Dineva Julieta, Wojtowicz Anna K, Augustowska Katarzyna, Vangelov Ivailo, Gregoraszczuk Ewa L, Ivanova Maria D
Institute of Biology and Immunology of Reproduction, Acad. K. Bratanov, Bulgarian Academy of Science, Sofia, Bulgaria.
Endocr Regul. 2007 Mar;41(1):11-8.
Our aim was to examine the expression patterns of ANP, the rate of apoptosis bcl-2 and p53 expression and caspase-3 activity and progesterone (P) production in porcine granulosa cells (pGCs) stimulated in vitro for luteinization and after treatment with leptin.
Freshly isolated prepubertal pGCs were cultured as monolayers for 24 h, subsequently FSH was supplemented for 24 h, and finally LH was added to a part of the cells for 24 h to induce luteinization. The effect of leptin on in vitro luteinized pGCs was tested by the addition of 10 ng/ml human recombinant leptin (hrL) 24 h after LH administration. Indirect immunofluorescence for ANP, bcl-2 and p53 expression was used, P production was assayed by direct enzyme immunoassay (EIA) and colorimetric AcDEVD-Pna assay for caspase-3 activity was applied.
Stimulation of prepubertal pGCs with FSH resulted in a moderate expression of ANP and elevation in P production. When FSH treatment was followed by LH, the pronounced expression of ANP was observed in all cells. Suppressive effect of FSH and LH on p53 expression and caspase-3 activity with parallel increase in bcl-2 expression and increased P production was observed. The treatment of in vitro luteinized (FSH/LH-stimulated) pGCs with leptin did not influence the expression of ANP. However, in FSH/LH plus leptin treated cells the concomitant increase in bcl-2 expression and parallel inhibitory effect on p53 expression and caspase-3 activity was noted, compared to control cells without any significant increase in P production.
The present data demonstrated the positive relationship between ANP expression and P production in pGCs stimulated for luteinization in vitro by FSH and LH, as well as their antiapoptotic role mediated presumably by cGMP accumulation in the luteinized pGCs. A direct anti-apoptotic effect of leptin on in vitro luteinized pGCs, without any significant modulation of P production, was documented.
我们的目的是检测在体外诱导黄体化以及用瘦素处理后的猪颗粒细胞（pGCs）中，心钠素（ANP）的表达模式、凋亡率、bcl-2和p53的表达以及半胱天冬酶-3（caspase-3）活性和孕酮（P）的产生情况。
将刚分离的青春期前pGCs单层培养24小时，随后补充促卵泡激素（FSH）24小时，最后向部分细胞中添加促黄体生成素（LH）24小时以诱导黄体化。在给予LH 24小时后，通过添加10 ng/ml人重组瘦素（hrL）来检测瘦素对体外黄体化pGCs的影响。采用间接免疫荧光法检测ANP、bcl-2和p53的表达，通过直接酶免疫测定法（EIA）检测P的产生，并应用比色法AcDEVD-Pna测定法检测caspase-3活性。
用FSH刺激青春期前pGCs导致ANP适度表达且P产生增加。当FSH处理后接着用LH处理时，在所有细胞中均观察到ANP的显著表达。观察到FSH和LH对p53表达和caspase-3活性有抑制作用，同时bcl-2表达平行增加且P产生增加。用瘦素处理体外黄体化（FSH/LH刺激）的pGCs对ANP的表达没有影响。然而，与未显著增加P产生的对照细胞相比，在FSH/LH加瘦素处理的细胞中，观察到bcl-2表达同时增加，对p53表达和caspase-3活性有平行抑制作用。
目前的数据表明，在体外经FSH和LH刺激黄体化的pGCs中，ANP表达与P产生之间存在正相关关系，以及它们可能通过黄体化pGCs中cGMP积累介导的抗凋亡作用。记录了瘦素对体外黄体化pGCs有直接的抗凋亡作用，而对P产生没有任何显著调节作用。
