ERα特异性抑制剂MPP降低小鼠2-细胞胚G1/S期过渡相关因子Nanog、cyclin D1和cyclin E水平

目的观察雌激素受体(ERa)特异性抑制剂甲基哌啶吡唑(methyl-piperidino-pyrazole,MPP)对小鼠2-细胞胚G1/S期过渡相关因子Nanog、cyclin D1和cyclin E表达水平的影响,阐明ERa对小鼠2-细胞胚G1/S期过渡的作用,为进一步探讨ERa在小鼠植入前胚合子基因组激活中的作用机制提供理论依据。方法收集昆明雌性小鼠与雄性小鼠交配所得的受精卵,分别置于无MPP的培养液和含20mmol/L MPP的培养液中培养,获取体外发育的2-细胞胚,采用免疫荧光染色技术检测细胞内Nanog、cyclin D1和cyclin E水平。结果免疫荧光染色结果显示,含20mmol/L MPP培养液培养的受精卵其体外发育2-细胞胚内Nanog、cyclin D1和cyclin E免疫反应性明显降低。结论 ERa可通过调控小鼠2-细胞胚内Nanog、cyclin D1和cyclin E水平,影响细胞从G1期到S期的过渡。     

p-CREB在小鼠植入前胚的表达及其与2-细胞阻滞的关系

目的观察p-CREB在小鼠植入前胚以及阻滞品系(昆明小鼠)与非阻滞品系(B6C3F1小鼠)2-细胞胚的分布与表达,初步探讨p-CREB在小鼠植入前胚中的作用以及与小鼠2-细胞阻滞的关系。方法利用激光扫描共聚焦显微镜检测p-CREB在B6C3F1小鼠植入前胚中的定位,比较不同品系小鼠体外培养以及B6C3F1小鼠体外培养与体内发育2-细胞胚内p-CREB的表达变化。结果 p-CREB的免疫阳性反应在小鼠1-细胞胚中主要分布于细胞质中;2-细胞胚及其后期植入前胚主要定位于细胞核内;昆明小鼠体外培养2-细胞胚核内荧光强度显著低于同一条件发育的B6C3F1小鼠,B6C3F1小鼠体外培养2-细胞胚核内荧光强度显著高于体内发育。结论 p-CREB在小鼠植入前胚发育中起重要作用,p-CREB在昆明小鼠2-细胞胚核内表达较低可能与小鼠2-细胞阻滞有关。     

雌激素受体α抑制剂MPP对小鼠合子型基因组激活的影响可能与S期启动相关

目的观察雌激素受体α(ERα)特异性抑制剂甲基哌啶吡唑(Methyl-Piperidino-Pyrazole,MPP)对小鼠早期胚胎发育的影响与合子型基因组激活(ZGA)发生的时间是否相关,并检测与小鼠ZGA密切相关的2-细胞胚DNA复制是否受MPP处理的影响,为进一步探讨ERα在小鼠ZGA过程中的作用机制提供实验基础。方法 1收集昆明小鼠1-细胞胚、2-细胞胚和4-细胞胚,以空白KSOM培养液(单纯型优化的胚胎培养基)为对照组,以KSOM中添加MPP为实验组;将各时间点收集的早胚培养3h后再移入新KSOM培养液中继续培养至囊胚阶段,并记录囊胚数;2收集昆明小鼠末期1-细胞胚,分别置于上述两组培养液中培养15h至2-细胞胚S期后期,用荧光显微镜技术检测2-细胞胚核像变化;Brd U标记法分析DNA复制情况。结果1于不同时间段经MPP 3h处理后,在1-细胞胚末期、2-细胞早期和2-细胞胚中后期,MPP处理对小鼠早胚发育具有显著的抑制作用,其阶段特异性影响与小鼠ZGA发生的时间一致。另外,MPP对2-细胞胚G1/S过渡期(36~39h)抑制明显,但是对S期中后期(39~42h)进程无显著影响。2与KSOM组相比,经MPP处理15h后,2-细胞胚核变圆;Brd U标记水平明显下降。结论MPP在小鼠早胚中的阶段性作用与ZGA有关;ERα可能通过促进S期启动来调控ZGA。     

M16添加硫氧还蛋白过氧化物酶Ⅱ无法克服昆明小鼠早胚体外发育2-细胞阻滞

目的观察硫氧还蛋白过氧化物酶Ⅱ(Peroxiredoxin Ⅱ,PrxII)是否可以克服昆明(Kunming)小鼠早胚体外发育2-细胞阻滞。方法取昆明小鼠1-细胞胚置于含PrxII蛋白的M16培养液中培养,观察PrxII对昆明小鼠早胚发育潜能和2-细胞胚内活性氧自由基(reactive oxygen species,ROS)水平的影响;同时比较昆明和B6C3F1小鼠1-细胞胚在M16中各自的发育情况;激光扫描共聚焦显微镜分别检测比较昆明与B6C3F1小鼠体外培养2-细胞胚内ROS水平以及昆明小鼠体外培养与体内发育2-细胞胚内ROS水平。结果M16培养液中添加PrxII蛋白(1nmol/L和100nMol/L)可以明显降低昆明小鼠体外培养2-细胞胚内ROS水平(P<0.05),但不能克服昆明小鼠体外发育2-细胞阻滞;昆明小鼠1-细胞胚在M16中培养存在2-细胞阻滞现象,而B6C3F1小鼠无2-细胞阻滞现象;昆明小鼠体外培养2-细胞胚内ROS水平显著低于体内发育2-细胞胚(P<0.05),亦略低于B6C3F1小鼠体外培养2-细胞胚内ROS水平(P>0.05)。结论M16培养液中添加PrxII可以明显降低2-细胞胚...     

用小鼠血液淋巴细胞构建核移植重构胚的研究

本实验用小鼠血液淋巴细胞为核供体进行了核移植研究。用淋巴细胞分离液(比重1．088)分离出小鼠血液中的淋巴细胞,直接用作核移植供体细胞,采用胞质内注射法成功构建的重构胚经常规培养2h后,SrCl2激活处理6h,然后添加mM16培养液和小鼠输卵管上皮细胞饲养层共培养。把发育至早期囊胚阶段的重构胚转移至小鼠胎儿成纤维细胞饲养层上,添加ES细胞培养液继续培养。对孵化出的内细胞团进行消化,然后接种培养。结果显示,小鼠血液淋巴细胞可以支持体细胞核移植重构胚的发育,核移植重构胚2-细胞率41．03％(128／312),桑葚胚和囊胚发育率分别为9．29％(29／312),1．92％(6／312)。重构囊胚在小鼠胎儿成纤维细胞饲养层上分离出2个内细胞团,分离率为0．64％(2／312)。实验证实利用小鼠血液淋巴细胞进行体细胞核移植是可行的,可用于深入研究。     

用小鼠血液淋巴细胞构建核移植重构胚的研究

本实验用小鼠血液淋巴细胞为核供体进行了核移植研究。用淋巴细胞分离液(比重1.088)分离出小鼠血液中的淋巴细胞,直接用作核移植供体细胞,采用胞质内注射法成功构建的重构胚经常规培养2h后,SrCl_2激活处理6h,然后添加mM16培养液和小鼠输卵管上皮细胞饲养层共培养。把发育至早期囊胚阶段的重构胚转移至小鼠胎儿成纤维细胞饲养层上,添加ES细胞培养液继续培养。对孵化出的内细胞团进行消化,然后接种培养。结果显示,小鼠血液淋巴细胞可以支持体细胞核移植重构胚的发育,核移植重构胚2-细胞率41.03%(128/312),桑葚胚和囊胚发育率分别为9.29%(29/312),1.92%(6/312)。重构囊胚在小鼠胎儿成纤维细胞饲养层上分离出2个内细胞团,分离率为0.64%(2/312)。实验证实利用小鼠血液淋巴细胞进行体细胞核移植是可行的,可用于深入研究。     

白血病抑制因子与哺乳动物的胚胎着床(英文)

胚胎着床是处于活化状态的胚泡与处于接受态的子宫相互作用,最后导致胚胎滋养层与子宫内膜建立紧密联系的过程。已证实白血病抑制因子(LIF)在哺乳动物胚胎着床过程中起着十分重要的调节作用。LIF通过其受体及信号传递亚单位gp130发挥其生物学功能。LIF对胚胎发育到胚泡阶段及以后内细胞团和滋养层细胞的生长和分化有明显的促进作用。 在小鼠中,LIF及其受体和gp130在着床期小鼠子宫内表达量最高,因此LIF可能在小鼠胚胎着床过程中起重要作用。在人中,LIF在子宫内膜中的表达与人胚胎着床的时间一致,提示LIF可能与人的胚胎着床紧密相关。此外,LIF在猪、羊、水貂、兔和臭鼬等动物胚泡着床前和着床期的子宫中也都有表达,并在着床期出现峰值。因此,LIF也可能在这些动物的胚胎发育和着床过程中有重要作用。LIF受体基因敲除小鼠表现为胎盘发育不全,这说明LIF对小鼠胎盘形成和胎盘的功能维持起重要作用。 小鼠子宫中LIF的表达可能受雌激素而上调。美洲长尾猴(绒)及兔子宫中LIF的表达则呈孕酮依赖性。然而孕酮可抑制人着床期子宫内膜腺上皮和蜕膜组织内LIF的表达。在不同种类的动物中,LIF在子宫中的表达有不同的调节机制。 胚泡在LIF基因敲除的雌鼠子宫内不能着床的原因并不是由于胚泡发育异常,而是由于雌鼠不能表     

一种非手术性小鼠胚胎移植技术

为了提高非手术性小鼠胚胎移植技术的成功率,利用塑料移植导管模拟非手术胚胎移植过程,通过观察染料在子宫角的分布而评估胚胎移植效果,并在此基础上将自然妊娠3.5 d的小鼠囊胚经子宫颈移植受体小鼠。结果表明:将CD-1小鼠囊胚移植假孕2.5 d小鼠单侧子宫角,平均70.9%的胚胎能够发育至成活新生仔鼠,建立了高效非手术性小鼠胚胎移植技术。该方法简便快捷、不易污染、费用低,无需专业的手术器械,且符合实验动物伦理原则,完全可以取代手术法胚胎移植技术,更重要的是,它为人类和其他大动物的胚胎移植提供了研究模型。     

小鼠卵母细胞体外培养成熟及“试管小鼠”的研究

本文研究建立了一个有效的小鼠体外受精系:t。将小鼠附覃尾精子在简单合成的培荞液中休外获 能后,与体内成熟的卵母细胞共同孵育受精,然后体外培养。结果发育至2一细胞期胚胎的百分率为75一 85 %。将这些2一细胞期胚胎移植到受体小鼠输卵管,得到11只“试管小鼠”。用小鼠卵巢中未成熟卵母 细胞,经体外培养成熟后,沐外受精培养,达2一细咆期胚胎的百分率为20-40%,经移植后得到6只“试 管小鼠,,。     

用卵移植法研究18-甲基三烯炔诺酮对着床的影响

为了分析18-甲基三烯炔诺酮对着床的影响,我们进行了2-4细胞期受精卵的输卵管移植和胚泡的子宫移植的实验。移植于输卵管的结果表现,由于药物的影响,使卵在输卵管运行的速度加快,干扰了卵与子宫发育的同步性,因而卵不能着床。在移植于子宫的实验中,卵的着床率为61.5%,对照组为65.2%,两组无明显的差别。但胚胎着床以后,在妊娠期中,大部分胚胎的发育受到阻碍,正常的胚胎只有20.8%,而对照组为92.9%,两组有明显的差别。可能是药物影响受卵兔子宫蜕膜细胞,致使不能满足胚胎进一步发育所需要的条件。     

金黄色葡萄球菌致小鼠慢性输卵管炎性不孕模型制作

目的通过金黄色葡萄球菌直接感染小鼠输卵管,建立炎症致不孕的动物模型。方法用1×109/mL的金黄色葡萄球菌接种小鼠,制作慢性输卵管炎症模型,观察输卵管病理炎性改变以及小鼠的受孕情况。结果造模术75 d后,模型组小鼠受孕率、输卵管通畅率显著低于对照组（P〈0.001）。模型组肉眼观察输卵管有不同程度积水积脓、僵硬,输卵管与周围组织均有不同程度的粘连;病理学观察输卵管管腔被异物肉芽组织完全阻塞,全层均见大量慢性炎细胞浸润。结论输卵管内接种浓度1×109/mL的金黄色葡萄球菌可以成功建立小鼠输卵管炎性不孕模型。     

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.     

Endoscopic approaches to manage in vitro and in vivo embryo development: Use of the bovine oviduct

The oviduct plays a major part in different reproductive processes providing the microenvironment for numerous steps in early embryogenesis. Consequently, there is a growing demand to perform comparative studies focusing on causal mechanisms related to embryo development within its environment including complex and holistic strategies. However, the routine flushing and transfer procedure of bovine embryos is limited to the morula and blastocyst stage. Additionally, the use of in vitro production of bovine embryos provides access to an extra amount of embryos at various stages. But the quality of these embryos does not reflect the quality of its ex vivo counterparts. For two decades our own studies have focused on use of the oviductal environment of different species to optimize early embryo development for different purposes. The current article briefly highlights some main characteristics of the fallopian tube and reviews the endoscopic approach to access the fallopian tube using the stepwise minimal invasive technique established in different species.     

PMSG和FSH同期发情处理对小鼠子宫、卵巢和输卵管中雌激素α受体分布的影响

目的从雌激素α受体（estrogen receptorα,ERα）的角度探讨孕马血清促性腺激素（pregnant mareserum gonadotropin,PMSG）和促卵泡激素（follicle-stimulating hormone,FSH）处理小鼠的卵巢、输卵管和子宫中,ERα分布是否有显著性差异。方法 10只8周龄母鼠,随机分为处理方式不同的两个组：PMSG组和FSH组,两组均在处理第48小时取其卵巢、输卵管和子宫样固定,采用免疫组织化学法分别观察组织中ERα分布情况。结果免疫组化结果显示,两个处理组小鼠卵巢、输卵管和子宫内膜的细胞中都有ERα表达;PMSG处理组卵巢中的初级卵泡和成熟卵泡上ERα阳性率和平均吸光度均显著高于FSH处理组;FSH处理组的输卵管中ERα阳性率和平均吸光度均高于PMSG处理组;FSH处理组子宫基质和腺上皮细胞中ERα的阳性率显著高于PMSG组,其中PMSG组基质中的平均吸光度显著高于FSH组,而子宫内膜上皮细胞的阳性率和平均吸光度两处理组间差异无显著性。结论 PMSG和FSH同期发情诱导由于其特性可不同程度地影响小鼠卵巢、输卵管和子宫中ERα的分布,使之在不同组织中产生差异性变化。     

Prolonged in vivo functional assessment of the mouse oviduct using optical coherence tomography through a dorsal imaging window

The oviduct (or fallopian tube) serves as an environment for gamete transport, fertilization and preimplantation embryo development in mammals. Although there has been increasing evidence linking infertility with disrupted oviduct function, the specific roles that the oviduct plays in both normal and impaired reproductive processes remain unclear. The mouse is an important mammalian model to study human reproduction. However, most of the current analyses of the mouse oviduct rely on static histology or 2D visualization, and are unable to provide dynamic and volumetric characterization of this organ. The lack of imaging access prevents longitudinal live analysis of the oviduct and its associated reproductive events, limiting the understanding of mechanistic aspects of fertilization and preimplantation pregnancy. To address this limitation, we report a 3D imaging approach that enables prolonged functional assessment of the mouse oviduct in vivo. By combining optical coherence tomography with a dorsal imaging window, this method allows for extended volumetric visualization of the oviduct dynamics, which was previously not achievable. The approach is used for quantitative analysis of oviduct contraction, spatiotemporal characterization of cilia beat frequency and longitudinal imaging. This new approach is a useful in vivo imaging platform for a variety of live studies in mammalian reproduction.      

Staging mouse preimplantation development in vivo using optical coherence microscopy

In mammals, preimplantation development primarily occurs in the oviduct (or fallopian tube) where fertilized oocytes migrate through, develop and divide as they prepare for implantation in the uterus. Studies of preimplantation development currently rely on ex vivo experiments with the embryos cultured outside of the oviduct, neglecting the native environment for embryonic growth. This prevents the understanding of the natural process of preimplantation development and the roles of the oviduct in early embryonic health. Here, we report an in vivo optical imaging approach enabling high‐resolution visualizations of developing embryos in the mouse oviduct. By combining optical coherence microscopy (OCM) and a dorsal imaging window, the subcellular structures and morphologies of unfertilized oocytes, zygotes and preimplantation embryos can be well resolved in vivo, allowing for the staging of development. We present the results together with bright‐field microscopy images to show the comparable imaging quality. As the mouse is a well‐established model with a variety of genetic engineering strategies available, the in vivo imaging approach opens great opportunities to investigate how the oviduct and early embryos interact to prepare for successful implantation. This knowledge could have beneficial impact on understanding infertility and improving in vitro fertilization. OCM through a dorsal imaging window enables high‐resolution imaging and staging of mouse preimplantation embryos in vivo in the oviduct.      

Morphological analysis of transportation pathways of microspheres after their introduction into the uterine horn cavity in cyclic pigs

Countercurrent transfer is thought to be one of the most important mechanisms involved in the transfer of substances between the uterus and oviduct. The present study was aimed at recognizing other putative transportation pathways from the uterine cavity through the oviduct onto the surface of and into internal ovarian structures. Microspheres (latex beads, 0.8 m in diameter) were introduced into the uterine horn cavity of pigs, for 30 min, at various days of the estrous cycle. The transportation pathways of the beads were then analyzed by light and electron microscopy. The transport of microspheres through the oviduct canal into ovarian tissues took place on each day of the estrous cycle. The largest numbers of microspheres passed through the tunica albuginea to the corpora lutea. Some of microspheres also reached the surface of the uterine ligament through the oviduct canal, where they attained the lumen of blood and lymphatic vessels, mainly of the vascular subovarian (VSP) and paraovarian lymphatic plexus (PLP), via the lymphatic stomata pathway. Transport of microspheres also took place simultaneously through the uterine and oviduct walls and from particular organs through blood and lymphatic vessels. Although the present results do not exclude the participation of countercurrent transfer between venous, lymphatic, and arterial vessels, they provide morphological evidence for the presence of direct transportation pathways of substances, released into the uterine lumen, into ovarian tissues through the oviduct canal.     

Differences in prolactin receptor (PRLR) in mouse and human fallopian tubes: evidence for multiple regulatory mechanisms controlling PRLR isoform expression in mice

The anterior pituitary-derived hormone prolactin (PRL) signals through the PRL receptor (PRLR) and is important for female reproductive function in mammals. In contrast to the extensive studies of PRLR expression and regulation in human and mouse ovary and uterus, the mechanisms controlling the regulation of PRLR isoform expression in the fallopian tube are poorly understood. Because dynamic interaction of hormonal signaling in gonadal tissue and the pituitary or in gonadal tissues themselves in mammals suggests endocrine or paracrine regulation of PRLR expression, we questioned whether differential regulation of PRLR isoforms by PRL ovarian-derived estrogen (E(2)) and progesterone (P(4)) exists in the fallopian tube and pituitary of prepubertal female mice. Western blot analysis showed distinct molecular separation of PRLR isoforms in mouse and human fallopian tubes, and cellular localization was found in mouse and human tubal epithelia but not in mouse tubal smooth muscle cells. These data support the concept of an isoform- and cell type-specific expression of PRLR in human and mouse fallopian tubes. Moreover, expression of the long form of PRLR decreased after PRL treatment and increased after blockage of endogenous PRL secretion by bromocriptine (an inhibitor of PRL secretion) in a time-dependent manner in mouse fallopian tube. The opposite regulation was observed in the pituitary. Treatment with exogenous E(2) or P(4) led to changes in PRLR expression in the fallopian tube similar to those of PRL treatment. However, E(2) and P(4) did not affect PRLR expression in the pituitary. Estrogen had no effect on the long form of PRLR expression, whereas P(4) regulated the long form of PRLR in the fallopian tube, as did PRL. Taken together, the data from our comparative study provide evidence that PRLR can be regulated by an interplay of two different mechanisms, PRL or ovarian steroid hormones independently or in combination in a tissue-specific manner. Furthermore, we found that ovarian steroid hormones selectively suppress the expression of PRLR isoforms in mouse fallopian tubes. These findings may contribute to our understanding of the mechanisms controlling PRLR isoform expression in the fallopian tube (in addition to ovary and uterus), with implications for female reproduction.     

Constructing complex 3D biological environments from medical imaging using high performance computing

Extracting information about the structure of biological tissue from static image data is a complex task requiring computationally intensive operations. Here, we present how multicore CPUs and GPUs have been utilized to extract information about the shape, size, and path followed by the mammalian oviduct, called the fallopian tube in humans, from histology images, to create a unique but realistic 3D virtual organ. Histology images were processed to identify the individual cross sections and determine the 3D path that the tube follows through the tissue. This information was then related back to the histology images, linking the 2D cross sections with their corresponding 3D position along the oviduct. A series of linear 2D spline cross sections, which were computationally generated for the length of the oviduct, were bound to the 3D path of the tube using a novel particle system technique that provides smooth resolution of self-intersections. This results in a unique 3D model of the oviduct, which is grounded in reality. The GPU is used for the processor intensive operations of image processing and particle physics based simulations, significantly reducing the time required to generate a complete model.     

The oviduct as a complex mediator of mammalian sperm function and selection

The fallopian tube, or oviduct, is no longer considered merely a conduit that joins the uterine horns and the ovaries, being recognised as a venue for the capacitation of spermatozoa and fertilisation. However, recent evidence has implicated the oviduct in the stringent selection of spermatozoa prior to fertilisation, sperm storage prior to fertilisation, the regulation of sperm motility and possibly the guidance of spermatozoa towards the egg. Moreover, the arrival of spermatozoa within the oviduct is now known to regulate gene expression in oviductal epithelial cells with the consequent up- and downregulation of various proteins. In this review, we examine the emerging significance of sperm-oviduct interactions, as they relate to both physiological functions and the likelihood that the oviduct has a role in post-copulatory sperm selection by females (cryptic female choice) under conditions of sperm competition. The mechanisms by which sperm selection might operate still remain a mystery, especially when the underlying rationale for such mechanism appears to require the recognition by the female tract of sperm qualities related to the intrinsic integrity and information content of the sperm DNA. The oviduct not only selects against spermatozoa containing fragmented DNA but also imposes selection related to the fitness or quality of individual males. This implies the existence of, as yet unrecognised, mechanisms for the detection and interpretation of sperm-surface markers that link phenotypic and genotypic qualities of each individual cell.