Author
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LANKFORD, SCOTT - UNIV OF CENTRAL MISSOURI |
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Weber, Gregory - Greg |
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Submitted to: Biology of Reproduction Abstracts
Publication Type: Abstract Only Publication Acceptance Date: 5/15/2008 Publication Date: 5/27/2008 Citation: Lankford, S., Weber, G.M. 2008. In vitro treatment with 17,20b-dihydroxy-4-pregnen-3-one regulates mRNA levels of transforming growth factor beta superfamily members in rainbow trout (Oncorhynchus mykiss) ovarian tissue. Biology of Reproduction Abstracts . Interpretive Summary: Technical Abstract: Transforming growth factor beta (TGFB) superfamily members are important paracrine/autocrine regulators of ovarian development and steroidogenesis in mammals, but their reproductive role in fishes is not well understood. Our objectives were 3-fold: to determine if key TGFB superfamily transcripts are regulated during the final stages of 17,20b-dihydroxy-4-pregnen-3-one (17,20bP)-induced oocyte maturation, to determine if this regulation is affected by ovarian development stage and to determine the steroid specificity of this regulation. In the first experiment we used real-time PCR to measure mRNA levels of key TGFB superfamily members in rainbow trout follicle-enclosed oocytes progressing through oocyte maturation and the resumption of meiosis. Briefly, ovarian tissue was removed from 6 fish and verified to be at similar developmental stages. After careful dissection, groups of isolated follicle-enclosed oocytes were placed in culture plates and incubated with either a control medium or 17,20bP, which is the maturation inducing hormone (MIH) in rainbow trout. The MIH-stimulated oocytes were monitored for progression through maturation and sampled at 3 different time points, overnight, yolk vesicle fusion, and germinal vesicle breakdown (GVBD), over a 96h period. Control samples were also collected at these times, although they were not progressing through maturation. While inhibin alpha and inhibin beta-B subunits did not significantly change with treatment or time period, inhibin beta-A (Inhba) mRNA levels increased throughout the 96h incubation in control tissues, but were drastically attenuated in 17,20bP-stimulated tissues. The inhibin beta subunits form either hetero- or homodimers to make activins. Furthermore, mRNA levels of bambi (bmp and activin membrane-bound inhibitor), a type 1 psuedoreceptor for bone morphogenetic proteins and activins, increased in control tissues throughout the 96h incubation, but were further increased in MIH-stimulated tissues. Ovarian development stage specificity of these responses was investigated in samples from female rainbow trout that were classified as late vitellogenic, non competent, or competent based on histology and responsiveness to MIH. The MIH-induced decrease of inhba and increase of bambi was corroborated at all stages. However, the response of bambi was greatly enhanced in the competent tissues, suggesting limited stage specificity. Lastly, the specificity of these responses to MIH was investigated by incubating competent ovarian fragments in the presence of serial diluted aliquots (29nM - 2.9uM) of estradiol (E2), testosterone (T), cholesterol, 17,20bP, 17 hydroxyprogesterone (17HP), and progesterone (P4). Stimulation with E2, T and cholesterol did not alter transcript levels in comparison with controls, while all of the progestins were effective at attenuating inhba and augmenting bambi in a dose-responsive manner. However, the effectiveness of MIH to alter inhba and bambi transcript levels was far superior to 17HP or P4, which were only effective at the highest dose (2.9uM). When taken together with previously reported data describing MIH-induced regulation of steroidogenic enzymes in rainbow trout and the functions of TGFB members in mammals, these results support a model for an MIH positive feedback system at the level of the ovary and therefore suggest the involvement of key TGFB members and inhibitors in the final stages of rainbow trout ovarian development. |
