Merkl M, Ertl R, Handschuh S, Aurich C, Schäfer-Somi S
Clinic for Obstetrics, Gynecology, and Andrology, Department for Small Animals and Horses, Vetmeduni Vienna, Vienna, Austria.
Vetcore Facility for Research, Vetmeduni Vienna, Vienna, Austria.
Theriogenology. 2016 Apr 1;85(6):1080-9. doi: 10.1016/j.theriogenology.2015.11.019. Epub 2015 Nov 30.
In the present study, we assessed the presence of the ATP-binding-cassette (ABC) transporter molecules ABCA1 in spermatozoa of adult stallions and in testicular and epididymal tissue of prepubertal and adult stallions. For this purpose, semen samples from six fertile Shetland pony stallions aged 4 to 19 years were collected. Semen was collected from each stallion on three consecutive days. Ejaculates were analyzed immediately after collection, and only ejaculates meeting minimal requirements for fertile stallions were further evaluated. ABCA1 immunosignal was localized after staining of semen smears with different antibodies and counterstaining with Fluorescein isothiocyanate (FITC)-peanut agglutinin (PNA) and 4',6-Diamidin-2-phenylindol (DAPI). In a total of three samples, capacitation and acrosome reaction were induced by means of capacitation medium and progesterone substitution, respectively. Testicular and epididymal tissues were obtained from five prepubertal stallions aged 8 to 12 months and five adult stallions aged 4 to 9 years. For quantitative RT-PCR (qPCR), testicular and epididymal tissue of another seven adult (aged 1.5-14.5 years) and five prepupertal stallions (6-8 months) was used. For immunohistochemistry, sections from the caput, corpus, and cauda of the testes and epididymes were stained with the same specific antibodies as for immunocytochemistry. In stallion spermatozoa, strong immunosignal for ABCA1 was detected in the acrosomal area, the equatorial zone, and the principle piece of the flagellum but not in the caudal part of the head and the midpiece. In damaged or acrosome-reacted spermatozoa the FITC-PNA signal vanished together with the ABCA1 signal in most spermatozoa. In testicular tissue, strong immunostaining for ABCA1 was mainly visible in the heads and flagella of round spermatids and weaker signals in late spermatids and released spermatozoa. No staining was assessed in the Sertoli cells and spermatogonia of adult stallions, whereas strong signals in Leydig cells were present in prepubertal stallions. In prepubertal stallions, the ABCA1 messenger RNA level in testicular tissue was significantly higher than in adult stallions. We conclude that the ABCA1 transport molecule is present in adult and prepubertal stallion spermatozoa as well as testicular and epididymal tissue. ABCA1 is supposed to contribute to cholesterol transport and to support capacitation; however, this remains to be proven by functional studies. Species-specific differences concerning the localization inside the spermatozoa membrane are alike.
在本研究中，我们评估了ATP结合盒（ABC）转运蛋白分子ABCA1在成年种马精子以及青春期前和成年种马的睾丸和附睾组织中的存在情况。为此，收集了6匹年龄在4至19岁的可育设得兰矮种马的精液样本。从每匹种马连续三天采集精液。采集后立即对射精样本进行分析，只有符合可育种马最低要求的射精样本才会进一步评估。在用不同抗体对精液涂片进行染色并用异硫氰酸荧光素（FITC）-花生凝集素（PNA）和4',6-二脒基-2-苯基吲哚（DAPI）进行复染后，定位ABCA1免疫信号。总共对三个样本分别通过获能培养基和孕酮替代诱导获能和顶体反应。从5匹年龄在8至12个月的青春期前种马和5匹年龄在4至9岁的成年种马获取睾丸和附睾组织。对于定量逆转录聚合酶链反应（qPCR），使用了另外7匹成年（年龄在1.5至14.5岁）和5匹青春期前种马（6至8个月）的睾丸和附睾组织。对于免疫组织化学，睾丸和附睾头部、体部和尾部的切片用与免疫细胞化学相同的特异性抗体进行染色。在种马精子中，在顶体区域、赤道区和鞭毛的主段检测到强烈的ABCA1免疫信号，但在头部尾部和中段未检测到。在受损或发生顶体反应的精子中，大多数精子的FITC-PNA信号与ABCA1信号一起消失。在睾丸组织中，ABCA1的强烈免疫染色主要在圆形精子细胞的头部和鞭毛中可见，在晚期精子细胞和释放的精子中信号较弱。在成年种马的支持细胞和精原细胞中未评估到染色，而在青春期前种马的睾丸间质细胞中存在强烈信号。在青春期前种马中，睾丸组织中的ABCA1信使核糖核酸水平显著高于成年种马。我们得出结论，ABCA1转运分子存在于成年和青春期前种马的精子以及睾丸和附睾组织中。ABCA1被认为有助于胆固醇转运并支持获能；然而，这仍有待功能研究证实。精子细胞膜内定位的物种特异性差异是相似的。
