Oocytic tumor necrosis factor alpha: localization in the neonatal ovary and throughout follicular development in the adult rat.
Oocytic tumor necrosis factor alpha: localization in the neonatal ovary and throughout follicular development in the adult rat.
复制标题
卵细胞肿瘤坏死因子α：定位于新生卵巢和成年大鼠的整个卵泡发育。
DOI：
10.1095/biolreprod50.6.1251
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发表时间：
1994
影响因子：
3.6
通讯作者：
Terranova,PF
中科院分区：
文献类型：
--
作者：
Marcinkiewicz,JL;Krishna,A;Cheung,CM;Terranova,PF
肿瘤坏死因子α（TNFα）以前已在小鼠卵母细胞内免疫定位。我们的第一个目标是研究TNFα在成年、胎儿和新生大鼠卵巢中的免疫定位。我们的第二个目的是通过北方印迹分析和逆转录聚合酶链反应（RT-PCR）检测卵巢和卵母细胞中的TNFα mRNA。我们的最终目的是确定卵母细胞是否含有生物活性TNFα。在成年大鼠的整个发情周期中收集卵巢和输卵管，在预期分娩前1天获得胎儿卵巢，在出生后2天收集新生儿卵巢。用生物素-抗生物素蛋白免疫细胞化学方法定位TNFα。免疫细胞化学结果表明，TNFα主要分布于卵母细胞的卵浆内。免疫染色存在于所有的卵母细胞在成人，包括输卵管内的排卵卵母细胞。新生儿卵母细胞内也存在卵细胞TNFα免疫染色;然而，胎儿卵母细胞不含免疫反应性TNFα。北方杂交结果显示，成年、新生和胎儿卵巢均含有TNFα mRNA。从排卵前卵泡收集的卵母细胞的RT-PCR分析产生了500 bp的cDNA条带，对应于扩增的TNFα cDNA的预测大小。随后的Southern印迹分析显示，500 bp的条带与TNFα探针杂交，表明排卵前卵母细胞含有TNFα mRNA。排卵前卵母细胞用于L929细胞的TNFα细胞毒性试验。卵母细胞中TNFα的生物活性与重组鼠TNFα相似。我们的结果为在大鼠卵母细胞中鉴定免疫反应性和生物活性TNFα提供了证据，这进一步被卵母细胞中TNFα mRNA的存在所支持。这些研究还表明TNFα可能在出生时左右出现在卵母细胞中。
Tumor necrosis factor alpha (TNFα) has previously been immunolocalized within mouse oocytes. Our first objective was to examine TNFα immunolocalization in ovaries of adult, fetal, and neonatal rats. Our second objective was to examine TNFα mRNA in ovaries by Northern blot analysis and in oocytes by reverse-transcriptase polymerase chain reaction (RT-PCR). Our final objective was to determine whether oocytes contained bioactive TNFα. Ovaries and oviducts were collected throughout the estrous cycle in adult rats, fetal ovaries were obtained 1 day before expected delivery, and neonatal ovaries were collected 2 days after birth. TNFα was localized in tissues by a biotin-avidin immunocytochemical procedure. Immunocytochemistry in adult ovaries showed that the ooplasm of the oocyte was the primary site of TNFα localization within the follicle. Immunostaining was present in all oocytes in the adult, including ovulated oocytes within the oviduct. Oocytic TNFα immunostaining was also present within oocytes in the neonate; however, fetal oocytes did not contain immunoreactive TNFα. Northern blots showed that ovaries in the adult, neonate, and fetus all contained TNFα mRNA. RT-PCR analysis of oocytes collected from preovulatory follicles generated a cDNA band of 500 bp, corresponding to the predicted size for amplified TNFα cDNA. Subsequent Southern blot analysis showed that the 500-bp band hybridized to the TNFα probe, indicating that preovulatory oocytes contain TNFα mRNA. Preovulatory oocytes were used in TNFα cytotoxicity assays with L929 cells. Oocytes contained TNFα bioactivity that was similar to that of recombinant murine TNFα in the bioassay. Our results provide evidence for the identification of immunoreactive and bioactive TNFα within oocytes in the rat, which is further supported by the presence of TNFα mRNA within the oocyte. These studies also indicate that TNFα may appear in the oocyte around the time of birth.