Functional aromatase expression in porcine adrenal gland and testis
Functional aromatase expression in porcine adrenal gland and testis
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DOI：
10.1095/biolreprod54.2.497
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发表时间：
1996-02-01
影响因子：
3.6
通讯作者：
Harada, N
中科院分区：
文献类型：
--
作者：
Conley, AJ;Corbin, CJ;Harada, N
研究了芳香化酶细胞色素P450（P450（arom））在胎猪和新生猪肾上腺、睾丸和胎盘中的表达。Western免疫印迹分析在这些组织以及已知表达P450（arom）的其他猪组织（包括第12天的管状孕体、内膜和颗粒）中检测到单个48 - 50-kDa蛋白条带。迁移的轻微差异表明，P450蛋白在睾丸中的表达大于在肾上腺中，这是，反过来，大于在胎盘，卵泡膜，和颗粒。与P450（arom）在这些组织中的表达一致，编码猪P450（arom）的cDNA与猪囊胚、胎盘、胎儿和新生儿肾上腺和睾丸以及排卵前卵泡的卵泡膜和颗粒组织的北方分析中的2.3 kb转录本杂交。组织间转录本大小无差异。根据RACE程序，通过对从猪胎儿肾上腺、睾丸和胎盘中扩增的部分cDNA克隆进行序列分析，证实了P450（arom）转录本的身份。肾上腺和睾丸克隆的序列是相同的，但不同于胎盘序列，其代表了猪P450（arom）的前85个氨基酸。具体而言，肾上腺和睾丸克隆表达的转录本类似于猪P450的卵巢亚型（arom），而不是猪胎盘亚型，预测两个氨基酸缺失和12个预测的氨基酸取代。P450（arom）活性进行了检查，以进一步确定在这些组织中的表达。肾上腺、睾丸和胎盘匀浆中的活性受到4-羟基雄烯二酮（4OH-A4）的抑制，而依托咪酯的抑制作用在肾上腺和睾丸匀浆中得到证实，但在胎盘匀浆中没有。新生猪肾上腺和睾丸中活性对依托咪酯抑制的敏感性与卵巢P450（arom）活性相似。P450（arom）活性水平在胎盘中最高，在肾上腺中最低，在两种组织中均未观察到胎儿性别的影响。免疫细胞化学研究定位P450（芳香族）在新生儿肾上腺皮质髓质交界处的细胞，并具有更大的强度，细胞周围的发展中的髓质小叶的表达。相同的细胞表达细胞色素P450 17 α-羟化酶，其表达也延伸到整个束状体。P450（arom）在睾丸间质细胞中表达，但在精子小管内的任何细胞中均未检测到表达。这些数据表明，胎儿和新生猪肾上腺和睾丸表达活性P450（arom），类似于卵巢中表达的同种型。肾上腺表达的定位表明在胎儿和新生猪的髓质成熟和功能中可能起作用。
The expression of aromatase cytochrome P450 (P450(arom)) in the adrenal glands, testes, and placentas of fetal and newborn pigs was investigated. Western immunoblot analysis detected a single 48-50-kDa protein band in these tissues as well as in other porcine tissues known to express P450(arom) including Day 12 tubular conceptuses, theca interna, and granulosa. Slight differences in migration suggested that the P450 protein expressed in the testis was larger than that in the adrenal gland, which was, in turn, larger than that in placenta, theca, and granulosa. Consistent with P450(arom) expression in these tissues, a cDNA encoding porcine P450(arom) hybridized to a 2.3-kb transcript in Northern analyses of porcine blastocysts, placentas, and fetal and newborn adrenal glands and testes, as well as in theca and granulosa tissues from preovulatory follicles. No differences in transcript size were detectable among tissues. The identity of P450(arom) transcripts was confirmed by sequence analysis of partial cDNA clones amplified from porcine fetal adrenal glands, testes, and placentas according to the RACE procedure. The sequences of the adrenal and testis clones were identical but differed from the placental sequence, which represented the first 85 amino acids of porcine P450(arom). Specifically, the adrenal and testis clones expressed transcripts that resembled the ovarian isoform of porcine P450(arom) rather than the porcine placental isoform, predicting a two-amino acid deletion and 12 predicted amino acid substitutions.P450(arom) activity was examined to further define expression in these tissues. Activity in adrenal, testis, and placental homogenates was inhibited by 4-hydroxyandrostenedione (4OH-A4) whereas inhibition by etomidate was demonstrated in the adrenal and testis homogenates but not in the placental homogenates. The sensitivity of activity in the newborn porcine adrenal glands and testes to inhibition by etomidate was similar to that of ovarian P450(arom) activity. The level of P450(arom) activity was highest in the placenta and lowest in the adrenal gland, and no effect of fetal sex was noted in either tissue. Immunocytochemical studies localized the expression of P450(arom) in the adrenal gland of newborns to cells at the corticomedullary junction and, with greater intensity, to cells around the developing medullary lobules. The same cells expressed cytochrome P450 17 alpha-hydroxylase, the expression of which also extended throughout the zona fasciculata. The interstitial cells were the site of P450(arom) expression in the testis, but no expression could be detected in any cells within the spermatic tubules. These data demonstrate that fetal and newborn porcine adrenal glands and testes express an active P450(arom) that resembles the isoform expressed in the ovary. The localization of adrenal expression suggests a possible role in medullary maturation and function in the fetal and newborn pig.