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Animal behavioral models are crucial for neurobiological research, allowing for the thorough investigation of brain pathogenesis to be performed. In both animals and humans, anxiety has long been linked to vestibular disorders. However, although there are many tests of anxiety and vestibular deficits, there are few protocols that address the interplay between these two domains. The Suok test and its light–dark modification presented here appear to be suitable for testing this pathogenetic link in laboratory rodents. This protocol adds a new dimension to previously used tests by assessing animal anxiety and balancing simultaneously, resulting in efficient, high-throughput screens for testing psychotropic drugs, phenotyping genetically modified animals, and modeling clusters of human disorders related to stress/anxiety and balancing.

Grooming is a commonplace, robust behavior in rodent species. It has been shown to be highly sensitive to a number of experimental factors, making it an ideal target for manipulation. The complex patterning of grooming in rodents, which usually proceeds in a cephalo-caudal direction and involves several distinct stages, can be dissected into its constituent parts and microstructures. Several grooming patterning analysis methods are described in the protocol that allow for an assessment of this behavior based on measurements of grooming activity and its sequencing. Additionally, grooming can be evaluated in reference to the regional distribution and syntax in which it occurs. Owing to the ever-increasing number of rodent models that have strong grooming phenotypes, this high-throughput in-depth analysis is becoming crucial for biomedical research.

Vitamin D is a seco-steroid hormone with multiple functions in the nervous system. We discuss clinical and experimental evidence of the role of vitamin D in normal and pathological brain functions, and analyze the relative importance of vitamin D-modulated brain mechanisms at different stages of life. We also outline perspectives for the use of vitamin D in clinical nutrition to prevent or treat various brain disorders.

Low vitamin D level may predict rickets, osteomalacia, or osteoporosis. We examined serum 25(OH)D concentration as a predisposing factor for bone stress fracture in 756 military recruits. The average serum 25(OH)D concentration was significantly lower in the group with fracture, suggesting a relationship between vitamin D and fatigue bone stress fracture.Introduction:Low vitamin D level may predict rickets, osteomalacia, or osteoporosis. Fatigue bone stress fracture is one of the most frequently seen types of overuse injuries in athletes and military recruits. An association was recently shown between vitamin D and BMC. A correlation has also been found between low femoral BMD and stress fractures. We measured serum 25(OH)D concentration in a population sample of military recruits to determine if vitamin D is a predisposing factor for fatigue bone stress fracture.Materials and Methods:We prospectively followed 800 randomly selected, healthy Finnish military recruits with a mean age of 19 years for developing stress fractures in homogenous circumstances. Blood for serum 25(OH)D concentration was drawn at entry into military service, and the weight, height, body mass index (BMI), muscle strength, and 12‐minute running were measured for all subjects. Serum 25(OH)D concentrations were measured with enzyme immunoassay. At end of the 90‐day follow‐up, 756 subjects completed the study. Subjects without fracture constituted controls.Results:Twenty‐two recruits with stress fracture were identified (2.9%), the incidence being 11.6 (95% CI: 6.8–16.5) per 100 person‐years. In the final multivariate analysis, the significant risk factor for stress fracture in conscripts was a below median serum 25(OH)D level (75.8 nM), OR being 3.6 (95% CI: 1.2–11.1). No significant associations between BMI (p= 0.255), age (p= 0.216), or smoking (p= 0.851) and bone stress fracture were found in this study population.Conclusions:A lower level of serum 25(OH)D concentration may be a generally predisposing element for bone stress fractures. Considering the obvious need of additional vitamin D in prevention of stress fractures, the effects of vitamin D fortification of foods and supplementation will be subjects of interest for future research.

Low vitamin D level may predict rickets, osteomalacia, or osteoporosis. We examined serum 25(OH)D concentration as a predisposing factor for bone stress fracture in 756 military recruits. The average serum 25(OH)D concentration was significantly lower in the group with fracture, suggesting a relationship between vitamin D and fatigue bone stress fracture.

Vitamin D is a steroid hormone with many important functions in the brain, mediated through the vitamin D nuclear receptor. Numerous human and animal data link vitamin D dysfunctions to various behavioural disorders. To examine this problem, we studied whether genetic ablation of vitamin D receptors in mice may be associated with altered emotional behaviours. Here we show that the receptor-deficient mice demonstrate increased anxiety-like behaviours when subjected to a battery of behavioural tests. These studies suggest that vitamin D and its receptors are an important factor in the brain, whose imbalance may significantly affect emotional behaviour.

The steroid hormone progesterone is known to have profound effects on growth and differentiation of normal and malignant breast epithelial cells. The biologic actions of progesterone are exerted through the nuclear progesterone receptor-mediated control of target gene transcription. We utilized differential display polymerase chain reaction (DD-RT-PCR) to identify genes whose expression is altered in response to progestins in cultured breast cancer cells. Here we report identification of a gene encoding a member of the MAGUK protein family, hDlg5 (also known as KIAA0583 and P-dlg), as being the primary progestin target gene in MCF-7 breast cancer cells. Quantitative real-time RT-PCR analysis showed a rapid and strong upregulation of hDlg5 mRNA in cells treated with synthetic progestin medroxyprogesterone acetate (MPA) in the presence of estrogen in MCF-7, T47D and ZR-75-1 cells. The induction was abrogated by antiprogestin RU486. hDlg5 mRNA was also upregulated by progesterone, R5020 and dexamethasone. Protein synthesis inhibitor cycloheximide failed to block progestin-mediated induction of the hDlg5 gene. hDlg5 is a member of the growing family of MAGUKs (membrane-associated guanylate kinase homologs) and is to our knowledge the first member of the family reported to be hormonally regulated. hDlg5 is one of the human homologs of the Drosophila gene dlg [lethal(1)discs-large], which was initially identified as a tumor suppressor gene. The Dlg has a well-established role in cell growth control and maintenance of cell adhesion and cell polarity. Domain profile analysis revealed that hDlg5 has 2 additional PDZ domains than previously reported. © 2002 Wiley-Liss, Inc.

Bisphosphonates inhibit the development of experimental atherosclerosis and decrease the intima-media thickness of human carotid artery. Since arterial macrophages have a key role in atherogenesis, we studied whether clodronate, an antiatherogenic bisphosphonate, will suppress the appearance of macrophages generated by atheromatous process in the rabbit aorta. The atherosclerosis was caused in rabbits by means of a high-cholesterol (1%) diet, and the animals were treated simultaneously with saline (n=11) or 25 mg/kg of clodronate disodium (n=12) intravenously twice a week for 6 to 12 weeks. The cholesterol diet for 6 weeks caused no visible atheromatous plaques in the aorta, but feeding for 6 more weeks produced progressively atheromatous lesions. Immunohistochemistry with specific antimacrophage antibody showed an intensive accumulation of macrophages in the subendothelial layer of the aorta in cholesterol-fed rabbits treated with saline or clodronate for 6 weeks. In the aorta of rabbits treated with cholesterol diet + saline for 12 weeks, the area of immunoreactive macrophages extended from the internal elastic lamina up to the luminal surface of the aorta. However, far less immunoreactive macrophages were present in the atheromatous regions of the aorta of rabbits medicated with clodronate for 12 weeks; in the clodronate-treated animals the macrophages were located closer to the luminal surface of the aorta than in controls on saline. No atheromatous lesions and macrophages appeared in the aorta of rabbits on standard diet (n=7). The results suggest that clodronate suppresses the appearance of cholesterol-phagocyting macrophages in arterial walls during atherogenesis.

Objective:To investigate the correlation between the early follicular phase serum inhibin B levels and other indicators of ovarian reserve. Study design:Seventy-four women aged 24–40 years (mean 32) with different infertility etiologies were investigated in the early follicular phase of a spontaneous mentrual cycle. The volume of the ovaries was measured and the total number of follicles <5 mm in size counted by ultrasound. Serum levels of FSH, estradiol (E2) and inhibin B were measured on the same day. In stepwise regression analysis inhibin B levels were correlated with age, body-mass-index, the ultrasound measurements, cause of infertility, parity, FSH and E2. Results:FSH, BMI and the number of follicles proved to be statistically significant independent predictive factors for the inhibin B levels, FSH and BMI correlating negatively and the number of follicles positively with inhibin B serum concentrations. Conclusion:The number of small follicles reflect the inhibin B production of the ovaries. BMI being as strong predictive factor of inhibin B levels as FSH could in part explain the impaired likelihood of conceiving in obese patients.

Objectives:The aim was to evaluate the association between serum vitamin D (25-hydroxyvitamin D) level and risk of prostate cancer. Methods:The nested case–control study was based on a 13-year follow-up of about 19,000 middle-aged men who attended the first screening visit within the Helsinki Heart Study and were free of clinically verified prostate cancer at baseline. Through record linkage with the files of the Finnish Cancer Registry, 149 prostate cancer cases were identified in the cohort. They were matched (1:4) to probability density sampled controls for age, time of sample retrieval, and residence. Serum levels of 25-hydroxyvitamin D (25-VD) at entry were measured for cases and controls. The relative risks of prostate cancer were derived using conditional logistic regression analysis. Results:Prostate cancer risk, analyzed by quartiles of the 25-VD levels, was inversely related to 25-VD. Men with 25-VD concentration below the median had an adjusted relative risk (OR) of 1.7 compared to men with 25-VD level above the median. The prostate cancer risk was highest among younger men (<52 years) at entry and low serum 25-VD (OR 3.1 nonadjusted and 3.5 adjusted). Among those younger men (<52 years), low 25-VD entailed a higher risk of non-localized cancers (OR 6.3). The mean age at diagnosis of the patients with 25-VD concentration above the median was 1.8 years higher than that of patients with vitamin D below the median (63.1 vs61.3 years). Conclusions: We conclude that low levels of 25-VD associated with an increased risk for subsequent earlier exposure and more aggressive development of prostate cancer, especially before the andropause.

Keratinocyte growth factor (FGF-7/KGF) is a secreted member of the fibroblast growth factor family, which functions primarily as an important paracrine mediator of cell growth and differentiation. Inhibitory pathways of vitamin D may also involve participation of some growth factors. To determine whether vitamin D may play a role in the expression of FGF-7, we investigated FGF-7 expression in human breast cancer cells treated with 1,25-dihydroxyvitamin D3, which inhibited the growth of the cells. By means of cDNA microarray, RT-PCR, and Western blot analysis, we have shown an increase in expression of FGF-7 on both mRNA and protein levels after vitamin D exposure. This is the first demonstration of vitamin D regulation of FGF-7 expression and its possible involvement in mediating growth and differentiation by vitamin D.

The data suggest that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and androgens are essential for regulation of growth and differentiation in, e.g., human reproductive tissues. We investigated the possible cross-talk between 1,25(OH)2D3 and androgens in the human ovarian cancer cell line OVCAR-3. Our data demonstrate that 1,25(OH)2D3 and androgen (dihydrotestosterone, DHT) regulate the growth of OVCAR-3 cells. Nine days' treatment of OVCAR-3 cells with 100 nM DHT resulted in 48% stimulation of growth, whereas growth inhibition (73%) was observed after treatment with 100 nM 1,25(OH)2D3. The combination of 1,25(OH)2D3 and DHT showed that 1,25(OH)2D3 clearly reduces the growth-stimulatory effect of DHT on OVCAR-3 cells. Moreover, Western blot analysis revealed that these cells contain receptors for 1,25(OH)2D3 (VDR) and androgen (AR). Expression of VDR and AR was up-regulated by their cognate ligands. Up-regulation of AR by 1,25(OH)2D3 and of VDR by DHT provides evidence of cross-talk between 2 signaling pathways in OVCAR-3 cells. We also studied the immuno-histochemical distribution of VDRs and ARs in rat ovaries and human ovarian cancer cases. In rat ovaries, VDRs were observed mainly in granulosa and theca cells and ARs in granulosa cells and surface epithelium. In the human ovarian cancer cases studied, 43% were VDR-positive and 64% AR-positive. Combining the results suggests that the growth of ovarian tissue might be regulated by 1,25(OH)2D3 and androgen. Int. J. Cancer 86:40–46, 2000. © 2000 Wiley-Liss, Inc.

Two novel antibodies against the mammalian progesterone receptor (PR) were raised and characterized to study the distribution of PR and the effect of estrogen on PR expression in various female murine tissues by immunohistochemistry. There were estrogen-independent constitutive PR expressions in the smooth muscle cells of uterus, uterine blood vessels, urinary bladder, duodenum, and jejunum of ovariectomized mice. Uterine stromal cells, capsular cells of kidney and adrenal gland, and the epithelial cells of submandibular gland expressed PR constitutively. PR expression was detected in some thymic cells and the number of PR-positive thymic cells increased markedly after estrogen treatment. Estrogen induced PR expression in the epithelial cells of uterus, vagina, urethra, and skin and the stromal cells of vagina, urethra, and pancreatic ducts, as well as the smooth muscle cells of some blood vessels. These results suggest cell-specific progesterone actions in the urinary tract, skin, and gastrointestinal organs, on the immune functions, and on the regulation of local blood flow.

Activin is a dimeric glycoprotein hormone that was initially characterized by its ability to stimulate pituitary FSH secretion and was subsequently recognized as a growth factor with diverse biological functions in a large variety of tissues. In the testis, activin has been implicated in the auto/paracrine regulation of spermatogenesis through its cognate cell membrane receptors on Sertoli and germ cells. In this study we provide evidence for intranuclear activin/inhibin βAsubunit and show its distribution in the rat seminiferous epithelium. We have shown by transient expression in HeLa cells of β-galactosidase fusion proteins that the βAsubunit precursor contains a functional nuclear localization signal within the lysine-rich sequence corresponding to amino acids 231-244. In all stages of the rat seminiferous epithelial cycle, an intense immunohistochemical staining of nuclear βAwas demonstrated in intermediate or type B spermatogonia or primary spermatocytes in their initial stages of the first meiotic prophase, as well as in pachytene spermatocytes and elongating spermatids primarily in stages IX–XII. In some pachytene spermatocytes, the pattern of βAimmunoreactivity was consistent with the characteristic distribution of pachytene chromosomes. In the nuclei of round spermatids, βAimmunoreactivity was less intense, and in late spermatids it was localized in the residual cytoplasm, suggesting disposal of βAbefore spermatozoal maturation. Immunoblot analysis of a protein extract from isolated testicular nuclei revealed a nuclear βAspecies with a molecular mass of approximately 24 kDa, which is more than 1.5 times that of the mature activin βAsubunit present in activin dimers. These results suggest that activin/inhibin βAmay elicit its biological functions through two parallel signal transduction pathways, one involving the dimeric molecule and cell surface receptors and the other an alternately processed βAsequence acting directly within the nucleus. According to our immunohistochemical data, βAmay play a significant role in the regulation of nuclear functions during meiosis and spermiogenesis.

In vivo autoradiography with [3H]1,25-dihydroxycholecalciferol (vitamin D, soltriol) and immuno-staining with antibodies to vitamin D receptor were applied to identify specific binding sites in the abdominal scent gland of male Siberian hamster (Phodopus sungorus). Nuclear concentration of radiolabeled hormone and receptor antibodies was observed in the corresponding cell types including basal cells of sebaceous glands, cells of the outer hair sheaths and hair bulbs, and also keratinocytes in the epidermis. Cells of the hair dermal papillae and fibroblasts of the dermis did not show nuclear labeling. There was good correspondence between the autoradiographic and immunohistochemical data. The results indicate the presence of receptors for vitamin D-soltriol and suggest a seasonal regulation of scent gland marking activities by this steroid hormone of sunlight in cooperation with the sex steroid testosterone.

Immature chicks were used for the experiments 1–2 days after hatching. A group of chicks were injected with 5 mg of progesterone and sacrificed 1 or 24 hr later. An other group of chicks were injected daily for 9 days with 5 mg of diethylstilbestrol and there-after with single injection of progesterone. Cryostat sections were incubated with rabbit anti-avidin serum and with fluorescein labelled antirabbit globulin for fluorescence histochemistry.

A new combination of freeze-drying, formaldehyde induced fluorescence method for catecholamines and autoradiography of water soluble substances was developed and tested with different tissues containing biogenic amines. The freeze-dried tissue pieces were fixed with vaporours paraformaldehyde and OsO4 before embedding procedure. The effect of OsO4-vapour on the formaldehyde induced fluorescence was tested. The method produces semi-thin sections in which the fluorescence is well preserved and the tissue lightly stained with OsO4 which also fixes the steroid hormones used for radioautography.

The chicken progesterone receptor (cPR), like its human counterpart (hPR), exists as two isoforms, PR-A and PR-B, displaying different biological activities depending upon cellular and promoter contexts. Here we show that the ratio of PR isoforms observed in the immature chicken oviduct is changed during estrogen-induced differentiation from PR-B dominancy to that of PR-A. This is the first report describing that the expression ratio of PR isoforms is altered by upregulation of PR-A by estrogen actionin vivo.This result provides a plausible explanation to the differences in oviduct's response to progesterone depending on hormonal and developmental status of the animal.

The distribution of all-trans-retinoic acid (RA) was investigated using whole body autoradiography of normal and partial vitamin A deficient (VAD) mice. Retinoic acid receptors (α, β, and γ) were also studied in normal mice using immunoblotting. Normal and VAD mice were injected with 5 μCi 11CRA. The distribution of RA was quantitatively studied using a computer-assisted image analysis system. 11CRA was incorporated 0.5 hr after RA administration in both normal and VAD mice, while the labelling peak was at 6 hr in most organs in normal and VAD mice. The most intense labeling was found in liver, kidney, intestine, lung, Harderian gland, and salivary gland at all time points. A band of M, 51K was found in all mouse tissues by immunoblotting using the polyclonal antibody RAR82 against total RARs or the RARα specific monoclonal antibody Rα13. In some tissue, an additional band of 55-58K was also found. Lung, large intestine, small intestine, testis, seminal vesicle, and spleen contained highest concentration of total RARs, while heart, lung, small intestine, spleen, salivary gland, and preputial gland had the highest concentration of RARα. The uptake of labeled RA correlated well with RAR or RARα concentration in the corresponding tissues.

One month after castration male rats were injected daily with 2 × 50 μg of testosterone. Starting before the highest mitotic activity of seminal vesicles, 3 injections of normal seminal vesicle extract were given into the lumen at 3 h intervals. The contralateral seminal vesicle was injected with rat liver extract. Tritiated thymidine was given intravenously. The labelling index of the seminal vesicles was determined by means of radioautography.The crude supernatant of seminal vesicle was the most effective to inhibit the mitotic activity of the seminal vesicles. The decrease was on an average 34 per cent. The liver extract did not influence the mitotic activity. When the seminal vesicle extract was fractionated by Sephadex G-25, the largest fraction (M. W. > 3000) showed the highest inhibitory activity, whereas the smallest fraction (M.W. < 1000) was ineffective. The inhibiton of mitosis can also be induced by an unspecific factor, since pronase showed slight inhibitory action on seminal vesicle mitosis. However, the seminal vesicle extract had no inhibitory action on the epidermal mitotic activity.

Oestrogen-primed immature chicks were injected with 5.0 mg of progesterone. Twenty hours after progesterone avidin mRNA was isolated and purified by a method including an antibody precipitation of the avidin polysomes, phenol extraction and nitrocellulose trapping. A water solution of mRNA (1 μg) with [14C]amino acids was instilled into the uterine horn of oestrous or dioestrous mice. Saline and [14C]amino acids were injected into the opposite control uterine horn. Avidin was assayed by an antibody method. A clear-cut avidin synthesis was found in the oestrous uterus, whereas the dioestrous showed only a slight avidin synthesis. This suggest that there are no absolute species-specific factors required at the translational level of uterine protein synthesis and that the mammalian uterus can translate foreign hormone-specific proteins at a rate dependent on the endogenous protein synthesis.

The present paper is the first report to demonstrate that measurable amounts of activin B are secreted into the saliva. The results show wide fluctuations in activin B concentrations during the menstrual cycle with peak values detected at the follicular phase. Estrogen replacement therapy was found to increase salivary activin B levels in post-menopausal subjects. The concentration in males was negligible. These data suggest that activin B concentrations in the human saliva may be under hormonal regulation and propose that salivary activin B measurements may prove useful in investigating the as yet less well defined local and/or physiological roles of activin B. The present paper reports, in addition, the immunohistochemical localization of activin/inhibin subunits in the duct systems of the rat submandibular, sublingual and parotid salivary glands.

Antibodies against synthetic peptides selected from the amino acid sequences of human inhibin α- and β β β A-subunits were used to examine the distribution of inhibin subunit immunoreactivity in chicken embryos during the first week of development. Inhibin α.-subunit immunoreactivity was localized in skeletal and smooth muscle myoblasts as well as developing cardiac muscle cells. In somites, immunostaining was seen exclusively in myotomes. The appearance of α.-subunit immunore-activity was correlated with myogenic differentiation; immunoreactivity was not seen in non-differentiated mesenchymal cells or in terminally differentiated adult muscle cells. In cardiac muscle, some immunopositive myocytes were seen also in the adult. In the adult heart, the Purkinje fibers were strongly immunoreactive, suggesting a possible role of the immunoreactive protein in the impulse-conducting function of these specialized cells. Inhibin α.-subunit immunoreactivity was also seen in the visceral and parietal cells of the Bowman’s capsule in both mesonephric and metanephric kidneys. In addition to mesodermal derivatives, α.-subunit immunoreactivity was localized in neuroepithelial cells and axons in the developing central nervous system. Immunoblotting with anti-α.(1-32) revealed two protein bands with Mr values of 50 000 and 32 000 in cytosol samples of whole embryos under nonreducing conditions. In reduced samples an approximately 14 000 Mr protein species was detected. Inhibin β A-subunit immunoreactivity was detected only in chondrocytes, suggesting that the immunoreactive protein might represent a chicken homologue of the various cartilage and bone morphogenetic proteins expressed in mammals.

The Dunning tumor, originally described as a carcinoma of the rat dorsal prostate, has for long been used as an experimental model of prostatic cancer. We have recently presented a number of morphological findings that are incompatible with the prostatic origin of the H-subline of the Dunning tumor. In this paper, biochemical and immunohistochemical markers of rat prostate and mammary gland are studied in the R-3327 Dunning H tumor. Pieces of the H tumor were inoculated in male or lactating female rats. The electrophoretic protein pattern of Dunning tumor extracts was more similar to that of the mammary gland than the dorsolateral prostate. Proteins selectively appearing after metabolic labeling in Dunning tumors grown in lactating rats corresponded to labeled proteins in mammary glands from the same animals. Secretory proteins typical of the lateral prostate (SVS II) and dorsal prostate (transglutaminase) could not be detected immunohistochemically in the Dunning tumor. Western blot studies of tumor extracts and slot blot analysis of RNA preparations from the tumor confirmed the absence of SVS II and prostate specific transglutaminase from the Dunning tumor. On the other hand, the presence of mammary gland proteins such as milk fat globule membrane proteins, lactoperoxidase and lactalbumin were detected in the Dunning tumor by immunohistochemistry and Western blotting, but were absent from the dorsolateral prostate. Transferrin-mRNA, expressed in the male urogenital tract and also in the liver and other tissues, was detected in the mammary gland and Dunning tumor, but not in the dorsolateral prostate. The absence of mammary gland secretory β-casein in the Dunning tumor was related to the elevated Ha-ras oncogene expression in the tumor, previously reported to suppress casein expression. The findings clearly demonstrate that the prostate cannot be the origin of the Dunning tumor, presently being used in prostatic cancer research. The designation prostatic adenocarcinoma for this tumor is therefore invalid. Furthermore, the data support our view that mammary gland might be the origin of the Dunning tumor, although the derivation from the bulbourethral or the parotid glands cannot strictly be excluded.

The effects of anti-inflammatory glucocorticoids and a membrane stabilizer (disodium cromoglycate) on avidin induction were studied. Chicks were primed for 0, 3 or 7 days with diethylstilboestrol (DES; 0·5 mg/day per animal, s.c.). Actinomycin D (0·2 mg/kg, i.p.) stimulated avidin production in the oviduct, intestine, lung and wing muscle of both DES-primed and unprimed chicks. Prednisolone (5 mg/animal, i.m.) given 1 h before actinomycin D reduced the avidin amounts to control levels in the unprimed oviducts and in non-oviductal tissues. The signs of inflammation (ascites, oedema) in chicks receiving actinomycin D also disappeared with prednisolone premedication. Cortisol (50 mg/animal, i.m. or 1, 5 or 50 μg/ml medium), prednisolone (5 mg/animal, i.m. or 0·5, 5, 10 or 50 μg/ml medium) and dexamethasone (1 mg/animal, i.m. or 0·05 or 0·5 μg/ml medium) stimulated avidin production in DES-primed oviducts in vivoand in vitro. Culture in vitroinduced avidin production in lung and oviduct but not in wing muscle. This avidin production in culture was not inhibited by cortisol, prednisolone or disodium cromoglycate. It is concluded that actinomycin D induces avidin production through its inflammatory effect and that the induction can be prevented by anti-inflammatory glucocorticoids. It is proposed that the 'spontaneous' avidin production in culture is not the effect of trauma during tissue preparation but rather a new form of avidin induction.

Photoperiodic modulation of androgen levels and androgen receptor (AR) expression in testes and accessory sex glands were studied in a seasonally breeding rodent, the bank vole. Juvenile voles subjected to long photoperiod (20L:4D) for 6–8 wk attained sexual maturity, which was associated with a prominent increase in testicular testosterone (T) levels and weight of testes and accessory sex glands. Pubertal development in short photoperiod-treated (6L:18D for 6–8 wk) juveniles was arrested, and subsequently reproductive regression set in with a marked decrease in testicular T levels and gonadal weight. In sexually active voles, strong AR immunostaining was detected in nuclei of epithelial, smooth muscle, and stromal cells of the epididymis, prostate, and seminal vesicles. In active testes, AR was present in nuclei of Sertoli cells, peritubular cells, Leydig cells, and vascular smooth muscle cells. In juveniles, strong to moderate nuclear immunoreactivity was encountered in epithelial and stromal cells of the epididymis and prostate, whereas a weaker reaction was discerned in seminal vesicles. In juvenile testes, AR was localized to vascular smooth muscle cells, peritubular, and interstitial cells. In sexually regressed animals, nuclear staining was almost absent in accessory sex glands, whereas in testes, moderate immunostaining was retained in all other cell types except the Sertoli cells. Western blots of active and regressed testes indicated a marked photoperiod-induced down-regulation of immunodetectable AR in the regressed gonad.

To determine if fertilization occurs unnoticed among Norplant users who are ovulatory.

The importance of monitoring luteinizing hormone (LH) secretion during gonadotropin stimulation remains controversial. In the present study, the authors evaluated the occurrence of spontaneous LH surges in 170 cycles stimulated by clomiphene citrate and human menopausal gonadotropin, and correlated the success rate of embryo cleavage to the time interval between the occurrence of the LH surge peak value and the time of human chorionic gonadotropin (hCG) administration. LH was quantitated from urine by an avidin-biotin enzyme immunoassay. The results indicated that a spontaneous LH surge occurred in 18% of the cycles. The number of oocytes recovered was not affected by the occurrence of a spontaneous LH surge. In 12% of all cases, the spontaneous LH surge occurred less than 12 hours before the administration of hCG, and in these cases embryo cleavage was not reduced. In 6% of all cases, the spontaneous LH surge occurred over 12 hours before hCG administration, and in these cases embryo cleavage was reduced significantly.

Androgens regulate the development and sexual dimorphism of rodent Harderian and submandibular glands. This effect is believed to be mediated by the androgen receptor. Immunohistochemistry and immunoblotting were carried out to study the receptor in normal, castrated and dihydrotestosterone-supplemented rat Harderian and submandibular glands. Immunohistochemically, the most intense nuclear staining was observed in the acinar cells of the submandibular glands, followed by intercalated duct cells. The granular convoluted tubules showed weak immunostaining and the striated ducts were negative. In the Harderian gland, nuclear staining was seen in both type I and II secretory cells. Castration and treatment had no effect on the expression of the androgen receptor protein in either gland. A 110 K androgen receptor signal was detected by immunoblotting in the Harderian gland but not in the submandibular gland. An experiment was designed to explore the possible effect of proteinases on the receptor protein in the homogenate of submandibular gland. Our results demonstrate the cell-specific location of the receptor in Harderian and submandibular glands, and show that the expression of the receptor protein is androgen-independent.

Photoperiodic and hormonal modulation of mRNAs for testicular inhibin/activin subunits and follistatin were studied in a seasonally breeding rodent, the bank vole (Clethrionomys glareolus). Photoperiod-induced testicular regression had no effect on the relatively low steady-state levels of follistatin mRNA. Inhibin α (Iα) and βB (IβB) mRNA levels were significantly higher in regressed than in active gonads, but inhibin βA was undetectable. The effect of gonadotropin administration on testicular weight and mRNA concentrations differed between the sexually active and quiescent voles. Neither FSH (1.2 U/kg; s.c. for 5 days) nor hCG (600 IU/kg; s.c. for 5 days) affected testicular weight in sexually active voles, whereas both gonadotropins significantly increased testicular weight in photo-regressed individuals. FSH had no effect on Iα or IβB mRNA concentrations in the active testes, whereas excessive hCG challenge induced a decrease in the steady-state levels of these mRNAs. FSH induced an increase in Iα mRNA concentrations in the regressed gonad, whereas both gonadotropins concomitantly down-regulated IβB mRNA levels. In conclusion, the high expression of Iα and IβB mRNA in the regressed testis imply autocrine and paracrine roles for inhibin/activin in the quiescent gonad of seasonal breeders. Inhibin α-subunit expression is at least partly under the control of FSH in the bank vole testis. J. Exp. Zool. 281:336–345, 1998. © 1998 Wiley-Liss, Inc.

The expression of progesterone receptor (PR) and its induction by oestradiol during the embryogenesis of the chick bursa of Fabricius (BF) were studied by immunohistochemistry using three different polyclonal antibodies to the chicken oviduct PR. Mesenchymal cells of the cloacal area surrounding the bursa primordium in controls (without exogenous oestrogen) express the PR between 9 and 11 days of incubation. In the same cells, PR was induced experimentally by oestradiol at 9 days. Mesenchymal cells in the bursa did not express PR after oestradiol treatment before the age of 11 days. The PR was not inducible in the bursal epithelium or in haemopoietic cells. None of the bursal cells expressed the PR to a detectable level during embryonic life without exogenous treatment. Some haemopoietic cells showed strong artefactual staining in their nuclei. It is concluded that (1) the embryonic bursa of Fabricius is a sex-steroid-sensitive organ, (2) exogenous oestradiol is able to induce progesterone receptor in the mesenchymal cells, but (3) the PR is not expressed without exogenous oestrogen. This indicates that the PR becomes oestrogen inducible well before it is naturally expressed during sexual maturation and that the level of endogenous oestrogen during embryonic life is not high enough to affect the bursa significantly.

Progesterone was administered to oestrogen-treated and untreated chicks, or inflammation in the abdominal cavity was caused by intestine and liver injury or intraperitoneal actinomycin D administration. Local injury to the pectoral muscle was also carried out. Chicks were killed 24–26 h after the treatment and the biotin-binding egg white protein, avidin, was assayed in a number of tissues using a [14C] biotin-binding method and radioimmunoassay. Ovalbumin was also assayed with a radioimmunoassay. Avidin was not found in the tissues of control chicks. Progesterone induced avidin only in the oviducts of oestrogen-treated chicks. After intestine and liver injury avidin was found, however, in all the tissues of oestrogen-treated and untreated chicks studied except for the brain. The concentrations were highest in the oviduct, lung, intestine and bursa of Fabricius. Actinomycin D (200 μg/kg) caused ascites and subcutaneous oedema in 40–60 % of the chicks, and avidin was found only in the tissues of these inflamed animals. Avidin production caused by the local muscular injury was restricted to the injured area. Tissue injury and inflammation did not induce ovalbumin in any tissue.The study shows that avidin can be induced besides the oviduct also in non-oviductal chick tissues, and it is proposed that there are both progesterone-dependent and -independent avidin induction mechanisms.

Little attention, at least in Finland, has been paid to possible emotional problems caused by the medical curriculum. Courses in human dissection are suspected to leave their mark on the subsequent physician-patient relationships, particularly in relating to dying patients. At the University of Tampere, courses on the dissection of cadavers have for the past 6 years been preceded by a few 3-hr preparatory lectures dealing with dissection and death and psychic reaction to them. In order to learn students' responses to lectures, they were asked to fill out a questionnaire. Students reported that these lectures have helped them in encountering an unwanted situation. Awareness of their emotional state may also have improved learning results in that energy need not be dissipated in the effort to overcome emotions. Students describe the dissection course as leading to a better understanding of themselves and of the diversity of emotional reactions of their fellow students. An anticipated long-term effect of the lectures might be a better future relationship of the doctor with patients. We conclude that lectures dealing with dying and death during dissection courses are not only appropriate, but demanded and necessary for medical students.

To study androgen receptors in normal and malignant laryngeal tissue, high affinity, limited capacity androgen receptors were assayed from the cytosol fraction of laryngeal carcinomas of 18 male patients and of nonneoplastic laryngeal tissue of 5 male patients. Immediately after removal, the samples were frozen with dry ice and stored at -70°C. a specific radioligand, [3H]methyltrienolone incubation and dextran-coated charcoal method was used. Five laryngeal carcinomas showed androgen receptors. the number of receptor sites varied from 1.6 to 4.2 fmoles/mg protein, and the dissociation coefficient (KD) from 2.6 to 0.7 10-9 M. Androgen receptors stood at - 70°C for at least 4-5 months. 13 laryngeal carcinomas were receptor negative. Nonneoplastic laryngeal tissue contained 1.0-10.5 fmoles androgen receptors/mg cytosol protein and their KD was 0.2-2.1 10-9 M. This study indicates that some laryngeal carcinomas contain androgen receptors and these cases might be amenable to antiandrogen therapy.

Four radioautographic methods for soluble substances were compared to each other. When using the wire-loop method of Miller et al. (1964) difficulties were met because detachment of emulsion from the object slide. Severe chemographic artifacts in the radioautograms hampered the results when using the method of Appleton (1964). The freeze-drying and paraffin embedding method of Hammarström et al. (1965) resulted an extensive diffusion of radioactivity. Therefore, a simple dry-fixation method for steroid radioautography was developed.

The uptake, incorporation and distribution of tritiated thymidine and uridine into visceral organs of immature female and male rats was studied. Radioautographs of various organs were prepared 5, 15 and 30 min after intraperitoneal administration. The distribution of the radioactivity was compared with sections obtained 30 min after intravenous injection of the same amount of nucleotides.

The cell renewal rate and mitotic cycles were studied in the ovarian follicles of normal adult females during dioestrus and oestrus as well as in neonatally androgenized females at the age of 8 to 9 months. Incorporation of tritiated thymidine into nuclear DNA was followed using radioautography. The mitotic index was higher during dioestrus than during oestrus, both in the granulosa and in the theca interna cells. The labelling index during dioestrus was 32.4 per cent for the granulosa and 17.7 per cent for the theca, but there was no incorporation of radioactive thymidine during oestrus. The theca externa did not show any DNA synthetizing activity in either of the cyclic phases. A cell cycle was complete during dioestrus within 17 hours in the granulosa and within 31 hours in the theca interna.The polycystic ovaries of the persistent oestrous rats showed practically no mitotic activity. In three out of 12 animals some synthesis of DNA was obtained, and in one of them the labelling index exceeded that of the normal dioestrous rats.

The effect of testosterone propionate on cell renewal rate and mitotic cycle of castrated mice was investigated. 0.25 mg of the hormone was given thrice at 24 h intervals and 50 μc of 3H-thymidine was injected simultaneously with the last hormone injection. After this the mice were killed at 1 to 2 h intervals. Ventral prostates and seminal vesicles were weighed and the thymidine incorporation into their epithelia determined in radioautograms. A piece of proximal small intestine was used as reference tissue.The labelling index in the castrated control animals was 1.3 for the seminal vesicle; 68 h after testosterone treatment it was 44. The figures for the ventral prostate were 1.9 and 37, and for the small intestine 44 and 57. The mitotic index in the prostate of the experimental animals and that in the intestine of all the animals was 0.5; in the seminal vesicle it was 0.4. The mitotic index in the accessory glands of the control mice was very low. The specific activity of DNA per nucleus was higher in the hormone treated than in the control animals.The cell cycle (TC) in the prostate was shortened from about 900 h to 20 h after testosterone. The synthetic phase (TS) was shortened from 16 to 7 h, presynthetic (TG1) phase from 900 to 10 h, whereas the postsynthetic phase (TG2) remained unchanged. The corresponding figures for the seminal vesicle were: TG2from 4.5 to 0.5 h, TS from 12 to 7 h, TG1from 900 to 10 h and TCfrom 900 to 18 h. The changes in the intestinal epithelium were less marked: TCfrom 22 to 16 h, TSfrom 8.5 to 8 h, TG1from 12 to 6 h, while TG2remained unchanged.

Male rats were injected subcutaneously with 2 mg of cyproterone daily from birth to the 14th postnatal day. At the age of 40 days the animals were polycystic and corpora lutea were absent. There was an increased 37% of the ovarian implants showed distinct corpora lutea indicating cyclic secretion of gonadotrophins; the implants in the control animals were polycystic and corpora lutea were absent. There was an increased interstitial cell stimulating hormone output from the pituitary gland as indicated by the increased amount of 3β-hydroxysteroid dehydrogenase active Leydig cells in the testes of the feminized animals.The in vitrouptake of tritiated testosterone and oestradiol of various tissues was tested on rats, age 4.5 months. The anterior hypothalamus, the rostral middle hypothalamus, the median eminence and the anterior pituitary of the feminized males retained significantly more oestradiol than the cerebral cortex. The oestradiol uptake in these areas also exceeded that of the control animals. Only the median eminence of the cyproterone treated rats concentrated more testosterone than the cerebral cortex. The anterior hypothalamus and cerebral cortex of the feminized male rats were less able to transform tritiated testosterone to dihydrotestosterone or tritiated oestradiol to oestrone, as compared to adult castrated male or female animals, respectively.

Male and female rat foetuses taken two days before birth, newly born or when 2 days old were injected subcutaneously with tritium labelled testosterone or oestradiol. One hour later the accumulation of radioactivity in the cerebral cortex, hypothalamus and anterior pituitary of the foetuses was studied by means of a microcombustion technique in order to process the samples for liquid scintillation spectrometry. The anterior pituitary accumulated the highest amount of radioactivity i. e. about twice the amount recovered in the brain. No difference was found in the radioactivity of the hypothalamus as compared to that of the cerebral cortex. Both steroids were accumulated equally in the tissues investigated. The retention of the radioactivity was higher in the neonatal than in adult rats. However, no sex difference was observed in the binding capacity of the hypothalamus or the pituitary gland of the newly born animals.

A sensitive method for avidin assay was devised. Tritiated biotin is bound to avidin and this complex is then bound to bentonite. Radioactivity is converted into a gas form by combustion and counted in an automatic proportional counter with 55% efficiency and background of 3.7c.p.m. The sensitivity is 1–2ng of avidin.

This report describes a prospective follow-up study on 907 infertile men presenting as partners of infertile couples.Couples who conceived were compared to those who remained infertile during a 3-year follow up. A univariate life table analysis showed that the duration of infertility, the age of both spouses, a history of male urethritis, the body mass index, the percentage of motile spermatozoa, the quality of motility, the motile sperm density, the total motile count, the sperm morphology, and the pH of semen were significant predictors of fertility. Another eleven socio-demographic and nine semen variables were not associated with fertility. Cox multiple regression analysis identified the duration of infertility, percentage total motility, the quality of motility combined, a history of male urethritis, the wifes age, and the morphology of the spermatozoa to be independent predictors of the reproductive outcome. A prognostic ‘risk’ ratio (chance for achieving pregnancy) was calculated as a function of these variables for each couple and a life table analysis was made between three classified subgroups of couples with different risk ratios. These life table curves make it possible to define the probability of an individual couple to achieve pregnancy at any given time.