Rodríguez C M, Day J R, Killian G J
Department of Dairy and Animal Science, Pennsylvania State University, University Park, PA16802, USA.
J Reprod Fertil. 2000 Nov;120(2):303-9. doi: 10.1530/jrf.0.1200303.
The aim of this study was to localize expression of the prostaglandin D synthase gene in the reproductive tracts of Holstein bulls using northern blotting and in situ hybridization. For northern blotting, a digoxigenin-labelled prostaglandin D synthase cDNA probe was used to probe blots containing RNA isolated from the testes, epididymides, vas deferens, ampullae, seminal vesicles, prostate and bulbourethral glands of bulls. The digoxigenin-labelled cDNA for the bovine homologue of prostaglandin D synthase hybridized to a single band (approximately 0.9 kb) to RNA samples from the caput, corpus and cauda epididymides, as well as RNA samples from the vas deferens and the ampulla. The probe also detected a single band in testis samples, although the transcript size was slightly larger (approximately 1.0 kb) than the transcript found in the other tissues. The highest expression of prostaglandin D synthase was observed in the testes and caput epididymides. Prostaglandin D synthase transcripts were not found in the seminal vesicles or the prostate or bulbourethral glands using northern blotting. For in situ hybridization, antisense and sense riboprobes were synthesized and used to hybridize to cryosections obtained from the reproductive tissues of bulls. In situ hybridization of bull testes showed that prostaglandin D synthase transcripts were present within the germ cells in the adluminal compartment of the seminiferous tubules containing round and elongated spermatids, indicating that expression varied with stage of development of the seminiferous tubules. Prostaglandin D synthase expression was observed in the epithelial cells of the epididymides with greatest expression occurring in the caput epididymidis. Some expression was also observed in the epithelial cells of the vas deferens and a few cells of some lobules in the prostate and bulbourethral glands. Expression of the prostaglandin D synthase gene was not detected in ampullae or seminal vesicles by in situ hybridization.
本研究的目的是利用Northern印迹法和原位杂交技术,定位前列腺素D合成酶基因在荷斯坦公牛生殖道中的表达。对于Northern印迹法,使用地高辛标记的前列腺素D合成酶cDNA探针,探测含有从公牛的睾丸、附睾、输精管、壶腹、精囊、前列腺和尿道球腺分离的RNA的印迹。地高辛标记的牛前列腺素D合成酶同源物的cDNA与来自附睾头、附睾体和附睾尾的RNA样品以及来自输精管和壶腹的RNA样品杂交,形成一条单一的条带(约0.9 kb)。该探针在睾丸样品中也检测到一条单一的条带,尽管转录本大小略大于在其他组织中发现的转录本(约1.0 kb)。前列腺素D合成酶的最高表达在睾丸和附睾头中观察到。使用Northern印迹法在精囊、前列腺或尿道球腺中未发现前列腺素D合成酶转录本。对于原位杂交,合成了反义和正义核糖探针,并用于与从公牛生殖组织获得的冰冻切片杂交。公牛睾丸的原位杂交显示,前列腺素D合成酶转录本存在于含有圆形和延长精子细胞的生精小管近腔室的生殖细胞内,表明表达随生精小管的发育阶段而变化。在附睾的上皮细胞中观察到前列腺素D合成酶的表达,在附睾头中表达最高。在输精管的上皮细胞以及前列腺和尿道球腺的一些小叶的少数细胞中也观察到一些表达。通过原位杂交在壶腹或精囊中未检测到前列腺素D合成酶基因的表达。