Does prepubertal testicular tissue vitrification influence spermatogonial stem cells (SSCs) viability?
Does prepubertal testicular tissue vitrification influence spermatogonial stem cells (SSCs) viability?
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DOI：
10.1007/s10815-013-0050-x
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发表时间：
2013-10-01
影响因子：
3.1
通讯作者：
Mohammadi-asl, Javad
中科院分区：
文献类型：
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作者：
Gholami, Mohammadreza;Hemadi, Masoud;Mohammadi-asl, Javad
癌症儿童化疗或放疗前的睾丸冷冻保存是保留生育能力的方法之一。然而，冷冻保存可能会对睾丸实质细胞造成损害。本研究的目的是探讨玻璃化对从玻璃化睾丸获得的精原干细胞（SSC）中细胞内LDH渗漏、细胞周期/凋亡反应和凋亡相关基因表达模式的影响。玻璃化和新鲜组的幼鼠（6日龄，BALB/c）的睾丸均用酶（胶原酶、DNaseI（TM）、胰蛋白酶-EDTA）消化以分散细胞。 A 型 SSC 通过 MACS 从其余睾丸细胞中分离出来。立即通过细胞毒性测定、流式细胞术测定和实时PCR评估SSC的损伤量。与新鲜的相比，从玻璃化睾丸中收获的SSC中细胞内LDH渗漏的报道较少。此外，从玻璃化睾丸中获得的凋亡和坏死的SSC的百分比低于从新鲜样品中获得的百分比。此外，从玻璃化睾丸中收集的SSC的凋亡相关基因的表达谱发生了变化，P53和BCL-2表达水平增加，Bax和Fas表达水平降低。研究表明，青春期前睾丸组织的玻璃化并没有增加睾丸SSC中凋亡相关基因（例如Bax和Fas）的表达谱，这与细胞凋亡/坏死反应减弱和细胞内LDH渗漏不增加一致。
Testicular cryopreservation prior to chemotherapy or radiotherapy in children with cancer is one of the ways to preserve fertility. However, cryopreservation may cause damage to the testicular parenchyma cells. The objective of this study was to investigate effects of vitrification on the intracellular LDH leakage, cell cycle/apoptotic responses and apoptosis-related gene expression patterns in the spermatogonial stem cells (SSCs) obtained from the vitrified testis.The testes of the mice pups (6-day-old, BALB/c) both vitrified and fresh groups were digested with enzymes (collagenase, DNaseI (TM), trypsin-EDTA) to disperse the cells. The SSCs, type A, were isolated from the rest of testicular cells by MACS. The amount of damage to the SSCs immediately was evaluated by Cytotoxicity assay, Flow cytometry assay and Real-time PCR.The intracellular LDH leakage in the SSCs,harvested from the vitrified testes, was less reported compared with the fresh ones. Moreover, the percentage of apoptotic and necrotic SSCs obtained from the vitrified testes was lower than that of yielded from the fresh samples. Also, the apoptosis-related genes of the SSCs,collected from the vitrified testes, changed their expression profile as increasing P53 and BCL-2 expression levels and decreasing Bax and Fas expression levels.The study indicates that vitrification of prepubertal testicular tissue does not increase the expression profile of apoptosis-related genes such as Bax and Fas in the testicular SSCs consistent with diminished cell apoptotic/necrotic responses and no increasing intracellular LDH leakage.