通过流式细胞术分离的大小型牛黄体细胞中孕酮生成的调控

Control of progesterone production in small and large bovine luteal cells separated by flow cytometry.

作者信息

Alila H W, Dowd J P, Corradino R A, Harris W V, Hansel W

机构信息

Department of Physiology, Baker Institute of Animal Health, New York State College of Veterinary Medicine, Cornell University, Ithaca 14853.

出版信息

J Reprod Fertil. 1988 Mar;82(2):645-55. doi: 10.1530/jrf.0.0820645.

DOI:10.1530/jrf.0.0820645

PMID:3163003

Abstract

Corpora lutea were collected from Holstein heifers on Days 10 and 12 of the oestrous cycle and the cells were dispersed with collagenase. The dispersed cells were separated into preparations of highly purified (90-99%) small (less than 20 microns) and large (greater than 25 microns) luteal cells by unit gravity sedimentation and fluorescence-activated cell sorting. Net progesterone accumulation by 1 x 10(5) small cells and 1 x 10(3) large cells during 2 and 4 h incubations, respectively, were measured after additions of LH, PGF-2 alpha, and phorbol esters, alone and in combination. Progesterone synthesis was increased (P less than 0.05) by phorbol dibutyrate (PBt2) or PGF-2 alpha (P less than 0.05) in small, but not in large, luteal cells (10.1 +/- 3.0 and 18.1 +/- 5.0 ng/10(5) cells for 0 and 50 nM-PBt2, and 19.9 +/- 3.2 and 44.2 +/- 9.3 ng/10(5) cells for 0 and 1 microgram PGF-2 alpha/ml). The previously reported stimulatory effects of PKC activation and PGF-2 alpha addition to total dispersed cell preparations are therefore entirely attributable to the small, theca-derived cells. Small cells responded to low levels of LH (9.1 +/- 1.1, 69.0 +/- 5.4 and 154.7 +/- 41.4 ng/10(5) cells for 0, 1 and 5 ng LH/ml, respectively, P less than 0.05), while large cells responded only to high levels of LH (1635 +/- 318, 2662 +/- 459 and 3386 +/- 335 pg/10(3) cells for 0, 100 and 1000 ng LH/ml, respectively, P less than 0.05). PGF-2 alpha inhibited LH-, 8-Br-cAMP- and forskolin-stimulated progesterone synthesis in the large cells (3052 +/- 380, 3498 +/- 418, 3202 +/- 391 pg/10(3) cells for 1 microgram LH/ml, and 0.5 mM-8-Br-cAMP, and 1 microM-forskolin respectively and 1750 +/- 487, 2255 +/- 468, 2165 +/- 442 pg/10(3) cells for PGF-2 alpha + LH, PGF-2 alpha + 8-Br-cAMP and PGF-2 alpha + forskolin, respectively), indicating that the inhibitory effect of PGF-2 alpha on progesterone synthesis in large cells occurs at a site distal to cAMP generation. These results suggest that the large cells are the targets of the luteolytic effects of PGF-2 alpha, while the small cells are responsible for the previously reported luteotrophic effect of PGF-2 alpha in vitro.

摘要

在发情周期的第10天和第12天从荷斯坦小母牛收集黄体，并用胶原酶分散细胞。通过单位重力沉降和荧光激活细胞分选将分散的细胞分离成高度纯化（90 - 99%）的小（小于20微米）和大（大于25微米）黄体细胞制剂。在添加LH、PGF - 2α和佛波酯单独及联合使用后，分别测定1×10⁵个小细胞和1×10³个大细胞在2小时和4小时孵育期间的孕酮净积累量。佛波二丁酸酯（PBt2）或PGF - 2α（P < 0.05）可增加小黄体细胞中的孕酮合成（对于0和50 nM - PBt2，分别为10.1±3.0和18.1±5.0 ng/10⁵个细胞；对于0和1微克PGF - 2α/ml，分别为19.9±3.2和44.2±9.3 ng/10⁵个细胞），但对大黄体细胞无此作用。因此，先前报道的PKC激活和PGF - 2α添加到总分散细胞制剂中的刺激作用完全归因于小的、来自膜的细胞。小细胞对低水平的LH有反应（对于0、1和5 ng LH/ml，分别为9.1±1.1、69.0±5.4和154.7±41.4 ng/10⁵个细胞，P < 0.05），而大细胞仅对高水平的LH有反应（对于0、100和1000 ng LH/ml，分别为1635±318、2662±459和3386±335 pg/10³个细胞，P < 0.05）。PGF - 2α抑制大细胞中LH - 、8 - Br - cAMP - 和福斯可林刺激的孕酮合成（对于1微克LH/ml、0.5 mM - 的8 - Br - cAMP和1 microM - 的福斯可林，分别为3052±380、3498±418、3202±391 pg/10³个细胞；对于PGF - 2α + LH、PGF - 2α + 8 - Br - cAMP和PGF - 2α + 福斯可林，分别为1750±487、2255±468、2165±442 pg/10³个细胞），表明PGF - 2α对大细胞中孕酮合成的抑制作用发生在cAMP产生的远端位点。这些结果表明，大细胞是PGF - 2α黄体溶解作用的靶点，而小细胞负责先前报道的PGF - 2α在体外的黄体营养作用。