Soñez M C, Soñez C A, Mugnaini M T, Haedo M, Romera S A, Lombardo D M, Delhon G A
Histology and Embryology, Faculty of Veterinary Science, University of Buenos Aires, Buenos Aires, Argentina.
Biotech Histochem. 2010 Dec;85(6):355-63. doi: 10.3109/10520290903368774. Epub 2009 Nov 13.
The aim of this work was to determine the effects of cGnRH I pulse frequencies on FSH and LH release and the changes in features and number of cultured laying hen FSH-cells and LH-cells in vitro. Primary adenohypophyseal cell cultures taken from laying hens were stimulated by four 5 min pulses using 1 or 10 nM cGnRH, administered with interpulses between pulses at 15, 30 or 60 min. Pulse frequencies and dose dependent effects were examined in six separate experiments including two controls. After the last interpulse time, the supernatants were collected and stored at -70° C until the performance of an indirect enzyme-linked immunosorbent assay (ELISA) using chicken LH and chicken FSH antisera at 1:1000 and 1:2000 dilutions, respectively. Supernatants were coated in duplicate on the inner surface of Immulon 2 plates and later blocked with the optimal solutions. They were incubated with each antiserum and subsequently with isotype-specific peroxidase-labeled anti-rabbit antibodies. Hydrogen peroxide/o-phenylenediamine was added as substrate/chromogen and the optical density (OD) was determined at 492 nm. The ABC immunocytochemical method was performed to characterize and re-count the gonadotropes employing anti-chicken FSH and anti-chicken LH as primary antibodies. The number of FSH-LH cells was obtained using stereological analysis and the data were statistically processed. The ODs obtained for each anti-hormone were compared with the control groups and with each other. Significant differences were found in number of aggregated-positive LH cells, which decreased with 1 nM cGnRH-I, 15 vs. 30 min pulses, increased with 30 vs. 60 min pulses, and also with 10 nM cGnRH-I, 30 vs. 60 min pulses. Aggregated positive FSH cells, however, did not show significant differences in percentage at any GnRH dose or pulse frequencies, but did show activity at low pulse frequencies of 15 and 30 min. The results suggest that LH cells varied in percentage in a dose dependent manner at higher pulse frequency (15 min) and were dose independent at low pulse frequency (60 min) and showed inactive features; while FSH cell numbers were unaffected showing features of activity at low pulse frequencies. High and moderate pulse frequencies of cGnRH-I (15-30 min) increased the FSH release in dose independent manner without changes in features or percentage of FSH cells. Low pulse frequency (60 min) of cGnRH-I increased LH release dose independently disminished LH cell percentage and showed changes in cells' features. These results in avian cells showed differences in responses to GnRH pulse frequencies from those reported earlier in mammals.
本研究旨在确定cGnRH I脉冲频率对促卵泡激素（FSH）和促黄体生成素（LH）释放的影响，以及体外培养的蛋鸡FSH细胞和LH细胞的特征和数量变化。从蛋鸡采集的垂体前叶原代细胞培养物，使用1或10 nM的cGnRH进行4次5分钟的脉冲刺激，脉冲间隔为15、30或60分钟。在包括两个对照组的六个独立实验中，研究了脉冲频率和剂量依赖性效应。在最后一个脉冲间隔时间后，收集上清液并储存于-70°C，直至分别使用稀释度为1:1000和1:2000的鸡LH和鸡FSH抗血清进行间接酶联免疫吸附测定（ELISA）。上清液一式两份包被在Immulon 2板的内表面，随后用最佳溶液封闭。将它们与每种抗血清孵育，随后与同型特异性过氧化物酶标记的抗兔抗体孵育。加入过氧化氢/邻苯二胺作为底物/显色剂，并在492 nm处测定光密度（OD）。采用ABC免疫细胞化学方法，以抗鸡FSH和抗鸡LH作为一抗，对促性腺激素细胞进行表征和重新计数。使用体视学分析获得FSH-LH细胞的数量，并对数据进行统计学处理。将每种抗激素获得的OD与对照组进行比较，并相互比较。发现聚集阳性LH细胞数量存在显著差异，1 nM cGnRH-I、脉冲间隔15分钟与30分钟相比数量减少，30分钟与60分钟相比数量增加，10 nM cGnRH-I、30分钟与60分钟相比数量也增加。然而，聚集阳性FSH细胞在任何GnRH剂量或脉冲频率下，百分比均未显示出显著差异，但在15分钟和30分钟的低脉冲频率下表现出活性。结果表明，在较高脉冲频率（15分钟）下，LH细胞百分比呈剂量依赖性变化，在低脉冲频率（60分钟）下呈剂量非依赖性变化，且表现出无活性特征；而FSH细胞数量不受影响，在低脉冲频率下表现出活性特征。cGnRH-I的高和中等脉冲频率（15 - 30分钟）以剂量非依赖性方式增加FSH释放，FSH细胞的特征或百分比无变化。cGnRH-I的低脉冲频率（60分钟）剂量依赖性增加LH释放，降低LH细胞百分比，并显示细胞特征变化。这些在禽类细胞中的结果表明，对GnRH脉冲频率的反应与早期在哺乳动物中报道的有所不同。
