| AKAP13, which is also known as Brx-1, AKAP-Lbc and proto-Lbc, is one of AKAP family. It is highly expressed in several organs and tissues, such as the spleen, thymus, peripheral leukocytes, skeletal muscles and testis, while it is moderately expressed in placenta and lung. AKAP13, however, is not or minimally expressed in the liver, brain, small intestine, colon and prostate. This characteristic tissue distribution of AKAP13may indicate that AKAP13plays important roles in regulation of the immune, reproductive and the musculoskeletal systems. AKAP13contains two importan protein modules including a PKA-binding domain and a GEF domain. Many intracellular signaling pathways are mainly mediated by PKA. So AKAP13maybe can integrate many signaling pathways by binding with PKA. AKAP13plays an important role in diverse physiological processes by GEF domain activating the small GTP-binding protein RhoA as RhoA controls fundamental cell properties such as cell cycle progression, gene expression, actin remodeling and cytokinesis. In addition, it’s reported that cAMP-dependent PKA-mediated signaling pathway is involved in the FSHR signaling pathway conduction. And studies have shown that AKAP13can enhance the ligand-dependent estrogen receptor alpha (ER a) and beta (ER β) and glucocorticoid receptor activity. Given that AKAP13is involved in signal transduction of a variety of hormones and the importance of bingding PKA and activating G protein RhoA activity in the glycoprotein hormone receptor signaling pathway, as well as it has been shown to influence LHR signal transduction, we assume that AKAP13is involved and affect FSH receptor signaling pathway transduction in mouse follicle.A polyclonal antiserum directed against the AKAP13protein was used to perform immunolocalization on sections from5normal ovaries. We found that AKAP13protein was localized to the cytoplasm of granulosa cells from mature graafian follicles, the corpus luteum and there is not positively staining in preantral follicles. It’s found that AKAP13protein is widely distributed in the cytoplasm of mouse granulosa cells in primary culture by immunocytochemistry and immunofluorescence staining. The distribution is along the cytoskeleton, which suggests the function of AKAP13may be related to cytoskeleton as previously reported.We then use the mouse ovarian granulosa cells in primary culture system to analyse the role of AKAP13in FSHR signaling pathway of mature mouse ovary. We found that:(1) the expression of LHR and Aromatase mRNA can be significantly induced48hours later after treatment with FSH (P=0.04and0.02resprctively). There was a trend towards induction of AKAP13expression at48hours, although the difference was not significant (p=0.06).(2) Compared with the control group, AKAP13expression levels can be reduced significantly and it’s11%of control group. The difference was statistically significant (p=0.001). Knockdown of AKAP13can reduce basal expression of Aromatase and LHR mRNA. Knockdown of AKAP13also reduced induction of LHR mRNA in response to FSH in a variety of conditions, including order of addition experiments, in granulosa cell cultures from inmature mice, and in granulosa cells cultured under different conditions.In order to clarify the specific role of AKAP13in vivo, we deleted the key sequence in GEF domain of AKAP13by homologous recombination gene targeting technology and constructed AKAP13gene knockout mouse model. The homozygous AKAP13knockout mice died at embryonic day10.5-11because of severe heart defect. After examination of these homozygous embryo of AKAP13gene knock out mice, we found that they were able to normal development before E9-9.5days, but after E10days later, homozygous null embryos developed slowly with forebrain reduced in size, tight neck area as well as the first arm arch hypoplasia compared with the wild-type mouse embryos. More importantly, homozygous embryos were abnormal during heart development with ventricular enlargement and pericardial effusion. HE staining of homozygous embryos from AKAP13gene knockout mice revealed that myocardium became thin as the reduction in trabecular formation. In order to study the mechanism of cardiac development arrest in mice, we first assessed markers of proliferation and apoptosis in the heart. There was no difference of intensity of staining for Ki67proliferation antigen between wide type and homozygous null embryos for Intensity of staining for the Ki67proliferation antigen. There was no increase in apoptosis in homozygous null embryos at E9.0-9.5by TUNEL assay, in line with the embryonic development arrest, but not due to the increase in programmed cell death caused embryonic development to stop. AKAP13protein was mainly distributed in the forebrain of mice limb buds, the first arm bow and developing heart by immunohistochemical staining of wild type and heterozygous embryos. We tried to amplify AKAP13gene from AKAP13gene knock out mice by RT-PCR method, but failed. This indicates that the mutant gene is not transcribed or mutant mRNA transcript is unstable. Since AKAP13knockout mouse embryonic death, we can only analyse the mechanism of AKAP13in follicular development indirectly by using heterozygous AKAP13gene knock out mice. In order to assess the impact of the AKAP13gene on the reproductive capacity in mice, the wild type male C57BL/6mice were mated with wild type female C57BL/6mice and heterozygous AKAP13gene knock out female mice respectively. Among19total litters, heterozygous mothers averaged1.6pups fewer than those of wild type females, although the difference was not significant. However, it’s still shown that the AKAP13knockout mice reproductive capacity is reduced.In conclusion, FSH can induce the expression of AKAP13in addition to the known LHR. Knock down AKAP13expression not only can reduce basal expression of LHR and Aromatase mRNA. but also impair FSH induction of LHR. Besides that, reproductive capacity of the AKAP13gene knock out mice is reduced. All this shows that AKAP13may participate in FSHR signaling transduction by acting on the downstream effectors molecules of FSHR signal pathway and then plays a crucial role in mouse follicular development. |
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