Ryan P L, Klonisch T, Yamashiro S, Renaud R L, Wasnidge C, Porter D G
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Canada.
J Reprod Fertil. 1997 Jul;110(2):329-38. doi: 10.1530/jrf.0.1100329.
Immunoreactive, chromatographic and molecular techniques were used to study the expression of relaxin in mare ovaries at different stages of the oestrous cycle. Relaxin in follicular fluid ranged from 1.6 to 2.5, from 1.4 to 5.2, from 1.2 to 6.7 and from 1.0 to 3.5 ng ml-1 in small (< or = 2 cm), medium (> 2 < or = 3 cm), medium-large (> 3 < or = 4 cm) and large (> 4 cm) follicles, respectively, and total content of fluid relaxin per follicle increased (P < 0.05) with follicular size. When subjected to reverse phase HPLC analysis, follicular fluid yielded absorbance profiles corresponding closely to those of purified relaxin, and immunoreactive peaks in follicular fluid fractions measured by radioimmunoassay matched peaks of the relaxin standard. While relaxin was localized immunocytochemically to granulosa and theca cells of preovulatory follicles, northern blot and reverse transcriptase-PCR followed by Southern blot analysis failed to detect a relaxin transcript in these tissues. A single relaxin transcript (428 bp) corresponding to mRNA encoding relaxin was identified in early, mid- and late stage corpora lutea but not in corpora haemorrhagica or albicantia. Northern blot analysis revealed a weakly expressed 1 kb transcript in total cellular RNA from mature corpora lutea. In situ hybridization studies localized the mRNA to the large luteal cells of mature corpora lutea and relaxin protein was detected by immunocytochemistry in the same tissue. This is the first report demonstrating relaxin in the equine ovary and its expression by luteal cells, thereby suggesting a role for relaxin in follicular or corpus luteum function in cyclic mares.
采用免疫反应、色谱分析和分子技术研究发情周期不同阶段母马卵巢中松弛素的表达。小卵泡（直径≤2cm）、中等卵泡（直径＞2cm≤3cm）、中等大卵泡（直径＞3cm≤4cm）和大卵泡（直径＞4cm）卵泡液中的松弛素含量分别为1.6至2.5、1.4至5.2、1.2至6.7和1.0至3.5 ng/ml，每个卵泡中卵泡液松弛素的总含量随卵泡大小增加（P＜0.05）。对卵泡液进行反相高效液相色谱分析时，其吸光度图谱与纯化的松弛素非常接近，通过放射免疫测定法测得的卵泡液组分中的免疫反应峰与松弛素标准品的峰相匹配。虽然通过免疫细胞化学方法将松弛素定位到排卵前卵泡的颗粒细胞和膜细胞，但Northern印迹法以及逆转录酶 - PCR随后的Southern印迹分析未能在这些组织中检测到松弛素转录本。在早期、中期和晚期黄体中鉴定出一个与编码松弛素的mRNA相对应的单一松弛素转录本（428bp），但在出血性黄体或白体中未检测到。Northern印迹分析显示成熟黄体的总细胞RNA中有一个弱表达的1kb转录本。原位杂交研究将mRNA定位到成熟黄体的大黄体细胞，并且通过免疫细胞化学在同一组织中检测到了松弛素蛋白。这是首次报道在马卵巢中发现松弛素及其由黄体细胞表达，从而提示松弛素在周期性母马的卵泡或黄体功能中发挥作用。
