Marks D L, Smith M S, Clifton D K, Steiner R A
Department of Physiology and Biophysics, University of Washington, Seattle 98195.
Endocrinology. 1993 Sep;133(3):1450-8. doi: 10.1210/endo.133.3.7689958.
Galanin is colocalized with GnRH, and its expression in these neurons is enhanced at proestrus, a time of activation of GnRH neurons. We tested the hypothesis that the expression of both the GnRH and galanin mRNAs in GnRH neurons decrease during lactation in the rat, a reproductive state characterized by reduced gonadotropin secretion. For double label in situ hybridization, GnRH mRNA was detected with an antisense cRNA probe labeled with the hapten digoxigenin, whereas galanin mRNA was detected with a cRNA probe labeled with 35S. The number of silver grains deposited over a digoxigenin-labeled cell body provided an index of galanin mRNA levels in GnRH cells. We observed a 60% reduction in signal (grains per cell) for galanin mRNA in GnRH neurons of lactating animals compared with those of diestrus animals (P < 0.004), with no difference in the number of GnRH neurons between groups. To compare cellular GnRH mRNA content between groups, we used single label in situ hybridization and image analysis. Signal levels (grains per cell) for GnRH mRNA were not different between diestrus and lactating animals in either an initial (diestrus, 121.4 +/- 5.9; lactation, 117.3 +/- 8.0; P > 0.7) or in a subsequent trial (diestrus, 184.0 +/- 10.4; lactation, 197.5 +/- 13.0; P > 0.7). To confirm and extend these findings, we used a RNAse protection assay to measure and compare the content of GnRH mRNA in hypothalamic fragments between diestrus and lactating animals. The concentration of GnRH mRNA (picograms of mRNA per 25 micrograms total RNA) was not different between the two groups (diestrus, 1.21 +/- 0.25; lactation, 1.25 +/- 0.13; P > 0.7). A determination of the total GnRH peptide content by RIA in a separate set of hypothalamic dissections revealed no difference between groups in the level of GnRH content (nanograms) per hypothalamus (diestrus, 6.0 +/- 0.6; lactation, 5.7 +/- 0.4; P > 0.4). We conclude that galanin mRNA expression in GnRH neurons of the rat is diminished during lactation, whereas GnRH expression continues unabated. This decrease in galanin gene expression associated with lactation may lead to decreased synthesis and secretion of galanin, which, in turn, could diminish the pulsatile secretion of GnRH or reduce its activity at the pituitary.
甘丙肽与促性腺激素释放激素（GnRH）共定位，并且在发情前期（GnRH神经元激活的时期）这些神经元中的表达增强。我们检验了这样一个假说：在大鼠哺乳期，GnRH神经元中GnRH和甘丙肽的mRNA表达均会下降，哺乳期是一个以促性腺激素分泌减少为特征的生殖状态。对于双重标记原位杂交，用标记有半抗原地高辛的反义cRNA探针检测GnRH mRNA，而用标记有35S的cRNA探针检测甘丙肽mRNA。沉积在一个地高辛标记的细胞体上的银颗粒数量提供了GnRH细胞中甘丙肽mRNA水平的一个指标。我们观察到，与处于动情间期的动物相比，哺乳期动物的GnRH神经元中甘丙肽mRNA的信号（每细胞颗粒数）减少了60%（P < 0.004），两组之间GnRH神经元的数量没有差异。为了比较两组之间细胞内GnRH mRNA的含量，我们使用了单标记原位杂交和图像分析。在初始试验（动情间期，121.4±5.9；哺乳期，117.3±8.0；P > 0.7）或随后的试验（动情间期，184.0±10.4；哺乳期，197.5±13.0；P > 0.7）中，动情间期和哺乳期动物之间GnRH mRNA的信号水平（每细胞颗粒数）均无差异。为了证实并扩展这些发现，我们使用核糖核酸酶保护测定法来测量和比较动情间期和哺乳期动物下丘脑片段中GnRH mRNA的含量。两组之间GnRH mRNA的浓度（每25微克总RNA中mRNA的皮克数）没有差异（动情间期，1.21±0.25；哺乳期，1.25±0.13；P > 0.7）。在另一组下丘脑解剖样本中通过放射免疫分析测定总的GnRH肽含量，结果显示两组之间每个下丘脑的GnRH含量（纳克）水平没有差异（动情间期，6.0±0.6；哺乳期，5.7±0.4；P > 0.4）。我们得出结论：大鼠哺乳期GnRH神经元中甘丙肽mRNA的表达减少，而GnRH的表达持续不变。与哺乳期相关的甘丙肽基因表达的这种下降可能导致甘丙肽的合成和分泌减少，这反过来可能会减少GnRH的脉冲式分泌或降低其在垂体的活性。
