Long-term label retaining cells localize to distinct regions within the female reproductive epithelium

The uterus is an extremely plastic organ that undergoes cyclical remodeling including endometrial regeneration during the menstrual cycle. Endometrial remodeling and regeneration also occur during pregnancy and following parturition, particularly in hemochorial implanting species. The mechanisms of endometrial regeneration are not well understood. Endometrial stem/progenitor cells are proposed to contribute to endometrial regeneration in both humans and mice. BrdU label retention has been used to identify potential stem/progenitor cells in mouse endometrium. However, methods are not available to isolate BrdU label-retaining cells (LRC) for functional analyses. Therefore, we employed a transgenic mouse model to identify H2B-GFP LRCs throughout the female reproductive tract with particular interest on the endometrium. We hypothesized that the female reproductive tract contains a population of long-term LRCs that persist even following pregnancy and endometrial regeneration. Endometrial cells were labeled (pulsed) either transplacentally/translactationally or peripubertally. When mice were pulsed transplacentally/translactationally, the label was not retained in the uterus. However, LRCs were concentrated to the distal oviduct and endocervical transition zone (TZ) following natural (i.e., pregnancy/parturition induced) and mechanically induced endometrial regeneration. LRCs in the distal oviduct and endocervical TZ expressed stem cell markers and did not express ERα or PGR, implying the undifferentiated phenotype of these cells. Oviduct and endocervical TZ LRCs did not proliferate during endometrial re-epithelialization, suggesting that they do not contribute to the endometrium in a stem/progenitor cell capacity. In contrast, when mice were pulsed peripubertally long-term LRCs were identified in the endometrial glandular compartment in mice as far out as 9 months post-pulse. These findings suggest that epithelial tissue of the female reproductive tract contains 3 distinct populations of epithelial cells that exhibit stem/progenitor cell qualities. Distinct stem/progenitor-like cells localize to the oviduct, endometrium, and cervix.     

Genistein promotes DNA demethylation of the steroidogenic factor 1 (SF-1) promoter in endometrial stromal cells

It has recently been demonstrated that genistein (GEN), a phytoestrogen in soy products, is an epigenetic modulator in various types of cells; but its effect on endometrium has not yet been determined. We investigated the effects of GEN on mouse uterine cells, in vivo and in vitro. Oral administration of GEN for 1 week induced mild proliferation of the endometrium in ovariectomized (OVX) mice, which was accompanied by the induction of steroidogenic factor 1 (SF-1) gene expression. GEN administration induced demethylation of multiple CpG sites in the SF-1 promoter; these sites are extensively methylated and thus silenced in normal endometrium. The GEN-mediated promoter demethylation occurred predominantly on the luminal side, as opposed to myometrium side, indicating that the epigenetic change was mainly shown in regenerated cells. Primary cultures of endometrial stromal cell colonies were screened for GEN-mediated alterations of DNA methylation by a high-resolution melting (HRM) method. One out of 20 colony-forming cell clones showed GEN-induced demethylation of SF-1. This clone exhibited a high proliferation capacity with continuous colony formation activity through multiple serial clonings. We propose that only a portion of endometrial cells are capable of receiving epigenetic modulation by GEN.     

子宫内膜蜕膜化的特征及其调节因素

子宫内膜向蜕膜的转化是正常着床和妊娠的一个重要特征,对于胚泡着床是必不可少的。在蜕膜化过程中,子宫内膜基质细胞在形态和生理等方面都发生了很大的变化。蜕膜化过程受多种因素的调节,包括cAMP、胰岛素样生长因子结合蛋白-1(IGFBP-1)、自然杀伤细胞、同源盒基因-10(HOXA10)、激活素等。但对蜕膜化的机制及调节等仍不清楚。     

Expression of estrogen receptors alpha and beta in the corpus luteum and uterus from non-pregnant and pregnant llamas

Because estrogen may be involved in maternal recognition of pregnancy and embryonic migration in llamas, expression of estrogen receptor subtypes alpha (ERalpha) and beta (ERbeta) was evaluated in corpus luteum (CL), endometrium, and uterus using relative RT-PCR. Tissues were recovered from sterile-mated (SM) and pregnant (PG) females during Days 7-11 and 7-13 (Day 0 = day of mating), respectively, and follicular phase and juvenile females. Luteal expression of ERalpha and beta was similar (P > 0.10) in SM and PG females and within Days 7-11, however, expression of ERalpha in ovarian tissue from follicular phase females was greater (P < 0.05) than Days 7 and 9 CL. Uterus expressed less ERalpha and beta compared to endometrium (P = 0.07 and P < 0.01, respectively). Expression of ERalpha was greater (P < 0.05) in Day 7 and follicular phase uteri than Days 9 and 11, Day 13 PG and juvenile uteri. Uterine ERbeta expression was greater (P = 0.09) in PG versus SM females and in mated compared to follicular phase females (P < 0.05). Endometrial expression of ERalpha and beta did not differ (P > 0.10) between SM and PG females or by day. The presence of luteal ER during this period may mean a role for estradiol in maternal recognition of pregnancy. Observed increases in uterine ER expression with no changes in endometrium suggest expression increased in myometrium and/or perimetrium. Upregulation of myometrial ERbeta in PG females may be involved in supporting uterine migration of the embryo.     

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as “the scraping method” and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed “the trypsin method.” This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).     

卵巢激素对小鼠围着床期子宫内膜Le^y寡糖表达的调控

研究表明Le^y寡糖介导了胚胎与子宫内膜之间的识别与粘附,在胚胎植入中起重要作用。其α1,2、α1,3岩藻糖基的合成分别与α1,2岩藻糖基转移酶（FUT1）、α1,3岩藻糖基转移酶（FUT4）的催化作用密切相关,应用Western印迹、免疫组化和半定量RT-PCR方法,观察小鼠妊娠早期、去卵巢后雌孕激素处理的子宫内膜Le^y寡糖抗原以及其合成相关的FUT1、FUT4基因的表达,分析卵巢激素对Le^y寡糖表达的调控,结果显示：妊娠早期,FUT1、FUT4基因的转录水平随孕激素水平上程式而呈下降的趋势,这与Le^y寡糖抗原表达一致。进一步观察发现,去卵巢后经孕激素处理,FUT1、FUT4基因及Le^y寡糖抗原表达均较对照组降低,雌激素处理组表达则明显升高;雌孕激素联合作用介于雌激素组和孕激素组之间,结果表明,孕激素能下调FUT1、FUT4基因的表达。雌激素对其有上调作用,两种激素之间表现为相互拮抗,提示雌孕激素可能通过FUT1、FUT4基因转录水平调控Le^y寡糖抗原在小鼠子宫内膜上皮的表达。     

黄体酮对子宫内膜异位症在位内膜预处理与IVF治疗结局关系

摘要 目的：研究黄体酮对子宫内膜异位症在位内膜预处理改善IVF结局。方法：男性因素并卵巢子宫内膜异位囊肿168例,分两组：（1）实验组：IVF前自然周期月经第12天地屈孕酮30 mg/日、14天,3个月;（2）对照组：IVF前无干预。IVF前测血CA125,黄体期长方案促排卵,排卵后一周（垂体降调日）再测CA125并取子宫内膜行ER、PR、HOXA-10mRNA检测。HCG日测子宫内膜厚度、形态、血流。比较临床资料及结局、症状疼痛评分。结果：实验组胚胎种植率、临床妊娠率高于对照组（P<0.05）;HCG日对照组子宫内膜厚于实验组。实验组子宫内膜A型血流比率高于对照组（P<0.05）。实验组A型子宫内膜比率高于对照组,但无明显差异（P>0.05）;IVF前两组CA125均高于参考值,但无明显差异（P>0.05）。垂体降调日复查CA125,实验组明显低于对照组,实验组治疗后低于治疗前;实验组子宫内膜ER、PR、HOXA-10 mRNA表达量高于对照组,实验组分泌期子宫内膜比率高于对照组（P<0.05）;治疗后两组各项症状疼痛评分均较治疗前改善,且实验组优于对照组（P<0.05）。结论：IVF治疗中合并卵巢子宫内膜异位囊肿用黄体酮预处理在位内膜,可以降低血CA125,有利于转变子宫内膜组织类型、减小子宫内膜厚度、增加子宫内膜血流、增加子宫内膜ER、PR、HOXA-10 mRNA表达,改善在位内膜容受性,缓解症状疼痛,提高临床妊娠率。