Expression and distribution of AP-1 transcription factors in the porcine ovary.
Expression and distribution of AP-1 transcription factors in the porcine ovary.
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AP-1转录因子在猪卵巢中的表达和分布。
DOI：
10.1095/biolreprod.102.013995
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发表时间：
2003
期刊：
影响因子：
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通讯作者：
LaVoie,HollyA
中科院分区：
文献类型：
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作者：
Rusovici,Raluca;LaVoie,HollyA
激活蛋白-1（AP-1）转录因子是细胞增殖和分化的重要调节因子。AP-1家族成员在猪卵巢中的发育分布以前没有研究过。我们研究了AP-1因子在猪卵泡、颗粒细胞和黄体不同发育阶段的表达。免疫印迹分析证实，c-Jun，JunD，JunB，c-Fos，Fra-1，Fra-2和FosB免疫反应蛋白存在于所有有腔卵泡和黄体中期黄体（CL）的全细胞提取物（WCE）以及从不同大小的有腔卵泡分离的颗粒细胞（GC）。与有腔卵泡相比，CL WCE中c-Jun和c-Fos蛋白条带的强度降低。在排卵前卵泡（8-10 mm）的颗粒细胞中，与较小窦卵泡的颗粒细胞相比，Fra-2表现出从43 kDa到46 kDa的变化。分离细胞质和细胞核提取物，以确定这些组分之间是否存在发育差异。大多数AP-1因子在核部分中占主导地位，但有明显的例外。c-Fos在GC和滤泡中主要分布在细胞核中，而在CL中主要分布在细胞质中。除了GC从1-2毫米的卵泡，其中表达是相似的馏分之间，Fos-B主要是在细胞质部分。Fra-1在所有组织的细胞质和细胞核组分之间表现出相似的表达。对猪卵巢切片进行免疫组织化学（IHC）分析，以确定这些因子在不同卵泡阶段的细胞分布，并对免疫阳性细胞核进行评价。在原始和初级单层卵泡中，除了FosB外，所有AP-1因子均在颗粒核中检测到。多层腔前卵泡的颗粒细胞核对所有因子均呈免疫阳性，FosB表达较低。除卵泡膜中的FosB外，窦卵泡的所有因子均显示GC和卵泡膜细胞核染色。黄体细胞表现出最强烈的核染色的JUND和Fra-2，而所有其他因素都存在于黄体细胞核，虽然在较小的程度。用FosB抗体进行的IHC主要产生细胞质染色，但仅产生弱的黄体核染色。在白色体中，所有AP-1因子均观察到低水平的染色。EMSA法检测这些因子在颗粒细胞和CL中的DNA结合能力。来自1-2-mm或8-10-mm窦卵泡的颗粒细胞的核提取物结合AP-1 DNA共有序列，复合物主要由c-Jun、JunD、JunB、c-Fos和Fra-2组成。在CL中，c-Jun、JunD、JunB和Fra-2存在于DNA结合复合物中，未检测到c-Fos结合。总之，我们的研究结果表明，表达和DNA结合活性的AP-1因子在卵泡结构的变化与黄体化。向黄体表型的分化涉及核c-Jun和c-Fos的减少以及JunD和Fra-2的优势。
The activator protein-1 (AP-1) transcription factors are important regulators of cell proliferation and differentiation. The developmental distribution of AP-1 family members in porcine ovary has not been previously investigated. We examined the expression of AP-1 factors in porcine ovarian follicles, granulosa cells, and corpora lutea at different stages of development. Immunoblot analyses confirmed that c-Jun, JunD, JunB, c-Fos, Fra-1, Fra-2, and FosB immunoreactive proteins were present in whole-cell extracts (WCE) of all antral follicles and midluteal phase corpora lutea (CL) as well as granulosa cells (GC) isolated from different-sized antral follicles. The intensities of c-Jun and c-Fos protein bands were decreased in CL WCE compared to antral follicles. In granulosa cells from preovulatory follicles (8–10 mm), Fra-2 exhibited a shift from 43 kDa to 46 kDa when compared to granulosa cells from smaller antral follicles. Separation of cytoplasmic and nuclear extracts was performed to determine if developmental differences between these fractions existed. Most AP-1 factors predominated in the nuclear fraction with notable exceptions. c-Fos predominated in the nucleus in GC and follicles but predominated in the cytoplasmic fraction of CL. With the exception of GC from 1–2-mm follicles, in which expression was similar between fractions, Fos-B was found predominantly in the cytoplasmic fraction. Fra-1 exhibited similar expression between cytoplasmic and nuclear fractions for all tissues. Immunohistochemical (IHC) analyses of porcine ovary sections were performed to determine the cellular distribution of these factors at different follicular stages, and immunopositive nuclei were evaluated. In primordial and primary unilaminar follicles, all AP-1 factors studied except for FosB were detected in granulosa nuclei. Granulosa cell nuclei of multilaminar preantral follicles were immunopositive for all factors, with lower expression of FosB. Antral follicles exhibited GC and thecal cell nuclear staining for all factors with the exception of FosB in theca. Luteal cells exhibited the most intense nuclear staining for JunD and Fra-2, whereas all other factors were present in luteal cell nuclei although to a lesser extent. IHC with FosB antibodies yielded mostly cytoplasmic staining but only weak luteal nuclear staining. In corpora albicantia, low levels of staining were seen for all AP-1 factors. The DNA-binding abilities of these factors in granulosa cells and CL were evaluated by EMSA. Nuclear extracts from granulosa cells from 1–2-mm or 8–10-mm antral follicles bound an AP-1 DNA consensus sequence and complexes consisted predominantly of c-Jun, JunD, JunB, c-Fos, and Fra-2. In CL, c-Jun, JunD, JunB, and Fra-2 were present in DNA-binding complexes, and c-Fos binding was not detected. In conclusion, our results suggest that expression and DNA-binding activity of AP-1 factors in follicular structures changes with luteinization. Differentiation to the luteal phenotype involves a reduction in nuclear c-Jun and c-Fos and a predominance of JunD and Fra-2.