摘要类无精症缺失基因(deleted in azoospermia-like gene, Dazl)在雄性动物生殖细胞分化过程中起重要调控作用，但对于不同物种或处于不同发育阶段的同一物种而言，其调控机制存在差异。为了探讨Dazl在绵羊(Ovis aries)不同发育期睾丸组织中的表达、定位及调控作用，本实验选取0、2、5、12和24月龄健康的绵羊15只，每组3只，采取睾丸、心脏、肝脏、脾脏、肺脏、肾脏和肌肉组织。运用实时荧光定量PCR(quantitative Real time PCR, qRT-PCR)检测了0、2、5、12和24月龄绵羊睾丸组织及12月龄绵羊的不同体组织(心脏、肝脏、脾脏、肺脏、肾脏和肌肉)中Dazl mRNA的表达量，利用Western blot技术对Dazl基因编码蛋白的表达量进行检测，并采用免疫组织化学染色方法检测Dazl蛋白的阳性信号在不同月龄睾丸组织中的分布情况。结果显示，对于不同月龄睾丸组织，从mRNA和蛋白水平均检测到了Dazl基因的表达，并且二者的趋势基本相似。在0、2和5月龄睾丸中Dazl mRNA及蛋白表达量较低，12月龄中表达量较高且极显著高于0、2和5月龄(P＜0.01)，而在24月龄中Dazl mRNA及蛋白的表达量均出现不同程度的下调现象。12月龄绵羊各组织的qRT-PCR检测结果显示，Dazl mRNA只在睾丸组织中高表达，在心脏中表达量很低，而在其他组织中不表达。免疫组织化学染色结果显示，Dazl蛋白在初情期前(0、2和5月龄)定位于睾丸间质细胞，而在12和24月龄定位于睾丸间质细胞、初级精母细胞、次级精母细胞和精子细胞。研究结果表明，Dazl基因在性成熟前公羊的睾丸间质细胞及成年羊的睾丸间质细胞、精母细胞和精细胞中表达，并且在他们的增殖过程中起调控作用。由此推测，在绵羊睾丸发育过程中，Dazl基因可能通过直接作用(调控精母细胞的成熟分裂)和间接作用(调控睾丸间质细胞的增殖)共同调控精子发生。本研究为进一步研究Dazl基因在雄性动物精子发生过程中的调控机制提供了科学依据和参考。
Abstract：Deleted in azoospermia-like gene (Dazl) is required for differentiation of male animal germ cells. However, the regulation mechanisms of Dazl gene are not exactly the same in broad variety of animals or in the same animal at different developmental stages. In order to investigate the expression patterns, cellular localization and regulatory mechanisms involving with spermatogenesis of the Dazl gene in sheep (Ovis aries)testes at different developmental stages, testis, heart, liver, spleen, lung, kidney and muscle tissues were collected from Small-tail Han sheep at 0, 2, 5, 12 and 24 months of age. The Dazl mRNA expression patterns in 0, 2, 5, 12, 24-month-old sheep testes and multiple somatic tissues, including heart, liver, spleen, lung, kidney and muscle of 12-month-old sheep were detected by quantitative real time PCR (qRT-PCR), and the Dazl protein expression and cellular localization in testes at different developmental stages were observed and measured by Western blot and immunohistochemistry, respectively. The qRT-PCR and western blot results revealed that Dazl genes were expressed in sheep testes throughout development at mRNA and protein levels, and their expression patterns were similar basically. The Dazl mRNA and protein were at a lower expression level in pre-pubertal sheep testes (0-, 2- and 5-month-old), and the expression of the Dazl mRNA and protein in 12-month-old were significantly higher than those in 0-, 2- and 5-month-old (P＜0.01), whereas their expression levels were decreased in 24-month-old sheep testis. The qRT-PCR results in various tissues at 12 months of age, including testis, heart, liver, spleen, lung, kidney and muscle tissues, showed that the Dazl mRNA was predominantly expressed in testis, and low-expressed in heart, whereas not expressed in other tissues. The immunohistochemical staining in testes at different developmental stages showed that the Dazl protein was localized to Leydig cells in pre-pubertal (0-,2 -and 5-month-old) sheep testes, while it was localized to Leydig cells, primary spermatocytes, secondary spermatocytes and sperm cells in 12- and 24-month-old sheep testes. These results suggested that the Dazl gene was highly expressed in testes, playing vital roles in the proliferation of Leydig cells in various age of sheep testes, spermatocytes and sperm cells in post-pubertal sheep testes. It is inferred that the Dazl gene is involved in the regulation of postnatal ovine spermatogenesis via direct effect (modulating the maturation and meiosis of spermatocytes) and indirect effect (modulating the proliferation of Leydig cells). The observation provides a scientific basis and reference for further research on the regulatory mechanism of Dazl gene during spermatogenesis.
