FUNCTION OF UTERINE CALCITONIN DURING IMPLANTATION

本研究计划的长期目标是探索降钙素（CT）调控胚胎着床的分子基础，降钙素是一种调节钙稳态的肽激素。CT在妊娠前期在大鼠子宫腺上皮中诱导表达，在植入完成后被关闭。人子宫内膜的CT表达仅限于月经周期的分泌中期（19-24天），与推测的着床窗口密切重叠。这些发现提示CT可能在植入过程中作为子宫的重要调节信号。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。将针对CT mRNA的反义寡脱氧核苷酸（ODNs）注入着床前子宫，可显著抑制子宫CT mRNA的稳态水平。这种干预还伴随着植入胚胎数量的严重减少。这些结果表明，植入障碍可能是由反义ODN阻断CT基因表达的直接表型结果。反义odn诱导的CT缺失对(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进行检验。2. 目的：探讨转化人子宫内膜石川细胞系和人子宫内膜上皮细胞原代培养中CT信号转导通路。CT通过特定的细胞表面受体作用于靶细胞。CT受体在着床前子宫内膜上皮的表达也明显升高。CT通过其受体激活的第二信使通路将在Ishikawa和子宫内膜上皮细胞的原代培养中进行研究。3. 鉴定介导CT细胞作用的基因。CT刺激人子宫内膜细胞c-fos mRNA的表达，同时抑制骨桥蛋白mRNA的表达。为了进一步了解CT如何影响胚胎-子宫相互作用，将通过减法克隆鉴定靶细胞中表达受该激素调节的其他基因，并确定其在月经周期中在人类子宫内膜中的时空表达。本研究将提供有价值的见解，了解将子宫腺CT瞬时表达与着床期间胚胎-子宫内膜相互作用控制联系起来的一系列事件的分子机制。

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.