目的:精确控制睾丸间质细胞的增殖和分化对性腺雄激素生成和精子发生具有重要意义。尽管细胞周期蛋白依赖性激酶抑制剂对细胞增殖和分化至关重要,它们在早期成年Leydig细胞(ALC)发育中的作用仍未得到答复。要了解ALC开发的机制,在小鼠的干Leydig细胞(SLC)和祖细胞Leydig细胞(PLC)中研究了p57KIP2(cdkn1c)的功能表达。
方法:p57KIP2在增殖中的作用,分化,凋亡,通过早期新生儿睾丸中的抗体和溴脱氧尿苷(BrdU)标记以及分离的SLC和PLC中的p57kip2siRNA研究了SLC和PLC中的类固醇生成。通过细胞培养物中孕酮和睾丸激素的产生来检查PLC的类固醇分化。
结果:从出生后第1天到第14天,睾丸间质中的p57KIP2()梭形细胞为α-平滑肌肌动蛋白(αSMA)(-),肾小管周围的肌样细胞标记,表明它们是SLC和PLC。此外,p57KIP2也在HSD3β(+)胎儿睾丸间质细胞中表达。从PND1到14,BrdU(+)/αSMA(-),Ki67(+)/p57KIP2(+),BrdU(+)/p57KIP2(+)纺锤形细胞逐渐减少。从PND1到14,αSMA(-)/p57KIP2(+)细胞中的p57KIP在PND7达到峰值,此后降低。在THY1(+)分离的SLC中,p57kip2siRNA显著增加ki67和pcnamRNA和pdgfrαmRNA,分化标记和减少的巢蛋白mRNA,SLC标记。p57kip2siRNA处理后,凋亡相关基因mRNA无明显差异。在HSD3β(+)PLC中,p57kip2siRNA增加促凋亡基因mRNA,膜联蛋白V(+)早期凋亡细胞。重要的是,p57kip2siRNA显着降低了hsd3β6和cyp17a1mRNA和孕酮的产生。
结论:p57KIP2可抑制SLC的增殖并支持其干性。在PLC中,p57KIP2可能抑制细胞凋亡并增强类固醇生成分化。
OBJECTIVE: Precise control of proliferation and differentiation of Leydig cells is important for gonadal androgenesis and spermatogenesis. Though cyclin-dependent kinase inhibitors are crucial for cell proliferation and differentiation, their role in the development of early adult Leydig cells (ALCs) remained unanswered. To understand mechanism for ALC development, functional expression of p57KIP2 (cdkn1c) was investigated in the stem Leydig cells (SLCs) and progenitor Leydig cells (PLCs) in mice.
METHODS: The roles of p57KIP2 in the proliferation, differentiation, apoptosis, and steroidogenesis in SLCs and PLCs were investigated by antibodies and bromodeoxyuridine (BrdU) labeling in the early neonatal testes and p57kip2 siRNA in the isolated SLCs and PLCs. Steroidogenic differentiation of PLCs was examined by progesterone and testosterone production in cell culture.
RESULTS: From postnatal day (PND) 1 to 14, p57KIP2(+) spindle-shaped cells in the testis interstitium were α-smooth muscle actin (αSMA)(-), a peritubular myoid cells marker, suggesting that they are SLCs and PLCs. Besides, p57KIP2 was also expressed in HSD3β(+) fetal Leydig cells. From PND1 to 14, BrdU(+)/αSMA(-), Ki67(+)/p57KIP2(+), and BrdU(+)/p57KIP2(+) spindle-shaped cells were gradually decreased. From PND1 to 14, p57KIP in the αSMA(-)/p57KIP2(+) cells was peaked at PND7 and decreased thereafter. In THY1(+) isolated SLCs, p57kip2 siRNA significantly increased ki67 and pcna mRNA and pdgfrα mRNA, a differentiation marker and decreased nestin mRNA, a SLC marker. No significant difference in apoptosis related genes mRNA was found after p57kip2 siRNA treatment. In HSD3β(+) PLCs, p57kip2 siRNA increased proapoptotic genes mRNA, annexin V(+) early-apoptotic cells. Importantly, p57kip2 siRNA significantly decreased hsd3β6 and cyp17a1 mRNA and progesterone production.
CONCLUSIONS: p57KIP2 may suppress proliferation and support stemness of SLCs. In PLCs, p57KIP2 may suppress apoptosis and potentiate the steroidogenic differentiation.