Physiology: Sperm numbers and distribution within the human Fallopian tube around ovulation

This study aimed to determine the number and distribution ofspermatozoa within the human Fallopian tubes around ovulation.Parous women, undergoing total abdominal hysterectomy for menorrhagia,were inseminated with either partner's semen (3/10) or donorsemen (7/10). Approximately 18 h later both Fallopian tubeswere ligatured into ampullary, isthmic and intramural regions.These were removed and assessed for sperm content by flushing,scanning electron microscopy (SEM) or homogenization. A medianof 251 spermatozoa were recovered (range, 79–1386). Thenumber of spermatozoa within each tube was not significantlydifferent. The ovulatory ampulla contained a significantly (P 0.01) larger percentage of spermatozoa than the non-ovulatoryampulla. The number of motile spermatozoa inseminated was notsignificantly correlated to the number of spermatozoa recovered,but a trend was identified. The time between the onset of theluteinizing hormone surge and hysterectomy was significantlycorrelated (P 0.01) to the number of spermatozoa within theintramural regions, but not to the tubal sperm distribution.Spermatozoa were not observed, by SEM, bound to the tubal epithelium.These data suggest that, after artificial insemination at least,sperm access to the human Fallopian tube may be controlled,but that ovulation does not affect the redistribution of spermatozoabetween tubal regions and that the isthmus does not appear toact specifically as a sperm reservoir.     

Morphology of human Fallopian tubes after infection with Mycoplasma genitalium and Mycoplasma hominis--in vitro organ culture study

BACKGROUND: Female infertility can be caused by scarring and occlusion of the Fallopian tubes. Sexually transmitted bacteria can damage the delicate epithelial layer of human Fallopian tubes (HFT). Genital mycoplasmas are associated with human reproductive failure. Yet, there is not enough evidence that mycoplasmas can cause tubal factor infertility. We analysed the effects of infections with Mycoplasma hominis and Mycoplasma genitalium on the HFT epithelium and compared them with the effects of infections with genital pathogens: Chlamydia trachomatis and Neisseria gonorrhoeae. METHODS: We used an in vitro model in which pieces of normal HFT were infected with different bacteria, and the outcome of the infections was analysed by scanning electron microscopy (SEM) and confocal microscopy. RESULTS: The presence of M. hominis did not cause any morphological changes of the epithelium of HFT. Noticeable changes in the morphology of the ciliated cells were observed in M. genitalium-infected tissue. Five days post-infection, the cilia were abnormally swollen and some of the ciliated cells fell off the epithelium. These effects could be inhibited by pre-incubation of M. genitalium with antibody directed against the C-terminal part of the adhesion protein MgPa before infection of HFT organ culture. CONCLUSION: We have shown that the presence of M. genitalium, but not M. hominis, in the HFT organ culture affected the epithelium and resulted in cilia damage. The effect of infection with M. genitalium on the HFT was, however, very moderate when compared with the extensive damage of the epithelium caused by N. gonorrhoeae or C. trachomatis.     

Immunocytochemical localization of growth factors and their receptors in human pre-embryos and Fallopian tubes

We utilized indirect immunocytochemistry to demonstrate thepresence of growth factors and their receptors in human pre-embryosand Fallopian tubes. In pre-embryos, only transforming growthfactor- (TGF-) and the intracellu-lar domain of epidermal growthfactor receptor (EGFR) were found at the 4-cell stage. In 8-to 14-cell pre-embryos, TGF-, the intracellular and extracellulardomains of EGFR, and insulin-like growth factor-I and its receptorwere found. Antibodies against TGF- stained all Fallopian tubespecimens, while the extracellular domain of EGFR was only foundin specimens from patients with either blood type A or AB. Theseresults suggest a cross-reactivity between the extracellulardomain of the EGFR and blood group antigens. Our novel demonstrationof growth factor receptor staining in human pre-embryos showsthat growth factor receptor localization is dependent on thedevelopmental stage of human pre-embryos. We have also establisheda potentially important link between the Fallopian tube whichsecretes growth factors and the localization of growth factorreceptors in pre-embryos. These findings are compatible withthe hypothesis that tubal secretions are embryotrophic for theearly development of the pre-embryo.     

输卵管在卵巢癌中的遗传学作用的研究进展

卵巢癌在三种妇科恶性肿瘤中恶性度最高,是妇科恶性肿瘤患者死亡的主要病因.高级别浆液性卵巢癌(HGSC)是最常见、恶性度最高的上皮性卵巢癌,在发现时一般已为晚期.HGSC可能与BRCA1以及BRCA2基因的突变有直接关系.长久以来,我们一直认为HGSC起源于卵巢上皮细胞,但是,最近越来越多的证据表明,无论是在高风险的妇女中,还是在一般人群中,绝大多数卵巢癌均起源于输卵管上皮细胞.浆液性上皮内癌(STIC)可能最终发展成卵巢癌或腹膜癌.目前,预防性保留卵巢的输卵管切除术越来越被人们所接受,成为高风险妇女在绝经前降低患卵巢癌风险的做法.     

Fallopian tube ciliary beat frequency in relation to the stage of menstrual cycle and anatomical site

BACKGROUND: The cyclical changes in ciliary structure and motion within the human Fallopian tube are well documented. Previous investigators have studied ciliary beat frequency (CBF) in relation to menstrual cycle and anatomical site, but with conflicting results. METHODS: Using a technique that records variations in light intensity, we have studied the changes in CBF in relation to the menstrual cycle and anatomical site. Fallopian tubes were collected from 26 women who underwent hysterectomy for benign conditions. Menstrual history, hormone profile and endometrial biopsy results were used to determine the stage of the cycle. Fourteen women were in the proliferative phase, and 12 women in the secretory phase. RESULTS: Mean CBF for all subjects was 5.3 plus minus 0.2 Hz. There was no significant difference in CBF in relation to anatomical site. In the fimbrial region the ciliary beat was faster in the secretory (5.8 plus minus 0.3 Hz) as compared with the proliferative phase (4.9 plus minus 0.2 Hz), P < 0.02. CONCLUSIONS: It is possible that this increase in fimbrial CBF may contribute to ovum retrieval and transport after ovulation. However, the reproductive significance of the changes in CBF in relation to the menstrual cycle needs further investigation.     

The effect of ovarian follicular fluid and peritoneal fluid on Fallopian tube ciliary beat frequency

BACKGROUND: The Fallopian tube undergoes well-recognized changes during the ovarian cycle. Ciliary beat frequency (CBF) increases during the secretory phase of the cycle. The stimulus is unknown, although CBF is known to be hormone responsive. At ovulation, follicular fluid is released into the peritoneal cavity and enters the Fallopian tube. We hypothesized that this fluid may provide the stimulus for the increase in CBF detected after ovulation. METHODS: Using a technique which records changes in light intensity, we have studied the effect of pre-ovulatory follicular fluid on CBF of Fallopian tube epithelial cells, and compared this with the effect of either peritoneal fluid or culture medium alone. Follicular fluid samples from 13 women undergoing IVF were collected by selective aspiration of individual follicles. Peritoneal fluid was collected from six women undergoing laparoscopic sterilization. Fallopian tubes were collected from 10 women who underwent hysterectomy for benign conditions. RESULTS: After 24 h incubation, there was a highly significant difference in CBF between the Fallopian tube samples bathed in follicular fluid (mean CBF +/- SEM: 6.34 +/- 0.02 Hz) compared with explants bathed in either medium (4.20 +/- 0.06 Hz) or peritoneal fluid (5.24 +/- 0.03 Hz) (P < 0.005). There was also a significant difference in CBF between tissues bathed in secretory (5.47 +/- 0.03 Hz) compared with proliferative phase peritoneal fluid (4.75 +/- 0.02 Hz) (P < 0.005). CONCLUSIONS: The increase in CBF detected after ovulation may aid ovum pick-up and transport along the Fallopian tube. Factor(s) within human follicular fluid and secretory phase peritoneal fluid may be responsible for this increase in CBF.     

人胚胎纹状体来源神经干细胞的体外培养

背景：目前神经干细胞多由动物获得,不适合人类临床移植治疗。 目的：探索体外环境下人胚胎纹状体来源神经干细胞的培养方法,同时观察其生物学特性。 方法：取经水囊引产的孕8-16周人胚胎纹状体,体外用无血清DMEM培养基进行培养,待细胞形成神经球后进行传代,并应用含体积分数10%胎牛血清的DMEM/ F12培养液进行诱导分化。 结果与结论：体外培养的人胚胎纹状体来源神经干细胞生长迅速,表达神经干细胞标志物nestin。克隆形成实验显示细胞克隆形成率为6.0%-7.0%;BrdU掺入实验显示细胞增殖率为37.9%。免疫荧光染色显示经诱导分化的细胞表达神经元标志物Ⅲ型β微管蛋白、星形胶质细胞标志物胶质纤维酸性蛋白及神经干细胞标志物nestin,但不表达少突胶质细胞标志物髓鞘碱性蛋白。可见人胚胎纹状体来源神经干细胞在体外无血清条件下可保持其生物学特点,具有自我更新能力,经胎牛血清诱导后可向神经元及星形胶质细胞分化。     

Effect of mifepristone on the expression of cytokines in the human Fallopian tube

Cytokines are believed to play a critical role as mediators between the oviduct and the developing embryo. A synchronous development of embryo and endometrium is essential to successful implantation. It seems to be beneficial for embryo development to rest for some time in the Fallopian tube. Expression of cytokines in the human Fallopian tube and the effect of mifepristone were investigated. Fourteen women with regular menstrual cycles and proven fertility, admitted to the hospital for tubal ligation, were randomly allocated to control or treatment groups. Mifepristone 200 mg was given on day LH+2. Surgery was performed on day LH+3 to LH+5. Biopsies were obtained from the ampullar and isthmic regions of the tubes. Expression of interleukin 8 (IL-8), tumour necrosis factor alpha (TNFalpha), transforming growth factor beta (TGFbeta) and leukaemia inhibitory factor (LIF) was analysed using immunohistochemistry. All cytokines except IL-8 showed the same staining intensity both in the ampullar and isthmic region, while IL-8 was more pronounced in the ampullar region in both epithelial and stromal cells. Exposure to mifepristone made the spatial difference in IL-8 disappear and increased the expression of TNFalpha in the epithelium of the isthmus, but had no effect on the expression of TGFbeta1 or LIF. Changes in cytokine expression in the Fallopian tube are likely to influence embryo development, which could contribute to the contraceptive effect of mifepristone.     

Secretory cell outgrowth,PAX2 and serous carcinogenesis in the Fallopian tube

The ‘p53 signature’ is a benign secretory cell outgrowth in the distal Fallopian tube that shares properties with ovarian serous cancer—including p53 mutations—and is a putative serous cancer precursor. We expanded the precursor definition to all secretory cell outgrowths (SCOUTs) of 30 or more cells and scored normal (N) and altered (A) expression of both p53 and PAX2, a gene down‐regulated in ovarian and endometrial cancer. SCOUTs were identified by BCL2/p73 staining in tubes from women with serous carcinoma, inherited mutations in BRCA1 or BRCA2 and controls. SCOUTs were prevalent in both proximal and distal tube and significantly associated with serous carcinoma versus the others (p < 0.001); 89% were PAX2 (A) and 26% were PAX2 (A)/p53 (A) (p53 signatures). PAX2 (A)/p53 (N) SCOUTs were free of p53 mutations; however, 12 of 13 p53 signatures were PAX2 (A). A tubal carcinoma and contiguous SCOUT were p53 (A)/PAX2 (A) and shared the same p53 mutation. SCOUTs are discretely localized alterations commonly containing altered expression of multiple genes within histologically benign tubal epithelium. Geographic distribution in the tube varies by genotype and immunophenotype, from regionally unrestricted (PAX2) to greater likelihood specific area (fimbria) of shared prevalence (PAX2 and p53). This study reveals, for the first time, an entity (SCOUT) that is associated with serous cancer, expands the topography of altered PAX2 expression in the female genital tract mucosa and highlights another potential pathway disturbance involved in early serous carcinogenesis in the Fallopian tube. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

体外诱导脂肪干细胞向内皮细胞及成血管的分化

背景：脂肪干细胞作为组织工程中种子细胞的选择,可以诱导为内皮细胞从而有效解决材料血管化困难的难题。 目的：体外观察脂肪干细胞经诱导向内皮细胞转分化的可能性、以及在立体培养基上的血管形成情况。 方法：切取人皮下脂肪,用酶消化法分离和培养脂肪干细胞,将传至第3代或第4代的脂肪干细胞用内皮细胞诱导液、同时在Matrigel三维培养基内诱导培养,观察细胞生长情况及变化。对脂肪干细胞和诱导细胞行免疫组织化学检查CD31的表达。 结果与结论：免疫组织化学检测脂肪干细胞的CD31表达阴性,诱导细胞CD31可见阳性表达。在三维立体培养基内诱导的细胞24 h逐渐迁徙成团,伸出伪足,诱导1周细胞形成网格样交叉,2周形成较长血管,后血管增粗,并出现分叉,CD31阳性表达。因此,脂肪干细胞体外可以被诱导向内皮细胞表型转分化,并形成血管,提示脂肪干细胞可以作为促进组织工程移植物血管化的种子细胞的理想选择。     

Placental protein 14 production by human Fallopian tube epithelial cells in vitro

We studied the in-vitro secretory function of non-polarized and polarized cultured Fallopian tube epithelial cells by measurement of the placental protein 14 (PP14) secretion in primary cultures and subcultures from Fallopian tubes obtained from eight premenopausal women in different phases of the ovarian cycle. Primary cultures were established in minimal essential medium in Earle's salts supplemented with fetal bovine serum and the cells were subcultured for six passages, in the polarized cell cultures, the cells being seeded on an extracellular matrix system. Cell freezing was carried out using 10% dimethyl sulphoxide. PP14 secretion into the culture media was measured by a radioimmunoassay using 125I-PP14 as label and rabbit anti-human PP14 serum. There was a large amount of PP14 secretion into the culture media in primary cultures, the secretion decreasing considerably after subculture 1. PP14 secretion after subculture 2 was not different from the control values. Polarized and non-polarized cells secreted similar amounts of PP14 and frozen-thawed cells did not appear to secrete PP14. Epithelial cells from Fallopian tubes obtained at different phases of the ovarian cycle did not appear to show any difference in PP14 secretion rates. Our data suggest that the in-vitro secretion of PP14 by human Fallopian tube epithelial cells is adversely affected by cell ageing and freezing.      

体外诱导骨髓间充质干细胞向软骨细胞的定向分化及其鉴定

目的: 体外诱导骨髓间充质干细胞(MSC)向软骨细胞定向分化, 并对分化后的细胞进行鉴定。方法: 由健康成人骨髓中分离出MSC, 取第 3代细胞进行实验。鉴定后, 将微小细胞团在TGF -β1、地塞米松(Dex)及维生素C(VitC)等诱导下分化。14d后, 细胞团经石蜡包埋、切片及HE染色后, 进行甲苯胺蓝染色及II型胶原(ColII)的免疫组化染色。采用Westernblot和RT- PCR, 分别检测诱导前后MSC中ColII及前ColIImRNA的表达。结果: HE染色显示, 诱导后细胞呈软骨细胞样形态; 甲苯胺蓝及ColII染色的细胞外基质呈阳性。Westernblot和RT PCR的结果显示, 诱导分化后的MSC可表达ColII和前ColII的mRNA。结论: MSC在TGF- β1、Dex及VitC等诱导后, 可分化为软骨细胞。     

星形胶质细胞瘤干细胞的体外分离、培养与鉴定

背景：肿瘤干细胞理论认为肿瘤中存在一小部分具有无限增殖潜能和自我更新能力,能够分化为成熟细胞表型的干细胞样细胞,对肿瘤发生、增殖、侵袭起关键作用。 目的：建立体外分离、培养与鉴定星形胶质细胞瘤干细胞的方法。 方法：采用直接培养法分离培养星形胶质细胞瘤干细胞。参照神经干细胞培养条件,进行体外培养。观察其增殖、分化并进行巢蛋白、CD133免疫细胞化学鉴定和诱导分化后神经元特异性烯醇化酶、胶质纤维酸性蛋白及O4免疫细胞化学鉴定。 结果与结论：培养7-10 d,可形成大量悬浮生长巢蛋白及CD133免疫阳性的神经球,经诱导分化后细胞呈神经元特异性烯醇化酶、胶质纤维酸性蛋白或O4免疫阳性。提示星形胶质细胞瘤中存在具有神经干细胞特性的肿瘤干细胞。CD133和巢蛋白是星形胶质细胞瘤干细胞重要的表面标记,可以用于星形胶质细胞瘤干细胞的分离。     

Prostaglandin E2 and F2alpha receptors in the human Fallopian tube before and after mifepristone treatment

Prostaglandins are associated with several reproductive processesin addition to their effects on the vascular system and muscularcontractility. The aim of this study was to gain informationabout the localization of the receptors for PGE₂ (EP1–EP4)and PGF₂ (FP) in the human Fallopian tube and their regulationfollowing treatment with mifepristone. Sixteen healthy fertilewomen received a single dose of 200 mg mifepristone or placeboimmediately after ovulation (LH+2). Laparoscopic sterilizationwas performed on days LH+4 to LH+6. Biopsies were taken fromthe Fallopian tubes bilaterally. The expression of EP1, EP2,EP3, EP4 and FP was analysed using immunohistochemistry andRT–PCR. The co-localization of prostaglandin receptorsand c-kit or e-nos was analysed using confocal microscopy. Theeffect of progesterone, mifepristone and prostaglandin on tubalcontractility was studied. The presence of EP1–EP4 andFP in the Fallopian tube was detected using immunostaining.The receptors were expressed in serosal cells, luminal epithelialcells, and the muscular wall and vessels of the Fallopian tube.Co-localization studies showed that the endothelial cells stainedpositive for EP1–EP4 and FP and that co-localization wasseen for EP4 and c-kit. Decreased contractility was seen afterprogesterone treatment, whereas increased contractility wasseen after PGF₂ and PGE₂ treatment. These data suggest thatboth the transport of the embryo and the communication betweenthe embryo and the Fallopian tube involve the action of prostaglandinsthrough EP and FP receptors in addition to the effect of prostaglandinson the vascular system and muscular contractility.     

用组织块法培养成年和老年鼠神经干细胞

目的　探索体外培养扩增成年及老年动物室管膜下区 (SVZ)神经干细胞的实用方法。方法　取 8、14及 2 4三个月龄SD大鼠SVZ组织块置含bFGF的DMEM/F12 +B2 7培养液培养 ,Nestin免疫组化法鉴定细胞表型。结果　各月龄的SVZ组织块均能长出Nestin阳性的神经小球及神经干细胞 ,培养 7～ 10天产生的神经球最多 ,神经干细胞状态最佳。结论　用含bFGF的培养基培养成年和老年大鼠SVZ组织块能产生较多的神经干细胞 ;此法是一种具有实用价值的培养、扩增神经干细胞的手段。     

改良组织块培养法体外培养人角膜上皮干细胞

文题释义：角膜上皮干细胞：属于单能干细胞,具有细胞周期长、低分化状态、增殖潜力大、不对称分裂等特点,定位于角膜缘基底细胞层,又称之为角膜缘干细胞,对角膜上皮细胞更新及维持角膜透明起着重要作用。角膜缘干细胞的体外培养方法：主要包括酶消化培养法和组织块培养法。酶消化培养法是利用DispaseⅡ酶破坏角膜缘上皮细胞与基底膜之间的半桥粒连接,然后剥取角膜缘上皮层,再使用胰酶将其消化为单个细胞进行培养。组织块培养法没有经过酶的双重消化,将剖取的角膜缘组织块进行贴壁,细胞游离出组织块进行贴壁生长,需要一个漫长的过程。  摘要背景：角膜上皮干细胞定位于角膜缘,又称之为角膜缘干细胞,临床上由于眼表严重热烧伤、化学性烧伤、慢性炎症等原因引起的角膜缘干细胞缺乏或功能障碍治疗较为棘手。目前利用组织工程技术体外培养角膜上皮干细胞并进行临床移植成为新型有效的治疗方向。目的：探讨在无血清培养条件下采用改良组织块培养法培养人角膜上皮干细胞的可行性。方法：人角膜缘组织来自河南省眼库,植片直径小于8 mm角膜移植术后的供者剩余眼球材料,手术显微镜下剖取角膜缘上皮层外2/3区域,采用2种方法培养人角膜上皮干细胞,常规组织块培养组是将组织块上皮面向上贴壁,加入K-SFM培养液后置于 37 ℃、体积分数为5%CO₂细胞培养箱中培养;改良组织块培养组是先将组织块浸泡于K-SFM培养液中,置于细胞培养箱中孵育12 h,然后组织块上皮面向下贴壁培养。组织块周边有细胞游离出贴壁生长记作“培养第1天”,每日相差显微镜下观察细胞生长变化。利用免疫荧光染色技术检测改良组织块培养第5,10,14天时原代细胞中p63及K3的表达。结果与结论：①改良组织块培养组出膜时间明显短于常规组织块培养组(P < 0.05),出膜率明显高于常规组织块培养组(P < 0.05);②改良组织块培养组细胞生长状态良好,培养第10天可见小体积细胞较多,聚集成灶状分布;培养第14天可见细胞克隆灶,克隆灶内细胞体积较小,形态均一;③培养第5天,K3表达量较多,p63表达量较少;培养第10天,K3和p63表达量均增多;培养第14天,K3表达量未见明显增多,p63表达量明显增多;④在无血清培养条件下,改良组织块培养法能显著促进角膜上皮干细胞的游离,提高体外培养细胞数量,为人角膜缘上皮组织片的构建提供种子细胞。ORCID: 0000-0001-8370-174X(许中中) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程