CATECHOLESTROGENS AND PROLACTIN IN REGULATING UTERINE GLYCOGEN METABOLISM, MINK

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 子宫糖原（GLY）储备是胚胎存活、发育和植入的潜在能量来源。雌二醇-17 <$（E2）以依赖于生殖周期的模式刺激GLY的合成，在人类的子宫分泌期和啮齿动物的发情前期和发情期之间最高。水貂是季节性繁殖者，表现出专性胚胎滞育，导致延迟着床，根据繁殖日期，囊胚在着床时可能为60天。子宫GLY积累和动员为葡萄糖可能是该物种囊胚存活、激活（即：滞育终止）和着床所必需的。水貂的血浆E2水平在交配诱导排卵当天达到峰值，并在接近着床时再次达到峰值。此外，子宫将E2代谢为儿茶酚雌激素（CE），如2-羟基雌二醇（2-OHE 2）和4-羟基雌二醇（4-OHE 2）。以前，我们已经表明，E2和CE增加子宫GLY浓度6倍卵巢切除（OVX）水貂。这些激素同时降低子宫糖原合成酶激酶-3 <$（GSK-3 <$$>）的表达，GSK-3 <$是一种磷酸化和失活糖原合成酶（GS）的酶。 因为GS在GLY合成中是限速的，所以GSK-3的表达减少，应该可以消除对GS的抑制，从而增加GLY的产生。E2和4-OHE 2均促进GSK-3的磷酸化和失活，进一步增强GLY合成。此外，4-OHE 2和2-OHE 2增加子宫GS表达，同时减少GLY分解代谢酶GLY-磷酸化酶（GP）的表达，这也增加子宫GLY积累。由于它们的半衰期非常短，CE的影响是局部的，在血液中检测不到。除了E2，催乳素（PRL）通过其促黄体作用增加循环中的孕酮（P4）水平，对水貂的着床至关重要。此外，在水貂子宫中发现了PRL受体，表明该激素的直接促子宫作用也可能促进胚泡着床。我们假设PRL增加GLY催化剂，为囊胚激活、着床提供葡萄糖，并有助于增加窝仔数。我们将在三个特定的目标来测试这个假设，旨在确定（1）E2，4-OHE 2和2-OHE 2对GS和GP磷酸化的影响;（2）4-OHE 2和2-OHE 2是否通过独立于E2的途径作用于子宫;（3）PRL是否在水貂子宫中具有糖原分解作用。免疫印迹将用于特定目标1。 对于目标2，将用雌激素受体拮抗剂ICN-182，780单独和与E2、4-OHE 2或2-OHE 2组合处理貂，并测量子宫GLY水平。对于目标3，将使用多巴胺能拮抗剂沙利度醇（HAL）和测量的子宫GLY浓度使水貂高泌乳素血症。 这些研究的数据将有助于我们了解水貂的高效繁殖。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Uterine glycogen (GLY) reserves are a potential source of energy for embryo survival, development, and implantation. Synthesis of GLY is stimulated by estradiol-17¿ (E2) in a reproductive cycle-dependent pattern, being highest during the uterine secretory phase in human's and between proestrous and estrous in rodents. Mink, are seasonal breeder's, that exhibit obligate embryonic diapause resulting in delayed implantation where, depending on the date of breeding, blastocysts may be 60 days old at implantation. Uterine GLY accumulation and mobilization to glucose may be requisite to blastocyst survival, activation (ie: termination of diapause), and implantation in this species. Plasma E2 levels in mink peak on the day of mating-induced ovulation, and again near the time of implantation. In addition, the uterus metabolizes E2 to catecholestrogens (CE) such as 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2). Previously, we have shown that E2 and CEs increase uterine GLY concentrations 6-fold in ovariectomized (OVX) mink. These hormones simultaneously reduced the expression of uterine Glycogen Synthase Kinase-3¿ (GSK-3¿), an enzyme that phosphorylates and inactivates Glycogen Synthase (GS). Because GS is rate limiting in GLY synthesis, the reduction in GSK-3¿ expression, should remove inhibition on GS resulting in increased GLY production. Both E2 and 4-OHE2 promote the phosphorylation and inactivation of GSK-3¿, further enhancing GLY synthesis. Also, 4-OHE2 and 2-OHE2 increased uterine GS expression while reducing expression of the GLY catabolizing enzyme GLY-Phosphorylase (GP), which also increases uterine GLY accumulation. Because of their very short half-live's, the effects of CE's are localized and not detectable in the blood. In addition to E2, prolactin (PRL) is essential to implantation in mink through its luteotropic actions increasing progesterone (P4) levels in the circulation. Moreover, PRL receptors have been identified in the mink uterus, suggesting that direct uterotropic actions by the hormone may also promote blastocyst implantation. We hypothesize that PRL increases GLY catabolism, providing glucose for blastocyst activation, implantation, and contribute to increased litter sizes. We will test this hypothesis in three specific aims designed to determine (1) the effects of E2, 4-OHE2, and 2-OHE2 on the phosphorylation of GS and GP; (2) if 4-OHE2 & 2-OHE2 act on the uterus through pathways independent from E2; and (3) if PRL has glycogenolytic actions in the mink uterus. Immunoblots will be used in specific aim 1. For Aim 2, mink will be treated with the estrogen receptor antagonist ICN -182,780, alone and in combination with E2, 4-OHE2, or 2-OHE2 and uterine GLY levels will be measured. For Aim 3, mink will be made hyperprolactinemic using the dopaminergic antagonisthaloperidol (HAL) and uterine GLY concentrations measured. Data from these studies will contribute to our understanding of efficient reproduction in mink.