Identification of Sertoli cell-specific transcripts in the mouse testis and the role of FSH and androgen in the control of Sertoli cell activity.

BACKGROUND: The Sertoli cells act to induce testis differentiation and subsequent development in fetal and post-natal life which makes them key to an understanding of testis biology. As a major step towards characterisation of factors involved in Sertoli cell function we have identified Sertoli cell-specific transcripts in the mouse testis and have used the data to identify Sertoli cell-specific transcripts altered in mice lacking follicle-stimulating hormone receptors (FSHRKO) and/or androgen receptors (AR) in the Sertoli cells (SCARKO). RESULTS: Adult iDTR mice were injected with busulfan to ablate the germ cells and 50 days later they were treated with diphtheria toxin (DTX) to ablate the Sertoli cells. RNAseq carried out on testes from control, busulfan-treated and busulfan + DTX-treated mice identified 701 Sertoli-specific transcripts and 4302 germ cell-specific transcripts. This data was mapped against results from microarrays using testicular mRNA from 20 day-old FSHRKO, SCARKO and FSHRKO.SCARKO mice. Results show that of the 534 Sertoli cell-specific transcripts present on the gene chips, 85% were altered in the FSHRKO mice and 94% in the SCARKO mice (mostly reduced in both cases). In the FSHRKO.SCARKO mice additive or synergistic effects were seen for most transcripts. Age-dependent studies on a selected number of Sertoli cell-specific transcripts, showed that the marked effects in the FSHRKO at 20 days had largely disappeared by adulthood although synergistic effects of FSHR and AR knockout were seen. CONCLUSIONS: These studies have identified the Sertoli cell-specific transcriptome in the mouse testis and have shown that most genes in the transcriptome are FSH- and androgen-dependent at puberty although the importance of FSH diminishes towards adulthood.

Identification of Leydig cell-specific mRNA transcripts in the adult rat testis.

The adult population of Leydig cells acts to secrete testosterone which is essential for reproductive health and fertility in the adult male. However, other physiological functions of these cells are uncertain, and to address this issue a cell ablation model has been used to identify Leydig cell-specific mRNA transcripts. Ethane dimethane sulphonate (EDS) was synthesised by a novel process and was used to ablate Leydig cells in adult male rats previously treated with butane dimethane sulphonate (busulphan) to delete the germ cell population. Levels of mRNA transcripts were measured in the testis using microarrays 1, 3, 5, 8 and 12 days after EDS injection. During this period, there was a significant change in the levels of 2200 different transcripts with a marked decline in the levels of canonical Leydig cell transcripts, such as Cyp11a1, Cyp17a1 and Insl3. A total of 95 transcripts showed a similar decline in expression after EDS treatment, suggesting that they have a Leydig cell-specific origin. Analysis of selected transcripts confirmed that they were expressed specifically in Leydig cells and showed that most had a late onset of expression during adult Leydig cell development. Apart from transcripts encoding components of the steroidogenic apparatus, the most common predicted function of translated proteins was endogenous and xenotoxicant metabolism. In addition, a number of transcripts encode acute-phase proteins involved in reduction of oxidative stress. Results show that, in addition to androgen secretion, Leydig cells may have a critical role to play in protecting the testis from damage caused by toxicants or stress.

Proteomic analysis of the sheep caruncular and intercaruncular endometrium reveals changes in functional proteins crucial for the establishment of pregnancy.

The expression and regulation of endometrial proteins are crucial for conceptus implantation and development. However, little is known about site-specific proteome profiles of the mammalian endometrium during the peri-implantation period. We utilised a two-dimensional gel electrophoresis/mass spectrometry-based proteomics approach to compare and identify differentially expressed proteins in sheep endometrium. Caruncular and intercaruncular endometrium were collected on days 12 (C12) and 16 (C16) of the oestrous cycle and at three stages of pregnancy corresponding to conceptus pre-attachment (P12), implantation (P16) and post-implantation (P20). Abundance and localisation changes in differentially expressed proteins were determined by western blot and immunohistochemistry. In caruncular endometrium, 45 protein spots (5% of total spots) altered between day 12 of pregnancy (P12) and P16 while 85 protein spots (10% of total spots) were differentially expressed between P16 and C16. In intercaruncular endometrium, 31 protein spots (2% of total spots) were different between P12 and P16 while 44 protein spots (4% of total spots) showed differential expression between C12 and C16. The pattern of protein changes between caruncle and intercaruncle sites was markedly different. Among the protein spots with implantation-related changes in volume, 11 proteins in the caruncular endometrium and six proteins in the intercaruncular endometrium, with different functions such as protein synthesis and degradation, antioxidant defence, cell structural integrity, adhesion and signal transduction, were identified. Our findings highlight the different but important roles of the caruncular and intercaruncular proteins during early pregnancy.

Steroidogenic enzyme expression in the human fetal liver and potential role in the endocrinology of pregnancy.

The human feto-maternal unit produces large amounts of steroid hormones, particularly estrogens, during the second and third trimesters. The fetal adrenal gland and the placenta are considered the principal tissues driving steroid production but the fetal liver is likely to play an essential role in this process. This study was designed to measure transcript expression of proteins involved in steroid synthesis, metabolism, conjugation and signalling in the human fetal liver and to examine sex differences and effects of maternal smoking. Liver samples were taken from 55 normal fetuses from women undergoing second trimester elective termination. Levels of 23 mRNA transcripts encoding steroid synthesis/metabolic/conjugation enzymes and steroid receptors were measured by real-time PCR. The expression of representative proteins was confirmed by western blotting and immunohistochemistry. The human fetal livers expressed high levels of CYP19A1, SULT2A1, SULT1E1, HSD17B2, SRD5A3 and CYP3A7. Lower levels of SULT1A1, STS, UGT2B17, GPER, AKR1C3, UGT2B15, AR, CYP11A1, CYP21A2, HSD17B3, HSD17B1 and SRD5A1 were also detectable. The expression of ESR, ESR2, CYP17A1 and HSD3B transcripts was undetectable in most fetal livers, although HSD3B was shown to be present by western blotting. Sex differences were limited to SRD5A3 (lower in females) and UGT2B17 (higher in females). Maternal smoking increased the expression of CYP19A1, SULT2A1, UGT2B17, HSD17B2 and AKR1C3 and reduced the expression of SRD5A3 in the male fetal liver. This study shows that the human fetal liver is likely to have an extensive effect on circulating steroid levels in the human fetus and mother. The most important of these effects will be alterations to the species, conjugation and availability of estrogens in the fetus. Maternal smoking is likely to reduce circulating androgen bioactivity in male fetuses.

Foetal and post-natal exposure of sheep to sewage sludge chemicals disrupts sperm production in adulthood in a subset of animals.

Exposure to ubiquitous, environmental chemicals (ECs) has been hypothesized as a cause for declining male reproductive health. Understanding the long-term effects of EC exposure on reproductive health in humans requires animal models and exposure to 'real life', environmentally relevant, mixtures during development, a life stage of particular sensitivity to ECs. The aim of this study was to evaluate the effects of in utero and post-natal exposure to environmentally relevant levels of ECs, via sewage sludge application to pasture, on the adult male sheep testis. Hormones, liver concentrations of candidate ECs and Sertoli and germ cell numbers in testes of adult rams that were exposed to ECs in sewage sludge in utero, and until weaning via maternal exposure, and post-weaning via grazing pastures fertilized with sewage sludge, were quantified. Evaluated as a single group, exposure to sludge ECs was without significant effect on most parameters. However, a more detailed study revealed that 5 of 12 sludge-exposed rams exhibited major spermatogenic abnormalities. These consisted of major reductions in germ cell numbers per testis or per Sertoli cell and more Sertoli cell-only tubules, when compared with controls, which did not show any such changes. The sludge-related spermatogenic changes in the five affected animals were significantly different from controls (p < 0.001); Sertoli cell number was unaffected. Hormone profiles and liver candidate EC concentrations were not measurably affected by exposure. We conclude that developmental exposure of male sheep to real-world mixtures of ECs can result in major reduction in germ cell numbers, indicative of impaired sperm production, in a proportion of exposed males. The individual-specific effects are presumed to reflect EC effects on a heterogeneous population in which some individuals may be more susceptible to adverse EC effects. Such effects of EC exposure in humans could have adverse consequences for sperm counts and fertility in some exposed males.

Effects of exposure to environmental chemicals during pregnancy on the development of the male and female reproductive axes.

There is a large body of literature describing effects of environmental chemicals (ECs), many of them anthropogenic with endocrine-disrupting properties, on development in rodent laboratory species, some of which lead to impaired reproduction and adverse health. This literature joins extensive human epidemiological data and opportunistic wildlife findings on health effects of ECs. In contrast, the effect of endocrine disruption on foetal development and reproductive performance in domestic species is less extensively documented. This applies both to domestic farm and to companion species even though the former is critical to food production and the latter share our homes and many aspects of the modern developed human lifestyle. In domestic species, the nature of chemicals exposure in utero and their consequences for animal health and production are poorly understood. A complication in our understanding is that the pace of development, ontogeny and efficiency of foetal and maternal hepatic and placental activity differs between domestic species. In many ways, this reflects the difficulties in understanding human exposure and consequences of that exposure for the foetus and subsequent adult from epidemiological and largely rodent-based data. It is important that domestic species are included in research into endocrine disruption because of their (i) wide variety of exposure to such chemicals, (ii) greater similarity of many developmental processes to the human, (iii) economic importance and (iv) close similarities to developed world human lifestyle in companion species.

Maternal and fetal tissue accumulation of selected endocrine disrupting compounds (EDCs) following exposure to sewage sludge-treated pastures before or after conception.

Liver concentrations of selected pollutant classes were determined in groups of sheep fetuses and their dams, at 55 (Experiment 1) and 110 (Experiment 2) days of gestation (term = 145 d) following exposure, throughout their breeding lives and after mating, to pasture treated with either inorganic fertiliser (control, CC) or with sewage sludge (treated, TT). In a unique study designed to separate the respective contributions of environmental sources and mobilised tissue to the available EDC burden, in additional groups of animals, pollutant burdens at 110 days gestation were assessed following exposure to the respective treatments, either throughout their breeding lives until mating, but not thereafter (TC), or only between mating and slaughter (CT) (Experiment 3). With very few exceptions, maternal and fetal liver concentrations of diethylhexyl phthalate (DEHP) and selected polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDE) and polycyclic aromatic hydrocarbons (PAHs) were not significantly affected by sludge exposure in any group. In some cases, maternal and fetal tissue EDC concentrations were different but the differences were not consistent, and maternal and fetal concentrations of none of the classes of chemical were significantly correlated. It was not possible to identify a single chemical, or class of chemical, that may be responsible for previously observed physiological effects of exposure to sludge-treated pastures. It is concluded that exposure of sheep to pastures fertilised with sewage sludge was not associated with increased liver concentrations of EDCs, irrespective of the stage of development at which they were measured and of maternal tissue mobilisation and EDC release during gestation. Thus, retrospective measurements of EDC tissue burdens could not be used to accurately assess earlier fetal EDC insults.

Genome-wide expression changes induced by bisphenol A, F and S in human stem cell derived hepatocyte-like cells.

The debate about possible adverse effects of bisphenol A (BPA) has been ongoing for decades. Bisphenol F (BPF) and S (BPS) have been suggested as "safer" alternatives. In the present study we used hepatocyte-like cells (HLCs) derived from the human embryonic stem cell lines Man12 and H9 to compare the three bisphenol derivatives. Stem cell-derived progenitors were produced using an established system and were exposed to BPA, BPF and BPS for 8 days during their transition to HLCs. Subsequently, we examined cell viability, inhibition of cytochrome P450 (CYP) activity, and genome-wide RNA profiles. Sub-cytotoxic, inhibitory concentrations (IC50) of CYP3A were 20, 9.5 and 25 µM for BPA, BPF and BPS in Man12 derived HLCs, respectively. The corresponding concentrations for H9-derived HLCs were 19, 29 and 31 µM. These IC50 concentrations were used to study global expression changes in this in vitro study and are higher than unconjugated BPA in serum of the general population. A large overlap of up- as well as downregulated genes induced by the three bisphenol derivatives was seen. This is at least 28-fold higher compared to randomly expected gene expression changes. Moreover, highly significant correlations of expression changes induced by the three bisphenol derivatives were obtained in pairwise comparisons. Dysregulated genes were associated with reduced metabolic function, cellular differentiation, embryonic development, cell survival and apoptosis. In conclusion, no major differences in cytochrome inhibitory activities of BPA, BPF and BPS were observed and gene expression changes showed a high degree of similarity.

Developmental changes in human fetal testicular cell numbers and messenger ribonucleic acid levels during the second trimester.

CONTEXT: Normal fetal testis development is essential for masculinization and subsequent adult fertility. The second trimester is a critical period of human testicular development and masculinization, but there is a paucity of reliable developmental data. OBJECTIVE: The objective of the study was to analyze second-trimester human testicular morphology and function. DESIGN: This was an observational study of second-trimester testis development. SETTING: The study was conducted at the Universities of Glasgow and Aberdeen. PATIENTS/PARTICIPANTS: Testes were collected from 57 morphologically normal fetuses of women undergoing elective termination of normally progressing pregnancies (11-19 wk gestation). MAIN OUTCOME MEASURE(S): Testicular morphology, cell numbers, and quantitative expression of 22 key testicular genes were determined. RESULTS: Sertoli cell and germ cell number increased exponentially throughout the second trimester. Leydig cell number initially increased exponentially but slowed toward 19 wk. Transcripts encoding Sertoli (KITL, FGF9, SOX9, FSHR, WT1) and germ (CKIT, TFAP2C) cell-specific products increased per testis through the second trimester, but expression per cell was static apart from TFAP2C, which declined. Leydig cell transcripts (HSD17B3, CYP11A1, PTC1, CYP17, LHR, INSL3) also remained static per cell. Testicular expression of adrenal transcripts MC2R, CYP11B1, and CYP21 was detectable but unchanged. Expression of other transcripts known or postulated to be involved in testicular development (GATA4, GATA6, CXORF6, WNT2B, WNT4, WNT5A) increased significantly per testis during the second trimester. CONCLUSIONS: The second trimester is essential for the establishment of Sertoli and germ cell numbers. Sertoli and Leydig cells are active throughout the period, but there is no evidence of changing transcript levels.

Timing of Maternal Exposure and Foetal Sex Determine the Effects of Low-level Chemical Mixture Exposure on the Foetal Neuroendocrine System in Sheep.

We have shown that continuous maternal exposure to the complex mixture of environmental chemicals (ECs) found in human biosolids (sewage sludge), disrupts mRNA expression of genes crucial for development and long-term regulation of hypothalamic-pituitary gonadal (HPG) function in sheep. The present study investigated whether exposure to ECs only during preconceptional period or only during pregnancy perturbed key regulatory genes within the hypothalamus and pituitary gland and whether these effects were different from chronic (life-long) exposure to biosolid ECs. The findings demonstrate that the timing and duration of maternal EC exposure influences the subsequent effects on the foetal neuroendocrine system in a sex-specific manner. Maternal exposure prior to conception, or during pregnancy only, altered the expression of key foetal neuroendocrine regulatory systems such as gonadotrophin-releasing hormone and kisspeptin to a greater extent than when maternal exposure was 'life-long'. Furthermore, hypothalamic gene expression was affected to a greater extent in males than in females and, following EC exposure, male foetuses expressed more 'female-like' mRNA levels for some key neuroendocrine genes. This is the first study to show that 'real-life' maternal exposure to low levels of a complex cocktail of chemicals prior to conception can subsequently affect the developing foetal neuroendocrine system. These findings demonstrate that the developing neuroendocrine system is sensitive to EC mixtures in a sex-dimorphic manner likely to predispose to reproductive dysfunction in later life.

Regulation of functional steroid receptors and ligand-induced responses in telomerase-immortalized human endometrial epithelial cells.

Information on the regulation of steroid hormone receptors and their distinct functions within the human endometrial epithelium is largely unavailable. We have immortalized human primary endometrial epithelial cells (EECs) isolated from a normal proliferative phase endometrium by stably transfecting the catalytic subunit (hTERT) of the human telomerase complex and cultured these hTERT-EECs now for over 350 population doublings. Active hTERT was detected in hTERT-EECs employing the telomerase repeat amplification assay protocol. hTERT-EECs revealed a polarized, non-invasive epithelial phenotype with apical microvilli and production of a basal lamina when grown on a three-dimensional collagen-fibroblast lattice. Employing atomic force microscopy, living hTERT-EECs were shown to produce extracellular matrix (ECM) components and ECM secretion was modified by estrogen and progesterone (P4). hTERT-EECs expressed inducible and functional endogenous estrogen receptor-alpha (ER-alpha) as demonstrated by estrogen response element reporter assays and induction of P4 receptor (PR). P4 treatment down-regulated PR expression, induced MUC-1 gene activity and resulted in increased ER-beta transcriptional activity. Gene activities of cytokines and their receptors interleukin (IL)-6, leukemia inhibitory factor (LIF), IL-11 and IL-6 receptor (IL6-R), LIF receptor and gp130 relevant to implantation revealed a 17 beta-estradiol (E2)-mediated up-regulation of IL-6 and an E2- and P4-mediated up-regulation of IL6-R in hTERT-EECs. Thus, hTERT-EECs may be regarded as a novel in vitro model to investigate the role of human EECs in steroid hormone-dependent normal physiology and pathologies, including implantation failure, endometriosis and endometrial cancer.

The conceptus regulates tryptophanyl-tRNA synthetase and superoxide dismutase 2 in the sheep caruncular endometrium during early pregnancy.

Conceptus-derived paracrine signals play crucial roles in the preparation of a uterine environment capable of supporting implantation and development of the conceptus. However, little is known about the regulation of endometrial tryptophanyl tRNA synthetase (WARS) and manganese superoxide dismutase (SOD2) protein expression by the implanting and post-implanting conceptus. We hypothesized that the conceptus-derived signals favourably influences uterine environment for implantation through regulation of WARS and SOD2 expression in ovine caruncular endometrium. To test this hypothesis, WARS and SOD2 protein and mRNA expression was determined in caruncular endometrial tissues of unilaterally pregnant ewes at implantation (day 16) and post-implantation (day 20) periods. WARS protein expression increased in caruncular tissues of the gravid uterine horns compared with the non-gravid uterine horns on days 16 and 20 of pregnancy. There were no changes in SOD2 protein expression between the gravid and non-gravid uterine horns, irrespective of the day of pregnancy. On day 16 of pregnancy, there were no differences in WARS and SOD2 mRNA expression between the gravid and non-gravid uterine horns but expression of both genes was higher in the gravid uterine horns when compared with the non-gravid uterine horns on day 20 of pregnancy. In conclusion, the use of the unilaterally pregnant ewe model provides for the first time firm evidence that the early implantation and post-implanting conceptus-derived signals up-regulate WARS protein expression within the caruncular endometrium. Further studies are necessary to identify these signalling molecules and to understand mechanisms whereby they exert paracrine action within the endometrium.

Role of progesterone and nonsteroidal ovarian factors in regulating gonadotropin-releasing hormone self-priming in vitro.

We investigated the effects of gonadotropin surge-attenuating factor (GnSAF), inhibin, and follistatin on GnRH self-priming and its augmentation by progesterone. Two GnRH challenges, 60 min apart, were administered to rat pituitary monolayers after 90-min exposure to medium alone (control), progesterone, GnSAF, inhibin, or follistatin. Inhibin-stripped follicular fluid from superovulated women was used as a source of GnSAF bioactivity. Under control conditions, the greater response to the second GnRH challenge (peak 2, 9.2 +/- 2.1; peak 1, 4.4 +/- 0.9 ng LH/mL; P < 0.01) demonstrated GnRH self-priming. None of the treatments significantly altered the first LH peak. Progesterone markedly increased GnRH self-priming (peak 2, 12.6 +/- 2.5 ng LH/mL; P < 0.01). However, GnSAF and RU486 significantly reduced GnRH self-priming (peak 2, 4.6 +/- 0.9 and 5.6 +/- 1.6 ng LH/mL, respectively; P < 0.01). The augmentation of self-priming induced by progesterone was completely abolished by coincubation with either GnSAF or RU486 (peak 2, 7.5 +/- 1.6 and 4.3 +/- 0.9 ng LH/mL, respectively; P < 0.01). Neither inhibin nor follistatin had any effect on GnRH self-priming or its augmentation by progesterone. The actions of RU486 in the presence and absence of progesterone demonstrate a nonprogestagenic effect of RU486 on the gonadotropes. In conclusion, the suppression of GnRH self-priming, with or without progesterone augmentation, supports the hypothesis that GnSAF acts by maintaining the pituitary in an unprimed state of reduced responsiveness to GnRH.

Human fetal testis: second trimester proliferative and steroidogenic capacities.

The period of Leydig cell hyperplasia (14-18 weeks gestation) in human fetal testis is crucial for normal gonad development. We have studied the spatio-temporal distribution of key developmental and functional markers in human fetal testis between 13-19 weeks gestation. Proliferating cell nuclear antigen-positive cells were immunolocalized to both interstitium and tubules. Image analysis confirmed an increase in positive interstitial cells during Leydig cell hyperplasia (P: < 0.05). c-Myc was localized to the interstitium with no gestational changes. The steroidogenic enzymes 3beta-hydroxysteroid dehydrogenase (protein) and cytochrome P450 17alpha-hydroxylase/C(17-20)-lyase (P450c17; messenger ribonucleic acid and protein) were confined to the Leydig cells. The number of immunopositive cells increased between 13 and 19 weeks (P: < 0.001). P450c17 mRNA (in situ hybridization) and protein were localized to the same population of interstitial Leydig cells. Androgen receptor and Bcl-2 protein (anti-apoptotic) were gradually restricted to the peritubular myoid cells as gestation progressed. Conversely, Bax protein (pro-apoptotic) was predominantly localized to the tubule Sertoli cells, whereas the germ cells were Bax immunonegative. In conclusion, human fetal Leydig cell hyperplasia is characterized by increasing numbers of proliferating cells and increased expression of steroidogenic enzymes. The Bcl-2-positive, Bax-negative status of the peritubular myoid cells may be a strategy for cell survival.

Changes in peripheral serum levels of total activin A during the human menstrual cycle and pregnancy.

The main objective of this study was to determine whether activin A concentrations in peripheral blood fluctuate during the normal human menstrual cycle and pregnancy. Blood samples were collected longitudinally from five regularly cycling volunteers (22-30 yr) throughout a spontaneous menstrual cycle and cross-sectionally from normal pregnant women attending the antenatal clinic (8-38 weeks gestation: 3-20 subjects/time point). Total (i.e. bound plus free) activin A concentrations were measured using a recently developed two-site enzyme immunoassay that employs an analyte denaturation/oxidation step to eliminate interference due to endogenous activin-binding proteins. During the menstrual cycle, mean serum activin A levels varied in a biphasic manner (by ANOVA, P = 0.02), with highest levels around midcycle (approximately 220 pg/mL) and the late luteal/early follicular phase (approximately 310 pg/mL) and nadirs in both midfollicular (approximately 125 pg/mL) and midluteal (approximately 120 pg/mL) phases. Between the mid- to late luteal phase, the activin A level increased progressively (approximately 2.5-fold; P < 0.05), whereas inhibin A, estradiol, and progesterone all decreased progressively (approximately 10-fold; P < 0.001). During pregnancy, serum activin A levels were much higher than those in nonpregnant subjects, with a value of 2.12 +/- 0.31 ng/mL recorded in week 8. Levels remained at approximately 2 ng/mL between weeks 8-24, but increased thereafter to reach 25.5 +/- 6 ng/mL by week 38, a value approximately 100 times greater than that during the normal menstrual cycle. Serum activin A levels during pregnancy were significantly correlated with inhibin A (r = 0.69; P < 0.001), estradiol (r = 0.55; P < 0.001), and progesterone (r = 0.74; P < 0.001) values. Gel permeation chromatography indicated that all of the detectable activin A in human follicular fluid, pregnancy serum, and term placental extract eluted with an apparent molecular mass between 70-200 kDa, indicating that little, if any, free activin (molecular mass, 25 kDa) is present in these samples. Although these results support a possible endocrine role for circulating activin A during the human menstrual cycle and pregnancy, the observation that all detectable activin A is associated with binding protein(s) raises questions about its relative bioavailability for action on peripheral target cells.

Pharmacokinetic and pharmacodynamic profiles of sumatriptan in migraine patients with headache recurrence or no response.

OBJECTIVES: Sumatriptan is effective in the acute treatment of migraine. However, about 15% of patients with migraine do not experience headache relief after sumatriptan, and up to 40% may experience recurrence of headache within 24 hours. We studied whether pharmacokinetic or pharmacodynamic differences may explain these different clinical effects. METHODS: We compared the pharmacokinetic profiles of subcutaneous sumatriptan in 14 patients who consistently had headache relief without headache recurrence, in 12 patients who had headache recurrence in every attack, and in six patients who did not have headache relief after sumatriptan. Because the antimigraine action of sumatriptan may be mediated through vasoconstriction of cranial blood vessels, we also compared in these patients changes in blood vessel diameter and blood velocity in the common, internal, and external carotid arteries. RESULTS: Despite sufficient power of the study, no important differences in pharmacokinetic and pharmacodynamic profiles between the three patient groups were detected. CONCLUSION: Headache recurrence and lack of headache relief after sumatriptan do not appear to be explained by pharmacokinetic or pharmacodynamic differences between patients, which may be an important finding for the development of novel antimigraine drugs.

Validation of the in vivo measurement of adipose tissue by magnetic resonance imaging of lean and obese pigs.

In vivo quantification of adipose tissue with magnetic resonance imaging (MRI) was validated with pigs. Thirteen transaxial MRI sections were collected, at intervals proportional to body length, from each pig, which was then killed, frozen, and sliced at the locations of the MRI sections. Adipose-tissue quantities were determined by dissecting each slice, and lipid contents of the dissected slices and of the tissue segments between slices were measured. Compared with dissection, MRI underestimated abdominal percent adipose tissue and overestimated cervical percent adipose tissue by less than 6%. When all 13 sections were used, MRI closely predicted percent lipid and dissected percent adipose tissue with small residual SDs (RSD = 1.9 and 2.1, respectively), which increased only slightly if two sections (4, upper thorax and 8, upper abdomen) were used (RSD = 2.3 and 2.6, respectively). In conclusion MRI accurately quantifies adipose tissue in vivo, matching values produced by dissection and chemical analysis.

Total and subcutaneous adipose tissue in women: the measurement of distribution and accurate prediction of quantity by using magnetic resonance imaging.

Total and subcutaneous adipose tissue in seven lean and seven obese women were quantified using magnetic resonance imaging (MRI). The distributions of adipose tissues along the body were closely correlated: subcutaneous with total, both within and between lean and obese groups. Lean women had proportionally less adipose tissue in the lower thorax and upper abdomen than did obese women. Reducing the number of MRI scans from 17 to 4 did not increase the residual SD of predicted body adipose tissue (2.9 percent) when body density was used as the reference measure. MRI gave an estimate of total-body adipose tissue significantly closer to the value for fat percent produced when the results from five other techniques (skinfold thickness, underwater weighing, 40K whole-body counting, isotopic water dilution, and tetrapolar bioelectrical impedance) were averaged than when any other technique was used alone. MRI-determined percent body adipose tissue in women is close to, and proportional to, estimates derived by underwater weighing.

The effects of steroidal and non-steroidal ovarian hormones on pituitary responsiveness to gonadotrophin surge-attenuating factor.

Primary pituitary cultures from adult female rats were used to investigate the effects of steroidal (oestradiol and progesterone) and non-steroidal (inhibin, follistatin) ovarian hormones on the suppressive actions of the ovarian factor gonadotrophin surge-attenuating factor (GnSAF) in the control of gonadotrophin secretion. The source of GnSAF was a chromatographic preparation from follicular fluid containing four distinct protein bands as resolved on SDS-PAGE. Oestradiol and progesterone added alone had no effect on gonadotrophin secretion but had a wide range of effects on the suppression of both LH and FSH secretion caused by the non-steroidal factors. Oestradiol, progesterone and oestradiol+progesterone enhanced the suppressive actions of GnSAF on GnRH-induced LH secretion (causing 19.3 +/- 5.2% (P < 0.05), 41.9 +/- 3.4% (P < 0.001) and 32.2 +/- 5.3% (P < 0.001) greater suppression than GnSAF alone). Progesterone and oestradiol+progesterone completely abolished the suppression of basal FSH secretion caused by inhibin (causing 157.1 +/- 22.2%, P < 0.001, and 160.9 +/- 11.3%, P < 0.001, stimulation compared with inhibin alone). Separately the steroids had no effect on the suppression of gonadotrophin secretion caused by follistatin. However, in combination, oestradiol+progesterone potentiated the suppressive actions of follistatin on GnRH-induced LH secretion causing 29.9 +/- 5.3% (P < 0.05) greater suppression than follistatin alone. In combination, high-dose follistatin and GnSAF caused 31.1 +/- 6.5% (P < 0.01) greater suppression than GnSAF alone. Thus in combination high-dose follistatin and GnSAF have additive effects on the suppression of GnRH-induced LH secretion. Recombinant human inhibin and GnSAF added in combination had little further effect compared with either alone suggesting that they may have a similar mechanism of action at the pituitary level. These results demonstrate that while FSH secretion in vitro is mainly controlled by inhibin and follistatin, LH secretion is affected by the presence of a whole range of factors. We have demonstrated that oestradiol and progesterone potentiate the suppressive actions of GnSAF in vitro. These data are compatible with the suggestion that in the late follicular phase it is falling levels of GnSAF that allow positive feedback of the steroids on the pituitary to elicit the LH surge, rather than increases in the stimulatory effects of the ovarian steroids overcoming GnSAF. The actions of GnSAF on the pituitary may be modulated by follistatin but it is unlikely that inhibin has any modulatory effects on the GnSAF-induced suppression of LH secretion.

Galactopoietic and mammogenic effects of long-term treatment with bovine growth hormone and thrice daily milking in goats.

Starting in mid-lactation, goats were treated daily for 22 weeks with 0.15 mg recombinant bovine GH (bGH)/kg, or an equivalent volume of vehicle. One gland of each goat was milked thrice daily throughout treatment, the other twice daily. Mammary differentiation was studied in biopsy samples obtained before treatment and after 3 and 22 weeks of treatment, by determination of in-vitro synthesis rates of milk constituents and measurement of enzyme activities. Mammary growth was measured using a whole-body imaging technique (magnetic resonance imaging; MRI). bGH caused an immediate and sustained increase in milk yield of approximately 23% overall, whilst the glands milked thrice daily produced approximately 14% more than the control glands milked twice daily. The effects of the combined treatment were additive, but not synergistic. A synergistic effect of the combined treatment resulted in a significant improvement in lactation persistency. A stimulatory effect of milking frequency on mammary enzyme activities was evident only in bGH-treated goats at 3 weeks, but in both groups at 22 weeks. Synthesis rates of casein and lactose were increased at 3 weeks only by the combined treatment. Thus bGH accelerated or augmented the differentiative response to thrice daily milking. Mammary parenchyma volume, estimated by MRI, increased significantly during the first 12 weeks of bGH treatment and remained higher throughout the rest of the treatment period. Cell number was estimated from parenchyma volume and DNA concentration; this decreased significantly in the controls between weeks 1 and 22, but remained constant in the bGH group. In nine of the ten goats, parenchyma volume and cell number increased in the gland milked thrice daily relative to the control gland milked twice daily during the course of the experiment. Thus bGH stimulated growth of the mammary gland over and above that induced by the frequent milking. The absence of any detectable increase in thymidine incorporation suggests that this growth consisted of cellular hypertrophy rather than hyperplasia.