FGFs/FGFRs及其介导信号通路基因的异常表达影响犏牛未分化精原细胞增殖活性

doi:10.11843/j.issn.0366-6964.2023.07.020

摘要/Abstract

摘要： 旨在研究FGFs （fibroblast growth factors）/FGFRs （fibroblast growth factor receptors）及其介导的Ras和MAPK信号通路基因在牦牛和犏牛未分化精原细胞的差异表达，以探讨犏牛生精停滞的原因。本研究采集健康的24月龄3头公麦洼牦牛和3头F1代公犏牛的睾丸组织，分为牦牛和犏牛两个样品组，每组3个生物学重复。采用HE染色检测牦牛和犏牛精子发生的差异；采用细胞群体倍增时间、CCK-8和EDU染色检测牦牛和犏牛未分化精原细胞增殖能力的差异；采用荧光定量PCR检测6种FGF家族成员、3种FGFR、FGFs/FGFRs介导的Ras和MAPK信号通路基因在牦牛和犏牛未分化精原细胞中的差异表达。与牦牛相比，犏牛生精小管发育异常，生殖细胞类型单一，未分化精原细胞增殖活性显著低于牦牛（P<0.05）。FGF2、FGF4、FGF5、FGF8、FGF9和FGF21在犏牛睾丸组织及FGFs受体FGFR1、FGFR2和FGFR3在犏牛未分化精原细胞的表达都显著低于牦牛（P<0.01）。FGFs/FGFRs介导Ras信号通路基因中，除HRas和ARaf以外，其余基因（Raf1、BRaf、MEK1和ERK）都是在犏牛未分化精原细胞中以更低的水平表达（P<0.01）。FGFs/FGFRs介导MAPK信号通路基因中，p38、MEKK3、MEKK6和ASK1也是在犏牛未分化精原细胞中低表达（P<0.01）。受Ras通路调控的与细胞增殖相关的基因中，只有SHISA6、ID4在犏牛未分化精原细胞中高表达（P<0.01），其余基因（TAF4B、Etv4、GFRα1和Ret）都是在牦牛未分化精原细胞中表达量高（P<0.01）。受Ras和MAPK通路共同调控的与细胞增殖相关的基因中，ETV5和BCL6B都是在牦牛未分化精原细胞中高表达（P<0.01）。受Ras通路调控的与细胞分化相关的基因中，除PIWIL4外，SOX3、NGN3和Stra8都在牦牛未分化精原细胞中高表达（P<0.01）。因此FGFs/FGFRs及其介导的Ras和MAPK通路基因在犏牛未分化精原细胞中的异常表达，降低了未分化精原细胞增殖活性，导致了犏牛没有足够数量的未分化精原细胞进入分化阶段。这可能是犏牛生精停滞的重要原因之一。

关键词: 成纤维细胞生长因子, 未分化精原细胞, 信号通路, 细胞增殖, 犏牛, 牦牛

Abstract: The aim of this study was to investigate the differential expression of FGFs (fibrolast growth factor)/FGFRs (fibrolast growth factor receptors) and genes in their mediated Ras and MAPK signaling pathways in undifferentiated spermatogonia of yak and cattleyak, in order to explore the causes of spermatogenesis arrest in cattleyak. In this study, testicular tissues of 3 healthy male Maiwa yaks and 3 F1 generation cattleyak aged 24 months were collected and divided into two sample groups, yak and cattleyak, with 3 replicates in each group. HE staining was used to detect the difference of spermatogenesis between yak and cattleyak. The difference of proliferation ability of undifferentiated spermatogonia of yak and cattleyak was detected by population doubling times, CCK-8 and EDU staining. The differential expressions of 6 FGF family members, 3 FGFRs, FGFs/FGFRs-mediated Ras and MAPK signaling pathway genes in undifferentiated spermatogonia of yak and cattleyak were detected by RT-qPCR. Compared with yak, cattleyak had abnormal seminiferous tubule development, single germ cell type, and the proliferation capacity of undifferentiated spermatogonia was significantly lower than that of yak (P<0.05). The expression of FGF2, FGF4, FGF5, FGF8, FGF9 and FGF21 in the testis of cattle-yak and the FGF receptors FGFR1, FGFR2 and FGFR3 in the undifferentiated spermatogonia of cattleyak were significantly lower than those of yak (P<0.01). In FGFs/FGFRs-mediated Ras signaling pathway genes, except for HRas and ARaf, the remaining genes (Raf1, BRaf, MEK1 and ERK) were all expressed at lower levels in cattleyak undifferentiated spermatogonia (P<0.01). p38, MEKK3, MEKK6 and ASK1 in the FGFs/FGFRs-mediated MAPK signaling pathway genes were lower expressed in the undifferentiated spermatogonia of cattleyak (P<0.01). Among the genes related to cell proliferation and regulated by the Ras pathway, only SHISA6, ID4 were highly expressed in cattleyak undifferentiated spermatogonia (P<0.01), while the remaining genes (TAF4B, ETV4, GFRα1 and Ret) were highly expressed in yak undifferentiated spermatogonia (P<0.01). Among the genes related to cell proliferation and co-regulated by the Ras and MAPK pathways, ETV5 and BCL6B were both highly expressed in undifferentiated spermatogonia of yak (P<0.01). Among the genes related to cell differentiation and regulated by the Ras pathway, except for PIWIL4, SOX3, NGN3 and stra8 were all highly expressed in yak undifferentiated spermatogonia (P<0.01). Therefore, the abnormal expression of FGFs/FGFRs and their mediated Ras and MAPK pathway genes in cattleyak reduces the proliferative activity of the undifferentiated spermatogonia, and leads to the insufficient number of undifferentiated spermatogonia entering the differentiation stage. This may be one of the important reasons for the stagnation of the reproduction of the cattleyak.

Key words: fibroblast growth factor, undifferentiated spermatogonia, signaling pathway, cell proliferation, cattleyak, yak

中图分类号:

张鹏, 王明秀, 敬科民, 彭巍, 田园, 李雨谦, 付长其, 舒适, 钟金城, 蔡欣. FGFs/FGFRs及其介导信号通路基因的异常表达影响犏牛未分化精原细胞增殖活性[J]. 畜牧兽医学报, 2023, 54(7): 2886-2897.

ZHANG Peng, WANG Mingxiu, JING Kemin, PENG Wei, TIAN Yuan, LI Yuqian, FU Changqi, SHU Shi, ZHONG Jincheng, CAI Xin. Abnormal Expression of FGFs/FGFRs and Their Mediated Signaling Pathway Genes Affect the Proliferative Activity of Undifferentiated Spermatogonia in Cattleyak[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2886-2897.

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