Development of a Methodology for and Assessment of Pulsatile Luteinizing Hormone Secretion in Juvenile and Adult Male Mice
Development of a Methodology for and Assessment of Pulsatile Luteinizing Hormone Secretion in Juvenile and Adult Male Mice
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DOI：
10.1210/en.2013-1502
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发表时间：
2013-12-01
期刊：
影响因子：
4.8
通讯作者：
Chen, C.
中科院分区：
文献类型：
--
作者：
Steyn, F. J.;Wan, Y.;Chen, C.
目前监测小鼠脉冲LH释放的方法受到测定灵敏度不足的限制，导致需要收集大量血液。因此，评估小鼠中的脉冲式LH分泌仍然具有高度挑战性，并且观察仅限于成年小鼠。为了解决这一问题，我们开发了一种高度灵敏的ELISA，用于评估小鼠全血中小部分LH浓度。我们证明，该测定是能够可靠地检测LH下降到理论极限为0.117毫微克/毫升在2 μ L的部分全血。使用已建立的频繁采血程序，我们通过测定早期成年（10周龄）C57 BL 6/J雄性小鼠的脉冲式LH分泌来验证该方法的准确性。数据表明LH有规律的脉冲释放，LH分泌峰值很少超过3 ng/mL。此外，在Gpr 54基因敲除小鼠中LH释放的评估表明，在Kisspeptin介导的青春期成熟丧失后，缺乏脉冲式LH释放。接下来，我们通过评估青春期前（28日龄）C57 BL 6/J雄性小鼠的LH分泌并在成年期（120日龄）对相同小鼠进行重复评估，确定了与年龄相关的脉动LH分泌变化。数据表明，小鼠青春期成熟后总LH分泌的增加沿着脉动LH分泌速率的总体增加。这与LH分泌事件（脉冲数）的数量显著增加相结合。此外，我们观察到清除率降低（半衰期增加）和LH释放规律性（近似熵）降低。该方法在生殖生物学领域具有广泛的通用性。
Current methodology to monitor pulsatile LH release in mice is limited by inadequate assay sensitivity, resulting in the need for collection of large blood volumes. Thus, assessment of pulsatile LH secretion in mice remains highly challenging, and observations are limited to adult mice. To address this, we developed a highly sensitive ELISA for assessment of mouse LH concentrations in small fractions of whole blood. We demonstrate that this assay is capable of reliably detecting LH down to a theoretical limit of 0.117 ng/mL in a 2-mu L fraction of whole blood. Using an established frequent blood collection procedure, we validated the accuracy of this method by determining the pulsatile LH secretion in early-adult (10 weeks old) C57BL6/J male mice. Data demonstrate regular pulsatile release of LH, with peaks in LH secretion rarely exceeding 3 ng/mL. Moreover, assessment of LH release in Gpr54 knockout mice demonstrates the lack of pulsatile LH release after the loss of kisspeptin-mediated pubertal maturation. We next determined age-associated changes in pulsatile LH secretion by assessment of LH secretion in prepubertal (28 days old) C57BL6/J male mice and repeated assessment in the same mice in adulthood (120 days old). Data demonstrate that the rise in total LH secretion in mice after pubertal maturation occurs along with an overall rise in the pulsatile LH secretion rate. This was coupled with a significant increase in the number of LH secretory events(number of pulses). In addition, we observed a decrease in the clearance (increased half-life) and a decrease in the regularity (approximate entropy) of LH release. This method will be of wide general utility within the field of reproductive biology.