Identification of Premeiotic, Meiotic, and Postmeiotic Cells in Testicular Biopsies Without Sperm from Sertoli Cell-Only Syndrome Patients
Identification of Premeiotic, Meiotic, and Postmeiotic Cells in Testicular Biopsies Without Sperm from Sertoli Cell-Only Syndrome Patients
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DOI：
10.3390/ijms20030470
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发表时间：
2019-02-01
影响因子：
5.6
通讯作者：
Huleihel, Mahmoud
中科院分区：
文献类型：
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作者：
Abofoul-Azab, Maram;Lunenfeld, Eitan;Huleihel, Mahmoud
只有支持细胞综合征（SCOS）影响约26.3-57.8%的无精子症男性，其曲细精管仅含有支持细胞。最近，据报道，非梗阻性无精子症（NOA）患者的睾丸活检含有生殖细胞，并且使用睾丸精子提取技术可以在20%的SCOS患者的小管中发现精子。由于睾丸活检中没有精子的患者没有治疗方法来帮助他们生育生物学上的孩子，因此从他们的睾丸中分离的精原干细胞（SSCs）的体外成熟是未来可能的不育治疗的新方法。最近，使用不同的培养系统证实了人和小鼠精原干细胞增殖和分化的诱导。我们的小组报告了使用3D琼脂和甲基纤维素（MCS）培养系统诱导小鼠、恒河猴和青春期前男孩癌症患者精原细胞增殖和分化至减数分裂和减数分裂后阶段。本研究的目的是确定SCOS患者无精子活检中存在的生精细胞类型，并检查使用3D MCS体外从这些活检的分离精原细胞诱导精子发生的可能性。我们使用了9例无精子的SCOS患者的活检组织，并通过PCR和特异性免疫荧光染色分析评估了生精标记物的存在。在存在含有不同生长因子的StemPro富集培养基的情况下，在MCS中培养分离的睾丸细胞，并在显微镜下检查集落/簇的发育。我们检查了在MCS中培养3-7周前后精子发生不同阶段的细胞的存在。我们的结果表明，这些活检显示存在减数分裂前标记物（2至7个标记物/活检），减数分裂标记物（9个活检，5个中检测到cAMP反应元件调节剂-1（CREM-1），5个中检测到乳酸脱氢酶（LDH），3个中检测到BOULE）和减数分裂后标记物（6个活检中检测到鱼精蛋白，3个中检测到顶体酶）。此外，我们能够诱导的减数分裂和/或减数分裂后阶段的精原细胞从三个活检分离的发展。因此，我们的研究首次表明，存在减数分裂和/或减数分裂后的细胞活检没有精子的SCOS患者。在体外培养条件下，来自这些活检中的一些的分离的细胞可以被诱导到减数分裂和/或减数分裂后阶段。
Sertoli cell-only syndrome (SCOS) affects about 26.3-57.8% of azoospermic men, with their seminiferous tubules containing only Sertoli cells. Recently, it was reported that testicular biopsies from nonobstructive azoospermic (NOA) patients contained germ cells, and that sperm could be found in the tubules of 20% of SCOS patients using testicular sperm extraction technology. Since the patients without sperm in their testicular biopsies do not have therapy to help them to father a biological child, in vitro maturation of spermatogonial stem cells (SSCs) isolated from their testis is a new approach for possible future infertility treatment. Recently, the induction of human and mice SSCs proliferation and differentiation was demonstrated using different culture systems. Our group reported the induction of spermatogonial cell proliferation and differentiation to meiotic and postmeiotic stages in mice, rhesus monkeys, and prepubertal boys with cancer using 3D agar and methylcellulose (MCS) culture systems. The aim of the study was to identify the type of spermatogenic cells present in biopsies without sperm from SCOS patients, and to examine the possibility of inducing spermatogenesis from isolated spermatogonial cells of these biopsies in vitro using 3D MCS. We used nine biopsies without sperm from SCOS patients, and the presence of spermatogenic markers was evaluated by PCR and specific immunofluorescence staining analyses. Isolated testicular cells were cultured in MCS in the presence of StemPro enriched media with different growth factors and the development of colonies/clusters was examined microscopically. We examined the presence of cells from the different stages of spermatogenesis before and after culture in MCS for 3-7 weeks. Our results indicated that these biopsies showed the presence of premeiotic markers (two to seven markers/biopsy), meiotic markers (of nine biopsies, cAMP responsive element modulator-1 (CREM-1) was detected in five, lactate dehydrogenase (LDH) in five, and BOULE in three) and postmeiotic markers (protamine was detected in six biopsies and acrosin in three). In addition, we were able to induce the development of meiotic and/or postmeiotic stages from spermatogonial cells isolated from three biopsies. Thus, our study shows for the first time the presence of meiotic and/or postmeiotic cells in biopsies without the sperm of SCOS patients. Isolated cells from some of these biopsies could be induced to meiotic and/or postmeiotic stages under in vitro culture conditions.