PTSD in our sample. These preliminary findings yield further encouragement for exploring multivariate strategies, using hormonal ratios or profiles, in an effort to increase the diagnostic sensitivity of neuroendocrine criteria in the assessment of psychiatric patients.

Sustained urinary norepinephrine and epinephrine elevation in post-traumatic stress disorder.

Urinary norepinephrine and epinephrine levels (microgram/day) were measured at two-week intervals during the course of hospitalization in the following patient groups: post-traumatic stress disorder (PTSD); major depressive disorder (MDD); bipolar I, manic (BP); paranoid schizophrenia (PS); and undifferentiated schizophrenia (US). The mean norepinephrine level during hospitalization was significantly higher in PTSD (76 +/- 10.4 micrograms/day) than in BP (60.6 +/- 8.4 micrograms/day), MDD (41.2 +/- 4.7 micrograms/day), PS (33.4 +/- 4.9 micrograms/day) and US (34.3 +/- 5.9 micrograms/day) groups, according to Duncan's multiple range test, (F(4,39) = 6.94, p less than 0.0003). The norepinephrine elevations in the PTSD group were sustained throughout hospitalization. The only other group to show mean levels in this range was the BP group in the first sample after hospital admission. This finding supports prior psychophysiological studies indicating increased sympathetic nervous system activity in PTSD patients. The mean epinephrine level during hospitalization was also significantly higher in PTSD (22.7 +/- 2.4 micrograms/day) than in MDD (13.6 +/- 1.7 micrograms/day), PS (14.7 +/- 2.4 micrograms/day), and US (18.9 +/- 1.8 micrograms/day), but not higher than in BP (21.5 +/- 2.7 micrograms/day). The relationship of epinephrine levels among diagnostic groups was sustained throughout hospitalization. It appears likely that the main underlying mechanisms for elevations of both hormones are psychological, but further work will be required to establish the exact nature of these mechanisms.