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Expression and function of brain-derived neurotrophin factor and its receptor, TrkB, in ovarian follicles from the domestic hen (Gallus gallus domesticus)

T. Jensen^* and A. L. Johnson

Department of Biological Sciences, PO Box 369, The University of Notre Dame, Notre Dame, IN 46556, USA
^* Present address: Center for Reproduction of Endangered Species, Zoological Society San Diego, PO Box 120551, San Diego, CA 92112-0551, USA

View larger version (54K): [in a new window]   Fig. 1. Representative northern blot of brain-derived neurotrophic factor (BDNF) mRNA in theca cells (A) reveals two transcripts of approximately 4.5kb and 1.5kb. Levels of mRNA were measured by densitometric scans of northern blots and were standardized to 18S rRNA (B). Data are expressed as fold difference compared with F1 follicle theca. Values are means + S.E.M. (N=3 replicate experiments). a and b denote significant differences (P<0.05) in levels of the 4.5kb transcript. Str, stromal tissue; 3–5 and 6–8 refer to the diameter (in mm) of prehierarchal follicles, while 9–12 (follicle diameter 9–12mm) and F3, F2 and F1 represent preovulatory follicles, with F1 representing the next follicle to ovulate.

View larger version (45K): [in a new window]   Fig. 2. Representative northern blot of trkB (the high-affinity neurotrophin receptor of brain-derived neurotrophic factor) mRNA in granulosa cells (A) reveals three transcripts of approximately 9, 6.3 and 5kb. Levels of mRNA from granulosa and stroma tissues (B) and theca (C) were standardized to 18S rRNA and are expressed as fold difference compared with F1 follicle granulosa. Values are means + S.E.M. (N=3 replicate experiments). a and b denote significant differences (P<0.05) in levels of the 9kb transcript. Abbreviations are as described in the legend to Fig.1.

View larger version (96K): [in a new window]   Fig. 3. Localization of brain-derived neurotrophic factor (BDNF) and trkB (the high-affinity neurotrophin receptor of brain-derived neurotrophic factor) mRNA and protein within the preovulatory follicle. In situ hybridization and immunocytochemical detection shows BDNF mRNA (A) and protein (B) expression, respectively, within the theca interna of the F1 follicle (arrows). By comparison, in situ hybridization shows trkB mRNA (C) localized within the theca interna and granulosa layers (arrows), while expression of the TrkB protein (D) is localized to the basement membrane side of the granulosa cell layer and to a lesser extent within the theca interna (arrows). (E) A negative control for in situ hybridization using a non-sense cDNA probe; (F) a negative control for immunolocalization using preimmune serum. The scale bars are graded in µm. Y, yolk; Gr, granulosa layer; BM, basement membrane (arrowhead); Ti, theca interna; Te, theca externa.

View larger version (24K): [in a new window]   Fig. 4. (A) Levels of the 9.0kb trkB (the high-affinity neurotrophin receptor of brain-derived neurotrophic factor) transcript in cultured prehierarchal follicle granulosa cells following overnight pre-culture and subsequent treatment for 18h without (control) or with vasoactive intestinal peptide (VIP; 1µmoll-1) or follicle-stimulating hormone (FSH; 100ngml-1) in the absence or presence of the phosphodiesterase inhibitor IBMX (0.01µmoll-1). (B) Levels of trkB mRNA in cultured preovulatory follicle granulosa cells following treatment for 18h without or with VIP or luteinizing hormone (LH) in the absence or presence of 3-isobutyl-1-methylxanthine (IBMX). trkB mRNA was standardized to 18S rRNA and expressed as fold difference compared with non-cultured (T0) cells. P<0.05 compared with T0 by a post-hoc paired t-test; *P< 0.05 compared with control; P<0.05 compared with the respective treatment in the absence of IBMX. Values are means + S.E.M. (N=3 replicate experiments).

View larger version (30K): [in a new window]   Fig. 5. (A) Progesterone (P4) production in preovulatory follicle granulosa cells following treatment with brain-derived neurotrophic factor (BDNF), k252a (100nmoll-1), BDNF (50ngml-1) plus the Trk (the high-affinity neurotrophin receptor of brain-derived neurotrophic factor) kinase activity inhibitor k252a or vehicle (control) for 18h following overnight preplating. Levels of progesterone are expressed as fold difference compared with the control. Values are means + S.E.M. (N=4 or 5 replicate experiments). a,bP<0.05; *P<0.05 for BDNF at 50ngml-1 compared with the control, as determined by a post-hoc paired t-test. (B) Progesterone production in preovulatory follicle granulosa cells following treatment with BDNF (50ngml-1), phorbol 12-myristate-13-acetate (PMA; 167nmoll-1), BDNF plus PMA or vehicle (control) for 18h after overnight preplating. Levels of progesterone, expressed as fold difference compared with control cells, are not different among the three treatment groups. Values are means + S.E.M. (N=3 replicate experiments). *P<0.025 compared with the control, as determined by a post-hoc two-tail paired t-test.