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  • 1.
    Berg, A. Håkan
    et al.
    Örebro University, Department of Natural Sciences.
    Thomas, Peter
    Olsson, Per-Erik
    Örebro University, Department of Natural Sciences.
    Biochemical characterization of the Arctic char (Salvelinus alpinus) ovarian progestin membrane receptor2005In: Reproductive Biology and Endocrinology, ISSN 1477-7827, E-ISSN 1477-7827, Vol. 3, p. 64-Article in journal (Refereed)
    Abstract [en]

    Membrane progestin receptors are involved in oocyte maturation in teleosts. However, the maturation-inducing steroid (MIS) does not appear to be conserved among species and several progestins may fulfill this function. So far, complete biochemical characterization has only been performed on a few species. In the present study we have characterized the membrane progestin receptor in Arctic char (Salvelinus alpinus) and show that the 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) receptor also binds several xenobiotics, thus rendering oocyte maturation sensitive to environmental pollutants. We identified a single class of high affinity (Kd, 13.8 +/- 1.1 nM), low capacity (Bmax, 1.6 +/- 0.6 pmol/g ovary) binding sites by saturation and Scatchard analyses. Receptor binding displayed rapid association and dissociation kinetics typical of steroid membrane receptors, with t1/2 s of less than 1 minute. The 17,20beta-P binding also displayed tissue specificity with high, saturable, and specific 17,20beta-P binding detected in ovaries, heart and gills while no specific binding was observed in muscle, brain or liver. Changes in 17,20beta-P binding during oocyte maturation were consistent with its identity as the oocyte MIS membrane receptor. Incubation of fully-grown ovarian follicles with gonadotropin induced oocyte maturation, which was accompanied by a five-fold increase in 17,20beta-P receptor binding. In addition, competition studies with a variety of steroids revealed that receptor binding is highly specific for 17,20beta-P, the likely maturation-inducing steroid (MIS) in Arctic char. The relative-binding affinities of all the other progestogens and steroids tested were less than 5% of that of 17,20beta-P for the receptor. Several ortho, para derivatives of DDT also showed weak binding affinity for the 17,20beta-P receptor supporting the hypothesis that xenobiotics may bind steroid receptors on the oocyte's surface and might thereby interfere with oocyte growth and maturation.

  • 2.
    Berg, Håkan
    et al.
    Örebro University, School of Science and Technology.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Liimatta, Harri
    Örebro University, School of Science and Technology.
    Hoffmann, Erik
    Karlsson, Johnny
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Characterization of antibodies for quantitative determination of spiggin protein levels in male and female three-spined stickleback (Gasterosteus aculeatus)2009In: Reproductive Biology and Endocrinology, ISSN 1477-7827, E-ISSN 1477-7827, Vol. 7, no 1, article id 46Article in journal (Refereed)
    Abstract [en]

    Spiggin is an adhesive glycoprotein produced in the kidney of sticklebacks during the breeding season and is subsequently secreted into the urinary bladder from where it is employed for nest building. Since the production of the protein has been shown to be under androgenic control, spiggin has been suggested to be a useful biomarker for androgenic substances in the environment. In this study, two polyclonal spiggin antibodies based on synthetic peptides and one polyclonal antibody directed against native spiggin have been characterized. The antibodies ability to identify spiggin was investigated by quantitative immunoassay. For both peptide antibodies the quantification range was determined to be between 1 and 80 ng spiggin and determination of renal spiggin levels from immature and mature males displayed a 15-fold increase in total spiggin content of the kidney resulting in a 6-fold increase in male kidney weight due to hypertrophy. The kidney somatic index (KSI) was found to correlate well with the total renal spiggin content and therefore it appears that KSI in sticklebacks could be used as an initial method to identify substances displaying androgenic effects. Furthermore, western blot analysis revealed that the polyclonal antibodies recognize different spiggin isoforms and that spiggin can be detected in the urinary bladder and kidney of both males and female sticklebacks. In order to develop a quantitative detection method for native spiggin it is necessary to produce a standard that can be used in a bioassay. Due to the adhesive and polymerization characteristics of spiggin the protein is difficult to use as a standard in bioassays. So far spiggin has been shown to exist in at least 14 isoforms, all of which contain polymerization domains. To overcome the solubility problem we have produced recombinant spiggin gamma, with only one polymerization domain, that can be expressed in E. coli. Western blot analysis demonstrated that the polyclonal antibodies were able to detect recombinant spiggin gamma protein in bacterial cell lysate, suggesting that it may be developed into a useful source of standard spiggin to be used for quantitative determination of androgen induced spiggin production in sticklebacks.

  • 3.
    Hoffmann, Erik
    et al.
    Department of Zoology, Stockholm University, Stockholm, Sweden.
    Walstad, Anders
    Örebro University, School of Science and Technology.
    Karlsson, Johnny
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Borg, Bertil
    Department of Zoology, Stockholm University, Stockholm, Sweden.
    Androgen receptor-beta mRNA levels in different tissues in breeding and post-breeding male and female sticklebacks, Gasterosteus aculeatus2012In: Reproductive Biology and Endocrinology, ISSN 1477-7827, E-ISSN 1477-7827, Vol. 10, article id 23Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Androgens induce male characters by activating androgen receptors (AR). Previous quantitative studies on AR in fishes have been limited to few tissues and/or a single season/reproductive state. The aim of this investigation was to study the possible role of AR-beta expression levels in the control of male traits in the three-spined stickleback. To that end, AR-beta expression levels in major tissues in breeding and post-breeding male and female sticklebacks were examined.

    METHODS: AR-beta mRNA levels were quantified in ten tissues; eye, liver, axial muscle, heart, brain, intestine, ovary, testis, kidney and pectoral muscle in six breeding and post-breeding males and females using reverse transcription quantitative PCR.

    RESULTS: Breeding in contrast to post-breeding males built nests and showed secondary sexual characters (e.g. kidney hypertrophy) and elevated androgen levels. Post-breeding females had lower ovarian weights and testosterone levels than breeding females. AR-beta was expressed in all studied tissues in both sexes and reproductive states with the highest expression in the gonads and in the kidneys. The kidney is an androgen target organ in sticklebacks, from which breeding males produce the protein spiggin, which is used in nest-building. There was also high AR-beta expression in the intestine, an organ that appears to take over hyperosmo-regulation in fresh water when the kidney hypertrophies in mature males and largely loses this function. The only tissue that showed effects of sex or reproductive state on AR-beta mRNA levels was the kidneys, where post-breeding males displayed higher AR-beta mRNA levels than breeding males.

    CONCLUSION: The results indicate that changes in AR-beta mRNA levels play no or little role in changes in androgen dependent traits in the male stickleback.

  • 4.
    Stygar, Denis
    et al.
    Division for Reproductive Endocrinology, Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden.
    Muravitskaya, Natalia
    Division for Reproductive Endocrinology, Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden.
    Eriksson, Britt
    Division for Reproductive Endocrinology, Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden.
    Eriksson, Håkan
    Division for Reproductive Endocrinology, Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden.
    Sahlin, Lena
    Division for Reproductive Endocrinology, Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden.
    Effects of SERM (selective estrogen receptor modulator) treatment on growth and proliferation in the rat uterus2003In: Reproductive Biology and Endocrinology, ISSN 1477-7827, E-ISSN 1477-7827, Vol. 1, article id 40Article in journal (Refereed)
    Abstract [en]

    Background: Selective estrogen receptor modulators (SERMs) have been developed in order to create means to control estrogenic effects on different tissues. A major drawback in treatment of estrogen receptor (ER) positive breast cancer with the antagonist tamoxifen (TAM) is its agonistic effect in the endometrium. Raloxifene (RAL) is the next generation of SERMs where the agonistic effect on the endometrium has been reduced.

    Methods: The aim of the present study was to determine the effect of SERM treatment on the uterus, as assessed by proliferation markers and several factors involved in uterine growth. Ovariectomized (ovx) rats were treated with estradiol (E2), tamoxifen (TAM), RAL, ICI182780 (ICI) or vehicle (OVX-controls). We studied the effects on mRNA levels of the growth hormone (GH) receptor, insulin-like growth factor-I (IGF-I), ERalpha and ERbeta. In addition, by immunohistochemistry the proliferation markers PCNA and Ki-67, as well as ERalpha and ERbeta, were detected.

    Results: The uterine weight of the rats treated with E2 or TAM was increased as compared to OVX-controls. The uterine GH-receptor mRNA level was highest in the E2 treated animals. In ICI treated rats no GH-receptor mRNA could be detected. The IGF-I mRNA level increased 16-fold in uteri of the TAM treated group and 9-fold in the E2 treated rats as compared to OVX-controls. The ERalpha mRNA level was increased in the E2 treated rats, while the ERbeta mRNA level was increased after TAM treatment. The proliferation, as assessed by PCNA, was lowest in ICI treated animals.

    Conclusions: The uterine wet weight, the LE height and the GH-receptor mRNA levels showed similar patterns, indicating that GH is involved in the regulation of uterine weight. Tamoxifen, which has been related to increased incidence of endometrial carcinoma in women, dramatically increased IGF-I mRNA levels in rat uterus. Since proliferation was not higher in TAM and E2 treated rats than in OVX controls, this assay of simple, early proliferation does not give the full explanation of why TAM should enhance the risk of developing endometrial cancer.

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