FUNCTION OF UTERINE CALCITONIN DURING IMPLANTATION

这项研究的长期目标是探索降钙素(CT)调节胚胎着床的分子基础，降钙素是一种调节钙稳态的多肽激素。CT的表达在妊娠着床前的大鼠子宫腺上皮细胞中被诱导，并在着床完成后被关闭。CT在人子宫内膜中的表达仅限于月经周期的分泌期中期(19-24天)，并与推测的着床窗口密切重叠。这些发现提示CT可能在子宫着床过程中作为重要的调节信号发挥作用。本建议的具体目的是：1.确定植入过程中CT调节的功能事件(S)。将针对CT基因的反义寡核苷酸(ODN)注入植入前子宫，可显著抑制子宫CT基因的稳定表达。这一干预还伴随着植入胚胎数量的严重减少。这些结果表明，植入的损伤可能是反义ODN阻断CT基因表达的直接表型结果。反义ODN诱导的CT缺陷对(A)子宫容受性和(B)胚胎植入能力的影响将通过胚胎移植实验来检验。2.探讨CT在转化的人子宫内膜细胞系Ishikawa和原代培养的人子宫内膜上皮细胞中的信号转导途径(S)。CT通过特定的细胞表面受体作用于靶细胞。CT受体在植入前子宫内膜上皮中的表达也明显升高。由CT通过其受体激活的第二信使通路将在石川和原代培养的子宫内膜上皮细胞中进行研究。3.确定介导CT细胞作用的基因。CT促进人子宫内膜细胞c-fos基因的表达，抑制骨桥蛋白基因的表达。为了进一步了解CT是如何影响胚胎-子宫相互作用的，将通过消减克隆来鉴定在靶细胞中表达受该激素影响的其他基因，并确定它们在月经周期中在人类子宫内膜中的时空表达。这项拟议的研究将为揭示子宫腺体中CT瞬时表达与着床期间胚胎-子宫内膜相互作用控制的一连串事件链背后的分子机制提供有价值的见解。

The long-term objective of this research proposal is to explore the molecular basis of the regulation of embryonic implantation by calcitonin (CT), a peptide hormone that regulates calcium homeostasis. The expression of CT is induced in the glandular epithelium of rat uterus in the preimplantation phase of gestation and is switched off once implantation is completed. CT expression in human endometrium is restricted to the mid- secretory phase (days 19-24) of the menstrual cycle, with closely overlaps with the putative window of implantation. These findings suggest that CT may function as an important regulatory signal in the uterus during implantation. The specific aims of this proposal are: 1. To determine the functional event(s) regulated by CT during implantation. Administration of antisense oligodeoxynucleotides (ODNs), targeted against CT mRNA, into the preimplantation phase uterus results in marked suppression of the steady-state level of uterine CT mRNA. This intervention is also accompanied by a severe reduction in the number of implanted embryos. These results suggest that the impairment of implantation could be a direct phenotypic consequence of the blockade of CT gene expression by the antisense ODN. The effect of antisense ODN-induced CT deficiency on (a) uterine receptivity and (b) the ability of the embryo to implant will be examined by embryo transfer experiments. 2. To elucidate the signal transduction pathway(s) of CT in transformed human endometrial cell line Ishikawa and in primary cultures of human endometrial epithelial cells. CT acts on target cells through specific cell surface receptors. The expression of the CT receptor is also markedly elevated in the preimplantation endometrial epithelium. The second messenger pathways that are activated by CT via its receptor will be investigated in Ishikawa and primary cultures of endometrial epithelial cells. 3. To identify the genes that mediate the cellular actions of CT. CT stimulates the expression of c-fos mRNA while it inhibits the expression of osteopontin mRNA in human endometrial cells. To further understand how CT influences the embryo-uterine interactions, additional genes whose expression in the target cells is modulated in response to this hormone will be identified by subtractive cloning and their spatio-temporal expression in human endometrium during the menstrual cycle will be determine. The proposed study will provide valuable insights into the molecular mechanisms underlying the chain of events that link the transient expression of CT in the uterine glands to the control of embryo-endometrial interactions during implantation.